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Brain concentrations of glutamate and GABA in human epilepsy: A review

  • Gabrielle L. Sarlo
    Affiliations
    Department of Psychology, Behavior, Cognition and Neuroscience Program, American University, Washington DC, United States
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  • Kathleen F. Holton
    Correspondence
    Corresponding author at: American University, 4400 Massachusetts Ave NW, McCabe 203, Washington DC 20016, United States.
    Affiliations
    Department of Health Studies, American University, Washington DC, United States

    Center for Behavioral Neuroscience, American University, Washington DC, United States
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Open ArchivePublished:June 28, 2021DOI:https://doi.org/10.1016/j.seizure.2021.06.028

      Highlights

      • Glutamate metabolites are elevated in epilepsy patients and epileptogenic regions.
      • Studies on GABA levels in epilepsy patients and epileptogenic regions are lacking.
      • More pediatric and high MRI Tesla strength research is needed.

      Abstract

      An imbalance between excitation and inhibition has been a longstanding proposed mechanism regarding ictogenesis and epileptogenesis. This imbalance is related to increased extracellular glutamate in the brain and/or reduction in GABA concentrations, leading to excitotoxicity, seizures, and cell death. This review aims to summarize the microdialysis and magnetic resonance spectroscopy (MRS) literature investigating glutamate and GABA concentrations in epilepsy patients, present limitations, and suggest future directions to help direct the search for novel epilepsy treatments. The majority of microdialysis studies demonstrated increased glutamate in epileptic regions either compared to control regions or to baseline levels; however, sample sizes were small, with some statistical comparisons missing. For the MRS research, two of six studies reported significant changes in glutamate levels compared to controls, though the results were mixed, with one reporting increased and the other reporting decreased glutamate levels. Eleven of 20 studies reported significant changes in Glx (glutamate + glutamine) or Glx ratios, with most reporting increased levels, except for a few epilepsy syndromes where reduced levels were reported. Few studies investigated GABA concentrations, with one microdialysis and four spectroscopy studies reporting increased GABA levels, and one study reporting decreased GABA in a different brain region. Based on this review, future research should account for medication use; include measurements of GABA, glutamate, and glutamine; use high-tesla strength MRI; and further evaluate the timing of microdialysis. Understanding the importance of brain glutamate and GABA levels in epilepsy may provide direction for future therapies and treatments.

      1. Introduction

      Epilepsy is a chronic neurological disorder characterized as two or more unprovoked seizures twenty-four hours apart, one unprovoked seizure with the probability of more seizures equivalent to the general recurrence risk, or an epilepsy syndrome diagnosis [
      • Fisher R.S.
      • Acevedo C.
      • Arzimanoglou A.
      • Bogacz A.
      • Cross J.H.
      • Elger C.E.
      • et al.
      ILAE Official Report: a practical clinical definition of epilepsy.
      ]. Epilepsy affects 50 million people worldwide [
      • Beghi E.
      • Giussani G.
      • Nichols E.
      • Abd-Allah F.
      • Abdela J.
      • Abdelalim A.
      • et al.
      Global, regional, and national burden of epilepsy, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016.
      ]. The prevalence rate of active epilepsy in the US is 1.2%, or about 3.4 million people [
      • Zack M.M.
      • Kobau R.
      National and State Estimates of the Numbers of Adults and Children with Active Epilepsy — United States, 2015.
      ]. Of those with epilepsy, about one third are refractory cases, meaning their seizures are not controlled despite the use of at least two appropriately tried anti-epileptic medications [
      • Stafstrom C.E.
      • Carmant L.
      Seizures and Epilepsy: an Overview for Neuroscientists.
      ]. For these patients, development of alternative treatment options is critical, which requires an in-depth understanding of the biological mechanisms associated with seizure occurrence.
      An imbalance between excitation and inhibition has been implicated in ictogenesis and epileptogenesis and underscores the importance of understanding the relationship between epilepsy and glutamate [
      • Treiman D.M.
      GABAergic Mechanisms in Epilepsy.
      ,
      • Bozzi Y.
      • Provenzano G.
      • Casarosa S.
      Neurobiological bases of autism-epilepsy comorbidity: a focus on excitation/inhibition imbalance.
      ,
      • Scharfman H.E.
      The Neurobiology of Epilepsy.
      ,
      • Barker-Haliski M.
      • White H.S.
      Glutamatergic Mechanisms Associated with Seizures and Epilepsy.
      ,
      • Coulter D.A.
      • Eid T.
      Astrocytic regulation of glutamate homeostasis in epilepsy.
      ,
      • Devinsky O.
      • Vezzani A.
      • Najjar S.
      • De Lanerolle N.C.
      • Rogawski M.A.
      Glia and epilepsy: excitability and inflammation.
      ]. Glutamate is the major excitatory neurotransmitter in the central nervous system and the most abundant amino acid in the mammalian brain [
      • Zhou Y.
      • Danbolt N.C.
      Glutamate as a neurotransmitter in the healthy brain.
      ]. Glutamate is essential for countless processes including, but not limited to, learning, memory, cognition and emotion [
      • Barker-Haliski M.
      • White H.S.
      Glutamatergic Mechanisms Associated with Seizures and Epilepsy.
      ,
      • Yalcin G.
      • Yalcin A.
      Mini Review Geriatric Medicine and Care Excitotoxicity as a molecular mechanism in Epilepsy.
      ]. All brain glutamate activity, including release, action, and reuptake, occurs in the extracellular space [
      • Zhou Y.
      • Danbolt N.C.
      Glutamate as a neurotransmitter in the healthy brain.
      ]. The biosynthesis, release, reuptake, and degradation of glutamate are all outlined in Fig. 1. Glutamate is released from the glutamatergic neuron's presynaptic terminal via a calcium dependent manner and enters the extracellular space. Once in the extracellular space, glutamate can act on ionotropic and metabotropic glutamate receptors [
      • Barker-Haliski M.
      • White H.S.
      Glutamatergic Mechanisms Associated with Seizures and Epilepsy.
      ]. Additionally, astrocytic and neuronal transporters work to prevent overexcitation by removing glutamate from the synaptic cleft via reuptake [
      • Zhou Y.
      • Danbolt N.C.
      Glutamate as a neurotransmitter in the healthy brain.
      ]. In astrocytes, glutamate is converted to glutamine via glutamine synthetase. Glutamine is then transported back to the glutamatergic neurons, where it is converted back to glutamate via glutaminase and made available for release, allowing the cycle to continue [
      • Rowley N.M.
      • Madsen K.K.
      • Steve White H.
      Glutamate and GABA synthesis, release, transport and metabolism as targets for seizure control.
      ]. Hence, glutamate regulation involves neurons and astrocytes, and any dysfunction and or/dysregulation within the system can cause an imbalance in excitation and inhibition.
      Fig 1
      Fig. 1Biosynthesis, transport, and degradation of Glutamate and GABA. Reprinted with permission from Elsevier
      [
      • Rowley N.M.
      • Madsen K.K.
      • Steve White H.
      Glutamate and GABA synthesis, release, transport and metabolism as targets for seizure control.
      ]
      .
      Astrocytes can also provide glutamine to GABAergic neurons, where glutamine is converted into glutamate and then into gamma-aminobutyric acid (GABA) via glutamate decarboxylase, before being packaged into vesicles for release [
      • Rowley N.M.
      • Madsen K.K.
      • Steve White H.
      Glutamate and GABA synthesis, release, transport and metabolism as targets for seizure control.
      ]. GABA is the major inhibitory neurotransmitter in the brain and ideally should be in balance with glutamate [

      Bromfield E.B., Cavazos J.E., Sirven J.I. Basic Mechanisms Underlying Seizures and Epilepsy. 2006 [cited 2020]; Available from: https://www.ncbi.nlm.nih.gov/books/NBK2510/.

      ]. Excess glutamate and/or inadequate GABA can lead to overexcitation in the CNS, leading to seizure occurrence [
      • Bozzi Y.
      • Provenzano G.
      • Casarosa S.
      Neurobiological bases of autism-epilepsy comorbidity: a focus on excitation/inhibition imbalance.
      ,
      • Scharfman H.E.
      The Neurobiology of Epilepsy.
      ,

      Bromfield E.B., Cavazos J.E., Sirven J.I. Basic Mechanisms Underlying Seizures and Epilepsy. 2006 [cited 2020]; Available from: https://www.ncbi.nlm.nih.gov/books/NBK2510/.

      ].
      Potential dysregulation in glutamatergic mechanisms in epilepsy include dysfunction of neuronal, glial, and/or neuronal-glial interactions. These include potential dysregulation or dysfunction of ionotropic or metabotropic receptors, abnormal expression of astrocytic glutamate transporters, and/or malfunction of neuronal or astrocytic enzymes [
      • Treiman D.M.
      GABAergic Mechanisms in Epilepsy.
      ,
      • Bozzi Y.
      • Provenzano G.
      • Casarosa S.
      Neurobiological bases of autism-epilepsy comorbidity: a focus on excitation/inhibition imbalance.
      ,
      • Scharfman H.E.
      The Neurobiology of Epilepsy.
      ,
      • Barker-Haliski M.
      • White H.S.
      Glutamatergic Mechanisms Associated with Seizures and Epilepsy.
      ,
      • Coulter D.A.
      • Eid T.
      Astrocytic regulation of glutamate homeostasis in epilepsy.
      ,
      • Devinsky O.
      • Vezzani A.
      • Najjar S.
      • De Lanerolle N.C.
      • Rogawski M.A.
      Glia and epilepsy: excitability and inflammation.
      ,
      • López-Pérez S.J.
      • Ureña-Guerrero M.E.
      • Morales-Villagrán A.
      Monosodium glutamate neonatal treatment as a seizure and excitotoxic model.
      ,
      • Eid T.
      • Lee T.W.
      • Patrylo P.
      • Zaveri H.P.
      Astrocytes and Glutamine Synthetase in Epileptogenesis.
      ,
      • Hanada T.
      Ionotropic Glutamate Receptors in Epilepsy: a Review Focusing on AMPA and NMDA Receptors.
      ,
      • Albrecht J.
      • Zielińska M.
      Mechanisms of Excessive Extracellular Glutamate Accumulation in Temporal Lobe Epilepsy.
      ]. More specifically, genetic mutations of N-methyl-d-aspartate (NMDA) receptors, including GRIN1, GRIN2B, and GRIN2D; as well as mutations of α-amino-3‑hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, leading to increased AMPA expression; are suspected to contribute to the disordered physiological processes of epilepsy via remodeling and rewiring of the neuronal network [
      • Barker-Haliski M.
      • White H.S.
      Glutamatergic Mechanisms Associated with Seizures and Epilepsy.
      ,
      • Hanada T.
      Ionotropic Glutamate Receptors in Epilepsy: a Review Focusing on AMPA and NMDA Receptors.
      ]. Glutamatergic dysregulation can lead to a buildup of glutamate in the synapse and overactivation of the glutamate receptors, resulting in excitotoxicity and ultimately, cell death [
      • Zhou Y.
      • Danbolt N.C.
      Glutamate as a neurotransmitter in the healthy brain.
      ,
      • Yalcin G.
      • Yalcin A.
      Mini Review Geriatric Medicine and Care Excitotoxicity as a molecular mechanism in Epilepsy.
      ]. The resulting hypothesis is that a reduction in extracellular brain glutamate concentration, and thereby a reduction in glutamate excitotoxicity, can diminish epileptogenesis [
      • Yalcin G.
      • Yalcin A.
      Mini Review Geriatric Medicine and Care Excitotoxicity as a molecular mechanism in Epilepsy.
      ].
      Additionally, a decrease in inhibitory GABA levels would also increase the risk of seizures [
      • Treiman D.M.
      GABAergic Mechanisms in Epilepsy.
      ]. As mentioned above, glutamatergic and GABAergic processes are heavily related to one another, as well as ictogenesis and epileptogenesis. The interconnectedness of these neurotransmitters both in synthesis and action demonstrates the importance of balance between these two neurotransmitters. As a result, it is important to investigate changes in both glutamate and GABA concentrations in those with epilepsy.
      The objectives of this paper are to review the current literature that has examined glutamate and GABA concentrations in people with epilepsy, summarize the findings and the limitations of this body of research, and suggest future directions which may help inform the search for novel epilepsy treatments.

      2. Methods

      The search engines PubMed and PsychInfo were used to identify articles of interest. The search terms were “epilepsy AND (glutamate or GABA)”, “epilepsy AND glutamate AND microdialysis”, and “epilepsy AND GABA AND microdialysis.” The search results were limited to humans and studies using magnetic resonance spectroscopy (MRS) or microdialysis. Articles were excluded if they were animal studies, in vitro, or ex vivo studies; were written in a language other than English; did not have an available abstract; were reviews, case studies, or MRS studies without controls; or were specifically focused on rare types of epilepsies.

      3. Results

      Thirty-three articles were identified which met inclusion criteria, and of these, eight articles were microdialysis studies and 25 articles were MRS studies.

      3.1 Microdialysis results

      Table 1 summarizes the literature on microdialysis studies which measured glutamate and/or GABA concentrations in epilepsy patients.
      Table 1Microdialysis studies measuring glutamate, glutamine, and/or GABA concentrations in epilepsy patients.
      Microdialysis Studies
      ReferenceSubjectsEpilepsy ClassificationSeizure PhaseASM UseDrug Effect CheckedGlu & GABA Metabolite(s) MeasuredRegion(s) of InterestResults
      During & Spencer (1991)
      • During M.J.
      In vivo neurochemistry of the conscious human brain: intrahippocampal microdialysis in epilepsy.
      8 Epilepsy; NRIntractable Complex Partial SeizuresPeri-ictal & ictalYes, but weaned during seizure occurrenceNoGlu, Gln, GABAHippocampusGlu significantly elevated 1.5 & 4.5 mins peri‑ictally compared to baseline. No difference in Gln.

