Localization of excitatory amino acid transporters EAAT1 and EAAT2 in human postmortem cortex: A light and electron microscopic study

Abstract

The process of glutamate release, activity, and reuptake involves the astrocyte, the presynaptic and postsynaptic neuron. Glutamate is released into the synapse and may occupy and activate receptors on both neurons and astrocytes. Glutamate is rapidly removed from the synapse by a family of plasma membrane excitatory amino acid transporters (EAATs), also localized to neurons and astrocytes. The purpose of the present study was to examine EAAT labeling in postmortem human cortex at the light and electron microscopic levels. Postmortem prefrontal cortex was processed for EAAT1 and EAAT2 immunohistochemistry. At the light microscopic level, EAAT1 and EAAT2 labeling was found in both grey and white matter. Most cellular labeling was in small cells which were morphologically similar to glia. In addition, EAAT1 labeled neurons were scattered throughout, some of which were pyramidal in shape. At the electron microscopic level, EAAT1 and EAAT2 labeling was found in astrocytic soma and processes surrounding capillaries. EAAT labeling was also found in small astocytic processes adjacent to axon terminals forming asymmetric (glutamatergic) synapses. While EAAT2 labeling was most prevalent in astrocytic processes, EAAT1 labeling was also present in neuronal processes including the soma, axons, and dendritic spines. Expression of EAAT1 protein on neurons may be due to the hypoxia associated with the postmortem interval, and requires further confirmation. The localization of EAATs on the astrocytic plasma membrane and adjacent to excitatory synapses is consistent with the function of facilitating glutamate reuptake and limiting glutamate spillover. Establishment that EAAT1 and EAAT2 can be measured at the EM level in human postmortem tissues will permit testing of hypotheses related to these molecules in diseases lacking analogous animal models.

GLAST But Not Least—Distribution, Function, Genetics and Epigenetics of l-Glutamate Transport in Brain—Focus on GLAST/EAAT1

“…Furthermore, they detected EAAT1 protein in neurons, where it was present in the soma, all parts of the axon, dendritic spines and in the postsynaptic density (PSD). This is at variance with most of the other studies which have located EAAT1 in astrocytes; the presence of an apparent EAAT1 antigen in neurons might have been caused by changes taking place in the tissue during the post-mortem delay [17,18] but there are other ways to explain the observed discrepancies, including the occurrence of splice variants. EAAT1 is now known to be subject to alternate splicing and to give rise to multiple variant proteins.…”

“…There are intriguing exceptions in the literature, though. For example, Roberts et al [17] reported that EAAT1 labelling in human brain was located on the plasma membrane of astrocytes but also in the soma and nucleus. Furthermore, they detected EAAT1 protein in neurons, where it was present in the soma, all parts of the axon, dendritic spines and in the postsynaptic density (PSD).…”

“…Thus it remains a possibility that an as yet unidentified EAAT2/GLT1 variant with structural characteristics capable of assuming suitable conformation and displaying Cl -permeability may act as the hypothetical putative ''inhibitory'' glutamate receptor. As to the other EAATs, it is unlikely that GLAST, EAAT3 or EAAT4 can be involved in such process as there is little evidence for presynaptic GLAST (see [17] above, though) but it cannot be ruled out that the highly Cl --permeable EAAT5 might contribute to the negative feedback control at glutamatergic synapses in some brain regions where it is present in significant amounts [38,39,42]. EAAT3 (EAAC1) is located in neurons but, overall, it is expressed at levels about two orders of magnitude lower then EAAT2/GLT1 [8] and is frequently expressed in postsynaptic locations rather than presynaptic elements.…”

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex

“…According to published electron microscopic studies, SLC1A2 is mainly present in astrocytic soma and processes surrounding capillaries and axon terminals mainly around glutamatergic synapses, consistent with its role in facilitating glutamate reuptake and limiting glutamate spillover (Roberts et al, 2014). SLC6A1…”

Glutamate Efflux at the Blood–Brain Barrier: Cellular Mechanisms and Potential Clinical Relevance

“…A study on rat capillaries revealed little or no EAAT staining on the abluminal surface of brain capillaries (7). However, a recent electron microscopy study by Roberts et al showed a clear EAAT-1 expression in brain capillary endothelial cells from human postmortem cortex samples (17).…”