Robert L. Macdonald

It's Time to Revise the Definition of Status Epilepticus

Generalized, tonic-clonic status epilepticus is well recognized as a common neurologic emergency requir- ing prompt treatment. The diagnosis is usually not diffi- cult, other than for patients with prolonged seizures, who often develop increasingly subtle clinical features (1,2). There also appears to be a consensus among physicians regarding treatment (3). Nonetheless, there is a major, persistent dilemma regarding status epilepticus: its defi- nition. Discussions concerning the precise definition of status epilepticus all too often result in agreement that current “textbook” definitions are either imprecise, at odds with clinical practice, or both. Here we propose a revised system for defining status epilepticus that ad- dresses these problems. References to status epilepticus prior to the mid- 19th century focused on cases in which seizures lasted many hours to days (4). In 1904, Clark and Prout (5) defined status epilepticus as a state in which seizures occur so frequently that ‘‘the coma and exhaustion are continuous between the seizures.” In his general textbook of neu- rology published in 1940, Kinnier Wilson (6) referred to status epilepticus as the severest form of seizures in which “the post-convulsive sleep of one attack is cut short by development of the next.” Aspects of these definitions were mirrored in the first International Clas- sification of Epileptic Seizures that was developed in 1964 by the International League Against Epilepsy (ILAE). Status epilepticus was defined as the situation in which “a seizure persists for a sufficient length of time or is repeated frequently enough to produce a fixed and enduring epileptic condition” (7). The same definition was retained in the revised classification published in 1970 (8), and it was modified slightly in 1981 to refer to the situation in which “a seizure persists for a sufficient length of time or is repeated frequently enough that re- covery between attacks does not occur” (9).

Antiepileptic Drug Mechanisms of Action

Summary: Clinically used antiepileptic drugs (AEDs) decrease membrane excitability by interacting with ion channels or neurotransmitter receptors. Currently available AEDs appear to act on sodium channels, GABAA receptors, or calcium channels. Phenytoin, carbamazepine, and possibly valproate (VPA) decrease high‐frequency repetitive firing of action potentials by enhancing sodium channel inactivation. Benzodiazepines and barbiturates enhance GABAA receptor‐mediated inhibition. Ethosuximide and possibly VPA reduce a low‐threshold calcium current. The mechanisms of action of AEDs currently under development are less clear. Lamotrigine may decrease sustained high‐frequency repetitive firing. The mechanisms of action of felbamate are unknown. Gabapentin (GBP) appears to bind to a specific binding site in the central nervous system with a restricted regional distribution, but the identity of the binding site and the mechanism of action of GBP remain uncertain.

Extrasynaptic GABAA Receptors: Form, Pharmacology, and Function

GABA is the principal inhibitory neurotransmitter in the CNS and acts via GABAA and GABAB receptors. Recently, a novel form of GABAA receptor-mediated inhibition, termed “tonic” inhibition, has been described. Whereas synaptic GABAA receptors underlie classical “phasic” GABAA receptor-mediated inhibition (inhibitory postsynaptic currents), tonic GABAA receptor-mediated inhibition results from the activation of extrasynaptic receptors by low concentrations of ambient GABA. Extrasynaptic GABAA receptors are composed of receptor subunits that convey biophysical properties ideally suited to the generation of persistent inhibition and are pharmacologically and functionally distinct from their synaptic counterparts. This mini-symposium review highlights ongoing work examining the properties of recombinant and native extrasynaptic GABAA receptors and their preferential targeting by endogenous and clinically relevant agents. In addition, it emphasizes the important role of extrasynaptic GABAA receptors in GABAergic inhibition throughout the CNS and identifies them as a major player in both physiological and pathophysiological processes.