      Of the 3 patients sampled during the ictal period, there was a pattern of a more prolonged and greater increase in Glu Glu-in the epileptogenic compared to the nonepileptogenic hippocampus. GABA release in the epileptogenic hippocampus was noted to appear weakened.
      Engstrom et al. (1992)
      • Ronne-Engström E.
      • Hillered L.
      • Flink R.
      • Spännare B.
      • Ungerstedt U.
      • Carlson H.
      Intracerebral Microdialysis of Extracellular Amino Acids in the Human Epileptic Focus.
      7 Epilepsy; Adults & ChildrenMedically intractable epilepsy (1 cortical dysplasia, 5 gliosis in the hippocampus or in the cortex, 1 gliomatous tumor)Interictal epileptiform activity, spontaneous seizure activity, electrically induced after discharges or seizure activityYesNoGluFrontal & temporal regionsGlu Glu- elevated between 1.8 & 16.2 times the basal levels for spontaneous & electrically induced seizures. Elevations within 2 mins of seizure onset & back to baseline within 6 mins. No statistical comparisons provided.
      During & Spencer (1993)
      • During D.
      • Spencer D.
      Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.
      6 Epilepsy; AdultsRefractory complex partial epilepsy (mesial temporal sclerosis)Preictal, ictal & postictalNRNoGlu, GABAHippocampusGlu significantly elevated in the epileptogenic hippocampus 1.5 mins preictally & 4.5–16.5 mins postictally compared to the non-epileptogenic hippocampus. Glu elevated during seizures in epileptogenic hippocampus. GABA elevated during & after the seizure in the epileptogenic & non-epileptogenic hippocampus. Significantly elevated GABA in non-epileptogenic hippocampus postictally.
      Wilson et al. (1996)
      • Wilson C.L.
      • Maidment N.T.
      • Shomer M.H.
      • Behnke E.J.
      • Ackerson L.
      • Fried I.
      • et al.
      Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy.
      12 Epilepsy; NRTemporal lobe epilepsyIctal & interictal spike activityNRNoGlu; GABATemporal lobe (hippocampus, amygdala, entorhinal cortex, parahippocampal gyrus, posterior cingulate gyrus)Several fold increase in Glu concentration-from baseline were observed over 6 seizures from 3 complex partial seizure patients. Three other seizures from an epileptogenic hippocampus did not result in a rise in Glu. No statistical comparisons provided. Seizures after 7 days of implantation frequently showed little or no change in Glu. Some patients showed no significant Glu change during any seizures. GABA elevated during a seizure in 1 patient.
      Thomas, Phillips, O'Connor (2004)
      • Thomas P.
      • Phillip J.
      • O'Connor W
      Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.
      7 Epilepsy; AdultsTemporal lobe epilepsyEpileptiform activityYesNoGlu, GABAMiddle temporal gyrus & anterior hippocampusNo significant difference between Glu & GABA levels in the minimally epileptiform lateral & ipsilateral medial temporal lobe. Glu & GABA were in greater concentrations in the vigorously epileptiform medial temporal lobe compared to the minimally epileptiform lateral & ipsilateral medial temporal lobe.
      Cavus et al. (2005)
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      38 Epilepsy; AdultsMedication resistant epilepsyInterictal: Seizure free ≥ 6 hYes (except 4)Yes, no difference.Glu, GlnHippocampus; Cortex (frontal, temporal, parietal, & occipital neocortices)Glu significantly elevated in epileptogenic compared to non-epileptogenic hippocampus & marginally elevated in epileptogenic compared to non-epileptogenic cortex.

      No difference in Gln. Significant reduction in Gln/Glu ratio in epileptogenic hippocampus compared to non-epileptogenic hippocampus & epileptogenic cortex. No difference between epileptogenic & non-epileptogenic cortex. Positive correlation between Glu & Gln levels in the epileptogenic hippocampus. No significant correlations between Glu & Gln in non-epileptogenic hippocampus or cortex or epileptogenic cortex.
      Pan, Cavus, Kim, Hetherington, Spencer (2008)
      • Pan J.
      • Cavus I.
      • Kim J.
      • Hetherington H.
      • Spencer D.
      Hippocampal extracellular GABA correlates with metabolism in human epilepsy.
      24 Epilepsy; AdultsControl: Neocortical epilepsy (11 temporal, 1 occipital). Hippocampal epilepsy (7 HS, 5 “paradoxical”)Interictal: Seizure free ≥ 6 hYesNo, but excluded patients on valproate.GABAHippocampusNo significant difference between the MTLE & neocortical groups.

      A significant inverse correlation was reported between GABA & NA/Cr in the MTLE group, while a significant positive correlation was reported in the non-MTLE group. A significant positive correlation was also reported between glial count & GABA in the MTLE group.
      Çavuş et al. (2016)
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      79 Epilepsy; AdultsMedication-refractory complex partial seizuresInterictal: Seizure free ≥ 6 hYesNoGlu, Gln, GABAHippocampus, cortex, nonlocalized, lesions (heterotopias, cortical tubers, dysplasia, encephalomalacias)Glu Glu-significantly elevated in epileptogenic, nonlocalized, & lesioned cortical sites compared to non-epileptogenic cortex. Glu significantly elevated in cortical lesion sites compared to epileptogenic sites & epileptogenic hippocampus compared to the non-epileptogenic hippocampus.

      No difference in Gln Gln-or GABA.
      ASM: anti-seizure medication; Glu: Glutamate; Gln: Glutamine; GABA: gamma-aminobutyric acid.

      3.2 Microdialysis results

      Microdialysis is an invasive surgical technique used for the sampling, collecting, and quantification of small molecular weight substances in the extracellular space. [
      • Chefer V.I.
      • Thompson A.C.
      • Zapata A.
      • Shippenberg T.S.
      Overview of Brain Microdialysis.
      ,
      • Darvesh A.S.
      • Carroll R.T.
      • Geldenhuys W.J.
      • Gudelsky G.A.
      • Klein J.
      • Meshul C.K.
      • et al.
      In vivo brain microdialysis: advances in neuropsychopharmacology and drug discovery.
      ]. The microdialysis technique involves the insertion of probes into tissue(s) of interest, which for the purposes of this paper, are different regions of the brain. At the tip of the probe is a semi-permeable membrane, which has both inflow and outflow tubing within the membrane. The inflow tubing is used to infuse perfusate, which is a solution that closely resembles the extracellular space. The perfusate is continuously and slowly infused during the sampling period. Because the membrane is semi-permeable, the substances of interest, such as glutamate and GABA, cross the membrane by diffusion. The process of diffusion allows the perfusate to carry glutamate and GABA through the outflow tubing, allowing for them to be collected at certain time points for analysis [
      • Chefer V.I.
      • Thompson A.C.
      • Zapata A.
      • Shippenberg T.S.
      Overview of Brain Microdialysis.
      ].
      Microdialysis does not come without its disadvantages. One major disadvantage is its limited spatial and temporal resolution, making it impossible to acquire real time changes in the neurotransmitters mentioned above [
      • Chefer V.I.
      • Thompson A.C.
      • Zapata A.
      • Shippenberg T.S.
      Overview of Brain Microdialysis.
      ]. Other potential issues include tissue damage and changes to the neurochemical milieu via probe implementation, perfusate, or anesthetics, which may modify baseline neurotransmitter levels; and inaccurate neurotransmitter measurements due to flow rate [
      • Chefer V.I.
      • Thompson A.C.
      • Zapata A.
      • Shippenberg T.S.
      Overview of Brain Microdialysis.
      ,
      • Westphalen R.I.
      • Hemmings H.C.
      Volatile anesthetic effects on glutamate versus GABA release from isolated rat cortical nerve terminals: basal release.
      ,

      Westphalen R.I., Hemmings H.C., Hemmings H.C. Selective Depression by General Anesthetics of Glutamate versus GABA Release from Isolated Cortical Nerve Terminals. 2002 [cited 2021]; 13. Available from: https://jpet.aspetjournals.org/content/jpet/early/2002/12/13/jpet.102.044685.full.pdf.

