Douglas A. Coulter

Selective changes in single cell GABA A receptor subunit expression and function in temporal lobe epilepsy

Temporal lobe epilepsy is the most prevalent seizure disorder in adults. Compromised inhibitory neurotransmitter function in the hippocampus contributes to the hyperexcitability generating this condition, but the underlying molecular mechanisms are unknown. Combining patch-clamp recording and single-cell mRNA amplification (aRNA) techniques in single dentate granule cells, we demonstrate that expression of GABAA receptor subunit mRNAs is substantially altered in neurons from epileptic rats. These changes in gene expression precede epilepsy onset by weeks and correlate with profound alterations in receptor function, indicating that aberrant GABAA receptor expression and function has an essential role in the process of epileptogenesis.

Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low-threshold current.

1. Calcium currents were recorded with whole‐cell voltage‐clamp procedures in relay neurones of the rat thalamus which had been acutely isolated by an enzymatic dissociation procedure. 2. Low‐threshold and high‐threshold Ca2+ currents were elicited by depolarizing voltage steps from holding potentials more negative than ‐60 mV. A transient current, analogous to the T‐current in sensory neurones, was activated at low threshold near ‐65 mV and was completely inactivating at command steps up to ‐35 mV. Voltage steps to more depolarized levels activated a high‐threshold current that inactivated slowly and incompletely during a 200 ms step depolarization. 3. The high‐threshold current contained both non‐inactivating and slowly inactivating components which were insensitive and sensitive to holding potential, respectively. 4. A ‘T‐type’ current was prominent in relay neurones, in both absolute terms (350 pA peak current average) and in relation to high‐threshold currents. The average ratio of maximum transient to maximum sustained current was greater than 2. 5. T‐current could be modelled in a manner analogous to that employed for the fast Na+ current underlying action potential generation, using the m3h format. The rate of activation of T‐current was voltage dependent, with a time constant (tau m) varying between 8 and 2 ms at command potentials of ‐60 to ‐10 mV at 23 degrees C. The rate of inactivation was also voltage dependent, and the time constant tau h varied between 50 and 20 ms over the same voltage range. With command potentials more positive than ‐35 mV, the inactivation of Ca2+ current could no longer be fitted by a single exponential. 6. Steady‐state inactivation of T‐current could be well fitted by a Boltzman equation with slope factor of 6.3 and half‐inactivated voltage of ‐83.5 mV. 7. Recovery from inactivation of T‐current was not exponential. The major component of recovery (70‐80% of total) was not very voltage sensitive at potentials more negative than ‐90 mV, with tau r of 251 ms at ‐92 mV and 23 degrees C, compared to 225 ms at ‐112 mV. A smaller, voltage‐sensitive component accounted for the remainder of recovery. 8. All kinetic properties, including rates of activation, inactivation, and recovery from inactivation, as well as the amplitude of T‐current, were temperature sensitive with Q10 (temperature coefficient) values of greater than 2.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular Actions of Topiramate: Blockade of Kainate-Evoked Inward Currents in Cultured Hippocampal Neurons

Purpose: This study was undertaken to evaluate the effects of topiramate (TPM) on excitatory amino acid‐evoked currents.

Selective induction of astrocytic gliosis generates deficits in neuronal inhibition

Reactive astrocytosis develops in many neurologic diseases, including epilepsy. Astrocytotic contributions to pathophysiology are poorly understood. Studies examining this are confounded by comorbidities accompanying reactive astrocytosis. We found that high-titer transduction of astrocytes with enhanced green fluorescent protein (eGFP) via adeno-associated virus induced reactive astrocytosis without altering the intrinsic properties or anatomy of neighboring neurons. We examined the consequences of selective astrocytosis induction on synaptic transmission in mouse CA1 pyramidal neurons. Neurons near eGFP-labeled reactive astrocytes had reduced inhibitory, but not excitatory, synaptic currents. This inhibitory postsynaptic current (IPSC) erosion resulted from a failure of the astrocytic glutamate-glutamine cycle. Reactive astrocytes downregulated expression of glutamine synthetase. Blockade of this enzyme normally induces rapid synaptic GABA depletion. In astrocytotic regions, residual inhibition lost sensitivity to glutamine synthetase blockade, whereas exogenous glutamine administration enhanced IPSCs. Astrocytosis-mediated deficits in inhibition triggered glutamine-reversible hyperexcitability in hippocampal circuits. Thus, reactive astrocytosis could generate local synaptic perturbations, leading to broader functional deficits associated with neurologic disease.

A Neuronal Glutamate Transporter Contributes to Neurotransmitter GABA Synthesis and Epilepsy

The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring–freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.

Enhanced Astrocytic Ca2+ Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

Status epilepticus (SE), an unremitting seizure, is known to cause a variety of traumatic responses including delayed neuronal death and later cognitive decline. Although excitotoxicity has been implicated in this delayed process, the cellular mechanisms are unclear. Because our previous brain slice studies have shown that chemically induced epileptiform activity can lead to elevated astrocytic Ca2+ signaling and because these signals are able to induce the release of the excitotoxic transmitter glutamate from these glia, we asked whether astrocytes are activated during status epilepticus and whether they contribute to delayed neuronal death in vivo. Using two-photon microscopy in vivo, we show that status epilepticus enhances astrocytic Ca2+ signals for 3 d and that the period of elevated glial Ca2+ signaling is correlated with the period of delayed neuronal death. To ask whether astrocytes contribute to delayed neuronal death, we first administered antagonists which inhibit gliotransmission: MPEP [2-methyl-6-(phenylethynyl)pyridine], a metabotropic glutamate receptor 5 antagonist that blocks astrocytic Ca2+ signals in vivo, and ifenprodil, an NMDA receptor antagonist that reduces the actions of glial-derived glutamate. Administration of these antagonists after SE provided significant neuronal protection raising the potential for a glial contribution to neuronal death. To test this glial hypothesis directly, we loaded Ca2+ chelators selectively into astrocytes after status epilepticus. We demonstrate that the selective attenuation of glial Ca2+ signals leads to neuronal protection. These observations support neurotoxic roles for astrocytic gliotransmission in pathological conditions and identify this process as a novel therapeutic target.

