Characterization of nicotinic acetylcholine receptors expressed in primary cultures of cerebellar granule cells

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 30 (1995) 17-28

Research report

Characterization of nicotinic acetylcholine receptors expressed in primary cultures of cerebellar granule cells M. Didier a,., S.A. Berman ~', J. Lindstrom b, S. Bursztajn

~'

~' Laboratories .fbr Molecular Neuroscience, Department of Psychiatry and Program in Neuroscience, Hart'ard Medical School / McLean Hospital. 115 Mill Street, Belmont, MA 02178, USA ~' Uniz'ersity of Pennsyh'ania Medical Center, 36th and Hamilton Walk. 217 Stemmler Hall, Philadelphia, PA 10104-6074. USA

Accepted 8 November 1994

Abstract Nicotinic acetylcholine receptors (nAChRs), like other calcium permeable channel receptors, may play a crucial role during neuronal development. We have characterized nAChRs in developing mouse cerebellar granule cells in primary culture. L-[3H]Nicotine, [3H]cytisine and [125I]a-bungarotoxin binding experiments revealed the presence of a single class of saturable and specific high affinity binding sites for each ligand. The expression of these nicotinic binding sites followed a developmental pattern reaching a maximum during the establishment of excitatory amino acid synaptic contacts. Immunolabeling with monoclonal antibodies to nAChR subunits revealed the presence of t~4 and 132 subunits in most neurons. Moreover, some neuronal cells displayed a somatic as well as a neuritic localization for the a7 subunit as shown by [1251]a-bungarotoxin autoradiography. The reverse transcription-polymerase chain reaction (RT-PCR) detected the presence of mRNAs for a3, a4, a5, a7, /32 and /34 nAChR subunits. Non-neuronal cells did not express nAChRs, as shown by [3H]nicotine and [125I]abungarotoxin binding, immunocytochemistry and PCR. Maximum Ca 2+ influx elicited by nicotine, and partly scnsitivc to a-bungarotoxin, was observed around 10-14 days after plating. This correlated with the time period at which the highest number of nicotine binding sites was detected. Sensitivity to several NMDA receptor antagonists as well as to removal of endogenous glutamate by pyruvate transaminase treatment revealed a glutamatergic component in the nicotine stimulated calcium influx. The time-dependent specific nAChR expression and the potential association between nAChRs and NMDA receptor activation suggest that nAChRs may regulate glutamatergic activity during synaptogenesis in cerebellar granule cells. Keywords." Nicotinic acetylcholine receptor; NMDA receptor; Cerebellar granule cell; Primary culture; Neuronal development;

45Ca2 + influx

Abbreviations: BTX, bungarotoxin; CNS, central nervous system: CPP, 3(( _+)-2-carboxypiperazin4-yl)propyl-l-phosphonate; DIV, days in vitro; EDTA, ethylenediaminetetraacetic acid; GPT, glutamate pyruvate transaminase; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HRP, horseradish peroxidase; mAb, monoclonal antibody; MKS0I, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate; nAChR, nicotinic acetylcholine receptors; NMDA, N-methyl-D-aspartate; PBS, phosphatebuffered saline; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; RT, reverse transcription; VSCC, voltagesensitive calcium channel * Corresponding author. 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 169-328X(94)00266-5

I. Introduction

Central cholinergic systems are known to control basic functions of the brain. Acetylcholine mediates synaptic transmission in the vertebrate central nervous system (CNS) through the activation of two major receptor subtypes, the muscarinic and nicotinic (nAChR) acetylcholine receptors. Until recently, muscarinic receptors have received more attention than nAChRs. However, the molecular cloning of several

