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Axonal site for impulse initiation and rhythmogenesis in Aplysia pacemaker neurons
Brain Research, 187 (1980) 201-205. © Elsevier/North-HollandBiomedicalPress
201
Axonal site for impulse initiation and rhythmogenesis in Aplysia pacemaker neurons
STEVEN N. TREISTMAN Department of Biology, Bryn Mawr College, Bryn Mawr, Pa. 19010 (U.S.A.)
(Accepted December 6th, 1979) Key words: Aplysia pacemaker cell -- impulse initiation -- rhythmogenesis-- axon spikes
Spontaneous pacemaker activity is currently receiving a great deal of attention in neurobiology. Pacemaker neurons in molluscs have been the focus of a large numbe~ of investigations into the nature and modification of bursting pacemaker activity~. Much of this work has been predicated on an understanding that the cell body was the site of the subthreshold oscillatory rhythm and spike initiation in these cells. This understanding is based upon indirect evidence which suggests that cell bodies separated from their axons, either by ligation1 or enzymatic dissociation4, retain their ability to generate oscillations and associated action potentials, and inferences of action potential origin drawn from single electrode somatic recordings 9. A cell body locus for spike initiation would be different from that found in non-pacemaker Aplysia neurons 7,t°, as well as neurons from non-molluscan tissue 5, where spike initiation occurs in the axon. Since such a difference is, potentially, of great importance in the basic biology of the neuron, as well as in the interpretation of data regarding pacemaker activity, the locus of spike initiation in intact pacemaker neurons of Aplysia californica was explored by simultaneous intracellular recordings from different regions of the spontaneously active cell. Abdominal ganglia from Aplysia were removed through an incision in the foot and pinned out through the connective tissue sheath. The connective tissue overlying a hemiganglion was removed by microdissection, and individual somata separated from neighboring cells by removing the surrounding cells with a glass needle. After penetration of the cell body with a micropipette electrode containing 3 M KC1, the region surrounding the cell body was probed with a second micropipette electrode until an axon was impaled which exhibited spiking time-locked to that occurring in the cell body. Recording was by conventional methods and intracellular stimulation was done with a bridge circuit. Voltage clamp data were obtained with independent voltage and current electrodes, by conventional means. Fig. 1A and B show traces from the cell body and axon of pacemaker neurons, in which it is clear that the onset of the action potential occurs first in the axon. Also evident is the faster rate-of-rise of the axon spike, indicating an electrode position closer to the site of origin.
202
Fig. 1. Simultaneous recordings from cell body and axon of pacemaker neurons. In all pairs of traces, cell body is upper and axon recording is the lower. A: recordings from a left upper quadrant cell. The cell was beating, and traces are of spontaneous action potentials. In top pair, both electrodes are in cell body. The middle pair of traces represent cell body and an axon impalement approximately 0.5 mm from medial edge of cell body. In the bottom pair, the axon electrode was moved to approximately 1.0 mm medial to edge of cell body. B : recordings from a bursting left upper quadrant cell, illustrating a prominent inflection in axonal action potential. Uppermost pair of traces are both from the cell body. Lower pair shows traces after axon electrode was moved to a point approximately 1.0 mm from base of cell body. Traces represent first spike in spontaneous bursts. C: recordings from a bursting left upper quadrant cell. Upper traces are from the cell body and axon, approximately 1.0 mm from border of cell body, during the first spike of a spontaneous burst. Bottom pair of traces showing the start of the action potential, are from the same electrode placements, during an antidromic spike initiated by stimulation of the genital-pericardial nerve. Apparent differences in amplitude between the spontaneous and antidromic spikes result from the loss of the slow depolarization leading to the spontaneous spike, from the picture. The shoulder on the axonal spike is larger and of longer duration following antidromic stimulation. Calibrations: (A) 20 mV, 2 msec; (B) top, 20 mV, 2 msec; bottom, 20 mV, 5 msec; (C) 20 mV, 2 msec.
