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Measurement of intracellular Ca2+ in neurons using a confocal laser-scanning microscope
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23. Membrane biophysics MEASUREMENT OF ~ c ~ r , T . U L A R Ca 2+ IN NEURC~S USING A OONFOCAL LASER-SCANNING MICROSCOPE. K~qJI KUBA, MITSUO NOHMI AND HUA SHAO-YING, DeF~rtment of Physiology, Saga Medical School, Nabesh--~-~, Saga 840-01, Japan. The suitability of a confocal laser-scanning microsqope (CLSM:MRC-500, Bior~d, Nikon XF-EFD2, CF Fluor 40X and Argon Laser 488nm) for intracellular^Ca z~ measurement with a CaZ+-probe, fluor-3, was examined. The intensity of the fluorescence (Caz+ signal) in a micro-pipette 0~ 1 urn) was reduced tQ less than half by lowering the stage by 1 urn, indicating a thin focal depth. Quenching of the CaZ+-probe by continuous scanning with a laser beam (0.3 roW) occurred more slowly than by continuous epifluorescence excitation to produce a similar fluorescence intensity. This was partly due to intermittent excitation joy the scanning beam and to better spatial discrimination requiring a weaker excitation energy. CaZ~-signals from either the cytoplasm or CaZ+-storing organelles were clearly distinguished in many cultured bullfrog sympathetic ganglion cells. Their relative intensities depended on the concentration of fluor-3 (AM) and the duration of loading. Raising the external K + to 50 n~M increased Ca z+ signals at both the cell soma and processes and occasional~ caused retraction of the latter. Caffeine (5 ~M) produced a transient rise or oscillatioD of Ca ~ signal (mainly of the cytoplasm~$ and occasionally altered the distribution of strong Ca z+ signal regions (of organelles). The Ca ~ signal from in situ sympathetic neurons increased in response to a postganglionic tetanus. Accordingly, the ~ is a powerful tool for measuring spatial changes in intracellular Ca z+. Although the speed of image acquisition with the ~ is slow (i or 4Hz), it could be improved by applying line scanning (512 Hz) at a fixed y-axis position.
CALCIUM CURRENT OF RETINAL BIPOLAR CELLS FROM THE MOUSE. MASAOTACHIBANA1 , AKIMICEI KANEKO AND
LAWRENCE H. PINTO"2, N a t i o n a l I n s t i t u t e for P h y s i o l o g i c a l S c i e n c e s , Okazaki 444 and ~Morthwestern U n i v e r s i t y , Evanstonp I1 60201, U. S. A. ( , P r e s e n t Address: Dept. of P s y c h o l o l y , Faculty of L e t t e r s , The U n i v e r s i t y of Tokyo, Hondo r Bunkyo-ku, Tokyo 113) Bipolar c e l l s were i s o l a t e d by enzymatic (papain) d i s s o c i a t i o n of mouse (C57bl/6J) r e t i n a . The c e l l s were v o l t a g e - c l a s p e d by a patch p i p e t t e in the w h o l e - c e l l clamp c o n f i g u r a t i o n . To a n a l y z e the p r o p e r t i e s of the Ca c u r r e n t , the c o u n t e r a c t i n g K c u r r e n t s were blocked by TEA and Cs in the s u p e r f u s a t e and by Cs in the recording p i p e t t e . In 10 mN [Ca2"]o, the Ca conductance was a c t i v a t e d at p o t e n t i a l s more p o s i t i v e than -65 mV ( h a l f maximum at approximately - 2 5 mV and f u l l a c t i v a t i o n at >30 mY), and was i n a c t i v a t e d at p o t e n t i a l s more p o s i t i v e than -80 mY ( t o 50Z at approximately -50 mV and to lOOm at >-30 mV). The Ca current was i n s e n s i t i v e to 50 # N Cd2 . , 10 ~M n i f e d i p i n e or 10 ~N Bay K 8644. These p r o p e r t i e s i n d i c a t e that t h i s current is of T type. Since both the perikaryon and the axon terminal generated Ca c u r r e n t s with s i m i l a r p r o p e r t i e s , t h i s current may p a r t i c i p a t e in synaptic transmission. The v o l t a g e range of the a c t i v a t e d Ca current i n c l u d e s the presumed