GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons.

Human mutations of the GRID1 gene encoding the orphan delta1 glutamate receptor-channel (GluD1) are associated with schizophrenia but the explicit role of GluD1 in brain circuits is unknown. Based on the known function of its paralog GluD2 in cerebellum, we searched for a role of GluD1 in slow glutamatergic transmission mediated by metabotropic receptor mGlu1 in midbrain dopamine neurons, whose dysfunction is a hallmark of schizophrenia. We found that an mGlu1 agonist elicits a slow depolarizing current in HEK cells co-expressing mGlu1 and GluD1, but not in cells expressing mGlu1 or GluD1 alone. This current is abolished by additional co-expression of a dominant-negative GluD1 dead pore mutant. We then characterized mGlu1-dependent currents in dopamine neurons from midbrain slices. Both the agonist-evoked and the slow postsynaptic currents are abolished by expression of the dominant-negative GluD1 mutant, pointing to the involvement of native GluD1 channels in these currents. Likewise, both mGlu1-dependent currents are suppressed in GRID1 knockout mice, which reportedly display endophenotypes relevant for schizophrenia. It is known that mGlu1 activation triggers the transition from tonic to burst firing of dopamine neurons, which signals salient stimuli and encodes reward prediction. In vivo recordings of dopamine neurons showed that their spontaneous burst firing is abolished in GRID1 knockout mice or upon targeted expression of the dominant-negative GluD1 mutant in wild-type mice. Our results de-orphanize GluD1, unravel its key role in slow glutamatergic transmission and provide insights into how GRID1 gene alterations can lead to dopaminergic dysfunctions in schizophrenia.

Nicotine consumption is regulated by a human polymorphism in dopamine neurons.

Smoking is the most important preventable cause of morbidity and mortality worldwide. Recent genome-wide association studies highlighted a human haplotype on chromosome 15 underlying the risk for tobacco dependence and lung cancer. Several polymorphisms in the CHRNA3-CHRNA5-CHRNB4 cluster coding for the nicotinic acetylcholine receptor (nAChR) α3, α5 and ß4 subunits were implicated. In mouse models, we define a key role in the control of sensitivity to nicotine for the α5 subunit in dopaminergic (DAergic) neurons of the ventral tegmental area (VTA). We first investigated the reinforcing effects of nicotine in drug-naive α5(-/-) mice using an acute intravenous nicotine self-administration task and ex vivo and in vivo electrophysiological recordings of nicotine-elicited DA cell activation. We designed lentiviral re-expression vectors to achieve targeted re-expression of wild-type or mutant α5 in the VTA, in general, or in DA neurons exclusively. Our results establish a crucial role for α5*-nAChRs in DAergic neurons. These receptors are key regulators that determine the minimum nicotine dose necessary for DA cell activation and thus nicotine reinforcement. Finally, we demonstrate that a single-nucleotide polymorphism, the non-synonymous α5 variant rs16969968, frequent in many human populations, exhibits a partial loss of function of the protein in vivo. This leads to increased nicotine consumption in the self-administration paradigm. We thus define a critical link between a human predisposition marker, its expression in DA neurons and nicotine intake.

Co-activation of VTA DA and GABA neurons mediates nicotine reinforcement.

Smoking is the most important preventable cause of mortality and morbidity worldwide. This nicotine addiction is mediated through the nicotinic acetylcholine receptor (nAChR), expressed on most neurons, and also many other organs in the body. Even within the ventral tegmental area (VTA), the key brain area responsible for the reinforcing properties of all drugs of abuse, nicotine acts on several different cell types and afferents. Identifying the precise action of nicotine on this microcircuit, in vivo, is important to understand reinforcement, and finally to develop efficient smoking cessation treatments. We used a novel lentiviral system to re-express exclusively high-affinity nAChRs on either dopaminergic (DAergic) or γ-aminobutyric acid-releasing (GABAergic) neurons, or both, in the VTA. Using in vivo electrophysiology, we show that, contrary to widely accepted models, the activation of GABA neurons in the VTA plays a crucial role in the control of nicotine-elicited DAergic activity. Our results demonstrate that both positive and negative motivational values are transmitted through the dopamine (DA) neuron, but that the concerted activity of DA and GABA systems is necessary for the reinforcing actions of nicotine through burst firing of DA neurons. This work identifies the GABAergic interneuron as a potential target for smoking cessation drug development.

