Injection Of Kainic Acid Research Articles

Homer expression in the Xenopus tadpole nervous system

Homer proteins are integral components of the postsynaptic density and are thought to function in synaptogenesis and plasticity. In addition, overexpression of Homer in the developing Xenopus retinotectal system results in axonal pathfinding errors. Here we report that Xenopus contains the homer1 gene, expressed as the isoform, xhomer1b, which is highly homologous to the mammalian homer1b. The mammalian homer1 gene is expressed as three isoforms, the truncated or short form homer1a and the long forms homer1b and -1c. For Xenopus, we cloned three very similar variants of homer1b, identified as Xenopus xhomer1b.1, xhomer1b.2, and xhomer1b.3, which display up to 98% homology with each other and 90% similarity to mammalian homer1b. Furthermore, we demonstrate that Xenopus also contains a truncated form of the Homer1 protein, which could be induced by kainic acid injection and is likely homologous to the mammalian Homer1a. xHomer1b expression was unaffected by neuronal activity levels but was developmentally regulated. Within the brain, the spatial and temporal distributions of both Homer isoforms were similar in the neuropil and cell body regions. Homer1 was detected in motor axons. Differential distribution of the two isoforms was apparent: Homer1b immunoreactivity was prominent at junctions between soma and the ventricular surface; in the retina, the Mueller radial glia were immunoreactive for Homer1, but not Homer1b, suggesting the retinal glia contain only the Homer1a isoform. Homer1b expression in muscle was prominent throughout development and was aligned with the actin striations in skeletal muscle. The high level of conservation of the xhomer1 gene and the protein expression in the developing nervous system suggest that Homer1 expression may be important for normal neuronal circuit development.

Transient changes in the limbic histaminergic system after systemic kainic acid-induced seizures

Increased brain histamine is reported to protect against convulsions. We used systemic kainic acid (KA) administration to study possible changes of the histaminergic system in rat brain in status epilepticus (SE). Robust increases in brain histamine concentrations and numbers of histamine-immunoreactive nerve fibers were detected in the piriform cortex (Pir) and amygdala after KA injection, suggesting a reactive increase, which is opposite to other published aminergic transmitter responses. These changes, lasting several weeks, might be coupled to a mechanism unrelated to the anticonvulsive function of histamine. Transient increases in mRNA expression of H(3) receptor isoforms with a full-length third intracellular loop, coupled to mitogen-activated protein kinase pathway, were detected first in the hippocampal CA3c area, followed by the Pir and amygdala and then the hippocampal CA1 area. These results suggest that histamine and H3 receptors, which also control the release of GABA and glutamate, might be involved in convulsive SE.

Clozapine, ziprasidone and aripiprazole but not haloperidol protect against kainic acid-induced lesion of the striatum in mice, in vivo: Role of 5-HT 1A receptor activation

Excessive activation of non-NMDA receptors, AMPA and kainate, contributes to neuronal degeneration in acute and progressive pathologies, possibly including schizophrenia. Because 5-HT 1A receptor agonists have neuroprotective properties (e.g., against NMDA-induced neurotoxicity), we compared the effects of the antipsychotics, clozapine, ziprasidone and aripiprazole, that are partial agonists at 5-HT 1A receptor, with those of haloperidol, which is devoid of 5-HT 1A agonist properties, on kainic acid (KA)-induced striatal lesion volumes, in C57Bl/6N mice. The involvement of 5-HT 1A receptors was determined by antagonist studies with WAY100635, and data were compared with those obtained using the potent and high efficacy 5-HT 1A receptor agonist, F13714. Intra-striatal KA lesioning and measurement of lesion volumes using cresyl violet staining were carried out at 48 h after surgery. F13714, antipsychotics or vehicle were administered ip twice, 30 min before and 3 1/2 h after KA injection. WAY100635 (0.63 mg/kg) or vehicle were given sc 30 min before each drug injection. Clozapine (2 × 10 mg/kg), ziprasidone (2 × 20 mg/kg) and aripiprazole (2 × 10 mg/kg) decreased lesion volume by 61%, 59% and 73%, respectively. WAY100635 antagonized the effect of ziprasidone and of aripiprazole but only slightly attenuated that of clozapine. In contrast, haloperidol (2 × 0.16 mg/kg) did not affect KA-induced lesion volume. F13714 dose-dependently decreased lesion volume. The 61% decrease of lesion volume obtained with F13714 (2 × 0.63 mg/kg) was antagonized by WAY100635. WAY100635 alone did not affect lesion volume. These results show that 5-HT 1A receptor activation protects against KA-induced striatal lesions and indicate that some atypical antipsychotic agents with 5-HT 1A agonist properties may protect against excitotoxic injury, in vivo.

