Katherine D. Holland

Identification of a Median Thalamic System Regulating Seizures and Arousal

Summary: This study better defines the way in which the thalamus controls expression of experimental generalized seizures. The effects of small intrathalamic injections of the direct GABA agonist muscimol on the thresholds of pentylenetetrazol (PTZ)‐induced seizures and on spontaneous behavior were determined in the rat and compared with the effects of injections of γ‐vinyl‐GABA (GVG), an irreversible inhibitor of GABA transaminase. Muscimol injections produced neuronal inhibition in a relatively small area of thalamus, whereas GVG injections produced inhibition in a much larger area. Muscimol injections in the midline thalamus in the vicinity of the paraventricular, paratenial, interanteromedial, intermediodorsal, and central medial nuclei facilitated PTZ myoclonic and clonic seizures and also produced sedation. These effects on seizure thresholds were attributable both to a lower PTZ threshold dose for initiation of electroencephalographic (EEG) seizure activity and to an increased probability of this EEG activity being expressed as behavioral seizures. Midline injections located more posteriorly in the thalamus also inhibited tonic seizures. Muscimol injections placed laterally, dorsally, or ventrally to this midline thalamic region had much less effect on behavior or seizures. In contrast, GVG injections in the anterior medial thalamus elevated the threshold for all PTZ seizure types and for associated EEG seizure activity but had little effect on spontaneous behavior. These findings demonstrate the existence of an important seizure regulatory system in the midline of the thalamus and a direct anatomic link between the mechanisms for regulating arousal and seizure production which may help explain the association between sleep and seizure facilitation in humans. The results of this study also show that inhibition of discrete thalamic regions modifies seizure expression, but that simultaneous inhibition of large portions of the thalamus is required for a general antiepileptic effect.

Relative anticonvulsant effects of GABAmimetic and GABA modulatory agents.

Summary: Anticonvulsant properties of compounds that enhance GABA‐mediated inhibition through modulatory sites on the GABAA receptor [phenobarbital (PB), clonazepam (CZP), α‐ethyl‐α‐methyl‐γ‐thiobutyrolactone (α‐EMTBL)] were compared with anticonvulsant effects of compounds believed to be antagonists at these modulatory sites (Ro 15‐ 1788 and a‐isopropyl‐α‐methyl‐γ‐butyrolactone γ‐IMGBL)] and to 4,5,6,7‐tetrahy‐droisoxazolo‐[4,5‐c]‐pyridin‐3‐ol (THIP, GABAA receptor agonist), (±) baclofen (GABAB receptor agonist), and γ‐vinyl GABA, a compound that increases endogenous GABA. The compounds were tested for their ability to block experimental seizures caused by maximal electroshock, pentylenetetrazol, picrotoxin, methyl‐6,7‐dirnethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM), bicuculline (BIC), aminophylline, strychnine, and τ‐butyl‐bicyclophosphorothionate (TBPS) in mice. CZP blocked all but strychnine seizures. PB was also highly effective, blocking all but TBPS seizures. α‐EMTBL, representing a new class of experimental anticonvulsant drugs, prevented all seizures except strychnine (STR)‐ and aminophylline‐induced seizures. The antagonists are effective only against one convulsant stimulus. Rol S‐1788 and α‐IMGBL prevented only DMCM‐ and pentylenetetrazol (PTZ)‐induced seizures, respectively. THIP and γ‐vinyl GABA both blocked only BIC and picrotoxin seizures. Baclofen had no anticonvulsant activity. These data demonstrate that compounds that increase neuronal inhibition by potentiating the action of GABA have a broader spectrum of anticonvulsant action than either antagonists or GABAmimetic agents or compounds that increase endogenous GABA.

Structure-activity studies of fluoroaljyl-substituted γ-butyrolactone and γ-thiobutyrolactone modulators of GABAA receptor function

