Kainate receptors in epilepsy and excitotoxicity.

Kainate (KA), an analog of glutamate, is a potent neurotoxin that has long been known to induce behavioral and electrophysiological seizures as well as neuropathological lesions reminiscent of those found in patients with temporal lobe epilepsy. More than a decade after the initial KA studies, molecular cloning of ionotropic glutamate receptors identified a family of receptors that binds KA with high affinity. The present review explores the links between the epileptogenic and excitotoxic actions of KA and the function of kainate receptors (KARs) in the activity of neuronal networks. We first present evidence that KARs are the main targets of KA to produce the epileptogenic and excitotoxic effects of KA and KA analogs, and we discuss the mechanisms of action of KA. Then the review evaluates the involvement of KARs activated by the endogenous agonist glutamate in the generation and propagation of epileptiform activity. Finally, we report recent findings proposing KARs as targets of antiepileptic drugs and neuroprotective agents.

Potentiation of nicotinic receptor response by external calcium in rat central neurons.

Nicotinic acetylcholine receptor (nAChR) responses of rat medial habenular neurons are potentiated up to 3.5-fold by increasing the concentration of external Ca2+ in the millimolar range. This effect, independent of voltage, is probably due to the binding of Ca2+ to an external site. External Ca2+ decreases nAChR single-channel conductance at negative but not positive potentials, and it markedly enhances the frequency of opening of acetylcholine activated channels. The potentiating effect of Ca2+ is mimicked by Ba2+ and Sr2+, but barely by Mg2+. These data support the existence of positively acting allosteric sites for Ca2+, distinct from those involved in the decrease of single-channel conductance. A model in which external Ca2+ changes the properties of activation of the nAChR appears consistent with these data.

Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation.

The Ca2+ permeability of a nicotinic acetylcholine receptor (nAChR) in the rat CNS was determined using both current and fluorescence measurements on medial habenula neurons. The elementary slope conductance of the nAChR channel was 11 pS in pure external Ca2+ (100 mM) and 42 pS in standard solution. Ca2+ influx through nAChRs resulted in the rise of cytosolic Ca2+ concentration ([Ca2+]i) to the micromolar range. This increase was maximal under voltage conditions (below -50 mV) in which Ca2+ influx through voltage-activated channels was minimal. Ca2+ influx through nAChRs directly activated a Ca(2+)-dependent Cl- conductance. In addition, it caused a decrease in the GABAA response that outlasted the rise in [Ca2+]i. These results underscore the physiological significance of Ca2+ influx through nAChR channel in the CNS.

Subunit composition of kainate receptors in hippocampal interneurons.

Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function.

Functional GluR6 kainate receptors in the striatum: indirect downregulation of synaptic transmission.

Kainate receptors (KARs) are abundantly expressed in the basal ganglia, but their function in synaptic transmission has not been established. In the present study, we show that the GluR6 subunit of KARs is expressed in both substance P- and enkephalin-containing GABAergic projection neurons of the mouse striatum. Using whole-cell voltage-clamp recordings in brain slices, we demonstrate the presence of functional KARs in the dorsal striatum activated by low concentrations of the AMPA/KAR agonist domoate in wild-type but not GluR6-deficient mice. Despite the abundance of KARs, we found no evidence for synaptic activation of these receptors after single or repetitive stimulation of glutamatergic afferents. Domoate induces a transient increase in the frequency of spontaneous IPSCs of small amplitude and a sustained depression of large IPSCs evoked by minimal electrical stimulation within the striatum in wild-type mice but not in GluR6-deficient mice. This depressant effect is inhibited in presence of adenosine A(2A) receptor antagonists, ZM-241385 and SCH-58261. These data strongly suggest that, in striatal neurons, KARs depress GABAergic synaptic transmission indirectly via release of adenosine acting on A(2A) receptors.

Spatial distribution of kainate receptor subunit mRNA in the mouse basal ganglia and ventral mesencephalon.

