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L-GlutamateThe King of NeurotransmissionDr.A.Balaji Sainath1st year PG student30-Oct-161

INTRODUCTIONFor over 50 years biogenic amines have dominated thinking about the role of neurotransmitters in the pathophysiology of psychiatric disorders.However over the last decade evidence from postmartem, brain imaging , genetic studies shows that amino acid neurotransmitters in particular GLUTAMATE & GABA play an important role in (if not central- in pathophysiology) a broad range of psychiatric disorders including Schizophrenia, BPAD, MDD, Alzheimers and Anxiety .

Amino acid neurotransmitter

GABA GlycineInhibitory neurotransmitterAt 15-20% of synapses

GlutamateAspartateExcitatory neurotransmitterAt 7580% of synapsesGLUTAMATE3

Glutamic Acid (or) GlutamateAcidic nonessential amino acid.Important as the building block of protein synthesis.As a neurotransmitter in CNS.Major excitatory neurotransmitter.Called king of neurotransmittersAlso called master switch of brainConcentration in brain is 10mM, the highest of all aminoacids and of all NT.

Synthesis of GlutamateGiven the excitatory effects of glutamate, it is excluded from the brain by BBB i.e, Blood Brain Barrier is impermeable to Glutamate.Thus, glutamate in the brain must be synthesised de novo from Glucose, TCA Alpha Ketoglutarate Glutamic acid (via transamination)Reuptake to storage vessels, 20% of glutamate turnover through glutamate transporter & 40% through glutamine cycle.

GLUTAMATE SYNTHESIS

Synthesis of glutamate cotransmitters( Glycine, D-serine)One of the key receptors for glutamate (NMDA) requires a cotransmitter in addition to glutamate to function.Glycine synthesized either from glycine neurons or from glia.Type 2 glycine transporter (GlyT2) present on glycine neurons.* (on glycine neurons)Type 1 glycine transporter (GlyT1) and glial SNAT (specific neutral amino acid transporter) present on glia. *Glycine synthesized in glia from amino acid L-serine with an enzyme called SHMT.

Contd..This enzyme works in both directions, ThusGlycine converted to L-serine in glia with an enzyme SHMT (serine hydroxymethyl transferase) which in turn converted to D-serine.L-serine to D-serine conversion occurred in glia with an enzyme called D-serine racemase.Thus D-serine can be derived either from glycine or from L-serine.Reuptake of D-serine is by glial D-serine transporter (D-SER-T).

Glutamate receptors & Transporters

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TransportersThe neuronal presynaptic reuptake pump (EAAT or excitatory amino acid transporter), Glutamate is transported across membranes of synapse by these Na++ dependent transporters.These are 5 typesEAAT1 AstrocyteEAAT2 Astrocytes, ForebrainEAAT3 Upper motor neuronsEAAT4 Cerebellar purkinje cellsEAAT5 RetinaOf these EAAT1 & 2 are involved in the reuptake and release of glutamate during glutamine cycleThe vesicular transporter for glutamate into synaptic vesicles (vGluT)

Glutamate fast neurotransmissionSynthesis, packaging, reuptake, degradation

(error - should be EAAT)

Glutamate receptors

12 Our understanding of the molecular structure of glutamate receptors began 10 years ago, with the cloning byfunctional expression of a glutamate receptor by Hollmann. This feat initiated the cloning of representativemembers of the various subtypes of glutamate receptors. Members of AMPA kainate, and NMDA receptor families have all been cloned. These families of receptors include multiple subunit types that may form homomeric (composed of only one subunit type) or heteromeric (composed of two or more different subunit types) receptors. Adding to the complexity is the fact that splice variants of subunit messenger ribonucleic acids (mRNAs) have been described for AMPA and NMDA receptor types. It is unclear at present which subunits within a family form functional receptor channel complexes in the intact animal, although recent evidence suggests that at least four subunits come together toform a functional receptor.

