Thyrotropin-releasing hormone gene expression and receptors are differentially modified in limbic foci by seizures.

Previous studies using two seizure paradigms, electroconvulsive shock and kindling, suggested potential sites of endogenous thyrotropin-releasing hormone (TRH) action in specific epileptogenic areas. We studied TRH gene expression and TRH receptors in rat limbic areas using the kindling model of epilepsy. Immunoassayable TRH increased 4- to 20-fold over control levels in specific subregions of the hippocampus 24 hours after a single stage 5 seizure. Concurrently, TRH receptor binding was significantly reduced in hippocampal (23-39%) and amygdaloid (21-22%) membranes. Dramatic temporal and spatial changes in prepro-TRH messenger RNA were visualized by in situ hybridization histochemistry in the hippocampal dentate gyrus, the piriform cortex, and the amygdala. Peak hybridization occurred 6 and 12 hours postictally in these loci and returned toward basal levels by 24 hours. These results are consistent with the hypothesis that TRH may have an important role in the pathophysiology epilepsy by modulating excitatory processes.

Estradiol replacement to female rats facilitates dorsal hippocampal but not ventral hippocampal kindled seizure acquisition.

Estradiol replacement facilitates amygdala and neocortical kindling. This study determined if estradiol also interacts with kindling of additional limbic sites, the dorsal (dH) and ventral (vH) hippocampus. During dH stimulations, ovariectomized female rats with estradiol (E) replacement required 29.7 +/- 3.5 trials to kindle and accumulated 1170 +/- 90 s of afterdischarge (AD), significantly less than the 40.6 +/- 3.7 trials and 1620 +/- 225 s in rats without estradiol (nE). E did not significantly alter the long series of partial limbic seizures preceding generalized seizures despite the early appearance of AD in the contralateral amygdala. E significantly advanced the onset of generalized seizures compared to nE (22.7 +/- 2.3 versus 27.9 +/- 3.2 trials), an event coincident with neocortical AD development. Following secondary seizure generalization, E rats rapidly completed late-kindled seizure acquisition. In contrast, nE rats required an almost twofold greater number of AD trials and AD s to complete late kindling compared to E rats. One factor in the slower late kindling of nE rats was the instability of generalized seizures which frequently regressed to focal or partial responses. In marked contrast to dH kindling, vH kindling was uneffected by E replacement. The results provide further experimental evidence for a role for estradiol in catamenial epilepsy and support our previous work suggesting that the focal origin of seizure development is critical to E facilitation of kindling and that secondary generalization of seizures is especially sensitive to estradiol.

Estradiol replacement facilitates the acquisition of seizures kindled from the anterior neocortex in female rats.

Estradiol replacement facilitates kindling from a limbic region, the amygdala. This study determined if estradiol also interacts with kindling from a non-limbic region, the anterior neocortex. Ovariectomized female rats with estradiol replacement required 24 +/- 1.6 trials to kindle and accumulated 434 +/- 28 sec of afterdischarge (AD), significantly less than the 38 +/- 2.2 trials and 840 +/- sec in rats without estradiol. Estradiol replacement did not significantly alter the long series of focal cortical seizures preceding generalized seizures in spite of the early appearance of AD in the contralateral amygdala. Estradiol significantly advanced the onset of generalized seizures compared to rats without estradiol (19 +/- 0.6 versus 24 +/- 1.9 trials). Following secondary seizure generalization, estradiol rats rapidly completed late kindled seizure acquisition. In contrast, late kindling in rats without estradiol was slower as reflected by a 3-fold greater number of AD trials and AD seconds to complete kindling compared to rats with estradiol. One factor in the slower late kindling of rats without estradiol was the instability of generalized seizures which frequently regressed to focal or partial responses. The results provide further experimental evidence for a role for estradiol in catamenial epilepsy and suggest that the process of secondarily generalization of seizures is especially sensitive to estradiol.

Evidence for a chronic loss of inhibition in the hippocampus after kindling: electrophysiological studies.

