Quantification of the density of cooperative neighboring synapses required to evoke endocannabinoid signaling.

The spatial pattern of synapse activation may impact on synaptic plasticity. This applies to the synaptically-evoked endocannabinoid-mediated short-term depression at the parallel fiber (PF) to Purkinje cell synapse, the occurrence of which requires close proximity between the activated synapses. Here, we determine quantitatively this required proximity, helped by the geometrical organization of the cerebellar molecular layer. Transgenic mice expressing a calcium indicator selectively in granule cells enabled the imaging of action potential-evoked presynaptic calcium rise in isolated, single PFs. This measurement was used to derive the number of PFs activated within a beam of PFs stimulated in the molecular layer, from which the density of activated PFs (input density) was calculated. This density was on average 2.8 µm(-2) in sagittal slices and twice more in transverse slices. The synaptically-evoked endocannabinoid-mediated suppression of excitation (SSE) evoked by ten stimuli at 200 Hz was determined from the monitoring of either postsynaptic responses or presynaptic calcium rise. The SSE was significantly larger when recorded in transverse slices, where the input density is larger. The exponential description of the SSE plotted as a function of the input density suggests that the SSE is half reduced when the input density decreases from 6 to 2 µm(-2). We conclude that, although all PFs are truncated in an acute sagittal slice, half of them remain respondent to stimulation, and activated synapses need to be closer than 1.5 µm to synergize in endocannabinoid signaling.

GABAergic synaptic communication in the GABAergic and non-GABAergic cells in the deep cerebellar nuclei.

The deep cerebellar nuclei (DCN) are the final integrative units of the cerebellar network. The strongest single afferent to the DCN is formed by GABAergic Purkinje neuron axons whose synapses constitute the majority of all synapses in the DCN, with their action strongly regulating the intrinsic activity of their target neurons. Although this is well established, it remains unclear whether all DCN cell groups receive a functionally similar inhibitory input. We previously characterized three types of mouse DCN neurons based on the expression of glutamic acid decarboxylase isoform 67 (GAD67), their active membrane properties and morphological features. Here we describe the GABAergic synapses in these cell groups and show that spontaneous GABAergic synaptic activity can be seen in all three cell types. Since the majority of DCN neurons fire action potentials spontaneously at high frequencies both in vivo and in vitro, we expected that spontaneous GABAergic synaptic activities mediated by intra-DCN synaptic connections could be uncovered by their sensitivity to TTX. However, TTX had little effect on spontaneous synaptic activity. It seems, therefore that functional GABAergic connectivity within the DCN is sparse and/or weak at least under our experimental conditions. Even though present in all cell types, the spontaneous GABAergic events showed significant differences between the cell types. The synaptic currents in GABAergic cells had lower amplitude, lower frequency and slower kinetics than those of non-GABAergic cells. These differences could not be sufficiently explained by considering only cell size differences or a differential GABA(A)-receptor alpha-subunit composition. Rather, the main differentiating factor appears to be the dendritic localization of GABAergic synapses in the GABAergic cells.

Subcellular localization of the voltage-gated potassium channels Kv3.1b and Kv3.3 in the cerebellar dentate nucleus of glutamic acid decarboxylase 67-green fluorescent protein transgenic mice.

Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD- and GAD+ dentate neurons of glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD- and GAD+ dentate neurons. The vast majority of GAD- somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD- axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD- somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD- cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD- and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD- and GAD+ neurons in the dentate nucleus.

Genetically encoded fluorescent sensors of membrane potential.

Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.

Two new non-competitive mGlu1 receptor antagonists are potent tools to unravel functions of this mGlu receptor subtype.

The validation of the selective, potent and systemically active non-competitive mGlu1 antagonists YM-298198 and JNJ16259685 in a physiological functional assay will facilitate elucidation of this receptor's role in brain function and as a potential drug target.

Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells.

