Sensitivity and density of glutamate receptor subtypes in the hippocampal formation are altered in pentylenetetrazole – kindled rats

Kindling induced by 13 intraperitoneal injections of 40 mg/kg pentylenetetrazole (PTZ) over a period of 4 weeks resulted in a significant long-lasting increase in both the convulsive susceptibility of animals to the convulsant and the density of the specific [³H]-l-glutamate binding sites in the hippocampus. The quisqualate- and kainate-sensitive [³H]-l-glutamate binding sites were increased 24 h after the final PTZ injection, whereas the N-methyl-d-aspartate (NMDA)-sensitive sites had only a tendency to be enhanced. Furthermore, we investigated [³H]-l-glutamate binding on metabotropic receptors and found a significant increase in the hippocampus following PTZ kindling. In addition, in hippocampal tissue of kindled rats (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated inositol phosphate formation is increased. It can be concluded that the increase in metabotropic glutamate receptor (mGluR) density may be the expression of a specific enhancement in susceptibility of the glutamatergic systems to this excitatory amino acid developing in the course of PTZ-induced kindling. Received: 15 September 1997 / Accepted: 5 March 1998     

Kainate and quisqualate receptor autoradiography in rat brain after angular bundle kindling

The kainate and quisqualate types of excitatory amino acid receptor were visualized autoradiographically in brain sections from rats kindled by stimulating the angular bundle. Kainate receptors were labeled with [3H]kainate and quisqualate receptors with L-[3H]glutamate. When assayed one day after the last evoked seizure, kainate receptor binding had declined by 24-29% in stratum lucidum of hippocampal area CA3 and by 12-14% in the inner third of the dentate molecular layer, but was unchanged in the neocortex and basolateral amygdala. Saturation binding curves revealed that, under the conditions of these experiments, [3H]kainate labeled a single class of binding sites with a KD of 33-36 nM. In stratum lucidum of area CA3, kindling reduced the density of kainate receptors without altering their affinity for kainate. At the same time, quisqualate receptor binding had declined by 20-35% in many layers of the hippocampal formation and neocortex, but remained unchanged in the basolateral amygdala. Repeated stimulation or repeated seizures were required to produce these effects, since both kainate and quisqualate receptor binding were unchanged one day after a single afterdischarge. These receptor changes largely or completely reversed during a 28-day period without further stimulation. Thus maintenance of the kindled state probably cannot be explained by a long-lasting change in the expression of kainate or quisqualate receptors. The transient, regionally-selective down-regulation of these receptors may represent a compensatory response of forebrain neurons to repeated stimulation or seizures.     

Autoradiographic visualisation of [3H]DTG binding to sigma receptors, [3H]TCP binding sites, and L-[3H]glutamate binding to NMDA receptors in human cerebellum.

The autoradiographic distributions of [3H]1,3-di-ortho-tolyguanidine ([3H]DTG), [3H]1-[1-(2-thienyl) cyclohexyl] piperidine ([3H]TCP) and L-[3H]glutamate were studied in the human cerebellum. [3H]DTG is a selective label for the sigma receptor, while L-[3H]glutamate binding was carried out under conditions selective for the N-methyl-D-aspartate (NMDA) receptor. [3H]TCP binding sites and sigma receptors showed marked enrichment in the Purkinje cell layer, while L-[3H]glutamate-labelled NMDA receptors showed virtually no binding in the Purkinje cell layer. The results confirm the existence of [3H]TCP binding sites which are not linked to NMDA receptors in the human cerebellum, having a distribution which is more similar to that of the haloperidol-sensitive sigma receptor.     

Pentylenetetrazole-induced seizures affect binding site densities for GABA, glutamate and adenosine receptors in the rat brain

