177
Brain Research, 133 (1977) 177-182 © Elsevier/North-Holland Biomedical Press
Tetrodotoxin-resistant divalent action potentials in an axon of
Aplysio
RICHARD HORN
Department of Anatomy, University of California, Los Angeles, Calif. 90024 (U.S.A.) (Accepted May 25th, 1977)
The replacement of Na ion with a relatively nonpermeant cation, such as Tris + or choline + ,usually abolishes action potentials of vertebrate and invertebrate axons 5. In several crustacean and molluscan neurons the somata are capable of producing Ca spikes in Na-free medium2,6-s,17, 21. Axon spikes of the same cells are abolished by Na-free and in most cases by tetrodotoxin (TTX)-containing solutions. Under special conditions Ca spikes can be elicited in squid axon la. These action potentials are TTXsensitive 22, and are believed to be due to Ca passing through the Na channel 14. However a TTX-insensitive Ca conductance has been described in certain invertebrate axonslS, ~s, and in presynaptic terminals 9-11. The soma of the giant neuron R2 of Aplysia californica is capable of producing all-or-none Ca spikes in the absence of Na (see ref. 2). The R2 axon spike is abolished by Na-free or TTX-containing mediumS, a, suggesting that it may be a pure Na spike. This report will describe the ability of R2 axon to produce conducted, TTX-insensitive, Ca spikes in the presence of intracellular tetraethylammonium (TEAl) and Ba spikes in the absence of TEA. The preparation of the visceral ganglion together with the right connective containing the axon of R2 has been described previously 6,s, and involves pretreatment with Pronase. Microelectrodes (2-15 Mg)) were used for somatic and axonal impalement. Extracellular recordings indicate that regenerative activity in the axon is abolished by Na-free medium as close as 250 #m from the axo-somatic junction s. Accordingly, all axonal impalements were made at least 1.5 mm from the soma. The axonal electrode was filled with 3 M KC1. Iontophoretic application of TEA was accomplished by passing current pulses (800 msec, 150-400 hA, 1/sec) between a 2.5 M TEA Br and a 3 M potassium acetate (KAc) electrode in the soma. Somatic membrane potential was recorded with the KAc electrode, or with a KC1 electrode in the studies of Ba spikes. Experiments were conducted at 21-23 °C. Solution changes were made by perfusing at least 20 times the chamber volume of each solution. Normal saline (NS) had the composition: NaC1 494 mM, KC1 11 mM, CaC12 11 mM, MgCl2 19 mM, MgSO4 30 mM, Tris.HC1 (pH = 7.7) 10 raM. Variations in concentrations of ions were achieved by substituting osmotically equivalent amounts of Tris.HCl (pK = 8.3).
178
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Fig. 1. Effect of TEAl on somatic and axonal action potentials. Somatic and axonal membrane potentials are depicted. Dashed lines indicate bath potentials. The axon was impaled 2.0 mm from the soma. Row A shows spikes in NS. Row B was recorded two hours after iontophoresis of TEA into the soma. B1 in is NS. B2 and 3 show that the axon spike is reduced 3 and 10 rain after exposure to Na-free medium. Row C was recorded 90 min after row B. C2 shows an axon spike after 15 min in Na-free medium.