Three first-generation fluorescent protein voltage sensitive probes (FP-voltage sensors) were characterized in mammalian cells. Flare, a Kv1.4 variant of FlaSh [Siegel MS, Isacoff EY. Neuron 1997;19(October (4)):735-41], SPARC [Ataka K, Pieribone VA. Biophys J 2002;82(January (1 Pt 1)):509-16], and VSFP-1 [Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T. Eur J Neurosci 2001;13(June (12)):2314-18] were expressed, imaged and voltage clamped in HEK 293 cells and in dissociated hippocampal neurons. We were unable to detect a signal in response to changes in membrane potential after averaging16 trials with any of the three constructs. Using the hydrophobic voltage sensitive dye, di8-ANEPPS, as a surface marker, confocal analyses demonstrated poor plasma membrane expression for Flare, SPARC and VSFP-1 in both HEK 293 cells and dissociated hippocampal neurons. Almost all of the expressed FP-voltage sensors reside in internal membranes in both cell types. This internal expression generates a background fluorescence that increases the noise in the optical measurement.

Behavioral motor dysfunction in Kv3-type potassium channel-deficient mice.

The voltage-gated potassium channels Kv3.1 and Kv3.3 are expressed in several distinct neuronal subpopulations in brain areas known to be involved in motor control such as cortex, basal ganglia and cerebellum. Depending on the lack of Kv3.1 or Kv3.3 channel subunits, mutant mice show different Kv3-null allele-dependent behavioral alterations that include constitutive hyperactivity, sleep loss, impaired motor performance and, in the case of the Kv3.1/Kv3.3 double mutant, also severe ataxia, tremor and myoclonus (Espinosa et al. 2001, J Neurosci 21, 6657-6665, Genes, Brain Behav 3, 90-100). The lack of Kv3.1 channel subunits is mainly responsible for the constitutively increased locomotor activity and for sleep loss, whereas the absence of Kv3.3 subunits affects cerebellar function, in particular Purkinje cell discharges and olivocerebellar system properties (McMahon et al. 2004, Eur J Neurosci 19, 3317-3327). Here, we describe two sensitive and non-invasive tests to reliably quantify normal and abnormal motor functions, and we apply these tests to characterize motor dysfunction in Kv3-mutant mice. In contrast to wildtype and Kv3.1-single mutants, Kv3.3-single mutants and Kv3 mutants lacking three and four Kv3 alleles display Kv3-null allele-dependent gait alterations. Although the Kv3-null allele-dependent gait changes correlate with reduced motor performance, they appear to not affect the training-induced improvement of motor performance. These findings suggest that altered cerebellar physiology in the absence of Kv3.3 channels is responsible for impaired motor task execution but not motor task learning.

Olfactory nerve stimulation-evoked mGluR1 slow potentials, oscillations, and calcium signaling in mouse olfactory bulb mitral cells.

Fast synaptic transmission between olfactory receptor neurons and mitral cells (MCs) is mediated through AMPA and NMDA ionotropic glutamate receptors. MCs also express high levels of metabotropic glutamate receptor 1 (mGluR1) whose functional significance is less understood. Here we characterized a slow mGluR1-mediated potential that was evoked by high-frequency (100-Hz) olfactory nerve (ON) stimulation in the presence of NBQX and D-APV, blockers of ionotropic glutamate receptors, and that was associated with a local Ca2+ transient in the MC dendritic tuft. High-frequency ON stimulation in the presence of NBQX and D-APV also evoked a slow, nearly 2-Hz oscillation of MC membrane potential that was abolished by the mGluR1 antagonist LY367385 (50 microM). Both mGluR slow potential and slow oscillation persisted in the presence of gabazine (10 microM), a GABA(A) receptor antagonist, and intracellular QX-314 (10 mM), a Na+ channel blocker. In contrast to a slow mGluR1 potential in cerebellar Purkinje neurons, the MC mGluR1 potential was not depressed by SKF96365 (< or =250 microM) and thus is likely not mediated by TRPC1 cation channels, nor was it potentiated by an elevation of intracellular Ca2+ level. Imaging with the Na+ indicator SBFI revealed a Na+ transient in the MC dendrite accompanying the mGluR1 slow potential. We conclude that the MC mGluR1 potential triggered by glutamate released from the ON supports oscillations and synchronizations of MCs associated within one glomerulus.