18

M. Didier et al. /Molecular Brain Research 30 (1995) 17-28

neuronal nAChR subunits has provided new tools to study these receptors and revealed the existence of multiple receptor subtypes which may be heterogeneously expressed in the brain [16,24,45,48]. The unique channel properties and cellular distribution of some nAChRs suggest that they could regulate intracellular calcium homeostasis in a complementary manner with voltage-sensitive calcium channels (VSCC) and N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors [40]. Regulation of the intraneuronal calcium level may be crucial during the development of the mamalian CNS. Both NMDA receptor and VSCCs have been shown to control neuronal differentiation in vivo as well as in vitro [5,10,12,17,28,36]. In most cases, these properties are accompanied by an increase of the neuronal expression of NMDA receptors [36]. Although much less evidence has been described, three observations suggest that it is plausible that nAChRs play an important role during the normal development of mamalian CNS. First, in the avian nervous system, nAChR number is also strictly regulated by innervation during the development of some neural structures [9,14,26,37,38]. Second, activation of postsynaptic nAChRs appears important in the regulation of neuronal survival in the the avian ciliary ganglion [39] and process outgrowth in some mammalian ceils [33,34,42]. Third, two reports have revealed the presence of nAChRs in several brain regions of the post-natal mouse [20,21]. Neuronal cell culture systems are potentially useful models for investigating the role of nAChRs in developing neuronal cells. Primary cultures of embryonic neuronal cells follow a maturation process with a progresive appearance of morphological and biochemical neuronal properties. To our knowledge, mamalian nAChRs have been identified in primary cultures only from the cortex [31,32], hippocampus and brain stem [1,4]. Here, we report evidence for the expression of nAChRs in primary cultures of cerebellar granule cells. These cultures have two major advantages for biochemical and molecular studies of nAChRs: (1) they provide a homogeneous system enriched in granule cells, and (2) the developmental patterns in vitro, including the expression of the muscarinic acetylcholine receptor [2], are well defined [5,10,17-19,47]. This is the first report that identifies and characterizes nAChR subtypes in cultured cerebellar granule cells using binding of radiolabeled ligands, monoclonal antibodies, the polymerase chain reaction (PCR) and calcium influx studies. Our study shows that nAChR expression follows a developmental pattern with a maximum level during the period of increased excitatory synaptogenesis [19,47]. Moreover, our results indicate that nAChRs may regulate NMDA receptor activity by modulating the release of endogenous glutamate.

2. Materials and methods

2.1. Primary culture of cerebellar granule and glia cells Cerebellar granule cells were cultured as described by Van-Vliet et al. [47]. Briefly, cerebella from 7-day-old CD1 mice (Charles River Breeding, USA) were mechanically dissociated in a serum containing culture medium after incubation in an EDTA solution (Versene 1/5000, from Gibco). Cells were plated (1.4-1.5 × 106 cells/ml; 2 ml/well) in poly-L-ornithine coated 35 mm diameter culture dishes (6-well clusters, Corning). Culture medium was composed of a 1:1 mixture of Dulbecco's minimal medium and F-12 nutrient (Gibco), supplemented with glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), HEPES buffer (5 mM), KC1 (25 mM), horse and fetal calf serum (5% each), penicillin (100 IU/ml) and streptomycin (100 p~g/ml). To prevent glial cell multiplication, 40 tzM cytosine arabinosine was added to the medium 48 h after plating. Glial cell primary cultures were obtained following a similar procedure except that cells were plated at a lower density and the culture medium was changed every 48 h without adding cytosine arabinosine. These cultures were used after 10 days in vitro.

2.2. Radioligand binding 2.2.1. Preparation of a membrane suspension from cultured cells Cultures were washed in ice-cold PBS containing 0.6% glucose. Cells were scraped into ice-cold Tris-HCl (5 mM containing EDTA 1.2 mM, leupeptin 100 /xM, PMSF 1 mM, pH 7.4) and then homogenized by passing the cell lysate several times through a Pasteur pipette. Membranes were centrifuged at 20,000 × g for 10 rain at 4°C. The resulting pellet was resuspended in a small volume of ice-cold Tris-HCl buffer (100/zl/6well culture cluster) and sonicated for 30 s. An aliquot was used for protein assay (BCA, Pierce). This preparation was used the same day for binding experiments.

2.2.2. L-[ 3H]Nicotine and [~H]cytisine binding Binding experiments were performed as described previously [22,30]. Membrane proteins (400-800 ~g) were incubated in a final volume of 200 /zl of assay buffer. In saturation experiments, concentrations of L-[3H]nicotine (Dupont NEN Research product, Boston MA; 72-85 Ci/mmol) and [3H]cytisine (Dupont NEN, 30.1 Ci/mmol) were 0.5 to 60 nM and 0.4 to 15 nM, respectively. Incubations were carried out at 0°C for 2 h since preliminary experiments showed that specific binding for 5 nM L-[3H]nicotine or 2 nM [3H]cytisine reached an equilibrium within 60 min and remained

M. Didier et al. /Molecular Brain Research 30 (1995) 17-28

stable for a least 2 h. Non-specific binding was determined using 10 IzM L-nicotine salicylate. After incubation, membranes were filtered through polyethyleneimine pretreated glass filters using a 48-well Brandel tissue harvester. Filters were washed with icecold assay buffer (3 times, 5 ml each) and air dried. The bound radioactivity was determined by scintillation counting at 50% efficiency. Scatchard analysis of saturation experiments were performed using a computer assisted program (Ligand).

