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Background and purpose: Specific and selective inhibitors for mGlu1 receptors are presently inadequate. A new generation of non-competitive mGlu1 antagonists with low nanomolar potencies is emerging. We evaluated two new compounds, YM-298198 and JNJ16259685, for effectiveness, potency and specificity for the first time in a brain slice preparation. Experimental approach: Patch-clamp recording of Purkinje neurones in cerebellar slices were obtained. The slow mGlu1mediated EPSP was used to establish a concentration-response curve. Fast excitatory synaptic inputs were tested for nonspecific effects. Key results: YM-298198 and JNJ16259685 inhibited the synaptic activation of mGlu1 in a concentration-dependent manner (IC50 values of 24 nM and 19 nM, respectively). The antagonists were slow to inhibit and to reverse on washout, probably due to their lipophilic nature. There were no non-specific effects on fast AMPA receptor-mediated synaptic transmission in the cerebellum. Conclusions and implications: These compounds are more than a thousand-fold more potent than previously available compounds. Their selectivity and specificity will be very useful for studying the role of mGlu1 receptors both in vitro and in vivo. British Journal of Pharmacology (2007) 151, 870–876; doi:10.1038/sj.bjp.0707286; published online 14 May 2007

CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) is frequently used to test for the involvement of mGlu1 receptors. Using whole-cell voltage recording from Purkinje cells in slices of rat cerebellum we find that CPCCOEt, at concentrations used to block mGlu1 receptors, causes an enhancement of the climbing fibre response. Application of alternative antagonists with activity at mGlu1 neither mimicked nor occluded the effects of CPCCOEt. Receptor antagonists demonstrated that this non-mGlu1 action of CPCCOEt was not mediated by other mGlu receptors or GABAB receptors. Voltage-clamped climbing fibre EPSCs are unaffected by CPCCOEt whilst application of a glutamate transport blocker did not occlude the CPCCOEt effect. This suggests that a postsynaptic voltage-dependent component of the complex climbing fibre response is the target. We have found no evidence for the involvement of the hyperpolarisation-activated current, Ih, and calcium-activated conductances. Voltage-gated sodium, calcium and potassium channels are possible targets with inhibition of a potassium channel the most likely. Awareness of this non-mGlu-mediated effect of CPCCOEt is likely to be important for the correct interpretation of its actions.

Excessive nitric oxide formation may contribute to the pathology occurring in diseases affecting central white matter. such as multiple sclerosis. The rat isolated optic nerve preparation was used to investigate the potential toxicity of the molecule towards such tissue. The nerves were exposed to a range of concentrations of different classes of nitric oxide donor for up to 23 h. with or without a subsequent period of recovery, and the damage assessed by quantitative histological methods. Degeneration of axons and macroglia occurred in a time- and concentration-dependent manner, the order of susceptibility being: axons > oligodendrocytes > astrocytes. Use of NONOate donors differing in half-life indicated that nitric oxide delivered in an enduring manner at relatively low concentration was more toxic than the same amount supplied rapidly at high concentration, The mechanism by which nitric oxide affects axons was studied using a donor [3-(n-propylamino)propylamine/NO adduct, PAPA/NO] with an intermediate half-life that produced selective axonopathy after a 2-h exposure (plus 2 h recovery). Axon damage was abolished if, during the exposure. Na+ or Ca2- was removed from the bathing medium or the sodium channel inhibitors tetrodotoxin or BW619C89 (sipatrigine) were added. In electrophysiological experiments, the donor elicited a biphasic depolarisation. The second. larger component (occurring after 7-10 min) was associated with a block of nerve conduction and could be inhibited by tetrodotoxin. Coincident with the secondary depolarisation was a reduction in ATP levels by about 50%, an effect that was also inhibited by tetrodotoxin.It is concluded that nitric oxide, in submicromolar concentrations, can kill axons and macroglia in white matter. The findings lend support to the hypothesis that nitric oxide may be of importance to white matter pathologies, particularly those in which inducible nitric oxide synthase is expressed. The axonopathy, at least when elicited over relatively short time intervals, is likely to be caused by metabolic inhibition. As in anoxia and anoxia/aglycaemia, nitric oxide-induced destruction of axons is likely to be caused by the Ca2+ overload that follows a reduction in ATP levels in the face of continued influx of Na+ through voltage-dependent channels. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Metabotropic glutamate (mGlu) receptors are located pre- and postsynaptically at central synapses. Activation of the receptors by exogenous agonists usually results in a reversible depression of fast glutamatergic neurotransmission. Evidence that synaptically released glutamate has such an action, however, is scarce. Sharp microelectrode recordings were used to investigate the modulatory role of mGlu receptors at a well-studied glutamatergic synapse, the one between parallel fibres and Purkinje cells in rat cerebellar slices. Brief, tetanic stimulation of the parallel fibres caused a depression of subsequent fast EPSPs. This posttetanic depression (PTD) reached its maximum 4.5 s after the tetanus. Measured at this point, PTD was frequency-dependent; 10 stimuli at 20 Hz produced no significant depression, whereas, at 100 Hz the same number of stimuli was maximally effective (~50% depression). The nonselective mGlu antagonist, (S)-a-methyl-4-carboxyphenylglycine 1 mM or the GABAB antagonist, CGP35348 (1 mM), both decreased the magnitude of the PTD. In the presence of CGP35348 the mGlu1 antagonist, 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (300 mM), inhibited PTD further. A group II/III mGlu antagonist had no effect. These observations indicate that synaptically activated mGlu1 receptors not only generate a slow EPSP and induce Ca 2+ mobilization in Purkinje cells, as reported previously, but also produce a transient depression of fast synaptic transmission. This short-term plasticity may be important for shaping the output of cerebellar circuits and/or it could provide a substrate for long-term depression when additional mechanisms are superimposed.

