F. Crepel

Excitation of rat prefrontal cortical neurons by dopamine: An in vitro electrophysiological study

The effects of dopamine (DA) on prefrontal pyramidal neurons were studied in vitro on rat cerebral cortex slices using intracellular recordings. Pyramidal neurons were first identified by Lucifer yellow and some of their basic bioelectrical properties were analysed. At resting potential, white matter stimulation mainly evoked depolarizing inhibitory postsynaptic potentials (IPSPs) which reversed between -60 and -50 mV and were almost totally abolished by bicuculline. Furthermore, pyramidal cells often exhibited spontaneous depolarizing IPSPs abolished by bicuculline. Under tetrodotoxin (TTX) this synaptic noise was partly blocked suggesting that it was due both to the spontaneous firing of presynaptic gamma-aminobutyric acid (GABA)ergic neurons and to a spontaneous quantal release from these afferent fibers. In pyramidal cells, DA enhanced the number of spikes evoked by depolarizing current pulses, and the input resistance was increased by 10-20%. DA also clearly increased the inhibitory synaptic noise. This effect was blocked by fluphenazine. In contrast, evoked IPSPs were not consistently affected by DA. Taken altogether, these results suggest, that in the prefrontal cortex, dopamine has a mild excitatory effect on both pyramidal cells and GABAergic interneurons impinging on them.

Protein kinases, nitric oxide and long-term depression of synapses in the cerebellum

We have analysed the effects of polymyxin B, a potent inhibitor of calcium-dependent protein kinases, of L-N-monomethylarginine, an inhibitor of nitric oxide synthesis, and of methylene blue which prevents activation of soluble guanylate cyclase, on long-term depression of parallel fibre-mediated EPSPs of rat cerebellar Purkinje cells in slices maintained in-vitro. In control conditions, a long-term depression of parallel fibre-mediated EPSPs was consistently induced by their pairing with calcium spikes directly elicited in the postsynaptic cells. This long-term change in synaptic strength was not observed in the presence of polymyxin B, of L-N-monomethylarginine, or of methylene blue, suggesting that calcium-dependent protein kinases and nitric oxide are both involved.

Dopamine modulation of synaptic transmission in rat prefrontal cortex: an in vitro electrophysiological study

The prefrontal cortex is innervated by a well-defined dopaminergic bundle originating from the brainstem and is a key structure in higher order mental processes. We have studied the effects of dopamine (DA) on layer V pyramidal cells of the prefrontal cortex using intracellular recording in rat brain slices maintained in vitro. Bath administration of DA (50-100 microM) had weak effects on membrane properties of these neurons. In contrast, DA markedly decreased all components of the synaptic responses evoked by electrical stimulation of layer I or VI, and in particular the monosynaptic excitatory postsynaptic potential (EPSP) which arises from activation of glutamatergic receptors. The afferents from layer VI seemed less affected by DA than those from layer I. The NMDA (N-methyl-D-aspartate) and AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazolepropionic acid) components of monosynaptic EPSPs were equally reduced by DA. The isolated fast gabaergic potential (IPSP) resulting from GABAA receptors activation was similarly reduced by DA. The suppressive effect of DA on glutamatergic transmission was partially mimicked by the D1 receptor agonist SKF 38393 (50 microM) whereas the D2 receptor agonist quinpirole (50 microM) was ineffective. Conversely, this effect was antagonized by the D1 receptor blocker SCH 23390 (100 microM) but not by the D2 receptor antagonist sulpiride (100 microM). These findings indicate that DA decreases both glutamatergic and gabaergic synaptic transmission in neurons located in layer V of rat prefrontal cortex. These results also suggest that D1 dopamine receptor is involved in the decrement of glutamatergic transmission. These interactions between DA and glutamate are important in regard to the suspected implications of both neurotransmitters in psychiatric diseases.

Long‐term Depression Requires Nitric Oxide and Guanosine 3′:5’Cyclic Monophosphate Production in Rat Cerebellar Purkinje Cells

In patch‐clamped Purkinje cells, bath application of the nitric oxide synthase inhibitor NG‐methyl‐l‐arginine consistently prevents the induction of long‐term depression (LTD) of parallel fibre‐mediated excitatory postsynaptic potentials (EPSPs) induced by their pairing with calcium spikes. On the other hand, bath application of nitric oxide donors and of 8‐bromoguanosine 3′:5’cyclic monophosphate is able to reproduce an LTD‐like phenomenon. LTD of parallel fibre‐mediated EPSPs also occurs when nitric oxide donors or guanosine 3′:5’cyclic monophosphate are directly dialysed into Purkinje cells, and this effect partially occludes LTD induced by pairing protocols. These results show that nitric oxide does play a role in LTD induction, and demonstrate for the first time that its site of action is probably the soluble guanylate cyclase of Purkinje cells.

