Unilateral Injection Of Kainic Acid Research Articles

Localizing the Seizure Onset Site Through Metabolic Imaging of GABA.

In Vivo Gamma-Aminobutyric Acid Increase as a Biomarker of the Epileptogenic Zone: An Unbiased Metabolomics Approach Hamelin S, Stupar V, Maziere L, et al. Epilepsia. 2021;62(1):163-175.Objective:Following surgery, focal seizures relapse in 20% to 50% of cases due to the difficulty of delimiting the epileptogenic zone (EZ) by current imaging or electrophysiological techniques. Here, we evaluate an unbiased metabolomics approach based on ex vivo and in vivo nuclear magnetic resonance spectroscopy (MRS) methods to discriminate the EZ in a mouse model of mesiotemporal lobe epilepsy (MTLE).Methods:Four weeks after unilateral injection of kainic acid (KA) into the dorsal hippocampus of mice (KA-MTLE model), we analyzed hippocampal and cortical samples with high-resolution magic angle spinning (HRMAS) MRS. Using advanced multivariate statistics, we identified the metabolites that best discriminate the injected dorsal hippocampus (EZ) and developed an in vivo MEGAPRESS MRS method to focus on the detection of these metabolites in the same mouse model.Results:Multivariate analysis of HRMAS data provided evidence that γ-aminobutyric acid (GABA) is largely increased in the EZ of KA-MTLE mice and is the metabolite that best discriminates the EZ when compared with sham and, more importantly, when compared with adjacent brain regions. These results were confirmed by capillary electrophoresis analysis and were not reversed by a chronic exposition to an antiepileptic drug (carbamazepine). Then, using in vivo noninvasive GABA-edited MRS, we confirmed that a high GABA increase is specific to the injected hippocampus of KA-MTLE mice.Significance:Our strategy using ex vivo MRS-based untargeted metabolomics to select the most discriminant metabolite(s), followed by in vivo MRS-based targeted metabolomics, is an unbiased approach to accurately define the EZ in a mouse model of focal epilepsy. Results suggest that GABA is a specific biomarker of the EZ in MTLE.

In vivo γ-aminobutyric acid increase as a biomarker of the epileptogenic zone: An unbiased metabolomics approach.

Following surgery, focal seizures relapse in 20% to 50% of cases due to the difficulty of delimiting the epileptogenic zone (EZ) by current imaging or electrophysiological techniques. Here, we evaluate an unbiased metabolomics approach based on ex vivo and in vivo nuclear magnetic resonance spectroscopy (MRS) methods to discriminate the EZ in a mouse model of mesiotemporal lobe epilepsy (MTLE). Four weeks after unilateral injection of kainic acid (KA) into the dorsal hippocampus of mice (KA-MTLE model), we analyzed hippocampal and cortical samples with high-resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS). Using advanced multivariate statistics, we identified the metabolites that best discriminate the injected dorsal hippocampus (EZ) and developed an in vivo MEGAPRESS MRS method to focus on the detection of these metabolites in the same mouse model. Multivariate analysis of HRMAS data provided evidence that γ-aminobutyric acid (GABA) is largely increased in the EZ of KA-MTLE mice and is the metabolite that best discriminates the EZ when compared to sham and, more importantly, when compared to adjacent brain regions. These results were confirmed by capillary electrophoresis analysis and were not reversed by a chronic exposition to an antiepileptic drug (carbamazepine). Then, using in vivo noninvasive GABA-edited MRS, we confirmed that a high GABA increase is specific to the injected hippocampus of KA-MTLE mice. Our strategy using ex vivo MRS-based untargeted metabolomics to select the most discriminant metabolite(s), followed by in vivo MRS-based targeted metabolomics, is an unbiased approach to accurately define the EZ in a mouse model of focal epilepsy. Results suggest that GABA is a specific biomarker of the EZ in MTLE.

