Voltage-dependent calcium signaling in rat cerebellar unipolar brush cells.

Unipolar brush cells (UBCs) are a class of excitatory interneuron found in the granule cell layer of the vestibulocerebellum. Mossy fibers form excitatory inputs on to the paint brush shaped dendrioles in the form of giant, glutamatergic synapses, activation of which results in prolonged bursts of action potentials in the postsynaptic UBC. The axons of UBCs themselves form mossy fiber contacts with other UBCs and granule cells, forming an excitatory, intrinsic cerebellar network that has the capacity to synchronize and amplify mossy fiber inputs to potentially large populations of granule cells. In this paper, we demonstrate that UBCs in rat cerebellar slices express low voltage activated (LVA) fast-inactivating and high voltage activated (HVA) slowly inactivating calcium channels. LVA calcium currents are mediated by T-type calcium channels and they are associated with calcium increases in the dendrites and to a lesser extent the cell soma. HVA currents, mediated by L-type calcium channels, are slowly inactivating and they produce larger overall increases in intracellular calcium but with a similar distribution pattern. We review these observations alongside several recent papers that examine how intrinsic membrane properties influence UBCs firing patterns and we discuss how UBC signaling may affect downstream cerebellar processing.

Unipolar brush cells form a glutamatergic projection system within the mouse cerebellar cortex.

Unipolar brush cells (UBCs) of the mammalian vestibulocerebellum receive mossy fiber projections primarily from the vestibular ganglion and vestibular nuclei. Recently, the axons of UBCs have been shown to generate an extensive system of cortex-intrinsic mossy fibers, which resemble traditional cerebellar mossy fiber afferents and synapse with granule cell dendrites and other UBCs. However, the neurotransmitter used by the UBC axon is still unknown. In this study, we used long-term organotypic slice cultures of the isolated nodulus (lobule X) from postnatal day 8 mouse cerebella to identify the neurotransmitter and receptors at synapses of the UBC axon terminals, relying on the notion that, in these cultures, all of the cortex-extrinsic fibers had degenerated during the first few days in vitro. Quantification of glutamate immunogold labeling showed that the UBC axon terminals have the same high gold-particle density as the glutamatergic parallel fiber varicosities. Furthermore, UBCs identified by calretinin immunoreactivity expressed the glutamate receptor subunits GluR2/3, NMDAR1, and mGluR2/3, like they do in the mature mouse cerebellum in situ. Evoked excitatory postsynaptic currents (EPSCs), spontaneous EPSCs, and burst discharges were demonstrated in UBCs and granule cells by patch-clamp recording. Both the evoked and spontaneous EPSCs were blocked by ionotropic glutamate receptor antagonists CNQX and D-AP5. We conclude that neurotransmission at the UBC axon terminals is glutamatergic. Thus, UBCs provide a powerful network of feedforward excitation within the granular layer, which may amplify vestibular signals and synchronize activity in clusters of functionally related granule cells which project vertically to patches of Purkinje cells.

Unipolar brush cell: a potential feedforward excitatory interneuron of the cerebellum.

Unipolar brush cells are a class of interneurons in the granular layer of the mammalian cerebellum that receives excitatory mossy fiber synaptic input in the form of a giant glutamatergic synapse. Previously, it was shown that the unipolar brush cell axon branches within the granular layer, giving rise to large terminals. Single mossy fiber stimuli evoke a prolonged burst of firing in unipolar brush cells, which would be distributed to postsynaptic targets within the granular layer. Knowledge of the ultrastructure of the unipolar brush cell terminals and of the cellular identity of its postsynaptic targets is required to understand how unipolar brush cells contribute to information processing in the cerebellar circuit. To investigate the unipolar brush cell axon and its targets, unipolar brush cells were patch-clamped in fresh parasagittal slices from rat cerebellar vermis with electrodes filled with Lucifer Yellow and Biocytin, and examined by confocal fluorescence and electron microscopy. Biocytin was localized with diaminobenzidine chromogen or gold-conjugated, silver-intensified avidin. Light microscopic examination revealed a single thin axon emanating from the unipolar brush cell soma that gave rise to 2-3 axon collaterals terminating in mossy fiber-like rosettes in the granular layer, typically within a few hundred microm of the soma. In some cases, axon collaterals crossed the white matter within the same folium before terminating in the adjacent granular layer. Electron microscopic examination of serial ultrathin sections revealed that proximal unipolar brush cell axons and axon collaterals were unmyelinated and devoid of synaptic contacts. However, the rosette-shaped enlargements of each collateral formed the central component of glomeruli where they were surrounded by dendrites of granule cells and/or other unipolar brush cells, with which they formed asymmetric synaptic contacts. A long-latency repetitive burst of polysynaptic activity was observed in granule cells in this cerebellar region following white matter stimulation. The unipolar brush cell axons, therefore, form a system of cortex-intrinsic mossy fibers. The results indicate that synaptic excitation of unipolar brush cells by mossy fibers will drive a large population of granule cells, and thus will contribute a powerful form of distributed excitation within the basic circuit of the cerebellar cortex.

