Persistent regional increases in brain-derived neurotrophic factor in the flurothyl model of epileptogenesis are dependent upon the kindling status of the animal.

Brain-derived neurotrophic factor (BDNF) appears to be both regulated by and a regulator of epileptogenesis. In the flurothyl (HFE) model of kindling mice exposed to successive flurothyl trials over 8 days express a rapid, long-lasting reduction in generalized seizure threshold and a more slowly evolving change in seizure phenotype in response to subsequent flurothyl exposure. The BDNF genotype of particular mouse strains appears to influence the epileptogenic progression in this model. Thus, we hypothesized that BDNF signaling pathways are altered by flurothyl-induced seizures. Following HFE kindling, fully kindled (eight seizures) adult male C57BI/6J mice had significantly elevated whole brain BDNF levels through at least 28 days after their final seizure. Mice that received only four HFE seizures (not kindled) had elevated BDNF levels, but only at 1 day post-seizure (DPSz), while BDNF levels were not significantly altered in mice receiving just one HFE seizure at any time point studied. Regional expression patterns of BDNF in the hippocampus, hypothalamus, and frontal cortex were also elevated by one DPSz and returned to control values by 14 DPSz in mice that received four HFE seizures. No changes were seen in the cerebellum, striatum, or piriform cortex. In contrast, fully kindled mice had significantly elevated BDNF levels within the hippocampus, hypothalamus, neocortex, and striatum that remained elevated through at least 14 DPSz, while levels were unchanged in the cerebellum and piriform cortex. Regional results were confirmed using anti-BDNF immunohistochemistry (IHC). Despite changes in BDNF levels following HFE kindling, we were unable to demonstrate alterations either in full-length tyrosine kinase receptor B (TrkB) expression (Western blot and IHC) or in truncated TrkB (IHC) expression levels. Together, these data suggest a model of a positive feedback loop involving seizure activity and seizure number and persistently modified BDNF signaling pathways that influences seizure phenotypes within the HFE kindling paradigm. Thus, long-term elevations in BDNF may be responsible in part for epileptogenic processes and the development of human refractory epilepsies.

Increased mitotic activity in the dentate gyrus of the hippocampus of adult C57BL/6J mice exposed to the flurothyl kindling model of epileptogenesis.

Repeated flurothyl-induced generalized forebrain seizures result in a progressive and permanent lowering of the generalized seizure threshold in mice and an increase in the percentage of animals expressing forebrain-brainstem seizures, when rechallenged with flurothyl, after a stimulation-free period. Since this seizure paradigm serves as an excellent model for examining changes in seizure threshold and seizure propagation, we were interested in examining mitotic activity in hippocampal progenitors following flurothyl-induced epileptogenesis. In the present studies, we investigated (1). the effect of one or eight flurothyl-induced seizures on mitotic activity in the hippocampal dentate gyrus of adult mice measured by 5-bromo-2'-deoxyuridine incorporation, (2). the time course of change in hippocampal mitotic activity, (3). the cellular phenotype of these mitotically active cells, and (4). the relationship of changes in mitotic activity to changes in seizure threshold and phenotype. Significant increases in hippocampal mitotic activity were observed in mice exposed to either one or eight flurothyl-induced seizures. Increases were observed at 1 and 3 days following one seizure, and at 0, 1, 3, and 7 days following eight seizures. Confocal analyses, using neuronal and glial markers, suggest that the majority of these mitotic cells are neurons. In addition, no correlation was observed between hippocampal mitotic activity and the final seizure type that animals expressed following incubation and flurothyl retest. A significant correlation was observed between hippocampal mitotic activity and seizure threshold in flurothyl-kindled mice. Overall, these results indicate that both one and eight flurothyl-induced seizures are potent inducers of hippocampal neurogenesis in adult mice. Results further suggest that the increases in hippocampal neurogenesis are not directly related to the processes that underlie the shift in behavioral seizure phenotype, but may be involved in either the establishment or the maintenance of seizure threshold in this flurothyl model of epileptogenesis.

Bidirectional transfer between electrical and flurothyl kindling in mice: evidence for common processes in epileptogenesis.

