Epilepsy: A Comprehensive Textbook
2nd Edition
© 2008 Lippincott Williams & Wilkins
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Chapter 23
GABAA and GABAB Receptor-Mediated Inhibitory Synaptic Transmission
Robert L. MacDonald
Istvan Mody
Introduction
The neurotransmitter γ-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the central nervous system (CNS). GABAergic inhibition is the primary form of fast inhibition in the forebrain, and can mediate both presyn- aptic and postsynaptic inhibition. GABA-mediated presynaptic inhibition occurs when GABAergic nerve terminals release GABA onto presynaptic nerve terminals, resulting in a reduction of neurotransmitter release. In contrast, GABAergic postsynaptic inhibition is mediated by the interaction of the neurotransmitters with specific postsynaptic receptors. GABA binds to two distinct types of GABA receptor, GABAA,C receptors and GABAB receptors, to produce neuronal inhibition. GABAA and GABAC receptors are ligand-gated chloride ion (Cl-) channels from the cys loop family of receptors, and GABAB receptors are seven-transmembrane domain receptors from the G-protein–coupled receptor (GPCR) family. Reduction of GABAergic inhibition because of mutation of GABAA receptors has been shown to be associated with several types of idiopathic generalized epilepsies, including childhood absence epilepsy, generalized epilepsy with febrile seizures plus, and juvenile myoclonic epilepsy.70,71 Enhancement of GABAergic inhibition is the basis of action of a number of antiepileptic drugs (AEDs).
γ-Aminobutyric Acid
GABA is synthesized in and released from the presynaptic terminals of GABAergic neurons. Glucose is the primary precursor of GABA, but pyruvate and other amino acids can also serve as precursors when metabolized in the Krebs cycle.79 The immediate precursor of GABA is glutamate, which can be produced from glutamine by glutamate synthetase or from α-ketoglutarate. Glutamic acid is then decarboxylated to form GABA. GABA is degraded by the enzyme GABA transaminase. GABA transaminase converts GABA into succinicsemialdehyde. This reaction involves removal of an amine group from GABA and transformation of α-ketoglutarate into glutamate. Therefore, as the GABA is degraded, its precursor molecule is formed, ensuring precursor availability for further synthesis. Succinicsemialdehyde is then degraded by the enzyme succinicsemialdehyde dehydrogenase (SSADH) to form succinic acid. This synthetic and catabolic pathway is called the GABA shunt. The shunt is thought to operate by an interaction with adjacent glia. GABA is released presynaptically and is taken up by high-affinity GABA transporters in nerve terminals and glia. In glia, GABA is degraded to succinicsemialdehyde, thus generating glutamate. Glutamate is then converted to glutamine by glutamine synthetase. It is believed that glutamine can then diffuse from the glia into nerve terminals, where it then again converted by glutaminase into glutamate, thus providing a substrate for GABA synthesis. GABA transaminase and SSADH are thought to be bound to mitochondria, whereas glutaminase and GAD are free in the terminal cytoplasm. Glutamine synthetase occurs free in the glial cytoplasm.
