Low-Flow Push-Pull Perfusion for Measuring Neurotransmitters with High Spatial and Temporal Resolution within the Living Brain.

Neuroscience Methods;Bioanalytical Chemistry;Microfluidics;Glutamate;Dopamine;Neurosciences;Chemistry;Science (General);Health Sciences;Science;Chemistry

Slaney, Thomas R.Kopelman, Raoul ;

University of Michigan

关键词: Neuroscience Methods;    Bioanalytical Chemistry;    Microfluidics;    Glutamate;    Dopamine;    Neurosciences;    Chemistry;    Science (General);    Health Sciences;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/97958/tslaney_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Low-flow push-pull perfusion is a technique for measuring neurotransmitters within the brain with ~200 μm resolution. Activity of neurotransmitters can vary on this size scale; therefore, low-flow push-pull may offer new insights into physiology. Flow rates used by this technique (50 nL/min) may present challenges for sample handling and assay sensitivity due to nL sample fractions. In this work, the temporal resolution of low-flow push-pull was advanced to 7 s in vivo, several different neurochemical assays were implemented, and gradients of neurotransmitters were mapped across sub-mm distances.To address collection and manipulation of 7 s fractions collected in vivo, push-pull samples were stored as 6 nL plugs in an oil carrier phase. A tee was developed to address each fraction discretely for reagent addition. L-glutamate was measured within the striatum of anesthetized rats by using a fluorogenic enzyme assay. Microinjection of a potassium solution at the probe tip evoked L-glutamate concentration transients that had maxima of 4.5 ± 1.1 μM and rise times of 22 ± 2 s.Nanospray ionization mass spectrometry was used to simultaneously measure three neurochemicals in plug samples. After microinjection of neostigmine at the push-pull probe tip, rapid extracellular concentration increases of neostigmine (14 ± 3 s), acetylcholine (35 ± 4 s) and a gradual decrease in choline (60 ± 13 s) were observed. This experiment highlights the ability of low-flow push-pull perfusion to observe drug-neurotransmitter dynamics in vivo. A GABA enzyme assay and capillary electrophoresis were demonstrated for analysis of push-pull perfusion plugs.A miniaturized push-pull probe was adapted for awake, freely moving animals and used to measure 13 neurotransmitters and metabolites.Concentration gradients were observed between proximate brain regions.For example, dopamine in the ventral tegmental area was 4.8 ± 1.5 nM, but in the red nucleus (200 µm apart) was 0.5 ± 0.2 nM. This collection of work illustrates that low-flow push-pull perfusion is a versatile tool for monitoring many different neurotransmitters within the brain with 200 μm spatial and 7 s or faster temporal resolution. Future research directions may include ms temporal resolution in vivo measurements and microfabricated probes.