GABAA receptor subunit γ2 and δ subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts

1 To determine their contributions to the rapid kinetic properties of GABAA receptor (GABAR) currents, α1 and β3 subunit subtypes without or with δ or γ2L subtypes were transiently coexpressed in mouse L929 fibroblasts to produce α1β3, α1β3δ, or α1β3γ2L GABAR isoforms. 2 Brief (2–3 ms) applications of 1 mM GABA to outside‐out membrane patches containing α1β3, α1β3δ, or α1β3γ2L isoforms elicited currents that activated rapidly with monophasic time courses and deactivated rapidly with biphasic time courses. α1β3γ2L currents exhibited a slower mean deactivation rate (76.1 ms) than α1β3 (34.1 ms) or α1β3δ currents (42.8 ms). 3 During 1 mM GABA applications, α1β3γ2L currents activated more rapidly (0.46 ms) than α1β3 currents (1.7 ms) or α1β3δ currents (2.4 ms). During 4000 ms GABA applications, α1β3 and α1β3γ2L currents desensitized with triphasic time courses to similar extents (α1β3, 94.6%; α1β3γ2L, 92.4%) and with similar mean rates (α1β3, 352 ms; α1β3γ2L, 462 ms). In contrast, α1β3δ currents desensitized only 55.6% with a biphasic time course and slower mean rate (1260 ms). 4 These experiments demonstrated that the α1β3 heterodimer formed a GABAR channel with rapid deactivation and rapid and nearly complete desensitization. Addition of the δ subunit did not alter the activation rate, but produced a receptor with slower and less complete desensitization. Addition of the γ2L subtype increased activation rate, prolonged deactivation and changed the pattern of rapid desensitization. 5 Rapid kinetic and steady‐state single‐channel data were used to construct kinetic models that predicted the behaviour of the α1β3γ2L and α1β3δ currents. These models represent a reconciliation of macroscopic and steady‐state single‐channel data for GABARs and provide a framework for systematically assessing the functional significance of different GABAR isoforms.

Neurosteroid regulation of GABAA receptor single-channel kinetic properties of mouse spinal cord neurons in culture.

1. Single‐channel kinetics of steroid enhancement of single gamma‐aminobutyric acidA (GABA) receptor currents obtained from somata of mouse spinal cord neurones in culture were investigated using the excised outside‐out patch‐clamp recording technique. GABA (2 microM) and GABA (2 microM) plus androsterone (5 alpha‐androstan‐3 alpha‐ol‐17‐one, AND, 10 nM‐10 microM) or pregnanolone (5 beta‐pregnan‐3 alpha‐ol‐20‐one, PRE, 100 nM‐10 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage clamped at ‐75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were increased in the presence of the steroids. 2. GABA receptor currents were recorded with at least two conductance levels, a predominant or main‐conductance level of about 28 pS (which contributed 96% of the current evoked) and a minor or sub‐conductance level of about 20 pS. The current amplitudes of the two conductance levels were unchanged by the steroids. The gating (opening and closing) kinetics of both of the conductance levels were analysed. Findings for the main‐conductance level are summarized below. 3. Both steroids increased the average GABA receptor channel open duration. Consistent with the increased GABA receptor channel average open duration, the steroids shifted frequency histograms of GABA receptor channel open durations to longer durations. Three exponential functions were required to fit best the frequency histograms of GABA open durations, consistent with at least three kinetic open states of the main‐conductance level. Time constants obtained from the GABA receptor channel open‐duration frequency histograms were unchanged in the presence of the steroids. The basis for the increased average GABA receptor channel open durations by the steroids was due to an increased relative proportion of the two longer open‐duration time constants. The GABA receptor channel average open durations were increased by AND and PRE in a concentration‐dependent manner by shifting the proportion of openings to the longer open time constants. At a concentration of 10 microM, the prolongation of the average open duration was decreased, suggesting that the GABA receptor channel was blocked by these steroids. 4. GABA receptor channel opening frequency was increased and average channel‐closed duration was decreased by AND or PRE. Consistent with this, areas of the frequency histograms of channel closed durations were shifted to shorter durations. Closed frequency distributions were fitted best with five to six exponential functions, suggesting that the channel had multiple kinetic closed states. The three briefest time constants were not greatly altered by the steroids.(ABSTRACT TRUNCATED AT 400 WORDS)

Pentylenetetrazol and penicillin are selective antagonists of GABA-mediated post-synaptic inhibition in cultured mammalian neurones