Glutamate transporters contribute to the time course of synaptic transmission in cerebellar granule cells.

Transporters are thought to assist in the termination of synaptic transmission at some synapses by removing neurotransmitter from the synapse. To investigate the role of glutamate transport in shaping the time course of excitatory transmission at the mossy fiber-granule cell synapse, the effects of transport impairment were studied using whole-cell voltage- and current-clamp recordings in slices of rat cerebellum. Impairment of transport by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) produced a prolongation of the decay of the AMPA receptor-mediated current after a repetitive stimulus, as well as prolongation of single stimulus-evoked EPSCs when AMPA receptor desensitization was blocked. PDC also produced a prolongation of both single and repetitive-evoked NMDA receptor-mediated EPSCs. Enzymatic degradation of extracellular glutamate did not reverse the PDC-induced prolongation of AMPA receptor-mediated current after a repetitive stimulus, suggesting that transporter binding sites participate in limiting glutamate spillover. In current-clamp recordings, PDC dramatically increased the total area of the EPSP and the burst duration evoked by single and repetitive stimuli. These data indicate that glutamate transporters play a significant role in sculpting the time course of synaptic transmission at granule cell synapses, most likely by limiting the extent of glutamate spillover. The contribution of transporters is particularly striking during repetitive stimulus trains at physiologically relevant frequencies. Hence, the structural arrangement of the glomerulus may enhance the contribution of transporters to information processing by limiting the extent of glutamate spillover between adjacent synapses.

Prolonged physiological entrapment of glutamate in the synaptic cleft of cerebellar unipolar brush cells.

The cellular mechanism underlying the genesis of the long-lasting alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor-mediated excitatory postsynaptic currents (EPSCs) at the mossy fiber (MF)-unipolar brush cell (UBC) synapse in rat vestibular cerebellum was examined with the use of whole cell and excised patch-clamp recording methods in thin cerebellar slices. Activation of MFs evokes an all-or-none biphasic AMPA-receptor-mediated synaptic current with a late component that peaks at 100-800 ms, which has been proposed to originate from an entrapment of glutamate in the MF-UBC synaptic cleft and is generated by the steady-state activation of AMPA receptors. Bath application of cyclothiazide, which blocks desensitization of AMPA receptors, produced a dose-dependent enhancement of the amplitude of the synaptic current (median effective dose 30 microM) and slowing of the rise time of the fast EPSC. N-methyl-D-aspartate-receptor-mediated EPSCs in UBCs were not potentiated in amplitude or time course by cyclothiazide (100 microM). The dose-response relations for the steady-state current evoked by glutamate acting at AMPA receptors in excised outside-out patches from UBC and granule somatic membranes was biphasic, peaking at 50 microM and declining to 50-70% of this value at 1 mM glutamate. When glutamate was slowly washed from patches to simulate the gradual decline of glutamate in the synapse, a late hump in the transmembrane current was observed in patches from both cell types. The delivery of a second MF stimulus at the peak of the slow EPSC evoked a fast EPSC of reduced amplitude followed by an undershoot of the subsequent slow current, consistent with the hypothesis that the peak of the slow EPSC reflects the peak of the biphasic steady-state dose-response curve. Estimates of receptor occupancy and glutamate concentration derived from the ratio of fast EPSC amplitudes, and the amplitude and polarity of the initial steady-state current in paired-pulse experiments, predict a slow decline of glutamate with a time constant of 800 ms, declining to ineffective concentrations at 5.4 s. Manipulation of cleft glutamate concentration by lowered extracellular calcium or delivery of brief stimulus trains abolished the slow EPSC and restored the undershoot to paired stimuli, respectively, in a manner consistent with a prolonged lifetime of glutamate in the cleft. The slow component of the EPSC was prolonged in duration by the glutamate reuptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting that glutamate transport contributes to the time course of the synaptic current in UBCs. The data support the notion that the MF-UBC synapse represents an ultrastructural specialization to effectively entrap glutamate for unusually prolonged periods of time following release from MF terminals. The properties of the postsynaptic receptors and constraints on diffusional escape of glutamate imposed by synaptic ultrastructure and glutamate transporters act in concert to sculpt the time course of the resulting slow EPSC. This in turn drives a long-lasting train of action potentials in response to single presynaptic stimuli.

