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Calbindin D-28k positive projection neurones and calretinin positive interneurones of the rat globus pallidus
Brain Research 929 (2002) 243–251 www.elsevier.com / locate / bres
Research report
Calbindin D-28k positive projection neurones and calretinin positive interneurones of the rat globus pallidus A.J. Cooper, I.M. Stanford* Department of Pharmacology, Division of Neuroscience, Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2 TT, UK Accepted 23 August 2001
Abstract Immunohistochemistry for three calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin revealed neuronal heterogeneity within the GP. Each neurone appeared to express either a single type of calcium binding protein or none at all. The co-localisation of calcium binding proteins was not observed. Combined immunohistochemistry and retrograde tract tracing using colloidal gold particles injected into the projection fields, the substantia nigra or subthalamic nucleus, revealed that projection neurones could be labelled with either calbindin or parvalbumin. These cells were of medium size (22312 mm), multipolar and moderate varicose dendritic trees. In contrast, calretinin-positive neurones were never retrogradely labelled, even in regions where neuronal colloidal gold deposits were numerous. This, combined with their rarity (,1%) and small size (1139 mm), suggests that calretinin may be a neurochemical marker for putative rat globus pallidus interneurones. Calcium-binding proteins are known to have unique buffering characteristics that may confer specific functional properties upon pallidal neurones. Indeed, differential calcium binding protein expression may underlie the electrophysiological heterogeneity observed in the rat globus pallidus. 2002 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing, and imaging techniques Keywords: Basal ganglia; Parvalbumin; Colloidal gold; Subthalamic nucleus; Substantia nigra
1. Introduction Until recently, the globus pallidus (GP: equivalent to the external segment of the primate globus pallidus) was viewed as a simple structure composed of a homogenous population of neurones which act as relays between the striatum and the subthalamic nucleus. With advances in techniques, this view has recently been challenged and the GP is now considered a heterogeneous structure which has a wide range of connections throughout the basal ganglia [41] including the internal segment of the globus pallidus [21] and the substantia nigra pars reticulata [40], the striatum [42], the reticular thalamic nucleus [39] and the pedunculopontine region [28]. Morphological studies using the Golgi technique [25,17] and intracellular staining combined with electrophysiology *Corresponding author. Tel.: 144-121-414-4529; fax: 144-121-4144509. E-mail address: [email protected] (I.M. Stanford).
[33,24] have revealed multiple GP neuronal subtypes. In vivo, multiple subtypes of rat GP neurone have been identified on the basis of firing pattern and waveform [23], while sharp microelectrode studies in vitro have indicated that the guinea pig GP contains three neuronal subtypes [29,30]. Our own patch clamp studies in slices of rat GP have identified two major populations of GP neurones which can be distinguished on the basis of membrane properties and morphology [45]. Differences in neuronal electrophysiological characteristics are often accompanied by differential neurochemical expression. At the macroscopic level, the GP exhibits a complementary pattern of parvalbumin (PV) and calbindin (CB) neuropil expression [10,36]. However, to date, only PV positive cell bodies have been observed in the rat GP [15,36] although CB positive [10,32] and calretinin (CR) positive neurones [13,32] have been observed in the GPe of the primate. The aim of the current study was to determine whether pallidal neurones could be identified on the basis of somal
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PV, CB or CR expression and whether there is evidence for the co-localisation CaBPs as in other areas of the basal ganglia such as substantia nigra pars compacta [38] and subthalamic nucleus [1]. Immunohistochemistry for CaBPs was coupled with retrograde tract tracing to determine whether their differential expression could be used as a marker neuronal phenotype in the GP.
2. Material and methods
2.1. Subjects Male Sprague–Dawley rats (110–140 g) were used throughout the study in order to permit comparisons with the results of in vitro electrophysiological studies being conducted in the laboratory [45]. The animals were group housed on a 12 h-light / dark cycle and given free access to food and water. All procedures were conducted in accordance with the Animals (Scientific Procedures) Act, 1986, UK.
2.2. Retrograde neuroanatomical labelling of globus pallidus neurones A colloidal gold tracer was stereotaxically injected into 18 animals, which had been anaesthetised with Domitor / Vetalar (i.p.). The tracer consisted of a complex of wheatgerm agglutinin and inactivated (apo) horseradish peroxidase (Sigma, UK) conjugated to colloidal gold particles (WGAapoHRP-Au). The colloidal gold particles were produced according to a modified version of the method of Basbaum and Menetrey [3] as previously described [9,27]. Bilateral injections were made into either the substantia nigra (SN, 200 nl per site) or subthalamic nucleus (STN, 100 nl per site). Co-ordinates for the injection sites were based on the atlas of Paxinos and Watson [34]. The tracer was injected over a period of 20 min using a 1-ml Hamilton syringe, which was left in place for 10 min after the tracer had been injected in order to prevent excessive spreading of the tracer within the tract. All animals survived for 3 days post-operatively, to allow the tracer to be retrogradely transported.
2.3. Tissue preparation All animals were transcardially perfused under terminal barbiturate anaesthesia. The initial perfusate consisted of 50 ml of 0.9% saline containing 50 IU Heparin (BDH, UK). This was followed by a cold fixative solution consisting of 4% paraformaldehyde (TAAB, UK) in 0.1 M phosphate buffer (pH 7.4). For some animals the fixative also contained 0.2% glutaraldehyde (BDH, UK). Brains were immediately removed and stored in 1% paraformaldehyde in 0.1 M phosphate buffer at 48C until sectioning. Coronal sections of 70 mm thickness were obtained using a
vibratome (General Scientific, UK) and stored in phosphate buffered saline (PBS, pH 7.4) at 48C until processing.
2.4. Antibodies Antibodies specific for each of the CaBPs were obtained commercially. The dilution of the three primary antibodies that gave optimal specific staining was determined by serial dilution as being 1:5000. CB was localised using a monoclonal antibody (Sigma, UK) which has previously been shown to have no cross-reactivity with other CaBPs [6]. CR was localised using a polyclonal rabbit antibody against guinea pig CR (Chemicon International Inc., USA) which has previously been shown to be specific for CR [48] and, in particular, showed no cross-reactivity with CB. PV was localised with a monoclonal antibody (Sigma, UK) which has been shown to be specific for PV [5].
