A review article I created for a class in 2012. The paper attempts to overview the roles of GABA(A) receptors [Including pharmacology, mutations, and developmental disorders] in causing or alleviating Temporal Lobe Epilepsy (TLE).

1. Brandon Turner Receptors & Channels May 10, 2012
Investigating the Roles of GABAergic Inhibitory Currents in Epilepsy
Introduction
Epilepsy is one of the more common neurological disorders in humans and is
characterized by the occurrence of repeated seizures. Seizures themselves result from an area of
the brain exhibiting large amounts of depolarizing currents/over activity. For this reason, past
research has focused on an up-regulation of excitatory neurotransmission, such as AMPA
receptors, to be the cause of such over activity (Rogawski, 2011). However, treatment of seizures
with anti-convulsant that target excitability has proved to be less effective than desired. More
recent research has discussed the role of inhibitory currents, notably those mediated by the γ-
amino butyric acid (GABAA) type A receptor, which is responsible for the majority of the fast
inhibitory signaling in the brain. The GABAA receptor exists as a heteropentamer usually
comprised of two pairs of α and β subunits and a variable subunit (γ, δ, ρ, π, ε), each of which
may have several subtypes (α [1-6], β [1-3], etc). With such subunit variability and differences of
subunit expression levels in specific brain regions, the pharmacology of the GABAA receptor has
a high degree of variance. The receptor binds several allosteric potentiating ligands, such as
benzodiazepines (BZDs), which require the γ subunit, and neurosteroids which bind directly to
the α subunit.
The involvement of such receptors in epilepsy is bound to be highly complex. Indeed,
many factors can contribute to epilepsy via GABAergic currents, such as environmental
intoxicants like RDX (Williams et al 2011), developmental defects involving time of onset of
seizures (Briggs SW and Galanopoulou AS, 2011), regulation of GABA transport both, synaptic

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and extrasynaptic (Chiu et al 2005), neuronal migration (Bozzi et al 2012), or inherent genetic
factors (Gurba et al 2012, Macdonald et al 2009). It has also been shown that GABA subunit
composition changes during the course of seizures and epileptogenesis (Loup et al 2000).
Despite these problems, neurosteroids and benzodiazepines are known to be effective
anticonvulsants if administered with proper timing, yet some types of epilepsy, such as temporal
lobe epilepsy (TLE), remain resistant to such treatment. Clearly the role of GABAA in epilepsy is
not straightforward and requires additional investigation if a more conclusive understanding of
the disease and its treatment are to be found. In this review, I will focus on changes in
GABAergic inhibitory current due to developmental changes, GABA concentration variations in
multiple forms of epilepsy, and on receptor specific changes in temporal lobe epilepsy in the
hopes of directing future research to develop anticonvulsants targeting these receptors that more
accurately despite the inherent complexity of their role in epileptogenesis.
Developmental Malformations in GABAergic Systems
Developmental miscues can result in a host of neurological disorders that include many
subtypes of epilepsy. Some of these include genetic defects that decrease the migration of
GABAergic neurons to their proper positions, including interneurons of the cortex and
hippocampus. Two of these factors, Dlx and Reelin, have been shown in knockout and mutation
studies to impair the migration of such inhibitory neurons to the cortex and proper layer in the
hippocampus, respectively (Bozzi et al 2012), and have been implicated several forms of
epilepsy. Many other factors have been shown to induce epilepsy in model animals as well, all
concerning cellular circuits and positioning in the brain. Notably, Velisek et al have recently
shown that haploinsufficiency of BRD2 (Bromodomain- containing gene 2) in mice contributes
to lack of GABAergic neurons in the neocortex and striatum and a deficiency in GABAergic