Phosphodiesterase type 2 and the homeostasis of cyclic GMP in living thalamic neurons.

The ubiquitous second messenger cyclic GMP (cGMP) is synthesized by soluble guanylate cyclases in response to nitric oxide (NO) and degraded by phosphodiesterases (PDE). We studied the homeostasis of cGMP in living thalamic neurons by using the genetically encoded fluorescence resonance energy transfer sensor Cygnet, expressed in brain slices through viral gene transfer. Natriuretic peptides had no effect on cGMP. Basal cGMP levels decreased upon inhibition of NO synthases or soluble guanylate cyclases and increased when PDEs were inhibited. Single cell RT-PCR analysis showed that thalamic neurons express PDE1, PDE2, PDE9, and PDE10. Basal cGMP levels were increased by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and BAY60-7550 but were unaffected by PDE1 or PDE10 inhibitors. We conclude that PDE2 regulates the basal cGMP concentration in thalamic neurons. In addition, in the presence of 3-isobutyl-1-methylxanthine (IBMX), cGMP still decreased after application of a NO donor. Probenecid, a blocker of cGMP transporters, had no effect on this decrease, leaving PDE9 as a possible candidate for decreasing cGMP concentration. Basal cGMP level is poised at an intermediate level from which it can be up or down-regulated according to the cyclase and PDE activities.

The endogenous somnogen adenosine excites a subset of sleep-promoting neurons via A2A receptors in the ventrolateral preoptic nucleus.

Recent research has shown that neurons in the ventrolateral preoptic nucleus are crucial for sleep by inhibiting wake-promoting systems, but the process that triggers their activation at sleep onset remains to be established. Since evidence indicates that sleep induced by adenosine, an endogenous sleep-promoting substance, requires activation of brain A(2A) receptors, we examined the hypothesis that adenosine could activate ventrolateral preoptic nucleus sleep neurons via A(2A) adenosine receptors in rat brain slices. Following on from our initial in vitro identification of these neurons as uniformly inhibited by noradrenaline and acetylcholine arousal transmitters, we established that the ventrolateral preoptic nucleus comprises two intermingled subtypes of sleep neurons, differing in their firing responses to serotonin, inducing either an inhibition (Type-1 cells) or an excitation (Type-2 cells). Since both cell types contained galanin and expressed glutamic acid decarboxylase-65/67 mRNAs, they potentially correspond to the sleep promoting neurons inhibiting arousal systems. Our pharmacological investigations using A(1) and A(2A) adenosine receptors agonists and antagonists further revealed that only Type-2 neurons were excited by adenosine via a postsynaptic activation of A(2A) adenosine receptors. Hence, the present study is the first demonstration of a direct activation of the sleep neurons by adenosine. Our results further support the cellular and functional heterogeneity of the sleep neurons, which could enable their differential contribution to the regulation of sleep. Adenosine and serotonin progressively accumulate during arousal. We propose that Type-2 neurons, which respond to these homeostatic signals by increasing their firing are involved in sleep induction. In contrast, Type-1 neurons would likely play a role in the consolidation of sleep.

Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons.

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit (GluR1-4) mRNAs expressed by single neurons in rat hippocampal cultures were quantified by single-cell RT-PCR using an internal standard RNA after whole-cell patch-clamp recording. The internal standard RNA, derived from GluR2 with a single nucleotide substitution, was reverse-transcribed and PCR-amplified with the same efficiency as GluR1-4 mRNAs. The mean mRNA numbers harvested in vitro from pyramidal-like neurons on day 9 were 1150 +/- 324 molecules of GluR1, 1080 +/- 273 molecules of GluR2, 100 +/- 20 molecules of GluR3, and 50 +/- 10 molecules of GluR4 (mean +/- SEM, n = 12). In a non-pyramidal neuronal population that expresses AMPA receptors characterized by high Ca(2+) permeability, the numbers of GluR1, GluR3 and GluR4 mRNA molecules harvested per cell were 354 +/- 64, 25 +/- 17 and 168 +/- 36, respectively (n = 8). The GluR2 mRNA was not detected in this cell type. The calculated ratio of AMPAR mRNA molecules per total mRNA molecules was 1/240 in pyramidal-like neurons (1/500 for GluR2), being in the range obtained with total RNA from rat forebrain and cerebellum (1/170 and 1/380, respectively). Finally, our results indicated that the proportion of GluR1-4 mRNA located in neurites reached approximately 60% in pyramidal-like neurons. However, we found no evidence of preferential subcellular distribution of a given subunit.

