Search

Purkinje cells (PCs) are spontaneously active neurons of the cerebellar cortex that inhibit glutamatergic projection neurons within the deep cerebellar nuclei (DCN) that provide the primary cerebellar output. Brief reductions of PC firing rapidly increase DCN neuron firing. However, prolonged reductions of PC inhibition, as seen in some disease states, certain types of transgenic mice, during optogenetic suppression of PC firing, and in acute slices of the cerebellum, do not lead to large, sustained increases in DCN firing. Here we test whether DCN neurons undergo spike frequency adaptation that could account for these properties. We perform current-clamp recordings at near physiological temperature in acute brain slices from mice of both sexes to examine how DCN neurons respond to prolonged depolarizations. DCN neuron adaptation is exceptionally slow and bidirectional. A depolarizing current step evokes large initial increases in firing that decay to ∼20% of the initial increase within ∼10 s. We find that spike frequency adaptation in DCN neurons is mediated by a novel mechanism that is independent of the most promising candidates, including calcium entry and Na(+)-activated potassium channels mediated by Slo2.1 and Slo2.2. Slow adaptation allows DCN neurons to gradually and bidirectionally adapt to prolonged currents but to respond linearly to current injection on rapid timescales. This suggests that an important consequence of slow adaptation is that DCN neurons respond linearly to the rate of PC firing on rapid timescales but adapt to slow firing rate changes of PCs on long timescales. SIGNIFICANCE STATEMENT Excitatory neurons in the cerebellar nuclei provide the primary output from the cerebellum. This study finds that these neurons exhibit very slow bidirectional spike frequency adaptation that has important implications for cerebellar function. This mechanism allows neurons in the cerebellar nuclei to adapt to long-lasting changes in synaptic drive while also remaining responsive to short-term changes in excitatory or inhibitory drive.

Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1. SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.

Golgi cells (GoCs) are inhibitory interneurons that influence the cerebellar cortical response to sensory input by regulating the excitability of the granule cell layer. While GoC inhibition is essential for normal motor coordination, little is known about the circuit dynamics that govern the activity of these cells. In particular, although GoC spontaneous spiking influences the extent of inhibition and gain throughout the granule cell layer, it is not known whether this spontaneous activity can be modulated in a long-term manner. Here we describe a form of long-term plasticity that regulates the spontaneous firing rate of GoCs in the rat cerebellar cortex. We find that membrane hyperpolarization, either by mGluR2 activation of potassium channels, or by somatic current injection, induces a long-lasting increase in GoC spontaneous firing. This spike rate plasticity appears to result from a strong reduction in the spike after hyperpolarization. Pharmacological manipulations suggest the involvement of calcium-calmodulin-dependent kinase II and calcium-activated potassium channels in mediating these firing rate increases. As a consequence of this plasticity, GoC spontaneous spiking is selectively enhanced, but the gain of evoked spiking is unaffected. Hence, this plasticity is well suited for selectively regulating the tonic output of GoCs rather than their sensory-evoked responses.

Within the dorsal lateral geniculate nucleus (dLGN) of the thalamus, retinal ganglion cell (RGC) projections excite thalamocortical (TC) cells that in turn relay visual information to the cortex. Local interneurons in the dLGN regulate the output of TC cells by releasing GABA from their axonal boutons and specialized dendritic spines. Here we examine the functional role of these highly specialized interneurons and how they inhibit TC cells in mouse brain slices. It was widely thought that activation of metabotropic glutamate receptor type 5 (mGluR5) on interneuron spines leads to local GABA release restricted to sites receiving active RGC inputs. We reexamined experiments that supported this view, and found that in the presence of TTX, mGluR5 agonists evoked GABA release that could instead be explained by interneuron depolarization and widespread intracellular calcium increases. We also examined GABA release evoked by RGC activation and found that high-frequency stimulation induces a long-lasting subthreshold afterdepolarization, persistent firing, or prolonged plateau potentials in interneurons and evokes sustained GABA release. mGluR5 antagonists virtually eliminated sustained spiking and the resulting widespread calcium-signals, and reduced inhibition by >50%. The remaining inhibition appeared to be mediated by a fraction of interneurons in which plateau potentials produced large and widespread calcium increases. Local calcium signals required for local GABA release were not observed. These findings indicate that, contrary to the previous view, RGC activation does not simply evoke localized GABA release by activating mGluR5, rather, synaptic activation of mGluR5 acts primarily by depolarizing interneurons and evoking widespread dendritic GABA release.