Fig. 1, row A, shows somatic a n d a x o n a l action potentials in N S generated by an i n t r a s o m a t i c c u r r e n t pulse. The b a t h potentials for the axon a n d s o m a electrodes are i n d i c a t e d by d a s h e d lines. The axon was i m p a l e d 2.0 m m f r o m the soma. Subsequent to the records in row A, 3.6 × 10 -4 C o f charge was passed over a 50 min p e r i o d between the T E A a n d K A c electrodes in the soma. The records in row B were filmed two hours after the i o n t o p h o r e t i c current was discontinued. D u r i n g this p e r i o d the d u r a t i o n o f the axon spike increased, indicating t h a t T E A diffuses d o w n the axon, as r e p o r t e d previously 1. B1 was r e c o r d e d in n o r m a l saline. B2 a n d 3 show action potentials rec o r d e d 3 a n d 10 min after the N a was replaced by Tris buffer. The s o m a c o n t i n u e d to fire, b u t the a x o n spike was greatly reduced. Because o f the electrotonic coupling between the s o m a a n d this p a r t o f the axon, it is n o t clear if the a x o n a l response h a d a regenerative c o m p o n e n t . C1 shows action potentials in N S r e c o r d e d 90 min after o b t a i n i n g record B3. The increased d u r a t i o n o f somatic a n d axonal a c t i o n potentials indicates t h a t at this time o u t w a r d current h a d further been decreased by the TEAI. C2 shows that the a x o n c o n t i n u e d to fire 15 min after N a was r e m o v e d f r o m the bath. The rate o f rise a n d c o n d u c t i o n velocity were b o t h decreased by the r e m o v a l o f Na, indicating t h a t N a
179 current contributes to the rising phase of the axon spike. C3 shows action potentials 10 min after immersion in NS. The axon spike in C2 was an active, not an electrotonic, event because (1) it had a greater amplitude and overshoot potential than the soma spike, and (2) an extracellular electrode at this location on the axon showed a negative potential during the rising phase o f the intracellular potential, indicating net inward current at this location. In the absence o f TEAl the negative extracellular potential of the axon is abolished by Na-free medium 6. The presence o f an action potential in Na-free medium at a given location on the axon apparently requires time for a sufficient concentration o f TEAi to diffuse to that location. The diffusion is slow enough in the course of an experiment that the spikes never c o n d u c t more than a few m m distal to the soma. In the absence o f TEAi, I was unable to elicit an axon spike in Na-free medium enriched with 100 m M T E A Br. At distances < 3 m m from the soma this concentration o f extracellular T E A causes prolonged axon spikes in the presence o f Na. A possible effect o f the anion, acetate, was dismissed after injecting 10-3 C between two K A c electrodes in the soma over a 2-h period. N o prolongation o f spikes was observed, n o r was the axon capable o f firing in the absence o f Na, as close as 250 # m from the soma. Fig. 2 shows that the axon spike in Na-free medium has several features characteristic of a Ca spike. I n this preparation, impaled at 1.6 m m f r o m the soma, an axon spike could be elicited in Na-free medium one h o u r after passing 2.5 × 10-4 C o f charge between the iontophoretic electrodes in the soma. Somatic and axonal potentials are indicated together with intrasomatic stimulating current. Fig. 2A is in NS one h o u r after injection o f TEA, and shows two axon spikes in response to a single somatic spike. Fig. 2B shows that the axon continued to fire in Na-free medium. Fig. 2C shows that the addition o f 3 0 / z M T T X failed to abolish the action potentials in b o t h the soma and axon. This concentration of T T X abolishes b o t h the axon spike and the N a c o m p o n e n t of the somatic spike in the absence o f TEAi (see refs. 2 and 8). In Na-free m e d i u m
A
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Fig. 2. Study of action potentials in Na-free medium. Membrane potentials and intrasomatic stimulating currents are shown. The axon was impaled 1.6 mm from the soma. All records were filmed one hour after iontophoretic application of TEA. A is in NS. B and C show that 30 pM TTX does not abolish the spikes in O Na. D and E show that neither the addition of 30 mM Co z+ nor the removal of Ca from the medium completely abolishes spikes. Addition of 1 mM EGTA to the Na- and Ca-free medium rapidly abolishes regenerative activity. G is in NS.
180 30 m M COC12 blocks the Ca spike in R2 somaL Fig. 2D shows that neither the soma spike nor the axon spike were completely blocked by 30 m M Co ~÷, although both were markedly reduced. Even the removal o f Ca f r o m the Na-free medium failed to prevent regenerative events in the soma and axon (Fig. 2E). However, the addition o f 1 m M ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), a Ca chelating agent, to the Na- and Ca-free medium rapidly abolished all regenerative activity (Fig. 2F). This result suggests that in a nominally Ca-free medium some Ca may be retained in extracellular sites near the membrane. Fig. 2G shows recovery in NS. The rising phase of the axon spike in NS precedes that of the soma, in agreement with earlier work showing a spike triggering zone on the proximal axon ~0. Subsequent experiments (in preparation) show that the overshoot potential of the axon spike in Na-free medium responds to changes in extracellular Ca concentration approximately in the manner predicted by the Nernst equation for a Ca electrode. These results strongly indicate that the axon spike in Na-free medium is a Ca spike and that, unlike the Ca spike in squid axon 14, inward current is passing t h r o u g h a Ca channel separate from the N a channel. Fig. 3 shows that, in the absence of TEA, Ba spikes can be elicited in the R2 axon. Ba is more permeable than Ca in Ca channels, and in some preparations blocks K efflux ~. In Fig. 3A, B and C spikes were generated by stimulating the cut end of the right connective. The axon was impaled 5.8 m m from the soma. The stimulus artifact is most obvious in Fig. 3A. The dashed line is the zero potential for both the somatic and axonal traces. N o t e the variation in sweep speeds. Fig. 3A is in NS. Fig. 3B shows that
A
B NS
.
