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. Dopamine, on the other hand, seems to have a complex role in subcortical modulation of epil eptogenic circuits ( 93 ). Alterations in dopamine pathways occur in ring chromosome 20 epi lepsy, temporal lobe epilepsy, juvenile myoclonic epilepsy, and auto- somal dominant noctur nal frontal lobe epilepsy (ADNFLE) ( 94 ). The thalamocortical pathways which underlie gene ralized epilep- sies are regulated by GABAergic projections from the thalamus; however, osc illatory pathways within the basal ganglia also have projections which modulate the thalamo cortical loops. This includes the dopaminergic nigrostriatal pathway originating in the sub stantia nigra. Based on this, many investigators have proposed that basal ganglia dysfuncti on has a signi \201 cant role in the patho- physiology of some epilepsies ( 95 , 96 ). Als o of potential interest is the dopaminergic mesocorticolimbic pathway from the ventral tegm ental area to the frontal cortex which might help explain both motor and behavioral symptom s seen in epilepsy. These \201 ndings have yet to inspire any signi \201 cant pursuit of dopaminergic drugs as antiepileptic agents. Nonetheless, further de \201 nition of the ro le of dopamine, especially in speci \201 c epileptic syndromes, remains an intriguing area of active research. Lastly, noradrenaline also has support as an antiepileptic neuro- transmitter. Of the multi ple noradrenergic brainstem nuclei, it is the most rostral pontine nucleus, the locus coeru leus (LC), which appears to have a role in epilepsy. The LC projects along two small ascend ing pathways, the dorsal bundle and the rostral limb of the dorsal periventricular pathway, before extensively branching to innervate the entire cerebrum. There are also descending p rojec- tions to the cerebellum, medulla, and spinal cord. Evidence for the anticonvulsant p roperties of noradrenaline stem from pharmaco- logic agonism of its receptors and from lesi onal studies of the LC in which seizure magnitude and associated pathological destruction w as heightened in both genetic and acquired animal models of epilepsy. Furthermore, when ani mal models of epilepsy are also exposed to common AEDs, including valproic acid, carbamazep ine, phenytoin, and phenobarbital, increased levels of noradrenaline can be measured in som e brain regions. In addition, noradrenaline is associated with the ef \201 cacy of vagal n erve stimulation (VNS), a treatment for refractory partial epilepsy thought to exert its ef fects through stimulation of the nucleus of the solitary tract and other neighboring brains tem nuclei that modulate the subcortical gating of epileptic circuits. In 1998, Krahl et al . ( 106 ) showed that bilateral LC lesions eliminated the seizure-suppressing ability of VN S. Finally, several other neurotransmitters are co-released with noradrenaline including ga lanin, neuropeptide Y, and adenosine which may also be contributory ( 107 ). Acetylcholine was the \201 rst identi \201 ed neurotransmitter, largely due to its promi nence in the parasympathetic nervous system and neuromuscular junction. Its discovery earne d Dale and Loewi the Nobel Prize in Physiology or Medicine in 1936. As a result, early stud ies of the neurochemical basis for epilepsy focused on acetyl- choline, even prompting Fost er in 1946 to conjecture that \200\230the physiological cause of epilepsy may be de \201 ned as abnormalities of acetylcholine metabolism \200\231 ( 97 ). However a major role for acetyl- choline in epilepsy remained controversial for decades. Obvious support stemmed fro m \201 ndings that either direct intrahippocam- pal or systemic treatment with muscarinic cholinergic agonists such as pilocarpine could provoke status epilepticus followed by chro nic seizures in rodents ( 98 \200\223100 ). Anatomically in humans, cholinergic projections to the hippocampus arise from cells in the medial septum and the diagonal band of Broca al ong the septohip- pocampal pathway which also carries GABAergic and glutamater- gic project ions ( 24 ). This pathway regulates hippocampal theta rhythms most prominent during wakeful ness and REM sleep, which are periods of decreased seizure likelihood in patients with epil epsy. This antiepileptic role is also further supported by the fact that the cholinergic ag onist carbachol induces hippocampal theta and protects against epileptogenesis in rodent mo dels of chronic epilepsy ( 101 ). The major cholinergic pathways in the brain begin in the basal nucleus of Meynert in the forebrain and project dif- fusely throughout the cortex wit h a signi \201 cant role in arousal. Some of these projections also terminate in the thala mic reticular nucleus, where acetylcholine has a modulatory effect on the tha- lamocortical loops ( 102 ). Separately, the neuronal nicotinic acetyl- choline receptor mutations CHRNA 4 ( 4 subunit) and CHRNB2 ( subunit) are implicated in ADNFLE types 1 and type 3, res pec- tively. The CHRNA4 mutation, in fact, was the \201 rst demonstration The neurochemical basis of antiepileptic drugs Given our modern understanding of the contri butions of