Pentylenetetrazole (PTZ) is a convulsant used to model epileptic seizures in rats. In the PTZ-model, altered heat shock protein 27 (HSP-27) expression highlights seizure-affected astrocytes, which play an important role in glutamate and GABA metabolism. This raises the question whether impaired neurotransmitter metabolism leads to an imbalance in neurotransmitter receptor expression. Consequently, we investigated the effects of seizures on the densities of seven different neurotransmitter receptors in rats which were repeatedly treated with PTZ (40 mg/kg) over a period of 14 days. Quantitative in vitro receptor autoradiography was used to measure the regional binding site densities of the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) receptors, the adenosine receptor type 1 (A₁), which is part of the system controlling glutamate release, and the γ-aminobutyric acid (GABA) receptors GABA_A and GABA_B as well as the GABA_A-associated benzodiazepine (BZ) binding sites in each rat. Our results demonstrate altered receptor densities in brain regions of PTZ-treated animals, including the HSP-27 expressing foci (i.e. amygdala, piriform and entorhinal cortex, dentate gyrus). A general decrease of kainate receptor densities was observed together with an increase of NMDA binding sites in the hippocampus, the somatosensory, piriform and the entorhinal cortices. Furthermore, A₁ binding sites were decreased in the amygdala and hippocampal CA1 region (CA1), while BZ binding sites were increased in the dentate gyrus and CA1. Our data demonstrate the impact of PTZ induced seizures on the densities of kainate, NMDA, A₁ and BZ binding sites in epileptic brain. These changes are not restricted to regions showing glial impairment. Thus, an altered balance between different excitatory (NMDA) and modulatory receptors (A₁, BZ binding sites, kainate) shows a much wider regional distribution than that of glial HSP-27 expression, indicating that receptor changes are not following the glial stress responses, but may precede the HSP-27 expression.     

Phencyclidine and related drugs bind to the activated N-methyl-D-aspartate receptor-channel complex in rat brain membranes

In functional studies, phenycyclidine (PCP) and similar drugs non-competitively antagonize neuronal responses to the excitatory amino acid, N-methyl-D-aspartate (NMDA). Here we show that, in crude postsynaptic densities from rat brain, the binding of [3H]TCP (a PCP analogue) was enhanced almost 4-fold by L-glutamate and NMDA, but not by quisqualate, kainate or gamma-aminobutyric acid. The potencies of excitatory amino acid agonists and antagonists in the [3H]TCP binding assay closely paralleled their affinities for NMDA-sensitive L-[3H]glutamate binding sites. In contrast, dissociative anaesthetics and sigma-opiates inhibited [3H]TCP binding (with a profile characteristic of PCP binding sites), but had no effect on L-[3H]glutamate binding. These data indicate that PCP binding sites are linked to NMDA receptors, and that PCP and related drugs bind preferentially to the activated configuration of the NMDA receptor channel complex.     

Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate

Summary The possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [³H]L-glutamate ([³H]GLU) and [³H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [³H]Glycine always labelled a single population of sites; densities of binding sites (B_max) in cortical, cerebellar and granule membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (K_d) in the same three preparations were 0.13, 0.31 and 1.9 M, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [³H]GLU was saturable and to a single population of sites: K_d 0.5–0.9 M and B_max 3.2–3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [³H]GLU was always L-aspartate>L-cysteate>L-cysteinesulphinate>L-serine-O-sulphate>ibotenate>L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [³H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations. Although the binding of [³H]GLU was relatively insensitive to NMDA itself and competitive NMDA antagonists, binding may be to a recognition site for NMDA-like agonists, since they fully inhibited specific binding. This excitatory amino acid recognition for NMDA agonists was conserved in the three membrane preparations. In cortical and granule membranes the B_max values for the binding of [³H]GLU and [³H]glycine had a stoichiometry of 1:1, whilst in cerebellar synaptic membranes this ratio was 4:1. Receptor autoradiography of NMDA-related [³H]GLU and [³H]glycine binding in tissue sections failed to reveal any differential labelling patterns in cerebral cortex and cerebellum. In the cerebellum, densities of silver grains found with both [³H]ligands were concentrated in the granule cell layer relative to the molecular layer, but the differences detected in membrane binding studies were not observed in cerebellum. Our findings suggest the existence of three types of heterogeneity for the glycine domain of the NMDA receptor: (1) differing affinities for glycine, (2) differing pharmacological profiles, and (3) differing stoichiometry in relation to the putative NMDA-like agonist site. Our evidence supports an hypothesis for the existence of multiple glycine domains which might differentially modulate NMDA-mediated neurotransmission.     

