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Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy☆
Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsyq G Sperk, A Wieselthaler-Hoelzl, and M Drexel, Medical University Innsbruck, Innsbruck, Austria Ó 2017 Elsevier Inc. All rights reserved.
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Introduction Background Methods Results Studies in Animal Models of Epilepsy Studies in the Human TLE Hippocampus Future Goals Acknowledgment Further Reading
Introduction g-Amino butyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts through two classes of receptors: the GABAA receptors that are ligand-operated ion channels, and the G-protein-coupled metabotropic GABAB receptors. GABAergic neurons are ubiquitously distributed and encompass a fundamental role in processing and integration of all brain functions. A great number of differently assembled GABAA receptors that are differentially distributed throughout the brain have been observed. It has been suggested that dysfunction of the GABAergic system may have a crucial role in the propagation of acute seizures and in the manifestation of epilepsy syndromes. Indeed, blockade of the fast inhibitory GABAA receptors by bicuculline, pentylenetetrazole, or picrotoxin causes severe motor seizures in experimental animals, and mutant mice lacking the synthetic enzyme for GABA production (glutamate decarboxylasedGAD) or expressing mutations of certain GABAA receptor subunits are prone to spontaneous epileptic seizures. GABAA receptors are central to GABAergic transmission. Their roles are seen not only at the synaptic level, but also in volume transmission, synchronization of neurons, and generation of network oscillations. The multiple molecular forms in which GABAA receptors are assembled in the membrane (discussed in the following sections) indicate highly diverse physiological properties of these receptors. There is growing evidence that mutations in certain GABAA receptor subunits can lead to altered brain functions, including epilepsies. Further, it has been proposed that an initial severe seizure event (status epilepticus) may alter the expression profile of individual GABAA receptor subunits, causing different assembly and thus altered function of GABAA receptors in certain brain areas. These changes could influence (support or inhibit) epileptogenesis. Acute or recurrent seizures may also influence the sensitivity of GABAA receptors to anticonvulsant drugs (such as barbiturates or benzodiazepines) known to act through the GABAA receptor. Changes in the expression of individual GABAA receptor subunits in subfields of the hippocampus may reflect altered receptor function in this brain areada region thought to be involved often in epileptogenesis or drug resistance. The cellular sites at which such changes take place may be critical for their roles in these processes.
Background The GABAA receptor is a member of a superfamily of ligand-gated ion channels that also includes the nicotinic acetylcholine receptor, the 5-hydroxytryptamine type 3 receptor, and the glycine receptor. GABAA receptors are composed of five subunits forming a chloride channel. So far, a total of six a, four b, three g, one d, one ε, one p, one q, and three r subunits of GABAA receptors have been identified in the mammalian nervous system. This subunit heterogeneity is further increased by alternative exon splicing of the premRNA, which generates two forms of the g2 subunit (g2S and g2L), from one gene, that are differently distributed in the brain. Splice variants have also been detected for other subunits, and subunit homologues have been identified in nonmammalian species. The majority of native GABAA receptors, however, appear to be composed of two a, two b, and one g subunit. A d subunit, or less frequently, an ε or p subunit, can replace the g subunit in the GABAA receptor, whereas the q subunit may be able to replace a b subunit. A given GABAA receptor complex may consist of various a and b subunits; for example, a functioning receptor may contain two a1 subunits, two b2 and one g2; or one a1, one a2, two b2 and one g2 subunit; or any pair of existing a subunits.
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Change History: November 2016. G Sperk revised Figure 1 of this article.
Reference Module in Neuroscience and Biobehavioral Psychology
http://dx.doi.org/10.1016/B978-0-12-809324-5.00229-7
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Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy
Also, the b subunits within a given GABAA receptor may originate from the same or from different genes. The most abundant subunits in the rodent brain are a1 to a5, b1 to b3, g2, and d. The binding site of GABA to the GABAA receptor is located at the interface of an a and a b subunit. Binding of benzodiazepines occurs at the interface of an a and a g subunit. Benzodiazepines bind with high affinity to receptors containing a1, and with somewhat lower affinity to those containing an a2, a3, or an a5 subunit (but not to a4 or a6 containing receptors), and only the g2 (no other g subunit or d) subunit can mediate the benzodiazepine-induced increase in affinity to GABA. Barbiturates exert their action after binding at the chloride channel pore, and seem to bind also to a4, a6, and/or d subunit containing receptors. GABAA receptors containing only a and b subunits have been described in hippocampal pyramidal cells. They do not respond to benzodiazepines but are highly sensitive to Zn2þ ions. The subunit composition of GABAA receptors also critically determines their physiological and pharmacological functions. GABA exerts its action by opening a chloride channel. Benzodiazepines and barbiturates can increase the affinity for GABA, thus increasing the frequency or duration of channel opening, respectively. The direction of the chloride flux is dependent on the chloride gradient at the cell membrane. In the mature brain, the chloride transporters (such as the potassium-dependent chloride transporter KCC2) extrude chloride from the cell, allowing in an inward chloride flux and a hyperpolarization after channel opening. In the immature brain, the expression level of KCC2 is low; instead, a sodium–potassium-dependent cotransporter NKCC1 is expressed that transports chloride into the neuron. The resulting high intracellular chloride concentration drives an outward chloride flux and depolarization of the cell upon stimulation of GABAA receptors. Altered chloride transporter expression or function has been associated with genetic forms of epilepsy. It has also been proposed that expression of KCC2 may be altered in certain neurons and lead to impaired inhibition. In the adult mammalian CNS, synaptic transmission through GABA synapses is characterized by fast and precisely timed inhibitory activity in the form of inhibitory postsynaptic currents that mediate phasic inhibition. A second form of inhibitory transmission, resulting from GABA diffusion out of the synaptic cleft and activating GABAA receptors located outside the synapse, has been characterized recently and is referred to as tonic inhibition. Tonic conductance has been found in a large variety of principal neurons and interneurons, including those found in the cerebellum, cortex, hippocampus, thalamus, and spinal cord. Depending on the brain region, certain subunits such as a4, a5, and d preferentially constitute such extrasynaptic receptors and mediate tonic inhibition.