PURPOSE: This study sought to determine whether there was a transfer of seizure susceptibility between two models of epileptogenesis, electrical kindling and a newly described model of flurothyl kindling. In this study, we determined the effects of preexposure to one kindling agent on the seizure responsiveness to the other. METHODS: Mice were divided into three groups: (a) six mice (FLK) were kindled with flurothyl, rechallenged with flurothyl after a 28-day incubation phase, implanted with olfactory bulb (OB) electrodes, and electrically kindled; (b) six mice (ELK) were implanted with OB electrodes, electrically kindled to six stage 5 seizures, and given one flurothyl trial 3 days later and a second flurothyl trial after a 28-day incubation period; and (c) six mice (IMP) were implanted with OB electrodes, tested with flurothyl at the same times as the ELK group, and later electrically kindled. RESULTS: Mice that were previously kindled with flurothyl (FLK) had significantly faster electrical kindling rates to one stage 5 seizure or to six stage 5 seizures compared with animals in the ELK and IMP groups. Mice that were previously exposed to either electrical kindling or flurothyl kindling had significantly diminished latencies to generalized seizure onset (flurothyl-induced seizure thresholds) either before or after a 28-day incubation period compared with the IMP control mice. In addition, both the FLK and ELK groups had significantly increased percentages of mice expressing forebrain-brainstem seizures, compared with the IMP group, following either rechallenge with flurothyl after a 28-day incubation or focal electrical kindling. CONCLUSIONS: These findings indicate a near-complete bidirectional transfer between these electrical and flurothyl kindling models. Mice that were previously exposed to either electrical or flurothyl kindling have increased seizure susceptibilities and altered seizure phenotypes when exposed to the other seizure paradigm. Overall, these studies indicate that previous seizures are the critical determinant of the bidirectional transfer of seizure susceptibility observed, and not the electrical or pharmacologic properties of the original kindling agent. Finally, the observation of near identity in transfer characteristics between electrical and flurothyl kindling models suggests that the proepileptogenic processes initiated by exposure to either model are similar.

The role of the ventromedial nucleus of the hypothalamus in epileptogenesis.

Flurothyl kindling initiates a time-dependent process that results in a facilitated propagation from the forebrain to the brainstem seizure system and in an increase in the complexity of behavioral seizure expression. We investigated the involvement of the ventromedial nucleus of the hypothalamus (VMH) in mediating this facilitated propagation between these seizure systems. Bilateral ibotenic acid lesions of the VMH, but not the dorsomedial nucleus of the hypothalamus (DMH), resulted in a disruption in the propagation of seizure activity from the forebrain to the brainstem. Moreover, VMH lesioned mice were able to express brainstem seizures following minimal corneal electroconvulsive shock (mECS). Together, our results indicate that the VMH is a critical substrate involved in propagating seizure activity between the forebrain and brainstem, but is not involved in the expression systems necessary for forebrain or brainstem seizure manifestations.

A role for the bilateral involvement of perirhinal cortex in generalized kindled seizure expression.

The perirhinal cortex (PRh) has been suggested as a substrate for the expression of generalized clonic seizures in the late stages of kindling development (stages 4-5). Using the induction of Fos as a marker of neuronal activation, the PRh region was investigated after kindling or nonkindling electrical stimulation. Nonkindling electrical stimulation of the PRh elicited stimulus-locked behaviors, without afterdischarge. These behaviors were characterized by rearing and bilateral forelimb clonus which were terminated upon electrical stimulus offset in half of the rats displaying this behavior (with the other half expressing self-sustained seizures). In these animals, Fos immunoreactivity was found throughout neocortical and subcortical structures in the hemisphere ipsilateral to the stimulating electrode. By contrast, Fos-immunoreactivity in the contralateral hemisphere was localized primarily in the PRh and frontal motor cortex. Likewise, similar patterns of Fos immunoreactivity were observed in both hemispheres of rats following kindling to one generalized clonic seizure from several limbic and paleocortical structures. These results suggest that the bilateral involvement of the PRh is critical in producing the bilateral behaviors associated with generalized clonic seizure expression. In support of this interpretation, infusion of 3 M KCl directly into the contralateral PRh of rats kindled to a single stage 4-5 (generalized clonic) seizure from the ipsilateral amygdala reduced seizure manifestations from a generalized clonic seizure (stage 4-5) to a unilateral clonic seizure (stage 3) without affecting measures of focal excitability. Taken together, these data indicate a role for the bilateral involvement of the PRh in generalized clonic seizure expression whether evoked from the naive or kindled state. These results further indicate that bilateral behaviors require the bilateral involvement of the structures necessary for the expression of these behaviors.