One n o t a b l e difference between action potential shape in the cell body a n d axon is a p r o m i n e n t inflection on the rising phase of the axonal spike. This inflection is more p r o m i n e n t in the axon of some cells t h a n others. F o r example, cell R15 typically has a less p r o m i n e n t inflection t h a n the axon of the left upper q u a d r a n t cells at a similar distance from the cell body. This inflection coincides with the rise of the cell body spike, a n d m a y reflect the loading effect of the cell body m e m b r a n e on the current generator in the axon t°. If this interpretation is correct, a geometry similar to that occurring spontaneously should result when the cell is activated antidromically, where the action potential definitely arises in the axon. We tested this by stimulating left u p p e r q u a d r a n t cells t h r o u g h a suction electrode o n the distal genital-pericardial nerve, which contains a n axon of these cells, while recording from the cell body a n d
203 m o r e p r o x i m a l axon, intracellularly. Fig. 1C shows t h a t a similar inflection is present on the rising phase o f the a x o n a c t i o n potential. It has been n o t e d in a n u m b e r o f m o l l u s c a n neurons, t h a t the a x o n a l a c t i o n p o t e n t i a l is m o r e N a + - d e p e n d e n t t h a n is the cell b o d y a n d / o r p r o x i m a l axon spike, which m a y be m o r e able to use C a 2+ as the charge carrier6,11. I n an a t t e m p t to shift the locus for spike i n i t i a t i o n t o w a r d s the cell body, the a b d o m i n a l ganglion was b a t h e d in artificial seawater in which N a + c o n c e n t r a t i o n was lowered by r e p l a c e m e n t with T H A M ( H y d r o x y m e t h y l ) a m i n o m e t h a n e ( F i s h e r Scientific). Fig. 2 A shows t h a t in lowered N a ÷, there is a distinct d i m i n u t i o n o f the shoulder on the rising phase o f the a x o n a l a c t i o n potential, a n d this is c o n c u r r e n t with a shift to simultaneous onset a n d similar rate-of-rise o f the spike in the two regions. Thus, it a p p e a r s t h a t u n d e r n o r m a l circumstances, the a x o n is the site for a c t i o n potential p r o d u c t i o n , b u t t h a t the m o r e p r o x i m a l a x o n or cell b o d y has the ability to generate a c t i o n potentials when the n o r m a l site is inhibited. The traces shown in Fig. 2B d e m o n s t r a t e t h a t spiking is initiated in the a x o n regardless o f whether a d e p o l a r i z i n g current is a p p l i e d to the cell b o d y or axon. This observation suggests t h a t the axon o f p a c e m a k e r cells has a lower
Fig. 2. A: effects of reduced sodium on left upper quadrant cell. Top traces show activity pattern of cell immediately prior to higher speed traces of an individual action potential, shown below. In these pairs of traces, cell body is on top and axon recording is below. 1, control, in normal artificial seawater; 2, after 10 rain in artificial seawater with 2 0 ~ of normal sodium content; 3, after 35 rain in reduced-sodium seawater; 4, 10 rain after return to normal artificial seawater. B • depolarization of cell body and axon of a weakly bursting left upper quadrant neuron. Cell body is upper and axon is shown in the lower trace. 1, first spike of a burst; 2, spike initiated by depolarization of axon during silent period; 3, spike initiated by depolarization of cell body during silent period. The cell body spike is of smaller amplitude because of depolarization induced by current injection, while the axonal spike is somewhat larger, presumably due to a decreased loading effect from the cell body. Calibrations: (A) top trace, 60 mV, 5 sec; bottom traces, 20 mV, 2 msec; (B) 20 mV, 2 msec.
