Fos Induction Following Systemic Kainic Acid: Early Expression in Hippocampus and Later Widespread Expression Correlated With Seizure

doi:10.1016/S0306-4522(96)00462-9

Neuroscience

Volume 77, Issue 2, 21 February 1997, Pages 379-392

https://doi.org/10.1016/S0306-4522(96)00462-9 Get rights and content

Abstract

We determined the distribution of Fos protein expression in a model of generalised epilepsy caused by excessive neuronal excitation. Fos immunoreactivity was mapped in forebrain in unrestrained rats, previously prepared with an indwelling venous catheter, after the intravenous administration of kainic acid (10 mg/kg). We determined cerebral activation following various periods of exposure to kainic acid by using intravenous administration of pentobarbitone to prevent further activation. Within a few minutes, kainic acid caused episodes of staring, sniffing, wet dog shakes, nodding and chewing. Fos induction occurred initially and simultaneously in hippocampus, subiculum, septum and entorhinal cortex as early as 9.5 min after kainate injection. After up to 40 min of staring, sniffing, wet dog shakes, nodding and chewing, Fos induction was not further increased above levels present within the first 9.5 min. After 56±6 min a motor convulsion occurred, initially affecting the jaw, head and tail and variably extending to the forelimbs, trunk or hindlimbs. Following the convulsive event, additional Fos was expressed in hippocampus, thalamus, caudate–putamen and other subcortical structures and in the cerebral cortex. Fos induction was sometimes asymmetric in entorhinal, visual, piriform, cingulum, parietal and frontal cortices and in amygdala and dorsal endopiriform area. Electroencephalographic recordings after a few minutes exposure to kainic acid revealed an increased amplitude of fast frequencies in hippocampus which appeared to correlate with Fos induction in this structure.

The findings are generally consistent with the reported distribution and slow development of kainic acid-induced seizure activity using electrophysiological and deoxyglucose methods. However, the Fos distribution suggests that (i) hippocampal, possibly dentate, activation precedes significant activation elsewhere, (ii) extensive involvement of other cerebral structures and cerebral cortex occurs simultaneously and correlates with motor seizures and (iii) brain structures can be recruited asymmetrically.

Section snippets

Animals

Male inbred Sprague–Dawley rats were obtained from the animal house of the Flinders Medical Centre. They are known not to spontaneously express spike and wave discharges.[33]Animals weighing 400–600 g were housed in individual cages at an ambient temperature of 22°C with a 12:12 h light:dark cycle and were given unrestricted access to food and water. Furthermore, because acute handling induces Fos in cortical neurons,[34]we prepared animals with a chronically-implanted venous catheter to enable

Behaviour

Kainic acid caused episodes of staring, wet dog shakes, nodding or chewing and finally in our experiments, a single bilateral motor convulsion affecting the jaw, head and tail, often the forelimbs and sometimes the trunk and hindlimbs, consistent with previous descriptions.1, 2, 17, 28Wet dog shakes occurred within a few minutes of kainic acid injection and these, as well as the other non-convulsive behaviours, increased in frequency until the motor convulsion occurred. The motor convulsions

Discussion

Use of the Fos method to reveal activated neurons in a model of seizures in which progression has been prevented by pentobarbitone, has provided additional information about the initial development of motor seizures induced by kainic acid.

As reviewed by Lothman et al.[16]2-deoxyglucose metabolic mapping studies after kainic acid administration revealed that activity first increased within the hippocampus, that there was a second phase of increased activity incorporating the entorhinal cortex,

Conclusions

This study has (i) strengthened the role of the hippocampus as the structure first activated by systemic kainic acid, in particular the role of the dentate gyrus, (ii) has supported EEG evidence that there is activation of cerebral cortex at the time of the first generalized motor convulsion and (iii) has suggested that spread of neuronal activity occurs in two phases, as an initial hippocampal event and then as a generalized seizure. The significance of our findings for primary generalized

Acknowledgements

Supported by grants from the National Health and Medical Research Council, the Australian Brain Foundation, the Flinders Medical Centre Research Foundation and the Thyne-Reid Education Trust for Research in Epilepsy. Ms Rachel Cale provided technical assistance.