Metabotropic glutamate receptor mediated long-term depression in developing hippocampus.

The effects of bath application of the metabotropic glutamate receptor (mGluR) agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10 microM) were studied at the Schaffer collateral-CA1 synapse in hippocampal slices from rats of 8-33 days postnatal age. In immature animals (8-12 days) ACPD induced a biphasic response characterized by an acute decrease in field EPSP slope (approximately 50-60% of baseline) in the presence of the agonist, followed by long-term depression (LTD, approximately 75-80% of baseline) after washout. In animals older than 20 days, ACPD induced a slow onset potentiation or minimal change. Both the acute depression and LTD were blocked by the mGluR antagonist alpha-methyl-4-carboxyphenyl glycine (MCPG). ACPD-induced LTD was blocked by the N-methyl-D-aspartate receptor (NMDAR) antagonists D(-)-2-amino-5 phosphopentanoic acid (AP5) and dizocilpine maleate (MK-801), and by ethanol. Glutamic pyruvic transaminase, an enzyme that selectively metabolizes endogenous extracellular glutamate, also blocked LTD suggesting that the requisite NMDA currents were tonically activated by extracellular rather than synaptically released glutamate. ACPD-induced LTD was blocked by staurosporine, indicating a requirement for serinethreonine kinase activation, and was unaffected by the L-type voltage sensitive calcium channel blocker nitrendipine and the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). Because mGluR-mediated LTD was observed only in immature CA1, mGluRs may play a role in hippocampal development, perhaps by contributing to synapse pruning in a temporally restricted fashion.

Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic glutamate receptor.

The application of the glutamate analog L-2-amino-4-phosphonobutyric acid (L-AP4) to neurons produces a suppression of synaptic transmission. Although L-AP4 is a selective ligand at a subset of metabotropic glutamate receptors (mGluRs), the precise physiological role of the L-AP4-activated mGluRs remains primarily unknown. To provide a better understanding of the function of L-AP4 receptors, we have generated and studied knockout (KO) mice lacking the mGluR4 subtype of mGluR that displays high affinity for L-AP4. The mGluR4 mutant mice displayed normal spontaneous motor activity and were unimpaired on the bar cross test, indicating that disruption of the mGluR4 gene did not cause gross motor abnormalities, impairments of novelty-induced exploratory behaviors, or alterations in fine motor coordination. However, the mutant mice were deficient on the rotating rod motor-learning test, suggesting that mGluR4 KO mice may have an impaired ability to learn complex motor tasks. Patch-clamp and extracellular field recordings from Purkinje cells in cerebellar slices demonstrated that L-AP4 had no effect on synaptic responses in the mutant mice, whereas in the wild-type mice 100 microM L-AP4 produced a 23% depression of synaptic responses with an EC50 of 2.5 microM. An analysis of presynaptic short-term synaptic plasticity at the parallel fiber-->Purkinje cell synapse demonstrated that paired-pulse facilitation and post-tetanic potentiation were impaired in the mutant mice. In contrast, long-term depression (LTD) was not impaired. These results indicate that an important function of mGluR4 is to provide a presynaptic mechanism for maintaining synaptic efficacy during repetitive activation. The data also suggest that the presence of mGluR4 at the parallel fiber-->Purkinje cell synapse is required for maintaining normal motor function.

Modification of NMDA receptor channels and synaptic transmission by targeted disruption of the NR2C gene.