Regional analysis of the spatial patterns of Fos induction in brain following flurothyl kindling.

We have recently demonstrated that eight, daily flurothyl-induced generalized clonic seizures, followed by a four week stimulus-free interval, results in a long-lasting reduction in generalized seizure threshold and a change in the type of seizure expressed in response to flurothyl from clonic to tonic. There is a progressive increase in the probability that a mouse will express a tonic seizure during the four week interval, suggesting that prior flurothyl seizures initiate a proepileptogenic process that requires time to develop. In this study, the immunohistochemical detection of the c-fos protein (Fos) was used to evaluate whether seizure-induced epileptogenesis resulted in regional differences in the degree of neuronal activation. Fos immunoreactivity was examined 1.5 h following either a single generalized seizure, the last of eight consecutive daily seizures or a retest seizure evoked two weeks after the last of eight seizures. In each condition, generalized seizure behaviours were elicited in C57BL/6 mice using flurothyl and classified as either "forebrain" (face and forelimb clonus) or "brainstem" (running/bouncing, treading, tonic extension). The spatial distribution of Fos induction was compared on the basis of the seizure phenotype and the seizure history. The predominant differences in Fos distribution were found to be related to the type of seizure expressed regardless of the seizure history. Furthermore, the different motor components that make up a "brainstem" seizure could not be distinguished by the pattern of Fos labelling suggesting that multiple convulsive behaviours are mediated by one anatomical system. Finally, Fos induction in the ventromedial hypothalamic nucleus preceded and predicted the change in seizure type from "forebrain" to "brainstem". These data support the concept that separate anatomical systems mediate the expression of the two generalized seizure phenotypes. In addition, the ventromedial nucleus of the hypothalamus may be a point of interaction between the systems and may play a role in seizure-induced neural reorganization.

Decreased brainstem seizure thresholds and facilitated seizure propagation in mice exposed to repeated flurothyl-induced generalized forebrain seizures.

We recently have described a novel model of epileptogenesis utilizing the inhalant chemoconvulsant, flurothyl (Applegate et al., 1997; Samoriski and Applegate, 1997). The hallmark feature of this model is a change in behavioral seizure phenotype from a forebrain seizure, observed during the initial flurothyl exposures, to a brainstem seizure, elicited by flurothyl, after a 28-day stimulation free incubation period. In this study, we sought to establish the basis for this change in behavioral seizure response. To this end, we examined the effects of exposure to this paradigm on the generalized brainstem seizure threshold and on the propagation of forebrain seizures onto the brainstem seizure substrate. Ten mice were given flurothyl-induced generalized forebrain seizures on 8 consecutive days (induction phase). The other ten mice were not exposed to the flurothyl induction paradigm and served as controls. Minimal corneal electroconvulsive shock (mECS--20 mA) was used to assay whether there was any change in the animals' generalized brainstem seizure thresholds at 3, 14 and 28 days following the last flurothyl seizure trial. Mice that were exposed to flurothyl exhibited a progressive increase in the percentage of animals having a mECS-induced brainstem seizure when tested at 3 (40%), 14 (70%) and 28 (90%) days following the last flurothyl seizure. Control mice rarely had a brainstem seizure at any of the three time points tested, mostly forebrain seizures were observed. These results suggest that there is a significant progressive lowering of the brainstem seizure threshold, during the incubation phase of the flurothyl paradigm, which is coincident with the previously reported time course of change in the behavioral seizure phenotype observed using this flurothyl model (Applegate et al., 1997; Samoriski and Applegate, 1997). Following mECS testing, mice were implanted with bipolar electrodes and kindled from the olfactory bulb (OB). Mice exposed to the flurothyl paradigm demonstrated significantly faster kindling rates, longer afterdischarge durations. and longer durations of and latencies to stage 5 seizures compared to controls. Furthermore, animals exposed to the flurothyl protocol demonstrated an increase in the expression of brainstem seizures after focally-elicited OB afterdischarges. These results suggest that there is an increased interaction between the forebrain and brainstem seizure systems after exposure to this model of epileptogenesis. Together, results indicate that the change in behavioral seizure phenotype observed following exposure to our flurothyl paradigm are promoted by both decreases in brainstem seizure thresholds and facilitated forebrain seizure propagation onto the brainstem seizure system.