IONOTROPIC GLUTAMATE RECEPTORS

AMPAKAINATENMDA

NMDANMethylDAspartic acid receptor.It is a hetero tetramer composed of NR1 sub unit that comprises channel and also has binding site glycine modulatory site & NR2 sub unit contains ligand binding site for agonist.At resting membrane potential channel blocked by Mg++It has been described as coincidence detector because 3 events must occur for the channel to openA sufficient amount of glutamate has been released and binds to receptorGlycine/D-serine release from astrocytes and binds to its siteThe synaptic membrane sufficiently depolarized to remove the Mg++ blockade

Contd..NR2 sub unit further divided into 4 types, 2A-2D.NR2A- corticolimbic regions in mature brainNR2B- immature cortexNR2C- cerebellumNR2D- cerebellum, midbrain Have prominent role in learning, excitotoxicity.Sub units also have binding sites for Zn++, H+ and a polyamine site.

AMPA-Amino 3Hydroxy 5Methyl 4-Isoxazole propionate (AMPA) receptors are broadly distributed in CNS.They are mediating most EPSPs in CNS.Contains 4 sub units, GluR1-4.Subunits 1,3,4 have Glutamine(Q) residue that results in high permeability to Ca++.Subunit 2 have Arginine(R) residue that restricts Ca++ passage and conducts only Na+.mRNA editing of GluR2 results in Arginine to Glutamine change and increases Ca++ conductance.

Contd..Ca++ acts as important second meesenger Activates intracellular cascades. Ca++ binds to calmodulin protein Activates protein kinases like CAM kinase.CAM Kinase effects AMPA receptors in 2 ways :-Phosphorylates AMPA receptors already present in dendritic spine membraneIncreasing their conductance to sodium ions&Promotes intracellular AMPA receptors to move to the membrane Making more receptors available to stimulate the spine(Trafficking) LONG TERM POTENTIATION

Kainite receptorsConsists of 5 sub units, GluR 5-7, KA1, KA2.These are less understood than AMPA, NMDA.GluR 5-7 sub units form glutamate gated cation channels.KA1, KA2 sub units aggregate with GluR 5-7 sub units to form high affinity kainite receptors.Their activation reduces glutamatergic neurotransmission because of presynaptic location.Eg:- GluR7 allelic variant associated with MDD.

Physiological or Pathological Roles

AMPA receptors Mediate most fast EPSPs in the CNS

Kainate receptorsRegulation of neuronal excitability Epilepsy, Excitotoxicity and Pain

NMDA receptors Mediate most fast EPSPs in the CNSAnaesthesiaLearning and memoryDevelopmental plasticityEpilepsyExcitotoxicity (eg :- Stroke)Schizophrenia

Metabotropic glutamate receptorsGroup 1Group 2 & 3mGluR 1,5Activate phospolipase CThrough Gq ProteinPredominantly located postsynaptically.mGluR 2,3 & mGluR 6,7,8Inhibit adenylyl cyclaseThrough Gi ProteinPredominantly located presynaptically as Auto receptors.

g

b

a

G-protein coupled receptorsCell membraneG-protein composed of one alpha, beta, and gamma subunit2 primary signaling cascades: cAMP or phosphatidylinositol pathwaysPathway activated depends on alpha subunit typeGDP bound to a when inactive

gbaGDP

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g

b

a

Contd..Cell membrane

When a ligand binds, the receptor changes conformation, allowing G-protein to be activated (GDP is exchanged for GTP)G-protein dissociates from receptor then subunits from each other.

GDPGTP

aGTP

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g

b

a

Contd..Cell membrane

GDPGTP

aGTP

Gs binds to Adenylate Cyclase (AC) and stimulates cAMP synthesis from ATP Gi binds to AC and inhibits cAMP synthesis

AC

ACATPcAMP

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Postsynaptic DensityThepostsynaptic density(PSD) is a protein densespecializationattached to thepostsynapticmembrane. The PSD is in close apposition to the presynapticactive zone and ensures that receptors are in close proximity topresynapticneurotransmitter release sites.The structure and composition of the PSD have been the focus of numerous molecular studies ofsynaptic plasticity, a cellular model of learning and memory.The PSD is a multiprotein complex that contains scaffolding proteins, cell adhesion molecules, and proteins for intercellular signalling pathways.PSDs vary in size and composition among brain regions and have been studied in great detail atglutamatergicsynapses.