A novel strain of mutant mouse has been generated with a deletion of the gene encoding the NR2C subunit of the NMDA receptor, which is primarily expressed in cerebellar granule cells. Patch-clamp recordings from granule cells in thin cerebellar slices were used to assess the consequences of the gene deletion. In granule cells of wild-type animals, a wide range of single-channel conductances were observed (19-60 pS). The disruption of the NR2C gene results in the disappearance of low-conductance NMDA receptor channels ( < 37 pS) normally expressed in granule cells during developmental maturation. The NMDA receptor-mediated synaptic current is markedly potentiated in amplitude, but abbreviated in duration (with no net difference in total charge), and the non-NMDA component of the synaptic current was reduced. We conclude that the NR2C subunit contributes to functional heteromeric NMDA receptor-subunit assemblies at the mossy fiber synapse and extrasynaptic sites during maturation, and the conductance level exhibited by a given receptor macromolecule may reflect the stochiometry of subunit composition.

Frequency dependent activation of a slow N-methyl-D-aspartate-dependent excitatory postsynaptic potential in turtle cerebellum by mossy fibre afferents.

The synaptic responses of turtle cerebellar Purkinje cells to stimulation of localized mossy fibre systems have been studied by use of intrasomatic and intradendritic recordings in a brainstem-cerebellum preparation in vitro. Activation of mossy fibre inputs from the spinocerebellar pathway evoked fast, disynaptic postsynaptic potentials which were graded in amplitude with stimulus intensity and elicited at latencies consistent with those reported for peripheral nerve stimulation. Repetitive activation (50-100 Hz, 2-10 stimuli) of both spinocerebellar and trigeminocerebellar pathways evoked a slow, long-lasting excitatory postsynaptic potential regardless of whether single stimuli resulted in excitatory, inhibitory, or no postsynaptic responses. This slow potential was capable of triggering dendritic pacemaker discharges in recorded Purkinje cells in addition to volleys of simple spikes when activated at or near resting membrane potential. The fast excitatory synaptic potentials evoked by spinocerebellar stimulation were blocked by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, consistent with the hypothesis that they are mediated by activation of ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methylisox-azole-4-proprionic acid subtype at the mossy fibre-granule cell synapse and the subsequent parallel fibre-Purkinje cell synapse. The slow excitatory synaptic potential evoked by repetitive stimulation of either the spinocerebellar tract or trigeminal nerve was blocked by DL-2-amino-5-phosphonvalerate, indicating that this potential is primarily dependent upon N-methyl-D-aspartate receptors at the mossy fibre-granule cell synapse for its expression. This slow potential was reversibly potentiated by L-2-amino-4-phosphonobutyrate and bicuculline; the metabotropic glutamate antagonist (+)-alpha-methyl-4-carboxyphenylglycine did not block this potentiation. The ability of mossy fibre inputs to drive long, slow excitatory events in Purkinje cells adds another dimension to the mechanisms by which various sensory modalities can be processed interactively in the cerebellar cortex. The ability of incoming systems to access a second, longer duration response of the cerebellar output neuron may be of significant consequence to our understanding of the manner in which this neural centre integrates sensory information from multiple sources.

Properties of transmission at a giant glutamatergic synapse in cerebellum: the mossy fiber-unipolar brush cell synapse.

1. The synaptic activation by mossy fibers (MFs) of unipolar brush cells (UBCs) in the vestibular cerebellum (nodulus and uvula) was examined using patch-clamp recording methods in thin, rat cerebellar slices with Lucifer yellow-filled pipettes for subsequent fluorescence microscopic verification of the cell morphology. 2. UBCs were distinguished from adjacent granule cells in thin cerebellar slices in the uvula and nodulus regions by their larger soma diameters and short dendritic brush, greater whole-cell capacitance, and a prolonged, biphasic excitatory postsynaptic current (EPSC) to stimulation of MFs. 3. Thin-section transmission electron micrographs of the MF-UBC synapse displayed an unusually extensive area of synaptic apposition estimated to measure 12-40 microns2. The majority of UBCs was innervated by a single MF. At high magnification, individual clusters of presynaptic vesicles could be discerned, separated by regions of presynaptic membrane lacking vesicles, but apposed to continuous regions of postsynaptic density. Thus, after release, transmitter diffusion from the synaptic cleft must traverse considerable stretches of postsynaptic membrane before escape into extracellular space. In contrast, MF-granule cell synapses in these cerebellar regions resembled glutamate synapses in other brain regions in that the total synaptic area measured < or = 4 microns2. These synaptic junctions were flanked by short stretches of unspecialized plasma membrane, providing a short (0.5 micron) diffusional path from the site of neurotransmitter release to a branch point of the extracellular space. 4. The MF-evoked EPSC in UBCs was composed of a fast (10-90% rise time: 0.70 ms) and slow (10-90% rise time: 395 ms; 10-90% decay time: 3.1 s) component. The fast component was blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and displayed linear current-voltage (I-V) relations in the presence or absence of external magnesium. 5. The slow EPSC was also mediated by glutamate receptors, but in most neurons both AMPA/KA and N-methyl-D-aspartate (NMDA) receptors contributed to the slow EPSC, with the contribution of NMDA receptors predominating in the majority of cells. Consequently, although all cells displayed linear I-V relations in Mg(2+)-free saline, cells in which the slow EPSC was predominently mediated by NMDA receptors exhibited voltage-dependent rectification in the presence of external Mg2+ (1 mM). 6. With increasing postnatal age (10-30 d), the contribution made to the slow EPSC by NMDA receptors declined, with a reciprocal increase in the contribution being made by AMPA/KA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

