Synaptic Vesicular Localization and Exocytosis ofl-Aspartate in Excitatory Nerve Terminals: A Quantitative Immunogold Analysis in Rat Hippocampus

Abstract

To elucidate the role of aspartate as a signal molecule in the brain, its localization and those of related amino acids were examined by light and electron microscopic quantitative immunocytochemistry using antibodies specifically recognizing the aldehyde-fixed amino acids. Rat hippocampal slices were incubated at physiological and depolarizing [K ϩ ] before glutaraldehyde fixation. At normal [K ϩ ], aspartate-like and glutamatelike immunoreactivities were colocalized in nerve terminals forming asymmetrical synapses on spines in stratum radiatum of CA1 and the inner molecular layer of fascia dentata (i.e., excitatory afferents from CA3 and hilus, respectively). During K ϩ depolarization there was a loss of aspartate and glutamate from these terminals. Simultaneously the immunoreactivities strongly increased in glial cells. These changes were Ca 2ϩ -dependent and tetanus toxin-sensitive and did not comprise taurine-like immunoreactivity. Adding glutamine at CSF concentration prevented the loss of aspartate and glutamate and revealed an enhancement of aspartate in the terminals at moderate depolarization.In hippocampi from animals perfused with glutaraldehyde during insulin-induced hypoglycemia (to combine a strong aspartate signal with good ultrastructure) aspartate was colocalized with glutamate in excitatory terminals in stratum radiatum of CA1. The synaptic vesicle-to-cytoplasmic matrix ratios of immunogold particle density were similar for aspartate and glutamate, significantly higher than those observed for glutamine or taurine. Similar results were obtained in normoglycemic animals, although the nerve terminal contents of aspartate were lower. The results indicate that aspartate can be concentrated in synaptic vesicles and subject to sustained exocytotic release from the same nerve endings that contain and release glutamate.

Vesicular Release of L- and D-Aspartate from Hippocampal Nerve Terminals: Immunogold Evidence~!2008-09-01~!2008-10-15~!2008-12-05~!

“…[1][2][3][4][5][6][7]). Strongly in favour of an exocytotic release mechanism are our previous immunocytochemical results showing that K + induced depolarisation elicited depletion of L-aspartate from nerve endings, which could be inhibited by low extracellular Ca 2+ -concentrations and tetanus toxin [8][9][10]. In addition, Daspartate has been shown to be subject to exocytotic release both from cultured neurons [11] There is ample evidence that excitatory nerve endings in the brain contain functional plasma membrane excitatory amino acid transporters (EAATs) [16][17][18][19][20].…”

“…Hippocampal slice experiments were performed as previously described [8,9,18]. In brief, in each experiment we…”

Reduced aspartate release from rat hippocampal synaptosomes loaded with Clostridial toxin light chain by electroporation: Evidence for an exocytotic mechanism

“…Aspartate immunoreactivity has been localized to synaptic vesicles of certain excitatory [10,11] and inhibitory [12] hippocampal pathways. In those pathways, aspartate immunoreactivity was associated with synaptic vesicles to the same degree as glutamate or GABA immunoreactivity.…”

“…Due to methodological differences between the two studies, however, further investigations are needed to substantiate the implied physical separation of aspartate and glutamate release sites. The colocalization of vesicular aspartate with vesicular glutamate in synaptic terminals of certain excitatory hippocampal pathways [10,11,13] and its colocalization with vesicular GABA in others [12] suggests a physiological role for aspartate different from that of other transmitter amino acids. We suggested that aspartate release at a distance from the presynaptic active zones should activate extrasynaptic NMDA receptors and that aspartate may indeed be a major endogenous agonist for these receptors [1].…”

“…Pretreatment of neocortical synaptosomes [17], whole brain synaptosomes [6] or hippocampal slices [10,11] with Clostridial toxins inhibited chemicallyevoked aspartate release, as expected for an exocytotic process, but either a high concentration of toxin was used or the possibility of release by heteroexchange with released glutamate was not excluded. In our study of rat hippocampal synaptosomes, pretreatment with Clostridial toxins reduced glutamate release markedly, but did not reduce the simultaneous release of aspartate from the same tissue samples [1].…”