The actions of reportedly group-selective metabotropic glutamate (mGlu) receptor agonists and antagonists on neurotransmission at parallel fibre-Purkinje cell synapses in the rat cerebellum have been characterised using sharp microelectrode recording and an in vitro slice preparation. Application of the group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) or the group III selective agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) depressed synaptic transmission in a reversible and concentration-dependent manner (EC(50)=18 and 5 microM, respectively). The depression produced by DHPG was unrelated to the depolarisation observed in some Purkinje cells. The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV, 1 microM) had no effect. The effects of DHPG were inhibited by the group I-selective antagonist 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), but not by the group II/III antagonist alpha-methyl-4-phosphonophenylglycine (MPPG). The effect of L-AP4 was inhibited by MPPG, but not by the group I/II antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG). By themselves, the antagonists did not affect the EPSPs, suggesting that neither receptor is activated during low frequency neurotransmission. It is concluded that, in addition to the excitatory role for group I receptors described previously, both group I and III (but not group II) mGlu receptors operate at this synapse to inhibit synaptic transmission. The specific receptor subtypes involved are likely to be mGlu1 and mGlu4.

In the classical view, a central neuron integrates incoming synaptic information by simple algebraic summation of the resultant bioelectrical signals that coincide in time. The voltage dependence of the NMDA (N-methyl-D-aspartate) type of ionotropic glutamate receptor endows neurons with an additional tool that allows one synaptic input to influence another, providing, again, that the two are active simultaneously. Here we identify a new mechanism by which non-coincident signals generated by different synaptic inputs are integrated. The device serves to regulate neuronal excitation through G-protein-coupled, metabotropic glutamate receptors (mGluRs) in a powerful and specific manner. We show that, in cerebellar Purkinje cells, a single activation of the climbing-fibre input markedly potentiates mGluR-mediated excitation at parallel-fibre synapses. The potentiation results from a transient rise in cytosolic Ca2+ which is 'memorized' in such a way that it promotes excitation through mGluRs for about two minutes. A Ca2+-transient is also effective if imposed up to two seconds after parallel-fibre stimulation. By allowing temporally and spatially dispersed synaptic signals to be assimilated, this mechanism adds a new element to the computational power of central neurons.