Contd..At glutamatergic synapses, major scaffolding protein is PSD-95. it contains several regions that bind other proteins.There are 3 PDZ (PSD-95/disc large/zona occludens-1) domains which contain approximately 90 AA that bind the C-terminus proteins.Attachments :-Neuroligin attachment to PDZ stabilizes the synapse.Palmitic acid attachment to PSD-95 links lipid rafts of plasma membrane. PSD-95 also binds to Alph-Actin.NR2 sub unit of NMDA receptor binds to PDZ domain.Effector enzymes nitric oxide synthase (NOS) and calmodulin activated protein kinase-2 (CAMK2) are also bound to PSD-95, keeping them in close proximity to the Ca++ permeable NMDA receptor channel.

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Contd..AMPA receptors indirectly attached to PSD-95 through intermediary filaments & TARPs (transmembrane AMPA receptor regulatory proteins).mGluR7 attaches to PSD-95 throgh 2 scaffolding proteins homer and schank.Mutations in proteins that comprise the PSD including neurexin, neuroligin, and schank have been implicated in Autism.

Plasticity in glutamatergic neurotransmissionLearning and memory involved use dependent changes in synaptic efficacy. (Hebb)A brief period of intense stimulation resulted in a subsequent persistent increase in efficacy of transmission known as long term potentiation (LTP).A period of stable low frequency stimulation results in a persistent reduced transmission known as long term depression (LTD).LTP requires activation of NMDA receptors, hence conditions results in blockade of LTP in hippocampus leads to impairment in acquisition of new memories.NMDA antagonists ketamine, PCP are causes blockade of LTP.AMPA receptor insertion or withdrawl from PSD decides LTP/LTD.* (insertion/withdrawl)

Contd..The extinction of conditioned fear mediated by activation of NMDA receptor in amygdala, hence NMDA receptor antagonists prevents this extinction (rats) & treatment with D-cycloserine (glycine modulatory site partial agonist) facilitates. a study in acrophobia placebo + CBT vs D-cycloserine + CBT for 3 months suggesting that pharmocologically augmenting neural plasticity may be used to bolster psychological interventions.Glutamate mediated synaptic plasticity is not only functional but also structural.Persistent activation of NMDA receptors results in dendritic spine maturation i.e, protein synthesis in individual spines (mice).Fragile X mental retardation protein (FMRP) also synthesized locally in dendritic spines during NMDA receptor activation. In fragile X syndrome number of dendritic spines are less.

Contd..Loss of FMRP exaggerates responses of mGluR5, which stimulates dendritic protein synthesis, hence treatment with mGluR5 antagonist reverses the fragile X phenotype.

ExcitotoxicityLarge amounts of glutamate causes neuronal degeneration.Direct injection of glutamate receptor agonists such as Kainic acid, Ibotenic acid, N-Methyl-D-Aspartic acid causes a pattern of neuronal degeneration with,In proximity to injection site :- Persistent and overwhelming activation of AMPA/Kainite receptors and NMDA receptors causes a tremendous influx of Ca++ and Na+ and secondary influx of H2O cellular edema narcotic cell death.In distant to injection site :- persistent elevation of Ca++ disrupts the mitochondria release cytochrome C and activate caspases Apoptotic cell death.