      ]. However, steps to minimize these issues can be taken, including waiting for a period of two to five days after probe implementation and use of the zero flow microdialysis method, developed in 2004, which allows for baseline concentration measurements in the extracellular space during steady state conditions [
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ,
      • Pan J.
      • Cavus I.
      • Kim J.
      • Hetherington H.
      • Spencer D.
      Hippocampal extracellular GABA correlates with metabolism in human epilepsy.
      ,
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      ]. Additionally, a major complication of microdialysis is that it is an unreliable indicator of exocytotic release of these transmitters [
      • Del Arco A.
      • Segovia G.
      • Fuxe K.
      • Mora F.
      Changes in dialysate concentrations of glutamate and GABA in the brain: an index of volume transmission mediated actions?.
      ,
      • Watson C.J.
      • Venton B.J.
      • Kennedy R.T.
      In vivo measurements of neurotransmitters by microdialysis sampling.
      ,
      • Timmerman W.
      • Westerink B.H.
      Brain microdialysis of GABA and glutamate: what does it signify?.
      ,
      • van der Zeyden M.
      • Oldenziel W.H.
      • Rea K.
      • Cremers T.I.
      • Westerink B.H.
      Microdialysis of GABA and glutamate: analysis, interpretation and comparison with microsensors.
      ]. However, changes during stimulating conditions, including chemical, electrical, and behavioral stimulation, have been proposed to be a result of neuronal activation, as well as reflecting alterations in neuron astrocyte network activation [
      • Chefer V.I.
      • Thompson A.C.
      • Zapata A.
      • Shippenberg T.S.
      Overview of Brain Microdialysis.
      ,
      • Del Arco A.
      • Segovia G.
      • Fuxe K.
      • Mora F.
      Changes in dialysate concentrations of glutamate and GABA in the brain: an index of volume transmission mediated actions?.
      ,
      • Watson C.J.
      • Venton B.J.
      • Kennedy R.T.
      In vivo measurements of neurotransmitters by microdialysis sampling.
      ,
      • Timmerman W.
      • Westerink B.H.
      Brain microdialysis of GABA and glutamate: what does it signify?.
      ,
      • van der Zeyden M.
      • Oldenziel W.H.
      • Rea K.
      • Cremers T.I.
      • Westerink B.H.
      Microdialysis of GABA and glutamate: analysis, interpretation and comparison with microsensors.
      ].
      Despite these disadvantages, microdialysis is one of only a few tools available for the quantification of extracellular amino acids, such as glutamate and GABA, in the human brain [
      • Chefer V.I.
      • Thompson A.C.
      • Zapata A.
      • Shippenberg T.S.
      Overview of Brain Microdialysis.
      ,
      • Darvesh A.S.
      • Carroll R.T.
      • Geldenhuys W.J.
      • Gudelsky G.A.
      • Klein J.
      • Meshul C.K.
      • et al.
      In vivo brain microdialysis: advances in neuropsychopharmacology and drug discovery.
      ]. However, its utility is limited by its invasiveness, with its use being reserved for patients that are undergoing evaluation for epilepsy surgery. The consequence of this is that there are a limited number of microdialysis studies in epilepsy, and the studies that do exist are confined to a small subset of patients. To date, there are only seven studies that have investigated glutamate concentration in epilepsy patients, three studies that investigated glutamine concentration, and six studies that have investigated GABA concentration.
      Seven studies reported elevations in glutamate concentration in the epileptogenic regions, and these elevations were observed during a variety of timepoints and conditions, including peri‑ictally, preictally, ictally, postictally, during electrically induced seizures, during epileptiform activity, and interictally [
      • During M.J.
      In vivo neurochemistry of the conscious human brain: intrahippocampal microdialysis in epilepsy.
      ,
      • Ronne-Engström E.
      • Hillered L.
      • Flink R.
      • Spännare B.
      • Ungerstedt U.
      • Carlson H.
      Intracerebral Microdialysis of Extracellular Amino Acids in the Human Epileptic Focus.
      ,
      • During D.
      • Spencer D.
      Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.
      ,
      • Wilson C.L.
      • Maidment N.T.
      • Shomer M.H.
      • Behnke E.J.
      • Ackerson L.
      • Fried I.
      • et al.
      Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy.
      ,
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ,
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      ,
      • Eid T.
      • Thomas M.J.
      • Spencer D.D.
      • Rundén-Pran E.
      • Lai J.C.K.
      • Malthankar G.V.
      • et al.
      Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.
      ]. The reported rise in glutamate concentration was compared to either non-epileptogenic regions, baseline measures in the epileptogenic region, or regions of minimal epileptiform activity [
      • During M.J.
      In vivo neurochemistry of the conscious human brain: intrahippocampal microdialysis in epilepsy.
      ,
      • Ronne-Engström E.
      • Hillered L.
      • Flink R.
      • Spännare B.
      • Ungerstedt U.
      • Carlson H.
      Intracerebral Microdialysis of Extracellular Amino Acids in the Human Epileptic Focus.
      ,
      • During D.
      • Spencer D.
      Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.
      ,
      • Wilson C.L.
      • Maidment N.T.
      • Shomer M.H.
      • Behnke E.J.
      • Ackerson L.
      • Fried I.
      • et al.
      Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy.
      ,
      • Thomas P.
      • Phillip J.
      • O'Connor W
      Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.
      ,
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ,
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      ,
      • Eid T.
      • Thomas M.J.
      • Spencer D.D.
      • Rundén-Pran E.
      • Lai J.C.K.
      • Malthankar G.V.
      • et al.
      Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.
      ]. However, some of these results were not statistically significant (see Table 1). Of those with significant results, two of the four had large sample sizes (38 and 79 patients) and had control regions [
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ,
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      ]. Studies that reported non-significant results had small sample sizes (ranging from three-12 patients) and two of the four compared changes in glutamate concentration against baseline measures, as opposed to control regions [
      • During M.J.
      In vivo neurochemistry of the conscious human brain: intrahippocampal microdialysis in epilepsy.
      ,
      • Ronne-Engström E.
      • Hillered L.
      • Flink R.
      • Spännare B.
      • Ungerstedt U.
      • Carlson H.
      Intracerebral Microdialysis of Extracellular Amino Acids in the Human Epileptic Focus.
      ,
      • Wilson C.L.
      • Maidment N.T.
      • Shomer M.H.
      • Behnke E.J.
      • Ackerson L.
      • Fried I.
      • et al.
      Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy.
      ,
      • Thomas P.
      • Phillip J.
      • O'Connor W
      Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.
      ].
      Interestingly, studies also noted a lack of change in glutamate concentration (1) in patients with minimal epileptiform activity, (2) in patients where seizures occurred a week after probe implantation, and (3) in some patients despite seizure activity [
      • Wilson C.L.
      • Maidment N.T.
      • Shomer M.H.
      • Behnke E.J.
      • Ackerson L.
      • Fried I.
      • et al.
      Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy.
      ,
      • Thomas P.
      • Phillip J.
      • O'Connor W
      Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.
      ]. Suggested reasons for some of these findings included minimal recovery and differences in tissue hyperexcitability [
      • Wilson C.L.
      • Maidment N.T.
      • Shomer M.H.
      • Behnke E.J.
      • Ackerson L.
      • Fried I.
      • et al.
      Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy.
      ,
      • Thomas P.
      • Phillip J.
      • O'Connor W
      Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.
      ]. Furthermore, elevations in glutamate concentration differed among studies during seizures. Wilson et al. (1996) reported lower elevations in glutamate concentration than a study by During & Spencer (1993), which the authors attributed to their inclusion of patients who did not have severe hippocampal sclerosis and who had seizures that became generalized [
      • Wilson C.L.
      • Maidment N.T.
      • Shomer M.H.
      • Behnke E.J.
      • Ackerson L.
      • Fried I.
      • et al.
      Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy.
      ]. It is possible that anatomical changes associated with prominent hippocampal sclerosis, such as neuronal loss and gliosis, and the spread of seizure activity in generalized seizures, may play a role in the degree of elevation of glutamate concentration in epilepsy patients during seizure activity [
      • Thom M.
      Review: hippocampal sclerosis in epilepsy: a neuropathology review.
      ].
      In addition to glutamate, three studies measured glutamine, the precursor to glutamate. One study reported no difference in glutamine during the peri‑ictal phase compared to baseline [
      • During M.J.
      In vivo neurochemistry of the conscious human brain: intrahippocampal microdialysis in epilepsy.
      ]. The other two studies reported no difference in glutamine in epileptogenic regions compared to non-epileptogenic regions, with one of the studies reporting a positive correlation between glutamate and glutamine in the epileptogenic hippocampus and a significant reduction in glutamine/glutamate ratio in the epileptogenic hippocampus compared to the non-epileptogenic hippocampus and epileptogenic cortex [
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ,
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      ].
      Elevated extracellular glutamate concentration, unaltered glutamine concentration, and a reduced glutamine/glutamate ratio lend evidence to the impaired glutamate-glutamine cycle hypothesis proposed by Eid and colleagues. Under normal conditions, extracellular glutamate is released by the presynaptic receptor and taken up by astrocytes, where it is converted to glutamine via glutamine synthase. Glutamine is shuttled from the astrocyte to the neuron, where it is converted back to glutamate via glutaminase. Glutamate is then packaged into vesicles and released by the neuron into the extracellular space to act on postsynaptic receptors, where the cyclical process continues [
      • Rose C.R.
      • Felix L.
      • Zeug A.
      • Dietrich D.
      • Reiner A.
      • Henneberger C.
      Astroglial Glutamate Signaling and Uptake in the Hippocampus.
      ]. The impaired glutamate-glutamine cycle hypothesis proposes that in certain types of epilepsies, such as focal structural epilepsies, there is impaired functioning of the glutamate-glutamine cycle, resulting in neurotoxic increases of extracellular glutamate [
      • Eid T.
      • Thomas M.
      • Spencer D.
      • Rundén-Pran E.
      • Lai J.
      • Malthankar G.
      • et al.
      Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.
      ]. The hypothesis was introduced by Eid et al. in 2004, where they reported reduced expression of glutamine synthetase by 40% and reduced activity by 38% in hippocampi of mesial temporal lobe epilepsy patients compared to non-mesial temporal lobe epilepsy hippocampi. Microdialysis studies support this theory [
      • Eid T.
      • Thomas M.J.
      • Spencer D.D.
      • Rundén-Pran E.
      • Lai J.C.K.
      • Malthankar G.V.
      • et al.
      Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.
      ]. The observed increase in glutamate levels was not accompanied by an increase in glutamine, resulting in a reduced glutamine to glutamate ratio in the epileptogenic hippocampus [
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ]. If the glutamate-glutamine cycle was functioning normally, one would expect increased levels of glutamine alongside equal or reduced levels of glutamate. Dysfunction in glutamatergic regulation, such as glutamine synthase dysfunction, could result in reduced glutamate clearance alongside a buildup of extracellular glutamate, leading to excitotoxicity and eventually, cell death [
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ]. Hence, results from the microdialysis studies reinforce the need for glutamatergic targeted therapies. Treatments focused on reducing extracellular glutamate concentration, whether that be from targeting glutamine synthase dysfunction, or from targeting the buildup of glutamate in the synaptic cleft, could be key in reducing seizure activity and excitotoxic consequences.
      In regards to the glutamate-glutamine cycle, GABA is an important metabolite to consider. As discussed above, while glutamate is the major excitatory neurotransmitter, GABA is the major inhibitory neurotransmitter [
      • Treiman D.M.
      GABAergic Mechanisms in Epilepsy.
      ]. Furthermore, glutamate is the precursor for GABA synthesis, demonstrating the interconnectedness and delicate balance that must exist between the two neurotransmitters [
      • Rowley N.M.
      • Madsen K.K.
      • Steve White H.
      Glutamate and GABA synthesis, release, transport and metabolism as targets for seizure control.
      ]. Of the eight studies, GABA concentration was measured in six. None of the studies reported significant differences in GABA except for one, which reported elevated GABA in the non-epileptogenic hippocampus postictally. Interestingly, this result was significant despite the same study reporting ictal and postictal elevations in GABA in the epileptogenic and non-epileptogenic hippocampus as well [
      • During D.
      • Spencer D.
      Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.
      ]. The finding that GABA was elevated in the non-epileptogenic hippocampus compared to the epileptogenic hippocampus postictally, despite rising GABA levels in both hippocampi, suggests a delay in the rise of GABA in the epileptogenic region [
      • During D.
      • Spencer D.
      Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.
      ]. This delayed rise, paired with the significant rise in glutamate preictally, produces an imbalance between excitation and inhibition. Since an imbalance between glutamate and GABA can result in overexcitation and seizures, further investigation is warranted. The implications from these studies are minimal, as those with non-significant results were a mix of comparisons including pre-post, comparisons between different epilepsy populations, and between epileptogenic and non-epileptogenic regions. Also worth noting, the study that compared the seizure onset zone in mesial temporal lobe epilepsy to the non-seizure onset zone in neocortical epilepsy (hippocampus) reported opposing correlational relationships between GABA and mitochondrial function, defined as the ratio of N-acetyl aspartate (NAA) to creatine, in the two groups. Those with mesial temporal lobe epilepsy had a negative correlation between GABA and NAA/Cr (mitochondrial function) and the neocortical group had a positive correlation [
      • Pan J.
      • Cavus I.
      • Kim J.
      • Hetherington H.
      • Spencer D.
      Hippocampal extracellular GABA correlates with metabolism in human epilepsy.
      ]. Such findings are of interest when considering current therapies for refractory patients, such as the ketogenic diet, which has been proposed to improve mitochondrial biogenesis, and may also be of interest for the development of novel therapies [

      Branco A.F., Ferreira A.E., Sim~ R.F., Aes-Novais M., Zehowski C., Cope E., et al. Ketogenic diets: from cancer to mitochondrial diseases and beyond. [cited 2021]; . Available from: www.ejci-online.com.

      ]. Future studies that investigate GABA concentration should include comparisons to other metabolites, consider different seizure timeframes (preictal, ictal, postictal, interictal), and use non-epileptogenic regions for comparison.
      When discussing glutamate and GABA measurements in microdialysis studies, it is also important to mention the potential confounding factor of anti-seizure medications. The goal of anti-seizure medications is to reduce excitation, increase inhibition, or both. Many anti-seizure medications work by directly targeting glutamatergic and GABAergic transmission, such as phenobarbital, primidone, stiripentol, and benzodiazepines including diazepam, lorazepam, clonazepam, midazolam, and clobazam, which all act on GABAA receptors. Topiramate, felbamate, cenobamate, and retigabine are also suspected to act on these receptors as well. In addition to those listed above, there are other medications which have GABAergic molecular targets such as the GAT-1 GABA transporter, GABA transaminase, and carbonic anhydrase. In terms of glutamatergic targets, perampanel is the only drug whose mechanism of action is non-competitive blocking of the AMPA receptors. However, there are other medications that, at least in part, have effects on glutamatergic transmission including felbamate, topiramate, levetiracetam, and phenobarbital. Additionally, there are a host of other anti-seizure medications that effect glutamatergic and GABAergic transmission through indirect mechanisms of action, including targeting voltage gated ion channels (sodium, calcium, potassium) and synaptic release via voltage gated calcium channels and synaptic vesicle glycoprotein 2A (SV2A) [
      • Sills G.J.
      • Rogawski M.A.
      Mechanisms of action of currently used antiseizure drugs.
      ].
      Six of the eight papers reported that patients were on anti-seizure medications, including a mix of mono and polytherapy [
      • During M.J.
      In vivo neurochemistry of the conscious human brain: intrahippocampal microdialysis in epilepsy.
      ,
      • Ronne-Engström E.
      • Hillered L.
      • Flink R.
      • Spännare B.
      • Ungerstedt U.
      • Carlson H.
      Intracerebral Microdialysis of Extracellular Amino Acids in the Human Epileptic Focus.
      ,
      • Thomas P.
      • Phillip J.
      • O'Connor W
      Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.
      ,
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ,
      • Pan J.
      • Cavus I.
      • Kim J.
      • Hetherington H.
      • Spencer D.
      Hippocampal extracellular GABA correlates with metabolism in human epilepsy.
      ,
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      ]. The remaining two papers did not mention anti-seizure medications at all in their publication, resulting in unknown information [
      • During D.
      • Spencer D.
      Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.
      ,
      • Wilson C.L.
      • Maidment N.T.
      • Shomer M.H.
      • Behnke E.J.
      • Ackerson L.
      • Fried I.
      • et al.
      Comparison of seizure related amino acid release in human epileptic hippocampus versus a chronic, kainate rat model of hippocampal epilepsy.
      ]. Of the six that reported medication use, two did not provide the specific medications the patients were on, and one had the patients wean their medication during the microdialysis period [
      • During M.J.
      In vivo neurochemistry of the conscious human brain: intrahippocampal microdialysis in epilepsy.
      ,
      • Ronne-Engström E.
      • Hillered L.
      • Flink R.
      • Spännare B.
      • Ungerstedt U.
      • Carlson H.
      Intracerebral Microdialysis of Extracellular Amino Acids in the Human Epileptic Focus.
      ]. Only one study checked for a medication effect and reported that medication given within 24 h of the study had no significant effect on metabolite levels among patients, though the authors noted this could be due to the small sample sizes for each medication [
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ]. Interestingly, while one study measuring GABA excluded patients on valproate due to its effect on GABA levels, others did not [
      • Thomas P.
      • Phillip J.
      • O'Connor W
      Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.
      ,
      • Pan J.
      • Cavus I.
      • Kim J.
      • Hetherington H.
      • Spencer D.
      Hippocampal extracellular GABA correlates with metabolism in human epilepsy.
      ,
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      ]. Due to the variety of mechanisms of action among anti-seizure medications, the fact that many patients were on a combination of medications, and that the goal of most anti-seizure medications is to reduce excitation and increase inhibition, it is important to consider the potential impact that these medications may have on the study results.
      In addition to medications, anesthetics can also impact glutamate and GABA, as research has shown that general anesthetics can reduce glutamate transmission and increase GABA transmission [
      • Westphalen R.I.
      • Hemmings H.C.
      Volatile anesthetic effects on glutamate versus GABA release from isolated rat cortical nerve terminals: basal release.
      ,

      Westphalen R.I., Hemmings H.C., Hemmings H.C. Selective Depression by General Anesthetics of Glutamate versus GABA Release from Isolated Cortical Nerve Terminals. 2002 [cited 2021]; 13. Available from: https://jpet.aspetjournals.org/content/jpet/early/2002/12/13/jpet.102.044685.full.pdf.

      ]. As mentioned previously, one way to combat the effects of anesthetics on metabolite levels is to institute a protocol that includes a period of a few days between surgery and microdialysis sampling, which newer papers have utilized [
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ,
      • Pan J.
      • Cavus I.
      • Kim J.
      • Hetherington H.
      • Spencer D.
      Hippocampal extracellular GABA correlates with metabolism in human epilepsy.
      ,
      • Çavuş I.
      • Romanyshyn J.C.
      • Kennard J.T.
      • Farooque P.
      • Williamson A.
      • Eid T.
      • et al.
      Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: microdialysis study of 79 patients at the yale epilepsy surgery program.
      ]. However, some of the earlier papers, prior to 2004, did not have this period to mitigate anesthetic effects, or only had one day recovery prior to sampling, which may have an effect on the results obtained [
      • During M.J.
      In vivo neurochemistry of the conscious human brain: intrahippocampal microdialysis in epilepsy.
      ,
      • Ronne-Engström E.
      • Hillered L.
      • Flink R.
      • Spännare B.
      • Ungerstedt U.
      • Carlson H.
      Intracerebral Microdialysis of Extracellular Amino Acids in the Human Epileptic Focus.
      ,
      • During D.
      • Spencer D.
      Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.
      ,
      • Thomas P.
      • Phillip J.
      • O'Connor W
      Hippocampal microdialysis during spontaneous intraoperative epileptiform activity.
      ].
      In summary, microdialysis studies were mostly limited to adult surgical candidates with temporal lobe epilepsy. Research expanding beyond this subset of patients, including pediatric patients, are necessary for additional insight into changes in glutamate and GABA concentration in epilepsy. As there are a variety of potential causes of changes in glutamate concentration in epilepsy, including dysregulation/dysfunction of glutamate receptors, glutamate transporters, and inflammation, all of which extend beyond astrocytic enzyme dysfunction, it is important to study glutamate concentration beyond focal structural epilepsies [
      • Treiman D.M.
      GABAergic Mechanisms in Epilepsy.
      ,
      • Bozzi Y.
      • Provenzano G.
      • Casarosa S.
      Neurobiological bases of autism-epilepsy comorbidity: a focus on excitation/inhibition imbalance.
      ,
      • Scharfman H.E.
      The Neurobiology of Epilepsy.
      ,
      • Barker-Haliski M.
      • White H.S.
      Glutamatergic Mechanisms Associated with Seizures and Epilepsy.
      ,
      • Coulter D.A.
      • Eid T.
      Astrocytic regulation of glutamate homeostasis in epilepsy.
      ,
      • Devinsky O.
      • Vezzani A.
      • Najjar S.
      • De Lanerolle N.C.
      • Rogawski M.A.
      Glia and epilepsy: excitability and inflammation.
      ,
      • López-Pérez S.J.
      • Ureña-Guerrero M.E.
      • Morales-Villagrán A.
      Monosodium glutamate neonatal treatment as a seizure and excitotoxic model.
      ,
      • Eid T.
      • Lee T.W.
      • Patrylo P.
      • Zaveri H.P.
      Astrocytes and Glutamine Synthetase in Epileptogenesis.
      ,
      • Hanada T.
      Ionotropic Glutamate Receptors in Epilepsy: a Review Focusing on AMPA and NMDA Receptors.
      ,
      • Albrecht J.
      • Zielińska M.
      Mechanisms of Excessive Extracellular Glutamate Accumulation in Temporal Lobe Epilepsy.
      ]. Furthermore, research with drug naïve populations would be extremely beneficial, as it would remove a large confounding factor in the research. However, the invasiveness of microdialysis limits its utility beyond its current use regarding expanding to other populations. Hence, an alternative technique is needed to investigate a wider variety of epilepsies, with one such option being magnetic resonance spectroscopy.