Classification of fusiform neocortical interneurons based on unsupervised clustering.

A classification of fusiform neocortical interneurons (n = 60) was performed with an unsupervised cluster analysis based on the comparison of multiple electrophysiological and molecular parameters studied by patch-clamp and single-cell multiplex reverse transcription-PCR in rat neocortical acute slices. The multiplex reverse transcription-PCR protocol was designed to detect simultaneously the expression of GAD65, GAD67, calbindin, parvalbumin, calretinin, neuropeptide Y, vasoactive intestinal peptide (VIP), somatostatin (SS), cholecystokinin, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, N-methyl-d-aspartate, and metabotropic glutamate receptor subtypes. Three groups of fusiform interneurons with distinctive features were disclosed by the cluster analysis. The first type of fusiform neuron (n = 12), termed regular spiking nonpyramidal (RSNP)-SS cluster, was characterized by a firing pattern of RSNP cells and by a high occurrence of SS. The second type of fusiform neuron (n = 32), termed RSNP-VIP cluster, predominantly expressed VIP and also showed firing properties of RSNP neurons with accommodation profiles different from those of RSNP-SS cells. Finally, the last type of fusiform neuron (n = 16) contained a majority of irregular spiking-VIPergic neurons. In addition, the analysis of glutamate receptors revealed cell-type-specific expression profiles. This study shows that combinations of multiple independent criteria define distinct neocortical populations of interneurons potentially involved in specific functions.

Selective excitation of subtypes of neocortical interneurons by nicotinic receptors.

The cellular mechanisms by which neuronal nicotinic cholinergic receptors influence many aspects of physiology and pathology in the neocortex remain primarily unknown. Whole-cell recordings and single-cell reverse transcription (RT)-PCR were combined to analyze the effect of nicotinic receptor agonists on different types of neurons in acute slices of rat neocortex. Nicotinic receptor agonists had no effect on pyramidal neurons and on most types of interneurons, including parvalbumin-expressing fast spiking interneurons and somatostatin-expressing interneurons, but selectively excited a subpopulation of interneurons coexpressing the neuropeptides vasoactive intestinal peptide (VIP) and cholecystokinin. This excitation persisted in the presence of glutamate, GABA, and muscarinic receptor antagonists and in the presence of tetrodotoxin and low extracellular calcium, suggesting that the depolarization was mediated through the direct activation of postsynaptic nicotinic receptors. The responses were blocked by the nicotinic receptor antagonists dihydro-beta-erythroidine and mecamylamine and persisted in the presence of the alpha7 selective nicotinic receptor antagonist methyllycaconitine, suggesting that the involved nicotinic receptors lacked the alpha7 subunit. Single-cell RT-PCR analysis indicated that the majority of the interneurons that responded to nicotinic stimulation coexpressed the alpha4, alpha5, and beta2 nicotinic receptor subunits. Therefore, these results provide a role for non-alpha7 nicotinic receptors in the selective excitation of a subpopulation of neocortical interneurons. Because the neocortical interneurons expressing VIP have been proposed previously to regulate regional cortical blood flow and metabolism, these results also provide a cellular basis for the neuronal regulation of cortical blood flow mediated by acetylcholine.

Properties of bipolar VIPergic interneurons and their excitation by pyramidal neurons in the rat neocortex.

