Francis Crépel

AMPA receptor subunits expressed by single purkinje cells

Several subunits of the glutamate receptor of the AMPA subtype have been cloned recently. These subunits, named GluR1, GluR2, GluR3, and GluR4, exist as two splicing variants (flip and flop). We have determined the subset of AMPA receptor subunits expressed by single cerebellar Purkinje cells in culture. This was achieved by combining whole-cell patch-clamp recordings and a molecular analysis, based on the polymerase chain reaction, of the messenger RNAs harvested into the patch pipette at the end of each recording. We found that each single cell expresses the messenger RNAs encoding the following five subunits: the flip and flop versions of GluR1 and GluR2 as well as GluR3flip, GluR2 being the most abundant. In addition, GluR3flop and GluR4flip were scarcely expressed in half of these neurons, and GluR4flop was never detected.

Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel

The diversity of known glutamate-gated channels has been markedly increased by the discovery of multiple subunits and their spliced and edited variants. 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 coupled patch-clamp recordings and reverse transcription followed by PCR amplification to correlate the presence of mRNAs for each subunit and the functional properties of native glutamate receptors at the single-cell level. In a homogeneous population of functionally identified hippocampal neurons (type II) in culture bearing a glutamate receptor of the AMPA subtype with a high calcium permeability, we found that, among the multiple subunits, only two, the flop forms of GluR1 and GluR4, were expressed. In particular, GluR2 was never detected. This composition explains the uncommon properties of AMPA receptors in type II neurons.

Cannabinoids decrease excitatory synaptic transmission and impair long-term depression in rat cerebellar Purkinje cells

1 CB‐1 cannabinoid receptors are strongly expressed in the molecular layer of the cerebellar cortex. We have analysed, in patch‐clamped Purkinje cells (PCs) in rat cerebellar slices, the effect of the selective CB‐1 agonists WIN55,212‐2 and CP55,940 and of the selective CB‐1 antagonist SR141716‐A on excitatory synaptic transmission and synaptic plasticity. 2 Bath application of both agonists markedly depressed parallel fibre (PF) EPSCs. This effect was reversed by SR141716‐A. In contrast, responses of PCs to ionophoretic application of glutamate were not affected by WIN55,212‐2. 3 The coefficient of variation and the paired‐pulse facilitation of these PF‐mediated EPSCs increased in the presence of WIN55,212‐2. 4 WIN55,212‐2 decreased the frequency of miniature EPSCs and of asynchronous synaptic events evoked in the presence of strontium in the bath, but did not affect their amplitude. 5 WIN55,212‐2 did not change the excitability of PFs. 6 WIN55,212‐2 impaired long‐term depression induced by pairing protocols in PCs. This effect was antagonized by SR141716‐A. The same impairment of LTD was produced by 2‐chloroadenosine, a compound that decreases the probability of release of glutamate at PF‐PC synapses. 7 The present study demonstrates that cannabinoids inhibit synaptic transmission at PF‐PC synapses by decreasing the probability of release of glutamate, and thereby impair LTD. These two effects might represent a plausible cellular mechanism underlying cerebellar dysfunction caused by cannabinoids.

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 μM 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 μM 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.

Control of Ca2+ influx by cannabinoid and metabotropic glutamate receptors in rat cerebellar cortex requires K+ channels

