The brain engages in an accurate and highly co-ordinated cell-to-cell signalling and communication through two main modes of transmission. In one mode, neurons secrete neurotransmitters, such as glutamate, to send excitatory signals. In the other mode, neurons release inhibitory neurotransmitters, such as gamma-Aminobutyric acid (GABA)(Figure 1), to prevent the continued propagation of these excitatory signals. Although the presence of GABAergic interneurons is much lower than those of the glutamatergic neurons, they play crucial inhibitory roles that allow the brain to function in a highly integrated fashion1. Defects in the GABAergic system can lead to serious neurological disorders, particularly epilepsy. Seizures are the outward manifestation of this disorder that arises from hypersynchronous or hyperexcitable action potentials2. Therefore, an imbalance between the excitatory and inhibitory signals, due to a defective GABAergic system, can have debilitating consequences to an epileptic individual.







Figure 1. Structural configuration of Gamma-Aminobutyric acid
Figure 1. Structural configuration of Gamma-Aminobutyric acid








1.The GABAergic System

There are two main types of GABA receptors: GABAA and GABAB receptors2.

1.1 GABA A Receptor

When a neuron binds to glutamate from a pre-synaptic membrane, the sum of these signals get translated into excitatory postsynaptic responses (EPSPs). EPSPs are the main mode of communication among neurons. When an EPSP reaches the end of an axon, it depolarizes it, causing the axon to release glutamate into the synapse in order for the signal to be passed on to the next neuron. However, for co-ordinated communication and optimal brain function, not all EPSPs should be propagated and passed on. Without an inhibitory regulatory system, the brain would be in a constant hyperactive state.

GABA is released by the GABAergic inhibitory interneurons into the synaptic cleft and binds to the GABAA receptors present on the post-synaptic membrane (Figure 2)3. When GABA binds, it opens chloride channels and chloride ions flow down the electrochemical gradient and into the cell3. The influx of chloride ions hyperpolarizes the cell and makes it more difficult for it to send an action potential, thereby inhibiting the neuron from firing.

pic.jpg
Figure 2. The production, release, action and degradation of GABA at a typical GABAergic synapse.





The chloride electrochemical gradient is established and maintained by the expression of the cation-chloride co-transporters (CCCs)4. CCCs establish this electrochemical gradient by acting as a symptorer to maintain low levels of chloride inside the cell. There are three types of these co-transporters, all of which are ion neutral4. Two of these types of CCCs are chloride importing, known as the sodium/potassium/chloride cotransporters (NKCCs) and the sodium/chloride cotransporters (NCCs)5. The third type of CCC exports chloride ions out of the cell, called the potassium/chloride co-transporters (KCCs)5. During the early developmental stage, immature neurons highly express the chloride-importing CCCs, such as the NKCC1 and NCC6. Therefore, during this state, there are high levels of chloride ions within the neuron. When GABA binds to the GABAA receptor, the channel opens and the chloride ions exit, down the electrochemical gradient. An efflux of ions depolarizes the neuron, reducing the threshold required to send an action potential.

On the other hand, a mature neuron tends to express more chloride exporting CCC, such as the KCC2, than do the chloride importing types7. Therefore, there is a net efflux of chloride ions, leaving low levels of chloride ions inside. The change in ratio of these types of CCC will be influenced by factors such as age, sex, and species. The CCC co-transporters, KCC2 and NKCC1, are particularly expressed among the neurons, whereas other subtypes can be found in other regions of the body8. In humans, it is estimated that this change in CCC shift occurs sometimes at or soon after birth. Therefore, impairments that deter this shift or mutations that affect the proper formation and function of the GABAA receptor will impair inhibitory functions and lead to hyperexcitability8.
1.2 GABA B Receptor

GABAB receptors are usually located on the pre-synaptic membranes of neurons9. When GABA binds to this receptor, it opens and allows the flow of potassium along the electrochemical gradient, and thus out of the cell9. This hyperpolarizes the axon terminal, requiring greater depolarization to reach the threshold level required to send the next action potential. In sum, by GABA binding to GABAB receptor, it prevents the release of glutamate from the pre-synaptic neuron into the cleft.

