S100 protein expression in subpopulations of neurons of rat brain

Neuroscience Vol. 67, No. 4. DD.977-991, 1995

Pergamon

0306-4522(94)00615-6

SlOO PROTEIN

El&r Science Ltd Copyright a 1995 IBRO Printed in Great Britain. All rights reserved 0306.4522195 $9.50 + 0.00

EXPRESSION IN SUBPOPULATIONS NEURONS OF RAT BRAIN M. RICKMANN*

Department

of Anatomy,

OF

and J. R. WOLFF University

of Gottingen,

Germany

Abstract-Available data are conflicting as regards the occurrence of Ca2+ and Zn*+ binding SIOO proteins in neurons of mammalian brain. Here the localization and expression of SlOO was re-investigated using several different antibodies and in situ hybridization. A map is provided for the distribution of two classes of SIOO-positive neuron populations in the adult rat CNS. “Persistently SlOO-positive” neurons had large size, were strongly immunoreactive and were mainly distributed in the nuclei of the lower brainstem and cerebellum. “Variably SlOO-positive” neurons were preferentially found in the forebrain of rats older than 90 days and were especially numerous in limbic regions. The SlOO-immunoreactivity in these neurons was moderately intense, occurred with high interindividual variation and appeared related to function as suggested by variations due to anesthesia. The expression of SlOO mRNA in neurons was re-investigated at high spatial resolution with non-radioactive in sifu hybridization using an oligonucleotide specific for SlOOb-mRNA. Expression of SlOO was demonstrated in astrocytes and in those neuron populations which were also strongly SlOO-immunoreactive. No expression of SlOOg message was seen in weakly immunoreactive neurons, but this may be due to low sensitivity of the techniques used. The data suggest that the SlOO proteins are synthesized in all astrocytes and in distinct subpopulations of neurons in rat brain. These neurons show a characteristic topography and vary in SlOO expression probably due to their function and maturation.

SlOO proteins belong to the group of Ca’+-binding proteins which are characterized by the so called EF-hand structure.3’ From the CNS mainly the dimers SlOOb and SlOOa have been isolated which have the subunit composition @ and o$ respectively. In the CNS of rat, the fraction of SlOOb amounts to more than 95% of total SlOO*’ which occurs at high concentrations (l-l.5 pg/mg soluble protein26). In contrast to Ca2+-binding proteins such as calbindin-D28k, parvalbumin and calretinin which are specifically expressed by certain types of neurons,* SlOO proteins predominantly occur in glial cells of the mammalian CNS. Among glial cells, SlOO immunoreactivity was found to be restricted to cells of the astroglial cell lineage in the adult mammalian cerebral cortex and cerebellum where virtually all astrocytes and Bergmann-glial cells have been labeled.7~‘3.27,38,39 In addition, there are some reports of S 100 localization in subpopulations of oligodendroglial cells9~” but SlOO has not been demonstrated in microglial or in endothelial cells. In contrast to astrocytes, the distribution of SlOO proteins and their presence in neurons is controver-

*To whom correspondence should be addressed at: Zentrum Anatomie, Kreuzbergring 36, D 37075 Giittingen, Germany. Abbreviations: BSA, bovine serum albumin; EDTA, ethylenediaminetetra-acetate; GFAP, glial fibrillary acidic protein; MOPS, 3-[N-morpholinolpropanesulphonic acid; SSC, sodium saline citrate; TBS, Tris-buffered saline. 977

sial. Available data present confusing patterns of interspecies and regional variations in neuronal SlOO immunoreactivity. Neuronal SlOO immunoreactivity has been demonstrated in the snail nervous system36 and in brains of the turtle, frog, fish and chick.4.‘4 Exclusive astroglial localization of SIOO has been shown for different brain regions in mammals However, SIOO immunoreactivity was only. 7,‘3*27.38,39 seen along the surface of neuronal membranes in the frontal cortex of rat and rabbit,16 and in the human hippocampus, subpopulations of neurons apparently contain S10O~r.‘~Faint staining of neurons was noticed in the cerebellar nuclei and the brainstem of cat, whereas in the cerebellum of chick, Purkinje cells were more intensely labeled than Bergmann-glial cellsi In the spinal cord of rat, SlOO/l- and SlOOcximmunoreactive motor neurons were reported.37 On the basis of this evidence, it is still unclear whether in the mammalian brain, SlOO can be expressed by specific types of neurons. Also, SIOO could occur in neurons only under certain conditions, and neurons might simply contain much less SlOO protein than astrocytes. Whether or not SlOO may be detected in neurons by immunocytochemical means seems to depend on unknown factors, which either influence immunoreactivity or regulate the presence of SlOO in neurons but not in astrocytes. In a previous study, we investigated influences of tissue preparation on SlOO immunoreactivity patterns in the rat brain33 and defined the conditions for maximum preservation of SlOO immunoreactivity.

M. Rickmann and J. R. Wolff

978

Under these circumstances, we have reinvestigated the distribution of SlOO occurring in neurons of the CNS. EXPERIMENTAL