multiple neurotransmitter systems that drive inhibition and excitation of the ep ileptic circuits, one might expect that the neurobiology has driven the drug designs. At ti mes, this has been true; however, the CHAPTER 3 neurochemistry of epilepsy 33 receptors, and, in fact, the most likely mechanism of action is via binding the 2 sub unit of voltage-gated calcium channels ( 117 ). However, there is also some evidence that g abapentin modulates GABA systems including increasing GABA synthesis and turnover as well a s possibly inhibiting or reversing GABA transportation. The second agent vigabatrin was fou nd to be an irreversible inhibitor of GABA transaminase, the enzyme which breaks down GABA. Without GABA catabolism, GABA levels increase, possibly by way of reversal of GAT. Approxi mately 10 years following its introduc- tion on the market, reports of visual \201 eld lo ss because of retinal side effects limited enthusiasm for continued use, especially in the US, although it is still used in many other countries around the world, though almost exclu sively for the treatment of infantile spasms. Finally, tiagabine is a GAT inhibitor which a lso effectively increases GABA levels ( 116 ). Rounding out the story, both fel- bamate and topiramate have some effects on GABA A receptors, and though the main mechanism of action of levetiracetam is likely related to binding to the synaptic vesicular protein SV2A which may inhibit calcium-dependent vesicular release ( 117 ), levetira- cetam reportedly also re verses an exogenous allosteric modulator of GABA A receptors ( 118 ). drug development evolved with our understanding of the biology such that most of the curren t AEDs which we use today were not borne out of newly identi \201 ed molecular targets. Fo r example, bro- mide salts were found to be effective antiseizure treatments in 1853, used for well over 50 years in humans, and are still used in veterinary medicine today. However, after discovery of safer agents, like phenobarbital in 1912 and phenytoin in 1937, their u se in humans ceased ( 108 ). The proposed GABAergic mechanism of action was not reported un til the 1980s ( 109 ), highlighting that rational drug design is often challenging because our observations outpace our understanding. A later chapter will detail the AEDs, but becau se the neurochemical basis of epilepsy offers us signi \201 cant insight into the use of m edications, a brief discussion is merited. The major mechanisms by which AEDs exert their effects can be subdivided into three categor ies as described by Rogawski and Loscher ( 104 ): ( 1 ) modulation of voltage-gated ion cha nnels including sodium and calcium, ( 2 ) enhancement of synaptic inhi- bition, and ( 3 ) i nhibition of synaptic excitation. The \201 rst category includes what clinicians often re fer to as the sodium channel block- ers, which includes the voltage-gated fast and the pers istent sodium currents. Modulation affects neuronal ability to respond to depo- larization with generation of an action potential. Relevant AEDs include phenytoin, carbamazepine, oxc arbazepine, lamotrigine, zonisamide, and lacosamide ( 110 ) as well as ru \201 namide, fel bamate, valproic acid, and topiramate. This effect on action potentials reduces downstream synaptic transmission, with evidence that glutamate release may be more affected than other neurotransmit- ters, such as GABA ( 111 , 112 ). Sodium channel blockers tend to be effect ive against partial and generalized tonic \200\223clonic seizures. The voltage-gated calciu m channel blockers include the high voltage- activated (HVA) (L-, R-, P/Q-, and N-types) an d the low voltage- activated (LVA) (T-type). HVA calcium channels are primarily involved in presynaptic neurotransmitter release, while LVA cal- cium channels regulate bursting and o scillations such as previously described in the thalamus. Probably the most recognizable of the AEDs that primarily affect calcium channels is ethosuximide which blocks T-type channe ls and disrupts the hypersynchronized tha- lamocortical loops responsible for absence epile psy. Other AEDs which interact with T-type channels include zonisamide and pos- sibly valpr oic acid, while the AEDs which have HVA calcium chan- nel activity include lamotrigine, fel bamate, topiramate, gabapentin, and phenobarbital. Levetiracetam at clinically relevant dos es reportedly also inhibits HVA (N-type) calcium channels ( 113 , 114 ). More recently, vol tage-gated potassium channels were recog- nized as another potential target for AEDs. In pa rticular, the KCNQ/Kv7 activating drug retigabine has shown signi \201 cant prom- ise in c linical trials ( 115 ). Finally, glutamate receptors are also molecular targets for AED development. In animal mode ls, antagonism of AMPA and NMDA is effective, but clinical trials have been disappointing d ue to sig- ni \201 cant side effects. The NMDA receptor blocker ketamine is sometimes bene \201 cial in the treatment of refractory status epilepti- cus. Furthermore, felbamate may somewhat selectively block NMDA receptors containing the NR2B subunit which is largely res tricted in its CNS distribution to the