Methods Using histochemical methods, we investigated changes in the expression of GABAA receptor subunits after systemic application of kainic acid (KA; 10 mg kg1, i.p.) in rats. In this model, an initial status epilepticus leads, after a “latent phase,” to epilepsy characterized by recurrent spontaneous seizure activity. In order to differentiate changes during the acute KA-induced status epilepticus from changes during epileptogenesis (i.e., during the latent phase) and in chronic epilepsy, we investigated GABAA receptor subunit expression at 24 h, 8 days, and 1–3 months after the initial kainate-induced status epilepticus. Expression was studied at the mRNA and protein level using in situ hybridization and immunohistochemistry, respectively. For in situ hybridization, we used 20 mm thick cryotome-cut sections from snap frozen brains. We incubated them with synthetic oligo-DNA probes labeled by reaction with 35S-dATP in presence of terminal deoxynucleotidyl transferase. For immunohistochemistry, we used 40 mm thick sections from 4% paraformaldehyde fixed brains and processed them by the indirect peroxidase method. Antibodies specific for individual GABAA receptor subunits were supplied by Prof. Werner Sieghart, Brain Research Center, Medical University Vienna. For semiquantitative evaluation of changes in the expression of individual subunits, we performed densitometry on film autoradiographs and on immunolabeled brain sections. Studies in human brain tissue were done in collaboration with Prof. Thomas Czech, Department of Neurosurgery, Medical University of Vienna, who supplied us with specimens collected from TLE patients during routine surgery (after approval of the Institutional board of the Medical University of Vienna). The specimens were either snap-frozen or immersion fixed in 4% paraformaldehyde. For in situ hybridization, the method described earlier was used, applying probes complementary to the respective human RNA sequences. For immunohistochemistry in the human specimens, we performed the slightly more sensitive avidin– biotin method (ABC-kit by Boehringer, Ingelheim, Germany) after treating the sections with target retrieval solution; the same antibodies used for investigating rat brains were also applied for the human tissue.
Results Studies in Animal Models of Epilepsy In the studies using the KA rat model of temporal lobe epilepsy (TLE), we observed marked losses in GABAA receptors that were obviously related to neuronal cell losses (mainly in sectors CA1 and CA3 of the Ammon’s horn, hilar interneurons, and the subiculum). This result is consistent with losses in GABAA receptor binding sites in the whole hippocampus. These prominent neurodegeneration-related changes suggest marked alterations in GABAA mediated transmission. In addition, there were also significant changes in GABAA receptor subunit expression in the molecular layer of the dentate gyrusda structure that remains relatively intact in this animal model. Most conspicuous in this region was a rapid decrease in subunit d (Fig. 1). Indeed, downregulation of
Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy
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Figure 1 Changes in immunoreactivites of GABAA receptor subunits in rats with spontaneous seizures, 3 months after status epilepticus induced by injection of kainic acid. Note the decrease in d subunit immunoreactivity, contrasted by increases in subunits a2, a4, b3, and g2. Apparent increase in subunit a1 immunoreactivity in the epileptic rats is mainly present in interneurons.
subunit d was a consistent observation in most animal models, including the kindling model, recurrent seizures induced by electrical stimulation, and the mouse model of pilocarpine-induced status epilepticus and epileptogenesis. Interestingly, expression of the a4 subunit (a frequent partner of the d subunit in the GABAA receptor complex) is not reduced but increased in the KA model of the rat (Fig. 1) and in the pilocarpine model of the mouse. We also observed a clear-cut reduction in the expression of subunit a5 in the dentate gyrus, as well as in the Ammon’s horndalthough these latter changes were obscured by the severe neurodegeneration in this area after kainate-induced status epilepticus. Houser et al. reported less severe damage of the pyramidal cell layer in the mouse pilocarpine model, and were able to demonstrate a reduction in the expression of subunit a5. They also discussed the possibility that these observed decreases in d subunit expression (and possibly in a5 subunit expression) are associated with reduced tonic inhibition in the epileptic mice. In contrast to receptors containing the d subunit, receptors containing the g2 subunit are primarily associated with synaptic or phasic inhibition. In addition, the g2 subunit mediates the sensitivity of the receptor to benzodiazepines, and the g2 subunit is importantly involved in the targeting of the GABAA receptor complex to the cell membrane. In our initial experiments, we observed a marked and rapiddbut transientdreduction in g2 mRNA levels in the dentate gyrus (6 h) after kainate injection. In the pyramidal layer, a persistent reduction (presumably related to cell damage) was seen. Using immunohistochemistry, we observed conspicuous labeling for subunit g2-immunoreactivity of dendrites of pyramidal cells and interneurons, lasting up to 1 month. We interpreted these data as a reflection of overexpression of this subunit. Such an increase in g2-immunoreactivity can also be seen in the dentate molecular layer up to 4 months after KA injection (Fig. 1). Recent studies by Kapur et al. and Wasterlain et al., have demonstrated rapid internalization of g2 subunit protein following status epilepticus. Using immunofluorescence, they demonstrated as early as 1 h after seizure induction, a translocation of subunit g2-immunoreactivity from the cell membrane to the cytoplasm of the respective neurons. Functionally, this translocation may have considerable consequences. First, it may result in receptors devoid of g2 subunit. Such receptors (if active at all in vivo) would have strongly altered physiological and pharmacological functions. As mentioned earlier, receptors consisting only of a and b subunits have high sensitivity to the ion Zn2þ that can be released from mossy fibers; this sensitivity to mossy