The N-methyl-D-aspartate (NMDA) receptor complex: a stoichiometric analysis of radioligand binding domains

A stoichiometric analysis of pharmacological domains within the N-methyl-D-aspartate (NMDA) receptor complex was made by evaluating the binding of L-[3H]glutamate, [3H]CPP, [3H]glycine and [3H]MK-801 to purified synaptic membranes isolated from rat telencephalon. The binding of all radioligands exhibited pharmacological and kinetic properties consistent with the labeling of homogeneous populations of sites associated with the NMDA receptor. However, strychnine-insensitive [3H]glycine binding sites were present at close to 2-fold the density of the other sites examined. These data, together with recent electrophysiological and receptor autoradiographic findings, are utilized as a basis for hypotheses regarding the ratio of transmitter recognition, allosteric and channel binding sites within the NMDA receptor complex.     

Excitatory amino acid projections to the nucleus accumbens septi in the rat: a retrograde transport study utilizing D[3H]aspartate and [3H]GABA

Afferents to the nucleus accumbens septi utilizing glutamate or aspartate have been investigated in the rat by autoradiography following injection and retrograde transport of D[3H]aspartate. Parallel experiments with the intra-accumbal injection of [3H]GABA were employed to establish the transmitter-selective nature of the retrograde labelling found with D[3H]aspartate. The topography of cortical and thalamic perikarya labelled by D[3H]aspartate was extremely precise. D[3H]Aspartate labelled perikarya were found in layer V of agranular insular cortex; bilaterally within prelimbic and infralimbic subareas perikarya, but predominantly ipsilaterally. Ipsilateral labelling was observed in dorsal, ventral and posterior agranular insular cortices, and in perirhinal cortex. Injections into ventral accumbens labelled perikarya in ipsilateral entorhinal cortex, while infusion of D[3H]aspartate into anterior caudate-putamen resulted in labelling of perikarya in ipsilateral cingulate and lateral precentral cortices. Following infusion of D[3H]aspartate, ipsilateral midline thalamic nuclei contained the highest density of labelled perikarya; infusions centred on nucleus accumbens resulted in heavy retrograde labelling of the parataenial nucleus, but labelling was sparse from a lateral site and not observed after injection into anterior caudate-putamen. Less prominent labelling of perikarya was seen in other thalamic nuclei (mediodorsal, central medial, rhomboid, reuniens and centrolateral), mostly near the midline. Perikaryal labelling was also found in the ipsilateral amygdaloid complex, particularly in basolateral and lateral nuclei. Only weak labelling resulted in ventral subiculum. Numerous labelled cells were present bilaterally in anterior olfactory nucleus, although perikarya were more prominent ipsilaterally. Labelled perikarya were not consistently observed in other regions (ventral tegmental area, medial substantia nigra, raphe nuclei and locus coeruleus) known to innervate nucleus accumbens. Presumptive anterograde labelling was detected in ventral pallidum/substantia innominata, ventral tegmental area and medial substantia nigra. [3H]GABA was generally not retrogradely transported to the same regions labelled by D[3H]aspartate; an exception being the anterior olfactory nucleus, where large numbers of labelled perikarya were found. [3H]GABA failed to label perikarya in thalamus and amygdala, and a topographic distribution of label was absent in neocortex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in rat hippocampal long-term slice cultures isolated from external afferents

The distribution of ionotropic glutamate receptors in transverse hippocampal sections and along the septotemporal hippocampal axis can be correlated to hippocampal connectivity, in particular to area- and layer-specific termination zones of afferents. However, in isolated organotypic hippocampal slice cultures developing without extrinsic afferent input no systematic studies exist about the distribution of glutamate receptors. In the present study we used receptor autoradiography to examine [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites in hippocampal slice cultures prepared from 6-day-old rats. After 24 days in vitro layer-specific concentrations of [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites were compared to age-matched hippocampi in situ (P30 rats). An obvious difference between hippocampal slice cultures and hippocampi in situ was a changed distribution of binding sites among the hippocampal areas showing a relative increase of [³H]MK-801 and [³H]AMPA binding sites in CA3 as compared to CA1 and to the dentate gyrus in the cultures. In CA1, however, the relative layer-specific distributions of [³H]MK-801 and [³H]AMPA binding sites were identical in hippocampal slice cultures and in hippocampi in situ. Interestingly, layer-specific binding of [³H]Kainate in the cultures exceeded that in the hippocampi in situ 3–5 times. Moreover, in the cultures the binding of the three ligands varied systematically showing gradients along the ”superficiomembranal’’ axis. Cultures taken from different positions along the hippocampal axis differed with respect to concentrations of [³H]MK-801 and [³H]Kainate binding sites, but not of [³H]AMPA binding sites. The results suggest a massive sprouting and reorganisation of intrinsic projections in long-term hippocampal slice cultures.     

Distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in rat hippocampal long-term slice cultures isolated from external afferents

The distribution of ionotropic glutamate receptors in transverse hippocampal sections and along the septotemporal hippocampal axis can be correlated to hippocampal connectivity, in particular to area- and layer-specific termination zones of afferents. However, in isolated organotypic hippocampal slice cultures developing without extrinsic afferent input no systematic studies exist about the distribution of glutamate receptors. In the present study we used receptor autoradiography to examine [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites in hippocampal slice cultures prepared from 6-day-old rats. After 24 days in vitro layer-specific concentrations of [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites were compared to age-matched hippocampi in situ (P30 rats). An obvious difference between hippocampal slice cultures and hippocampi in situ was a changed distribution of binding sites among the hippocampal areas showing a relative increase of [³H]MK-801 and [³H]AMPA binding sites in CA3 as compared to CA1 and to the dentate gyrus in the cultures. In CA1, however, the relative layer-specific distributions of [³H]MK-801 and [³H]AMPA binding sites were identical in hippocampal slice cultures and in hippocampi in situ. Interestingly, layer-specific binding of [³H]Kainate in the cultures exceeded that in the hippocampi in situ 3–5 times. Moreover, in the cultures the binding of the three ligands varied systematically showing gradients along the ”superficiomembranal’’ axis. Cultures taken from different positions along the hippocampal axis differed with respect to concentrations of [³H]MK-801 and [³H]Kainate binding sites, but not of [³H]AMPA binding sites. The results suggest a massive sprouting and reorganisation of intrinsic projections in long-term hippocampal slice cultures. Accepted: 6 February 2001     

Cortical afferents of the nucleus accumbens in the cat,studied with anterograde and retrograde transport techniques

The cortical afferentation of the nucleus accumbens in the cat was studied with the aid of retrograde tracing techniques. Retrograde experiments were carried out with horseradish peroxidase or one of the fluorescent tracers Bisbenzimid, Nuclear Yellow and Fast Blue. In the anterograde experiments [³H]leucine and [³⁵S]methionine were used as tracers.Following injections in the nucleus accumbens, retrogradely-labelled cells were found in the medial frontal cortex, the anterior olfactory nucleus, the posterior part of the insular cortex, the endopiriform nucleus, the amygdalo-hippocampal area, the entorhinal and perirhinal cortices and the subiculum of the hippocampal formation. In the medial frontal cortex most of the labelled cells were found in layers III and V of the prelimbic area (area 32 of Brodmann), but retrogradely-filled neurons were also present in the infralimbic area and in the caudoventral part of the lateral bank of the proreal gyrus. Retrogradely-labelled cells in the entorhinal and perirhinal cortices were located in the deep cellular layers. Following large injections in the nucleus accumbens, retrograde labelling in the subiculum extended from the most dorsal, septal pole to the most ventral, temporal pole.Injections of anterograde tracers were placed in the frontal cortex, the entorhinal and perirhinal cortices and the hippocampal formation. The prelimbic area was found to project via the internal capsule to mainly the rostral half of the nucleus accumbens, whereas in the caudal half of the nucleus only a lateral region receives frontal cortical fibres. Following injections in the infralimbic area only fibres passing through the nucleus accumbens were labelled. Afferents from the entorhinal and perirhinal cortices reach the nucleus accumbens by way of the external capsule and terminate mainly in a ventral zone of the nucleus accumbens.Afferents from the entorhinal area are distributed to the entire accumbens, whereas the termination field of the perirhinal afferents is largely restricted to the lateral part of the nucleus accumbens. Both the frontal cortex and the entorhinal and perirhinal cortices appear to project also to the nucleus caudatus and the tuberculum olfactorium. These cortical areas also project to the contralateral striatum.Both anterograde and retrograde tracing experiments demonstrated a topographical relationship between the subiculum and the nucleus accumbens. The ventral pole of the subiculum projects via the fornix to the medial part of the caudal half of the nucleus accumbens and to a small dorsomedial area in its rostral half. Successively more dorsal portions in the subiculum project to successively more ventrolateral parts in the rostral nucleus accumbens. The projection from the hippocampus was found to extend also to the tuberculum olfactorium. The results of the present study do not provide unambiguous criteria for the delimitation of the nucleus accumbens in the cat.     

