Calbindin D-28k and parvalbumin in the rat nervous system

0306-4522/90$3.00+ 0.00 Pergamon Press plc 0 1990IBRO

,VeuroscienceVol. 35, No. 2, pp. 375..475,1990 Printed in Great Britain

CALBINDIN

D-28k AND PARVALBUMIN NERVOUS SYSTEM* M. R.

IN THE RAT

CELIOt

Institute of Anatomy, University of Kiel, Olshausenstrasse 40, D-2300 Kiel, F.R.G. Abstract-This paper describes the distribution of structures stained with mono- and polyclonal antibodies to the calcium-binding proteins calbindin D-28k and parvalbumin in the nervous system of adult rats. As a general characterization it can be stated that calbindin antibodies mainly label cells with thin, unmyelinated axons projecting in a diffuse manner. On the other hand, parvalbumin mostly occurs in cells with thick. mvelinated axons and restricted. focused nroiection fields. The distinctive staining with antibodies against these two proteins can be ‘observed throughout the nervous system. Calbindin D-28k is primarily associated with long-axon neurons (Go&$ type I cells) exemplified by thalamic projection neurons, strionigral neurons, nucleus basalis Meynert neurons, cerebellar Purkinje cells, large spinal-, retinal-, cochlear- and vestibular ganglion cells. Calbindin D-28k occurs in all major pathways of the limbic system with the exception of the fornix. Calbindin D-28k is, however, also found in some short-axon cells (Golgi type II), represented by spinal cord interneurons in layer II and interneurons of the cerebral cortex. It is also detectable in some ependymal cells and abundantly occurs in vegatative centres of the hypothalamus. The “paracrine core” of the nervous system and its adjunct (1985, Nieuwenhuys, Chemoarchifecrure o.the Brain. Springer, Berlin) is very rich in calbindin D-28k. The dist~bution of calbindin D-28k-positive neurons is very similar to that of the dihydro~rydine subtype of calcium channels. Most of the cells containing calbindin D-28k are vulnerable to neurodegenerative processes. Pa~albumin-immunoreactive neurons have a different, and mostly complement~y dist~bution compared with those which react with calbindin D-28k antisera, but in a few cases (Purkinje cells of the cerebellum, spinal ganglion neurons), both calcium-binding proteins co-exist in the same neuron. Many pa~albu~n-immunoreactive cells in the central nervous system are intemeurons (Golgi type II) and, to a lesser extent, long-axon cells (Golgi type I), whereas conditions are vice versa in the peripheral nervous system. Intrinsic parvalbuminic neurons are prominent in the cerebral cortex, hippocampus, cerebellar cortex and spinal cord. Long-axon ~~albumin-immuno~active neurons are, for example, the Purkinje cells, neurons of the thalamic reticular nucleus, globus pallidus, substantia nigra (pars reticulata) and a subpopulation among large spinal-, retinal-, cochlear- and vestibular ganglion cells. Parvalbumin is rich in cranial nerve nuclei related to eye movements. In addition to nervous elements, pa~album~n immunoreactivity occurs in a few ependymal cells and in some pillar cells of the organ of Corti. In contrast to calbindin D-28k, parvalbumin is virtually absent from vegetative centres and pathways. Parvalbumin co-exists with GABA in cortical neurons and is, in general, prefe~ntially associated with “fast-tiring” neurons. Whole functional systems are revealed by either parvalbumin or calbindin D-28k immunohistochemistry. Pa~albumin, for example, occurs in the whole chain of neurons of the epicritic sensibility in the somatosensory system. Calbindin D-28k, on the other hand, occurs in the whole taste pathway. With antibodies against calbindin D-28k and parvalbumin it is possible to reveal at least two as yet undescribed brain nuclei: one in the hypothalamus ~a~albumin I), the second in the medulla (~lbindin D-28k 1). Calbindin D-28k and parvalbumin are evenly distributed within the various domains of the same neuron, but in some instances their levels vary between soma and cell processes. The immunostaining with calbindin D-28k and pa~albumin antisera show gradations in intensity among neurons belonging to different populations, a phenomenon compatible with the presence of varying concentrations of these proteins. Calbindin D-28k and pa~albumin are excellent new neuroanatomi~l markers which can be utilized to selectively visualize certain neurons and pathways in the central nervous system and peripheral nervous system.

Calcium ions were discovered by Ringeti3’ to be a necessary component in the bathing fluid to preserve myocardial contractibility in explanted frog hearts. *Dedicated with love to my wife Monica. tPresent address: Institute of Histology,

University

of

Fribourg. CH-1700 Fribourp. Switzerland. Abbreviah.&:

CaBP, calbind:; D-28k; CAMP, cyclic adenosine-3’,5’-monophosphate; DAB, 3,3’-diaminobenzidine; EGTA, ethyIeneglycolbis(aminoethylether)tetra-acetate; IR, immunoreactive; PAP, peroxidaseantiperoxidase; PBS, phosphate-buffered saline; PV, parvalbumin; SDA, sexually dimorphic area; TBS, Tris-

buffered saline.

Ten years later Locket63 found that neuromuscular transmission in frog skeletal muscles subsided in the absence of extracellular calcium. In the following 60 years a large number of observations on the crucial role of calcium in the most distinct fields of biology accumulated.‘47 A major conceptual advance was the gradual reaiization that Ca*’ elicited it’s effects intracellularly.3Q Of further relevance were the suggestions that Ca2+ couples stimulation and contraction in skeletal

muscles,237 and takes part in stimulation and exocytosis in endocrine glands and nervous tissue.77.2” 375

376

M. R. CELIO

Ca2+ is indispensable for axoplasmic transport”‘~‘*’ and may even be involved in leaming’37 and memory formation.*0~‘67 On the other hand, un~ontroiied elevation of the intracellular Ca2+ concentration leads to excessive cell activation, injury and, ultimately, cell death.39 Based on the observation that Ca*+ selectively triggers a wide variety of biological responses, Rasmussenzz4 advanced the idea that Ca*+ may serve as a universal second messenger, analogous to cyclic adenosine-3’S’-monophosphate (CAMP). Although Rasmussen’s224 article fails to explain how the wide variety of Ca*+ effects can be performed by one and the same messenger, the concept of “transmembrane information transfer” by means of Ca’+ is an established hypothesis in modern biology. The discovery of a specific, high-a~nity intracellular acceptor protein, troponin-C,78,79 which binds Ca2+ to induce skeletal muscle contraction, initiated a new epoch in the field of catcium research. But for a period of time, the interaction of Ca*+ with an intracellular calcium-binding protein was regarded as exclusive for skeletal muscles. With the isolation of a widely occurring calciumbinding protein, ~almodulin,% the general importance of calcium-binding proteins became evident. Caimodulin has been found to bind Ca*+ and to transduce--through graded conformational changes-the signal carried by this ion in at least 15 different biologically selective effects,“’ leading to its designation as a “trigger protein”.66 How calmodulin alone may give rise to all these effects is still not completefy understood. The problem of the specificity of the Ca2+-signal has been moved to another level, but in principle, it remains unresolved. Perhaps the differential intracellular distribution or the combinations and ~~utations of many different calcium-binding proteins in a given cell and their subtle competition for CaZ+ help to establish an ordered sequence of reactions. In addition to calmodulin and troponin-C, some other proteins are known to bind Ca*+ with highaffinity; these are the vitamin D-dependent calciumbinding proteinz7’ (now called calbindin D-28k, CaBP), S-1003* and parvalbumin (PV).“6,“7,209New calcium-binding proteins are being discovered in rapid suceession,‘77~199~202~2”~276 and most of them belong to a single family of proteins, which have evolved from a common precursor.‘47 The function of these calcium-binding proteins is less versatile than that of calmodulin; PV and CaBP either act in calcium transport or as intracellular calcium butTers,14’ so called “transport/buffer” proteins.& PV in fast muscle Gbres is thought to transiently bind Ca*+ and to shuttle them back to the sarcoplasmic reticulum, thus increasing the speed of muscle reiaxation.43.96*209 On the same line of thought, CaBP may be involved in the transI~ation of Ca2 + through the intestinal mucosa.“4,277 Up to now all calcium-binding proteins, except

troponin-C’3~233 have been isolated from the brain of various species. While S-100 occurs in astrocytes,sg~‘” calmodulin 27~159~247uZVBl~~42.47 and ~a~p’6,‘7,‘8,19”.W.9’~~30,2W66

are

ali

present

in

neurons,

Calmodulin is ubiquitous”’ and occurs in all neurons (see, however, Refs 159,247), while CaBP and PV only occur in certain subsets of neurons. Information published on the anatomical distribution of the calcium-binding proteins are still fragmentary and sometimes controversial. Therefore, the aim of the present investigation is to analyse in detail the distribution of CaBP and PV immunoreactivities in cell bodies, processes and pathways in the adult rat nervous system by sensitive and specific immunohistological methods. This morphological study is a necessary step to allow future physiological, biochemical and pha~aco~o~~l studies of calciumbinding proteins in the brain. It was undertaken in order to gain insight into the role of these two calcium-binding proteins in the nervous system and to exploit the antibodies against ~lcium-binding proteins as new neuroanatomical markers. This report is complete but not exhaustive and shall help to raise interest in these two proteins. The results are presented according to to~~aphical aspects, while the discussion is organized according to functional entities. EXFRRIM~TAL

PROCEDURRS

The brain of 67 adult rats (42 Wistar, 20 ZUR-Siv, four Sprague-Dawley, one Long-Evans) of both sexes (37 males and 30 females), the spinal cord and the spinal ganglia and various peripheral nerves of five more animals, the eyes of four animals and the inner ear of three other creatures were used for this study. In addition we studied 10 incomplete series of rat brains-and other tissues as well as single sections of the brain of other rats. For Fig. 12 we employed a CB 57 mouse, processed by perfusion fixation with‘lO% formalin “embedded” in 50% bovine serum albumin. For Figs 7 1, 72 and 73, a seven-day-old rat was used, processed by perfusion with Bouin fluid. In addition we studied sections of the neurological mice mutants “staggerer”24* and “quaking”X8 (Jackson Laboratories, U.S.A.). All animals were kept under a constant dark-Tight schedule (7 h light on, 19 h light off) with food pellets (NAFAG, St Gallen, Switzerland) and tap water provided ad libitunz. They weighed between 250 and 350 g at the time of the tissue collection, which took place at different times of the day.

Fifty microlitres of colchicine solution in phosphatebuffered saline (PBS) (1 mg/ml) were injected intraventricularly to nine rats (Sprague-Dawley and Wistar). After two days the paraplegic animals were perfused and processed by perfusion with 4% (w/v) pa~fo~~dehyde in 0.1. M phosphate buffer, pH 7.4. Tissue processing

Unless stated otherwise, the animals were perfused through the ascending aorta with c. 300 ml of fixative. The perfusion was followed by excision of the tissue and 2 h nostfixation. The followina fixatives were used. (1) Perfusion with Bouin fluid [saturated aqueous picric acid; forrnalin 40% (Merck); concentrated acetic acid, 15: 5:2 by volume]. The tissue was dehyd~ted and embedded in paraffin foliowing routine methods. For the inner ear, the whole petrosal bone was decalcified with 5% formic acid in water for two

Calcium-binding proteins in the rat brain days previous to embedding. (2) Perfusion fixation with 10% formalin (made from 40%. Merck) in 0.1 M cacodylate buffer pH 7.4. The tissue was either: (a)“‘embedded” in -50% bovine serum albumin for Vibratome sectioning; or (b) soaked in 18% sucrose (w/v) for 24 h at 4°C and frozen on pulverized dry ice for cryostat sections. (3) Perfusion with 4% (w/v) paraformaldehyde (Merck or Aldrich) in 0.1 M phosphate-buffer, pH 7.4. The tissue was processed as in 12). (41 Perfusion with 2.5% alutaraldehyde (v/v) (25% amp&l&, Polysciences), 2.5%- (w/v) paraformaldehyde (Aldrich) in 0.1 M cacodylate buffer pH 7.4 (+ 50 mM CaCl,). For the detection of PV and GABA on consecutive sections, 1 g/l Na-metabisuhite was added to the lixative which was prepared at pH 7.8. Alternatively, blocks were embedded in ~Araldite. and sections depolymerixed with Na-alcoholate.‘76 (5) Banid free&a of 4-5-mm-thick dimes of unfixed tissue in isopentane, co&d by liquid nitrogen, freeze substitution with acetone at -70°C for one week; freeze fixation at the same temperature with a mixture containing acetone (90 ml), formalin 40% (4 ml), concentrated acid (1 ml), distilled water (5 ml) for one week; embedding in paraplast. The thicker (50 urn) Vibratome or cryostat sections l(2) and (3) above] were used for the survey and counts of the relative and absolute cell body density and fibre systems and for electron microscopic pi-e-embedding staining, while the 6-pm-thin paraliln sections [( 1) above] provided finer details of perikaryal and dendritic morphology. Method (5) above was only included to provide evidence that no artificial redistribution of CaBP and PV took place before embedding and immunostaining (see Ref. 268, on this subject). It was, however, not used as a routine because of the laborious procedure.. The spinal cord was at best immunostained after processing with methods (I), (2b) and (3) above, whereas the eye and particularly the inner ear could only be studied after using method (I), because of obvious technical constraints. Method (4) was used to test the resistance of the antigens studied to the effects of strong fixation and also, slightly modified, to permit the visualization of GABA and PV on consecutive sections. The pa&in (5-pm-thick) and cryostat (16~pm-thick) sections were collected on chrome-alum gelatine-coated slides, dewaxed with xylol and mhydrated (for paraffin processed for immunohistochemistry and sections), mounted in Et&i@. The Vibratome (50 pm) sections were incubated floating in diluted antiserum solution on a shaker, collected on coated slides and mounted in Eukitt’a after short dehydration in ethanol and xylol. In three cases the Vibratome sections were mounted undehydrated with glycerine gelatine (1: 1). Four rat brains (all males) were processed by method (1) (Bouin/paratBn) and cut serially in coronal (twice), longitudinal or horizontal sections, respectively. Every 100 sections, three consecutive sections were collected on three different slides. All three were immunostained; two were counterstained for cells with Cresyl Violet (1% in acetate buffer pH 3.4 for 20 min at 4OC), respectively for fibres with Luxol Fast Blue (12 h at 60°C). .I

_

Antibodies Three different polyclonal antibodies against rat muscle PV (identical to brain PV25) were used first. They satisfy various criteria of specificity as demonstrated by Ouchterlony immunodiffusion and preadsorption experimentsz6”*“’ and immunoblotting. ‘w’~ In addition we used three well characterized monoclonal antibodies against carp PV (235, 239, 267) reacting with mammalian PV.x’ The polyclonal antibodies against chicken intestinal CaBP have been characterized by radioimmunoassays7 and have been used by various groups for the localization of the antigen in various tiss~es.~*~~~~~~~*‘~ Later we used two well characterized monoclonal antibodies against chicken gut CaBP (nos 300, 316) reacting

311

with mammalian calbindin.sl Their specificity has been tested in immunoblots. No cross-reaction between PV and CaBP antibodies is evident according to cross-adsorption tests They also do not recognize calmodulin, oncomodulin’73 and S-100. The antiserum agaist GABA (coupled to albumin) was purchased from Immunonuclear Corporation, Stillwater, MN, U.S.A. and the monoclonal antibody against GABA was kindly provided by Dr Streit, Ziirich.‘75 The specificity of the antiserum was independently proven by various tests!’ PrearLrorption tests The CaBP and PV antibodies used in this study have been preadsorbcd with their respective antigens: high performance liquid chromatography, purified rat muscle PV and chicken intestinal CaBP (I-10pm). The adsorption was carried out in the following manner: the given amount of antigen was mixed with 100 ~1 of antibodies diluted 1: 100 to 1:50,000. Tubes containing exactly the same dilutions of antibodies but without any addition of antigen were incubated in parallel and served as controls. The adsorbed and unabsorbed antibodies were incubated with the paraffin sections for 48 h at 4°C and further processed with the standard peroxidase-antiperoxidase (PAP)-technique (see above). Immunohistochemistry A synthesis of the method used is given in a separate publication.“* The sections on the slides (respectively floating) were incubated for 48-72 h at 4°C in a moist chamber with the primary antibodies diluted 1: 1000 to 1: 50,000 in Ca2+- and M$+-free Tris-buffered saline (TBS). The best signalto-noise ratio was at a dilution of I:2000 to 1: 10,000 for PV and 1: 5000 to 1: 20,000 for the CaBP antibodies. For the detection of PV and GABA on consecutive sections, the antibodies were diluted in TBS with 1 mg/ml Nametabisulfite and 38Omg/l Na-borohydride. After 3 x 5 min, TBS rinsing (3 x 15 min for the Vibratome sections) the sections were incubated with goat-anti-rabbit IgG (Miles), 1: 200 for 30 min (1: 200 for 2 b) at room temperature. After a further wash with TBS (3 x 5 min and 3 x 15 min. resoectively) the sections were incubated with rabbit PAP’complex (Stemberger-Meyer Inc., Jarretsville Pike, MD, U.S.A.) 1: 500 for 30 min (1: 500 for 4 h) at room temperature. The locations of the antibody-bound peroxidase were then visualized by incubation with the substrate 3,3’-diaminobenzidine (DAB)-HCl-hydrogen peroxide under visual control. The monoclonal antibodies were localized by an indirect procedure which involved the use of an affinity purified goat-anti-mouse IgG coupled to peroxidase (Miles, 1: 500), or by using the avidin-biotin method (Vector Laboratories, U.S.A.). Some sections, particularly those treated according to methods (2a) and (2b) above, were osmicated with 0.01% osmium tetroxide (0~0,) after completion of the immunostaining. Quant$caiion of immunoreactive neurons The size of the immunostained perikarya in selected areas was determined by measuring the major and minor diameter in the mid nucleolus plane by means of a calibrated eyepiece grid (see Table 2). Bouin’s flxation followed by paratlin embedding is known to produce a 20% shrinkage of the tissue.206With the exception of the small cells in the external plexiform layer of the olfactory bulb and of spinal ganglion cells, cell measurements have therefore been performed on Vibratome sections [method (2a) above] mounted in glycerine gelatine. The total number of PV-immunoreactive (-IR) cells in the fascia dentata of two animals were counted in complete series of Vibratome and paraflin sections, those in the caudatoputamen by counting PV+ neurons in every second Vibratome section of a whole sagittal series.

M. R.. CELIO

378 Camera lucida drawings

Drawings were performed using a drawing tubus and a x 40 plan or a x 100 planapo (oil) Zeiss objective. The size of the cells under study was determined using a calibrated eye-piece grid. The r~onst~~tion could only be carried out on Vibratome sections (50 ,um) which permit visualization of the dendritic network; all sections of this series were dehydrated and embedded in Eukitt’s. RESULTS*

General

remarks

This paper deals with the distribution of CaBP and PV in the normal adult rat central nervous system, the specialized primary sensory end organs and the peripheral nervous system.

*Terminology and summary of the most important physiological and biochemical data regarding the ions and the proteins discussed in this paper

PV immunoreactive, PV-IR, PV-positive structure, PV+, parvalbuminic are used as synonymes, as are calbindin-positive CaBP+, CaBP-IR, CaBP-immunoreactive. Extracellular Ca*+ and Mg’+ in the central nervous

svstem value to 1-2 mM.‘95 ’ Ca*+ and Mg2+ : free in~a~llular

calcium and magnesium, as measured with ion selective microelectrodes are 1.7 x lo-‘M, 6.6 x 10w4M in invertebrate giant neurons, respectively. r3 Values of free intracellular Ca2+ for vertebrate neurons are of the order of lo-’ M.‘** Rat PV (molecular weight of 12,000, isoelectric point 4.9): this designation reflects the small size, the high solubilitv in water and the high electrophoretic mobility of this protein (high diffusion constant and low vik cositvj.“3J’6 Besides Ca*+. PV also binds M~z*+.~’At physiolo~~al levels of Mgi+ (1 mM) and K+ @X0 mM), and at levels of Ca2+ corresponding to those of resting cells (approximately lo-’ M;see below), 1 mol PV binds 2 mol of Me2+ and none of Ca*+. A rise in intracellular calcium level causes calcium binding, which is accompanied by a release of Mr$+.‘r’ PV has an af%nity for ?a*+ of the order of lO’M&d for Mg*+ of about l@ M. PV miaht reeulate the Ca*+ (and Ma*+ ) fluxes in the cell (“bu~er/t&sport”) and participa; in Ca*+ and/or Mg2+ activated processes.66 The synthesis of PV in muscles is neurally regu---.I

lat&153J89

CaBP, the new name for the vitamin D-dependent calcium-binding protein,2’7~278~279~2a’ (molecular weight 28,000; isoelectric point 4.8): CaBP seems to influence the efficiency of the Ca*+ transport mechanism in the chicken intestine. It’s synthesis in the chicken gut, kidney and peripheral nervous system’” is dependent on the presence of vitamin D.‘97~2”~280 In the brain, however: such a vitamin D-dependence could not be found.‘S~242**67 CaBP binds four Ca*+ with high affinity f& = 2 x lo6 M), not to be confused with the 9000 mol. wt CaBP, isolated from rat intestine,279 which is not detectable in nervous tissue (Ref. 269 and own unpublished observation).

In general terms, no obvious differences can be found between the pattern of immunostaining due to various procedures of tissue fixation and embedding. In accordance with the observations of others2@ with CaBP, no immunoreaction can be carried out with unfixed tissue sections. We also suppose that PV, as soluble protein not anchored to any subcellular organelle (at least in muscles99) can be easily displaced and redistributed. In the following description, dot-like structures will be referred to as nerve terminals (boutons terminaux) and the smooth, immunoreactive fibres as axons. Sometimes, especially in the thalamus and reticular formation, the distinction between axons and nerve terminals is difficult at light microscopic level and our interpretation has to be assumed as only tentative. In some cases the intra~llular dist~bution of CaBP and PV varies reproducibly between the various parts of a given neuron. The soma in general contains the highest density of CaBP- and PV-IR sites as seen in the antibody dilution tests and as inferred from the resistance of its staining towards the action of strong fixatives. There are instances, however, where PV occurs only in the axon and terminals and CaBP only in the perikaryon. Examples for the first case are the neurons of the deep cerebellar nuclei, for the second pyramidal cells of the hippocampal CAl-region. In various regions of the brain, the CaBP antibodies produce an homogeneous and diffuse reaction as if all neural elements present in the tissue section, neurons and all their processes, had been stained. Colchicine application does not change the staining pattern of cell bodies reacting with PV antibodies in cerebral cortex, hippocampus, thalamus and cerebellar cortex. However, interference with axoplasmic transport produces an accumulation of PV in certain long-axon neurons. Examples include the deep cerebellar nuclei, the vestibular nuclei, the retinal ganglion cells. This aspect of the distribution of PV is described in a separate publication’ but the most important exceptions are included in Table 6. Control sections of brains incubated in preimmune serum or antigen (1O-9 M) adsorbed antiserum do not exhibit specific immunostaining. Oniy unequivocally stained structures are included in the description and discussion; certain obviously negative, but important exceptions will be pointed out from time to time. The description of the results for CaBP in regions (e.g. cerebellum, hippocampus) already well described by other groups’8X90*95 will be kept at a minimum.

Figs l-76. Many of the following figures have been obtained with the technique of “histography”. By this method the sections on the slide are directly projected on photographic paper, which is subsequently developed and fixed. The image, therefore, is a negative of the original and the immunos~~n~ structures (brown in the original) appear white. This old technique produces sharper images compared with methods which make use of an internegative. For abbreviations see Table 6.

C~cium-binding

proteins in the rat brain

Fig. 1. (A) Coronal section of the olfactory bulb at the level of Fig. 83 incubated with calbindin antiserum. A prominent terminal field is seen in the central half of the accessory olfactory bulb (AOB). Immunoreactive perikarya are very numerous in the glomerular layer (IGr), but also occur in all other layers of the main olfactory bulb. Notice the stripe of thin terminals in the inner half of the external plexiform layer (EPL). No axons course in the lateral olfactory tract (lo). The labelling with the monoclonal antibody against CaBP differs in not showing the terminal field in the AOB, which, therefore, probably derives from the cross-reaction of the antiserum with calretinin. x 50. (B) Consecutive section to (A) incubated with PV antibodies. Terminal fields are virtually absent. Interneurons occupy the external plexiform layer (EPL) and some are scattered in the inner granular layer (I&). x 50. Fig. 2. (A) Photograph of a portion of a coronal section of the olfactory bulb labelled with a CaBP antiserum. A band of strong immunoreactive cell bodies with short processes, representing periglomerular cells, is seen in the upper third of the figure (GL). Scattered neurons are found in the external plexiform layer (EPL) at the boundary between mitral (M) and internal plexiform layer (IPL) and in the granular layer (bottom of the figure). Some of the positive neurons at the boundary between mitral and internal plexiform layer are drawn with the camera lucida Fig. 77A. Notice bundles of thin axons (arrowheads) in the olfactory nerve layer (ON). One of this bundle impinges upon a ~omer~um (star). x 120. (B) Section adjacent to that of Fig. lA, but incubated with a PV antibody. Neurons predominantly in the external plexiform layer (EPL) are tagged. These cells have slender cell processes radiating in all directions, but respecting the laminar borders. Some of these cells are drawn in Fig. 778. Few periglomerular neurons are tagged in the glomerular layer (arrows). Notice axons in the granular layer (arrowhead). Abbreviations as in Fig. 1A. x 120.

