RAPID COMMUNICATION Electrophysiological Properties of Cultured Neonatal Rat Dorsal Horn Neurons Containing GABA and Met-Enkephalin-Like Immunoreactivity

Jo, Y. H., M. E. Stoeckel, and R. Schlichter. Electrophysiological which preserves the peptide content of the cells and the properties of cultured neonatal rat dorsal horn neurons containing presence of met-enkephalin (met-ENK) and/or glutamic GABA and met-enkephalin-like immunoreactivity. J. Neurophysacid decarboxylase (GAD)-like immunoreactivities was reiol. 79: 1583–1586, 1998. We have developed a culture of neurons vealed in the electrophysiologically identified neurons by dissociated from the most superficial laminae of the neonatal rat using a double immunofluorescence labeling technique. spinal cord dorsal horn (DH). By using the perforated patch-clamp technique, we distinguished four types of neurons based on their M E T H O D S firing properties in response to intracellular injection of 900 ms lasting current pulses. Type 1 neurons were characterized by a tonic Cell culture firing. Type 2 neurons displayed marked spike accommodation and fired brief (õ500 ms) bursts of action potentials, whereas type 3 Primary cultures of spinal DH neurons were prepared from 3neurons fired a single spike. Type 4 neurons exhibited different to 4-day-old Wistar rats. After decapitation under deep diethyltypes of firing patterns, but all of them possessed a time-dependent ether anesthesia, a laminectomy was performed and the dorsal third inwardly rectifying current activated by membrane hyperpolarizaof the spinal cord was cut with razor blade and incubated at 377C tion. Met-enkephalin-like immunoreactivity (met-ENK-LI) and in oxygenated divalent-free Earle’s balanced salt solution (EBSS, glutamic acid decarboxylase-like immunoreactivity (GAD-LI) Gibco) containing papain (20 U/ml, Sigma). After 45 min, the were colocalized in 42% of the neurons (n Å 59), which were enzymatic digestion was stopped by adding 3 ml EBSS containing previously identified electrophysiologically. Type 1–4 neurons bovine serum albumin (BSA, 1 mg/ml, Sigma), trypsine inhibitor represented respectively 4, 64, 20, and 12% of the population of (10 mg/ml, Sigma), and DNAse (0.01%, Sigma). After trituration neurons colocalizing met-ENK-LI and GAD-LI. We conclude that with a 1-ml plastic pipette, the homogenate was deposited on top the electrophysiological properties of DH neurons present in our of 4 ml of a solution of similar composition to that described cultures are similar to those described in acute slice or hemisected above, except that the concentration of BSA was increased to 10 spinal cord preparations and that met-ENK-LI and GABA-LI are mg/ml. After centrifugation (5 min at 500 rpm), the supernatant preferentially colocalized in type 2 neurons. was removed and replaced with culture medium composed of: MEM-alpha (Gibco), fetal calf serum (5% vol/vol, Sigma), heatinactivated horse serum (5% vol/vol, Gibco), penicillin and strep-