In an attempt to gain knowledge of the possible functions of kainate receptors, we have used in situ hybridization to examine the regional and cellular expression patterns of glutamate receptor subunits GluR5-7, KA1 and KA2 in the adult mouse basal ganglia, known to play a pivotal role in the translation of motivation into actions. Kainate receptor subunits were found to be differentially expressed in the circuitry forming the basal ganglia. They differ from each other in expression levels and their spatial localization. GluR6 appeared as the key subunit for the descending gamma-aminobutyric acid (GABA)ergic-glutamatergic pathways, with highest message levels in the caudate putamen, globus pallidus and subthalamic nucleus as well as in the nucleus accumbens and olfactory tubercle. GluR7 exhibited highest expression in the ascending nigrostriatal and mesolimbic dopaminergic neurons. GluR5 had a restricted distribution pattern, with high expression in the ventral pallidum, the islands of Calleja and pars compacta of the substantia nigra. KA2 was usually coexpressed with GluR6, although with a generally lower level of expression. Finally, KA1 mRNA was barely detectable in these neuronal circuits. These data suggest that kainate receptors in general may be involved in the functions associated with the basal ganglia, with a key role in the control of the central dopaminergic transmission. Thus, they might be implicated in the neurodegenerative and psychic disorders associated with an impairment of the basal ganglia.

Absence of spindle oscillations in the cat anterior thalamic nuclei.

Intracellular recordings were performed in cat anterior thalamic nuclei (anterior ventral, medial and dorsal) which, according to a recent anatomical study, do not receive afferents from the reticularis thalami nucleus. Neurons of anterior nuclei did not display spontaneous membrane potential oscillations of the spindle type and such oscillations could neither be evoked in these cells by cortical stimulation. The absence of spontaneous and evoked spindling activity was observed despite that anterior thalamic cells displayed intrinsic membrane properties similar to those of thalamocortical cells in other nuclei. Electrographic recordings from cortical areas connected to anterior thalamic nuclei were also free of spindle activity. Taken together with the evidence that thalamic relay neurons deprived from their reticularis input by sections or kainic acid lesions lose their spindling activity, our results point to the essential role of the reticularis complex in the genesis of thalamic spindle waves.

The effects of QX314 on thalamic neurons.

The effects of QX314 were studied in cat thalamic neurons recorded in vivo. Besides blocking Na+ spike electrogenesis, QX314 transformed spindle oscillations into a single long-lasting period of hyperpolarization that terminated with a rebound Ca2+ spike. Large sustained injections of the drug resulted in the occurrence of numerous fast prepotentials that had the characteristics of attenuated dendritic Ca2+ spikes. These effects are interpreted as resulting from the blockage by QX314 of a persistent Na+ current and a delayed rectifier K+ current which currents have already been disclosed in thalamic neurons recorded in vitro.

Peripheral maps and synapse elimination in the cerebellum of the rat. I. Representation of peripheral inputs through the climbing fiber pathway in the posterior vermis of the normal adult rat.

The present study gives a detailed description of the functional characteristics and of the topographic distribution of responses, mediated through the climbing fiber pathway and elicited by electrical stimulation of several peripheral inputs, in a circumscribed region of the posterior vermis of the rat cerebellum. Experiments were carried out on normal adult rats under urethane anaesthesia. Purkinje cells (PCs) which responded to the electric stimulation of the contralateral snout, of the ipsilateral and contralateral hindlimb or forepad, and of the tail were recorded in an area extending 1000 microns laterally to the midline in the vermal part of lobules VII and VIII. Using precise micromapping techniques and computer analysis, we located the cells on the map of the unfolded PC layer. Taking into account the mean latency of the responses and the probability of discharge of the PCs, restricted areas of projection were found for the snout, the forepads and the tail. Zones of short-latency responses form compact patches of less than 1 mm2. There was some overlap of projection zones from tail and snout and from forepads and snout. In these zones, there was a convergence of several peripheral inputs on some of the PCs tested. No precise projection of the hindlimbs could be detected in the same lobules. These results fit well with the hypothesis already proposed that the representation of peripheral inputs through the climbing fiber pathway is fractured into a mosaic of patches, which are partly overlapping, and in which remote parts of the body are represented in adjacent areas.

