Isolation of medullary dorsal horn neurons and characteristics of membrane currents

Abstract

Medullary dorsal horn (MDH) neurons receive noxious inputs from primary afferent fibers in the orofacial area, integrate modulatory influences from descending and local circuits and send the information to the thalamus and somatosensory cortex. In addition, this region contains many putative neurotransmitters, including excitatory and inhibitory amino acids and peptides. Because of the importance of MDH neurons in orofacial pain transmission, the modulation of activities of these cells is very interesting. In this study, we tried to establish a method for isolating and preparing MDH neurons for the purpose of studying the pain modulation mechanism in future, and identify if the acutely isolated cells preserve their electrophysiological activities using patch-clamp and fura-2-based microfluoroetry techniques. Thransverse slices (400㎛) of lower brainstem, 2-3 mm caudal to the obex, from neonatal rats (1-2 weeks old) were sequentially digested with two proteases (pronase 1 mg/ 5ml, and thermolysin 1 mg/5 ml) at 32℃ for 20 min. the MDH region was then dissected out and mechanically dissociated under a stereomicroscope using a series of fire-polished glass pipettes with a variety of orifice sizes. Single isolated cells were used for the experiment. This method yields multipolar, bipolar, unipolar, ellipse and pyramidal cells. These cells seem to be marginal cells of lamina I, stalked cells or islet cells in lamina II and pyramidal cells in lamina III, respectively. Fura-2-based microfluorometry showed that the intracellular Ca2+ concentration([Ca2+]) increased in response to the membrane depolarization induced by high K+, and the increase of [Ca2+] was caused by the influx of extracellular Ca2+, demonstrating the expression of voltage-sensitive Na+ and Ca2+ currents were directly recorded, the results were similar to those studies in vivo or slice presparations These facts indicate that electrophysiological activities are well preserved in the acutely isolated neurons, and these cells can be effectively used for biophysical and pharmacological studies, including understanding the pain modulation mechanism mediated by VSCC in the MDH.