Neuroprotective effects of brain-derived neurotrophic factor in seizures during development.

Although the immature brain is highly susceptible to seizures, it is more resistant to seizure-induced neuronal loss than the adult brain. The developing brain contains high levels of neurotrophins which are involved in growth, differentiation and survival of neurons. To test the hypothesis that neurotrophins may protect the developing brain from seizure-induced neuronal loss, brain-derived neurotrophic factor up-regulation was blocked by intracerebroventricular infusion of an 18mer antisense oligodeoxynucleotide sequence to brain-derived neurotrophic factor in 19-day-old rats using micro-osmotic pumps. Control rats were infused with sense or missense oligodeoxynucleotide. Status epilepticus was induced by intraperitoneal administration of kainic acid 24 h after the start of oligodeoxynucleotide infusion. Seizure duration was significantly increased in the antisense oligodeoxynucleotide plus kainic acid group compared to groups that received kainic acid alone or kainic acid plus sense or missense oligodeoxynucleotide. There was no difference between groups in the latency to forelimb clonus. A twofold increase in brain-derived neurotrophic factor levels was observed in the hippocampus 20 h following kainic acid-induced seizures. This kainic acid-induced increase was absent in animals receiving infusion of antisense oligodeoxynucleotide to brain-derived neurotrophic factor at time of seizure induction. Hippocampi of rats in this group (antisense oligodeoxynucleotide plus kainic acid) showed a loss of CA1 and CA3 pyramidal cells and hilar interneurons. This neuronal loss was not dependent upon seizure duration since animals injected with diazepam to control seizure activity in the antisense plus kainic acid group also showed similar neuronal loss. Administration of kainic acid or infusion of antisense alone did not produce any cell loss in these regions. Induction of seizures at postnatal day 20, in the presence or absence of antisense oligonucleotide, did not produce an impairment in learning and memory when tested 15 days later in the Morris water maze. The hippocampi of these animals did not show any synaptic reorganization as assessed by growth-associated protein-43 immunostaining and Timm staining. Our findings confirm prior studies demonstrating that seizures in the immature brain are associated with little, if any, cell loss. However, when seizure-induced increase in brain-derived neurotrophic factor is blocked, seizures do result in neuronal loss in the developing brain. Thus, brain-derived neurotrophic factor appears to provide protection against kainic acid seizure-induced neuronal damage in the developing brain.     

Different sensitivity of hippocampal neurons to kainic acid in the rabbit

The toxic effect of kainic acid, a cyclic analogue of glutamic acid, was investigated in the rabbit hippocampus. Using histological methods it was found that intrahippocampal injection of one or two doses of 5 nm of kainic acid produced cell losses in different cell layers. The cells with the highest sensitivity to kainic acid were the cornu ammonis pyramidal cells, whereas those with the lowest sensitivity were the dentate granular cells.These findings suggest that the sensitivity to kainic acid is different in discrete cell areas of the hippocampus.     

Ibotenic acid-induced neuronal degeneration: A morphological and neurochemical study

Summary Possible neurotoxic actions of intracerebral injections of ibotenic acid, a conformationally restricted analogue of glutamic acid, have been evaluated in rat brain and compared with those of kainic acid.Light microscopical analysis revealed that ibotenic acid produced a marked disappearance of nerve cells in all areas studied, namely striatum, the hippocampal formation, substantia nigra and piriform cortex. Lesions in areas distant to the injection site were not seen. Axons of passage and nerve terminals of extrinsic origin did not seem to be damaged, since, e.g., no apparent degeneration of the dopaminergic terminals in the neostriatum was observed except for a small area surrounding the cannula. In the neostriatum, enkephalin immunoreactive neuronal cell bodies as well as nerve terminals disappeared after injection of ibotenic acid into this nucleus. After injection into the substantia nigra tyrosine hydroxylase immunoreactive cell bodies in the zona compacta disappeared, whereas no certain effect could be seen on the enkephalin immunoreactive nerve fibers.In vitro experiments, conducted with striatal synaptosomal and membrane preparations, showed that ibotenic acid differed from kainic acid by being devoid of a significant inhibitory effect on high affinity glutamate uptake and by having a low affinity for ³H-kainic acid binding sites. Furthermore, ibotenic acid did not interfere with the binding of a number of radioligands for other transmitter receptors.As compared to kainic acid, ibotenic acid has the advantage of being less toxic to the animals and of producing more discrete lesions, possibly due to faster metabolism and/or other fundamental biochemical differences. Because of these special features, ibotenic acid seems to represent a valuable new tool in the morphological and functional analysis of central neuronal systems.     

