Role of the vesicular transporter VGLUT3 in retrograde release of glutamate by cerebellar Purkinje cells.

In the cerebellum, retrograde release of glutamate (Glu) by Purkinje cells (PCs) participates in the control of presynaptic neurotransmitter release responsible for the late component of depolarization-induced suppression of excitation (DSE), as well as for depolarization-induced potentiation of inhibition (DPI). It might also participate in the depolarization-induced slow current (DISC) in PCs, although this contribution was later challenged. We also know that both DPI and DISC are soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent processes, although the molecular nature of the vesicular transporter was not determined. In PCs, VGLUT3 is the only known vesicular glutamate transporter identified and is expressed during the same developmental frame as when DPI, DISC, and the Glu-dependent component of DSE are observed. We therefore tested the hypothesis that all these processes depend on the presence of VGLUT3 by comparing the Glu-dependent component of DSE, DPI, and DISC in nearly mature (2- to 3-wk-old) wild-type and VGLUT3 knockout mice. Our data demonstrate that, in nearly mature mice, the slow component of DSE occurs through vesicular release of Glu that involves VGLUT3. This Glu-dependent component of DSE is no longer present in fully mature mice. This study also establishes that, in nearly mature mice, DPI also depends on the presence of VGLUT3, whereas this is not the case for DISC. Finally, the unusually large basal paired-pulse facilitation observed in nearly mature VGLUT3(-/-) mice but not in adult ones suggests that some basal retrograde release of Glu occurs during development and contributes to basal concentrations of extracellular Glu.

Role of presynaptic kainate receptors at parallel fiber-purkinje cell synapses in induction of cerebellar LTD: interplay with climbing fiber input.

Until recently, except for A1 adenosine, N-methyl-d-aspartate, and cannabinoid receptors, little effort has been made to unravel possible roles of parallel fiber (PF) presynaptic receptors in long-term depression (LTD) of synaptic transmission at PF-Purkinje cell (PC) synapses. Presynaptic kainate (KA) receptors are also present on PFs and might also influence LTD induction by modulating glutamate (Glu) release at PF-PC synapses. This hypothesis was tested by comparing the efficacy of two pairing protocols in inducing LTD in adult wild-type and knock-out mice for the Glu receptor 6 (GluR6) subunit of KA receptors. Activation of presynaptic KA receptors was unnecessary for LTD induction when PF inputs were paired with climbing fiber (CF) stimulation but became crucial when CF input was replaced by direct depolarization of PCs. By comparing basal paired-pulse facilitation of PF-excitatory postsynaptic currents (EPSCs) and depolarization-induced suppression of excitation in adult wild-type and GluR6 knock-out mice, it was shown that the participation of PF presynaptic KA receptors in LTD induction was likely to mainly result from their tonic activation by basal extracellular Glu, rather than from their activation by retrograde release of Glu by PCs during pairing protocols. Finally, this study suggests that, in adult mice, CFs not only participate in LTD induction by depolarizing postsynaptic cells but also by activating postsynaptic mGluR1alpha metabotropic glutamate receptors at CF-PC synapses.

Developmental changes in agonist-induced retrograde signaling at parallel fiber-Purkinje cell synapses: role of calcium-induced calcium release.

