P.C. Jobe

Serotonergic abnormalities in the central nervous system of seizure-naive genetically epilepsy-prone rats

Seizure predisposition in Genetically Epilepsy-Prone Rats (GEPRs) is characterized by abnormal sensitivity to a number of seizure provoking stimuli. The GEPR model is composed of two independently derived colonies with each exhibiting a characteristic convulsive pattern. In response to a standardized sound stimulus, GEPR-3s exhibit moderate or clonic convulsions while GEPR-9s exhibit more severe tonic extensor convulsions. In order to further characterize the neurochemical abnormalities that underlie seizure predisposition in GEPRs, the current study examined serotonin concentrations in 14 discrete brain areas of controls, GEPR-3s and GEPR-9s. In all areas examined, serotonin concentrations were lower in either one or both GEPR types than in seizure resistant controls. In 6 of the 14 areas both GEPR-3s and GEPR-9s had levels significantly lower than controls. In an additional 7 areas GEPRs had serotonin concentrations of similar magnitude which were significantly lower than control when the GEPR values were combined. In cerebellum, GEPR-3s had significantly lower serotonin concentration than either controls of GEPR-9s while in the striatum, GEPR-9s had significantly lower serotonin levels than either GEPR-3s or controls. In summary, GEPRs have widespread deficits in serotonin concentration and that these abnormalities appear to contribute to the seizure predisposition that characterizes these animals.

Comparative fos immunoreactivity in the brain after forebrain, brainstem, or combined seizures induced by electroshock, pentylenetetrazol, focally induced and audiogenic seizures in rats

To help discern sites of focal activation during seizures of different phenotype, the numbers of Fos immunoreactive (FI) neurons in specific brain regions were analyzed following brainstem-evoked, forebrain-evoked and forebrain/brainstem combination seizures induced by a variety of methods. First, pentylenetetrazol (PTZ, 50 mg/kg) induced forebrain-type seizures in some rats, or forebrain seizures that progressed to tonic/clonic brainstem-type seizures in other rats. Second, minimal electroshock induced forebrain seizures whereas maximal electroshock (MES) induced tonic brainstem-type seizures in rats. Third, forebrain seizures were induced in genetically epilepsy-prone rats (GEPRs) by microinfusion of bicuculline into the area tempestas (AT), while brainstem seizures in GEPRs were induced by audiogenic stimulation. A final set was included in which AT bicuculline-induced forebrain seizures in GEPRs were transiently interrupted by audiogenic seizures (AGS) in the same animals. These animals exhibited a sequence combination of forebrain clonic seizure, brainstem tonic seizure and back to forebrain clonic seizures. Irrespective of the methods of induction, clonic forebrain- and tonic/clonic brainstem-type seizures were associated with considerable Fos immunoreactivity in several forebrain structures. Tonic/clonic brainstem seizures, irrespective of the methods of induction, were also associated with FI in consistent brainstem regions. Thus, based on Fos numerical densities (FND, numbers of Fos-stained profiles), forebrain structures appear to be highly activated during both forebrain and brainstem seizures; however, facial and forelimb clonus characteristic of forebrain seizures are not observable during a brainstem seizure. This observation suggests that forebrain-seizure behaviors may be behaviorally masked during the more severe tonic brainstem seizures induced either by MES, PTZ or AGS in GEPRs. This suggestion was corroborated using the sequential seizure paradigm. Similar to findings using MES and PTZ, forebrain regions activated by AT bicuculline were similar to those activated by AGS in the GEPR. However, in the combination seizure group, those areas that showed increased FND in the forebrain showed even greater FND in the combination trial. Likewise, those areas of the brainstem showing FI in the AGS model, showed an even greater effect in the combination paradigm. Finally, the medial amygdala, ventral hypothalamus and cortices of the inferior colliculi showed markedly increased FND that appeared dependent upon activation of both forebrain and brainstem seizure activity in the same animal. These findings suggest these latter areas may be transitional areas between forebrain and brainstem seizure interactions. Collectively, these data illustrate a generally consistent pattern of forebrain Fos staining associated with forebrain-type seizures and a consistent pattern of brainstem Fos staining associated with brainstem-type seizures. Additionally, these data are consistent with a notion that separate seizure circuitries in the forebrain and brainstem mutually interact to facilitate one another, possibly through involvement of specific transition mediating nuclei.

