None of the NT discussed so far mediate fast excitatory transmission in the CNS…glutamate accounts for 85% of synapses in the brain. All cells produce glutamate and about 80% of neurons use glutamate as a NT. All cells in the brain can respond to glutamate (excitatory response). Repolarization of the membrane is the main reason the brain utilizes so much glucose and oxygen.

Projection neurons of cortex and hippocampus Primary sensory pathways Output of thalamus and amygdala Cerebellar granule neurons

Glutamate is an amino acid-not accepted as a bona fide NT until 1980’s Glutamate is an amino acid-not accepted as a bona fide NT until 1980’s. Very hard to demonstrate--need several different types of evidence to implicate glutamate or other amino acid as a NT. Measure levels, look at Ca2+ dependent release, uptake, and now, expression of the vesicle transporter. Use of radiolabeled ligands (aspartate and glutamate) to demonstrate uptake and K+ evoked release.

Correlation of glutamate level, uptake, and receptor binding Main points: glutamate levels are pretty constant, glutamate and aspartate uptake are very similar in all regions though receptor binding varies

Roles of glutamate Learning is the main one and most studied Plasticity Long term potentiation or depression of synaptic efficacy (strength) Memory formation, language, “thought” Purposeful movement, quality of stimuli, process and understand sensations

Long term potentiation

Produced in the brain, not brought in from circulation Produced in the brain, not brought in from circulation. Glucose is transported from circulation and is the major precursor of glutamate. Immediate precursor is a-ketoglutarate. Transaminase reaction: transfer of an amino group from one amino acid to an a-ketoacid. Branched chain amino acids like leucine, isoleucine, valine are important amino donors. GDH-glutamate dehydrogenase removes the amino group from glutamate, converting it back into a-ketoglutarate where it can reenter the TCA cycle. Another TCA intermediate, oxaloacetate can accept an amino group and be converted into aspartate.

Recycling of glutamate-key to not depleting glucose and maintaining the amino group in the brain. Readily taken up into astrocytes, where it reacts with ammonia to produce glutamine via glutamine synthase. GS only expressed in glia. Glutamine is taken up by neurons and converted to glutamate by glutaminase. 40-60% of glucose turnover is through this pathway (almost half of glutamate released by neurons is recycled through glia!) This process helps to avoid a drain on TCA cycle intermediates which are necessary for energy production.

Evidence for importance of glutamine cycle Blocking GS leads to rapid impairment of glutamate transmission Incubate with 3H-glutamine, observe 3H-glutamate released in a Ca2+ dependent manner Similar experiments have been carried out with 13C-acetate

Vesicular glutamate--a different compartment, a different function distinguishing glutamatergic neurons from all cells (which make and use glutamic acid for metabolism and protein synthesis). Expression of a vesicular glutamate transporter is a defining feature of neurons that use transmitter glutamate. Purpose is to concentrate glutamate in the vesicles (about 50 fold). VGLUTs are proton driven and ATP dependent like the monoamine and serotonin transporters. Glutamate is negatively charged..estimates that a proton is present in the vesicle about 10% of the time means that this positive charge must be enough to attract molecules of glutamate. Cl- in the cytoplasm helps drive the charge gradient too.

3 VGLUTs have been cloned 3 VGLUTs have been cloned. VGLUT3 is expressed in neurons using monoamines, GABA, and Ach as NT. Co-release? Unique expression patterns of VGLUT1 and 2 raise questions about whether all transmitter glutamate is the same…or all glutamate containing vesicles? VGLUT 1 or 2 gene knockout is lethal. VGLUT1 may be important for neuronal plasticity--more glutamate per vesicle? VGLUT1 vs 2 pattern not correlated to firing rate.

Regulation of glutamate transporter levels by activity Bic=bicuculine GABA antagonist (excites cells) TTX=tetrodotoxin-blocks vesicle fusion and therefore NT release Opposite actions may have homeostatic value--normalize loading and quantal capacity of vesicles?

VGLUTs are found in astrocytes: some evidence that glutamate can be released in a Ca2+ and SNARE dependent manner from astrocytes. Age related decline in all 3 VGLUTs in astrocytes

Stimulation evoked glutamate release from astrocytes Rose Bengal=VGLUT inhibitor, most potent known. Ki in low nM range. BAPTA=Ca2+ chelator

How to regulate glutamate release How to regulate glutamate release? Use for so many things, hard to say exactly. May be level of cytoplasmic glutamate from reuptake, activity of EAAT2, amount/activity of VGLUTs. Inactivation of glutamate transmission: big picture. EAAT2-excitatory amino acid transporter 2 Prevent receptor desensitization and prevent excitotoxicity

Co transport with Na+, one molucule of glutamate per transport cycle.

Glutamate as a co-transmitter with DA Stimulation of DA neurons in culture leads to EPSPs that are blocked with glutamatergic antagonists. DA neurons express VGLUT2. VGLUT positive terminals of TH+ neurons are synaptic, while other non-VGLUT positive terminals are not synaptic. Raphe neurons may use VGLUT3, based on expression.