2.5. Single and double immunohistochemistry A 1:4 series of GP sections was processed by standard immunohistochemical methods using one of the antibodies (six animals per antibody). All antibodies were diluted in PBS containing 1% normal serum (Sigma, UK) and all washes between incubations were performed using PBS. Briefly, after permeabilisation in 0.3% Triton X-100 in PBS and blocking of non-specific binding sites with normal serum, sections were incubated in primary antibody overnight at 48C with agitation. The following day, sections were incubated in an appropriate biotinylated secondary antibody (1:400; goat anti-mouse for CB and PV and goat anti-rabbit for CR; Sigma, UK) for 2 h at room temperature. This was followed by incubation in ABC (Vector Laboratories, UK) for 2 h at room temperature, which was pre-prepared according to the manufacturer’s instructions. The signal was revealed using a chromogen solution consisting of 3,39-diaminobenzidine (DAB; 0.025%), ammonium nickel sulphate (0.35%) and H 2 O 2 (0.0006%) for approximately 10 min. The sections were thoroughly washed before being air-dried on to gelatin subbed slides, dehydrated through a series of ethanols and permanently mounted from xylene with Eukitt (BDH, UK). Some sections were processed as above but with the omission of the primary antibody to serve as controls for the specificity of the immunostaining. Two to three sections from each animal were also processed for double immunohistochemistry for all combinations of antibodies. The process was as given for single staining with the following exceptions. After the first chromogenic reaction, any residual peroxidase activity was blocked by incubation in 0.1% H 2 O 2 . Sections were then sequentially incubated with the components of an avidin / biotin blocking kit (Vector Laboratories, UK) to prevent cross-reactivity between the second application of ABC
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and any unbound biotin residues on the first secondary antibody. Three chromogens were used to reveal the antigens, namely nickel-enhanced DAB (as described above) which produced a blue–black reaction product, DAB alone (0.05%10.003% H 2 O 2 ) which produced a brown reaction product and VIP [25] (Vector Laboratories, UK) which produced a purple reaction product. In pilot studies, all possible combinations of chromogens were used to reveal the two antigens in order to establish which combination gave the best colour contrast.
2.6. Co-processing of sections for gold particles and immunohistochemistry A 1:4 series of sections through the GP and sections which included the injection site for each of the animals were co-processed to both reveal gold particles and for immunohistochemistry as previously described [9]. Retrogradely transported gold particles were visualised first using a modified Danscher silver technique [11]. Briefly, sections were incubated in citrate buffer (pH 6) before incubation for 2 h in a developer solution containing silver lactate. Sections were washed overnight in PBS before processing for single immunohistochemistry for one of the CaBPs as described above.
2.7. Topographical distribution, quantitative analysis and image production The boundaries of the GP were determined following staining with cresyl violet acetate (Sigma, UK). The topographical distributions of CB and CR positive neurones were determined by plotting the location of each immunopositive neurone on to stylised line drawings of the GP based on the atlas of Paxinos and Watson [34]. To assess the relative proportion of neurones expressing CB or CR, the number of CB / CR positive neurones per section was determined for each of 40 GP sections which had been processed for single / double immunohistochemistry or combined retrograde tract tracer and immunohistochemistry. An estimate of the mean (6S.E.) number of CB / CR positives per section of GP was then calculated. Estimates of the mean (6S.E.) somal dimensions for 100 neurones for each of the three CaBPs were made for comparison purposes. The long axis (maximum diameter) and short axis (perpendicular to the long axis) were measured with the 340 objective using an eyepiece graticule. Sections were viewed using a standard Olympus BX microscope with a high-resolution video camera attached. Images were captured by means of software from Inspector, UK and viewed in Adobe Photoshop, where annotations were added and adjustments in brightness made. Colour prints were obtained using a high-resolution inkjet printer.
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3. Results
3.1. Distribution of calcium binding protein immunoreactivity At a macroscopic level, CB positive neuropil staining was highest in the medial parts of the GP, although a thin band of staining was also seen adjacent to the striatal border. CB positive cell bodies (8.3560.74 cell bodies per GP section; mean6S.E.M.) were observed throughout the GP (Fig. 1A). Cell bodies were multipolar, bipolar or fusiform in shape (mean dimensions 19.9260.553 11.860.24 mm) with smooth or moderately varicose processes (Fig. 2A). Many of the bipolar neurones lay parallel to the border with the striatum and their processes could often be followed for up to 500 mm within the GP (Fig. 3A). A network of fine, varicose CR positive fibres was visible throughout the GP, with the highest level in the medial part of the structure. CR positive neurones were not restricted to any particular region of the GP (Fig. 1B) (1.5360.22 cell bodies per GP section). These cells were small (11.4960.2238.8260.11 mm) and oval in shape. The somata were either multi- or bipolar with branching, highly varicose proximal processes (Fig. 2B). In agreement with previous studies, many cell bodies and fibres were immunopositive for PV throughout the GP [15,36]. Both cell bodies and fibres showed a gradient in staining density along the medial / lateral axis with the highest levels being present in the lateral parts of the GP, i.e. in a complementary pattern to the CB staining. With regard to morphology, PV positive neurones were heterogeneous, being either fusiform or multipolar although they appeared of similar size to CB positive neurones (22.6660.5312.2760.24) (Fig. 2C). The proximal dendrites of the PV positive cells were infrequently branched and predominantly smooth. PV positive boutonlike structures were visible throughout the GP, although at greatest density in the lateral region.
3.2. Double immunohistochemical staining Double immunohistochemistry enabled direct comparisons of cell body and dendritic morphology to be made in a single section. The greatest contrast between chromogens was given by the combination of nickel-intensified DAB (blue–black), followed by DAB alone (brown, Fig. 3A,B). All combinations of the three primary antibodies were examined. In no instance was a neurone observed which appeared to be double-labelled as evidenced by ‘colourmixing’ of chromogens. Moreover, reversing the sequence of chromogen addition had no effect on the numbers of labelled cells observed for each CaBP. Indeed, the distribution and level of labelling for each CaBP, following a double labelling procedure, mimicked that seen after single immunohistochemistry implying that: (1) GP neurones do
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Fig. 1. (A) Distribution of calbindin D-28k positive neurones in the rat globus pallidus. All calbindin D-28k positive neurones observed in a 1:4 series of sections from one representative animal plotted on to stylised drawings of the rat globus pallidus. Note that calbindin D-28k positive neurones were found in all axes of the globus pallidus. Some neurones were double-labelled with calbindin D-28k immunoreactivity and colloidal gold particles, which had been retrogradely transported following injection of the neuronal tract tracer into the subthalamic nucleus (filled circles). Other neurones were labelled with calbindin D-28k immunoreactivity alone (open circles). (B) Distribution of calretinin positive neurones in the rat globus pallidus. All calretinin positive neurones observed in a 1:4 series of sections from one representative animal plotted on to stylised line drawings. All neurones observed were labelled for calretinin immunoreactivity alone (open circles). In no instance did neurones appear co-labelled for calretinin immunoreactivity and colloidal gold particles, following injection of the tracer into the subthalamic nucleus. Bar51 cm.
not co-express CaBPs; (2) the double immunohistochemical processing did not result in the deterioration of the second antigen and (3) the chromogens were of equivalent sensitivity.