19

formaldehyde for 10 min and rinsed with PBS and dehydrated. Cultures were exposed to NTB-3 emulsion at 4°C for 10 days and developed with D19 developer.

2.5. RNA isolation and RT-PCR

Cells grown on plastic Aclar coverslips were rinsed in cold PBS containing 0.6% glucose and fixed with 4% paraformaldehyde for 5 min. Coverslips were washed 3 times for 10 rain with PBS containing 10% rabbit serum prior to the first incubation with antibody. Antibodies against the nAChR subunits a 4 (mAb 299) and /32 (mAb 270) were diluted to final concentrations of 6.5 and 10 nM, respectively, in PBS-10% serum. The first incubation was carried out at 4°C for 12 h. After 3 washes with PBS containing serum, fixed cultures were incubated with a second biotinylated antibody (rabbit anti-rat IgG) at room temperature for 1 h. Then, cells were rinsed with PBS and incubated either with streptavidin-Texas red or streptavidin-horseradish peroxidase (HRP) for 1 h at room temperature. Cells were washed and sealed under a coverslip. To reveal the H R P labeling, cells were first incubated with a Tris buffer solution containing 0.6 m g / m l diaminobenzidine and 0.03% H 2 0 2. Photomicroscopy was carried out with a Zeiss fluorescence microscope.

Cultured cells (10 days in vitro for both granular neuron and glial cell cultures) were washed twice with sterile ice-cold PBS containing 0.6% glucose and total RNA was prepared by the guanidinium/cesium chloride. Cultured cells or neural structures from rat and mouse brains were lysed and homogenized in 4 M guanidium thiocyanate solution. The homogenate was then centrifuged on a cesium chloride layer (5.7 M) at 200,000 × g for 18 h at 22°C. The resultant pellet was washed with 70% ethanol, dissolved, and phenol-chloroform extracted twice. After precipitation, the RNA was dissolved in D E P C treated water and subjected to a DNAse I treatment. Total RNA (2 /xg) was used as template for first strand synthesis by the random primer method using reverse transcriptase (BRL superscript kit). cDNAs were then amplified by PCR (35 cycles of 94°C, 0.5 min; 55°C, 0.5 rain; 72°C, 1 rain) using appropriate primers. The primers used are as follows: rat a 2 (exon 5): forward A A G G C T C A C C T C T T C T T C A C (position 45 to 64), reverse T T C T T C C T C C T C T T C C T C T G (position 827 to 836); rat a3: forward C A C T T G A G T A G C T G T G C T T C (position - 6 7 to - 4 8 ) , reverse G T T G T T G T A C A G T A C G A T G T (position 342 to 361); rat a4: forward T G C T A G C A G C C A C A T A G A G A (position - 2 9 to - 1 0 ) , reverse A A C T T C A T G G T A C A G T T C T G (position 414 to 433): rat a5: forward G C T G C G C T G C T C T T G A T G G T (position 87 to 106), reverse C G T A T G T C C A C G A G C C G A A T (position 617 to 636); rat c~7: forward A C A A G G A G C T G G T C A A G A A C (position 88 to 107), reverse A A A G C G C T C A T C A G C A C T G T (position 346 to 365); rat /32: forward T C C A A C T C A A T G G C G C T G T T ( p o s i t i o n 25 to 44), r e v e r s e G A G C G A A A C T T C A T G G T G C A (position 514 to 533): rat /33: forward T T C A T C A G G C A G C T G G T C A C (position 32 to 51), reverse G T C G T A A G T C C A G G A T C CAA (position 717 to 736); rat /34: forward A G A G T G C C T G C A A G A T T G A G (position 100 to 119), reverse A G C T G A C T G C A G A C T I ' A G G A (position 867 to 886); mouse cyclophiline: forward T I ' G C T G C A G C C A T G G T C A A (position 31 to 49), reverse GAGCTG T C C A C A G T C G G A A A (position 514 to 533).

2.4. [ I25I]a-BTX autoradiography

2. 6. 45Ca 2 + influx

A similar procedure to the [~25I]a_BTX binding protocol described above was followed except that cells were incubated with 10 nM [125I]a-BTX (Amersham IL, 2,000 Ci/mmol). After washing, cells were fixed with 4% paraformaldehyde solution containing 0.5%

At 5, 10 or 30 days in vitro, cells were rinsed twice with 1 ml of Locke's buffer/well (6 well-cluster culture plates). After 30 min preincubation in the same solution, the medium was replaced with MgCl2-free Locke's buffer containing 1 ixCi 45CaC12/ml (Dupont NEN