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The aim of the present study was to examine the role of ionotropic glutamate receptors in the cerebellum during generalized seizures. Epileptic neuronal activation was evaluated through the immunohistochemical detection of c-fos protein in the cerebellar cortex. Generalized seizures were precipitated by the intraperitoneal injection of 4-aminopyridine. The animals were pretreated with the NMDA receptor antagonists MK-801 (2 mg/kg), amantadine (50 mg/kg), and the AMPA receptor antagonist GYKI 52466 hydrochloride (50 mg/kg). Two hours after 4-aminopyridine injection, the number of c-fos immunostained cell nuclei was counted in serial immunohistochemical sections of the cerebellar vermis. The number of c-fos immunostained cell nuclei in the granular layer decreased significantly in animals pretreated with the glutamate receptor antagonists compared to the untreated animals having convulsion. We can conclude that mossy fiber stimulation exerts its seizure-generating action mainly through the ionotropic glutamate receptors of the mossy fiber synapses. Both NMDA and AMPA receptor antagonists are effective in reducing glutamate-mediated postsynaptic effects in the cerebellar cortex.
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There is ample literature describing neuronal c-Fos induction following kindling and other seizure models (Barone et al., 1993; Bozzi et al., 2011; Chiasson et al., 1995; Dragunow and Faull, 1989; Dragunow and Robertson, 1988; Morgan and Curran, 1991a, 1991b; Motte et al., 1997). Furthermore, several studies have demonstrated a correlation between the expansion of c-Fos induction from limbic areas to the cerebral cortex and the progression of epileptic seizures (Andre et al., 1998; Clark et al., 1991; Ebert and Loscher, 1995; Hiscock et al., 2001; Simler et al., 1999; Szyndler et al., 2009; Willoughby et al., 1997). These studies and others have capitalized on the following advantages of c-Fos as a marker of neuronal activity: ease of detection, good sensitivity following neuronal stimulation, cellular mapping that enables the examination of all neurons in a particular network spanning multiple brain regions, and the ability to combine c-Fos labeling with other immunohistochemical markers and/or retrograde axonal tracing for connectivity studies.
Loss of glutamine synthetase (GS) in hippocampal astrocytes has been implicated in the causation of human mesial temporal lobe epilepsy (MTLE). However, the mechanism by which the deficiency in GS leads to epilepsy is incompletely understood. Here we ask how hippocampal GS inhibition affects seizure phenotype and neuronal activation during epilepsy development (epileptogenesis). Epileptogenesis was induced by infusing the irreversible GS blocker methionine sulfoximine (MSO) unilaterally into the hippocampal formation of rats. We then used continuous video-intracranial electroencephalogram (EEG) monitoring and c-Fos immunohistochemistry to determine the type of seizures and spatial distribution of neuronal activation early (1–5 days postinfusion) and late (16–43 days postinfusion) in epileptogenesis. Early in epileptogenesis, seizures were preferentially mild (stage 1–2), activating neurons in the entorhinal-hippocampal area, the basolateral amygdala, the piriform cortex, the midline thalamus, and the anterior olfactory area. Late in epileptogenesis, the seizures were generally more severe (stages 4–5) with neuronal activation extending to the neocortex, the bed nucleus of the stria terminalis, the mediodorsal thalamu\s, and the central nucleus of the amygdala. Our findings demonstrate that inhibition of GS focally in the hippocampal formation triggers a process of epileptogenesis characterized by gradual worsening of seizure severity and involvement of progressively larger neuronal populations over a period of several weeks. Knowledge about the underlying mechanism of epileptogenesis is important because such knowledge may result in more specific and efficacious treatments of MTLE by moving away from large and poorly specific surgical resections to highly targeted surgical or pharmacological interventions of the epileptogenic process.
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Conversely, in saline-treated animals, c-fos mRNA hippocampal staining was more intense in En1Cre/+; tOtx2ov/+ than control mice. The pattern of c-fos mRNA induction observed in our control mice perfectly matches that observed in inbred or mutant mouse strains (Bozzi et al., 2000; Bozzi and Borrelli, 2013), as well as in the rat (Willoughby et al., 1997). It is generally accepted that c-fos induction after seizures is a reliable marker of neuronal hyperactivity; however, several findings indicate that c-fos induction may not correlate with seizure severity (Labiner et al., 1993; Fabene et al., 2004).