Therefore, the GABAergic interneurons reduce excitation by affecting EPSPs at the pre-synaptic and post-synaptic membranes through two different types of receptors. Impairment in their levels or capacity to function optimally would reduce the brain’s ability to control excitatory signals, potentially leading to seizures.
1.3 GABAergic Interneurons

Afferent neurons send signals to GABAergic interneurons, in addition to passing the signal to post-synaptic neurons. This dual communication modulates the neural signal in order to filter out noise and to ensure that only relevant and salient stimuli are processed and propagated. Depending on when the interneuron gets activated, the signalling will be classified as feedforward or feedback10. Specifically, in feedforward signalling, the interneuron gets activated before or at the same time as the post-synaptic neuron10. The interneuron then passes an IPSP signal to the post-synaptic neuron, preventing it from depolarizing and propagating an action potential.

The feedback pathway is involved in preventing a neuron from repetitively being active and passing its signal off to nearby cells11. Currently, a set of neurons is thought to be present between the axon of the incoming neuron and the dendrite of the GABAergic interneuron. For example, in the dentate gyrus, researchers concluded that the mossy cells found in the hilar polymorphic region were crucial in supporting the inhibitory neurons perform their function11. In fact, when the mossy cells are not present, it is very difficult to activate the GABAergic system11.


The glutamic acid decarboxylase(GAD) isoforms GAD65 and GAD67 are responsible for the production of GABA in the brain12. They are responsible for establishing stores of GABA ready to be used upon need at the pre-synaptic axon terminal. Having this abundance of GABA readily available to be released upon tonic stimulation is important establishing inhibition. In mouse knockout studies of GAD, where there was a lack of these reserve pools, the mice had a reduced threshold for seizures during the presence of an antagonist, compared to mice with functioning GAD13, 14. Specifically, mutations of GAD co-factors have been indirectly implicated in increased seizure susceptibility in humans.

2. Seizures

Epilepsy is a complex neurological disorder and seizures can arise as a result of a number of different problems. Defects in the GABAergic system are just one set of possible abnormalities that can lead to this outward manifestation. These GABAergic problems can be broadly categorized as insufficient inhibition or excessive inhibition (Figure 3)15. Insufficient inhibition can result from a lack of sufficient GABA or GABAA/GABAB receptors in the brain. Inhibitory levels will also be influenced by the type of GABA receptor combination present at appropriate locations. Furthermore, if GABA abnormally functions to depolarize the cell, instead of hyperpolarizing it, or if GABAergic interneurons miswire, causing GABA to be released in the wrong synaptic cleft, it can lead to the failure of the inhibitory function15. On the other hand, excessive inhibition can result when GABAergic interneurons are inhibited, allowing the primary neurons to continue firing, or when the inhibition causes the epileptic focal point to fire in synchronized fashion15.
Figure 3. Schematic depiction of simple models through which dysregulation of GABAA receptor-mediated inhibition can increase the activity of neuronal networks, potentially generating seizures. GABA inhibition can fail when GABA or GABAA receptor expression is low, when GABA depolarizes neurons, or when miswiring and mistargeting of synapses occur. Excessive GABA inhibitionmay trigger seizures by disinhibiting target cells, or via excessive synchronization of the neurons in the epileptogenic focus (Briggs and Galanopoulou, 2011).
Figure 3. Schematic depiction of simple models through which dysregulation of GABAA receptor-mediated inhibition can increase the activity of neuronal networks, potentially generating seizures. GABA inhibition can fail when GABA or GABAA receptor expression is low, when GABA depolarizes neurons, or when miswiring and mistargeting of synapses occur. Excessive GABA inhibitionmay trigger seizures by disinhibiting target cells, or via excessive synchronization of the neurons in the epileptogenic focus (Briggs and Galanopoulou, 2011).




A number of epidemiological studies have revealed that infants and young children are at a greater risk of having seizures than older individuals16. One of the major explanations for this phenomenon is the result of the presence of the immature GABAergic system that leaves young children with a higher susceptibility to seizures17. As discussed earlier, children have a different GABAA composition than do adults. Males are at a higher risk than females because the maturation of the GABAergic system is delayed. In addition, the delayed development of the substantia nigra pars reticulata (SNR), which is a subcortical region of a group of GABAergic neurons also involved in mediating this hyperactivity16.