Olfactory nerve stimulation-induced calcium signaling in the mitral cell distal dendritic tuft.

Olfactory receptor neuron axons form the olfactory nerve (ON) and project to the glomerular layer of the olfactory bulb, where they form excitatory synapses with terminal arborizations of the mitral cell (MC) tufted primary dendrite. Clusters of MC dendritic tufts define olfactory glomeruli, where they involve in complex synaptic interactions. The computational function of these cellular interactions is not clear. We used patch-clamp electrophysiology combined with whole field or two-photon Ca2+ imaging to study ON stimulation-induced Ca2+ signaling at the level of individual terminal branches of the MC primary dendrite in mice. ON-evoked subthreshold excitatory postsnaptic potentials induced Ca2+ transients in the MC tuft dendrites that were spatially inhomogeneous, exhibiting discrete "hot spots." In contrast, Ca2+ transients induced by backpropagating action potentials occurred throughout the dendritic tuft, being larger in the thin terminal dendrites than in the base of the tuft. Single ON stimulation-induced Ca2+ transients were depressed by the NMDA receptor antagonist D-aminophosphonovaleric acid (D-APV), increased with increasing stimulation intensity, and typically showed a prolonged rising phase. The synaptically induced Ca2+ signals reflect, at least in part, dendrodendritic interactions that support intraglomerular coupling of MCs and generation of an output that is common to all MCs associated with one glomerulus.

Functional topology of the mossy fibre-granule cell--Purkinje cell system revealed by imaging of intrinsic fluorescence in mouse cerebellum.

We report an activity-induced green fluorescence signal observed when mouse cerebellar slices were illuminated with blue light and parallel fibre-Purkinje cell synapses were activated. The optical signal consisted of an initial increase in fluorescence that peaked within 1-2 s after the onset of stimulation, followed by a long lasting (40 s) transient decrease in fluorescence. Single or tetanic electrical stimuli applied to the molecular layer elicited 'beam-shaped' fluorescence changes along the trajectory of parallel fibres. These signals reported activation of Purkinje cells as they were depressed by antagonists of ionotropic and metabotropic glutamate receptors at Purkinje cells and correlated with Purkinje cell spiking activity. Optical responses induced by direct pharmacological activation of glutamate receptors were reduced by a calcium-free extracellular medium, consistent with the hypothesis that they reflect metabolic activity due to an increased intracellular calcium load associated with neuronal activation. We used these intrinsic fluorescence signals to address the question of whether granule cells excite Purkinje cells only locally via the ascending branches of their axons, or more widespread along the parallel fibre trajectory. White matter stimulation of the mossy fibres also elicited a beam-like fluorescence change along the trajectory of parallel fibres. Simultaneous imaging and extracellular recording demonstrated the association between the beam-like fluorescence signal and Purkinje cell spiking. This non-invasive imaging technique supports the notion that parallel fibre activity, evoked either locally or through the mossy fibre-granule cell pathway, can activate postsynaptic Purkinje cells along more than 3 mm of the parallel fibre trajectory.

Subcellular localization of the voltage-dependent potassium channel Kv3.1b in postnatal and adult rat medial nucleus of the trapezoid body.

A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held-globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held-principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.

Glutamate uptake controls expression of a slow postsynaptic current mediated by mGluRs in cerebellar Purkinje cells.