Longitudinal imaging of Microglia-Astrocyte activation in mouse mesial temporal lobe epilepsy with TSPO PET to identify the best therapeutic time windows

Objectives Mesiotemporal lobe epilepsy (MTLE) is the most common type of drug-resistant partial epilepsy with a specific history which begins by a status epilepticus due to a neurological insult followed by a silent period before the recurrent seizures begin. Once established, these seizures can become resistant to medications. Along this period a specific lesion is often found, described as a unilateral hippocampal sclerosis (HS). Currently it is still challenging to determine when and which drugs can be most effective along the constitution of this epileptic process. Neuroinflammation has been reported to play an important role in pathophysiological mechanisms in epilepsy, and therefore brain inflammation probes can be potent epilepsy biomarkers. The Translocator Protein 18 kDa (TSPO) expression is associated with neuroinflammatory processes involving reactive astrocytes and microglia. A unilateral intrahippocampal kainate injection mouse model can reproduce the defining features of human temporal lobe epilepsy with unilateral hippocampal sclerosis and the pattern of chronic pharmacoresistant temporal seizures. We hypothesized that TSPO PET imaging using 18F-DPA-714 TSPO-targeted PET could be a highly potent longitudinal biomarker of epilepsy and could be of interest to determine the therapeutic windows in epilepsy and follow response to treatment in the mouse model of MTLE. Methods The model was induced by unilateral injection of kainic acid into the right dorsal hippocampus of male C57/Bl6 mice. μPET/CT scanning was performed before model induction and along the development of the HS at 7 days, 14 days, 1 month and 6 months. In vitro autoradiography and immunohistofluorescence were performed on additional mice at each time point. Results TSPO PET uptake reached the peak at 7 days and mostly correlated with microglial activation whereas after 14 days, reactive astrocytes were shown to be the main cells expressing TSPO which would be related to the increased PET uptake. Our studies suggest that mTOR signal pathways play an important role in the modulation of astrocyte activities. Our hypothesis is that the neurotoxicity during ictogenesis (aggravation phase) could be due to persistent mTOR reactive signaling in astrocytes. Conclusion : on this knowledge of TSPO expression over time in the MTLE model, TSPO PET imaging could be a valuable tool to evaluate efficacy of mTOR treatments.

A guinea pig model of mesial temporal lobe epilepsy following nonconvulsive status epilepticus induced by unilateral intrahippocampal injection of kainic acid

Models of temporal lobe epilepsy are commonly utilized to study focal epileptogenesis and ictogenesis. The criteria that define animal models representative of human mesial temporal lobe may vary in different laboratories. We describe herein a focal epilepsy model of mesial temporal (hippocampal) origin that relies on the analysis of interictal and ictal electroencephalography (EEG) patterns and on their correlation with seizure symptoms and neuropathologic findings. The study is based on guinea pigs, a species seldom utilized to develop chronic epilepsy models. Young adult guinea pigs were bilaterally implanted under isoflurane anesthesia with epidural electrodes over somatosensory cortex and depth electrodes in CA1 hippocampal region. A stainless steel guide cannula was positioned unilaterally in the right dorsal hippocampus to inject 1 μl of 0.9% NaCl solution containing 1 μg kainic acid (KA). One week after surgery, continuous 24 h/day video-EEG monitoring was performed 48 h before and every other week after KA injection, for no <1 month. EEG data were recorded wide-band at 2 kHz. After video-EEG monitoring, brains were analyzed for thionine and Timm staining and glial fibrillary acid protein (GFAP) immunostaining. Unilateral injection of KA in dorsal hippocampus of guinea pigs induces an acute nonconvulsive status epilepticus (SE) that terminates within 24 h (n = 22). Chronic seizures with very mild motor signs (undetectable without EEG monitoring) and highly variable recurrence patterns appear in 45.5% (10 of 22) KA-treated animals, with variable delays from the initial SE. In these animals interictal events, CA1 cell loss, gliosis, and altered Timm staining pattern were observed. The induction of a chronic condition did not correlate with the duration of the nonconvulsive acute SE, but correlated with the extension and quality of neuropathologic damage. We demonstrate that a model of hippocampal (mesial temporal lobe) epilepsy can be developed in the guinea pig by intrahippocampal injection of KA. Seizure events in this model show little behavioral signs and may be overlooked without extensive video-EEG monitoring. The establishment of a chronic epileptic condition correlates with the extension of the hippocampal damage (mainly cell loss and gliosis) and not with the intensity of the initial SE.

