Isolation and electrical characterization of the rat spinal dorsal horn neurons

The spinal dorsal horn is the area where primary afferent fibers terminate and cutaneous sensory information is processed. A number of putative neurotransmitter substances, including excitatory and inhibitory amino acids and peptides, are present in this region. In this study, single neurons of the spinal dorsal horn were acutely isolated and the properties of whole cell current and responses to excitatory and inhibitory neurotransmitters were studied by patch clamp technique. Transverse slice (300 μm) of lumbar spinal cords from young rata (7 ~ 14 days) were sequentially treated with two proteases (pronase 0.75 mg/ml and thermolysin 0.75 mg/ml), then single neurons were mechanically dissociated. These neurons showed near-intact morphology such as multipolar, ellipsoidal and bipolar, and pyramidal cells and we recorded the typical whole cell currents of K⁺, Ca²⁺ and ligand-operated channels from these neurons. Glutamate (30 μM) and N-methyl-D-aspartate (NMDA, 30 μM) induced inward currents of 117 ± 12.4 pA (n= 5) and 49 ± 6.9 pA (n = 3), respectively. Glycine (1 μM) potentiated glutamate-induced currents 4 ~ 5 times and NMDA-induced currents 8 ~ 10 times. In addition, glycine (30 μM) induced inward current (31 ± 6.1 nA, n = 2), which was rapidly desensitized after the peak to a new steady-state level. However, the inward currents induced by γ-amino butyric acid (GABA, 1 μM) decreased continuously after the peak (226 ± 41.6 pA, n = 3) nder the similar experimental condition. The ionic currents and pharmacological responses of isolated neurons in this work were similar to those observed in vivo or in vitro spinal cord slice, indicating that acutely isolated neurons could be effectively used for further pharmacological studies.