Kindling and electrode effects on the benzodiazepine receptors density of olfactory bulb and hippocampus after olfactory bulb kindling.

The olfactory bulb (OB) kindling is a model of limbic secondary generalized epilepsy. Ten days after the completion of OB kindling, we have studied the long term effects of both electrode insertion and kindling on the binding of [3H]diazepam to crude mitochondrial fractions. On the one hand, we have shown that electrode implantation in sham-operated controls induced an obvious increase in benzodiazepine (BZD) receptor density (Bmax) only at the site of the electrode in comparison to sham-unoperated rats. These results might indicate an additional mechanism extending earlier observations reported by others, who have shown that prolonged electrode implantation induced changes in sham-operated and kindled rats. On the other hand, the long lasting effect of OB kindling on the binding parameters of [3H]diazepam was examined in the focus and in the hippocampus. The results indicate a bilateral increase of BZD receptors in the OB and an ipsilateral increase in the hippocampus. These changes might be a regulation phenomenon in response to a hyperexcitability state and to focal stimulations.     

Diazepam restores the increased [3H]glutamate binding to hippocampal synaptic membranes in the amygdaloid-kindled rat

[3H]Glutamic acid binding to hippocampi was increased after amygdaloid kindling in rats, and diazepam inhibited this increased binding, without any effect on the enhanced binding by CaCl2 or the binding in control rats. By inducing kindling in the same way as that used in the binding experiment, the inhibiting effects of diazepam on kindled seizures, the afterdischarge and the development of kindling were observed.     

Retrograde transport of D-[3H]-aspartate injected into the monkey amygdaloid complex

Summary The possibility that certain of the afferents of the primate amygdaloid complex use an excitatory amino acid transmitter was evaluated by injecting D-[³H]-aspartate into the amygdala of twoMacaca fascicularis monkeys. The distribution of D-[³H]-aspartate labeled neurons was compared with those labeled with the nonselective retrograde tracer WGA-HRP injected at the same location as the isotope. Retrogradely labeled cells of both types were observed in a variety of cortical and subcortical structures and in discrete regions within the amygdala. D-[³H]-aspartate labeled neurons were observed in layers III and V of the frontal, cingulate, insular and temporal cortices. In the hippocampal formation, heavily labeled cells were observed in the CA1 region and in the deep layers of the entorhinal cortex. Of the subcortical afferents, the claustrum and the midbrain peripeduncular nucleus contained the greatest number of D-[³H]-aspartate labeled cells. Subcortical afferents that are not thought to use excitatory amino acids, such as the cholinergic neurons of the basal nucleus of Meynert, did not retrogradely transport the isotope. Within the amygdala, the most conspicuous labeling was in the paralaminar nucleus which forms the rostral and ventral limits of the amygdala. When the D-[³H]-aspartate injection involved the basal nucleus, many labeled cells were also observed in the lateral nucleus. Retrograde transport of D-[³H]-aspartate injected into the amygdala, therefore, appears to demonstrate a subpopulation of inputs that may use an excitatory amino acid transmitter.     

Age-related changes in arginine and its metabolites in memory-associated brain structures