fiber-released zinc could result in impaired GABA-mediated inhibition. Receptors lacking the g2 subunit are also resistant to treatment with benzodiazepines. And, since the g2 subunit is important for anchoring the GABAA receptor complex in the cell membrane, internalization of the subunit g2 may also limit the survival of the whole receptor in the cell membrane. This translocation phenomenon does not affect all GABAA receptors and/or is not permanent, since even 3 month after kainateinduced status epilepticus we observed high levels of g2-immunoreactivty in granule cells and pyramidal cells (Fig. 1). Consistent with these findings, Houser et al. also observed an increase in g2-immunoreactivty in the chronic phase after pilocarpine-induced status epilepticus in the mouse. In the normal molecular layer of the dentate gyrus of the rat, the a2 subunit is thought to be the most prevalent. The a1 subunit appears to be preferentially expressed in interneurons of the hippocampus. Expression levels of subunit a4 appear to be low in the rat hippocampus (compared with the thalamus for example), but more abundant in the dentate molecular layer than in the strata oriens and radiatum of the hippocampus proper. In contrast to the human hippocampus, there is no a3 subunit expressed in rat
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Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy
dentate granule cells, and little in interneurons. Subunit a5 is more extensively expressed in pyramidal cells than in granule cells of the rat. Again, this pattern is somewhat different from the expression pattern in humans, where equally high levels of subunit can be seen in dendrites of granule cells and pyramidal cells. Expression patterns of individual GABAA receptor subunits are also slightly different in the mouse. As noted earlier, subunits a4 and a5 are presumably related to tonic inhibition. The subunit a4, expressed only slightly in normal brain, is slightly upregulated and subunit a5 downregulated in the dentate gyrus, notable at the later intervals after kainate injection. At this time, in situ hybridization and immunohistochemistry also show increased expression of subunits a1 and a2 in rats treated with KA or after electrically induced status epilepticus. All three b subunits are expressed in principal cell layers of the rat hippocampus. Expression of subunit b3 appears to be the strongest, that of b1 weakest. Subunit b2 is even more abundant in hippocampal interneurons (coexistent with subunit a1 and g2, less with d). In most TLE models, notably in the KA model, there is some upregulation of all three b subunits (notably of b2 and b3). Taken together, these data indicate increased expression of GABAA receptors in the dentate gyrus following kainate-induced status epilepticus; however, there is a clear shift from receptors associated with tonic inhibition to receptors associated with phasic inhibition. Increased concentrations of GABAA receptors in the dentate hilus were reported more than 20 years ago (using ligands for either GABA or benzodiazepine-binding sites)deven in the face of neurodegeneration related receptor losses in Ammon’s horn). Why subunits related to phasic inhibition are preferentially lost remains to be determined. One possibility is that GABA produced at high concentrations in granule cells may be released extrasynaptically onto granule cell dendrites, and may then contribute to downregulation of extrasynaptic receptors.
Studies in the Human TLE Hippocampus There are only a few studies on the expression of GABAA receptor subunits in specimens removed by surgery in TLE patients. Difficulties experienced in such studies include the availability of valid antibodies (specifically detecting the human subunits) and difficulties in comparing fresh (surgically removed) tissue with post mortem control tissue (in which mRNA and proteins may be degraded to varying extents). The first extensive study was published by Loup et al., who showed upregulation of subunits a1, a2, b2/3, and g2 subunits in granule cells and granule cell dendrites, downregulation of these subunits in the subgranular zone, and loss of a1 subunit immunoreactivity in interneurons of the hippocampus proper. Using in situ hybridization and immunohistochemistry, we also observed increases in all three b subunits, notably b1 and b3, in the molecular layer, the hippocampus proper
Figure 2 Changes in immunoreactivites of GABAA receptor subunits in TLE patients. Note the increased expression of subunit b3 mRNA (B vs. A) and immunoreactivity (D vs. C) in the dentate molecular layer and in the subiculum and the increase in a3 immunoreactivity in the subiculum (F vs. E).
Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy
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and subiculum. In the subiculum (which is highly preserved in human TLE), we observed increases in subunits a3 and g2, but losses in subunits a1, a3, and g2 in Ammon’s horn (Fig. 2). Taken together, these data indicate that in human TLE there are marked changes in the expression and assembly of GABAA receptors, suggesting remodeling of GABAergic transmission. Subfields of the hippocampus that are less vulnerable to seizure-induced neurodegeneration, such as the subiculum and dentate granule cell layer, display upregulation of various subunits, indicating facilitation of GABAergic transmission. Striking and difficult to explain is the especially pronounced expression of b subunitsdwhich could result in imbalanced assembly of GABAA receptors (such as receptors containing three b and two a and no g subunit, or three b, one a and one g2 or d subunit). Studies with recombinant receptors indicate that they would not bind GABA with high affinity or capacitydand if they lacked g2, they may not be affected by benzodiazepines but would be sensitive to Zn2þ. Due to the difficult comparison with postmortem controls, we have so far been unable to obtain evidence for downregulation of subunits d or a5 in the human tissue, which would (in analogy to the rat) be indicative of reduced extrasynaptic tonic inhibition in the epileptic brain. Appropriate human controls are essential since, as mentioned earlier, the distribution (and thus the function) of certain GABAA receptor subunits is different in the human hippocampus compared to that in the rat.