COGNITIVE FUNCTION AND VASCULAR EFFECTS ON WHITE MATTER IN SCHIZOPHRENIA

Mesial temporal lobe epilepsy (MTLE) is the most prevalent form of epilepsy, characterized by recurrent complex partial seizures and hippocampal sclerosis. The pathophysiology underlying this disorder remains unidentified. While a loss of benzodiazepine binding sites is a key diagnostic feature of MTLE, experimental studies have shown enhanced inhibitory transmission and increased expression of GABAA-receptors, suggesting that compensatory mechanisms are operative in epileptic hippocampus. In the present study, changes in the expression and cellular distribution of major GABAA-receptor subunits were investigated in the hippocampus of pilocarpine-treated rats during the phase of spontaneous recurrent seizures. A uniform decrease in GABAA-receptor subunit-immunoreactivity was observed in regions of extensive neuronal death (i.e. CA1, CA3, hilus), whereas a prominent increase occurred in the dentate gyrus (DG). Most strikingly, the increase was largest for the α3- and α5-subunits, which are expressed at very low levels in the DG of control rats, suggesting the formation of novel GABAA-receptor subtypes in epileptic tissue. Furthermore, an extensive loss of interneurons expressing the α1-subunit, representing presumptive basket cells, was seen in the DG. These changes were very similar to those reported in a novel mouse model of MTLE, based on the unilateral injection of kainic acid into the dorsal hippocampus (Bouilleret et al., 1999). This indicates that the regulation of GABAA-receptor expression is related to chronic recurrent seizures, and is not due to the extrahippocampal neuronal damage affecting pilocarpine-treated rats. These results allow causal relationships in the induction and maintenance of chronic recurrent seizures to be distinguished. The loss of a critical number of interneurons in the DG is a possible cause of seizure initiation, whereas the long-lasting upregulation of GABAA-receptors in granule cells represents a compensatory response to seizure activity.

ALTERED AGEING IN SCHIZOPHRENIA: META-ANALYSIS OF CELL DENSITY STUDIES IN SCHIZOPHRENIA INDICATES DIRECTION OF CHANGE DEPENDS ON AGE OF SUBJECTS

Mesial temporal lobe epilepsy (MTLE) is the most prevalent form of epilepsy, characterized by recurrent complex partial seizures and hippocampal sclerosis. The pathophysiology underlying this disorder remains unidentified. While a loss of benzodiazepine binding sites is a key diagnostic feature of MTLE, experimental studies have shown enhanced inhibitory transmission and increased expression of GABAA-receptors, suggesting that compensatory mechanisms are operative in epileptic hippocampus. In the present study, changes in the expression and cellular distribution of major GABAA-receptor subunits were investigated in the hippocampus of pilocarpine-treated rats during the phase of spontaneous recurrent seizures. A uniform decrease in GABAA-receptor subunit-immunoreactivity was observed in regions of extensive neuronal death (i.e. CA1, CA3, hilus), whereas a prominent increase occurred in the dentate gyrus (DG). Most strikingly, the increase was largest for the α3- and α5-subunits, which are expressed at very low levels in the DG of control rats, suggesting the formation of novel GABAA-receptor subtypes in epileptic tissue. Furthermore, an extensive loss of interneurons expressing the α1-subunit, representing presumptive basket cells, was seen in the DG. These changes were very similar to those reported in a novel mouse model of MTLE, based on the unilateral injection of kainic acid into the dorsal hippocampus (Bouilleret et al., 1999). This indicates that the regulation of GABAA-receptor expression is related to chronic recurrent seizures, and is not due to the extrahippocampal neuronal damage affecting pilocarpine-treated rats. These results allow causal relationships in the induction and maintenance of chronic recurrent seizures to be distinguished. The loss of a critical number of interneurons in the DG is a possible cause of seizure initiation, whereas the long-lasting upregulation of GABAA-receptors in granule cells represents a compensatory response to seizure activity.