GABAA Receptors
GABAA receptors are macromolecular proteins from the cys loop family of ligand-gated ion channels that include the nicotinic acetylcholine receptor (nAChR), glycine receptor, and serotonin 5–HT3 receptor. GABAA receptors contain multiple specific binding sites including sites for GABA, antagonists such as bicuculline and picrotoxin, AEDs including barbiturates and benzodiazepines, and the anesthetic steroids. GABAA receptors form a Cl--selective channel.73,90
GABAA Receptor Molecular Biology
GABAA receptors are hetero-oligomeric pentamers that form a transmembrane Cl-channel. Seven GABAA receptor subunit families (α, β, γ, δ, π, ∊, θ) have been identified,90 and each subunit family is composed of one or more subtypes: (α1–α6), (β1–β3), (γ1– γ3), (δ1), (∊1), (π1), and (θ1). In addition, several splice variants have been reported. An additional subunit, the ρ subunit, is highly homologous to the GABAA receptor subunits but primarily forms retinal receptors that have been classified as GABAC receptors. Members of the same GABAA receptor family share approximately 70% to 80% sequence homology, whereas members of different families share 30% to 40% sequence identity. The number of potential GABAA receptor isoforms that could be formed from the 12 α, β, and γ subtypes alone is staggering. However, it has been demonstrated that not all potential subtype combinations assemble to create functional GABAA receptors,3,4 and the α1β2γ2 receptor is likely the dominant native benzodiazepine-sensitive isoform. The stoichiometry of native GABAA receptors has shown to be 2α2β1γ, with an assembly pattern of γ-β-α-β-α, as seen from the synaptic cleft.6,26,57,110
GABAA Receptor Structure
GABAA receptor subunits are about 450 amino acids in length and have a large N-terminus of approximately 200 amino acids that is shaped by the signature cysteine disulfide bridge and is likely an extracellular domain. The GABA binding site is at the two α/β subunit interfaces.101 Hydropathy analysis predicted four transmembrane-spanning domains (M1–M4) of about 20 amino acids in length, which have the highest degree of amino acid sequence homology across subunit
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families and across the cys loop family in general. The M2 domain is thought to form the GABAA receptor channel pore.26 A short (eight amino acids) cytoplasmic segment (M1–M2 linker) connects M1 and M2, and a slightly longer extracellular segment (M2–M3 linker) connects M2 to M3. Also, a highly variable large cytoplasmic loop spans between M3 and M4 and contains consensus sequences for protein phosphorylation and protein–protein interaction that vary among subtypes. This domain is important for receptor trafficking and surface clustering through interactions with cytoplasmic proteins.
GABA Binding Site
Detailed structural information is available for the extracellular domain of cys loop receptors based on homology with the acetylcholine binding protein (AChBP),100 which has been crystallized and solved.21 The acetylcholine-binding pocket is formed at AChR subunit interfaces by three loops (A–C) from the positive side and three β-strands (D–F) from the negative side of the protomer. Using mutagenesis and the substituted cysteine accessibility method (SCAM) approach,1,15,48,86,120 the GABAA receptor binding pocket has been shown to be similar to that of the AChBP binding pocket.
GABAA Receptor Conduction Pathway
M2 likely lines the channel, and the amino acids lining the pore are likely α helical126 as is the nAChR M2.82 Using electron microscopy of crystalline postsynaptic membranes, the nAChR channel has been shown to be tapered, as viewed from the synaptic cleft, and is formed by five α-helical segments.82 The pore has a minimum diameter in the middle of M2 at residues L251 and V255, and this region has been identified as the channel gate.82 GABAA receptors also have a 9′L in the middle of M2, and mutation of the M2 9′L causes destabilization of the closed state,13 similar to the nAChR. Thus, the GABAA receptor gate is likely to be similar in location to that of the nAChR.
Coupling of GABA Binding to Channel Gating
Recent insights have emerged into the coupling of binding of GABA to GABAA receptors and subsequent gating of the channel. The extracellular N-terminal domain is directly connected to the membrane by M1; however, evidence from several sources has suggested that the N-terminal domain also interacts with the extracellular M2–M3 linker. Several disease mutations in the M2–M3 linker have been identified. In the nAChR, these mutations are associated with congenital myasthenic syndromes38; in the GABAA receptor, they are associated with a generalized epilepsy7,14; and in the glycine receptor, these mutations are associated with hyperekplexia.61 All these mutations decreased receptor current, suggesting a disruption in the coupling of agonist binding to channel gating. Based on the AChBP, it was suggested that loop 2 (and possibly loop 7) interact with the M2–M3 linker to couple GABA binding to gating.54 Using SCAM, gating was shown to induce a conformational change in and/or around the N-terminal half of the M2–M3 linker.9 Using mutagenesis of pairs and triplets of amino acids in the nAChR α subunit, a network of interacting amino acid residues have been shown to connect the preM1 region, loop 2, and the M2–M3 loop.64 Thus, coupling of agonist binding to channel gating may involve both direct (pre M1) and indirect (loop 2) pathways that connect to the M2–M3 loop. The gating process has been further clarified using electron microscopy of nAChRs.82,116 Loop 2 of both α subunits is positioned such that it contacts the distal M2, just before the beginning of the M2–M3 loop. Binding of ACh induces both loop 2s rotate by 15 degrees about an axis passing through the disulfide bridge, normal to the membrane. The loop 2 rotations are associated with M2 rotations, which are translated down M2 to the gate, presumably causing the gate to open. Thus the major transduction of binding to gating appears to pass in the α subunits from loop 2 to the distal M2 and then to the M2 gate.