Bcl-2, Bax and Bcl-x expression following kainic acid administration at convulsant doses in the rat.

Neuronal death was produced in the CA1 and CA3 areas of the hippocampus, amygdala, and piriform and entorhinal cortices after intraperitioneal administration of kainic acid at convulsant doses to adult rats. To assess the involvement of members of the Bcl-2 family in cell death or survival, immunohistochemistry, western and northern blotting to Bcl-2, Bcl-x and Bax, and in situ hybridization to Bax were examined at different time-points after kainic acid treatment. Members of the Bcl-2 family were expressed in the cytoplasm of pyramidal neurons in the hippocampus, and in a subset of neurons of the piriform and the entorhinal cortices, amygdala and neocortex in the normal adult brain. Dying neurons in the pyramidal cell layer of CA1 and CA3 areas, entorhinal and piriform cortices, and amygdala also expressed Bcl-2, Bax and Bcl-x following excitotoxicity, although many dying cells did not. In addition, a number of cells in the affected areas showed Bax immunoreactivity in their nuclei at 24-48 h following kainic acid administration, thus indicating Bax nuclear translocation in a subset of dying cells. Western blots disclosed no modifications in the intensity of the bands corresponding to Bcl-2, Bcl-x and Bax, between control and kainic acid-treated rats. No modifications in the intensity of the bcl-2 messenger RNA band on northern blots was observed in kainic acid-treated rats. However, a progressive increase in the intensity of the bax messenger RNA band was found in kainic acid-treated rats at 6 h, 12 h and 24 h following kainic acid administration. Interestingly, a slight increase in Bax immunoreactivity was observed in the cytoplasm of neurons of the dentate gyrus at 24-48 h, a feature which matches the increase of bax messenger RNA in the same area, as shown by in situ hybridization at 12-24 h following kainic acid injection. The present results suggest that cell death or survival does not correlate with modifications of Bcl-2, Bax and Bcl-x protein, and messenger RNA expression, but rather that kainic acid excitotoxicity is associated with Bax translocation to the nucleus in a subset of dying cells.     

Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive

Argyrophilic dark neurons (DNs) reflect the early histopathological state of neurons following various brain insults. We examined the fate of DNs, about to either die or recover, following two types (heavy and light damage) of brain insult. Wistar rats were injected ibotenic acid unilaterally into the hippocampal CA1 region (ibotenic acid [IA] injection) or were forced to swim (SWIM). Argyrophil III (DNs)-, activated caspase-3 immuno-, TUNEL- and hematoxylin-eosin (H-E)-staining and ultrastructural examinations were then performed. One to three hours after IA injection, typical DNs (argyrophilic both in somata and dendrites) with corkscrew-like dendrites were densely packed in the pyramidal cell layer of hippocampal CA1 around the injection site. After 12-24 h, DNs were argyrophilic only in the somata and proximal dendrites but absent in distal dendrites in the CA1 region. However, at this time typical DNs were found in remote areas. At 3 h, caspase-3 activation was detected at the injection site, which increased to a peak level after 12 h. Three to 7 days after injection, TUNEL positive cells were detected in the CA1 pyramidal cell layer. Immediately following SWIM, "brown" rather than "dark" neurons were detected in the various areas and most frequently in the CA1 pyramidal cell layer. No typical DNs were detected over the first 3 days. Some activation of caspase-3 was detected in a few CA3 pyramidal cells but no TUNEL-positive cells were detected. Ultrastructural examination revealed a diffuse distribution of aggregated silver particles in the dendrites and cytoplasm of pyramidal cells at the sites of IA injection. After SWIM, silver particles were detected mainly on mitochondria of affected cells. These data suggest that DNs provide a measure of neuronal damage: typically dark neurons with broad damage to the cytoskeleton of dendrites would die, while non-typical brown neurons, that may have a disturbance in mitochondria, predominantly survive.     