PROCEDURES

Animals

Adult Sprague-Dawley rats of both sexes were obtained from Charles River (Sulzfeld, Germany) or bred in the local animal quarters. Rats were kept at a normal diurnal rhythm and had access to food and water ad libitum. In 47 animals ranging in age from 90 to 180 days, SIOO immunoreactivity was investigated in the CNS. Developmental changes of the SlOOprotein distribution were studied in one animal at each of the postnatal days (PD) 6, 10, 16 and 24, in two animals at PD40 and four animals at PD60. For in situ hybridization rats were used at the age of PD60 to PD65, because the gross distribution of hybridization signal in these brains could be compared with that previously reported after radioactive in situ hybridization in animals of the same age.” Most animals were deeply anesthetized with ether prior to fixation. A few animals were anesthetized for periods of 10min or 1 h by intraperitoneal injections of urethane (1.25 g/kg body weight), Ketavets/Rompt& (mixture of 113.7 mg ketamine hydrochloride and 9.2 mg 2,6-dihydro-2(2,6-xylidino)-4H-1,3-thiazine hydrochloride per kg body weight) or pentobarbital (30mg/kg initially and 24mg/kg body weight after 30 min). Immunohistochemistry Tissue preparation. For transcardial perfusion fixation, the vascular system was flushed with buffer until the jugular veins had cleared (about 2 min). Then a fixative containing the same buffer as the rinsing solution was infused for 10 min at room temperature. Optimally perfused and fixed brains were removed from the skull and immersed for 2 to 6 h in the same fixative. The fixatives contained 4% paraformaldehyde, 0.3% glutaraldehyde. Two different buffers were alternatively used, 0.1 M Na-phosphate or 0.1 M Na-cacodylate supplemented with 0.1% CaCl,, both at pH 7.3. Vibratome sections were cut at 50pm thickness under TBS (50 mM Tris, 150 mM NaCl, pH 7.6) or blocks of fixed tissue were soaked overnight in 20% sucrose before cutting 40-pm-thick frozen sections. Afinity pkjication of antibodies. A commercially available polyclonal antibody to SlOO (DAK0 2311, Lot 026) was fractionated in three cycles of loading, washing and elution. Either SlOOb (Sigma, S-8390, Lot 47F7813) or SlOOa (Sigma, S-8265, Lot 47F7812) was coupled to Eupergit-C@ (Riihm Pharma, Weiterstadt), and excess oxirane groups were blocked with glycin according to the manufacturer’s instructions. Determination of protein concentrations (Coomassie Blue techniques) revealed a coupling efficiency for SlOOb and SlOOa of 72% and 56%, respectively. The antiserum was diluted 1: 1 with double strength washing buffer (0.1 (MOPS) at pH 7.4, 1 mM CaCl,). Except for this addition of Ca*+-ions during antibody binding and washing, the purification method was standard” using 1 M pronrionic acid for elution. ultrafiltration for dialvsis and concentration, and absorbance at 280 nm for measuring protein concentrations. The following purification scheme was applied: (1) To obtain antibody enriched in anti-SlOOb-reactivity the antiserum was applied to immobilized SlOOb. The eluate was used for immunocytochemistry. (2) Antibodies washed out before elution in (1) were reapplied to the regenerated SlOOb-column to remove anti-SlOOb-reactivity. (3) Protein from the washing in (2) was purified on immobilized SlOOa. From the eluate a very small amount of antibody enriched in anti-SlOOa-reactivity was recovered and used for immunohistochemistry.

Incubation of sections. For immunocytochemical demonstration of SIOOthe rabbit antiserum against SlOO (DAKO) was diluted 1: 2000, the optimal dilution in a test series. The affinity purified anti-SlOOu- or SlOOP-antibodies were used at 1.5 pg/ml concentration. Monoclonal G12B8-anti-SIOOantibodyI in ascites fluid was kindly provided by the authors and was diluted 1: 1000. Monoclonal SG-Bl-antibody (specific for SlOOg, Sigma S2532) was applied in 1: 1000 dilution. Negative controls were run either with normal rabbit serum or with control ascites used at the same concentrations as the specific antibodies. For positive controls, rabbit anti glial fibrillary acidic protein (GFAP) antibody (DAKO) or monoclonal SM131 or SM132 antibodies recognizing neurofilaments (Sternberger) were applied to sections of the same series at 1: 1000 dilution. The ABC method” was applied to free floating sections using the following sequence of incubation steps at room temperature. (1) Blocking solution consisting of 0.1 M DLlysine, 1% BSA (bovine serum albumin), 1: 10 normal goat serum, 0.02% sodium azide in TBS for 2-4 h. (2) Primary antibodies diluted in blocking solution for 12 h. (3) Three rinses in TBS, 20 min each. (4) Biotinylated goat-anti-rabbit or goat-anti-mouse antibody (DAKO) diluted 1:500 in blocking solution for 1 h. (5) Three rinses in TBS, 20 min each. (6) ABC-complex which was mixed 45 min before usage from 1:250 biotinylated peroxidase and I :250 streptavidin (DAKO) in TBS containing 1% BSA for I h. (7) Three rinses in TBS and one rinse in 0.1 M Na-phosphate at pH 7.2, 20 min each. (8) Histochemical peroxidase reaction was carried out for 10min in a heavy metalsupplemented DAB solution’ consisting of 0.025% diaminobenzidine hydrochloride, 0.025% CoCI,, 0.02% (NH,SO,),Ni,xH,O, 0.01% H,O, in 0.1 M Na-phosphate at pH 7.2, (9) followed by four rinses in 0.1 M Na-phosphate at pH 7.2, 5 min each. Endogenous peroxidase activity was checked by omitting steps (5) and (6) and found negligible in perfusion fixed brain tissue. Preparation for light und electron microscopy. For direct observation Vibratome or frozen sections were transferred to 0.5% gelatin in 40% ethanol, mounted on microscope slides and thoroughly air dried. Dehydration in ethanol and xylene was followed by coverslipping in Entellan (Merck). For light microscopic observation of semithin sections and for electron microscopy the thick sections were postfixed with 2% 0~0, in 0.1 M Na-phosphate at pH 7.2 for 1 h, dehydrated, infiltrated with Spurr’s epoxy medium and embedded absolutely flat between microscope slides and separating polypropylene sheaths. Selected sections were polymerized to the flat surface of an epoxy block, detached from the slide and resectioned at 1-2-pm-thickness. Optimally stained semithin sections were selected for a second resectioning process into ultrathin sections which were stained with uranyl acetate and lead citrate4 or bismuth oxynitrate” before electron microscopic observation.

In situ hybridization Preparation of oligonucleotide probes. A 30-base antisense oligonucleotide probe specific for the S100/3-mRNA (R,5R-R7P7: ___ _I. AG GAA AUC AAA GAG CAG GAA GUG GUG GAC A, i.e. R,,, to R,,, of the coding region*r) was svnthesized bv W. Brvsch and K. H. Schlinnensienen6 and kindly provided for these experiments. A sense probe of the Na-channel II6 with the same GC-content served for controls. The probes were labeled with digoxigenin-dUTP using the 3’-end tailing kit from Boehringer, Mannheim. The tailing solution was mixed according to the manufacturer’s instructions using the probes at 2.5 nmol/ml concentration. The reaction was performed for 2 h at 37°C and stopped by chilling. No further purification of labeled probes was done.” Tissue preparation. Sprague-Dawley rats (60-65 days of age) were fixed by transcardial perfusion of the following solutions. (1) 0.1 M Na-phosphate buffer at pH 7.3 for

SIOO protein expression in neurons 2 min; (2) 4% paraformaldehyde in 0.1 M Na-phosphate buffer at pH 7.3 for 10 min; (3) 20% sucrose in 0.1 M