The homeobox-containing transcription factor Otx2 controls the identity, fate and proliferation of mesencephalic dopaminergic (mesDA) neurons. Transgenic mice, in which Otx2 was conditionally overexpressed by a Cre recombinase expressed under the transcriptional control of the Engrailed1 gene (En1^Cre/+; tOtx2^ov/+), show an increased number of mesDA neurons during development. In adult mice, Otx2 is expressed in a subset of neurons in the ventral tegmental area (VTA) and its overexpression renders mesDA more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Here we further investigated the neurological consequences of the increased number of mesDA neurons in En1^Cre/+; tOtx2^ov/+ adult mice. Immunohistochemistry for the active, glycosylated form of the dopamine transporter (glyco-Dat) showed that En1^Cre/+; tOtx2^ov/+ adult mice display an increased density of mesocortical DAergic fibers, as compared to control animals. Increased glyco-Dat staining was accompanied by a marked hypolocomotion in En1^Cre/+; tOtx2^ov/+ mice, as detected in the open field test. Since conditional knockout mice lacking Otx2 in mesDA precursors (En1^Cre/+; Otx2^floxv/flox mice) show a marked resistance to kainic acid (KA)-induced seizures, we investigated the behavioral response to KA in En1^Cre/+; tOtx2^ov/+ and control mice. No difference was observed between mutant and control mice, but En1^Cre/+; tOtx2^ov/+ mice showed a markedly different c-fos mRNA induction profile in the cerebral cortex and hippocampus after KA seizures, as compared to controls. Accordingly, an increased density of parvalbumin (PV)-positive inhibitory interneurons was detected in the deep layers of the frontal cortex of naïve En1^Cre/+; tOtx2^ov/+ mice, as compared to controls. These data indicate that Otx2 overexpression results in increased DAergic innervation and PV cell density in the fronto-parietal cortex, with important consequences on spontaneous locomotor activity and seizure-induced gene expression. Our results strengthen the notion that Otx2 mutant mouse models are a powerful genetic tool to unravel the molecular and behavioral consequences of altered development of the DAergic system.
Intrahippocampal cholinesterase inhibition induces epileptogenesis in mice without evidence of neurodegenerative events
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The mechanisms of epileptogenesis remain largely unknown and are probably diverse. The aim of this study was to investigate the role of focal cholinergic imbalance in epileptogenesis. To address this question, we monitored electroencephalogram (EEG) activity up to 12 weeks after the injection of a potent cholinesterase (ChE) inhibitor (soman) at different doses (0.53, 0.75, 1, 2, 2.8, 4 and 11 nmol) into the right dorsal hippocampus of C57BL/6 mice. Different parameters were used to choose the dose for a focal model of epileptogenesis (mainly electrographic patterns and peripheral ChE inhibition). The pattern of neuronal activation was studied by Fos immunohistochemistry (IHC). Brain damage was evaluated by hemalun-phloxin, neuronal nuclei antigen IHC and silver staining. Glial fibrillary acidic protein IHC was used to evaluate astroglial reaction. Finally, long-term behavioral consequences were characterized. At the highest dose (11 nmol), soman quickly evoked severe signs, including initial seizures and promoted epileptogenesis in the absence of tissue damage. With lower doses, late-onset seizures were evidenced, after 1–4 weeks depending on the dose, despite the absence of initial overt seizures and of brain damage. Only a weak astroglial reaction was observed. Following injection of 1 nmol, Fos changes were first evidenced in the ipsilateral hippocampus and then spread to extrahippocampal areas. A selective deficit in contextual fear conditioning was also evidenced two months after injection. Our data show that focal hypercholinergy may be a sufficient initial event to promote epilepsy and that major brain tissue changes (cellular damage, edema, neuroinflammation) are not necessary conditions.
Increased susceptibility to kainic acid-induced seizures in Engrailed-2 knockout mice
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A precise correlation exists between the occurrence of generalized seizures and the induction pattern of the IEGs c-fos and c-jun following KA. Pre-convulsive behaviors (stages 1–3 of the Racine's scale) induce IEGs mainly in the hippocampus and other limbic areas (hippocampus, amygdala, entorhinal and pyriform cortices) whereas continuous generalized seizures (stages 4–6) result in a widespread expression in several brain areas (Willoughby et al., 1997; Bozzi et al., 2000). Accordingly, WT mice experienced no or very brief generalized seizures after KA, showing IEGs mRNA induction restricted to the hippocampus and other limbic areas.