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signaling in other regions of the brain associated with idiopathic generalized epilepsies. Clearly,
developmental factors and genetic regulation can play into developing epilepsy by removing
inhibition in different brain regions.
Other changes in normal neuronal function, most notably seizures at an early age, can
induce epilepsy in adult animal models. Depending on age, GABAA currents can be either
depolarizing (early age) or hyperpolarizing. Recently, this has been shown to be linked to the
concentration of Cl- within the cell, which affects GABAA subunit compositions (Succol et al
2012). The authors used both electrophysiological and immunostaining to show that changes in
intracellular chloride concentrations affected EGABA depending on the expression levels of
KCC2, a potassium/chloride cotransporter. Consistent with this data, it has been shown that
disruption of KCC2 funciton can contribute to early life seizures and that these seizures
themselves can alter GABAA subunit composition as well (Reviewed by Briggs SW,
Galanopoulou AS, 2011). It may be that a rapid influx of chloride ions due to compensatory
inhibition during seizures may contribute to the changes in GABAA receptor subunit
composition, or that changes in chloride trafficking may result in a reversion to immature-like
expression and function of GABAA receptors, causing them to be depolarizing rather than
inhibitory. Further research into the temporal allocation of shifts in chloride gradients,
transporter activity, and subunit composition during the ictal period of seizures may provide
insight into epileptogenesis.
Changes in Extracellular and Synaptic GABA Concentrations
Changes in GABA concentrations at the synapse due to disruptions in transport or
synthesis could contribute to altered levels of GABAA signaling, which could, in turn, contribute

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to epileptic phenotypes. As reviewed by Pavlov et al, 2012, “Reversal of GABA transport in
certain subpopulations of neurons or individual cells in epileptic tissue cannot be ruled out. Some
interneurons in chronic epilepsy may become metabolically more active, express more GAD and
have elevated intracellular GABA concentrations.” This speaks the idea that overexpression of
GAD (Glutamate Decarboxylase), which converts Glutamate to GABA, could raise intracellular
GABA and reverse transportation, which is largely dependent on voltage and concentration
gradients (Pavlov et al, 2012). Consistent with this, impaired GABA uptake by GABA
transporter deficiency has been shown to cause tremors and ataxia in mice (Chiu et al 2005).
Despite the possible negative roles of GAT impairment in epilepsy, it has been shown that GAT
impairment functions to increase tonic inhibition in epileptic rats (Frahm et al, 2003) by
increasing available GABA to the extrasynaptic, high affinity and slow desensitizing receptors.
In this sense, GABA transporters could serve as a possible drug target for anticonvulsants should
allosteric up-regulation be possible.
Much of the regulation of extrasynaptic GABA concentrations is dependent on the
function of GABA transporters in astrocytes. GAT-3, which is primarily found in astrocytes,
contributes substantially to extracellular GABA levels and can induce increased tonic inhibition
in the presensce of glutamate (Heja et al 2009). In vivo studies have also shown that uptake of
glutamate in hippocampal astrocytes is necessary for GABA release by GAT-2,3 and can convert
excess amounts of glutamate excitatory signaling to inhibition in an epileptic model (Heja et al
2012). Due to increased variability of GABAA subunits during seizures, increased tonic GABA
can initiate a negative feedback loop and prevent the spread of excess excitation to other brain
regions without the need of specifically designed drugs for the variant receptors. However, this
may not serve as an effective treatment for epilepsy, as past studies have demonstrated in vitro

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that increased extra-cellular GABA concentrations are not enough to mitigate the hyper-
excitability of hippocampal neurons in an epileptic model (Yeh et al 2005).
Since extracellular GABA levels and tonic inhibition seem to be inefficient at preventing
seizures, investigation of vesicular GABA transporters (VGAT) may provide insight into
mechanisms for preventing seizures. It has recently been shown that epileptic neurons, as
induced by excess exposure to glutamate, express a truncated form of VGAT that accumulates at
non-synaptic sites (Gomes et al 2011). However, removal of VGAT from vesicles contributes to
increased synaptic GABA concentrations, suggesting that VGAT truncation may serve as a
mechanism to increase inhibition in their model of temporal lobe epilepsy rather than furthering
the progression of the disease. Given that synaptic GABAA receptors desensitize more rapidly
than extrasynaptic receptors, it would seem that enhancing GABA at the synapse may also prove
to be ineffective at alleviating epileptogenesis. Since the level of GABA transport appears to be
providing negative feedback in epileptic models, their dysfunction could contribute to
epileptogenesis. However, due to their regulation by both voltage and concentration gradients,
modifying these transporters would prove difficult at the least, suggesting that changes in
receptor expression and subunit concentration could provide more insight into understanding
epileptogenesis and drug design.
GABA Receptor Expression and Composition Prior to and Following Seizures
Temporal lobe epilepsy (TLE) is among the most common forms of epilepsy in adults
and is mainly thought to arise from alterations in excitation/inhibition in the hippocampus (Joshi
et al 2011). Those with the disease normally display a loss of both CA1 and CA3 pyramidal
neurons and changes in receptor expression in dentate gyrus cells (DGCs). These changes have

