Simultaneous efflux of endogenous D-ser and L-glu from single acute hippocampus slices during oxygen glucose deprivation

Abstract

D-serine and L-glutamate play crucial roles in excitotoxicity through N-methyl-D-aspartate receptor coactivation, but little is known about the temporal profile of efflux during cerebral ischemia. We utilized a newly designed brain slice microperfusion device coupled offline to capillary electrophoresis laser-induced fluorescence to monitor dynamic efflux of endogenous D-ser and L-glu in response to oxygen glucose deprivation (OGD) in single acute hippocampus slices. Efflux profiles with 2-min temporal resolution in response to 24-min OGD show that efflux of D-ser slightly precedes efflux of L-glu by one 2-min sampling interval. Thus both coagonists are available to activate NMDA receptors by the time when glu is released. The magnitude of D-ser efflux relative to baseline values is, however, less than that for L-glu. Peak efflux during OGD, expressed as pre-OGD baseline values, was as follows: D-ser 254% +/- 24%, L-glu 1,675% +/- 259%, L-asp 519% +/- 128%, and L-thr 313% +/- 33%. L-glutamine efflux was shown to decrease significantly in response to OGD. The microperfusion/CE-LIF approach shows several promising attributes for studying endogenous chemical efflux from single, acute brain slices.

Fig. 1

Custom microperfusion chamber for single acute brain slices showing polycarbonate microperfusion chamber (a), schematic diagram of major components of microchamber (b; actual chamber dimensions L × W × H ∼9 × 5 × 0.7 mm, with 0.3-mm deep microchannel support), magnification of microchamber well illustrating microchannel support designed to increase fluid flow around slice (c), and schematic of microperfusion setup from syringe pump to fraction collection (d).

Fig. 2

OGD treatment profile determined by using trace D-glu perfusion across slices. A stepwise change in D-glu levels (0.5 μM switched to 3.0 μM) occurs in ∼4.5 min, which establishes the turnover rate of the microchamber and allows for precise monitoring of the treatment profile.

Fig. 3

Comparison of typical electropherograms from basal (left) and OGD (right) perfusate samples demonstrating changes in analyte concentrations relative to internal standard (IS). Basal sample shown is from perfusate collected 10 min prior to OGD treatment, whereas OGD sample shown was collected 24 min into OGD treatment. Both basal and OGD samples were obtained from the same acute slice experiment.

Fig. 4

Efflux of D-ser occurs in parallel with efflux of L-glu during OGD in acute hippocampal slices. Percentage change in endogenous amino acid concentrations relative to average of last 10 pretreatment samples are shown: D-ser (a), L-glu (b), L-asp (c), L-thr (d), and L-gln (e). Sampling and analysis were carried out using the newly developed microperfusion/CE-LIF strategy. Temperature was maintained at 36°C throughout perfusion. In control experiments, perfusate treatment was administered by switching to standard oxygenated aCSF medium. Data points represent average ± SEM of n slices as indicated. ^★P < 0.05, OGD vs. CTL (Tukey post hoc comparison).