Lawrence N. Eisenman

Slow Actions of Neuroactive Steroids at GABAA Receptors

Neuroactive steroids are potent and efficacious modulators of GABAA receptor activity and are potent sedatives and anesthetics. These positive modulators of GABAA receptors both potentiate the actions of GABA at the receptor and, at higher concentrations, directly gate the channel. The contribution of direct gating to the cellular and behavioral effects of neuroactive steroids is considered of little significance because it has been generally found that concentrations well above those needed for anesthesia are required to gate channels. By studying solitary glutamatergic neurons devoid of synaptic GABA input, we show that direct gating occurs and significantly alters membrane excitability at concentrations ≤100 nm. We propose that the relevance of direct gating has been overlooked partly because of the extremely slow kinetics of receptor activation and deactivation. We show that slow deactivation of directly gated currents does not result from an inherently tight ligand-receptor interaction because the slow deactivation is markedly accelerated by γ-cyclodextrin application. We hypothesize that steroids access the relevant GABAA receptor site from a non-aqueous reservoir, likely the plasma membrane, and that it is slow reservoir accumulation and departure that accounts for the slow kinetics of receptor gating by neuroactive steroids.

The sticky issue of neurosteroids and GABAA receptors

Endogenous neurosteroids and their synthetic analogs (neuroactive steroids) are potent modulators of GABA(A) receptors. Thus, they are of physiological and clinical relevance for their ability to modulate inhibitory function in the CNS. Despite their importance, fundamental issues of neurosteroid actions remain unresolved. Recent evidence suggests that glutamatergic principal neurons, rather than glia, are the major sources of neurosteroid synthesis. Other recent studies have identified putative neurosteroid binding sites on GABA(A) receptors. In this Opinion, we argue that neurosteroids require a membranous route of access to transmembrane-domain binding sites within GABA(A) receptors. This has implications for the design of future neuroactive steroids because the lipid solubility and related accessibility properties of the ligand are likely to be key determinants of receptor modulation.

Impaired and facilitated functional networks in temporal lobe epilepsy.

How epilepsy affects brain functional networks remains poorly understood. Here we investigated resting state functional connectivity of the temporal region in temporal lobe epilepsy. Thirty-two patients with unilateral temporal lobe epilepsy underwent resting state blood-oxygenation level dependent functional magnetic resonance imaging. We defined regions of interest a priori focusing on structures involved, either structurally or metabolically, in temporal lobe epilepsy. These structures were identified in each patient based on their individual anatomy. Our principal findings are decreased local and inter-hemispheric functional connectivity and increased intra-hemispheric functional connectivity ipsilateral to the seizure focus compared to normal controls. Specifically, several regions in the affected temporal lobe showed increased functional coupling with the ipsilateral insula and immediately neighboring subcortical regions. Additionally there was significantly decreased functional connectivity between regions in the affected temporal lobe and their contralateral homologous counterparts. Intriguingly, decreased local and inter-hemispheric connectivity was not limited or even maximal for the hippocampus or medial temporal region, which is the typical seizure onset region. Rather it also involved several regions in temporal neo-cortex, while also retaining specificity, with neighboring regions such as the amygdala remaining unaffected. These findings support a view of temporal lobe epilepsy as a disease of a complex functional network, with alterations that extend well beyond the seizure onset area, and the specificity of the observed connectivity changes suggests the possibility of a functional imaging biomarker for temporal lobe epilepsy.

Diverse voltage-sensitive dyes modulate GABAA receptor function

Voltage-sensitive dyes are important tools for assessing network and single-cell excitability, but an untested premise in most cases is that the dyes do not interfere with the parameters (membrane potential, excitability) that they are designed to measure. We found that popular members of several different families of voltage-sensitive dyes modulate GABAA receptor with maximum efficacy and potency similar to clinically used GABAA receptor modulators. Di-4-ANEPPS and DiBAC4(3) potentiated GABA function with micromolar and high nanomolar potency, respectively, and yielded strong maximum effects similar to barbiturates and neurosteroids. Newer blue oxonols had biphasic effects on GABAA receptor function at nanomolar and micromolar concentrations, with maximum potentiation comparable to that of saturating benzodiazepine effects. ANNINE-6 and ANNINE-6plus had no detectable effect on GABAA receptor function. Even dyes with no activity on GABAA receptors at baseline induced photodynamic enhancement of GABAA receptors. The basal effects of dyes were sufficient to prolong IPSCs and to dampen network activity in multielectrode array recordings. Therefore, the dual effects of voltage-sensitive dyes on GABAergic inhibition require caution in dye use for studies of excitability and network activity.

