Wade G. Regehr

Retrograde Inhibition of Presynaptic Calcium Influx by Endogenous Cannabinoids at Excitatory Synapses onto Purkinje Cells

Brief depolarization of cerebellar Purkinje cells was found to inhibit parallel fiber and climbing fiber EPSCs for tens of seconds. This depolarization-induced suppression of excitation (DSE) is accompanied by altered paired-pulse plasticity, suggesting a presynaptic locus. Fluorometric imaging revealed that postsynaptic depolarization also reduces presynaptic calcium influx. The inhibition of both presynaptic calcium influx and EPSCs is eliminated by buffering postsynaptic calcium with BAPTA. The cannabinoid CB1 receptor antagonist AM251 prevents DSE, and the agonist WIN 55,212-2 occludes DSE. These findings suggest that Purkinje cells release endogenous cannabinoids in response to elevated calcium, thereby inhibiting presynaptic calcium entry and suppressing transmitter release. DSE may provide a way for cells to use their firing rate to dynamically regulate synaptic inputs. Together with previous studies, these findings suggest a widespread role for endogenous cannabinoids in retrograde synaptic inhibition.

Calcium control of transmitter release at a cerebellar synapse

The manner in which presynaptic Ca2+ influx controls the release of neurotransmitter was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory postsynaptic currents were measured using whole-cell voltage clamp, and changes in presynaptic Ca2+ influx were determined with the Ca(2+)-sensitive dye furaptra. We manipulated presynaptic Ca2+ entry by altering external Ca2+ levels and by blocking Ca2+ channels with Cd2+ or with the toxins omega-conotoxin GVIA and omega-Aga-IVA. For all of the manipulations, other than the application of omega-Aga-IVA, the relationship between Ca2+ influx and release was well approximated by a power law, n approximately 2.5. When omega-Aga-IVA was applied, release appeared to be more steeply dependent on Ca2+ (n approximately 4), suggesting that omega-Aga-IVA-sensitive channels are more effective at triggering release. Based on interactive effects of toxins on synaptic currents, we conclude that multiple types of Ca2+ channels synergistically control individual release sites.

Interplay between Facilitation, Depression, and Residual Calcium at Three Presynaptic Terminals

Synapses display remarkable alterations in strength during repetitive use. Different types of synapses exhibit distinctive synaptic plasticity, but the factors giving rise to such diversity are not fully understood. To provide the experimental basis for a general model of short-term plasticity, we studied three synapses in rat brain slices at 34°C: the climbing fiber to Purkinje cell synapse, the parallel fiber to Purkinje cell synapse, and the Schaffer collateral to CA1 pyramidal cell synapse. These synapses exhibited a broad range of responses to regular and Poisson stimulus trains. Depression dominated at the climbing fiber synapse, facilitation was prominent at the parallel fiber synapse, and both depression and facilitation were apparent in the Schaffer collateral synapse. These synapses were modeled by incorporating mechanisms of short-term plasticity that are known to be driven by residual presynaptic calcium (Cares). In our model, release is the product of two factors: facilitation and refractory depression. Facilitation is caused by a calcium-dependent increase in the probability of release. Refractory depression is a consequence of release sites becoming transiently ineffective after release. These sites recover with a time course that is accelerated by elevations of Cares. Facilitation and refractory depression are coupled by their common dependence on Cares and because increased transmitter release leads to greater synaptic depression. This model captures the behavior of three different synapses for various stimulus conditions. The interplay of facilitation and depression dictates synaptic strength and variability during repetitive activation. The resulting synaptic plasticity transforms the timing of presynaptic spikes into varying postsynaptic response amplitudes.

Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Developmental Remodeling of the Retinogeniculate Synapse

Anatomical rearrangement of retinogeniculate connections contributes to the refinement of synaptic circuits in the developing visual system, but the underlying changes in synaptic function are unclear. Here, we study such changes in mouse brain slices. Each geniculate cell receives a surprisingly large number of retinal inputs (>20) well after eye-specific zones are formed. All but one to three of these inputs are eliminated over a 3-week period spanning eye opening. Remaining inputs are strengthened approximately 50-fold, in part through an increase in quantal size, but primarily through an increase in the number of release sites. Changes in release probability do not contribute significantly. Thus, a redistribution of release sites from many inputs to few inputs at this late developmental stage contributes to the precise receptive fields of thalamic relay neurons.