      3.3 Magnetic resonance spectroscopy group comparison results

      Interestingly, compared to the microdialysis studies, the MRS literature yielded mixed results on glutamate concentrations in epilepsy (Table 2).
      Table 2In vivo magnetic resonance spectroscopy studies on glutamate, glutamine and/or GABA concentrations.
      ReferenceSubjectsEpilepsy Classification(s)Time Since Last SeizureASM UseDrug Effect CheckedMRI Tesla Strength & Glu & GABA Metabolite(s) EvaluatedRegion(s) of InterestSignificant Results Reported
      Pan, Venkatra, Vives, Spencer (2006)
      • Pan J.
      • Venkatraman T.
      • Vives K.
      • Spencer D.
      Quantitative glutamate spectroscopic imaging of the human hippocampus.
      5 Epilepsy, 10 ControlsUnilateral hippocampal epilepsy w/ MTS (4 lateralized abnormal MRI; 3 bilateral seizures)NRNRNo4T; Glu, Glu/NAAHippocampusGlu significantly reduced in ipsilateral hippocampus compared to controls.
      Riederer et al. (2006)
      • Riederer F.
      • Bittsanský M.
      • Schmidt C.
      • Mlynárik V.
      • Baumgartner C.
      • Moser E.
      • et al.
      1H magnetic resonance spectroscopy at 3 T in cryptogenic and mesial temporal lobe epilepsy.
      21 Epilepsy, 22 Controls; Adults12 mTLE (HS, atrophy); 9 cTLE (MRIN)Seizure free ≥ 5 hYesNo3T; Glx, GluMedial & lateral temporal lobeGlx significantly higher in lateral than medial voxels in the diagnostic groups. No difference in Glx between mTLE, cTLE, & control groups. Glu significantly elevated in lateral compared to medial voxels in mTLE patients on the ipsilateral side.
      Doelken et al. (2010)
      • Doelken M.
      • Mennecke A.
      • Stadlbauer A.
      • Kecskeméti L.
      • Kasper B.
      • Struffert T.
      • et al.
      Multi-voxel magnetic resonance spectroscopy at 3 T in patients with idiopathic generalised epilepsy.
      18 Epilepsy, 25 Controls; AdultsGTCS (MRIN)Free of tonic-clonic seizures > 14 daysYes (except 1)Yes, for Glx. No difference among patients with differing medications.3T; Glx, GluPrecuneus, cingulum, frontal & parietal gray/white matter, thalamus, putamen, insular cortex, hippocampusGlx significantly elevated in the right thalamus. Glx elevated broadly in both hemispheres.

      Glu significantly elevated in central region white matter & pronounced in the left putamen & both insular cortices. A significant correlation based on tNAA & Glx was reported between the thalamus & the central region, cingulum, putamen.

      Significant correlation of Glx between the thalamus & the medial frontal cortex.
      Hattingen et al. (2014)
      • Hattingen E.
      • Lückerath C.
      • Pellikan S.
      • Vronski D.
      • Roth C.
      • Knake S.
      • et al.
      Frontal and thalamic changes of GABA concentration indicate dysfunction of thalamofrontal networks in juvenile myoclonic epilepsy.
      15 Epilepsy, 15 Controls; AdultsJME (MRIN)Seizure free ≥ 24 hYes (except 1)Yes, increase in Gln in thalamus of valproate group compared to the no-valproate group. No difference for Glu or GABA.3T; Glu, Gln, GABAThalamus, frontal lobe, motor cortexNo difference in Glu.

      Gln significantly elevated in the frontal lobe. GABA significantly increased in the frontal region. Significant decrease in GABA in the thalamus.
      Gonen et al. (2020)
      • Gonen O.
      • Moffat B.
      • Desmond P.
      • Lui E.
      • Kwan P.
      • O'Brien T
      Seven-tesla quantitative magnetic resonance spectroscopy of glutamate, γ-aminobutyric acid, and glutathione in the posterior cingulate cortex/precuneus in patients with epilepsy.
      35 Epilepsy, 10 Controls; Adults19 TLE, 16 IGEShort-term seizure freedom (w/n 3 months prior to scan) 11/19 TLE (10/18 in final analysis) & 6/16 IGEYesYes, a reduction in the Glu concentration in TLE patients treated with valproate.7T; Glu, GABA, Glu/GABAPCC/ precuneusNo significant differences. Multiple regression of GABA with age & epilepsy duration showed a significant reduction in GABA concentration with age in the TLE group. Glu concentration was a predictor of short-term seizure freedom. Average Glu concentration was higher in patients who reported short-term seizure freedom compared to those that did not.
      Lundberg, Weis, Eeg-Olofsson, Raininko (2003)
      • Lundberg S.
      • Weis J.
      • Eeg-Olofsson O.
      • Raininko R.
      Hippocampal region asymmetry assessed by 1H-MRS in rolandic epilepsy.
      13 Epilepsy, 15

      Controls;

      Children
      Electroclinically typical RE (MRIN)Seizure free ≥ 1 monthYes, except 4 without treatment for ≥ 1 year, & 3 treatment naïveNo1.5T; Glx/tCr, AisHippocampusNo difference in epilepsy patients compared to controls.
      Simister, McLean, Barker, Duncan (2003)
      • Simister R.J.
      • McLean M.A.
      • Barker G.J.
      • Duncan J.S.
      A Proton Magnetic Resonance Spectroscopy Study of Metabolites in the Occipital Lobes in Epilepsy.
      30 Epilepsy, 15 Controls; AdultsIGE (15) (JME (3), JAE (2), eyelid myoclonic with absences (1), GTCS on awakening (1), unclassified (8)); OLE (15)

      All MRIN except 1 OLE with porencephalic cyst in frontal parietal region)
      Convulsive seizure free for a median duration of 45 daysYes, except 6Yes, no difference.1.5T; Glx, Glx/Ins, Glx/Cr, Glx/GABA+, Glx/NAAt, GABA+Occipital lobeGlx & GABA+ significantly elevated in the IGE group compared with the control group, but not significant after reanalysis with covariable of gray matter content. Glx elevated in the IGE & OLE groups compared to the control group. Positive correlation between Glx & NAAt in the OLE group. Post hoc significant variation of Glx/Ins between OLE group & the control group, but not significant on covariate analysis.
      Flügel, McLean, Simister, Duncan (2006)
      • Flügel D.
      • McLean M.A.
      • Simister R.J.
      • Duncan J.S.
      Magnetisation transfer ratio of choline is reduced following epileptic seizures.
      10 Epilepsy, 10 Controls; AdultsRefractory focal epilepsy (7 Temporal, 1 Extra-temporal, 2 Uncertain); 9 positive MRI findings including brain damage, HS, lesion, glioma, atrophy, gliosis, Rasmussen's encephalitis, cortical damage, & abnormal signal L)Interictal: seizure free ≥ 12 h

      Postictal: as soon as possible post -seizure
      YesYes, but not for Glx.1.5T; GlxFrontal lobeNo difference between patients & controls.
      Doelken et al. (2008)
      • Doelken M.
      • Stefan H.
      • Pauli E.
      • Stadlbauer A.
      • Struffert T.
      • Engelhorn T.
      • et al.
      (1)H-MRS profile in MRI positive- versus MRI negative patients with temporal lobe epilepsy.
      26 Epilepsy, 23 Controls;

      Adults
      Unilateral TLE (17 unilateral HS, 9 MRIN)Seizure free ≥ 3 daysNRNo1.5T; GlxHippocampusNo difference between unilateral HS group, MRIN group, or control group.
      Simister, McLean, Salmenpera, Barker, Duncan (2008)
      • Simister R.
      • McLean M.
      • Salmenpera T.
      • Barker G.
      • Duncan J.
      The effect of epileptic seizures on proton MRS visible neurochemical concentrations.
      10 Epilepsy, 10 Controls; Adults2 FLE (1 MCD), 1 OLE (HT), 1 PLE (MCD), 5 TLE (3 HS), 1 X-TLE (white matter lesions)Post-ictal & interictal. Post-ictal: immediately following seizure; Interictal: seizure free ≥ 7 hYesNo1.5T; Glx, Glx/CrFrontal lobeGlx/Cr significantly elevated in the post-ictal scan compared to the interictal scan. No difference between epilepsy patients & controls.
      Simister, McLean, Barker, Duncan (2009)
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton MR spectroscopy of metabolite concentrations in temporal lobe epilepsy and effect of temporal lobe resection.
      16 Epilepsy (10 after anterior temporal lobe resection), 15 controls (19 for re-test); AdultsTLE w/ unilateral HSHS patients were seizure free for a median duration of 5 days, surgical HS patients were seizure free for a median duration of 6 daysYesData from patients taking GABAergic medications known to increase GABA levels were not included in the GABA+ analysis.1.5T; Glx, Glx/Cr, GABA+/CrTemporal LobeNo difference before or after surgery compared to controls. GABA+/Cr was highest in the ipsilateral temporal lobe in TLE patients, but this was not significant.
      Cevick et al. (2016)
      • Cevik N.
      • Koksal A.
      • Dogan V.
      • Dirican A.
      • Bayramoglu S.
      • Ozturk M.
      • et al.
      Evaluation of cognitive functions of juvenile myoclonic epileptic patients by magnetic resonance spectroscopy and neuropsychiatric cognitive tests concurrently.
      20 Epilepsy, 20 Controls; AdultsJME (MRIN)No grand mals in the last week;no absence & myoclonics in the last dayYesNo1.5T; Glx, Glx/CrPrefrontal cortex, thalamusNo difference between epilepsy patients & controls.
      Leite, Valente, Fiore, Maria Otaduy (2017)
      • Leite C da C.
      • Valente K.D.R.
      • Fiore L.A.
      • Otaduy M.C.G.
      Proton spectroscopy of the thalamus in a homogeneous sample of patients with easy-to-control juvenile myoclonic epilepsy.
      21 Epilepsy, 14 Controls; AdultsJME (MRIN)All patients in total remissionYesNo1.5T; Glx, Glx/CrThalamusNo difference between epilepsy patients & controls.
      Woermann et al. (1999)
      • Woermann F.
      • McLean M.
      • Bartlett P.
      • Parker G.
      • Barker G.
      • Duncan J.
      Short echo time single-voxel 1H magnetic resonance spectroscopy in magnetic resonance imaging-negative temporal lobe epilepsy: different biochemical profile compared with hippocampal sclerosis.
      30 Epilepsy, 15 Controls; AdultsMedically intractable epilepsy: 15 unilateral TLE w/ unilateral HS, 15 unilateral TLE w/ MRINHS patients seizure free for a median of 5 days, MRI-negative patients seizure free for a median of 3 daysYesYes, no difference found.1.5T; Glx, NAA/GlxHippocampusGlx significantly elevated in MRIN hippocampi ipsilateral to seizure onset compared to both hippocampi in the HS group. Significantly lower NAA/ Glx levels in sclerotic hippocampi compared to control & contralateral hippocampi in patients.

      NAA/Glx significantly reduced in MRIN hippocampi ipsilateral to the epileptic focus compared to controls & hippocampi contralateral to HS.
      Savic et al. (2000)
      • Savic I.
      • Thomas A.
      • Ke Y.
      • Curran J.
      • Fried I.
      • Engel J.
      In vivo measurements of glutamine + glutamate (Glx) and N-acetyl aspartate (NAA) levels in human partial epilepsy.
      28 Epilepsy, 10 Controls; Adults & 1 child18 Partial mTLE, Partial NCE (2 temporal neocortex, 2

      occipital lobe, 2 motor cortex, 1

      SMA, 1 parietal lobe & 2 in the

      anterior cingulate cortex.

      MRI findings: temporal & occipital lobe dysplasia; hamartoma; occipital lobe amygdala, & hippocampal atrophy; parietal hemangioma; HS; temporal gliosis
      Time since last seizure ranged from 0 to 90 daysYesNo systematic variation seen.1.5T; Glx/Cr, Glx/NAAEpileptic region (amygdala, hippocampus, parahippocampus gyrus, superior & middle temporal gyrus, motor cortex, left occipital lobe, right SMA, right anterior cingulate, left parietal lobe)Glx/Cr & Glx/NAA significantly elevated in the epileptogenic region compared to homologous region. No difference between non-epileptogenic regions & controls.

      Using the 95% confidence interval of controls, elevated Glx/NAA identified 23/30 epileptogenic regions (28 patients), elevated Glx/Cr ratio identified 16 epileptogenic regions (16 patients). Five patients had elevated Glx/Cr & Glx/NAA ratios in the homologous region. Using the 95% confidence interval of controls, elevated Glx/NAA &/or Glx/Cr ratios identified 8/10 neocortical epileptogenic regions & 17/20 MTLE epileptogenic regions (18 patients).