At the cerebellar parallel fiber-Purkinje cell synapse, isolated presynaptic activity induces fast excitatory postsynaptic currents via ionotropic glutamate receptors while repetitive, high-frequency, presynaptic activity can also induce a slow excitatory postsynaptic current that is mediated by metabotropic glutamate receptors (mGluR1-EPSC). Here we investigated the involvement of glutamate uptake in the expression of the mGluR1-EPSC. Inhibitors of glutamate uptake led to a large increase of the mGluR1-EPSC. D-aspartate (0.4 mM) and L(-)-threo-3-hydroxyaspartate (0.4 mM) increased the mGluR1-EPSC approximately 4.5 and approximately 9-fold, respectively, while dihydrokainic acid (1 mM), had no significant effect on the mGluR1-EPSC. D-aspartate (0.4 mM) shifted the concentration-response curve of the depression of the mGluR1-EPSC by the low-affinity mGluR1 antagonist (S)-a-Methyl-4-carboxyphenylglycine [(S)-MCPG] to higher concentrations and decreased the stimulus intensity and the number of necessary stimuli to evoke an mGluR1-EPSC. Depression of the mGluR1-EPSC by rapid pressure application of (S)-MCPG at varying time intervals after tetanic stimulation of the parallel fibers indicated that the glutamate concentration in the peri- and extrasynaptic space decayed with time constants of 36 and 316 ms under control conditions and with inhibition of glutamate uptake, respectively. These results show that expression of the slow mGluR-mediated excitatory postsynaptic current is controlled by glutamate transporter activity. Thus in contrast to fast glutamatergic synaptic transmission, metabotropic glutamate receptor-mediated transmission is critically dependent on the activity and capacity of glutamate uptake.

Imaging postsynaptic activities of teleost thalamic neurons at single cell resolution using a voltage-sensitive dye.

Optical recording of neuronal activities using voltage-sensitive dyes (VSDs) is a useful method for simultaneous multi-site recording. However, it has been rather difficult to distinguish optical signals from individual, identified cells. We applied the optical recording technique using a high-speed charge coupled device (CCD) imaging system to a teleost thalamic nucleus, corpus glomerulosum (CG) which has a well-defined histological organization and large postsynaptic dendrites. Patch-like dye (di-4-ANEPPS) signals were observed in the dendritic layer of the CG in response to afferent nerve stimulations. These responses were completely blocked by an alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA) receptor antagonist, did not propagate, and the size of the patches were close to that of a single dendritic tip of the 'large cell'. Thus, we found that these patch-like VSD signals most likely represent postsynaptic potentials at individual dendritic tips of the large cells.

Functional characterization of permuted enhanced green fluorescent proteins comprising varying linker peptides.

New variants of green fluorescent protein (GFP) can be engineered by circular permutation of their amino acid sequence. We characterized a series of permuted enhanced GFP (PEGFP) with new termini introduced at N144-Y145 and linkers of 1, 3, 5 and 6 residues inserted between G232 and M1, as well as a variant with an extended 7-residues linker between K238 and M1. A minimum linker length of 3 residues was necessary for a functional chromophore to be formed, and linkers exceeding 4 residues yielded almost the same fluorescence quantum yield as enhanced GFP (EGFP). PEGFP exhibited dual-wavelength absorption and fluorescence excitation with peaks at 395 and 490 nm but single-wavelength emission at 512 nm. Fluorescence emission increased with increasing pH for all excitation wavelengths with a pKa of 7.7. Between the pH values of 6 and 8 optical absorption showed an isobestic point at 445 nm. PEGFP rapidly denatured in urea between 50 and 60 degrees C. Renaturation proceeded with a short (approximately 29 s) and a longer (> 150 s) time constant. Transient transfection of HEK293 and HeLa cells revealed the expression dynamics of PEGFP to be similar to that of EGFP. Laser-scanning microscopy of HeLa cells demonstrated that the PEGFP are particularly well suited as fluorescent indicators in two-photon imaging.

Characterization of the mGluR(1)-mediated electrical and calcium signaling in Purkinje cells of mouse cerebellar slices.