forebrain, perhaps explain- ing why its side effect pro \201 le is better. Phenobarbital also blocks AMPA receptors, and topiramate antagonize s both AMPA and kai- nate receptors. Recall that kainate may be an especially intriguing ta rget for blockade as it causes both postsynaptic excitation and presynaptically inhibits GA BA release. Most recently, the \201 rst-in- class, highly selective, non-competitive AMPA receptor blocker peramapanel (investigation compound E2007) is currently being evaluated f or clinical use. Clinical complexities of the neurochemistry of epilepsy The development of multiple antiepi leptic medications with differ- ent mechanisms of action has led to the concept of \200 \230rational polyp- harmacy \200\231 for the treatment of refractory epilepsy. The idea is that combining medications with different mechanisms of action may produce synergistic effe cts that improve ef \201 cacy. Unfortunately, there is surprisingly little clinical data t o guide treatment ( 119 ) and despite our ever increasing understanding of the neurochemist ry of epilepsy, it is still necessary to assess combinations of medica- tions with differen t proposed mechanisms of action in clinical trials in patients with epilepsy. One trial, de signed as a \200\230 \201 rst add- on \200\231 study, compared effects of tiagabine to ph enytoin and car- bamazepine, using a parallel design. Because the study allowed comparison of drugs with different proposed mechanisms and highlights the complexities of \200\230rat ional polypharmacy \200\231, the study will be presented in detail. Potentiation of GABA systems has been the major AED mecha- nism since effective treatments became recognized. Despite that excitatory synapses in epileptic regions often signi \201 cantly outnum- ber inhibitory ones, the ability of GABA to inhibit feedforward positive loo ps is critically important and a potent strategy for sei- zure treatment. Both benzodiazepi nes and phenobarbital accentu- ate the effects of GABA at the GABA A receptor through diffe rent mechanisms. Valproic acid increases both GABA synthesis and turnover. The \201 rst e xample of rational drug design focused on GABA and produced three agents in use today. Gaba pentin, a lipophilic 3-cyclohexyl analog of GABA, was synthesized in an attempt to create a GABA agonist, but it does not activate GABA Patients with complex partial seizures with or without secondary generalization, which were not satisfactorily controlled on pheny- toin or carbamazepine monotherapy, were randomized to either a oxford textbook of epilepsy and epileptic seizures 34 References tiagabine or carbamazepine/phenytoin add-on treatment group. Ef \201 cacy determinations w ere based on seizure frequency. The add-on AED was titrated to effect or unacceptable adver se effects to permit patients to \201 nd the best tolerated and effective dose and provid e a fair comparison between the study drugs ( 120 ). 1 . Van Rijn C , Meinardi H . Neurochemistry and epileptology . Epilepsia 2009 ; 50(Suppl 3) : 17 \200\223 29 . 2 . Kravitz EA , Kuf \202 er SW , Potter DD . Gamma-aminobutyric acid and other blocking compounds in crustacea. Iii. Their relative concentrations in separated moto r and inhibitory axons . J Neurophysiol 1963 ; 26 : 739 \200\223 51 . Eight tests of mental abilities and three of mood and adjustment were administered prior to assignment of add-on treatment and after up to 16 weeks of add-on treatment. Overall, add- on tiagab- ine showed few or no differences in comparison with add-on car- bamazepine and a dd-on phenytoin ( 121 ). Slightly more side effects were reported with the addition of eith er phenytoin or car- bamazepine compared to addition of tiagabine. Measuring the median sei zure reduction of the two \200\230combined \200\231 groups, which compared the average of the add-on carbamazepine and add-on phenytoin groups with the average of the tiagabine add- on groups, showed a statistically signi \201 cant difference in favor of the car- bamazepi ne/phenytoin groups which was attributed to the car- bamazepine add-on to phenytoin group ( 122 , 123 ). 3 . Otsuka M . 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This well-designed study showed better tolerability with add-on tiagabine, few differences in effects on cognitive ability, mood, and adjustment between the groups, and better ef \201 cacy with add-on carbamazepine/phenytoin. The concept of \200\230rational polypharmac y \200\231 was not supported by this trial since add-on therapy with tiagabine (GABAergic m echanism) was less effective than add-on therapy with carbamazepine/phenytoin. Improved ef \201 cacy in the car- bamazepine add-on group may well re \202 ect mechanisms of action o f carbamazepine beyond sodium channel modulation ( 124 ), dif- ferential ef \201 cacy betw een carbamazepine, phenytoin, and tiagab- ine, an effect of the trial design or some combin ation of factors. One other unexplained outcome is the observation that the addi- tion of c arbamazepine to phenytoin was apparently superior to the addition of phenytoin to carbamaze pine. 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