Sample collection and amino acids analysis of extracellular fluid of mouse brain slices with low flow push–pull perfusion

Abstract

Brain tissue slices are a common neuroscience model that allows relatively sophisticated analysis of neuronal networks in a simplified preparation. Most experimental methodology utilizes electrophysiological tools to probe these model systems. The work here demonstrates the adaptation of low-flow push-pull perfusion sampling (LFPS) to a brain slice system. LFPS is used to sample from the hippocampus of mouse brain slices. Perfusate amino acid levels are quantified following sampling with capillary electrophoresis. Glutamate was measured from the CA1 region of the hippocampus in slices taken from a cystine-glutamate transporter deletion mutant, xCT(-/-), and the background strain C57BL/6J. Sampling is performed over up to 6.5 h with standard tissue slice preparation and experimentation methods. Four amino acids were quantified to demonstrate the ability to perform LFPS and show good agreement with published literature. Perfusate glutamate levels are found to be significantly lower with xCT(-/-) slices (1.9(±0.5) μM) relative to controls (4.90(±1.1) μM). But, experiments with control slices show a significant decrease in glutamate over the 6 h sampling period that are not seen with xCT(-/-) slices. Increasing the LFPS sample collection rate during the first 90 min of sampling did not show a sampling artifact in perfusate glutamate content. Sampling immediately following slicing did not show an early increasing glutamate level that would be indicative of a significant contribution from blood or tissue damage. The data presented here show a complementarity to electrophysiological studies of tissue slices. The ability to characterize extracellular fluid chemical content with LFPS in these slices provides an alternative data stream for probing neurochemical signaling networks in brain tissue slices.

Analytical Techniques in Neuroscience: Recent Advances in Imaging, Separation, and Electrochemical Methods

“…LFPS has been used to investigate sex differences in amino acid concentrations in the mouse brain with detection limits around 190 nM. 86,87 A new form of this probe is the push-pull theta pipette which has a diameter as small as 1.2 µm. Sample was pulled through one barrel of the theta pipette while perfluorodecalin was pushed through the other.…”

“…However, the absence of a size selective membrane allows enzymes and other macromolecules to be collected as well, which may require additional sample manipulation prior to separation and analysis. LFPS has been used to investigate sex differences in amino acid concentrations in the mouse brain with detection limits around 190 nM. , A new form of this probe is the push–pull theta pipet which has a diameter as small as 1.2 μm. Sample was pulled through one barrel of the theta pipet while perfluorodecalin was pushed through the other.…”

Development of μ-Low-Flow-Push–Pull Perfusion Probes for Ex Vivo Sampling from Mouse Hippocampal Tissue Slices

“…probe was utilized for necessary mechanical strength of insertion into a hippocampal slice. 25 This o.d. is still quite large relative to individual neurons and synapses, thereby limiting spatial resolution.…”

Recent advances in amino acid analysis by capillary electromigration methods: June 2015–May 2017

“…Similar sampling problems exist for brain slices. A low‐flow push‐pull perfusion sampling (LFPS) was adapted to a brain slice system . The sample collection was performed over 6.5 h. Glu was examined under the scope.…”