Future Goals It will be crucial to continue work in the human brain, since the physiological properties of single GABAA receptor may vary much depending on the circuitry in which they are integrated and less critically on their actual subunit composition. Detailed knowledge about the pathology of altered receptor expression will be important for developing appropriate drugs targeting individual GABAA receptor subtypes (e.g., acting through the benzodiazepine binding site). A limitation of our studies is that although we have learned something about changes in the GABAA receptor subunit distribution, we have yet to determine the details about changes in actual receptor composition. We are currently performing neurochemical studies, using subtractive purification methods in microdissected hippocampal subfields from KA rats, to approach this question.
Acknowledgment The work was supported by the Austrian Science Fund (projects P19464, P26680) and by the EC contract number LSH-CT-2006–037315 (EPICURE) FP6dThematic priority LIFESCIHEALTH.
Further Reading Bowery, N.G., 1993. GABAB receptor pharmacology. Annu. Rev. Pharmacol. Toxicol. 33, 109–147. Farrant, M., Nusser, Z., 2005. Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat. Rev. Neurosci. 6, 215–229. Glykys, J., Mody, I., 2007. Activation of GABAA receptors: views from outside the synaptic cleft. Neuron 56, 763–770. Goodkin, H.P., Joshi, S., Mtchedlishvili, Z., Brar, J., Kapur, J., 2008. Subunit-specific trafficking of GABAA receptors during status epilepticus. J. Neurosci. Off. J. Soc. Neurosci. 28, 2527–2538. Houser, C.R., Esclapez, M., 2003. Downregulation of the alpha5 subunit of the GABAA receptor in the pilocarpine model of temporal lobe epilepsy. Hippocampus 13, 633–645. Huberfeld, G., Wittner, L., Clemenceau, S., et al., 2007. Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy. J. Neurosci. Off. J. Soc. Neurosci. 27, 9866–9873. Loup, F., Wieser, H.G., Yonekawa, Y., Aguzzi, A., Fritschy, J.M., 2000. Selective alterations in GABA A receptor subtypes in human temporal lobe epilepsy. J. Neurosci. Off. J. Soc. Neurosci. 20, 5401–5419. Mody, I., De Koninck, Y., Otis, T.S., Soltesz, I., 1994. Bridging the cleft at GABA synapses in the brain. Trends Neurosci. 17, 517–525. Naylor, D.E., Liu, H., Wasterlain, C.G., 2005. Trafficking of GABAA receptors, loss of inhibition, and a mechanism for pharmacoresistance in status epilepticus. J. Neurosci. Off. J. Soc. Neurosci. 25, 7724–7733. Noebels, J.L., 2003. The biology of epilepsy genes. Annu. Rev. Neurosci. 26, 599–625. Nusser, Z., Hajos, N., Somogyi, P., Mody, I., 1998. Increased number of synaptic GABAA receptors underlies potentiation at hippocampal inhibitory synapses. Nature 395, 172–177. Palma, E., Amici, M., Sobrero, F., et al., 2006. Anomalous levels of Cl transporters in the hippocampal subiculum from temporal lobe epilepsy patients make GABA excitatory. Proc. Natl. Acad. Sci. U.S.A. 103, 8465–8468. Peng, Z., Huang, C.S., Stell, B.M., Mody, I., Houser, C.R., 2004. Altered expression of the delta subunit of the GABAA receptor in a mouse model of temporal lobe epilepsy. J. Neurosci. Off. J. Soc. Neurosci. 24, 8629–8639. Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., Sperk, G., 2000. GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815–850. Pirker, S., Schwarzer, C., Czech, T., et al., 2003. Increased expression of GABAA receptor beta-subunits in the hippocampus of patients with temporal lobe epilepsy. J. Neuropathol. Exp. Neurol. 62, 820–834. Schwarzer, C., Tsunashima, K., Wanzenbock, C., Fuchs, K., Sieghart, W., Sperk, G., 1997. GABAA receptor subunits in the rat hippocampus II: altered distribution in kainic acidinduced temporal lobe epilepsy. Neuroscience 80, 1001–1017. Sieghart, W., Sperk, G., 2002. Subunit composition, distribution and function of GABAA receptor subtypes. Curr. Top. Med. Chem. 2, 795–816. Sperk, G., 1994. Kainic acid seizures in the rat. Prog. Neurobiol. 42, 1–32. Sperk, G., Furtinger, S., Schwarzer, C., Pirker, S., 2004. GABA and its receptors in epilepsy. Adv. Exp. Med. Biol. 548, 92–103. Tsunashima, K., Schwarzer, C., Kirchmair, E., Sieghart, W., Sperk, G., 1997. GABAA receptor subunits in the rat hippocampus III: altered messenger RNA expression in kainic acidinduced epilepsy. Neuroscience 80, 1019–1132. Zhang, N., Wei, W., Mody, I., Houser, C.R., 2007. Altered localization of GABAA receptor subunits on dentate granule cell dendrites influences tonic and phasic inhibition in a mouse model of epilepsy. J. Neurosci. Off. J. Soc. Neurosci. 27, 7520–7531.