Global increases in seizure susceptibility in mice lacking 5-HT2C receptors: a behavioral analysis.

Previous studies have shown that mice bearing a targeted disruption of the 5-HT2C receptor gene exhibit an epilepsy syndrome associated with sporadic spontaneous seizures that occasionally result in death. In this study, we have defined the seizure susceptibility profiles of these 5-HT2C receptor mutant mice backcrossed onto a C57BL/6 background. Wild-type and mutant animals were either electrically kindled from the olfactory bulb, exposed to corneal electroshock, or tested with the chemoconvulsant, flurothyl. In all paradigms, mice lacking the 5-HT2C receptor were significantly more seizure susceptible than wild-type controls. Results indicate that mutants have lower focal seizure thresholds, increased focal seizure excitability, and facilitated propagation within the forebrain seizure system. Mutants also exhibit lower generalized seizure thresholds for the expression of both generalized clonic and generalized tonic seizures. Importantly, the 5-HT receptor antagonist, mesulergine (2 or 4 mg/kg), administered prior to electroshock testing, recapitulated the mutant phenotype in wild-type mice. Together, these data strongly implicate a role for serotonin and 5-HT2C receptors in the modulation of neuronal network excitability and seizure propagation globally, throughout the CNS.

Repeated generalized seizures induce time-dependent changes in the behavioral seizure response independent of continued seizure induction.

This study examined both the acute and long-lasting changes in seizure susceptibility that occur in response to the repeated induction of generalized seizure activity. Daily flurothyl-induced generalized clonic seizures resulted in a progressive decrease in both the generalized seizure threshold and the latency to the first myoclonic jerk. The threshold reduction was significant as early as the second trial and was maximal by trial 5. However, a minimum of eight seizures was necessary for the maximal reduction to be long-lasting. The present study also examined the effects of the number of seizures and the duration of the stimulation-free interval on the type of generalized seizure expressed. During the induction phase of the experiment, only generalized clonic seizures ("forebrain seizures") were expressed. If, however, the animal was retested after a 1, 2, 3, or 4 week stimulation-free interval, a progressive increase in both the proportion of animals expressing "brainstem seizure" behaviors and the median seizure score was observed. The progression of flurothyl-induced generalized seizure behaviors was significantly altered if (1) a minimum of eight generalized clonic seizures had been expressed, and (2) a minimum of a 2 week stimulation-free interval followed. Fewer generalized clonic seizures failed to reliably produce changes in seizure phenotype, even after extended stimulus-free intervals. These data indicate that specific kindling processes are initiated during the interval of repeated seizure induction and evolve in the absence of continued seizure induction. Furthermore, these mechanisms of epileptogenesis were found to be manifest predominantly as a change in the seizure phenotype expressed and to proceed independent of changes in the generalized seizure threshold.

Effects of valproate, phenytoin, and MK-801 in a novel model of epileptogenesis.

PURPOSE: We have developed and characterized a novel model of epileptogenesis based on the convulsive actions of flurothyl in mice. The hallmark feature of this model is a reliable change in the type of seizure expressed in response to flurothyl from generalized clonic to generalized tonic seizures. The purpose of our study was to evaluate the effects of chronic administration of valproate (VPA), phenytoin (PHT), and MK-801 on the change in seizure phenotype observed in our model system. METHODS: Male C57BL/6J mice received flurothyl seizures on 8 consecutive days. Two hours after the last generalized seizure, chronic drug or vehicle was administered twice daily at 12-h intervals for 28 days. The drugs evaluated were VPA (250 mg/kg), PHT (30 mg/kg), and MK-801 (0.5 mg/kg). After a 7-day drug washout period, mice were retested with flurothyl. RESULTS: Among uninjected or vehicle-injected control mice, there was a significant increase in the proportion of animals expressing tonic seizures after the 28-day stimulation-free interval. Chronic administration of VPA or MK-801, but not PHT, blocked the characteristic change in seizure type from clonic to tonic. CONCLUSIONS: The change in seizure phenotype observed after exposure to our paradigm indicates a fundamental reorganization in the propagation of flurothyl-initiated seizures. As in electrical kindling, VPA and MK-801 are effective at blocking or retarding the reorganization, whereas PHT is not. The concordance in pharmacologic profiles between kindling and our model suggests that the processes underlying changes in seizure susceptibility in these two models share mechanisms in common.