Impairment of dentate gyrus neuronal progenitor cell differentiation in a mouse model of temporal lobe epilepsy

Unilateral intrahippocampal injection of kainic acid (KA) in adult mice induces an epileptic focus replicating major histopathological features of temporal lobe epilepsy (TLE). In this model, neurogenesis is impaired in the lesioned dentate gyrus, although cell proliferation transiently is increased bilaterally in the subgranular zone (SGZ). To investigate further the relationship between epileptogenesis and neurogenesis, we compared the differentiation of cells born shortly before and after KA injection. Immunohistochemical staining for doublecortin and PSA-NCAM, two markers of young neurons, revealed a rapid downregulation of both markers ipsilaterally, whereas they were increased transiently on the contralateral side. To determine whether KA treatment directly affects neural progenitors in the SGZ, dividing cells were prelabeled with 5'-bromo-2'deoxyuridine (BrdU) treatment before unilateral injection of KA. Double staining with the proliferation marker PCNA showed that prelabeled BrdU cells survived KA exposure and proliferated bilaterally. Unexpectedly, the neuronal differentiation of these cells, as assessed after 2 weeks with doublecortin and NeuN triple-staining, occurred to the same extent as on the contralateral side. Only 5% of pre-labeled BrdU cells were GFAP-positive within the lesion. Therefore, SGZ progenitor cells committed to a neuronal phenotype before KA treatment complete their differentiation despite the rapid down-regulation of doublecortin and PSA-NCAM. These findings suggest impaired fate commitment and/or early differentiation of proliferating cells in the lesioned dentate gyrus. Loss of neurogenesis in this TLE model likely reflects an irreversible alteration of the SGZ germinal niche during development of the epileptic focus and may therefore be relevant for human TLE.

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.

Spatio-temporal changes in neurofilament proteins immunoreactivity following kainate-induced cerebellar lesion in rats.

1. Spatio-temporal changes in phosphorylated (pNFP) and nonphosphorylated (npNFP) neurofilament proteins were assessed immunocytochemicaly in adult rat cerebellum, 2-30 days following unilateral injection of kainic acid (KA) or physiological saline (s.c.). 2. Analysis of the staining intensity and pattern demonstrated that injection of both KA and physiological saline elicited significant and long-lasting increase of pNFP and npNFP immunoreactivity, at the ipsilateral, and to lesser extent at the contralateral side of lesion. 3. Kainate intoxication induced abundant expression of pNFP and npNFP in cerebellar white matter, as well as in all layers of perilesioned cortex. Higher pNFP expression was evidenced in the Purkinje cell layer, particularly at cell bodies, initial segments, and proximal dendrites, which normally do not contain pNFP. In addition, synaptophysin immunocytochemistry was used as a marker of synaptogenesis and plasticity. 4. Spatio-temporal pattern of NFP and synaptophysin expression suggests that perilesioned cortex undergoes dynamic changes following brain demage and possess a reparative capacity to abridge the consequences of brain trauma.

Overexpression of adenosine kinase in epileptic hippocampus contributes to epileptogenesis.

