Glutamate and GABA

Glutamate

Glutamate Synthesis, Release, and Inactivation

• Glutamate and aspartate are amino acid neurotransmitters that have potent excitatory effects on neurons throughout the brain and spinal cord. Although glutamate is contained within all cells because of its multiple biochemical functions, glutamatergic neurons are thought to possess higher glutamate concentrations than other cells and to segregate their neurotransmitter pool of this amino acid.
• Many of the glutamate molecules that are released synaptically are synthesized from glutamine in a chemical reaction catalyzed by the enzyme glutaminase.
• Glutamate is packaged into vesicles by the vesicular transporters VGLUT1, VGLUT2, and VGLUT3. In some neurons, these transporters are expressed with markers for other neurotransmitters such as 5-HT, DA, ACh, and GABA, indicating that glutamate is a co-transmitter in these cells. Some evidence suggests that the DA–glutamate coexpressing neurons of the ventral tegmental area release the two transmitters from separate vesicles at different kinds of axon terminals.
• After they are released, glutamate molecules are removed from the extracellular fluid by five different excitatory amino acid transporters, designated EAAT1 to EAAT5. Uptake of glutamate into astrocytes by EAAT2 is especially important for glutamate clearance, as can be seen by the fact that mutant mice lacking this transporter exhibit seizures, neuronal cell death, and a shortened life span. The major neuronal glutamate transporter is EAAT3, which is thought to be located postsynaptically rather than presynaptically.
• Glutamate taken up by astrocytes is converted to glutamine by the enzyme glutamine synthetase.
• This glutamine subsequently can be transported from the astrocytes to the glutamatergic neurons, where it is transformed back into glutamate by the enzyme glutaminase and then reutilized. This constitutes an important metabolic interplay between glutamatergic nerve cells and their neighboring glial cells.