PENTYLENETETRAZOL (PTZ) and penicillin (PCN) are potent convulsants commonly used to produce focal or generalised epileptiform discharges in the mammalian central nervous system1. Investigations of the cellular basis for their con-vulsant effects have been performed in various vertebrate and invertebrate preparations wtih two basic hypotheses dominating the literature: (1) that the convulsant effect is produced by pharmacological actions on synaptic transmissions to reduce inhibition2–8 and/or to enhance excitation9, or (2) that the convulsants directly enhance the excitability of neuronal membranes apart from effects on synaptic transmission10–16. We have studied the mechanism of their convulsant action in a mammalian tissue culture system and report here that both PTZ and PCN selectively antagonise post-synaptic responses to γ-aminobutyric acid (GABA), a putative inhibitory neurotransmitter17. The results presented here provide direct evidence derived from the mammalian nervous system that the convulsant action of PTZ and PCN may be due ito a specific pharmacological action on GABA-mediated inhibitory synaptic transmission.

Cyclic AMP-dependent protein kinase decreases GABAA receptor current in mouse spinal neurons

GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.

Kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture.

1. The kinetic properties of the main conductance state of gamma‐aminobutyric acidA (GABA) receptor channels from somata of mouse spinal cord neurones in cell culture were investigated using patch clamp techniques. 2. Whole‐cell GABA receptor currents increased in a concentration‐dependent manner from 0.5 to 5 microM. 3. Single‐channel currents were recorded with a main conductance state of 27.2 pS and a less frequent conductance state of 15.9 pS. The main conductance state opened singly and in bursts of several openings. 4. Mean open times of GABA receptor main conductance currents were increased and open‐time frequency histograms were shifted to longer times as GABA concentration was increased from 0.5 to 5 microM. Three exponential functions were required to fit the histograms at all GABA concentrations, suggesting that the channel opened into at least three open states (O1, O2 and O3). The three functions had the same time constants (1.0 +/‐ 0.2, 3.7 +/‐ 0.4 and 11.3 +/‐ 0.5 ms; mean +/‐ S.D.) at each concentration. The increase in long open times with concentration was due to a shift in relative frequency of occurrence of openings from the shortest (O1) to the two longest (O2 and O3) open states. 5. Closed‐time distributions of closures between main conductance state openings were fitted with multiple exponential functions, suggesting that the channel had several closed states. The two shortest time constants (0.24 +/‐ 0.03 and 2.0 +/‐ 0.3 ms) were concentration independent (0.5 to 5 microM). Three longer time constants decreased as concentration increased. 6. Bursts were defined as groups of openings surrounded by closures greater than a critical closed time (tc = 5 ms). Mean burst durations were increased and burst duration frequency histograms were shifted to longer times as GABA concentration was increased from 0.5 to 5 microM. Burst‐duration frequency histograms were best fitted with three exponential functions. The time constants were concentration independent and were 1.0 +/‐ 0.2, 5.5 +/‐ 0.2 and 29.8 +/‐ 1.6 ms. The increase in burst duration with concentration was due to a relative shift from short duration bursts to longer duration bursts. 7. The shortest burst time constant was similar to the shortest open time constant suggesting that there was a population of single openings of short duration. The two longest burst time constants were longer than the two longest open time constants, suggesting that the bursts from the two longest burst components were composed of two or more openings.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzodiazepine and beta‐carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture.