380

M. R.

Telencephalon Olfactory bulb (Figs 82 and 83) C~~b~d~n D-28k. A lot of cells in the ~omeru1~

layer are intensely stained as are scattered neurons at the boundary between the mitral and inner plexiform layer (Figs 1A, 77A). The neurons in the periglomerular region have one, or seldom, two cell processes entering one glomerulus (see Table 2 for sizes). An exact arborization pattern of the dendrites of single cells is difficult to discern because of intermingling cell processes. The inner half of the external plexiform layer shows an homogeneous band-like staining (Fig. lA, EPL) but terminals cannot be individually discerned. The neurons in the inner plexiform layer are of two types, as described in the legend to Fig. 77A. At higher antiserum concentration CaBP+ neurons also appear in the external plexiform layer and in the internal granular layer (Figs 1A and 2A). Giant, faintly CaBPf neurons with coronally oriented cell processes are frequently seen around the olfactory ventricle. With polyclonal antibodies, cross-reacting with calretinin on immunoblots, CaBP immunoreactivity also occurs in the axons of the Jacobson’s nerve innervating the vomeronasal organ. The CaBP+ terminal fields in the accessory bulb (Fig. 1A) form a sharply demarcated sphere of interlacing axons and te~inals. No CaBP+ neurons are found. Fascicles of extremely thin, probably unmyelinated axons impinge upon some glomerula (five to six in a 50+mthick section) of the main olfactory bulb (Fig. 2A). My observations with mon~lon~ ~ti~dies are in complete agreement with those of some authors,‘* but differ from those of others,95 who probably used CaBP antibodies cross-reacting with calretinin. Some glomerula probably receive calretinin-positive axons. Perhaps these fibres carry odour-rn~~ity specific info~atio~. Therefore, our observation gives further support to the spatial pattern hypothesis of olfactory processing.* The cells stained in the periglomerular layer preponderantly are periglomerular cellist’ because of their size (Table 2) and their typical dendritic r~ifi~tion pattern. Some larger cell bodies (15 x 10pm) may belong to external tufted cells. Neurons 1, 3, 4 at the boundary between mitral cell layer and inner plexiform layer (Fig. 77A may all represent horizontal cells; Fig. 2, cells 15 and 16)24’whereas cell 2 in the camera lucida drawing in Fig. 77A resembles more the Cajal cells (Fig. 2, cells 9, 10 of the aforementioned paper). Cell 4 actually resembles the cell depicted on the right of the photomontage of Fig. 4 in Ref. 241; this cell is called “superficial short-axon cell”, but according to these authors only occurs in the externat plexiform layer. It is not

CELIO

possible to depict neurons of deep layers with the drawing tubus but I have the impression that representatives of all interneuronszi6 are immunolabelled. The general staining pattern of CaBP in the olfactory bulb resembles that seen with ~et~nk~halin antisera.‘% Parualbumin. In the rostra1 part (Figs 1B and 2B) small (c. 12pm) PV-IR neurons are limited to the external plexiform layer. The soma are preferentially located at the inner and outer periphery of the external plexiform layer (railroad track) and the cell processes are mostly directed towards the centre of the external plexiform layer. The immunoreactive cell processes arborize in close proximity to the cell body and are typically fairly contorted (Figs 2B and 77B). Scattered PV-IR cell bodies can also be found in the mitral cell layer and in the inner plexiform layer, but their processes ramify in the external plexiform layer. Single, very smalf immunoreactive cells (approx. 10 pm) are situated parallel to the tractus olfactorius lateralis, but only in its rostra1 part. In addition, ~~glomer~ar cells in the glomerular layer and a few elongated neurons, squeezed against the convex medial surface of the accessory olfactory bulb, are stained with the PV antibodies (Fig. 2B). PV + axons, running horizontally in coronal sections, can be seen in the internal granular layer and, seldomly, in the external plexiform layer. No PV+ axons can he discerned in the lateral olfactory tract (Figs 1B, 3B). The neurons in the external pIexiform Iayer, immunostained with PV antibodies, belong to various classes, Cell 3 in Fig. 77B presumably represents a Van Gehuchten cell (Fig. 3, cells 17 and 18).*4’ Cells 1, 2, 447 represent superficial short-axon cells (Fig. 3, cells 20-23).*“’ At this point I have to correct a statement made in our previous communication,42 namely that al1 GABAergi@ periglomerular cells are PV-IR. As born out later, I misinterpreted the layering of the olfactory bulb and located PV+ cells in the periglomerular region instead of the external piexiform layer. No~iths~nding, a subpopulation of ~~glomeNJar c& indeed display PV. Hama’s group’45 also detected PV immunoreactivity in the external plexiform layer. In the literature I found no remarks on the electrophysiology or chemistry of intemeurons of the external plexiform layer. Anterior olfactory nucleus (Figs 84 and 85) Ca~bindin D-28k. CaBP+

subdivisions

of the anterior Parvalbumin. Large cells antibodies in the polymorph of the ventral, lateral and

neurons are found in all olfactory nucleus. are stained by the PV layer of the rostra1 part dorsal divisions of the

Fig. 3. Series of eight consecutive coronal sections through the basal ganglia and the amygdala from rostra1 to caudal, alternatively incubated with CaBP (A, C, E, G) and PV antibodies (B, D, F, H). The section plane corresponds to: (A, B) Fig. 86, (C, D) Fig. 88, (E, F) Fig. 89 (G, H) Fig. 90. Notice the striosomes in the striatum (arrows in A) and the continuity between caudatoputamen and olfactory tubezcle (cell bridges, CB) in the CaBP-incubated sections. CaBP+ strands are inserted between unlabelled portions of the olfactory tubercle. The amygdala is parcellated and is poor in CaBP in the basolateral (BL) and intercalated cell mass (I), whereas these zones are rich in PV (E, F). PV-IR axons are prominent in the optic nerve (F, H). Notice drop-like unla~lled regions in the lateral nucleus (La) of the amygdaia (G; island of Calleja?). An, as yet undescribed, PVt neuronal aggregation media1 to the optic tract (opt) and embedded in axons of the median forebrain bundle (ml%) is encircled (PRVI) (H), cf. Fig. 318. x 20.

Calcium-binding

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anterior olfactory nucleus, but not in the medial division (Fig. 84). At more caudal levels however, the medial anterior olfactory nucleus contains parvalbuminic cells. Preco~i~~urai hippocampus (tenia recta] indusium griseum (Figs 85 and 86) Calbindin D-28k. A subpopulation of neurons of the indusium griseum and tenia tecta is slightly

stained with calbindin antibodies. The neurons have beaded dendrites, spanning the whole width to the medial surface of the brain (Fig. 29A). Parua[bum~~. Interneurons displaying PV immunoreactivity are numerous in the indusium griseum (both tenia tecta, and tenia tecta, d~visions~~). Their axons impinge upon the soma of pyramidal cells and give an appearance similar to that of the hippocampus proper to this layer (Fig. 29B). Endopirifurm nucleus (Figs 86-90) Calbindin D-28k. Only a few, small neurons with short processes can be visualized. Within the endopiriform nucleus CaBP-~mmunostaining shows fluffy and intensely immunostained terminals (Fig. 3G). P~~a~~urn~~. Only a few immuno~active cell bodies and processes occur in the endopiriform nucleus, particularly at caudal levels. The neurons have a slender tree of dendrites running in all directions, but the axons cannot be discerned. The endopiriform nucleus is delimited from the surrounding by its paleness due to the absence of a terminal field (Fig. 3H).

Primary olfactory cortex (piriform cortex)

(Figs 85-90) Ca~b~~d~~ D-28k. A multitude of scattered CaBPi-

perikarya with slender spineless processes are seen in the depth of the polymo~h layer (Figs 3C-G, 4A, 7A). The plexiform layer lacks CaBP+ cell bodies and most, if not all, faint CaBPi neurons are detectable in the pyramidal ceil layer. The neuropil of the poiymorph and of the pyramidal cell layer is occupied by a large amount of extremely thin terminals, which give an homogeneous appearance to this region. Parvalbumin. Positive neurons are numerous, of varying sizes and have divergent, spineless dendritic processes. They are mostly located in the upper

and lower part of the polymorphic layer or at the boundary between polymorphic- and pyramidal cell layer (Figs 3B,D,F,H, 4B, 7Bf. Few, smaller PV-IR cells occur in the plexiform layer and single neurons in the pyramidal layer. PV+ cell processes (axons?) deriving from these cells engulf the perikarya of pyramidal cells. Extensive dendritic arborizations are detectable in the polymorphic cell layer (Fig. 7B). The richness in forms, sizes and locations of CaBP- and PV-IR cells in the piriform cortex is overwhelming. Comparing my slides with the Golgi drawings,‘0s*‘79it becomes apparent that both, CaBP and PV, occur in representatives of ail subsets of smooth dendritic neurons. No particular difference in the morphology between CaBP+ and PV+ neurons can be detected, although there are si~ifi~ut~~ more CaBPf than PV+ neurons. The majority ofimmunoreactive cells are found in the potymorph cell layer and resemble the neurons depicted in Figs 15 and 16 of Ref. 108. inhibitory interneurons with high discharge frequencies in layer III (polymorph layer) of the rabbit piriform cortex (Table 3) have been described,238 but unfortunately the authors do not provide us with the morphology of the impaled cells. Most of these neurons display glutamate decarboxylase immunoreactivity.‘*” ~~ppo~ampus a& dentate gyros (Figs 89-92) Co~~ind~nD-28k. The CaBP antibodies stain with moderate intensity all portions of the granule cells of the dentate gyrus (soma, dendrites, axon, te~inals). Therefore, overall (Fig. 4A), the dentate gyrus and the mossy fibres converging to the CA3 subfield of the hippocampus are selectively visualized. The granular layer of the dentate gyrus is stained stronger than the molecular layer and the mossy fibres (arrows in Fig. 6A,C,E). The labelling of the molecular layer is homogeneous and trilaminar, with the middle portion being more coloured. This middle band of terminals coalesces with a band of terminals in the lacunosum-molecuIare layer of CA3 (Figs 4A, arrows in 6A,C,E). An unreactive zone is observed just subjacent to the granular cell layer. In Wistar rats we only see a infrapyramidal mossy fibre projection (Figs 4A, 6A,C,E), whereas both infra- and suprapyramidal mossy fibre projections are characteristic for Long-Evans rats. Pyramidal neurons located in the inner half of the CA1 and CA2 pyramidal cell layers have their soma and, less intensely, the stem dendrites moderately stained with the CaBP antibodies (Fig. 8A) but pyramidal neurons in the CA3 and CA4 subfieids remain unreactive (Figs 4A, BA,C,E). A band of

Fig. 4. Horizontal section of the whole rat brain at the level of Figs 58-59 of a rat brain atlas.“% Overview of the staining pattern from the olfactory bulb to the cerebellum. (A) incubated with calbindin antibody; (B) with PV antibody. This figure instructively demonstrates the complementary nature of the staining pattern of CaBP and PV in the basal ganglia (CPU and GP), in the thaiamus, in the medial genie&ate body (MC) and in other brain regions. Other areas are rich in both CaBP and PV, e.g. the cortical mantle, hippocampus, cerebellum (Ce) and vestibular area (Ve). The second point of interest of these two images is that both proteins respect quite accurately cytoarchitectonic boundaries and selectively visualize certain brain portions (not always the case for CaBP). Notice the stratification of labelling in the neocortex and hippocampus. The star in the caudatoputamen of A marks the laterodorsal zone of lower CaBP immunoreactivity. The pial labelhng in both sections is artefactual. x 12.

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Fig. 5. Low-power histography of two consecutive cross-sections at the level of Fig. 27 in Ref. 208 incubated with CaBP (A) and PV antibodies (B). Notice the thalamic and hypothalamic labelling which are virtually complementary in the two sections. The hypothalamic labelling with PV antibodies (B) is probably artefactual. x 12.

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Fig. 6. Sequence of sections through the hippocampus and adjoining cortex at various levels from ventral to dorsal incubated with antibodies against CaBP (A, C, E) and PV (8, D, F). C and D correspond to the level depicted in Fig. 2 of a review article. 263Notice the patchy distribution of labelling in layer 2 of the entorhinal cortex (Ent) with both antisera and the sharp boundaries between CAI, subiculum (S), presubiculum (Prs), entorhinal (Ent) and perirhinal cortex (PRh). In the hippocampus, on the other hand, only CaBP reveals the cytoarchitectonic boundaries, whereas PV fairly similarly stains CAI-CA4. The white arrow in A, C and E marks the mossy fibre projection. The open arrow marks the probable projection from the entorhinal cortex to the molecular layer of the dentate gyrus (L‘perforant path”). x 15.

axons and terminals, lying parallel to the pyramidal cell layer, is seen in the stratum lacunosummoleculare of CAl, CA2 and CA3 (Figs 4A, arrows in 6A-C). It might represent part of the projection to the dentate gyrus from the entorhinal cortex. Bundles of extremely thin CaBP+axons course in the alveus CaBP+

and ventral hippocampal commissure and a few in the fimbria hippocampi. Single, intensely stained cells are regularly dispersed in the various layers of CA1 (Fig. 8A). These “interneurons” cluster at the CA2-CA3 border, (Fig. 8C) whereas they are extremely rare in the dentate gyrus and CA4.

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Fig. 7. (A) Picture of a coronal section of the primary olfactory cortex incubated with CaBP antibodies. Positive multipolar cells are concentrated in the polymorph layer (PO]). Note the homogeneous terminal labelling in all layers with the exception of a juxtapyramidal band in the plexiform layer (Pie). x 120. (B) The PV antisera reveals populations of somewhat larger multi~lar neurons, located more su~~cially in the polymorph (Pol) and in the pyramidal cell (Py) layer. These cells have dendrites ascending in the plexiform layer (Ple). Note the punctate labelling. x 120.

My observations in ammons horn are consistent with over work published the years by some arouns lb.l7.18~19.9O.~3O~22l~222.25~ but not with those of others95 who do not find pyramidal cell staining. In general, I have nothing to add to the descriptions of the aforementioned authors, with the small exception that CaBP-IR interneurons are often observed at the CAlCA3 border and that they form a population distinct from the PV-IR interneurons (not shown). Some CaBP+ neurons in the molecular layer probably correspond to cell b of Fig. 477 in Ref. 37. The thin CaBP- infragranular band corresponds to the adenosine triphosphatase-rich zone of the hippocampus.2R2 The band of terminals in the lower part of the stratum lacunosum-moleculare is in size and location comparable with that seen in acetylcholinesterase27’ and in Timm-stained material (Lipp, persona1 communication). It might derive from neurons of the nucleus reuniens thalami.‘” Most of the terminal field in the molecular layer of the dentate gyrus arises in the entorhinal cortex, containing CaBPf projection neurons in layers II and III. Electronhysiologically, it is well known that pyramidal cells of the -hippocampus display dendritic CaZ+ spikesz4’ a nronertv bv the CaBP+ Purkinje cells . . . * also dismayed and inferior olivary neurons.-I” Like the granule cells of the dentate gyrus, the pyramidal cells show long-term potentiation which may be a Ca?+-dependent phenomenon.*” The seizure threshold of cells in the hippocampus directly correlates with the amount of CaBP? the fascia dentata shows the highest threshold and the highest CaBP content. The CA2CA3 region has the lowest threshold4,*” and no CaBP. Ca2+-conductances are less prominent in the dentate gyrus.“’ Pur~albu~j~. PV+ cell bodies are detected in the stratum oriens, stratum pyramidale, and stratum radiatum of the various hippocampal subfields and in

the stratum granulare, moieculare and hylus (str. polymorphe) of the dentate gyrus (Figs 4B, 6B,D,F, SB). The cell bodies are mostly polygonal and belong to two different size classes (see Table 2), in both the hippocampus and in the fascia dentata (Figs 8B, 78). The processes (axons) emanating from the neurons enmesh the cell bodies of pyramidal and granule cells. The largest PV+ cells of the dentate gyrus have slender, delicate dendrites penetrating the molecular layer. The punctate structures abutting on pyramidal and granule cells are thin and extremely closely packed (Fig. 8D) and give to the lamina pyramidalis and granularis a darker and diffuse look overall (Figs 4B, 6B,D,F, 8B). The lacunosum-moleculare layer of the hippocampus is occupied by a variety of beaded and straight, coarse and thin PV+ cell processes, mainly coursing ~r~ndicularly to the pyramidal cell layer (Fig. 79). The density of PV+ neurons in the hippocampus is differing in the various subfields (e.g. dorsoposterior CA 1 has less than other parts of CA1) but a quanti~cation is not attempted. Most of the PV+ cells in the fascia dentata have their perikarya situated at the boundary between granular layer and hylus; fewer occur embedded in the granular layer, and PV+ cefl bodies in the molecular layer are an exception. A camera iucida drawing of the different cell types in the hilar region of the hippocampus and dentate gyrus is presented in Fig. 78. In the molecular layer of the DG the PV + processes are coarse and smooth, and beaded dendrites are rarely observed. The ratio of PV-IR cells to granule cells

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Fig. 8. (A) Immunohistochemical demonstration of CaBP in a longitudinal section of the adult rat hippocampus (CAl-field). The soma and dendrites of pyramidal cells (Py) are lightly labelled, whereas single neurons lying in the striatum oriens (Or), radiatum (Rad) and lacunosum-mole~lare (LMol) are intensely labelled in their entirety. Notice that only the innermost row of pyramidal cells displays CaBP, whereas the outer pyramidal cells are unstained. CaBP+ axons run in the alveus (alv) and in the stratum lacunosum-moleculare (black arrow). x 120. (8) lmmunohistochemical demonstration of PV in a coronal section of the adult rat hippocampus. The bipartite dark, fluffy layer, occupying the middle part of the picture correponds to the pyramidal cell layer (Py), which is ensheathed by innumerable fibres and terminals. Perikarya are labelled in the stratum oriens (Or), pyramidale (Py) and radiatum (Rad) and an array of processes perpendicular to the pyramidal cell layer can be seen in the stratum oriens (Or) radiatum (Rad) and lacunosum-moleculare (LMol). Some of the processes are beaded (see Fig. 79). x 190. (C) CAZ-3 border in a Vibratome section incubated with CaBP antisera. The mossy fibre (MF) staining tapers out (arrow). Various interneurons are concentrated at this point in the stratum radiatum (Rad). In the upper part of the figure, the CaBP+ pyramidal cells of the CA2 region are visible (arrowheads). x 120. (D) High magnification of a semithin (1 hrn) cryo-section incubated with PV antibodies. The surface of pyramidal cells (Py) is tapestried with PV+ terminals. A PV-IR interneuron is marked by an arrow. x 750.

within the dentate gyrus is about 1: 200, with regional variations. The absolute number of parvalbuminic cells in the fascia dentata is of approx. 4000 + 500 as determined in series through two rat brains. Single PV+ axons are observed in the fimbria hip~campi and in the dorsal hippocampal commissure but none in the ventral hippocampal commissure. PV+ cell processes and cells are found in the septohippocampal nucleus too (Figs 4B, 29B). The cells staining with the PV antibodies in the rat ammons horn evidently represent interneurons. This statement is based on several lines of evidence; firstly, virtually no axons are seen leaving with the fimbria hippocampi, thus PV-IR cells are intrinsic to the ammons horn. Secondly, only basket cells have their cell bodies in the stratum oriens.

Thirdly, the pyramidal cell perikarya are ensheathed by a plexus of PV-IR terminals, obviously representing the basket-like endings of the axis cylinder collaterals of basket cells. Fourthlv. the number of PV+ cells (at least in the fascia dentat;) is comparable with that of interneurons counted according to pure mo~hologi~l criteria by others.246 The morphology of PV-IR cells in the rat hippocampus corresponds closely to those of the interneurons depicted in a classical paper on the mouse ammons horn’65 (Fig. 6, nos 1. 2 and 3, Fig. 7, no. 4. Fig. 8, no. 2). The PVC neurons in the dentate-gyrus corres&nd to representatives of most cells de&ted in Ficl. 28 of another oaoer’ (cf. Fie. 78 of this paper).‘Therefore, >V is a selective’marke; for iiterneurons in the hippocampus of adult rats. Preliminary evidence suggests, however, that only a minor subpopulation of interneurons is PV + In consecutive sections GABA and PV, as well as GABA and CaBP, co-exist in the same basket cell, but there are more GABA than PV or CaBP neurons.

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Hama’s group has recently published an interesting series of papers ‘38~‘39.‘40.‘41 in which they demonstrate occurrence of PV in 20% of the GABA cells. These authors are also inclined to interpret PV+ neurons as representing basket cells. Additional publicatio&’ confirm and extend these observations. Interneurons are also known to display immunoreactivity towards glutamate decarboxylase’88,“9 and various peptides’84.‘6’.‘75 and to be cytochrome-oxidase-

positive.“’ Table 3 summarizes the scanty data about the known electrophysiology of hippocampal interneurons. They are characterized by a high firing rate similar to many other PV+ cells. Interestingly, their action potential duration is half that of pyramidal cells243and they seem to be a primary target for excitatory (oxytocin + vasopressin)“’ or inhibitory

(opioid

peptides)“’

peptide

effects.

Subiculur comp1e.u (Fig. 92) Both, CaBP and PV, allow a sharp demarcation of the boundaries between the various parts of the subicular complex (Fig. 6). Calbindin D-28k. The immunolabelling varies somewhat between dorsal and ventral (see Fig. 6A,C,E). The pyramidal cell layers and the neuropil in str. lacunosum-moleculare of the prosubiculum are more strongly immunoreactive (Fig. 6A,C,E) than those in CAI and in the subiculum. Between subiculum and presubiculum there is a CaBP-poor band. The presubiculum displays a strong CaBP-labelling of the superficial, densely packed small cells, whereas the parasubiculum contains only scattered CaBP + elements in its whole thickness. Parralbumin. The distribution of PV+ neurons in the prosubiculum does not deviate from the description given above for the CAI region of the hippocampus. In the subiculum. the PV+ cells are loosely packed and form a wider band (Figs 4B, 6B,D,F). PV labels innumerable neurons in the superficial and lower layers of the presubiculum. The presubiculum lower layer is stuffed with PV+ neurons and terminals, whereas the superficial layer is so to a lesser degree. Neocortex (Figs 84-93) A parcellation and stratification according to cortical areas is evident in the distribution of elements containing PV and, less so, in those containing CaBP. A detailed description of the “cytoarchitectonics according to calcium-binding proteins” goes beyond the scope of this paper. Therefore, a general account is given, limited to the parietal cortex and Fig. 9 depicts representative views of some cortical regions. described in the rat brain.‘” Virtually all neurons displaying CaBP and PV immunoreactivity are GABAergic4’ In the somatosensory cortex some rare cases of co-existence of both PV and CaBP with GABA in the same neuron can be determined by studying consecutive cryo-sections. Although the CaBP+ and PV+ neurons constitute the majority of GABAergic neurons, a minor subpopulation of GABA cells remains unreactive towards CaBP and PV antisera.47 It is as yet unknown

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if this subpopulation corresponds to a particular cell type. It is worth noting that GABA+ neurons in layer I never contain calcium-binding proteins. Calbindin D-28k. The immunostaining with the CaBP antibodies in the cerebral cortex shows a fairly homogeneous and strong labelling of the upper three layers (I, II and III) and a moderate homogeneous staining of layer V (Fig. 91,H). In these layers most, but not all pyramidal cells, interneurons and the neuropil, are stained. Interneurons in the upper layers are strongly immunoreactive and are of the bipolar and bitufted type (Fig. 1 IA). Layers V and VI of the cortex display single, scattered, multipolar CaBP-IR interneurons. whereas IV is poor in CaBP+ elements. The interneurons in layer VI are spaced enough to permit identification of their spineless dendritic ramifications. They represent mainly interneurons, probably those having ascending axons (Fig. 73. cells 19, 20, 21 in Ref. 164). All CaBP+ interneurons of the cerebral cortex are GABAergic, but represent a minor subpopulation. Often CaBP-IR dendrites come in close contiguity to each other, or to CaBP+ perikarya. Positive axons are difficult to discern because of their thinness, but they leave the cortical mantle, e.g. through the corpus callosum (Fig. 9A). The total number of CaBP+ neurons in the cerebral cortex is impossible to ascertain because of the diffuse neuropil labelling in the upper three layers. The number of strongly stained. scattered interneurons is less than 5%. The substrate for the homogeneous staining of the upper cortical layers, also seen in monkey?* and cat”“’ visual cortex remains as yet undetermined and only immunoelectron microscopy may shed light on this problem. The homogeneous labelling suggests a diffuse CaBP+ cortical innervation. possibly originating in the intralaminar thalamic nuclei. in the nucleus basalis of Meynert”” and in the dorsal raphe nuclei. region The “barrel-field ” ,zxxa well-defined koniocortical of the rodent cerebral cortex is characterized by “puffs” of intense. homogeneously immunoreactive terminal fields (“barrels”). demarcated by calbindin-poor “septa”.

Purralbumin. Relative number and size of PV-IR cells vary considerably throughout the depth of the cortex and from area to area (Fig. 9). These neurons are mainly located in layers II and IV, but are present in all cortical layers, except for layer I. Layer I appears as a white band in low-power pictures (Fig. 4). At higher magnification only some randomly dispersed immunoreactive cell processes can be seen. PV-reaction products appear within somata, dendrites. axons of neurons and within punctate structures. Neurons of different morphologies and sizes contain PV. The PV+ cell body is mostly multipolar and, less often, bitufted (Fig. I IB). In this last case, the cell axis is perpendicular to the pia mater: a few neurons with horizontal cell axis are observed. Some PV+ perikarya, particularly in the frontal cortex. have the shape of an inverted pyramid. However. dendritic spines are never seen on PV + cortical cells. In most layers the PV+ cells are packed in such a

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Fig. 10. Co-existence of PV and GABA in some interneurons of the somatosensory cortex. Consecutive, adjacent sections incubated with antibodies against GABA (A} and PV (8). Arrow points out the cell harbouring the two substances. The slight variation in cell morphology is due to differentiai stretching of the frozen sections during the thawing process. Asterisks mark two landmark blood vessels. x 750. Fig. I I. Interneurons of the cerebra1 cortex reveaied with CaBP (A, B) and PV (C, D) antibodies. (A) Notice the gradations in staining intensity between different CaBP+ neurons. The strongly stained represent interneurons, probably of the bipolar (Bip) and bitufted (Bit) sort. The moderately labelled ones are small pyramidal cells. Photograph was taken at the boundary between layers II and III. x 300. (B) Semithin cryo-section (0.5-l pm) of a region similar to A. The pyramidal shape of the moderately CaBP-labelled cells is evident (arrows). Both the cytoplasm and nuclei are immunoreactive. Intemeurons are marked by arrowheads. Dorsal is left. x 300. (C) PV-IR multipolar neurons at the boundary between layers I and II and in layer II. Notice the terminal field in layer II. x 300. (D) PVt multipolar neurons in lower cortical layers. Both cytoplasm and nuclei (without nucleoli) are labelled. Notice the absence of spines from dendrites. x 300. 390

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Fig. 12. Low-power view of a tangential section through layer IV of the barrel-held of the rat somatosensory cortex incubated with PV antibodies. The basic organization in barrels (B) and septal meanders (S), characteristic of this region, is visualized. The black dots are PV+ interneurons. x 120. Fig. 13. Higher magnification of a field-of-view of the rat basal ganglia showing a large immunoreactive neuron, a delicate meshwork of Iibres and terminals and an immunola~ll~, sohtary axon, coursing in the bundles of the internal capsule (arrow). x 480. (B, C) Multipolar, PV + interneurons are clustered together in the dorsolateral part of the caudatoputamen. Fibres of the internal capsule (arrows). Notice a discrete background labelling of terminals in the neuropil. x 120.

density as to make a ~~onst~ct~on of their dendritic tree impossible. In layers IV and lower V, terminals

impinge upon the surface of almost every perikaryon. They are so numerous, that they appear to form a continuous sheet around the soma.” PV+ terminals can also be found abutting on PV+ cell bodies and proximal dendrites. Roughly estimated, PV+ neurons in the parietal cortex represent approximately 10% of the total neuronal number (see, however, Ref. 29). The size, shape and morphology of PV+ neurons are similar to those of interneurons as described in Golai36~37~‘~~i66~Z~ and electron microsconic studiesztO The absence of PV+ fibres in the corpus callosum, anterior commissure and crura cerebri suggests that PV occurs mainly in neurons intrinsic to the rat neocortex.” However, PV + fibres course in the bundles of the internal capsule through the caudatoputamen, while others perforate the corpus callosum. A precise association of PV + neurons to Go&i-classified intemeuronal types cannot be readily achieved because PV-immunost~n~ cells are not spatially isolated, and therefore interfere with the reconstruction of PV-IR cell

processes. However, with the exception of short-axon interneurons located in layer I (Figs 71, IOF, IlA, UK),‘@ representatives of all other types of intemeurons,‘~,~~ with the possible exception of bipolar cells, seem to display PV immunoreactivity. In the “barrel-field” the neuropil is subdivided in nearly circular PV-rich zones of approximately lOO-pm diameter (“barrels”) and in PV-poor “septal”-meanders, Small, multipolar, spineless PV neurons are scattered at the barrel side or in the septa, and their cell processes are polarized towards the centre of the barrel (“hollow”) (Fig. 12). The PVC neurons in the SMl barrel-field are spine-free and may correspond to those described as type II non-pyramidal cell~.*~~~al~oco~i~~ tibres extensively terminate on the soma and proximal dendrites of these smooth stellate cells in the barrels.283J*4These neurons are thought to be the “substrate for the fast-spike units, characterized by the rapid course of their bioelectric waveforms, their high rates of spontaneous activity and their ability to respond to higher freouencv stimulation of the vibrissae”249(see also Table 3). The d&ibution of PV-IR neurons has been compar& with the published localizations of neuropeptides’~s~28152~‘8s~2’3 or neurotmnsmitter synthesizing enzymes (glutamate decarboxylase). The best match in the distribution of PV is found with the inhibitor neurotransmitter GABA as revealed by GAD-immunohistochemistry. ‘*8~22’~228~229 In consecutive sec-