Limbic seizures cause pronounced changes in the expression of neurokinin B in the hippocampus of the rat.

Immunohistological and in situ hybridization techniques were used to study the influence of kainic acid-induced seizures and of pentylenetetrazol kindling on neurokinin B immunoreactivity and neurokinin B mRNA in the rat hippocampus. Pronounced increases in neurokinin B immunoreactivity were observed in the terminal field of mossy fibres 10-60 days after intraperitoneal injection of kainic acid. These slow but persistent increases in immunoreactivity were accompanied by markedly enhanced expression of neurokinin B mRNA in the granule cells and in hilar interneurons adjacent to the granule cell layer. These changes were preceded by transient increases in neurokinin B mRNA and immunoreactivity in CA1 pyramidal cell layer two and 10 days after kainic acid, which, however, subsided later on. Pentylenetetrazol kindling caused similar increases in neurokinin B mRNA expression in granule cells and in CA1 pyramidal cells, but not in hilar interneurons. In CA1, increased neurokinin B message was present two days after termination of the kindling procedure but not after 10 days. Sixty days after kainic acid injection, neurokinin B immunoreactivity extended to the inner-third of the molecular layer of the dentate gyrus. After pentylenetetrazol kindling, a neurokinin B-immunoreactive band was observed in the infrapyramidal region of CA3. Lesions of the dentate granule cells by local injection of colchicine in kainic acid-treated rats abolished the supragranular neurokinin B-positive staining, whereas it was almost unchanged after transection of the ventral hippocampal commissure. These observations suggest that neurokinin B immunoreactivity may be located in ipsilateral mossy fibres undergoing collateral sprouting to the inner molecular layer or to the infrapyramidal region in CA3, respectively. Preprotachykinin A mRNA, which encodes for neurokinin A and substance P, and substance P immunoreactivity were not changed in the hippocampus of epileptic rats compared with untreated animals. The observed changes in neurokinin B immunoreactivity and mRNA indicate that specific functional and morphological changes may be induced in hippocampal neurons by recurrent limbic seizures.     

Kainic acid-mediated increase of preprotachykinin-A messenger RNA expression in the rat hippocampus and a region-selective attenuation by dexamethasone.

The hippocampus contains the highest number of glucocorticoid-sensitive neurons in the rat brain and excessive exposure to glucocorticoids can cause damage to hippocampal neurons and impair the capacity of the hippocampus to survive neuronal insults. In this study in situ hybridization combined with quantitative image analysis was used to study preprotachykinin-A mRNA levels after administration of a toxic dose of kainic acid in animals pretreated with glucocorticoids. Kainic acid was injected into dorsal hippocampus CA3 region in animals pretreated with the synthetic glucocorticoid receptor agonist dexamethasone and in control animals. Preprotachykinin-A mRNA was not detected in the hippocampus of untreated animals or in animals analysed 30 min after a kainic acid injection. However, 4 h after injection of kainic acid, the level of preprotachykinin-A mRNA increased to 20-times above the detection limit both in the dentate gyrus and the CA3 region of the hippocampus. Treatment of kainic acid-injected animals with dexamethasone 30 min before and 2 h after the injection attenuated the increase in the granule cells of the dentate gyrus by 50%. In contrast, dexamethasone pretreatment had no significant effect on the kainic acid-induced increase of preprotachykinin-A mRNA in pyramidal cells in regions CA3 or CA1. These results show that an excitatory stimulus within the hippocampus causes a substantial increase in the level of preprotachykinin-A mRNA in hippocampal granule and pyramidal cells and suggest that in granule cells of the dentate gyrus this increase can be modulated by glucocorticoids.     