Electroshock- and pentylenetetrazol-induced seizures in genetically epilepsy-prone rats (GEPRs): differences in threshold and pattern

Using facial and forelimb (F&F) clonus (a proposed forebrain marker) and running-bouncing (R/B) clonus and tonus (proposed brain-stem markers), the responsiveness of forebrain and brain-stem to electroshock or pentylenetetrazol seizures was assessed in GEPRs. The most striking finding was the failure of GEPR-9s to display F&F clonus in response to transcorneal electroshock at any stimulus intensity. Indeed, GEPR-9s displayed only R/B clonus or tonus indicative of brain-stem seizure discharge. GEPR-3s and normal rats, on the other hand, displayed F&F clonus in response to the least effective electroshock stimulus, and R/B clonus and tonus at higher stimulus intensities. After treatment with phenytoin (50 mg/kg) to inhibit the tonic seizure, the least effective electroshock stimulus also produced F&F clonus in GEPR-9s. These findings suggest that the threshold for triggering brain-stem seizure discharge by electroshock is lower than that for triggering forebrain seizure discharge in GEPR-9s, whereas the reverse relationship is true in normal rats and GEPR-3s. The rank ordering of the electroshock thresholds was: normals greater than GEPR-3s greater than GEPR-9s. Both GEPR-3s and GEPR-9s were found to be hyper-responsive to pentylenetetrazol as evidenced by shorter latency for the tonic seizure and a greater seizure severity than normal rats. The rank ordering of seizure severity in response to pentylenetetrazol was: GEPR-9 greater than GEPR-3 greater than normal rats.

Noradrenergic Abnormalities in the Central Nervous System of Seizure‐Naive Genetically Epilepsy‐Prone Rats

Summary: Norepinephrine (NE) concentrations were measured in 15 discrete areas of the central nervous system of two types of genetically epilepsy‐prone rats (GEPRs) and in nonepileptic controls. Both moderate‐seizure (GEPR‐3) and severe‐seizure (GEPR‐9) animals had extensive abnormalities in brain NE concentration. Deficits of equal magnitude in GEPR‐3 s and GEPR‐9s were found in the spinal cord, midbrain minus the inferior colliculus, inferior colliculus, hypothalamus, amygdala, hippocampus, occipital + parietal cortex, frontal cortex, and olfactory septum. Because both types of GEPRs share these deficits and share seizure susceptibility, we hypothesize that these areas are candidates for regulation of seizure susceptibility in GEPRs. In addition, because GEPR‐9s have more severe seizures than GEPR‐3s and because GEPR‐9s had greater NE deficits in several brain areas (cerebellum, pons‐medulla, thalamus, and possibly the temporal cortex and olfactory bulbs), we hypothesize that these areas may be important in regulation of seizure severity in GEPRs. All animals used in these experiments had been protected from seizure‐provoking stimuli and were naive to seizures. Because the abnormalities in NE concentration were present in seizure‐predisposed animals that were protected from seizures, we conclude that these abnormalities are important components of the seizure‐predisposition characteristic of GEPRs and do not result from seizure experience.

Effect of precollicular transection on audiogenic seizures in genetically epilepsy-prone rats