Organization and Function of the Glutamatergic System

• Glutamate is the workhorse for fast excitatory signaling in the nervous system. There are numerous glutamatergic pathways in the brain, including the projections of the pyramidal neurons of the cerebral cortex, the parallel fibers of the cerebellar cortex, and several excitatory pathways within the hippocampus.
• AMPA, kainate, and NMDA receptors constitute the three subtypes of ionotropic glutamate receptors. Each is named for an agonist that is relatively selective for that subtype. All of these receptors permit Na⁺ ions to cross the cell membrane, thereby producing membrane depolarization and an excitatory postsynaptic response. NMDA receptors also conduct Ca²⁺ ions and can trigger Ca²⁺-dependent second-messenger actions within the postsynaptic cell.
• Most NMDA receptors are composed of two GluN1 subunits and two GluN2 subunits.
• AMPA and kainate receptors possess different protein subunits that give them somewhat different electrophysiological and pharmacological properties. Behavioral functions of AMPA receptors have been revealed through the use of the antagonist NBQX. Administration of high doses of this compound to rodents leads to sedation, ataxia, deficient rotarod performance, and protection against seizures, indicating involvement of this receptor subtype in locomotor activity, coordination, and brain excitability.
• NMDA receptors are distinct from the AMPA and kainate receptor subtypes in several ways, in addition to the difference in ionic conductances. First, the opening of NMDA receptor channels requires a co-agonist in addition to glutamate. This co-agonist may be either glycine or d-serine. Second, NMDA receptors possess a binding site for Mg²⁺ ions within the receptor channel. When the cell membrane is at the resting potential, this site is occupied and the channel is blocked even if the receptor has been activated by agonists. However, depolarization of the membrane reduces Mg²⁺ binding, thus allowing the channel to open. Consequently, for the NMDA receptor to function, some other synaptic input must excite the cell at the same time that glutamate and either glycine or d-serine bind to the receptor. Third, NMDA receptors also possess a channel binding site that recognizes PCP, ketamine, memantine, and MK-801. These compounds act as noncompetitive antagonists of the NMDA receptor.
• There are also eight different metabotropic receptors for glutamate, designated mGluR1 to mGluR8. Group I metabotropic glutamate receptors, consisting of mGluR1 and mGluR5, are located postsynaptically and mediate excitatory responses by stimulating the phosphoinositide second-messenger system. Group II receptors, consisting of mGluR2 and mGluR3, and group III receptors, consisting of mGluR4 and mGluR6–8, are typically located presynaptically where they reduce transmitter release by inhibiting cAMP formation.
• Compounds acting at various mGluRs are being tested for their possible usefulness in treating a variety of neuropsychiatric disorders, including schizophrenia. One potential application is in fragile X syndrome, a leading genetic cause of intellectual disability and autistic symptoms. This syndrome is caused by mutations in the fragile X mental retardation 1 gene, which codes for the fragile X mental retardation protein (FMRP). Researchers proposed a metabotropic glutamate receptor theory of fragile X syndrome, in which loss of FMRP causes exaggerated group I mGluR–related functions, leading to dendritic spine abnormalities and elevated rates of LTD. However, clinical trials with mavoglurant, an mGluR5 antagonist, failed to produce significant symptom improvement in patients with fragile X syndrome.
• NMDA and AMPA receptors are believed to play an important role in learning and memory. NMDA receptor antagonists impair the acquisition of various learning tasks. Activation of this receptor is necessary for the induction of hippocampal LTP, a mechanism of synaptic strengthening. The Ca²⁺-activated enzyme CaMKII has an obligatory role in LTP induction. LTP expression, which is independent of NMDA receptors, involves increased trafficking of AMPA receptors into the postsynaptic membrane. LTP can be divided into two phases: E-LTP, which has just been described and only persists for a few hours, and the longer-lasting L-LTP, which involves protein synthesis, release of the neuropeptide BDNF, activation of atypical PKCs, and dendritic spine growth. LTP may occur in the human brain, particularly a form of LTP induced by theta burst stimulation.
• Excessive exposure to glutamate and other excitatory amino acids can damage or even kill nerve cells through a process of depolarization-induced excitotoxicity. This process is usually mediated primarily by NMDA receptors, with some contribution from AMPA and/or kainate receptors. One type of excitotoxic cell death occurs via rapid necrosis, which involves cellular swelling and eventual lysis. Additionally, there are slower programmed forms of cell death that either have some of the features of necrosis or are mediated by apoptosis, which involves disruption of the cell nucleus and breakdown of DNA.
• In humans, excitotoxic cell death can be caused by ingestion of food contaminated with the algal toxin domoic acid. Excitotoxicity is also thought to be a major contributory factor to the brain damage that occurs in the penumbra of a focal ischemic event (e.g., stroke) and in some kinds of traumatic brain injury. This type of excitotoxic cell death is mediated by Ca²⁺ entry through extrasynaptic NMDA receptors containing GluN2B subunits. A slower form of excitotoxicity may be a contributing factor in the loss of motor neurons in ALS and the loss of hippocampal neurons in refractory temporal lobe epilepsy. Therapeutic approaches to reduce excitotoxic cell death include blockade of glutamate receptors, inhibition of glutamate release (e.g., by riluzole), and enhancement of glutamate uptake (e.g., by EAAT2 up-regulation).

GABA

GABA Synthesis, Release, and Inactivation

• GABA is the major inhibitory amino acid neurotransmitter in the brain. This transmitter is synthesized from glutamate in a single biochemical reaction catalyzed by GAD, an enzyme found only in GABAergic neurons.
• Because of the widespread inhibitory effects of GABA on neuronal excitability, treatment with drugs that inhibit GABA synthesis by blocking GAD leads to seizures.
• GABA is taken up into synaptic vesicles by the vesicular transporter VGAT (also known as VIAAT because the same vesicular transporter is used by glycine).
• After release into the synaptic cleft, GABA is removed from the cleft by three different transporters designated GAT-1, GAT-2, and GAT-3. Astrocytes express all three of these transporters and therefore must play a significant role in GABA uptake. GAT-1 is also found in GABAergic neurons, and the GAT-1 inhibitor tiagabine (Gabitril) is used clinically in the treatment of some patients with epilepsy.
• GABA is co-expressed and coreleased with several other classical neurotransmitters, including glycine, acetylcholine, dopamine, and glutamate. This is accomplished, in part, by co-expression of the vesicular transporters for multiple transmitters in the same neuron. Corelease of glutamate is of particular interest, as it involves a combination of inhibitory and excitatory neurotransmitter signaling.
• In addition to uptake, the other process that regulates GABAergic transmission is GABA metabolism. The key enzyme in GABA breakdown is GABA-T, which is present in both GABAergic neurons and astrocytes. A by-product of the reaction catalyzed by GABA-T is glutamate, which is the precursor of GABA. Hence, GABA breakdown in neurons or in glial cells may involve a recycling process that assists in the formation of new GABA molecules.
• Vigabatrin (Sabril) is an irreversible inhibitor of GABA-T and thereby elevates GABA levels in the brain. Like tiagabine, vigabatrin has been licensed for the treatment of certain types of epilepsy. However, there are reports that repeated vigabatrin use can lead to visual system abnormalities in adults and children; therefore, caution should be exercised when this compound is administered to patients.