range of membrane p o t e n t i a l s accompanying l i g h t - e v o k e d r e s p o n s e s . Therefore, the t r a n s i e n t c h a r a c t e r of the Ca current may help to shape t r a n s i e n t r e s p o n s e s of g a n g l i o n c e l l s in the mouse r e t i n a . TIGHT-SEAL WHOLE-CELL RECORDING OF POTASSIUM CURRENTS IN MOTONEURONS VISUALLY IDENTIFIED IN THIN SLICES OF RAT SPINAL CORD. TOMOYUKI TAKAHASHI, Department of Physiology r Kyoto University~ Faculty of Medicine~ Kyoto 606, Japan. Potassium currents were recorded from motoneurons of newborn rat lumbar spinal cord by the whole-cell voltage clamp method. Motoneurons were retrogradely pre-labeled with a fluorescent dye, Evans Blue, and visually identified in slices under fluorescent and Nomarski optics. Outward potassium currents evoked by membrane depolarization were reversibly diminished in Ca-free solution. Ca-dependent potassium currents, revealed by subtraction, had transient as well as steady components. Most of the steady currents observed in Ca-free solution were abolished by application of tetraethylammonium (30 mM) but a transient outward current remained. This current showed steady-state inactivation by depolarizing prepulses with a half inactivation voltage of -77 mV and its magnitude was markedly but reversibly suppressed by 4-aminopyridine (4-AP, 4 mM), suggesting that it is the A current (IA). Current clamp recordings of action potentials showed that replacement of external Ca by Mg prolonged the repolarizing phase and abolished the slow afterhyperpolarization. When 4-AP was applied to the Ca-free solution the repolarizing phase was further prolonged. It is suggested that both transient Ca-dependent and I A potassium currents contribute to action potential repolarization and that steady Ca-dependent potassium currents produce the slow afterhyperpolarization in rat spinal motoneurons.
23. Membrane biophysics MEASUREMENT OF ~ c ~ r , T . U L A R Ca 2+ IN NEURC~S USING A OONFOCAL LASER-SCANNING MICROSCOPE. K~qJI KUBA, MITSUO NOHMI AND HUA SHAO-YING, DeF~rtment of Physiology, Saga Medical School, Nabesh--~-~, Saga 840-01, Japan. The suitability of a confocal laser-scanning microsqope (CLSM:MRC-500, Bior~d, Nikon XF-EFD2, CF Fluor 40X and Argon Laser 488nm) for intracellular^Ca z~ measurement with a CaZ+-probe, fluor-3, was examined. The intensity of the fluorescence (Caz+ signal) in a micro-pipette 0~ 1 urn) was reduced tQ less than half by lowering the stage by 1 urn, indicating a thin focal depth. Quenching of the CaZ+-probe by continuous scanning with a laser beam (0.3 roW) occurred more slowly than by continuous epifluorescence excitation to produce a similar fluorescence intensity. This was partly due to intermittent excitation joy the scanning beam and to better spatial discrimination requiring a weaker excitation energy. CaZ~-signals from either the cytoplasm or CaZ+-storing organelles were clearly distinguished in many cultured bullfrog sympathetic ganglion cells. Their relative intensities depended on the concentration of fluor-3 (AM) and the duration of loading. Raising the external K + to 50 n~M increased Ca z+ signals at both the cell soma and processes and occasional~ caused retraction of the latter. Caffeine (5 ~M) produced a transient rise or oscillatioD of Ca ~ signal (mainly of the cytoplasm~$ and occasionally altered the distribution of strong Ca z+ signal regions (of organelles). The Ca ~ signal from in situ sympathetic neurons increased in response to a postganglionic tetanus. Accordingly, the ~ is a powerful tool for measuring spatial changes in intracellular Ca z+. Although the speed of image acquisition with the ~ is slow (i or 4Hz), it could be improved by applying line scanning (512 Hz) at a fixed y-axis position.