The En2 gene, coding for the homeobox-containing transcription factor Engrailed-2 (EN2), has been associated to autism spectrum disorder (ASD). Due to neuroanatomical and behavioral abnormalities, which partly resemble those observed in ASD patients, En2 knockout (En2^−/−) mice have been proposed as a model for ASD. In the mouse embryo, En2 is involved in the specification of midbrain/hindbrain regions, being predominantly expressed in the developing cerebellum and ventral midbrain, and its expression is maintained in these structures until adulthood. Here we show that in the adult mouse brain, En2 mRNA is expressed also in the hippocampus and cerebral cortex. Hippocampal En2 mRNA content decreased after seizures induced by kainic acid (KA). This suggests that En2 might also influence the functioning of forebrain areas during adulthood and in response to seizures. Indeed, a reduced expression of parvalbumin and somatostatin was detected in the hippocampus of En2^−/− mice as compared to wild-type (WT) mice, indicating an altered GABAergic innervation of limbic circuits in En2^−/− mice. In keeping with these results, En2^−/− mice displayed an increased susceptibility to KA-induced seizures. KA (20 mg/kg) determined more severe and prolonged generalized seizures in En2^−/− mice, when compared to WT animals. Seizures were accompanied by a widespread c-fos and c-jun mRNA induction in the brain of En2^−/− but not WT mice. Long-term histopathological changes (CA1 cell loss, upregulation of neuropeptide Y) also occurred in the hippocampus of KA-treated En2^−/− but not WT mice. These findings suggest that En2^−/− mice might be used as a novel tool to study the link between epilepsy and ASD.
Activation of brain metabolism and fos during limbic seizures: The role of Locus Coeruleus
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Therefore, Fos immunostaining is widely used as a tool for functional brain mapping after different physiological stimuli. By the same token, the evaluation of brain regional expression of Fos has been performed in a number of animal models of limbic seizures and SE (Dragunow and Robertson, 1987; Dragunow et al., 1992; Gass et al., 1992; Le Gal La Salle, 1988; Morgan et al., 1987; Willoughby et al., 1997). The pattern of protein expression affected by Fos varies, depending on the brain region and neuronal type involved (Hedegen and Leah, 1998).
The noradrenergic nucleus Locus Coeruleus (LC) densely innervates limbic structures. In rats, the damage to LC by the neurotoxin DSP-4, converts episodic limbic seizures induced by bicuculline infusion in the anterior piriform cortex (APC) into self-sustaining status epilepticus (SE). SE induced by this approach is similar to SE induced by co-infusing cyclothiazide and bicuculline into APC in rats bearing an intact LC. As opposed to other commonly used rat SE models (e.g. systemic kainate or pilocarpine), this approach allows one to analyze the effects of SE on brain regions which are solely due to spreading of seizure activity, rather than to direct effect of systemic chemoconvulsant.
We evaluated the expression of Fos protein (an immediate early gene product), and the local cerebral metabolic rates for [¹⁴C] 2-deoxyglucose (lCMRglc), in rats following SE induced either by cyclothiazide+bicuculline or by DSP-4+bicuculline.
We demonstrated that regional Fos expression after SE does not parallel the increase in lCMRglc, in LC-lesioned rats. In DSP-4+bicuculline rats there is an overall lower expression of the protein as compared with the cyclothiazide+bicuculline or bicuculline alone groups; even more, such a difference co-exists with an higher lCMRglc in the DSP-4+bicuculline-treated rats in some regions, as compared with the other groups.
These data show that LC neurons play an important role in determining immediate early genes expression even in conditions of strong pathological activation, such as limbic SE. This might have relevant effects in the plastic mechanisms related with epileptogenesis.

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Fos Induction Following Systemic Kainic Acid: Early Expression in Hippocampus and Later Widespread Expression Correlated With Seizure

Abstract

Section snippets

Animals

Behaviour

Discussion

Conclusions

Acknowledgements

Brain Res.

Neuroscience

Neurosci. Lett.

Molec. Brain Res.

Neuroscience

Expl Neurol.

Neurosci. Lett.

Meth. Neurosci.

Prog. Neurobiol.

Brain Res.

Brain Res.

Life Sci.

Brain Res.

Prog. Neurobiol.

Epilepsy Res.

Molec. Brain Res.

Brain Res.

Nucleus basalis and thalamic control of neocortical activity in the freely moving rat

J. Neurosci.