NICOLETA NEACŞU, MARIA-LUISA FLONTA
Department of Biophysics & Physiology, Faculty of Biology, University of Bucharest,
91–95, Splaiul Independenţei, Bucharest 050095, Romania
Abstract. This work proposes an in vitro model to study different types of ionic channels in primary sensory neurons involved in nociception. Using dorsal root ganglia (DRG) slices from newborn rats maintained in culture on three types of substrates, we have tested neuronal viability by two distinct methods: a stereological (physical dissector) and an electrophysiological method (patch-clamp technique, whole cell configuration). After applying viability tests, we have chosen to maintain DRG slices in culture on collagen-coated Petri dishes. This substrate preserved a high (80–90%) neuronal viability after 10 days in culture, and was easy to prepare. For subclassification of DRG neurons using current signature protocols, slices were maintained in culture for either 2 or 5 days. In addition, to differentiate neurons belonging to nociceptive classes, we analyzed some characteristics of action potentials (duration at 0 mV, at return to baseline, at –60 mV, and at 80% recovery from afterhyperpolarization (AHP 80%)). Following the criteria of Petruska et al. , we found after 2 days culture only types 1, 3, 4, 5, 7, 8 and 9 neurons. Cultures at 5 days lacked the types 2, 5, 6, 7 and 8 of cells. The magnitude of hyperpolarization-activated currents (HACs) obtained by us in DRG neurons from newborn rats was small (–94 ± 1.1 pA), confirming that primary sensory neurons at this developmental age feature low levels of HAC expression.
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