Endocannabinoids Control the Induction of Cerebellar LTD

The long-term depression (LTD) of parallel fiber (PF) synapses onto Purkinje cells plays a central role in motor learning. Endocannabinoid release and LTD induction both depend upon activation of the metabotropic glutamate receptor mGluR1, require postsynaptic calcium increases, are synapse specific, and have a similar dependence on the associative activation of PF and climbing fiber synapses. These similarities suggest that endocannabinoid release could account for many features of cerebellar LTD. Here we show that LTD induction is blocked by a cannabinoid receptor (CB1R) antagonist, by inhibiting the synthesis of the endocannabinoid 2-arachidonyl glycerol (2-AG), and is absent in mice lacking the CB1R. Although CB1Rs are prominently expressed presynaptically at PF synapses, LTD is expressed postsynaptically. In contrast, a previously described transient form of inhibition mediated by endocannabinoids is expressed presynaptically. This indicates that Purkinje cells release 2-AG that activates CB1Rs to both transiently inhibit release and induce a postsynaptic form of LTD.

Brief presynaptic bursts evoke synapse-specific retrograde inhibition mediated by endogenous cannabinoids

Many types of neurons can release endocannabinoids that act as retrograde signals to inhibit neurotransmitter release from presynaptic terminals. Little is known, however, about the properties or role of such inhibition under physiological conditions. Here we report that brief bursts of presynaptic activity evoked endocannabinoid release, which strongly inhibited parallel fiber–to–Purkinje cell synapses in rat cerebellar slices. This retrograde inhibition was triggered by activation of either postsynaptic metabotropic or ionotropic glutamate receptors and was restricted to synapses activated with high-frequency bursts. Thus, endocannabinoids allow neurons to inhibit specific synaptic inputs in response to a burst, thereby dynamically fine-tuning the properties of synaptic integration.

Participation of multiple calcium channel types in transmission at single climbing fiber to Purkinje cell synapses

The sensitivity of synaptic transmission to antagonists of different calcium channels was examined at the powerful climbing fiber synapse between neurons from the inferior olive and cerebellar Purkinje cells. In rat brain slices, climbing fibers were activated with extracellular electrodes, and synaptic currents were recorded with whole-cell patch clamp. Dihydropyridines did not discernibly affect synaptic strength. omega-Conotoxin GVIA, a potent antagonist of N-type calcium channels, reduced synaptic currents by an average of 29%. omega-Agatoxin-IVA, a high affinity blocker of P-type calcium channels, reduced synaptic strength by an average of 77%. Together, the two toxins virtually eliminated synaptic transmission (91% inhibition). These results indicate that omega-agatoxin-IVA-sensitive calcium channels play an important role in transmission at the climbing fiber synapse. They also suggest that in single climbing fibers, release is evoked by at least two pharmacologically distinct calcium currents, one sensitive to omega-agatoxin-IVA, the other to omega-conotoxin GVIA.

Short-term forms of presynaptic plasticity

Synapses exhibit several forms of short-term plasticity that play a multitude of computational roles. Short-term depression suppresses neurotransmitter release for hundreds of milliseconds to tens of seconds; facilitation and post-tetanic potentiation lead to synaptic enhancement lasting hundreds of milliseconds to minutes. Recent advances have provided insight into the mechanisms underlying these forms of plasticity. Vesicle depletion, as well as inactivation of both release sites and calcium channels, contribute to synaptic depression. Mechanisms of short-term enhancement include calcium channel facilitation, local depletion of calcium buffers, increases in the probability of release downstream of calcium influx, altered vesicle pool properties, and increases in quantal size. Moreover, there is a growing appreciation of the heterogeneity of vesicles and release sites and how they can contribute to use-dependent plasticity.

Quantal events shape cerebellar interneuron firing.

Many small synaptic inputs or one large input are needed to influence principal cell firing, whereas individual quanta exert little influence. However, the role of a quantum may be greater for small interneurons with high input resistances. Using dynamic clamp recordings, we found that individual quanta strongly influence rat cerebellar stellate cell firing. When the frequency of synaptic inputs was low, the timing of recent spikes regulated the influence of excitatory quanta. In contrast, when input frequency was high, spike timing was less important than interactions with other inputs. Inhibitory quanta rapidly terminated firing, whereas small numbers of coincident excitatory quanta reliably and rapidly triggered firing. Our results suggest that stellate cells achieve temporal precision through coincidence detection and disynaptic inhibition, despite their high resistances and long membrane time constants. Thus, we propose that small interneurons can process synaptic inputs in a fundamentally different way from principal cells.