Efficient measurement of endogenous neurotransmitters in small localized regions of central nervous systems in vitro with HPLC

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Abstract

High performance liquid chromatography (HPLC) is widely used to determine neurotransmitter concentrations in the central nervous system (CNS). Finding the optimal methods to sample from CNS tissue poses a challenge for neuroscientists. Here, we describe a method that allows assay of neurotransmitters (or other chemicals) in small regions (down to 180 μm in diameter) in in vitro preparations concurrently with electrophysiological recordings. The efficiency for measuring small amounts of chemicals is enhanced by a sample collecting pipette with filter paper at the tip that makes close contact with the target region in CNS tissue. With a wire plunger in the calibrated pipette controlled by a microsyringe pump, there is virtually no dead volume. Samples in a volume of 10 μL (taken, e.g., at 2 μL/min over 5 min) can be injected into a HPLC machine with microbore columns. We demonstrate the effectiveness of this method by measuring acetylcholine (ACh) in the ventral horn and its surrounding areas of the spinal cord in en bloc brainstem–spinal cord preparations. In control conditions, endogenous ACh levels in these regions were detectable. Application of neostigmine (an inhibitor of acetylcholinesterases (AChEs)) increased ACh concentrations, and at the same time, induced tonic/seizure-like activity in efferent motor output recorded from cervical ventral nerve roots. Higher ACh concentrations in the ventral horn were differentiated from nearby regions: the lateral and midline aspects of the ventral spinal cord. In addition, ACh in the preBötzinger Complex (preBötC) and the hypoglossal nucleus in medullary slice preparations can also be measured. Our results indicate that the method proposed in this study can be used to measure neurotransmitters in small and localized CNS regions. Correlation between changes in neurotransmitters in target regions and the neuronal activities can be revealed in vitro. Our data also suggest that there is endogenous ACh release in spinal ventral motor columns at fourth cervical (C4) level that regulates the respiratory-related motor activity.

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.

References (25)

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