1. The effects of the benzodiazepine receptor agonist, diazepam (DZ), and the inverse agonist, methyl‐6,7‐dimethoxy‐4‐ethyl‐beta‐carboline‐3‐carboxylate (DMCM), on gamma‐aminobutyric acid (GABAA) receptor single channel currents were characterized. Outside‐out patches were obtained from somata of cultured mouse spinal cord neurones and voltage clamped at ‐75 mV (ECl = 0 mV). 2. GABA (2 microM) alone or with DZ (20‐1000 nM) or DMCM (20‐100 nM) was applied to patches by pressure ejection from blunt micropipettes. DZ enhanced GABAA receptor currents with an inverted U‐shaped concentration‐response curve. Mean steady‐state currents were increased by low concentrations of DZ (20‐50 nM). At higher concentrations of DZ, the enhancement was diminished. Mean steady‐state currents were decreased by DMCM at all concentrations. 3. GABAA receptor channels opened most frequently to a 27 pS main conductance level and less frequently to a 19 pS subconductance level. Neither DZ nor DMCM altered the proportion of time spent at either of the conductance levels. The kinetic properties of the main conductance level were studied. 4. Neither DZ nor DMCM altered the mean GABAA receptor channel open or burst durations. Sums of three exponential functions were required to fit best open and burst duration‐frequency histograms for GABA alone or with DZ or DMCM. No significant changes in the three time constants or areas of the three exponential functions for open or burst duration histograms were produced by DZ or DMCM. 5. With increasing concentrations of DZ up to 50 nM, GABA evoked an increased frequency of channel openings and bursts. With higher DZ concentrations, the magnitudes of the increase in channel opening and burst frequencies were reduced. At all concentrations of DMCM, GABA evoked a decreased frequency of channel openings and bursts. 6. Closed duration‐frequency histograms for GABA alone or with DZ or DMCM were best fitted by sums of at least six exponential functions. The three shortest closed duration time constants were unchanged by DZ or DMCM. The three longest closed duration time constants were altered by DZ and DMCM, consistent with alterations in opening frequency. 7. DZ increased and DMCM decreased steady‐state GABAA receptor current by increasing or decreasing channel opening frequency without altering mean channel open duration. We propose that DZ and DMCM alter GABAA receptor current by acting reciprocally to increase or decrease only, respectively, the apparent agonist association rate at the first of two proposed GABA binding steps without altering channel gating.(ABSTRACT TRUNCATED AT 400 WORDS)

Barbiturate regulation of kinetic properties of the GABAA receptor channel of mouse spinal neurones in culture.

1. Barbiturate regulation of the kinetic properties of gamma‐aminobutyric acidA (GABA) receptor channel chloride currents from somata of mouse spinal cord neurones were investigated using whole‐cell and excised outside‐out patch‐clamp recording techniques. 2. GABA (2 microM), GABA (2 microM) plus phenobarbitone (PhB) (500 microM) and GABA (2 microM) plus pentobarbitone (PB) (50 microM), applied by pressure ejection from blunt perfusion micropipettes, evoked inward chloride currents when neurones or patches were voltage clamped at ‐75 mV and the chloride equilibrium potential was 0 mV. GABA receptor channel currents were increased by PhB and PB. 3. Single GABA receptor channel currents were recorded with a main conductance state of 27 pS and a less frequent subconductance state of 16.5 pS. The conductances of the two states were unchanged by the barbiturates. 4. The main conductance state kinetics were analysed. GABA alone or with the barbiturates gated the channel open singly and in groups of openings. 5. The barbiturates increased GABA receptor channel mean open time and shifted frequency histograms of channel open times to longer times. 6. Three exponential functions were required to fit the frequency histograms of GABA receptor channel open times, suggesting that the channel has at least three open states (O1, O2, O3). The time constants for the exponential functions (0.9, 2.7 and 7.8 ms, respectively) were unchanged by the barbiturates. The increases in mean open times and the shifts of the open‐time frequency histograms by the barbiturates were due to a reduction in relative frequency of occurrence of the two short open states (O1 and O2) and to an increase in the relative frequency of occurrence of the longest open state (O3). 7. Frequency histograms of GABA receptor channel closed times were fitted with five exponential functions, suggesting that the channel has multiple closed states. None of the time constants nor areas of the exponential functions were significantly changed by the barbiturates. 8. For analysis, a burst was defined as openings surrounded by closures greater than a critical closed time, tc, of 5 ms. For GABA (2 microM), frequency histograms of GABA receptor channel bursts were fitted with three exponential functions, suggesting that the channel has three burst states (B1, B2, B3). The B1 burst state was probably a single opening to the O1 open state while the B2 and B3 burst states were probably composed of multiple openings to the O2 and O3 open states.(ABSTRACT TRUNCATED AT 400 WORDS)