GABAA Receptor Desensitization
Receptor desensitization occurs when agonist-evoked current declines during continued agonist application. No clear structural basis exists for desensitization of cys loop receptors. Desensitization of nAChRs has been studied using the SCAM technique.124 The “desensitization gate” was suggested to include the closed gate, but to extend further (more toward the extracellular portion) into M2. GABAA receptor subunit composition influences current desensitization rates.37,97,117 In glycine receptors, desensitization is influenced by amino acids at the distal end of M1, leading to the hypothesis that this region and the M1–M2 linker served as a hinge involved in the gating of the channel.69 Using subunit chimeras constructed with N-terminal δ sequence spliced to γ2L sequence at various points within M1 and M2, and using rapid agonist application, fast desensitization was demonstrated to depend on the structure of M1 and proximal N terminus.12
GABAA Receptor Pharmacology
GABAA receptor pharmacology has been shown to be quite complex.73,90 To fully activate the receptor, two molecules of GABA are required to bind to two independent sites on the GABAA receptor channel at the two α/β interfaces. In addition to GABA, a number of GABA analogs can activate the receptor, including the plant alkaloid muscimol and the synthetic GABA agonist tetrahydroisoxazolopyridinol (THIP). GABAA receptor currents can be competitively antagonized by the convulsant drug bicuculline and noncompetitively antagonized by the convulsant drug picrotoxin. GABAA receptor current can also be noncompetitively reduced by the convulsant antibiotic penicillin, which enters the GABAA receptor channel to produce a fast open-channel block. The antiepileptic benzodiazepines and barbiturates enhance GABAA receptor currents, but through different binding sites on the GABAA receptor. In contrast, the convulsant β-carbolines, such as methyl 6,7–dimethoxy-4–ethyl-β-carboline-3–carboxylate (DMCM), decrease GABAA receptor current. A number of compounds, including CL 218872, inverse agonist β-carbolines, and imidazolpyridines (zolpidem), bind to the benzodiazepine site. In addition to binding benzodiazepine receptor agonists, such as diazepam, and inverse agonists, such as DMCM, the receptor binds a benzodiazepine receptor antagonist, flumazenil, which binds to the receptor and antagonizes the actions of benzodiazepine receptor antagonists and inverse agonists but has little intrinsic activity. In addition to the preceding allosteric regulators, certain anesthetic and naturally occurring neuro- steroids, including alfaxalone, pregnenolone, and androsterone, alter GABAA receptor current. Most of these anesthetic steroid compounds bind to a specific site on the GABAA receptor to enhance current.
GABAA Receptor Physiology
GABA binds to GABAA receptors and evokes openings that occur in bursts.74 When the channel opens, it opens to a main conductance level of 27 pS and to two smaller, subconductance levels.16 GABAA receptor channels are permeable to a number of anions and exhibit a conductance sequence of CI->Br->SCN->F-. GABAA receptor channels are almost exclusively Cl selective, with a permeability ratio of potassium (K) to Cl ions smaller than 0.05. The permeability sequence for large polyatomic anions suggests that open
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GABAA receptor channels have effective pore diameters of about 5.6 Å.