1 In the rodent cerebellum, both presynaptic CB1 cannabinoid receptors and presynaptic mGluR4 metabotropic glutamate receptors acutely depress excitatory synaptic transmission at parallel fibre‐Purkinje cell synapses. Using rat cerebellar slices, we have analysed the effects of selective CB1 and mGluR4 agonists on the presynaptic Ca2+ influx which controls glutamate release at this synapse. 2 Changes in presynaptic Ca2+ influx were determined with the Ca2+‐sensitive dyes fluo‐4FF AM or fluo‐3 AM. Five stimulations delivered at 100 Hz or single stimulations of parallel fibres evoked rapid and reproducible transient increases in presynaptic fluo‐4FF or fluo‐3 fluorescence, respectively, which decayed to prestimulus levels within a few hundred milliseconds. Bath application of the selective CB1 agonist WIN55,212‐2 (1 μm) markedly reduced the peak amplitude of these fluorescence transients. This effect was fully reversed by the selective CB1 antagonist SR141716‐A (1 μm). 3 Bath application of the selective mGluR4 agonist l‐AP4 (100 μm) also caused a transient decrease in the peak amplitude of the fluorescence transients evoked by parallel fibre stimulation. 4 Bath application of the potassium channel blocker 4‐AP (1 mm) totally prevented both the WIN55,212‐2‐ and the l‐AP4‐induced inhibition of peak fluorescence transients evoked by parallel fibre stimulation. 5 The present study demonstrates that activation of CB1 and mGluR4 receptors inhibits presynaptic Ca2+ influx evoked by parallel fibre stimulation via the activation of presynaptic K+ channels, suggesting that the molecular mechanisms underlying this inhibition involve an indirect inhibition of presynaptic voltage‐gated Ca2+ channels rather than their direct inhibition.

Mechanisms underlying cannabinoid inhibition of presynaptic Ca2+ influx at parallel fibre synapses of the rat cerebellum

Activation of CB1 cannabinoid receptors in the cerebellum acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses by decreasing the probability of glutamate release. This depression involves the activation of presynaptic 4‐aminopyridine‐sensitive K+ channels by CB1 receptors, which in turn inhibits presynaptic Ca2+ influx controlling glutamate release at these synapses. Using rat cerebellar frontal slices and fluorometric measures of presynaptic Ca2+ influx evoked by stimulation of parallel fibres with the fluorescent dye fluo‐4FF, we tested whether the CB1 receptor‐mediated inhibition of this influx also involves a direct inhibition of presynaptic voltage‐gated calcium channels. Since various physiological effects of CB1 receptors appear to be mediated through the activation of PTX‐sensitive proteins, including inhibition of adenylate cyclases, activation of mitogen‐activated protein kinases (MAPK) and activation of G protein‐gated inwardly rectifying K+ channels, we also studied the potential involvement of these intracellular signal transduction pathways in the cannabinoid‐mediated depression of presynaptic Ca2+ influx. The present study demonstrates that the molecular mechanisms underlying the CB1 inhibitory effect involve the activation of the PTX‐sensitive Gi/Go subclass of G proteins, independently of any direct effect on presynaptic Ca2+ channels (N, P/Q and R (SNX‐482‐sensitive) types) or on adenylate cyclase or MAPK activity, but do require the activation of G protein‐gated inwardly rectifying (Ba2+‐ and tertiapin Q‐sensitive) K+ channels, in addition to 4‐aminopyridine‐sensitive K+ channels.

Retrograde modulation of transmitter release by postsynaptic subtype 1 metabotropic glutamate receptors in the rat cerebellum

1 The aim of the study was to elucidate the mechanisms underlying the depressant effect of the group I/II metabotropic glutamate receptor (mGluR) agonist 1S,3R‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) on parallel fibre (PF) to Purkinje cell (PC) synaptic transmission. Experiments were performed in rat cerebellar slices using the whole‐cell patch‐clamp technique and fluorometric measurements of presynaptic calcium variation 2 Analysis of short‐term plasticity, fluctuation of EPSC amplitude and responses of PCs to exogenous glutamate showed that depression caused by 1S,3R‐ACPD is presynaptic. 3 The effects of 1S,3R‐ACPD were blocked and reproduced by group I mGluR antagonists and agonists, respectively. 4 These effects remained unchanged in mGluR5 knock‐out mice and disappeared in mGluR1 knock‐out mice. 5 1S,3R‐ACPD increased calcium concentration in PFs. This effect was abolished by AMPA/kainate (but not NMDA) receptor antagonists and mimicked by focally applied agonists of these receptors. Thus, it is not directly due to mGluRs but to presynaptic AMPA/kainate receptors indirectly activated by 1S,3R‐ACPD. 6 Frequencies of spontaneous and evoked unitary EPSCs recorded in PCs were respectively increased and decreased by mGluR1 agonists. Similar results were obtained when mGluR1s were activated by tetanic stimulation of PFs. 7 Injecting 30 mm BAPTA into PCs blocked the effects of 1S,3R‐ACPD on unitary EPSCs. 8 In conclusion, 1S,3R‐ACPD reduces evoked release of glutamate from PFs. This effect is triggered by postsynaptic mGluR1s and thus implies that a retrograde messenger, probably glutamate, opens presynaptic AMPA/kainate receptors and consequently increases spontaneous release of glutamate from PF terminals and decreases evoked synaptic transmission.