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tions we have shown that both substances co-exist in a subpopulation ofintemeurons.47 However, there are cortical GABA cells, which lack PV. It is not astonishing that a calcium-binding protein, which has the property of ~ntroll~g the ex~i~biiity of a neuron, is particularly accumulated in inhibitory neurons, which are in charge of controlling the excitability of other nerve cells. Basal gangiia Clauslrum (Figs 86-89) Cafbindin D-28k. The claustrum itself is almost devoid of CaBP (Fig. 3C, 14A). On the other hand, the claustrocortex is extremely rich in positive tern+ nals which occupy the upper layers (Fig. 3C). This zone is easily seen in coronal sections and delimits the piriform cortex from the parietal cortex. Pa~albumi~. Immunoreactive eel1 bodies and processes occur in the whole extent of the claustrum (Figs 3B,D, 14B). The cells have a slender tree of dendrites running in all directions, but the axon cannot be discerned. PV+ neurons in the most rostra1 regions of the claustrum have highly beaded cell processes. The claustrum is delimited from the surroundings by its fluffy, diffuse immunostaining, deriving from terminal fields (Fig. 14B). The claustrocortex is defined by a sharp band of immunoreactive neurons and terminals, probably located in layer V (Figs 3B,D, 14B). Caudatoputame~ (Figs 86-90) Calbindin D-28k. The immunostaining in the caudatoputamen and nucleus accumbens is one of the most impressive observations made wrth these antibodies. The CaBP-labelhng is homogeneously distributed in the innermost core of the neostriatum, but leaves a dorsolaterally crescent-shaped shell, virtually unstained {Figs 3C, stars in 4A, 29A). This portion contains the highest concentration of PV + terminals (see later). Particularly strong is the staining in the fundus striati (Fig. 3E,G). In the medial caudatoputamen only the fan-shaped fibre bundles of the internal capsule and the anterior commissure remain untagged by the CaBP antibodies. It is worth noting that small oval islands of tissue (striosomes) oriented parallel or perpendicularly to the course of the internai capsule, lack CaBP immunoreactivity (arrows in Fig. 3A). At higher magnification, the immunostain-

ing is observed in great numbers of densely packed, medium-sized, spiny neurons and in a fine texture of cell processes, terminals and fibres. The CaBPimmunostained region gradually merges with the relatively unstained dorsolateral portion of the caudatoputamen, but ends sharply at the boundary with the globus pallidus (Figs 3E,G, 4A, 14A, 29A). The dorsolateral portion of the striatum has no CaBP+ cell bodies, but harbours a moderate density of terminals. In parasagittal sections, a tongue-like protrusion, directly behind the external capsule, displays higher CaBP immunoreactivity than the surroundings. From the neostriatum and accumbens

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arises a very consistent CaBP+ projection to the mediodorsal substantia nigra (pars reticulata) (Fig. 14A). Although the size of CaBP+ cells in the caudatoputamen is very homogeneous (15 x IS pm), some larger neurons (20 x 20 pm) can be seen, The striking immunostaining in the caudatoputamen has been depicted schematically,gO and exactly described by Gerfen et al.” CaBP antibodies visualize a very prominent strionigral pathway, and in this respect they resemble antibodies against calcineurinto2 and DARRP-32, a neuronal phosphoprotein, occurring in the whole caudatoputamenu4 Yet the caudatoputamen staining with CaBP antisera is of further interest because of the visuali~tion of “striosome”like patches,97J05~‘fo which may correspond to the zones of low acetylchohnesterase activity, being avoided by the afferent terminals from the intralaminar nuclei.10s Although drawing of the CaBP-immunostained cells in the striatum cannot be performed because of the interlacing of ceil processes, it is assumed that they are medium spiny neurons, which represent about 95% of striatal neurons.r4r Parvalbumin. Spatially separated, strongly PV-IR neurons of various sizes are observed in the caudatoputamen, mainly in its dorsolateral, CaBP-poor part (Figs 3B,D, 4B, 14B). The cell bodies of the larger neurons have a smooth surface and polygonal or fusiform shapes (Figs 13A,B,C, 80,81). From here a tree of spine-free dendrites radiates over long distances (200pm) and intermingles with the processes, belonging to adjacent PV + cells, The axons of these cells are difficult to visualize, but they appear to ramify locally. In a few cases, axons entering the bundles of the internal capsule heading toward the globus pallidus can be seen. A terminal field, which can be best traced at low magnification (Figs 4B, 5B), lies in the same area where PV + neurons are located. The morphology of the largest cells at best resemble that of non-spiny type I large neurons$) and the “spine lose makroneurone”.67.**’ Their number and location do not match precisely that described by the above and other authors with the Golgi-method. but we have to keep in mind that PV antibodies may only stain a subpopulation of these neurons and that the capricious Golgi-method may deliver erroneous distributions. Large cells in the striatum have been shown by others to be acetylchotinesterase- and cholineacetyItransferase-1R.‘5 The smaller PV + neurons of the neostriatum appear cytologically identical to the larger cells in not having somatic or dendritic spines and only a few beaded dendrites; the axon has never been traced. The smaller PV+ neurons in the caudatoputamen of the rat at best correspond to type III medium neurons of Chang et al.” and may represent projection neurons. In the gray matter of the neostriatal dorsocaudal region, a higher density of fibres and terminals is seen. Single PV+ axons are detected in the fibre bundles

of the internal capsule, running through the caudatoput~en; their number increases laterally and in rising proximity to the globus pallidus. PV-IR cells account for less than 2% of caudatoputamen neurons as inferred from counting the total number of neurons in adjacent Cresyl Violetstained sections. Their number in a 40-urn-thick sagittal section is approx. 480 k 30 (n = 12).

Calcium-binding proteins in the rat brain

393

Pig. 14. Horizontal section through the substantia nigra, visualizing the whole extent of the CaBP-IR striatonigral pathway arising in the “matrix” compartment of the caudatoputamen (CPU) (A). Notice the clustering of CaBP striatal terminals in the medial SNR and of PV+ neurons (B) in the lateral SNR. At this level the SNC has no CaBP+ neurons (cf. Fig. 32A). Striosomes are evident in the caudatoputamen of A (arrows). Another complementary feature of labelling is found in the subthalamic nucleus (S’lh), which has many PV+ terminals (B), probably deriving from the globus pallidus, but no CaBP-IR structures (A). x 10.

Accumbens (Figs 86 and 87) Calbindin D-28k. The general morphology is similar to that described for the caudatoput~en. Again there are zones of decreased immunoreactivity, particularly mediodorsally (shell of the aceumbens; arrows in Fig. 3A). Rostra1 to the anterior commissure, the CaBP+ accumbens is sharply demarcated basally but displays “finger-like” protrusions of CaBP+ neurons (cell bridges) penetrating the olfactory tubercle and reaching the inferior pial surface (Fig.

3A). The morphology of the CaBP+ neurons of the accumbens and of the caudatoputamen is identical. Par~alb~min. The dist~bution and ~oncent~tion of PV+ neurons in the accumbens is similar to that seen in the medial caudatoputamen. However, only the small cell type occurs. @Ifactory tubercle (Figs 86-88) Calbindin D-28k. “Bridges” of CaBP+ neurons descend perpendicularly from the accumbens and

394

M. R.

span the whole width of the olfactory tubercle (cell bridges in Fig. 3A). These protrusions of labelled neurons alternate regularly with CaBP-unstained portions of the olfactory tubercle. The CaBP + neurons in the olfactory tube&e cannot be differentiated morphologically from those in the caudatoputamen or accumbens (Fig. 15A). P~~aIb~~. The large, multipolar PV+ neurons are mostly found in the polymorph layer and resemble pallidal neurons (Fig. 15B). Smaller PV+, multipolar cells occur in the pyramidal cell layer of the olfactory tubercle, and only a few in the plexiform layer. Islands of Calleja Parvalbumin. Large PV+

cells, in their morphology resembling pallidal neurons, are dispersed between the Islands of Calleja. Their shape is somewhat obscured by the large amount of thick PV-t axons, coursing in the medial forebrain bundle. Globus pallidus (Figs 88 and 89) and entopeduncular nucleus (Fig. 90) Calbindin D-28k. Only a sheet of the outermost and a funnel-shah (in parasagittal sections) portion of the innermost part of the lentiform nucleus are slightly CaBP+ (Figs 4A, 14A). These terminal fields probably arise from collaterals of CaBP+ striatonigral fibres. The large central portion of the globus palhdus is poor in such terminals and may receive a separate input from the dorsolaterally located, CaBPstriatal region. This further chemical subdivision of the globus pallidus has not been seen either with substance P,“’ or d~orphin. ” In contrast to the ~udatoputamen, perikarya of the globus pallidus and entopeduncular nucleus never exhibit CaBP immunoreactivity. Pawalbumin. A great number of neurons of the globus pallidus (Figs 4B, 14B) and ento~un~uiar

CELIO

nucleus (Fig. 14B) display PV-IR, associated with their soma and processes. The cell bodies are polymorph, but mostly quadrangular (Fig. 18) and belong to two different size classes (see Table 1). The PV+ dendrites form a coarse network traversed by the unstained fibres of the internal capsule. PV+ axons leave the GP with the ansa lenticularis, lenticular fasciculus and stria medullars thalami. Pallidal neurons exhibit phasic, high frequency, firingT2 (see also Table 3). Ventral pallidus (Fig. 88) ~albindin D-28k. Against the background

of the caudatoputamen, the ventral pallidus looks rather pale with only a few terminals. In coronal sections these terminals are clustered in a “coma-shaped” lateral region. Parvalbumin. PV+ neurons are similar to those described in the globus pallidus, but occur in lower numbers. Most of the pallidal, entopeduncular and ventral pallidus neurons are GABAergictBS and some probably contain substance P.‘u Basal nucleus of Meynert (Fig. 89) Calbindin D-28k. The rat nucleus basalis is populated by large CaBP+ multipolar neurons with elongated soma (20-30 x 10pm) and thick and widely branching dendrites (Fig. 16). Earlier we described the presence of CaBP in the monkey basal nucleus.46 Subthalamic nucleus C~lb~nd~~ D-28k.

CaBP+ fibres and terminals avoid the subthalamic nucleus (Fig. 14A). Parvalbumin. The nucleus is crowded with a high number of thin PV+ axons and terminals, particularly in the lateral part (Fig. 14B). In adequate

Fig. 15. Horizontal section through the olfactory tubercle. The distribution of CaBP+ (A) and PV+ (B) elements is simiIar; both antigens mark neurons in the polymorph layer (Pol). The PV-staining (B) is analogous to the one observed in the globus pallidus. Notice in both cases the homogeneous staining of the pyramidal cell layer (Py) (stronger in B) and the absence of staining in the plexiform layer (Pie). Nomarsky optics. x 200. Fig. 16. Horizontal section of the nucleus basalis of Meynert (B) at the level of Fig. 57 in a brain atlasZoH CaBP+ neurons (arrows) have their perikarya and their coarse dendrites ordered parallel to the posterior border of the globus pallidus (GP). x 120. Fig. 17. Horizontal section of the olfactory tubercle at the level of Fig. 54 in Ref. 208. PV-immunoIabelling. Notice the positive fibres of the medial forebrain bundle and the unstained islands of Calleja (arrows). x 120. Fig. 18. Horizontal section of the globus pallidus (GP), incubated with PV antibodies. Large, triangular perikarya with coarse, interlacing cell processes are visualized. Nomarsky optics. x 350. Fig. 19. Horizontal section of the reticular nucleus of the thaiamus (Rt), incubated with PV antibodies. The intensity of staining is higher than in the GP (Fig. 18). Axons (arrows) leave the reticular nucleus of the thalamus in the direction of ventroposterolateral thalamic nucleus (VPL). Nomarsky optics. x 350. Fig. 20. Horizontal section of the amygdala incubated with CaBP antibodies. Large, multipolar neurons are visualized in the basolateral amygdaloid group (BL). x 200. Fig. 2 1.Horizontal section of the amygdala incubated with PV antibodies. The morphology of the positive neurons in the basolateral amygdaloid group (BL) is very similar to that of Fig. 20. x 200.

Calcium-binding proteins in the rat brain

Figs 15-21.

395

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M. R.

CELIO

Table I. Relative staining intensities of some selected neurons towards calbindin D-28k and parvalbumin antibodies CaBP -_._ ~__._._..... . -.____ . ...________ Purkinje cells +++-I_ Basket- and stellate cells of the cerebellum Interneurons of the cerebral cortex +++ Periglomerular cells +++ _ Cells in the lamina plex. ext. Cells around the olfactory ventricle ++ Fascia dentata granule cells ++* CA1 pyramidal cells +* Hippocampal intemeurons ++e Medial habenular nucleus ++++ ~audatoputamen +++/++* _ Giobus palhdus Substantia nigra (pars reticulata) (pars compacta) +++ Thalamic nuclei ++* Retina +++ Inner hair cells (Corti’s organ) t++ Spinal ganglion ceils +++;++ Plexus myentericus +++ Plexus submucosus +++

PV __~_._ ++++ +++ +++i++ - (+) fff _ fff +++ -I+,/+ fff +++ +++ -I-i--t-i++ _ _

*Diffuse staining. + t + + ~very high; + + + , high; + + , moderate: +, low; -, absent.

Table 2. Size (minor and major diameter in microns) of pa~albuminand calbindin D-28k-immunoreactive cells in selected brain regions

Olfactory bulb Cortex cerebri Cortex cerebelli (Purkinje cells) Hippocampus (interneurons) Fascia dentata (interneurons) Nucleus reticularis thalami Caudatoputamen Globus pallidus Colliculus inferior Spinal ganglia (Ll) Ggl. spirale cochleae Nucleus basalis Meynert

CaBP

PV

10 x 10 15x24 35 x 35

12.5 x 10 12 x 30118 x 35 35 x 35

15x25

17 x 23 17 x 2312.3x 35 20x30/15x 15 17x30/20x20

15x15/20x20

30 x 50

35 x 47* 30 X 55120 X 20 15 x I5

20(30Q x 10

*Other different classes. tThe coarse primary dendrites make a delimitation of the somatic boundary dihicult. The tissue has been treated with (CaBP) or without (PV) albumin embedding and coverslipped in glycerine gelatin (PV) or Eukitt after dehydration (CaBP). The only exceptions are the spinal ganglia, which have been treated with Bouin + paraffin.

Table 3. El~tropbysiological

characteristics of some pa~~bum~n-positive

Effect _-_______--._.__I_ ______ .-...-. __ ___._,__” ____I__ inhibitory Layer III lnterneurons in pyriform cortex Barrels (IV layer) Inhibitory Neurons in SMI cortex inhibitory Nucleus reticularis thalami Inhibitory CA1 pyr. layer Hippocampus (basket-cells) Inhibitory Purkinje cells cerebellum Localization

Renshaw ceils Pallidal neurons and neurons in the SNR

Spinal cord

Inhibitory Inhibitory

cells in the brain Discharge rate and type -l_l___l_-l__ --.. Repetitivez3* Repetitive”’ S~ntaneous: 120/s (bursts of 3 s?” Spontaneous: .50/s”” 70/s quietly sitting monkey’“‘-‘*’ 400/s during movements (3060/s spontaneous)“2 Gery high-frequency~3’ Phasic, high frequency firing”

Calcium-binding proteins in the rat brain

397

sections, the projection is seen to originate from the globus pallidus. Many moderately ~mmunoreactive neurons populate the subthalamic nucleus as well.

in subregions. In general the content of CaBP is very high, whereas PV is moderately rich in the amygdala.

Substantiu nigra (Figs 91 and 92)

Calbindin D-28k. The medial amygdaloid group is occupied by an extremely rich complement of CaBP+ neurons, embedded in a rich network of CaBP+ fibres and terminals (Fig. 3E,G). The posteromedial cortical amygdaloid nucleus is rich in neurons and endings which merge dorsally with the amygdalohippocampal transition area. Parvalhumin. The amygdalohippocampal transition area has neurons and terminals which are ordered similarly as in the hippocampus.

Calbind~ D-28k. A massive terminal field contain-

ing CaBP, is found in the mediodorsal portion of the substantia nigra, pars reticulata (Figs 14A, 22A). The staining is less pronounced or even absent in the laterodorsal aspect of the substantia nigra, pars reticulata (Figs 14A, 22A). The dorsal sheet of the substantia nigra, pars compacta, the substantia nigra, pars lateralis, the retrorubral area and the peripeduncular nucleus are populated by a moderate number of CaBP-IR neurons. In the substantia nigra, pars compacta, those located rostroventrally (Fig. 32A) are smaller, clustered and more numerous than those located distally. In the peripeduncular nucleus, CaBP+ neurons are embedded in a rich background of thin CaBP+ terminals and fibres. Parvalbumin. PV antibodies label many neurons in the substantia nigra, pars reticulata, in the substantia nigra, pars lateralis and in the ~~~duncular nucleus and in the pars compacta (Figs 14B, 22B). In the substantia nigra, pars reticulata, parvalbuminic neurons are of various sizes, but mainly large (20 x 25 pm) and unevenly distributed between the various quadrants. Coarse PV+ processes are intermingled with PV+ neurons in the more lateral portions, whereas the medial part of the SNR contains fewer PV+ neurons and thinner fibres. The immunostained cells have slender somata and coarse dendrites and resemble in shape those of the globus pallidus. The nigral cells merge with other PV+ neurons, belonging to the peri~duncular nucleus. PV + axons leave the substantia nigra in mediodorsal and rostrolateral direction. Ventral tegmental area (Figs 91 and 92) Caibind~n D-28k.

Widely spaced, small CaBP+ neurons are typical for the ventral tegmental area and adjacent portions of the ventral tegmentum (Fig. 22A). Others have first described these CaBP+ neurons and proved them to be dopaminergic.97 The linear raphe nucleus contains nothing but fibres and terminals. In addition to having a complement of small neurons, the interfascicular nucleus displays a dense, homogeneous terminal field as well. Parvalbumin. The ventral tegmental area is pierced by an innumerable quantity of PV + , coarse axons, but virtually does not display PV+ neurons (Fig. 22B). Robust parvalb~ini~ fibre tracts, surrounding the ventral tegmental area are the medial lemniscus, the medial longitudinal fascicle and the mammillary peduncle. Amygdala (Figs 89 and 90)

The use of CaBP- and PV-immunohistochemistry results in a sharp discrimination of the topography of the amygdala. Subnuclei are being further subdivided

Medial amygdaloid group

Basolateral amygdaloid group Calbindin D-28k. The lateral nucleus possesses a fair amount of terminals and displays CaBP+ neurons as well (Fig. 3E). On the other hand the rostra1 portion of the basolateral and basomedial show up as rather pale nuclei, surrounded by a dense terminal field region (Fig. 3E). They harbour a few, spatially separated, multipolar neurons with slender, nonspiny, cell processes. At more caudal levels (Figs 3G, 20, 30A) the basolateral amygdaloid group, particularly its lateral half, the basomedial and the ventral basolateral amyg~loid group are intensely tagged by CaBP antisera. The intercalated nuclei (I) have neither cells nor terminals inside their boundaries (Fig. 3E). Parvalbum~n. The drop-shaped basolateral and lateral nuclei are selectively visualized by the presence of a fine meshwork of PV+ fibres (Fig. 3F). Large neurons with slender, spineless cell processes occupy the basolateral (Figs 21, 30B) (ventral and dorsal) and the lateral amygdaloid nuclei. The PV+ neurons in the ventral basolateral ventral nucleus intermingle with those of the polymorph layer of the primary olfactory cortex (Fig. 3F,H). PV+ fibres arising in the globus pallidus enter the mediocranial aspect of the basolateral nucleus. The distribution of PV+ neurons and terminals is uneven as if a parceilation existed. The intercalated amygdaloid nuclei are devoid of PV immunoreactivity. Central amygdaloid group Caibindin D-28k.

The central nucleus shows a parcellation in a mediocranial region, very rich in calbindin-positive neurons and terminals and a lateral region with only a few cells and terminals (Fig. 3). Olfactory amygdala Calbindin D-28k, The anterior cortical amygdaloid

nucleus has only a few CaBP+ neurons and moderate terminal labelling. The posterolateral cortical amygdaloid nucleus is occupied by innumerable small, weakly labelled neurons embedded in a strong terminal field. The amygdalopi~fo~ transition area

398

M. R. CELIA

is characterized by a huge terminat field but relatively few strongly labelled neurons. Septal compiex (Figs 87 and 88) Calbindin D-28k. Probably all ceils of the triangu-

lar septal, (Fig. 28A) of the septohippocampal (Fig, 29A) and most of those in the lateral septal (with exception of the dorsal portion} nuclei, (Figs 28A, 29A) are CaBP+. The ventral and intermediate portion of the lateral septum are further parcellated into subzones by the presence or absence of CaBP+ multipolar neurons and terminals (Figs 28A, 29A). CaBP + terminals form an oblique band from dorsomedial to ventrolateral. The CaBP+ triangular septal cells infiltrate the ventral hippocampal commissure, the fornix and the stria medullaris thalami and reach as far as the fimbria hippocampi (Fig. 28A). The vertical limb of the diagonal band contains a few CaBP+ neurons. The septohippocampal nucleus shows a high density of CaBP f terminals and many CaBP+ axons (Fig. 29A). CaBP+ axons in the fornix are sharply segregated to the posterior third. Thin CaBPf axons course in the anteromedial aspect of the stria medullaris thalami. The neurons of the septofimbrial and medial septal nuclei do not express CaBP (Fig. 4A). Parvalbumin. In general the labelling is very discrete (Fig. 4A), or even absent (Fig. 28B). The occurrence of a few multipolar PV + neurons in the medial septal nucleus and subpially in clusters of two

to three in the lateral septal nuclei is regular (Fig. 4A). PV -I- cells in the triangular and in the septofimbrial nucleus can rarely be seen. Some PV + axons leave the septum in a dorsomedial direction and proceed to the stria medullaris thalami. Some large neurons, oriented sagittally, occupy the most medial portion of the nucleus of the diagonal band. Bed nucleus of the anterior commissure Parvalbumin. A few. medium-sized PV + cells with their cell processes inte~ingl~ are invariably found in the bed nucleus of the anterior commissure (Fig. am/ L3).

Bed nucleus of the stria terminalis (Fig. 88) Calbindin D-28k. The immunolabelling is very similar to that observed in the lateral septum: a myriad of small neurons embedded in a rich fibre and terminal matrix (Fig. 3C). The lateral and ventral nuclei have less positive sites than the medial nuclei.

Diencephalon Epithalamus Habenular nucIei (Fig. 90) Calbindin D-28k. Cell bodies in the lateral division of the medial (and a few in the ventral medial) habenular nucleus display a strong immunoreactivity towards CaBP antibodies. In a coronal section of the diencephalon this is the most striking staining (Fig. 27A). The nucleus is also more or less homogeneously and diffusely stained by the antibody, but the terminals and cell processes never attain the staining intensity reached by the soma. CaBP+ axons course in the habenular commissure. The habenulointerpeduncular tract’*’ is known to consist of substance P and acetylcholinestera~-containing fibres’j’ and is possible to represent the only efferent connection of the medial habenula. The medial habenula is another location, beyond the nucleus basalis of Meynert,46 where CaBP occurs in a cholinergic cell group, although cholinergic neurons are more common in the ventral medial habenular nucleus (see Fig. 4 in Ref. 253). Parvalbumin. PV+ fibres in the stria medullaris thalami form a rim in its lateral portion, enter the lateral habenular nucleus and impinge upon its neurons. PV + terminals are detectable only in the lateral habenular nucleus, also displaying some immunoreactive neurons (Fig. 27B). ~ha~omu~*6*1~~,‘33 (Figs 89-91)

The complementary nature of staining between PV and CaBP antibodies can be best appreciated in the thalamus. PV primarily occurs in “specific” nuclei, whereas CaBP is rich in “unspecific” thalamic nuclei. The rich, diffuse and homogeneous CaBPimmunostaining creates a peculiar chemical parcellation of the rostra1 thalamus. As already pointed out by others,” the distribution of CaBP “does not strictly correspond to defined nuclear groups”. Interestingly, neurons, located at the boundaries between classical thalamic nuclei, express calbindin immunoreactivity (e.g. Figs 5A, 22A). On the other hand, the PV immunoreactivity is dominated by axons and terminals and respects nuclear boundaries (e.g. Figs 5B, 22B). Perikaryal iabelling is a rare exception. Notwithstan~ng the lack of PV in the cell body, some axons deriving from thalamic cells and projecting to the cortex through the capsula interna, show PV immunoreactivity. Thus, in the rat, some thaiamic neurons behave like other long projecting neurons by segregating this protein to axon and terminals.

Nuclei of the diagonal band (Fig. 87) ~a~bindin D-2&k. In the horizontal

limb of the nuclei of the diagonal band CaBP is poorly represented. Parvulbumin. In the horizontal and vertical limb of the diagonal band the antibodies against PV stain some large neurons with few thick dendrites. These neurons are often clustered together.

Anterior nuclear group Calbindin D-28k. The anteroventrai shows terminals and scattered positive neurons. The labelling fades gradually from lateral to medial (Figs 4A, 29A). Parv~~~~~n. The anterodorsal, anteroventral and anteromedial nuclear groups dispiay a rich complement of terminals and axons (Figs 4B, 29B) which are

399

Calcium-binding proteins in the rat brain sharply demarcated from the ventrolateral nucleus by a thin, unlabelled band (arrow in Fig. 29B). Of the three nuclei the anterodorsal nuclear group is the most strongly labelled.

and corresponds to the nucleus of the optic tract, and in a triangular medioventral extension of the posterior nucleus which points in the direction of the retroflex fascicle (arrow in Fig. 22A).

Medial nuclei

rntralami~ar nuclei

Calbindin D-28k. The mediodorsal nucleus has many CaBP+ polymorph neurons at its circumference, clustered as a shell around an unstained central core (Figs 5A, 29A). The CaBP+ neurons merge with similar positive neurons in the paracentral and in the intermediodorsal nuclei (Fig. 29A).

Calbindin D-28k. The central medial, paracentral and central lateral nuclei are consistently stained by antisera against calbindin (Figs 4A, SA, 29A). The small CaBP neurons (15 x 15 pm) merge caudally with the periventricular gray. Within the intralaminar nuclei, the neurons are immunostained all over including the afferent terminals. Future immunoelectron microscopic investigation will shed light on this phenomenon.