AP5 blocks LTP in developing rat dentate gyrus and unmasks LTD.

The hippocampal dentate gyrus undergoes active neuronogenesis as well as growth and regression of neuronal elements and connections during the early postnatal period. In some brain regions, most notably in the visual system, both activity-dependent synaptic plasticity and NMDA receptor activation are candidate mechanisms by which neuronal architecture may be refined during brain maturation. To investigate whether similar mechanisms might obtain in developing dentate, we studied the effects of tetanic stimulation before and after NMDA receptor blockade in hippocampal slices from rats at 7-33 days. Field potentials were recorded in the suprapyramidal granule cell layer in response to stimulation of the medial perforant path. Robust long-term potentiation (LTP) of population spike amplitude (approximately 200% of baseline) was produced by a single tetanus (100 Hz, 2 s, 200 microseconds) at all ages studied. Application of 10 microM AP5 depressed population spike amplitude only in the younger slices (approximately 81% of baseline at 8-15 days; approximately 86% of baseline at 16-24 days), suggesting that the NMDA receptor-mediated component of normal synaptic transmission is higher in early development and decreases with maturation. AP5 prevented or significantly diminished LTP at all ages, establishing the NMDA dependence of LTP induction in the medial perforant path throughout development. AP5 also unmasked tetanus-induced homosynaptic long-term depression (62-75% of baseline) in the younger slices (8-24 days). Thus, prominent NMDA receptor-mediated activity and the capacity for bidirectional synaptic plasticity are characteristic of immature dentate. These processes may influence dentate morphogenesis by contributing to the growth, regression, and stabilization of neuronal elements.

The synaptic activation of N-methyl-D-aspartate receptors in the rat medial vestibular nucleus.

1. We examined the synaptic activation of N-methyl-D-aspartate (NMDA) receptors by stimulation of primary vestibular afferent projections to second-order neurons in the medial vestibular nucleus (MVN) using whole cell patch-clamp recording methods in rat brain stem slices maintained in vitro. 2. Stimulation of the vestibular nerve (nVIII) evoked monosynaptic excitatory postsynaptic potentials (EPSPs) in second-order MVN neurons. Bath application of the gamma-aminobutyric acid receptor antagonist bicuculline (10 microM) revealed a late, slow EPSP that was blocked by the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5; 50 microM) and displayed a voltage-dependent reduction at hyperpolarized potentials in the presence of external magnesium (1 mM). The early component of the nVIII-evoked EPSP in the presence of bicuculline was blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 microM) and displayed linear current-voltage relations in the presence of external magnesium. 3. In some cells both components of the EPSP were blocked by DNQX, whereas only the late component was sensitive to D-AP5, indicating that NMDA receptors also mediate excitation via intrinsic pathways within MVN. 4. The NMDA receptor-mediated excitatory postsynaptic current (EPSC) evoked by nVIII stimulation was recorded in voltage-clamped MVN neurons in a magnesium-free saline containing bicuculline (10 microM) and DNQX (10 microM). At -80 mV the NMDA receptor-mediated EPSC (latency = 2.7 ms) displayed a slow rise time (10-90%, 5.8 ms) and exhibited a biexponential decay [time constant of fast component of decay (tau s) = 27.6 ms, time constant of slow component of decay (tau s) = 147.4 ms].(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous network of endoplasmic reticulum in cerebellar Purkinje neurons.

Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions.

The developmental onset of NMDA receptor-channel activity during neuronal migration.