Functional changes in neuropeptide Y- and somatostatin-containing neurons induced by limbic seizures in the rat

The influence of sustained epileptic seizures evoked by intraperitoneal injection of kainic acid on the gene expression of the neuropeptides somatostatin and neuropeptide Y and on the damage of neurons containing these peptides was studied in the rat brain. Injection of kainic acid induced an extensive loss of somatostatin and, though less pronounced, of neuropeptide Y neurons in the inner part of the hilus of the dentate gyrus. Neuropeptide Y-immunoreactive neurons located in the subgranular layer of the hilus, presumably pyramidal-shaped basket cells, were spared by the treatment. Although neuropeptide Y messenger RNA was not detected in granule cells of control rats, it was found there after kainic acid seizures at all time intervals investigated (12 h to 90 days after injection of kainic acid). High concentrations of neuropeptide Y messenger RNA were especially observed 24 h after injection of kainic acid. At this time neuropeptide Y messenger RNA was also transiently observed in CA1 pyramidal cells. Neuropeptide Y synthesis in granule cells in turn gave rise to an intense immunoreactivity of the peptide in the terminal field of mossy fibers which persisted for the entire time period (90 days) investigated. In addition, neuropeptide Y messenger RNA concentrations were also drastically elevated in presumptive basket cells located at the inner surface of the granule cell layer, especially at the “late” time intervals investigated (30–90 days after kainic acid).

These data support the concept that extensive activation of granule cells by limbic seizures contributes to the observed neuronal cell death in CA3 pyramidal neurons and interneurons of the hilus. Consecutively, basket cells containing neuropeptide Y and presumably GABA might be activated and participate in recurrent inhibition of granule cells. Neuropeptide Y-immunoreactive fibers observed in the inner molecular layer at “late” time intervals after kainic acid may result either from collateral sprouting of mossy fibers or from basket cells extensively expressing the peptide.

It is speculated that neuropeptide Y synthesized and released at a high rate from granule cells and basket cells may exert a protective action against seizures.     

Fcγ receptors contribute to pyramidal cell death in the mouse hippocampus following local kainic acid injection

Recent studies have demonstrated the contribution of the gamma subunit of the Fc receptor of IgG (FcRγ) to neuronal death following ischemic injury and Parkinson's disease. We examined the role of FcRγ in hippocampal pyramidal cell death induced by kainic acid (KA). FcRγ-deficient mice (FcRγ−/−) and their FcRγ+/+ littermates (wild type, B6) received an injection of KA into the dorsal hippocampus. Pyramidal cell death was quantified 24 and 72 h after the injection. The number of survived pyramidal cells was significantly larger in FcRγ−/− mice than in B6 mice in both the CA1 and CA3. Immunohistochemical and immunofluorescent studies detected FcγRIIB protein in parvalbumin neurons, whereas FcγRIII and FcγRI proteins were detected in microglial cells. No activated microglial cells were detected 24 h after the KA injection in FcRγ−/− mice, whereas many activated microglial cells were present in B6 mice. The production of nitrotyrosine as well as of the inducible nitric oxide synthase and cyclooxygenase-2 proteins, increased by 16 h after the KA injection in B6 mice. In addition, tissue plasminogen activator and metalloproteinase-2 proteins increased. By contrast, the magnitude of oxidative stress and the increase in protease expression were mild in FcRγ−/− mice. Co-injection of a neutralizing antibody against FcγRll and FcγRlll with KA abolished pyramidal cell death and microglial activation. In addition, the neutralizing antibody reduced oxidative stress and expression of proteases. These observations suggested a role for FcγRllB in parvalbumin neurons as well as FcRγ in microglia in pyramidal cell death.     

Differences of neuronal sensitivity to amino acids and related compounds in the rat hippocampal slice

The perfusion of slices of rat hippocampus with solutions containing N-methyl-DL-aspartic acid (NMA), kainic acid, ibotenic acid or quinolinic acid produced a reduction in the size of antidromically evoked population spikes in the CA1 pyramidal cells or dentate gyrus granule cells. The relative potencies of these compounds on CA1 cells compared with granule cells were kainate 3.65, quinolinate 3.46, NMA 2.19 and ibotenate 1.50. Since the former two compounds are known to show a degree of selective toxicity towards the CA1 cells, whereas NMA and ibotenate do not, these results are consistent with the excitotoxic hypothesis that excitation and neurotoxicity are related.     

