Endocannabinoids Differentially Modulate Synaptic Plasticity in Rat Hippocampal CA1 Pyramidal Neurons

Background

Hippocampal CA1 pyramidal neurons receive two excitatory glutamatergic synaptic inputs: their most distal dendritic regions in the stratum lacunosum-moleculare (SLM) are innervated by the perforant path (PP), originating from layer III of the entorhinal cortex, while their more proximal regions of the apical dendrites in the stratum radiatum (SR) are innervated by the Schaffer-collaterals (SC), originating from hippocampal CA3 neurons. Endocannabinoids (eCBs) are naturally occurring mediators capable of modulating both GABAergic and glutamatergic synaptic transmission and plasticity via the CB1 receptor. Previous work on eCB modulation of excitatory synapses in the CA1 region largely focuses on the SC pathway. However, little information is available on whether and how eCBs modulate glutamatergic synaptic transmission and plasticity at PP synapses.

Methodology/Principal Findings

By employing somatic and dendritic patch-clamp recordings, Ca²⁺ uncaging, and immunostaining, we demonstrate that there are significant differences in low-frequency stimulation (LFS)- or DHPG-, an agonist of group I metabotropic glutamate receptors (mGluRs), induced long-term depression (LTD) of excitatory synaptic transmission between SC and PP synapses in the same pyramidal neurons. These differences are eliminated by pharmacological inhibition with selective CB1 receptor antagonists or genetic deletion of the CB1 receptor, indicating that these differences likely result from differential modulation via a CB1 receptor-dependent mechanism. We also revealed that depolarization-induced suppression of excitation (DSE), a form of short-term synaptic plasticity, and photolysis of caged Ca²⁺-induced suppression of Excitatory postsynaptic currents (EPSCs) were less at the PP than that at the SC. In addition, application of WIN55212 (WIN) induced a more pronounced inhibition of EPSCs at the SC when compared to that at the PP.

Conclusions/Significance

Our results suggest that CB1 dependent LTD and DSE are differentially expressed at the PP versus SC synapses in the same neurons, which may have an impact on synaptic scaling, integration and plasticity of hippocampal CA1 pyramidal neurons.     

Endocannabinoid-mediated long-term plasticity requires cAMP/PKA signaling and RIM1alpha

Endocannabinoids (eCBs) have emerged as key activity-dependent signals that, by activating presynaptic cannabinoid receptors (i.e., CB1) coupled to G(i/o) protein, can mediate short-term and long-term synaptic depression (LTD). While the presynaptic mechanisms underlying eCB-dependent short-term depression have been identified, the molecular events linking CB1 receptors to LTD are unknown. Here we show in the hippocampus that long-term, but not short-term, eCB-dependent depression of inhibitory transmission requires presynaptic cAMP/PKA signaling. We further identify the active zone protein RIM1alpha as a key mediator of both CB1 receptor effects on the release machinery and eCB-dependent LTD in the hippocampus. Moreover, we show that eCB-dependent LTD in the amygdala and hippocampus shares major mechanistic features. These findings reveal the signaling pathway by which CB1 receptors mediate long-term effects of eCBs in two crucial brain structures. Furthermore, our results highlight a conserved mechanism of presynaptic plasticity in the brain.     

The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptor-stimulated cannabinoid CB1 receptor activity

The cannabinoid CB1 receptor (CB1R) is a G protein-coupled receptor, which couples to the Gi/o family of heterotrimeric G proteins. The receptor displays both basal and agonist-induced signaling and internalization. Although basal activity of CB1Rs is attributed to constitutive (agonist-independent) receptor activity, studies in neurons suggested a role of postsynaptic endocannabinoid (eCB) release in the persistent activity of presynaptic CB1Rs. To elucidate the role of eCBs in basal CB1R activity, we have investigated the role of diacylglycerol lipase (DAGL) in this process in Chinese hamster ovary (CHO) cells, which are not targeted specifically with eCBs. Agonist-induced G protein activation was determined by detecting dissociation G protein subunits expressed in CHO cells with bioluminescence resonance energy transfer (BRET), after labeling the alpha and beta subunits with Renilla luciferase and enhanced yellow fluorescent protein (EYFP), respectively. Preincubation of the cells with tetrahydrolipstatin (THL), a known inhibitor of DAGLs, caused inhibition of the basal activity of CB1R. Moreover, preincubation of CHO and cultured hippocampal neurons with THL increased the number of CB1Rs on the cell membrane, which reflects its inhibitory action on CB1R internalization in non-simulated cells. In CHO cells co-expressing CB1R and angiotensin AT1 receptors, angiotensin II-induced Go protein activation that was blocked by both a CB1R antagonist and THL. These data indicate that cell-derived eCB mediators have a general role in the basal activity of CB1Rs in non-neural cells and neurons, and that this mechanism can be stimulated by AT1 receptor activation.     