3. Antiepileptic Drugs


A number of drug therapies have been synthesized to compensate for these abnormal functions, ranging from channel blockers to neurotransmitter agonists. Some of the most common antiepileptic drugs (AEDs) fall into the following classes: GABAA agonists, GABA reuptake inhibitors, and GABA-acting AEDs. However, many AEDs lack target specificity, therefore, their efficacy varies from patient to patient. This is one of the major reasons why one third of patients who take AEDs do not receive any reduction in seizure frequency or severity, despite having taken various drugs or combinations of drugs. In addition, many of these drugs come with serious side effects.

3.1 GABA A Agonists


Benzodiazepines are group psychoactive drugs that act as GABAA receptor agonists18. When they bind to this receptor, they facilitate the opening of chloride channels, which in turn, allow the influx of chloride ions into the cell18. This hyperpolarizing effect prevents an action potential from being propagated. The four common benzodiazepines used to treat epilepsy are lorazepam, diazepam, clonazepam, and clobazam19. Lorazepam and diazepam are used to treat immediate and urgent seizure presentations in order to stop an individual from convulsing19. However, they are not prescribed as a regular treatment because there is a high risk of patients developing a tolerance to these drugs. Clobazam and Clonazepam are more commonly used and vary slightly in their structural composition compared to most benzodiazepines, in order to minimize the adverse effects seen in this family of drugs, like a reduction in anxiety and sedation20. However, the trade off involves modified GABAA binding affinity and half-life properties, potentially affecting their efficacy. These types of drugs still also adverse effects.

3.2 GABA Reuptake Inhibitors


Tiagabine and Nipecotic acid are AEDs that prevent GABA from being removed from the synaptic cleft21. They perform their function by directly inhibiting the function of the GABA transporter-1 (GAT-1). Although these drugs have shown some level of efficacy at preventing seizures, they also have a number of side affects. They include dizziness, the increased risk of depression onset, tremor and diarrhea21.

3.3 GAD Enhancers


GAD is an enzyme that converts glutamate into GABA. Valproate (VPA) and Gabapentin (GBP) enhance GAD’s ability to produce GABA22, 23. Furthermore, VPA has also been found close sodium channels during repetitive firing, thus enhancing the overall inhibitory effect24. VPA was often used against many different seizure types25. However, its serious side effects, such as insulin resistance, bone marrow suppression, changes in sex hormone levels and the risk of hepatic failure have led physicians to more cautious before prescribing VPA as an AED25. GBP, on the other hand, has been shown to weakly inhibit the function of GAT-1, thereby supporting the increased presence of GABA in the synaptic and the over all IPSP effect. GBP has minor side effects26.

3.4 AED Efficacy


Although a number of antiepileptic drugs have been synthesized, their efficacy varies and very few have been effective at permanent seizure control. Defects at multiple different levels of the GABAergic system as well as other neuronal systems have added to the complexity of synthesizing an effective drug. Physicians have attempted to address this concern by prescribing different combinations of AEDs in different doses. Furthermore, there is a move towards personalized epileptic treatment, achieved primarily by genetic analysis to find mutations that impair specific aspects of the GABAergic system as well as other neuronal systems important for this co-ordinated brain signalling. However, drug treatments for epileptic seizures have a long way to go in order to achieve optimal, desired effects. In addition to achieving improved efficacy, the problem of accompanying negative side effects of AEDs need to addressed.