Patch-clamp recordings of granule cells in thin slices of developing rat cerebellum maintained in vitro displayed spontaneous single-channel activity mediated via activation of N-methyl-D-aspartate (NMDA) receptors. The frequency of tonic single-channel activity was reversibly inhibited by the NMDA receptor/channel antagonists D-2-amino-5-phosphonovalerate (D-AP5), 7-chloro-kynurenate (7-Cl-Kynu) and MgCl2, potentiated by glycine, and unaffected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or tetrodotoxin (TTX). Tonic channel activity was also reversibly inhibited by enzymatic degradation of endogenous glutamate by glutamate pyruvate transaminase, which did not affect the NMDA sensitivity of granule cells. Both the frequency of spontaneous channel activity and the NMDA sensitivity were low in premigratory cells of the external germinal layer (EGL), with large increases observed in migrating cells in the molecular layer (ML) and in postmigratory cells within the internal granule cell layer (GCL). Tonic channel activity was enhanced by the glutamate uptake inhibitor L-alpha-aminoadipate (L-alpha-AA), the degree of enhancement being greater in the EGL than the GCL. The results demonstrate that a dramatic increase in the tonic NMDA receptor-channel activity occurs during the stages of granule cell differentiation, migration and synaptogenesis, which is driven by endogenous glutamate release and regulated by NMDA receptor density and local glutamate uptake.

Metabotropic glutamate receptors and calcium signalling in dendrites of hippocampal CA1 neurones.

We have combined patch-clamp recording with confocal microscopy to investigate how the synaptic activation of metabotropic glutamate receptors (mGluRs) may participate in the modulation of intracellular free calcium (Ca2+) in the dendrites of single CA1 pyramidal neurones, within hippocampal slices. Tetanic stimulation (100 Hz, 1 sec) of the Schaffer collateral-commissural pathway led to a transient rise in Ca2+ in the dendrites of neurones voltage- clamped at -35 mV, as determined using the fluorescent indicator fluo-3. The specific mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), applied at a concentration of 250 or 500 microM, reduced the size of the Ca2+ transient whilst either producing a small reduction or, more commonly, having no effect on the synaptic current evoked by the tetanus. These data suggest that the synaptic activation of mGluRs can contribute to Ca2+ signalling in hippocampal neurones.

Activation of metabotropic glutamate receptors induces long-term depression of GABAergic inhibition in hippocampus.

1. The long-term enhancement of synaptic excitability in CA1 hippocampal pyramidal neurons produced by activation of metabotropic glutamate receptors (mGluRs) was studied in rabbit hippocampal slices in vitro. 2. Bath application of the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3- dicarboxylic acid (1S,3R-ACPD) (5-20 microM) for 20 min produced a reversible depolarization of membrane potentiatil, blockade of spike accommodation, and increase in input resistance of CA1 neurons. However, a long-lasting increase in synaptic excitability was observed: single stimuli applied to the Schaffer collateral commisural fiber pathway evoked epileptiform discharges in the presence of 1S,3R-ACPD and after the washout of 1S,3R-ACPD, persistent paroxysmal depolarization shifts (PDSs) were evoked by afferent stimulation. A long-lasting enhancement of synaptic excitability was also observed in the presence of the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), which blocked the stimulation-evoked PDS and associated afterdischarges. 3. When biphasic, monosynaptically evoked inhibitory post-synaptic potentials (IPSPs) were recorded in the presence of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-15 microM) and D-AP5 (20 microM), the bath application of 1S,3R-ACPD produced a significant reduction (approximately 50%) of both components of the IPSP, which persisted after the washout of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiation of NMDA receptor-mediated transmission in turtle cerebellar granule cells by activation of metabotropic glutamate receptors.