Rapid application of GABA to outside-out patches from hippocampal neurons or to HEK293 cells expressing α1β3γ2L receptors evokes rapidly rising (1–2 msec) currents that then decay over three to four phases of desensitization.14,25,45 Following GABA application, the currents deactivate over two phases. GABAA receptor subunit composition influences current desensitization rates.14,37,97,117 For example, addition of the δ subunit to α1 and β3 subtypes significantly reduced both the rate and extent of desensitization relative to α1β3γ2L receptor currents.45
Ensemble currents (whole cell or macropatch currents, inhibitory postsynaptic currents [IPSCs]) observed experimentally represent the summed behavior of individual channels. A complete mechanistic understanding of GABAergic inhibition requires analysis at the level of single channels. GABAA receptor single channels exhibit complex patterns of activity. GABA increases the probability of channel opening and, after the channel opens, it closes and rapidly reopens in bursts of openings. Increasing GABA concentration increases average open and burst durations. Detailed kinetic analyses of channel behavior have been obtained for native74,87,115,122 and recombinant23,39,45 GABAA receptors, demonstrating multiple open and closed states occurring in bursts. Kinetic models were proposed to account for these properties, with connections of discreet kinetic states (Markov modeling) and incorporating two sequential GABA binding sites, three open states, and ten closed states.115
Recombinant α1βχγ2 receptors also exhibit multiple open and closed states.3,39,45 However, α1β1δ GABAA receptors have different gating properties, with only two open states that are similar to the two briefest open states of native and α1βχγ2 GABAA receptors. The model was used to interpret actions of modulators of GABAA receptor function, including benzodiazepines, barbiturates, neurosteroids, picrotoxin, penicillin, and β-carbolines.111,112,113,114 Using rapid application of GABA to outside-out patches from hippocampal neurons, Celentano and Wong25 demonstrated three desensitization phases. Jones and Westbrook52 demonstrated that GABAA receptors entered long closed states and subsequently reopened, suggesting that desensitized states prolong currents evoked by brief, synaptic-like GABA applications. The combination of single-channel properties and macroscopic kinetic properties, not easily extractable from single-channel recordings (such as activation, desensitization, deactivation), led to a comprehensive GABAA receptor model.45
Channel gating and desensitization have also been shown to affect GABA binding. GABA is locked onto GABAC and GABAA receptors by channel opening27 and, once bound to α1β3γ2L GABAA receptors, GABA is “trapped” by open, preopen, and desensitized states.14 This trapping of GABA on the receptor prolongs currents following brief GABA application, as occurs during IPSCs.52 In contrast, it has been reported that ACh is not locked on nAChRs by open states.43
GABAA Receptor Anatomic Distribution and Development
The widespread and overlapping distribution of GABAA receptor subunit cDNAs identified using in situ hybridization65,125 and the large number of subunits suggests that GABAA receptors exist in vivo in multiple isoforms. However, the colocalization of subtype mRNAs does not provide information on subunit assembly. Immunoprecipitation has been used to determine subtypes that coassemble in vivo.50,89,94,95,108 In the cerebellum, the most likely isoforms are α1βχγ2, α6βχγ2, α1α6βχγ2, α6βχ2δ, and α1α6βχδ receptors. The δ subunit is likely to combine with the α6 subtype in the cerebellum and with the α4 subtype in the thalamus, and based on coexpression, it may also combine with the α1 subtype in the cortex, thalamus, hippocampus, and olfactory bulb. Characterization of GABAA receptor subunit expression patterns using in situ hybridization has been reported in the developing63 and adult62,125 rodent brain. A detailed immunohistochemical study identified general anatomic patterns as well as cell-type specificity within regions and gross subcellular localization (soma versus neurite membranes).94
GABAB Receptors
GABAB receptors are seven-transmembrane proteins and are coupled to calcium (Ca) or K ion channels via guanosine triphosphate (GTP) binding proteins.17,18,19 GABAB receptors are located on presynaptic terminals and on postsynaptic membranes. When activated by GABA presynaptically, GABAB receptors reduce synaptic transmitter release by decreasing presynaptic calcium entry; when activated postsynaptically, GABAB receptors produce slow postsynaptic inhibition by increasing K conductance.
GABAB Receptor Molecular Biology, Structure, and Trafficking
Two different GABAB receptor subunits have been cloned, the GABAB1 and GABAB2 subunits.51,56,59,80,88,123 These subunits have an approximate length of 950 residues, and the GABAB2 subunit has two splice variants, GABAB1a and GABAB1b.55 At the amino acid level, GABAB1 and GABAB2 subunits have approximately 35% identity and 55% similarity, and GABAB receptors are heterodimers composed of both GABAB1 and GABAB2 subunits. GABAB receptors are members of the GPCR superfamily; they have seven membrane-spanning domains and a long extracellular chain at the N terminus. Heterodimerization is required for successful trafficking of GABAB receptors to the cell surface and for effective agonist-induced activation.24,42,77,92 The GABAB1 subunit appears to be necessary for agonist activation, whereas the GABAB2 subunit is involved in trafficking of the heterodimer to the cell surface. When exposed, a C-terminal arginine-based endoplasmic reticulum (ER) retention/retrieval signal, RSRR, results in the retention of unassembled GABABB1 subunits in the ER and prevents surface trafficking of heteromeric receptors. Interaction of C-terminal coiled-coil domains mask the RSRR retention signal motif in the GABAB1 subunit and permit cell surface trafficking.10,77,92 Agonist binds to the GABAB1 subunit and produces a conformational change that permits the GABAB2 subunit to activate G-protein–coupled signaling.