Disrupted Dentate Granule Cell Chloride Regulation Enhances Synaptic Excitability during Development of Temporal Lobe Epilepsy

GABAA receptor-mediated inhibition depends on the maintenance of intracellular Cl− concentration ([Cl−]in) at low levels. In neurons in the developing CNS, [Cl−]in is elevated, EGABA is depolarizing, and GABA consequently is excitatory. Depolarizing GABAergic synaptic responses may be recapitulated in various neuropathological conditions, including epilepsy. In the present study, rat hippocampal dentate granule cells were recorded using gramicidin perforated patch techniques at varying times (1–60 d) after an epileptogenic injury, pilocarpine-induced status epilepticus (STEP). In normal, non-epileptic animals, these strongly inhibited dentate granule cells act as a gate, regulating hippocampal excitation, controlling seizure initiation and/or propagation. For 2 weeks after STEP, we found that EGABA was positively shifted in granule cells. This shift in EGABA altered synaptic integration, increased granule cell excitability, and resulted in compromised “gate” function of the dentate gyrus. EGABA recovered to control values at longer latencies post-STEP (2–8 weeks), when animals had developed epilepsy. During this period of shifted EGABA, expression of the Cl− extruding K+/Cl− cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked EGABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on EGABA in granule cells, and on gatekeeper function of the dentate gyrus. This suggests a shared mechanism, reduced KCC2 function. These findings demonstrate that decreased expression of KCC2 persists for weeks after an epileptogenic injury, reducing inhibitory efficacy and enhancing dentate granule cell excitability. This pathophysiological process may constitute a significant mechanism linking injury to the subsequent development of epilepsy.

Specific petit mal anticonvulsants reduce calcium currents in thalamic neurons

Low-threshold calcium current (LTCC) in thalamic neurons is important in generation of normal thalamocortical rhythms, and may be involved in the genesis of abnormal activities such as spike-wave discharges that characterize petit mal epilepsy. Ethosuximide and dimethadione, anticonvulsants effective in petit mal, reduced the LTCC when applied to thalamic neurons at clinically relevant concentrations. Therapeutic concentrations of phenytoin and carbamazepine, drugs ineffective in the control of petit mal, had minimal effects on calcium conductances. Reduction in LTCC may be an important mechanism of action by which specific petit mal anticonvulsants depress spike-wave activity.

Dynamic Regulation of Synaptic GABA Release by the Glutamate-Glutamine Cycle in Hippocampal Area CA1

Vesicular GABA and intraterminal glutamate concentrations are in equilibrium, suggesting inhibitory efficacy may depend on glutamate availability. Two main intraterminal glutamate sources are uptake by neuronal glutamate transporters and glutamine synthesized through the astrocytic glutamate-glutamine cycle. We examined the involvement of the glutamate-glutamine cycle in modulating GABAergic synaptic efficacy. In the absence of neuronal activity, disruption of the glutamate-glutamine cycle by blockade of neuronal glutamine transport with α-(methylamino) isobutyric acid (MeAIB; 5 mm) or inhibition of glutamine synthesis in astrocytes with methionine sulfoximine (MSO; 1.5 mm) had no effect on miniature IPSCs recorded in hippocampal area CA1 pyramidal neurons. However, after a period of moderate synaptic activity, application of MeAIB, MSO, or dihydrokainate (250 μm; an astrocytic glutamate transporter inhibitor) significantly reduced evoked IPSC (eIPSC) amplitudes. The MSO effect could be reversed by exogenous application of glutamine (5 mm), whereas glutamine could not rescue the eIPSC decreases induced by the neuronal glutamine transporter inhibitor MeAIB. The activity-dependent reduction in eIPSCs by glutamate-glutamine cycle blockers was accompanied by an enhanced blocking effect of the low-affinity GABAA receptor antagonist, TPMPA [1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid], consistent with diminished GABA release. We further corroborated this hypothesis by examining MeAIB effects on minimal stimulation-evoked quantal IPSCs (meIPSCs). We found that, in MeAIB-containing medium, moderate stimulation induced depression in potency of meIPSCs but no change in release probability, consistent with reduced vesicular GABA content. We conclude that the glutamate-glutamine cycle is a major contributor to synaptic GABA release under physiological conditions, which dynamically regulates inhibitory synaptic strength.

Mechanisms of epileptogenesis: a convergence on neural circuit dysfunction

Epilepsy is a prevalent neurological disorder associated with significant morbidity and mortality, but the only available drug therapies target its symptoms rather than the underlying cause. The process that links brain injury or other predisposing factors to the subsequent emergence of epilepsy is termed epileptogenesis. Substantial research has focused on elucidating the mechanisms of epileptogenesis so as to identify more specific targets for intervention, with the hope of preventing epilepsy before seizures emerge. Recent work has yielded important conceptual advances in this field. We suggest that such insights into the mechanisms of epileptogenesis converge at the level of cortical circuit dysfunction.