Depolarization of presynaptic terminals that arises from activation of presynaptic ionotropic receptors, or somatic depolarization, can enhance neurotransmitter release; however, the molecular mechanisms mediating this plasticity are not known. Here we investigate the mechanism of this enhancement at the calyx of Held synapse, in which presynaptic glycine receptors depolarize presynaptic terminals, elevate resting calcium levels, and potentiate release. Using knock-out mice of the calcium-sensitive PKC isoforms (PKCCa), we find that enhancement of evoked but not spontaneous synaptic transmission by glycine is mediated primarily by PKCCa. Measurements of calcium at the calyx of Held indicate that deficits in synaptic modulation in PKCCaknock-out mice occur downstream of presynaptic calcium increases. Glycine enhances synaptic transmission primarily by increasing the effective size of the pool of readily releasable vesicles. Our results reveal that PKCCacan enhance evoked neurotransmitter release in response to calcium increases caused by small presynaptic depolarizations.

R-type calcium channels in postsynaptic spines signal through functional calcium microdomains to regulate a calcium/calmodulin-sensitive potassium channel that in turn regulates postsynaptic hippocampal long-term potentiation (LTP). Here, we ask whether R-type calcium channels in presynaptic terminals also signal through calcium microdomains to control presynaptic LTP. We focus on presynaptic LTP at parallel fiber to Purkinje cell synapses in the cerebellum (PF-LTP), which is mediated by calcium/calmodulin-stimulated adenylyl cyclases. Although most presynaptic calcium influx is through N-type and P/Q-type calcium channels, blocking these channels does not disrupt PF-LTP, but blocking R-type calcium channels does. Moreover, global calcium signaling cannot account for the calcium dependence of PF-LTP because R-type channels contribute modestly to overall calcium entry. These findings indicate that, within presynaptic terminals, R-type calcium channels produce calcium microdomains that evoke presynaptic LTP at moderate frequencies that do not greatly increase global calcium levels.

Activity-dependent elevation of calcium within presynaptic boutons regulates many aspects of synaptic transmission. Here, we examine presynaptic residual calcium (Cares) transients in individual presynaptic boutons of cerebellar granule cells at near-physiological temperatures using two-photon microscopy. Properties of Caresunder conditions of zero-added buffer were determined by measuring Carestransients while loading boutons to a steady-state indicator concentration. These experiments revealed that, in the absence of exogenous calcium buffers, a single action potential evokes transients of Caresthat vary widely in different boutons both in amplitude (400–900 nm) and time course (25–55 ms). Variation in calcium influx density, endogenous buffer capacity, and calcium extrusion density contribute to differences in Caresamong boutons. Heterogeneity in Careswithin different boutons suggests that plasticity can be regulated independently at different synapses arising from an individual granule cell. In a given bouton, Caressignals were highly reproducible from trial to trial and failures of calcium influx were not observed. We find that a factor contributing to this reliability is that an action potential opens a large number of calcium channels (20–125) in a bouton. Presynaptic calcium signals were also used to assess the ability of granule cell axons to convey somatically generated action potentials to distant synapses. In response to pairs of action potentials or trains, granule cell boutons showed a remarkable ability to respond reliably at frequencies up to 500 Hz. Thus, individual boutons appear specialized for reliable calcium signaling during bursts of high-frequency activation such as those that are observedin vivo.

When the synaptic connection between two neurons consists of a small number of release sites, the ability to maintain transmission at high frequencies is limited by vesicle mobilization and by the response of postsynaptic receptors. These two properties were examined at single release sites between granule cells and stellate cells by triggering bursts of quantal events either with alpha-latrotoxin or with high-frequency trains of presynaptic activity. Bursts and evoked responses consisted of tens to hundreds of events with frequencies of up to hundreds per second. This indicates that single release sites can rapidly supply vesicles from a reserve pool to a release-ready pool. In addition, postsynaptic AMPA receptors recover from desensitization with a time constant of approximately 5 ms. Thus, even for synapses composed of a single release site, granule cells can effectively activate stellate cells during sustained high-frequency transmission because of rapid vesicle mobilization and fast recovery of AMPA receptors from desensitization.