Distinct Mechanistic Roles of Calpain and Caspase Activation in Neurodegeneration as Revealed in Mice Overexpressing Their Specific Inhibitors

Enzymatic proteolysis has been implicated in diverse neuropathological conditions, including acute/subacute ischemic brain injuries and chronic neurodegeneration such as Alzheimer disease and Parkinson disease. Calcium-dependent proteases, calpains, have been intensively analyzed in relation to these pathological conditions, but in vivo experiments have been hampered by the lack of appropriate experimental systems for a selective regulation of the calpain activity in animals. Here we have generated transgenic (Tg) mice that overexpress human calpastatin, a specific and the only natural inhibitor of calpains. In order to clarify the distinct roles of these cell death-associated cysteine proteases, we dissected neurodegenerative changes in these mice together with Tg mice overexpressing a viral inhibitor of caspases after intrahippocampal injection of kainic acid (KA), an inducer of neuronal excitotoxicity. Immunohistochemical analyses using endo-specific antibodies against calpain- and caspase-cleaved cytoskeletal components revealed that preclusion of KA-induced calpain activation can rescue the hippocampal neurons from disruption of the neuritic cytoskeletons, whereas caspase suppression has no overt effect on the neuritic pathologies. In addition, progressive neuronal loss between the acute and subacute phases of KA-induced injury was largely halted only in human calpastatin Tg mice. The animal models and experimental paradigm employed here unequivocally demonstrate their usefulness for clarifying the distinct contribution of calpain and caspase systems to molecular mechanisms governing neurodegeneration in adult brains, and our results indicate the potentials of specific calpain inhibitors in ameliorating excitotoxic neuronal damages.

Neuroprotective effect of citicoline against KA-induced neurotoxicity in the rat retina

We examined whether citicoline has neuroprotective effect on kainic acid (KA)-induced retinal damage. KA (6 nmol) was injected into the vitreous of rat eyes. Rats were injected intraperitoneally with citicoline (500 mg kg −1, i.p.) twice (09:00 and 21:00) daily for 1, 3 and 7 days after KA-injection. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers. In addition, immunohistochemistry was conducted to elucidate the expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH). Morphometric analysis of retinal damage in KA-injected eyes showed a significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at the 1, 3 and 7 days after KA injection, but not in the outer nuclear layers (ONL). At 1 and 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of ChAT and TH. The immunoreactivities of ChAT and TH had almost disappeared in the retina after 7 days of KA injection. However, prolonged citicoline treatment for 7 days significantly attenuated the reduction of retinal thickness and immunoreactivities of ChAT and TH. The present study suggests that treatment with citicoline has neuroprotective effect on the retinal damage due to KA-induced neurotoxicity.

Epileptogenesis and chronic seizures in a mouse model of temporal lobe epilepsy are associated with distinct EEG patterns and selective neurochemical alterations in the contralateral hippocampus

Major aspects of temporal lobe epilepsy (TLE) can be reproduced in mice following a unilateral injection of kainic acid into the dorsal hippocampus. This treatment induces a non-convulsive status epilepticus and acute lesion of CA1, CA3c and hilar neurons, followed by a latent phase with ongoing ipsilateral neuronal degeneration. Spontaneous focal seizures mark the onset of the chronic phase. In striking contrast, the ventral hippocampus and the contralateral side remain structurally unaffected and seizure-free. In this study, functional and neurochemical alterations of the contralateral side were studied to find candidate mechanisms underlying the lack of a mirror focus in this model of TLE. A quantitative analysis of simultaneous, bilateral EEG recordings revealed a significant decrease of theta oscillations ipsilaterally during the latent phase and bilaterally during the chronic phase. Furthermore, the synchronization of bilateral activity, which is very high in control, was strongly reduced already during the latent phase and the decrease was independent of recurrent seizures. Immunohistochemical analysis performed in the contralateral hippocampus of kainate-treated mice revealed reduced calbindin-labeling of CA1 pyramidal cells; down-regulation of CCK-8 and up-regulation of NPY-labeling in mossy fibers; and a redistribution of galanin immunoreactivity. These changes collectively might limit neuronal excitability in CA1 and dentate gyrus, as well as glutamate release from mossy fiber terminals. Although these functional and neurochemical alterations might not be causally related, they likely reflect long-ranging network alterations underlying the independent evolution of the two hippocampal formations during the development of an epileptic focus in this model of TLE.