Ventral nuclei Calbindin D-28k. The ventromedial nucleus is rich in positive neurons and endings, whereas the other ventral nuclei display clusters of stained neurons, fibres and terminals (arrows in Fig. 29A). They are sharply demarcated medially, but not laterally (Fig. 29A). Particularly striking are bands of CaBP-reactive neurons located in the ventromedial and at the boundaries between ventrolateral and ventroposterolateral (Fig. 29A, left), ventrolateral and the posterior thalamic nuclei, as well as between the ventroposterolateral and posterior thalamic nuclei. Calbindin-IR fibres enter the thalamus from the tegmental tracts. Pa~u~bumin. A rich plexus of PV+ axons and terminals of various calibers is typical for the ventromedial, ventrolateral, ventroposterolateral and ventroposteromedial complex (Figs 4B, 5B, 29B). The highest density of PV+ fibres and terminals is found in the ventroposteromedial nucleus (Fig. 29B). The axon staining can be traced back to the medial lemniscus. The dist~bution of PV+ terminals in the ventroposteromedial nucleus is patchy (Fig. 29B), reflecting perhaps the terminal fields occupied by single whisker afferences. The major iibre tracts ending in the ventral thalamus are rich in strongly PV-IR axons: medial lemniscus, trigeminal tract, superior cerebellar peduncle, ansa lenticularis (see also under fibre tracts). Lateral nuclear group Caibindin D-28k. The lateral nuclear group has a

large number of moderately stained CaBP+ neurons, where only the perikaryon is fabelled. Pa~albumin. The lateral nuclear group possesses the highest density of PV+ terminals following the ventral group. No perikarya are labelled (Fig. SB). Posterior nucleus Calbindin D-28k. A unique band with patches of

calbindin-IR neurons occupies the boundaries of the posterior nucteus (Figs 22A, 29A). At more caudal levels (Fig. 22A), this antiserum reacts diffusely with neurons and processes of the posterior, lateroposterior and suprageniculate thalamic nuclei. The staining in the dorsomedial lip, is particularly intense

The intralaminar nuclei receive afferences from various brain regions and from the inner segment of the pallidus”’ and project to layers I and VI of the cortex and to the striatum. In the cortex, we actually observe terminal fields in layers I -III and V, although the intralaminar nuclei may take part in the projection to layer I, other projection systems (i.e. from the nucleus basalis of Meynert) converge to the same site. The thalamic intralaminar nuclei project to the caudatoputamen4i and this correlates with the homogeneously CaBP-labelfed central core of the caudatoputamen. Parvalbumin. Light terminal fields occur in central medial, paracentral and parafascicular cleus. PV-t neurons are not detected except in parafascicular nucleus. These terminal fields do respect nuclear boundaries.

the nuthe not

Midline nuclei Calbindin D-28k. The rhomboid nucleus is calbindin-negative throughout and its boundaries are clearly delineated. The reuniens, interanteromedial, intermediodorsal and paratenial nuclei contain a large amount of CaBP+ neurons embedded in strongly labelled terminal fields. The paraventricular nucleus is very rich in calbindin-positive terminals (Fig. 29A). Parvalbumin. PV+ fibres cross the midline in the region of the rhomboid nucleus. Terminal fields, axons and single PV+ neurons are found in the paratenial nucleus (Fig. 29B). Ventroposterior nuclei Medial geni~late

complex

Calbindin D-28k. The boundaries of the medial geniculate complex ,are sharply demarcated because of the strong iabelling in the posterior thalamic group (Figs 22A, 29A) and due to an island of neurons in the ventral geniculate nucleus. The ventral medial geniculate complex has many perikarya, being moderately stained. The medial portion has lightly stained, and the dorsal portion stronger, but diffusely stained neurons (Fig. 29A) with enmeshed axons and terminals. The lamella of white matter, covering the medial geniculate complex has many CaBP+ thin axons, running in the frontal plane,

400

M. R. CELIO

Par~o~b~rni~. Ail three subdivisions of the medial geniculate complex display a rich plexus of thin terminals and axons (Figs 22B, 29B). The meshwork is more dense in the medial and ventral portion, as well as in the intergeniculate nucleus. No celI bodies are PV+. Lateral geniculate nucleus Calbindin D-28k. The dorsal lateral geniculate nucleus and the magnocellular ventral lateral geniculate nucleus show similar, moderate amounts of positive neurons and terminals, which are markedly concentrated in the outer rim of these two regions (Fig. 22A). They are continuous with the strong labelling of the intergeniculate leaflet nucleus, located at their boundaries (Pig. 22A). The terminals in the dorsal lateral geniculate nucleus stem from a subgroup of CaRP+ axons in the optic tract. They are filiform, beaded or punctated. Terminal fields form “puffs” of more intense immunoreactivity, separated by areas with fewer immunoreactive processes. Pawalbumin. Positive axons deriving from the optic tract, as well as terminals, are more numerous in the dorsal than in the ventral lateral genicuiate body (Fig. 223), and the two regions are sharply demarcated by the unstained intergeniculate leaflet. Moderately labelled neurons are observed in the most lateral part of the magnocellular ventral dorsal geniculate body (Fig. 4A).

The terminals found in the ventral lateral geniculate nucleus may represent collaterals of small retinal axons (W cells) destined to reach the ventral lateral geniculate nucleus.i’0 The identity of the small PV i- cells in the ventral lateral geniculate nucleus is unknown but their size is compatible with that of interneurons (see also Ref. 258).

mingled PV+ fibres and punctuate structures are found (Fig. 19). The PV+ processes are mostly ordered perpendicular to the course of the thalamocortical and ~orticothalamic radiations traversing the reticular thalamic nucleus. The axons leaving the reticufar thalamic nucleus can be followed to the dorsal and other nuclei of the thalamus, where they end in a profuse way. The endings in the thalamic nuclei do not delineate the shape of thalamic neurons; therefore, they are probably not preferentially located on the soma. Rarely have I seen PV-IR processes piercing the external capsule and projecting rostrally towards the striatum. PV-IR axons deriving from the globus pallidus and coursing in the ansa lenticularis pierce the ventromedial reticular thalamic nucleus. Our observations on the reticular thalamic nucleus and the course of its axons completefy support the careful analysis of others.“‘*239All reticular thalamic nucleus nenr_-. ons stain with PV antisera, within which two different size classes can be distinguished (see Table 2). Some authorszss are even able to classify reticular thalamic nucleus neurons in three different classes by means of Golgi jmpregnation. From a physiological point of view reticular thalamic nucleus neurons belong to two distinct categories of cells’” and they have been found to have a very high firing rate and (see also Table 3). The reticubursting activity rhythmus far thalamic nucleus is immunoreactive towards antisera against the GABA cells marker enzyme glutamate decarboXy~ase’2s.‘88.2” and, in the cat, the “inhibitory” neuropeptide somatostatin. ‘&The targets of the reticular thalamic ._._ nucleus in the thalamus disnlav very high densities of GARA receptors2” The reticular nucleus ii po
Ventral thalam~ Reticular thulumic nucleus Calbindin D-28k. A few, fairly coarse axons and boutons can be found in the ventral blade of the reticular thalamic nucleus whereas only a very faint, homogeneous coloration of the dorsal blade can be seen (Fig. 29A). Pawalbumin. PV is observed in all neurons of the nucleus reticularis thalami. The intense immunostaining delimits this crescent-shaped nucleus from the adjacent internal capsule, and the mediocaudally located thalamic nuclei (Figs 4B, 29B) and the zona incerta. PV-IR neurons are detectable in all parts of the reticular thalamic nucleus as shown by studying horizontal, longitudinal and coronal sections. At the caudomedial extent of the reticular thalamic nucleus, PV-IR neurons form a distinct boundary with the adjacent thalamic nuclei, whereas ventromedially they coalesce with the zona incerta. The PV-IR neurons in the reticular thalamic nucleus are mostly fusiform in shape and occur in two different size classes (see Table 2). In addition to the stained neuronal cell bodies, a large number of coarse, inter-

~alb~~din D-28k. A few, widely dispersed, large, multipolar neurons of the posterior zona incerta display CaBP-IR (Fig. 22A). Par~aZbumin. Neurons in the latero-rostro-ventral aspect of the zona incerta (nucleus ventralisi4’) are immunoreactive towards PV antibodies. Hypothalamus (Figs 88-91)

In general, the hypothalamus is extremely rich in CaBP+ terminal fields and has many calbindinpositive perikarya of various sizes. The distinction of the classical nuclear aggregations is not always possible, and therefore, the description hinges upon general entities. On the other hand, PV is scarce in the hypothalamus. Sex and individual variations are noticed and shall be described in later studies. Peri~e~tric~~ar hypothalamus Cafbindin D-28k.

fibres and terminals Identification of the intensity of labelling varies. A plexus

Neuronal perikarya and thin are prominent (Figs 31A, 32A). various subnuclei is difficult. The of the mostly fusiform perikarya of thin fibres occupies the

401

Calcium-binding proteins in the rat brain subependymal layer. The cell body’s density does not change from the preoptic to the mammillary level. Vascular organ of the lamina terminalis

The vascular organ is occupied by densely packed, small CaBP+ cells, embedded in a dense network of fibres and terminals (Figs 3lA, 33). Many tanycytes, lining the third ventricle anteriorly, are CaBP+ (T in Fig. 33).

the lateral hypothalamic area (Figs 3% 31A). The central-most region of the nucleus has the highest staining intensity, declining gradually towards the borders.

Calbindin D-28k.

Median preoptic nucleus Calbindin D-28k. This nucleus displays small biand multipolar calbindin-positive neurons within a dense terminal plexus (Figs 3lA, 32A). The sexually dimorphic area (SDA) is furred with CaBP + neurons (Figs 32A, 34). Suprachiasmatic

Premammillary The

Tuberal magnocellular nucleus Calbindin D-28k. This nucleus stands out as an unlabelled island in the medial zone (Figs 3lA, 36). Lateral zone

Calbindin D-28k. It has lighter calbindin

Paraventricular

D-28k.

nucleus

terminal fields than the surrounding anterior hypothalamus. Some strongly stained multipolar neurons occur at the medial and lateral periphery of the nucleus.

nuclei

dorsal premammillary nucleus is practically devoid of CaBP immunoreactivity and appears as an unlabelled bilateral stripe in horizontal sections (Figs 30A, 31A). On the other hand, the ventral premammillary nucleus is rich in CaBP + elements. Calbindin

Supraoptic nucleus Calbindin D-28k. Most supraoptic neurons are CaBP+. The axons projecting to the posterior lobe of the pituitary are also positive (see Ref. 83).

nucleus

Both the parvo-, as well as the magnocellular portion, have CaBP + neurons (Fig. 35). The projection from the paraventricular nucleus to the posterior lobe of the pituitary is calbindin-IR (see also Ref. 83). Parvalbumin. Some PV+ neurons are seen in the paraventricular nucleus, particularly in the magnocellular division (Fig. 3 1B).

Lateral preoptic area

Calbindin D-28k.

Calbindin D-28k. Scattered positive neurons occur in much lower concentration than in the adjoining hypothalamic nuclei (Fig. 31A). Parvalbumin. A few large, multipolar neurons are embedded in a meshwork of coarse axons, which run longitudinally. Lateral hypothalamus

Bed nucleus of the stria terminalis (preoptic)

Immunoreactive cells are multipolar in shape and have fairly coarse proximal dendrites, which stand out clearly against a background of terminals. Calbindin D-28k.

Medial zone

In this zone a parcellation with CaBP antibodies is possible. The anterior hypothalamic area and the compact part of the dorsomedial nucleus have low concentration of CaBP elements. The medial preoptic, tuberal, as well as the nuclei at the mammillary level show intense labelling.

Many large, calbindin-positive multipolar perikarya occupy the lateral hypothalamus (Figs 30A, 31A). The terminal fields are much less pronounced than in the medial and periventricular zone. Parvalbumin. A few, large PV+ multipolar neurons are scattered in the lateral hypothalamus, intermingled with PV+ fibres of the medial forebrain bundle. One constant cluster of PV+ neurons is named PRVl (see Figs 3H, 25, 3lB). The large neurons of the tuberomammillary nucleus are PV+ (Figs 26, 31B) and receive PV+ terminals. Calbindin

D-28k.

Mammillary Dorsomedial

nucleus

The dorsomedial nucleus has a rich complement of CaBP+ multipolar neurons. Parvalbumin. It contains neurons similar to those in the lateral hypothalamus. Calbindin D-28k.

Perifornical

nucleus

Calbindin D-28k. CaBP magnocellular elements with slender dendrites occupy this nucleus (Fig. 32A). Ventromedial

hypothalamic

nucleus

Calbindin D-28k. It is extremely rich in CaBP+ terminals and neurons and merges indistinctly with

nuclei (Fig. 91)

Calbindin D-28k. Many neurons of the medial mammillary group of nuclei and of the supramammillary nucleus are calbindin-positive. Particularly intense is the staining of the median mammillary nucleus and of the lateral part of the medial nucleus (Fig. 22A). The posterior part of the medial mammillary nucleus, and the lateral mammillary nucleus, are practically devoid of calbindin-IR sites (Fig. 30A). The gemini-nuclei are strongly CaBP+. Parvalbumin. This is detectable in all mammillary nuclei, but with a decreasing concentration from medial to lateral. Terminals are present mainly in the medial and posterior nuclei (Figs 22B, 30B, 31 B).

Fig. 22. Coronal section of the caudal dien~phaion incubated with CaBP (A) and PV (B) antibodies. (A) Notice ttte bilateral, inverted, night cap-shaped, CaBP-rich zone and particularly the richness in iabelled fibres at the medial tip (arrows). The labelled neurons transgress the boundaries between different thalamic nuclei (cf. B) in the lateral posterior thalamic (LP) and the posterior thalamic (PO) nuciei. It is worth noting the large number of neurons, fibres and terminals in the various mammillary nuclei and in the supramammillary decussation (sumx). The olivary pretectal nucleus (OPT) is characteristically composed of CaBP+ neurons. Widely scattered neurons colonize the Zona incerta (ZI). x 20. (8) The image of the PV-incubated section is dominated by terminals and axons. The PV-immunola~iling allows an exact definition of the cytoarchitectonic boundaries between thaiamic nuclei, Notice the ring of positive neurons around the olivary pretectal nuclei (OPT). The posterior commissure (pc) is devoid of positive axons. x 20. 402

Calcium-binding proteins in the rat brain

Fig. 23. PV-i- neurons and interlacing fibres in the bed nucleus of the anterior commissure (BAC). x 300. Fig. 24. Zone of absent PV immunoreactivity in the auditory cortex of an adult rat (Tel). PV neurons (black dots) are detectable only in the lower half of the picture, whereas terminals occur, with decreasing intensity, also in the upper half. The cause for this phenomenon is unknown, but could be related to disuse of the pathway. x 120. Fig. 25. The PRVl nucleus revealed as a new entity by the PV antibodies. A small number of compact, small neurons between the fibres of the medial longitudinal fascicle (mfb). See also Figs 3H and 31B. Nomarsky optics. x 200. Fig. 26. Neurons of the tuberomammillary nucleus (TM) revealed by the PV antibodies. Large scattered neurons with clumsy dendrites infdtrate the fibres of the medial longitudinal fascicle (mlf). Compare with Fig. 31B. Nomarsky optics. x200. Fig. 27. Two consecutive horizontal sections of the habenula. (A) CaBP label the medial habenula (MHb) and a cluster of cells in the lateral habenular nucleus medial (LHbM). Nomarsky optics. x 200. (B) PV + terminals occur at this level in the lateral habenular nucleus. Nomarsky optics. Dorsal is on the left. x 200.

403

M. R. CELIO

404

PV+ cell bodies occur mainly in the medial parts of the medial nuclei and less so in the lateral mammillary nuclei (Fig. 22B). The mammillary peduncle is composed of PV+ axons (Fig. 32B).

Purvalbumin. Many, strongly positive coarse axons, probably deriving from the entopeduncular nucleus, form a rim in the lateral portion of the stria medullaris thalami (Figs 4B, 29B).

Pretectum (Fig. 91)

Fasciculus retroJexus (Fig. 91)

Nucleus of the optic tract Calbindin 28k. This nucleus is homogeneously and strongly stained with the CaBP antiserum. It is sharply demarcated towards the dorsal anterior pretectal area medially and merges laterally with the lateral posterior nucleus (Fig. 22A). Olivary pretectal nuclei

Calbindfn D-28k. CaBP+ fibres, arising from the medial habenular nucleus, occupy the posterolateral quadrant of the retroflex fascicle (Figs 4A, 14A, 29A). Caudally they take a marginal position (Fig. 22A). Cingulum (Figs 87-9 1) Cafbfnd~n D-28k.

CaBP+

The cingulum thin axons (Fig. 9A). Anterior commissure

Calbindin D-28k. The olivary pretectal nucleus is

rich in CaBP-IR cells (Fig. 22A). Purvafbumin. Immunoreactive cells form a ring around an unstained core (Fig. 22B).

is built up of

The anterior commissure lacks both CaBPf and PV + elements (Figs 3C,D, 14A,B). Axons can, however, be found in the “quaking” mice.

Anterior preie~tuf area Cafbindin D-28k. This is one of the regions poorest in CaBP immunoreactivity in the whole rat central nervous system. Parvafbum~n. PV+ small neurons populate the most rostra1 aspect, whereas the ventral part is occupied by widely dispersed, multipolar neurons. The anterior pretectal area contains a large amount of coarse axons and terminals (Figs 22B, 29B). Fibre systems of the ,forebrain

~edfaf forebrain bundle (Figs 86 and 87) Calbindin D-28k. This antibody

labels diffusely a large portion of the medial forebrain bundle. Parvalbumin. Coarse, probably myelinated axons, deriving from the giobus pallidus compose the overwhelming majority of the medial forebrain bundle, particularly its lateral portion (lateral forebrain bundle)‘72 (Figs 3B,D, 17, 25, 26, 31B, 32B). In cross-section, the field, occupied by the medial forebrain bundle, has trapezoid shape and invades the horizontal limb of the diagonal band and the multiform layer of the olfactory tubercule (Figs 3B,D). Fornix

The fornix consistently lacks CaBP + and PV+ elements, In “quaking mice”, lacking myelin sheaths, the fornix is immunoreactive (see also Table 6). Stria terminafis (Fig. 89) Calbtndin D-28k. The stria terminalis

contains a Large number of extremely thin axons, probably coming from the amygdala (Figs 4A, 5A, 29A). Stria meduffaris thafami (Fig. 89) Cafbind~n D-28k.

Thin axons probably coming from the triangular septal nudei occupy the medial aspect of the stria medullaris thalami.

Calbindin D-28k. The ventral hippocampal commissure displays some bundles of CaBP+ axons, whereas the dorsal lacks CaBP immunoreactivity (Fig. 9A). Corpus calfosum (Figs 86-91) Calbindin D-28k. An extremely rich and compact

contingent of thin CaBP-t axons course in the inner two-thirds of the corpus callosum (Fig. 9A) (except genu corporis callosi, Fig. 28A). The outer one-third harbours loosely arranged CaBP+ axons (Fig. 9A). Suprauptic decussation (Fig. 89) Calbindin D-28k. A large number of axons in the supraoptic decussation contain calbindin-JR (Fig. 3E,G). A large portion seems to derive from the caudatoputamen. Parvalbumin. There is a fair amount of PV f axons (Fig. 3F), probably arising from within the globus pallidus. Mummillothafamic tract (Figs 89, 90) Culbindin D-28k. The mammillothalamic tract i\ composed of thin CaBP+ axons (Fig. 32A). ~a~albumin. A large number of thin PV-t- ;tsonq also course in this tract (Fig. 32B). Mesencephalon Coificufus superior (Figs 92, 93)

The labelling for CaBP is similar to that for PV in the upper two layers and complementary in the lower four layers. This pattern of being complementary is reminiscent of a similar observation, made in the monkey visual cortex“* and adds further support to a morphologic compartmentalization in certain layers of the rat superior collicuius. There are no differences in labelling in the anteroposterior or mediolateral extent.

405

Calcium-binding proteins in the rat brain

Fig. 28. Horizontal sections through the septal region. (A) Richness of CaBP neurons in the lateral (LSI and LSD) and triangular septal (TS) nuclei. Few neurons are visualized in the subfomical organ (SFO). The staining of the ventricular surface is artefactual (see also B). x 20. (3) Few positive neurons in the triangular septal nuclei are PV-IR (arrows). The round, white spots are light-scattering artefacts due to the embedding medium. x 20. Ccdbindin D-28k. Homogeneous, intense immunostaining of the neuropil is found in the zonal and superficial gray layers, as well as in the deep gray

layer (Figs 37A, 38A). Strands of immunoreactive neuropil containing some CaBP+ neurons connect the superficial gray layer with the deep gray layer (Fig. 37A). These strands are regularly spaced (arrows in Fig. 38A) and alternate with the PV + regions (arrows in Fig. 388 see below). The CaBP-immunolabelled cells are embedded in a matrix of enmeshed terminals and dispIay various shapes and sizes (around 12 x 12 pm) and are widely dispersed in both layers. Slightly larger multipolar neurons with slender cell processes are densely packed in the optic nerve layer (Figs 37A, 38A). At least two different size classes (15 x IS/20 x 20 pm) can be differentiated. The optic nerve layer contains many longitudinally oriented CaBP+ axons, whereas the intermediate white matter has only single CaBP-IR axons (Fig. 37A). The ~lb~ndin-positive neurons in the superficial gray layer of the superior coliiculus cannot be drawn. Nevertheless, most types, depicted by others”’ are recognizable. With a few exceptions,“’ neurons in the optic nerve layer have received scarce attention in the Golgi literature, and a classification of the CaBP+ elements therefore is impossibIe. The intensive staining of the unmyelinat~3’ axons of the zonal layer, which may derive from a subpopulation of retinal ganglion cells is interesting.2M Parvalbumin. PV-rich zones are the superficial-, intermediate- and deep gray layers and, to a lesser extent, the optic nerve layer (Figs 37B, 38B). PVC neurons in the superficial gray layer are small and

widely scattered and stain with different intensities. The cell processes are difficult to visualize. Some PV+ neurons in the optic nerve layer and in the intermediate gray layer are strongly immunoreactjve, have bipolar shape and are oriented parallel to the pia mater of the superior colliculus. PV-t- neurons in the deep gray layer are quite large but only their cell body is tagged. The terminal field in the superficial layer is homogeneous, whereas in the deep and particularly in the intermediate gray layer it forms irregular bands, alternating with stripes of lower PV immunoreactivity (Fig. 37B). In horizontal sections the exact nature of these inhomogeneities can best be appreciated (Fig. 38B). The PV+ bands are the products of a confluent network of PV-rich zones. The PV-poor zones coincide with the ~albindin-rich stripes (see Fig. 38A above). The optic nerve layer, the intermediate and the deep gray layers display many PV+ axons; those in the two upper layers are oriented longitudinally. Additionally, PV+ axons are found in the brachium colliculus superius and in the commissure of the inferior colliculus (Fig. 38B). Inferior collicuh

(Figs 93, 94)

Calbindin D-28k. In low power pictures the inferior colliculus is sharply demarcated towards the superior colliculus and central gray region by the paucity of calbindin immunoreacti~ty (Figs 37A, 38A). The immunostaining is limited to an annular peripheral rim, comprising layer I of the dorsal and external cortex (Figs 37A, 38A, 43), which is confluent medioventrally with the central gray (Fig. 37A). In

scattered positive neurons of the trnchiear nu&us

ticethe strong iat& (4 in 8). x 12.

Calcium-binding proteins in the rat brain

Figs 30-31.

407

408

M. R.

CELIO

Fig. 32. Two consecutive sections at the level of Fig. S of Ref. 208, therefore c. 0.5 mm dorsal to Pig. 3 I. (A) The hypothalamic labelling with the CaBP antibody is evident in its richness. Laterally it is bound by the CaBPt tibres of the strionigral pathway (curved, large arrows). Notice the cluster of intensely immunostained neurons of the SDA in the anterior preoptic region (arrow) and the labelled neurons of the basal substantia nigra, compact partfr, retrogex fascicle. (B) PV antibodies visualize the mammillothalamic tract (mt), the stria medullaris thalami (sm) and the mamillary peduncle (mp). The moderate labelling of the surface of the third ventricle is artefactual. Notice the positive axons of the oculomotor nerve (3n, see also Pig. 41). x 20.

Fig. 30. Two cons~utive horizontal sections through the hypo~aIamus at the level of Fig. 53 of Ref. 208. The hypothalamus occupies the centre of the figure and is rich in CaBP (A), but low in PV (B). PV only occurs in the posterior shce, containing the mamm~lla~ nuclei (MP). The anterior portion of the figure demonstrates the complementary nature between CaBP- and PV-immunolabelling, CaBP labels strands of tissue to the pial surface (arrows, A). These oblong isiands are spared by PV antibodies (B), which only colour multipolsr, large neurons. The four small arrows at the top of (A} mark four tongues of immunorea~tivity, belonging to the cellular bridges (CB) (A). The large arrows in (A) mark four regions of diminished CaBP immunoreactivity in the central nucleus of the amygdala. x 20. Fig. 31. Horizontal section at the level of Fig. 54 of Ref. 208 therefore c. 0.5 mm dorsal to Fig. 30. (A) The hypothalamus is somewhat parcellated with the CaBP antibody, but boundaries between various nuclei cannot be discerned at ease. Islands of strongly labelled neurons occur scattered in some hypothalamic nuclei. Notice the unlabelled fornix and the finger-like protrusions of the basal ganglia in direction of the olfactory tubercle (Tu). The dorsal premammillary (PMD) and the tuberal magnocelhtlar (TMC) nucleus lack CaBP. The large arrow on the right lower portion marks the region harbouring the PRVl cluster (see B). The small arrow in the middle upper portion marks the hair of my technician enclosed with the section! (B) PV is virtually absent from the hypothalamus. Few, moderately labelled, magnocellular elements occupy the paraventricuiar nucleus (Pa), but are barely visible at this magnification. Interesting is the ovoidal cluster of small cells embedded in the fibres of the medial forebrain bundle (mfb). This nucleus has, to my knowledge, never been described and receives the denomination PRW. The somewhat larger contingent of widely scattered neurons represents the tu~romamilla~ nucleus (TM) (compare also with Figs 3H, 25 and 26). x 20.

~~ci~-b~~din~

Fig. 33. Higher ma@&ttion

409

proteins in the rat brain

of the rostra1 h~~thaI~mus

in Fig. %A. Nomarsky optics. x 200.