2.2.3. [ 125I]oz-BTX binding Cultured cerebellar cells were washed twice with Locke's buffer (128 mM NaC1, 5 mM KC1, 2.7 mM CaCI2, 1.2 mM Na2HPO4, 1.2 mM MgCI2, 10 mM glucose and 20 mM HEPES, pH 7.35, O 2 / C O 2 9 5 / 5 % ) and incubated for 1 h at 37°C with various concentrations of [I25I]a-BTX (0.5 to 15 nM) (NEN, Boston MA, 125 Ci/mmol). Cells were then washed at least three times with 4 ml of Locke's solution. Non-specific [125I]a-BTX binding was revealed by incubating cultures with 50 nM unlabeled a - B T X during 1 h preincubation and incubation periods. Cells were lysed with N a O H 0.1 N containing 1% Triton X-100 and radioactivity was measured using a 3,-counter.

2.3. lrnmunocytochemistry

20

M. Didier et al. /Molecular Brain Research 30 (1995) 17-28

research product, Boston) with NMDA or nicotinic drugs. Incubations were carried out for the indicated times and then washed twice with ice-cold Locke's buffer. Cells were dissolved in 1.25 ml of NaOH (1 N)/Triton X-100 (0.5%) and 1 ml aliquots were neutralized with 10 ~1 of 10 N acetic acid and counted in a liquid scintillation counter. A 0.25 ml aliquot was used for protein determination by the BCA method (Pierce) using serum albumin as a standard.

3. Results

3.1. Characterization of [3H]nicotine binding and localization of nAChR subunits in cultured cerebellar granule cells

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Cerebellar granule ceils in vitro exhibit morphological, biochemical, and functional maturation [5,1719,47]. In particular, the vesicular release of endogenous glutamate which appears in the first days after plating, reaches a plateau around day 11, suggesting the formation of synaptic contacts at this culture stage [47]. It has been reported that at this developmental period the number or the activity of neurotransmitter receptor types, such as NMDA and muscarinic AChRs, increases in this culture [2,18,19]. In the first approach to the study of nAChRs, we assayed 10-day-old cerebellar granule cells for the expression of specific binding sites for three nicotinic ligands, namely nicotine, cytisine and a-BTX. Preliminary experiments revealed that L-[3H]nicotine binding was linear when the protein concentration varied from 400 to 1000 ~g. The specific binding of L-[3H]nicotine and [3H]cytisine, displaced by 10/zM nicotine on cerebellar granule cell membranes, varied from 80 to 50 and 50 to 25%, respectively depending on the concentration of the radioligand used (0.5 to 60 nM for nicotine and 0.4 to 15 nM for cytisine). Scatchard plots of the data, calculated from saturation experiments, were consistently linear as expected for a single class of binding sites (Fig. 1). The apparent equilibrium dissociation constants were of the same order as described previously for cortical culture or brain membrane extracts (2-4 nM for nicotine and 1 nM for cytisine). The total number of binding sites (Bmax) ranged from 12 to 18.5 and 10 to 13 fmol/mg of protein after 10 days in culture for L-[3H]nicotine and [3H]cytisine (Fig. 1). Nicotine and cytisine are thought to share the same high affinity binding sites on nAChRs mostly constituted by the a4 and /32 subunits [22]. We therefore investigated the binding of another ligand known to bind a7 a n d / o r a8 nAChR subunits [27]. [~esI]cPBTX binding experiments showed that a-BTX binds on a single class of sites on cerebellar granule cells with the same K o as for the avian immunoisolated ce7 subunit [27] (Fig. 1). Scatchard plot analysis also

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Fig. 1. Binding of t--[3H]nicotine (a,b), [3H] cytisine (c,d) or [125I]o'bungarotoxin (e,f) to cerebellar granule cells. Figures show saturation experiments and represent total (e) and non-specific ( - ) binding, respectively. Non-specific binding was determined in presence of 10 /xM t.-nicotine for t--[3H]nicotine and [3H]cytisine or 50 nM unlabeled a-BTX for [125I]a-BTX. t--[3H]nicotine and [3H]cytisine binding experiments were performed on membranes from 10-day-old cerebellar cultures. [125I]ot-BTX binding was measured on living neuronal cells cultured for 10 days. Scatchard plots were calculated from the specific binding data shown to the left. Results represent typical experiments which have been repeated 3 times with similar results.

revealed that cultured cells contained around 15 fmol/mg protein of a-BTX binding sites (Fig. 1). Although glial cells represent no more than 5% of the total cell number in these cerebellar cultures [47], the contribution of these cells to the binding of [3H]nicotine and [t25I]a-BTX was investigated by incubating membranes from primary glial cell cultures in the presence of these radioligands. Glial membrane extracts incubated with [3H]nicotine or [t25I]a-BTX showed no detectable binding (data not shown). Moreover, NMDA-induced neurotoxicity dramatically decreased the specific binding of [3H]nicotine on granule

