145 . Suls A , Mullen SA , Weber YG , Verhaert K , Ceulemans B , Guerrini R , et al . Early -onset absence epilepsy caused by mutations in the glucose transporter GLUT1 . Ann Neurol 2 009 ; 66 : 415 \200\223 19 . 126 . Guerrini R , Mei D , Sisodiya S , Sicca F , Harding B , Takahashi Y , et al . Germlin e and mosaic mutations of FLN1 in men with periventricular heterotopia . Neurology 2004 ; 6 3 : 51 \200\223 6 . 146 . Mullen SA , Marini C , Suls A , Mei D , Della Giustina E , Buti D , et al . 127 . Parrini E , Rivas IL , Toral JF , Pucatti D , Giglio S , Mei D , et al . In-frame del etion in FLNA causing familial periventricular heterotopia with skeletal dysplasia in males . Am J Med Genet A 2011 ; 155A : 1140 \200\223 6 . Glucose transporter 1 de \201 ciency as a treatable cause of myoclonic astatic epilepsy . Arch Neurol 2011 ; 68 : 1152 \200\223 5 . oxford textbook of epilepsy and epileptic seizures 26 regions and genes in unexplained mental retardation . Neurology 2007 ; 68 : 721 \200\223 2 . 147 . Singh R , McKinlay Gardner RJ , Crossland KM , Scheffer IE , Berkovic SF . Chromosoma l abnormalities and epilepsy: a review for clinicians and gene hunters . Epilepsia 2002 ; 4 3 : 127 \200\223 40 . 151 . Kim HS , Yim SV , Jung KH , Zheng LT , Kim YH , Lee KH , et al . Altered DNA copy num ber in patients with different seizure disorder type: by array-CGH . Brain Devel 2007 ; 29 : 639 \200\223 43 . 148 . Guerrini R , Battaglia A , Carrozzo R , Gobbi G , Parrini E , Pramparo T , et al . Ch romosomal abnormalities . In: Engel J Jr . , Pedley TA (eds) Epilepsy: A Comprehensive Text book (2nd edn) , pp. 2589 \200\223 601 . Philadelphia, PA : Lippincott Williams and Wilkin s , 2008 . 149 . Ledbetter DH . Cytogenetic technology: genotype and phenotype . 152 . de Kovel CG , Trucks H , Helbig I , Mefford HC , Baker C , Leu C, et al . Recurrent m icrodeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies . Bra in 2010 ; 133 : 23 \200\223 32 . N Engl J Med 2008 ; 359 : 1728 \200\223 30 . 153 . Cotton RG , Auerbach AD , Axton M , Barash CI , Berkovic SF , Brookes AJ , et al . GE NETICS: the Human Variome Project . Science 2008 ; 322 : 861 \200\223 2 . 150 . Engels H , Brockschmidt A , Hoischen A , Landwehr C , Bosse K , Walldorf C , et al . DNA microarray analysis identi \201 es candidate CHAPTER 3 Neurochemistry of Epilepsy B. Keith Day, Lawrence Eisenman, and R. Edward Hogan Introduction Epileptic seizures emerge from complex biochemical processes in the human brai n. Physicians and scientists engaged in the study of this disease process have gained signi \201 cant knowledge about the neurochemical basis for epilepsy. The earliest studies at t he turn of the 20th century focused attention and subsequent theories on changes in chemica ls detected in the urine, serum, and cerebrospi- nal \202 uid, many of which we today rec ognize as non-speci \201 c associ- ated biochemical changes ( 1 ). However, with advances in studies of the human brain on cellular and molecular levels and the evolu- tion of the p harmacology of epilepsy, the early idea that excitatory and inhibitory in \202 uences in t he brain existed in balance that could be tipped toward excitation or away from inhibition to produce seizures was supported in numerous types of epilepsies and seizure-related condi tions. With this simple yet elegant view in mind, studies advancing our knowledge of the ne urochemistry of epilepsy continue to aid our understanding of the pathophysiology of epilep tic disorders as well as our approach to their diagnosis and treatment. Having reviewed the neurochemistry of the neurotransmitter sys- tems, we next turn to the n eurochemistry of antiepileptic drugs (AEDs). Finally, we conclude with a review of a clinic al trial of a drug speci \201 cally designed to modulate the GABA system that highlights s ome of the complexities of applying our knowledge of neurochemistry to patient care. GABA GABA is the major inhibitory molecule in the mammalian brain. GABA was recognized as a neurochemical in 1950 and as the amino acid in the inhibitory nerves of crustaceans by 196 3 ( 2 ). It was later accepted as satisfying criteria as a neurotransmitter in the mam- mal ian brain in 1973 ( 3 ). The idea that reduction of GABAergic inhibition results in epileps y while its potentiation is antiepileptic became known as the GABA hypothesis of epilepsy a nd continues to withstand experimental scrutiny ( 4 , 5 ). As discussed in more detail late r, its role in epilepsy became viewed as so important that designer drugs (including vigaba trin, tiagabine, and gabapentin) were created in hopes of mimicking the effects of GABA. The focus of this chapter will be on synaptic neurotransmission and its role in epilepsy. T he main excitatory and inhibitory neurochemicals in the human brain are glutamate and gamma -aminobutyric acid (GABA), respectively, which have gained