Sparing of cholinergic neurons following quinolinic acid lesions of the rat striatum

A combination of immunocytochemical and enzyme histochemical methods have been used to study those neurons which survive lesions of the rat striatum, produced by low doses of the excitotoxin quinolinic acid. Nissl-stained sections revealed that following injection of this toxin many large neurons remained within areas of extensive cell loss. These large cells were found to express both the enzyme acetylcholinesterase and choline acetyltransferase-like immunoreactivity. The surviving cells did not contain the enzyme reduced nicotinamide adenine dinucleotide phosphate or the peptides, somatostatin and neuropeptide Y. This pattern of selective cell sparing was also found following lesions induced by low doses of the toxins ibotenic acid and kainic acid. The survival of large neurons indicates that the excitotoxin-lesioned rat striatum shares common features with the pattern of cell loss found in the caudate-putamen in Huntington's disease. The major difference between these two examples of striatal nerve cell degeneration is, however, the selective preservation of somatostatin/neuropeptide Y/nicotinamide adenine dinucleotide phosphate-diaphorase-containing neurons found in Huntington's disease but not observed following quinolinic acid lesions.     

Perforant path transections protect hippocampal granule cells from kainate lesion

Stereotaxic injection of nmol quantities of kainic acid (KA) into the rat hippocampus induced selective degeneration of nerve cell bodies in this brain region. Vulnerability to the neurotoxin varied markedly between the various hippocampal cell groups: Hilus cells Ca3/CA4 > CA1 > granule cells > CA2. Combined transections of perforant path and commissural fibers 3–5 days prior to injections of kainic acid prevented the degeneration of granule but not of pyramidal cells. The possible role of glutamic acid in degeneration and protection phenomena observed after kainic acid treatment, is discussed.     

Behavioural, biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnocellularis of rats

Lesions of the nucleus basalis magnocellularis in rats have been used to investigate functions of the extrinsic cortical cholinergic system which originates from these neurons. These lesions also produce extensive non-specific subcortical damage and associated regulatory and neurological impairments, causing doubt about the specificity of consequent functional impairments. Here, nucleus basalis magnocellularis lesions made with four different neurotoxic amino acids (kainic acid, ibotenic acid, N-methyl-D-aspartate, and quisqualic acid) have been compared. Quisqualic acid produced less subcortical damage and lesser neurological and regulatory impairments than the other toxins at doses that produced comparable cholinergic deafferentation of the neocortex, as assessed both histologically and biochemically. This suggests that these impairments are non-specific rather than specific consequences of cholinergic cell loss. The effects on learning a spatial navigation task were more ambiguous, suggesting the involvement of both cholinergic and non-cholinergic systems. Impairment of a passive shock avoidance task was as great following quisqualic acid as the other neurotoxins, which may suggest a more direct relationship specifically with the decline in cortical cholinergic activity. It is concluded that in the absence of availability of a specific cholinergic neurotoxin, quisqualic acid produces less non-specific neuroanatomical and neurological side effects than the more widely used toxins N-methyl-D-aspartate, kainic acid or ibotenic acid.     

Opposite turning effects of dainic and ibotenic acid injected in the rat substantia nigra.

Unilateral intranigral administration of kainic and ibotenic acid, two putative stimulants of glutamatergic mechanisms, elicited turning behaviour starting from doses of 10 ng. While the turning produced by kainic acid was ipsilateral, that produced by ibotenic acid was contralateral to the injected side. Previous destruction of dopaminergic neurons on the side of the intranigral injection failed to reduce the turning behaviour. Peripheral treatment with picrotoxin did not reduce the turning in response to ibotenic acid. The results might suggest the existence of excitatory and inhibitory glutamate receptors which control nigral non-dopaminergic neurons mediating turning-behaviour.     

Intrahippocampal kainic acid,seizures and local neuronal degeneration: Relationships assessed in unanesthetized rats