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Introduction Background Methods Results Studies in Animal Models of Epilepsy Studies in the Human TLE Hippocampus Future Goals Acknowledgment Further Reading
Introduction g-Amino butyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts through two classes of receptors: the GABAA receptors that are ligand-operated ion channels, and the G-protein-coupled metabotropic GABAB receptors. GABAergic neurons are ubiquitously distributed and encompass a fundamental role in processing and integration of all brain functions. A great number of differently assembled GABAA receptors that are differentially distributed throughout the brain have been observed. It has been suggested that dysfunction of the GABAergic system may have a crucial role in the propagation of acute seizures and in the manifestation of epilepsy syndromes. Indeed, blockade of the fast inhibitory GABAA receptors by bicuculline, pentylenetetrazole, or picrotoxin causes severe motor seizures in experimental animals, and mutant mice lacking the synthetic enzyme for GABA production (glutamate decarboxylasedGAD) or expressing mutations of certain GABAA receptor subunits are prone to spontaneous epileptic seizures. GABAA receptors are central to GABAergic transmission. Their roles are seen not only at the synaptic level, but also in volume transmission, synchronization of neurons, and generation of network oscillations. The multiple molecular forms in which GABAA receptors are assembled in the membrane (discussed in the following sections) indicate highly diverse physiological properties of these receptors. There is growing evidence that mutations in certain GABAA receptor subunits can lead to altered brain functions, including epilepsies. Further, it has been proposed that an initial severe seizure event (status epilepticus) may alter the expression profile of individual GABAA receptor subunits, causing different assembly and thus altered function of GABAA receptors in certain brain areas. These changes could influence (support or inhibit) epileptogenesis. Acute or recurrent seizures may also influence the sensitivity of GABAA receptors to anticonvulsant drugs (such as barbiturates or benzodiazepines) known to act through the GABAA receptor. Changes in the expression of individual GABAA receptor subunits in subfields of the hippocampus may reflect altered receptor function in this brain areada region thought to be involved often in epileptogenesis or drug resistance. The cellular sites at which such changes take place may be critical for their roles in these processes.
Background The GABAA receptor is a member of a superfamily of ligand-gated ion channels that also includes the nicotinic acetylcholine receptor, the 5-hydroxytryptamine type 3 receptor, and the glycine receptor. GABAA receptors are composed of five subunits forming a chloride channel. So far, a total of six a, four b, three g, one d, one ε, one p, one q, and three r subunits of GABAA receptors have been identified in the mammalian nervous system. This subunit heterogeneity is further increased by alternative exon splicing of the premRNA, which generates two forms of the g2 subunit (g2S and g2L), from one gene, that are differently distributed in the brain. Splice variants have also been detected for other subunits, and subunit homologues have been identified in nonmammalian species. The majority of native GABAA receptors, however, appear to be composed of two a, two b, and one g subunit. A d subunit, or less frequently, an ε or p subunit, can replace the g subunit in the GABAA receptor, whereas the q subunit may be able to replace a b subunit. A given GABAA receptor complex may consist of various a and b subunits; for example, a functioning receptor may contain two a1 subunits, two b2 and one g2; or one a1, one a2, two b2 and one g2 subunit; or any pair of existing a subunits.
q
Change History: November 2016. G Sperk revised Figure 1 of this article.
Reference Module in Neuroscience and Biobehavioral Psychology
http://dx.doi.org/10.1016/B978-0-12-809324-5.00229-7
1
2
Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy
Also, the b subunits within a given GABAA receptor may originate from the same or from different genes. The most abundant subunits in the rodent brain are a1 to a5, b1 to b3, g2, and d. The binding site of GABA to the GABAA receptor is located at the interface of an a and a b subunit. Binding of benzodiazepines occurs at the interface of an a and a g subunit. Benzodiazepines bind with high affinity to receptors containing a1, and with somewhat lower affinity to those containing an a2, a3, or an a5 subunit (but not to a4 or a6 containing receptors), and only the g2 (no other g subunit or d) subunit can mediate the benzodiazepine-induced increase in affinity to GABA. Barbiturates exert their action after binding at the chloride channel pore, and seem to bind also to a4, a6, and/or d subunit containing receptors. GABAA receptors containing only a and b subunits have been described in hippocampal pyramidal cells. They do not respond to benzodiazepines but are highly sensitive to Zn2þ ions. The subunit composition of GABAA receptors also critically determines their physiological and pharmacological functions. GABA exerts its action by opening a chloride channel. Benzodiazepines and barbiturates can increase the affinity for GABA, thus increasing the frequency or duration of channel opening, respectively. The direction of the chloride flux is dependent on the chloride gradient at the cell membrane. In the mature brain, the chloride transporters (such as the potassium-dependent chloride transporter KCC2) extrude chloride from the cell, allowing in an inward chloride flux and a hyperpolarization after channel opening. In the immature brain, the expression level of KCC2 is low; instead, a sodium–potassium-dependent cotransporter NKCC1 is expressed that transports chloride into the neuron. The resulting high intracellular chloride concentration drives an outward chloride flux and depolarization of the cell upon stimulation of GABAA receptors. Altered chloride transporter expression or function has been associated with genetic forms of epilepsy. It has also been proposed that expression of KCC2 may be altered in certain neurons and lead to impaired inhibition. In the adult mammalian CNS, synaptic transmission through GABA synapses is characterized by fast and precisely timed inhibitory activity in the form of inhibitory postsynaptic currents that mediate phasic inhibition. A second form of inhibitory transmission, resulting from GABA diffusion out of the synaptic cleft and activating GABAA receptors located outside the synapse, has been characterized recently and is referred to as tonic inhibition. Tonic conductance has been found in a large variety of principal neurons and interneurons, including those found in the cerebellum, cortex, hippocampus, thalamus, and spinal cord. Depending on the brain region, certain subunits such as a4, a5, and d preferentially constitute such extrasynaptic receptors and mediate tonic inhibition.