GABAB Receptor Pharmacology
GABAB receptors were initially defined as sites that were GABA sensitive but were not blocked by the GABAA receptor antagonist bicuculline.17 Although GABA is undoubtedly the natural ligand for this receptor, a drug used to treat spasticity, β-p-chlorophenyl GABA (baclofen) was shown to be virtually inactive at GABAA receptors but stereo- specifically active at GABAB receptors. The active form of baclofen is the minus isoform. Since the identification of baclofen as a specific GABAB receptor agonist, a number of closely related analogs of baclofen and GABA also have been shown to be agonists. These compounds include β-hydroxy GABA, muscimol, 3–aminopropopolyphosphinic acid (3APPA), 3–aminopropyl (methyl)-phosphinic acid (3AMPA), and CGP44532. The phosphinic acid analogs are the most potent GABAB receptor agonists, often having affinities 10 to 100 times higher than baclofen.4 Because GABAA receptor agonists
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such as bicuculline and picrotoxin were inactive at GABAB receptors, it became important to develop specific GABAB receptor antagonists to study the effects of synaptic GABA interaction with GABAB receptors. The first GABA antagonist was the phosphonic derivative of baclofen, phaclofen. Although phaclofen is a very weak GABAB receptor antagonist, it is selective. In addition, a number of other GABAB receptor antagonists have been introduced (CGP 35348, CGP 36742, CGP 55845A, CGP 62349, and SCH50911).
GABAB Receptor Physiology
GABAB receptors have several different effector mechanisms, including the adenylate cyclase system and Ca and K channels. The actions of GABAB receptor agonists on these effectors are blocked by pertussis toxin, suggesting that GABAB receptors couple to their channel targets via a Giα- or Goα-type G protein (especially Gi2α). GABAB agonists inhibit basal and forskolin-stimulated adenylate cyclase in the brain through a G-protein–dependent pathway to reduce intracellular cyclic adenosine monophosphate (cAMP). GABAB receptor agonists also decrease voltage-gated Ca channels. Multiple high-threshold and low-threshold Ca channels have been reported.40,81 High-threshold channels include N-type, L-type, P-type, and R-type channels. The low-threshold channel is a T Ca channel. These channels differ in the voltage range of activation and in rates of inactivation. GABAB receptor agonists have been demonstrated to reduce current flow through P/Q- and N-type Ca channels.81 If this effect occurred at presynaptic terminals, this would result in decreased presynaptic Ca entry and, therefore, decreased release of neurotransmitter. The effect appeared to provide a basis for GABA-mediated presynaptic inhibition. These effects of GABAB receptor agonists have been shown in the peripheral nervous system and have been shown to inhibit excitatory synaptic transmission between hippocampal neurons.98,103,109,127 It is likely that the effect of GABAB receptor agonists on reducing high-threshold transient Ca currents accounts for the reduction in synaptic transmission produced by these compounds. Activation of GABAB receptors has been shown to reduce the excitatory synaptic transmission presumably mediated by glutamate, but GABAB receptors have also been identified on presynaptic terminals of GABAergic neurons, and thus produce autoinhibition of GABA release from GABAergic nerve terminal. Thus GABAB receptors appear to be involved in the regulation of GABA release from nerve terminals and to affect the regulation of release of both excitatory and inhibitory neurotransmitters. In addition to regulation of presynaptic Ca channels, GABAB receptor agonists have postsynaptic action to enhance Kir3–type K conductance.68 In a number of brain regions, including the hippocampus, cerebral cortex, thalamus, septum, and medulla, activation of GABAergic neurons produces biphasic IPSPs. A rapid early bicuculline-sensitive component is followed by a phaclofen- and CGP 35348–sensitive slow component. The fast component of the IPSP has been shown to be Cl-mediated, whereas the late slow phase is K-mediated.