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been shown by S. Joshi et al to lower neurosteroid sensitivity, which they attribute to changes in
seizure susceptibility of women dependent on the menstrual cycle (known to cause alterations in
the levels of endogenous neurosteroids in the brain). Indeed, recent studies have shown using
radiolabeled ligand binding that rats with temporal lobe epilepsy show decreased binding in the
cerebral cortex and further show that this corresponds to a decrease in expression of α1, γ, δ,
GABAB receptors, and GAD, the enzyme that converts glutamate to GABA (Mathew et al
2012). Past studies have also shown that changes in CA1 and CA3 cells include a marked
decrease in α1 subunit expression and an increase in α2/3 expression (Loup et al 2000). Also,
Rajasekaran et al had recently shown that the low affinity of neurosteroids for epileptic GABAA
receptors is likely due to lower incorporation of the δ subunit, which is down-regulated in TLE.
How intracellular mechanisms contribute to subunit regulation is currently not well understood,
but recent studies have shown that δ subunit incorporation is largely dependent on intracellular
[Cl-]. Increased intracellular [Cl] in the presence of a KCC2 knockdown decreased the amount of
δ subunit incorporated into receptors, as shown by immunostaining, present at the membrane, as
well as increasing the incorporation of α3 subunits and decreasing the incorporation of α1
subunits (Succol et al 2012). The authors focused on this shift of subunit incorporation primarily
to elucidate the shift of GABAA from depolarizing to hyperpolarizing during neuronal
development, which they showed to be mediated by KCC2 up-regulation. However, this could
also point to a method of altered subunit expression in epileptic animals, but whether the KCC2
ion transporter is involved is not known.
In addition to differential regulation of GABAA subunits during epilepsy, there is also a
loss of synaptic GABAA receptors that may be due to changes in gephyrin and collybistin
scaffolding. Past studies have shown that gephyrin preferentially binds to α2 and α3 subunits,

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while collybistin binds preferentially to α2 only. They assert that the subunit and the scaffolding
proteins form a trimeric complex via co-immunoprecipitation and that disruption of this
interaction may lead to epilepsy, as seen in a genetic mutation causing a change in the SH3
domain of collybistin which is known to cause mental disability and seizures (Saiepour et al
2010). More recent studies have also shown that gephyrin is able to bind to α1 subunits as well,
also using immunoprecipitation (Mukherjee et al 2011). Since α1-3 containing receptors are the
primary receptors found at synaptic sites, changes in gephyrin binding, expression, or
localization may contribute to loss of these receptors at the synapse and change the
pharmacological properties of these sites during epilepsy. Congruent with this idea, other studies
have shown that induction of status epilepticus alters gephyrin and neuroligin-2 expresssion, but
these did not have an effect on GABAA clustering . Mechanisms underlying the lack of
correlation between the two remain unexplained, but other studies have shown that such a
relationship does exist. Forstera et al had recently demonstrated that splicing of gephyrin exons
is altered in brain regions undergoing TLE or cellular stress which corresponded to a loss of α2
subunit containing receptors at the synapse. It is possible that a decrease of gephyrin binding and
the reported up-regulation of α2 GABAA subunits could contribute to an unnatural clustering of
these receptors at extra-synaptic sites, which normally contain α4 α6 subunits, specifically.
However, Forstera et al report that no concurrent mutations in gephyrin are concurrent with
temporal lobe epilepsy in humans, which may indicate that collybistin-gephyrin clustering, along
with their association with GABAA receptors is complex and requires additional study. Whether
these changes in protein scaffolding are reflect potential feedback mechanisms to stop
epileptogenesis or a method in which seizures are generated will require additional research and