Coactivation of metabotropic glutamate receptors and of voltage-gated calcium channels induces long-term depression in cerebellar Purkinje cells in vitro.

SummaryUsing an in vitro slice preparation, we studied the effects, on parallel fiber (PF)-mediated EPSPs, of coactivation of metabotropic-glutamate receptors and of voltage-gated calcium (Ca) channels of Purkinje cells (PCs) by bath application of 50 μM trans-1-aminocyclopentyl-1,3-dicarboxylate (trans-ACPD) and by direct depolarization of the cells, respectively. These effects were compared with changes in synaptic efficacy obtained when α-amino-3hydroxy-5-methylisoxalone-4-propionate (AMPA) receptors of PCs were also activated through stimulation of PFs during the pairing protocol, as well as when similar experiments were performed without trans-ACPD in the bath. In a control medium, pairing for 1 min of PF-mediated EPSPs evoked at 1 Hz with Ca spikes evoked by steady depolarization of PCs (n = 13) led to LTD of synaptic transmission in 9 cases whereas for the others EPSPs were not affected. No LTD occurred in 9 out of 10 other cells tested when PF stimulation was omitted during the 1 min period of Ca spike firing of PCs. Bath application of 50 μM trans-ACPD, in conjunction with the same pairing protocol as before (n = 8), led to a significantly larger LTD of PF-mediated EPSPs after washing out of this drug. Moreover, a clearcut LTD of PF-mediated EPSPs was also observed in 5 of the 8 other cells, when PF stimulation was omitted during Ca spike firing in the presence of trans-ACPD. As trans-ACPD alone induced fully reversible depressions of EPSPs, coactivation of metabotropic-glutamate receptors and of voltage-gated Ca channels is therefore likely to be sufficient to induce LTD of PF-mediated EPSPs.

RECEPTORS AND SECOND MESSENGERS INVOLVED IN LONG-TERM DEPRESSION IN RAT CEREBELLAR SLICES IN VITRO : A REAPPRAISAL

In patch‐clamped Purkinje cells (PCs), bath application of the ionotropic glutamate receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) prevents induction of long‐term depression (LTD) of parallel fibre (PF)‐mediated EPSPs by a pairing protocol between Ca2+ spike firing and PF stimulation whereas bath application of (RS)‐α‐methyl‐4‐carboxyphenylglycine (MCPG), a metabotropic glutamate (mGLU) receptor antagonist, does not. On the other hand, LTD can be also induced by pairing direct depolarization of PCs with activation of mGLU receptors by 1 S,3R‐aminocyclopentyl‐dicarboxylate (1S, 3R‐ACPD), even in the presence of CNQX. In this case, LTD induction is not consistently blocked by bath application of the nitric oxide synthase inhibitor, NG‐methyl‐l‐arginine (l‐NMMA), whereas it is strongly blocked when the protein kinase C inhibitor peptide 19‐36 is dialysed into PCs. These results are at variance with LTD induced by a pairing protocol between Ca2+ spikes and PF‐mediated EPSPs which depends to the same extent on both cascades. Finally, thapsigargin, which depletes most intracellular Ca2+ pools, does not block induction of LTD by a pairing protocol between Ca2+ spikes and PF‐mediated EPSPs whereas it prevents the induction of LTD depending on strong mGLU receptor activation.

Effects of ACPD and AP3 on parallel-fibre-mediated EPSPs of Purkinje cells in cerebellar slices in vitro

SummaryThe effects of trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD) and of DL-2-amino-3-phosphonopropionic acid (AP3), i.e. selective agonist and antagonist of metabotropic quisqualate receptors respectively, on parallel fibre (PF)-mediated EPSPs of Purkinje cells (PCs) were studied in an in vitro slice preparation. Bath application of 500 μM trans-ACPD in conjunction with PF stimulation at 0.2 or 1 Hz depending on the cell always induced a marked depression of PF-mediated EPSPs, which was fully reversible in most cases after wash-out of this compound. Trans-ACPD also often induced a transient depolarization of PCs which induced calcium spike firing in these cells and which again no longer persisted after wash-out of trans-ACPD. Even in cells which were depolarized by trans-ACPD, the decrease in amplitude of PF-mediated EPSPs started before the appearence of calcium spikes, lasted longer than the transient depolarizing effect of trans-ACPD, and was accompanied by no variation in input resistance of the cells when they were manually clamped at their initial resting potential. Bath application of 600 μM DL-AP3 had no effect on PF-mediated EPSPs or the bioelectrical activities of PCs. Moreover, it did not prevent the effects of trans-ACPD mentioned before. The present results are not consistent with the view that coactivation of ionotropic and metabotropic quisqualate receptors of PCs is sufficient to induce a long-term depression of PF-mediated EPSPs.