wide acceptance as the major neu rochemical in \202 uences in epilepsy. However, other neu- rotransmitters such as aspartat e and glycine remain chemicals of interest as excitatory and inhibitory amino acids. Also, the roles of acetylcholine, serotonin, and the catecholamines (dopamine and noradrenaline) in epilepsy have been studied. We will review the basic chemistry of neurotransmission for the major neurotransmit- ters glutamate and GABA to provide a substrate for further discus- sion. Next, we brie \202 y consider the contribution of selective neuronal circuits to se izure generation. Animal models of seizures and epi- lepsy have been a critical source of k nowledge of the neurochemis- try of epilepsy, and we briefly review relevant data from seve ral such models with a focus on microdialysis experiments. We turn back to human studies, a gain focusing on microdialysis exper- iments. We also review magnetic resonance spectroscop y and the emerging technology of microelectrode arrays as newer methods that are complement ary to microdialysis. We then address potential contributions of the other neurotransmitter systems noted earlier. GABA is biochemically synthesized in presynaptic terminals with the removal of a carboxyl g roup from the alpha carbon of L-glutamate by the enzyme glutamic acid decarboxylase (GAD) ( 6 ). Depolarization of GABA-producing presynaptic terminals leads to voltage-gated calcium channel activation. Calcium entering the terminals facilitates vesicular docking and relea se of GABA into the extracellular space at the synaptic cleft where it is able to interact with two recognized receptor types, GABA A and GABA B . GABA A receptors are ligand-gated i on channels. When GABA binds GABA A receptors in the adult brain, a conformational change i n the shape of the receptor allows chloride to \202 ow along its concen- tration gradient into the postsynaptic cell increasing the negative membrane potential and hyperpolarizing the cell. GABA B recep- tors are metabotropic receptors that are coupled to G-proteins whic h activate adenylate cyclase leading to changes in cyclic adeno- sine monophosphate (cAMP) signaling to alter the conductance of a potassium channel. This, in turn, releases potassiu m from within the cell leading again to hyperpolarization. Both GABA receptor types make it more dif \201 cult for excitatory in \202 uences to depolarize the cell body to the poin t of creating new action poten- tials. GABA is actively removed from the extracellular spac e by oxford textbook of epilepsy and epileptic seizures 28 but reduce the activities of other excitatory and inhibitory ion channels. Overall, in addi tion to an important role in positive feed- back inhibiting further glutamate release, mGlu Rs modulate the postsynaptic cell \200\231s response to both excitatory and inhibitory in \202 u- ences ( 14 , 15 ). GABA transporters (GAT-1 through GAT-4). GAT is present on perisynaptic glia and the presyn aptic cell. Coupled to the uptake of one GABA molecule is the in \202 ux of two sodium ion s and one chlo- ride ion ( 7 , 8 ). The reversal of GATs may serve as a non-synaptic mechan ism for GABA receptor activation as well ( 9 ). Glutamate is actively removed from the extracellular space by glial and postsynaptic neuron al uptake through \201 ve subtypes of excitatory amino acid transporters (EAATs) each wit h a predomi- nant neuroanatomical distribution: EAAT1 (cerebellar glia), EAAT2 (forebrain g lia), EAAT3 (cortical neurons), EAAT4 (cere- bellar Purkinje neurons), and EAAT5 (retina) ( 16 , 17 ). Glutamate is not taken back up into the presynaptic terminal, so to replenish v esicles, the glutamate must be shuttled back to the original cell. This is accomplished thr ough the glutamate-glutamine cycle in which glial intracellular glutamate is converted to g lutamine by glutamine synthetase. Glutamine is released by the glia and taken up by the pre synaptic cell where it is converted back to glutamate by the enzyme glutaminase. Glutamate is actively transported along with protons into vesicles by the vesicular glutamate transpo rters (VGLUTs). Whereas EAATs will transportation aspartate, VGLUTs will not ( 16 ). Revers al of glutamate uptake may also contribute to extracellular release with both potential phy siological and path- ologically effects ( 18 ). Glutamate Glutamate is the major excitatory neurotransmitter in the mam- malian brain. It i s also one of the 20 amino acids used to synthesize proteins in humans and is readily avail able through dietary con- sumption of L-glutamic acid salts (such as monosodium gluta- mate ) and protein. However, most intracellular glutamate originates from glucose through glycol ysis and the Krebs cycle. The Krebs cycle intermediate -ketoglutarate is converted to gl utamate through transamination. Because of its ubiquitous nature in the body and very high concentrations