Immunocytochemical analysis of glutamate and GABA in hippocampus of genetic absence epilepsy rats (GAERS)

doi:10.1016/S0006-8993(03)03349-3

Brain Research

Volume 988, Issues 1–2, 24 October 2003, Pages 180-188

https://doi.org/10.1016/S0006-8993(03)03349-3 Get rights and content

Abstract

In the present study, we used an immunocytochemical technique at the electron microscopic level to determine if there are changes in the glutamate and GABA neurotransmitter content of the hippocampus of genetic absence epilepsy rats from Strasbourg (GAERS). We also investigated if there was mossy fiber reorganization. After perfusion fixation, brains were removed and cryostat sections were stained according to the neo-Timm’s procedure. High-resolution electron microscopy was used for ultrastructural examination of the hippocampus of GAERS and non-epileptic control Wistar animals. For ultrastructural and immunocytochemical studies, ultrathin-cut sections were obtained and immunolabeled with anti-glutamate and anti-GABA antibodies. The number of gold particles per nerve terminal was counted and the area of the nerve terminal was determined using the program NIH Image Analysis. No mossy fiber sprouting was detected in the hippocampus of GAERS. GABA and glutamate immunoreactivity were observed in the mossy fiber terminals of both the control and GAERS groups. Glutamate density in the CA3 region of GAERS hippocampus was found to be significantly increased compared to the control group. However, there was no difference in the GABA density of nerve terminals and in areas of GABAergic and mossy terminals between GAERS and the control group. The difference in glutamate level may merely be due to strain differences between the GAERS strain and the original Wistar strain or it is also possible that it appears after seizures have started.

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Acknowledgements

The authors thank Cynthia Moore for assistance during routine tissue preparation and immunolabeling for electron microscopy. The authors also thank the Brain Research Society (Beyin Arastirmalari Dernegi—BAD, Istanbul, Turkey) for providing a bursary. CKM was supported by the Department of Veterans Affairs Merit Review Program.