Rats were kindled with either of 2 protocols: (1) a rapidly recurring hippocampal seizure (RRHS) paradigm in which 10 sec stimulus trains were delivered every 5 min through hippocampal electrodes; and (2) a traditional approach in which 1 sec stimulus trains were given to the amygdala once daily. Three groups of kindled rats were prepared: (1) one of amygdala-kindled rats that had experienced 9-15 seizures; (2) one of RRHS-kindled rats that had experienced 96 seizures; and (3) one of RRHS-overkindled rats that had experienced 144-336 seizures. After a 1 month seizure-free period, the animals were anesthetized with urethane and measurements were made on the potency of paired pulse inhibition in the CA1 region of the hippocampus. All groups of kindled animals were found to have significantly less paired pulse inhibition than control rats. This decrement was confined to interpulse intervals less than or equal to 70 msec. The amount of inhibition lost correlated with the number of seizure that had occurred. The GABAergic agonist muscimol restored paired pulse inhibition in kindled animals for interpulse intervals less than or equal to 70 msec towards normal values. These results indicate that not only RRHS, but also other modes of kindling, reduced GABAergic inhibition in the CA1 region of the hippocampus and that this diminution was long-lasting, if not permanent.

Acquisition of amygdala-kindled seizures in female rats: relationship between the effect of estradiol and intra-amygdaloid electrode location.

Ovariectomized, adult female rats, with or without estradiol replacement, were kindled by daily amygdala stimulation. Kindling acquisition varied with the intra-amygdala site of stimulation. During stimulation of the medial (AME) or central (ACE) nucleus, the only effect of estradiol replacement (E), compared to non-replaced rats (nE), was to significantly decrease the number of trials with afterdischarge (AD) during early kindling (stage 0). In rats receiving stimulation of the cortical nucleus (ACO) or the baso-lateral group of nuclei (ABL), a similar effect of estradiol was extended through stage 1. In addition, nE rats with ACO or ABL electrodes required significantly more trials with AD and accumulated more than twice the sec of AD during the late stages of kindling, compared to E rats and regressed to lower stage responses between the first stage 4 and last stage 5 responses; regressed responses never occurred in E rats. Estradiol also significantly decreased the prekindling AD threshold of the AME and ACE. These results indicate that estradiol accelerates early stage kindling, likely by proconvulsive properties to increase excitability within immediate amygdala projections. During late kindling stages, estradiol may participate in reinforcing or sustaining the convulsive readiness of kindling circuits established during bilateral recruitment. The site of action for this latter effect of estradiol may reside within circuits accessed by stimulation of the ACO or ABL, and not the AME or ACE.

Postictal events in amygdala-kindled female rats with and without estradiol replacement.

This study investigated the effect of estradiol on ictal and postictal events in ovariectomized female rats kindled by daily amygdala stimulation during the presence or absence of estradiol replacement. Although ictal components of kindled seizures were not altered by estradiol, several postictal events such as myoclonic jerks were significantly increased by estradiol. Twelve days after kindling, estradiol-replaced rats with electrodes in the basal, lateral, or cortical amygdala nuclei developed brief postictal "bursts" of behavioral and EEG activity which developed into a secondary seizure in some rats. Rats without estradiol never displayed these postictal events. Pretreatment of rats with pentylenetetrazol, 20 mg/kg, 15 days after kindling, resulted in a further increase in the incidence of bursts and secondary seizures in the presence of estradiol; these events were most prominent in rats kindled in the absence of estradiol. Pentylenetetrazol also caused a significant increase in myoclonic jerks in estradiol-replaced rats, and postictal spikes in all rats. A series of five consecutive generalized seizures at 48-h intervals caused an increase in bursts in rats with estradiol replacement, but not in rats without estradiol. The results indicate that estradiol exerts excitatory effects during the postictal period following amygdala-kindled seizures and suggest that multiple regions mediate the postictal effects of estradiol, perhaps depending on the intraamygdala site of kindling stimulations.

Alterations in beta-adrenergic receptor binding in partially and fully amygdala-kindled juvenile and adult rats.

Twenty-eight-day-old (juvenile) rats were kindled with hourly stimulations to partial or fully kindled status. Adult rats were stimulated with hourly or daily stimulations. Alterations in [3H]dihydroalprenolol binding were determined 3 weeks after the last stimulation. We found that partially kindled, hourly stimulated juvenile rats showed a significant increase in the dissociation constant (Kd), with no change in maximal binding values. Fully kindled juvenile rats showed no change in Kd or Bmax. Partially kindled, hourly stimulated adult rats showed a significant decrease in Kd, with no change in Bmax. There was no change in Kd or Bmax values in fully kindled, hourly stimulated adult rats. Fully kindled, daily stimulated adult rats showed a decrease in maximal binding, with no change in Kd values. These findings indicate that kindling-induced beta-adrenergic receptor alterations were influenced by the age of the animal and the kindling parameters used, as well as the extent to which the animals were kindled.