Although all GABAB receptors appear to be heterodimers composed of GABAB1 and GABAB2 subunits, the role of the GABAB1 splice variants has been unclear. Recently this issue has been clarified by the demonstration of differential compartmentalization and functions of dimers containing either the GABAB1a or GABAB1b splice variant. In hippocampal CA3–to-CA1 and layer 1 somatosensory cortical dendritic synapses on layer 5 pyramidal neurons, presynaptic glutamate release is inhibited by activation of GABAB receptors containing the GABAB1a splice variant, whereas activation of GABAB receptors containing the GABAB1b splice variant mediates postsyn- aptic inhibition.93,119
GABAB Receptor Anatomic Distribution
GABAB receptors have a widespread distribution in the central and peripheral nervous systems.17 As has been described earlier, GABAB receptors exist on the synaptic terminal of excitatory/inhibitory neurons and on the cell bodies of neurons. In the CNS, an uneven distribution of receptors occurs, as measured by receptor autoradiography. This regional heterogeneity undoubtedly is of great importance for understanding the function of GABAB receptors. The location of GABAB receptors does not correlate with the presence of GABAA receptors. There are locations where primarily GABAA receptors are present and other locations where primarily GABAB receptors are present. The highest density of GABAB receptor binding sites appears to occur in the cerebral cortex, certain thalamic nuclei, cerebellum molecular layer, interpeduncular nucleus, and spinal cord dorsal horn.20,29 Moderate densities of GABAB receptor sites are also present throughout the hippocampal formation. In addition, GABAB receptor binding sites have been located in the globus pallidus, habenula nucleus, superior colliculus, and amygdala. In the hippocampus, there is moderate density of binding in the stratum radiatum, dentate gyrus molecular layer, and stratum pyramidale in CA1. In contrast, there is a low level of binding in the subiculum. In the cerebral cortex, a nonhomogeneous pattern of binding is found, with high levels of binding in layers 1 to 3, and moderate levels of binding in layer 4. Thalamic binding is high in the medial geniculate and dorsal lateral geniculate, and low in the ventral lateral geniculate. In the superior colliculus, high binding occurs in the superficial gray layer and low binding in the intermediate gray layer.
Frequency-Dependent Plasticity at Inhibitory Synapses
The magnitude of the conductance change produced by a neurotransmitter or the amplitude of the synaptic potential is referred to as the strength of synaptic transmission. In general, the strength of synaptic transmission at most synapses is not invariant; synaptic strength may increase or decrease, commonly because of previous synaptic activity.75,128 Alterations in the strength can be short-term increases (facilitation) or decreases (depression), or they can be long-term increases (long-term potentiation [LTP]) or decreases (long-term depression [LTD]). GABAergic synaptic inhibition is no exception, except that primarily use-dependent depression is seen at inhibitory synapses. For example, in hippocampus, inhibitory synaptic strength decreases during repetitive stimulation.8 The basis for use-dependent depression in inhibitory synapses is unclear but could be multifactorial. The most likely mechanisms may involve postsynaptic desensitization of GABAA receptors, reduction in driving force due to Cl redistribution, and a loss of the transmembrane Cl gradient or feedback inhibition of GABA release mediated by presyn- aptic GABAB receptor autoreceptors. When modest stimulation protocols are employed, such as paired pulse stimulation, it is thought that the primary mechanism of GABAergic synaptic depression is due to feedback inhibition of GABA release mediated by presynaptic GABAB autoreceptors.35,84 However, it is unlikely that the activation of GABAB autoreceptors is the sole explanation of synaptic depression at inhibitory synapses because not all GABAergic terminals contain GABAB autoreceptors.11,60 For example, in inhibitory synapses in area CA3 of the rat hippocampus, both GABAB autoreceptor-dependent and autoreceptor-independent components of paired-pulse depression have been described. Activation of GABAB autoreceptors produces fast paired-pulse
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depression that lasts for less than 1 second, whereas a second GABAB autoreceptor-independent mechanism persists for several seconds. The GABAB receptor-dependent fast paired-pulse depression was shown to require activation of several presynaptic inhibitory neurons, whereas GABAB receptor-independent paired-pulse depression occurred at single inhibitory synapses.60 Thus, although GABAergic inhibition is essential for maintaining an appropriate level of excitability in synaptic circuits, under those conditions in which multiple GABAergic pathways are stimulated, GABAergic synaptic strength can be decreased by GABAB receptor-dependent as well as GABAB receptor-independent mechanisms, thus producing frequency-dependent depression. This phenomenon undoubtedly has significance in the acute disinhibition that may precede the development of epileptiform discharges.