In cerebellar Purkinje cells (PCs), activation of postsynaptic mGluR1 receptors inhibits parallel fiber (PF) to PC synaptic transmission by retrograde signaling. However, results were conflicting with respect to whether endocannabinoids or glutamate (Glu) is the retrograde messenger involved. Experiments in cerebellar slices from 10- to 12-day-old rats and mice confirmed that suppression of PF-excitatory postsynaptic currents (EPSCs) by mGluR1 agonists was entirely blocked by cannabinoid receptor antagonists at this early developmental stage. In contrast, suppression of PF-EPSCs by mGluR1 agonists was only partly blocked by cannabinoid receptor antagonists in 18- to 22-day-old rats, and the remaining suppression was accompanied by an increase in paired-pulse facilitation. This endocannnabinoidindependent suppression of PF-EPSCs was potentiated by the Glu uptake inhibitor D-threo-beta-benzyloxyaspartate (D-TBOA) and blocked by the desensitizing kainate (KA) receptors agonist SYM 2081, by nonsaturating concentrations of 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX) [but not by GYKI 52466 hydrochloride (GYKI)] and by dialyzing PCs with guanosine 5'-[beta-thio]diphosphate (GDP-betaS). An endocannnabinoid-independent suppression of PF-EPSCs was also present in nearly mature wild-type mice but was absent in GluR6(-/-) mice. The endocannnabinoid-independent suppression of PF-EPSCs induced by mGluR1 agonists and the KA-dependent component of depolarization-induced suppression of excitation (DSE) were blocked by ryanodine acting at a presynaptic level. We conclude that retrograde release of Glu by PCs participates in mGluR1 agonist-induced suppression of PF-EPSCs at nearly mature PF-PC synapses and that Glu operates through activation of presynaptic KA receptors located on PFs and prolonged release of calcium from presynaptic internal calcium stores.

Developmental changes in retrograde messengers involved in depolarization-induced suppression of excitation at parallel fiber-Purkinje cell synapses in rodents.

At parallel fiber (PF) to Purkinje cell (PC) synapses, depolarization-induced suppression of excitation (DSE) and suppression of PF-excitatory postsynaptic currents (EPSCs) by activation of postsynaptic mGluR1 glutamate (Glu) receptors involve retrograde release of endocannabinoids. However, Levenes et al. suggested instead that Glu was the retrograde messenger in this latter case. Because the study by Levenes et al. was performed in nearly mature rats, whereas most others were performed in juvenile animals, DSE was re-investigated in juvenile versus nearly mature rats and mice. Indeed, DSE was preferred here to agonist-induced suppression of PF-EPSCs, to avoid possible indirect effects in this latter case. In 10- to 12-day-old rats, DSE of PF-EPSCs was entirely mediated through retrograde release of endocannabinoids. In 18- to 22-day-old-rats, DSE was partly resistant to CB1 cannabinoid receptor antagonists. The remaining component was potentiated by the Glu uptake inhibitor d-threo-beta-benzyloxyaspartate (d-TBOA) and blocked by the desensitizing kainate (KA) receptor agonist (2S,4R)-4-methylglutamic acid (SYM 2081). This SYM-2081-sensitive component of DSE was accompanied by a paired-pulse facilitation increase that was also potentiated by d-TBOA and blocked by SYM 2081. In nearly mature wild-type and GluR6 -/- mice, results fully confirmed the presence of an endocannabinoid-independent component of DSE that involves retrograde release of Glu and activation of presynaptic KA receptors including GluR6 receptor subunits. Therefore retrograde release of Glu by PCs participates to DSE at PF-PC synapses in nearly mature rodents but not in juvenile ones, and Glu probably operates through activation of presynaptic KA receptors that include GluR6 receptor subunits.

Synapses between parallel fibres and stellate cells express long-term changes in synaptic efficacy in rat cerebellum.

Various forms of synaptic plasticity underlying motor learning have already been well characterized at cerebellar parallel fibre (PF)-Purkinje cell (PC) synapses. Inhibitory interneurones play an important role in controlling the excitability and synchronization of PCs. We have therefore tested the possibility that excitatory synapses between PFs and stellate cells (SCs) are also able to exhibit long-term changes in synaptic efficacy. In the present study, we show that long-term potentiation (LTP) and long-term depression (LTD) were induced at these synapses by a low frequency stimulation protocol (2 Hz for 60 s) and that pairing this low frequency stimulation protocol with postsynaptic depolarization induced a marked shift of synaptic plasticity in favour of LTP. This LTP was cAMP independent, but required nitric oxide (NO) production from pre- and/or postsynaptic elements, depending on the stimulation or pairing protocol used, respectively. In contrast, LTD was not dependent on NO production but it required activation of postsynaptic group II and possibly of group I metabotropic glutamate receptors. Finally, stimulation of PFs at 8 Hz for 15 s also induced LTP at PF-SC synapses. But in this case, LTP was cAMP dependent, as was also observed at PF-PC synapses for presynaptic LTP induced in the same conditions. Thus, long-term changes in synaptic efficacy can be accomplished by PF-SCs synapses as well as by PF-PC synapses, suggesting that both types of plasticity might co-operate during cerebellar motor learning.