8. 8
holds promise in understanding how spatial localization of inhibitory receptors may contribute to
TLE.
Most studies have alluded to the involvement of hippocampal neurons in the pathogenesis
of TLE, but, due to the aberrant shuffling of subunits and inherent neuro-plasticity that
accompanies seizures many forms of TLE have remained resistant to anti-convulsants. As
previously described, GABAA receptors in the hippocampus display lowered sensitivity to both
benzodiazepines and neurosteroids, possibly due to increases in α4 containing receptors, which
are insensitive to BZDs, and a decrease in δ containing receptors, which have a high sensitivity
to neurosteroids (Joshi et al 2011). A recent study has shown that other brain regions may be
involved in the propagation of excitatory signaling from the hippocampus and are involved in the
development of seizures. The thalamus, particularly the Thalamic Parafascicular Nucleus, has
been shown to be directly involved in the development of seizures (Langlois et al 2010).
Specifically, they show in vivo that inhibition of NMDA receptors and/or the presence of
GABAA antagonists injected into the TPN was sufficient to prevent behavioral effects following
the creation of an epileptic center in the hippocampus. In agreement with these findings, similar
data was published using muscimol to activate GABAA receptors in the mediodorsal region of
the thalamus and similar results were observed, most notably being the attenuation of seizure
duration (Sloan et al 2011). Although changes in receptor signaling and expression may be
present in the thalamus as well, it provides a novel target for preventing TLE behavioral effects
that circumvents the complex problem of receptor subunit regulation, shuffling, or changes in
ligand concentrations. Until the generation of seizures is fully understood, the thalamus may be
the best option for treating patients with TLE and provides a novel target for specific drugs that
could serve as anti-convulsants for TLE patients.

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Conclusion
Comprehending epilepsy from the molecular to the physiological level is a difficult gap
to bridge, but the numerous studies already done have shown many changes at both levels, both
before and after the occurrence of seizures. The myriad of developmental factors that could
contribute to the onset of epilepsy are not well understood beyond their number, yet the
numerous in ways in which they affect the nervous system, as per neuronal development and
migration or receptor/transporter mutations that cause aberrant signaling in mature neurons. This
significantly narrows the mechanisms in which developmental disorders can contribute to
epileptogenesis, but correcting neuronal migration in the brain is unlikely, while finding methods
that can target malformed regions and restore them to their normal inhibitory function by anti-
convulsant drugs is much more likely. GABA transporters, however, seem to serve a role as
increasing GABAergic current in epileptic areas of the brain and their selective modulation by
allosteric drugs would also prove arduous due to their voltage sensitivity and dependence on
intra/extra cellular GABA concentrations. It is possible that an increase in GAD activity may in
neurons or astrocytes may be able to shunt the concentration of GABA to favor increased
exportation, but it has been shown that increased ambient GABA at the synapse or in extra-
synaptic regions have been ineffective at preventing seizures. A more novel target would be
selective modulation of GABAA receptors to increase tonic and phasic inhibition, but the regions
affected by seizure generation display irregular GABAA subunit composition and pharmacology,
further complicating the effect of anti-convulsants. Also, TLE is markedly insensitive to anti-
convulsant drugs, perhaps due to this or the inability of such drugs to act in the epileptic centers.
Yet new research suggests that inhibiting the progression of seizures to other areas of the brain
via increased inhibition or decreased excitation in the thalamus may be an effective way of

10. 10
mitigating seizure genesis at a behavioral level. Although working in the hippocampus to
discover the origin of TLE genesis, preventing the spread of seizures from here by increased
inhibition in the thalamus may be the most effective direction to work with until the exact
mechanism of the marked increase in excitability of hippocampal neurons is discovered and
more specific drugs can be developed.

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