Intrahippocampal infusion of nanogram amounts of the neurotoxin kainic acid were used to investigate possible relationships between the convulsive and the local neurodegenerative properties of the amino acid. Bilateral hippocampal depth electrodes and cortical leads were employed to provide simultaneous and continuous electroencephalographic records following kainate injection in unanesthetized freely-behaving rats. In every animal, morphological analysis was performed 3–5 days after administration of kainic acid and attempts were made to correlate neuronal destruction with electroencephalographic patterns. Doses as low as 500 pg kainate led to behavioral sequelae consisting of grooming, scratching and enhanced locomotor activity. In a roughly dose-dependent fashion (range 500 pg-250 ng), these behaviors increased in frequency and at the highest doses the rats also displayed wet-dog shakes, stereotype mouth movements and occasional facial myoclonus. Apart from these automatisms, generalized motor seizures were never seen. Following kainic acid, a spectrum of electroencephalographic changes could occur consisting of one or more of the following: high voltage fast activity, slow and fast high voltage spiking, paroxysmal bursts, spindle bursts or postictal depression periods. The combination of any two of these changes were defined as an ictal episode if they occurred in all four leads simultaneously. Upon morphological examination, only the highest dose used (250 ng) resulted reliably in the degeneration of CA₃, CA₄ and, partly, CA₁ pyramidal cells on the injected side. While the duration of electroencephalographic changes at this dose was significantly higher than at any of the lower doses, the number of seizures or the total time spent in seizures was not different at 250 ng from that at 50 ng. At the latter dose, however, only marginal cell damage could be found.Our data indicate that very low doses of kainic acid directly applied to hippocampal CA₃ neurons, can elicit bilateral changes in the electroencephalogram indicative of repetitive limbic seizures which are not necessarily accompanied by neuronal degeneration. At higher doses (250 ng), kainic acid treatment results in both seizure activity and nerve cell death but the two effects appear mechanistically unrelated. While there is no clear-cut dose-response relationship between neuronal damage and seizures, extended electroencephalographic changes of a 15–30 Hz fast activity or simple spiking phenomena may be instrumental for the degenerative process. This dissociation between convulsive and neurodegenerative properties of kainic acid, however, does not argue against a role of an endogenous substance related to kainic acid in the etiology of temporal lobe seizure disorders.     

Subicular lesions cause dendritic atrophy in CA1 and CA3 pyramidal neurons of the rat hippocampus

The subiculum is a major source of output projections from hippocampus to cortical and subcortical regions. Our previous studies have demonstrated the selective loss of CA1 pyramidal neurons of the hippocampus, and operant and spatial learning impairment in subicular lesioned rats [Govindaiah et al. (1997) Brain Res. 745, 121-126; Laxmi et al. (1999) Brain Res. 816, 245-148]. In the present study, the effect of ibotenate lesions of the subiculum on the dendritic morphology of CA1 and CA3 pyramidal neurons of the hippocampus was investigated in 30-day-old male Wistar rats. The ventral subiculum was lesioned bilaterally with multiple injections of ibotenic acid, stereotaxically. The dendritic branching points and intersections were studied in apical and basal dendrites up to 320 and 160 microm, respectively, in Golgi-impregnated CA1 and CA3 pyramidal neurons of the hippocampus. The results revealed a significant (P<0.001) decrease in the number of dendritic branching points, intersections and total number of dendrites in both apical and basal dendrites of CA1, as well as CA3 pyramidal neurons of the hippocampus. It is surprising that the subicular lesions caused dendritic atrophy of CA3 neurons without affecting the cell density.The results of the present study demonstrate the dendritic atrophy of hippocampal neurons following selective subicular lesions. This might be responsible for the impairments in operant and spatial learning tasks in these rats as observed in our earlier studies. In addition, hippocampal damage is also associated with an impairment in the process of the active monitoring of movements in space, rather than place learning per se [Whishaw (1998) Neurosci. biobeh. Rev. 22, 209-220]. Accordingly, further studies are required to correlate the differential effect of subicular lesions on impairments in learning and movement in space in rats.     

Decrease in hippocampal [3H]vinylidene kainic acid binding in genetically epilepsy-prone rats

