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Voltage-gated ionic currents in isolated retinal ganglion cells of the cat
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OBSERVATION OF INTRAMEMBRANE PARTICLES IN PLASMA MEMBRANES OF THE OUTER SEGMENT, CONNECTING CILIUM, AND INNER SEGMENT OF RETINAL RODS IN THE RAT. KATSUYUKI MIYAGUCttI AND PAULO H. tIASHIMOTO, Department of Anatomy, Osaka U n i v e r s i t y ~'iedical School, 3-57 Nakanoshima 4, Kitaku, Osaka 530, J a p a n . To clarity how opsin molecules are c o n v e y e d from the i n n e r segment to the disk membaranes, we o b s e r v e d plasma membranes of basal rod o u t e r segment (ROS), c o n n e c t i n g cilium (CC), and rod i n n e r segment (RIS) of the f r e s h rat r e t i n a with r a p i d - f r e e z e and d e e p - e t c h t e c h n i q u e , i?e also examined u p t a k e of ttRP into the isolated r e t i n a , and localization of opsin using a n t i - o p s i n antibodies and WGA. Intramembrane p a r t i c l e s (IMP) on the P-face of the CC and basal ROS (10nm in mean diameter) varied in size and s h a p e , s u g g e s t i n g t h e s e membranes consist of o p s i n molecules, while that of the RIS (8nm in mean diameter) does n o t . On the e x t r a c e l l u l a r s u r f a c e (ES) of the CC, t r a n s v e r s e l y a r r a n g e d s t r a n d s of particles ( l l - 1 5 n m in diameter) were d e t e c t e d , in c o n t r a s t with the smooth a p p e a r a n c e of t h a t of the basal ROS. Such p a r t i c l e s a p p e a r e d to c o r r e s p o n d to IMP of ciliary necklaces on the E-face (8nm in d i a m e t e r ) . T h e s e large n o n - o p s i n p r o t r u s i o n s on the ES may account for little labeling with a n t i - o p s i n a n t i b o d y at the CC s u r f a c e reported by others. HRP was d e t e c t e d in the most basal disk and dilated c i s t e r n s below it at 15rain i n c u b a t i o n . T h e s e c i s t e r n s , which were more numerous in the ROS of developing r a t s t h a n in a d u l t s , were visible in the f r e s h r e t i n a s with r a p i d - f r e e z e and d e e p - e t c h i n g , indicating t h a t t h e y are not p r o d u c t s of chemical fixation. T h u s , opsin molecules may b e c o n v e y e d to the basal ROS plasma membrane via the plasma membrane of the CC and e n d o c y t o s e d in the form of dilated c i s t e r n s , t h a t might c h a n g e to the d i s k .
VOLTAGE-GATED IONIC CURRENTS IN ISOLATED RETINAL GANGLION CELLS OF THE CAT. MAKOTO KANEDA AND AKIMICHI KANEKO, Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan. Retrogradely labelled ganglion cells were dissociated from the cat retina and identified by their fluorescent image under an inverted microscope after the dissociation. Six types of voltage-gated ionic currents were separated under voltage-clamp conditions using a patch pipette in the whole cell configuration. They were voltage-gated sodium (INn), calcium (Iea), potassium (calcium-dependent: IK(C,), delayed rectifier: Igiv), A-current: IN(A)) and h-current (Ih). IN, was activated by depolarization of the cell from the holding potential (Vn) of -95 mV to more positive voltages than -45 mV. I~-, was identified by the fast activation and inactivation time course, and the reversible blockage by either tetrodotoxin or removal of extracellular No. Ic, was activated by depolarization of the cell from Vn of -97 mV to more positive voltages than -57 mV. Ic, was identified to be the L-type by the slow inactivation time course and similarity of I-V curves recorded at Vn of -97 mV and -57 inV. IKlca) was separated as a Cadependent sustained outward current using Co. Ia'(~} was observed as a sustained outward current and suppressed by TEA. The amplitude of Ig(~) was the largest among the three types of K currents. IK(a) was observed as a transient outward current and was inactivated within 200 msec after the command onset. IK(A) was sensitive to 4-AP and inactivated by holding the cell at -40 mV. In was activated by membrane hyperpolarization of the cell from V n of -60 mV to more negative voltages than -90 mY. Activation of In was slow and did not reach a steady state level until 2 sec. In was carried by Na and K.
Na CURRENTS IN X AND W GANGLION CELLS ISOLATED FROM THE ADULT CAT RETINA. AKIMICHI KANEKO AND MAKOTO KANEDA, Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan. Ganglion ceils were labeled by injecting DiI into the lateral genieulate nucleus or the superior colliculus 2 3 days prior to the experiment. Ganglion cells were dissociated from the peripheral retina (>2 m m from the area centralis) by mechanical trituration after incubating in the medium containing papain (5 - 6 U/ml) and cysteine. X- and W-type ganglion cells were identified by their projection target and by the soma size (X cells, approximately 20/Jm; W cells, approximately 15 ~m). Both types of ganglion cells had voltage-gated Na current (IN~). IN~ was blocked by 10 -s M TTX reversibly. The reversal potential of I~v~ was dependent on the extracellular Na concentration ({Na+],) with a relation of 58 mV for a 10-fold change in [Na+],. IN~ flowed only transiently; it was inactivated within a few re.see after the onset of the command depolarization. After being inactivated, INa recovered by holding the cell membrane hyperpolarized. The time course of recovery was fitted by a double exponential function and was dependent on the cell type; X-cells showed a rapid recovery, while W-cells showed a slow recovery. Since the speed of recovery from inactivation of INs is one of the factors determining the rate of the action potential train, the present finding seems to provide a reasonable interpretation to the generation of brisk and sluggish spike patterns in X and W cells, respectively.