Peripheral maps and synapse elimination in the cerebellum of the rat. II. Representation of peripheral inputs through the climbing fiber pathway in the posterior vermis of X-irradiated adult rats.

The present study gives a detailed description of the functional characteristics and of the topographic distribution of responses mediated through the climbing fiber (CF) pathway in the cerebellum of adult rats which have been repeatedly irradiated from birth so that the Purkinje cells (PCs) in the adult remained innervated by several climbing fibers instead of one. Experiments were carried out under urethane anaesthesia. After electrical stimulation of the contralateral snout, the ipsilateral and contralateral hindlimb or forepad, and the tail, CF-EPSPs were recorded from PCs in an area extending 1000 microns laterally to the midline in the vermal part of lobules VII and VIII. The stepwise variation of CF-EPSP amplitude demonstrated the multiple innervation of the PCs by CFs in these animals. The responses of a given PC through separate CFs were analyzed separately and, using precise micromapping techniques and computer analysis, cells were located on the map of the unfolded PC layer. Taking into account the mean latency of the responses and the probability of discharge of the PCs, restricted areas of projection were found for the snout, the forepads and the tail. Zones of short-latency responses formed compact patches of approximately 1 mm2. Their disposition was very similar to that found in normal rats. The responses evoked through the different CFs converging on a given PC were in general very homogeneous and very similar to those recorded in control rats. There was some overlap of projection zones from tail and snout and from forepads and snout. In this latter case, there was a convergence of several peripheral inputs on some of the PCs tested. No precise projection of the hindlimbs could be detected in the same lobules. These results suggest that, with the type of stimulation used, the representation of peripheral inputs through CF pathway is roughly conserved even though multiple innervation of PCs by CFs is maintained until adulthood.

Early stages of myelination in the spiral ganglion cells of the kitten during development.

Myelinated cell bodies of spiral ganglion in kittens during development can first be distinguished by light microscopic observation at the end of the first postnatal week. By mean of electron microscopic observation, the first signs of myelinated perikarya can be observed around the time of birth. Myelination is preceded by the ensheathment of the ganglion cell body by two or three layers of Schwann cell cytoplasmic processes. At birth, the first sign of myelination is visible at the basal part of the cochlea with thin thickenings which contain two or three major dense lines. At this stage, these thickenings cover only a small perikaryal area. Later on, the thickness and the number of thickenings increase and cover the major part of the cell body. The onset of myelination of the processes seems to precede the perikaryal myelination.

Kainate receptor-interacting proteins and membrane trafficking.

Kainate receptors are composed of several subunits and splice variants, but the relevance of this diversity is still not well understood. The subunits and splice variants show great divergence in their C-terminal cytoplasmic tail region, which has been identified as a region of interaction with a number of protein partners. Differential trafficking of kainate receptors to neuronal compartments is likely to rely on interactions with distinct subsets of protein partners. This review summarizes our knowledge of the regulation of trafficking of kainate receptors and focuses on the identification and characterization of functions of interacting partners.

Potentiation of GABAergic synaptic transmission by AMPA receptors in mouse cerebellar stellate cells: changes during development.