Activation-dependent properties of pregnenolone sulfate inhibition of GABAA receptor-mediated current.

Sulfated steroids like pregnenolone sulfate (PS) are found endogenously in the central nervous system where they may modulate GABAA receptors. Understanding the mechanism of steroid inhibition is important for understanding the conditions under which endogenous steroids modulate GABAA receptor function, assessing their potential clinical utility, and for evaluating sulfated steroids as probes of receptor behaviour. Some previous studies suggest that sulfated steroid inhibition exhibits activation dependence, whilst other studies suggest only slow, time‐dependent inhibition, perhaps reflecting slow PS association with receptors. We tested activation dependence in several ways. Steroid potency increased 2‐ to 3‐fold with ≈10‐fold change in GABA concentration. PS inhibition of saturating partial agonist responses suggested that the level of channel activation, rather than receptor occupancy by agonist, is important for PS inhibition. Inhibition by sulfated steroids exhibited weak or no voltage dependence. Responses to rapid applications of exogenous GABA differed little whether PS was pre‐applied or simply co‐applied with GABA, consistent with the hypothesis that the actions of PS are facilitated by receptor activation. PS applied during steady‐state GABA responses exhibited slow onset and offset rate constants. The offset, rather than onset, was significantly slowed by elevated GABA concentration. At hippocampal synapses, large, multiquantal IPSCs were inhibited more effectively by a fixed concentration of PS than small quantal content IPSCs, consistent with known ‘pooling’ of transmitter following multiquantal release. Picrotoxinin, although superficially similar to PS in its activation dependence, was dissimilar from PS in a number of details. In summary, PS inhibition exhibits activation dependence that may be explained by activation‐dependent binding and altered desensitization.

The Influence of Neuroactive Steroid Lipophilicity on GABAA Receptor Modulation: Evidence for a Low-Affinity Interaction

Anesthetic steroids with actions at gamma-aminobutyric acid type A receptors (GABA(A)Rs) may access transmembrane domain binding site(s) directly from the plasma cell membrane. Accordingly, the effective concentration in lipid phase and the ability of the steroid to meet pharmacophore requirements for activity will both contribute to observed steady-state potency. Furthermore, onset and offset of receptor effects may be rate limited by lipid partitioning. Here we show that several GABA-active steroids, including naturally occurring neurosteroids, of different lipophilicity differ in kinetics and potency at GABA(A)Rs. The hydrophobicity ranking predicted relative potency of GABA(A)R potentiation and predicted current offset kinetics. Kinetic offset differences among steroids were largely eliminated by gamma-cyclodextrin, a scavenger of unbound steroid, suggesting that affinity differences among the analogues are dwarfed by the contributions of nonspecific accumulation. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-tagged fluorescent analogue of the low-lipophilicity alphaxalone (C17-NBD-alphaxalone) exhibited faster nonspecific accumulation and departitioning than those of a fluorescent analogue of the high-lipophilicity (3alpha,5alpha)-3-hydroxypregnan-20-one (C17-NBD-3alpha5alphaA). These differences were paralleled by differences in potentiation of GABA(A)R function. The enantiomer of C17-NBD-3alpha5alphaA, which does not satisfy pharmacophore requirements for steroid potentiation, exhibited identical fluorescence kinetics and distribution to C17-NBD-3alpha5alphaA, but was inactive at GABA(A)Rs. Simple simulations supported our major findings, which suggest that neurosteroid binding affinity is low. Therefore both specific (e.g., fulfilling pharmacophore requirements) and nonspecific (e.g., lipid solubility) properties contribute to the potency and longevity of anesthetic steroid action.