Na-phosphate buffer for 10min. Brains were dissected, immediately frozen, sectioned on a freezing microtome at 40 pm thickness and collected in 0.1 M Na-phosphate, pH 7.3. All following incubations were done on free floating sections. Before hybridization they were simultaneously treated for 20 min with 0.1% DEPC (freshly dissolved in 0.1 M Na-phosphate at pH 7.3) followed by a rinse in 50% formamide in 2 x SSC (1 x SSC: 150 mM NaCl, 15 mM Na-citrate, pH 7.0). Hybridizntion. The hybridization solution contained 75% QuickHyb* (Stratagene), 0.5 mg/ml salmon sperm DNA (Sigma D1626), 0.5mg/ml digested herring sperm DNA (crude oligonucleotides, Sigma D3 159), 1mg/ml yeast tRNA, 12.5% formamide. Prehybridization was done for 4 h and hybridization with 200 ng/ml probe overnight, both at 37°C. After hybridization, the sections were rinsed twice in 2 x SSC for 30 min each. Then two incubations in 0.25 x SSC with 50% formamide and one in 0.1 x SSC were applied for 1 h each. These washes were performed at room temperature. The sections were transferred to TBS, ready for further processing. Detection of digoxygenin labeled probes. To detect the digoxygenin of the hybridized probes we used a monoclonal mouse anti-digoxygenin antibody (Boehringer) at 1: 5000 dilution. Incubations were carried out as detailed above for the immunohistochemical detection of SIOO proteins. None of the solutions contained Na-azide, instead they were sterile filtered. For the final detection of peroxidase, a different nickel-intensified DAB reaction was used which did not produce background staining at long incubation times. This incubation medium consisted of 0.05% diaminobenzidine hydrochloride, 0.15% NiCl,, 0.005% H,O, in 0.2 m TBS at pH 7.6’ Sections were reacted for 15 min at room temperature and were washed three times for 5 min in TBS. Silver enhancement. At this stage, sections had turned faintly blue, but structural detail could not yet be observed. Therefore, staining was enhanced in most sections by intensifying the reaction product with a physical silver developer” which was mixed from equal volumes of two solutions immediately before use. Solution A consisted of freshly prepared 5% Na,CO,. Solution B contained 0.2% AgNO,, 0.2% NH,NO,, 1% (Bl, rapid) or 7% (B7, slow) tungsto-silicic acid (Merck) and 0.5% formalin (0.5 ml of 37% stock solution for lOOmI). Usually a developer was used with 2% tungsto-silicic acid (5*Bl + l*B7), and the reaction was stopped when control sections had turned grey. The sections were passed through the following incubation temperature: (1) 1% Na-acetate steps at room (CH,COONa*3H,O) for 1min, (2) IOmM CuSO, for 10 min, (3) three changes of 1% Na-acetate for 1min, (4) 3% H,O, in 1% Na-acetate for less than 10 min, (5) four changes of 1% Na-acetate for 1 min, (6) silver developer for up to 20 min, (7) two changes of 1% acetic acid for 5 min, (8) 0.1% auric acid for 2 min, (9) 1% Na-acetate for 1 min, (10) 1% Na-&O,*SH,O for 3 min, (11) two changes of 1% Na-acetate for 1min. Finally, sections were mounted on microscope slides, or they were flat embedded in epoxy resin and resectioned into semithin sections, The same procedures were used as described for immunohistochemistry of SlOO protein. RESULTS

Immunohistochemistry

Our material confirms that astrocytes regularly contained SlOO in all parts of the rat brain and spinal cord. In addition, SlOO-immunoreactive cells were found which did not resemble astrocytes but dis-

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played morphological characteristics of neurons (Fig. 1A). Unlike astrocytes, SIOO-positive neurons showed smooth outlines of both cell bodies and processes. These cell bodies were relatively large, included substantial amounts of perikaryal cytoplasm and gave rise to dendrites that were also SlOO-positive at least in their proximal parts (Fig. 1B). In various brain regions, the morphology of these cells corresponded to that of characteristic neuronal cell classes, especially the larger ones. Electron microscopy revealed that among SlOO-positive cells there were neurons making contact with numerous presynaptic profiles that were SlOO-negative (Fig. lC, D). Distribution of S IOO-immunoreactive neurons. Two groups of SlOO-positive neurons could be distinguished on the basis of different staining characteristics and topographic distribution: “persistently positive” and “variably positive” neurons. In none of the brain regions of the rat were all neurons SlOO-immunoreactive. Wherever neurons were SlOO-positive, they tended to belong to the largest class of neuron in the given brain region. Persistently

S lOO-positive neurons

Light microscopic appearance of these neurons was characterized by SlOO-immunoreactivity which was relatively strong but restricted to the cell body and proximal dendrites. Staining intensity was lower than that in astrocytes. In adult rats, the distribution patterns of persistently SlOO-positive neurons did not show interindividual variability in various brain regions and were similar in rats and guineapigs (data not shown). Moreover, different kinds of anesthesia and tissue preparation did not influence the appearance of these neurons. Only when EDTA was added to the fixing solutions was SIOO-immuno-staining lost in neurons as in astrocytes (for details see Ref. 33). A mapping of SlOO-immunoreactive neurons in rat brain revealed that persistently labeled neurons (dots in Fig. 2) were more numerous in caudal than in more rostra1 portions of the rat CNS (spinal cord z rhombencephalon > diencephalon > telencephalon, cf. Fig. 2A-I). Numerous persistently SlOO-positive neurons were found among motor neurons, i.e. in the spinal ventral horn, the facial and trigeminal motor nuclei (Fig. 3B, D). In these brain regions, the proportion of motor neurons containing SlOO was high, as was the fraction of SlOO-positive axons in the respective nerve roots. A lower density of SlOO-containing neurons was found in the oculomotor and hypoglossal nuclei, while SIOO-positive motoneurons were absent from the dorsal, vagal motor nucleus and from the nucleus ambiguus. Thus, many but not all motoneurons appeared as persistently SlOO-positive. The density of SlOO-positive neurons also differed among sensory brain regions. The highest number was found in all four cochlear nuclei and the mesencephalic trigeminal nucleus. Their number was