204
A
B
C
Fig. 3. Axonal spiking in a voltage-clamped left upper quadrant cell. A: recording from the cell body prior to voltage clamping. B: voltage (upper) and current traces recorded from cell body in response to a depolarizing voltage clamp step from a holding potential of --40 mV. Following initial inward current surge (downward deflection represents inward current) are two smaller downward deflections, representing spiking in an undamped axonal region. C: voltage (upper) and current traces when cell is voltage clamped to a holding potential of--29 mV. Spontaneous axonal spikes similar to those seen in B are evident in the current trace. D: demonstration of isopotentiality in voltage-clamped cell body. Electrodes marked I and VI were used for voltage clamp. An independent voltage-sensing electrode was inserted first at site marked V2 and then at site marked V3. Voltage excursions resulting from identical depolarizing clamp steps were equivalent at all 3 sites. There was no sign of action potential voltage deflection at any of the sites, although these steps elicited large inward transient currents. Representation of neuron is diagrammatic. Relative positions of elements are accurate, but axon actually dipped straight into ganglion. Calibrations: (A) 50 mV, 20 sec; (B) 50 mV (top), 1/~A (bottom), 40 msec; (C) 50 mV (top), 500 nA (bottom), 20 sec; (D) 50 mV, 40 msec. threshold t h a n the cell body, and therefore pacemaker neurons resemble nonpacemaker cells in this respect7,10. In order to examine whether the axons o f burster pacemaker neurons were capable o f generating bursting patterns, voltage excursions o f the somatic membrane were blocked by voltage clamping the cell body. Delayed transient inward currents iesulting from action potentials generated in unclamped axonal regions are evident during depolarizing voltage clamp steps under these conditions 2. Fig. 3B illustrates this phenomenon. W h e n the cell b o d y of a majority o f endogenous bursters was voltage clamped at a steady holding potential between - - 3 0 and - - 2 5 mV, patterned action potential currents continued to be seen (Fig. 3C), indicating that the cell body was not necessary for their generation. The spike frequency within bursts as well as the
205 burst frequency were usually decreased when the cell body was clamped, suggesting that they may interact with the axonal site in the unmanipulated cell. An independent voltage-sensing electrode was inserted into various regions of the cell body to confirm that it was relatively well space-clamped, assuring that the action potential currents seen were actually arising from axonal regions (Fig. 3D). N o t every burster neuron examined showed axonal spiking when the cell body was voltage clamped. It is impossible to tell, in these cases, whether the cell body was the site of rhythmogenesis, or whether this site was in the axon with a large space constant between it and the cell body. When coupled with previous findings that the isolated cell body is capable of spike initiation and rhythmogenesisl, 4, these results suggest that different parts of the neuron are capable of expressing the characteristic electrical activity pattern of the cell. It is possible that the situation in rhythmogenic neurons is similar to that in the vertebrate heart, where more than one region is capable of pattern generation 8, and one region normally dominates. The possibility of an axonal site for rhythmogenesis may be important in the interpretation of data pertaining to the modulation of pacemaker activity. The recent findings of Wilson and Wachtel 1~ which show that a reduction of the negative slope resistance characteristic of bursting neurons is abolished by iontophoresis of transmitter substances onto the axon, whereas they are without effect when applied to the soma, are compatible with the presence of an axonal rhythmogenic site. This work was supported by Research Grant BNS 77-01548 from the National Science Foundation and G r a n t NS 15195-01 from National Institutes of Health. Part of the w o r k was conducted at the Marine Biological Laboratory, Woods Hole, Mass. 02543, U.S.A. 1 Alving, B. O., Spontaneous activity in isolated somata of Aplysia pacemaker neurons, J. gen. Physiol., 51 (1968) 29-45. 2 Barker, J. L. and Smith, T. G., Peptide regulation of neuronal membrane properties, Brain Research, 103 (1976) 167-170. 3 Chalazonitis, N. and Boisson, M. (Eds.), Abnormal Neuronal Discharges, Raven Press, New York, 1978. 4 Chen, C. F., Von Baumgarten, R. and Takeda, R., Pacemaker properties of completely isolated neurones in Aplysia californica, Nature New Biol., 233 (1971) 27-29. 5 Eccles, J. C., The Physiology of Synapses, Springer-Verlag, Berlin, 1964. 6 Junge, D. and Miller, J., Different spike mechanisms in axon and soma of molluscan neurones, Nature (Lond.), 252 (1974) 155-156. 7 Kado, R. T., Aplysia giant cell: soma-axon voltage clamp current differences, Science, 182 (1973) 843-845. 8 Marshall, J. M. In V. B. Mountcastle (Ed.), Medical Physiology, 1Iol. 1, C. V. Mosby, St. Louis, 1968. 9 Rasmussen, H. H., Change in spike initiation site in Helix pacemaker neurones, Comp. biochem. Physiol., 54 (1976) 315-318. 10 Tauc, L., Site of origin and propagation of spike in giant neuron of Aplysia, J. gen. Physiol., 45 (1962) 1099-1115. 11 Wald, F., Ionic differences between somatic and axonal action potentials in snail giant neurones, J. PhysioL (Lond.), 220 (1972) 267-281. 12 Wilson, W. A. and Wachtel, H., Prolonged inhibition in burst firing neurons: synaptic inactivation of slow regenerative inward current, Science, 202 (1978) 772-775.