Previous studies have demonstrated that generalized tonic-clonic seizures (GTCS) consisting of running/bouncing clonic and tonic extension can still be elicited in rats after brain transections which separate forebrain from brain stem, showing that forebrain circuitry is not required for GTCS. Inasmuch as sound-induced generalized tonic-clonic seizures in rodents are characterized by running-bouncing clonic and tonic convulsions, we have hypothesized that these are brain stem seizures that can occur independently of the forebrain. To test this hypothesis, we examined the response of two strains of genetically epilepsy-prone rats (GEPR-3s and GEPR-9s) to seizure-evoking auditory stimuli 3 h after a precollicular transection or sham surgery performed under ether anesthesia. In addition, the effect of a precollicular transection on audiogenic seizures was evaluated in normal rats made susceptible to such seizures by infusing NMDA into the inferior colliculus. Following the transection 58% of GEPR-9s displayed a sound-induced tonic-clonic convulsion and the remaining 42% exhibited a sound-induced seizure when subjected to stimulation 5 min after a subconvulsant dose of pentylenetetrazol (PTZ). While sham surgery and the precollicular transection both reduced sound-induced seizure severity in GEPR-3s, the full seizure response could be elicited by sound stimulation following a subconvulsant dose of PTZ. Moreover, the audiogenic seizures in normal rats rendered susceptible by NMDA were unaltered by the precollicular transection. These findings show that the anatomical circuitry required for generalized tonic-clonic seizures evoked by sound stimulation in rodents resides within the brain stem.

Noradrenergic mechanisms for the anticonvulsant effects of desipramine and yohimbine in genetically epilepsy-prone rats: studies with microdialysis

A large body of evidence suggests that the seizure-prone state of genetically epilepsy-prone rats (GEPRs) results, in part, from deficits in central nervous system noradrenergic function. In order to link the synaptic concentration of norepinephrine (NE) to seizure behavior, we evaluated the effects of both desipramine and yohimbine on convulsions and on extracellular NE and serotonin (5-HT) concentrations in the thalamus of severe seizure GEPRs (GEPR-9s). Under anesthesia, guide cannulae were stereotaxically placed over thalami. After recovery from surgery, dialysis probes were inserted and the animals were placed individually into a plexiglass chamber where they were allowed to move about freely. Artificial CSF was perfused and samples were collected for analysis on HPLC with electrochemical detection. Either desipramine (10 and 20 mg/kg) or yohimbine (10 mg/kg) was administered i.p. after a stable baseline of NE or 5-HT was established. Significant increases in the extracellular NE concentration were seen after injection of both drugs. Temporal linkage exists between the maximum NE increase and the maximum decrease in audiogenic response score (ARS) for these two agents. No significant increases in the extracellular 5-HT concentration occurred after administration of either desipramine or yohimbine at a dose of 10 mg/kg. We conclude that these two drugs are effective anticonvulsants in GEPRs at least partially because they enhance noradrenergic transmission.

A microdialysis study of amino acid concentrations in the extracellular fluid of the substantia nigra of freely behaving GEPR-9s: relationship to seizure predisposition

Substantia nigra (SN) is known to play an important role in seizure generalization. Both excitatory and inhibitory neurotransmitters can modulate this role of SN. Previous studies have shown that GABA as well as aspartate and glutamate participate in seizure regulation through this site. Evidence for such a role comes from studies on the genetically epilepsy-prone rat (GEPR) and other seizure models. In the GEPR, bilateral microinjections of NMDA receptor antagonists in SN block or reduce seizure severity. In order to further evaluate which neurotransmitters are specifically involved at the SN level of seizure regulation in the GEPR, we undertook a microdialysis study of K+ stimulated release of amino acids in the SN of GEPR-9s- and non-epileptic controls. A 1 mm loop-type microdialysis probe was inserted through pre-implanted guides into the SN of awake and freely moving rats (seven GEPR-9s and four non-epileptic controls), and used to perfuse a 100 mM K+ (high K+) solution for 2 h. Four 30 microliters samples were collected prior to high K+ stimulation (basal release), during high K+ perfusion, and after high K+ infusion. After precolumn derivatization with phenylisothiocyanate, levels of aspartic (ASP) and glutamic (GLU) acids, glycine (GLY), taurine (TAU) and GABA were measured by reversed phase high performance liquid chromatography. Two hours after the initiation of high K+ infusion, the increases relative to basal were, for non-epileptic controls, 35%, 74%, 68%, 847% and 283% respectively for ASP, GLU, GLY, TAU and GABA. Corresponding increases for GEPR-9s were 14%, 10%, 41%, 505% and 123% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acids, monoamines and audiogenic seizures in genetically epilepsy-prone rats: effects of aspartame