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M. Rickmann Abbreviations

2-$1-9 Acb ACg ACo AHi AOB AOE AOL AOP Arc BL BLV BM BST CAl-4 CeL CeM CG cg CICDM CICVL CL Cl CM CPU cu CxA DA DCo DG DLL DM DR Dsc EIc En Ent FrPaM FrPaSS G Gi GrA GrCo I ICj IGr InC InG Int La LatC LD LH LHb LRt LSD LSI LSV LTz

layers of Rexed for spinal cord accumbens nu ant cingulate cortex ant cortical amygdaloid nu amygdalohippocampal area accessory olf bulb ant olf nu, ext ant olf nu, lat ant olf nu, post arcuate hy nu basolateral amygdaloid nu basolateral amygdaloid nu, vent basomedial amygdaloid nu bed nu stria terminalis fields CAl-4 of ammons horn central amygdaloid nu, lat central amygdaloid nu, med central gray cingulum central nu inf colliculus, dorsomedial central nu inf colliculus, ventrolateral centrolateral th nu claustrum central med thu nu caudate putamen cuneate nu cortex amygdala transition zone dors hy area dors cochlear nu dentate gyrus dors nu lat lemniscus dorsomedial hy nu dors raphe nu lamina disseccans entorhinal cortex ext nu inf colliculus endopiriform nu entorhinal cortex frontoparietal cortex, motor area frontoparietal cortex, somatosensory area gelatinosus thu nu gigantocellular reticular nu granule cell layer, accessory olf bulb granule cell layer cochlear nu intercalated nu amygdala ilands of Calleja int granule cell layer, olf bulb insterstitial nu of Cajal intermediate grey layer sup colliculus interpositus cerebellar nu lat amygdaloid nu lat cerebellar nu laterodors th nu lat hy area lat habenular nu lat reticular nu lat septal nu, dors lat septal nu, intermediate lat septal nu, vent lat nu trapezoid body

and J. R. Wolff used in the figures lat vestibular nu mediodors th nu reticular nu medulla, dors reticular nu medulla, vent med amygdaloid nu nu mesencephalic tract trigeminal nerve med geniculate nu, dors MGD MGV med geniculate nu, vent mitral cell layer, olf bulb Mi MIA mitral cell layer, accessory olf bulb med septal nu MS med nu trapezoid body MTz N12 hypoglossal nu N7 facial nu parasubiculum PaS PC paracentral th nu post cingulate cortex PCg Pe periventricular hy nu PGi paragigantocellular reticular nu PLCO posterolateral cortical amygdaloid nu PMCo posteromedial cortical amygdaloid nu PnO pontine reticular nu, oral PO post th nuclear group PO primary olf cortex PPT post pretectal nu PrS presubiculum R red nu reuniens th nu Re Rh rhomboid th nu RSpl retrosplenial cortex Rt reticular th nu RtTg reticulotegmental nu pons subiculum S SHi septohipoocampal nu substantia nigra SN so1 sup olive SPSC nu spinal tr trigeminal nerve, caudal sp50 nu spinal tr trigeminal nerve, oral Strl7, 18, 18a striate cortex areas SuVe sup vestibular nu TeAud temporal cortex, auditory Tu olf tubercle TuPl olf tubercle, plexiform layer TuPo olf tubercle, polymorph layer TuPy olf tubercle, pyramidal layer vco vent cochlear nu VDBD nu ventral limb diagonal band, dors VDBV nu ventral limb diagonal band, vent VL ventrolateral th nu VLL vent nu lat lemniscus VM ventromedial th nu VMH ventromedial hy nu VP vent pallidum VPL ventoposterior th nu, lat VPM ventroposterior th nu, med vent tegmental nu VTg ZI zona incerta LVe MD MdD MdV Me Me5

Fig. 1. SlOO-immunoreactivity in the lateral vestibular nucleus. (A) In the lateral vestibular nucleus (1) most Deiters neurons are strongly SlOO-positive whereas astroglial labeling is relatively sparse. Some neurons are SlOO-positive also in the superior (s) but not in the medial (m) vestibular nucleus in which astroglial staining dominates. Labeled neurons (arrows), vestibular nerve root (8). (B) Deiters neurons (labeled nuclei, stars; unlabeled nucleus, asterisk), their proximal dendrites (arrows), astrocytes (arrowheads) and astroglial processes (open arrows) are immunoreactive. (C, D) The perikaryon (n in C) and dendrite (d in D) of labeled Deiters neurons are almost completely covered by numerous SlOO-negative presynaptic elements (asterisks). Arrow head (D) points to a type-11 and arrow (D) to a reciprocal synapse. Astroglial processes (stars) and lamellae (open arrows) show variable staining intensity. A: x 75, B: x 510, C: x 14,900, D: x 27,300.

SlOO protein expression in neurons

Fig. 1.

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breama tbrj +5.7 mm

A

br+0.2

br -2.8

v

--

-

2mm br -13.8 Fig. 2. Distribution of SlOO-immunoreactive neurons in the CNS of rat. The distribution of labeled neurons was mapped to the left halves of the drawings (transverse planes and nomenclature according to Paxinos and Watson”‘). Persistently SlOO-positive neurons were symbolized by filled circles (0, ca. two cells per symbol in one Vibratome section). For variably positive neurons (+) data were collected from 14 animals, and the maximal density for each brain region was mapped (ca. eight cells per symbol in one Vibratome section). At the most frontal levels (A, B), only variable SlOO-positive neurons were found. The ventral pallidum (C) was the most rostra1 location of persistently SlOO-positive neurons. Note the absence of any SlOO-positive neurons from the thalamic nuclei VPM and VPL (D) but the presence of variable positive neurons in the medial geniculate body (E). In the nuclei of the trapezoid body (F) and the cochlear nuclei (G), SlOO-positive neurons were constantly found but in highly variable numbers (compare text). Further caudally in the myelencephalon (H) and the spinal cord (I), only persistently SlOO-positive neurons

I I

SlOO protein expression in neurons

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Fig. 3. Brain regions showing persistently SlOO-positive neurons. Some of the ceil bodies of SlOO-positive neurons (arrows), stained axons (bent arrows) and astrocytes (arrow heads) are indicated. (A) Population of large neurons in spinal trigeminal nucleus. Immunoreactivity is also found in some perpendicularly cut axons (b) of adjacent tract. (B) Motor trigeminal nucleus showing labeled motor neurons and axons. (C) In the facial nucleus, nuclear immunoreactivity dominates in most neurons (arrows) but a few showing label only in the cytoplasm (open arrow). (D) Red nucleus showing labeled neurons in its magnocellular part. It is traversed by axon bundles of the oculomotor nerve a few of which are SlOO-positive (bent arrow). (E) Cluster of labeled neurons in gigantocellular reticular formation. SlOO-immunoreactivity was detected by polyclonal (A, C, D) and monoclonal G12B8 (B, E) antibodies. x 178. smaller in the superior and lateral vestibular nuclei and the medial part of lamina IV of the spinal cord.

