Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies Phasic GABAA-Mediated Inhibition Enrico Cherubini Neurobiology Department, International School for Advanced Studies (SISSA), Via Bonomea 265, 34136 Trieste, Italy Abstract Phasic inhibition consists of fast GABAA-mediated inhibitory postsynaptic potentials (IPSPs) regulating point to point communication between neurons. In physiological conditions it exerts a powerful control on cell excitability and network oscillations thought to be associated with high cognitive functions. GABAA receptors facing the presynaptic release sites are exposed, for a brief period of time, to high concentration of GABA released by exocytosis of presynaptic vesicles. Once released, GABA diffuses throughout the neuropil before being taken up by selective plasma membrane transporters which contribute to its clearance. In this Chapter different steps contributing to shape synaptic currents, including vesicle exocytosis, dynamics of GABA transient in the synaptic cleft, postsynaptic receptors and GABA transporters will be highlighted. Particular attention will be paid to the regulation of GABA release by the calcium-induced calcium release process and by presynaptic GABAA and/or GABAB receptors. Finally, the heterogeneity of GABAA receptor subunits will be discussed in relation to their ability to give rise to a variety of functional GABAA receptors, differently regulated by endogenous or exogenous molecules useful for the treatment of severe neurological and psychiatric disorders. γ-Aminobutiric acid (GABA) is the main inhibitory neurotransmitter in the adult mammalian CNS. It inhibits neuronal firing by activating two different classes of receptors, GABAA and GABAB. Unlike GABAA receptors which form integral ion channels, GABAB receptors are coupled to ion channels via guanine nucleotide-binding proteins and second messengers. GABAA receptors mediate two distinct forms of inhibition, phasic and tonic. The first consists of fast inhibitory postsynaptic potentials (IPSPs), regulating point-to-point communication between neurons. The second consists of a persistent inhibitory conductance that plays a crucial role in regulating the membrane potential and network excitability. 1 In the case of phasic inhibition, synaptic GABAA receptors, facing presynaptic release sites, are activated by a brief exposure to a high concentration of GABA released by exocytosis of presynaptic vesicles. Once released, GABA diffuses throughout the neuropil before being taken up by selective plasma membrane transporters, which contribute to the clearance of the neurotransmitter. 2 In the case of tonic inhibition, extrasynaptic GABAA receptors, localized away from the synapses, are persistently exposed to low concentration of “ambient” GABA. This review will focus on GABAA-mediated phasic inhibition which, in physiological conditions, exerts a powerful control on cell excitability and network oscillations thought to be associated with higher cognitive functions. 3 An impairment of fast GABAergic signaling is involved in various psychiatric and neurological disorders including epilepsy. 4 GABAA-MEDIATED IPSCs: PRESYNAPTIC REGULATION Source of GABA GABA is released from GABAergic interneurons which comprise ~ 15 % of the total neuronal population. It is synthesized from glutamate by the enzyme Glutamic Acid Decarboxylase (GAD65 and GAD67) and in particular, by GAD65 which is preferentially localized at GABAergic nerve terminals. GABAergic interneurons constitute a heterogeneous group of Page 2 cells which have been characterized on the basis of their firing patterns, molecular expression profiles and selective innervation of different domains of principal cells. 5 They provide feedback and feed-forward inhibition. 6, 7 In addition, the spatio-temporal dynamics between Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies the activity of interneurons, with extensive axonal arborizations, and that of pyramidal cells leads to coherent network oscillations crucial for information processing. 5, 8 In the majority of cases, the release of GABA from GABAergic interneurons occurs via vesicle exocytosis, the process by which a single vesicle fuses with the presynaptic plasma membrane and releases its content into the synaptic cleft. However, the release of GABA may also occur in a non-vesicular way through the reversal of GABA transporters. 9 Thus, in pathological conditions (e.g. in temporal lobe epilepsy), bursting-induced membrane depolarization and increase in intracellular [Na+] would enhance GAT-1 reversal (GAT-1 is the isoform of GABA transporters localized almost exclusively on axon terminals) and GAT-1-mediated GABA release. 