in the central nervous system, its role as a neurotransmitter was doubted fo r many decades. However, by the mid to late 1970s, investigators were demonstrating bio- ch emical pathways of glutamate synthesis, specialized uptake by glia, and pharmacological ant agonism of neuronal excitation by compounds which prevented glutamate binding ( 10 \200\223 13 ). Depolarization at glutamatergic presynaptic terminals leads to vesicular release of glutama te at the synaptic cleft where it can act on multiple receptor types, both ionotropic and m etabotropic. Ligand-gated ion channels activated by glutamate include alpha- amino-3-hydrox yl-5-methyl-4-isoxazole-propionate (AMPA), N-methyl-D-aspartate (NMDA), and kainate recepto rs. AMPA receptors permit sodium to rapidly enter the postsynaptic cell accompanied by a sm aller ef \202 ux of intracellular potassium, with the net result being depolarization. Kai nate receptors act very simi- larly to AMPA receptors although the duration of the open ion channel is shorter. Kainate receptors are also found presynaptically on some GABAergic ter minals facilitating the release of GABA and, thus, can have an inhibitory effect at times. NMDA receptors require glycine as a co-ligand for activation and also have divalent magnesi um ions blockading the channels. The depolarization of the cell membrane by AMPA activation leads to expulsion of mag- nesium ions, so that movement of sodium and potassium can occur . Although there is also a small calcium conductance through AMPA and kainate receptors, NM DA receptors are the main gluta- mate ionotropic receptors that allow the passage of calciu m into the cell. The overall result is depolarization of the postsynaptic cell ( 14 ). Meta botropic glutamate receptors are G-protein coupled recep- tors that can be divided into thr ee groups: Group I (mGluR1 and 5), Group II (mGluR2 and 3), and Group III (mGluR4, 6, 7, an d 8). Group I receptors activate phospholipase C via G q proteins leading to phospholipid-r elated cell signaling through inositol 1,4,5- triphosphate (IP3) and diacyl glycerol (DAG), the latter of which can increase protein kinase C activity. Though they can be found presy naptically, Group I receptors are primarily postsynaptic. Experimentally, Group I receptor activation is both excitatory and inhibitory with capacity to modulate sodium, potassium, a nd volt- age-gated calcium channels, inhibit glutamate release, and enhance NMDA receptor a ctivity. Group II and III receptors coupled to G 0 proteins inhibit adenylate cyclase activ ity and decrease cAMP con- centrations. These receptors are primarily presynaptic and inhib it further release of glutamate. Postsynaptic ligand-bound Group II and III receptors do no t directly change the membrane potential GABA, glutamate, and epileptic circuitry GABA and glutamate neurotransmission are integral components of how seizures emerge, spread, and recur, and modulation of their effects is on e of the mechanisms by which many of our current AEDs prevent and abort seizures. In locali zation-related epilepsy, prolonged depolarization of neurons in the seizure focus leads to \201 ring of multiple sodium-dependent action potentials, a phenom- enon referred to as t he paroxysmal depolarization shift (PDS). The PDS is also commonly referred to as the intra cellular correlate of the epileptiform discharges seen with electroencephalographic recordi ngs. The PDS is typically followed by GABA-mediated hyperpolarization. If GABAergic inhibit ion fails, the bursting behavior of the PDS may precipitate continuous high-frequency neuro nal \201 ring which can propagate locally, spread through longer circuits to affect large r brain regions, or generalize to the entire brain ( 19 ). A typical seizure will end when the affected brain repo- larizes, and the postictal state is characterized by hyperpolariza tion. In generalized epilepsies, where many or all brain areas begin \201 ring abnormally simultaneously, the normal oscillatory rhythms of the thalamocortical loops are dysfunctio nal. The thalamic nucleus reticularis controls GABAergic thalamic relay neurons via T-type calcium channels. The thalamic relay neurons in \202 uence the activ- ity of glutamatergic pyramidal cortical neurons ( 20 , 21 ). Seizure creation results from an imbalance in exci tatory and inhibitory in \202 uences within these focal and generalized networks with glut a- mate and GABA playing dominant roles. The hippocampus within the temporal lobe is a region of extraor- dinary importance in epile psy research and clinical practice. Temporal lobe epilepsy (TLE) represents about 40 % of a dult onset epilepsies. The hippocampus has been extensively studied micro- structurally dat ing back to the initial studies of Ram n Y Cajal in the late 1800s, and with more advanced techniques, we now also understand much of the neurochemistry complementing the