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Citation Excerpt :
In addition, the basal extracellular levels of glutamate in the hippocampus are higher in GAERS than in normal controls (Richards et al., 2000). Similarly the density of glutamate immunolabeling in the CA3 and hilar regions of adult GAERS hippocampus was found to be affected (Sirvanci et al., 2003, 2005). Immunohistochemical analysis of all hippocampal subfields of 6-month-old WAG/Rij rats showed an increased expression of neuronal mGlu2/3 receptors compared to 6 month-old control rats (Ngomba et al., 2005).
Typical and atypical seizures of absence epilepsy are thought to be generated by a rhythmogenic interplay between the cortex and the thalamus. However, the question remains as to which other subcortical and extrathalamic structures are involved in the pathophysiology of typical and atypical absence epilepsy. Limbic structures are not thought to be involved in typical absence seizures, since in animal models and human patients there is no evidence for the occurrence of spike-and-wave discharges of absence seizures in the limbic regions. However, there are a number of observations from animal models of absence epilepsy that point to a possibly important link between absence seizure mechanisms and limbic structures. Atypical absence seizures are distinct in many ways from typical absence seizures although they bear considerable clinical, EEG, and pharmacological resemblance to typical absence seizures. The differences between typical and atypical seizures of absence epilepsy appear to be circuitry dependent. While both typical and atypical absence seizures involve the cortico-thalamo-cortical circuitry, they each engage different neuronal networks within that circuitry. This review examines the involvement of limbic structures in typical and atypical absence seizures, shows that limbic circuitry forms an integral component of the absence epilepsy network and concludes that further knowledge of this component is important for understanding the complex relationships involved in absence epilepsy.
NMDA receptors in the medial prefrontal cortex and the dorsal hippocampus regulate methamphetamine-induced hyperactivity and extracellular amino acid release in mice
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These evidences suggested that the stimulant of glutamatergic system in the mPFC plays a key role in the addictive drugs-induced psychological dependence and the whole mPFC regulated the process of the glutamatergic projection. The DHC contains many pyramidal neurons, including glutamatergic and GABAergic neurons [24–26], and is also crucial to psychostimulant-induced hyperactivity and CPP [16,27–29]. The DHC is likewise reported to regulate MAP-induced ascorbic acid (AA) release in the NAc and striatum [30].
The medial prefrontal cortex (mPFC) and the dorsal hippocampus (DHC) play significant roles in stimulant-induced neurobehavioral effects. Methamphetamine (MAP)-induced hyperactivity has been reported to be involved in the regulation of the glutamatergic system. The present study examined whether the glutamatergic and GABAergic systems in the mPFC and DHC were involved in MAP-induced hyperactivity in mice. A combined kainic acid (KA) or N-methyl-d-aspartate (NMDA) lesion and microdialysis technique targeting both the mPFC and DHC were used. The results showed that both KA- and NMDA-induced lesions of the mPFC facilitated MAP-induced hyperactivity, while neither KA- nor NMDA-induced lesions of the DHC had a similar effect. MAP increased the extracellular glutamate (Glu) levels in the mPFC and reduced Glu levels in the DHC. GABA levels in both of these regions were reduced. A KA or NMDA lesion of the mPFC inhibited the Glu reduction in the DHC, and the same lesion of the DHC inhibited the Glu increase in the mPFC induced by MAP. A NMDA lesion of the mPFC blocked GABA reduction in the DHC, but a lesion of DHC enhanced the GABA decrease in the mPFC induced by MAP. Furthermore, a NMDA lesion of DHC increased the vesicular glutamate transporter-2 (VGLUT2) expression in the mPFC following MAP-administration. These findings indicate that glutamatergic as well as GABAergic systems in these two regions are involved in MAP-induced hyperactivity. Moreover, there may be an inhibitory role in these two regions, especially mediated by NMDA receptors, in MAP-induced abnormal behavior and neurotransmission responses.
Ultrastructural GABA immunocytochemistry in the mossy fiber terminals of Wistar and genetic absence epileptic rats receiving amygdaloid kindling stimulations
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The existence of absence epilepsy and temporal lobe epilepsy in the same patient is not common in clinical practice. The reason why both types of seizures are rarely seen in the same patient is not well understood. Therefore, we aimed to investigate kindling in a well known model of human absence epilepsy, genetic absence epilepsy rats from Strasbourg (GAERS). In the present study, we analyzed whether the GABA content of GAERS that received kindling stimulations was altered in the hippocampal mossy fiber terminals compared to non-epileptic control (NEC) Wistar rats. For this purpose, we used an immunocytochemical technique at the ultrastructural level. Ultrathin sections were immunolabeled with anti-GABA antibody and transmission electron microscopy was used for the ultrastructural examination. The number of gold particles per nerve terminal was counted and the area of the nerve terminal was determined using NIH image analysis program. The GABA density was found to be higher in sham-operated GAERS than sham-operated Wistar rats. The density was increased in kindling Wistar group compared to sham-operated Wistar and kindling GAERS groups. No statistical difference was observed between sham-operated GAERS and kindling GAERS groups. The increase in GABA levels in stimulated Wistar rats may be a result of a protective mechanism. Furthermore, there may be strain differences between Wistar rats and GAERS and our findings addressing different epileptogenesis mechanisms in these strains might be a basis for future experimental studies.