Status epilepticus facilitated by pilocarpine in amygdala-kindled rats.

A novel model of status epilepticus based on the kindling model of epilepsy is described. The model involves the administration of a small dose of pilocarpine (20 mg/kg) to rats that have been previously kindled. Stimulation of these pretreated rats produces seizures which continue uninterrupted for approximately 4 h before spontaneous termination. The electroencephalographic discharge pattern showed characteristic changes in polarity and amplitude throughout the duration of status epilepticus. Behaviorally, the animals showed motor seizures which varied between stages I through IV, with evidence of extensive bilateral hemispheric involvement through much of the seizure episode. Animals that had been partially kindled to stage II seizures did not develop status epilepticus after stimulation when pretreated with pilocarpine, indicating that prior kindling is integral to the development of status epilepticus in this model. Administration of scopolamine was ineffective in terminating the condition when it had begun, suggesting that cholinergic stimulation is necessary for the initiation, but not the maintenance, of status epilepticus. This model holds promise for the study of status epilepticus because the condition develops in a seizure-prone (kindled) rat, and the seizures are self-sustaining, without the presence of exogenous chemicals or neurotoxins.

Estrogen alters the acquisition of seizures kindled by repeated amygdala stimulation or pentylenetetrazol administration in ovariectomized female rats.

Ovariectomized female rats received estradiol (E2) replacement by means of subcutaneous silastic capsules (10% E2 in cholesterol). E2-replaced rats required fewer daily amygdala stimulations to develop fully kindled seizures as compared with ovariectomized rats implanted with cholesterol-only capsules. Other rats were injected with pentylenetetrazol (PTZ), 40 mg/kg i.p., every 48 h. E2-replaced rats showed a progressive increase in convulsive severity from minimal responses after the first injection to tonic convulsions after eight injections. Most rats without E2 replacement failed to progress past clonic convulsive responses after 22 injections. The results support a marked influence of E2 on seizure processes as demonstrated in two different models of seizure acquisition.

Amygdala kindling in juvenile rats following neonatal administration of 6-hydroxydopamine.

The role of catecholamines in mediating the acquisition of amygdala-kindled seizures was investigated in juvenile rats administered intracisternal injections of 6-hydroxydopamine (6-OHDA) on postnatal days 1 and 2. Amygdala kindling was initiated on day 28, using stimulations delivered each hour through two consecutive stage V seizures. The 6-OHDA treatment resulted in a 53% increase in the overall rate of kindling in juvenile rats. This acceleration was confined primarily to the early phases of kindling in that the 6-OHDA-treated rats skipped the early kindling stages, and the later stages of kindling were unaffected. These findings support evidence from adult rats that catecholamines play a role in initially limiting the spread of seizure activity during kindled seizure acquisition; however, when the seizures have begun to generalize, the ability of catecholaminergic systems to inhibit seizure spread diminishes.

Methylazoxymethanol as a developmental model of neurotoxicity.

Behavioral consequences of exposure to toxic chemicals during development often depend on critical periods of exposure. The present study employed the antimitotic agent methylazoxymethanol (MAM) to interfere with neuron proliferation during specific periods of brain development. Behavioral profiles of neonatal rats were obtained after MAM administration on gestational days 15, 19 (G15 or G19) or postnatal day 1 (PN 1). MAM administration on G15 produced neonates with decreased electroconvulsive shock thresholds (TT50) on postnatal day 8 and increased locomotor activity levels from postnatal day 18 through 29. Treatment on G19 resulted in an increased TT50 and a delay in the developmental locomotor activity curve. Pups treated on PN1 displayed decreased levels of activity on postnatal days 12 through 16 and impaired performance on a rotorod at 29 days of age. MAM treatment on G15 produced decreases in forebrain and cortical mass to 51 and 40% of respective control values. MAM administration on PN1 resulted in small but significant decreases in forebrain, cortex and hindbrain and a 35% decrease in cerebellar size. No changes in regional brain weights were measurable in the G19 treatment group. The results indicate that neonatal behavior can be altered in a relatively specific manner by perturbation of neuronal development at critical time periods and that the functional tests employed clearly distinguish among treatment groups. The findings of this study are interpreted in light of previously described neurochemical and morphological effects of antimitotic treatment.

Convulsive thresholds and severity and the anticonvulsant effect of phenobarbital and phenytoin in adult rats administered 6-hydroxydopamine or 5,7-dihydroxytryptamine during postnatal development.