3.3. Colloidal gold-labelled projection neurones Injections of colloidal gold tracer into the STN produced injection sites that filled a large proportion of this structure (Fig. 4A) and resulted in the retrograde transport of gold particles to many neurones throughout all axes of the GP. In contrast, injections into the SN resulted in injections localised to a region of the structure (Fig. 4B) and consequently produced a more restricted distribution of retrogradely labelled neurones in the GP, reflecting the topography of the GP to SN projection [46]. As the distribution and extent of retrogradely labelled neurones are a function of both the site and size of the injection, any attempt to quantify the relative proportions of CB, CR and
PV projection neurones would give a gross underestimation and therefore was not undertaken.
3.4. CaBPs in projection neurones The majority of PV positive neurones were doublelabelled with gold particles following injection into either the STN or SN (Fig. 4C). Those PV positive neurones that were not double-labelled were usually located in regions exhibiting low numbers of gold-labelled cells, and were more likely to come from animals in which the tracer had been injected into the SN. Gold-labelled neurones which were not double-labelled for PV immunoreactivity were also observed, often intermingled with double-labelled neurones, suggesting that PV is not a marker for all GP projection neurones (Fig. 4C). Colloidal gold-labelled CB positive neurones were observed following injection into either the STN or SN (Fig. 4D). These double-labelled neurones were not restricted to any one region of the structure but were found
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in all areas where retrogradely labelled neurones were numerous. In contrast, no CR and gold particle doublelabelled cells were seen in any of the series of sections (taken from six animals) following injection into either the STN (Fig. 4E) or SN (Fig. 4F), even in regions of the GP containing a high density of gold particle-labelled neurones.
4. Discussion The results demonstrate that both CB and CR immunopositive neurones were distributed throughout the GP. In keeping with previous studies, many PV positive cell bodies were observed the density of which was greatest in the lateral parts of the structure [4,15,22,36]. Sequential double immunohistochemical processing suggests that these CaBPs be uniquely expressed with no more than one CaBP being localised in a single cell. Furthermore, combined immunohistochemistry and retrograde tract-tracing technique revealed at least two subpopulations of pallidal projection neurones that express either CB or PV and population non-projection CR positive cells. A summary of the results is presented in Table 1.
4.1. Extent of CaBP distribution Approximately eight CB positive and 1–2 CR positive neurones per section were observed. As the total number of neurones in the rat GP has been estimated, using nonbiased stereological techniques, to be 46 000 [31], it can be deduced that the proportion of CB / CR positive GP cells constitutes approximately 2% of the total number of GP neurones which is less than the 6–15% of CR positive neurones estimated in both segments of the primate pallidum [13]. The proportion of GP neurones expressing PV has not been estimated here, but has previously been reported to be around 66% of the neuronal population [23]. Hence, it is concluded that there is a subpopulation of GP neurones, which expresses none of these CaBPs.
4.2. Morphology of GP neurones
Fig. 2. Monochrome photomicrographs of neurones in the rat globus pallidus showing calbindin D-28k, calretinin and parvalbumin immunoreactivity. (A) A typical calbindin D-28k immunopositive bipolar neurone in the medial region of the globus pallidus. (B) Calretinin immunopositive cell in the medial portion of the globus pallidus with varicose proximal processes emanating from a small, round cell body. (C) Intensely stained parvalbumin positive bipolar and multipolar cell bodies surrounded by a dense plexus of immunopositive fibres in the lateral region of the GP. Bar520 mm.
Due to the extensive CaBPs labelling, estimates of soma size and description of dendritic morphology could be made and comparisons made with previous Golgi / cell filling studies. CB positive and PV positive GP cells were of a comparable size being 20312 and 23312 mm, respectively. These values are broadly in keeping with the major population of medium-sized GP neurones described previously [26,30], while small (11–12314–16 mm) and highly varicose CR positive neurones described here are similar to those small cells with varicose dendrites described in the same studies.
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Fig. 3. Double immunohistochemical staining for calbindin D-28k / parvalbumin and calretinin / parvalbumin. (A) A single calbindin D-28k positive neurone (blue–black) in the lateral globus pallidus with long, branched dendrites running parallel to the striatal border, which is delineated by a dashed line. A band of calbindin D-28k positive neuropil can also be seen adjacent to the striatal border. Many parvalbumin positive globus pallidus neurones (brown) in the lateral part of the globus pallidus were seen. Note the high level of calbindin D-28k staining (blue–black) in the striatal neurones. (B) A typical small, round calretinin positive neurone (blue–black) close to a large bipolar parvalbumin positive neurone (brown). Bar in A560 mm; bar in B510 mm.
4.3. Projection neurones and interneurones
4.4. Origins of CaBP neurophil
The retrograde tract-tracing technique combined with immunohistochemistry identified two populations of projection neurones that expressed either PV or CB. These PV and CB positive neurones could be labelled following injection into either STN or SN suggests that these cells are typical projection neurones in that they send collaterals to both structures [24]. No CR positive and gold-labelled neurones were observed even in areas in which there are large numbers of labelled neurones (Fig. 4E,F) suggesting that CR may be a neurochemical marker for a subpopulation of interneurones in the rodent GP. However, this population may extend projections to the striatum or entopeduncular nucleus, which were not targeted in this study. The possible existence of a sparse interneurone population of small diameter GP cells has been previously proposed in the rat on the basis of electrophysiological [30], morphological [26] and neurochemical data [43]. Indeed, the NMDAR2D glutamate receptor subunit, a marker for interneurones in many regions of the brain [44] is expressed in the GP. The significance of such a population largely depends on the connectivity with other neurones. In the striatum, small populations of interneurones have a profound effect on output and physiological function [18]. Striatal and GP CR positive cells represent a sparse population in rodents. However in the primate striatum CR interneurones are the most abundant interneurones outnumbering PV positive cells by 2–3:1 [49]. Thus, it is possible that a similar situation may occur in the primate GP.