M. Didieret al. /Molecular Brain Research 30 (1995) 17-28 20"

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Fig. 2. Binding of L-[3H]nicotine, [3H]cytisine and [125I]abungarotoxin binding during the development of cerebellar granule cells in primary culture. Binding experiments using 30 nM L[3H]nicotine, 10 nM [3H]cytisine and 5 nM [125I]a-BTX were performed on membranes extracted from cultured granule neurons or living cells (for [1251]a-BTX) at different days in vitro (DIV). Results are means + S.E.M. of experiments performed on at least 3 different cultures for each day.

cell c u l t u r e s ( d a t a n o t shown). T a k e n t o g e t h e r , t h e s e o b s e r v a t i o n s s u g g e s t e d t h a t the m a j o r i t y of n A C h R s e x p r e s s e d in c u l t u r e d c e r e b e l l a r cells w e r e l o c a t e d on neuronal components. T h e t e m p o r a l p a t t e r n of [3H]nicotine, [3H]cytisine a n d [125I]a-BTX b i n d i n g sites was e x a m i n e d d u r i n g n e u r o n a l m a t u r a t i o n b e t w e e n 6 a n d 14 days in vitro (Fig. 2). O u r results s h o w e d an i n c r e a s e in all t h e t h r e e b i n d i n g sites up to day 10, which c o r r e l a t e s with t h e p e r i o d o f s y n a p t o g e n e s i s of c u l t u r e d g r a n u l e cells [47]. A t 14 days in vitro, a significant d e c r e a s e in cytisine

21

b i n d i n g sites was o b s e r v e d . This was also a c c o m p a n i e d by a slight r e d u c t i o n in the n u m b e r of n i c o t i n e b i n d i n g sites. In contrast, t h e a m o u n t of [~25I]a-BTX b i n d i n g was stable at least at 10 a n d 14 days. T o f u r t h e r indentify n A C h R s u b u n i t localization on c e r e b e l l a r n e u r o n s , we u s e d two d i f f e r e n t m o n o c l o n a l a n t i b o d i e s , which a r e t h o u g h t to recognize the a 4 ( m A b 299) and /32 ( m A b 270) subunits, as well as [~25I]a-BTX which b i n d s on a 7 subunits. W e f o u n d specific i m m u n o l a b e l i n g with b o t h m o n o c l o n a l a n t i b o d ies on n e u r o n a l cells but not on n o n - n e u r o n a l ceils (Fig. 3). In g e n e r a l , the labeling o b t a i n e d with m A b 2 9 9 was s t r o n g e r t h a n t h a t o b s e r v e d with m A b 2 7 0 and s h o w e d a s o m a t i c as well as a neuritic localization o f n A C h R s (Fig. 4a,b). N o i m m u n o s t a i n i n g was seen in p r i m a r y glial cell c u l t u r e s ( d a t a not shown). Most but n o t all cells showing g r a n u l e cell m o r p h o l o g y were d e n s e l y i m m u n o s t a i n e d (89 _+ 4%; Fig. 4c). [125I ] a - B T X clearly b o u n d to the s o m a a n d the n e u r i t e s of some n e u r o n s (32 +_ 6 % of total n e u r o n a l cells; Fig. 5a,b). This b i n d i n g was d i s p l a c e d by an excess of u n l a b e l e d a - B T X (Fig. 5c).

3.2. Detection by RT-PCR of nAChR m R N A s expressed in cultured cerebellar granule cells T h e m o l e c u l a r n a t u r e of n A C h R s e x p r e s s e d in cereb e l l a r c u l t u r e s was i d e n t i f i e d using R T - P C R experiments. P r e l i m i n a r y studies s h o w e d t h a t c D N A sizes, o b t a i n e d a f t e r a m p l i f i c a t i o n of c D N A from rat brain, w e r e i d e n t i c a l to t h o s e e x p e c t e d for the specific clone s e g m e n t s a m p l i f i e d ( d a t a not shown). Similar sizes for

Fig. 3. Cellular localization of nAChR subunits. Cerebellar granule cells grown for 10 days in vitro were fixed and labelled with monoclonal antibodies (mAb) raised against the a4 and /32 nAChR subunits. The control experiment performed in absence of a mAb. Immunostaining by mAb 299 to a4 and mAb 270 to /32 was revealed using a second biotinylated antibody coupled to a streptavidin-horse radish peroxidase. Glial cells are indicated by a G; Arrows indicate some immunostained neuronal cells. Horizontal bar = 10 tzm.