Rats were administered intracisternal 6-hydroxydopamine (6-OHDA) or 5,7-dihydroxytryptamine (5,7-DHT) within the first three postnatal days, at several ages centered on the third postnatal week or on postnatal day 180. When the rats were 210-days-old, maximal electroshock convulsive thresholds and responses and the anticonvulsant effect of phenobarbital and phenytoin were determined. All 5,7-DHT treatments resulted in an approximate 21% decrease in the tonic convulsive threshold and increased the incidence of tonic hindlimb extension (HLE). Only the 5,7-DHT treatment at 180 days was associated with a more severe HLE response (shortened onset and prolonged duration). All neonatal 6-OHDA treatments were associated with no change in the tonic threshold, but increased the incidence and severity of HLE. The latter effect depended on the postnatal age of 6-OHDA-treatment: treatment at postnatal days 14 and 15 resulted in the greatest increase in severity (52% decrease in onset and 48% increase in duration). The 6-OHDA treatment to 180-day-old rats increased the incidence and duration of HLE but had no influence on the tonic threshold or onset of extension. The effectiveness of both phenobarbital and phenytoin to block HLE was variably decreased by all neurotoxin treatments. The results suggest that interference with the postnatal maturation of monoaminergic influences on seizure processes can have a long-lasting influence on the ability of the brain to limit the generation and spread of seizure activity and on the effectiveness of anticonvulsant drugs.

Activation of liver tryptophan oxygenase by hydrocortisone, hematin and tryptophan in streptozotocin-diabetic rats.

This study compared changes in liver tryptophan oxygenase (TPO) activity in response to hydrocortisone, hematin and tryptophan administration to non-diabetic and diabetic (streptozotocin) rats. Hydrocortisone caused similar increases in apoenzyme (inactive), holoenzyme (heme-saturated) and total (holoenzyme + apoenzyme) TPO activities in non-diabetic and diabetic rats. The ability of hematin to increase total TPO activity was significantly less in diabetic rats. The largest differences between diabetic and non-diabetic rats were found with tryptophan which increased total TPO and holoenzyme activities 300% and 650% respectively in non-diabetic rats. However, tryptophan increased both apoenzyme (unchanged in non-diabetic rats) and holoenzyme activities by 300% in diabetic rats. These results indicate that in the diabetic state, the TPO-heme conjugation process is impaired, especially substrate mediated TPO-heme saturation.

Tonic convulsive thresholds and responses during the postnatal development of rats administered 6-hydroxydopamine or 5,7-dihydroxytryptamine within three days following birth.

The maturation of the electroshock tonic convulsive pattern and threshold was investigated in rats between the ages of 4 and 30 days following intracisternal injections of 6-hydroxydopamine (6-OHDA) on postnatal days 1 and 2; or 5,7-dihydroxytryptamine (5,7-DHT) after desipramine on postnatal day 3. In 6-OHDA treated rats decreases in brain norepinephrine (mean values of 55% of control) and dopamine (mean values of 17% of control) were associated with a large reduction in the convulsive threshold and intensification of the pattern on postnatal day 4. Whereas the reduction in catecholamine concentrations and the intensification of the pattern were still evident on postnatal day 30, the last day of testing, the threshold effect was not evident by postnatal day 15. Although 5,7-DHT reduced brain serotonin concentrations (mean values of 59% of control) as early as postnatal day 4, the pattern was not intensified until postnatal day 8, and the threshold was not reduced until postnatal day 21. These effects were still evident on postnatal day 30. The results demonstrate a sequential maturation of monoaminergic regulation in seizure susceptibility and severity, with an apparent transition from catecholaminergic to serotonergic regulation of the tonic threshold during the third postnatal week.

Effect of streptozotocin-induced diabetes on tryptophan oxygenase activity and brain tryptophan levels in rats.

