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123 Electrophysiological and morphological development of cultured spinal neurons
S26
123 Department
ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL DEVELOPMENT OF CULTURED SPINAL NEURONS of Physiology, Osaka City University Medical School, l-4-54, Abeno-ku, Osaka 545, Japan
FUSAO NAKAMURA, MIYUKI KUNO, HIROMU SAKAI, HIROKAZU MORIHATA, SHIUSHI MATSUURA We investigated electrophysiological and morphological development of spinal neurons in vitro. The cells were obtained from rat embryo (Wistar rats, E13-14) and were cultured for up to 4 weeks. The area of soma was smaller th& 300 pm2 within 1 week in vitro and the number of larger cells (>300 ,um2) gradually increased. Most cells exhibited voltage-dependent outward and inward conductances in the whole cell clamp configuration within 3 days in vitro. The outward current consists of two components, 4-aminopyridine-sensitive and TEA-sensitive Kf currents. The transient inward current was activated at around -4OmV and was blocked by TTX, suggesting that the current was mediated by Na +. The Na+ currents tended to be larger in later culture period. Glycine (O.Ol-1mM) increased a conductance with the reversal potential at -40 N -5OmV. These results suggest that most spinal cells developed in vitro exhibit voltage-dependent Na+ and K+ channels and respond to glycine.
124
Voltage-dependent Ca 2+ channels distributed on the cereal giant interneuron in the cricket Kawachi biofhght Project, ERATO, JST, Tokyo 153, Japan’, Neuro-Cybern. Lab., Res. Inst. for Electronic Science, Hokkaido Univ., Sapporo 060, Japan2, Dept. of Mech. Engin., Fat. of Sci. and Technol., Keio Univ., Yokohama 223, Japan3 Hiroto Ogawa’ , Yoshichika Baba2, Kotaro Oka We found that depolarization of the wind-sensitive interneurons in the cricket could induce dendritic Ca accumulation, using a fluorescent imaging technique (Ogawa et al., 1996). To estimate the cellular mechanism underlying dendritic processing of the cricket giant interneurons, we further examined the dendritic Ca dynamics and simultaneous recordings of membrane potential. When each cereal nerve was separately stimulated, the Ca responses in several dendritic arborizations showed different directional sensitivity in peak amplitude and rising velocity. A train of subthreshold depolarization as EPSP could induce no pronounced Ca elevation. Bath-application of not only a mixture 1 mM each of Ni‘2+, Co2+ and Cd2+ but also l-micro M TTX suppressed Ca elevation induced by an antidromic stimulus of connective nerves. These results suggest that the back-spread Na+ spikes initiated at different neurite positions induce the dendritic Ca influx through the voltage-dependent channels.
125
WHOLE-CELL PATCH-CLAMP ANALYSIS OF OUTWARD CURRENTS LYING PACEMAKER POTENTIALS OF TERMINAL NERVE (TN)-GnRH
MISAKI MARINE BIOLOGICAL STATION, SCHOOL OF SCIENCE, MISAKI, MIURA, KANAGAWA 238-02, JAPAN
UNIVERSITY
UNDERCELLS.
OF TOKYO,
Hideki Abe, Yoshitaka Oka Endogenous pacemaker activities play important roles in the putative neuromodulator functions of the GnRHimmunoreactive TN cells. To study the underlying outward currents which should be involved in the repolarizing phase of pacemaker potentials (PPs), we carried out whole-cell patch-clamp recordings from TN-GnRH cells in in vitro whole-brain preparation of a small fish brain. Outward currents were isolated pharmacologically by blocking Na+ currents, and Ca2+ currents, and were separated using K+ current blockers. Three components could be identified; l)TEA-sensitive component, 2)4AP-sensitive transient component which were inactivated at physiological membrane potentials, and 3)TEA- and 4AP-resistant component which consist of two distinct currents on the basis of kinetic properties. From the result of voltage and current clamp experiments, we suggest that the TEA-sensitive outward current plays an important role in the repolarizing phase of PPs.