The metabotropic glutamate receptor 1 (mGluR(1)) plays a fundamental role in postnatal development and plasticity of ionotropic glutamate receptor-mediated synaptic excitation of cerebellar Purkinje cells. Synaptic activation of mGluR(1) by brief tetanic stimulation of parallel fibers evokes a slow excitatory postsynaptic current and an elevation of intracellular calcium concentration ([Ca2+](i)) in Purkinje cells. The mechanism underlying these responses has not been identified yet. Here we investigated the responses to synaptic and direct activation of mGluR(1) using whole cell patch-clamp recordings in combination with microfluorometric measurements of [Ca2+](i) in mouse Purkinje cells. Following pharmacological block of ionotropic glutamate receptors, two to six stimuli applied to parallel fibers at 100 Hz evoked a slow inward current that was associated with an elevation of [Ca2+](i). Both the inward current and the rise in [Ca2+](i) increased in size with increasing number of pulses albeit with no clear difference between the minimal number of pulses required to evoke these responses. Application of the mGluR(1) agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) by means of short-lasting (5-100 ms) pressure pulses delivered through an agonist-containing pipette positioned over the Purkinje cell dendrite, evoked responses resembling the synaptically induced inward current and elevation of [Ca2+](i). No increase in [Ca2+](i) was observed with inward currents of comparable amplitudes induced by the ionotropic glutamate receptor agonist AMPA. The 3,5-DHPG-induced inward current but not the associated increase in [Ca2+](i) was depressed when extracellular Na+ was replaced by choline, but, surprisingly, both responses were also depressed when bathing the tissue in a low calcium (0.125 mM) or calcium-free/EGTA solution. Thapsigargin (10 microM) and cyclopiazonic acid (30 microM), inhibitors of sarco-endoplasmic reticulum Ca2+-ATPase, had little effect on either the inward current or the elevation in [Ca2+](i) induced by 3,5-DHPG. Furthermore, the inward current induced by 3,5-DHPG was neither blocked by 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy] ethyl-1H-imidazole, an inhibitor of store operated calcium influx, nor by nimodipine or omega-agatoxin, blockers of voltage-gated calcium channels. These electrophysiological and Ca2+-imaging experiments suggest that the mGluR(1)-mediated inward current, although mainly carried by Na+, involves influx of Ca2+ from the extracellular space.

Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein.

Optical imaging of electrical activity has been suggested as a promising approach to investigate the multineuronal representation of information processing in brain tissue. While considerable progress has been made in the development of instrumentation suitable for high-speed imaging, intrinsic or extrinsic dye-mediated optical signals are often of limited use due to their slow response dynamics, low effective sensitivity, toxicity or undefined cellular origin. Protein-based and DNA-encoded voltage sensors could overcome these limitations. Here we report the design and generation of a voltage-sensitive fluorescent protein (VSFP) consisting of a voltage sensing domain of a potassium channel and a pair of cyan and yellow emitting mutants of green fluorescent protein (GFP). In response to a change in transmembrane voltage, the voltage sensor alters the amount of fluorescence resonance energy transfer (FRET) between the pair of GFP mutants. The optical signals respond in the millisecond time-scale of fast electrical signalling and are large enough to allow monitoring of voltage changes at the single cell level.

The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1.

G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or beta-adrenergic receptor kinase (beta ARK, GRK2), no receptor substrate for GRK4 has been so far identified. Here we show that GRK4 is expressed in cerebellar Purkinje cells, where it regulates mGlu(1) metabotropic glutamate receptors, as indicated by the following: 1) When coexpressed in heterologous cells (HEK293), mGlu(1) receptor signaling was desensitized by GRK4 in an agonist-dependent manner (homologous desensitization). 2) In transfected HEK293 and in cultured Purkinje cells, the exposure to glutamate agonists induced internalization of the receptor and redistribution of GRK4. There was a substantial colocalization of the receptor and kinase both under basal condition and after internalization. 3) Kinase activity was necessary for desensitizing mGlu(1a) receptor and agonist-dependent phosphorylation of this receptor was also documented. 4) Antisense treatment of cultured Purkinje cells, which significantly reduced the levels of GRK4 expression, induced a marked modification of the mGlu(1)-mediated functional response, consistent with an impaired receptor desensitization. The critical role for GRK4 in regulating mGlu(1) receptors implicates a major involvement of this kinase in the physiology of Purkinje cell and in motor learning.

Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex.