l-arginine is metabolised by nitric oxide synthase (NOS) and arginase to form l-citrulline and nitric oxide, and l-ornithine and urea, respectively. The present study investigated NOS and arginase activities, and the levels of l-arginine, l-citrulline and l-ornithine, as well as glutamate and γ-aminobutyric acid (GABA), in memory-related brain structures in 4, 12 and 24 months old rats. Significantly increased NOS and arginase activities with age were found across the CA1, CA2/3 and dentate gyrus (DG) sub-regions of the hippocampus and the prefrontal, entorhinal, perirhinal, postrhinal and temporal cortices in a region-specific manner. For l-arginine, there were age-related increases in CA1 and the perirhinal and temporal cortices, and decreases in the entorhinal and postrhinal cortices. l-citrulline levels were decreased with age in the prefrontal, postrhinal and temporal cortices. There were age-related decreases in l-citrulline/l-arginine molar ratio in CA1 and CA2/3 and the prefrontal and temporal cortices, but an increase in the entorhinal cortex (EC). Increased l-ornithine levels and l-ornithine/l-arginine molar ratios with age were found in most of the brain regions examined. Glutamate levels were significantly decreased with age in the prefrontal, entorhinal, perirhinal and temporal cortices, whereas GABA level was largely unchanged except for age-related increase in CA1. There were significantly decreased glutamate/GABA molar ratios with age in six brain regions. Correlational analyses revealed no inverse relationship between NOS and arginase activities, and no positive correlations between the activities of the two enzymes and the tissue concentrations of their products. Interestingly, there were significant positive correlations between glutamate and GABA, and l-arginine and its metabolites in many brain regions. These results demonstrate that the aging process has dramatic effects on the NOS and arginase metabolic pathways of l-arginine and the glutamatergic neurotransmitter system. Since l-arginine metabolism is complex, there is a need to determine its metabolomic profile in vivo in the future.     

Retention of maze performance inversely correlates with N-methyl-D-aspartate receptor number in hippocampus and frontal neocortex in the rat

The N-methyl-D-aspartate (NMDA) receptor may play a critical role in learning and memory. In the present study, a significant correlation was found between the number of NMDA-displaceable, Na+-independent L-[3H]glutamate binding sites in the hippocampus and neocortex of young rats and the mean number of errors during retention, but not acquisition, in a 14-unit T-maze.     

THE ROLE OF THE PIRIFORM CORTEX IN KINDLING

In epilepsy research, there is growing interest in the role of the piriform cortex (PC) in the development and maintenance of limbic kindling and other types of limbic epileptogenesis leading to complex partial seizures, i.e. the most common type of seizures in human epilepsy. The PC (“primary olfactory cortex”) is the largest area of the mammalian olfactory cortex and receives direct projections from the olfactory bulb via the lateral olfactory tract (LOT). Beside the obvious involvement in olfactory perception and discrimination, the PC, because of its unique intrinsic associative fiber system and its various connections to and from other limbic nuclei, has been implicated in the study of memory processing, spread of excitatory waves, and in the study of brain disorders such as epilepsy with particular emphasis on the kindling model of temporal lobe epilepsy with complex partial seizures. The interest in the kindling model is based primarily on the following observations. (1) the PC contains the most susceptible neural circuits of all forebrain regions for electrical (or chemical) induction of limbic seizures. (2) During electrical stimulation of other limbic brain regions, broad and large afterdischarges can be observed in the ipsilateral PC, indicating that the PC is activated early during the kindling process. (3) The interictal discharge, which many consider to be the hallmark of epilepsy, originates in the PC, independent of which structure serves as the kindled focus. (4) Autoradiographic studies of cerebral metabolism in rat amygdala kindling show that, during focal seizures, the area which exhibits the most consistent increase in glucose utilization is the ipsilateral paleocortex, particularly the PC. (5) During the commonly short initial afterdischarges induced by stimulation of the amygdala at the early stages of kindling, the PC is the first region that exhibits induction of immediate-early genes, such as c-fos. (6) The PC is the most sensitive brain structure to brain damage by continuous or frequent stimulation of the amygdala or hippocampus. (7) Amygdala kindling leads to a circumscribed loss of GABAergic neurons in the ipsilateral PC, which is likely to explain the increase in excitability of PC pyramidal neurons during kindling. (8) Kindling of the amygdala or hippocampus induces astrogliosis in the PC, indicating neuronal death in this brain region. Furthermore, activation of microglia is seen in the PC after amygdala kindling. (9) Complete bilateral lesions of the PC block the generalization of seizures upon kindling from the hippocampus or olfactory bulb. Incomplete or unilateral lesions are less effective in this regard, but large unilateral lesions of the PC and adjacent endopiriform nucleus markedly increase the threshold for induction of focal seizures from stimulation of the basolateral amygdala (BLA) prior to and after kindling, indicating that the PC critically contributes to regulation of excitability in the amygdala. (10) Potentiation of GABAergic neurotransmission in the PC markedly increases the threshold for induction of kindled seizures via stimulation of the BLA, again indicating a critical role of the PC in regulation of seizure susceptibility of the amygdala. Microinjections of NMDA antagonists or sodium channel blockers into the PC block seizure generalization during kindling development. (11) Neurophysiological studies on the amygdala-PC slice preparation from kindled rats showed that kindling of the amygdala induces long-lasting changes in synaptic efficacy in the ipsilateral PC, including spontaneous discharges and enhanced susceptibility to evoked burst responses. The epileptiform potentials in PC slice preparations from kindled rats seem to originate in neurons at the deep boundary of PC. Spontaneous firing and enhanced excitability of PC neurons in response to kindling from other sites is also seen in vivo, substantiating the fact that kindling induces long-lasting changes in the PC comparable to abnormalities seen in primary foci. Taken together, these observations indicate that the PC might be part of an epileptic network which is pivotal in the genesis of kindling, facilitating and intensifying the spread of seizures from a focus in amygdala or hippocampus to cortical and subcortical regions along pathways that also are utilized in normal movements. Although direct evidence implicating the PC in the pathogenesis of human epilepsy is not yet available, the experimental data reviewed in this paper should initiate clinical studies on the potential role of this brain structure as a pacemaker or secondary focus in TLE and other types of epilepsy. Copyright © 1996 Elsevier Science Ltd.     