Contd..The neuronal degeneration of ischemic stroke is due to excitotoxicity. Local hypoxia decreased ATP production collapse of sodium gradient reversal of sodium dependent glutamate transporter massive release of glutamate.Drugs that block ionotropic glutamate receptors reduces the amount of neuronal damage. (no clinical trail proved effective)Excitotoxicity also been implicated in alzheimers disese. 1)Beta-amyloid fibrils depolarize neurons loss of Mg++ blockade enhance NMDA receptor sensitivity to glutamate. 2)Fibrils also impair glutamate transport into astrocytes increasing extracellular glutamate.Thus, non competitive inhibitor of NMDA receptor, Memantine recently approved for mild to moderate alzheimers. (It reduces tonic sensitivity of NMDA receptors but not alter phasic transmission)

Glutamate in Schizophrenia

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Dopamine theoryTRAID OF DA THEORYDrugs that increase dopamine, such as amphetamine and cocaine, can cause psychosis.Antidopaminergic drugs can improve psychosis.Mechanism overactivity in the mesolimbic dopamine pathway could be the mediator of positive symptoms of schizophrenia such as delusions and hallucinations. PROBLEMS OF DA THEORYIt explains only part of schizophrenia (positive symptoms not negative symptoms).Anti-dopamInergic drugs usually: make negative symptoms worse in patients.induce negative symptoms in healthy people.Atypical antipsychotic drugs e.g. Clozapine (with weaker anti-dopaminergic activity) are better anti-schizophrenic drugsUnder activity in the mesocortical dopamine pathway is hypothesized to be the mediator of negative symptoms of schizophrenia: This indicates that reduced dopamine activity is the problem rather than dopamine overactivity.DA theory is a psychosis theory more than it is a schizophrenia theory

NMDA receptor Hypofunction hypothesis of schizophrenia Ketamine and PhencyclidineFaulty NMDA synapses on GABA interneurons in prefrontal cortexLinking with dopamine hypothesis of schizophrenia- positive symptoms and negative symptoms

Ketamine and PCPThe idea of a glutamatergic abnormality in schizophrenia was first proposed by Kim and colleagues in 1980 based on their findings of low cerebrospinal fluid (CSF) glutamate levels in patients with schizophrenia.When NMDA receptors are made hypofunctional by NMDA receptor antagonists (ketamine & PCP), this produces a psychotic condition in normal humans similar to schizophrenia.Amphetamine, cocaine which release dopamine also produces a psychotic condition of delusions and hallucinations similar to positive symptoms of schizophrenia.NMDA receptor antagonists also mimic cognitive, negative and affective symptoms of schizophrenia. It can also explain the dopamine hypothesis as a downstream consequence of hypofunctioning NMDA receptors.

Faulty NMDA synapses on GABA interneurons in prefrontal cortexThis is a part of neurodevelopmental hypothesis which results in NMDA hypofunction at specific sites.This is due to neurodevelopmental abnormalities in formation of glutamate receptors in specific sites.At certain GABA interneurons in cerebral cortex genetic programming goes wrong calcium binding protein called parvalbumin cantaining GABA interneurons in prefrontal cortex these are faulty post synaptic partners to incoming glutamate input defective NMDA receptor containing synaptic connections.This dysconnectivity may be genetically programmed from a variety of faulty genes.

Contd..As a conseqence of this dysconnectivity they also have deficits in the enzyme (Glutamic acid decorboxylase-GAD67) that makes their own GABA, causing a compensatory increase in post synaptic 2 sub unit of GABAA receptors.Parvalbumin containing GABA interneurons fail to function properly do not adequately inhibit key glutamatergic pyramidal neurons in prefrontal cortex glutamate neurons become hyperactive

NMDA hypofunction hypothesis vs Dopamine hypothesis Positive symptomsDysconnectivity in upstream glutamate neurons Hyperactivity (disinhibition) of the downstream mesolimbic dopamine pathwaysPositive symptoms

Contd..A four neuron circuit,The dysconnected and defective hippocampal parvalbumin containing GABA interneuron The hippocampal glutamate neuron projecting to nucleus accumbens, then that neuron pojecting into 2 spiny neuronsFirst to globus pallidusSecond from globus pallidus to VTA Hyperactivity (disinhibition) of the downstream mesolimbic dopamine pathwaysPositive symptoms

30-Oct-1645Role of Glutamate in the Mesolimbic System

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NMDA hypofunction hypothesis vs Dopamine hypothesis Negative symptomsCortico brainstem glutamate neuronsInnervates GABA interneuronsInnervates mesocortical dopamine neuronsNegative and Cognitive symptoms