Neurons release endocannabinoids from their dendrites to trigger changes in the probability of transmitter release. Although such retrograde signaling has been described for principal neurons, such as hippocampal pyramidal cells and cerebellar Purkinje cells (PCs), it has not been demonstrated for local interneurons. Here we tested whether inhibitory interneurons in the cerebellum, stellate cells (SCs) and basket cells, regulate the strength of parallel fiber (PF) synapses by releasing endocannabinoids. We found that depolarization-induced suppression of excitation (DSE) is present in both SCs and basket cells. The properties of retrograde inhibition were examined more thoroughly for SCs. Both DSE and synaptically evoked suppression of excitation (SSE) triggered with brief PF bursts require elevations of postsynaptic calcium, are blocked by a type 1 cannabinoid receptor (CB1R) antagonist, and are absent in mice lacking the CB1R. SSE for SCs is similar to that described previously for PCs in that it is prevented by BAPTA and DAG lipase inhibitors in the recording pipette; however, unlike in PCs, NMDA receptors (NMDARs) play an important role in SSE for SCs. Although SCs express CB1Rs postsynaptically, neither high-frequency firing of SCs nor PF bursts lead to autocrine suppression of subsequent SC activity. Instead, PF bursts decrease the amplitude of disynaptic inhibition in PCs by evoking endocannabinoid release that transiently reduces the ability of PF synapses to trigger spikes in SCs. Thus, local interneurons within the cerebellum can release endocannabinoids through metabotropic glutamate receptor- and NMDAR-dependent mechanisms and contribute to use-dependent modulation of circuit properties.

Changes in synaptic strength during ongoing activity are often mediated by neuromodulators. At the synapse between cerebellar granule cell parallel fibers (PFs) and Purkinje cells (PCs), brief bursts of stimuli can evoke endocannabinoid release from PCs and GABA release from interneurons that both inhibit transmission by activating presynaptic G-protein-coupled receptors. Studies in several brain regions suggest that synaptic activity can also evoke calcium signals in astrocytes, thereby causing them to release a transmitter, which acts presynaptically to regulate neurotransmitter release. In the cerebellum, Bergmann glia cells (BGs) are intimately associated with PF synapses. However, the mechanisms leading to calcium signals in BGs under physiological conditions and the role of BGs in regulating ongoing synaptic transmission are poorly understood. We found that brief bursts of PF activity evoke calcium signals in BGs that are triggered by the activation of metabotropic glutamate receptor 1 and purinergic receptors and mediated by calcium release from IP3-sensitive internal stores. We found no evidence for modulation of release from PFs mediated by BGs, even when endocannabinoid- and GABA-mediated presynaptic modulation was prominent. Thus, despite the fact that PF activation can reliably evoke calcium transients within BGs, it appears that BGs do not regulate synaptic transmission on the time scale of seconds to tens of seconds. Instead, endocannabinoid release from PCs and GABA release from molecular layer interneurons provide the primary means of feedback that dynamically regulate release from PF synapses.

At many central synapses, endocannabinoids released by postsynaptic cells inhibit neurotransmitter release by activating presynaptic cannabinoid receptors. The mechanisms underlying this important means of synaptic regulation are not fully understood. It has been shown at several synapses that endocannabinoids inhibit neurotransmitter release by reducing calcium influx into presynaptic terminals. One hypothesis maintains that endocannabinoids indirectly reduce calcium influx by modulating potassium channels and narrowing the presynaptic action potential. An alternative hypothesis is that endocannabinoids directly and selectively inhibit N-type calcium channels in presynaptic terminals. Here we test these hypotheses at the granule cell to Purkinje cell synapse in cerebellar brain slices. By monitoring optically the presynaptic calcium influx (Cainflux) and measuring the EPSC amplitudes, we found that cannabinoid-mediated inhibition arises solely from reduced presynaptic Cainflux. Next we found that cannabinoid receptor activation does not affect the time course of presynaptic calcium entry, indicating that the reduced Cainfluxreflects inhibition of presynaptic calcium channels. Finally, we assessed the classes of presynaptic calcium channels inhibited by cannabinoid receptor activation via peptide calcium channel antagonists. Previous studies established that N-type, P/Q-type, and R-type calcium channels are all present in granule cell presynaptic boutons. We found that cannabinoid activation reduced Cainfluxthrough N-type, P/Q-type, and R-type calcium channels to 29, 60, and 55% of control, respectively. Thus, rather than narrowing the presynaptic action potential or exclusively modulating N-type calcium channels, CB1 receptor activation inhibits synaptic transmission by modulating all classes of calcium channels present in the presynaptic terminal of the granule cell to Purkinje cell synapse.

