Carole Levenes

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.

Cellular mechanisms of cerebellar LTD

In the past decade there have been advances in understanding the cellular mechanisms of the long-term depression (LTD) of synaptic transmission at parallel fiber-Purkinje cell synapses in the cerebellum. This review first summarizes current views on mechanisms involved in LTD induction, from activation of voltage-gated Ca2+ channels, of ionotropic (AMPA) and metabotropic (mGluRI) glutamate receptors, to stimulation of protein kinase C and nitric oxide formation. Second, we will focus on recent findings that point towards the involvement of Ca2+ release from internal stores in LTD induction, localize the sources and targets of nitric oxide and indicate a postsynaptic site for LTD expression. Finally, a role for LTD in motor learning is now well supported by recent experiments on transgenic mice.

NMDA RECEPTOR CONTRIBUTION TO THE CLIMBING FIBER RESPONSE IN THE ADULT MOUSE PURKINJE CELL

Among integrative neurons displaying long-term synaptic plasticity, adult Purkinje cells seemed to be an exception by lacking functional NMDA receptors (NMDA-Rs). Although numerous anatomical studies have shown both NR1 and NR2 NMDA-R subunits in adult Purkinje cells, patch-clamp studies failed to detect any NMDA currents. Using more recent pharmacological and immunodetection tools, we demonstrate here that Purkinje cells from adult mice respond to exogenous NMDA application and that postsynaptic NMDA-Rs carry part of the climbing fiber-mediated EPSC (CF-EPSC), with undetectable contribution from presynaptic or polysynaptic NMDA currents. We also detect NR2-A/B subunits in adult Purkinje cells by immunohistochemistry. The NMDA-mediated CF-EPSC is barely detectable before 3 weeks postnatal. From the end of the third week, the number of cells displaying the NMDA-mediated CF-EPSC rapidly increases. Soon, this EPSC becomes detectable in all the Purkinje cells but is still very small. Its amplitude continues to increase until 12 weeks after birth. In mature Purkinje cells, we show that the NMDA-Rs contribute to the depolarizing plateau of complex spikes and increase their number of spikelets. Together, these observations demonstrate that mature Purkinje cells express functional NMDA receptors that become detectable in CF-EPSCs at ∼21 d after birth and control the complex spike waveform.

Purkinje Cell NMDA Receptors Assume a Key Role in Synaptic Gain Control in the Mature Cerebellum

A classic view in cerebellar physiology holds that Purkinje cells do not express functional NMDA receptors and that, therefore, postsynaptic NMDA receptors are not involved in the induction of long-term depression (LTD) at parallel fiber (PF) to Purkinje cell synapses. Recently, it has been demonstrated that functional NMDA receptors are postsynaptically expressed at climbing fiber (CF) to Purkinje cell synapses in mice, reaching full expression levels at ∼2 months after birth. Here, we show that in the mature mouse cerebellum LTD (induced by paired PF and CF activation), but not long-term potentiation (LTP; PF stimulation alone) at PF to Purkinje cell synapses is blocked by bath application of the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV). A blockade of LTD, but not LTP, was also observed when the noncompetitive NMDA channel blocker MK-801 was added to the patch-pipette saline, suggesting that postsynaptically expressed NMDA receptors are required for LTD induction. Using confocal calcium imaging, we show that CF-evoked calcium transients in dendritic spines are reduced in the presence of D-APV. This observation confirms that NMDA receptor signaling occurs at CF synapses and suggests that NMDA receptor-mediated calcium transients at the CF input site might contribute to LTD induction. Finally, we performed dendritic patch-clamp recordings from rat Purkinje cells. Dendritically recorded CF responses were reduced when D-APV was bath applied. Together, these data suggest that the late developmental expression of postsynaptic NMDA receptors at CF synapses onto Purkinje cells is associated with a switch toward an NMDA receptor-dependent LTD induction mechanism.