201
Axonal site for impulse initiation and rhythmogenesis in Aplysia pacemaker neurons
STEVEN N. TREISTMAN Department of Biology, Bryn Mawr College, Bryn Mawr, Pa. 19010 (U.S.A.)
(Accepted December 6th, 1979) Key words: Aplysia pacemaker cell -- impulse initiation -- rhythmogenesis-- axon spikes
Spontaneous pacemaker activity is currently receiving a great deal of attention in neurobiology. Pacemaker neurons in molluscs have been the focus of a large numbe~ of investigations into the nature and modification of bursting pacemaker activity~. Much of this work has been predicated on an understanding that the cell body was the site of the subthreshold oscillatory rhythm and spike initiation in these cells. This understanding is based upon indirect evidence which suggests that cell bodies separated from their axons, either by ligation1 or enzymatic dissociation4, retain their ability to generate oscillations and associated action potentials, and inferences of action potential origin drawn from single electrode somatic recordings 9. A cell body locus for spike initiation would be different from that found in non-pacemaker Aplysia neurons 7,t°, as well as neurons from non-molluscan tissue 5, where spike initiation occurs in the axon. Since such a difference is, potentially, of great importance in the basic biology of the neuron, as well as in the interpretation of data regarding pacemaker activity, the locus of spike initiation in intact pacemaker neurons of Aplysia californica was explored by simultaneous intracellular recordings from different regions of the spontaneously active cell. Abdominal ganglia from Aplysia were removed through an incision in the foot and pinned out through the connective tissue sheath. The connective tissue overlying a hemiganglion was removed by microdissection, and individual somata separated from neighboring cells by removing the surrounding cells with a glass needle. After penetration of the cell body with a micropipette electrode containing 3 M KC1, the region surrounding the cell body was probed with a second micropipette electrode until an axon was impaled which exhibited spiking time-locked to that occurring in the cell body. Recording was by conventional methods and intracellular stimulation was done with a bridge circuit. Voltage clamp data were obtained with independent voltage and current electrodes, by conventional means. Fig. 1A and B show traces from the cell body and axon of pacemaker neurons, in which it is clear that the onset of the action potential occurs first in the axon. Also evident is the faster rate-of-rise of the axon spike, indicating an electrode position closer to the site of origin.
202
Fig. 1. Simultaneous recordings from cell body and axon of pacemaker neurons. In all pairs of traces, cell body is upper and axon recording is the lower. A: recordings from a left upper quadrant cell. The cell was beating, and traces are of spontaneous action potentials. In top pair, both electrodes are in cell body. The middle pair of traces represent cell body and an axon impalement approximately 0.5 mm from medial edge of cell body. In the bottom pair, the axon electrode was moved to approximately 1.0 mm medial to edge of cell body. B : recordings from a bursting left upper quadrant cell, illustrating a prominent inflection in axonal action potential. Uppermost pair of traces are both from the cell body. Lower pair shows traces after axon electrode was moved to a point approximately 1.0 mm from base of cell body. Traces represent first spike in spontaneous bursts. C: recordings from a bursting left upper quadrant cell. Upper traces are from the cell body and axon, approximately 1.0 mm from border of cell body, during the first spike of a spontaneous burst. Bottom pair of traces showing the start of the action potential, are from the same electrode placements, during an antidromic spike initiated by stimulation of the genital-pericardial nerve. Apparent differences in amplitude between the spontaneous and antidromic spikes result from the loss of the slow depolarization leading to the spontaneous spike, from the picture. The shoulder on the axonal spike is larger and of longer duration following antidromic stimulation. Calibrations: (A) 20 mV, 2 msec; (B) top, 20 mV, 2 msec; bottom, 20 mV, 5 msec; (C) 20 mV, 2 msec.