CALCIUM CURRENT OF RETINAL BIPOLAR CELLS FROM THE MOUSE. MASAOTACHIBANA1 , AKIMICEI KANEKO AND
LAWRENCE H. PINTO"2, N a t i o n a l I n s t i t u t e for P h y s i o l o g i c a l S c i e n c e s , Okazaki 444 and ~Morthwestern U n i v e r s i t y , Evanstonp I1 60201, U. S. A. ( , P r e s e n t Address: Dept. of P s y c h o l o l y , Faculty of L e t t e r s , The U n i v e r s i t y of Tokyo, Hondo r Bunkyo-ku, Tokyo 113) Bipolar c e l l s were i s o l a t e d by enzymatic (papain) d i s s o c i a t i o n of mouse (C57bl/6J) r e t i n a . The c e l l s were v o l t a g e - c l a s p e d by a patch p i p e t t e in the w h o l e - c e l l clamp c o n f i g u r a t i o n . To a n a l y z e the p r o p e r t i e s of the Ca c u r r e n t , the c o u n t e r a c t i n g K c u r r e n t s were blocked by TEA and Cs in the s u p e r f u s a t e and by Cs in the recording p i p e t t e . In 10 mN [Ca2"]o, the Ca conductance was a c t i v a t e d at p o t e n t i a l s more p o s i t i v e than -65 mV ( h a l f maximum at approximately - 2 5 mV and f u l l a c t i v a t i o n at >30 mY), and was i n a c t i v a t e d at p o t e n t i a l s more p o s i t i v e than -80 mY ( t o 50Z at approximately -50 mV and to lOOm at >-30 mV). The Ca current was i n s e n s i t i v e to 50 # N Cd2 . , 10 ~M n i f e d i p i n e or 10 ~N Bay K 8644. These p r o p e r t i e s i n d i c a t e that t h i s current is of T type. Since both the perikaryon and the axon terminal generated Ca c u r r e n t s with s i m i l a r p r o p e r t i e s , t h i s current may p a r t i c i p a t e in synaptic transmission. The v o l t a g e range of the a c t i v a t e d Ca current i n c l u d e s the presumed range of membrane p o t e n t i a l s accompanying l i g h t - e v o k e d r e s p o n s e s . Therefore, the t r a n s i e n t c h a r a c t e r of the Ca current may help to shape t r a n s i e n t r e s p o n s e s of g a n g l i o n c e l l s in the mouse r e t i n a . TIGHT-SEAL WHOLE-CELL RECORDING OF POTASSIUM CURRENTS IN MOTONEURONS VISUALLY IDENTIFIED IN THIN SLICES OF RAT SPINAL CORD. TOMOYUKI TAKAHASHI, Department of Physiology r Kyoto University~ Faculty of Medicine~ Kyoto 606, Japan. Potassium currents were recorded from motoneurons of newborn rat lumbar spinal cord by the whole-cell voltage clamp method. Motoneurons were retrogradely pre-labeled with a fluorescent dye, Evans Blue, and visually identified in slices under fluorescent and Nomarski optics. Outward potassium currents evoked by membrane depolarization were reversibly diminished in Ca-free solution. Ca-dependent potassium currents, revealed by subtraction, had transient as well as steady components. Most of the steady currents observed in Ca-free solution were abolished by application of tetraethylammonium (30 mM) but a transient outward current remained. This current showed steady-state inactivation by depolarizing prepulses with a half inactivation voltage of -77 mV and its magnitude was markedly but reversibly suppressed by 4-aminopyridine (4-AP, 4 mM), suggesting that it is the A current (IA). Current clamp recordings of action potentials showed that replacement of external Ca by Mg prolonged the repolarizing phase and abolished the slow afterhyperpolarization. When 4-AP was applied to the Ca-free solution the repolarizing phase was further prolonged. It is suggested that both transient Ca-dependent and I A potassium currents contribute to action potential repolarization and that steady Ca-dependent potassium currents produce the slow afterhyperpolarization in rat spinal motoneurons.