In the spinal and principal trigeminal nuclei (Fig. 3A) and the cuneate nucleus, the density was lower. Very few SlOO-positive neurons were detected in the medial vestibular nucleus, the solitary tract nucleus and the nucleus gracilis. Hence, also neurons in sensory relay nuclei may or may not react with SlOO-antibodies. Other brain regions with a high density of persistently SlOO-positive neurons were the cerebellar nuclei and the magnocellular part of nucleus ruber (Fig. 3D). Intermediate densities of these neurons were found throughout the rhomb- and mesencephalon, especially in the nuclei of the reticular formation (Fig. 3E) and tegmentum extending into regions medial of the spinal and principal trigeminal nuclei and lateral lemniscus, lateral to the raphe nuclei, dorsal to the facial nucleus and trapezoid body and ventrolateral to the central grey. Rostra1 to the mesencephalon, persistently SlOO-positive neurons were restricted to the reticular thalamic nucleus, zona incerta and ventral pallidum. A common feature of persistently SlOO-positive neurons of the CNS was their dense innervation of

perikaryon and proximal dendrites. This property was confirmed in functionally different nuclei, such as the lateral vestibular nucleus, lateral superior olive and facial nucleus. Persistently SIOO-immunoreactive neurons also occurred in sensory ganglia (data not shown). We investigated the trigeminal ganglion in detail where SlOO was found in many large ganglion cells while smaller ones were spared. The immunoreactivity extended into peripheral and central neurites. As a result the majority of large axons of the trigeminal nerve and tract (Fig. 3A) were SlOO-positive. The majority of neurons in the maxillary part of the ganglion belonged to the population of large SlOOpositive cells, whereas the mandibular part mainly contained smaller ganglion cells being SlOO-negative. Variably

Some only in neurons cephalic was to positive

S 100-positive neurons

populations of neurons were SlOO-positive a fraction of the brains investigated. These were mainly distributed in di- and telebrain regions, i.e. the distribution pattern some extent the reverse of the persistently neurons. These neurons were not only

Fig. 4. Variably SIOO-positive neurons in the hippocampus (A-E) and in the neocortex (F--I). Rats were anesthetized with ether (A-G) or urethane (H, I) and perfused with fixatives buffered with Na-phosphate (A, D, E, H, I) or with Ca*+-supplemented Na-cacodylate (B, C, F, G). Neurons (arrows), astrocytes (arrowheads), strata oriens (o), pyramidale (p), radiatum (r) and lacunosum moleculare (Im). cortical white (w) and gray (g) matter. (A) and (B) exemplify extreme differences of SIOO immunoreactivity in pyramidal cells of region CA1 detected with polyclonal antiserum. (A) In the hippocampus with exclusive astroglial SlOO immunoreactivity, unstained pyramidal cell somata and dendrites are outlined by densely stained processes of astrocytes. (B) Maximally labeled somata and apical dendrites of pyramidal cells and reactivity in interneurons (open arrow) obscure stained astrocytes. (C) Maximal SlOO immunoreactivity of G12B8 antibody in pyramidal cells showing lighter dendritic staining than in (B). SlOO in astrocytes appears restricted to cell bodies and large processes when compared to (A). (D, E) Aligned fields of view of adjacent sections stained with polyclonal (D) or SH-Bl-antibody (E) show transition between regions positive (right) and negative (left) for SIOO immunoreactivity in pyramidal cells. Dendritic staining with SH-Bl is faint but present (arrows). (F, G) Maximum neuronal (F) and almost exclusive astroglial (G) St00 immunoreactivity in parietal cortex recognized by polyclonal antiserum. (H, I) Compare the difference of SlOO-labeling with polyclonal antiserum in the frontal cortex of two female litter mates seen after 10 min (H) and I h (I) of urethane anesthesia. A--C: x 184, D, E: x 97, F. G: x 32, H, I: x 377. 984

SlOO protein expression in neurons

Fig. 5. SIOO immunoreactivity in the hippocampus with affinity purified antibodies. The micrographs in (A) to (C) were taken from serial Vibratome sections at 100 pm distance. They show aligned fields of view of the stratum pyramidale (p), radiatum (r) and lacunosum moleculare (Im) of region CAI. (A) With polyclonal antiserum pyramidal cell (arrows) somata, apical dendrites and astrocytes (arrowheads) arc detected. Open arrow: interneuron. (B) After affinity purification on SlOOb cloudy staining of small, presumably astroglial processes is reduced. (C) Enrichment of S lOOcI-reactive antibody suppresses S IO0 immunoreactivity in neurons, especially in their dendrites, more than in astrocytes. In (C) staining of the section was distinctly lighter than in (A) and (B). x 219.

variably stained (varying in amount from one rat to the other) but were also less intensely stained than the astrocytic cell bodies and the persistently SlOO-positive neurons. In some cases, immunoreactivity appeared at the limits of detection. Although variably S I 00-positive neurons were on average smaller than the persistently positive ones, they usually belonged to the largest neuron population of the respective brain region, For each brain region, the maxima1 density of variably SIOO-positive neurons observed in all animals investigated has been included in the map (stars in Fig. 3). These regions also showed most frequently SIOO-positive neurons in the set of animals studied. Such brain regions were the hippocampus, amygdala, entorhinal, piriform and cingulate cortex, septal nuclei, striatum and claustrum. In the hippocampus proper, pyramidal cells and also interneurons lying in the stratum radiatum and adjacent to the pyramidal cell layer could be SlOOpositive (Fig. 4A-E). Pyramidal cells were labeled to a variable degree. The least labeled ones showed only weak nuclear staining, whereas maximally labeled cells were also immunoreactive in perikaryon and apical dendrites (Fig. 4B,D). In every section, there

were also completely negative pyramidal cells. Both positive and negative neurons were clustered. Hence, there were stretches of the pyramidal cell layer populated with SlOO-positive and -negative cells. respectively (Fig. 4D, E). In serial sections, these patterns could be followed up to one 1 mm and apparently did not match with the hippocampal fields CA] to CA4. CA3 was the most probable part to contain SIOOpositive pyramidal cells, but no predilection site was detected along the septocaudal extent of the hippocampus. The S l OO-positive interneurons appeared evenly distributed at low density and also occurred in zones populated by SlOO-negative pyramidal cells. The dentate gyrus contained fewer SIOO-positive neurons than the hippocampus proper. Some granule cells and large hilar neurons contained SIOO. While immunoreactivity extended into the proximal dendrites of the hilar cells, it was strictly limited to the cell body in granule cells. The granule cell layer showed a partition into patches either populated predominantly by SlOO-positive or SIOO-negative cells. On average the neocortex (Fig. 4F, H) showed fewer positive neurons than the hippocampus. As depicted in Fig. 4F there were, however, area1 and