One n o t a b l e difference between action potential shape in the cell body a n d axon is a p r o m i n e n t inflection on the rising phase of the axonal spike. This inflection is more p r o m i n e n t in the axon of some cells t h a n others. F o r example, cell R15 typically has a less p r o m i n e n t inflection t h a n the axon of the left upper q u a d r a n t cells at a similar distance from the cell body. This inflection coincides with the rise of the cell body spike, a n d m a y reflect the loading effect of the cell body m e m b r a n e on the current generator in the axon t°. If this interpretation is correct, a geometry similar to that occurring spontaneously should result when the cell is activated antidromically, where the action potential definitely arises in the axon. We tested this by stimulating left u p p e r q u a d r a n t cells t h r o u g h a suction electrode o n the distal genital-pericardial nerve, which contains a n axon of these cells, while recording from the cell body a n d
203 m o r e p r o x i m a l axon, intracellularly. Fig. 1C shows t h a t a similar inflection is present on the rising phase o f the a x o n a c t i o n potential. It has been n o t e d in a n u m b e r o f m o l l u s c a n neurons, t h a t the a x o n a l a c t i o n p o t e n t i a l is m o r e N a + - d e p e n d e n t t h a n is the cell b o d y a n d / o r p r o x i m a l axon spike, which m a y be m o r e able to use C a 2+ as the charge carrier6,11. I n an a t t e m p t to shift the locus for spike i n i t i a t i o n t o w a r d s the cell body, the a b d o m i n a l ganglion was b a t h e d in artificial seawater in which N a + c o n c e n t r a t i o n was lowered by r e p l a c e m e n t with T H A M ( H y d r o x y m e t h y l ) a m i n o m e t h a n e ( F i s h e r Scientific). Fig. 2 A shows t h a t in lowered N a ÷, there is a distinct d i m i n u t i o n o f the shoulder on the rising phase o f the a x o n a l a c t i o n potential, a n d this is c o n c u r r e n t with a shift to simultaneous onset a n d similar rate-of-rise o f the spike in the two regions. Thus, it a p p e a r s t h a t u n d e r n o r m a l circumstances, the a x o n is the site for a c t i o n potential p r o d u c t i o n , b u t t h a t the m o r e p r o x i m a l a x o n or cell b o d y has the ability to generate a c t i o n potentials when the n o r m a l site is inhibited. The traces shown in Fig. 2B d e m o n s t r a t e t h a t spiking is initiated in the a x o n regardless o f whether a d e p o l a r i z i n g current is a p p l i e d to the cell b o d y or axon. This observation suggests t h a t the axon o f p a c e m a k e r cells has a lower
Fig. 2. A: effects of reduced sodium on left upper quadrant cell. Top traces show activity pattern of cell immediately prior to higher speed traces of an individual action potential, shown below. In these pairs of traces, cell body is on top and axon recording is below. 1, control, in normal artificial seawater; 2, after 10 rain in artificial seawater with 2 0 ~ of normal sodium content; 3, after 35 rain in reduced-sodium seawater; 4, 10 rain after return to normal artificial seawater. B • depolarization of cell body and axon of a weakly bursting left upper quadrant neuron. Cell body is upper and axon is shown in the lower trace. 1, first spike of a burst; 2, spike initiated by depolarization of axon during silent period; 3, spike initiated by depolarization of cell body during silent period. The cell body spike is of smaller amplitude because of depolarization induced by current injection, while the axonal spike is somewhat larger, presumably due to a decreased loading effect from the cell body. Calibrations: (A) top trace, 60 mV, 5 sec; bottom traces, 20 mV, 2 msec; (B) 20 mV, 2 msec.