30-Oct-1647Role of Glutamate in the Mesocortical System

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48Neurodevelopmental Theory of Schizophrenia Schizophrenia could be the result of an early brain insult, whichaffects brain development leading to abnormalities in the maturebrain (Murray et al, 1992).The theory has been postulated since Kraepelin in the early 20th century. The cause of the brain lesion could beeither:Abnormal genes, which impair brain development.Some foetal or neonatal adversity.30-Oct-16

49Contd..During adolescence, brain changes normally include: Decrease in delta sleepDecrease in membrane synthesis Decreased volume of cortical grey matterDecreased prefrontal metabolismIn schizophrenia, there are more pronounced decrements in the same parameters.This supports the possibility of an exaggeration of the normal process of synaptic pruning that occurs in schizophrenia during adolescence . (Fienberg,1983)30-Oct-16

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50Neurodevelopmental Theory of Schizophrenia: ModelsThe early neurodevelopmental model: Fixed lesion from early life interacts with normal neurodevelopment occurring later, lying dormant until the brain matures sufficiently to call into operation the damaged systems (Murray & Lewis, 1987).The late neurodevelopmental model: schizophrenia may result from an abnormality in peri-adolescent synaptic pruning (Feinberg, 1983).30-Oct-16

51Contd..Keshavan and Hogarty (1999): Maldevelopment in schizophrenia takes place during 2 critical time points (early brain development and adolescence),Early developmental insults may lead to dysfunction of specific neural networks that would account for premorbid signs.At adolescence, excessive synaptic pruning and loss of plasticity may account for the emergence of symptoms.30-Oct-16

52Glutmate and Neurodevelopmental Theory of SchizophreniaNMDA receptors are a critical component of developmental processes during adolescence. This includes: development of neural pathwaysNeural migration Neural survivalNeural plasticity Neural pruning of cortical connections30-Oct-16

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53Contd..Stahl (2009): suggests that Glutamate excitotoxicity first facilitates the neurodevelopmental disorder in adolescence.Later, this results in a chronic state of Glutamate hypofunctioning which maintains the schizophrenic pathology in later stages. 30-Oct-16

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Glutamate and Neurodegenerative Model of SchizophreniaKraeplin and others believed that Schizophrenia is caused by a form of progressive neuronal degeneration characterized by earlier onset than that seen with previously described entities, such as Huntington's disease or Alzheimer's disease > Dementia praecox However, the neurodegenerative theory was opposed by the neurodevelopmental theory: Most of the brain pathology in schizophrenia starts in early adulthoodNo evidence of necrosis There is no neurochemical explanation for neurodegenerationTheory was later supported by the discoveries about apoptosis and glutamate system. 5430-Oct-16

55Contd..The neurostructural changes in schizophrenia have led to the hypothesis that apoptosis may contribute to the pathophysiology of schizophrenia. Such changes include:Reduced neuropils (region between neuronal cell bodies in the gray matter) and reductions of neurons. Neuroimaging data > progressive loss of cortical grey matter in schizophrenia. Postmortem studies: markers of apoptosis and levels of apoptotic proteins indicate > increased apoptotic vulnerability. 30-Oct-16

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56Contd..Again, glutamate is the main factor involved in apoptosis (Stahl, 2009):High concentrations of glutamate accumulate in the brain are thought to be involved in the aetiology of a number of neurodegenerative disorders including Alzheimer's disease. A number of in vitro studies > at high concentrations, glutamate is a potent neurotoxin capable of destroying neurons by apoptosis.30-Oct-16

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57Conclusion of Glutmate role in SchizophreniaBoth glutamate hypo activity as well as hyper activity contribute to the pathology of schizophrenia (Stahl, 2009). Gupta & Kulhara (2010) suggested that:Schizophrenia cannot be explained by a single process of development or degeneration. Research evidence exists for degeneration as well as developmental disorders. The glutamatergic hypothesis bridges the gap between development and neurodegeneration in schizophrenia > two hit hypothesis (Keshavan, 1999). 30-Oct-16