Selected Papers: Laboratory Studies

Abstract *†Soichi Obara, *Shin-ichi Imamura, ‡Koichi Akaike, §Shigeya Tanaka, †Ikuro Maruyama, and *Jun-ichi Kuratsu Departments of *Neurosurgery , †Laboratory and Molecular Medicine and ‡Neuropsychiatry, University of Kagoshima, Faculty of Medicine , and §Tanaka Neurosurgical Clinic, Kagoshima, Japan . Purpose: Prior studies indicated that 2-arachidonoglycerol (2-AG), an endogenous cannabinoid, might have neurophysiologic effects on the hippocampus, possibly by activating cannabinoid receptor-1 (CB1). This study was designed to clarify the effect of 2-AG on hippocampal neuronal damages caused by kainic acid (KA)-induced limbic seizures. Methods: Twenty-four rats underwent stereotactic implantation of electrodes in the left amygdala (LA), left hippocampus (LH), and left sensorimotor cortex (LCx). A stainless steel cannula also was introduced into the LA and LH. The animals then were divided into four groups according to the pretreatment agent infused into LH: sham control [dimethyl sulfoxide (DMSO); 1 μl, n = 6], KA control (DMSO; 1 μl, n = 6), group A (2-AG; 100 μM, n = 6), and group B [2-AG (100 μM) + SR-141716A (a CB-1 antagonist; 10 μM), n = 6]. After 10 min, all rats except the sham-treated group received 1 μg of kainic acid (KA) via the cannula. Sham-treated rats received phosphate-buffered saline (PBS) instead. After electroclinical observation for 7 days, histologic examination and statistical analysis were performed. Results: In KA controls, immediate multiple spike discharges in LA propagated concurrently to the LH. The propagation involved the LCx, with development of status epileptics 1–2 h after KA injection. Seizures were characterized by mastication, salivation, facial twitching, forelimb clonus, and sometimes rearing and falling, and lasted 1–2 days. Microscopic examination revealed severe neuronal cell damage in the LA and LH. In group A, seizure discharges were eliminated overall and neuronal cell damage in LH was reduced compared with the KA controls. Rats in group A only showed behavioral changes such as wet-dog shake. In group B, seizure discharges and neuronal cell damage in LH were virtually the same as in controls. Sham-treated rats showed no electroclinical and histologic changes. Conclusions: These results indicate that the 2-AG exhibits a neuroprotective effect against limbic seizures by activating CB1.

Up‐regulation of neuropeptide Y levels and modulation of glutamate release through neuropeptide Y receptors in the hippocampus of kainate‐induced epileptic rats

Kainate-induced epilepsy has been shown to be associated with increased levels of neuropeptide Y (NPY) in the rat hippocampus. However, there is no information on how increased levels of this peptide might modulate excitation in kainate-induced epilepsy. In this work, we investigated the modulation of glutamate release by NPY receptors in hippocampal synaptosomes isolated from epileptic rats. In the acute phase of epilepsy, a transient decrease in the efficiency of NPY and selective NPY receptor agonists in inhibiting glutamate release was observed. Moreover, in the chronic epileptic hippocampus, a decrease in the efficiency of NPY and the Y(2) receptor agonist, NPY13-36, was also found. Simultaneously, we observed that the epileptic hippocampus expresses higher levels of NPY, which may account for an increased basal inhibition of glutamate release. Consistently, the blockade of Y(2) receptors increased KCl-evoked glutamate release, and there was an increase in Y(2) receptor mRNA levels 30 days after kainic acid injection, suggesting a basal effect of NPY through Y(2) receptors. Taken together, these results indicate that an increased function of the NPY modulatory system in the epileptic hippocampus may contribute to basal inhibition of glutamate release and control hyperexcitability.

