Glutamate transporters around the tripartite synapse

Glutamate is the major excitatory neurotransmitter in the mammalian nervous system. It is inactivated by cellular uptake catalyzed by a family of glutamate transporter proteins (GLT1, GLAST, EAAC1, EAAT4 and EAAT5). The main aim of the present thesis was to determine the contributions of the individual glutamate transporter subtypes to the total glutamate uptake around hippocampal synapses focusing on the EAAC1 subtype and on the mysterious transporter responsible for nerve terminal uptake of glutamate. The first step on this endeavor was to make antibodies to EAAC1. As outlined in Paper I, it turned out to be more difficult to make good antibodies to EAAC1 than to the other glutamate transporters. Specificity testing using tissue from EAAC1 knockout mice as negative controls revealed highly specific interactions with unrelated proteins. Paper II summarizes of the lessons learnt about immunocytochemical specificity testing, and Paper III illustrates how the antigen pre-adsorption test can be misleading. After having overcome methodological problems, we were in position to address the original question. In Paper IV a new procedure for immunoisolation of EAAC1 was developed, and known amounts of pure EAAC1 protein was used as standard to quantify EAAC1 concentrations in brain tissue extracts. EAAC1 was found to be present at 13 μg per gram hippocampal protein. This is 100 times less than GLT1 and argues against a significant contribution of EAAC1 to rapid transmitter activation. EAAC1is selectively expressed in neuronal somata and dendrites throughout the brain, and thereby in a total surface area similar to that of astrocytes. In Paper V we show that nerve terminal glutamate uptake fully depends on GLT1, and that about 10% of hippocampal GLT1 protein is expressed terminals. This also explains why high levels of GLT1 mRNA is present in CA3 pyramidal cells. In Paper VI we describe antibodies to GLT1 splice variants and show that GLT1a represents about 90 % of total hippocampal GLT1, while GLT1b and GLT1c represented 6 and 1 %, respectively. We also provide the first data on the distribution of the GLT1b and show that this variant does not contribute to nerve terminal uptake.