Medial prefrontal cortex endocannabinoid system modulates baroreflex activity through CB(1) receptors

Neural reflex mechanisms, such as the baroreflex, are involved in the regulation of cardiovascular system activity. Previous results from our group (Resstel LB, Correa FM. Medial prefrontal cortex NMDA receptors and nitric oxide modulate the parasympathetic component of the baroreflex. Eur J Neurosci 23: 481-488, 2006) have shown that glutamatergic synapses in the ventral portion of the medial prefrontal cortex (vMPFC) modulate baroreflex activity. Moreover, glutamatergic neurotransmission in the vMPFC can be modulated by the endocannabinoids system (eCBs), particularly the endocannabinoid anandamide, through presynaptic CB(1) receptor activation. Therefore, in the present study, we investigated eCBs receptors that are present in the vMPFC, and more specifically whether CB(1) receptors modulate baroreflex activity. We found that bilateral microinjection of the CB(1) receptor antagonist AM251 (100 or 300 pmol/200 nl) into the vMPFC increased baroreflex activity in unanesthetized rats. Moreover, bilateral microinjection of either the anandamide transporter inhibitor AM404 (100 pmol/200 nl) or the inhibitor of the enzyme fatty acid amide hydrolase that degrades anandamide, URB597 (100 pmol/200 nl), into the MPFC decreased baroreflex activity. Finally, pretreatment of the vMPFC with an ineffective dose of AM251 (10 pmol/200 nl) was able to block baroreflex effects of both AM404 and URB597. Taken together, our results support the view that the eCBs in the vMPFC is involved in the modulation of baroreflex activity through the activation of CB(1) receptors, which modulate local glutamate release.     

Subcellular compartment-specific molecular diversity of pre- and post-synaptic GABAB-activated GIRK channels in Purkinje cells

Activation of G protein-gated inwardly-rectifying K⁺ (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABA_B) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABA_B receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABA_B receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post-synaptic association of GIRK subunits with GABA_B receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABA_B receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABA_B receptors. The association of GIRK channels and GABA_B receptors with excitatory synapses at both post- and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.     

Functional diversity on synaptic plasticity mediated by endocannabinoids

Endocannabinoids (eCBs) act as modulators of synaptic transmission through activation of a number of receptors, including, but not limited to, cannabinoid receptor 1 (CB1). eCBs share CB1 receptors as a common target with Δ⁹-tetrahydrocannabinol (THC), the main psychoactive ingredient in marijuana. Although THC has been used for recreational and medicinal purposes for thousands of years, little was known about its effects at the cellular level or on neuronal circuits. Identification of CB1 receptors and the subsequent development of its specific ligands has therefore enhanced our ability to study and bring together a substantial amount of knowledge regarding how marijuana and eCBs modify interneuronal communication. To date, the eCB system, composed of cannabinoid receptors, ligands and the relevant enzymes, is recognized as the best-described retrograde signalling system in the brain. Its impact on synaptic transmission is widespread and more diverse than initially thought. The aim of this review is to succinctly present the most common forms of eCB-mediated modulation of synaptic transmission, while also illustrating the multiplicity of effects resulting from specializations of this signalling system at the circuital level.     

Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18‐1

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal‐regulated kinase (ERK) that mediates CB1R‐ and mGluR2/3‐induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock‐induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18‐1. Mimicking constitutive phosphorylation of Munc18‐1 results in a drastic decrease in synaptic transmission. ERK‐mediated phosphorylation of Munc18‐1 ultimately leads to degradation by the ubiquitin–proteasome system. Conversely, preventing ERK‐dependent Munc18‐1 phosphorylation increases synaptic strength. CB1R‐ and mGluR2/3‐induced synaptic inhibition and depolarization‐induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK‐dependent Munc18‐1 phosphorylation is blocked. Thus, ERK‐dependent Munc18‐1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.     