Hippocampal cell loss and propagation of abnormal discharges accompanied with the expression of tonic convulsion in the spontaneously epileptic rat
2010, Brain Research
Citation Excerpt :
It is well known that the cerebral cortex and thalamus interact to form spike-wave complexes (Danober et al., 1998; Luhmann et al., 1995). In some genetic epilepsy models, the hippocampus displays certain abnormal characteristics (Qiao and Noebels, 1993; Sirvanci et al., 2003), and atypical findings in the limbic system may be attributable to the development of absence seizures (Tolmacheva et al., 2004). From these reports, each seizure in SER could influence and probably potentiate neuronal activities of the hippocampus.
Spontaneously epileptic rats (SER) are double mutants with both tonic convulsion and absence-like seizures from the age of 8 weeks. Hippocampal CA3 neurons in SER display a long-lasting depolarizing shift accompanied by repetitive firing (attributed to abnormalities of the Ca²⁺ channels) with a single stimulation of the mossy fibers. In the present investigation, we examined if the seizure discharges of SER were correlated with the hippocampal abnormality of SER using electrophysiological and histological methods. In CA1 neurons of seizure-susceptible mature SER, higher-voltage (< 8–11 V) stimulations induced a long depolarization shift (in 25% of neurons) with repetitive firing (in 12.5% of neurons). However, the tremor rat, one of the parent strains of SER, did not exhibit such abnormal firing in the CA3 region of the hippocampus. The number of CA3 neurons in SER was significantly (p < 0.01) lower than that in tremor rats and Wistar rats, although no significant difference was established in the hilus. Sprouting of mossy fiber was observed in the dentate of mature SER; however, negligible staining was spotted in the dentate of both mature tremor and Wistar rats. Interestingly, expression of the brain-derived neurotrophic factor was higher in the hilus, CA3, and granular cell layer of dentate gyrus in SER than normal Wistar rats. The expression levels of TUNEL, bax, and Caspase-3 did not show significant changes between the SER and Wistar rats. SER exhibited hippocampal sclerosis-like changes which did not have enough potential for epileptogenesis. Repetitive tonic seizures and vulnerable CA3 neurons of SER could be involved in the induction of sclerosis-like changes in the hippocampus.
Elevated forebrain excitatory l-glutamate, l-aspartate and d-aspartate in the Naples high-excitability rats
2009, Behavioural Brain Research
The Naples high-excitability (NHE) rats are thought to model the mesocortical variant of attention-deficit hyperactivity disorder (ADHD). The aim of this study was to investigate forebrain level of l-glutamate, l-aspartate and d-aspartate, in NHE vs. Naples random bred (NRB) control rats. Thus, prepuberal NHE and NRB rats were daily handled in the 5th and 6th week of postnatal life. Then rats were exposed to two spatial novelties i.e. a Làt and a Olton maze for 10 min. Amino acids were detected by HPLC in the prefrontal cortex (PFC), striatum (STR), hippocampus (HPC) and hypothalamus (HYP). Results indicate that all amino acids were higher in NHE than in NRB rats.
This, in turn, may explain the behavioural hyperactivity and attention deficit of this animal model of ADHD.
Glutamate and GABA immunocytochemical electron microscopy in the hippocampal dentate gyrus of normal and genetic absence epilepsy rats
2005, Brain Research
Citation Excerpt :
There was no difference in the density of GABA in GABAergic nerve terminals between non-epileptic control and GAERS groups in the present study. Likewise, in our previous study we showed that GABA density in GAERS was not significantly different from that of control in CA3 region [25]. Consistent with our findings, no difference in extracellular GABA was found in the hippocampus between the GAERS and the control group [23].
It is generally accepted that absence epilepsy results from the impairment of GABAergic and glutamatergic neurotransmission. In particular, besides excessive GABA mediation within the thalamo-cortico-thalamic circuit in absence epilepsy, neuronal networks of the hippocampus have recently received attention. In the present study, we examined the density of glutamate and GABA neurotransmitter immunolabeling in the dentate gyrus of the hippocampus of genetic absence epilepsy rats from Strasbourg (GAERS) compared to the control group. GABA and glutamate were found to exist in synaptic vesicles of the mossy fiber terminals of the control and GAERS groups. The density of glutamate immunolabeling within the mossy fiber terminals in the hilar region of GAERS hippocampus was found to be significantly decreased compared to the control group. There was no difference in the density of immunolabeling within GABA nerve terminals between GAERS and control group. The findings of this study suggest that mechanisms underlying absence seizures in GAERS may also manifest themselves in other brain regions such as the hippocampus. The presence of GABA within synaptic vesicles of mossy fiber terminals, as revealed by high resolution ultrastructural immunocytochemistry, has provided additional evidence to the possible modulatory role of GABA on synaptic transmission between the mossy fiber and the target cell.

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Short communicationImmunocytochemical analysis of glutamate and GABA in hippocampus of genetic absence epilepsy rats (GAERS)

Abstract

Section snippets

Acknowledgements

Brain Res.

J. Neurosci. Methods

Prog. Neurobiol.

Brain Res.

Epilepsy Res.

Epilepsy Res.

Neuropharmacology

Neuroscience