Fig. 34. Higher magnification of the SDA of Fig. 32A. Nomarsky optics. x 200. Fig. 3.5. Higher magnification of the anterior hypothalamic area in Fig. 32A. Nomarsky optics. x 200. Fig. 36. Higher magnification of the caudal periventricular hypothalamus of Fig. 3 IA. Nomarsky optics. x 200.

these layers, neurons are labelled in various intensities; most have bipolar shape with their long axis parallel to the pial surface (Fig. 43). These calbindinpositive cells are embedded in a strongly stained neuropil. The extemd nuchs regularly shows two to three large CaBP+ neurons in layer 3 at the border to the superior colliculus. Layer 3 of the external nucleus and the central nucleus are infiltrated with a light meshwork of thin CaBP-IR axons, displaying many “boutons terminaux” (Fig. 43). ~~~~~~~~~. In low power pictures, the immunostaining with PV is complementary to that of calbindin. Layer I of the dorsal and external cortex’? remains unreactive and PV is concentrated in the central nucleus as well as in the pericentral nucleus and in the lower layers of the external cortex. In these two locations, large amounts of PV + cells of various diameters (some giant in the centra1 nucleus, with sizes of 35 x 47 pm) are intermingled with a profuse network of coarse, PV-IR processes (Figs 3X3, 38B). Patches of clustered, small, moderately immunoreac-

tive cells and intermingled axons are found in layer 3 of the pericentrai nucleus and of the external cortex (arrowhead in Fig. 44). Probably due to the particular o~eutation ofthe dendritic tree, it seems as if only the somata stain. The central nucleus shows a hint of lamination in the distribution of PV+ cells. Punctate structures occur on the soma of PV- neurons and, rarely, on PV+ perikarya. Many “boutons” are also present in the neuropil (Fig. 44). The variety of PV-IR cells possibly represents projection neurons, while some may be intemeurons. PV+ neurons of the central nucleus, a relay station in the ascending auditory pathway, project to the medial geniculate nucleus. Many PV+ axons cross the midline in the commissure of the inferior collicle (Figs 38B, 39). Prerubral

Jipld (Fig. 9 1)

Pff~ul~urni~. Large, multipolar neurons canfer a reticulated pattern to the prerubral field (Fig. 22B).

Fig.- 37. Sag&al,

strands of immunoreactivr! cells bath, in the intermediate,

and deep gray layers of the superior colkulws (A, 3). x 25.

411

Calcium-binding proteins in the rat brain Red nucleus (Fig. 92) Calbindin D-28k. Large, CaBP+ neurons with spherical perikarya dominate the caudal part of the red nucleus. Only the proximal portion of dendrites contains calbindin (Fig. 45A) and the axons cannot be traced. Parvalbumin. Coarse parvalbuminic axons impinge upon the neurons of the parvo- and magnocellular portion of the nucleus ruber (Fig. 45B). A portion of these fibres continues rostrolaterally in the direction of the ventral thalamic group. A few, polygonal PV + neurons are present. Their number, particularly in the magnocellular portion, increases dramatically after colchicine application (see Table 6). Oculomotor nucleus (Fig. 92) Calbindin D-28k. Scattered neurons, located particularly at the posterior end of the oculomotor nucleus are positive (Fig. 29A). Parvalbumin. This nucleus is crowded with a large amount of fibres and terminals (Figs 29B,49). The perikarya are only faintly reactive, but the axons are highly PV + and leave the brain with the oculomotor nerve (Figs 32B, 41). Colchicine application produces an accumulation of PV in the cell bodies.3 During ontogeny, the perikarya of the oculomotor nucleus are one of the first systems to express PV immunoreactivity.‘83,254

calbindinic neurons are grouped between the locus coeruleus and the laterodorsal tegmental nucleus (Figs 46A, 47). This cell cluster may correspond to the “Barrington nucleus”.208The presence of CaBP in the locus coeruleus of the rat was reported,95but the same group could not confirm this result in human materiaL9’

Nucleus raphe dorsalis (Figs 93, 94) Calbindin D-28k. The rhombencephalic

portion of the raphe dorsalis harbours CaBP+ neurons (Fig. 58C), whereas the mesencephalic portion is devoid of CaBP. The neurons are strongly calbindin-IR and are of medium size (20 x 20 grn) with dorsoventrally oriented cell processes. Most CaBP+ neurons are segregated in the ventral portion of the nucleus. Other raphe nuclei Calbindin D-28k.

The nucleus raphe pontis is strongly immunoreactive populated by many, neurons, which merge laterally with the pontine reticular nucleus. The median raphe nucleus is filled with many thin terminals and has a few, moderately immunoreactive neurons. In both, the caudal and rostra1 linear nuclei, calbindin-positive neurons are predominant.

Trochlear nucleus

Interpeduncular nuclei (Fig. 92)

Calbindin D-28k. Compared with the oculomotor,

the trochlear nucleus displays more positive neurons and terminals (Fig. 29A). Parvalbumin. The trochlear nucleus has fewer terminals than the oculomotor nucleus, but the PV+ neurons are better delineated (Fig. 29B). They have slender cell processes and are of the bipolar type. PV+ axons course in the trochlear nerve. The ontogenyzS4 and the reaction to colchicine application3 are similar to that found in the oculomotor nuclei. Tegmental nuclei (Fig. 94)

The immunolabelling is complementary for the two calcium-binding proteins. Calbindin D-28k. Antibodies tag terminals in the medial portion of the ventral tegmental nucleus and widely scattered neurons in the lateral part of the dorsal tegmental nucleus (Fig. 46A). Parvalbumin. Immunoreactive neurons and terminals occupy the anterior tegmental nucleus, the lateral portion of ventral tegmental nucleus and the medial aspect of dorsal tegmental nucleus. The intensity of staining is higher in ventral tegmental nucleus than in dorsal tegmental nucleus (Fig. 46B). Axons project to the mammillary bodies through the mammillary peduncle. Locus coeruleus (Fig. 94) Calbindin D-28k. In the locus coeruleus itself there are no immunoreactive neurons (Fig. 59A), but many

The distribution of CaBP and PV does not match the distribution of classical neurotransmitters.“’ Calbindin D-28k. Some interpeduncular nuclei have an homogeneous but light reaction, which is incremented in the paramedian and apical interpeduncular nucleus to a high degree of punctate staining. The central nucleus (Fig. 32A) is devoid of CaBP. Moderately immunoreactive neurons and fibres are seen in the whole extent of the outer posterior subnucleus (Fig. 51). Most positive fibres enter the interpeduncular nuclei with the habenulointerpeduncular tract. Paroalbumin. The PV antibodies immunostain the neurons of the interpeduncular nuclei quite homogeneously. Those in the central nucleus mostly have an elongated bipolar form and are oriented sagittally (Figs 32B, 52). Nucleus

Darkschewitsch

and

interstitialis

Cajal

(Fig. 91) Calbindin D-28k. In both locations, CaBP immunoreactivity occurs in only a few multipolar neurons and in terminals (Fig. 29A). Parvalbumin. Pavalbuminic cell bodies occur in both neuronal aggregates; fibres and terminals are abundant, particularly in and around the Darkschewitsch nucleus (Fig. 29B). This latter is separated from the oculomotor nucleus by an oblique, lightly stained septum (Fig. 29B).

412

M. R. CELIO Central gray (Figs 92, 93fibY

Calbindin D-28k. This antibody strongly but diffusely labels the central gray region (Figs 4A, 29A). On this background, many calbindinic neurons further subdivide the central gray. The neurons are polymorphic, small and have a fairly constant diameter of 20 x 20 pm. The dorsal central region has less CaBP+ terminals and cells. The neurons of the Edinger-Westphal nucleus are stained with CaBP antibodies and this nucleus also displays a higher terminal density. Parvalbumin. Immunoreactivity is detectable in the large soma and proximal dendrites of the neurons in the mesencephalic trigeminal nucleus (Figs 37B, 46B). With the exception of some axons, running in all directions, the central gray matter is characterized as being one of the regions with the lowest concentration of parvalbuminic sites within the whole rat nervous system (Figs 4B, 29B). Formatio reticularis Calbindin D-28k. A cluster of CaBP+ neurons with contorted dendrites occurs in the medial part of the gigantocellular nucleus. Other clusters are seen in the parvocellular part (Fig. 59A) and one accumulation of CaBP+ neurons probably coincides with the linear nucleus of the reticular formation (not shown). Other distinct CaBP+ cell ciusters are seen Iaterodorsally in the reticular formation but cannot be related to any known nuclei. In cross-sections through the rostra1 medulla, many large neurons with dorsoventral axis are scattered in the lateral aspect of the media1 longitudinal fascicle (Figs 53, 54, 58C, 95A). In cytoarchitectural, histochemical and functional respects, the presence of a nucleus in this location has never been described in the literature, but some of these neurons probably correspond to adrenaline cells. I24This agglomerate of calbindinpositive neurons is called CaBPl. At mesencephalic levels, the cuneiform nucleus is composed of calbindinic neurons and terminals, which merge medially with the central gray. Parvalbumin. In general, there are many more fibres and neurons stained with the PV antiserum, than with the CaBP antiserum (Figs 57, 59B). PV+ cell bodies occur in all portions of the reticular formation, and the staining of the neurons of the gigantocellular and parvocellular part is particularly intense (Fig, 59B). The paragigantocellular part is pierced by innumerable, coarse PV+ axons. Area postrema (Fig. 97) Calbindin D-28k. In a cross-section

of the lower medulla, the area postrema stands out as a strongly positive disk (Figs 48, 56). At higher magnification this corresponds to the labelling of cells, fibres and terminals. The size of the bipolar calbindin-positive neurons varies. but mostly they are small (8%l0pm) (Fig. 48).

Parua~b~min. The area postrema displays few PV+ neurons at its inferior periphery. They have thin, delicate dendritic ramifications, some of which penetrate the area postrema and some the solitary complex. Trigeminal nuclei (Figs 94-97) Calbindin D-28k. Only a few neurons in the principal sensory and in the spinal trigeminal nuclei are calbindin-IR (Figs 58A, 59A). The motor trigeminal nucleus is negative for this antigen (Fig. 58A). The spinal tract of the trigeminal nerve harbours some coarse CaBP+ axons (Fig. 59A). Parvalbumin. PV+ are the large cells which form a narrow stripe in the lateral margin of the periacqueductai gray, and represent the mesencephalic trigeminal nucleus (Figs 37B, 46B). Not only the cell soma, but also the diverging cell processes contain PV (Fig. 55). The peripheral branch is known to innervate the muscle spindles of the muscle of mastication,’ the central branch impinges upon the neurons of the motor trigeminal nucleus. In the principal sensory trigeminal nucleus, PV-immunostaining forms isfands of immunoreactivity, separated by strands deprived of immunoreactive terminals (Fig. 58B,D). The PV+ neurons are small. In the nucleus of the spinal tract (Sp50, Sp51, and SpSC) the situation is identical (Fig. 59B). The spinal and mesencephalic tract of the trigeminal nucleus are made up of thin PV-t- axons. ~‘estibu~arnuclei complex (Figs 95, 94)

Calcium-binding proteins are not helpful markers in delineating the subnuclei of the vestibular complex. In fact, variations in the terminal density in various parts of the vestibular nuclei are just discrete. With the exception of the endings of the vestibular nerve and the Purkinje cell terminals on neurons of the lateral vestibular nucleus, the derivation of ail other axons and terminals is impossible to discern. The perikarya of vestibular nuclei neurons are negative for both PV and CaBP (with a few exceptions) but accumulates PV after colchicine application. PV-mRNA can be detected in the perikaryon of vestibular nuclei neurons.” PV+ fibres can be found in the vestibulospinal tract. Ca~bj~d~~zR-28k. All vestibular nuclei harbour some CaBP+ fibres and terminals (Figs 4A, 58A,C). The superior vestibular nuclei display a delicate meshwork of thin, beaded terminals (Fig. 59A). The giant neurons of the lateral vestibular nuclei are contacted by many CaBP+ terminals, impinging upon their surface. The spinal vestibular nuclei has the highest terminal density in its lateral portion and is crossed by transverse bundles of CaBP+ fibres. The medial vestibular nuclei has longitudinal bundles of thin CaBP+ fibres. Parvalbumin. All vestibular nuclei contain an enormous concentration of thin PV+ terminals and axons (Figs 4B, 58B,D, 59B). The lateral (Deiter’s) vestibular nucleus is traversed by innumerable

413

Calcium-binding proteins in the rat brain

axons of Purkinje cells, which also contact the perikarya and proximal dendrites of its giant (60 x 60 pm) neurons. These terminals are so tightly packed that they define the contour of the soma. The spinal and lateral vestibular nuclei are pierced by longitudinal bundles of PV+ fibres. The medial vestibular nuclei have bundles of thin lon~tudinal and the internal portion of the spinal vestibular nuclei transverse tibre bundles. Some immunost~ned perikarya occur in the medial vestibular nuclei.

coarse

The characteristic distribution of PV in the vestibular system has recently been reportedIs (see also Ref. 254). Cochleur nuclei (Figs 94, 95)28’ Calbind~n LL28k. A few CaBP-IR terminals are observed in the dorsal and ventral cochlear nuclei (Fig. ,58A,C). Quite often solitary CaBP+ neurons resembling Purkinje cells can be observed in a subpial position of the ventral cochiear nucleus. The pasterior ventral cochlear nucleus harbours an island, composed of a group of calbindin-~sitive neurons (arrowhead in Fig. 58A). These cells are round and large and a few thick stem dendrites diverge from the cell body. Parvulbumin. The ventral anterior cochlear nucleus is characterized by intermingted bundles of coarse PV+ axons and terminals (Figs %B,D), The posterior cochlear nucleus shows a similar pattern, but the axons are more numerous and of smaller diameter. PV+ “boutons terminaux” are observed on the soma of PV- ventroposterior cochlear nucleus neurons. Very strong PV-IR neurons in the ventral nuclei are also found in subpial location, embedded in a thin rim of delicate cell processes, pa~icularly in the posterior aspect of the nucleus. Many, strongly PV+ neurons are interspersed in the anterior aspect (Fig. 58B). In the subpial convex region of the dorsal cochlear nucleus, small PV+ perikarya represent only a minority of the neurons. The whole extent of the dorsal cochlear nucleus is invaded by numerous, beaded axons and terminals (Fig. 58B,D). Abducens nucleus Ca~bindin D-28k.

A drop-like immunoreactive area, lateroventral of the ~lbindin-positive medial lon~tudinal fascicle contain a wealth of extremely thin ~lbi~din-positive terminals. Only a few immunoreactive cell bodies, however, can be visualized, Parvalbumin. All neurons of the abducens nucleus are moderately PV-IR. The axons cross to the contralateral side and leave the brainstem basally, Many PV+ terminals “light up” this nucleus in crosssection (Fig. 50). The ontogenyzs4 and the reaction to colchicine application3 are similar to what is found with the oculomotor nuclei.

inferior olivary nucleus rather intensely (Fig. 56). Axons crossing to the contralateral side and projecting to the cerebellum are visualized by the calbindin antisera, too. However, terminals cannot be discerned in the cerebellum. The subdivisions of the superior olivary nucleus as well as the periolivary nuclei have a high concentration of coarse axons and terminals (Fig. 59A). The oliv~ochlear bundie is CaBP- . Parvalbumin. The amount of terminals and fibres in the medial, superior and lateral olivary nuclei is alike (Fig. 59B). They coalesce with those in the superior and ventral periolivary nuclei and form a compact oblong mass. The neuronai cell bodies are faintly PV+ compared with the strong staining in the adjacent medial trapezoid nucleus (Fig. 59B). Nucleus of the trapezoid body (Fig. 94)

The neurons of the nucleus of the trapezoid body are intensely stained with both, the CaBP as well as the PV antisera (Fig. 59A,B). Innumerable PV-IR (but a few CaBP-IR) axons course in the trapezoid body (Fig. 59A,B). Nucleus tractus soiitarii (Figs 95-97) Ca~bindin D-28k. The nucleus of the solitary tract is labelled by the CaBP antibodies in an homogeneous and diffuse manner, particularly in its medial aspect (Fig. 56). The immunoreactive boundaries are, medially and dorsally, sharply demarcated but merge indistinc~y with the reticular fo~ation ventrofaterally. Immunoreactive cell bodies are mostly bipolar and small in the medial and gelatinous nucleus of the solitarius, and large, multipolar in the lateral solitarius. The solitary tract harbours CaBP+ axons. A clustering of terminal fields in the nucleus of the solitary tract suggests a further subdivision of this nucleus (Fig. 56). Pa~albumin. PV is absent from the nucleus tractus solitarii, but a delicate meshwork of PV-IR “boutons” separates the nucleus of the solitary tract from the dorsal motor nucleus of the vagus (arrow in Fig. 57). Dorsal motor nucIeus of the vagus and nucleus ambiguus (Figs 96, 97) Calbindin D-28k. Calbindin

Parvalbumin. A few neurons in the anterolateral aspect of the dorsal motor nucleus are PV-IR and give off processes, which inte~ingie with neighbouring axons (Fig. 57). PV+ neurons of similar size are also found in the ambiguus, although they are stained less intensely. In the ambiguus there are also many PV+ terminals.

O&vary u~d perioliuary nuclei (Figs 94, 96-97) ~a~bi~din D-28k. The calbindin

ably stains all neurons NSC 35/2-H

antiserum probin all subdivisions of the

is absent from both

nuclei.

~emnis~al nuclei (Fig. 93f Ca~bi~di~ D-28k.

homogeneous

CaBP-IR cell bodies of fairly shape and size, as well as moderate

M. R.. CELIO

414

coarse processes occupy the paralemniscal nucleus, and the dorsal and ventral Iemniscal nuciei and spherical nucfeus of the dorsal lateral lemniscus (Figs 4A, 29A, 464). A larger contingent of strongly labelled neurons embraces the ventral Iemniscal nucleus medio~ranially (Fig. 58A,C). The paralemnis~al nucleus is crowded with terminals. Parvalbumin. In the ventral and dorsal nuclei of the lemniscus lateralis all neurons and their processes are strongly PV+ and confer a reticulated pattern on them (Figs 48,29B, 46B). The intensity of staining is higher in the axons of the lateral lemniscus and in the terminal arborization, than in the neurons of the nucleus. The ventral lemniscal nucleus displays many more terminals than the dorsal lemniscai nucleus (Fig. 58B,D). Parabrachiul nuclei (Fig. 94) C~lbindin D-28k. The dorsal parabrachial nucleus has a huge terminal field, in which many immunoreactive neurons are embedded (Fig. 46A). The ventral parabrachial nucleus consists of a posterior portion rich in CaBP+ neurons and an anterior, unlabelled part (Fig. 46A). Prepositus hypoglossal nucleus (Fig. 95) Pa~album~n. Many immunoreactive cells occur in the prepositus hypoglossal nucleus embedded in a matrix of terminals (Fig. 58D). Dorsal column nuclei (Figs 96, 97) Calbindin D-28k. Immunoreactive axons travel with the cuneate and gracilis fascicles and end in the respective nuclei (Fig. 56). The gracile nucleus also ha&our some strongly positive, small neurons. The CaBP+ neurons in the cuneate nucleus are polymorph. The external cuneate nucleus is devoid of immunoreactivity. Pa~albumin. The lateral and medial cuneate nuclei, as well as the gracilis and Z-nuclei receive a large amount of PV+ terminals (Fig. 57). Faintly immunoreactive perikarya are detected in the nucleus gracilis and in the cuneatus medialis, and lateralis. Their form and size is variable.

Cerebellum (Fig. 96)

The majority of Purkinje cells displays at least three different calcium-binding proteins in coexistence: calmodulin,‘s9 CaBP95*‘SS and PV.42 Culbindin D-28k. Purkinje cells in all folia of the cerebellum are labelled by the CaBP antibodies (Fig. 60). The entire extent of the cell, in&ding nuclei, dendritic spines (Fig. 61) and axon terminals in the deep cerebellar nuclei, is immunostained. The Purkinje cell staining is defective in certain cerebeilar folia of some animals, but this phenomena has not been quantified or particularly mapped. CaBPf Golgi cells are never seen (see, however, Ref. 95). The only other structure, tagged by the CaBP antibodies, are axons of at least two different calibers, bundled in the white matter and in the granular layer. Some of these axons are beaded and have collaterals forming a meshwork subjacent to the Purkinje cell layer. In lobe “X” of the cerebellum this “juxta-Purkinje plexus” oriented parallel to the surface of the cerebellum is denser than in other lobes. Beaded terminals also delineate the contours of fusiform neurons in the upper granular cell layer. These neurons, probably “Lugaro” cells, have their cell body oriented parallel to the pia mater. No CaBP+ climbing fibre afferents are seen in the molecular layer. Parualbum~n. All folia of the cerebellum stain in exactly the same manner. The most conspicuous staining is represented by the Purkinje cells, which are labelled in their completeness, including nuclei, soma, dendritic arborizations and dendritic spines, axons and terminals (Figs 62-63). Time and again, in some specimens the PV labelling of some Purkinje cell bodies is defective. In the stratum moleculare, the basket and stellate cells are completely PV+. No perikarya are stained in the granular layer. Purkinje-, basket- and stellate cells are all GABAergic neurons. Golgi cells are also supposed to use GABA as their neurotransmitter,‘~~ but do not show PV immunoreactivity. Basket-cells in the cerebellum were the first inhibitory interneurons in the mammalian brain to be recognized electrophysiologically.” Of interest for our hypothesis on the role of PV in the brain (see later and Ref. 47) is that typical discharge frequency of basket-cells is around 700/s -._-

Fig. 39. Crossing of PV+ axons in the commissure of the inferior colliculus (CIC) in a coronal section (see also Fig. 38). Dorsal is on the left. x 120. Fig. 40. Crossing of PV+

axons in the “decussatio pedunculorum superiorum” (xscp). Rostra] is up. x 120.

Fig. 41. Bundfes (arrows) of PV-t axons of &heoculomotor nerve (3n). See aIso Fig. 328. x 120. Fig. 42. Ectopic “Purkinje cells” in the superior colliculus of a CBS7 mouse (arrow). x 120. Fig. 43. Section of the inferior colliculus labelled with CaBP antibodies. The labelling is mainly confined in the external nucleus (EIC). x 120. Fig. 44. Consecutive section to Fig. 43 incubated with PV antibodies. Notice the richness of PV in tbe central nucteus (Cn) and the island of neurons in the external nucleus (EIC) (arrow). x 120. Fig. 45. (A) Horizontal section through the red nucleus incubated with CaBP antibodies. Magnocellular elements are visualized. x 120. (B) Consecutive section to A incubated with PV antibodies. Very dense concentration of terminals and axons. x 120.

Calcium-binding proteins in the rat brain

Figs 39-45.

415

416

M. R. CELIO

Fig. 46. Horizontal section at the level of the locus coeruleus (LC). Notice the absence of CaBP (A) and PV immunola~lling (B) in the locus coeruleus but a well defined cluster of CaISP-IR neurons (arrow) between locus coeruleus and the laterodorsal tegmental nucleus (LDTg) (arrow). This conglomerate probably represents the Barrington nucleus (Bar). The dorsal raphe nucleus (DR) displays CaBP+ neurons. The labelling of ependymal cells is absent in controls. Notice the strong labelhng of the parabrachial nuclei with CaBP antibodies (DPB, VPB). x 20.

and that they show great excitatory afferent convergenceiz9 (see also Table 3). Golgi cells, on the other hand, are slow firing neuron@ (c. 15 spikes/s). The CaBP and PV-IR axons of Purkinje cells travel individually in the molecular layer and then are bundled in the white matter (Fig. 63). Here, they are geometrically stratified with unstained lamellae of axons. The CaBP+ and

PV+ axons of Purkinje cells impinge upon the soma and proximal dendrites of neurons in the various cerebellar nuclei. No afferents are discerned ending in glomerula of the granular layer. However, a bimodal distribution of axon calibers is seen in the white matter. In the paraflocculus, some of these fibres represent axons coming directly from the vestibular organ (vide infra).

Fig. 47. Magnification of the locus coeruleus (LC) demonstrating the virtual absence of stained perikarya in the nucleus itself but the occurrence of a cranial cap of positive neurons (Bar, Barrington nucleus). Horizontal section. Nomarsky optics. x 200. Fig. 48. Coronal section of the area postrema (AP). Notice the terminals labelled in the nucleus tractus sohtarius (Sol) and the positive neurons in the AP. CaBP antibody. x 120. Fig. 49. Cross-section through the oculomotor nucleus (3) showing some PV + perikarya. x 200. Fig. 50. Cross-section through the abducens nucleus showing the PV + perikarya. G7, genu of the facial nerve. x 200. Fig. 51. Horizontal section of the inte~duncuiar

central nucleus. CaBP. Nomarsky optics. x200.

Fig. 52. Following section to 51 incubated with PV antibodies. Nomarsky optics. x200. Fig. 53. A “new” nucleus in the rat rhom~n~phalon is revealed by the CaBP antibodies (large arrows, CaBPl, see also Figs 58C and 95A). Coronal section. x 120. Fig. 54. The same nucleus can also be detected in the mouse brain (large arrow). x 120. Fig. 55. Higher magnification of the perikarya and proximal dendrites of PV+ mesencephalic trigeminal nucleus. Nomarsky optics.x 500.

neurons in the

Calcium-binding proteins in the rat brain

Figs 47-55.

417

418

M. R.

CELICI

Fig. 56. Coronal section of the upper medulla. The antibody against CaBP lights up the dorsally located area postrema (AP), the arc-shaped inferior olive (IO) and the solitary nucleus (Sol}. All subnuclei of the inferior olive are labelled with similar intensity. With the exception of the inferior olive-staining this picture resembles substance P labelling at the same brain level (see Fig. SD in Ref. 63). Fig. 57. The PV antibody lights up cross-cut axons in the whole coronal section. The only axon-free region are the pyramidal tract (py) and the nucleus of the solitary tract (Sol). The labelling of the AP is background. x 20.

