Regional distribution of sodium-dependent excitatory amino acid transporters in rat spinal cord

Abstract

Background/objective: The excitatory amino acid transporters (EAATs), or sodium-dependent glutamate transporters, provide the primary mechanism for glutamate removal from the synaptic cleft. EAAT distribution has been determined in the rat brain, but it is only partially characterized in the spinal cord.

Methods: The regional anatomic distribution of EAATs in spinal cord was assessed by radioligand autoradiography throughout cervical, thoracic, and lumbar cord levels in female Sprague-Dawley rats. EAAT subtype regional distribution was evaluated by inclusion of pharmacologic transport inhibitors in the autoradiography assays and by immunohistochemistry using subtype-specific polyclonal antibodies to rat GLT1 (EAAT2), GLAST (EAAT1), and EAAC1 (EAAT3) rat transporter subtypes.

Results: [3H]-D-Aspartate binding was distributed throughout gray matter at the 3 spinal cord levels, with negligible binding in white matter. Inclusion of pharmacologic transport inhibitors indicates that the EAAT2/ GLT1 subtype represents 21% to 40% of binding. Both EAAT1/GLAST and EAAT3/EAAC1 contributed the remainder of binding. Immunoreactivity to subtype-specific antibodies varied, depending on cord level, and was present in both gray and white matter. All 3 subtypes displayed prominent immunoreactivity in the dorsal horn. EAAT3/EAAC1 and to a lesser extent EAAT1/GLAST immunoreactivity also occurred in a punctate pattern in the ventral horn.

Conclusions: The results indicate heterogeneity of EAAT distribution among spinal cord levels and regions. The presence of these transporters throughout rat spinal cord suggests the importance of their contributions to spinal cord function.

Figure 1. Representative autoradiograms of ³H-D-aspartate binding in 8-μm tissue sections from naïve Sprague-Dawley rats from (left to right) cervical, thoracic, and lumbar cord levels. Circumscribed areas on the left half of the gray matter indicate the dorsal (D) and ventral (V) regions analyzed; those on the right indicate “full” (F) gray matter measures. Note the slightly higher density in dorsal than ventral regions in cervical and lumbar compared to thoracic spinal cord.

Figure 2. Pharmacologic differentiation of glutamate transporter subtypes in Sprague-Dawley spinal cord by inclusion of transport inhibitors in autoradiographic assays. Inhibitors were included at 10 times their Km or Ki values for uptake in other expression systems (DHK, 100 μmol/L; 2,4-PDC, 500 μmol/L; 2,3-PDC, 100 μmol/L). Binding density was quantitated bilaterally in each section and then averaged from duplicate sections at 2 cervical (C4, C6), 4 thoracic (T3, T5, T8, T10), and 2 lumbar levels (L1, L3) from each rat—from 6 rats for DHK and from 12 to 15 rats for 2,4-PDC and 2,3-PDC. Inhibitor binding is represented as the percent of total binding for each region analyzed. * = P, 0.05; ** = P < 0.01 compared to control.

Figure 3. Spinal cord glutamate transporter subtype regional distribution in Sprague-Dawley rats as determined by immunohistochemical detection of EAAT2/GLT1 (A, B, C), EAAT1/GLAST (D, E, F), and EAAT3/EAAC1 (G, H, I) in cervical (A, D, G), thoracic (B, E, H), and lumbar (C, F, I) cord. Duplicate, 10-μm spinal cord cryostat cross sections (post-fixed with paraformaldehyde) were incubated with primary antibodies to GLT1 (1:3,000), GLAST (1:5,000), or EAAC1 (1:8,000). Immunoreactivity was greatest in gray compared to white matter. All 3 transporter subtypes were expressed heavily in the dorsal horn, particularly in cervical and thoracic sections. Dorsal horn binding intensity was greatest in with EAAT2/GLT1 and EAAT1/GLAST in laminae II (A–F). Dorsal horn EAAT3/EAAC1 immunoreactivity, which was less intense than that of EAAT2/GLT1 and EAAT1/GLAST, included laminae I at all 3 cord levels (G,H,I) and laminae III at the lumbar level (I). EAAT2/GLT immunoreactivity was moderately intense in other cord laminae. EAAT3/EAAC1 immunoreactivity was also characterized by a punctate pattern in the ventral horn, which was most pronounced at the cervical (G) and lumbar (I) cord levels. EAAT1/GLAST immunoreactivity produced a less intense punctate pattern in the ventral horn (D, E, F). Antibodies to all 3 rat glutamate transporter subtypes produced a radial pattern of immunoreactivity in white matter. Images were visualized at 4×. The arrows within circumscribed regions highlight the punctate immunoreactivity with EAAT1/GLAST (D, E, F) and EAAT3/EAAC1 (G, H, I).