neuroanato my. The major circuit within the hippocampus is the CHAPTER 3 neurochemistry of epilepsy 29 system, while NMDA receptor activation is needed to initiate seizures in the area tempestas and non-NMDA receptors (AMPA and KA) are required for the seizures to spread into temporal regions ( 35 ). trisynaptic pathway. Glutamatergic projections from the entorhi- nal cortex perforate acros s a fold of the spiral hippocampal struc- ture to synapse on the dendrites of the granule c ells of the dentate gyrus creating the perforant pathway. Glutamatergic projections from th e granule cells project to the CA3 region. These axons are called mossy \201 bres due to their microstructural appearance. The third type of synapse of the pathway arises from glut amatergic projections from CA3 to CA1 pyramidal neurons called Schaffer collaterals. There are also recurrent pathways including dentate gyrus and CA1 to entorhinal cortex which are excitatory. Inhibitory control of this feedforward positive loop is provided by GABAergic i nterneurons found in the hilum ( 22 ). Finally, there is also subcor- tical modulation of t he system along the septohippocampal path- way which broadly projects cholinergic connectio ns throughout the hippocampus and well as speci \201 c GABAergic projections onto the GABA interneurons which would disinhibit the trisynaptic loop ( 23 ). More recently, glutamater gic projections have also been identi \201 ed in the septohippocampal pathway with a lesse r known role ( 24 ). Lessons from animal models of epilepsy In 1989, R.S. Fisher ( 36 ) published a review which described over 50 different animal models of epilepsy, several of which are still used tod ay. Many of the neuroactive chemicals that were found to produce seizures have both histori cal relevance and academic importance in epilepsy. For example, penicillin was found to ind uce seizures during neurosurgical procedures when it was applied directly to the cortex for antibiotic prophylaxis. Later, while conducting intracellular recordings of cat neocortex follow- ing focal penicillin administration, the PDS was \201 rst described ( 37 , 38 ). The chemical pentylenetetrazol (PTZ) was so reliable that it was used in British mental hos pitals to provoke seizures to treat psychiatric illness until 1939 when it was supplanted b y electrocon- vulsive therapy. For a time, it was also thought of as the prototypi- cal epi lepsy-invoking agent because of its ability to induce spike-wave or polyspike discharges on EEG. It even became an industry standard in AED screening; the ability to prevent PTZ- pro voked seizures was associated with a higher chance of prevent- ing primary generalized seiz ures, especially absence seizures. Much later, PTZ was found to work in part through the GA BA A receptor although the exact mechanism by which it provokes seizures remains unclear. I n fact, with the exception of the bromides and phenobarbital, every AED used today was \201 rst studied using animal models of epilepsy ( 36 ). Of special note, use of the maxima l electro- shock model to screen barbiturate derivatives led to the discovery of phenytoin in the late 1930s ( 39 ). The pathological structural correlate of mesial temporal lobe epilepsy (MTLE) is mesial tem poral sclerosis (MTS). MTS is the most common postsurgical histopathological \201 nding f rom patients with refractory epilepsy who undergo epilepsy surgery ( 25 , 26 ). It was \201 rst grossly recognized in the early 1800s followed by extensive study by Sommer in 188 0 ( 27 ). MTS became widely recognized as a common correlate of temporal lobe epilepsy in t he 1970s ( 28 ) and was histopathologically de \201 ned in a modern sense in the 1980s ( 2 9 , 30 ). MTS has the same pathological \201 ndings as the very closely related Ammon \200\231s horn sclerosis but extends into other neighbor- ing regions including the entorhi nal cortex and amygdala. Pathologically there is signi \201 cant segmental neuronal loss a ccom- panied by astrogliosis. Speci \201 cally, this damage is at minimum observed in Somm er \200\231s sector made up of the CA1 and presubicu- lum. Grossly, the tissue appears shru nken and hardened which can be appreciated with brain magnetic resonance imaging (MRI) and the corresponding metabolic dysfunction can be observed with positron emission tomography a nd single-positron emission com- puted tomography ( 31 , 32 ). Additional histopathological changes in MTLE include loss of hilar mossy cells, hilar somatostatin- containing GABAergi c interneurons, and CA1 pyramidal neurons with associated dispersion of the granule cell la yer and aberrant sprouting from the granule cells, spared GABAergic interneurons, and remai ning CA1 pyramidal neurons ( 26 ). The resulting altera- tions in glutamate and GABA transm ission are widely assumed to contribute to the pathophysiology of MTLE and remain areas of intense