Disinhibition and Epilepsy
Epilepsy is associated with hypersynchronous activation of large populations of neurons, and considerable interest has been focused on the possibility that reduction of inhibition, or disinhibition, is associated with the pathogenesis of some forms of epilepsy. The primary genesis for the hypothesis that modified GABAergic inhibition produces epileptiform discharge has been based on the experimental evidence that drugs that block GABAergic inhibition produced paroxysmal bursting in isolated neurons and produced partial seizures in experimental animals when the convulsants are applied topically to the cortex or hippocampus.99 Application of GABA antagonists, including penicillin, picrotoxin, or bicuculline, produces large depolarizations because of combined giant excitatory postsynaptic potentials, thus producing paroxysmal depolarization shifts (PDSs), which are the interictal manifestation of an epileptiform event. It is thought that production of these epileptiform events requires a combination of reduced GABAergic inhibition, feed-forward excitation, and bursting properties of individual neurons.125 It is likely that local paroxysmal bursting can spread to involve large areas of the hippocampus or generalize to the cortex when inhibition is further weakened and when other synchronizing factors occur, such as altered extracellular K and Ca concentration.
The ultimate form of disinhibition in epilepsy may be considered to be the conversion of a well-behaved GABA-mediated hyperpolarizing inhibitory event into a malicious depolarizing excitatory potential. Depolarizing or shunting inhibition has long been known to exist is neurons2 and, depending on the threshold for action potential generation of the innervated cell, it can be inhibitory or excitatory.106 The latter action is mainly confined to the passive phase of the synaptic potential.44 The sign of the voltage change evoked by GABA is solely dependent on the reversal potential of the permeant ions (Cl- and HCO3-) through the channel. Early on during development, most cells are loaded with Cl-, causing GABA effects to be depolarizing91 and thus playing a major role in shaping the development of the neurons by activating the influx of the second-messenger cation Ca2+. The depolarizing action of GABA does not last into the adulthood because, at a given stage in development, the activation of various pumps and exchangers make sure that permeant ions become extruded from the cell.96 However, adult epilepsy is an exception. It seems that, perhaps because of the malfunctioning of some of the anion extrusion mechanisms in the epileptic brain, some principal cells become depolarized by GABA to the extent at which interictal spiking can be induced as if GABA were an excitatory transmitter.31
Conclusive evidence that alteration of GABAergic inhibition is involved in the pathogenesis of idiopathic generalized seizures has come from the identification of several GABAA receptor subunit point mutations in the α1, γ2, and δ subunits; these reports have been reviewed.70,71 The γ2 subunit mutations include γ2(R43Q) associated with febrile seizures (FS) and childhood absence epilepsy (CAE),121 γ2(K289M) associated with generalized epilepsy with febrile seizures plus (GEFS+,5, γ2(Q351X) associated with GEFS+,47 γ2(Q1X) associated with severe myoclonic epilepsy of infancy (SMEI),47 and γ2(IVS6 + 2T → G) associated with GEFS+53. The α1 subunit mutation, α1(A322D), is associated with juvenile myoclonic epilepsy (JME).32 Two δ subunit variants, δ(R220H) and δ(E177A), were identified as susceptibility genes associated with GEFS+ and JME.36
Inhibition and Synchrony
It would be extremely simplistic to regard GABAergic neurons in the brain as only “inhibitory.” It has become exceedingly clear over the past decade and a half that the vast anatomic heterogeneity of these cells corresponds to an equal functional diversity.41,78,83,104 It is also evident that the activity of highly specific subsets of interneurons can entrain various oscillatory rhythms in neuronal networks.23,41,58,104 Does this action of interneurons amount to “inhibition”? Certainly not.