In the rat neocortex, a subset of GABAergic interneurons express the neuropeptide vasoactive intestinal peptide (VIP). Previously, we demonstrated that a population of VIPergic interneurons could be accurately identified by their irregular spiking (IS) pattern and their bipolar morphology. IS interneurons were studied in neocortical slices from 16-22-day-old rats using whole-cell recordings, intracellular labelling and single-cell RT-PCR. In response to a depolarizing pulse, IS interneurons typically discharged a burst of action potentials followed by spikes emitted at an irregular frequency. Several seconds of depolarization, micromolar concentrations of 4-aminopyridine, and nanomolar concentrations of either dendrotoxin I or K converted this irregular pattern to a sustained discharge, suggesting the involvement of an ID-like K+ current. The main glutamate receptor subunits detected in IS cells were GluR1 flop and GluR2 flop, GluR5 and GluR6, and NR2B and NR2D for the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartic acid (NMDA) subtypes, respectively. Paired whole-cell patch-clamp recordings indicated that pyramidal neurons provide intracortical glutamatergic inputs onto IS interneurons. Most connections had high probabilities of response and exhibited frequency-dependent paired pulse depression. Comparison of the amplitude distribution of paired responses suggested that most of these connections consisted of multiple functional release sites. Finally, two discrete subpopulations of IS cells could be identified based on the duration of the initial burst of action potentials and the differential expression of calretinin and choline acetyltransferase.

Subunit composition, kinetic, and permeation properties of AMPA receptors in single neocortical nonpyramidal cells.

Native AMPA receptors (AMPARs) were investigated in neocortical fast-spiking (FS) and regular-spiking nonpyramidal (RSNP) cells. The onset of and recovery from desensitization as well as current rectification and single-channel conductance were studied by using fast glutamate application to outside-out patches. The GluR1-4 subunit, flip/flop splicing, and R/G editing expression patterns of functionally characterized cells were determined by single-cell reverse transcription-PCR to correlate the subunit composition of native AMPARs with their functional properties. Our sample, mostly constituted by RSNP neurons, predominantly expressed GluR3 flip and GluR2 flop. In individual cells, flip/flop splicing of each subunit appeared to be regulated independently, whereas for R/G editing all subunits were either almost fully edited or unedited. We confirmed that the relative GluR2 expression controls the permeation properties of native AMPARs, whereas none of the single molecular parameters considered appeared to be a key determinant of the kinetics. FS neurons displayed AMPARs with relatively homogeneous functional properties characterized by fast desensitization, slow recovery from desensitization, marked inward rectification, and large single-channel conductance. In contrast, these parameters varied over a wide range in RSNP neurons, and their combination resulted in various AMPAR functional patterns. Indeed, in different cells, fast or slow desensitization was found to be associated with either slow or fast recovery from desensitization. Similarly, fast or slow kinetics was associated with either strong or weak rectification. Our results suggest that kinetic and permeation properties of native AMPARs can be regulated independently in cortical neurons and probably do not have the same molecular determinants.

Molecular and physiological diversity of cortical nonpyramidal cells.

The physiological and molecular features of nonpyramidal cells were investigated in acute slices of sensory-motor cortex using whole-cell recordings combined with single-cell RT-PCR to detect simultaneously the mRNAs of three calcium binding proteins (calbindin D28k, parvalbumin, and calretinin) and four neuropeptides (neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin). In the 97 neurons analyzed, all expressed mRNAs of at least one calcium binding protein, and the majority (n = 73) contained mRNAs of at least one neuropeptide. Three groups of nonpyramidal cells were defined according to their firing pattern. (1) Fast spiking cells (n = 34) displayed tonic discharges of fast action potentials with no accommodation. They expressed parvalbumin (n = 30) and/or calbindin (n = 19) mRNAs, and half of them also contained transcripts of at least one of the four neuropeptides. (2) Regular spiking nonpyramidal cells (n = 48) displayed a firing behavior characterized by a marked accommodation and presented a large diversity of expression patterns of the seven biochemical markers. (3) Finally, a small population of vertically oriented bipolar cells, termed irregular spiking cells (n = 15), fired bursts of action potentials at an irregular frequency. They consistently co-expressed calretinin and vasoactive intestinal polypeptide. Additional investigations of these cells showed that they also co-expressed glutamic acid decarboxylase and choline acetyl transferase. Our results indicate that neocortical nonpyramidal neurons display a large diversity in their firing properties and biochemical patterns of co-expression and that both characteristics could be correlated to define discrete subpopulations.

Correlation between kinetics and RNA splicing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in neocortical neurons.