Methods Using histochemical methods, we investigated changes in the expression of GABAA receptor subunits after systemic application of kainic acid (KA; 10 mg kg1, i.p.) in rats. In this model, an initial status epilepticus leads, after a “latent phase,” to epilepsy characterized by recurrent spontaneous seizure activity. In order to differentiate changes during the acute KA-induced status epilepticus from changes during epileptogenesis (i.e., during the latent phase) and in chronic epilepsy, we investigated GABAA receptor subunit expression at 24 h, 8 days, and 1–3 months after the initial kainate-induced status epilepticus. Expression was studied at the mRNA and protein level using in situ hybridization and immunohistochemistry, respectively. For in situ hybridization, we used 20 mm thick cryotome-cut sections from snap frozen brains. We incubated them with synthetic oligo-DNA probes labeled by reaction with 35S-dATP in presence of terminal deoxynucleotidyl transferase. For immunohistochemistry, we used 40 mm thick sections from 4% paraformaldehyde fixed brains and processed them by the indirect peroxidase method. Antibodies specific for individual GABAA receptor subunits were supplied by Prof. Werner Sieghart, Brain Research Center, Medical University Vienna. For semiquantitative evaluation of changes in the expression of individual subunits, we performed densitometry on film autoradiographs and on immunolabeled brain sections. Studies in human brain tissue were done in collaboration with Prof. Thomas Czech, Department of Neurosurgery, Medical University of Vienna, who supplied us with specimens collected from TLE patients during routine surgery (after approval of the Institutional board of the Medical University of Vienna). The specimens were either snap-frozen or immersion fixed in 4% paraformaldehyde. For in situ hybridization, the method described earlier was used, applying probes complementary to the respective human RNA sequences. For immunohistochemistry in the human specimens, we performed the slightly more sensitive avidin– biotin method (ABC-kit by Boehringer, Ingelheim, Germany) after treating the sections with target retrieval solution; the same antibodies used for investigating rat brains were also applied for the human tissue.
Results Studies in Animal Models of Epilepsy In the studies using the KA rat model of temporal lobe epilepsy (TLE), we observed marked losses in GABAA receptors that were obviously related to neuronal cell losses (mainly in sectors CA1 and CA3 of the Ammon’s horn, hilar interneurons, and the subiculum). This result is consistent with losses in GABAA receptor binding sites in the whole hippocampus. These prominent neurodegeneration-related changes suggest marked alterations in GABAA mediated transmission. In addition, there were also significant changes in GABAA receptor subunit expression in the molecular layer of the dentate gyrusda structure that remains relatively intact in this animal model. Most conspicuous in this region was a rapid decrease in subunit d (Fig. 1). Indeed, downregulation of
Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy
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Figure 1 Changes in immunoreactivites of GABAA receptor subunits in rats with spontaneous seizures, 3 months after status epilepticus induced by injection of kainic acid. Note the decrease in d subunit immunoreactivity, contrasted by increases in subunits a2, a4, b3, and g2. Apparent increase in subunit a1 immunoreactivity in the epileptic rats is mainly present in interneurons.
subunit d was a consistent observation in most animal models, including the kindling model, recurrent seizures induced by electrical stimulation, and the mouse model of pilocarpine-induced status epilepticus and epileptogenesis. Interestingly, expression of the a4 subunit (a frequent partner of the d subunit in the GABAA receptor complex) is not reduced but increased in the KA model of the rat (Fig. 1) and in the pilocarpine model of the mouse. We also observed a clear-cut reduction in the expression of subunit a5 in the dentate gyrus, as well as in the Ammon’s horndalthough these latter changes were obscured by the severe neurodegeneration in this area after kainate-induced status epilepticus. Houser et al. reported less severe damage of the pyramidal cell layer in the mouse pilocarpine model, and were able to demonstrate a reduction in the expression of subunit a5. They also discussed the possibility that these observed decreases in d subunit expression (and possibly in a5 subunit expression) are associated with reduced tonic inhibition in the epileptic mice. In contrast to receptors containing the d subunit, receptors containing the g2 subunit are primarily associated with synaptic or phasic inhibition. In addition, the g2 subunit mediates the sensitivity of the receptor to benzodiazepines, and the g2 subunit is importantly involved in the targeting of the GABAA receptor complex to the cell membrane. In our initial experiments, we observed a marked and rapiddbut transientdreduction in g2 mRNA levels in the dentate gyrus (6 h) after kainate injection. In the pyramidal layer, a persistent reduction (presumably related to cell damage) was seen. Using immunohistochemistry, we observed conspicuous labeling for subunit g2-immunoreactivity of dendrites of pyramidal cells and interneurons, lasting up to 1 month. We interpreted these data as a reflection of overexpression of this subunit. Such an increase in g2-immunoreactivity can also be seen in the dentate molecular layer up to 4 months after KA injection (Fig. 1). Recent studies by Kapur et al. and Wasterlain et al., have demonstrated rapid internalization of g2 subunit protein following status epilepticus. Using immunofluorescence, they demonstrated as early as 1 h after seizure induction, a translocation of subunit g2-immunoreactivity from the cell membrane to the cytoplasm of the respective neurons. Functionally, this translocation may have considerable consequences. First, it may result in receptors devoid of g2 subunit. Such receptors (if active at all in vivo) would have strongly altered physiological and pharmacological functions. As mentioned earlier, receptors consisting only of a and b subunits have high sensitivity to the ion Zn2þ that can be released from mossy fibers; this sensitivity to mossy fiber-released