study. Fortunately, interest in developing animal models of epilepsy over decades has provided fur ther insight into the role of GABA in epilepsy. In addition to PTZ, there are numerous addi tional GABA antagonists that have been shown to be effective in producing recurrent seizure s in animals, including bemegride (GABA A antag- onist), picrotoxin (non-competitive GABA A antagonist that acts by blocking the ion channel itself and not the receptor binding site) , and bicuculline (competitive GABA A antagonist). In 1974, Meldrum et al. ( 40 ) showed th at allylglycine, which inhibits gluta- mate decarboxylase (GAD) and prevents GABA synthesis , caused prolonged status epilepticus in monkeys. Treiman ( 41 ) outlined multiple GABAergi c mechanisms found in animal models of epi- lepsy including alterations in GABA levels, rec eptor binding, and uptake as well as the antiepileptic properties of drugs which enhance GA BA neurotransmission. The animal models also support a signi \201 cant role for glutamate in epilepsy. Most anim al models of complex partial seizures utilize enhanced excitation either through genetic ma nipulation, kindling, or the administration of excitatory chemicals. Both glutamate and its structural analog kainate, \201 rst discovered in red algae in 1953 ( 42 ), were known t o be potent excitotoxins. Kainate became one of the most widely used neuroactive chemicals to induce seizures in animals starting in the late 1970s and 1980s ( 43 ). These animals al so show structural changes in the brain which are very similar to human MTS. Studies show t hat mice genetically designed to be de \201 cient in glutamate uptake are susceptible to s eizures ( 44 ). Those strains without the ability to post-transcriptionally edit glutamate receptor subunits (Q/R GluR-B subunit), causing poorly regulated A lesser known epileptic region in which glutamate and GABA are important is the area tempe stas, a structure in the forebrain \200\231s deep prepiriform cortex. It has been identi \201 ed as a particularly epi- leptogenic region with connections to the temporal lobes. In itial studies showed that injections directly into this region with chemi- cals such as kai nate, glutamate, aspartate, and NMDA as well as bicuculline produce seizures. At the same t ime, treatment with the NMDA receptor antagonist 2-amino-phosphonoheptanoic acid (2APH) or the GABA A agonist muscimol was found to prevent sei- zures ( 33 , 34 ). Further studies at tempted to elucidate speci \201 c neuro- transmitter-receptor pathways through which seizu res could propagate from the area tempestas into temporal regions. They found that GABA inh ibition was suf \201 ciently antiepileptic throughout the oxford textbook of epilepsy and epileptic seizures 30 amino acid changes sampled using microdialysis between the rat kainate model of chronic par tial epilepsy and twelve patients with TLE. At least 90 days elapsed between intrahippocamp al kainate injections and microdialysis sampling in the rat group. For both groups, they us ed electrographic monitoring and sampled micro- dialysate every 30 minutes interictally and every 5 minutes for an hour at the time of a seizure. Ictally in the patients, there was a signi \201 cant elevation of hippocampal glutamate, aspartate, GABA, and taurine, while d uring seizures in the rat hippocampus, all of these were elevated except taurine. More rece ntly, Meurs et al. ( 58 ) published the results of their own microdialysis studies of three animal models of acute epilepsy using intrahippocampal reverse dialysis to apply pilocarpi ne (cholinergic muscarine agonist), picrotoxin (GABA A antagonist), and a Group I metabotro pic glutamate antagonist, all of which had previously been shown to provoke seizures. Durin g seizures, both glutamate and GABA were signi \201 cantly increased in all three models, especially glutamate following picrotoxin administration. AMPA-related calcium in \202 ux, are useful models of temporal lobe epilepsy. Kindling to the point of status epilepticus is associated with elevated glutamate levels as well as inc reased NMDA receptor number and activity and other glutamate receptor changes ( 26 , 45 ). Microdialysis: animal studies In the last few decades, investigators have developed techniq ues to measure extracellular levels of neurochemicals in animal models of epilepsy, resecte d human tissue, and patients with epilepsy, both ictally and interictally. To date, microdi alysis, in which a small probe with a dialysis membrane at its tip is inserted into a brain region of interest, has been the most widely used of these technolo- gies. Fluid can run t hrough the probe in a contained loop, allowing chemicals to pass from the extracellular spa ce across the membrane into the \202 uid for sampling, or in some cases, chemicals can be directly applied in the area of the probe for experimental purposes. Measurements