CaBP- and PV-IR Purkinje cells are not uncommon in extracerebellar localizations, for example, in the superior colliculus {Fig. 42) and in the dorsal cochlear nucleus (see also Ref. 68). In “quaking” mice, a neurological mutant with a primary myehn defect,24Rthe number of these misplaced Purkinje cells is higher (unpublished observation). Deep cerebellar nuclei (Fig. 95)

Neither CaBP-, nor PV-IR neurons can be seen in the medial, anterior and posterior interposed and dentate nuclei. These nuclei have a large amount of terminals associated with the surface of their perikarya. Nevertheless, immunorea~tivities appear in the somata after coichicine application.’ See also Table 6. Fibre tracts

CaBP-IR peduncles.

axons are absent

from all cerebellar

PV+ axons form the majority of axons in the inferior and superior cerebellar peduncles and in the decussatio pedunculorum superiorum (Fig. 40). The middle cerebellar peduncle is devoid of PV-IR axons. Spinal cord (Fig. 98) Calbindin D-28/c. Perikarya, terminals and axons in both the gray and white substance of the spinal cord are tagged by the CaBP antiserum (Fig. 64A,C). CaBP + terminals are randomly dispersed in the gray matter, with particular aggregation around the central canal. The largest concentration of CaBP-lneurons is in Rexed layers I and II, where a myriad of small cells together with their processes are labelled (Fig. 64A,C). Some of these cells have longitudinally oriented cell processes. The CaBP+ neurons in layer I may represent subpopulations of those described by other authorsts8 Other clusters of a few cells are

Fig. 58. Four consecutive horizontal sections through the vestibular and cochlear nuclei incubated with CaBP (A, C) and PV (B, D) antibodies. The upper pictures are more dorsal than the lower. The vestibulocochlear pathway is obviously richer in PV (B, D) than in CaBP (A, C). Only a few, scattered neurons are labelled with the CaBP antibody (arrow in A). Notice the strong PV+ terminal field in the ventral nucleus of the lateral lemniscus (VLL). The genu of the facial nucleus (g7) stands out as an unlabelled island with both antibodies. Notice ependymal labelling (arrow in A) particularly in the calbindin-incubated sections and the CaBPl nucleus (see also Figs 53, 54 and 95A). x 20. 419

420

M. R. CELIO

Fig. 59. Consecutive sections incubated with CaBP (A) and PV antibodies (B). Notice the richness in cross-cut axons in the PV-incubated section and the presence of many labelled neurons in the gigantocellular nucleus (Gi). Notice the circumscript ependymal (arrows). x 20.

found in layers IV (small cells) and V (large cells) and in layers VIII and IX, intermingled with unstained neurons. These latter CaBP+ neurons are sometimes of large diameter (20 x 50 pm) and have widely branching dendrites piercing through the axon bundles of the anterolateral funiculi. Contrary to observations in the fish spinal cord’I we do not find labelled motoneurons. Single, large and slightly immunostained cells in the area of lamina VIII give rise to

processes which cross to the contralateral side. All funiculi exhibit some CaBP-IR axons of various diameters. Particularly rich in CaBPf axons are the dorsal aspect of the dorsal (Fig. 64C) and the anterior funiculi. CaBP+ neurons, fibres and terminals are abundant in the lateral cervical nucleus, whereas the spinal lateral nucleus only displays terminals. CaBP+ cells are sometimes found in subpial location in the dorsal funiculi. In the lumbosacral spinal cord

421

Calcium-binding proteins in the rat brain bipolar large neurons of layers III-IV have a dendritic field oriented obliquely to the axis of the spinal cord. The l&lling of dorsal horn lamina II in the human spinal cord has been reported by others.” Pu~~l~u~~. In general, the PV-immunostaining in the spinal cord is much more abundant than that observed with CaBP antisera (Fig. 64B,D). PV-IR structures are evident in both gray and white matter of all segments of the spinal cord. In the gray matter, positive perikarya, fibres and terminals are found in the dorsal and ventral horn. In the white matter, parvalbuminic axons are seen in all funiculi. The neuropil throughout the spinal cord exhibits PV+ structures in different concentrations. In the dorsal horn, punctate immunoreactive sites intermingled with small ovoidal or spindle-shaped cells are limited to a convex-curved band running parallel to the posterior surface of the dorsal horn (Fig. MB). In comparison with adjacent Cresyl Violet stained sections, this band corresponds to the inner lamina II (IIb) and to a small portion of outer III of Rexed. This kind of PV immunoreactivity in the dorsal horn gray matter is constantly found along the whole extent of the spinal cord, from the lower coccygeal segments up to the cervical segments and is also detectable in the spinal trigeminal tract (pars caudalis). The PV-IR cells in Rexed lamina IIb can be confidently diagnosed as representing islet cells. In fact, their typical fusiform shape in longitudinal sectionsX*lO’ and classical location in the innermost part of the Roland0 substance” are “morphognomonic”. The punctate structures seen in cross-sections represent perpeadiculariy sectioned tufts of the lon~tu~~~y oriented dendritic and axonal trees. A participation of terminals of thin primary afferents to this labelling in layer IIb seems unlikely, but cannot be ruled out. Islet cells belong to the class of short axon cells and have axo-axonic contacts with incoming primary afferences. Neurot~sin-positive cell bodies in the dorsal horn of the spinal cord* have a similar distribution as compared with those containing PV. However, the neurotensin punctate reaction product is disseminated in the whole substantia gelatinosa Rolandi. The neurophysiological behaviour of isletceilshas not been studied by intra~llular recordings.

Laminae I and IIa are devoid of immunoreactive except for PV+ fibres entering from the dorsal root, or running from the dorsal funiculus to deeper layers in the ventral horn (vide infra). The remaining dorsal horn (laminae III-V) and the medial aspect of the intermediate gray (lamina VII) shows moderately strong concentrations of punctate PV + reaction products. The area around the central canal (layer X), the nucleus cervicalis centralis, lamina VI in the cervical spinal cord and the motoneuron region (lamina IX) exhibit a high terminal density. Besides the small neurons in layer IIb, accumulations of PV-IR cell bodies are observed in the nucleus cervicalis centralis of the cervical spinal cord, and some in the Clarke-Stilling nucleus of the thoracic spinal cord. Single cells of various diameters are scattered across layers III-IV and V of the dorsal horn in the thoracal segments. PV-IR arcuated cells can also be seen in the dorsal gray commissure of the thoracal segments (dorsal commissural nucleus). In lamina VII of the cervical and the lumbal segments, some PV-IR cells are situated dorsomedially, with respect to the motor nuclei. Somewhat larger perikarya appear in lamina VIII of the cervical and lumbal enlargements. At all levels of the spinal cord, very small, intensely PV+ neurons are observed at the border between gray and white matter in lamina VII (and VIII) and rarely even in lamina IX, intermingled with motoneurons. Many neurons, particularly in the Clarke-Stilling nucleus, in the central cervical nucleus, and the motoneurons in the ventral gray matter have punctuate reaction products associated with their somata. The PV-IR cells in other areas of the spinal cord gray matter are more difficult to classify, because only a few cells are identified anatomically and physiologically. structures,

The cells in the Clarke-Stilling column and nucleus intermediomedialis are small and may represent interneurons. The larger PV-IR cells positioned dorsomedially to the motor nuclei (lamina VII) can represent Ia inhibitory intemeurons, and the smaller ones at the border between gray and white matter in layers VII and VIII, Renshaw cells. The only combined morphophysiologic study on Renshaw cells,‘3’ showed that in the cat these cells have a diameter of 1CH5 ,um and are located at the medioventral border of the

Table 4. Selected examples for the relation of cell body density and axon terminal density CaBP Cell bodies high/terminals low

Glomeruli olfactory bulb Septal nuclei

Cell bodies low/te~nals

Subst. nigra pars compacta Deep cerebellar nuclei

high

Cell bodies high/terminals high Cell bodies low/terminals low

Caudatoputamen Periacqueductal gray Hypo~~amic nuclei Inferior colliculus Anterior pretectal area

PV Nucleus reticularis thalami Globus pallidus,/nucl. entopeduncularis Sub@. nigra p. reticulata Dorsal thalamic nuclei Deep cerebellar nuclei Vestibular nuclei Cerebral cortex Hippocampal pyramidal layer Inferior collicuius Periacqueductal gray

The discrimination between regions rich in cell bodies and regions with many terminals may be important for the correlation with receptor mapping studies.

422

M. R.

motor nuclei. Renshaw cells utilize glycine as their neurotransmitter (or GABA6’) and display the highest firing rate of all neurons studied to date (see aiso Table 3). la inhibitory interneurons are also characterized by their high discharge frequency, which can reach ~OOHZ.‘“~The various P?‘+ neurons scattered in the ventral spinal gray matter of layer VII, probably represent propriospinal interneurons, although the presence of PV-IR large axons in the anterior commissure points to the existence of projection neurons, giving rise to crossed ascending fibres.

In the white matter, all funiculi contain large numbers of immunoreactiv~ axons, with the exception of the central part and of a wedge-shaped dorsal portion of the dorsal funiculus. Strong, homogeneous immunoreaction in the axoplasm of large axons is observed in the fasciculus gracilis and cuneatus (Fig. 64D) as well as in the medial part of the central funiculus. The diameter of the immunoreactive flbres in the funiculus lateralis and ventralis is of medium size, whereas in the lateral part of lamina V in the thoracal segments the PV antisera tag very thin axons. PV-IR bundles of axons enter the dorsal horn coming from the dorsal funiculus at its superiormedial border at various points, thus crossing a varying amount of laminae and coursing in the direction of the motor nuclei (lamina IX) and of the intermediate gray matter. Other scattered bundles of a few PV-IR fibres and single axons are also seen entering (or leaving} the ventral horn from the fasciculus lateralis. Many PV+ fibres intermingle in all directions, mostly in the frontal plane. Immunoreactive fibres are often seen in the ~ommissura alba and crossing the ventral and dorsal gray commissure.

CELIO

The raphe spinal system and the reticulospinal system both end in layers I and II.*r Since CaBP+ cells in these hindbrain regions are common, we infer that terminals in layers I-II of the spinal cord may partly derive from these nuclei. The inverse line of thought can be used for the corticospinal tract,76 which lacks both calbindin and PV. The PV- corticospinal tract terminates in layers III-VI and outer VII (see Fig. 10.8 in Ref. 34) therefore the PV+ terminals, demonstrable in these localizations, cannot belong to these descending fibre systems. Dorsal and venfral roots Culbindin D-28k. Coarse, CaBP-IR axons are regularly seen in the dorsal root (Fig. 64,65), whereas the ventral root is devoid of positive axons. Farvalb~~in. The same holds true for PV. However, the number of PV+ axons in the dorsal root is larger than those displaying CaBP-IR (Fig. 65B). Again, the ventral root is free of PV. Spinal ganglia

Calbindin D-28k, This antigen is expressed in some large spinal ganglion cells and in their peripheral and central processes (Fig. 66A). A subpopulation expresses CaBP and PV concomitantly (Fig. 66). Parvalbumin. The overwhelming majority of labelled cells in the spinal and trigeminal ganglia belongs to a class of large cells (Fig. 66B, Table 2). However, there is a subpopulation of small PV+ ganglion cells as well. The ganglia are crossed by a large number of immunoreactive fibres; in adequate sections, the contorted initial segments of PV+ pseudounipolar ganglion cells are seen to be PV-IR. Descending pathwaysz7’ from the brain and medulla In the peripheral axons, PV immunoreactivity is not oblongata terminate in discrete regions of the spinal gray matter, respecting the tamination pattern in most cases.285 increased at the Ranvier node (Fig. 67).

Fig. 60. Semithin cryo-section of the cerebellar cortex incubated with CaBP antibody. Notice the la~lling of the whole Purkinje cell (PU), including nucleus and dendritic spines (see Fig. 61). The white strands in the perikaryon represents cracks due to water crystal formation during freezing. Axons leave the Purkinje cell basally (arrow) and form a plexus. Curved arrows mark cell bodies of untagged basket cells. Phase contrast picture. x 300. Fig. 61. Higher magnification of the molecular iayer of the same section as in Fig. 60, incubated with CaBP antibodies. Stem dendrites (large arrows) give rise to smaller dendrites which are tapestried with dendritic spines (innumerable black dots). The thinner, horizontal arrow marks the pia at the boundary between two cerebellar folia. Phase contrast. x 750. Fig. 62. Semithin cryo-section of the cerebellum incubated with PV antibodies. In addition to Purkinje, also basket and stellate cells are immunoreactive in the molecular layer. Notice again the staining of the whole Purkinje cell. The beard-like appendages at the base of the Purkinje cell (arrows) are the terminal baskets deriving from the basket cells of the molecular layer. x 300. Fig. 63. White matter of a cerebellar fohum incubated with PV antibodies. The great majority of cross-sectioned axons is immunoreactive. The white band around the immunoteactive axoplasm represents myelin extracted during the histological procedure. Some oligodendrocytes are pointed out with an arrow. Star marks empty blood vessels. x 300. Fig. 64. Cross-sections through the cervical dorsal horn of the spinal cord; (A, C) incubated with the CaBP antibody; (B, D) incubated with the PV antibody. Layers I and II are completely blackened by reaction products associated to local circuit neurons and possibly also to CaBP+ terminals. Notice a few axons in the dorsal root, Layer III harbours some scattered nerve cells. Notice the intense staining of the reticular spinal nucleus (Rns). x 120. (B,D) Only the innermost rim of layer II (IIb) displays consistent labelling of intermingled perikarya, cell processes and possibly also terminals. Notice the richness of axons in the dorsal root, in the cuneate fascicle (cu) and in the lateral funicuhrs (lfu). x 120.

Calcium-binding proteins in the rat brain

Figs 60-64.

423

424

M. R.

CELIO

Fig. 65. Semithin section of a lumbal dorsal root incubated with CaBP antibodies. The CaBP+ axons (arrows) are less frequent than the PV+ ones (B) and exclusively belong to a large-size class of as yet unknown origin. (B) Semithin section of a lumbal dorsal root incubated with PV antibodies. The antibody sticks to large-calibre axons (arrows) but also to a subpopulation of thinner axons (arrowheads). Some axons are faintly labelled, but this may only be a dilution effects. x 300. Fig. 66. Two following sections through a lumbal spinal ganglion incubated with CaBP (A) and PV (B) antibodies. Some ganglion cells (large arrows) express both antigens. Others harbour only CaBP (open arrow in A) or PV (open arrow in B). Semithin araldite section. x 300. Fig. 67. PV in a peripheral nerve (ischiadicus). The paranodal (small arrows) region of the Ranviers’s node (arrow) is slightly less immunoreactive than the rest of the axoplasm. Notice variations in the intensity of immunolabelling between different ftbres. x 500.

The smaller neurons have been shown to be heterogeneous with respect to their content of neuropeptides.‘23 On the other hand, the larger, ~~ouni~lar spinal ganglion cells are not characterized by the presence of a particular neurotransmitter,236 although o-aspartate and GABA have

been suggested as possible candidates.@ The only peculiar chemistry of these large neurons seems to be their high ~r~anhydra~ activity, which has been correlated with their intense metabolic activity.2s6 Some authors’30 surprisingly missed CaBP-immuno-

Calcium-binding proteins in the rat brain

425

Fig. 68. (a) A section of the retina, incubated with polyclonal CaBP antibodies reveals richness of cells in the inner granular layer (Igl) (horizontal and amacrine cells), some immunoreactive cells in the ganglion cell layer (arrows) and a trilaminated pattern of staining in the inner plexiform layer (IpI). This complex staining might partly derive from the cross-reaction of the antiserum used in this case with cahetinin,231 a new, calbindin-like CaBP of the retina. The monoclonal antibody 300 stains only horizontal cells. x 250. (b) In the retina PV occurs in two rows of cells at the inner (amacrine cells) and outer ~o~~nt~ cells) borders of the inner granular layer (Igl). The neurons located at the inner border protrude processes into the inner plexiform layer. The neurons at the outer border have horizontal processes, A subpopulation of large ganglion cells (or displaced amacrine cells) stains with this antibody (arrows). Seven-day-old rat. x 250. Fig. 69. In the cochlea, the calbindin antibody only visualizes the inner hair cells (IHC). OHC, outer hair cells; MT, tectorial membrane. Adult rat. x 320. (b) In the lower cochlear turns, PV only occurs in the inner (IHC) sensory cells. MT, tectorial membrane. x 320.

staining in the spinal ganglia. With a few exceptions,2’2 other authors working with this protein never examined the peripheral nervous system. A powerful mechanism for the removal of cytoplasmicfree calcium in spinal ganglion cells has beep discoveredm (large cells; MacBumey, personal communication). It will therefore be interesting to see, if direct correlations can be found between the distribution of calcium-binding proteins and these suggestive physiological results. Sensory organs Retina ~a~~~~ O-28k. CaBP occurs in the majority of horizontal cells as well as in some amacrine cells and

a subpopulation

of ganglion cells (Fig. 68a).

This pattern has already been reported by othersms In primates (our own ovation), and in humans, cones are also calbindin-IR. Using the polyclonal antibody, the inner

plexiform layer is characterized by a tripartite line of immunoreactive terminals whose middle shows the highest intensity. One groupuo reported only horizontal cells being labelled, the additional cells observed by us and others might correspond to those containing ~l~ti~n,23’ a protein recognized by the polyclonal CaBP antibody. The monoclonal antibody 300 only detects horizontal cells. Parvalbtmin. Immunoreactive cell bodies in the retina are restricted to two discrete layers: the inner part of the inner granular layer and the multipolar ganglion cell layer (Fig. 68b). PV-IR cells of the inner granular layer are round and small (10 pm diameter) and have thin immunoreactive processes penetrating the inner plexiform layer. Some processes project to the ganglion cell layer. Additionally, a subpopulation of cells in the ganglion cell layer is PV + . These cells display processes penetrating deeply into the optic nerve layer and represent displaced amacrlne cells or

M. R. CELIO

426

ganglion cells. This last staining is capricious, and often less obvious than that depicted in Fig. 68b. No differences are detected between the various retinal quadrants. Rat retinal ganglion cells have been reported not to contain PV immunoreactivity;8s232(personal communication). In the cat retina, on the other hand, “apparently all ganglion cells could be labelled by anti-parvalbumin sera”.2’2Since the rat optic nerve is overcrowded with PV+ axons, retinal ganglion cells may segregate PV intracellularly. This has been confirmed by colchicine application and in situ hybridization.’ Optic nerve, tract and chiasm Calbindin D-28k. The CaBP+ axons are less coarse than those containing PV, but occur in higher numbers. A cluster of large diameter axons (3 pm) is observed dorsonasally. Parvalbumin. The optic nerve, chiasm and tract have a large number of coarse, PV+ axons. The diameter of the axons varies between 0.2 and 0.4 ,um. Cochlear system Calbindin D-28k. The largest ganglion cells of the spiral ganglion of the cochlea are immunostained, but they are fewer in number than those stained with PV antisera. Their central and peripheral processes are also calbindin-positive. The peripheral process contacts the inner hair cell, which is also strongly immunoreactive towards calbindin antisera, but without polarization towards the apex of the cell (Fig. 69a). Supposing cells and the outer hair cells are unreactive.

With the exception of this last statement our observations agree with others’0~‘56J98~219 (see also Ref. 83). Fa~a~bumin. Perikarya of ganglion cells stain with varying degrees of intensity and have diameters around 15 pm. A few, unstained small cell bodies are

intermingled in between. The central as well as the peripheral processes of these bipolar neurons are PV+ . Stained peripheral processes are seen to contact the inner hair cells of the Corti’s organ. The nerve fibres, which pass through the tunnel of Corti and impinge upon the outer hair cells are unlabelled. The inner hair cell of the lower cochlear turns are PV+ (Fig. 69b). In the upper turns, not only the inner, but also the outer hair cells display PV immunoreactivity, in some cases even the inner and outer pillar cells of the lower turns. The immunostaining of the inner hair cells is often more intense at the apical end (cuticular plate). The central process of the ganglion cells remains PV+ until the brainstem targets are reached. Vestibular system Calbindin D-28k. The majority of somata in the vestibular ganglion are CaBP+ . The peripheral processes make bowl-shaped contacts with type I hair cells of the various vestibular end organs. The type II receptors are not stained, but the type I hair cells react strongly with the antiserum (Fig. 70a,b,c). Similar observations are reported for the human fetu~.69.70.?i

Parvalbumin. All cell bodies in the vestibular ganglion are PV + , regardless of their size. The labelling is often granular and more intense at the periphery of the cell body than perinuclear. The peripheral and central axonal processes are also immunostained. Some “Chianti flask”-shaped cells (type I sensory cells) in the epithelia of the sacculus and utriculus display PV immunoreactivity. The immunoreaction is very intense at the mushroom-shaped luminal border of the cytoplasm (cuticular plate). Staining is absent from the sterociha, but is seen in a few examples of adequately preserved kinocilia. Type II sensory cells

Fig. 70. Section through a crista ampullaris (CA} of the inner ear. The cell bodies of some receptor ceils as well as nerve fibres below them are immunolabelled with the calbindin antibody. The arrow points to nerve fibre bundles of the vestibular nerve. x 120. (b) Ma&a statica (MS) of the vestibular apparatus incubated with CaBP antibodies. Some cells in the sensory epithelium and the nerve libres contacting them, are immunolabelled (arrows). x 120. (c) Higher magnification than b. Most CaBP-IR cells have “Chianti-flask”-form (long arrow) and may belong to the type I sensory cells. Notice beading of the sensory fibres (short arrows). x 300. Fig. 71. Higher magnification of a CaBPf cell in the submucous plexus of the duodenum. A tree of processes is seen on one side of the soma and a single fibre leaves the other pole of the cell. Nomarsky optics. x 240. Fig. 72. Immunostaining of the duodenum with antibodies against CaBP. Both the myenteric (My) and the submucous plexus (Su) contain labelled cells. long, longitudinal muscle fibtes; circ, circular muscle fibres; Br, Brunner’s glands. Nomarsky optics. x240. Fig. 73. Detection of PV immunorea~tivity in a longitudinally sectioned muscle spindle of the extensor digitorum longus of a seven-day-old rat. A single intrafusal fibre is ensheathed in a network of immunostained thin nerve fibres. Notice the heavy immunolabeiling of extrafusal muscle fibres. x 350. Fig. 74. In a cross-section of a muscle spindle of the soleus muscle, the CaBP antiserum tag three intrafusal fibres, probably the two chain- and the Bag l-fibre. Notice the absence of staining in the extrafusal muscle fibres. The two arrows mark the position of the two PV+ fibres of the next figure. x 300. Fig. 75. Following section incubated with PV antibodies. In addition to the Rag I- and Bag 2-intrafusal tibres two extrafusal fibres express this protein too (arrows). Notice the striated appearance of the infrafusal labelling.

Calcium-binding proteins in the rat brain

421

.

Figs 70-75.

428

M. R.

remain unlabelled, although PV+ sensory terminals are seen contacting them.

CELIO

Table 5. Presence of axons immunoreactive towards calbindin D-28k and parvalbumin antibodies in various nerves

Other locations in the central nervous system

CaBP and PV are absent from the pineal gland (see, however, Ref. 148) and from the anterior and intermediate lobes of the pituitary gland. Calbindin immunoreactivity, however, is found in the posterior pituitary lobe. 83 CaBP and PV occur in ependymal cells in circumscribed regions of the cerebral ventricles. CaBP is expressed in many ependymal cells of all four ventricles (Figs 29A, 46A, 58A, 59A). Other groups90,95 observed ependymal staining in the rat whereas in humans this was not the case.93 This is in accordance with the absence of calbindin mRNA from these cell~.~~’The distribution of PV is more restricted in comparison with CaBP, being confined to discrete sites in the dorsolateral ventricular wail and in the roof of the calamus scriptorius at the entrance in the central canal of the spinal cord. At the light microscopic level, the CaBP+ and PV+ ependymal cells cannot be differentiated morphologically from neighbouring ependymal cells. No immunoreactive processes are seen leaving the basal margin, nor special differentiations observed at the luminal side. Circumventricular organs Calbindin D-28k.

Immunoreactive elements are evident and numerous in the subfornical organ but absent from the subcommissural organ. Unnamed regions with a high density of calbindin D -28k - and/or parvalbumin -positive sites

CaBP+ and PV + neurons occur in several discrete regions which, at present can only be qualified by their topographic relationship. This situation is particularly common in the periaqueductal gray (CaBP) and reticular formation (PV). Some of these “new”, definite cell clusters have been named CaBP or PV, followed by a number. The CaBPl (Figs 53, 54, 58C) nucleus is located subependymally in the distal medulla oblongata. Its cells have a diameter of 25 pm and often surround bundles of fibres of the fasciculus longitudinalis medialis. The CaBPl nucleus consists of triangular neurons with long dendrites, directed ventrally or laterally. Dendrites can reach a length of a few millimetres. The dendrites never cross the midline and have a beaded appearance. The axons reach the contralateral side. Autonomic nervous system In adult animals we get specific immunostaining for both antigens in excised pieces of the cervical vagus nerve but not of sympathicus ganglia (Table 5). In whole embedded heads of fetal and newborn material, we have observed an obvious CaBP labelling of cells in the jugular ganglion of the glossopharyngeus and in the nodose ganglions3 but not in the superior cervical ganglion.

Nervus hypoglossus Nervus saphenus Nervus lingualis Nervus ischiadicus Nervus vagus (cervical) Nervus vagus (abdominal) Jugular and nodose ganglion Sympathetic ganglia (cervicalis superior)

CaBP

PV

-

_

(Y)

(t’f

(T) _ + _

t+f _ + _

(+), few; + , some; + + , many. The staining was performed on semithin, deplasticized Araldite-sections.

Enteric nervous system Calbindin D-28k. A subpopulation of ganglion cells in the myenteric and submucous plexuses are immunolabelled by the CaBP antiserum (Figs 71,72). Neurons of the submucous plexus display long cellular processes (Fig. 71). 0thers35~zz5~u6 have published reports on the localization of CaBP in the mammalian gut. Neurons of the myenteric plexus have been shown to possess Ca*+ spikes.rz2 Peripheral nervous system

Table 5 summarizes the results obtained by incubating sections of different peripheral nerves with CaBP and PV antibodies. Calbindin D-28k. Axons of unknown origin are observed in various peripheral nerves. Parvalbumin. PV-IR axons are seen entering in intimate contact with muscle spindles and coil themselves around intrafusal muscle fibres (Fig. 73). (This last example stems from a seven-day-old animal; in adults such intensive staining was never seen.) A few immunoreactive fibres were seen time and again in the thorium of the skin, in the vicinity of probable Merkel cells. Peripheral sensory receptors Calbindin D-28k. The chain and Bag 2 fibres are CaBP+, for example, in the extensor digitorum longus of the adult rat (Fig. 74). Parvalbumin. The polar region of Bag 1 and Bag 2 are positive, for example, in the extensor digitorum longus of the adult rat (Fig. 75). Nuclear staining The nuclei (but not the nucleoli), of most CaBPand PV-IR Purkinje cells, spinal ganglion cells, interneurons in the cerebral cortex and those of scattered cells in other areas are also labelled (not shown). A similar observation has been reported for CaBP”’ and for PV.2s7 I disagree with the interpretation that the above represents an artefactual binding of antibodies257 and postulate that it is an indication of th;: possible role of calcium-binding proteins in the regulation of gene expression. In this connection it is interesting that calmodulin has been found to play a role in cell proliferation and DNA repair.”