Background: The biological actions of NO depend critically on its concentration, which is very difficult to measure. Results: Recorded using the most sensitive detectors yet described, NO levels synthesized by activated brain neurons generally were in the picomolar range but varied regionally. Conclusion: NO operates physiologically at the subnanomolar concentrations that selectively target its guanylyl cyclase-linked receptors. Significance: Physiological NO levels are far lower than commonly supposed., Nitric oxide (NO) is a widespread signaling molecule with potentially multifarious actions of relevance to health and disease. A fundamental determinant of how it acts is its concentration, but there remains a lack of coherent information on the patterns of NO release from its sources, such as neurons or endothelial cells, in either normal or pathological conditions. We have used detector cells having the highest recorded NO sensitivity to monitor NO release from brain tissue quantitatively and in real time. Stimulation of NMDA receptors, which are coupled to activation of neuronal NO synthase, routinely generated NO signals from neurons in cerebellar slices. The average computed peak NO concentrations varied across the anatomical layers of the cerebellum, from 12 to 130 pm. The mean value found in the hippocampus was 200 pm. Much variation in the amplitudes recorded by individual detector cells was observed, this being attributable to their location at variable distances from the NO sources. From fits to the data, the NO concentrations at the source surfaces were 120 pm to 1.4 nm, and the underlying rates of NO generation were 36–350 nm/s, depending on area. Our measurements are 4–5 orders of magnitude lower than reported by some electrode recordings in cerebellum or hippocampus. In return, they establish coherence between the NO concentrations able to elicit physiological responses in target cells through guanylyl cyclase-linked NO receptors, the concentrations that neuronal NO synthase is predicted to generate locally, and the concentrations that neurons actually produce.

Nitric oxide (NO) functions as a diffusible transmitter in most tissues of the body and exerts its effects by binding to receptors harboring a guanylyl cyclase transduction domain, resulting in cGMP accumulation in target cells. Despite its widespread importance, very little is known about how this signaling pathway operates at physiological NO concentrations and in real time. To address these deficiencies, we have exploited the properties of a novel cGMP biosensor, named δ-FlincG, expressed in cells containing varying mixtures of NO-activated guanylyl cyclase and cGMP-hydrolyzing phosphodiesterase activity. Responsiveness to NO, signifying a physiologically relevant rise in cGMP to 30 nM or more, was seen at concentrations as low as 1 pM, making cells by far the most sensitive NO detectors yet encountered. Even cells coexpressing phosphodiesterase-5, a cGMP-activated isoform found in many NO target cells, responded to NO in concentrations as low as 10 pM. The dynamics of NO capture and signal transduction was revealed by administering timed puffs of NO from a local pipette. A puff lasting only 100 ms, giving a calculated peak intracellular NO concentration of 23 pM, was detectable. The results could be encapsulated in a quantitative model of cellular NO-cGMP signaling, which recapitulates the NO responsiveness reported previously from crude cGMP measurements on native cells, and which explains how NO is able to exert physiological effects at extremely low concentrations, when only a tiny proportion of its receptors would be occupied.

Amino acid levels were measured in perfusates from biplanar slices of rat cerebellum installed in a Krebs-filled three-compartment chamber. The two lateral compartments housed the white matter and a section containing parallel fibres respectively. The central compartment housed cortical structures, including the Purkinje cell and granule cell bodies. This arrangement allows selective electrical stimulation of the parallel fibre or mossy fibre pathways, recording of the evoked responses to such stimulation and collection of the perfusion medium passing through the central chamber for amino acid analysis using high-pressure liquid chromatography (HPLC). Both, 2-Hz and 5-Hz stimulation of white matter caused a delayed increase in arginine levels in the perfusate. Since L-arginine is the physiological precursor of nitric oxide, a neuronal messenger in the brain, the data suggest that physiological stimuli can result in the release of this precursor, possibly to supply the nitric oxide synthase.

