Craig D. Applegate

Global Increases in Seizure Susceptibility in Mice Lacking 5-HT2CReceptors: 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.

Differential effects of perinatal hypoxia and anoxia on long term seizure susceptibility in the rat

We have previously demonstrated that hypoxia is acutely epileptogenic in the immature rat but not in the adult. The window during which hypoxia induces seizures in the rat ranges from postnatal day (P) 5-17, with the most severe seizures occurring at P10-12. Perinatal hypoxia resulted in significantly more acute seizure activity than perinatal anoxia. The present study evaluates the long term effects of perinatal hypoxia versus anoxia. Animals were exposed to hypoxia (3%O2) or anoxia (0%O2) at P10 and challenged later in adulthood (P55-60) with administration of pentylenetetrazol (PTZ) (45 mg/kg subcutaneously). Compared to normal littermate controls, the animals which had been exposed to perinatal hypoxia had a significantly higher frequency of generalized convulsions (GC) and a significantly shorter latency to the first myoclonic jerk (MJ) after PTZ. In contrast, perinatal anoxia did not alter long term seizure susceptibility. These results are discussed in context of previous studies which have shown variable long term effects using different models of perinatal hypoxia and/or ischemia.

Kindling antagonism : effects of norepinephrine depletion on kindled seizure suppression after concurrent, alternate stimulation in rats

The concurrent, alternate electrical stimulation of the entorhinal cortex and septal nucleus results in the development of fully generalized seizures at one electrode site and the suppression of seizure development at the other. We have labeled this phenomenon kindling antagonism. Selective, whole-brain depletion of norepinephrine (NE) virtually eliminates the development of kindling antagonism such that fully generalized seizures develop at both sites in a majority of animals. This effect occurs in the absence of appreciable changes in kindling characteristics of these animals compared with either untreated or vehicle-treated controls. These results suggest that the suppression of seizure development observed in the kindling antagonism model is normally maintained by a NE-dependent mechanism. Our results support those of earlier studies using single-site kindling paradigms in which NE depletion facilitates the rate of kindled seizure development. We suggest that the NE-dependent mechanism responsible for the seizure suppression observed to follow concurrent, alternate stimulation and the suppression of seizure development using single-site kindling paradigms may be the same. The nature of this NE-dependent seizure suppression mechanism and the anatomic locus or loci critical for this effect remain questions for future research.

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.

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.

Effects of Valproate, Phenytoin, and MK‐801 in a Novel Model of Epileptogenesis

Summary: 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.

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.

Evidence for the interaction of brainstem systems mediating seizure expression in kindling and electroconvulsive shock seizure models

Amygdala kindling was observed to increase significantly the proportion of rats that exhibited tonic hindlimb extension in response to corneal electroshock stimulation. Mechanical brainstem lesions which abolished electroshock-induced tonic hindlimb extension failed to alter either the expression of fully generalized kindled seizures or the development of amygdala kindled seizures. Results suggest that while kindling can alter the sensitivity of brainstem systems involved in the expression of tonic hindlimb extension, these same systems are not necessary for either the development or expression of amygdala kindled seizures.

Microinjections of GABA agonists into the amygdala complex attenuates kindled seizure expression in the rat

The effects of intraamygdala injections of either gamma-vinyl GABA or muscimol on the behavioral and electrographic expression of stable, fully generalized, kindled seizures were assessed. Results suggest that intraamygdala administration of GABA agonists preferentially attenuates the behavioral, but not the focal, electrographic expression of kindled seizures elicited from either the insular or entorhinal cortex. These results, in conjunction with those of others, suggest that the amygdala becomes an integral and necessary structure for the expression of seizures kindled from a variety of forebrain areas.

Kindling antagonism: a role for hindbrain norepinephrine in the development of site suppression following concurrent, alternate stimulation

The concurrent, alternate electrical stimulation of the septal nucleus and the entorhinal cortex results in the development of fully generalized seizures at one site (dominant site) and the lack of development of kindled seizures at the other (suppressed or antagonized site). We have labeled this phenomenon kindling antagonism. Previous work from our laboratory has demonstrated that the whole brain depletion of norepinephrine (NE) eliminates the development of kindling antagonism. In the present study animals were treated with the neurotoxin 6-hydroxydopamine (6-OHDA) as neonates. The neonatal administration of 6-OHDA produced robust increases in brainstem and cerebellar NE levels and depletions of forebrain NE levels when assayed at maturity. Striatal dopamine levels were spared by this treatment. Neonatal 6-OHDA did not alter the development of the kindling antagonism phenomenon which is typically observed following concurrent, alternate stimulation of the septal nucleus and entorhinal cortex. Neonatal 6-OHDA treatment significantly facilitated the rate of kindled seizure development at dominant sites but failed to alter thresholds for the elicitation of afterdischarges (AD) or patterns of development of AD durations. Other characteristics of kindling antagonism were similarly unaffected by 6-OHDA treatment. These data suggest that brainstem and/or cerebellar NE are sufficient to mediate the development of kindling antagonism in the relative absence of forebrain NE.