:AC Bar BL BLV

SE Aq Arc ATg AV

2: APir APit

zb ACo AD AHi AHY al alv AM Amb AOB

2n 3 3a 4 4n 6 6n 7 7n 8n 10 12 12n AA

Abbreviations -_-

optic nerve oculomotor nucleus oculomotor nerve trochlear nucleus trochlear nerve abducens nucleus root of abducens nerve facial nucleus facial nerve or root of facial nerve vestibulocochlear nerve dorsal motor nucleus of vagns hypoglossal nucleus root of hypoglossal nerve anterior amygdaloid area anterior commissure aanimbens nucleus anterior cortical amygdaloid nucleus anterodorsal thalamic nucleus amygdalohippocampal transition area anterior hypothalamic area ansa lenticularis alveus of the hippocampus anteromedial thalamic nucleus ambiguus nucleus accessory olfactory bulb anterior olfactory nucleus area postrema amygdalopyriform transition area anterior lobe of the pituitary anterior pretectal area, dorsal part anterior pretectal area, ventral part cerebral aqueduct (Sylvius) arcuate hypothalamic nucleus anterior @mental nucleus anteroventral thalamic nucleus cells of the basal nucleus of Meynert bed nucleus of the anterior commissure Barrington nucleus basolateral amygdaloid nucleus basolateral amygdaloid nucleus, ventral part

Brain region --

Table 6. Abbreviations and corresponding denominations -

5

3 O-l 2-3

2 3 O-1 4 &I 1-Z

4-S

1

5

5 4-5

I-2

3

ot

l-2

l-2 l-2

;: 2.-3

I

o-l

l-2 O-1 O-l 0 2-3 2-3

:

1

0 3 l-2 2

2

0

Continued overleaf

4-5 4

l-2 2 0 3

4 4

3 1-2

34 3 0

t&l

0

5

0

2

2 0 3-4 1

0

0

5 3 5

3

Density of axons

34

4-5

5

0

4-5 4

3-4 4.5

l-2 34 3 O-l

5

3

4-s 1

4

0 0

0

5*

3

4

4

Density of nerve terminals

PV

(l-2) s

&I

2-3

Density of cell bodies

(l-2) 5*

1__-

Density of axons

2

Density of nerve terminals

-

1

Density of cell bodies

CaBP

of the individual brain regions in alphabetical orde?‘s

D

E

cu

CSC

CLi CM CP CPU

Cl

ChP CIC tic CL

%

cg2

CG

z

cc

BM BST CaBPl Ce CA1 CA2 CA3 CA4 CB Cb

Abbreviations

Brain region -~ .__~_ -._-._ basomedial amygdaloid nucleus bed nucleus of the stria terminahs CaBPl nucleus central amygdaloid nucleus field CA1 of ammons horn field CA2 of ammons horn field CA3 of ammons horn field CA4 of ammons horn cell bridges between caudate-putdmen and olfactory tubercle cerebellum -molecular layer -Purkinje cells -granular layer -white matter central canal corpus callosum central amygdaloid nucleus central (periaquaeductal) gray cingulate cortex, area 2 cingulum choroid plexus central nucleus of the inferior colliculus commiatmre of the inferior colliculus centrolateral thalamic nucleus claustrum caudal (central) linear nucleus of the raphe central medial thalamic nucleus cerebral peduncle, basal part caudate-putamen (striatum) commissure of the superior colliculus cuneate nucleus cuneate fascicle cerebral cortex Layer I Layer I1 Layer III Layer IV Layer V Layer VI dorsal nucleus (Clarke--Stilling)

CaBP

5

45

3-4 3-4 l--2 l-2 l--2

1

0 1-2 1-2 1-2 1-2 1-2

1

2

3-4

4 1 4 4

4-5 0 4-5 4-5

o-1

0

3-4 5 3

o--I

0

2-4 223 o-1 2-3 2-3 S

Density of nerve terminals -_ 2-3 4-5

0

l-3 l-3 2

0 4-S

l-2 3-5 5 1-s 3 1-2 1-2 I--2 3

Density of cell bodies

Table 6-Conrinued

3-5

0

3

2 5 0

o-4

l-2 l-2

0

4-5

Density of axons

~._.___

2 2-3

1-2 (4-s*)

1

o-1 2 2 3-4 2 2 4

4

O-l

1

34

4 1-2

0

3

2 0

0 0

3-4

0

34 2-3

3

Density of nerve terminals

PV

0

5 4-5

1-2 1-2 1-2 l-2

l-2

Density of cell bodies

3-s

3

0 O-I

4 2-3

0

O-l

0

0

l-2 1-2

Density of axons

DC0

Hi

HDB

%A

:; GP Gr

Em

dfu DG Gr MOP HiP dhc Dk DLG DLL DM DPB DPG DpMe DpWh DR dr D’fg dtgx E ECU EIC En Ent EP EPl f FF ii FR fr Frl FStr G

dorsal cochlear nucleus dorsal funiculus dentate gyms granular layer molecular layer hilus dorsal hippocampal commissure nucleus of Darkschewitsch dorsal lateral geniculate nucleus dorsal nucleus lateral lemniscus mediodorsal hypothalamic nucleus dorsal parabrachial nucleus deep gray layer Sup. coil. deep mesencephalic nucleus deep white layer of the superior colliculus dorsal raphe nucleus dorsal root of spinal nerve dorsal tegmental nucleus (Gudden) dorsal tegmental decussation ependyma and subependymal layer external cuneate nucleus external nucleus of the inferior colliculus endopiriform nucleus entorhinal cortex entopeduncular nucleus external plexiform layer of the olfactory bulb fomix fields of Fore1 fimbria of the hippocampus formatio reticularis fasciculus retroflexus (habenulointerpeduncular tract) frontal cortex (primary motor) fundus striati gelatinosus nucleus of the thalamus genu of the corpus callosum gemini nuclei gigantocellular reticular nucleus glomerular layer of the olfactory bulb globus pallidus gracile nucleus gracile fasciculus granule cell layer of the accessory olfactory bulb nucleus of the horizontal limb of the diagonal band hipp~mpus Or oriens layer Py pyramidal cell layer O-l l-5

l-2 O-l

5 l-5

1

l-2 2 3-4

4

3 5 o-1

2 5 0 5 l-2 4-5 0 0

2

l-2

1

2

1

3 0 1 2-3 2 3

34 l-2 3 4-5 5 3

l-2 l-2 2-3 4-5 4 l-2 l-2 l-2

5 5 4

2

5 &--I O-1

o-1

2

0 0

4 3

0t

It

3

3

1

2-3

4 cr-If

I

l---2

3 3

1-2

4 l-2

l--2

1-2 (5*) O-I 5 l-2 (4-5*)

2

l-2 (4*)

1

1 1-2 O-1 1-2 2 5 2-3

O-1 5

1

O-l 4-5

3

2-3

2

0 3 o-1

4

3-4

4 4 4

l---2 O-l 4-5 O-l

1

4 0 O-l

2-3

l--2 O-l 4

0-I

Continued

2

2 o-1

0

o--l

o--l 0 2 0-l

o-l

3-4

2-3

4

o-l 3-4

3 5

overleaf

&

LH LHb Li II LM 1Q

It-Ii

IAM IC ic ICj icp IG IGr ILN IMD InC InfS InG Int InWh IO IPA IPC IPIP wit IPI IPGP La Lat LC LD LDTg

HY I

Abbreviations

Brain region ___I._-

0

1-2

3-4

0 1-2

0 (4;)

I

1 0 O-l 1

0

3-4

1 3-4 5 O-l

0

4 0

3 4-s

l-2 3-4s 5 1-2

1 2 3-4 4 4

4 l-2

5 5 5 0

0

3

5

0

3

2-3 4 l-2

5

4-5

1-2 0

2 5

0-l

o-1 &l

I

l-2 1 0 O-l 1 I-2 0 (5’) 0

O-l

2-3

I 4-5 5 0 4-5

3-4 1 0

0

3-4

3-4 l-2 2

4

5

3

3 4 1-2

3

0-I

4-5

4 O-3

Density of axons

0 (59; Cap Kooy nucleus

3-4 4-5

3-4

1-2 1-2 34f

5

5

0

0-l O-l

2-3

0

4.5

2-3 2-3 1 0

Density of nerve terminals

Density of cell bodies

Density of nerve terminals Density of axons

PV

CaBP

1 2-3 34 4-5 1-2

0

O-l

o-1 o-l 1-5 0 4-S

-~-~-“. Density of cell bodies

&-Continued

I&d stratum radiatum LMol lacunosum moleculare layer ltypothalamus intercalated nuclei of the amygdala interanteromedial thalamic nucleus inferior colliculus internal capsule islands of Calleja inferior cerebehar peduncle induseum griseum internal granular layer of the olfactory bulb intergeniculate leaflet intermediodorsaI thalamic nucleus interstitial nucleus of Cajal infundibular stem intermediate gray layer of the superior colhculus interpositus (intermediate) cerebellar nucleus intermediate white layer of the superior colliculus inferior olive interpeduncular nuclens, apical part interpeduncular nucleus, central part interpeduncular nucleus, inner part of the posterior subnucleus intermediate lobe of the pituitary internal plexiform layer of the olfactory bulb briar nucleus, outer part of the posterior subnucleus lateral amygdaloid nucleus lateral (dentate) cerebellar nucleus locus coeruleus laterodorsal thalamic nucleus lateral tegmental nucleus lateral funiculus of the spinal cord lateral hypothalamic area lateral habenuIar nucleus linear ntius of the medulla lateral kmniscus lateral mammillary nucleus lateral olfactory tract

______

Table

%T opt OT Pa Pal Pa2

ogj,

F& mt mtg MTU MVe Gc

Zf MM MMn MnR Mo5 MP mp MPG

ZL

m5 mcp MCPC MD MDL Me Me5 me5 Med mfb MG MHb

LVe

LSD LSI LSG LSV

lateral posterior thalamic nucleus (pulvinar) lateral pmoptic area lateral septal nucleus, dorsal part iateral septal nucleus, intermediate part lateral superior olive lateral septal nucleus, ventral part lateral vestibular nucleus motor root of the trigeminal nerve middle cerebellar peduncle magnocelhrlar nucleus posterior commissure mediodorsal thalamic nucleus mediodorsal thalamic nucleus, lateral part medial amygdaloid nucleus nucleus of the mesencephalic tract of the trigeminal nerve mesetwphalic tract of the trigeminal nerve medial (fastigial) cerebellar nucleus medial forebrain bundle medial geniculate nucleus medial habemdar nucleus mitral cell layer of the olfactory bulb medial mammiflary nucleus, lateral part medial lemniscus medial longitudinal fasciculus medial mammiky nucleus, medial part medial mammillary nucleus, median part median raphe (superior central) nucleus motor trigeminal nucleus medial mammillary nucleus, posterior part mammillary peduncte medial preoptic area medial septal nucleus medial superior olive mammillothalamic tract ~Uote~en~l tract medial tuberal nucleus medial vestibular nucleus 1B occipital cortex, area 1 binocular 1M occipital cortex, area 1 monocular olfactory nerve layer optic nerve layer of the superior colliculus olivary pretectal nucleus optic tract nucleus of the optic tract paraventricular h~thalamic nucleus parietal cortex, area 1 parietal cortex, area 2 4 4-5 4-5

3-4 4-5

l-2 4-5

3-4 4 3 5 5

3-4 4-5

2 2

O-l 2

4-5

2-3 3

2 2

3-4

4-S 0

O-l

4 4 4

5

0

5 5 l-2 0 o-l

4 4 3-4

5 4 3-4 4

2 1

22

O$) 0

l-2 l-2 4-s 3-4

3 3

2

3 l-2 3-4

:

4

4-5

4

5

0

4 3

4-5 4 4

3

3

3-4 O-l O-l

4 2

S O-l

3 4

4-5

0

4

Continued

overleaf

O-l (only magnocellular part) S O-l O-l 5 :

l--2 l-2

&&) 2 2

O-l 1 2-3

4

0 l-2 O-l O-l

3-4 3

2

4

5

2

5

3-4

O-l O-l 4 4 1-2 l-2

0 u*)

5

2

0 (57 0

O-l

o-l

RSA RSG Rt S

z RLi Rns RPn

k&C PCRt Pe PeF PF PH Pi Pir Pmco PMD PMV PO PO PP PPit PR Prs Prh PRh PrS PRVI PT PVN PY R RCh

Pa.9 PC

Abbreviations ____I_ -~

~_.__

Brain region

.____I___

parasubiculum paracentral thalamic nucleus posterior commissure pericentral nucleus of the inferior collicuius parvoeellular reticular nucleus periventricular hypothalamic nucleus perifarnical hypothalamic nucleus parafascicular thalamic nucleus posterior h~othaiamic nucleus pineal gland piriform cortex posteromedial corticaf amygdaloid nucleus premammillary nucleus, dorsal part premammillary nucleus, ventral part primary olfactory (piriform) cortex posterior thalamic nuclear group peripeduncular nucleus posterior lobe of the pituitary prerubral field principal trigeminal nucleus prepositus hypoglossi nucleus perirhinal area presubiculum parvalbumin- 1-nucleus paratenial thalamic nucleus paraventricular thalamic nucleus pyramidal tract red nucleus retrochiasmatic area reuniens thalamic nucleus rhomboid thalamic nucleus rostra1 linear nucleus of the raphe reticular nucleus spinal cord raphe pontis nucleus agranular retrospleniat cortex granular retrosplenial cortex reticular thalamic nucleus subiculum

I.--

4 4

4-5

3-5

2-3

5 0 3

I 5

4 5

2 5

l-2

4

5

3-4 3 2

2-3 3-4

3

4

Density of nerve

CaBP

4-s

3 0 2-4 3-4 0 3-4 2-1 2-3

2 2 4-5 O-1

Densit) of cell bodies

Table &---Continued

3

0

O-1

0

Density of axons -.--

2 5

2

2 5 1-2

3

2

0 (4*)

2 4 5 3 2

4

0

Cl

4-5 3-4 2-3 2 2 l-2 5 @I

3

2

1

1

2

Density of nerve terminals ..- _._ .-._.__

2

l-3

0

(2)

2

Density of cell bodies

PV

&l

:

3

3

0

2

I

0

0

Density of axons

Tz tz vco VDB

s;mNC SNL SNR so so1 Sol sol sox SP5 SDS SpiGl SpVe st stg STh SuG SUM sumx SuVe TC Tel Te2 TM TMC TS T-r TU

SI

SHY

SF0 SGi SHi

SC SCh SC0

55

sensory root of the trigeminal nerve superior colliculus suprachiasmati~ nucleus subcommissural organ superior cerebellar peduncle (brachium conjunctivum) septofimbrial nucleus subfornical organ suprageni~ulate thalamic nucleus septohippocampal nucleus septohypothalamic nucleus substantia innominata stria medullaris of the thalamus substantia nigra, compact part substantia nigra, lateral part substantia nigra, reticular part supraoptic hypothalamic nucleus superior olive nucleus of the solitary tract solitary tract supraoptic decussation spinal tract of the trigeminal nerve nucleus of the spinal tract of the trigeminal nerve spinal ganglia spinal vestibular nucleus stria terminalis stigmoid hypothalamic nucleus subthalamic nucleus (Luys) super&&l grey layer of the superior eolliculus supramam~lla~ nucleus supramammillary decussation superior vestibular nucleus tuber cinereum temporal cortex, area 1 temporal cortex, area 2 tuberomammillary nucleus tuberal magnocellular hypothalamic nucleus triangular septal nucleus taenia tecta (anterior hippocampal rudiment) olfactory tubercle plexiform layer polymorph layer pyramidal layer nucleus of the trapezoid body trapezoid body ventral cochlear nucleus nucleus of the vertical limb of the diagonal band (Broca) 0 O-1

3-4

0

3-5

0

0 5

2 2

4-5

4 0 1-2 2-3

2

3 O-l

4

2-4 2-4 2-4 O-l 3-s O-l 3-4 2-4 2-4 2-4

4 4 3

3-4

4 4

4-5

4

: 34

2 O-l

0 3

2 2 3

3 1-2

2-3 1-2

$3

1-2 2-3 4-S

o-1 2

2

3 3

3

34

2---3 3-4 5

3-4 4

3

3 24

2-3

0

3-4 4 4 4

4

0

2

3-4

l-2 l-2

I-2

3-4 3-4 5 O-l 4 45

2-3 2-3 0 4

2 4-5 4

1 2-3 5 4 45

C-l

o--l

l-2

0

2-3 0

3 2-4

l-2 O-1 0

Continued overleaf

3-4

5

3

34

2-3 3-4

3 4

4-S

2-3

ventral funiculus of the spinal cord ventral hippocampal commissure ventrolateral thalamic nucleus ventral lateral geniculate nucleus ventral nucleus of the lateral lemniscus ventromedial thalamic nucleus ventromedial hypothalamic nucleus vomeronasal nerve layer vomeronasal nerve vascular organ of the lamina terminalis ventral pallidum ventral (medial) parabrachial nucleus ventroposterior thalamic nucleus, lateral part ventroposterior thalamic nucleus, medial part ventral root of spinal nerve ventral reuniens thalamus nucleus ventral tegmental area (Tsai) ventral tegmental nucleus ventral tegmental decussation decussation of the superior cerebellar peduncle Z nucleus zona incerta zonal layer of the superior colliculus

Brain region ~~-

4 2

45 2-3

5

3 2-3 2-3

34 l-2 I-2

o-1 4-5

4

4 5 51

2 1

4

I o-1 CL1 45 5

Density of nerve terminals

CaBP

0

3f 4 2

l-2

2 223

Density of axons

TCaBP-IR axons can be observed in “quaking fNot seen with the monoclonal antibody 300.

mice”.

4, many; 5, aft.

5 2-3 5

3

1-2 45

0

2

5 5

4-5 2 4 4-5

Density of nerve terminals

PV

0

334

S-1 (47 4-5

Density of cell bodies

The density of immunoreactive sites is graded according to the following density scores: 0, absent; 1, few; 2, some; 3, a large proportion; *Cell body density scored after colchicine pre-treatment of the animal.

zo

ZI

xscp Z

&c VTA VTg vtgx

t;“o VP VPB VPL VPM

vfu vhc VL VLG VLL VM VMH VN

Abbreviations

Density of cell bodies

Table &Continued

3 5

334

4 5 0

45 2-3 34

Density of axons

Calcium-binding proteins in the rat brain

Fig. J6A.

431

M. R. CELIO

Fig.

76B.

Fig, 76. Schematic drawing of neurons and pathways displaying CaBP (A) and PV (IS)immunoreactivities in the normal rat, superimposed in a picture of the human brain according to Ref. 196. The schema is not complete, Filled cells and continuous lines represent positive structures identified with certainty. Circfes represent PV - neurons giving rise to a PV+ pathway. PV+ pathways in the spinal cord have been omitted since their origin is uncertain.

Calcium-binding proteins in the rat brain

Fig. 77. Figs 77-81. The drawings of PV- and CaBP-IR cells are strictly limited to the stained cell body and the cytoplasmic processes. This obviously implies that the real shape of the cell in question may differ from the depicted one. The drawings are limited to neurons of those few brain regions, in which the spatial separation of immuno~ctive cells made a precise r~onst~ction possible. Axons can seldomly be traced with certainty to their parent perikarya or dendrites. Thin, smoothly immunostained processes which can represent axons are, however, often encountered in the neuropil and have been depicted (e.g. for PV in the h~pp~ampus) (Fig. 78). Fig. 77. (A) Camera lucida drawing of four CaBP-IR neurons at the border between mitral and inner plexiform layer of the olfactory bulb. The perikarya of the positive cells are partly (cells 1 and 4) or totally {cell 2) embedded in the mitral cell layer(M). Some others are found in the internal plexiform layer (IPL). Two major types of CaBP+ neurons are observed: one has a fan-shaped tree of dendrites, opened towards the inner plexiform layer (e.g. neuron 3). The other type has dendrites parallel to the mitral cell layer (e.g. neuron 4). The clumsy stem dendrites give rise to thinner branches, which ramify in the mitral cell layer or internal plexiform layer or even in the granular layer. Appendages are seen in form of discrete varicosities on cells 1 and 4. The arrow marks the possible axon of cell 3, which is traced until the mitral cell layer. (B) Drawing of 21 PV-IR neurons in the coronal plane of the olfactory bulb in their precise topographical location in the external plexiform layer. This drawing represents the synthesis of different visual fields of various sections. Most cells have their perikaryon and major dendritic processes confined to the external plexiform layer (EPL). Cells 1, 3,6 and 8 have part of their dendritic arborization ordered perpendicularly, while cells 2, 3, 7 and 9 have dendrites parallel to the mitral cell layer. Note some large varicosities on the dendrites of cells 5 and 8 and a few small ones on the dendrite of cell 9. Cells 10 and 11, located in the glomerular layer (Gl) correspond to periglomerular, respectively tufted cells.

Abbreviahms

al A cc Cer CG ccg co CPU cx DC DCN DH EP f GP Hi HNL HY

ansa lenticularis amygdala corpus callosum cerebellum central gray matter cingulum cochlear nuclei caudatoputamen cortex cerebri dorsal column nuclei deep cerebellar nuclei dorsal horn, spinal cord entopeduncular nucleus fornix globus pallidus hippocampus hypophyseal neural lobe hypothalamus

used in Fig. 76

IC IO IP M Me5 MHb B 0 08 SC S sm SO1 ?H

T TZ Ve

inferior colliculus inferior olive interpeduncular nucleus mammillary body mesencephalic trigeminal nucleus medial habenula nucleus basalis of Meynert olfactory nuclei olfactory bulb superior colliculus septum stria medulla& thalami nucleus of the solitary tract stria terminalis thalamus tegmental nuclei trapezoid body vestibular nuclei

439

M. R. CELIO

Fig. 78. Drawing of 32 PV+ neurons of the hylus of the dentate gyms in their topographical location. Composite drawing obtained by su~~mpo~tion of 17 sections made in lon~tudinal and coronal planes. PV+ neurons and known Golgi-types may be tentatively compared; some neurons are tagged with numbers, which correspond to those of similar cells on picture 28 of Ref. 9. 2, pyramidal basket-cell; 4, giant aspiny stellate eeii; 14, dentate basket-cell; 16, type two dentate basket-cells. One of these cell types is common in the molecular layer of the dentate gyrus and has been baptized “Hantel cell” (arrows).

Other pecuiiarit~e~ of the immunostoin~ng In some animals portions of the cerebral cortex, particularly the auditory cortex, lack PV-IR ceil bodies (Fig. 24). A similar situation has been observed in the monkey visual cortex (unpublished observations). The cause of this phenomenon is unknown, but we suspect local functional abnormalities. Immunostaining in neurological mutant animals “Quaking” mice have been studied since they have defective myelin sheaths, thus allowing a better penetration of the antibodies in tissue sections. With CaBP antibodies we observed a labelling very similar to that of the normal rat but, in addition a staining in the anterior commissure, fomix, dorsal hippocampal commissure and fimbria hippocampi (see Table 6). “Staggering” mice lack Ca*+ spikes in their Purkinje cells6* Since calcium-binding proteins could be involved in the regulation of Ca*+ spikes we have looked for their occurrence in the cerebella of these mutants. Both CaBP and PV are normally expressed in their Purkinje cells. DISCUSSION

General considerations of immunohistochemistr~ Our undertaking

has been simplified by the fact

that even strong fixatives can be utilized for anchoring the CaBP and PV molecules to their compartments, while their antigenicity remains unaltered. The immunohistological localization of calcium-binding proteins in the central nervous system is in fact not affected by harsh fixation or embedding procedures. Even strong fixatives (glutaraldehyde 2.5%), and embedding in Epon or Araldite do not destroy their antigenicity, if enough CaCl, is added to the fixative. This “abnormal” behaviour of calciumbinding proteins may reside in peculiarites in their primary structure, since they are poor in the amino acids tyrosine, tryptophan, cysteine and proline, respectively.25 Cross -reactions of the poly - and monoclonal antibodies The distribution of CaBP-IR sites in the rodent brain is distinct from that of PV, as seen in comparing consecutive sections (see Figs 3, 4). S-100 antibodies only tag astroglial ceils; calmodulin antisera stain all neurons and troponin-C is undectable in the brain. From this palette of staining patterns, cross-reactions are highly improbable and the cross-absorption and immunoblot ex~~ments26.so confirm this impression. The polyclonal antibody against CaBP, used at the beginning, cross-react with calretinin, a new caiciumbinding protein,2’4,23’ whereas the monoclonal antibody (no. 300) does not.”

Calcium-binding proteins in the rat brain

441

Fig. 79.

Fig. 81. Fig. 79. High power drawing of a single field of observation of the hippocampal lacunosum-moleculare layer (CAI) incubated with PV antibodies; a rich profusion of beaded and smooth dendrites is visible. Most of them represent dendrites of PV+ interneurons located in the pyramidal or oriens layer. The thinnest fibres probably represent axons of these same intemeurons, as they often bend and return to the pyramidal cell layer. Roughly two kinds of cell processes can be distinguished: with (1) or without (2) varicosities (“Perlschnurketten”). These two categories can be further subdivided in thin (2a) or thick (2b) smooth fibres and fibres with small (la) or large (lb) varicosities. Eventually, a further subdivision can be performed by taking into consideration the intervaricose distance. x 800. Fig. 80. Drawing of three PV+ cells (from a parasagittal section) of the caudatoputamen in their exact topographic relationships. Such clusters of PV-IR cells are common in the rat caudatoputamen. The cell processes come in very close spatial ~lationship, although a contiguity cannot be detected at this level of resolution. All three neurons have similar size (20 x 20 pm), shape and kind of dendritic arborization. The dendrites are slender and have few varicosities. The arrow points to the corkscrew-shaped dendrite; this kind of “malfo~tion” is often seen, but can represent a cutting artefact. These three cells are located

in the ventrolateral part of the caudatoputamen. Dorsal is left and lateral is up. Fig. 81. Drawing of a particularly well “impregnated” neuron in a 50-pm-thick coronal section of the CPU incubated with PV antibodies. The perikaryon displays six stem dendrites, which give out multiple branches. These are mostly smooth and regular, but few display varicosities (arrows). Dorsal is up, lateral is right.

The specificity of the immunoreaction is a hotly debated topic in immunohistochemis~.zls Dilution curves, preadsorption and “blotting” experiments after SDS-gel electrophoresis, as well as RIA determinations are all necessary but not sufficient to prove the specificity of the staining. The definitive confirmation of the synthesis of licit-binding proteins in all the neurons revealed by immunocytochemistry await “in situ” hybridization” techniques. We, and others have established the validity of the immuno-

histochemical localization of PV and CaBP by in situ hybridization.3,24s Colchicine experiments The intraventricular application of colchicine has subtle consequences on the distribution of perikarya immunoreactive towards PV antibodies. In fact, many long projecting neurons a~umulate PV in their soma after interruption of axoplasmic transport (see also Table 6). The most obvious examples are the deep cerebellar nuclei, the vestibular nuclei, dorsal

M. R. CELIO

442

Terminals density

moderate low

CasP

PV Fig. 82

Fig. caption p. 464

PV Fig. X3.

Fig. caption p. 464

Calcium-binding proteins in the rat brain

CaBp

PV Fig 84.

column nuclei and retinal ganglion cells. Thus, in the intact neuron PV is rapidly transported in the axon and terminals of these nerve cells.

In general, the distribution of CaBP- and PV-IR sites has been found to be virtualIy identical in all strains of rats studied, and appears to be largely independent of sex with the possible exception of the hypothalamic staining. A subtle difference is detected in the hippocampus of the Long-Evans rat, stained with CaBP antibodies. This animal has both, a supraand an infrapyramidal mossy fibre projection, whereas in the Wistar and the Sprague-Dawley rats only a suprapyramidal projection is detectable. Such differences in staining pattern reflect a primary neuroanatomic variability’@ and no capriciousness of labelling. Age and sex differences exist and are partly the subject of separate communications.s3,25~ The intensity of labelling within a category of cells is comparable: so all the Purkinje cells react identically with both CaBP and PV antisera, basket cells in the cerebellum stain with the same intensity as do islet cells in the spinal cord layer 2b. However, the staining intensity varies from every cell population to another. For example, Purkinje cells stain more heavily for PV, than basket or stellate cells; basket cells of the cerebral cortex are more immunoreactive than bitufted cells. Therefore, some cells appear to har-

Fig. caption p. 464

bour more immunoreactive material than others (see also Table 1). In a given cell, in most cases the labelling is homogeneously dist~buted across the various cell processes and the soma. This is best visualized in the Purkinje cell, which is reactive with both antisera from the finest dendritic spines through the soma and axon to the terminals. Although this seems to be the most frequent case, it is by no means a general rule. Sometimes the PV-immunolabelling is distributed differentially between various domains of the same neuron, This phenomenon is only noticed in Golgi type I cells (long-axon cells), and, interestingly, mainly in neurons in the sensory chain to the brain. The cell body is unlabelled (or lightly labelled) and only the axons and the terminals belonging to retinal ganglion cells, nucleus gracilis and cuneatus cells, ncl. cochl. ventr. and dorsalis cells, neurons of the various vestibular nuclei and of deep cerebellar nuclei display PV-IR. It is likely that the soma of other, as yet unidentified neurons, behaves in a similar manner. Deep cerebellar nuclei receive excitatory afferent collaterals and a strong inhibitory input from Purkinje cells. Since inhibition does not alter Ca2+ fluxes, these perikarya are exposed to moderate Ca2+ transients, which are perhaps managed by other Ca2+ buffering systems. This situation of intracellular segregation of calcium-binding proteins is remini~nt of a similar observation, made with the mitochondrial enzyme cytochrome-C-oxidase. The level of this enzyme varies between different segments of the same

444

M.