Axonal sodium channel distribution shapes the depolarized action potential threshold of dentate granule neurons.

Intrinsic excitability is a key feature dictating neuronal response to synaptic input. Here we investigate the recent observation that dentate granule neurons exhibit a more depolarized voltage threshold for action potential initiation than CA3 pyramidal neurons. We find no evidence that tonic GABA currents, leak or voltage‐gated potassium conductances, or the expression of sodium channel isoform differences can explain this depolarized threshold. Axonal initial segment voltage‐gated sodium channels, which are dominated by the NaV1.6 isoform in both cell types, distribute more proximally and exhibit lower overall density in granule neurons than in CA3 neurons. To test possible contributions of sodium channel distributions to voltage threshold and to test whether morphological differences participate, we performed simulations of dentate granule neurons and of CA3 pyramidal neurons. These simulations revealed that cell morphology and sodium channel distribution combine to yield the characteristic granule neuron action potential upswing and voltage threshold. Proximal axon sodium channel distribution strongly contributes to the higher voltage threshold of dentate granule neurons for two reasons. First, action potential initiation closer to the somatodendritic current sink causes the threshold of the initiating axon compartment to rise. Second, the proximity of the action potential initiation site to the recording site causes somatic recordings to more faithfully reflect the depolarized threshold of the axon than in cells like CA3 neurons, with distally initiating action potentials. Our results suggest that the proximal location of axon sodium channels in dentate granule neurons contributes to the intrinsic excitability differences between DG and CA3 neurons and may participate in the low‐pass filtering function of dentate granule neurons.

Indistinguishable synaptic pharmacodynamics of the N-methyl-D-aspartate receptor channel blockers memantine and ketamine.

Memantine and ketamine, voltage- and activation-dependent channel blockers of N-methyl-d-aspartate (NMDA) receptors (NMDARs), have enjoyed a recent resurgence in clinical interest. Steady-state pharmacodynamic differences between these blockers have been reported, but it is unclear whether the compounds differentially affect dynamic physiologic signaling. In this study, we explored nonequilibrium conditions relevant to synaptic transmission in hippocampal networks in dissociated culture and hippocampal slices. Equimolar memantine and ketamine had indistinguishable effects on the following measures: steady-state NMDA currents, NMDAR excitatory postsynaptic current (EPSC) decay kinetics, progressive EPSC inhibition during repetitive stimulation, and extrasynaptic NMDAR inhibition. Therapeutic drug efficacy and tolerability of memantine have been attributed to fast kinetics and strong voltage dependence. However, pulse depolarization in drug presence revealed a surprisingly slow and similar time course of equilibration for the two compounds, although memantine produced a more prominent fast component (62% versus 48%) of re-equilibration. Simulations predicted that low gating efficacy underlies the slow voltage–dependent relief from block. This prediction was empirically supported by faster voltage-dependent blocker re-equilibration with several experimental manipulations of gating efficacy. Excitatory postsynaptic potential–like voltage commands produced drug differences only with large, prolonged depolarizations unlikely to be attained physiologically. In fact, we found no difference between drugs on measures of spontaneous network activity or acute effects on plasticity in hippocampal slices. Despite indistinguishable synaptic pharmacodynamics, ketamine provided significantly greater neuroprotection from damage induced by oxygen glucose deprivation, consistent with the idea that under extreme depolarizing conditions, the biophysical difference between drugs becomes detectable. We conclude that despite subtle differences in voltage dependence, during physiologic activity, blocker pharmacodynamics are largely indistinguishable and largely voltage independent.

Levetiracetam Reduces Spike–Wave Density and Duration during Continuous EEG Monitoring in Patients with Idiopathic Generalized Epilepsy

Levetiracetam reduces spike-wave density and duration during continuous EEG monitoring in patients with idiopathic generalized epilepsy.

Self‐reported Seizure Frequency and Time to First Event in the Seizure Monitoring Unit

Summary:  Purpose: To compare seizure frequency reported in the clinic with time to first diagnostic event during video‐EEG monitoring. The effect of the artificial environment of the monitoring unit on self‐reported seizure frequency was explored.