.
.
L .
.
.
.
.
.
!
50 ms
Is
OJs
50 ms
Fig. 3. Ba spikes in the soma and axon. Somatic and axonal potentials are indicated. The dashed line is bath potential for both the somatic and the axonal electrode. Note changes in sweep speed. Axon impaled 5.8 mm from the soma. In A, B and C spikes were generated by stimulating the end of the connective with a hook electrode 2.5 cm distal (rostral) to the axon microelectrode. A is in NS. B and C are in 60 mMBa 2+ (see text), and show a single antidromic axon spike followed by a somatic action potential and a train of orthodromic axon spikes. D shows that 6 mM Ca 2+ inhibits the Ba spike.
181 a x o n spikes can be g e n e r a t e d in a Na-free m e d i u m in which all o f the C a a n d M g are replaced by a n e q u i m o l a r c o n c e n t r a t i o n o f Ba (60 m M ) . A single a n t i d r o m i c axon spike was generated at the distal end o f the right connective a n d i n v a d e d the soma, causing a 3.5-sec somatic spike (Fig. 3C), which in turn caused repetitive o r t h o d r o m i c spikes in the axon. The a d d i t i o n o f 6 m M Ca e+ effectively p r e v e n t e d the c o n d u c t i o n o f the a n t i d r o m i c a x o n spike ( n o t shown). Fig. 3D shows t h a t in this solution the d u r a t i o n a n d a m p l i t u d e o f the somatic a c t i o n potential, generated by i n t r a s o m a t i c stimulation, decreased. The decreased rate o f rise o f the a x o n spike suggests t h a t it decremented. C a m a y have two a c t i o n s : (1) it m a y c o m p e t i t i v e l y inhibit Ba c u r r e n t 3, a n d / o r (2) it m a y activate p o t a s s i u m c o n d u c t a n c O , 12A3, which shunts the i n w a r d current. A s r e p o r t e d a b o v e for the Ca spike, 30 # M T T X has little or no influence on a c t i o n p o t e n t i a l s in 60 m M Ba. A divalent channel, distinct f r o m the N a channel a n d c a p a b l e o f passing a net i n w a r d current, exists in the a x o n o f R2. This channel m a y be c o m p a r a b l e with the voltage-sensitive Ca channel o f the s o m a o f this cell 2, a n d with that o f p r e s y n a p t i c terminals 9-11. T E A i m a y b l o c k the early o u t w a r d K efflux 15 which usually counteracts i n w a r d Ca current. I t h a n k D. Junge for the facilities a n d his help, a n d M. Brodwick, R. Eckert, a n d S. H a g i w a r a for c o m m e n t s o n the manuscript.
