The acidic amino acids are the predominant excitatory neurotransmitters in the mammalian central nervous system (CNS). Most evidence indicates that Na+-dependent high affinity transporters are responsible for the clearance and regulation of extracellular excitatory amino acids (EAA). Pharmacological, biochemical, and molecular biological studies indicate that there are several subtypes of Na+-dependent glutamate (Glu) transporters that are expressed in different brain regions and on different cell types, including the astroglial transporters GLT-1 and GLAST, and the neuronal transporters EAAC1 and EAAT4. This project represents a collaborative effort between two laboratories who have defined the biochemical, pharmacological and pathophysiological properties of Glu transport. The goal of the investigators is to increase understanding of the predominant mechanism that normally protects the brain from excitotoxicity. This information should improve our understanding of the failure of these systems during acute and chronic insults to the CNS, including stroke and head trauma. The investigators will use in vitro and in vivo preparations to test the functional significance of our observations. The investigators have previously established the pharmacology of transporter activity in vitro and in vivo preparations. The investigators hypothesize that the cloned transporters recapitulate the properties observed in these preparations. This hypothesis will be tested by expressing the individual transporters in xenopus oocytes. In addition to this correlative strategy, the investigators propose to use antisense oligonucleotides to """"""""knock-out"""""""" subtypes of the transporters to examine the relative contributions of each transporter to activity observed in astrocyte-enriched cultures and in synaptosomes prepared from different brain regions. The investigators hypothesize different transporters contribute to the regulation of extracellular Glu in the pre- and early post-natal period. The investigators propose to study the level of expression and ultrastructural localization of the transporter subtypes during development. Based on the preliminary data, the investigators hypothesize that neurons regulate expression of glial Glu transporters. In vivo and in vitro systems will be used to examine this regulation. The investigators propose that cAMP through activation of protein kinase A is one of the factors that controls expression of the GLT-1 subtype of glial transporter. This will be tested in astrocyte-enriched cultures. The effects of selective antisense """"""""knock-out"""""""" of subtypes of transporters on excitotoxicity and the accumulation of extracellular Glu will be examined in vitro. The investigators also propose to study the effects of excitotoxic insults on the expression of subtypes of transporters.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurological Sciences Subcommittee 1 (NLS)
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Jacobs, Tom P
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Children's Hospital of Philadelphia
United States
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