In agreement with previous studies [15,36], CB and PV were largely expressed in a complementary pattern, with CB staining highest in the medial region and PV immunoreactivity highest in the lateral regions of the GP. In addition a band of CB positive fibres lying adjacent to the striatal border was also observed as previously described by Rajakumar et al. [36]. Anatomical tracing studies of the striatopallidal pathway have described two sites of terminal arborisation, one lying adjacent to the striatal border and the other more medially [47]. As the striatal output neurones are known to express high levels of CB [14], these two regions of fine CB fibres may correspond to the regions of the GP with a high density of striatal inputs. The origin of the fine CR positive fibre network is less clear. It is unlikely to be derived from the striatal input as CR positive neurones of the striatum are considered to be interneurones [12]. Similarly, these fibres are unlikely to originate in the STN as no CR positive cells have been identified in this structure in the rat [37].
4.5. Functional considerations The CaBPs are related proteins belonging to the EFhand family [35]. It has been proposed that the CaBPs act to buffer intracellular calcium ion concentrations, which are involved in a functionally diverse range of neuronal processes [20]. However, an excessive intracellular concentration of Ca 21 ions is neurotoxic [8]. Hence, maintenance of intracellular calcium ion levels within a range is
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Fig. 4. Combined calcium binding protein immunohistochemistry and retrograde tracing. (A) Injection site in the subthalamic nucleus (STN) which is delineated by a dashed line; ic, internal capsule. (B) Injection site in the substantia nigra (SN) which is delineated by a dashed line. (C) Retrogradely transported silver-enhanced gold particles (black) in the globus pallidus following injection of the tracer into the STN. Note that two of the neurones were also parvalbumin immunopositive (brown), whilst one cell was not co-labelled for parvalbumin. (D) Calbindin D-28k immunopositive neurone (brown) which was double-labelled with retrogradely transported silver-enhanced gold particles (black) following injection of the tracer into the SN. (E, F) Calretinin (brown) positive neurones in the globus pallidus were not labelled with retrogradely transported silver-enhanced gold particles (black) following injection into either the STN (E) or SN (F). Bar in A, B55 mm; bar in C, D510 mm.
critical for normal neuronal functioning. Although the ability of the CaBPs to bind and thereby buffer intracellular calcium levels is accepted [2], the precise function of
the individual CaBPs in neurones remains undetermined. CaBP expression does not appear to be linked with a particular neurotransmitter since, for example, PV is
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Table 1 Summary of results
Location of Cell bodies Neuropil Relative abundance of cell bodies Size of cell bodies Long axis (mm) Short axis (mm) Proximal dendrites Projection neurones
Calbindin D-28K
Calretinin
Parvalbumin
Uniform Medial1adjacent to striatal border ,2%
Uniform Ventromedial
Lateral Lateral
,1%
Numerous
19.9260.55 11.860.24 Smooth or varicose Yes
11.4960.22 8.8260.11 Varicose No
22.6660.5 12.2760.24 Smooth Yes
expressed in GABAergic, glycinergic and glutamatergic neurones (reviewed by Baimbridge et al. [2]). However, electrophysiological studies have linked the expression of parvalbumin with fast-spiking activity [19]. In-keeping with this, electrophysiological studies have identified cells that exhibit fast-spiking activity in the GP [33,45]. In contrast, no correlation between physiological activity and expression of either CB or CR has been found, although both have been suggested to have a neuroprotective role [16,7]. Future studies linking electrophysiological properties and expression of a particular CaBP will without doubt be instructive in defining the role that GP neurones play in basal ganglia circuitry.
Acknowledgements The authors wish to thank Dr I.J. Mitchell for use of the photomicroscope system and for invaluable assistance in criticising the manuscript. This work was supported by the Wellcome Trust, UK (Grant No. 050196 / Z / 97 / Z) and a Research Project Grant from the Faculty of Medicine and Dentistry, University of Birmingham.
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A. J. Cooper, I.M. Stanford / Brain Research 929 (2002) 243 – 251 [24] H. Kita, S.T. Kitai, The morphology of globus pallidus projection neurones in the rat: an intracellular staining study, Brain Res. 636 (1994) 308–319. [25] J.L. Lanciego, P.H. Goede, M.P. Witter, F.G. Wouterlood, Use of peroxidase substrate vector VIP for multiple staining in light microscopy, J. Neurosci. Methods 74 (1997) 1–7. [26] O.E. Millhouse, Pallidal neurones in the rat, J. Comp. Neurol. 254 (1986) 209–227. [27] I.J. Mitchell, S. Lawson, B. Moser, S.M. Laidlaw, A.J. Cooper, G. Walkinshaw, C.M. Waters, Glutamate-induced apoptosis results in a loss of striatal neurones in the parkinsonian rat, Neuroscience 63 (1994) 1–5. [28] T. Moriizumi, T. Hattori, Separate neuronal populations of the rat globus pallidus projecting to the subthalamic nucleus, auditory cortex and pedunculopontine tegmental area, Neuroscience 46 (1992) 701–710. [29] A. Nambu, R. Llinas, Electrophysiology of globus pallidus neurones in vitro, J. Neurophysiol. 72 (1994) 1127–1139. [30] A. Nambu, R. Llinas, Morphology of globus pallidus neurones: its correlation with electrophysiology in guinea pig brain slices, J. Comp. Neurol. 377 (1997) 85–94. [31] D.E. Oorschott, Total number of neurones in the neostriatal, pallidal, subthalamic and substantia nigral nuclei of the rat basal ganglia: a stereological study using the Cavalieri and optical disector methods, J. Comp. Neurol. 366 (1996) 580–599. ˆ ´ F. Cicchetti, Calcium-binding [32] A. Parent, M. Fortin, P.Y. Cote, proteins in primate basal ganglia, Neurosci. Res. 25 (1996) 309– 334. [33] M.R. Park, W.M. Falls, S.T. Kitai, An intracellular HRP study of the rat globus pallidus. I. Responses and light microscopic analysis, J. Comp. Neurol. 211 (1982) 284–294. [34] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Co-ordinates, 2nd Edition, Academic Press, New York, 1986. [35] A. Persechini, N.D. Moncrief, R.H. Kretsinger, The EF-hand family of calcium-modulated proteins, Trends Neurosci. 12 (1989) 462– 467. [36] N. Rajakumar, W. Rushlow, C.C.G. Naus, K. Elisevich, B.A. Flumerfelt, Neurochemical compartmentalisation of the globus pallidus in the rat: an immunocytochemical study of calciumbinding proteins, J. Comp. Neurol. 346 (1994) 337–348. [37] A. Resibois, J.H. Rogers, Calretinin in rat brain; an immunohistochemical study, Neuroscience 46 (1992) 101–134.