Endogenous adenosine in the brain is thought to prevent the development and spread of seizures via a tonic anticonvulsant effect. Brain levels of adenosine are primarily regulated by the activity of adenosine kinase. To establish a link between adenosine kinase expression and seizure activity, we analyzed the expression of adenosine kinase in the brain of control mice and in a kainic acid-induced mouse model of mesial temporal lobe epilepsy. Immunohistochemical analysis of brain sections of control mice revealed intense staining for adenosine kinase, mainly in astrocytes, which were more or less evenly distributed throughout the brain, as well as in some neurons, particularly in olfactory bulb, striatum, and brainstem. In contrast, hippocampi lesioned by a unilateral kainic acid injection displayed profound astrogliosis and therefore a significant increase in adenosine kinase immunoreactivity accompanied by a corresponding increase of enzyme activity, which paralleled chronic recurrent seizure activity in this brain region. Accordingly, seizures and interictal spikes were suppressed by the injection of a low dose of the adenosine kinase inhibitor 5-iodotubercidin. We conclude that overexpression of adenosine kinase in discrete parts of the epileptic hippocampus may contribute to the development and progression of seizure activity.

Kainic acid lesion-induced nigral neuronal death

Parkinson's disease (PD) is characterized by progressive death of dopamine (DA) neurons in the substantia nigra pars compacta. We report a rat model that exhibits progressive death of nigral neurons following unilateral injection of kainic acid in the striatum. In situ end-labeling revealed significant numbers of dying nigral neurons ipsilateral to the lesion during the first 3 weeks following injection. An indication of the gradual nature of death was that similar small numbers of cells were detected at each time point. These early morphological markers of neuronal death led to a significant reduction (20%) at 5 months of tyrosine hydroxylase-positive neurons and total number of neurons in the ipsilateral substantia nigra compared with the contralateral control. To examine the role of nigrostriatal DA metabolism in the observed nigral neuronal death, we manipulated DA metabolism during the initial 2 weeks following kainic acid lesion. Neurons in the ventral tier of the substantia nigra pars compacta were protected from death by treatment with 2,4-diamino-6-hydroxy-pyrimidine (DAHP), an inhibitor of GTP cyclohydrolase, the initial enzyme in the synthesis of the tyrosine hydroxylase co-substrate, tetrahydrobiopterin (BH 4). Neurons in both the dorsal and ventral tier of substantia nigra pars compacta were protected from death by treatment with DAHP and l-DOPA. These experiments suggest that intrastriatal kainic acid lesion is an in vivo model of trophic support withdrawal. This experimental procedure is useful for studying mechanisms underlying protracted death of nigral DA neurons and may provide valuable mechanistic information relevant to understanding the etiology of PD.

Evolution of hippocampal epileptic activity during the development of hippocampal sclerosis in a mouse model of temporal lobe epilepsy

Unilateral intrahippocampal injection of kainic acid in adult mice reproduces most of the morphological characteristics of hippocampal sclerosis (neuronal loss, gliosis, reorganization of neurotransmitter receptors, mossy fiber sprouting, granule cell dispersion) observed in patients with temporal lobe epilepsy. Whereas some neuronal loss is observed immediately after the initial status epilepticus induced by kainate treatment, most reorganization processes develop progressively over a period of several weeks. The aim of this study was to characterize the evolution of seizure activity in this model and to assess its pharmacological reactivity to classical antiepileptic drugs. Intrahippocampal electroencephalographic recordings showed three distinct phases of paroxystic activity following unilateral injection of kainic acid (1 nmol in 50 nl) into the dorsal hippocampus of adult mice: (i) a non-convulsive status epilepticus, (ii) a latent phase lasting approximately 2 weeks, during which no organized activity was recorded, and (iii) a phase of chronic seizure activity with recurrent hippocampal paroxysmal discharges characterized by high amplitude sharp wave onset. These recurrent seizures were first seen about 2 weeks post-injection. They were limited to the injected area and were not observed in the cerebral cortex, contralateral hippocampus or ipsilateral amygdala. Secondary propagation to the contralateral hippocampus and to the cerebral cortex was rare. In addition hippocampal paroxysmal discharges were not responsive to acute carbamazepine, phenytoin, or valproate treatment, but could be suppressed by diazepam. Our data further validate intrahippocampal injection of kainate in mice as a model of temporal lobe epilepsy and suggest that synaptic reorganization in the lesioned hippocampus is necessary for the development of organized recurrent seizures.