In the rodent primary somatosensory cortex, the configuration of whiskers and sinus hairs on the snout and of receptor-dense zones on the paws is topographically represented as discrete modules of layer IV granule cells (barrels) and thalamocortical afferent terminals. The role of neural activity, particularly activity mediated by NMDARs (N-methyl-D-aspartate receptors), in patterning of the somatosensory cortex has been a subject of debate. We have generated mice in which deletion of the NMDAR1 (NR1) gene is restricted to excitatory cortical neurons, and here we show that sensory periphery-related patterns develop normally in the brainstem and thalamic somatosensory relay stations of these mice. In the somatosensory cortex, thalamocortical afferents corresponding to large whiskers form patterns and display critical period plasticity, but their patterning is not as distinct as that seen in the cortex of normal mice. Other thalamocortical patterns corresponding to sinus hairs and digits are mostly absent. The cellular aggregates known as barrels and barrel boundaries do not develop even at sites where thalamocortical afferents cluster. Our findings indicate that cortical NMDARs are essential for the aggregation of layer IV cells into barrels and for development of the full complement of thalamocortical patterns.

Elevation of intradendritic sodium concentration mediated by synaptic activation of metabotropic glutamate receptors in cerebellar Purkinje cells.

Cerebellar Purkinje cells express both ionotropic glutamate receptors and metabotropic glutamate receptors. Brief tetanic stimulation of parallel fibers in rat and mouse cerebellar slices evokes a slow excitatory postsynaptic current in Purkinje cells that is mediated by the mGluR1 subtype of metabotropic glutamate receptors. The effector system underlying this mGluR1 EPSC has not yet been identified. In the present study, we recorded the mGluR1 EPSC using the whole-cell patch-clamp technique in combination with microfluorometric recordings of the intracellular sodium concentration ([Na+]i) by means of the fluorescent sodium indicator SBFI. The mGluR1 EPSC was induced by local parallel fibre stimulation in the presence of the ionotropic glutamate receptor antagonists NBQX and D-APV and the GABAA receptor antagonists bicuculline or picrotoxin. The mGluR1 EPSC was associated with an increase in [Na+]i that was restricted to a specific portion of the dendritic tree. The mGluR1 EPSC as well as the increase in [Na+]i were inhibited by the mGluR antagonist S-MCPG. In the presence of NBQX, D-APV, pictrotoxin and TTX, bath application of the selective mGluR agonist 3,5-DHPG induced an elevation in [Na+]i which extended over the whole dendritic field of the Purkinje cell. This finding demonstrates that the mGluR1-mediated postsynaptic current leads to a significant influx of sodium into the dendritic cytoplasm of Purkinje cells and thereby provides a novel intracellular signalling mechanism that might be involved in mGluR1-dependent synaptic plasticity at this synapse.

Selective blockade of mGlu5 metabotropic glutamate receptors protects rat hepatocytes against hypoxic damage.

Western blot analysis of protein extracts from rat liver revealed the presence of the mGlu5 receptor, one of the G-protein-coupled receptors activated by glutamate (named "metabotropic glutamate receptors" or mGlu receptors). mGlu5 expression was particularly high in extracts from isolated hepatocytes, where levels were comparable with those seen in the rat cerebral cortex. The presence of mGlu5 receptors in hepatocytes was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) analysis, immunohistochemistry in neonate or adult rat liver, as well as by immunocytochemical analysis in HepG2 hepatoma cells, where the receptor appeared to be preferentially distributed in cell membranes. Interestingly, mGlu1 receptors (which are structurally and functionally homologous to mGlu5 receptors) were never found in rat liver or hepatocytes. In hepatocytes exposed to anoxic conditions for 90 minutes, glutamate, (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD) and quisqualate, which all activate mGlu5 receptors, accelerated the onset and increased the extent of cell damage, while 4-carboxy-3-hydroxyphenylglycine (4C3HPG), an agonist of mGlu2/3 receptors, was inactive. 2-methyl-6-(2-phenyl-1-ethynyl)-pyridine (MPEP), a novel, noncompetitive, highly selective mGlu5 receptor antagonist, not only abolished the toxic effect of 1S,3R-ACPD, but, unexpectedly, was protective by itself against anoxic damage. This suggests that hepatocytes express mGlu5 receptors and that activation of these receptors by endogenous glutamate facilitates the development of anoxic damage in hepatocytes.