Incomplete regression of multiple climbing fibre innervation of cerebellar Purkinje cells in mGluR1 mutant mice

RECENT reports have suggested the existence of a causal relationship between impaired regression of multiple climbing fibre innervation and impaired motor coordination in protein kinase Cγ subunit (PKCγ) mutant mice. In the present patch-clamp study, performed in thin cerebellar slices prepared from adult mutant mice deficient in metabotropic glutamate receptors of the mGluR1 subtype, only 15% of Purkinje cells remained multiply innervated by climbing fibres, but motor coordination was largely impaired in these animals. The present results do not preclude the existence of a causal relationship between impairement of regression of multiple innervation during development and improper motor coordination in the adult.

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.

Inositol-1,4,5-trisphosphate-mediated rescue of cerebellar long-term depression in subtype 1 metabotropic glutamate receptor mutant mouse

Recent reports have outlined that cerebellar long-term depression requires the activation of subtype 1 metabotropic glutamate receptors, since long-term depression is impaired in subtype 1 metabotropic glutamate receptor (mGluR1) knockout mice. In order to better define the role of mGluR1-activated signal transduction pathways, we attempted to rescue cerebellar long-term depression in mGluR1 knockout mice by direct activation of subsequent intracellular cascades. The present results demonstrate that the inositol-1,4,5-trisphosphate signal transduction pathway remains functional in mGluR1 knockout mice, that calcium release from internal stores evoked by the combined photolytic release of inositol- 1,4,5-trisphosphate/pairing protocol is sufficient to rescue long-term depression in these mutants, and that this long-term depression is sensitive to a protein kinase C inhibitor. Therefore, our results provide compelling evidence that the impairment of long-term depression observed in mGluR1 knockout mice is not a consequence of developmental abnormalities, but is directly due to mGluR1 gene inactivation.

Novel protective effect of mifepristone on detrimental GABAA receptor activity to immature Purkinje neurons

Immature Purkinje neurons are particularly vulnerable cells. They survive in cerebellar slice cultures under treatment by the synthetic steroid mifepristone (RU486) that depolarizes them at this age. The present study aims at understanding the mechanism underlying this neuroprotective effect. In the developing cerebellum, the role of γ‐aminobutyric acid (GABA) in neuron survival is unknown. In 3‐d‐old mouse cerebellar slice cultures, we show that GABAA receptor activation is depolarizing and excitatory. Antagonists of GABAA receptors rescue Purkinje neurons, demonstrating that GABA is endogenously released in this preparation and is toxic. Mifepristone likely protects these neurons by reversing GABAA receptor‐mediated chloride fluxes and reducing their driving force. Neuroprotection by mifepristone is dose‐dependently decreased by the agonist of GABAA receptors muscimol and by caffeine, an agonist of internal calcium store release. Moreover, the survival induced by neomycin, an inhibitor of calcium release, is partially reversed by muscimol. The p38 mitogen‐activated protein kinase (MAPK) inhibitor SB239063 also rescues Purkinje neurons. In summary, we propose that when GABA is depolarizing, mifepristone protects Purkinje neurons by shunting GABA responses and probably chloride fluxes, by inhibiting p38 MAPK activity and likely internal calcium store release. A new and nonhormonal effect of mifepristone is thus revealed.—Rakotomamonjy, J., Levenes, C., Baulieu, E. E., Schumacher, M., Ghoumari, A. M. Novel protective effect of mifepristone on detrimental GABAA receptor activity to immature Purkinje neurons. FASEB J. 25, 3999–4010 (2011). www.fasebj.org

Death and survival of heterozygous Lurcher Purkinje cells In vitro

The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the δ2 glutamate receptor (GluRδ2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRδ2+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRδ2+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRδ2+/+ and GluRδ2+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRδ2+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRδ2+/Lc Purkinje cell dendrites were stunted and the number of surviving GluRδ2+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1‐naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRδ2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRδ2+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival.

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.