Defects in Mouse Cortical Glutamate Uptake Can Be Unveiled In Vivo by a Two-in-One Quantitative Microdialysis

Abstract

Extracellular glutamate levels are maintained low by efficient transporters, whose dysfunction can cause neuronal hyperexcitability, excitotoxicity, and neurological disease. While many methods estimate glutamate uptake in vitro/ex vivo, a limited number of techniques address glutamate transport in vivo. Here, we used in vivo microdialysis in a two-in-one approach combining reverse dialysis of isotopic glutamate to measure uptake ability and zero-flow (ZF) methods to quantify extracellular glutamate levels. The complementarity of both techniques is discussed on methodological and anatomical basis. We used a transgenic mouse model of human disease, expressing low levels of the EAAT-2/GLT1 glutamate transporter, to validate our approach in a relevant animal model. As expected, isotopic analysis revealed an overall decrease in glutamate uptake, while the ZF method unveiled higher extracellular glutamate levels in these mice. We propose a sensitive and expedite two-in-one microdialysis approach that is sufficiently robust to reveal significant differences in neurotransmitter uptake and extracellular levels through the analysis of a relatively low number of animals.

DM1 Transgenic Mice Exhibit Abnormal Neurotransmitter Homeostasis and Synaptic Plasticity in Association with RNA Foci and Mis-Splicing in the Hippocampus

“…The ratio between the two radio-labelled molecules increased during the wash-out period, peaking 4–6 min after the end of isotopic infusion ( Figure 1 B). Lower glutamate/mannose ratios were found in DMSXL animals, when compared with WT controls, suggestive of defective glutamate uptake ability in the hippocampus, as previously shown in the frontal cortex, another brain region very relevant for DM1 neuropsychological manifestations [ 30 ]. To confirm that the DMSXL traces were indicative of decreased glutamate uptake in the hippocampus, we administered an uptake blocker (dihydrokainate, DHK) concomitantly with the two radio-labelled molecules and measured the glutamate/mannose ratio during the perfusion and wash-out periods.…”

“…As expected, the unspecific blockade of the glial transporters by DHK decreased the wash-out glutamate/mannose ratio in WT mice to levels that were undistinguishable from those found in the DMSXL hippocampus ( Figure 1 B). We used the area under the curve (AUC) during the wash-out period as an estimate of glutamate uptake ability in the different mouse groups [ 30 ] and confirmed that glutamate uptake was significantly reduced in the DMSXL hippocampus relative to WT controls ( Figure 1 C).…”

“…Western blot quantification of GLT1 steady-state levels revealed a significant decrease in the glial glutamate transporter by 50% ( p < 0.001) ( Figure 1 A). Glutamate uptake ability was challenged by administrating exogenous glutamate [ 27 , 28 ], simultaneously to nonmetabolizable mannitol [ 29 ] as previously described [ 30 ] ( Supplementary Figure S1 ). The ratio between the two radio-labelled molecules increased during the wash-out period, peaking 4–6 min after the end of isotopic infusion ( Figure 1 B).…”

“…We then determined the net extracellular concentration of glutamate in DMSXL mice, using quantitative microdialysis in vivo, as recently described [ 30 ]. Notwithstanding the reduced glutamate uptake found in the DMSXL hippocampus, the overall extracellular concentration of glutamate remained unaltered, compared with WT controls ( Figure 2 A).…”

“…Using microdialysis, we showed that glutamate uptake was significantly reduced in the whole DMSXL hippocampus, relative to WT controls. However, this sampling technique failed to detect higher levels of extracellular glutamate in the hippocampus, unlike the frontal cortex of DMSXL mice, where we previously reported increased levels of extracellular glutamate in association with GLT1 downregulation [ 30 ]. Despite the unchanged global glutamate levels found in the DMSXL hippocampus by microdialysis, it is conceivable that the spatial resolution of this sampling technique is not sufficient to target specific subregions of the hippocampus (DG vs. CA1).…”