Many types of neurons release endocannabinoids from their dendrites in response to elevation of intracellular calcium levels. Endocannabinoids then activate presynaptic cannabinoid receptors, thereby inhibiting neurotransmitter release for tens of seconds. A crucial step in understanding the physiological role of this retrograde signaling is to determine its sensitivity to elevations of postsynaptic calcium. Here we determine and compare the calcium dependence of endocannabinoid-mediated retrograde inhibition at three types of synapses onto cerebellar Purkinje cells. Previous studies have shown that Purkinje cell depolarization results in endocannabinoid-mediated retrograde inhibition of synapses received from climbing fibers, granule cell parallel fibers, and inhibitory interneurons. Using several calcium indicators with a range of affinities, we performed a series ofin situandin vitrocalibrations to quantify calcium levels in Purkinje cells. We found that postsynaptic calcium levels of ∼15 μM are required for half-maximal retrograde inhibition at all of these synapses. In contrast, previous studies had suggested that endocannabinoid release could occur with slight elevations of calcium above resting levels, which implies that inhibition should be widespread and continuously modulated by subtle changes in intracellular calcium levels. However, our results indicate that such small changes in intracellular calcium are not sufficient to evoke endocannabinoid release. Instead, because of its high requirement for calcium, retrograde inhibition mediated by calcium-dependent endocannabinoid release from Purkinje cells will occur under more restricted conditions and with greater spatial localization than previously appreciated.

Postsynaptic AMPA receptor desensitization leads to depression at some synapses. Here we examine whether desensitization occurs at mossy fiber to granule cell synapses and how synaptic architecture could contribute. We made whole-cell voltage-clamp recordings from granule cells in rat cerebellar slices at 34°C, and stimulated mossy fibers with paired pulses. The amplitude of the second EPSC was depressed by 60% at 10 msec and recovered with τ ∼30 msec. This fast component of recovery from depression was reduced by cyclothiazide and enhanced when release probability was increased, suggesting that it reflects postsynaptic receptor desensitization. We evaluated the importance of synaptic ultrastructure to spillover and desensitization by using serial electron microscopy to reconstruct mossy fiber glomeruli. We found that mossy fiber boutons had hundreds of release sites, that the average center-to-center distance between nearest release sites was 0.46 μm, and that these sites had an average of 7.1 neighbors within 1 μm. In addition, glia did not isolate release sites from each other. By contrast, desensitization plays no role in paired-pulse depression at the cerebellar climbing fiber, where glial ensheathment of synapses is nearly complete. This suggests that the architecture of the mossy fiber glomerulus can lead to desensitization and short-term depression. Modeling indicates that, as a consequence of the close spacing of release sites, glutamate released from a single site can desensitize AMPA receptors at neighboring sites, even when the probability of release (pr) is low. Whenpris high, desensitization would be accentuated by such factors as glutamate pooling.

Cholinergic innervation of the hippocampus has been implicated in memory formation and retrieval. Here we study cholinergic modulation of excitatory transmission in the CA3 area of the rat hippocampus. We used a combination of optical measurements of presynaptic calcium and electrophysiological measurements of synaptic currents to study associational–commissural (A/C) and mossy fiber (MF) synapses in brain slices. Direct synaptic modulation mediated by ACh receptors is only evident at the A/C synapse, where synaptic inhibition primarily reflects presynaptic calcium channel inhibition mediated by muscarinic receptors. MF synapses can, however, be indirectly modulated by muscarinic receptor activation. Muscarine elevates the firing rate of inhibitory cells, which increases GABA release and inhibits MF synapses by activating presynaptic GABABreceptors. Muscarine also depolarizes dentate granule cells and elevates their rate of firing. This leads to synaptic enhancement when combined with the use-dependent facilitation of MF synapses. In addition we were unable to evoke an increase in presynaptic calcium levels in MF boutons with local application of nicotinic receptor agonists. This finding does not support a leading hypothesis for MF modulation in which activation of presynaptic nicotinic receptors enhances transmission directly by elevating presynaptic calcium levels. However, indirect synaptic modulation could arise from nicotinic excitation of inhibitory neurons. Thus, to understand cholinergic modulation within the CA3 region, it is necessary to take into account secondary actions on synapses arising from other chemical messengers released by other cell types and to consider effects on firing patterns of presynaptic cells, which in turn influence release via use-dependent synaptic plasticity.