4. GABAergic Neuronal Precursor Grafting – A New Mode of Cell-Based, Regenerative Therapy


Because of the problems with pharmaceutical treatments for epilepsy, research has focused on alternative therapies. An exploration into grafting embryonic stem cells and fetal hippocampus cells to replace the dysfunctional inhibitory cells has been attempted over the last decade27. It was hypothesized that these cells may integrate into the local structure, differentiate and mitigate the role of the abnormal GABAergic system28. Although these experiments showed some therapeutic effect in mice, this treatment posed a number of limitations, which hinder their continued use for such purposes. For example, these cells showed low tissue distribution, some embryonic stem cells showed potential to become teratocarcinomas, and the benefits were short-lived28. In addition, the lack of understanding of how these new cells integrated and functioned within the existing network at an electrophysiological level make it difficult to predict their action in a human being.
Due to the malfunction of the GABAergic system under which many seizures arise, another line of research pursued is the grafting of GABAergic interneuron precursor cells from the medial ganglionic eminence (MGE) region29. In normal brains, GABAergic interneurons precursor cells migrate from the MGE to appropriate regions in the cortex and the hippocampus. In fact, grafting these precursor cells in the mouse neonatal brain has already been shown to be effective. Cells migrated to the cortex and hippocampus, differentiated and integrated into the existing brain regions30.
Recently, the effectiveness of this type of grafting has been tested in an epileptic mouse model. MGE-precursor cells from a neonatal mouse were grafted into the hippocampus of epilepsy model mice, which had their GABAergic interneurons in the hippocampus destroyed30. The results of the experiment showed a similar effect to what was seen in the neonatal mice31. These precursor cells migrated, differentiated and functioned much like the normal GABA interneurons30, 31. The grafted cells were shown survive to one year and there was no indication to suggest that these grafted cells increased tumour susceptibility31. However, different mice and rat models of epilepsy showed different levels of efficacy. The grafted cells were also seen to differentiate differently under disease condition versus normal conditions31. Furthermore, the exact functional effects of these grafted interneurons are unknown31. Therefore, although this type of therapy is showing early promise, much work is yet to be done before it is introduced into the clinical setting.

References

1. Roth, F.C. & Draguhn, A. GABA Metabolism and Transport: Effects on Synaptic Efficacy. Neural Plasticity (2012).

2. Briggs, S.W. & Galanopoulou, A.S. Altered GABA Signaling in Early Life Epilepsies. Neural Plasticity (2011).

3. Farrant, M. & Kaila, K. in Gaba and the Basal Ganglia: from Molecules to Systems (eds. Tepper, J.M., Abercrombie, E.D. & Bolam, J.P.) 59-87 (2007).

4. Blaesse, P., Airaksinen, M.S., Rivera, C. & Kaila, K. Cation-Chloride Cotransporters and Neuronal Function. Neuron 61, 820-838 (2009).

5. Mortensen, M., Patel, B. & Smart, T.G. GABA potency at GABA(A) receptors found in synaptic and extrasynaptic zones. Frontiers in Cellular Neuroscience 6 (2012).

6. Dzhala, V.I. et al. NKCC1 transporter facilitates seizures in the developing brain. Nature Medicine 11, 1205-1213 (2005).

7. Viitanen, T., Ruusuvuori, E., Kaila, K. & Voipio, J. The K plus -Cl- cotransporter KCC2 promotes GABAergic excitation in the mature rat hippocampus. Journal of Physiology-London 588, 1527-1540 (2010).

8. Kaila, K. & Miles, R. Chloride homeostasis and GABA signaling in temporal lobe epilepsy. Epilepsia 51, 52-52 (2010).

9. Benarroch, E.E. GABA(B) receptors Structure, functions, and clinical implications. Neurology 78, 578-584 (2012).

10. Sebe, J.Y. & Baraban, S.C. The Promise of an Interneuron-Based Cell Therapy for Epilepsy. Developmental Neurobiology 71, 107-117 (2011).

11. Sandler, R. & Smith, A.D. COEXISTENCE OF GABA AND GLUTAMATE IN MOSSY FIBER TERMINALS OF THE PRIMATE HIPPOCAMPUS - AN ULTRASTRUCTURAL-STUDY. Journal of Comparative Neurology 303, 177-192 (1991).

12. Errichiello, L., Striano, S., Zara, F. & Striano, P. Temporal lobe epilepsy and anti glutamic acid decarboxylase autoimmunity. Neurological Sciences 32, 547-550 (2011).

13. Walls, A.B. et al. Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine. Journal of Cerebral Blood Flow and Metabolism 31, 494-503 (2011).