      Glx ratios also identified 5/6 MRI negative MTLE epileptogenic zones.
      Simister et al. (2002)
      • Simister R.J.
      • Woermann F.G.
      • McLean M.A.
      • Bartlett P.A.
      • Barker G.J.
      • Duncan J.S.
      A Short-echo-time Proton Magnetic Resonance Spectroscopic Imaging Study of Temporal Lobe Epilepsy.
      20 Epilepsy, 10 Controls; AdultsIntractable epilepsy: 10 TLE w/ unilateral HS, 10 TLE w/ MRINHS seizure free for a median duration of 3 days, MRIN seizure free for a median duration of 5 daysYesNo1.5T; GlxMTL, LTLGlx significantly elevated in MTL in control & temporal lobes of the MRIN groups compared to HS group. MRIN contralateral Glx significantly elevated in the anterior voxel compared to the control, MRIN ipsilateral, & hippocampal sclerosis contralateral & ipsilateral groups.
      Helms, Ciumas, Kyaga, Savic (2006)
      • Helms G.
      • Ciumas C.
      • Kyaga S.
      • Savic I.
      Increased thalamus levels of glutamate and glutamine (Glx) in patients with idiopathic generalised epilepsy.
      43 Epilepsy, 38 Controls;

      Adults
      IGE (23 JME, 20 GTCS)Seizure free same year of seizure onset

      Yes (except 2)Yes. Normal Glx levels in the occipital cortex. No difference in thalamic Glx levels for different ASMs.1.5T; GlxThalamus, Occipital cortex (reference region)Glx significantly elevated in the right thalamus.
      Hammen et al. (2006)
      • Hammen T.
      • Kerling F.
      • Schwarz M.
      • Stadlbauer A.
      • Ganslandt O.
      • Keck B.
      • et al.
      Identifying the affected hemisphere by (1)H-MR spectroscopy in patients with temporal lobe epilepsy and no pathological findings in high resolution MRI.
      22 Epilepsy, 30 Controls; AdultsTLE w/ MRINNRNRNo1.5T; GlxHippocampusGlx significantly elevated contralateral to EEG focus in TLE patients compared to controls. Glx elevated in ipsilateral, but not

      significant.
      Simister, McLean, Barker, Duncan (2007)
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton magnetic resonance spectroscopy of malformations of cortical development causing epilepsy.
      15 Epilepsy, 15 Controls; AdultsRefractory focal epilepsy associated with MCD. Positive MRI findings include 9 HT, 6 PMGSeizure free 1 dayYesYes, but not for Glx. Data from 6 patients taking Gabapentin & Topiramate were not included in the GABA analyses.1.5T; Glx, Glx/NAAt, GABA+, GABA+/CrMajor visible MCD (fronto-parietal/ parieto-occipital)Glx, & Glx/NAAt significantly elevated in MCD. HT group had significantly elevated Glx & Glx/NAAt compared to controls.

      GABA+/Cr significantly elevated in the patient group compared to controls. GABA+ levels showed only a trend to elevation.
      Lin et al. (2009)
      • Lin K.
      • Carrete Jr H.
      • Lin J.
      • Peruchi M.M.
      • de Araújo Filho G.M.
      • Guaranha M.S.B.
      • et al.
      Magnetic resonance spectroscopy reveals an epileptic network in juvenile myoclonic epilepsy.
      60 Epilepsy, 30 Controls;

      Adults
      JME (MRIN)Seizure-free ≥ 48 hYesYes, but results not reported for Glx/Cr.1.5T; Glx/CrMPC, PMC, thalamus, striatum, cingulum, insular, parietal, & occipital corticesSignificantly reduced Glx/Cr in bilateral PMC & MPC & right cingulum. Significantly elevated Glx/Cr in left insula & striatum.
      De Araújo Filho et al. (2009)
      • de Araújo Filho G.M.
      • Lin K.
      • Lin J.
      • Peruchi M.M.
      • Caboclo L.O.S.F.
      • Guaranha M.S.B.
      • et al.
      Are personality traits of juvenile myoclonic epilepsy related to frontal lobe dysfunctions? A proton MRS study.
      41 Epilepsy, 16 Epilepsy with cluster B PDs, 30 Controls; AdultsJME (MRIN)Seizure free at least 72 h before psychiatric evaluationYesNo1.5T; Glx/CrThalamus, insula, cingulate gyrus, medial & lateral primary motor, medial & lateral supplementary motor, striatum, & parietal & occipital lobesGlx/Cr significantly elevated in left insula & striatum for both JME groups compared to controls. Glx/Cr significantly elevated in the right medial primary motor & left lateral primary motor regions in the JME group with PDs.
      Shen et al. (2009)
      • Shen J.
      • Zhang L.
      • Tian X.
      • Liu J.
      • Ge X.
      • Zhang X.
      Use of short echo time two-dimensional 1H-magnetic resonance spectroscopy in temporal lobe epilepsy with negative magnetic resonance imaging findings.
      32 Epilepsy, 10 Controls; Children and AdultsTLE w/ (MRIN)NRNRNo3T; Glx/Cr-PCrHippocampusGlx/Cr–PCr ratio significantly lower on the contralateral side compared with controls & the epileptogenic side.
      Mori et al. (2011)
      • Shen J.
      • Zhang L.
      • Tian X.
      • Liu J.
      • Ge X.
      • Zhang X.
      Use of short echo time two-dimensional 1H-magnetic resonance spectroscopy in temporal lobe epilepsy with negative magnetic resonance imaging findings.
      4 Epilepsy, 10 Controls; ChildrenTSCNRYesNo3T; Glu, Gln, GABAEpilepsy: Epileptogenic tuber in parietal or temporal lobe; Control: Right parietal lobe; Control region for epilepsy patients: contralateral brain region to the tuberNo difference in Glu between epilepsy patients & controls/control regions of the epilepsy patients.

      No difference in Gln between epilepsy patients & controls/control regions of the epilepsy patients (with the exception of 1 patient).

      GABA significantly elevated in cortical tubers compared to controls/control regions of the epilepsy patients.
      Jansen et al. (2014)
      • Jansen J.
      • van der Kruijs S.
      • Vlooswijk M.
      • Majoie H.
      • Hofman P.
      • Aldenkamp A.
      • et al.
      Quantitative MR and cognitive impairment in cryptogenic localisation-related epilepsy.
      35 Epilepsy, 21 Controls; AdultsCLRE (temporal & frontal lobe)NRYesYes, no significant effect of drug load.3T; GlxHippocampus, frontal & temporal lobeGlx significantly elevated in the left frontal lobe compared to controls.
      Chowdhury et al. (2015)
      • Chowdhury F.A.
      • O'Gorman R.L.
      • Nashef L.
      • Elwes R.D.
      • Edden R.A.
      • Murdoch J.B.
      • et al.
      Investigation of Glutamine and GABA Levels in Patients With Idiopathic Generalized Epilepsy Using MEGAPRESS.
      13 Epilepsy, 16 Controls; AdultsIGE (7 GTCS, 2 JAE, 1 CAE, 1 JME, 1 unclassified, 1 absences with eyelid myoclonia)Seizure free ≥ 1 dayYes (except 4), 6 seizure free ≥ 1 yearYes, but only for valproate dose. No effect found.3T; Gln, GABALeft DLPFCGln & GABA significantly elevated in IGE group compared to controls.