It has been suggested that aspartame facilitates seizures in man and animals because phenylalanine, one of its major metabolites, interferes with brain transport of neurotransmitter precursors and alters the synthesis of monoamine neurotransmitters such as norepinephrine, dopamine and/or serotonin. This facilitation is purportedly more likely in subjects predisposed to seizures. One test of this hypothesis would be to administer a wide range of aspartame doses to subjects whose seizure predisposition is dependent on abnormalities in monoaminergic function. Genetically epilepsy-prone rats (GEPRs) have a broadly based seizure predisposition that is based, in part, on widespread central nervous system noradrenergic and serotonergic deficits. Further reductions in the functional state of these neurotransmitters increases seizure severity in GEPRs. Thus, GEPRs appear ideally suited for testing the hypothesis that aspartame facilitates seizures by interfering with central nervous system monoamines. Oral administration of acute (50-2000 mg/kg) or sub-chronic (up to 863 mg/kg/day for 28 days) doses of aspartame did not alter seizure severity in either of two types of GEPRs. Not surprisingly, acute aspartame doses produced dramatic changes in plasma and brain amino acid concentrations. Hypothesized alterations in monoamine neurotransmitter systems were largely absent. Indeed, increases in norepinephrine concentration, rather than the hypothesized decreases, were the most evident alterations in these neurotransmitter systems. We conclude that aspartame does not facilitate seizures in GEPRs and that convincing evidence of seizure facilitation in any species is lacking.

Fetal raphe transplants reduce seizure severity in serotonin-depleted GEPRs.

THIS study investigated whether transplantation of fetal raphe tissue into genetically epilepsy-prone rats (GEPR- 3s) would reduce the severity of seizures previously exacerbated by depletion of brain serotonin. Mild-seizure GEPR-3s were depleted of brain serotonin by 5,7-dihy- droxytryptamine (DHT) and evaluated for seizure severity. Rats then received 15-day fetal raphe tissue, fetal neocortical tissue or were sham grafted. GEPR-3s treated with 5,7-DHT showed increased seizure severity following depletion of serotonin and subsequent reductions in severity as a result of fetal raphe transplantation. Sham- or neocortex-grafted rats maintained elevated seizure severity scores throughout the study. Prominent raphe or cortical grafts were observed within the third ventricle of GEPRs at autopsy. These findings show that transplantation of fetal raphe tissue promotes lasting reductions in increased seizure severity resulting from depletion of serotonin in the GEPR brain.

A comprehensive electrographic and behavioral analysis of generalized tonic-clonic seizures of GEPR-9s

This study records noise-free intracerebral EEG of the genetically epilepsy prone rat (GEPR-9), along with behavioral correlates, during a seizure on unanesthetized freely behaving unrestrained animals. The GEPR-9 exhibits acoustically triggered generalized tonic-clonic seizures, and often times the EEG, recorded with conventional techniques, has resulted in data with imbedded movement artifact. For noise-free video-EEG recordings, we used a previously developed system that consists of a head connector with a FET preamplifier and battery, signal conditioning device (5000x gain, 1 Hz-100 Hz filters), A/D converter and video/PC-PC/video computer boards for recording image data. Each animal was implanted with three monopolar/referential electrodes chosen among the following areas: cortex, inferior colliculus, reticular formation and caudal medulla. The video-EEG data were quite similar for all recorded animals: (1) basal desynchronized EEG before sound stimulus; (2) increase in EEG frequency after stimulus and before seizure onset; (3) high-amplitude polyspikes during massive myoclonic thrusts with or without a very fast running episode; (4) an electrodecremental response during tonic extension; (5) wave and spike complex during forelimb and hindlimb tonic rigidity and posttonic clonus; (6) low-amplitude EEG during postictal depression. Time sequenced spectral analysis also highlights the epileptiform EEG pattern during seizure with high reproducibility between animals. While testing seizure naive GEPR-9s, there was a clear evolution from modest epileptiform EEG activity on the first acoustic stimulation to progressively higher amplitude, duration and frequency epileptiform EEG activity throughout seizure repetition.