R.

CELIO

CaBP

Pv Fig. 86.

Fig. caption p. 464

AN /

AID

h

447

448

M. R. CELIO

Fig. 88B.

neuron in response to varying levels of excitatory synaptic inputs, received by that segment.“’ There is no evidence for an alternative way of intracellular segregation of PV as for example exclusively in perikarya, terminals, axons, or in dendrites or any combination between these. It is much more difficult to detect a phenomenon of intracellular redist~bution in Golgi type II (short-axon) neurons because of the intricate staining pattern in areas rich in these kind of ceils. Gross non-uniformities are also seen in the distribution of CaBP, as exemplified by the pryramidal cells of the hippocampus. They have soma and stem dendrites labelled by CaBP antibodies, but not the axon leaving through the fornix. As a matter of fact, this bizarre intra~llular compartmentalization of calcium-binding proteins makes the interpretation of the mapping more difficult.

Fig. captionp. 444

One group2@ reported a preferential staining for CaBP near the plasma membrane of Purkinje cells. This could be an interesting observation since the CaZf buffering activity in the neurons of the invertebrate Apfysia caiijknica is higher near the plasma membrane.270 However, using semithin and ultrathin (unpublished information) cryo-sections, we cannot confirm their imm~oh~st~he~cal results (Fig. 60). The subplasmalemmal staining may be an artefact due to penetration problems of the antibodies, or to diffusion and adsorption of the diaminobenxidine reaction product.44 The identification of CaBP and PV+ cells is-with a few exceptions (e.g. interneurons in the spinal cord)--easy (e.g. Purkinje cells). Various morphological criteria (shape, dimension, location) and multiple morphological techniques (Nissl, Khiver-Barrera staining, electron microscopy) have been used in this

Calcium-binding proteins in the rat brain

449

450

M. R. CELIO

Fig. 89B.

study to identify immunoreactive structures. The course of immunoreactive fibres often coincide with classical neuroanatomical pathways (e.g. ansa lenticularis; fasciculus cuneatus and gracilis for PV, striatonigral pathway for CaBP). However, lesion studies combined with colchicine application are required to investigate the origin and termination of many CaBP+ and PV + neurons. The labelling for PV and CaBP generally respect cytoarchitectonic boundaries between the nuclei and cortical areas. There are some exceptions particularly in the distribution of CaBP. In the thalamus, for example, CaBPt cells transgress established boundaries. One of the most important contributions, which can be fulfilled by immunohistochemistry of neuronal markers, is the exact quantification of labelled cells in defined areas. This permits one to follow the fate of

Fig. caption

p. 464

these cells after experimental manipulations, or to detect morphological abnormalities in mutant animals or in diseased subjects. The questions still remain as to whether all celfs, belonging to a certain defined morphological class (i.e. hippocampal basketcells, islet cells in lamina II\, of the spinal cord, periglomerular cells, horizontal cells of the retina). or only a subpopulation are recognized by GaBP or PV antibodies. Unfortunately, no quantitative data on the number of these cells are available from classical neuroanatomical studies. The exception is shown by the basket-cells in the fascia dentata. which have been counted in semithin sections according to some structural and positional characteristics.‘“’ The amount of “basket-cells” in the dentate gyrus according to these authors is of some 4000; a number well in accordance with that of PV+ cells.

Calcium-binding proteins in the rat brain

451

452

M. R. CELIO

Fig. 90B.

Co-existence

of both proteins in the same neuron

The co-existence of PV and CaBP has been directly observed in the following locations: Purkinje cells, spinal ganglion cells, neurons of the medial trapezoid nuclei. Additional co-expression of CaBP and PV is probable in certain neurons of the olfactory tubercle and in the basolateral nucleus of the amygdala. Comparison of the results with those of earlier reports

There is a general agreement between authors studying the distribution of CaBP in the cerebellum, hippocampus, cerebral cortex, and olfactory bulb. A detailed comparison with reports by colleagues has been made in the appropriate sections of the results. Here 1 limit myself to a comparison of my observa-

Fig. caption p. 464

tions with comprehensive reports, which studied the whole central nervous system of the rat. Our results on CaBP are similar to those of the most comprehensive publication.95 The few important divergences are probably due to the use of different labelling techniques. In our hands the lateral olfactory tract does not contain CaBP+ axons, and the vestibular nuclei, medial septal nuclei as well as the locus coeruleus are devoid of CaBP neurons. Other minor divergences concern the presence of fibres (terminals) in certain brain nuclei. The interested reader might compare Table I of Ref. 95 with our Table 6. Of neuroanatomi~al interest is another paper”’ in which the striatal staining with CaBP antisera is reported first. These authors limited their study to

Calcium-binding proteins in the rat brain

453

h

Fig. 9lB.

the forebrain and schematicalty depict the iabelling pattern. UthersJ3’ also give an account of the ~st~butio~ of CaBP in the chicken and rat brain. Curiously, they do not observe ~mmunoreactive neurons in the spinal cord and spinal ganglia. Contrary to CaBP, the distribution of PV has been studied by only a few groups. Its presence in the retina and in endocrine glands has been reported.84**5 We have not observed PV either in the pituitary gland (this study) or in the thyroid and adrenal glands (unpublished observations). In 1987 a series of papers on the localization of PV in the olfactory bulb, hippocampus and cerebral cortex has been published.143*L44*145 The association of PV with GABA neurons47 has been confirmed. has performed elegant Scheich’s group 3n~31,293

Fig. caption p. 464

studies in the zebra finch brain, comparing the PV distribution with cytochrome-oxidase labelling and ~~4CJdeoxyg~ucose uptake. Although a comparison between regions of birds and mammals is sometimes difficult, there is good correlation between our data. Indeed, these authors also come to the conclusion that: “Parvalbumin . . . v(is) present in neuronal systems that can reach high levels of electrical (and metabolic) activity”.”

CaBP and PV occur in each of the consecutive neuronal links of a variety of functiona chains (see Fig. 76). This is exemplified by PV in the somatosensory system and CaBP in the basal ganglia. Neuromediators, on the other hand, are seldomly present in

Calcium-binding proteins in the rat brain a chain of neurons towards the brain.‘% At best cholecystokinin occurs in the viscerosensory projection to the brain,“’ and leutinizing hormone releasing hormone in the accessory olfactory system.22 Another relevant difference between neurotransmitters and calcium-binding proteins is found in the fidelity, with which calcium-binding proteins respect nuclear and area1 boundaries (with the exception of CaBP in the thalamus). Neurotransmitters, on the other hand, tend to transgress the cytoarchitectonic borders and to be more diffusely dispersed.262 Co-localization of calcium-binding neuropeptides and neurotransmitters

proteins

with

It has to be said that at the moment we have only compared directly the distribution of PV, CaBP and the inhibitory neurotransmitter GABA (see Fig. 10).

455

It is established that the co-existence of PV with GABA4’ is a widespread’43~‘U~‘45*2s* although not general (this study) phenomenon. PV only lights up the subpopulation of GABA neurons with high metabolism and electrical activity.30,3’,4SA relationship with GABA transmission per se is unlikely.2’3 From the localization of CaBP+ and PV+ elements, other co-existences can be inferred. So, for example, CaBP and somatostatin in layer II of the cerebral cortex, CaBP and GABA in the strionigral projection and CaBP and choline acetyltransferase in the nucleus basalis of Meynert. CaBP has been co-localized with enzymes of dopamine metabolism in the ventral tegmental area.97 Studies of co-localization are particularly interesting in the cerebral cortex and hippocampus, where the GABA neuron family can be dissected in various

CaBP Fig. 92A.

Fig. caption p. 464

456

M.

R.

CELIO

CaBP

Fig. 93A.

PV

Fig. caption p. 464

458

M. R. CELIO

Fig. 93B.

groups by neuropeptide immunohistochemistry. A co-existence of PV with somatostatin or cholecystokinin in the cerebral cortex has been excluded.‘” In summary, it can be stated that a complete match in the dist~bution of the regutatory chain-binding proteins and neurotransmitters or neuromodulators cannot be detected. The co-existence is fortuitous and points to the fact that caIcium-binding proteins may not play a direct role in neurotransmission. Circuits involving calbindin D -28k and parvalbumin neurons

Certain subdivisions of the brain, based on cytological and histological data, are strongly supported by the sharp distribution of these two calcium-binding proteins. A glance at Fig. 29 reveals the

Fig. captionp. 464

boundaries of thalamic nuclei; Fig. 3 immediately visualize subnuclei of the amygdala etc. Furthermore, functional entities, which have been defined after difficult hodologic studies, are immediately confirmed by the immunohistochem~cal localization of calcium-binding proteins. The zone II’“* consisting of the caudatoputamen, nucleus accumbens and tuberculus oifactorium lights up in a CaBP immunostained section. In the same line of thought, zone III,192 namely globus pallidus, nucleus entopeduncularis, pallidus ventrale and the substantia nigra (pars reticulata) are PV-rich. The “chemoarchitectonic according to calci~-binding proteins” even suggest the addition to this last group of the basolateral nucleus of the amygdala, which harbours PV neurons of similar shape and diameter as those in the globus pallidus.

h

II

m 8

460

M. R.

In certain functional systems the chain of nerve cells from the periphery to the brain contain CaBP or PV (see Fig. 76). So for example the somatosensory system displays PV in the receptors (muscle spindle fibres), in the IA afferences and in the medial lemniscus up to the thalamus and in terminals in layer IV of the somatosensory cortex. Furthermore, PV is expressed in all neurons involved in the control and execution of eye movements. On the other hand, CaBP is rich in the whole taste pathway: from the tongue over the chorda tympani to the nucleus tractus solitarius. From here to the parabrachial nuclei and further to the hypothalamus, respectively to the ventromedial thalamic nucleus.

CELIO

Other examples are schematically depicted in Fig. 76A,B. We infer that calcium-binding proteins enable or enhance a peculiar physiological function in a chain of functionally related neurons. Therefore, calcium-binding proteins are morphological tags for certain functional systems; information which can be further exploited for experimental manipulations. New nuclei revealed by antibodies against calciumbinding proteins During this study aggregates of neurons, never been described (PRVl) is depicted in

Fig. 95A.

I have observed at least two which, to my knowledge, have before. The PVf aggregate Figs 3H, 25, 31B and consist of

Fig. caption p. 464

Calcium-binding proteins in the rat brain

461

CaEP

PV Fig. 96.

a

F

0

Calcium-binding proteins in the rat brain

463

CaEP

PV Fig. 98.

Figs 82-98. The distribution and density of PV + and CaBP+ cell bodies, fibres and terminals in the adult rat brain are presented on slightly modified schematic drawings, copied from available atlases.M8~2S+~’ T’he mapping is based on frontal, horizontal and sagittal sections of the brain, and transverse and longitudinal sections of the spinal cord. It represents a synthesis based on observations of a large number of animals. The map is precise but the subtleties of the immunolabelling can only be appreciated in the histographies and in the pictures. Densities of cell bodies, axons and terminals are graded subjectively and visualized by the proximity of the labels to each other. Dendrites are not included in the map. The mapping of neurons of the cortical mantle and of the cerebellum is limited to selected examples. The cell bodies are represented by stars. One star represents l&20 PV (respectively CaBP) perikarya. The cell bodies can be grouped in intensely, moderately or lightly stained, according to the staining at very low (1: 20,000), moderate (1: 10,000) or only at high (1:2000) antibody concentrations. Gradations in somatal staining intensity and differences in the size of the cell bodies are summarized in Tables I and 2. Three grading steps have been used for terminals: high, moderate and low (see, also Table 1). For abbreviations see Table 6. 464

alar-bindjng

465

proteins in the rat brain

inte~ngled with the fibres of the media1 forebrain bundle. The CaBP+ “new” aggregate (CaBPl) is embedded in the fibres of the medial lon~tudinal fascicle (Figs 53, 54, 58C). The CaBPl neurons coincide somewhat with the adrenalinecontaining neurons of group Al, but their density is much higher in this region. The connections of these “new” nuclei are unknown but will be the object of future studies. Not only “new” nuclei, but also as yet unknown cell types like the “Hantel cells” of the dentate gyrus are revealed by antibodies against ~lci~rn-bin~ng proteins (Fig. 78). The~fore, beyond being magnifi~nt markers for known pathways, antibodies against ca1cium-binding proteins even contribute to increase our knowledge of the brain by revealing “new” nuclei and “new” neurons.

neurons

Hormones of matrix homeost~is and ~alci~-biding proteins Vitamin D ~tabo~ite~ and ea~&i~ -binding proteins. It remains to be dete~ined by experiments combining automdiography with immunohistochemistry, whether the CaBP+ and PV+ cells are targets for vitamin D or one of its metabolites. Previous studiesz~.2~,26’ have revealed a high concentration of receptors for I,25 (OH,) vitamin D, in the nucleus centralis amygdala, bed nucleus of the stria terminalis, nucleus ~~vent~e~~s, nucleus parataenia~s and rhomboideus, area postrema, nucleus tractus solitarius, caudal spinal trigeminal nucleus, as we11as lamina II of the spina cord. A11these regions abound in CaBP+ neurons and mostly lack PV neurons. Su~~singly, however, the richest store of CaBP, the cerebellum, has no vitamin D recep tors.s*z5g*2W Recent work,261 however, has reveaied additional brain targets of 1,25 (OH,) vitamin D,, some of which are rich in PV (e.g. nucleus reticularis thalami). In conclusion, therefore, the relationship between 1,25 (OH,) vitamin D, receptors and expression of calcium-binding proteins is not directly evident. It has been shown that the brain CaBP concentration can be raised by long-tetm chole~l~iferol administration in chronically rachitic chicksz6r Acutely rachitic rats, on the other hand, do not show abnormalities in the CaBP con~ntration of the cerebellum.242 Chronic application of ove~hysioIogi~1 doses of vitamin D to normal rats do not affect the CaBP ~on~ntration in the rat brain but change the ~n~nt~tion of PV in the ~udatoput~en,73 In the enteric nervous system, on the other hand, CaBP expression is under control of 1,25 (OH,) vitamin D, and even Ca2C?‘8-2go

Parath~rm~ne and caIcitoni~ There are no reports in the literature about correlations between the effect of paratho~one, calcitonin or the ~alcitonin-re1ated gene product on the int~celluiar ~lcium-bin~ng proteins.

Calcitonin and ~cito~n-gene related products9* as we11as ~n~ng sites for ca1citonini’9 occur in the brain. Although these studies are not detailed enough to permit a direct ~rnp~~n, the available evidence suggests some correlation with the ~s~bution of CaBP. ~a~~i~ ch~ne~s and calcium -binditzg pr~iei~ The entry of Ca’+ into neurons and other cell types is regulated by either re~ptor-o~rat~ channels or by voltage-de~ndent Ca*+ channels.2” There are probably at least three different types of voltagedependent Ca 2f channels, which can be differentiate by electrophysiolo~cal~ and pha~acolo~cal means. The organic Ca*+ antagonist are able to interact with these channels to modify Ca2+ fluxes. By labelling them radioactively, it is now possible to study their pro~rties*2,*~3 and to study the distribution of the binding sites~~1w~218 in the brain. The binding sites for the calcium antagonjst 1,4-dihydropy~dine are hetergeneoudy dist~buted in the brain and mainly concentrated on the granule cells of the dentate gyrus, in su~~~ia1 layers of the cerebral cortex, in the striatum, in the cerebellum and in the external plexiform layer of the olfactory bulb. Unfortunately, techniques do not permit a cellular resolution, but the general pattern of dist~bution matches fairly well the ~st~bution of immunorea~ti~ty towards CaBP antibodies (with the exception of the external plexiform layer of the olfactory bulb). Cal+ currents and &alci~-birding

proteins

All neurons seem to posses Cat+ currents,‘~ which permit a rapid modi~cation of the intra~llu1ar calcium concentration. These Ca*+ currents are, however, not sufficient in amplitude to generate calcium action potentials. ‘46An exception are some neurons, which develop tetrodotoxin resistant, Ca*+-dependent spikes. ‘~*‘6’,‘62,2~ These neurons are the Purkinje cells of the cerebellum, hip~ampal pyra~dal cells, granule cells of the dentate gyrus, inferior olive cells, 5% of the spinal ganglion cells, myenteric plexus neuronsizt and muscle spindle afferences.‘28 Again, the ~st~bution of cells showing calcium spikes matches best with those displaying CaBP immunoreactivity, and not with cells showing PV immunostaining. Nevertheless, the neurologicaf mutant mouse “staggerer’“, in which the Purkinje cells do not develop Ca*+ spikes,” displays normal CaRP and PV immunoreactivities (unpublish~ ob~rvation). ~ypothes~ on the role o~~~~&i~-b~~~protei~

irt the brain

~aicium-binding proteins like ~lm~ulin, which occurs in all cells, may be involved in a general housekeeper function, common to all neurons (i.e. neurot~smission,” or axopiasmic transport’n). Proteins like PV and CaBP, which are selectiveiy associated only with certain neurons, are probabty executing a more specific role. Both PV- and CaBP-IR neurons have different shapes

366

M R.

(bipolar. pseudoumpolar, multrpolar) sizes (from 10 x 10 LO x 35 pm diameter), localizations and connections. They also fall into both categories of tiolgi type I (projection neurons, “AssoziationmIlen, Knmmissurenzellen. Strangzellen”) and Golgi I1 (interneurons,“Binnenzellen, Sehaltzellen”) neurons. PV mostly occurs m inhibitory but also in cxcltatory interneurons (primary afferent neurens). C’aBP nntihndiei mainly stain excitatory neurons, but also some inhibitory ones (Purkinje cells ol the cerebellum). At first sight tt is therefore dilticull to find a common denominator among this variety nf cell typesreacting with PY and/or with CaBP, which could give the clue fol understandmg their role in the nervous system It can Only be stated Ihal neurons containing PV and/or CaBP are privileged iu the handling of Ca:+ and/or Mg?ions in a way which is precluded to other neurons. PV+ and!or CaBP-t- neurons may share physiological or biochemical characteristics, which distinguish them from other neurons. Like oalmoduhn, PV and C&P may be responsible for the cantrob of multiple processes inslde a cell. Although the “exercise book” for these two calcium-binding proteins may be well defined, it may not be completely executable m all neurons, if olhrr proteins, competing for the same ion, are present. Thus the difiirentlal intrawllular distribution and the combinations and permutations of different calciumbmding proteins in a given neuron, as well as their subtle competition for calcium,‘magnesium ions may establish the scqucnce of reactions. Furthermore, the presence of at least three calcium channels& and of a pulent calcium pump (Ca-ATFasel, dXerentially distributed in various neurons. may confer additional regulatory passlhilities. The only?&1 cstablishcd biochemical function of PV is to bind Mg=+ in the resting state and to exchange it against Ca2+ upon cell activation (see, however, Refs 100, 149) CaBP, on the other hand, map occur in a @+-free form and bind Ca” upon raising the intracellular level. According to this basic biochemical function, CaBP and PV rrprrseni an intracellular buffering mechanism. They limit, redistrlhok. and restore the physiologic intracellular Ca’+ !Mg’+ concentrations, perhaps by shuttling Ca*+, as they do in muscles43~Jh~20”m the endoplasmic reticulum, which may function as a cdl&m sink”, ” or in the newly discovered “calciosomes”.2Y4 The mapping study inspires hypotheses on the possible physiological and pathological role of these two proteins in the brain. We discuss them separakly. 50

I note a very close similarity between the distribution of CaBP and of the dihydroperydin subtype of calcium channels, mapped radioauto@rdphically.” ‘90~2’*Cells which display calcium spikes arc preferentially labelled by CaBP antibodies too and CaBP+ regions are rich in calcitonin, parathormone and vitamin D receptors. 0thers2”.‘Rn,‘5’ have noticed an inverse association between the concentration of CaBP and cellular excitability. However, none of these associations is compelling and their sigmficancc remains obscure. Intercstmgly. several of the systems conraining CaBP have been imphcated in the pathology of neurodegenerative disorders. The nucleus basolis of Meyncrt, the dorsal Raphe nucleus, hippocampal pyramidal cells, pyramidal cells of the upper cortical layersla6 (particularly those in the temporal lobe) are vulnerable to the neurodegeneration of the Alzheimer-type and are rich in C&P. The striatonigral pathway, affected by Huntington’s disease and the nigrostriatal projection, destroyed in Parkinson’s disease, are two of the most prominent CaBP systems. Thus, seemingly disparate elements ofneurodegenerative pathology all share an important chemical marker, CaBP, a protein, which presumably is decisively important in these neurons for intreneuronal homeustatic mechanisms involving calcium.

CELKI

Malfunctioning or interference with the function oLCaBP may render these cells more susceptible tn intermlttent calcium fluctuations. and perhaps more prone KI accumulate intolerable quantities of calcium.db~49 The uncontrolled elevation of the intracellular Ca2+ concentration leads to protein denaturation, mineral precipitation and cell death.fg the three landmarks of neurodegenerative disorders. In this context it is worth noting that a defect in the brain mineral metabolism was consIdered responsible for related neurodegenerative disorders, as seen in certain populations of the Western Pacific.” Thus, my working hypolhesir is that CaBP serves as 2, common molecular denominator, underlying several diffcrent manifestations of neuradegenerative pathology.$q

The mapping of PV largely supports the contention that PV IS a marker for fast neurons.45 In fact, the PV+ Cc&i type I neurons (l.c. large spinal ganglion cells, retinal ganglion ce!ls etc.) all belong to receptors with a slow adaptation. These receptors are characterized by their ability 10 maintain a high firing rdte for as long as the stimulus is applied. Sustained bursts of activity are also typical for the motoneurom in the oculomotor, trochlear and abducens nuclei. Thus, a high finng rate, sustained aver a long period of time, is a characteristic of Golgi type 1 neurons which display PV immunoreactivity. On the other hand, axons contacring peripheral receptors with rapid adaptation (i.e. Pacinian corpuscles) are devoid of PV immunoreactivity. The overwhelming majority of P:‘-IR neurons in the central nervous system are interneurons (GO@ type II cells). It seems as if most cortical PVC cells are iahibi;&y and use GABA as their neurotransmitter. 4’,‘4425RElectroohvsloloelcal data on Golgi type II cells are scarce in th; Iikrature, because of the difficulties in their impalement and identification. A study of Table 3 indicates that these neurons, all bring PV + . have a high firing rute and great convergence of afferent excitatory inputs. One group”” directly demonstrated that fast sptking cells in the CAI region of the hlppocampus contain PV, thus supporting the original jdea.J0,31.45.0 In helping decreasing the Ca” concentration, PV shortens the Ca*+-dependent K+ outflow, which is reponsible for the cell after hyperpolarization and the accomodation of firing. “’ The K+-current tends to hold the membrane in the hypcrpolarized state and is involved in spacing the action potentials. Hart~r: the role of PV is to shorten this relntive iefractory period. An analogous situation is mimicked by intracellular iniection of EGTA or BAPTA ICaL+ chelators with an affinity slightly higher than &BP and PV) inlo neurons.“’ Such experiments indeed show a dramatic shortening of the refractory time of neurons, a decrease in the si7e of postsynaptic potentials and a decrease in the duration of the post-tetanic potentiation.‘37 In this context it is noteworthy that a modulation of the Ca2 ’ -dependent K+-current by caltnodulin has been reported.“’ The increase in the concentration of FV, observed in epileptic mice.21 may represent a homeostatic mechanism to preserve intact the discharge properties of “defective” ncuron5 In concentrating on the possible functions of the calcium-PV interaction. ir should not he neglected that m binding Ca:+. PV releases Mg2+. And this may be more than an irrelevant side effect. because Mg’+ is a potent enzyme activator At least 100 enzymes are said to be magnesiumdependent or activated enzymes and several are involved in key steps in intermediary metabolism and phosphorylation.” These enzymes are important for providing energy for transport and for regulation of various processes in the cell and at the cell membrane. Magnesium also participates in protein and DNA synthesis. DNA and RNA transcription and translation of messenger RNA.‘O Furthermore, ATP mostly occw-R in the cell as a complex of Mg’.L--ATP.“’

Calcium-binding proteins in the rat brain One could object that, if PV has a role restricted to the control of the excitability of a neuron, then accordingly its localization should be close to the electrically relevant structures (plasma membrane). It is not difficult to integrate the presence of PV immunoreactivity in cytoplasm and even nuclei, if we assume that the rate at which a neuron recovers from a train of action potentials, depends on the metabolic state of the cell. A very active neuron, undergoing many depolarizations per minute, must have a higher metabolism,54 which in turn is probably dependent on Ca2+ (and/or Mg?+ ) for its synchronization and regulation. It is noteworthy that in many focahzations the dist~bution of PV matches well that of the enzyme cytochrome-oxidase30.3’ a “marker” for active neurons.“s The mechanisms by which the protein synthesis is coupled with the functional state of a cell remains controversial. Yet one attractive possibility is that Ca*+ ions serve as coupling factors between cellular excitation and the rate of protein synthesisJ3 Thanks to its extremely high diffusion constant s*.*’PV may therefore not only buffer incoming Ca*+ but also “facilitate” the transport of Ca*+ and Mg*+ to places of need in other parts of the neuron. We have no evidenceis for a loss of PV neurons in Aizheimer’s disease, as claimed by othersi In conclusion, PV is preferentially associated with the more active (electrically and metabolically) neurons in a given fun~tiona1 system. It may act primarily as a stabilizer of the intracellular Ca*+ concentration, thus inhibiting the Ca2+-dependent K+-conductance. At the same time, PV may directly or indirectly stimulate metabolism by the

461

concomitant release of M$+, which selectively triggers the enzymatic machinery of the cell. This hypothesis may be directly tested by injecting PV or antibodies against PV in giant neurons of Aplysia californica, some of which display an intense PV-like immunoreactivity.~’ Acknowledgements-I thank all colleagues and friends who helped me to identify immunostained structures on the original slides, on photographs and in drawings and those who read and commented on parts of the various versions of the manuscript. For kindly supplying part of the antisera and antigens used in this study I am indepted to Drs Heizmann, and K&i, Zurich (parvalbumin); Dr A. W. Norman, Riverside (calbindin D-2&); Dr B. Boss, La Jolla and Dr D. Cocchia, Milan fS-1001: Drs M. Berchtold and A. Means. Houston (calmodulin);’ br Shimada, Tokyo (troponin-C). MS Chr, Hemmerle, H. Tedaldi, H. Weber, E. Schiirer, G. Weinbrenner, E. Schiingarth, J. Schlahn and Mr W. BaierKustermann gave excellent technical, photographic and secretarial assistance during various phases of this work. The original research presented in this manuscript was supported partly by grants from the following foundations: Hartmann Milller-Stiftung, EMDO-Stiftung, Ciba-GeigyStiftung, R~he-Foundation, Emil-Barrell-Stiftung, Julius Klaus-Stiftung, Hermann Klaus-Stiftung, Ziircher Hochschulverein, NF 3559.083, DFG CE/23/1-1, WanderPharma AC, Sander-Stiftung, Sandoz-foundation.

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