Divisive Gain Modulation with Dynamic Stimuli in Integrate-and-Fire Neurons

The modulation of the sensitivity, or gain, of neural responses to input is an important component of neural computation. It has been shown that divisive gain modulation of neural responses can result from a stochastic shunting from balanced (mixed excitation and inhibition) background activity. This gain control scheme was developed and explored with static inputs, where the membrane and spike train statistics were stationary in time. However, input statistics, such as the firing rates of pre-synaptic neurons, are often dynamic, varying on timescales comparable to typical membrane time constants. Using a population density approach for integrate-and-fire neurons with dynamic and temporally rich inputs, we find that the same fluctuation-induced divisive gain modulation is operative for dynamic inputs driving nonequilibrium responses. Moreover, the degree of divisive scaling of the dynamic response is quantitatively the same as the steady-state responses—thus, gain modulation via balanced conductance fluctuations generalizes in a straight-forward way to a dynamic setting.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

Anandamide content and interaction of endocannabinoid/GABA modulatory effects in the NTS on baroreflex-evoked sympathoinhibition

Cannabinoids have been shown to modulate central autonomic regulation and baroreflex control of blood pressure (BP). The presence of cannabinoid CB(1) receptors on fibers in the nucleus tractus solitarius (NTS) suggests that some presynaptic modulation of transmitter release could occur in this region, which receives direct afferent projections from arterial baroreceptors and cardiac mechanoreceptors. This study, therefore, was performed to determine the mechanism(s) of effects of microinjection of an endocannabinoid, arachidonylethanolamide (anandamide, AEA), into the NTS on baroreflex sympathetic nerve responses produced by phenylephrine-induced pressure changes in anesthetized rats. AEA prolonged reflex inhibition of renal sympathetic nerve activity (RSNA), suggesting an increase in baroreflex sensitivity. This effect of AEA was blocked by prior microinjection of SR-141716 to block cannabinoid CB(1) receptors. To determine whether this baroreflex enhancement by AEA involved a GABA(A) mechanism, the baroreflex response to AEA was tested after prior blockade of postsynaptic GABA(A) receptors by bicuculline, which would eliminate any effects due to modulation of GABA activity. After bicuculline, which alone prolonged the baroreflex inhibition of RSNA, AEA shortened the duration of RSNA inhibition, suggesting a possible presynaptic inhibition of glutamate release previously obscured by a more dominant GABA(A) effect. To support a possible physiological role for AEA, AEA concentration in the NTS was measured after a phenylephrine-induced increase in BP. AEA content in the NTS was increased significantly over that in normotensive animals. These results support the hypothesis that AEA content is increased by brief periods of hypertension and suggest that AEA can modulate the baroreflex through activation of CB(1) receptors within the NTS, possibly modulating effectiveness of GABA and/or glutamate neurotransmission.     

Presynaptic specificity of endocannabinoid signaling in the hippocampus

Endocannabinoids are retrograde messengers released by neurons to modulate the strength of their synaptic inputs. Endocannabinoids are thought to mediate the suppression of GABA release that follows depolarization of a hippocampal CA1 pyramidal neuron-termed "depolarization-induced suppression of inhibition" (DSI). Here, we report that DSI is absent in mice which lack cannabinoid receptor-1 (CB1). Pharmacological and kinetic evidence suggests that CB1 activation inhibits presynaptic Ca2+ channels through direct G protein inhibition. Paired recordings show that endocannabinoids selectively inhibit a subclass of synapses distinguished by their fast kinetics and large unitary conductance. Furthermore, cannabinoid-sensitive inputs are unusual among central nervous system synapses in that they use N- but not P/Q-type Ca2+ channels for neurotransmitter release. These results indicate that endocannabinoids are highly selective, rapid modulators of hippocampal inhibition.     

Agonist selective modulation of tyrosine hydroxylase expression by cannabinoid ligands in a murine neuroblastoma cell line

Functional interactions between catecholamines and cannabinoid transmission systems could explain the influence of Delta(9)-tetrahydrocannabinol on several central activities. Hence, the presence of cannabinoid CB(1) receptors in tyrosine hydroxylase (TH) containing cells has been suggested, providing clue for a direct control of catecholamines synthesis. In the present study, we evidenced the constitutive expression of functional cannabinoid CB(1) receptors in N1E-115 neuroblastoma and reported on the use of this model to examine the influence of diverse cannabinoid ligands on TH expression. Exposure of the cells to the high-affinity agonist HU 210 (5 h) resulted in a significant decrease in TH content (pEC(50): 6.40). In contrast, no change was observed after a similar treatment with the structurally unrelated agonist CP 55,940. Besides, the use of a luciferase reporter assay revealed that these two agonists showed opposite influences on TH gene promoter activity. Thus, in cells expressing pTH-luc constructs, inhibition and induction of luciferase activity were respectively observed with HU 210 (pEC(50): 8.95) and CP 55,940 (pEC(50): 9.09). Pharmacological characterisation revealed that these reciprocal responses were both related to the specific activation of cannabinoid CB(1) receptor, suggesting an agonist-dependent modulation of distinct signalling pathways. While these data points out the possible pharmacological manipulation of TH expression by cannabinoid ligands, such approach should take into account the existence of agonist selective trafficking of cannabinoid CB(1) receptor signalling.     