Transient Sensitivity of Rat Cerebellar Purkinje Cells to N‐methyl‐D‐aspartate during Development. A Voltage Clamp Study in in vitro Slices

In vitro sagittal slices of immature rat cerebellum were used to study the development of the sensitivity of Purkinje cells (PC) to L‐glutamate (Glu) and N‐methyl‐D‐aspartate (NMDA). In 8‐day‐old animals, all PCs recorded in magnesium‐free medium responded to iontophoretic applications of both agonists by transient and dose dependent inward currents which, in both cases, were heavily contaminated by a Glu and NMDA‐induced synaptic noise. When 5 × 10−6 M tetrodotoxin (TTX) was added to the perfusing medium, this evoked synaptic noise was completely abolished in most cells whereas clear‐cut inward currents induced in PCs by Glu and NMDA applications on their dendrites were still visible. These responses were selectively antagonized by the non‐NMDA glutamate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and by the NMDA receptor antagonist D‐2‐aminophosphono‐5‐valeric acid (2APV) respectively. Excitatory responses induced by aspartate in 8– 10‐day‐old PCs were also markedly antagonized by CNQX. At this stage, the sensitivity of PCs to NMDA was about one order of magnitude less than that to Glu. In 15–20‐day‐old animals, all PCs were still responsive to Glu whereas only 70% of them were still excited by NMDA in the presence of TTX in the bath. Furthermore, the sensitivity of PCs to Glu was higher than at 8 days of age, whereas that to NMDA was significantly lower, even when considering only those cells which still responded to this agonist. This trend was still accentuated later on since at 2 months of age, only 25% of PCs were excited by NMDA whereas their sensitivity to Glu was similar to that observed in 15–20‐day‐old animals. Therefore, the present results are fully consistent with the view that PCs have a transient expression of NMDA receptors during development.

Blockade of NMDA receptors unmasks a long-term depression in synaptic efficacy in rat prefrontal neurons in vitro

SummaryAll the experiments were carried out in slices of rat prefrontal cortex maintained in vitro. The effect of 2-amino-5-phosphonovalerate (APV) was tested on the postsynaptic potential (PSP) recorded in layer V pyramidal cells, in response to single or high frequency stimulation of the superficial layers I–II. Wash-out of Mg2+ increased the amplitude and duration of the PSPs. This effect resulted from activation of N-methyl-D-aspartate (NMDA) receptors since it was suppressed by bath application of APV. Furthermore, in every cell tested in Mg2+ containing medium (N=16), exposure to APV reversibly reduced both mono- and polysynaptic components of the PSPs, indicating that, even in the control solution, activation of NMDA-coupled channels contributed to these synaptic events. Finally, the anomalous voltage-dependence of the EPSP in the presence of Mg2+ and its sensitivity to APV suggests that at least a fraction of the NMDA receptors are postsynaptically located. Tetanization was applied to the afferents of cells bathed in control- or APV-medium. Long-term potentiation (LTP) or long-term depression (LTD) is defined as an increase or a decrease respectively, of the PSPs peak amplitude or initial slope, lasting 20 min. In the control medium, LTP in synaptic efficacy was observed in 34% of the cells and LTD in 48% (N=23). When exposed to APV, none of the cells tested (N=16) showed LTP of the response. In contrast, the tetanus induced a LTD of the PSP amplitude or slope in 14 out of these 16 cells. The percentage of cells showing LTD in synaptic efficacy (87%) when the NMDA receptors activation was blocked was significantly higher than that in control-medium.

Properties of glutamate receptors are modified during long-term depression in rat cerebellar Purkinje cells

Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses occurs when these synapses are activated in conjunction with direct activation of voltage-gated calcium (Ca2+) channels of PCs. In the present study, we have used Aniracetam to test whether the expression of LTD at PF-PC synapses is due to a genuine modification of properties of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors of these neurons. Whole-cell recordings of PF-mediated EPSCs were performed in thin slices taken from 16-22-day-old rats. In all tested cells, bath application of Aniracetam potentiated PF-mediated EPSCs and prolonged their decay without notably changing their rising phase. On the other hand, Aniracetam prevented the induction of LTD by a pairing protocol with Ca2+ spikes and, conversely, the nootropic compound had a larger potentiating effect on PF-mediated EPSCs during expression of LTD than normally, when this change in synaptic efficacy had been induced prior to Aniracetam application. These data strongly suggest that LTD involves a desensitization of postsynaptic AMPA receptors at PF-PC synapses, or, at least, a change in their functional characteristics.