Dopaminergic modulation of long-term synaptic plasticity in rat prefrontal neurons.

In rat prefrontal cortex (the prelimbic area of medial frontal cortex), the induction of long-term depression (LTD) and long-term potentiation (LTP) of glutamatergic synapses is powerfully modulated by dopamine. The presence of dopamine in the bathing medium facilitates LTD in slice preparations, whereas in the anesthetized intact brain, dopamine released from dopaminergic axon terminals in the prefrontal cortex facilitates LTP. Dopaminergic facilitation of LTD is at least partly achieved by postsynaptic biochemical mechanisms in which enzymatic processes triggered by dopamine receptor activation cooperate with those triggered by glutamate metabotropic receptor activation. Evidence suggests that dopamine facilitates LTP also in the slice condition. In this case, dopamine receptors must be pre-stimulated ('primed') before the application of high-frequency stimuli in the presence of dopamine. This procedure may mimic baseline stimulation of dopamine receptors that occurs under physiological conditions.

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.

Long-term potentiation in rat prefrontal slices facilitated by phased application of dopamine.

We have previously shown that coupling bath application of dopamine with 50 Hz tetani induces long-term depression in rat prefrontal slices [Neuroscience 85 (1998) 669]. Here, we report a reliable protocol for inducing long-term potentiation in the same preparation. Long-term potentiation was induced by the same dopamine-tetani coupling protocol when the coupling was preceded (approximately 30 min) by a single bath application of dopamine. We suggest that metaplastic processes triggered by the first application of dopamine might underlie the LTP induction.

In Vitro Development of Rat Cerebellar Neurons of Early Embryonic Origin. An Anatomical and Electrophysiological Study.

The development of the major morphological and electrophysiological properties of presumptive Purkinje cells (PCs) was studied in primary cultures of rat cerebellum dissociated on the 14th embryonic day, when PCs are minimally differentiated and migrate in vivo. PCs were identified with a specific antibody to calbindin D-28K (CaBP), which allowed visualization of the different morphological types of PCs between 3 and 29 days in vitro (DIV). CaBP-immunopositive cells were first detected at 3 DIV. Thereafter, the shape of these cells resembled some of those described in vivo. After 20 DIV, 95% of the CaBP-immunopositive cells had characteristic PC dendritic trees, although they were very atrophic. Glial cells immunopositive for the glial fibrillary acidic protein (GFAP) were first seen at 3 DIV. Thereafter GFAP-immunopositive cells resembled Bergmann cells or velate astrocytes. Neurons regarded as PCs were studied electrophysiologically using the patch-clamp whole-cell configuration. Voltage-dependent, tetrodotoxin-sensitive fast inward currents were virtually absent at 2 - 4 DIV, but increased between 7 and 14 DIV to reach two-thirds of the amplitude obtained after 15 DIV. These currents were large enough to give rise to overshooting spikes as early as 7 DIV in the current-clamp mode. This time schedule is in keeping with that of PCs developed in situ. The tetraethylammonium-sensitive, slowly inactivating outward currents had reached two-thirds of the amplitude obtained after 15 DIV by 3 - 4 DIV. Their amplitude remained stable between 4 and 7 DIV, and increased to their maximal value during 7 - 14 DIV, with a marked shortening of action potentials. 4-Aminopyridine-sensitive, fast-inactivating outward currents might also be associated with development, since they were present in 66% of the cells between 7 and 14 DIV but in only 39% from 15 to 29 DIV; however, their amplitude did not vary with time. Presumptive PCs bore l-glutamate-activated receptors, which preceded the emergence of kynurenate-sensitive, spontaneous synaptic currents at 7 DIV. These currents were sometimes intermingled with inhibitory currents, although presumptive PCs were sensitive to gamma-aminobutyrate at 7 DIV. The present model represents some unequivocal features of PC development, although the PCs used had undergone minimal differentiation in vivo and were cultured in a very disturbed cellular environment.