1. The effects of low concentrations of domoate, an agonist at both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and kainate receptors (AMPARs and KARs, respectively), were investigated in stellate cells in slices of mouse cerebellum at two developmental stages (postnatal day (PN) 11-13 and PN21-25). 2. Low concentrations of domoate enhanced the frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin (TTX) at PN11-13 but not at PN21-25. 3. The effects of low concentrations of domoate on synaptic activity were probably mediated by the activation of AMPARs and not KARs, since they were blocked by GYKI 53655 (LY300168), a selective AMPAR antagonist. 4. Domoate increased mIPSC frequency in part by activation of presynaptic voltage-dependent Ca2+ channels since potentiation was reduced by 60 % in the presence of Cd2+. AMPARs in stellate cells were found to be permeable to Ca2+. The residual potentiation in the presence of Cd2+ could thus be due to a direct entry of Ca2+ through AMPAR channels. 5. In the presence of TTX, potentiation of synaptic activity by focal application of domoate was not restricted to the region of the cell body, but was observed within distances of 120 micro(m). These experiments also revealed a strong spatial correlation between the location of the presynaptic effects of domoate and the activation of postsynaptic AMPARs. 6. Our data show a developmentally regulated presynaptic potentiation of synaptic transmission between cerebellar interneurones mediated by AMPARs. We discuss the possibility that the developmental switch could be due to a shift in the localization of AMPARs from the axonal to the somato-dendritic compartment.

A novel type of nicotinic receptor in the rat central nervous system characterized by patch-clamp techniques.

We present a functional characterization of a neuronal nicotinic receptor in the CNS using patch-clamp techniques and a preparation of acutely isolated neurons from the medial habenular nucleus of 10- to 20-d-old rats. The salient pharmacological and electrophysiological properties of this nicotinic response are (1) its association with a channel that is relatively nonselective for cations and has a unitary conductance of 26.2 (+)+/- 5pS at room temperature; (2) its insensitivity to alpha-bungarotoxin and to neuronal bungarotoxin; (3) its activation by the ganglionic nicotinic agonists nicotine, 1,1-dimethyl-4-phenylpiperazinium and cytisine and its blocking by several nicotinic antagonists, mecamylamine, hexamethonium, d-tubocurarine, and dihydro-beta-erythroidine. The combination of these properties has not been reported for any other known type of nicotinic receptor.

Morphology and electrophysiological properties of reticularis thalami neurons in cat: in vivo study of a thalamic pacemaker.

Reticularis thalami neurons (RE neurons) were identified morphologically, and their electrophysiological properties were studied in cat under barbiturate anesthesia. Intracellular HRP injections showed that RE neurons possessed very long dendrites bearing numerous filopodia-like appendages and that their axons were directed toward main thalamic nuclei. As a rule, small axonal branches were also emitted within the RE nucleus itself. At rest, the membrane potential of RE neurons displayed 2 types of oscillations: a slow 0.1-0.2 Hz oscillation and fast 7-12 Hz oscillations occurring on the positive phase of the former. Episodes of spindle (7-12 Hz) waves lasted for 2-3 sec and were characterized by rhythmic depolarizations and burst discharges. Intracellular injections of QX314 and current pulse analyses revealed the presence in RE cells of 2 distinct inward currents: a persistent current that promoted tonic firing and a low-threshold current deinactivated by hyperpolarization that generated burst discharges. The low-threshold current deinactivated with large somatic hyperpolarizations (up to 30 mV) and produced depolarizing responses that lasted for about 70 msec. In addition, low-threshold responses appeared rhythmically at intervals of about 150 msec after recovery of the membrane potential from hyperpolarization. Because of their duration, voltage dependence, and persistence after intracellular injections of QX314, it is suggested that these responses resulted from activation of a low-threshold Ca2+ current at the dendritic level. In QX314-injected cells, selective components of spontaneous oscillations were abolished, among them the positive phase of the slow oscillation and late depolarizing humps that followed burst discharges within spindle sequences. However, the rhythmic occurrence of spindle episodes at 0.1-0.2 Hz was never affected by DC currents or by QX314 or Cl- injections, suggesting that oscillations within a particular RE neuron partly reflected the oscillatory behavior of a network of cells. On the basis of these electrophysiological results and the known morphological and neurochemical features, a new hypothesis is proposed to account for the rhythmicity of RE neurons.

Abolition of spindle oscillations in thalamic neurons disconnected from nucleus reticularis thalami.