204
A
B
C
Fig. 3. Axonal spiking in a voltage-clamped left upper quadrant cell. A: recording from the cell body prior to voltage clamping. B: voltage (upper) and current traces recorded from cell body in response to a depolarizing voltage clamp step from a holding potential of --40 mV. Following initial inward current surge (downward deflection represents inward current) are two smaller downward deflections, representing spiking in an undamped axonal region. C: voltage (upper) and current traces when cell is voltage clamped to a holding potential of--29 mV. Spontaneous axonal spikes similar to those seen in B are evident in the current trace. D: demonstration of isopotentiality in voltage-clamped cell body. Electrodes marked I and VI were used for voltage clamp. An independent voltage-sensing electrode was inserted first at site marked V2 and then at site marked V3. Voltage excursions resulting from identical depolarizing clamp steps were equivalent at all 3 sites. There was no sign of action potential voltage deflection at any of the sites, although these steps elicited large inward transient currents. Representation of neuron is diagrammatic. Relative positions of elements are accurate, but axon actually dipped straight into ganglion. Calibrations: (A) 50 mV, 20 sec; (B) 50 mV (top), 1/~A (bottom), 40 msec; (C) 50 mV (top), 500 nA (bottom), 20 sec; (D) 50 mV, 40 msec. threshold t h a n the cell body, and therefore pacemaker neurons resemble nonpacemaker cells in this respect7,10. In order to examine whether the axons o f burster pacemaker neurons were capable o f generating bursting patterns, voltage excursions o f the somatic membrane were blocked by voltage clamping the cell body. Delayed transient inward currents iesulting from action potentials generated in unclamped axonal regions are evident during depolarizing voltage clamp steps under these conditions 2. Fig. 3B illustrates this phenomenon. W h e n the cell b o d y of a majority o f endogenous bursters was voltage clamped at a steady holding potential between - - 3 0 and - - 2 5 mV, patterned action potential currents continued to be seen (Fig. 3C), indicating that the cell body was not necessary for their generation. The spike frequency within bursts as well as the
205 burst frequency were usually decreased when the cell body was clamped, suggesting that they may interact with the axonal site in the unmanipulated cell. An independent voltage-sensing electrode was inserted into various regions of the cell body to confirm that it was relatively well space-clamped, assuring that the action potential currents seen were actually arising from axonal regions (Fig. 3D). N o t every burster neuron examined showed axonal spiking when the cell body was voltage clamped. It is impossible to tell, in these cases, whether the cell body was the site of rhythmogenesis, or whether this site was in the axon with a large space constant between it and the cell body. When coupled with previous findings that the isolated cell body is capable of spike initiation and rhythmogenesisl, 4, these results suggest that different parts of the neuron are capable of expressing the characteristic electrical activity pattern of the cell. It is possible that the situation in rhythmogenic neurons is similar to that in the vertebrate heart, where more than one region is capable of pattern generation 8, and one region normally dominates. The possibility of an axonal site for rhythmogenesis may be important in the interpretation of data pertaining to the modulation of pacemaker activity. The recent findings of Wilson and Wachtel 1~ which show that a reduction of the negative slope resistance characteristic of bursting neurons is abolished by iontophoresis of transmitter substances onto the axon, whereas they are without effect when applied to the soma, are compatible with the presence of an axonal rhythmogenic site. This work was supported by Research Grant BNS 77-01548 from the National Science Foundation and G r a n t NS 15195-01 from National Institutes of Health. Part of the w o r k was conducted at the Marine Biological Laboratory, Woods Hole, Mass. 02543, U.S.A. 1 Alving, B. O., Spontaneous activity in isolated somata of Aplysia pacemaker neurons, J. gen. Physiol., 51 (1968) 29-45. 2 Barker, J. L. and Smith, T. G., Peptide regulation of neuronal membrane properties, Brain Research, 103 (1976) 167-170. 3 Chalazonitis, N. and Boisson, M. (Eds.), Abnormal Neuronal Discharges, Raven Press, New York, 1978. 4 Chen, C. F., Von Baumgarten, R. and Takeda, R., Pacemaker properties of completely isolated neurones in Aplysia californica, Nature New Biol., 233 (1971) 27-29. 5 Eccles, J. C., The Physiology of Synapses, Springer-Verlag, Berlin, 1964. 6 Junge, D. and Miller, J., Different spike mechanisms in axon and soma of molluscan neurones, Nature (Lond.), 252 (1974) 155-156. 7 Kado, R. T., Aplysia giant cell: soma-axon voltage clamp current differences, Science, 182 (1973) 843-845. 8 Marshall, J. M. In V. B. Mountcastle (Ed.), Medical Physiology, 1Iol. 1, C. V. Mosby, St. Louis, 1968. 9 Rasmussen, H. H., Change in spike initiation site in Helix pacemaker neurones, Comp. biochem. Physiol., 54 (1976) 315-318. 10 Tauc, L., Site of origin and propagation of spike in giant neuron of Aplysia, J. gen. Physiol., 45 (1962) 1099-1115. 11 Wald, F., Ionic differences between somatic and axonal action potentials in snail giant neurones, J. PhysioL (Lond.), 220 (1972) 267-281. 12 Wilson, W. A. and Wachtel, H., Prolonged inhibition in burst firing neurons: synaptic inactivation of slow regenerative inward current, Science, 202 (1978) 772-775.