The mechanism of ischaemic injury to white matter axons was studied by transiently depriving rat optic nerves in vitro of oxygen and glucose. Light and electron microscopic analysis showed that increasing periods of oxygen/glucose deprivation (up to 1 h) caused, after a 90-min recovery period, the appearance of increasing numbers of swollen axons whose ultrastructure indicated that they were irreversibly damaged. This conclusion was supported by experiments showing that the damage persisted after a longer recovery period (3 h). To quantify the axonal pathology, an automated morphometric method, based on measurement of the density of swollen axons, was developed. Omission of Ca2+ from the incubation solution during 1 h of oxygen/glucose deprivation (and for 15 min either side) completely prevented the axonopathy (assessed following 90 min recovery). Omission of Na+ was also effective, though less so (70% protection). The classical Na+ channel blocker, tetrodotoxin (1 microM), provided 92% protection. In view of this evidence implicating Na+ channels in the pathogenesis of the axonal damage, the effects of three different Na+ channel inhibitors, with known neuroprotective properties towards gray matter in in vivo models of cerebral ischaemia, were tested. The compounds used were lamotrigine and the structurally-related molecules, BW619C89 and BW1003C87. All three compounds protected the axons to varying degrees, the maximal efficacies (observed at 30 to 100 microM) being in the order: BW619C89 (>95% protection) > BW1003C87 (70%) > lamotrigine (50%). At a concentration affording near complete protection (100 microM), BW619C89 had no significant effect on the optic nerve compound action potential. Experiments in which BW619C89 was added at different times indicated that its effects were exerted during two distinct phases, one (accounting for about 50% protection) was during the early stage of oxygen/glucose deprivation itself and the other (also about 50%) during the first 15 min of recovery in normal incubation solution. The results are consistent with a pathophysiological mechanism in which Na+ entry through tetrodotoxin-sensitive Na+ channels contributes to Na+ loading of the axoplasm which then results in a lethal Ca2+ overload through reversed Na(+)-Ca2+ exchange. The identification of BW619C89 as a compound able to prevent oxygen/glucose deprivation-induced injury to white matter axons without affecting normal nerve function opens the way to testing the importance of this pathway in white matter injury in vivo.

The N-methyl-D-aspartate (NMDA) receptor-mediated component of the synaptic response of cerebellar granule cells to mossy fibre stimulation was selectively inhibited by the nitric oxide (NO) donor, nitroprusside (nitroferricyanide). The direct depolarisation of granule cells induced by perfusion of NMDA was similarly reduced. However, these effects are unlikely to be caused by NO because, inter alia, they could not be replicated by other NO donors whereas ferricyanide was similarly effective.

The role of metabotropic glutamate receptors (mGluRs) in the mossy fibre-granule cell pathway in rat cerebellum was studied using slice preparations and electrophysiological techniques. Application of the group I selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) evoked, in a concentration-dependent manner (EC50 = 33 microM), a depolarising/hyperpolarising complex response from granule cells which was preferentially inhibited by the group I selective antagonist (S)-4-carboxyphenylglycine (4CPG). The group III selective agonist L-amino-4-phosphonobutyrate (AP4) evoked a hyperpolarising response (EC50 = 10 microM) which was inhibited by the group II/III selective antagonist (S)-alpha-methyl-4-phosphonophenylglycine (MPPG). The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine (DCG-IV) elicited no measurable voltage change. The amplitude of the synaptically-mediated mossy fibre response in granule cells was unaffected during application of AP4, was reduced by DHPG and was enhanced by DCG-IV (EC50 = 80 nM). These effects were inhibited by the group selective antagonists 4CPG and (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-4), respectively. Further investigation using patch-clamp recording revealed that DCG-IV potently inhibited spontaneous GABAergic currents. We conclude that group I and III (but not group II) mGluRs are functionally expressed by granule cells, whereas unexpectedly group II or III mGluRs do not appear to be present presynaptically on mossy fibre terminals. Group II mGluRs are located on Golgi cell terminals; when activated these receptors cause disinhibition, a function which may be important for gating information transfer from the mossy fibres to the granule cells.

Unlike certain other brain areas, notably the hippocampal formation, synaptic transmission in the cerebellum, a 3-dimensional anatomical structure, is not ideally studied in a 2-dimensional slice preparation. We describe a method for cutting a cerebellar slice that incorporates two planes of section: one sagittal (in the plane of the Purkinje cell dendrites and of afferent and efferent fibres running in the white matter), and the other in the plane of the parallel fibres (granule cell axons). Details are given of how neurotransmission in the parallel fibre-to-Purkinje cell pathway and in the mossy fibre-to-granule cell pathway can be studied by applying a specially designed grease-gap recording technique to the biplanar slice. We further demonstrate the utility of the slice for intracellular recording.