123 Department
ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL DEVELOPMENT OF CULTURED SPINAL NEURONS of Physiology, Osaka City University Medical School, l-4-54, Abeno-ku, Osaka 545, Japan
FUSAO NAKAMURA, MIYUKI KUNO, HIROMU SAKAI, HIROKAZU MORIHATA, SHIUSHI MATSUURA We investigated electrophysiological and morphological development of spinal neurons in vitro. The cells were obtained from rat embryo (Wistar rats, E13-14) and were cultured for up to 4 weeks. The area of soma was smaller th& 300 pm2 within 1 week in vitro and the number of larger cells (>300 ,um2) gradually increased. Most cells exhibited voltage-dependent outward and inward conductances in the whole cell clamp configuration within 3 days in vitro. The outward current consists of two components, 4-aminopyridine-sensitive and TEA-sensitive Kf currents. The transient inward current was activated at around -4OmV and was blocked by TTX, suggesting that the current was mediated by Na +. The Na+ currents tended to be larger in later culture period. Glycine (O.Ol-1mM) increased a conductance with the reversal potential at -40 N -5OmV. These results suggest that most spinal cells developed in vitro exhibit voltage-dependent Na+ and K+ channels and respond to glycine.
124
Voltage-dependent Ca 2+ channels distributed on the cereal giant interneuron in the cricket Kawachi biofhght Project, ERATO, JST, Tokyo 153, Japan’, Neuro-Cybern. Lab., Res. Inst. for Electronic Science, Hokkaido Univ., Sapporo 060, Japan2, Dept. of Mech. Engin., Fat. of Sci. and Technol., Keio Univ., Yokohama 223, Japan3 Hiroto Ogawa’ , Yoshichika Baba2, Kotaro Oka We found that depolarization of the wind-sensitive interneurons in the cricket could induce dendritic Ca accumulation, using a fluorescent imaging technique (Ogawa et al., 1996). To estimate the cellular mechanism underlying dendritic processing of the cricket giant interneurons, we further examined the dendritic Ca dynamics and simultaneous recordings of membrane potential. When each cereal nerve was separately stimulated, the Ca responses in several dendritic arborizations showed different directional sensitivity in peak amplitude and rising velocity. A train of subthreshold depolarization as EPSP could induce no pronounced Ca elevation. Bath-application of not only a mixture 1 mM each of Ni‘2+, Co2+ and Cd2+ but also l-micro M TTX suppressed Ca elevation induced by an antidromic stimulus of connective nerves. These results suggest that the back-spread Na+ spikes initiated at different neurite positions induce the dendritic Ca influx through the voltage-dependent channels.
125
WHOLE-CELL PATCH-CLAMP ANALYSIS OF OUTWARD CURRENTS LYING PACEMAKER POTENTIALS OF TERMINAL NERVE (TN)-GnRH
MISAKI MARINE BIOLOGICAL STATION, SCHOOL OF SCIENCE, MISAKI, MIURA, KANAGAWA 238-02, JAPAN
UNIVERSITY
UNDERCELLS.
OF TOKYO,
Hideki Abe, Yoshitaka Oka Endogenous pacemaker activities play important roles in the putative neuromodulator functions of the GnRHimmunoreactive TN cells. To study the underlying outward currents which should be involved in the repolarizing phase of pacemaker potentials (PPs), we carried out whole-cell patch-clamp recordings from TN-GnRH cells in in vitro whole-brain preparation of a small fish brain. Outward currents were isolated pharmacologically by blocking Na+ currents, and Ca2+ currents, and were separated using K+ current blockers. Three components could be identified; l)TEA-sensitive component, 2)4AP-sensitive transient component which were inactivated at physiological membrane potentials, and 3)TEA- and 4AP-resistant component which consist of two distinct currents on the basis of kinetic properties. From the result of voltage and current clamp experiments, we suggest that the TEA-sensitive outward current plays an important role in the repolarizing phase of PPs.