M. Didier et al. / Molecular Brain Research 30 (1995) 17-28

22

amplified DNA were also obtained when cDNA templates came from various mouse neural structures (Fig. 6a). Amplification of cDNAs synthesized from total RNA showed the presence of a3, a4, a5, a7,/32 and /34 in a 10-day-old cerebellar granule cell culture (Fig. 6b). No signal was detected for either a2 or/33 nAChR subunits. Reactions performed with initial extracted RNA without synthetizing cDNA did not yield nAChR DNA. Glial cells in our culture represent less than 3-5% of the total ceils. However, the PCR technique can amplify and detect very low levels of gene expression. We verified that the detected nAChR RNAs were expressed by neuronal cells by performing PCR experiments on cerebellar glial cells in primary culture. We did not observe any neuronal-like ceils in this culture after 10 days in vitro. No signals were detected for any nAChR subunit (Fig. 6b). However, we were still able to detect cyclophilin expression, as was also observed for neuronal cultures (Fig. 6b).

3.3.

45Ca2 +

influx stimulated by nicotine treatment

Application of nicotine increased the 45Ca2+ influx in 10-day-old cerebellar granule cells in a time (Fig. 7a)

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and dose-dependent manner (Fig. 7b). Maximum stimulation induced by 200 IzM nicotine was reached after 2.5 rain incubation (Fig. 7a). The ECs0 of nicotine in inducing 45Ca2+ influx was of the order of 120 p~M (Fig. 7b). In some experiments, desensitization of the nicotine response was observed with a nicotine concentration > 400 /xM. Binding experiments showed an increase in the number of nicotinic binding sites during cerebellar granule cell maturation (Fig. 2). We investigated the nicotine-induced calcium flux at different culture stages. A transient pattern in the nicotine response was observed (Fig. 7c). At 5 days in culture, 200 #M nicotine did not stimulate the flux of 45Ca2+ into the cultured cells. A significant nicotine effect was detectable at 12 days, but this response decreased in later stages of cell culture. The nicotine stimulatory effect can be induced by other specific agonists for nAChR. Application of 100 /xM cytisine was also able to induce a 4 5 C a 2 + influx comparable to that stimulated by 400 p~M nicotine (Fig. 8). The nicotine response was inhibited by dtubocurarine (100 /xM) but not by a concentration of atropine (1 /xM) known to inhibit specifically muscarinic AChRs. In a preliminary experiment, Mg 2+ (1.2 mM), a channel blocker for the nAChR and

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Fig. 4. SubceUular and neuronal localization of a 4 n A C h R subunits. T h e binding of m A b 299 was revealed on 10-day-old cultures using a second biotinylated antibody coupled to streptavidin-texas red (a) or streptavidin-horse radish peroxidase (b and c). Arrows show the neuritic immunostaining. Plus and minus show positively labeled and unlabelled neurons, respectively. Horizontal bar = 10 ~ m .

M. Didier et al. / Molecular Brain Research 30 (1995) 17-28

NMDA receptors, was shown to completely supress the stimulatory nicotinic effect. We used the NMDA antagonists CPP (100/zM) and MK801 (1/xM). A micromolar concentration of MK801 is known to totally

23

block the NMDA receptor channel without interacting with nAChR activation [3]. Both reduced the nicotinic induced 45CaZ+ influx in neuronal cultures, suggesting a NMDA receptor activation component in the nico-

Fig. 5. [lzs]a-BTX autoradiography on cerebellar cultures. 10-day-old cultures were incubated with 10 n M [lZSI]a-BTX in the absence (A and B) or the presence (C) of 50 nM unlabeled a-BTX. A and B pictures represent the same field observed with phase contrast (A) to show the cells or with bright field (B) optics to show autoradiography grains. Large and small black arrows show specific signals observed on neuronal cell soma and neurites, respectively. Empty arrows indicate some unlabeled neurons. Horizontal bar = 10/xm.

24

M. Didier et al. / Molecular Brain Research 30 (1995) 17-28

tinic effect (Fig. 8). The effect of a - B T X on the nicotine response was also assayed because [t25I]a-BTX autoradiography and P C R experiments supported the presence of a BTX-binding subunits in cerebellar neurons. Pretreatment of cultures with 100 nM a - B T X for 1 h was able to reduce nicotine-stimulated 45Ca2+ cellular entry by 71 + 9% (n = 4). However, such a treatment did not affect the extensive N M D A - e v o k e d calcium influx (Fig. 9). N M D A receptor activation could be mediated by the release of endogenous glutamate after n A C h R stimulation. To test this hypothesis, we performed experiments combining extensive cellular washing and treatment with glutamate pyruvate transaminase (GPT), an enzyme which transforms glutamate into a-ketoglutarate in the presence of pyruvate [18]. Cultures were prewashed at least 6 times (5 min each) with 1 U / m l of G P T plus 2 mM pyruvate. The enzyme treatment was also performed during the nicotine stimulation. This treatment has been shown to suppress fully the extracellular glutamate released by cerebellar cultures after a K + depolarization [18]. Under this condition, the stimulatory effect of nicotine on cellular calcium influx was decreased by at least 95% and the remaining nicotine response became CPP-insensitive (Fig. 10).