1. The effects of metabotropic glutamate receptor (mGluR) agonists on excitatory postsynaptic potentials (EPSPs) evoked by stimulation of mossy fibers (MF) and parallel fibers (PF) were examined in turtle cerebellar Purkinje cells. 2. The mGluR agonist 1S,3R-ACPD (1-25 microM) reversibly potentiated the amplitude of the MF-evoked EPSPs, but was without effect on PF-evoked EPSPs. The potentiation of MF-evoked EPSPs was dose-dependent, with a median effective dose (ED50) of approximately 4.4 microM. At higher doses (15-25 microM) 1S,3R-ACPD produced a direct depolarization of Purkinje cells in 58% of cells examined. 3. The enhancement of MF EPSPs by 1S,3R-ACPD was mimicked by 1S,3S-ACPD (50 microM) and blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5), but not by the mGluR antagonist L-2-amino-3-phosphonopionic acid (L-AP3; 1 mM), or the 1R,3S isomer of ACPD (25-500 microM). 4. Quisqualate (1 microM) produced a biphasic effect on MF EPSPs, producing an initial blockade of the EPSP followed by a D-AP5-sensitive potentiation. 5. The potentiation of MF EPSPs by 1S,3R-ACPD was not blocked by prior exposure to the protein kinase C activator phorbol 12-myristate 13-acetate (10 microM), the protein kinase C inhibitor calphostin C (1 microM), the adenylate cyclase activator forskolin (25 microM), or the nitric oxide donator sodium nitroprusside (1 mM). Preincubation of the tissue for 24-48 h in pertussis toxin also failed to prevent the ability of 1S,3R-ACPD to potentiate the NMDA receptor-mediated component of the MF EPSP. PF EPSPs were also not significantly affected by these agents. 6. The results demonstrate that the mGluR agonists 1S,3R-ACPD, 1S,3S-ACPD, and quisqualate produce a potent, stereospecific potentiation of NMDA receptor-mediated transmission at the MF-granule cell synapse. Agents that modulate the intracellular messengers protein kinase C, adenylate cyclase, nitric oxide, or pertussis toxin-sensitive G proteins failed to mimic or block this effect. This would suggest that the potentiation of NMDA receptor-mediated transmission at this synapse is not mediated via these systems, and reflects a different site of action of mGluR agonists on the NMDA receptor. The observed interaction between mGluR and NMDA receptors in granule cells provides a means for activity-dependent modulation of synaptic transmission, which may play a role in synaptic integration at the MF-granule cell synapse.

Role of metabotropic glutamate (ACPD) receptors at the parallel fiber-Purkinje cell synapse.

1. The role of metabotropic glutamate receptors at the parallel fiber (PF)-Purkinje cell synapse in cerebellum was studied by examining the actions of the active stereoisomer (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [1S,3R-ACPD (25-50 microM)] on fura-2-loaded, patch-clamped rat Purkinje cells in thin slices. 2. The bath application of 1S,3R-ACPD evoked a direct post-synaptic depolarization that readily desensitized during prolonged (> 1 min) applications of the drug. This depolarizing response to 1S,3R-ACPD differed from the slow depolarization to 1S,3R-ACPD observed in cortical neurons mediated via closure of potassium channels in that it was not associated with an obvious change in membrane conductance and was not blocked by external barium. Similarly, slow inward rectifier currents were not affected during the 1S,3R-ACPD-induced depolarization. 3. The direct depolarization induced by 1S,3R-ACPD was not mediated by N-methyl-D-aspartate (NMDA) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid kainate (AMPA)-KA excitatory amino acid (EAA) receptor subtypes, because the response was not blocked in the presence of antagonists of these receptors. 4. The EAA antagonist L-2-amino-3-phosphonopropionic acid, which blocks 1S,3R-ACPD-induced inositide synthesis in other cell types, had no effect on the depolarizing response. 5. Fura-2 measurements of somatic [Ca2+]i revealed that [Ca2+]i was not elevated during the 1S,3R-ACPD-induced depolarization unless the cell fired calcium-dependent action potentials. 6. In addition to the direct depolarization induced by 1S,3R-ACPD, the amplitude of PF-evoked excitatory postsynaptic potentials (EPSPs) was profoundly and reversibly reduced. This effect was observed in all cells regardless of whether a direct depolarization was produced by 1S,3R-ACPD. This reduction of the PF EPSP generally preceded the onset of depolarizing responses, did not desensitize during prolonged applications of 1S,3R-ACPD, and was reversible. 7. The reversible reduction of the PF EPSP by 1S,3R-ACPD was not related to a postsynaptic blocking action of the drug, because responses of Purkinje cells to AMPA, an agonist of the EAA receptor subtype mediating the EPSP, were reversibly potentiated in the presence of 1S,3R-ACPD. 8. The nitric oxide synthesis promoter sodium nitroprusside (1-3 nM) had no effect on the amplitude of PF EPSP or the membrane properties of Purkinje cells.(ABSTRACT TRUNCATED AT 400 WORDS)