of the c hemicals of interest are typically per- formed off-line using highly sensitive techniques, such as high- performance liquid chromatography (HPLC). Because multiple samples can be tak en serially, the time course of chemical changes in the brain can be studied ( 46 ). Microdialysis: human studies In contrast to \201 ndings in animal models, intracerebral m icrodialy- sis studies of epileptic patients are more consistent. Prior to extra- cellular studies of resected human epileptic and non-epileptic tissue, whole tissue studies showed e vidence of increased glutamate levels and increased glutamate receptor binding. This was co upled with decreased GABA binding without changes in whole tissue levels or number of hippo campal GABAergic synapses despite clear loss of some of the hilar GABAergic interneurons ( 59 ). In 1981, Perry and Hansen ( 60 ) studied the amino acid composition of resected epile ptic foci from 35 patients and found no change in total GABA or aspartate concentrations bu t a signi \201 cant increase in glutamate, including levels greater than two standard devi ations above the mean in six patients, and postulated a possible aetiologi- cal role for gl utamate in epilepsy. However, the ability to measure extracellular amino acids in real time while monitoring patients having seizures was a breakthrough in understanding the neuro- c hemistry of epilepsy. In 1992, Ronne-Engstrom et al. ( 61 ) reported amino acid levels from seven patients undergoing epilepsy surgery in whom they collected samples via intraoperati ve in vivo microdi- alysis capturing \201 ve spontaneous and \201 ve electrically-induc ed sei- zures. Compared to basal levels measured before seizures, aspartate, glycine, gluta mate, and serine were signi \201 cantly elevated during the seizures. GABA levels were not analyzed. Experience with using intracerebral microdialysis to study epi- leptic and non-epileptic br ain regions in animal models of epilepsy revealed the signi \201 cant roles of neuroactive amino acids in the brain, especially glutamate and GABA. Zhang et al. ( 47 ) studied amino acid changes in the ventral hippocampus before and during kindling using electrical stimul ation of the deep prepiriform cortex which showed that both glutamate and glycine increased during kindling, with elevated basal glutamate levels for up to 30 days thereafter. Millan et al. ( 48 ) examined amino acid levels in the hip- pocampus in rats \201 nding signi \201 cant increases in aspartate and glutamate after kindling. They also found if pilocarpi ne was used to induce seizures in kindled animals, both glutamate and aspar- tate concentra tions rose prior to the onset of seizures. This effect of kindling has been shown by others as well ( 49 ). In 2001, a study using the rat kainate model of partial epilepsy showed de creased protein and messenger RNA levels for the main rodent glutamate transporters GLAST a nd GLT-1 as well as changes in potassium- evoked neurotransmitter release (increased glutam ate, decreased GABA) interictally in freely-moving animals which they inter- preted as a re sult of impaired glutamate uptake ( 50 ). In a rat model of absence epilepsy, which likely involves pathophysiological changes in GABAergic regulation of the glutamatergic thalamocor - tical loops, GABA B antagonists are highly anticonvulsant. Using in vivo microdialysis, i nvestigators were able to measure an increased basal level of GABA in this animal model, al though treat- ment with GABA B antagonist baclofen or GAT inhibitor tiagabine did not signi \201 cantly change GABA levels ( 51 ). Lacking, minimal, or con \202 icting microdialysi s neuroactive amino acid \201 ndings in animal models of epilepsy, as demonstrated in thi s study, are unfortunately common ( 52 \200\22356 ). Shortly thereafter During and Spencer ( 62 ) reported changes in glutamate and GABA in six patients who had bilaterally implanted hippocampal microdialysis probes. In the period prio r to their epi- lepsy surgeries, they were electrographically and behaviourally monitored f or seizures. They found that increased extracellular glutamate preceded seizure onset by ov er a minute and during this period there was also relatively lower extracellular GABA. Duri ng seizures, glutamate levels increased signi \201 cantly bilaterally and persisted much l onger on the side of seizure onset. GABA also rose to a lesser degree, and interestingly wa s signi \201 cantly higher on the contralateral side. This work was followed in 1995 by an investiga- tion focused on GABA. Previous work showed diminished GABA inhibition in the hu man epileptic hippocampus, despite relatively preserved GABAergic interneurons and receptor s. They compared potassium- and glutamate-evoked GABA release in the bilateral Several investigators have attempted to reconcile the con \202 icting results by conductin g their own potentially