4. Discussion Our results indicate an early expression of nAChRs early during the in vitro development of cerebellar granule cells. This expression displays a developmental pattern correlated with the period of synaptogenesis [47]. Although the exact molecular nature of these nAChRs is unknown, binding experiments, immunocytochemical and autoradiographic experiments show the presence of a4, a 7 and/32 subunits on granule cell membranes. Moreover, the polymerase chain reaction reveals the additional presence of a3, a5, and /34 m R N A s in these neurons after 10 days in culture. In the present study, we found a single class of binding sites with a high affinity for nicotine and cytisine c o r r e s p o n d i n g to the previously r e p o r t e d immuno-isolated n A C h R from avian nervous system [49], as well as in vitro and in vivo rodent models [8,22,30] and human brain [11]. A similar L-[3H]nico tine binding site has also been described in membranes prepared from adult cerebellum [43]. High affinity nicotine and cytisine binding is likely to reflect the presence of n A C h R comprised of a 4 and /32 [22,50]. Immunolabeling clearly supports the presence of these proteins in most neuronal cells. However, the presence of these subunits does not eliminate the possibility that

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Fig. 6. Detection of nAChR subunit mRNAs by polymerase chain reaction (PCR). Total RNA (2 txg), isolated from either mouse neural structures (hippocampus, medial habenula, thalamus and interpeduncular nucleus), 10-day-oldcerebellar neuronal cell (N) or cerebellar glial cell (G) cultures were used as template for first strand synthesis, cDNAs were then amplified by PCR using appropriate primers for a and/3 nAChR subunits and cyclophilin. PCR assay performed without cDNA synthesis did not reveal amplifications. A cyclophilin signal was detected for both neuronal (N) and glial (G) cultures.

M. Didier et al. /Molecular Brain Research 30 (1995) 17-28

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Fig. 8. Effect of various cholinergic and N M D A antagonists on nicotine-stimulated 4SCa2 ÷ influx. Calcium influx in cerebel[ar cultures induced by 400 tzM nicotine (nico) or 200 izM cytisine (cyti). The nicotine response was inhibited by 10ll /xM d-tubocurarine (d-tubo), 1 ~ M M K 8 0 1 , 1 0 0 / x M C P P a n d I mM Mg 2' but not by 1 /~M atropine (AT). Results are the mean values+S.E.M, of 3 independent experiments.Statistical significance was (a) P < 0.01, (b) P < 0.05 when compared to the stimulation by nicotine (Student's t test).

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sure o~-BTX binding in membranes extracted from this culture displaying an affinity closely related to the one reported for the ~7 subunit [27]. Secondly, using RTPCR we detected, mRNAs for the c~3, c~5, c~7 and /34 subunits. Further experiments need to be conducted to determine the subunit compositions of the AChR subtypes present and their relative amounts and cellular localizations. Heterogeneity in the nAChR subunit expression may reflect the presence of different cell types in our culture. We did not detect any nAChR protein or m R N A in glial cells, however Golgi interneurons present in cerebellar cultures are known to respond to nicotine in vivo [15]. Although they represent less than 3% of total cells in vitro, PCR may significantly amplify

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other nAChR subtypes are expressed in our model. In fact, we present evidences for the expression of additional A C h R subtypes. Firstly, we were able to mea-

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M. Didier et al. / Molecular Brain Research 30 (1995) 17-28