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30-Oct-1658Glutamate Linked Treatments of Schizophrenia Three classes of medications: NMDA partial antagonists (early stage schizophrenia)NMDA partial agonists (later stage schizophrenia) Glycine co-agonistsGlycine transporter inhibitorsNMDA modulatorsmGlu autoreceptors co-agonistsMinocycline

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30-Oct-1659 NMDA Partial AntagonistsTo treat excitotoxicity in early stage.They include:PCP and Ketamine: highly schizophrenogenic NMDA partial antagonists e.g. Memantine (already used in Alzheimer)Drugs which block presynaptic release of glutamate e.g. Lamotrigine, Gabapentin and Pregabalin.Anti-free radicals drugs e.g. vitamin E and experimental agents called lazaroids.

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30-Oct-1660 NMDA Partial Agonists Glycine co-agonists To treat glutamate hypofunctioning in later stages of schizophrenia.They act as agonists at the allosteric glycine receptor site of the NMDA complex (glycine co-agonists) as a way to avoid causing glutamate neurotoxicity.

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30-Oct-1661Glycine agonists to activate glycine site on the NMDA receptors as indirect way to potentiate the glutamte effect. e.g. glycine, d-serine, d-alanine and d-cycloserine. Provisional studies are promising. Research is still going on, using stronger agonists.

Glycine transporters inhibitors (GlyT1 inhibitirs): e.g. sarcosine promising remedy for negative symptoms of schizophreniat al, 2009) (Stahl, 2009).

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30-Oct-1662NMDA Modulators mGlu autoreceptors co-agonistsmGlu receptors are the ideal target for medication (co-agonists) e.g. methionine amide.Mechanisms of action are not quite clear.mGluR2/3 are mainly autoreceptors that prevent glutamate release. The final result is enhancing glutamate activity (?????).They reverse the effects of PCP and Ketamine in animals (Stahl, 2009)A RCT after four weeks of treatment, an agonist for the mGluR2/3 (LY404039 ) has similar efficacy as Olanzapine in ameliorating positive and negative symptoms of schizophrenia .

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30-Oct-1663NMDA Modulators (Minocycline) Second-generation tetracycline with a broad spectrum of antimicrobial activities and anti-inflammatory properties.Latest studies suggest that it is related to the glutamatergic system: minocycline reversed several NMDA antagonist effects in animal studies and showed good results in the treatment of patients with schizophrenia.Has neuroprotective effects in several animal and human models of neurological diseases, including Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and ischemia.

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Anxiety and DepressionGlutamatergic dysfunction been implicated in depression.Glutamate dysfunction is involved in the limbic and prefrontal circuits of depressed individuals.NMDA receptor antagonists have anti depressant effects.Ketamine can produce a rapid, substantial, and persistent reduction in symptoms in MDD.

Addiction disordersThe mesocorticolimbic dopamine system is central to our understanding of addiction.Glutamate modulates dopamine activity and is involved in the development and maintenance of drug dependence, particularly in reinstatement to drug-seeking behavior.

AlcoholismEthanol at intoxication levels has dual action of enhancing GABAergic receptor function and attenuating NMDA receptor function.Persistent abuse and dependecy results in downregulation of GABAA and upregulation of NMDA receptors.So acute discontinuation results in hyperexcitable state as delirium temens.Furthermore, supersensitive NMDA receptors in thiamine deficiency contribute to excitotoxic neuron degeneration of Wernicke-Korsakoff Syndrome.Acamprostate is a derivative of homotaurine first developed thought that acts via GABAA receptors as taurine has resembelence to GABA, but studies shown that it altetrs NMDA receptors.

Fetal alcohol syndromeMicroencephaly associated with fetal alcohol exposure results from inhibition of NMDA receptor function wide spread neuronal apoptosis in immature cortex.Hence NMDA receptor activation is essential for neuronal survival and differentiation.

Thank you

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