Laminar and subcellular heterogeneity of GLAST and GLT‐1 immunoreactivity in the developing postnatal mouse hippocampus

Abstract

Astrocytes express two sodium-coupled transporters, glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), which are essential for the maintenance of low extracellular glutamate levels. We performed a comparative analysis of the laminar and subcellular expression profile of GLAST and GLT-1 in the developing postnatal mouse hippocampus by using immunohistochemistry and western blotting and employing high-resolution fluorescence microscopy. Astrocytes were identified by costaining with glial fibrillary acidic protein (GFAP) or S100β. In CA1, the density of GFAP-positive cells and GFAP expression rose during the first 2 weeks after birth, paralleled by a steady increase in GLAST immunoreactivity and protein content. Upregulation of GLT-1 was completed only at postnatal days (P) P20-25 and was thus delayed by about 10 days. GLAST staining was highest along the stratum pyramidale and was especially prominent in astrocytes at P3-5. GLAST immunoreactivity indicated no preferential localization to a specific cellular compartment. GLT-1 exhibited a laminar expression pattern from P10-15 on, with the highest immunoreactivity in the stratum lacunosum-moleculare. At the cellular level, GLT-1 immunoreactivity did not entirely cover astrocyte somata and exhibited clusters at processes. In neonatal and juvenile animals, discrete clusters of GLT-1 were also detected at perivascular endfeet. From these results, we conclude there is a remarkable subcellular heterogeneity of GLAST and GLT-1 expression in the developing hippocampus. The clustering of GLT-1 at astrocyte endfeet indicates that it might serve a specialized functional role at the blood-brain barrier during formation of the hippocampal network.

Action Potential Firing Induces Sodium Transients in Macroglial Cells of the Mouse Corpus Callosum

“…For both GLT-1 and GLAST several distinct bands (60,130, 180 kd and 55, 130, 150, 190 kd, respectively) were detected in all age groups most likely due to monomeric and multimeric fractions of the transporters ( Figure 7A), confirming earlier studies indicating that both exist as homomultimers [45,46]. Numerical analysis of GLT-1 expression (60, 130, 180 kDa) revealed a steady increase in relative protein content during postnatal development (N = 4 per age group) as shown before for mouse hippocampus [43]. At the neonatal stage (P5), GLT-1 expression was low (10.4%) compared to P25 (100%).…”

“…While the GLAST protein expression level significantly rose from P5 to P10 (87.6%), there was no further significant increase at P15 (74.0%) as compared to P25 (N = 3 per age group) ( Figure 7B). Again, this general expression profile was similar to that reported from early postnatal hippocampus [43].…”

“…To address the involvement of glutamate transporter subtypes GLT-1 and GLAST, we performed immunohistochemical stainings and immunoblotting, demonstrating expression of both transporters at the protein level during the first three weeks after birth in corpus callosum. Their expression levels increased between P5 and P25, which is similar to hippocampus and neocortex [43,77].…”

“…Grey matter astrocytes express two different subtypes of sodium-dependent glutamate transporters, GLT-1 and GLAST, with the former being dominant after the first postnatal week [8,9,43]. To address the specific involvement of GLAST and GLT-1 in the generation of sodium signals in macroglia of the corpus callosum, we studied the effect of different pharmacological blockers on sodium transients evoked by puff application of glutamate (1 mM/250 ms).…”

Development of a simple in vitro mature mixed glial model to investigate differential expression of cell markers on glial cells and their potential as targets in multiple sclerosis

“…GLT-1 also has several known isoforms, which have been found to be differentially distributed at the regional, cellular and subcellular level in astrocytes in the hippocampus, lateral septum, cerebral cortex and striatum, in neurons, in photoreceptors of the retina, and in the liver (Patel and Klein, 2011;Jitoku et al, 2011;Magnotti et al, 2011;Lehre et al, 1995;Schmitt et al, 2002;Schreiner et al, 2014;Rauen et al, 2004). Several studies have reported GLT-1 expression by mature oligodendrocytes (DeSilva et al, 2009;Domercq et al, 2005;Werner et al, 2001), although that has been disputed by other investigators, who have reported that GLT-1 expression is lost in mature oligodendrocytes.…”