Apoptotic and necrotic cell death following kindling induced seizures.

The study was designed to determine which type of cell death occurs following kindling induced seizures, and to determine which neurons die. For this purpose seizures were kindled from the entorhinal cortex. Following a range of 5-85 stage 5 seizures, rats were sacrificed, and the tissue was prepared for analysis. The TUNEL and silver impregnation methods were used to identify apoptotic or necrotic cell death, respectively. These methods were subsequently combined with immunocytochemistry, to determine if diseased neurons expressed somatostatin or the NMDA receptor (NMDAR1). The tissue analysis demonstrated that following kindling induced seizures, 1) hippocampal and extrahippocampal neurons die, 2) some neurons die through apoptosis, others through necrosis, and 3) some of the diseased neurons express somatostatin, others the NMDAR1 and that both subpopulations of neurons are present at hippocampal and extrahippocampal sites.

Differential spatial patterns of Fos induction following generalized clonic and generalized tonic seizures.

The expression of generalized clonic and generalized tonic seizures has been suggested to result from the activation of different and independent neuronal circuits. Using the induction of the c-fos protein (Fos) as a marker of neuronal activity, we identified brain structures that are differentially associated with the expression of electroconvulsive shock-induced generalized clonic and generalized tonic seizures. Expression of either seizure phenotype resulted in a similar bilaterally symmetrical increase in Fos immunoreactivity in many forebrain structures, including the bed nucleus of the stria terminalis, hippocampal dentate gyrus, amygdala, and piriform cortex, compared to controls. However, following tonic hindlimb extension (THE), the degree of labeling in specific thalamic, hypothalamic, and brain stem areas was significantly greater than that of either controls or animals exhibiting clonic seizures. While a greater number of neurons in the hypothalamus (e.g., ventromedial nucleus), subparafascicular thalamic nucleus, peripeduncular area, deep medial superior colliculus, dorsal and lateral central gray, and paralemniscal nuclei were robustly labeled following THE, noticeably fewer cells were immunoreactive following face and forelimb clonic seizure behaviors. These differences were also found to be independent of the stimulus magnitude. In animals stimulated with the same current intensity but expressing either of the two seizure phenotypes, the pattern of Fos induction was consistent with the seizure phenotype expressed. These results demonstrate that specific subsets of neurons are differentially activated following the expression of different generalized seizure behaviors and that activity in discrete mesencephalic and diencephalic structures is more frequently associated with the expression of generalized tonic seizures than with the expression of generalized clonic seizures.

The effects of neonatal hypoxia on kindled seizure development and electroconvulsive shock profiles.

PURPOSE: Our previous research indicated that the exposure of rat pups to an hypoxic environment during a discrete developmental period (postnatal days 10-15) produces short-term seizures and confers an enduring increase in susceptibility to pentylenetetrazol- and flurothyl-induced seizures. In this study, we evaluated the effects of hypoxic insult in this neonatal period of susceptibility to electrical kindling and corneal electroconvulsive shock. METHODS: Ten-day-old rat pups were exposed to a 3% O2 environment, as previously described, and were either kindled or exposed to corneal electroshock at adulthood (70 days old). RESULTS: Neither kindled seizure development from the septal nucleus or amygdala nor electroconvulsive shock profiles were significantly altered by hypoxic pretreatment. CONCLUSIONS: Results indicate that hypoxia produces increases in seizure susceptibility that are observable in only some experimental seizure models but not in others. This outcome serves to target some anatomic systems more than others in the mechanisms involved in hypoxia-induced neural reorganization.