zinc could result in impaired GABA-mediated inhibition. Receptors lacking the g2 subunit are also resistant to treatment with benzodiazepines. And, since the g2 subunit is important for anchoring the GABAA receptor complex in the cell membrane, internalization of the subunit g2 may also limit the survival of the whole receptor in the cell membrane. This translocation phenomenon does not affect all GABAA receptors and/or is not permanent, since even 3 month after kainateinduced status epilepticus we observed high levels of g2-immunoreactivty in granule cells and pyramidal cells (Fig. 1). Consistent with these findings, Houser et al. also observed an increase in g2-immunoreactivty in the chronic phase after pilocarpine-induced status epilepticus in the mouse. In the normal molecular layer of the dentate gyrus of the rat, the a2 subunit is thought to be the most prevalent. The a1 subunit appears to be preferentially expressed in interneurons of the hippocampus. Expression levels of subunit a4 appear to be low in the rat hippocampus (compared with the thalamus for example), but more abundant in the dentate molecular layer than in the strata oriens and radiatum of the hippocampus proper. In contrast to the human hippocampus, there is no a3 subunit expressed in rat
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Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy
dentate granule cells, and little in interneurons. Subunit a5 is more extensively expressed in pyramidal cells than in granule cells of the rat. Again, this pattern is somewhat different from the expression pattern in humans, where equally high levels of subunit can be seen in dendrites of granule cells and pyramidal cells. Expression patterns of individual GABAA receptor subunits are also slightly different in the mouse. As noted earlier, subunits a4 and a5 are presumably related to tonic inhibition. The subunit a4, expressed only slightly in normal brain, is slightly upregulated and subunit a5 downregulated in the dentate gyrus, notable at the later intervals after kainate injection. At this time, in situ hybridization and immunohistochemistry also show increased expression of subunits a1 and a2 in rats treated with KA or after electrically induced status epilepticus. All three b subunits are expressed in principal cell layers of the rat hippocampus. Expression of subunit b3 appears to be the strongest, that of b1 weakest. Subunit b2 is even more abundant in hippocampal interneurons (coexistent with subunit a1 and g2, less with d). In most TLE models, notably in the KA model, there is some upregulation of all three b subunits (notably of b2 and b3). Taken together, these data indicate increased expression of GABAA receptors in the dentate gyrus following kainate-induced status epilepticus; however, there is a clear shift from receptors associated with tonic inhibition to receptors associated with phasic inhibition. Increased concentrations of GABAA receptors in the dentate hilus were reported more than 20 years ago (using ligands for either GABA or benzodiazepine-binding sites)deven in the face of neurodegeneration related receptor losses in Ammon’s horn). Why subunits related to phasic inhibition are preferentially lost remains to be determined. One possibility is that GABA produced at high concentrations in granule cells may be released extrasynaptically onto granule cell dendrites, and may then contribute to downregulation of extrasynaptic receptors.
Studies in the Human TLE Hippocampus There are only a few studies on the expression of GABAA receptor subunits in specimens removed by surgery in TLE patients. Difficulties experienced in such studies include the availability of valid antibodies (specifically detecting the human subunits) and difficulties in comparing fresh (surgically removed) tissue with post mortem control tissue (in which mRNA and proteins may be degraded to varying extents). The first extensive study was published by Loup et al., who showed upregulation of subunits a1, a2, b2/3, and g2 subunits in granule cells and granule cell dendrites, downregulation of these subunits in the subgranular zone, and loss of a1 subunit immunoreactivity in interneurons of the hippocampus proper. Using in situ hybridization and immunohistochemistry, we also observed increases in all three b subunits, notably b1 and b3, in the molecular layer, the hippocampus proper
Figure 2 Changes in immunoreactivites of GABAA receptor subunits in TLE patients. Note the increased expression of subunit b3 mRNA (B vs. A) and immunoreactivity (D vs. C) in the dentate molecular layer and in the subiculum and the increase in a3 immunoreactivity in the subiculum (F vs. E).
Temporal Lobe Epilepsy: Altered GABAA Receptor Subunit Composition in Temporal Lobe Epilepsy
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and subiculum. In the subiculum (which is highly preserved in human TLE), we observed increases in subunits a3 and g2, but losses in subunits a1, a3, and g2 in Ammon’s horn (Fig. 2). Taken together, these data indicate that in human TLE there are marked changes in the expression and assembly of GABAA receptors, suggesting remodeling of GABAergic transmission. Subfields of the hippocampus that are less vulnerable to seizure-induced neurodegeneration, such as the subiculum and dentate granule cell layer, display upregulation of various subunits, indicating facilitation of GABAergic transmission. Striking and difficult to explain is the especially pronounced expression of b subunitsdwhich could result in imbalanced assembly of GABAA receptors (such as receptors containing three b and two a and no g subunit, or three b, one a and one g2 or d subunit). Studies with recombinant receptors indicate that they would not bind GABA with high affinity or capacitydand if they lacked g2, they may not be affected by benzodiazepines but would be sensitive to Zn2þ. Due to the difficult comparison with postmortem controls, we have so far been unable to obtain evidence for downregulation of subunits d or a5 in the human tissue, which would (in analogy to the rat) be indicative of reduced extrasynaptic tonic inhibition in the epileptic brain. Appropriate human controls are essential since, as mentioned earlier, the distribution (and thus the function) of certain GABAA receptor subunits is different in the human hippocampus compared to that in the rat.
