Quantitative Microdialysis using Stable-isotope Labeled Neurotransmitters Improves Efficiency and Accuracy of Neurochemical Measurements.

Abstract

The brain controls many functions including memory, learning, and addiction. Being able to measure neurochemical changes associated with these processes can elucidate and provide insight into the neural mechanisms that are responsible for each function. The main technique used to sample from the central nervous system is microdialysis. Active tissue processes can affect the recovery of microdialysis probes which can be problematic when quantifying absolute neurotransmitter concentrations. In this work, we develop a novel approach to calibrating microdialysis probes in vivo based on infusing stable-isotope labeled (SIL) neurotransmitters and measuring loss of SIL standard. This allows transport across the membrane and probe recovery to be calculated enabling quantitative measurements. Additionally, the use infusion of SIL compounds allows flux and metabolism of these neurotransmitters by different pathways to be monitored by measuring downstream metabolites. Quantitative microdialysis is performed on dopamine (DA), glutamate (Glu), and the primary glutamate precursor, glutamine (Gln). By infusing 13C6-DA and 13C5-Glu through the probe, the extraction fraction and the apparent concentration (Capp) were shown to equal that of no-net-flux (NNF), an established but slower quantitative microdialysis technique. This SIL microdialysis method is also demonstrated to be applicable under transient conditions unlike NNF. This new microdialysis technique was used to measure the effect of acute leptin on DA in the nucleus accumbens of rats. Capp for DA in the nucleus accumbens decreased by 30 ± 5% after an injection of leptin but did not decrease after a vehicle injection. The cause of this decrease was further investigated by administering raclopride, a D2 antagonist, to rats that received vehicle or leptin. These experiments provide evidence that leptin increases uptake. SIL microdialysis was also used to measure neuronal Glu release by monitoring the formation of 13C5-Glu from 13C5-Gln. Glu is preferentially formed from Gln in the neuron so the 13C5-Glu produced should be neuronally derived. Administration of tetrodotoxin and metabotropic glutamate receptors decreased the 13C5-Glu by 60 ± 7% and 56 ± 10%, respectively. An increase in 13C5-Glu was detected when a stressor (tail pinch) was applied to the animal. These tests indicate that the 13C5-Glu is neuronally derived.