26

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one subunit expressed only in these inhibitory interneurons. Furthermore, granule neurons themselves also display such receptor diversity, as shown by immunocytochemistry and autoradiography experiments. nAChR function was first investigated by nicotine stimulation of 4 5 C a 2 + influx. A significant calcium influx was evoked by nicotine application, with an ECs0 comparable to that reported for Na + flux and Rb + effiux in PC12 and TE671 cells, respectively [13,35]. This concentration exceeded by several orders of magnitude the concentration needed to inhibit the high affinity radiolabelled nicotine binding. This sort of observation usually reflects the difference between the high agonist concentrations necessary to activate a resting nAChR and the lower concentrations necessary to bind at equilibrium to desensitized nAChRs [35]. The nicotine effect on the 45Ca2+ cellular influx was highly sensitive to a - B T X which inhibits a 7 containing nAChRs. Therefore, it is likely that nicotine response was mediated by the activation of a 7 AChRs rather than a4f12 AChRs. Nicotine-stimulated 4 5 C a 2 + influx appeared to be the consequence of the indirect activation of the N M D A receptors for three reasons. First, nicotine effects were sensitive to non-competitive as well as competitive N M D A antagonists. Second, using an enzymatic treatment to remove glutamate from the extracellular medium during the nicotinic stimulation, we clearly observed that most calcium influx measured resulted from an endogenous glutamate release and a resultant N M D A receptor activation. Thirdly, preliminary experiments showed a 60% increase of the extracellular glutamate concentration after nicotine treatment (L. Passani, J.T Coyle, M.D, S.A.B and S.B., in preparation). It is possible that nAChRs could mediate the release of endogeneous glutamate at a presynaptic

level. In this, many studies support the hypothesis of a nerve terminal localization of nAChRs in several brain areas modulating the n e u r o t r a n s m i t t e r release [6,7,29,51]. Interestingly, in our study [~25I]a-BTX autoradiography clearly showed a neuritic localization for a 7 nAChR subunits. The presence of such a subunit, known to form a Ca 2*-permeable nAChR, on the neuronal processes may play a crucial role in the control of neurotransmitter release from granule neurons. Taken together, our results suggest that nAChRs could also modulate the glutamate release from cerebellar granule cell nerve terminals, at least during the formation of parallel fibers. However, further experiments in vivo are required to verify this hypothesis. A developmental pattern of nAChR expression was observed in the cerebellar granule cell culture. Between the first and second weeks in vitro, the nAChR expression increased and seemed to decrease thereafter for at least nicotine and cytisine binding sites. Fielder et al. [20,21] also described a high amount of nicotine binding in the post-natal mouse brain. In some brain structures such as the cerebellum, the nAChR or a-BTX binding sites decrease thereafter to reach adult level. We have also observed a reduction of the a4 m R N A expression in the granule cell layer of the developing mouse cerebellum (Bix, S.A.B, S.B, M.D., submitted). At this stage, potential regulatory factors controling nAChR expression in cerebellar neurons have not been indentified. Cholinergic innervation is thought to initiate and regulate the nAChR expression in the avian nervous system [9,14,26,37,38]. In the mouse brain, the loss of nicotine as well as a-BTX binding correlated also with the increase in cholinergic enzymes [20]. However, such regulations are unlikely to occur in our cerebellar cultures since no choline acetyl transferase containing neurons have been detected (M.D., S.A.B, S.B., unpublished results). Therefore, nAChRs may be subject to a heteroregulation mediated by endogenous glutamatergic activity. Indeed, the peak in the nAChR number occurred during the period of intensive excitatory synaptogenesis in culture [47]. In addition, sustained N M D A receptor activation down-regulates the a 7 nAChR subunit in cultured granule neurons (M.D., S.A.B., S.B., in preparation). What could be the physiological relevance of nAChR expression in cerebellar granule cells? Some reports have attributed critical roles to nAChRs during neuronal development. These include control of neuritic outgrowth in rat retinal ganglion cells in culture [33] as well as neuronal survival in the avian ciliary ganglion [39]. Beside these major effects on cholinoceptive neurons, it is likely that nAChR activation has a less prominent role in neuronal cells receiving a minor cholinergic innervation. In the cerebellum, choline acetyltransferase immunoreactivity is localized on some mossy fibers, golgi neurons as well as granule cells and

M. Didier et al. /Molecular Brain Research 30 119951 17-28

their parallel fibers [25,41]. Except for Golgi cells, most of these neurons and fibers use excitatory amino acids as neurotransmitters [46]. However, modulation of the excitatory activity may be of great importance at particular periods of granular cell development [10] and nAChR activation by acetylcholine could represent one factor controling the NMDA receptor activity by stimulating glutamate release. Parallel activation of muscarinic acetylcholine receptors may also play an additive post-synaptic role in enhancing the reactivity of neurons to glutamate as described in hippocampal cells [44]. Interestingly, muscarinic receptor appearance was time-correlated with nAChRs in developing granule neurons [2].

Acknowledgements We would like to thank Min Xu for his technical assistance. M.D. was supported by a postdoctoral fellowship from the Human Frontier Science Organization; S.A.B. and S.B. by grants from NIH (NS24377) and the William Randolph Hearst Fund.

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