Activation of oxytocin-containing neurons of the paraventricular nucleus (PVN) following generalized seizures.

Due to the complex nature of generalized limbic seizures, marked disturbances in physiological homeostasis occur. Accompanying the motor manifestations which characteristically are associated with generalized limbic seizures, alterations in neuroendocrine, behavioral, and autonomic functions may be observed. The paraventricular nucleus (PVN) of the hypothalamus is known to play a significant role in such neuronal responses to stressful stimuli; however, the effect of seizures on hypothalamic neurons is unknown. We have used the immunocytochemical detection of the Fos protein to anatomically identify neurons in the PVN which are activated following generalized limbic seizures. To induce seizures, rats received intraperitoneal injections of kainic acid or were kindled from the entorhinal cortex. We have demonstrated that elicitation of generalized limbic seizures induces a dramatic number of neurons in the PVN to express the Fos protein. Numerous Fos-immunolabeled neurons were identified in both the parvicellular and magnocellular component of the PVN. In the latter, this study clearly reveals a preferential and selective activation of oxytocin-containing neurons, and it extends and supports the hypothesis that oxytocin plays a role in the body's response to specific stress paradigms. Data suggest that an activation of the oxytocin neuronal system may be part of the adaptive mechanism that enables the hypothalamus to modulate and maintain an adequate response to stressors (e.g., generalized seizures) to regain homeostasis.

Oxytocin and vasopressin mRNA expression in rat hypothalamus following kainic acid-induced seizures.

In this study, the regulation of hypothalamic oxytocin and vasopressin messenger RNA expression following the induction of seizures was investigated by in situ hybridization. Following kainic acid-induced seizures, a significant increase in oxytocin messenger RNA in the paraventricular nucleus was demonstrated at 1.5 h, one and two weeks; its level decreased at three weeks and was significantly increased again at four weeks; at eight weeks the messenger RNA level still remained higher than that of controls. Vasopressin messenger RNA in the paraventricular nucleus was increased significantly only at 1.5 h following induction of seizures. The oxytocin messenger RNA level in the supraoptic nucleus was also increased early at 1.5 h and later at four weeks following seizures; however, these increases did not last as long as those in the paraventricular nucleus. Vasopressin messenger RNA in the supraoptic nucleus was also increased after the initial seizures; however, its messenger RNA level vacillated up and down throughout the post-seizure times studied. The earliest significant increase of vasopressin messenger RNA was at one week after seizures, and there was a late significant increase of vasopressin messenger RNA at three weeks after seizures. The present study demonstrates that following kainic acid-induced seizures both, the oxytocin and vasopressin messenger RNA expressions, were up-regulated and these up-regulations were long-term events. The increase of oxytocin messenger RNA in the paraventricular nucleus was more persistent than the others. The pattern of messenger RNA up-regulation was different for oxytocin and vasopressin, and different in the paraventricular nucleus and supraoptic nucleus. These different patterns of messenger RNA elevations suggest that the different components of the rat hypothalamus were regulated differentially by kainic acid-induced seizures.

The kindling-activated neuronal network: recruitment of somatostatin-synthesizing neurons.

The present study demonstrates the anatomical extent of the kindling-activated neuronal network in general, and specifically the recruitment of extrahippocampal somatostatin (SST)-synthesizing neurons into this network. It has been known that SST neurons of the hippocampal formation are activated during episodes of seizure, however, it was not known if this activation was a local event or extended to other areas in the brain. We were therefore interested in determining if and which SST neurons outside the hippocampal formation might be recruited into this seizure-activated neuronal network. Using the kindling model of seizure elicitation, expression of the Fos protein in activated, depolarized neurons was utilized to identify seizure-activated neurons. Subsequently, the mRNA for SST was identified through in situ hybridization in the same tissue section, allowing the identification of seizure-activated, SST-synthesizing neurons. The results show that: (a) the majority of SST-synthesizing neurons in the forebrain and diencephalon became activated during the kindling development; (b) their recruitment into the kindling-activated neuronal network occurred progressively; and, (c) these SST-synthesizing neurons represented a component of the kindling-activated neuronal network throughout the development of kindling-induced seizures.

The substantia nigra pars reticulata, seizures and Fos expression.