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M. Rickmann and J. R. Wolff

laminar differences. Small neurons within lamina VI and pyramidal cells of laminae V and III were most probable to show SlOO immunoreactivity. Often all neurons of one type appeared SlOO-negative. In spite of variations in the relative number of cortical neurons showing SlOO-immunoreaction, never were all neurons of what ever type SlOO-positive. Frontal areas tended to contain more SlOO-positive neurons than occipital areas. In the olfactory bulb, deep granule cells and mitral cells could show SlOO immunoreactivity, but often did not. There were always parts of the mitral cell layer devoid of positive neurons, indicating that the population of mitral cells was inhomogeneous with respect to SlOO immunoreactivity. In diencephalic brain regions, SlOO immunoreactivity varied in neurons of all hypothalamic and most non-specific thalamic nuclei. Specific, thalamic nuclei were poor in SlOO-positive neurons, e.g. SlOO-positive neurons were never found in the somatosensory nuclei (VPM, VPL). In one of 34 animals, some neurons of the lateral geniculate nucleus contained SlOO (see below for conditions), while a moderate number of variably SlOO-positive neurons was found in the medial geniculate body. Also further caudally, populations of variably SlOO-positive neurons were detected mainly in regions connected to the acoustic system. Neurons stained at variable density occurred in the inferior colliculus, the nuclei of the lateral lemniscus and the trapezoid body and the cochlear nuclei. In these brain regions, it was impossible to distinguish between groups of persistently and variably SlOO-positive neurons on the basis of neuronal cell morphology. Most persistently positive neurons were found in the cochlear nuclei (see above) and most variably stained ones appeared in the lateral superior olive. In the inferior colliculi, superficial layers showed much more variability in the number of stained neurons than the deeper parts. In the cerebellar cortex, Purkinje cells very rarely showed SlOO immunoreactivity. Such cells were preferentially located in the vermis and flocculus. Also the pontine nuclei contained a few variably SlOO-positive neurons. Conditional factors influencing SlOO-immunoreactivity in neurons. Among all types of aldehyde fixation

only brains fixed by immersion showed a distinct decrease in SlOO-immunoreactive neurons. Perfusion fixation with EDTA containing fixatives decreased SlOO staining not only in neurons but also in the majority of astrocytes. Various concentration of aldehydes did not visibly influence the SlOO immunoreactivity in SlOO-positive neurons. In contrast to aldehyde concentration, the buffer of the fixative influenced the number of variably SlOOpositive neurons. At pH 7.3, fixatives containing 0.1 M Na-cacodylate buffer and 0.1% CaCl, showed more SlOO-positive neurons than Ca-free fixatives, e.g. buffered with 0.1 M Na-phosphate. Especially in

the hippocampus differences became apparent. In many brains perfused with phosphate buffer, pyramidal cells and granule cells were completely SlOO-negative (Fig. 4A). Only isolated interneurons around the pyramidal cell layer maintained SlOO immunoreactivity. In other brain regions influence of the fixation buffer was less pronounced. SlOO immunoreactivity in neurons was age dependent. Up to postnatal day (P) 60, there were no SlOO-positive neurons in those brain regions, which however, contained variably positive neurons in our main collective of 90-180 day old rats. In persistently SlOO-positive neurons, SlOO appeared much earlier. By P6, the mesencephalic trigeminal nucleus already contained strongly SlOO-positive neurons. In the motor trigeminal nucleus, neurons became SlOO positive next, and by P16, superior and lateral vestibular and reticular nuclei contained large, strongly SlOO-positive neurons. Neurons in the principal and spinal trigeminal nuclei were the latest to acquire SlOO-immunoreactivity (by P40). In all these brain regions astrocytes had their full complement of SlOO immunoreactivity before the first positive neurons appeared. We also checked the influence of anesthesia on SlOO immunoreactivity in neurons. No differences were seen for SlOO immunoreactivity in persistently SlOO-positive neurons. After short (< 10 min) anesthesia with ketavet/rompun and urethane, respectively, variably SlOO-positive neurons did not show changes in distribution compared to ether anesthesia, which was routinely used. Also the mitral cells of the olfactory bulb did not show differences between animals which either had inhaled ether or were injected intraperitoneally with the other anesthetics. However, after 1 h of anesthesia marked differences were seen in the distribution of variably SlOOpositive neurons (female litter mates compared). In the frontal cortex and striatum, the fraction of stained neurons had decreased after urethane and barbiturate (Fig. 3H, I). In other brain regions, more variable results were obtained. Barbiturate anesthesia resulted in a general decrease in the number of variably SlOO-positive neurons, while urethane seemed to modify the distribution patterns. Surprisingly, a fair number of SlOO-positive neurons appeared after long lasting urethane anesthesia in the lateral geniculate body, where positive neurons could not be observed after short periods of any anesthesia. The pontine nuclei were another region with increased neuronal SlOO immunoreactivity after longer period of urethane but not with barbiturate anesthesia. Intracellular distribution of SlOO-immunoreaction in neurons. In SlOO-positive neurons, staining prob-

ability was higher in cell nuclei than in perikarya, proximal dendrites and axons. Peripheral dendrites mostly remained unstained, even in otherwise strongly stained neurons. Among the variably SlOOpositive neurons, there were cells which had only their

Fig. 6. In situ hybridization of SlOO/I-mRNA using digoxygenin-labeled oligonucleotides showing cerebellum (AX) and lower brainstem (A, B, D, E) and hippocampus (F-I). Gross distribution of hybridization signal after incubation with control (A) and SlOO/I-specific probe (B). In the cerebellar cortex (C), Bergmann glial cells (arrowheads) show signal but not the intervening Purkinje cells (lines). In the lateral vestibular nucleus (D) and the gigantocellular reticular formation (E), somata of large neurons (arrows) are labeled in addition to presumably astroglial elements in neuropil. In the hippocampus (F), the signal was maximal around the pyramidal cell layer (p) and around the hippocampal fissure, i.e. in the stratum lacunosum moleculare (Im) and the molecular layer (m) of the dentate gyrus. At low magnification (G), the distribution of hybridization signal is diffuse in the stratum oriens (0). pyramidale (p) and radiatum (r) except for one distinctly labeled interneuron (arrow). At high magnification (HJ) of 1.5pm-thick semithin sections, SlOOp-mRNA is not located in pyramidal cells (arrows) but in the interposed neuropil, in perikarya of interneurons (open arrows) and in structures resembling astroglial perikarya (arrowheads). A, B: x 2, C, D, E: x 202, F: x 19, G: x 153, H, I: x 306. 987