Dihydro-2(3H)-furanones (γ-butyrolactones) and dihydro-2(3H)-thiophenones (γ-thiobutyrolactones) containing fluoroalkyl groups at positions C-3, C-4, and C-5 of the heterocyclic rings were prepared. The anticonvulsant/convulsant activities of the compounds were evaluated in mice. Brain concentrations of the compounds were determined and the effects of the compounds on [35S]-tert-butylbicyclophosphorothionate ([35S]TBPS) binding to the picrotoxin site on GABAA receptors were investigated. The effects of the compounds on GABAA receptor function were studied using electrophysiological methods and cultured rat hippocampal neurons. Fluorination at C-3 results in either subtle or pronounced effects on the pharmacological activity of the compounds. When hydrogen are replaced with fluorines at the methylene carbon of an ethyl groups, as in 3-(1,1-difluoroethyl)dihydro-3-methyl-2(3H)-furanone (1), the anticonvulsant actions of the compound are not much changed from those found for the corresponding alkyl-substituted analogue. In marked contrast, fluorination and the methyl carbon of the ethyl group, as in dihydro-3-methyl-3-(2,2,2-trifluoroethyl)-2(3H)-furanone (3), produces a compound having convulsant activity. This convulsant activity seems to be due to an increased affinity of the compound for the picrotoxin site on GABAA receptors caused by an interaction that involves the trifluoromethyl group. Results obtained with γ-butyrolactones containing either a 3-(1-trifluoromethyl)ethyl or a 3-(1-methyl-1-trifluoromethyl)ethyl substitutent indicate that the interactions of the trifluoromethyl group with the picrotoxin binding site are subject to both stereochemical and steric constraints. Sulfur for oxygen heteroatom substitution, as in the corresponding γ-thiobutyrolactones, affects the type (competitive, non-competitive, etc.) of binding interactions that these compounds have with the picrotoxin site in a complex manner. Fluorination of alkyl groups at the C-4 and C-5 positions of γ-butyrolactones having convulsant activity increases convulsant potency.

Alkyl-substituted γ-butyrolactones act at a distinct site allosterically linked to the TBPS/picrotoxinin site on the GABAA receptor complex

Effects of alkyl-substituted gamma-butyrolactones and gamma-thiobutyrolactones on [35S]t-butylbicyclophosphorothionate (35S-TBPS) dissociation from the picrotoxinin receptor were studied. Unlike picrotoxinin, these lactones accelerated the dissociation rate of 35S-TBPS. Thus, previous reports that these lactones change the Kd but not the Bmax of 35S-TBPS in equilibrium binding experiments is explained not by competitive inhibition, but by an allosteric interaction with the 35S-TBPS binding site. These results indicate that modulatory effects of alkyl-substituted gamma-butyrolactones may result from their action at a distinct site on the gamma-aminobutyric acid (GABA)A receptor.

Comparison of the Anticonvulsant Activities of Ethosuximide, Valproate, and a New Anticonvulsant, Thiobutyrolactone

Summary: Anticonvulsant properties of α‐ethyl‐α‐methyl–γ‐thiobutyrolactone (α‐EMTBL) were compared with those of the antiepileptic drugs ethosuximide (ESM) and valproate (VPA) by testing their ability to block seizures in mice caused by maximal electroshock (MES), pentylenetetrazol (PTZ), picrotoxin (PICRO), bicuculline (BIC), methyl‐6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM), N‐methyl‐D,L‐aspartate (NMDA), aminophylline (AMPH), strychnine (STR), β‐ethyl‐β‐methyl‐γ‐thiobutyrolactone (P‐EMTBL), and t‐butylbicyclophosphorothionate (TBPS). ESM was able to prevent PTZ‐, PICRO‐, DMCM‐, and β‐EMTBL‐induced seizures. In contrast, VPA and α‐EMTBL blocked all of these plus MES‐, TBPS‐, and BIC‐induced convulsions. Only VPA prevented AMPH‐induced seizures. None of the anticonvulsants blocked STR or NMD A seizures. Rotorod testing for acute neurotoxicity demonstrated that ESM was the least toxic and α‐EMTBL and VPA were equivalent. Animals treated daily with high doses of α‐EMTBL for a 2‐week period appeared healthier and had a higher survival rate than animals treated with VPA in the same manner. After a single intraperitoneal (i.p.) injection, the duration of anticonvulsant action of α‐EMTBL was 1.3 and 4 times longer than that of ESM and VPA, respectively. These results indicate that α‐EMTBL has a wide spectrum of anticonvulsant action like VPA but may be less toxic and longer acting. We suggest that α‐EMTBL is a compound worthy of further testing and development as an antiepileptic drug (AED).

Physiological modulation of the GABA receptor by convulsant and anticonvulsant barbiturates in cultured rat hippocampal neurons

The actions of convulsant and sedative barbiturates on responses to gamma-aminobutyric acid (GABA) application and on inhibitory postsynaptic currents were compared using voltage-clamp techniques in cultured rat hippocampal neurons. The convulsant barbiturates, 5-ethyl-5-(3-methylbut-2-enyl) barbituric acid (3M2B), and (+)-5-ethyl-5-(1,3-dimethylbutyl) barbituric acid [+)-DMBB), and the sedative barbiturate, 5-ethyl-5-(3-methylbutyl) barbituric acid (3MB), all potentiated GABA-mediated chloride currents. In addition, these compounds prolonged the duration of GABAergic inhibitory postsynaptic currents. The similarity between the action of convulsant and sedative barbiturates suggests that the convulsant activity of 3M2B and (+)-DMBB are not mediated by their actions at GABAergic synapses.