Inhibition by prostaglandin E2 of neurotransmission in rabbit but not human bronchus--a calcium-related mechanism?

Prostaglandins have been implicated in the development of airway hyperresponsiveness, and this may be mediated via modulation of neurotransmission. We compared the effects of prostaglandin E2 on the contractile response to electrical field stimulation in rabbit and human bronchus. Prostaglandin E2 produced marked inhibition in rabbit bronchus (mean % inhibition 35 +/- 17, P less than 0.05) but was without effect in human bronchus. The inhibition in rabbit bronchus was not the result of a direct effect on muscle tone and the site of action is likely to be pre-synaptic since prostaglandin E2 had only minor effects on exogenous acetylcholine. Since prostaglandins are known to affect calcium mobilization, we compared the dependence of cholinergic stimulation on the calcium voltage dependent channel (VDC) in the two species. Cholinergic stimulation was dependent on the VDC in rabbit but not human bronchus and this may be an explanation for the different effects of prostaglandin E2 in the two species.     

Cannabinoid-induced cell death in endometrial cancer cells: involvement of TRPV1 receptors in apoptosis

Among a variety of phytocannabinoids, Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most promising therapeutic compounds. Besides the well-known palliative effects in cancer patients, cannabinoids have been shown to inhibit in vitro growth of tumor cells. Likewise, the major endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), induce tumor cell death. The purpose of the present study was to characterize cannabinoid elements and evaluate the effect of cannabinoids in endometrial cancer cell viability. The presence of cannabinoid receptors, transient receptor potential vanilloid 1 (TRPV1), and endocannabinoid-metabolizing enzymes were determined by qRT-PCR and Western blot. We also examined the effects and the underlying mechanisms induced by eCBs and phytocannabinoids in endometrial cancer cell viability. Besides TRPV1, both EC cell lines express all the constituents of the endocannabinoid system. We observed that at concentrations higher than 5 μM, eCBs and CBD induced a significant reduction in cell viability in both Ishikawa and Hec50co cells, whereas THC did not cause any effect. In Ishikawa cells, contrary to Hec50co, treatment with AEA and CBD resulted in an increase in the levels of activated caspase ?3/?7, in cleaved PARP, and in reactive oxygen species generation, confirming that the reduction in cell viability observed in the MTT assay was caused by the activation of the apoptotic pathway. Finally, these effects were dependent on TRPV1 activation and intracellular calcium levels. These data indicate that cannabinoids modulate endometrial cancer cell death. Selective targeting of TPRV1 by AEA, CBD, or other stable analogues may be an attractive research area for the treatment of estrogen-dependent endometrial carcinoma. Our data further support the evaluation of CBD and CBD-rich extracts for the potential treatment of endometrial cancer, particularly, that has become non-responsive to common therapies.     

Cannabinoid modulation of sensory neurotransmission via cannabinoid and vanilloid receptors: roles in regulation of cardiovascular function

Capsaicin-sensitive sensory nerves are widely distributed in the cardiovascular system. They are activated by a variety of physical and chemical stimuli, characteristically by capsaicin acting via the vanilloid receptor VR1, and have a role in the regulation of peripheral vascular resistance and maintenance of homeostasis via their afferent and efferent functions. Cannabinoids, a recently discovered family of extracellular signalling molecules, can act at cannabinoid (CB) receptors expressed on sensory nerves, to cause inhibition of sensory neurotransmitter release. There is recent evidence, however, that anandamide, an endogenous cannabinoid, can activate VR1, coexpressed with CB receptors on the same sensory nerve terminals, causing a release of sensory neurotransmitter, vasorelaxation and hypotension. Hence, anandamide can elicit opposite actions, inhibition via CB receptors and excitation via VR1, on sensory neurotransmission. The possible biological significance of this is discussed.     