de \201 nitive studies. Wilson et al. ( 57 ) posited that prior st udies showing little to no change in neuroactive amino acids might be because the investiga - tors had not allowed enough time for pathophysiological changes to occur in the animal mo dels of chronic epilepsy. Their study was unique insofar as they compared measurements of h ippocampal CHAPTER 3 neurochemistry of epilepsy 31 MRS can detect glutamate at much lower \201 eld strengths ( 71 ). The best use of MRS in epilepsy is not yet clear. Studies over the last two decades have investigated metabolic an d energy abnormalities in generalized epilepsies, Rasmussen \200\231s encephalitis, and tem poral lobe epilepsy ( 72 \200\22375 ), as well as lateralization of seizure onset when MRI was inconclusive ( 76 , 77 ). Clearly, applications for research- ing the role of GABA have been the most proli \201 c to date, including predicting poorer seizure control in one st udy ( 78 ). However, the primary use has been investigation of GABA levels as they relate t o the mechanisms of action of multiple AEDs ( 79 \200\22381 ). There are also MRS studies o f glutamate and the glutamate-glutamine cycle in temporal lobe epilepsy ( 66 , 82 ). hippocampi and found increased potassium-evoked release and diminished glutamate-evoked rel ease on the epileptogenic side. Unlike potassium-evoked release, glutamate-evoked release w as calcium-independent (i.e. non-synaptic), and they speculated that the high concentration s of glutamate causing depolarization might reverse the sodium-dependent GATs. In parallel studies in amy- gdala-kindled rat hippocampus, they reported a 48 % reduction in GAT, sugge sting that decreased extracellular GABA might be related to decreased GAT density ( 63 ). Since these landmark studies, investigators have continued to study additional aspects of g lutamate and GABA in human epilep- togenic brain. Starting in 2003, O \200\231Connor \200 \231s group published a series of papers highlighting increased glutamate, aspartate, and G ABA intraoperatively in spontaneously epileptiform human hip- pocampus ( 64 ). In 2005, the metabolism of glutamate was exam- ined by Cavus et al. ( 65 ) using microdialysis and dept h electrodes interictally in 38 patients with both TLE and cortical epilepsy. In the epilep togenic hippocampus, they measured elevated basal glutamate and high lactate as well as poo r glucose utilization and a low glutamine-to-glutamate ratio raising the possibility of imp aired glutamate uptake and glutamate-glutamine cycling in association with inef \201 cient regional energy use. In epileptogenic cortex, gluta- mate was only marginally increased wh ich might indicate a differ- ent regional pathophysiology. In 2008, related groups publishe d multiple papers on the epileptogenic hippocampus, linking higher glutamate levels with di minished tissue volumes, energy impair- ment, and hyperexcitability and correlating GABA le vels with neu- rometabolism ( 66 \200\22368 ). Of particular note, Eid et al. ( 69 ) review ed the previous data and advanced the concept of the \200\230glutamate hypothesis \200\231 in MTLE tying together the importance of astrogliosis in MTS with the special role that as trocytes play in extracellular glutamate regulation. Astrocytes control extracellular gluta mate levels through rapid uptake and the glutamate-glutamine cycle, but they noted a 35 \200\22340 % decrease in glutamine synthetase (GS) and approximately 40 % decrease in activ ity of glutamine synthetase in MTLE and proposed that astrocytic glutamate release through uptake reversal may play a signi \201 cant role in its pathophysiology. Microelectrode arrays: animal studies and clinical potential A major limitation of whole ti ssue levels, MRS, and even microdi- alysis is temporal resolution. Whole tissue and MRS pro vide interictal snapshots of chemical pro \201 les. The vast majority of microdialysis-bas ed techniques cannot sample more quickly than every few minutes because higher \202 ow ra tes would disrupt tissue and confound measurements. A typical seizure lasts less than one m inute, and the kinetics of the neurochemical changes that occur during a seizure are likely to occur over the time course of seconds to minutes, especially with respect to extracellu lar neurochemicals with high af \201 nity uptake systems like glutamate and GABA. One intr iguing approach to address this problem is the use of implant- able microelectrode arrays w hich can provide second-by-second electrochemical detection of extracellular neurochemicals . Using an enzyme coating coupled with voltammetric techniques for iden- tifying speci \201 c neurochemicals, Dr Greg Gerhardt \200\231s group has re \201 ned a platinum-based, glutamate oxidase-coated microeletrode array capable of measuring basal and stimulated rele ase of gluta- mate in anesthetized and awake mice, rats, and monkeys ( 