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[38] J.H. Rogers, A. Resibois, Calretinin and calbindin D-28k in rat brain — patterns of partial colocalisation, Neuroscience 51 (1992) 843– 865. [39] S.J. Shammah-Lagnado, G.F. Alheid, L. Heimer, Efferent connections of the caudal part of the globus pallidus in the rat, J. Comp. Neurol. 376 (1996) 489–507. [40] Y. Smith, J.P. Bolam, Neurones of the substantia nigra pars reticulata receive a dense GABA-containing input from the globus pallidus in the rat, Brain Res. 493 (1989) 160–167. [41] Y. Smith, M.D. Bevan, E. Shink, J.P. Bolam, Microcircuitry of the direct and indirect pathways of the basal ganglia, Neuroscience 86 (1998) 353–387. [42] W.A. Staines, S. Atmadja, H.C. Fibiger, Demonstration of a pallidostriatal pathway by retrograde transport of HRP-labelled lectin, Brain Res. 206 (1981) 446–450. [43] D.G. Standaert, C.M. Testa, A.B. Young, J.B. Penney, Organisation of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat, J. Comp. Neurol. 343 (1994) 1–16. [44] D.G. Standaert, G.B. Landwehrmeyer, J.A. Kerner, J.B. Penney, A.B. Young, Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurones in the rat neostriatum, neocortex and hippocampus, Mol. Brain Res. 42 (1996) 89–102. [45] I.M. Stanford, A.J. Cooper, Electrophysiological and morphological characterisation of rat globus pallidus neurones in vitro, J. Physiol. 527 (2) (2000) 291–304. [46] D. Van der Kooy, T. Hattori, K. Shannak, O. Hornykiewicz, The pallido-subthalamic projection in rat: anatomical and biochemical studies, Brain Res. 204 (1981) 253–268. [47] C.J. Wilson, K.D. Phelan, Dual topographic representation of neostriatum in the globus pallidus of rats, Brain Res. 243 (1982) 354–359. [48] L. Winsky, H. Nakata, B.M. Martin, D.M. Jacobowitz, Isolation, partial amino acid sequence and immunohistochemical localisation of a brain-specific calcium-binding protein, Proc. Natl. Acad. Sci. USA 86 (1989) 10139–10143. [49] Y. Wu, A. Parent, Striatal interneurons expressing calretinin, parvalbumin or NADPH-diaphorase: a comparative study in the rat, monkey and human, Brain Res. 863 (1–2) (2000) 182–191.
Research report
Calbindin D-28k positive projection neurones and calretinin positive interneurones of the rat globus pallidus A.J. Cooper, I.M. Stanford* Department of Pharmacology, Division of Neuroscience, Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2 TT, UK Accepted 23 August 2001
Abstract Immunohistochemistry for three calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin revealed neuronal heterogeneity within the GP. Each neurone appeared to express either a single type of calcium binding protein or none at all. The co-localisation of calcium binding proteins was not observed. Combined immunohistochemistry and retrograde tract tracing using colloidal gold particles injected into the projection fields, the substantia nigra or subthalamic nucleus, revealed that projection neurones could be labelled with either calbindin or parvalbumin. These cells were of medium size (22312 mm), multipolar and moderate varicose dendritic trees. In contrast, calretinin-positive neurones were never retrogradely labelled, even in regions where neuronal colloidal gold deposits were numerous. This, combined with their rarity (,1%) and small size (1139 mm), suggests that calretinin may be a neurochemical marker for putative rat globus pallidus interneurones. Calcium-binding proteins are known to have unique buffering characteristics that may confer specific functional properties upon pallidal neurones. Indeed, differential calcium binding protein expression may underlie the electrophysiological heterogeneity observed in the rat globus pallidus. 2002 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing, and imaging techniques Keywords: Basal ganglia; Parvalbumin; Colloidal gold; Subthalamic nucleus; Substantia nigra
1. Introduction Until recently, the globus pallidus (GP: equivalent to the external segment of the primate globus pallidus) was viewed as a simple structure composed of a homogenous population of neurones which act as relays between the striatum and the subthalamic nucleus. With advances in techniques, this view has recently been challenged and the GP is now considered a heterogeneous structure which has a wide range of connections throughout the basal ganglia [41] including the internal segment of the globus pallidus [21] and the substantia nigra pars reticulata [40], the striatum [42], the reticular thalamic nucleus [39] and the pedunculopontine region [28]. Morphological studies using the Golgi technique [25,17] and intracellular staining combined with electrophysiology *Corresponding author. Tel.: 144-121-414-4529; fax: 144-121-4144509. E-mail address: [email protected] (I.M. Stanford).
[33,24] have revealed multiple GP neuronal subtypes. In vivo, multiple subtypes of rat GP neurone have been identified on the basis of firing pattern and waveform [23], while sharp microelectrode studies in vitro have indicated that the guinea pig GP contains three neuronal subtypes [29,30]. Our own patch clamp studies in slices of rat GP have identified two major populations of GP neurones which can be distinguished on the basis of membrane properties and morphology [45]. Differences in neuronal electrophysiological characteristics are often accompanied by differential neurochemical expression. At the macroscopic level, the GP exhibits a complementary pattern of parvalbumin (PV) and calbindin (CB) neuropil expression [10,36]. However, to date, only PV positive cell bodies have been observed in the rat GP [15,36] although CB positive [10,32] and calretinin (CR) positive neurones [13,32] have been observed in the GPe of the primate. The aim of the current study was to determine whether pallidal neurones could be identified on the basis of somal
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03263-2
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PV, CB or CR expression and whether there is evidence for the co-localisation CaBPs as in other areas of the basal ganglia such as substantia nigra pars compacta [38] and subthalamic nucleus [1]. Immunohistochemistry for CaBPs was coupled with retrograde tract tracing to determine whether their differential expression could be used as a marker neuronal phenotype in the GP.
2. Material and methods
2.1. Subjects Male Sprague–Dawley rats (110–140 g) were used throughout the study in order to permit comparisons with the results of in vitro electrophysiological studies being conducted in the laboratory [45]. The animals were group housed on a 12 h-light / dark cycle and given free access to food and water. All procedures were conducted in accordance with the Animals (Scientific Procedures) Act, 1986, UK.