14. Walls, A.B. et al. GAD65 is essential for synthesis of GABA destined for tonic inhibition regulating epileptiform activity. Journal of Neurochemistry 115, 1398-1408 (2010).

15. Margineanu, D.G. Epileptic hypersynchrony revisited. Neuroreport 21, 963-967 (2010).

16. Chudomel, O., Herman, H., Nair, K., Moshe, S.L. & Galanopoulou, A.S. AGE- AND GENDER-RELATED DIFFERENCES IN GABA(A) RECEPTOR-MEDIATED POSTSYNAPTIC CURRENTS IN GABAergic NEURONS OF THE SUBSTANTIA NIGRA RETICULATA IN THE RAT. Neuroscience 163, 155-167 (2009).

17. Jansen, L.A., Peugh, L.D., Roden, W.H. & Ojemann, J.G. Impaired maturation of cortical GABA(A) receptor expression in pediatric epilepsy. Epilepsia 51, 1456-1467 (2010).

18. Sankar, R. GABA(A) Receptor Physiology and Its Relationship to the Mechanism of Action of the 1,5-Benzodiazepine Clobazam. Cns Drugs 26, 229-244 (2012).

19. Abend, N.S., Gutierrez-Colina, A.M. & Dlugos, D.J. Medical Treatment of Pediatric Status Epilepticus. Seminars in Pediatric Neurology 17, 169-175 (2010).

20. Pizzol, A.D. et al. Impact of the chronic use of benzodiazepines prescribed for seizure control on the anxiety levels of patients with epilepsy. Epilepsy & behavior : E&B 23, 373-6 (2012).

21. Lile, J.A., Kelly, T.H. & Hays, L.R. Separate and combined effects of the GABA reuptake inhibitor tiagabine and Delta(9)-THC in humans discriminating Delta(9)-THC. Drug and alcohol dependence 122, 61-9 (2012).

22. Thome-Souza, S. & Valente, K.D. Valproate and lamotrigine in children and adolescents with drop attacks: Follow-up after the first year. Epilepsia 52, 2139-2139 (2011).

23. Lal, R. et al. Clinical Pharmacokinetics of Gabapentin After Administration of Gabapentin Enacarbil Extended-Release Tablets in Patients With Varying Degrees of Renal Function Using Data From an Open-Label, Single-Dose Pharmacokinetic Study. Clinical Therapeutics 34, 201-213 (2012).

24. Rejdak, K. et al. Analysis of ventricular late potentials in signal-averaged ECG of people with epilepsy. Epilepsia 52, 2118-2124 (2011).

25. Shearer, P. & Riviello, J. Generalized Convulsive Status Epilepticus in Adults and Children: Treatment Guidelines and Protocols. Emergency Medicine Clinics of North America 29, 51-+ (2011).

26. Berner, B., Hou, S.Y.E. & Gusler, G.M. (Depomed Inc, 2012).

27. Maisano, X. et al. Differentiation and Functional Incorporation of Embryonic Stem Cell-Derived GABAergic Interneurons in the Dentate Gyrus of Mice with Temporal Lobe Epilepsy. Journal of Neuroscience 32, 46-61 (2012).

28. Shetty, A.K. Progress in Cell Grafting Therapy for Temporal Lobe Epilepsy. Neurotherapeutics 8, 721-735 (2011).

29. Anderson, S.A. & Baraban, S.C. Cell therapy for epilepsy using GABAergic neural progenitors. Epilepsia 51, 94-94 (2010).

30. Hattiangady, B., Shuai, B., Parihar, V. & Shetty, A.K. Medial Ganglionic Eminence Precursor Cell Grafting Diminishes Spontaneous Seizures and Improves Memory Function in a Rat Model of Temporal Lobe Epilepsy. Cell Transplantation 20, 562-563 (2011).

31. Zipancic, I., Calcagnotto, M.E., Piquer-Gil, M., Mello, L.E. & Alvarez-Dolado, M. Transplant of GABAergic Precursors Restores Hippocampal Inhibitory Function in a Mouse Model of Seizure Susceptibility. Cell Transplantation 19, 549-564 (2010).