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cell nuclei stained. This fraction prevailed in the frontal cortex after 1 h of anesthesia where neuronal SlOO staining was generally reduced. Restrictions of SlOO immunoreactivity to the cytoplasm was relatively rare among neurons and could occur in all brain regions. Certain regions of white matter contained large nerve fibers with strong SlOO-immunoreactivity (see, e.g. Fig. 3). This staining was prominent in the trigeminal nerve tract, the central part of the facial nerve, superior and middle cerebellar peduncles indicating that persistently SlOO-positive neurons projecting through these tracts were transporting SlOO into their axons. In electron micrographs of SlOO-positive neurons, the immunoreactivity was predominantly localized in the cytosol (Fig. lC, D). Similar to astrocytes, the interior of neuronal organelles was devoid of immunoreaction product with the exception of a few profiles of smooth endoplasmic reticulum (found in hippocampal pyramidal dendrites) and the intermembrane space of mitochondria. This mitochondrial staining cannot be interpreted as an effect of mitochondrial catalase activity, because this staining was completely restricted to cells which also showed cytosolic SlOO immunoreactivity (see Fig. 1D). The distribution of SIOO within the neurons was uneven. Cytoplasm surrounding the Golgi apparatus or the endoplasmic reticulum was sparsely immunoreactive. Very strong reaction was found along the surfaces of mitochondria and microtubules. The reaction product lining microtubules showed periodical accumulations at distances of approx. 30 nm. Few postsynaptic thickenings showed immunoreactivity above average. These did not account for the total number of type-1 synapses. Neuronal SlOO-immunoreactivity with diSferent antibodies. The amount of SlOO-positive neurons ob-

served in our material was higher than had been expected from published data. To rule out unspecific antibody reactions affinity purified antibodies (affi-/I: affinity purified on bovine SlOOb, affi-a: enriched for SlOOa) and monoclonal antibodies (G12B8 recognizing SlOOc( and S100/I,‘5 SH-Bl: specific for SlOO/, Sigma S2532) were applied in parallel with a polyclonal antiserum. Persistently SlOO-positive neurons were recognized at equal staining intensity and topographical distribution by polyclonal, affi-fl, G12B8 and SH-Bl antibodies. The overall staining intensity of sections was less with affi-cc than with affi-8 antibody although both were used at a concentration of 1.5 pg/ml. Compared to sections reacted with polyclonal antiserum, the enrichment for SIOO-cc antibodies indicated less immunoreactivity in persistently SlOO-positive neurons than in astrocytes. This was most clearly seen in the spinal trigeminal nucleus. Variably SlOO-positive neurons were stained by all antibodies though at difference intensities and numbers. Nearly indistinguishable were the results obtained with polyclonal and affi-/I antibodies

(Fig. 5A, B). Monoclonal antibodies SH-Bl and G12B8 and affi-cc antibody showed weaker overall staining than the polyclonal antiserum. The G12B8 antibody reacted particularly poorly with variably SlOO-positive neurons (Fig. 4C). Affi-a antibody showed exceptionally weak immunoreaction in the cytoplasm of variably SlOO-positive neurons, whereas immunostaining of neuronal cell nuclei and of astrocytes was less diminished by the purification for SlOOc(-reactivity (Fig. 5C). Although G12B8 antibody is not specific for SlOOc(, its immunoreactivity pattern in fixed tissue was similar to that of affi-a antibody (cf. Figs. 4C and 5C). SlOOB-in situ hybridization Here, neuronal SlOO-synthesis has not been investigated by radioactive in situ hybridization, because this technique has a spatial resolution too low for distinguishing between astrocytes and neurons. Instead we tried a digoxygenin-labeled oligonucleotide specifically made for the antisense sequence of SlOO,!?, i.e. the subunit contained in SlOOa as well as in SlOOb proteins. As controls, parallel sections were incubated either with a control nucleotide (sense probe of the Na-channel II) or without any oligonucleotide (Fig. 6A, B). A further criterion for specificity was the presence of the hybridization signal in Bergman glial cells, but its absence from Purkinje cells (Fig. 6C). At low magnification, the distribution pattern of the hybridization signal was in accordance with that found by Landry et a1.24 after radioactive in situ hybridization. For better comparison, we restricted our hybridization experiments to brains from 60 days old rats, the oldest stage used by these authors. In the rhombencephalon, our materials showed SlOOfi-mRNA in the perikarya of magnocellular neurons of various nuclei in brainstem (Fig. 6D, E) and cerebellum. The location and numerical density of labeled neurons were comparable to those of neurons found to be persistently immunoreactive with SlOO-antibodies. However, labeling intensities were different. For example, neurons in the medial nucleus of the trapezoid body appeared extremely rich in SlOO/I-mRNA while these neurons were only weakly stained by immunocytochemistry. In spite of some neuronal labeling, the bulk of SlOO-mRNA appeared in a cell population, which according to size and distribution, was identified as astroglial cells in which the signal was mainly seen in the cytoplasm and cell processes. Accordingly, most of the label was seen in the neuropil, and only a fraction of astroglial cells could be identified by perikaryal labeling. In the forebrain, the hybridization signal was persistently found in SlOO-positive neurons of the reticular thalamic nucleus and the basal palhdum. In regions typically populated with variably immunopositive neurons, the in situ hybridization method labeled only fery few neuronal perikarya. The bulk of the hybridization signal was located in the neuropil and in astrocytic perikarya. The

SlOOprotein expression in neurons hippocampus showed maximal staining around the hippocampal fissure and over the pyramidal cell layer (Fig. 6F, G). Here, the label was localized predominantly in cell processes around, but not within, the pyramidal neurons. In addition, the hippocampus proper regularly showed some neurons positive for SlOOfi-hybridization signal. They corresponded to the population of S 100-immunoreactive interneurons and were predominantly localized adjacent to the pyramidal cell layer and in the stratum radiatum (Fig. 6G, H, I). In the neocortex, labeling appeared restricted to astrocytes. It was maximal in the barrel field and in lamina I of entorhinal and cingulate regions. This pattern was different from that of immunohistochemistry, i.e. astroglial SlOO immunoreactivity showed a more even laminar distribution and no regional differences within the somatosensory area. In conclusion, the in situ hybridization using a digoxygenin-labeled oligonucleotide unequivocally showed that at least some classes of neurons in various parts of the rat brain can produce SlOO. In cases where no SlOOg signal was detected in neurons, this may have been due to limits of the technique. Therefore, we cannot exclude that all neurons can express SlOO under certain conditions or that certain neuron classes are incapable of SlOO-expression. DISCUSSION

