Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

37-POS Board 19 NMDA Receptors and Large-Conductance Calcium-Activated Potassium Channels in Enkephalinergic Neurons of Mouse Spinal Superficial Dorsal Horn Eiko Kato , Yuuichi Hori. Dokkyo Medical University, Tochigi, Japan. Enkephalin (Enk)-containing neurons are distributed at a high density in the superficial dorsal horn (SDH) of the spinal cord. In the present experiments, we analyzed whether Enk-containing neurons in the SDH express the N -methyl- D -aspartate receptor (NMDAR) 2B (NR2B) subunit and large-conductance calcium-activated potassium (BK) channels, and how the activity of Enk- containing neurons is regulated by NMDAR and BK channels. The experiments were performed on 5- to 8-week-old male ICR mice. The lumbar spinal cord was dissected, and transverse slices were prepared. Tight-seal whole-cell recordings were obtained from neurons in the SDH. After the recordings, the neurons were collected, and single- cell real-time RT-PCR was performed to analyze the expression profile of genes in each SDH neuron. Puff application of L-glutamate evoked an inward current at a holding potential of -70 mV. Upon depolarizing the holding potential to 0 mV, outward current of long duration appeared after initial inward current. The NR2B-selective NMDAR antagonist ifenprodil reduced the outward current. The outward current was also abolished by the selective BK channels antagonist iberiotoxin. Single-cell real-time RT-PCR analysis revealed that single SDH neurons expressing the preproenkephalin mRNA also expressed the BK channel α-subunit, NR1, and NR2B subunit mRNAs. Additionally, the neurons generating the outward current showed a significant tendency to express BK channels α-subunit mRNA, which is consistent with the pharmacological results of a BK channels antagonist. Our experiments suggest that combining real-time RT-PCR with whole-cell recordings provides a useful tool to analyze the functions of genetically characterized central nervous neurons at the single-cell level and that Ca 2+ influx through NMDAR may activate BK channels and hyperpolarize Enk-containing neurons in the SDH.

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