OBSERVATION OF INTRAMEMBRANE PARTICLES IN PLASMA MEMBRANES OF THE OUTER SEGMENT, CONNECTING CILIUM, AND INNER SEGMENT OF RETINAL RODS IN THE RAT. KATSUYUKI MIYAGUCttI AND PAULO H. tIASHIMOTO, Department of Anatomy, Osaka U n i v e r s i t y ~'iedical School, 3-57 Nakanoshima 4, Kitaku, Osaka 530, J a p a n . To clarity how opsin molecules are c o n v e y e d from the i n n e r segment to the disk membaranes, we o b s e r v e d plasma membranes of basal rod o u t e r segment (ROS), c o n n e c t i n g cilium (CC), and rod i n n e r segment (RIS) of the f r e s h rat r e t i n a with r a p i d - f r e e z e and d e e p - e t c h t e c h n i q u e , i?e also examined u p t a k e of ttRP into the isolated r e t i n a , and localization of opsin using a n t i - o p s i n antibodies and WGA. Intramembrane p a r t i c l e s (IMP) on the P-face of the CC and basal ROS (10nm in mean diameter) varied in size and s h a p e , s u g g e s t i n g t h e s e membranes consist of o p s i n molecules, while that of the RIS (8nm in mean diameter) does n o t . On the e x t r a c e l l u l a r s u r f a c e (ES) of the CC, t r a n s v e r s e l y a r r a n g e d s t r a n d s of particles ( l l - 1 5 n m in diameter) were d e t e c t e d , in c o n t r a s t with the smooth a p p e a r a n c e of t h a t of the basal ROS. Such p a r t i c l e s a p p e a r e d to c o r r e s p o n d to IMP of ciliary necklaces on the E-face (8nm in d i a m e t e r ) . T h e s e large n o n - o p s i n p r o t r u s i o n s on the ES may account for little labeling with a n t i - o p s i n a n t i b o d y at the CC s u r f a c e reported by others. HRP was d e t e c t e d in the most basal disk and dilated c i s t e r n s below it at 15rain i n c u b a t i o n . T h e s e c i s t e r n s , which were more numerous in the ROS of developing r a t s t h a n in a d u l t s , were visible in the f r e s h r e t i n a s with r a p i d - f r e e z e and d e e p - e t c h i n g , indicating t h a t t h e y are not p r o d u c t s of chemical fixation. T h u s , opsin molecules may b e c o n v e y e d to the basal ROS plasma membrane via the plasma membrane of the CC and e n d o c y t o s e d in the form of dilated c i s t e r n s , t h a t might c h a n g e to the d i s k .
VOLTAGE-GATED IONIC CURRENTS IN ISOLATED RETINAL GANGLION CELLS OF THE CAT. MAKOTO KANEDA AND AKIMICHI KANEKO, Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan. Retrogradely labelled ganglion cells were dissociated from the cat retina and identified by their fluorescent image under an inverted microscope after the dissociation. Six types of voltage-gated ionic currents were separated under voltage-clamp conditions using a patch pipette in the whole cell configuration. They were voltage-gated sodium (INn), calcium (Iea), potassium (calcium-dependent: IK(C,), delayed rectifier: Igiv), A-current: IN(A)) and h-current (Ih). IN, was activated by depolarization of the cell from the holding potential (Vn) of -95 mV to more positive voltages than -45 mV. I~-, was identified by the fast activation and inactivation time course, and the reversible blockage by either tetrodotoxin or removal of extracellular No. Ic, was activated by depolarization of the cell from Vn of -97 mV to more positive voltages than -57 mV. Ic, was identified to be the L-type by the slow inactivation time course and similarity of I-V curves recorded at Vn of -97 mV and -57 inV. IKlca) was separated as a Cadependent sustained outward current using Co. Ia'(~} was observed as a sustained outward current and suppressed by TEA. The amplitude of Ig(~) was the largest among the three types of K currents. IK(a) was observed as a transient outward current and was inactivated within 200 msec after the command onset. IK(A) was sensitive to 4-AP and inactivated by holding the cell at -40 mV. In was activated by membrane hyperpolarization of the cell from V n of -60 mV to more negative voltages than -90 mY. Activation of In was slow and did not reach a steady state level until 2 sec. In was carried by Na and K.
Na CURRENTS IN X AND W GANGLION CELLS ISOLATED FROM THE ADULT CAT RETINA. AKIMICHI KANEKO AND MAKOTO KANEDA, Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan. Ganglion ceils were labeled by injecting DiI into the lateral genieulate nucleus or the superior colliculus 2 3 days prior to the experiment. Ganglion cells were dissociated from the peripheral retina (>2 m m from the area centralis) by mechanical trituration after incubating in the medium containing papain (5 - 6 U/ml) and cysteine. X- and W-type ganglion cells were identified by their projection target and by the soma size (X cells, approximately 20/Jm; W cells, approximately 15 ~m). Both types of ganglion cells had voltage-gated Na current (IN~). IN~ was blocked by 10 -s M TTX reversibly. The reversal potential of I~v~ was dependent on the extracellular Na concentration ({Na+],) with a relation of 58 mV for a 10-fold change in [Na+],. IN~ flowed only transiently; it was inactivated within a few re.see after the onset of the command depolarization. After being inactivated, INa recovered by holding the cell membrane hyperpolarized. The time course of recovery was fitted by a double exponential function and was dependent on the cell type; X-cells showed a rapid recovery, while W-cells showed a slow recovery. Since the speed of recovery from inactivation of INs is one of the factors determining the rate of the action potential train, the present finding seems to provide a reasonable interpretation to the generation of brisk and sluggish spike patterns in X and W cells, respectively.