2.2. Retrograde neuroanatomical labelling of globus pallidus neurones A colloidal gold tracer was stereotaxically injected into 18 animals, which had been anaesthetised with Domitor / Vetalar (i.p.). The tracer consisted of a complex of wheatgerm agglutinin and inactivated (apo) horseradish peroxidase (Sigma, UK) conjugated to colloidal gold particles (WGAapoHRP-Au). The colloidal gold particles were produced according to a modified version of the method of Basbaum and Menetrey [3] as previously described [9,27]. Bilateral injections were made into either the substantia nigra (SN, 200 nl per site) or subthalamic nucleus (STN, 100 nl per site). Co-ordinates for the injection sites were based on the atlas of Paxinos and Watson [34]. The tracer was injected over a period of 20 min using a 1-ml Hamilton syringe, which was left in place for 10 min after the tracer had been injected in order to prevent excessive spreading of the tracer within the tract. All animals survived for 3 days post-operatively, to allow the tracer to be retrogradely transported.
2.3. Tissue preparation All animals were transcardially perfused under terminal barbiturate anaesthesia. The initial perfusate consisted of 50 ml of 0.9% saline containing 50 IU Heparin (BDH, UK). This was followed by a cold fixative solution consisting of 4% paraformaldehyde (TAAB, UK) in 0.1 M phosphate buffer (pH 7.4). For some animals the fixative also contained 0.2% glutaraldehyde (BDH, UK). Brains were immediately removed and stored in 1% paraformaldehyde in 0.1 M phosphate buffer at 48C until sectioning. Coronal sections of 70 mm thickness were obtained using a
vibratome (General Scientific, UK) and stored in phosphate buffered saline (PBS, pH 7.4) at 48C until processing.
2.4. Antibodies Antibodies specific for each of the CaBPs were obtained commercially. The dilution of the three primary antibodies that gave optimal specific staining was determined by serial dilution as being 1:5000. CB was localised using a monoclonal antibody (Sigma, UK) which has previously been shown to have no cross-reactivity with other CaBPs [6]. CR was localised using a polyclonal rabbit antibody against guinea pig CR (Chemicon International Inc., USA) which has previously been shown to be specific for CR [48] and, in particular, showed no cross-reactivity with CB. PV was localised with a monoclonal antibody (Sigma, UK) which has been shown to be specific for PV [5].
2.5. Single and double immunohistochemistry A 1:4 series of GP sections was processed by standard immunohistochemical methods using one of the antibodies (six animals per antibody). All antibodies were diluted in PBS containing 1% normal serum (Sigma, UK) and all washes between incubations were performed using PBS. Briefly, after permeabilisation in 0.3% Triton X-100 in PBS and blocking of non-specific binding sites with normal serum, sections were incubated in primary antibody overnight at 48C with agitation. The following day, sections were incubated in an appropriate biotinylated secondary antibody (1:400; goat anti-mouse for CB and PV and goat anti-rabbit for CR; Sigma, UK) for 2 h at room temperature. This was followed by incubation in ABC (Vector Laboratories, UK) for 2 h at room temperature, which was pre-prepared according to the manufacturer’s instructions. The signal was revealed using a chromogen solution consisting of 3,39-diaminobenzidine (DAB; 0.025%), ammonium nickel sulphate (0.35%) and H 2 O 2 (0.0006%) for approximately 10 min. The sections were thoroughly washed before being air-dried on to gelatin subbed slides, dehydrated through a series of ethanols and permanently mounted from xylene with Eukitt (BDH, UK). Some sections were processed as above but with the omission of the primary antibody to serve as controls for the specificity of the immunostaining. Two to three sections from each animal were also processed for double immunohistochemistry for all combinations of antibodies. The process was as given for single staining with the following exceptions. After the first chromogenic reaction, any residual peroxidase activity was blocked by incubation in 0.1% H 2 O 2 . Sections were then sequentially incubated with the components of an avidin / biotin blocking kit (Vector Laboratories, UK) to prevent cross-reactivity between the second application of ABC
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and any unbound biotin residues on the first secondary antibody. Three chromogens were used to reveal the antigens, namely nickel-enhanced DAB (as described above) which produced a blue–black reaction product, DAB alone (0.05%10.003% H 2 O 2 ) which produced a brown reaction product and VIP [25] (Vector Laboratories, UK) which produced a purple reaction product. In pilot studies, all possible combinations of chromogens were used to reveal the two antigens in order to establish which combination gave the best colour contrast.
2.6. Co-processing of sections for gold particles and immunohistochemistry A 1:4 series of sections through the GP and sections which included the injection site for each of the animals were co-processed to both reveal gold particles and for immunohistochemistry as previously described [9]. Retrogradely transported gold particles were visualised first using a modified Danscher silver technique [11]. Briefly, sections were incubated in citrate buffer (pH 6) before incubation for 2 h in a developer solution containing silver lactate. Sections were washed overnight in PBS before processing for single immunohistochemistry for one of the CaBPs as described above.
2.7. Topographical distribution, quantitative analysis and image production The boundaries of the GP were determined following staining with cresyl violet acetate (Sigma, UK). The topographical distributions of CB and CR positive neurones were determined by plotting the location of each immunopositive neurone on to stylised line drawings of the GP based on the atlas of Paxinos and Watson [34]. To assess the relative proportion of neurones expressing CB or CR, the number of CB / CR positive neurones per section was determined for each of 40 GP sections which had been processed for single / double immunohistochemistry or combined retrograde tract tracer and immunohistochemistry. An estimate of the mean (6S.E.) number of CB / CR positives per section of GP was then calculated. Estimates of the mean (6S.E.) somal dimensions for 100 neurones for each of the three CaBPs were made for comparison purposes. The long axis (maximum diameter) and short axis (perpendicular to the long axis) were measured with the 340 objective using an eyepiece graticule. Sections were viewed using a standard Olympus BX microscope with a high-resolution video camera attached. Images were captured by means of software from Inspector, UK and viewed in Adobe Photoshop, where annotations were added and adjustments in brightness made. Colour prints were obtained using a high-resolution inkjet printer.