      Gln significantly elevated in JME subgroup compared to controls. GABA significantly elevated in GTCS subgroup compared to controls. A trend toward elevated Gln was also reported in the GTCS subgroup compared to controls.
      ASM: anti-seizure medication; Glu: Glutamate; GABA: gamma-aminobutyric acid; JME: juvenile myoclonic epilepsy; Gln: glutamine; MRIN: negative MRI; PCC: posterior cingulate cortex; GTCS: generalized tonic clonic seizures; Glx: glutamate + glutamine; MTS: mesial temporal sclerosis; NAA: N-acetyl aspartate; mTLE: mesial temporal lobe epilepsy; HS: hippocampal sclerosis; cTLE: cryptogenic temporal lobe epilepsy; RE: Rolandic epilepsy; tCR: total creatine; AIs: asymmetry indices; IGE: idiopathic generalized epilepsy; JAE: juvenile absence epilepsy;; OLE: occipital lobe epilepsy; HT: heterotopia; Ins: myoinositol; CR: creatine; GABA+: GABA plus homocysteine; NAAt: N-acetyl aspartate plus N-acetylaspartylglutamate; TLE: temporal lobe epilepsy; FLE: frontal lobe epilepsy; MCD: malformation of cortical development; PLE: parietal lobe epilepsy; X-TLE: non-localized extra temporal lobe epilepsy; IGE: idiopathic generalized epilepsy; NCE: neocortical epilepsy; SMA: supplementary motor area; LTL: lateral temporal lobe; NR: not reported; MPC: medial prefrontal cortex; PMG: polymicrogyria; PMC: primary motor cortex; PD: personality disorder; Cr-PCr: creatine plus phosphocreatine; TSC: Tuberous sclerosis complex; CLRE: Chronic localization-related epilepsy; DLPFC: dorsolateral prefrontal cortex.
      Unlike microdialysis, magnetic resonance spectroscopy (MRS) is a non-invasive technique used to measure glutamate concentrations in the brain [
      • Ramadan S.
      • Lin A.
      • Stanwell P.
      Glutamate and Glutamine: a Review of In Vivo MRS in the Human Brain.
      ]. Due to its non-invasiveness, the potential utility of MRS is much more widespread and its use is not limited to patients who are potential surgical candidates. As a result, MRS can be used to study a variety of epilepsy syndromes and diagnoses. However, it does not come without its limitations. One limitation of MRS is that this technique makes it more difficult to quantify glutamate concentration separately from glutamine concentration, with the literature commonly reporting these together as Glx. Due to the molecular structural similarity of glutamate and glutamine, these two metabolites often contaminate the measure of one another since separation of the peaks for glutamate and glutamine is difficult to discern, especially when using MRS with low-strength tesla MRI [
      • Ramadan S.
      • Lin A.
      • Stanwell P.
      Glutamate and Glutamine: a Review of In Vivo MRS in the Human Brain.
      ,
      • Zhang Y.
      • Shen J.
      Simultaneous quantification of glutamate and glutamine by J-modulated spectroscopy at 3 Tesla.
      ,
      • An L.
      • Li S.
      • Murdoch J.B.
      • Araneta M.F.
      • Johnson C.
      • Shen J.
      Detection of glutamate, glutamine, and glutathione by radiofrequency suppression and echo time optimization at 7 tesla.
      ]. Hence, an accurate and sole measurement of glutamate is not always possible, nor is a more in-depth examination of differences between glutamate and glutamine, without high-strength tesla MRIs.
      All studies reviewed herein had a Tesla strength of 1.5, except for nine studies, seven of which used a Tesla strength of 3T, 1 used a Tesla strength of 4T, and 1 used a Tesla strength of 7T [
      • Pan J.
      • Venkatraman T.
      • Vives K.
      • Spencer D.
      Quantitative glutamate spectroscopic imaging of the human hippocampus.
      ,
      • Riederer F.
      • Bittsanský M.
      • Schmidt C.
      • Mlynárik V.
      • Baumgartner C.
      • Moser E.
      • et al.
      1H magnetic resonance spectroscopy at 3 T in cryptogenic and mesial temporal lobe epilepsy.
      ,
      • Doelken M.
      • Mennecke A.
      • Stadlbauer A.
      • Kecskeméti L.
      • Kasper B.
      • Struffert T.
      • et al.
      Multi-voxel magnetic resonance spectroscopy at 3 T in patients with idiopathic generalised epilepsy.
      ,
      • Hattingen E.
      • Lückerath C.
      • Pellikan S.
      • Vronski D.
      • Roth C.
      • Knake S.
      • et al.
      Frontal and thalamic changes of GABA concentration indicate dysfunction of thalamofrontal networks in juvenile myoclonic epilepsy.
      ,
      • Gonen O.
      • Moffat B.
      • Desmond P.
      • Lui E.
      • Kwan P.
      • O'Brien T
      Seven-tesla quantitative magnetic resonance spectroscopy of glutamate, γ-aminobutyric acid, and glutathione in the posterior cingulate cortex/precuneus in patients with epilepsy.
      ,
      • Shen J.
      • Zhang L.
      • Tian X.
      • Liu J.
      • Ge X.
      • Zhang X.
      Use of short echo time two-dimensional 1H-magnetic resonance spectroscopy in temporal lobe epilepsy with negative magnetic resonance imaging findings.
      ,
      • Mori K.
      • Mori T.
      • Toda Y.
      • Fujii E.
      • Miyazaki M.
      • Harada M.
      • et al.
      Decreased benzodiazepine receptor and increased GABA level in cortical tubers in tuberous sclerosis complex.
      ,
      • Jansen J.
      • van der Kruijs S.
      • Vlooswijk M.
      • Majoie H.
      • Hofman P.
      • Aldenkamp A.
      • et al.
      Quantitative MR and cognitive impairment in cryptogenic localisation-related epilepsy.
      ,
      • Chowdhury F.A.
      • O'Gorman R.L.
      • Nashef L.
      • Elwes R.D.
      • Edden R.A.
      • Murdoch J.B.
      • et al.
      Investigation of Glutamine and GABA Levels in Patients With Idiopathic Generalized Epilepsy Using MEGAPRESS.
      ]. Of those nine studies, six measured glutamate. One study reported no difference in glutamate concentration in cortical tubers compared to controls and control regions [
      • Mori K.
      • Mori T.
      • Toda Y.
      • Fujii E.
      • Miyazaki M.
      • Harada M.
      • et al.
      Decreased benzodiazepine receptor and increased GABA level in cortical tubers in tuberous sclerosis complex.
      ]. Of the five remaining, one study reported reduced glutamate in the epileptogenic hippocampus compared to controls, two studies reported no difference in glutamate concentration in the posterior cingulate cortex/precuneus or in the thalamus, frontal lobe, or motor cortex of cases compared to controls, one reported significantly elevated glutamate in lateral compared to medial voxels on the ipsilateral side of the epileptic focus, and the last study reported elevations in glutamate in central region white matter of cases relative to controls [
      • Pan J.
      • Venkatraman T.
      • Vives K.
      • Spencer D.
      Quantitative glutamate spectroscopic imaging of the human hippocampus.
      ,
      • Riederer F.
      • Bittsanský M.
      • Schmidt C.
      • Mlynárik V.
      • Baumgartner C.
      • Moser E.
      • et al.
      1H magnetic resonance spectroscopy at 3 T in cryptogenic and mesial temporal lobe epilepsy.
      ,
      • Doelken M.
      • Mennecke A.
      • Stadlbauer A.
      • Kecskeméti L.
      • Kasper B.
      • Struffert T.
      • et al.
      Multi-voxel magnetic resonance spectroscopy at 3 T in patients with idiopathic generalised epilepsy.
      ,
      • Hattingen E.
      • Lückerath C.
      • Pellikan S.
      • Vronski D.
      • Roth C.
      • Knake S.
      • et al.
      Frontal and thalamic changes of GABA concentration indicate dysfunction of thalamofrontal networks in juvenile myoclonic epilepsy.
      ,
      • Gonen O.
      • Moffat B.
      • Desmond P.
      • Lui E.
      • Kwan P.
      • O'Brien T
      Seven-tesla quantitative magnetic resonance spectroscopy of glutamate, γ-aminobutyric acid, and glutathione in the posterior cingulate cortex/precuneus in patients with epilepsy.
      ]. Hence, just among those six studies, the results were mixed. It is important to note that the study reporting elevated glutamate levels in lateral compared to medial voxels on the side of the epileptic focus was not compared to a control group, thereby limiting the conclusions that can be drawn [
      • Riederer F.
      • Bittsanský M.
      • Schmidt C.
      • Mlynárik V.
      • Baumgartner C.
      • Moser E.
      • et al.
      1H magnetic resonance spectroscopy at 3 T in cryptogenic and mesial temporal lobe epilepsy.
      ]. It is possible that small sample sizes and heterogeneity among epilepsy syndromes could account for the varied results, as well as differences in the region of interest. For example, the only study that used a Tesla strength of 7T focused on the default mode network, as opposed to the epileptogenic network, which could have been a potential contributor to their lack of significant differences [
      • Gonen O.
      • Moffat B.
      • Desmond P.
      • Lui E.
      • Kwan P.
      • O'Brien T
      Seven-tesla quantitative magnetic resonance spectroscopy of glutamate, γ-aminobutyric acid, and glutathione in the posterior cingulate cortex/precuneus in patients with epilepsy.
      ]. In regards to differences in syndromes, it is also possible that the role of glutamate in these syndromes differ, adding to the complexity of epilepsy as a single disease, and perhaps suggesting the importance of research focusing on individual epilepsy syndromes.
      Of the 25 studies, 15 measured Glx [
      • Riederer F.
      • Bittsanský M.
      • Schmidt C.
      • Mlynárik V.
      • Baumgartner C.
      • Moser E.
      • et al.
      1H magnetic resonance spectroscopy at 3 T in cryptogenic and mesial temporal lobe epilepsy.
      ,
      • Doelken M.
      • Mennecke A.
      • Stadlbauer A.
      • Kecskeméti L.
      • Kasper B.
      • Struffert T.
      • et al.
      Multi-voxel magnetic resonance spectroscopy at 3 T in patients with idiopathic generalised epilepsy.
      ,
      • Simister R.J.
      • McLean M.A.
      • Barker G.J.
      • Duncan J.S.
      A Proton Magnetic Resonance Spectroscopy Study of Metabolites in the Occipital Lobes in Epilepsy.
      ,
      • Flügel D.
      • McLean M.A.
      • Simister R.J.
      • Duncan J.S.
      Magnetisation transfer ratio of choline is reduced following epileptic seizures.
      ,
      • Doelken M.
      • Stefan H.
      • Pauli E.
      • Stadlbauer A.
      • Struffert T.
      • Engelhorn T.
      • et al.
      (1)H-MRS profile in MRI positive- versus MRI negative patients with temporal lobe epilepsy.
      ,
      • Simister R.
      • McLean M.
      • Salmenpera T.
      • Barker G.
      • Duncan J.
      The effect of epileptic seizures on proton MRS visible neurochemical concentrations.
      ,
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton MR spectroscopy of metabolite concentrations in temporal lobe epilepsy and effect of temporal lobe resection.
      ,
      • Cevik N.
      • Koksal A.
      • Dogan V.
      • Dirican A.
      • Bayramoglu S.
      • Ozturk M.
      • et al.
      Evaluation of cognitive functions of juvenile myoclonic epileptic patients by magnetic resonance spectroscopy and neuropsychiatric cognitive tests concurrently.
      ,
      • Leite C da C.
      • Valente K.D.R.
      • Fiore L.A.
      • Otaduy M.C.G.
      Proton spectroscopy of the thalamus in a homogeneous sample of patients with easy-to-control juvenile myoclonic epilepsy.
      ,
      • Woermann F.
      • McLean M.
      • Bartlett P.
      • Parker G.
      • Barker G.
      • Duncan J.
      Short echo time single-voxel 1H magnetic resonance spectroscopy in magnetic resonance imaging-negative temporal lobe epilepsy: different biochemical profile compared with hippocampal sclerosis.
      ,
      • Savic I.
      • Thomas A.
      • Ke Y.
      • Curran J.
      • Fried I.
      • Engel J.
      In vivo measurements of glutamine + glutamate (Glx) and N-acetyl aspartate (NAA) levels in human partial epilepsy.
      ,
      • Simister R.J.
      • Woermann F.G.
      • McLean M.A.
      • Bartlett P.A.
      • Barker G.J.
      • Duncan J.S.
      A Short-echo-time Proton Magnetic Resonance Spectroscopic Imaging Study of Temporal Lobe Epilepsy.
      ,
      • Helms G.
      • Ciumas C.
      • Kyaga S.
      • Savic I.
      Increased thalamus levels of glutamate and glutamine (Glx) in patients with idiopathic generalised epilepsy.
      ,
      • Hammen T.
      • Kerling F.
      • Schwarz M.
      • Stadlbauer A.
      • Ganslandt O.
      • Keck B.
      • et al.
      Identifying the affected hemisphere by (1)H-MR spectroscopy in patients with temporal lobe epilepsy and no pathological findings in high resolution MRI.
      ,
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton magnetic resonance spectroscopy of malformations of cortical development causing epilepsy.
      ,
      • Jansen J.
      • van der Kruijs S.
      • Vlooswijk M.
      • Majoie H.
      • Hofman P.
      • Aldenkamp A.
      • et al.
      Quantitative MR and cognitive impairment in cryptogenic localisation-related epilepsy.
      ]. Twelve studies measured Glx as a ratio with other metabolites, including creatine (Cr), N-acetyl aspartate (NAA), N-acetyl aspartate plus N-acetylaspartylglutamate (NAAt) and myoinositol (Ins) [
      • Lundberg S.
      • Weis J.
      • Eeg-Olofsson O.
      • Raininko R.
      Hippocampal region asymmetry assessed by 1H-MRS in rolandic epilepsy.
      ,
      • Simister R.J.
      • McLean M.A.
      • Barker G.J.
      • Duncan J.S.
      A Proton Magnetic Resonance Spectroscopy Study of Metabolites in the Occipital Lobes in Epilepsy.
      ,
      • Simister R.
      • McLean M.
      • Salmenpera T.
      • Barker G.
      • Duncan J.
      The effect of epileptic seizures on proton MRS visible neurochemical concentrations.
      ,
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton MR spectroscopy of metabolite concentrations in temporal lobe epilepsy and effect of temporal lobe resection.
      ,
      • Cevik N.
      • Koksal A.
      • Dogan V.
      • Dirican A.
      • Bayramoglu S.
      • Ozturk M.
      • et al.
      Evaluation of cognitive functions of juvenile myoclonic epileptic patients by magnetic resonance spectroscopy and neuropsychiatric cognitive tests concurrently.
      ,
      • Leite C da C.
      • Valente K.D.R.
      • Fiore L.A.
      • Otaduy M.C.G.
      Proton spectroscopy of the thalamus in a homogeneous sample of patients with easy-to-control juvenile myoclonic epilepsy.
      ,
      • Savic I.
      • Thomas A.
      • Ke Y.
      • Curran J.
      • Fried I.
      • Engel J.
      In vivo measurements of glutamine + glutamate (Glx) and N-acetyl aspartate (NAA) levels in human partial epilepsy.
      ,
      • Simister R.J.
      • Woermann F.G.
      • McLean M.A.
      • Bartlett P.A.
      • Barker G.J.
      • Duncan J.S.
      A Short-echo-time Proton Magnetic Resonance Spectroscopic Imaging Study of Temporal Lobe Epilepsy.
      ,
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton magnetic resonance spectroscopy of malformations of cortical development causing epilepsy.
      ,
      • Lin K.
      • Carrete Jr H.
      • Lin J.
      • Peruchi M.M.
      • de Araújo Filho G.M.
      • Guaranha M.S.B.
      • et al.
      Magnetic resonance spectroscopy reveals an epileptic network in juvenile myoclonic epilepsy.
      ,
      • de Araújo Filho G.M.
      • Lin K.
      • Lin J.
      • Peruchi M.M.
      • Caboclo L.O.S.F.
      • Guaranha M.S.B.
      • et al.
      Are personality traits of juvenile myoclonic epilepsy related to frontal lobe dysfunctions? A proton MRS study.
      ,
      • Shen J.
      • Zhang L.
      • Tian X.
      • Liu J.
      • Ge X.
      • Zhang X.
      Use of short echo time two-dimensional 1H-magnetic resonance spectroscopy in temporal lobe epilepsy with negative magnetic resonance imaging findings.
      ]. Measurements of Glx as a ratio with other metabolites, such as Glx/N-acetyl aspartate (NAA) and Glx/creatine (Cr), are used as indicators of neuronal function, neurotoxicity, and/or to normalize/scale the data for statistical analyses [
      • Lundberg S.
      • Weis J.
      • Eeg-Olofsson O.
      • Raininko R.
      Hippocampal region asymmetry assessed by 1H-MRS in rolandic epilepsy.
      ,
      • Rosso I.M.
      • Crowley D.J.
      • Silveri M.M.
      • Rauch S.L.
      • Jensen J.E.
      Hippocampus Glutamate and N-Acetyl Aspartate Markers of Excitotoxic Neuronal Compromise in Posttraumatic Stress Disorder.
      ].
      Nine of the 20 studies that investigated Glx or Glx ratios reported no significant differences in Glx concentration or Glx ratios in epilepsy patients as compared to controls [
      • Riederer F.
      • Bittsanský M.
      • Schmidt C.
      • Mlynárik V.
      • Baumgartner C.
      • Moser E.
      • et al.
      1H magnetic resonance spectroscopy at 3 T in cryptogenic and mesial temporal lobe epilepsy.
      ,
      • Lundberg S.
      • Weis J.
      • Eeg-Olofsson O.
      • Raininko R.
      Hippocampal region asymmetry assessed by 1H-MRS in rolandic epilepsy.
      ,
      • Simister R.J.
      • McLean M.A.
      • Barker G.J.
      • Duncan J.S.
      A Proton Magnetic Resonance Spectroscopy Study of Metabolites in the Occipital Lobes in Epilepsy.
      ,
      • Flügel D.
      • McLean M.A.
      • Simister R.J.
      • Duncan J.S.
      Magnetisation transfer ratio of choline is reduced following epileptic seizures.
      ,
      • Doelken M.
      • Stefan H.
      • Pauli E.
      • Stadlbauer A.
      • Struffert T.
      • Engelhorn T.
      • et al.
      (1)H-MRS profile in MRI positive- versus MRI negative patients with temporal lobe epilepsy.
      ,
      • Simister R.
      • McLean M.
      • Salmenpera T.
      • Barker G.
      • Duncan J.
      The effect of epileptic seizures on proton MRS visible neurochemical concentrations.
      ,
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton MR spectroscopy of metabolite concentrations in temporal lobe epilepsy and effect of temporal lobe resection.
      ,
      • Cevik N.
      • Koksal A.
      • Dogan V.
      • Dirican A.
      • Bayramoglu S.
      • Ozturk M.
      • et al.
      Evaluation of cognitive functions of juvenile myoclonic epileptic patients by magnetic resonance spectroscopy and neuropsychiatric cognitive tests concurrently.
      ,
      • Leite C da C.
      • Valente K.D.R.
      • Fiore L.A.
      • Otaduy M.C.G.
      Proton spectroscopy of the thalamus in a homogeneous sample of patients with easy-to-control juvenile myoclonic epilepsy.
      ]. It is important to note that only one of the studies was in a pediatric population [
      • Lundberg S.
      • Weis J.
      • Eeg-Olofsson O.
      • Raininko R.
      Hippocampal region asymmetry assessed by 1H-MRS in rolandic epilepsy.
      ]. The only other pediatric study was the study on tuberous sclerosis, which only had 4 patients [
      • Mori K.
      • Mori T.
      • Toda Y.
      • Fujii E.
      • Miyazaki M.
      • Harada M.
      • et al.
      Decreased benzodiazepine receptor and increased GABA level in cortical tubers in tuberous sclerosis complex.
      ]. Two pediatric studies among a total of 33 studies, including microdialysis articles, indicates that research on glutamate, glutamine, and GABA in pediatric epilepsy patients is severely lacking. Without pediatric studies, there is no way to examine potential differences in glutamate concentration between pediatrics and adults with epilepsy, or between pediatric epilepsy patients and age-matched controls. Furthermore, without knowing these potential differences, development of age-specific treatments and therapies are limited.
      As previously mentioned, differing regions of interest may also be a major factor in these varying results. For example, five of the MRS studies examined juvenile myoclonic epilepsy [