Calcium and synaptic dynamics underlying reverberatory activity in neuronal networks

Persistent activity is postulated to drive neural network plasticity and learning. To investigate its underlying cellular mechanisms, we developed a biophysically tractable model that explains the emergence, sustenance and eventual termination of short-term persistent activity. Using the model, we reproduced the features of reverberating activity that were observed in small (50-100 cells) networks of cultured hippocampal neurons, such as the appearance of polysynaptic current clusters, the typical inter-cluster intervals, the typical duration of reverberation, and the response to changes in extra-cellular ionic composition. The model relies on action potential-triggered residual pre-synaptic calcium, which we suggest plays an important role in sustaining reverberations. We show that reverberatory activity is maintained by enhanced asynchronous transmitter release from pre-synaptic terminals, which in itself depends on the dynamics of residual pre-synaptic calcium. Hence, asynchronous release, rather than being a 'synaptic noise', can play an important role in network dynamics. Additionally, we found that a fast timescale synaptic depression is responsible for oscillatory network activation during reverberations, whereas the onset of a slow timescale depression leads to the termination of reverberation. The simplicity of our model enabled a number of predictions that were confirmed by additional analyses of experimental manipulations.     

Modulation of the dynamics of cerebellar Purkinje cells through the interaction of excitatory and inhibitory feedforward pathways

The dynamics of cerebellar neuronal networks is controlled by the underlying building blocks of neurons and synapses between them. For which, the computation of Purkinje cells (PCs), the only output cells of the cerebellar cortex, is implemented through various types of neural pathways interactively routing excitation and inhibition converged to PCs. Such tuning of excitation and inhibition, coming from the gating of specific pathways as well as short-term plasticity (STP) of the synapses, plays a dominant role in controlling the PC dynamics in terms of firing rate and spike timing. PCs receive cascade feedforward inputs from two major neural pathways: the first one is the feedforward excitatory pathway from granule cells (GCs) to PCs; the second one is the feedforward inhibition pathway from GCs, via molecular layer interneurons (MLIs), to PCs. The GC-PC pathway, together with short-term dynamics of excitatory synapses, has been a focus over past decades, whereas recent experimental evidence shows that MLIs also greatly contribute to controlling PC activity. Therefore, it is expected that the diversity of excitation gated by STP of GC-PC synapses, modulated by strong inhibition from MLI-PC synapses, can promote the computation performed by PCs. However, it remains unclear how these two neural pathways are interacted to modulate PC dynamics. Here using a computational model of PC network installed with these two neural pathways, we addressed this question to investigate the change of PC firing dynamics at the level of single cell and network. We show that the nonlinear characteristics of excitatory STP dynamics can significantly modulate PC spiking dynamics mediated by inhibition. The changes in PC firing rate, firing phase, and temporal spike pattern, are strongly modulated by these two factors in different ways. MLIs mainly contribute to variable delays in the postsynaptic action potentials of PCs while modulated by excitation STP. Notably, the diversity of synchronization and pause response in the PC network is governed not only by the balance of excitation and inhibition, but also by the synaptic STP, depending on input burst patterns. Especially, the pause response shown in the PC network can only emerge with the interaction of both pathways. Together with other recent findings, our results show that the interaction of feedforward pathways of excitation and inhibition, incorporated with synaptic short-term dynamics, can dramatically regulate the PC activities that consequently change the network dynamics of the cerebellar circuit.     

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.     

Depression of excitatory transmission at PF-PC synapse by group III metabotropic glutamate receptors is provided exclusively by mGluR4 in the rodent cerebellar cortex

In the rodent cerebellum, pharmacological activation of group III pre-synaptic metabotropic glutamate receptors (mGluRs) by the broad spectrum agonist l -2-amino-4-phosphonobutyric acid, acutely depresses excitatory synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses. Among the group III mGluR subtypes, cerebellar granule cells express predominantly mGluR4, but also mGluR7 and mGluR8 mRNA. Taking into account that previous functional and pharmacological studies have used group III mGluR broad spectrum agonists that do not differentiate between these various subtypes, their relative contribution to the modulation of glutamatergic transmission at PF-PC synapses remains to be elucidated. In order to clarify this issue, we applied conventional whole-cell patch-clamp recordings and pre-synaptic calcium influx measurements, combined with pharmacological manipulations to rat and mice cerebellar slices. With the use of (1 S ,2 R )-1-amino-2-phosphonomethylcyclopropanecarboxylic acid, a new and selective group III mGluR agonist, N -phenyl-7-(hydroxylimino)cyclopropa[b]-chromen-1a-carboxamide, the specific positive allosteric modulator of mGluR4, ( S )-3,4-dicarboxyphenylglycine, a selective mGluR8 agonist, and mGluR4 knock-out mice, we demonstrate that the inhibitory control of group III mGluRs on excitatory neurotransmission at PF-PC synapses of the rodent cerebellar cortex, is totally because of the activation of pre-synaptic mGluR4 autoreceptors.