Cleavage of Bid May Amplify Caspase-8-Induced Neuronal Death Following Focally Evoked Limbic Seizures

The mechanism by which seizures induce neuronal death is not completely understood. Caspase-8 is a key initiator of apoptosis via extrinsic, death receptor-mediated pathways; we therefore investigated its role in mediating seizure-induced neuronal death evoked by unilateral kainic acid injection into the amygdala of the rat, terminated after 40 min by diazepam. We demonstrate that cleaved (p18) caspase-8 was detectable immediately following seizure termination coincident with an increase in cleavage of the substrate Ile-Glu-Thr-Asp (IETD)-p-nitroanilide and the appearance of cleaved (p15) Bid. Expression of Fas and FADD, components of death receptor signaling, was increased following seizures. In vivo intracerebroventricular z-IETD-fluoromethyl ketone administration significantly reduced seizure-induced activities of caspases 8, 9, and 3 as well as reducing Bid and caspase-9 cleavage, cytochrome c release, DNA fragmentation, and neuronal death. These data suggest that intervention in caspase-8 and/or death receptor signaling may confer protection on the brain from the injurious effects of seizures.

Chronic epileptogenesis requires development of a network of pathologically interconnected neuron clusters: a hypothesis.

The "silent period" is a characteristic of human localization-related symptomatic epilepsy. In mesial temporal lobe epilepsy (MTLE), it follows an initial precipitating injury, and in animal models of MTLE in which brain damage is artificially created, there is also a prolonged interval between injury and the onset of spontaneous seizures. The neuronal reorganization responsible for epileptogenesis presumably takes place during this silent interval; however, the functional correlates of this process are poorly understood. We have previously described high-frequency (250 to 500 Hz) oscillations, called fast ripples (FR), in the hippocampus and entorhinal cortex (EC) of intrahippocampal kainic acid (KA)-injected rats and patients with MTLE that are confined to the region of spontaneous seizure generation. We have proposed, therefore, that FR reflect the mechanisms responsible for epileptogenesis. If this is the case, they should appear during the process of epileptogenesis, before the appearance of spontaneous seizures. The purpose of the present study was to record continuously from rats after KA injection to compare the temporal development of FR with spontaneous seizures. Additional goals were to determine in these rats after spontaneous seizures begin (a) the volume of tissue in which FR can be recorded in hippocampus and EC, (b) the multiple-unit and field potential correlates of FR oscillations, and (c) whether there is an association of FR with mossy fiber sprouting. After unilateral KA injection in the posterior hippocampus, interictal field epileptic activity and single-unit activity were recorded from freely moving animals using multiple-contact microelectrodes in dentate gyrus (DG) and EC. One group of animals underwent continuous recording to determine the time of onset of both FR oscillations and spontaneous seizures. A second group was implanted after behavioral seizures began to measure the area within which FR could be recorded as well as their unit and field potential correlates. The neo-Timm method was used to reveal mossy fiber sprouting, and gray value analysis was used to measure the intensity of sprouting in the inner molecular layer of DG. In KA-injected rats, FR were observed in hippocampal areas adjacent to the lesion and in the ipsilateral EC 11 to 14 days after injection, whereas spontaneous behavioral seizures occurred 2 to 4 months after injection. Analysis of depth profiles of interictal FR in the DG and EC showed that they were generated in local areas with a volume of about 1.0 mm3, and unit recordings indicated that they reflected fields of hypersynchronous action potentials. FR were found in areas of DG with more intensive mossy fiber sprouting. However, the correspondence was not absolute. The electrophysiological and anatomical data are consistent with the participation of FR oscillations, within small neuronal assemblies, in the development of chronic epileptogenesis. It is hypothesized that small clusters of pathologically interconnected neurons develop after focal hippocampal injury and that these clusters are capable of generating powerful hypersynchronous bursts of action potentials, which initiate epileptogenesis via a kindling effect. As the silent period progresses, a network of such clusters is formed that allows the development of discharges that spread throughout the limbic system. When this network engages brain areas that control motor activity, clinical seizures occur and the silent period ends.