Specific [3H]vinylidene kainic acid binding to the kainate-sensitive subtype of glutamate receptor was studied in brain of 31-day-old non-epileptic Sprague-Dawley control and two colonies of genetically epilepsy-prone rats using in vitro autoradiographic techniques. At 37.5 nM [3H]vinylidene kainic acid, specific [3H]vinylidene kainic acid binding was reduced significantly by 18 and 22% in dorsal and ventral hippocampal formation stratum lucidum of 31-day-old genetically epilepsy-prone-9 rats compared with non-epileptic controls. Hippocampal [3H]vinylidene kainic acid binding was reduced in genetically epilepsy-prone-3 rats by 15 and 18%, but these reductions were not statistically significant. Saturation of [3H]vinylidene kainic acid binding studies indicated that the total number of ventral hippocampal [3H]vinylidene kainic acid binding sites was decreased by 21% in genetically epilepsy-prone-3 rats and 28% in genetically epilepsy-prone-9 rats. The reduction in ventral hippocampal [3H]vinylidene kainic acid binding in genetically epilepsy-prone rats resembles the reduction in ventral hippocampal [3H]vinylidene kainic acid binding sites observed in perinatal hypothyroid rats. As genetically epilepsy-prone rats are hypothyroid during the neonatal period, the reduction in hippocampal [3H]vinylidene kainic acid binding in the genetically epilepsy-prone rats may be a consequence of a hypothyroid-induced defect in the development or maturation of the hippocampal mossy fiber projection in genetically epilepsy-prone rats. An alternative hypothesis is that the putative occurrence of spontaneous limbic seizures in genetically epilepsy-prone rats may lead secondarily to a reduction in hippocampal [3H]vinylidene kainic acid binding sites.     

Uncoupling of local blood flow and metabolism in the hippocampal CA3 in kainic acid-induced limbic seizure status

Limbic seizure status was induced by microinjection of kainic acid into a unilateral amygdala in rats. Two hours after kainic acid injection, distant neuronal cell damage was produced, especially in the hippocampal CA3 on the kainic acid-injected side. In order to elucidate the mechanism of this neuronal cell damage, local cerebral glucose utilization and local cerebral blood flow were studied by means of an autoradiographic method using [14C]2-deoxyglucose and [14C]iodoantipyrine during kainic acid-induced limbic seizure status. These studies were performed 2 h after kainic acid microinjection into a unilateral amygdala. Both local cerebral glucose utilization and local cerebral blood flow were remarkably increased in the limbic system, ventrobasal complex of the thalamus, septal nucleus, nucleus accumbens, caudate nucleus, substantia nigra and hypothalamus on the kainic acid-injected side. In the hippocampus, local cerebral glucose utilization increased 2.6 times control in CA1 and 4.1 times in CA3, whereas the rates of increase in local cerebral blood flow were similarly low in CA1 and CA3: 1.2 and 1.4 times control, respectively. The results demonstrated that the degree of uncoupling of local cerebral glucose utilization and local cerebral blood flow were higher in CA3 than in CA1, and also suggested that relative hypoxia occurred in CA3 in this high degree of uncoupling, resulting in pyramidal cell damage in CA3 in kainic acid-induced limbic seizure status.     

Induction of somatostatin by kainic acid in pyramidal and granule cells of the rat hippocampus.

Seizures were induced in rats by systemic administration of kainic acid and, 1.5-12 h after, expression of preprosomatostatin and c-fos mRNAs in 9 hippocampal areas and in the cerebral perirhinal cortex was investigated using in situ hybridization histochemistry. Immunohistochemistry was also performed to study somatostatin peptide. In the control animals preprosomatostatin mRNA was expressed in some cells in the dentate hilus, the stratum oriens and the stratum radiatum of Ammon's horn, the subiculum and the cortex. Starting 3 h after kainic acid administration preprosomatostatin mRNA was expressed in a subpopulation of granule and pyramidal cells which did not normally express it. Preprosomatostatin mRNA-positive cells were markedly increased in the subiculum. Immunohistochemical examination confirmed that preprosomatostatin mRNA in granule and pyramidal cells was translated into peptide. In contrast, c-fos mRNA was induced in most hippocampal and cortical neurons starting 1.5 h after the kainic acid injection. When diazepam was injected to suppress the generalized seizures, preprosomatostatin mRNA was still expressed in pyramidal and subicular cells but not in granule cells.     

Propidium iodide in vivo: an early marker of neuronal damage in rat hippocampus

We have investigated the use of the fluorescent exclusion dye propidium iodide as a marker for acutely degenerating cells in vivo, and report here that combined injection of kainic acid and propidium iodide into the lateral cerebral ventricle results in labelling of CA3 pyramidal cells 1 and 6 h after injection. Alternate sections stained with thionin at these early times revealed little evidence of histologically detectable cell damage.