Activity-dependent processes dynamically regulate synapses on the time scale of milliseconds to seconds. Here, we examine the factors governing synaptic strength during repetitive stimulation, both in control conditions and during presynaptic inhibition. Field recordings of presynaptic volleys, optical measurements of presynaptic calcium, and voltage-clamp recordings of postsynaptic currents were used to examine parallel fiber to Purkinje cell synapses in cerebellar brain slices at 34°C. In control conditions, regular stimulus trains (1–50 Hz) evoked up to a 250% peak synaptic enhancement, whereas during irregular stimulation, a threefold variability in EPSC amplitude was observed. When initial EPSC amplitudes were reduced by 50%, either by lowering external calcium or by activating adenosine A1or GABABreceptors, the peak enhancement during regular trains was 500%, and synaptic variability during irregular trains was nearly sixfold. By contrast, changes in fiber excitability and calcium influx per pulse were small during trains. Presynaptic calcium measurements indicated that by pulse 10, stimulus-evoked calcium influx had increased by ∼15%, which on the basis of the measured relationship between calcium influx and release corresponds to an EPSC enhancement of 50%. This enhancement was the same in all experimental conditions, even in the presence ofN6-cyclopentyladenosine or baclofen, suggesting that repetitive stimulation does not relieve the G-protein inhibition of calcium channels by these modulators. Therefore, for our experimental conditions, changes in synaptic strength during trains are primarily attributable to residual calcium (Cares)-dependent short-term plasticities, and the actions of neuromodulators during repetitive stimulation result from their inhibition of initial calcium influx and the resulting effects on Caresand calcium-driven processes.

At fast chemical synapses the rapid release of neurotransmitter that occurs within a few milliseconds of an action potential is followed by a more sustained elevation of release probability, known as delayed release. Here we characterize the role of calcium in delayed release and test the hypothesis that facilitation and delayed release share a common mechanism. Synapses between cerebellar granule cells and their postsynaptic targets, stellate cells and Purkinje cells, were studied in rat brain slices. Presynaptic calcium transients were measured with calcium-sensitive fluorophores, and delayed release was detected with whole-cell recordings. Calcium influx, presynaptic calcium dynamics, and the number of stimulus pulses were altered to assess their effect on delayed release and facilitation. Following single stimuli, delayed release can be separated into two components: one lasting for tens of milliseconds that is steeply calcium-dependent, the other lasting for hundreds of milliseconds that is driven by low levels of calcium with a nearly linear calcium dependence. The amplitude, calcium dependence, and magnitude of delayed release do not correspond to those of facilitation, indicating that these processes are not simple reflections of a shared mechanism. The steep calcium dependence of delayed release, combined with the large calcium transients observed in these presynaptic terminals, suggests that for physiological conditions delayed release provides a way for cells to influence their postsynaptic targets long after their own action potential activity has subsided.

Short-term depression is a widespread form of use-dependent plasticity found in the peripheral and central nervous systems of invertebrates and vertebrates. The mechanism behind this transient decrease in synaptic strength is thought to be primarily the result of presynaptic "depletion" of a readily releasable neurotransmitter pool, which typically recovers with a time constant of a few seconds. We studied the mechanism and dynamics of recovery from depression at the climbing fiber to Purkinje cell synapse, where marked presynaptic depression has been described previously. Climbing fibers are well suited to studies of recovery from depression because they display little, if any, facilitation (even under conditions of low-release probability), which can obscure rapid recovery from depression for hundreds of milliseconds after release. We found that recovery from depression occurred in three kinetic phases. The fast and intermediate components could be approximated by exponentials with time constants of 100 msec and 3 sec at 24 degrees C. A much slower recovery phase was also present, but it was only prominent during prolonged stimulus trains. The fast component was enhanced by raising extracellular calcium and was eliminated by lowering presynaptic calcium, suggesting that, on short time scales, recovery from depression is driven by residual calcium. During regular and Poisson stimulus trains, recovery from depression was dramatically accelerated by accumulation of presynaptic residual calcium, maintaining synaptic efficacy under conditions that would otherwise deplete the available transmitter pool. This represents a novel form of presynaptic plasticity in that high levels of activity modulate the rate of recovery as well as the magnitude of depression.