One of the most elegant demonstrations of synchronization through inhibition was done in recordings from two pyramidal cells innervated by a single inhibitory basket cell.30 If the basket cell was activated during the random and asynchronous firing of the two pyramidal cells, the two cells were both simultaneously inhibited, but started firing together for a few cycles upon their recovery from the inhibition.30 More recent findings show that depolarizing (shunting) inhibition can serve as a “homogenizer” of diverse firing rates, thus greatly enhancing oscillatory activity in the γ-frequency range.118 In light of such synchronizing action of interneurons, the role of inhibition in epilepsies becomes much more complex than initially thought. Depending on which inhibitory interneuron survives in the epileptic tissue, and which GABA receptors suffer from plasticity, the outcome of the inhibitory changes can be pro- or antiepileptic. The general consensus seems to be that perisomatic inhibition, the type of inhibition generally thought of as being responsible for the synchronization of principal cells, is preserved in epileptic brain.76 In contrast, the interneurons innervating the dendrites of principal cells and of other interneurons show considerably more morphologic plasticity. These GABAergic cells, which also contain various neuropeptide, are lost, sprout, and alter their synaptic contacts, thus leading to a much altered diversity of innervation in epilepsy.76 The functional significance of these plastic changes are not well understood; one also needs to keep in mind that the morphologic plasticity is accompanied by altered GABAA receptor expression and molecular composition in epileptic brain.67,105
If inhibitory postsynaptic events can lead to synchrony, it is not surprising to find enhanced synaptic activation of GABAA receptors in some models of epilepsy.7,33,89 In some epilepsy models, such as FS, the synchronizing effect of an enhanced inhibition is augmented by an increase in other voltage-gated conductances. The parallel increase in the hyperpolarization activated Ih current helps the simultaneous recovery of the principal cells from inhibition and promotes their bursting.28
Antiepileptic Drug Action and GABAA Receptors
There are a number of AEDs whose actions are mediated by an interaction with GABA receptor system.72 Postsynaptic GABAA receptor currents are enhanced by barbiturates and benzodiazepines. Presynaptic GABA release is likely modified by the AED vigabatrin. The uptake of GABA has been shown to be modified by compounds such as tiagabine. Finally, valproic acid has been suggested either to enhance the
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release of GABA or to enhance postsynaptic GABA responses. Gabapentin was designed specifically to be a GABA analog that penetrated into the CNS. Although there are some suggestions that gabapentin may react with the GABA system to enhance GABAergic inhibition, evidence remains uncertain. Blockage of the low-threshold T Ca channel–mediated Ca spike evoked by GABAergic IPSPs from the nucleus reticularis thal- ami (NRT) may be a mechanism of action of many drugs effective against generalized absence seizures.34 Spike-and-wave discharges in experimental animals are blocked by GABAB receptor antagonists,49,66,102 but the relevance of this observation to treatment of generalized absence seizures in humans remains uncertain. Finally, felbamate has been demonstrated to enhance GABAergic inhibition, although this result requires confirmation.
Summary and Conclusions
It is quite clear that a focal reduction of GABA-mediated inhibition will produce partial epilepsy and, when produced systemically, will cause generalized seizures. Conversely, drugs that enhance GABAergic inhibition are antiepileptic. Alterations in GABAergic inhibition may be produced by several mechanisms. First, changes could occur in the morphology, axonal arborization, number, excitability, or innervation of inhibitory interneurons. Second, there could be modification of GABA release produced from individual synaptic terminals due to alterations in GABA stores or modifications of GABA-synthesizing or GABA-metabolizing enzymes. Third, a change could occur in the number, distribution, or composition or properties of postsynaptic GABAA receptors, and of the reversal potential of the permeant ions through these receptors. Fourth, mutations may be present in GABAA receptor subunits that alter the trafficking or and/or function of GABAA receptors. Finally, modified forms of GABAergic inhibition are present during development, suggesting the possibility that altered GABAergic inhibition not only is important in epilepsy but may have differential importance during development. The functions of GABAergic system are complex; they include inhibition, excitation, and synchrony. Therefore, in situ, it is difficult to predict how specific alterations in the function of GABAergic mechanisms, some of them genetically determined,85,107 will produce epilepsy. Nevertheless, newly developed techniques for studying the specific identity of GABA receptors, properties of native GABA receptors, and behavior of GABAergic neurons and synapses should allow a more detailed understanding of the role of GABA-mediated synaptic transmission in epilepsies.
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