In the cortex fast excitatory synaptic currents onto excitatory pyramidal neurons and inhibitory nonpyramidal neurons are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors exhibiting cell-type-specific differences in their kinetic properties. AMPA receptors consist of four subunits (GluR1-4), each existing as two splice variants, flip and flop, which critically affect the desensitization properties of receptors expressed in heterologous systems. Using single cell reverse transcription PCR to analyze the mRNA of AMPA receptor subunits expressed in layers I-III neocortical neurons, we find that 90% of the GluR1-4 in nonpyramidal neurons are flop variants, whereas 92% of the GluR1-4 in pyramidal neurons are flip variants. We also find that nonpyramidal neurons predominantly express GluR1 mRNA (GluR1/GluR1-4 = 59%), whereas pyramidal neurons contain mainly GluR2 mRNA (GluR2/GluR1-4 = 59%). However, the neuron-type-specific splicing is exhibited by all four AMPA receptor subunits. We suggest that the predominance of the flop variants contributes to the faster and more extensive desensitization in nonpyramidal neurons, compared to pyramidal cells where flip variants are dominant. Alternative splicing of AMPA receptors may play an important role in regulating synaptic function in a cell-type-specific manner, without changing permeation properties.

Functional and molecular analysis of glutamate-gated channels by patch-clamp and RT-PCR at the single cell level.

In the central nervous system (CNS) rapid excitatory neurotransmission is mainly mediated by ligand gated, cationic channels activated by glutamate. Three main subtypes of glutamate-gated channels have been characterized by pharmacological studies. They have been named according to their preferred agonist, N-methyl-D-aspartate (NMDA), high affinity kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Furthermore, a large diversity within each class of glutamate-gated channels has been revealed by the molecular cloning of multiple subunits and their spliced and edited variants (for review see Wisden and Seeburg, 1993). These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have combined patch-clamp recording, reverse transcription (RT) and PCR to correlate, at the single cell level, the pattern of subunits expression with the functional properties of native glutamate receptors. We describe here results obtained on the AMPA receptors of hippocampal neurones and on the NMDA receptors of cerebellar granule cells which show that the subunit composition of these two types of receptors explains some of their functional properties. Furthermore, our data also indicate that the expression of NMDA receptor subunits during the postnatal development of cerebellar granule cells is regulated by an activity-dependent mechanism.

Neuronal activity differentially regulates NMDA receptor subunit expression in cerebellar granule cells.

Reverse-transcription PCR assays were used to measure levels of NMDA receptor (NR) subunit mRNAs encoding splice variants of NR1 (NR1a, -exon 5; NR1b, +exon 5) and the major NR2 subunits (NR2A, NR2B, and NR2C) in dissociated cerebellar granule cell cultures. Cultures chronically exposed to 25 mM KCl or 100 microM NMDA/15 mM KCl, which promote survival by stimulating Ca2+ influx through voltage-sensitive Ca2+ channels or NRs, were compared with 5 mM KCl culture conditions, which results in limited cell survival attributable to a lower level of NR stimulation by ambient glutamate. In situ granule-cell maturation is associated with downregulation of NR2B and increases both of NR2A and NR2C and in the ratio of NR1b/NR1a mRNAs. In culture, 25 mM KCl or NMDA rapidly induced NR2A and downregulated NR2B, followed by gradual induction of NR2C. In 5 mM KCl, a similar, rapid increase in NR2A was observed, but disappearance of NR2B occurred over a longer time course. By 9-12 d in vitro in 5 mM KCl, the relative proportions of all three NR2 mRNAs in surviving cells were not significantly different from cells cultured in 25 mM KCl. NR1a mRNA predominated at every stage of culture in 25 mM KCl or NMDA, however, whereas gradual induction of the mature-form NR1b was observed in 5 mM KCl. Although using high potassium- or NMDA-containing media enhanced granule cell survival, it did not reproduce the pattern of expression of NR mRNAs observed in situ, whereas this pattern was observed in granule cells surviving in 5 mM KCl.

Diversity of glutamate receptors in neocortical neurons: implications for synaptic plasticity.

The biochemical and functional characteristics of the AMPA subtype of the glutamate receptors expressed by pyramidal and non-pyramidal neurons of the neocortex have been studied in acute slices by means of single-cell RT-PCR and fast applications of glutamate on outside-out patches. Our results suggest that the predominant expression of the flop splice variants of the GluR1-4 AMPA subunits contributes to the faster desensitization of these receptors in non-pyramidal neurons compared to pyramidal cells where flip variants of GluR1-4 are dominant. Alternative splicing of AMPA receptors may therefore play an important role in regulating synaptic function in a cell-type specific manner.