Sequential changes of [3H]forskolin, [3H]cyclohexyladenosine and [3H]PN200‐110 binding sites in the brain of 6‐hydroxydopamine‐lesioned rats

Receptor autoradiographic technique was studied to investigate sequential changes in adenylyl cyclase, adenosine A₁ receptors and L ‐type calcium channels in the striatum and substantia nigra 1–8 weeks after unilateral 6‐hydroxydopamine injection of the medial forebrain bundle in rats. [³H]Forskolin, [³H]cyclohexyladenosine (CHA) and [³H]PN200‐110 were used to label adenylyl cyclase, adenosine A₁ receptors and L ‐type calcium channels, respectively. The degeneration of the nigrostriatal pathway caused a significant increase in [³H]forskolin binding in the striatum of both the ipsilateral and contralateral sides from 2 to 4 weeks post‐lesion. The ipsilateral substantia nigra showed a transient increase in [³H]forskolin binding 4 weeks post‐lesion. In contrast, [³H]CHA binding showed no significant change in most brain areas after lesioning. On the other hand, a conspicuous decrease in [³H]PN200‐110 binding was observed in the dorsolateral striatum of ipsilateral side 4 weeks post‐lesion. Thereafter, the striatum of both the ipsilateral and contralateral sides showed a significant decrease in [³H]PN200‐110 binding 8 weeks post‐lesion. These results demonstrate that unilateral 6‐hydroxydopamine into the medial forebrain bundle of rats can experimentally cause a significant increase in adenylyl cyclase binding sites in the striatum and substantia nigra, whereas no conspicuous change in adenosine A₁ receptors is observed in these areas during post‐lesion. In contrast, L ‐type calcium channels were progressively damaged in the striatum after unilateral 6‐hydroxydopamine treatment. These findings suggest that adenylyl cyclase and calcium system may contribute to the degeneration processes of the dopaminergic neurones.     

Transient postnatal increases in excitatory amino acid binding sites in rat ventral mesencephalon.

The developmental profile of three sub-types of excitatory amino acid (EAA) binding sites was determined in the ventral mesencephalon and the striatum of rats from prenatal day 19 to adult (3 months) using membrane binding assays. In the ventral mesencephalon, there was a transient increase of EAA receptor binding sites beyond adult levels, which peaked at postnatal day 7 (P7) for [3H]glutamate binding to NMDA receptors and at P14 for [3H]AMPA and [3H]kainate binding. In the striatum, [3H]glutamate/NMDA and [3H]kainate binding reached adult levels during the early postnatal period, stabilizing at this level with no transient overexpression beyond adult levels. [3H]AMPA binding also showed an increase above adult levels at P14 in the striatum. These results raise the possibility that the transient overexpression of EAA receptors in the ventral mesencephalon may affect the developmental fate of dopaminergic and other neurons in this region.