Organization and Function of the GABAergic System

• Many brain areas, including the cerebral cortex, hippocampus, substantia nigra, cerebellum, striatum, globus pallidus, and olfactory bulbs, are rich in GABA. GABAergic neurons may function as interneurons, as in the cortex and hippocampus, or they may function as projection neurons, as in pathways originating in the striatum and in the cerebellar Purkinje cells.
• There are two general GABA receptor subtypes: ionotropic GABA_A receptors and metabotropic GABA_B receptors.
• GABA_A receptors conduct Cl^– ions into the postsynaptic cell, causing membrane hyperpolarization and an inhibitory effect on cell excitability. Each receptor is composed of five subunits, usually including two α subunits, two βs, and one γ. A small number of GABA_A receptors contain a δ subunit instead of γ.
• The most common synaptic GABA_A receptor composition is (α1)₂(β2)₂(γ2). There are also extrasynaptic receptors containing two α4 or two α6 subunits, along with two β subunits and a δ subunit. Such receptors are sensitive to low GABA concentrations and mediate a mild tonic inhibitory effect on cell firing.
• Muscimol is a GABA_A receptor agonist derived from the mushroom Amanita muscaria (fly agaric). Ingestion of this mushroom or of pure muscimol causes hallucinations (including macroscopia) and other behavioral and physiologi-cal effects similar to those associated with LSD.
• GABA_A receptor antagonists include the competitive antagonist bicuculline and the noncompetitive inhibitors pentylenetetrazol (Metrazol) and picrotoxin, all of which are seizure inducing.
• BDZs, barbiturates, ethanol, anesthetics, and neurosteroids all act as positive allosteric modulators of the GABA_A receptor, which means that they enhance the action of GABA on the receptor. At high doses, barbiturates, anesthetics, and neurosteroids can open the receptor channel in the absence of GABA. Functionally, all of these compounds exert a CNS depressant effect that is manifested behaviorally as anxiolytic, sedative–hypnotic, and anticonvulsant properties.
• BDZs bind to a specific site on the GABA_A receptor complex that is considered to be a BDZ receptor. BDZ sensitivity requires the presence of a γ subunit (usually γ2), any β subunits, and an α1, α2, α3, or α5 subunit. Specific behavioral effects of BDZs and other allosteric modulators can be attributed to receptors with a particular α subunit composition: α1 for BDZ-mediated sedation, α2 for BDZ-mediated anxiety reduction, and α5 for anesthetic-mediated amnesia.
• Inverse agonists at the BDZ receptor also require the presence of GABA, but such compounds reduce instead of enhance the effectiveness of GABA in activating the GABA_A receptor. BDZ inverse agonists produce behavioral effects opposite to those produced by BDZ agonists, namely, anxiety, arousal, and increased susceptibility to seizures.
• The metabotropic GABA_B receptor is composed of two different subunits and is located both post- and presynaptically. Postsynaptic GABA_B receptors inhibit neuronal firing by stimulating K⁺ channel opening, whereas presynaptic GABA_B receptors (acting as either autoreceptors or heteroreceptors) inhibit neurotransmitter release by inhibiting Ca²⁺ channel opening. The receptors additionally inhibit cAMP formation.
• GABA_B receptors contribute to many behavioral functions, including learning and memory, anxiety- and depression-like behaviors, and responses to drugs of abuse. This information has been obtained from studies of knockout mice lacking one of the receptor subunits and from pharmacological studies involving the selective agonist baclofen (Lioresal), which is used clinically as a muscle relaxant and an antispastic agent, or the competitive antagonists saclofen or 2-hydroxysaclofen.