Pattern of Glial Fibrillary Acidic Protein Expression Following Kainate-Induced Cerebellar Lesion in Rats

In the present study glial fibrillary acidic protein (GFAP) expression was assessed following intravermian injection of kainic acid (KA) or physiological saline to adult rat cerebellum. After 2- to 30-day recovery period, free-floating sections cut with a microtome were obtained and were proccessed for immunocytochemistry against GFAP. Injection of both kainate and physiological saline elicited significant astrogliotic reaction, i.e. in the area around the lesion thick GFAP-positive Bergmann fibers with typical orientation appeared in the molecular and hypertrophied astrocytes abundantly appeared in the granular layer. However, following kainate intoxication lesion was not surrounded by typical demarcation glial scar during 30-day recovery period in contrast to the appearance of usual glial scar in the group injected with physiological saline, as early as 7-day postlesion. Preserved spatial organization of Bergmann fibers and the absence of typical demarcating glial scar after kainate-induced cerebellar lesion suggest distinct pattern of astrogliosis that presents an interesting model system to study the importance of glial scar in the recovery after ischemic brain insults.

Glutamate Receptor Antagonists and Benzodiazepine Inhibit the Progression of Granule Cell Dispersion in a Mouse Model of Mesial Temporal Lobe Epilepsy

Unilateral intrahippocampal injection of kainic acid (KA) in adult mice induces the progressive dispersion of dentate granule cells, one of the characteristic pathologic changes of mesial temporal lobe epilepsy. However, little is known about the mechanisms that trigger this dispersion. In this study, the possible involvement of glutamatergic and gamma-aminobutyric acid (GABA)ergic neurotransmissions in the development of granule cell dispersion (GCD) was examined in this model. Antagonists of N-methyl-d-aspartate (NMDA) receptor (MK-801) and non-NMDA receptor (GYKI52466), and an agonist of benzodiazepine-GABA(A) receptor (midazolam) were injected before and after KA in various ways, and the morphologic changes of the hippocampus, especially GCD, were examined. MK-801 (5 mg/kg, i.p.) did not reduce GCD when injected 2 h before KA injection but inhibited GCD almost completely for <or=14 days, when injected 4 h after KA. However, mild to moderate dispersion was observed at 28 days, indicating that MK-801 may delay the progression of GCD. Similarly, daily treatment with MK-801 (2 x 1 mg/kg i.p./day) for the first 26 days after KA significantly reduced GCD. In contrast, GYKI52466 (30 mg/kg, s.c.) was effective only when it was injected before KA. A significant reduction of GCD was also observed after continuous administration of midazolam (10 mg/kg/h) after KA. These data show that GCD in this mouse model is triggered by either the stimulation of the NMDA receptor or reduction of GABA(A)-mediated inhibition after intrahippocampal injection of KA. It is suggested that the increased excitation or the reduced inhibition or both could be one of the factors triggering or maintaining or both the process of GCD.

Up-regulation of peripherin is associated with alterations in synaptic plasticity in CA1 and CA3 regions of hippocampus

Peripherin is a type III intermediate filament protein normally undetectable in most brain neurons. Here, we report a similar pattern of peripherin expression in the brains of both mice treated with systemic injections of kainic acid (KA) and in peripherin transgenic mice (Per mice) over-expressing the normal peripherin gene under its own promoter. Double-immunofluorescence labeling revealed a partial co-localization of peripherin with the microtubule-associated protein MAP2, but not with neurofilament proteins. Electrophysiological studies revealed that synaptic plasticity was markedly altered in Per mice: in CA1, long-term potentiation (LTP) was decreased in Per slices (+29 +/- 2.0%, vs. +58 +/- 5.4%, in WT); while in CA3, LTP was increased in Per (+63 +/- 3.5% vs. +43 +/- 2.4.0%). In the hippocampus of Per mice, the levels of MAP2 were decreased, though synaptophysin and PSD95 remained unchanged. These intriguing findings suggest a role of peripherin in the alteration of hippocampal synaptic plasticity.