Early loss of interneurons and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy.

Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999; 89:717-729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1-120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABA(A)-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABA(A)-receptor alpha1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DC and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7-15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABA(A)-receptor staining. In contrast, DC granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABA(A)-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of pyramidal cells and granule cell dispersion may reflect distinct responses to recurrent seizures. Finally, GABA(A)-receptor upregulation in the DG may represent a compensatory response persisting for several months in epileptic mice.

Early loss of interneurons and delayed subunit‐specific changes in GABAA‐receptor expression in a mouse model of mesial temporal lobe epilepsy

Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999;89:717–729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1–120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABAA-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABAA-receptor α1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DG and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7–15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABAA-receptor staining. In contrast, DG granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABAA-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of pyramidal cells and granule cell dispersion may reflect distinct responses to recurrent seizures. Finally, GABAA-receptor upregulation in the DG may represent a compensatory response persisting for several months in epileptic mice. Hippocampus 10:305–324, 2000 © 2000 Wiley-Liss, Inc.

GABAergic neurons and GABA A-receptors in temporal lobe epilepsy

Mesial temporal lobe epilepsy (MTLE) is the most prevalent form of epilepsy, characterized by recurrent complex partial seizures and hippocampal sclerosis. The pathophysiology underlying this disorder remains unidentified. While a loss of benzodiazepine binding sites is a key diagnostic feature of MTLE, experimental studies have shown enhanced inhibitory transmission and increased expression of GABA A-receptors, suggesting that compensatory mechanisms are operative in epileptic hippocampus. In the present study, changes in the expression and cellular distribution of major GABA A-receptor subunits were investigated in the hippocampus of pilocarpine-treated rats during the phase of spontaneous recurrent seizures. A uniform decrease in GABA A-receptor subunit-immunoreactivity was observed in regions of extensive neuronal death (i.e. CA1, CA3, hilus), whereas a prominent increase occurred in the dentate gyrus (DG). Most strikingly, the increase was largest for the α3- and α5-subunits, which are expressed at very low levels in the DG of control rats, suggesting the formation of novel GABA A-receptor subtypes in epileptic tissue. Furthermore, an extensive loss of interneurons expressing the α1-subunit, representing presumptive basket cells, was seen in the DG. These changes were very similar to those reported in a novel mouse model of MTLE, based on the unilateral injection of kainic acid into the dorsal hippocampus ( Bouilleret et al., 1999). This indicates that the regulation of GABA A-receptor expression is related to chronic recurrent seizures, and is not due to the extrahippocampal neuronal damage affecting pilocarpine-treated rats. These results allow causal relationships in the induction and maintenance of chronic recurrent seizures to be distinguished. The loss of a critical number of interneurons in the DG is a possible cause of seizure initiation, whereas the long-lasting upregulation of GABA A-receptors in granule cells represents a compensatory response to seizure activity.

Adenosine involvement in amitriptyline effect on glutamate and aspartate release in rat prefrontal cortex: K. Gołembiowska. Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smétna 12, PL 31–343 Kraków, Poland