Publisher Summary Glutamate receptors can be broadly classified into the ionotropic glutamate receptors (iGluRs) and the metabotropic glutamate receptors (mGluRs). This chapter discusses the metabotropic glutamate receptors, their physiological functions, and their possible pathological roles as well. The metabotropic glutamate receptors are characterized by the observation that they exert their effects via a G-protein. Quisqualic acid remains the most potent agonist. Phenylglycines have been widely reported as antagonists at metabotropic glutamate receptors. Till date trans-1-arninocyclopentane-1,3-dicarboxylic acid (trans-ACPD) remains the only broad-spectrum mGluR agonist with no activity at iGluRs. The mGluRs have been divided into two main groups, according to the principal mode of second-messenger coupling identified in mammalian cells, expressing the respective mGluR sub-types. It is becoming increasingly clear that antagonist activity at mGluRs can often be achieved by the addition of an α-alkyl group to the amino acid moiety of a known agonist. The availability of cloned complementary DNA (cDNA) sequences for distinct receptor subtypes, via their expression in cell lines, has enabled the full characterization of mGluR ligands. In accordance with their ability to detect high frequency firing, mGluRs have been shown to participate in synaptic plasticity in several brain regions. It is early days for a full understanding of the capabilities of mGluRs in brain function, but already it is clear that they are an interesting target for the development of therapeutic compounds. However, the malfunctioning glutamate transmission has been implicated in many disorders of the nervous system, including epilepsy, ischemia, chronic pain, and slow degenerative disorders. The breadth of involvement of mGluRs in pathological functions that has already been elucidated can certainly fuel the search for new ligands.

Glutamate receptors of both the ionotropic (ion channel-linked) and metabotropic (enzyme-linked) categories are abundantly expressed by Purkenje cells in the cerebellum but the functional significance of the latter receptors is unknown. We have tested the possibility that they are activated by the parallel fibre input by recording from Purkinje cells within a biplanar cerebellar slice preparation using the grease-gap technique. Under conditions where ionotropic (NMDA and non-NMDA) glutamate and GABA receptors were blocked pharmacologically, electrical stimulation of parallel fibres gave rise to two very slow potentials. The first peaked about 400 msec from the start of stimulation and was depolarising. It was not evident with single stimuli but reached maximum amplitude after 6 shocks delivered at 50 Hz. The wave was abolished when the slices were perfused with Ca2+-free solution or with drugs that inhibit synaptic transmission, but it was resistant to blockade of GABAB receptors, acetylcholine receptors and adrenergic receptors. Next came a slow hyperpolarising potential that peaked about 30 sec after stimulation and which was also Ca2+-dependent. The sequence of potentials was replicated by perfusion of an exogenous agonist acting selectively on metabotropic glutamate receptors. We conclude that parallel fibre-to-Purkinje cell synaptic transmission involves not only fast signals generated through ionotropic non-NMDA receptors but also much slower potentials that are likely to be mediated by metabotropic glutamate receptors. These potentials are likely to be significant both for short-term (seconds to minutes) Purkinje cell excitability as well as for the induction of longer-term synaptic plasticity.

Binding studies indicate that the molecular layer of the cerebellum has a high concentration of gamma-aminobutyric acid type B (GABAB) receptors. In order to elucidate the function of these receptors we have recorded from Purkinje cells in biplanar slices of immature (14-day-old) and adult rat cerebellum using a low-noise, non-invasive, gap technique. The responses of Purkinje cells to parallel fibre stimulation in slices from both immature and adult rats contained a wave that could be inhibited by the GABAA antagonist, bicuculline. In slices from immature animals, application of 30 - 50 microM bicuculline revealed a slow (400 ms to peak) and very long-lasting (up to 1 s) hyperpolarizing wave which was inhibited by GABAB antagonists. Activation of GABAB receptors on Purkinje cells with an exogenous agonist, baclofen, also generated a hyperpolarization. Baclofen additionally inhibited the synaptic potentials generated in Purkinje cells on stimulating parallel fibres, an effect which could be reversed by GABAB antagonists. The potency of baclofen in this respect was similar in adult and immature tissue but another excitatory pathway in the cerebellar cortex, the mossy fibre to granule cell synapse, proved to be much less sensitive. We conclude that, at least in the immature rat, there are GABAB receptors on Purkinje cell dendrites and that these receptors can be activated following parallel fibre stimulation; there are also GABAB receptors on presynaptic terminals within the molecular layer of immature and adult cerebellum that, when stimulated, inhibit transmitter release.

The mGluR-mediated EPSP at parallel fibre-Purkinje cell synapses in the cerebellum was blocked concentration-dependently and reversibly by antagonists acting selectively on group-I mGluRs but not by an inhibitor of group-III receptors. The results provide pharmacological evidence that the receptor type responsible for the mGluR-EPSP is mGluR1.