Neuropeptide Y expression in mouse hippocampus and its role in neuronal excitotoxicity1

To investigate neuropeptide Y (NPY) expression in mouse hippocampus within early stages of kainic acid (KA) treatment and to understand its role in neuronal excitotoxicity. NPY expression in the hippocampus within early stages of KA intraperitoneal (ip) treatment was detected by immunohistochemistry (IHC) and in situ hybridization (ISH) methods. The role of NPY and Y5, Y2 receptors in excitotoxicity was analyzed by terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) assay. Using IHC assay, in granule cell layer of the dentate gyrus (DG), NPY positive signals appeared 4 h after KA injection, reached the peak at 8 h and leveled off at 16 and 24 h. In CA3, no positive signal was found within the first 4 h after KA injection, but strong signal appeared at 16 and 24 h. No noticeable signal was detected in CA1 at all time points after KA injection. Using the ISH method, positive signals were detected at 4, 8, and 16 h in CA3, CA1, and hilus. In DG, much stronger ISH signals were detected at 4 h, but leveled off at 8 and 16 h. TUNEL analysis showed that intracerebroventricularly (icv) infusion of NPY and Y5, Y2 receptor agonists within 8 h after KA insult with proper dose could remarkably rescue pyramidal neurons in CA3 and CA1 from apoptosis. NPY is an important anti-epileptic agent. The preceding elevated expression of NPY in granule cell layer of DG after KA injection might partially explain its different excitotoxicity-induced apoptotic responses in comparison with the pyramidal neurons from CA3 and CA1 regions. NPY can not only reduce neuronal excitability but also prevent excitotoxicity-induced neuronal apoptosis in a time- and dose-related way by activation of Y5 and Y2 receptors.

Neuroprotective Effect of Citicoline on Retinal Cell Damage Induced by Kainic Acid in Rats

To examine whether citicoline has a neuroprotective effect on kainic acid (KA)-induced retinal damage. KA (6 nmol) was injected into the vitreous of rat eyes. Citicoline (500mg/kg, i.p.) was administered to the rats once before and twice a day after KA-injection for 3- and 7-day intervals. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers using hematoxylin-eosin (H&E) staining. In addition, immunohistochemistry was conducted to elucidate the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS). Morphometric analysis of retinal damage in KA-injected eyes showed significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at 3 and 7 days after KA injection, but not in the outer nuclear layer (ONL). At 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of eNOS and nNOS. The immunoreactivities of eNOS and nNOS increased in the retina at 7 days after the KA injection. However, prolonged treatment for 7 days significantly attenuated the immunoreactivities and the reduction of thickness. The results indicate that citicoline has a neuroprotective effect on KA-induced neurotoxicity in the retina.

Cortical control for mastication in cats: Changes in masticatory movements following lesions in the masticatory cortex

In a previous paper (Hiraba and Sato ) we reported that an accurate mastication might be executed by the cortical processing in bilateral masticatory area (MA)and motor cortices. The aim of this study was to determine if cats with lesion of either unilateral or bilateral MA showed changes in mastication. After exploring mechanoreceptive fields and motor effects of mastication-related neurons (MRNs) in MA using the single unit recording and intracortical microstimulation methods, we made various lesions in MAs with injections of kainic acid (0.1%, 2.0 µl). Since the MA was divided into facial (F) and intraoral (I) projection areas as reported in the previous paper, cats with the unilateral lesion in F or I, and with the bilateral lesion in F & F, I & I or F & I (F on one side and I on other side) were prepared. Cats with unilateral lesion in F or I and with bilateral lesion in F & I showed no changes in mastication except for prolongation of the food intake and masticatory periods. Cats with bilateral lesion into F & F, or I & I showed wider jaw-opening during mastication. Particularly, the latter group showed enormous jaw-opening, delay in the start of mastication and difficulty in manipulating food on the tongue. In all cats with lesions of each type, masticatory and swallowing rhythms remained normal. These findings suggest that accurate mastication is executed by the close integration between F & F and I & I of the bilateral MA.

Ro5-4864, a peripheral benzodiazepine receptor ligand, reduces reactive gliosis and protects hippocampal hilar neurons from kainic acid excitotoxicity

The peripheral-type benzodiazepine receptor (PBR) is a critical component of the mitochondrial permeability transition pore, which is involved in the regulation of cell survival. Different forms of brain injury result in induction of the expression of the PBR in the areas of neurodegeneration, mainly in reactive glial cells. The consequences of induction of PBR expression after brain injury are unknown. To test whether PBR may be involved in the regulation of neuronal survival after injury, we have assessed the effect of two PBR ligands, Ro5-4864 and PK11195, on neuronal loss induced by kainic acid in the hippocampus. Systemic administration of kainic acid to male rats resulted in the induction of a reactive phenotype in astrocytes and microglia and in a significant loss of hilar neurons in the dentate gyrus. Administration of Ro5-4864, before the injection of kainic acid, decreased reactive gliosis in the hilus and prevented hilar neuronal loss. In contrast, PK11195 was unable to reduce reactive gliosis and did not protect hilar neurons from kainic acid. These findings suggest that the PBR is involved in control of neuronal survival and gliosis after brain injury and identify this molecule as a potential target for neuroprotective interventions.