Utilizing optimal conditions of tissue preparation,‘j results were obtained which are compatible with the notion that certain neuron populations in the mammalian brain show SlOO-like immunoreactivity. Similar results were obtained with different primary antibodies against SlOO proteins, and by using a relatively sensitive immunohistochemical method (see Ref. 20). The polyclonal antiserum from DAKO, which was used in the present study, has proven to be specific for SlOO proteins in immunoblots.” We further decreased the probability of falsely positive immunoreactivity by affinity purification of this antiserum. In the same populations of neurons, SlOO immunoreactivity was confirmed with these affinitypurified antibodies and with two different monoclonal antibodies. It is difficult to completely rule out the possibility that we detected a protein very closely related to SlOOc(and SlOOg. Present knowledge about the family of SlOO-genes’.‘” points out calcyclin as the relatively closest protein for which the gene products has been demonstrated in the CNS.” These authors found cerebellar granule cells immunoreactive for calcyclin. In our experiments, these neurons were always SIOO-negative. Thus, crossreactivity of all the primary antibodies used in the present study is an unlikely explanation for the SlOO-like immunoreactivity observed in neurons. Diffusion of SlOO from astrocytes into neurons during fixation has never been reported for brains NSC 67.‘4I

989

which were fixed by perfusion with aldehyde fixatives (see e.g. Ref. 13). Such artificial translocation would not be compatible with numerous SlOO-negative neurons, which are sometimes located side by side with SlOO-positive neurons. Hence, our immunohistochemical data are proof that distinct amounts of SlOO proteins occur in certain subpopulations of neurons in the brain of developing and adult rats. The number of SlOO-containing neurons is probably even underestimated by superposition with heavily stained astrocytes, especially when sections are permeabilized by detergent to improve access of antibodies. Then astrocytic processes and lamellae, which are ubiquitous in brain tissue and extremely rich in SlOO, overcast any structural detail as in frozen sections.33 Nevertheless, it can be excluded that all neurons contain SlOO protein. For the cerebral cortex SlOO protein concentration in astrocytes can be estimated to 250pg/ml from biochemicalZh and morphometric data. 32 Although concentrations may not be as high in neurons as in astrocytes, SlOO may have unexpected functions; e.g. much lower concentrations of SlOOb (125 ng/ml) may infuence neurite elongation.** However, intracellular functions of SlOO proteins in neurons remain to be elucidated. The staining results with the SlOOfi-specific SH-Blantibody suggest that neurons contain SlOOb. Also the affinity-purified antibodies showed more S 1OOgthan SlOOc(-reactivity in the cytoplasm of neurons. These findings are supported by the fact that more than 95% of the SlOO proteins of the rat brain consist of SlOOb.*’ However, not all neurons may contain SlOOb, since the relative amounts of total SlOOa and SlOOb show high interspecies variation.*” Hence, these findings do not contradict the exclusive S 100~immunoreactivity which has been reported by pyramidal cells in the human brain.” Here, the origin of SIOO protein in neurons received special consideration. The present non-radioactive in situ hybridization was designed for good spatial resolution and utilized oligonucleotides labeled with digoxygenin to distinguish between synthesis of SlOO in neurons and astrocytes. The oligonucleotide used was specific for SlOO/J-mRNA (last checked by FASTA at EMBL, Heidelberg on 14.9.94) and thus did not distinguish between SlOOa (@) and SlOOb (BP). Our results strongly suggest that subpopulations of neurons synthesize SlOO proteins. In the lower brainstem and cerebellum, the correlation between hybridization signal and immunoreactivity was good enough to localize SlOO synthesis to SlOO-immunoreactive neurons. This interpretation is supported by the cerebellar cortex, where both methods showed SlOO almost exclusively in Bergman glial cells, and the brainstem, where astroglia and magnocellular neurons were labeled by both methods. This is in agreement with the distribution patterns of SlOOp-mRNA which were observed after radioactive in situ hybridization24 at relatively low spatial resolution. The latter report

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and our electron microscopic data indicate that their perikarya receive an unusually dense synaptic innervation. These correlates may indicate high energy requirements for tonic neuronal activity4’ in persistently SIOO-positive neurons. Mainly in the forebrain, neuron populations were found which were variably labeled with SlOO-antibodies. We cannot exclude that there are neurons which cannot be induced to become SlOO-positive under whatever conditions. However, even in brain sections with maximum of neuronal S 100 immunoreactivity preserved, a distinct number of SlOO-negative neurons existed. Our observations suggest that a large fraction of neurons express SlOO proteins at least transiently. Changes in staining intensity were seen after one hour of anesthesia and variably SlOO-positive neurons preferentially occur in rats older than three months. This finding corresponds to biochemical data suggesting that the SlOOb-concentration increases with age especially in the cerebral cortex, but this increase has not been seen in the cerebellum or brainstem.*’

showed a maximum of hybridization signal over the pyramidal cell layer of the hippocampus of 60-dayold rats. Our data are comparable at low magnification but the higher resolution suggested that SlOOP-mRNA is predominantly localized in astroglial cells and processes located in or near the pyramidal cell layer. Again this distribution pattern was similar to the distribution of SlOO immunoreactivity in this brain region. Only in older rats did some pyramidal cells also show SlOO-immunoreactivity. However, one cannot exclude that at least some of the SlOO protein found in neurons was synthesized in astrocytes. SlOO can be released from astrocytes in culture4’ and in situ 3s by a mechanism which is still unknown. And neurons can take up protein-like substances from the extracellular space such as horseradish peroxidase etc. when used as anterograde or retrograde tracers of axonal transport. Such intravital translocation of SlOO protein would not necessarily contradict the cytosolic localization of SlOO in neurons. Also accumulated tracers may be distributed in the cytosol of neurons and then may lead to the well known diffuse, intracellular staining patterns. At present, however, the quantitative significance of SlOO-release is unclear. Therefore, the finding that SlOO-synthesis can be shown in neurons strongly labeled by SlOO-antibodies but not in many weakly labeled neurons, may be based on insufficient sensitivity of the in situ hybridization technique used rather than SlOO-translocation from astrocytes. Two different classes of SlOO-positive neuron populations could be distinguished by staining characteristics and topographical distribution. Most persistently SlOO-positive neurons had a larger size than non-reactive ones and were predominantly located in the lower brainstem and spinal cord. High cytochrome oxidase activity in the perikaryon was described for several of these neuron populations,43

CONCLUSION

In conclusion, our data suggest that the Ca*+- and Zn*+-binding SlOO proteins although present in all astrocytes are restricted to subpopulations of neurons. In rat brain, SlOO-immunoreactive neurons show a characteristic topography that corresponds to patterns of SlOO-expression. Acknowledgemenrs-The authors wish to express their gratitude for donation of the Gl2B8 antibody by Dr Barbara D. Boss; oligonucleotide preparation and donation by Drs W. Brysch and K. H. Schlingensiepen; excellent technical assistance of S. Freyer (in situ hybridization), H. Kern and H. Biittcher (immunohistochemistry), R. Dungan and B. Riilke (photography). This work was supported- by the German Science Foundation (DFG Wo 279/6-2).

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