The effect of changes in the shape of the presynaptic action potential on neurotransmission was examined at synapses between granule and Purkinje cells in slices from the rat cerebellum. Low concentrations of tetraethylammonium were used to broaden the presynaptic action potential. The presynaptic waveform was monitored with voltage-sensitive dyes, the time course and amplitude of presynaptic calcium entry were determined with fluorescent calcium indicators, and EPSCs were measured with a whole-cell voltage clamp. Spike broadening increased calcium influx primarily by prolonging calcium entry without greatly affecting peak presynaptic calcium currents, indicating that the majority of calcium channels reach maximal probability of opening in response to a single action potential and that spike broadening increases the open time of these channels. EPSCs were exquisitely sensitive to elevations of calcium influx produced by spike broadening; there was a high power relationship between calcium influx and release such that a 23% increase in spike width led to a 25% increase in total calcium influx, which in turn doubled synaptic strength.The finding that even small changes in spike width influence neurotransmitter release suggests that altering the presynaptic waveform may be an important means of modifying the strength of this synapse. Waveform changes do not, however, contribute significantly to presynaptic modulation via activation of adenosine A1or GABABreceptors. Furthermore, greatly reducing presynaptic calcium influx did not alter the presynaptic waveform, indicating that calcium channels and calcium-activated channels do not participate in shaping the presynaptic waveform.

Augmentation and posttetanic potentiation--two forms of short-term synaptic enhancement produced by repetitive presynaptic action potentials--are dependent on the buildup and decay of nerve terminal residual calcium that occurs on the seconds to minutes time scale. With the goal of providing a quantitative understanding of these kinetics, we measured the buildup and decay of calcium ions in nerve terminals at the crayfish neuromuscular junction under a variety of intracellular buffer conditions and stimulation paradigms. The calcium extrusion process in the terminals was characterized by analysis of calcium levels reached during long stimulus trains as a function of action potential frequency. The extrusion was linearly dependent on the free calcium ion concentration. Using this result, we developed a mathematical model and computer simulation of the residual calcium kinetics. The model demonstrates the experimentally observed dependence of decay rate on exogenous calcium buffer concentration, and can be explicitly solved to provide an expression for the limiting exponential time course of calcium decay following trains in terms of calcium buffer and extrusion characteristics. Methods to determine the calcium influx per action potential, characteristics of endogenous buffer, and the rate of calcium extrusion are suggested by our analysis and demonstrated experimentally.

Depolarization of cerebellar Purkinje neurons transiently suppresses IPSCs through a process known as depolarization-induced suppression of inhibition (DSI). This IPSC suppression occurs presynaptically and results from an unknown retrograde signal released from Purkinje cells. We recorded IPSCs from voltage-clamped Purkinje cells in cerebellar brain slices to identify the retrograde signal for cerebellar DSI. We find that DSI persists in the presence of the broad-spectrum metabotropic glutamate receptor antagonist LY341495 and the GABA(B) receptor antagonist CGP55845, suggesting that the retrograde signal is not acting through these receptors. However, an antagonist of the cannabinoid CB1 receptor AM251 completely blocked cerebellar DSI. Additionally, the cannabinoid receptor agonist WIN55,212-2 suppressed IPSCs and occluded any additional IPSC reduction by DSI. These results indicate that cannabinoids released from Purkinje cells after depolarization activate CB1 receptors on inhibitory neurons and suppress IPSCs for tens of seconds. Cerebellar DSI thus shares a common retrograde messenger with DSI in the hippocampus and depolarization-induced suppression of excitation in the cerebellum, suggesting that retrograde synaptic suppression by endogenous cannabinoids represents a widespread signaling mechanism.