ELECTRO-ACUPUNCTURE IMPROVES EPILEPTIC SEIZURES INDUCED BY KAINIC ACID IN TAURINE-DEPLETION RATS

Electro-acupuncture (EA) partially inhibited epilepsy with great success. The biological basis underlying EA anti-convulsion remained uncertain, which resulted in limited application and slow improvement of acupuncture. Our previous study indicated that taurine may play an inhibitory role against epilepsy as an inhibitory amino acid in the central nervous system and EA may inhibit epilepsy via up-regulating the expression of taurine transporter to increase the release of taurine. Involvement of taurine in kainic acid (KA)-induced epilepsy and anti-convulsion of EA was further addressed on taurine deficiency animal in the present work. We instituted endogenous taurine-deficiency model by supplementation of beta-alanine (3%) in drinking water for continuous 10 days initially, injected KA into lateral cerebral ventricle to induce epileptic seizure, and performed EA treatment on DU26 "RenZhong" and K "YongQuan" acupoints by an EA apparatus (Model G6805-2) using successive waves with the frequency 64Hz and the current intensity 0.8-1.0 mA for 30 minutes in Sprague-Dawley (SD) rats. Taurine levels markedly decreased in cortex, hippocampus, striatum and cerebellum of rats after beta-alanine administration by fluore-HPLC measurement. EA alleviated epileptic activity in rats at 3.5 h time point after KA injection, whereas beta-alanine-induced taurine depletion rendered rats more susceptible to KA-induced epilepsy. Taurine transporter level increased after EA treatment. These results suggested that taurine participated in epileptogenesis and EA may be related to taurine in controlling epileptic seizure.

7-nitroindazole, a selective neuronal nitric oxide synthase inhibitore in vivo, prevents kainate-induced intrahippocampal neurotoxicity

We investigated the effects of 7-nitroindazole (7-NI), a selective neuronal nitric oxide synthase inhibitor in vivo, on nitrite concentration after kainic acid injection unilaterally into the CA3 region of the rat hippocampus. The accumulation of nitrite, the stable metabolite of NO, was measured by the Griess reaction at different times in hippocampus, forebrain cortex, striatum, and cerebellum homogenates. 7-nitroindazole can effectively inhibit NO synthesis in rat brain after kainate-induced neurotoxicity and suppressed nitrite accumulation. The present results suggest that neuronal NO synthase inhibitors may be useful in the treatment of neurological diseases in which excitotoxic mechanisms play a role. &lt;br&gt;&lt;br&gt;&lt;font color="red"&gt;&lt;b&gt; This article has been retracted. Link to the retraction &lt;u&gt;&lt;a href="http://dx.doi.org/10.2298/ABS160412036E"&gt;10.2298/ABS160412036E&lt;/a&gt;&lt;u&gt;&lt;/b&gt;&lt;/font&gt;