The spontaneous and depolarization-evoked release of radiolabeled d-aspartic acid, previously taken up by rat striatal slices, was studied by using a superfusion system. Veratridine (10–50 μM), electrical field stimulation (20 Hz, 1.0 V, 60 sec), and potassium (53 mM) markedly potentiated the release of d-[3H]aspartate from striatal slices. The release of l-[3H]glutamate was also increased by veratridine, according to a pattern and time course of release similar to that of d-[3H]aspartate. However, the ratio of d-[3H]aspartic acid release evoked by veratridine over spontaneous levels of release was much higher when compared to that of radiolabeled l-glutamate. Omission of calcium from the superfusion medium almost completely suppressed d-[3H]aspartate release evoked by veratridine or by electrical stimulation whereas high K+-evoked release of the [3H]amino acid was only slightly reduced. However, increasing Mg2+ concentration to 12 mM in the superfusion medium did substantially block d-[3H]aspartate release induced by K+-depolarization. Additional experiments showed that tetrodotoxin (1 μM), a blocker of voltage-dependent Na+ channels, totally abolished veratridine-evoked release of d-[3H]aspartate from striatal slices. Finally, lesion studies showed that unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the high-affinity uptake of striatal d-[3H]aspartate and by a large and parallel loss from striatal slices in d-[3H]aspartate release evoked by either veratridine or high K+. In contrast, unilateral injection of kainic acid into the striatum did not influence depolarization-evoked release of d-[3H]aspartate from striatal slices. The findings reported suggest that d-[3H]aspartic acid may be taken up preferentially and then released, in a Ca2+-dependent manner, by veratridine and electrical stimulation from nerve terminals belonging to the cortico-striatal pathway. In addition, the results provide further support for the view that excitatory amino acids may act as neurotransmitters at the cortico-striatal nerve fibers.

Localization of ionotropic glutamate receptors in caudate‐putamen and nucleus accumbens septi of rat brain: Comparison of NMDA, AMPA, and kainate receptors

Changes in binding of selective radioligands at NMDA ([3H]MK-801), AMPA ([3H]CNQX), and kainate ([3H]kainic acid) glutamate (GLU) ionotropic receptors in rat caudate-putamen (CPu) and nucleus accumbens (NAc) were examined by quantitative autoradiography following: 1) unilateral surgical ablation of frontal cerebral cortex to remove descending corticostriatal GLU projections, 2) unilateral injection of kainic acid (KA) into CPu or NAc to degenerate local intrinsic neurons, or 3) unilateral injections of 6-hydroxydopamine (6-OH-DA) into substantia nigra to degenerate ascending nigrostriatal dopamine (DA) projections. Cortical ablation significantly decreased NMDA receptor binding in ipsilateral medial CPu (20%), and NAc (16%), similar to previously reported losses of DA D4 receptors. KA lesions produced large losses of NMDA receptor labeling in CPu and NAc (both by 52%), AMPA (41% and 45%, respectively), and kainate receptors (40% and 45%, respectively) that were similar to the loss of D2 receptors in CPu and NAc after KA injections. Nigral 6-OH-DA lesions yielded smaller but significant losses in NMDA (17%), AMPA (12%), and kainate (11%) receptor binding in CPu. The results indicate that most NMDA, AMPA, and kainate receptors in rat CPu and NAc occur on intrinsic postsynaptic neurons. Also, some NMDA, but not AMPA or kainate, receptors are also found on corticostriatal projections in association with D4 receptors; these may, respectively, represent excitatory presynaptic NMDA autoreceptors and inhibitory D4 heteroceptors that regulate GLU release from corticostriatal axons in medial CPu and NAc. Conversely, the loss of all three GLU receptor subtypes after lesioning DA neurons supports their role as excitatory heteroceptors promoting DA release from nigrostriatal neurons. Synapse 30:227–235, 1998. © 1998 Wiley-Liss, Inc.

Effects of hippocampal lesions on striatolimbic ionotropic glutamatergic receptors

Changes in ionotropic N-methyl- d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate glutamatergic (Glu) receptor binding in rat caudate putamen (CPu) and nucleus accumbens (NAc) and hippocampal CA 1 and CA 3 regions were examined after unilateral injections of kainic acid (KA) into hippocampus to degenerate local intrinsic neurons and remove the glutamatergic afferents projecting to CPu and NAc. KA injections produced large losses of NMDA, AMPA and kainate receptors in hippocampal CA 1 and CA 3 regions. In addition, NMDA and kainate, but not AMPA, receptors were significantly reduced in NAc and CPu. These results suggest that a large proportion of NMDA, AMPA and kainate receptors in rat hippocampus arise on intrinsic postsynaptic neurons, and that some NMDA and kainate, but not AMPA, receptors also occur on presynaptic terminals of hippocampostriatal afferents innervating the CPu and NAc.