      • Hattingen E.
      • Lückerath C.
      • Pellikan S.
      • Vronski D.
      • Roth C.
      • Knake S.
      • et al.
      Frontal and thalamic changes of GABA concentration indicate dysfunction of thalamofrontal networks in juvenile myoclonic epilepsy.
      ,
      • Cevik N.
      • Koksal A.
      • Dogan V.
      • Dirican A.
      • Bayramoglu S.
      • Ozturk M.
      • et al.
      Evaluation of cognitive functions of juvenile myoclonic epileptic patients by magnetic resonance spectroscopy and neuropsychiatric cognitive tests concurrently.
      ,
      • Leite C da C.
      • Valente K.D.R.
      • Fiore L.A.
      • Otaduy M.C.G.
      Proton spectroscopy of the thalamus in a homogeneous sample of patients with easy-to-control juvenile myoclonic epilepsy.
      ,
      • Lin K.
      • Carrete Jr H.
      • Lin J.
      • Peruchi M.M.
      • de Araújo Filho G.M.
      • Guaranha M.S.B.
      • et al.
      Magnetic resonance spectroscopy reveals an epileptic network in juvenile myoclonic epilepsy.
      ,
      • de Araújo Filho G.M.
      • Lin K.
      • Lin J.
      • Peruchi M.M.
      • Caboclo L.O.S.F.
      • Guaranha M.S.B.
      • et al.
      Are personality traits of juvenile myoclonic epilepsy related to frontal lobe dysfunctions? A proton MRS study.
      ]. Of those five, two reported no significant differences compared to controls in the thalamus or prefrontal cortex [
      • Cevik N.
      • Koksal A.
      • Dogan V.
      • Dirican A.
      • Bayramoglu S.
      • Ozturk M.
      • et al.
      Evaluation of cognitive functions of juvenile myoclonic epileptic patients by magnetic resonance spectroscopy and neuropsychiatric cognitive tests concurrently.
      ,
      • Leite C da C.
      • Valente K.D.R.
      • Fiore L.A.
      • Otaduy M.C.G.
      Proton spectroscopy of the thalamus in a homogeneous sample of patients with easy-to-control juvenile myoclonic epilepsy.
      ]. One study reported no change in glutamate, but significant elevation of glutamine in the frontal lobe [
      • Hattingen E.
      • Lückerath C.
      • Pellikan S.
      • Vronski D.
      • Roth C.
      • Knake S.
      • et al.
      Frontal and thalamic changes of GABA concentration indicate dysfunction of thalamofrontal networks in juvenile myoclonic epilepsy.
      ]. The other two studies reported Glx/Cr elevation in the left insula and striatum and significant reductions in the medial prefrontal cortex, primary motor cortex, and the right cingulum [
      • Lin K.
      • Carrete Jr H.
      • Lin J.
      • Peruchi M.M.
      • de Araújo Filho G.M.
      • Guaranha M.S.B.
      • et al.
      Magnetic resonance spectroscopy reveals an epileptic network in juvenile myoclonic epilepsy.
      ,
      • de Araújo Filho G.M.
      • Lin K.
      • Lin J.
      • Peruchi M.M.
      • Caboclo L.O.S.F.
      • Guaranha M.S.B.
      • et al.
      Are personality traits of juvenile myoclonic epilepsy related to frontal lobe dysfunctions? A proton MRS study.
      ]. Differing results among epilepsy syndromes suggest that glutamate concentration in epilepsy patients can vary depending on the region of interest being investigated; and this should be kept in mind when drawing conclusions from a single study on a single epilepsy subpopulation.
      There were 11 studies that reported significant changes in Glx or Glx ratios, a little more than one third of which examined the medial and/or lateral temporal lobe or specifically the hippocampus in temporal lobe epilepsy (TLE) patients. Major findings from the TLE articles were that (1) Glx was significantly elevated in the MRI negative (normal MRI) TLE group compared to the hippocampal sclerosis group, (2) NAA/Glx ratio was significantly reduced in sclerotic hippocampi and hippocampi ipsilateral to seizure onset compared to contralateral hippocampi and controls, (3) contralateral Glx in MRI negative patients was significantly elevated compared to controls, and (4) contralateral Glx/Cr-PCr was significantly lower compared to the epileptogenic side and controls [
      • Woermann F.
      • McLean M.
      • Bartlett P.
      • Parker G.
      • Barker G.
      • Duncan J.
      Short echo time single-voxel 1H magnetic resonance spectroscopy in magnetic resonance imaging-negative temporal lobe epilepsy: different biochemical profile compared with hippocampal sclerosis.
      ,
      • Simister R.J.
      • Woermann F.G.
      • McLean M.A.
      • Bartlett P.A.
      • Barker G.J.
      • Duncan J.S.
      A Short-echo-time Proton Magnetic Resonance Spectroscopic Imaging Study of Temporal Lobe Epilepsy.
      ,
      • Hammen T.
      • Kerling F.
      • Schwarz M.
      • Stadlbauer A.
      • Ganslandt O.
      • Keck B.
      • et al.
      Identifying the affected hemisphere by (1)H-MR spectroscopy in patients with temporal lobe epilepsy and no pathological findings in high resolution MRI.
      ,
      • Shen J.
      • Zhang L.
      • Tian X.
      • Liu J.
      • Ge X.
      • Zhang X.
      Use of short echo time two-dimensional 1H-magnetic resonance spectroscopy in temporal lobe epilepsy with negative magnetic resonance imaging findings.
      ]. Taken together, these findings suggest that there are reductions in Glx in hippocampal sclerosis and epileptogenic zones of MRI negative patients, apart from Shen et al. (2009), which reported lower Glx/Cr-PCr concentrations on the contralateral side compared to the epileptogenic side. Furthermore, one of the microdialysis studies reported a significant reduction in the glutamine/glutamate ratio in the epileptogenic hippocampus compared to the non-epileptogenic hippocampus [
      • Cavus I.
      • Kasoff W.S.
      • Cassaday M.P.
      • Jacob R.
      • Gueorguieva R.
      • Sherwin R.S.
      • et al.
      Extracellular metabolites in the cortex and hippocampus of epileptic patients.
      ]. Slow rates of glutamate-glutamine cycling, reduced glutamine levels and relative increase of glutamate levels has been reported in resected epileptogenic hippocampi, as has significant elevations in interictal glutamate and no change in glutamine or GABA in atrophic hippocampi compared to non-atrophic epileptogenic hippocampi [
      • Cavus I.
      • Pan J.W.
      • Hetherington H.P.
      • Abi-Saab W.
      • Zaveri H.P.
      • Vives K.P.
      • et al.
      Decreased hippocampal volume on MRI is associated with increased extracellular glutamate in epilepsy patients.
      ,
      • Petroff O.A.C.
      • Errante L.D.
      • Rothman D.L.
      • Kim J.H.
      • Spencer D.D.
      Glutamate-glutamine Cycling in the Epileptic Human Hippocampus.
      ]. Hence, future MRS studies on hippocampal epilepsy should focus on quantification of glutamate, glutamine, and GABA separately, in order to be able to tease apart differences among these metabolites.
      The other three studies with homogenous samples included juvenile myoclonic epilepsy (JME) patients and patients with refractory focal epilepsy associated with malformations of cortical development. The studies on JME reported significant Glx/Cr elevation in the left insula and striatum and significantly reduced Glx/Cr concentration in the primary motor cortex, medial prefrontal cortex, and right cingulum compared to controls [
      • Lin K.
      • Carrete Jr H.
      • Lin J.
      • Peruchi M.M.
      • de Araújo Filho G.M.
      • Guaranha M.S.B.
      • et al.
      Magnetic resonance spectroscopy reveals an epileptic network in juvenile myoclonic epilepsy.
      ,
      • de Araújo Filho G.M.
      • Lin K.
      • Lin J.
      • Peruchi M.M.
      • Caboclo L.O.S.F.
      • Guaranha M.S.B.
      • et al.
      Are personality traits of juvenile myoclonic epilepsy related to frontal lobe dysfunctions? A proton MRS study.
      ]. The study on patients with refractory focal epilepsy associated with malformations of cortical development reported significantly elevated Glx and Glx/NAAt in malformations of cortical development and specifically in the heterotopia patients with malformations of cortical development compared to controls [
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton magnetic resonance spectroscopy of malformations of cortical development causing epilepsy.
      ].
      The remaining studies included multiple epilepsy syndromes, which, alongside small sample sizes, makes it impossible to assess changes in Glx or Glx ratios on a syndromic basis. Generally, partial mesial temporal lobe epilepsy and partial neocortical epilepsy had significant elevations of Glx/Cr and Glx/ NAA in the epileptic region compared to the homologous region, frontal and temporal lobe epilepsy patients had significant Glx elevation in the left frontal lobe, and idiopathic generalized epilepsy patients had significant Glx elevation in the right thalamus [
      • Doelken M.
      • Mennecke A.
      • Stadlbauer A.
      • Kecskeméti L.
      • Kasper B.
      • Struffert T.
      • et al.
      Multi-voxel magnetic resonance spectroscopy at 3 T in patients with idiopathic generalised epilepsy.
      ,
      • Savic I.
      • Thomas A.
      • Ke Y.
      • Curran J.
      • Fried I.
      • Engel J.
      In vivo measurements of glutamine + glutamate (Glx) and N-acetyl aspartate (NAA) levels in human partial epilepsy.
      ,
      • Helms G.
      • Ciumas C.
      • Kyaga S.
      • Savic I.
      Increased thalamus levels of glutamate and glutamine (Glx) in patients with idiopathic generalised epilepsy.
      ,
      • Jansen J.
      • van der Kruijs S.
      • Vlooswijk M.
      • Majoie H.
      • Hofman P.
      • Aldenkamp A.
      • et al.
      Quantitative MR and cognitive impairment in cryptogenic localisation-related epilepsy.
      ]. However, despite the heterogeneity of the samples and regions of interest, all of the studies reported significant elevations in Glx or Glx ratio. However, without separate measures of glutamate and glutamine, we are unable to assess whether or not glutamine synthetase dysfunction could be implicated in these studies. Additionally, differences among voxel placement and/or the MRS acquisition technique add further challenges when attempting to compare studies to one another [
      • Chowdhury F.A.
      • O'Gorman R.L.
      • Nashef L.
      • Elwes R.D.
      • Edden R.A.
      • Murdoch J.B.
      • et al.
      Investigation of Glutamine and GABA Levels in Patients With Idiopathic Generalized Epilepsy Using MEGAPRESS.
      ]. Furthermore, without simultaneous EEG monitoring, studies with interictal or seizure free patients cannot exclude the presence of subclinical or EEG seizures, adding another layer of complexity when trying to draw conclusions [
      • Savic I.
      • Thomas A.
      • Ke Y.
      • Curran J.
      • Fried I.
      • Engel J.
      In vivo measurements of glutamine + glutamate (Glx) and N-acetyl aspartate (NAA) levels in human partial epilepsy.
      ].
      As previously mentioned, analysis of GABA alongside glutamate is important due to their relation to one another. However, of the 25 MRS studies, only seven included GABA analyses, and only four reported statistically significant results [
      • Hattingen E.
      • Lückerath C.
      • Pellikan S.
      • Vronski D.
      • Roth C.
      • Knake S.
      • et al.
      Frontal and thalamic changes of GABA concentration indicate dysfunction of thalamofrontal networks in juvenile myoclonic epilepsy.
      ,
      • Gonen O.
      • Moffat B.
      • Desmond P.
      • Lui E.
      • Kwan P.
      • O'Brien T
      Seven-tesla quantitative magnetic resonance spectroscopy of glutamate, γ-aminobutyric acid, and glutathione in the posterior cingulate cortex/precuneus in patients with epilepsy.
      ,
      • Simister R.J.
      • McLean M.A.
      • Barker G.J.
      • Duncan J.S.
      A Proton Magnetic Resonance Spectroscopy Study of Metabolites in the Occipital Lobes in Epilepsy.
      ,
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton MR spectroscopy of metabolite concentrations in temporal lobe epilepsy and effect of temporal lobe resection.
      ,
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton magnetic resonance spectroscopy of malformations of cortical development causing epilepsy.
      ,
      • Mori K.
      • Mori T.
      • Toda Y.
      • Fujii E.
      • Miyazaki M.
      • Harada M.
      • et al.
      Decreased benzodiazepine receptor and increased GABA level in cortical tubers in tuberous sclerosis complex.
      ,
      • Chowdhury F.A.
      • O'Gorman R.L.
      • Nashef L.
      • Elwes R.D.
      • Edden R.A.
      • Murdoch J.B.
      • et al.
      Investigation of Glutamine and GABA Levels in Patients With Idiopathic Generalized Epilepsy Using MEGAPRESS.
      ]. One reported a significant increase in GABA in the frontal region and significant decrease in the thalamus for patients with juvenile myoclonic epilepsy [
      • Hattingen E.
      • Lückerath C.
      • Pellikan S.
      • Vronski D.
      • Roth C.
      • Knake S.
      • et al.
      Frontal and thalamic changes of GABA concentration indicate dysfunction of thalamofrontal networks in juvenile myoclonic epilepsy.
      ]. This was also only one of two studies to assess glutamate, glutamine, and GABA, which interestingly reported elevations in glutamine and GABA in the frontal lobe, with no change in glutamate concentration compared to controls. The other study that included glutamate, GABA, and glutamine reported significant elevations in GABA in cortical tubers of patients with tuberous sclerosis complex, no change in glutamate, and one of four patients had an elevation in glutamine [
      • Mori K.
      • Mori T.
      • Toda Y.
      • Fujii E.
      • Miyazaki M.
      • Harada M.
      • et al.
      Decreased benzodiazepine receptor and increased GABA level in cortical tubers in tuberous sclerosis complex.
      ]. These results reflect the importance of being able to separate glutamate and glutamine measurements, alongside the importance of measuring GABA alongside glutamate metabolites, as capturing these differences are critical in understanding potential dysfunction and underlying mechanisms of epilepsy. The third study investigated both glutamine and GABA, and reported a significant elevation of both metabolites in idiopathic generalized epilepsy compared to controls [
      • Chowdhury F.A.
      • O'Gorman R.L.
      • Nashef L.
      • Elwes R.D.
      • Edden R.A.
      • Murdoch J.B.
      • et al.
      Investigation of Glutamine and GABA Levels in Patients With Idiopathic Generalized Epilepsy Using MEGAPRESS.
      ]. In support of the importance of studying epilepsies by subtype, this study also reported subgroup differences, despite the study not being designed for subgroup comparisons. Subgroup results included a significant elevation in glutamine in the juvenile myoclonic epilepsy subgroup compared to controls, and a significant elevation in GABA in the generalized tonic clonic subgroup compared to controls, though sample sizes were very small [
      • Chowdhury F.A.
      • O'Gorman R.L.
      • Nashef L.
      • Elwes R.D.
      • Edden R.A.
      • Murdoch J.B.
      • et al.
      Investigation of Glutamine and GABA Levels in Patients With Idiopathic Generalized Epilepsy Using MEGAPRESS.
      ]. Again, in regards to precision therapies, understanding these differences among epilepsies are key in developing the most effective treatment for patients. The fourth study reported an elevation in the GABA plus homocysteine (GABA+)/Cr ratio in patients with refractory focal epilepsy associated with malformations of cortical development compared to controls. However, GABA+ levels showed only a trend toward elevation [
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton magnetic resonance spectroscopy of malformations of cortical development causing epilepsy.
      ]. The other three studies included temporal lobe epilepsy, a mix of idiopathic generalized epilepsies and occipital lobe epilepsy, and a combination of temporal lobe epilepsy and idiopathic generalized epilepsy [
      • Gonen O.
      • Moffat B.
      • Desmond P.
      • Lui E.
      • Kwan P.
      • O'Brien T
      Seven-tesla quantitative magnetic resonance spectroscopy of glutamate, γ-aminobutyric acid, and glutathione in the posterior cingulate cortex/precuneus in patients with epilepsy.
      ,
      • Simister R.J.
      • McLean M.A.
      • Barker G.J.
      • Duncan J.S.
      A Proton Magnetic Resonance Spectroscopy Study of Metabolites in the Occipital Lobes in Epilepsy.
      ,
      • Simister R.
      • McLean M.
      • Barker G.
      • Duncan J.
      Proton MR spectroscopy of metabolite concentrations in temporal lobe epilepsy and effect of temporal lobe resection.
      ]. Worth noting, one of these studies investigated the default mode network (posterior cingulate cortex/precuneus) and not the epileptogenic network, which may have been a contributing factor to the results, or lack thereof [
      • Gonen O.
      • Moffat B.
      • Desmond P.
      • Lui E.
      • Kwan P.
      • O'Brien T
      Seven-tesla quantitative magnetic resonance spectroscopy of glutamate, γ-aminobutyric acid, and glutathione in the posterior cingulate cortex/precuneus in patients with epilepsy.
      ]. Overall, the limited amount of studies on GABA, in concurrence with the variety of syndromes both between and within studies, makes it difficult to draw generalized conclusions.
      The paucity of studies measuring GABA could be partially due to challenges in GABA detection, including the low concentration of GABA and overlap of its peak with peaks of other neurotransmitters, such as creatine [
      • Mullins P.G.
      • McGonigle D.J.
      • O'Gorman R.L.
      • Puts N.A.J.
      • Vidyasagar R.
      • Evans C.J.
      • et al.
      Current practice in the use of MEGA-PRESS spectroscopy for the detection of GABA.
      ]. Specialized pulse sequences are needed to quantify GABA, with the standard technique being Meshcher-Garwood Point Resolved Spectroscopy (MEGA-PRESS) [
      • Mullins P.G.
      • McGonigle D.J.
      • O'Gorman R.L.
      • Puts N.A.J.
      • Vidyasagar R.
      • Evans C.J.
      • et al.
      Current practice in the use of MEGA-PRESS spectroscopy for the detection of GABA.
      ]. It is important in terms of future directions that studies use higher power tesla strength MRIs and include detection and quantification of GABA to be able to tease apart the relation between glutamate, glutamine, and GABA.
      As mentioned in the microdialysis section, it is important to consider confounding factors that may be at play, such as the influence of anti-seizure medications on glutamatergic and GABAergic neurotransmission. Unlike microdialysis, since MRS is non-invasive, studies investigating glutamate and GABA concentrations in newly diagnosed drug naïve patients is not outside the realm of possibility. Having such research would be profoundly beneficial in understanding changes in these metabolites prior to any short- or long-term effects of pharmacological management.
      Finally, it is important to underscore that the information evaluated in this paper, measures of glutamate and GABA concentrations in the human epileptic brain, do not provide the whole picture for glutamate and GABA in epilepsy. The levels of glutamate and GABA are not representative of processes related to these metabolites, including biosynthesis, transport, or degradation, and do not present information on potential dysfunction or dysregulation that may be occurring within these processes. Furthermore, the results presented do not speak to potential compensatory mechanisms that could be at play.