Molecular biology and physiology at the single-cell level.

The functional characterization of the many complex proteins expressed in the CNS is a daunting task. The development of nucleic acid amplification techniques has provided a powerful tool to tackle this challenging enterprise. Recently, these techniques have been successfully used to correlate the functional properties of various CNS proteins with their specific mRNA expression patterns in the brain.

Kainate receptor subunits expressed in single cultured hippocampal neurons: molecular and functional variants by RNA editing.

To determine the kainate receptor subunits that are found in native kainate receptors, we have applied a multiplex PCR of cDNAs reverse transcribed from mRNA harvested from single cultured hippocampal neurons after electrophysiological recording. We found that all the cells showing rapidly desensitizing currents in response to kainate express the GluR6 subunit mRNA, and that some of them also express the GluR5 subunit mRNA. No GluR7, KA-1, or KA-2 subunit mRNAs were detected. Analysis of the editing sites of the GluR6 mRNA demonstrated that the three editing sites present in these subunits are edited to a different extent. Predominant expression of the unedited variant (Q) was observed, but edited and unedited variants may coexist in the same cell. In addition, we show that the Q/R site from the GluR6 subunit controls functional properties of native kainate receptors.

Single cell RT-PCR proceeds without the risk of genomic DNA amplification.

We have previously described a method for detection of mRNAs expressed in single cells after patch-clamp recordings. The method, termed single cell RT-PCR, involves aspiration of the cell content, a reverse transcription (RT) step, and a polymerase chain reaction (PCR) using specific primers. Since the nucleus is frequently harvested together with the cytosol, genomic DNA may generate false positive results. Thus, we demonstrated that dilutions containing a few copies of plasmid could be detected by PCR in a range which, according to the Poisson law, suggests that the PCR method can amplify from the two genomic alleles. We performed single cell RT-PCR of intronless GluR2 or GluR5 fragments by comparing cerebellar cell types where these mRNAs are known to be present or absent. For each cell the nucleus was harvested together with the cytosol. Following RT-PCR with GluR5 primers, all Purkinje cells (n = 6) yielded the expected PCR product, whereas it was not generated from any of the granule cells (n = 5). In corresponding experiments with GluR2 primers, we obtained the GluR2 product from all Purkinje cells (n = 5), but not from any of the glial cells (n = 5). These results are in agreement with the known cellular expression of GluR2 and GluR5 mRNAs. We conclude that the single cell RT-PCR method does not amplify the genomic DNA when the nucleus is aspirated together with the cytosol. We suggest that genomic DNA amplification is avoided, because the genomic alleles are not exposed during the procedure.

Activity-dependent regulation of N-methyl-D-aspartate receptor subunit expression in rat cerebellar granule cells.

The glutamate receptor channels of the N-methyl-D-aspartate (NMDA) subtype are composed of different subunits named NR1 and NR2A-D. These subunits can combine in different oligomers with diverging properties and their expression is developmentally regulated. We have used rat cerebellar slice cultures to test the involvement of bioelectrical activity and synaptic transmission in the changes in NR2A-C expression observed in developing granule cells. A correlation between the functional properties of the NMDA receptors and expression of the NR2A-C mRNAs was obtained in single granule cells by coupling patch-clamp recording and reverse transcription followed by polymerase chain reaction. Granule cells grown under standard culture conditions expressed mainly NR2A mRNA when examined after 15-40 days in vitro. Consistent with this observation, their responses to NMDA were only weakly reduced by 3 microM ifenprodil, a non-competitive antagonist which discriminates between NR2A and NR2B subunits in expression systems. In cerebellar cultures chronically exposed to tetrodotoxin to eliminate spontaneous electrical activity, granule cells maintained a predominant expression of NR2B subunits and their responses to NMDA were largely inhibited by 3 microM ifenprodil. These results provide evidence that the expression of the NR2A and B subunits is regulated through an activity-dependent mechanism leading to the formation of NMDA receptors with different pharmacological properties. Finally, the NR2C subunit, abundantly expressed in vivo by adult granule cells, was only rarely detected in slice cultures, even when excitatory synapses were formed between granule cells and fibres originating from co-cultured brainstem explants. These data suggest that the induction of NR2C expression observed in vivo requires an additional factor(s) that remains to be identified.