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

Abstract

Abstract:Glutamate is established as the most important excitatory transmitter in the brain. The transmitter status of aspartate is debated. There is evidence that aspartate is released from nerve terminals by exocytosis. However, release through excitatory amino acid transporters (EAATs) could be an alternative mechanism. We further investigated this by use of light and quantitative electron microscopic immunocytochemistry. The nerve terminal localisation of aspartate was compared to that of glutamate using antibodies specifically recognising the amino acids. Rat hippocampal slices were incubated under normal (3 mM) and depolarising (55 mM) concentrations of K + with and without the excitatory amino acid transporter inhibitor threo-beta-benzyloxyaspartate (TBOA). If aspartate is released either through reversal of the EAATs or through exchange with synaptically released glutamate, we would expect that TBOA would block the depolarisation induced release of aspartate. We found, however, that there was a substantial depletion of aspartate, as well as of glutamate, from hippocampal nerve terminals during K + induced depolarisation in the presence of TBOA. The possibility that aspartate is released through exocytosis from synaptic vesicles was further investigated by the use of a D-aspartate uptake assay, including exposure of the slices to exogenous D-aspartate and the use of D-aspartate immunogold cytochemistry to localise D-aspartate in the fixed tissue. We found that D-aspartate taken up into the terminals was concentrated in synaptic vesicles as opposed to in the cytoplasmic matrix. This is in line with the presence in synaptic vesicles of the recently identified vesicular transporter for aspartate.Keywords: Synaptic vesicles, immunocytochemistry, amino acids, electron microscopy, transport.The release of glutamate at synapses in the brain is well studied. Glutamate is released upon depolarisation of the presynaptic nerve terminal, leading to opening of voltage gated Ca 2+ channels and Ca 2+-dependent fusion of glutamate filled synaptic vesicles with the plasma membrane. The mechanism of release of L-aspartate at central synapses remains less defined. L-aspartate has been shown to be released in a Ca 2+ and clostridium toxin sensitive manner from various in vitro brain preparations (e.g. [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]. The previous aspartate release data do not clearly distinguish between Laspartate release via exocytosis and via these EAATs, which could release as...

Vesicular uptake and exocytosis of L‐aspartate is independent of sialin

“…As this release can be blocked by inhibiting the EAATs (36–38, 40, 42–44), we incubated the slices in the presence of the nontransportable EAAT blocker TFB‐TBOA (45) to determine whether EAATs contribute to the release of l ‐aspartate from nerve terminals during depolarization. As shown previously (23), addition of TBOA per se at high K + did not significantly reduce the depletion of gold particles signaling l ‐aspartate from excitatory nerve terminals ( Fig. 5 A–D ), suggesting that l ‐aspartate is not released via EAATs during membrane depolarization.…”

Aspartate Release and Signalling in the Hippocampus

“…This result, in concert with the distinct pharmacologies of aspartate and glutamate release processes, excluded the possibility that aspartate was released by heteroexchange with released glutamate. In support of this conclusion, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (TBOA), another competitive but non-transportable inhibitor of excitatory amino acid transport, also did not reduce aspartate release [23].…”

“…These results confirm that aspartate is released by exocytosis in rat hippocampus (see also Refs. [21,23]). They are also consistent with the hypothesis that the inability of relatively low concentrations of extracellularly-applied Clostridial toxins to inhibit aspartate release reflected limited access of the enzymatically-active, cytoplasmicallyreleased light chain to the aspartate release sites.…”

Postsynaptic response to stimulation of the Schaffer collaterals with properties similar to those of synaptosomal aspartate release

“…It is accumulated (Fleck et al, 2001b; Miyaji et al, 2008) and stored (Gundersen et al, 1998, 2001, 2004) in synaptic vesicles, released in a Ca 2+ -dependent manner by exocytosis (Wang and Nadler, 2007; Holten et al, 2008; Cavallero et al, 2009), and activates postsynaptic NMDA receptors selectively (Patneau and Mayer, 1990; Curras et al, 1992). There is no evidence to suggest that aspartate serves as the principal transmitter of any pathway in the brain.…”

“…Sialin, a lysosomal H + /sialic acid cotransporter, accumulates aspartate and glutamate in synaptic vesicles by a proton- and voltage-dependent mechanism independent of sialic acid transport (Miyaji et al, 2008). Accordingly, synaptic vesicles in Schaffer collateral terminals exhibit intense immunoreactivity for both aspartate and glutamate (Gundersen et al, 1998, 2001; Holten et al, 2008). These terminals also release aspartate, along with glutamate, in a Ca 2+ -dependent manner (Nadler et al, 1976; Burke and Nadler, 1988, 1989; Zhou et al, 1995; Gundersen et al, 1998).…”