      4. Future epilepsy biomarker research directions

      Future research should consider the addition of complementary techniques, such as molecular neuroimaging, which would provide insight into other mechanisms regarding glutamate and GABA in the epileptic brain, beyond concentration level. Two such techniques are positron emission tomography (PET) and single-photon emission computed tomography (SPECT). These techniques utilize radiotracers, or small amounts of radioactive material, to assess tissue function. There are a variety of tracers available that measure different outcomes, depending on the question being asked. Of interest are radiotracers used for in vivo imaging of glutamate and GABA receptor availability in the human epileptic brain.
      Flumazenil (PET) and Iomazenil (SPECT) are radiotracers used to measure the availability of GABAA receptors that are benzodiazepine sensitive, since the tracers work by blocking the benzodiazepine site [
      • Hodolic M.
      • Topakian R.
      • Pichler R.
      18)F-fluorodeoxyglucose and (18)F-flumazenil positron emission tomography in patients with refractory epilepsy.
      ]. These techniques can be used in conjunction with MRS, providing a more complete picture of what is occurring in the epileptic region. For example, the study on tuberous sclerosis patients reported increased GABA levels in cortical tubers, which the authors proposed was compensatory in nature as reduced GABAA receptor function was also reported using SPECT [
      • Mori K.
      • Mori T.
      • Toda Y.
      • Fujii E.
      • Miyazaki M.
      • Harada M.
      • et al.
      Decreased benzodiazepine receptor and increased GABA level in cortical tubers in tuberous sclerosis complex.
      ]. Furthermore, GABAA receptor binding has been reported to be reduced in temporal and extratemporal lobe epileptic regions and lesions [
      • Juhász C.
      • Mittal S.
      Molecular Imaging of Brain Tumor-Associated Epilepsy.
      ,
      • Szelies B.
      • Weber-Luxenburger G.
      • Pawlik G.
      • Kessler J.
      • Holthoff V.
      • Mielke R.
      • et al.
      MRI-guided flumazenil- and FDG-PET in temporal lobe epilepsy.
      ,
      • Debets R.M.
      • Sadzot B.
      • van Isselt J.W.
      • Brekelmans G.J.
      • Meiners L.C.
      • van Huffelen A.O.
      • et al.
      Is 11C-flumazenil PET superior to 18FDG PET and 123I-iomazenil SPECT in presurgical evaluation of temporal lobe epilepsy?.
      ,
      • Goethals I.
      • Wiele C.
      • Boon P.
      • Dierckx R.
      Is central benzodiazepine receptor imaging useful for the identification of epileptogenic foci in localization-related epilepsies?.
      ,

      Niu N., Xing H., Wu M., Ma Y., Liu Y., Ba J., et al. Performance of PET imaging for the localization of epileptogenic zone in patients with epilepsy: a meta-analysis. [cited 2021]; Available from: https://doi.org/10.1007/s00330-020-07645-4.

      ,
      • Fujimoto A.
      • Okanishi T.
      • Kanai S.
      • Sato K.
      • Itamura S.
      • Baba S.
      • et al.
      Double match of 18F-fluorodeoxyglucose-PET and iomazenil-SPECT improves outcomes of focus resection surgery.
      ,
      • Horky L.L.
      • Treves S.T.
      PET and SPECT in brain tumors and epilepsy.
      ]. This type of research may offer potential theories as to why GABA levels may appear unchanged or even increased in epileptic regions in MRS studies.
      Compared to GABA, the use of PET for measuring glutamate receptor availability in human epilepsy is scarce. Previous research on the mGluR5 receptor in cortical focal dysplasia reported reduced receptor availability at the epileptogenic site, as well as areas outside of the epileptogenic zone [
      • DuBois J.M.
      • Rousset O.G.
      • Guiot M.-.C.
      • Hall J.A.
      • Reader A.J.
      • Soucy J.-.P.
      • et al.
      Metabotropic Glutamate Receptor Type 5 (mGluR5) Cortical Abnormalities in Focal Cortical Dysplasia Identified In Vivo With [11C]ABP688 Positron-Emission Tomography (PET) Imaging.
      ]. A follow-up retrospective study investigating the mGluR5 network reported large scale network abnormalities, such as reduced network integration represented by reduced global efficiency and resilience, compared to controls [
      • DuBois J.M.
      • Mathotaarachchi S.
      • Rousset O.G.
      • Sziklas V.
      • Sepulcre J.
      • Guiot M.-.C.
      • et al.
      Large-scale mGluR5 network abnormalities linked to epilepsy duration in focal cortical dysplasia.
      ]. The progress in mGluR5 radiotracers and imaging noted in other work may have potential utility in epilepsy [
      • Kim J.-.H.
      • Marton J.
      • Ametamey S.M.
      • Cumming P.
      A Review of Molecular Imaging of Glutamate Receptors.
      ].
      Development of radiotracers in PET imaging for AMPA receptors has been severely limited due to issues with reduced brain clearance, quick clearance of the compound, and low binding specificity in rodent studies. Until recently, there were no radiotracers for AMPA receptor imaging in the living human brain. Miyazaki et al. developed an AMPA receptor radiotracer that in an exploratory study in temporal lobe epilepsy observed increased radiotracer uptake in the epileptogenic focus. Furthermore, in the surgical specimens of the same people the increased uptake was associated with the local AMPA receptor protein distribution [
      • Lam J.
      • DuBois J.M.
      • Rowley J.
      • González-Otárula K.A.
      • Soucy J.-.P.
      • Massarweh G.
      • et al.
      In vivo metabotropic glutamate receptor type 5 abnormalities localize the epileptogenic zone in mesial temporal lobe epilepsy.
      ]. This development is a major milestone in the potential to study AMPA receptor availability in vivo in human epilepsy.
      Similar to AMPA receptor radiotracers, the use of radiotracers for NMDA receptors in humans has been met with limited success. Investigations on NMDA radiotracers, such as 18F-GE-179, are newly emerging. Current studies have not been conducted in humans, and at least one animal study has reported issues with specificity [
      • Zhou W.
      • Bao W.
      • Jiang D.
      • Kong Y.
      • Hua F.
      • Lu X.
      • et al.
      [18F]-GE-179 positron emission tomography (PET) tracer for N-methyl-d-aspartate receptors: one-pot synthesis and preliminary micro-PET study in a rat model of MCAO.
      ,
      • Schoenberger M.
      • Schroeder F.A.
      • Placzek M.S.
      • Carter R.L.
      • Rosen B.R.
      • Hooker J.M.
      • et al.
      In Vivo [18F]GE-179 Brain Signal Does Not Show NMDA-Specific Modulation with Drug Challenges in Rodents and Nonhuman Primates.
      ,
      • Vibholm A.K.
      • Landau A.M.
      • Alstrup A.K.O.
      • Jacobsen J.
      • Vang K.
      • Munk O.L.
      • et al.
      Activation of NMDA receptor ion channels by deep brain stimulation in the pig visualised with [18F]GE-179 PET.
      ,
      • Vibholm A.K.
      • Landau A.M.
      • Møller A.
      • Jacobsen J.
      • Vang K.
      • Munk O.L.
      • et al.
      NMDA receptor ion channel activation detected in vivo with [18F]GE-179 PET after electrical stimulation of rat hippocampus.
      ,
      • López-Picón F.
      • Snellman A.
      • Shatillo O.
      • Lehtiniemi P.
      • Grönroos T.J.
      • Marjamäki P.
      • et al.
      Ex Vivo Tracing of NMDA and GABA-A Receptors in Rat Brain After Traumatic Brain Injury Using 18F-GE-179 and 18F-GE-194 Autoradiography.
      ]. However, recent research has described the development of a process for radiosynthesis that would allow the radiotracer to be appropriate for clinical studies [
      • Khan I.
      • Berg T.C.
      • Brown J.
      • Bhalla R.
      • Wilson A.
      • Black A.
      • et al.
      Development of an automated, GMP compliant FASTlabTM radiosynthesis of [18 F]GE-179 for the clinical study of activated NMDA receptors.
      ]. Additionally, development of radiotracers specific to NMDAR subtypes have emerged in recent years, with the first human study for a radiotracer for the GluNB2 subtype currently underway [
      • Tamborini L.
      • Chen Y.
      • Foss C.A.
      • Pinto A.
      • Horti A.G.
      • Traynelis S.F.
      • et al.
      Development of Radiolabeled Ligands Targeting the Glutamate Binding Site of the N-Methyl-d-aspartate Receptor as Potential Imaging Agents for Brain.
      ,
      • Mu L.
      • Krämer S.D.
      • Ahmed H.
      • Gruber S.
      • Geistlich S.
      • Schibli R.
      • et al.
      Neuroimaging with Radiopharmaceuticals Targeting the Glutamatergic System.
      ]. In short, the continued development and exploration of glutamate receptor radiotracers will be of significant relevance, and when paired with MRS or microdialysis, may provide additional critical insight into understanding the complex role of glutamate in epilepsy.

      5. Conclusion

      In summary, microdialysis studies and some MRS studies support the presence of increased extracellular glutamate levels in patients with epilepsy. To better understand the role that glutamate plays in epilepsy, it will be important for future studies to use high strength Tesla MRIs, expand studies to the pediatric population, consider effects of concomitant anti-seizure medication, and increase sample sizes among homogeneous epilepsy populations. The investigation of the relationships between glutamate, glutamine, and GABA levels, as well as their associations with markers of mitochondrial function, such as NAA/Cr, should be included in future research. Finally, the addition of complimentary techniques, such as PET, would offer the opportunity to investigate distinct biomarkers in epilepsies, while also providing a greater understanding of the complex roles of glutamate and GABA in epilepsy.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      Declaration of Competing Interest

      Declarations of interest: none

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