Evidence for a Slowly Inactivating K+ Current in Prefrontal Cortical Cells

The in vitro slice preparation of rat prefrontal cortical cells was used to analyse the presence and characteristics of a slowly inactivating outward current and its effect on the delayed integration of synaptic inputs. Pyramidal cells were identified as regular firing or bursting cells. In a fraction of these cells a depolarizing current pulse to –40 mV from a holding potential of –95 mV evoked the fast outward IA current followed by a slower outward current (IKs) which inactivated slowly during the 3‐s pulse. This slowly inactivating outward current was completely inactivated at holding potentials near –40 mV and was fully deinactivated by large hyperpolarizing pulses of 1 s duration. It was sensitive to micromolar concentrations of 4‐aminopyridine and to 10 mM tetraethylammonium. In current clamp experiments, when the cells were maintained at –80 mV, they responded to subliminal depolarizing current pulses by a slow rising depolarization which reached threshold for spike firing after a delay of several seconds. This delay was considerably reduced either by maintaining the cell at less hyperpolarized potentials or by bath application of 40 μM 4‐aminopyridine, or by repeated application of depolarizing pulses. The inactivation of IKs by the last procedure also led to plateau depolarization of the cell. These results suggest that the activation of the slowly inactivating outward current IKs can shunt excitatory inputs, preventing the cell from reaching spike threshold as long as it is not largely inactivated.

Direct and indirect interactions between cannabinoid CB1 receptor and group II metabotropic glutamate receptor signalling in layer V pyramidal neurons from the rat prefrontal cortex

At proximal synapses from layer V pyramidal neurons from the rat prefrontal cortex, activation of group II metabotropic glutamate receptors (group II mGlu) by (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl) glycine (DCG IV) induced a long‐lasting depression of excitatory postsynaptic currents. Paired‐pulse experiments suggested that the depression was expressed presynaptically. Activation of type 1 cannabinoid receptors (CB1) by WIN 55,212‐2 occluded the DCG IV‐induced depression in a mutually occlusive manner. At the postsynaptic level, WIN 55,212‐2 and DCG IV were also occlusive for the activation of extracellular signal‐regulated kinase. The postsynaptic localization of active extracellular signal‐regulated kinase was confirmed by immunocytochemistry after activation of CB1 receptors. However, phosphorylation of extracellular signal‐regulated kinase in layer V pyramidal neurons was dependent on the activation of N‐methyl‐d‐aspartate receptors, consequently to a release of glutamate in the local network. Group II mGlu were also shown to be involved in long‐term changes in synaptic plasticity induced by high frequency stimulations. The group II mGlu antagonist (RS)‐alpha‐methylserine‐O‐phosphate monophenyl ester (MSOPPE) favoured long‐term depression. However, no interaction was found between MSOPPE, WIN 55,212‐2 and the CB1 receptor antagonist SR 141716A on the modulation of long‐term depression or long‐term potentiation and the effects of these drugs were rather additive. We suggest that CB1 receptor and group II mGlu signalling may interact through a presynaptic mechanism in the induction of a DCG IV‐induced depression. Postsynaptically, an indirect interaction occurs for activation of extracellular signal‐regulated kinase. However, none of these interactions seem to play a role in synaptic plasticities induced with high frequency stimulations.

mGluR1 mutant mice as a tool to study calcium signalling and multiple innervation in the cerebellum

The present study reports that calcium signalling through voltage-gated calcium channels and release from internal stores is impaired in Purkinje cells of mutant mice lacking in GluR1 receptors and that the absence of these receptors also leads to an incomplete regression of multiple innervation in the cerebellum of these animals.