Alterations in brain tryptophan levels and the rate of hepatic tryptophan metabolism by tryptophan oxygenase (TPO) were studied in male Sprague-Dawley rats rendered diabetic by intravenous administration of streptozotocin (STZ), 65 mg/kg. Determinations were made at the early onset of diabetes (1-4 days of glucosuria) and 8-12 days following STZ injection. Rats were considered diabetic if their serum glucose exceeded 250 mg percent. Tryptophan brain levels decreased by 17% after four days of diabetes, decreased by 22% on day 5, and by 27% 8-12 days after STZ. Brain 5-hydroxyindoleacetic acid levels were significantly decreased by 27% on day 5, but returned to control levels by 8-12 days. Serotonin concentration in the brain remained at control values. The initial appearance of a significant increase in total TPO activity coincided with the onset of a change in brain tryptophan. Total TPO activity increased by 60% after 4 days of diabetes. The increase was caused by an increase in apoenzyme activity since holoenzyme activity remained unaltered. Holoenzyme activity was increased by 37% after 8-12 days, and accounted for the change in total TPO activity. Insulin treatment reversed the STZ-induced alterations. The results are compatible with the hypothesis that diabetes increases hepatic TPO activity that in turn results in decreased plasma tryptophan levels and decreased availability of tryptophan for brain uptake. However, compensatory changes appear to maintain a stable serotonin concentration in the brain. The early and later changes in TPO activity during diabetes are apparently caused by different regulatory events.

Age-dependent reduction in maximum electroshock convulsive threshold associated with decreased concentrations of brain monoamines.

This study investigated modification of the tonic convulsive threshold to maximum electroshock in 15- and 30 day old rats treated with drugs which reduce steady-state concentrations of monoamines. On postnatal day 15, reduction of central catecholamine concentrations by 6-hydroxydopamine or of central serotonin concentrations by 5,7-dihydroxytryptamine or p-chloroamphetamine did not alter the tonic convulsive threshold. However, simultaneous depletion of catecholamines and serotonin by tetrabenazine was associated with a significant decrease in the tonic threshold. This effect could be reversed partially by simultaneous administration of the catecholamine and serotonin precursors, L-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. On postnatal day 30, reduction of brain serotonin concentration, but not catecholamine concentrations, was associated with a significant decrease of the tonic convulsive threshold. In a previous study, in which 7-8 day old rats were used, a tetrabenazine-induced decrease in the tonic convulsive threshold prevented by L-dihydroxyphenylalanine but not 5-hydroxytryptophan. Furthermore, intracisternal 6-hydroxydopamine, but not 5,7-dihydroxyhyptamine, decreased the threshold on postnatal day 8. Therefore, the results of the present day study involving 15- and 30 day old rats, together with the earlier findings in 7-8 day old rats, [28] suggest an apparent developmental transition from catecholaminergic to serotonergic dominance in regulation of the tonic convulsive threshold during the first postnatal month.

Hippocampal seizure discharge produced by systemic digitoxigenin is antagonized by reserpine or p-chlorophenylalanine.

EEG was recorded from several brain regions during the i.v. infusion of digitoxigenin to conscious, freely-moving rats. Two seizure episodes were recorded from the ventral hippocampus with the second, more severe seizure characterized by low-frequency (3-6/sec), high-amplitude (0.9-1.5 mV) discharges. Secondary activity was recorded from the mesencephalic and reticular formations. Pretreatment of rats with p-chlorophenylalanine or reserpine prevented the first seizure and markedly delayed the second seizure to appear after 1.85 mg or 4.66 mg digitoxigenin/kg, respectively, compared to 0.79 mg/kg in untreated rats. The pretreatments did not alter the frequency of the discharges but reduced their amplitude to 0.4 -0.75 mV. The results suggest a role for monoamines, and in particular serotonin, in the effect of digitoxigenin to cause hippocampal seizure discharge.

Regional brain 3H-digitoxigenin and monoamine content during the development of convulsions produced by system digitoxigenin.

The uptake of 3H-digitoxigenin (3H-DIGT), administered by intravenous infusion to conscious, unrestrained rats, increased with time in the medulla-pons, cerebellum, cerebral cortex, corpus striatum, hippocampus, hypothalamus, and midbrain coincident with the development of behavioral and motor disruption. At four minutes of infusion, during clonic convulsions, uptake appeared to follow reported differences in regional blood flow, ranging from 0.48 microgram/gm (hypothalamus) to 0.67 microgram/gm (cerebellum). After 5 1/2 minutes of infusion, rats still in clonic convulsions had no further increase in uptake; rats entering tonic convulsions had two- to three-fold increases in 3H-DIGT uptake, ranging from 1.00 microgram/gm (hypothalamus) to 1.65 microgram/gm (cerebellum). Electroshock-induced tonic convusions at 2 minutes of infusion increased 3H-DIGT uptake two- to three-fold in all regions, suggesting that the increased neuronal activity associated with the tonic convulsion enhanced the uptake of 3H-DIGT into all regions. The only significant monamine alteration was a slightly decreased medulla-pons serotonin level after 5 1/2 minutes of infusion.