The induction of the proto-oncogene c-fos has been used extensively to identify spatially distributed neural systems activated by seizures. The substantia nigra pars reticulata (SNpr) has been implicated as a critical structure in neural networks involved in the modulation of seizure expression, yet the SNpr has not been reported to express Fos following seizures induced in a variety of seizure paradigms. In this study we determined whether (1) the temporal characteristics of Fos induction in the SNpr were different than those of other brain areas following kindled seizures, (2) neurons in the SNpr possess the cellular machinery to express Fos, (3) Fos can be induced in SNpr by direct electrical stimulation, and (4) Fos expression is induced in the SNpr following kainate or pilocarpine-induced status epilepticus. Results indicate that Fos is not induced in SNpr at any time point (1-12 h) after kindled seizures, and that serum response factor, a constitutively expressed nuclear protein necessary for Fos expression, is present in SNpr neurons. Results further indicate that Fos expression in the SNpr is induced following either direct electrical stimulation or pilocarpine status, but not status elicited by kainate. We conclude that, in so far as the SNpr represents a critical structure for modulating seizure expression, seizure activity does not represent a sufficient stimulus to induce Fos in SNpr neurons. Further, the neural networks defined by Fos expression following seizure may be incomplete, and should be interpreted conservatively.

Activation of somatostatin-synthesizing neurons in the hippocampal formation through kindling-induced seizures.

The present study was designed to determine if and to what extent somatostatin (SST) synthesizing neurons of the hippocampal formation are activated during seizures, elicited through kindling of the perforant pathway. Tissue was used and analyzed from animals which had experienced a single after discharge, or a stage 3 or stage 5 seizure. The protein expression of the oncogene c-fos in activated, depolarizing neurons was utilized to identify seizure-activated SST-synthesizing neurons. Combined immunocytochemical and in situ hybridization methods were used to identify these double-labeled, Fos protein, and SST mRNA-containing neurons. The results were quantified and compared across seizure stages. The resulting data demonstrate that at every stage of seizure development, a majority of SST-synthesizing neurons is activated, but that these activated SST mRNA-containing neurons represent only a minority of all seizure-activated, Fos-expressing neurons in the hippocampal formation. The data further reveal a numerical hierarchy in which the majority of double-labeled neurons is present in the hilus of the dentate, followed by the stratum oriens of CA1. It is concluded that SST-synthesizing neurons represent an integral component of the kindling activated neuronal network and, since the SST synthesizing neurons represent the minority of all seizure-activated neurons in the hippocampal formation, that this neuronal network is likely to be of considerable neurochemical complexity.

Seizure-induced activation of enkephalin- and somatostatin-synthesizing neurons.

The extent of the neuronal network that is activated by kainic acid-induced seizures was anatomically identified and neurochemically characterized. Seizure-activated neurons were identified through the immunocytochemical demonstration of Fos protein in neuronal nuclei. These seizure-activated neurons were characterized by determining if they contained the mRNA for somatostatin or enkephalin, using in situ hybridization procedures. The results demonstrate that a majority of enkephalin- and somatostatin-synthesizing neurons expressed the Fos protein following seizures and that they represent a major component of the kainic acid-induced, seizure-activated neuronal network.

Persistent increases in dopamine D2 receptor mRNA expression in basal ganglia following kindling.

Amygdala kindling resulted in significant increases in the expression of D2 receptor mRNA in the nucleus accumbens and striatum 30 days following the last kindling stimulation. Densitometric analyses of tissue sections incubated in the presence of an oligonucleotide probe directed against D2 receptor cDNA indicated a 20-35% increase in D2 receptor mRNA in these regions following kindling. Kindling from the amygdala followed by piriform cortical kindling in the transfer paradigm (overkindling) resulted in significant further increases in D2 receptor mRNA expression in both the accumbens (150% increase) and striatum (120% increase). There were no observed hemispheric asymmetries in D2 receptor mRNA in either kindled or overkindled animals. The data indicate an enduring upregulation of extrapyramidal D2 receptor mRNA following the kindling process. How this change may relate to kindling-induced alterations in seizure susceptibility or behaviors mediated by limbic dopaminergic pathways are questions for future studies.