Efficient measurement of endogenous neurotransmitters in small localized regions of central nervous systems in vitro with HPLC
Introduction
Quantitative measurement of neurotransmitters, hormones or their metabolites in specific regions in CNS provides important information linking regionally specific chemical events to CNS functions. In this regard, HPLC is extensively used to determine the concentrations of various neurotransmitters, including ACh, amino acids and dopamine (Bianchi et al., 2003, Gobert et al., 2003, Hernandez et al., 2003, Kapoor et al., 1990, Potter et al., 1983) in CNS. Determining the optimal sampling methods from CNS tissue poses a significant challenge. Sampling by microdialysis is one method used in vivo (Delgado et al., 1984, Fillenz, 2005, Kapoor et al., 1990). However, the relatively large size of the microdialysis probe (although the diameter of these probes can be made as small as 0.24 mm, the length of the membrane for chemical exchanges is typically ≥1 mm (CMA microdialysis Inc., MA, USA)) puts a significant limit on the spatial resolution of sampling. In addition, significant dead space volume in the microdialysis system limits the efficiency and accuracy of measurements. Alternatively, push–pull perfusion can be used and is reported to improve the recovery rates for dopamine and its metabolites from rat brain in vivo (Myers et al., 1998). Under in vitro conditions, samples from brain slices can be collected from the outflow of a perfusion system (Bianchi et al., 1999, Greer et al., 1992) concurrently with electrophysiological recordings or other physiological experiments. However, this method cannot provide information about the regional distribution of molecules of interest. The push–pull method can be used in in vitro conditions to allow sampling from and drug application to small regions of a brain slice in an interface chamber (West et al., 1992). However, the peristaltic pump connected to the push–pull cannula for application of drugs and sampling of fluid introduces a significant dead volume. An alternative approach (Roisin et al., 1991) uses a cannula positioned above the target region of a slice with which the spatial localization and the efficiency of sampling are limited by the distance between the tip of the cannula and the slice surface (∼100 μm), and also by the dead volume.
Here we describe a method that allows assay of neurotransmitters or other chemicals in regions down to 180 μm in diameter in vitro that can be done concurrently with electrophysiological recordings. The efficiency for measuring small amounts of neurotransmitters is enhanced by close contact of the collection pipette tip with the region of interest and by a sample collecting system with virtually no dead volume. To demonstrate the application of this method, we used an en bloc brainstem–spinal cord preparation from neonatal rats which contains the preBötC (the proposed primary site for respiratory rhythm generation) (Feldman and Del Negro, 2006, Smith et al., 1991) in the rostroventral medulla and the phrenic nucleus (phrenic motoneurons innervate the diaphragm) in the ventral horn of upper cervical spinal cord. We also use a medullary slice preparation which contains the preBötC and hypoglossal nucleus. These preparations generate respiratory rhythm which can be recorded from the ventral roots of spinal nerves (Smith and Feldman, 1987, Suzue, 1984) and from the hypoglossal nerve (XIIn) (Smith et al., 1991) in vitro. We measured the endogenous ACh levels under control conditions or following inhibition of AChEs by pharmacological agents and correlated the changes in ACh levels to simultaneously recorded neuronal activities. We also measured the ACh levels in a few neighboring regions to illustrate if the proposed method can differentiate the ACh levels in these regions in the spinal cord in vitro.
Section snippets
En bloc brainstem–spinal cord and slice preparations
All animal experiment protocols were in accordance with The National Institute of Health (USA) Guide for Care and Use of Laboratory Animals and approved by the UCLA Institutional Animal Care and Use Committee. Neonatal Sprague–Dawley rats (P0–P3) were anesthetized with isoflurane and then promptly decerebrated. The cerebellum was removed and the brainstem–spinal cord was isolated. For the en bloc brainstem–spinal cord preparation, transverse cuts were made rostrally at the pontomedullary
Results
To demonstrate the usefulness and effectiveness of the proposed method, we examined endogenous ACh release in the phrenic nucleus that may modulate respiratory motor output. We recorded respiratory rhythmic activity from one of the C2–C4 ventral roots and collected fluid samples from the surface of the ventral horn of the C4 spinal cord from the en bloc brainstem–spinal cord preparation (Fig. 3A). Fig. 3Aa shows a standard HPLC trace of 100 fmol/10 μL ACh and Ch (10 nM each in 10 μL of standard
Discussion
We developed a sampling system that collects fluid samples efficiently from small and localized CNS regions in vitro for HPLC measurement of neurotransmitters (or other chemicals). This method can be used concurrently with other physiological techniques such as stimulation, electrophysiological recording and pharmacological agent applications. Thus, this method can provide information regarding correlations between regional chemical events and changes in neuronal activity. This method is
Acknowledgements
This work was supported by Tobacco-Related Disease Research Program (California) Grant 13QT-0164 and NIH Grant HL40959.
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