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3. Results
3.1. Distribution of calcium binding protein immunoreactivity At a macroscopic level, CB positive neuropil staining was highest in the medial parts of the GP, although a thin band of staining was also seen adjacent to the striatal border. CB positive cell bodies (8.3560.74 cell bodies per GP section; mean6S.E.M.) were observed throughout the GP (Fig. 1A). Cell bodies were multipolar, bipolar or fusiform in shape (mean dimensions 19.9260.553 11.860.24 mm) with smooth or moderately varicose processes (Fig. 2A). Many of the bipolar neurones lay parallel to the border with the striatum and their processes could often be followed for up to 500 mm within the GP (Fig. 3A). A network of fine, varicose CR positive fibres was visible throughout the GP, with the highest level in the medial part of the structure. CR positive neurones were not restricted to any particular region of the GP (Fig. 1B) (1.5360.22 cell bodies per GP section). These cells were small (11.4960.2238.8260.11 mm) and oval in shape. The somata were either multi- or bipolar with branching, highly varicose proximal processes (Fig. 2B). In agreement with previous studies, many cell bodies and fibres were immunopositive for PV throughout the GP [15,36]. Both cell bodies and fibres showed a gradient in staining density along the medial / lateral axis with the highest levels being present in the lateral parts of the GP, i.e. in a complementary pattern to the CB staining. With regard to morphology, PV positive neurones were heterogeneous, being either fusiform or multipolar although they appeared of similar size to CB positive neurones (22.6660.5312.2760.24) (Fig. 2C). The proximal dendrites of the PV positive cells were infrequently branched and predominantly smooth. PV positive boutonlike structures were visible throughout the GP, although at greatest density in the lateral region.
3.2. Double immunohistochemical staining Double immunohistochemistry enabled direct comparisons of cell body and dendritic morphology to be made in a single section. The greatest contrast between chromogens was given by the combination of nickel-intensified DAB (blue–black), followed by DAB alone (brown, Fig. 3A,B). All combinations of the three primary antibodies were examined. In no instance was a neurone observed which appeared to be double-labelled as evidenced by ‘colourmixing’ of chromogens. Moreover, reversing the sequence of chromogen addition had no effect on the numbers of labelled cells observed for each CaBP. Indeed, the distribution and level of labelling for each CaBP, following a double labelling procedure, mimicked that seen after single immunohistochemistry implying that: (1) GP neurones do
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Fig. 1. (A) Distribution of calbindin D-28k positive neurones in the rat globus pallidus. All calbindin D-28k positive neurones observed in a 1:4 series of sections from one representative animal plotted on to stylised drawings of the rat globus pallidus. Note that calbindin D-28k positive neurones were found in all axes of the globus pallidus. Some neurones were double-labelled with calbindin D-28k immunoreactivity and colloidal gold particles, which had been retrogradely transported following injection of the neuronal tract tracer into the subthalamic nucleus (filled circles). Other neurones were labelled with calbindin D-28k immunoreactivity alone (open circles). (B) Distribution of calretinin positive neurones in the rat globus pallidus. All calretinin positive neurones observed in a 1:4 series of sections from one representative animal plotted on to stylised line drawings. All neurones observed were labelled for calretinin immunoreactivity alone (open circles). In no instance did neurones appear co-labelled for calretinin immunoreactivity and colloidal gold particles, following injection of the tracer into the subthalamic nucleus. Bar51 cm.
not co-express CaBPs; (2) the double immunohistochemical processing did not result in the deterioration of the second antigen and (3) the chromogens were of equivalent sensitivity.
3.3. Colloidal gold-labelled projection neurones Injections of colloidal gold tracer into the STN produced injection sites that filled a large proportion of this structure (Fig. 4A) and resulted in the retrograde transport of gold particles to many neurones throughout all axes of the GP. In contrast, injections into the SN resulted in injections localised to a region of the structure (Fig. 4B) and consequently produced a more restricted distribution of retrogradely labelled neurones in the GP, reflecting the topography of the GP to SN projection [46]. As the distribution and extent of retrogradely labelled neurones are a function of both the site and size of the injection, any attempt to quantify the relative proportions of CB, CR and
PV projection neurones would give a gross underestimation and therefore was not undertaken.
3.4. CaBPs in projection neurones The majority of PV positive neurones were doublelabelled with gold particles following injection into either the STN or SN (Fig. 4C). Those PV positive neurones that were not double-labelled were usually located in regions exhibiting low numbers of gold-labelled cells, and were more likely to come from animals in which the tracer had been injected into the SN. Gold-labelled neurones which were not double-labelled for PV immunoreactivity were also observed, often intermingled with double-labelled neurones, suggesting that PV is not a marker for all GP projection neurones (Fig. 4C). Colloidal gold-labelled CB positive neurones were observed following injection into either the STN or SN (Fig. 4D). These double-labelled neurones were not restricted to any one region of the structure but were found
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in all areas where retrogradely labelled neurones were numerous. In contrast, no CR and gold particle doublelabelled cells were seen in any of the series of sections (taken from six animals) following injection into either the STN (Fig. 4E) or SN (Fig. 4F), even in regions of the GP containing a high density of gold particle-labelled neurones.
4. Discussion The results demonstrate that both CB and CR immunopositive neurones were distributed throughout the GP. In keeping with previous studies, many PV positive cell bodies were observed the density of which was greatest in the lateral parts of the structure [4,15,22,36]. Sequential double immunohistochemical processing suggests that these CaBPs be uniquely expressed with no more than one CaBP being localised in a single cell. Furthermore, combined immunohistochemistry and retrograde tract-tracing technique revealed at least two subpopulations of pallidal projection neurones that express either CB or PV and population non-projection CR positive cells. A summary of the results is presented in Table 1.
4.1. Extent of CaBP distribution Approximately eight CB positive and 1–2 CR positive neurones per section were observed. As the total number of neurones in the rat GP has been estimated, using nonbiased stereological techniques, to be 46 000 [31], it can be deduced that the proportion of CB / CR positive GP cells constitutes approximately 2% of the total number of GP neurones which is less than the 6–15% of CR positive neurones estimated in both segments of the primate pallidum [13]. The proportion of GP neurones expressing PV has not been estimated here, but has previously been reported to be around 66% of the neuronal population [23]. Hence, it is concluded that there is a subpopulation of GP neurones, which expresses none of these CaBPs.
4.2. Morphology of GP neurones
Fig. 2. Monochrome photomicrographs of neurones in the rat globus pallidus showing calbindin D-28k, calretinin and parvalbumin immunoreactivity. (A) A typical calbindin D-28k immunopositive bipolar neurone in the medial region of the globus pallidus. (B) Calretinin immunopositive cell in the medial portion of the globus pallidus with varicose proximal processes emanating from a small, round cell body. (C) Intensely stained parvalbumin positive bipolar and multipolar cell bodies surrounded by a dense plexus of immunopositive fibres in the lateral region of the GP. Bar520 mm.