Free Communications: Experiment 2–Kainic Acid

Abstract *KatsunariYoshida, *KiyotakaHashizume, and *TatsuyaTanaka *Department of Neurosurgery, Asahikawa Medical College, Asahikawa, Japan Purpose: It is thought that neurons are generated during the embryonic and postnatal periods but not after adolescence. We have found that pyramidal cell loss and pyknosis occur in a kainic acid–induced limbic seizure rat model. In this study, bromodeoxyuridine (BrdU), a marker of cell proliferation, was used to determine the relation between seizure-induced hippocampal degeneration and neurogenesis. Methods: Male Wistar rats (250–300 g) were stereotaxically injected with kainic acid (0.5 (g) into the unilateral amygdala. All of the rats received a single injection of BrdU (100 mg/kg) intraperitoneally (i.p.) before killing. Under deep sodium pentobarbital anesthesia (60 mg/kg i.p.), each rat was transcardially perfused with saline and then with 4% paraformaldehyde. Animals were killed on days 3, 7, 14, and 28 (three to six animals each day) after seizure induction. The brains were embedded in paraffin and cut in 4-μm sections. For preliminary examination, newborn cells positive for BrdU were counted in the dentate gyrus and pyramidal cell layers by diaminobenzidine (DAB) staining. Then double-labeled immunofluorescence staining was performed by using primary antibodies to BrdU and (-tubulin III (TuJ1) as immature neuronal markers and conjugated fluorescein isothiocyanate (FITC) and rhodamine as secondary antibodies. Results: In kainic acid–induced limbic seizure models, the number of newborn neuronal cells increased significantly in the dentate gyrus from days 3 to 7 compared with those in controls. Several newborn cells were observed in the pyramidal cell layer (CA3), where cell loss and pyknosis were evident. In another investigation using cryostat sections, mossy fiber reorganization in the dentate inner molecular layer was observed by Timm staining 2 weeks after kainic acid injection. Conclusions: The rate of neurogenesis in the dentate granule cell layer during the early postnatal period and adulthood appears to be influenced by factors such as aging, environmental stimulation, glucocorticoid hormone levels, and glutamatergic input to the dentate gyrus. Recent investigations using a variety of experimentally induced pathologies, including seizures, cerebral ischemia, and toxin injection, showed alterations in dentate neurogenesis after brain injury. Repetitive seizure activities induce not only neuronal cell loss but also seizure-induced neurogenesis in the dentate gyrus and CA3. The dentate gyrus is positioned to be able to control hippocampal throughput, and it has been suggested that the dentate gyrus functions as a gate for controlling the propagation of seizures. Increase of hippocampal neurogenesis appeared in association with neuronal damage. Generation of new neurons and mossy fiber sprouting in the hippocampus may be an important neurobiologic element underlying the clinical effects of kainic acid–induced limbic seizure.

High-frequency oscillations after status epilepticus: epileptogenesis and seizure genesis.

To investigate the temporal relation between high-frequency oscillations (HFOs) in the dentate gyrus and recurrent spontaneous seizures after intrahippocampal kainite-induced status epilepticus. Recording microelectrodes were implanted bilaterally in different regions of hippocampus and entorhinal cortex. A guide cannula for microinjection of kainic acid (KA) was implanted above the right posterior CA3 area of hippocampus. After recording baseline electrical activity, KA (0.4 microg/0.2 microl) was injected. Beginning on the next day, electrographic activity was recorded with video monitoring for seizures every day for 8 h/day for > or = 30 days. Of the 26 rats studied, 19 revealed the appearance of sharp-wave activity and HFOs in the frequency range of 80 to 500 Hz in the dentate gyrus ipsilateral to the KA injection. In the remaining seven rats, no appreciable activity was noted in this frequency range. In some rats with recurrent seizures, HFOs were in the ripple frequency range (100-200 Hz); in others, HFOs were in the fast ripple frequency range (200-500 Hz), or a mixture of both oscillation frequencies was found. The time of detection of the first HFOs after status epilepticus varied between 1 and 30 days, with a mean of 6.3 +/- 2.0 (SEM). Of the 19 rats in which HFO activity appeared, all later developed recurrent spontaneous seizures, whereas none of the rats without HFOs developed seizures. The sooner HFO activity was detected after status epilepticus, the sooner the first spontaneous seizure occurred. A significant inverse relation was found between the time to the first HFO detection and the subsequent rate of spontaneous seizures. A strong correlation was found between a decreased time to detection of HFOs and an increased rate of spontaneous seizures, as well as with a decrease in the duration of the latent period between KA injection and the detection of spontaneous seizures. Two types of HFOs were found after KA injection, one in the frequency range of 100 to 200 Hz, and the other, in the frequency range of 200 to 500 Hz, and both should be considered pathological, suggesting that both are epileptogenic.

Circadian rhythms in the retina of rats with photoreceptor degeneration.

Previous studies have demonstrated that the mammalian retina contains a circadian clock system that controls several retinal functions. In mammals the location of the retinal circadian clock is unknown whereas, in non-mammalian vertebrates, earlier work has demonstrated that photoreceptor cells contain the circadian clock. New experimental evidence has suggested that in mammals the retinal circadian clock may be located outside the photoreceptor cells. In this study we report that circadian rhythms in Aa-nat mRNA (in vivo) and melatonin synthesis (in vitro) are still present in the retina of rats lacking photoreceptors. The circadian pacemaker(s) controlling such rhythms is probably located in kainic acid sensitive neurons in the inner retina since kainic acid injections abolished the rhythmicity. These data are the first direct demonstration that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors and the suprachiasmatic nuclei of the hypothalamus.