83 \200\22387 ). In animal studies, potassium-evoked release of glutamate has very fast kinetics and is complet ely cleared back to baseline within sec- onds ( 88 ). Efforts are ongoing to complete re \201 nements of a com- bined depth electrode and glutamate-sensitive microelectrode array f or human use. The details of potential microelectrode-based epilepsy therapy were published in 2008 ( 89 ). Magnetic resonance spectroscopy: human studies Nuclear magnetic resonance spectroscopy (MRS ) is an attractive tool for both clinical and investigational use in epilepsy given its rea dy availability at most academic medical centers, safety, and non-invasive nature. MRS meas ures the resonance frequencies of naturally occurring radioisotopes, such as 1 H, 31 P, and 13 C, to identify and quantitate a broad number of neurochemicals. These include N-acetyl aspartate (NAA, primarily neuronal), creatine- and choline-containing compounds (primarily glial), and lactate, which in combination can provide evidence of neuronal damage and energ y de \201 ciency. Other neurochemicals of special interest in epilepsy which can be measur ed include GABA, glutamate, and glutamine ( 59 , 70 ) which allows MRS to provide complemen tary data to that obtained using microdialysis. MRS is limited primarily insofar as it prov ides a measurement of a total level and cannot dif- ferentiate between intra- and extracell ular pools. GABA was \201 rst detected with 1 H MRS in 1984, whereas glutamate was detect ed initially in 1985 but required an 8.5 Tesla magnet. Currently 13 C Other neurotransmitters and epilepsy There are studies of other neurotransmitters in epilep sy. As neu- roactive amino acids, both aspartate and glycine show variable changes during s eizures as previously detailed. The excitatory neu- rochemical aspartate is a pervasive non -essential amino acid which may be co-released with glutamate. Its importance as a neurotra ns- mitter is questionable considering that, other than NMDA recep- tors which bind glutama te more tightly than aspartate, there are no currently identi \201 ed aspartate receptors. Aspartate can be removed from the extracellular space by glutamate transporters, but is no t packaged into vesicles by VGLUTs. On the other hand, the inhibi- tory neurotransmitter gl ycine exerts its effects through speci \201 c glycine receptors which are chloride ion cha nnels, like GABA A receptors, although GABA cannot activate them. The glycine recep- tor is found predominantly in the brainstem and spinal cord and plays a role in motor re \202 ex circuits and nociception as well as the rare neurological disorder hyperekplexia, or start le disease ( 90 ). oxford textbook of epilepsy and epileptic seizures 32 However, glycine receptors are also present in hippocampal neu- rons where activation by ex trasynaptic glycine may provide tonic inhibition ( 91 ). The NMDA receptor also has a glyci ne binding site giving glycine a role in excitation as well. To date, there are no studies reporting signi \201 cant pathophysiologic roles in epilepsy for either aspartate or glyci ne although glycine encephalopathy (non- ketotic hyperglycinemia), an inborn error of glyci ne metabolism, is associated with refractory seizures ( 92 ). that a heritable ion channel mutation can cause epilepsy ( 103 , 104 ). Overall, acetylchol ine has a signi \201 cant modulatory role in multiple types of epilepsy as well as an etio logical role in one type. However, beyond the potential bene \201 ts of future gene therap y for ADNFLE, cholinergic modulation has not shown ef \201 cacy in treating seizures, and its importance as target for further AED development remains unclear. Support for serotonin \200\231s contribution to epilepsy has been grow- ing since the mid 1 980s. Serotonergic projections originate in the midbrain raphe nuclei and make diffuse conn ections throughout the brain, including most well-known epileptic brain regions including t he cortex, hippocampus, and thalamus. Bagdy has sum- marized several key points ( 105 ). In animal models of epilepsy, increased serotonergic activity tends to be antiepileptic where as decreased activity is proconvulsant. Furthermore, provoked or genetic mouse models of ep ilepsy with knockout for 5-HT 1A or 5-HT 2C receptors tend to have more seizures. There is some evi- dence of changes in serotonin neurotransmission in resected tumors and epileptoge nic tissue. Among the multitude of serot- onin receptors, the 5-HT 2C receptor appears to b e of particular importance in epilepsy. Some currently used antiepileptic medica- tions inc luding valproic acid, lamotrigine, carbamazepine, pheny- toin, and zonisamide, when adminis tered experimentally in animals, are associated with increased extracellular serotonin leve ls. Altogether, there is some sentiment that serotonin should be more closely investigated for possible AED development.