Due to the extensive CaBPs labelling, estimates of soma size and description of dendritic morphology could be made and comparisons made with previous Golgi / cell filling studies. CB positive and PV positive GP cells were of a comparable size being 20312 and 23312 mm, respectively. These values are broadly in keeping with the major population of medium-sized GP neurones described previously [26,30], while small (11–12314–16 mm) and highly varicose CR positive neurones described here are similar to those small cells with varicose dendrites described in the same studies.
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Fig. 3. Double immunohistochemical staining for calbindin D-28k / parvalbumin and calretinin / parvalbumin. (A) A single calbindin D-28k positive neurone (blue–black) in the lateral globus pallidus with long, branched dendrites running parallel to the striatal border, which is delineated by a dashed line. A band of calbindin D-28k positive neuropil can also be seen adjacent to the striatal border. Many parvalbumin positive globus pallidus neurones (brown) in the lateral part of the globus pallidus were seen. Note the high level of calbindin D-28k staining (blue–black) in the striatal neurones. (B) A typical small, round calretinin positive neurone (blue–black) close to a large bipolar parvalbumin positive neurone (brown). Bar in A560 mm; bar in B510 mm.
4.3. Projection neurones and interneurones
4.4. Origins of CaBP neurophil
The retrograde tract-tracing technique combined with immunohistochemistry identified two populations of projection neurones that expressed either PV or CB. These PV and CB positive neurones could be labelled following injection into either STN or SN suggests that these cells are typical projection neurones in that they send collaterals to both structures [24]. No CR positive and gold-labelled neurones were observed even in areas in which there are large numbers of labelled neurones (Fig. 4E,F) suggesting that CR may be a neurochemical marker for a subpopulation of interneurones in the rodent GP. However, this population may extend projections to the striatum or entopeduncular nucleus, which were not targeted in this study. The possible existence of a sparse interneurone population of small diameter GP cells has been previously proposed in the rat on the basis of electrophysiological [30], morphological [26] and neurochemical data [43]. Indeed, the NMDAR2D glutamate receptor subunit, a marker for interneurones in many regions of the brain [44] is expressed in the GP. The significance of such a population largely depends on the connectivity with other neurones. In the striatum, small populations of interneurones have a profound effect on output and physiological function [18]. Striatal and GP CR positive cells represent a sparse population in rodents. However in the primate striatum CR interneurones are the most abundant interneurones outnumbering PV positive cells by 2–3:1 [49]. Thus, it is possible that a similar situation may occur in the primate GP.
In agreement with previous studies [15,36], CB and PV were largely expressed in a complementary pattern, with CB staining highest in the medial region and PV immunoreactivity highest in the lateral regions of the GP. In addition a band of CB positive fibres lying adjacent to the striatal border was also observed as previously described by Rajakumar et al. [36]. Anatomical tracing studies of the striatopallidal pathway have described two sites of terminal arborisation, one lying adjacent to the striatal border and the other more medially [47]. As the striatal output neurones are known to express high levels of CB [14], these two regions of fine CB fibres may correspond to the regions of the GP with a high density of striatal inputs. The origin of the fine CR positive fibre network is less clear. It is unlikely to be derived from the striatal input as CR positive neurones of the striatum are considered to be interneurones [12]. Similarly, these fibres are unlikely to originate in the STN as no CR positive cells have been identified in this structure in the rat [37].
4.5. Functional considerations The CaBPs are related proteins belonging to the EFhand family [35]. It has been proposed that the CaBPs act to buffer intracellular calcium ion concentrations, which are involved in a functionally diverse range of neuronal processes [20]. However, an excessive intracellular concentration of Ca 21 ions is neurotoxic [8]. Hence, maintenance of intracellular calcium ion levels within a range is
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Fig. 4. Combined calcium binding protein immunohistochemistry and retrograde tracing. (A) Injection site in the subthalamic nucleus (STN) which is delineated by a dashed line; ic, internal capsule. (B) Injection site in the substantia nigra (SN) which is delineated by a dashed line. (C) Retrogradely transported silver-enhanced gold particles (black) in the globus pallidus following injection of the tracer into the STN. Note that two of the neurones were also parvalbumin immunopositive (brown), whilst one cell was not co-labelled for parvalbumin. (D) Calbindin D-28k immunopositive neurone (brown) which was double-labelled with retrogradely transported silver-enhanced gold particles (black) following injection of the tracer into the SN. (E, F) Calretinin (brown) positive neurones in the globus pallidus were not labelled with retrogradely transported silver-enhanced gold particles (black) following injection into either the STN (E) or SN (F). Bar in A, B55 mm; bar in C, D510 mm.
critical for normal neuronal functioning. Although the ability of the CaBPs to bind and thereby buffer intracellular calcium levels is accepted [2], the precise function of
the individual CaBPs in neurones remains undetermined. CaBP expression does not appear to be linked with a particular neurotransmitter since, for example, PV is
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Table 1 Summary of results
Location of Cell bodies Neuropil Relative abundance of cell bodies Size of cell bodies Long axis (mm) Short axis (mm) Proximal dendrites Projection neurones
Calbindin D-28K
Calretinin
Parvalbumin
Uniform Medial1adjacent to striatal border ,2%
Uniform Ventromedial
Lateral Lateral
,1%
Numerous
19.9260.55 11.860.24 Smooth or varicose Yes
11.4960.22 8.8260.11 Varicose No
22.6660.5 12.2760.24 Smooth Yes
expressed in GABAergic, glycinergic and glutamatergic neurones (reviewed by Baimbridge et al. [2]). However, electrophysiological studies have linked the expression of parvalbumin with fast-spiking activity [19]. In-keeping with this, electrophysiological studies have identified cells that exhibit fast-spiking activity in the GP [33,45]. In contrast, no correlation between physiological activity and expression of either CB or CR has been found, although both have been suggested to have a neuroprotective role [16,7]. Future studies linking electrophysiological properties and expression of a particular CaBP will without doubt be instructive in defining the role that GP neurones play in basal ganglia circuitry.
Acknowledgements The authors wish to thank Dr I.J. Mitchell for use of the photomicroscope system and for invaluable assistance in criticising the manuscript. This work was supported by the Wellcome Trust, UK (Grant No. 050196 / Z / 97 / Z) and a Research Project Grant from the Faculty of Medicine and Dentistry, University of Birmingham.
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