The effects of depriving thalamic relay and intralaminar nuclei from their reticularis thalami (RE) inputs were investigated in acute and chronic experiments on cat. In acutely prepared animals, two (frontal and parasagittal) thalamic transections were made; extracellular and intracellular recordings were performed in RE-disconnected thalamic nuclei. In chronic experiments, the RE nuclear complex was lesioned by means of kainic acid injections; the activity of RE-deprived thalamocortical neurons was extracellularly studied during wakefulness and synchronized sleep. Two features distinguish RE-deprived nuclei from normal thalamic nuclei: absence of spindle-wave rhythmicity and all-burst activity of neurons. The abolition of spindle-related rhythms (sequences of 7- to 14-Hz waves recurring periodically with a rhythm of 0.1-0.2 Hz) in RE-disconnected thalamic nuclei and ipsilateral neocortical areas contrasted with normal spindling rhythmicity in contralateral EEG leads. Spontaneously occurring, rhythmic, long-lasting inhibitory postsynaptic potentials (IPSPs), as observed in intact preparations, were no longer observed in RE-disconnected thalamic neurons. The remaining inhibitory events consisted of short-duration IPSPs. The possibility that RE nucleus is a pacemaker for spindling rhythms, imposing them through inhibitory projections to target thalamic areas, is supported by our concurrent experiments that indicate RE neurons preserve their rhythmicity after disconnection from their major (cortical and thalamic) input sources. RE-deprived thalamocortical neurons exclusively exhibit high-frequency spike bursts whose intrinsic structure is identical to that of intact thalamic relay cells. Instead of the spindle-related sequences of bursts seen in normal animals, the bursts of RE-disconnected thalamocortical neurons are single events, with a dramatic rhythmicity at 1-2 Hz. The presumed mechanism of this rhythmicity is the periodic activation of a low-threshold somatic conductance whose deinactivation is brought about by temporal integration of short-lasting IPSPs. It is known that high-frequency spike bursts of thalamic relay neurons result from hyperpolarization of cell membrane. We blocked the underlying inhibitory events by bicuculline and reversibly changed the all-burst activity of RE-disconnected neurons into a tonic mode. Since the only activity of RE-deprived thalamocortical neurons consists of burst discharges, we hypothesize that local-circuit GABAergic neurons are released from inhibition after RE disconnection or lesion.

Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system.

Neuronal nicotinic ACh receptors (nAChRs) are present in the rat medial habenula (MHB) and interpeduncular nucleus (IPN), two brain regions connected through the fasciculus retroflexus (FR). The goal of the present study was to compare the electrophysiological and pharmacological characteristics of nAChRs located at pre- and postsynaptic sites within the MHB-IPN system. nAChRs located on the soma of IPN neurons were studied using patch-clamp techniques and a preparation of acutely isolated neurons. Whole-cell currents evoked by Ach and nicotine showed an intense rectification at positive membrane potentials. nAChR channels were relatively nonselective for cations, had a unitary conductance of 35 pS, and were activated by several nicotinic agonists with the following rank order: cytisine greater than ACh greater than nicotine greater than dimethylphenylpiperazinium (DMPP). They were blocked by mecamylamine, hexamethonium, curare, and dihydro-beta-erythroidine (DHBE), but were insensitive to alpha-bungarotoxin and neuronal bungarotoxin. In contrast, nAChRs recorded on the soma of MHB neurons under equivalent experimental conditions exhibited different characteristics for single-channel conductance and agonist and antagonist sensitivity. The pharmacological properties of presynaptic nAChRs in the IPN were analyzed in a rat brain slice preparation. Stimulation of the FR produced a presynaptic afferent volley recorded in the rostral subnucleus of the IPN. Nicotinic agonists decreased the amplitude of the afferent volley with different efficacies: nicotine greater than cytisine greater than ACh greater than DMPP. The action of nicotine was insensitive to alpha-bungarotoxin and to neuronal bungarotoxin, but was blocked by mecamylamine, hexamethonium, curare, and DHBE, with IC50 values different from those reported for IPN postsynaptic nAChRs. This study thus demonstrates the functional diversity of nAChRs in the rat CNS.