Future Goals It will be crucial to continue work in the human brain, since the physiological properties of single GABAA receptor may vary much depending on the circuitry in which they are integrated and less critically on their actual subunit composition. Detailed knowledge about the pathology of altered receptor expression will be important for developing appropriate drugs targeting individual GABAA receptor subtypes (e.g., acting through the benzodiazepine binding site). A limitation of our studies is that although we have learned something about changes in the GABAA receptor subunit distribution, we have yet to determine the details about changes in actual receptor composition. We are currently performing neurochemical studies, using subtractive purification methods in microdissected hippocampal subfields from KA rats, to approach this question.
Acknowledgment The work was supported by the Austrian Science Fund (projects P19464, P26680) and by the EC contract number LSH-CT-2006–037315 (EPICURE) FP6dThematic priority LIFESCIHEALTH.
Further Reading Bowery, N.G., 1993. GABAB receptor pharmacology. Annu. Rev. Pharmacol. Toxicol. 33, 109–147. Farrant, M., Nusser, Z., 2005. Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat. Rev. Neurosci. 6, 215–229. Glykys, J., Mody, I., 2007. Activation of GABAA receptors: views from outside the synaptic cleft. Neuron 56, 763–770. Goodkin, H.P., Joshi, S., Mtchedlishvili, Z., Brar, J., Kapur, J., 2008. Subunit-specific trafficking of GABAA receptors during status epilepticus. J. Neurosci. Off. J. Soc. Neurosci. 28, 2527–2538. Houser, C.R., Esclapez, M., 2003. Downregulation of the alpha5 subunit of the GABAA receptor in the pilocarpine model of temporal lobe epilepsy. Hippocampus 13, 633–645. Huberfeld, G., Wittner, L., Clemenceau, S., et al., 2007. Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy. J. Neurosci. Off. J. Soc. Neurosci. 27, 9866–9873. Loup, F., Wieser, H.G., Yonekawa, Y., Aguzzi, A., Fritschy, J.M., 2000. Selective alterations in GABA A receptor subtypes in human temporal lobe epilepsy. J. Neurosci. Off. J. Soc. Neurosci. 20, 5401–5419. Mody, I., De Koninck, Y., Otis, T.S., Soltesz, I., 1994. Bridging the cleft at GABA synapses in the brain. Trends Neurosci. 17, 517–525. Naylor, D.E., Liu, H., Wasterlain, C.G., 2005. Trafficking of GABAA receptors, loss of inhibition, and a mechanism for pharmacoresistance in status epilepticus. J. Neurosci. Off. J. Soc. Neurosci. 25, 7724–7733. Noebels, J.L., 2003. The biology of epilepsy genes. Annu. Rev. Neurosci. 26, 599–625. Nusser, Z., Hajos, N., Somogyi, P., Mody, I., 1998. Increased number of synaptic GABAA receptors underlies potentiation at hippocampal inhibitory synapses. Nature 395, 172–177. Palma, E., Amici, M., Sobrero, F., et al., 2006. Anomalous levels of Cl transporters in the hippocampal subiculum from temporal lobe epilepsy patients make GABA excitatory. Proc. Natl. Acad. Sci. U.S.A. 103, 8465–8468. Peng, Z., Huang, C.S., Stell, B.M., Mody, I., Houser, C.R., 2004. Altered expression of the delta subunit of the GABAA receptor in a mouse model of temporal lobe epilepsy. J. Neurosci. Off. J. Soc. Neurosci. 24, 8629–8639. Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., Sperk, G., 2000. GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815–850. Pirker, S., Schwarzer, C., Czech, T., et al., 2003. Increased expression of GABAA receptor beta-subunits in the hippocampus of patients with temporal lobe epilepsy. J. Neuropathol. Exp. Neurol. 62, 820–834. Schwarzer, C., Tsunashima, K., Wanzenbock, C., Fuchs, K., Sieghart, W., Sperk, G., 1997. GABAA receptor subunits in the rat hippocampus II: altered distribution in kainic acidinduced temporal lobe epilepsy. Neuroscience 80, 1001–1017. Sieghart, W., Sperk, G., 2002. Subunit composition, distribution and function of GABAA receptor subtypes. Curr. Top. Med. Chem. 2, 795–816. Sperk, G., 1994. Kainic acid seizures in the rat. Prog. Neurobiol. 42, 1–32. Sperk, G., Furtinger, S., Schwarzer, C., Pirker, S., 2004. GABA and its receptors in epilepsy. Adv. Exp. Med. Biol. 548, 92–103. Tsunashima, K., Schwarzer, C., Kirchmair, E., Sieghart, W., Sperk, G., 1997. GABAA receptor subunits in the rat hippocampus III: altered messenger RNA expression in kainic acidinduced epilepsy. Neuroscience 80, 1019–1132. Zhang, N., Wei, W., Mody, I., Houser, C.R., 2007. Altered localization of GABAA receptor subunits on dentate granule cell dendrites influences tonic and phasic inhibition in a mouse model of epilepsy. J. Neurosci. Off. J. Soc. Neurosci. 27, 7520–7531.