10 Recently, using a heterologous expression system, Wu et al. 9 have provided evidence that the membrane potential at which the GABA transporter GAT-1 reverses is close to the normal resting potential of neurons and that the reversal can occur rapidly enough to release GABA during action potentials. This is consistent with the hypothesis that transporter- mediated GABA release can contribute to maintain phasic GABAA-mediated inhibition also in physiological conditions as in the case of vesicular transmitter failure during high frequency firing, thus acting as a brake to prevent runaway excitation. 10 The release of GABA from presynaptic nerve terminals is influenced by the probability of release P and by the number of release sites N. Large P and/or large N can be used to distinguish strong from weak GABAergic synapses. Strong connections produce large IPSPs with few transmitter failures. Weak synapses with high failure rates exhibit paired-pulse facilitation (facilitation of a second response evoked at a short interval from the first), while strong synapses with low failures rate exhibit paired-pulse depression (depression of a second response occurring at a short interval from the first). Regulation of GABA Release by Presynaptic Intracellular Calcium Stores Interestingly, P can be affected by spontaneous calcium signals (reminiscent of “calcium sparks”), 11 as those detected at cerebellar interneurons-Purkinje cells synapses. 12 These events (sensitive to ryanodine) depend on the release of calcium from intracellular calcium stores following action potential-dependent increase in intracellular calcium rise. 13 Ryanodine-sensitive calcium stores are likely to mediate large amplitude miniature inhibitory postsynaptic currents (mIPSCs) due to the synchronous release of GABA from several release sites. At the same synapses, presynaptic calcium stores also regulate the evoked release of GABA as demonstrated by ryanodine-induced decrease in paired-pulse ratio and increase in the coefficient of variation, 14 defined as the ratio between the standard deviation and the mean. P can also be also modified in a synapse-selective manner by receptors localized on presynaptic terminals, whose activation up- or down-regulates the release of GABA. In addition, synapses can also differ in the number of functional release sites, N, which can vary from one to tens. For instance, at individual synaptic contacts among stellate cells in the cerebellum, presynaptic action potentials generate synaptic currents presumably arising from single or multiple release sites. 15 In case of multiple release sites, decreasing the probability of GABA release (by modifying the calcium/magnesium ratio) decreases the average number of release sites and shifts the amplitude distribution towards smaller values. In contrast, at single release sites, the amplitude distribution of the evoked currents whose potency (successes without failures) is unaffected by decreasing the release probability can be fitted with a Gaussian function. 16, 17 Some GABAergic synapses are also characterized by multivesicular release. This occurs when the content of several vesicles is released simultaneously by an action potential at a presynaptic active zone leading to fluctuations of inhibitory synaptic events which vary in magnitude Phasic GABAA-Mediated Inhibition Page 3 according to the degree of receptor saturation. For instance, at cerebellar stellate-basket cell synapses, more than one containing vesicle, released slightly asynchronously, leads to a very small increase in the peak amplitude of the synaptic currents, because, at these synapses, Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies receptors occupancy is high and close to saturation. 6,17,18 It is noteworthy that the functionally determined numbers of release sites is usually larger than those structurally defined at electron microscopic level, because a single presynaptic active zone is often made by a large number of functional release sites. 19 Regulation of GABA Release by Presynaptic GABAA and GABAB Receptors The release of GABA is up- or down-regulated by a variety of different receptors localized on GABAergic terminals in proximity of the release sites. Of particular interest are GABAA and GABAB autoreceptors, which can be activated by spillover of GABA from axon terminals or by “ambient” GABA present in the extracellular medium. While high P synapses more likely undergo activity-dependent depression of synaptic transmission, low P synapses undergo activity-dependent facilitation. A decrease in P may result from the shunting effect of GABA, due to the increase in conductance produced by presynaptic receptor activation. This in turn would produce a decrease in amplitude of presynaptic