1 Bryant, H. L. and Weinreich, D., Monosynaptic connexions among Aplysia neurones examined by the intracellular application of TEA, J. PhysioL (Lond.), 244 (1975) 181-195. 2 Geduldig, D. and Junge, D., Sodium and calcium components of action potentials in the Aplysia giant neurone, J. PhysioL (Lond.), 199 (1968) 347-365. 3 Hagiwara, S., Ca-dependent action potential. In G. Eisenman (Ed.), Membranes, .4 Series of Advances, VoL 3, Dekker, New York, 1975, pp. 359-381. 4 Heyer, C. B. and Lux, H. D., Control of the delayed outward potassium currents in bursting pacemaker neurones of the snail, Helix pomatia, J. PhysioL (Lond.), 262 (1976) 349-382. 5 Hodgkin, A. L., The ionic basis of electrical activity in nerve and muscle, Biol. Rev., 26 (1951) 339-409. 6 Horn, R. and Miller, J. J., A prolonged, voltage-dependent calcium permeability revealed by tetraethylammonium in the soma and axon of Aplysia giant neuron, J. NeurobioL, (1977) in press. 7 Iwasaki, S. and Satow, Y., Sodium- and calcium-dependent spike potentials in the secretory neuron soma of the X-organ of the crayfish, J. gen. PhysioL, 57 (1971) 216-238. 8 Junge, D. and Miller, J., Different spike mechanisms in axon and soma of molluscan neurone, Nature (Lond.), 252 (1974) 155-156. 9 Katz, B. and Miledi, R., The release of acetylcholine from nerve endings by graded electric pulses, Proc. roy. Soc. B, 167 (1967) 23-38. 10 Katz, B. and Miledi, R., Tetrodotoxin-resistant electric activity in presynaptic terminals, J. Physiol. (Lond.), 203 (1969) 459-487. 11 Llimis, R., Steinberg, I. Z. and Walton, K., Presynaptic calcium currents and their relation to synaptic transmission: voltage clamp study in squid giant synapse and theoretical model for the calcium gate, Proc. nat. Acad. Sci (Wash.), 73 (1976) 2918-2922. 12 Meech, R. W., Intracellular calcium injection causes increased potassium conductance in Aplysia nerve cells, Comp. Biochem. PhysioL, 42A (1972) 493-499. 13 Meech, R. W. and Standen, N. B., Potassium activation in Helix aspersa neurones under voltage clamp: a component mediated by calcium influx, J. Physiol. (Lond.), 249 (1975) 211-239. 14 Meves, H. and Vogel, W., Calcium inward currents in internally perfused giant axons, J. Physiol. (Lond.), 235 (1973) 225-265.
182 15 Neher, E. and Lux, H. D., Differential action of TEA on two K-current components of a molluscan neurone, Pfliigers Arch. ges. Physiol., 336 (1972) 87-100. 16 Orchard, I., Calcium dependent action potentials in a peripheral neurosecretory cell of the stick insect, J. comp. Physiol., 112 (1976) 95-102. 17 Standen, N. B., Calcium and sodium ions as charge carriers in the action potential of an identified snail neurone, J. Physiol. (Lond.), 249 (1975) 241-252. 18 Suzuki, J., Calcium activation in the giant axon of the crayfish. In J. P. Reuben, D. P. Purpura, M. V. L. Bennett and E. R. Kandel (Eds.), Electrobiology of Nerve, Synapse, and Muscle, Raven, New York, 1976, pp. 27-35. 19 Tasaki, I., Watanabe, A. and Singer, I., Excitability of squid giant axons in the absence of univalent cations in the external medium, Proc. nat. Acad. Sci. (Wash.), 56 (1966) 1116-1122. 20 Tauc, L., Site of origin and propagation of spike in the giant neuron of Aplysia, J. gen. Physiol., 45 (1962) 1077-1097. 21 Wald, F., Ionic differences between somatic and axonal action potentials in snail giant neurones, J. Physiol. (Lond.), 220 (1972) 267-281. 22 Watanabe, A., Tasaki, I., Singer, I. and Lerman, L., Effect of tetrodotoxin on excitability of squid giant axons in sodium-free media, Science, 155 (1967) 95-97.
Brain Research, 133 (1977) 177-182 © Elsevier/North-Holland Biomedical Press
Tetrodotoxin-resistant divalent action potentials in an axon of
Aplysio
RICHARD HORN
Department of Anatomy, University of California, Los Angeles, Calif. 90024 (U.S.A.) (Accepted May 25th, 1977)
The replacement of Na ion with a relatively nonpermeant cation, such as Tris + or choline + ,usually abolishes action potentials of vertebrate and invertebrate axons 5. In several crustacean and molluscan neurons the somata are capable of producing Ca spikes in Na-free medium2,6-s,17, 21. Axon spikes of the same cells are abolished by Na-free and in most cases by tetrodotoxin (TTX)-containing solutions. Under special conditions Ca spikes can be elicited in squid axon la. These action potentials are TTXsensitive 22, and are believed to be due to Ca passing through the Na channel 14. However a TTX-insensitive Ca conductance has been described in certain invertebrate axonslS, ~s, and in presynaptic terminals 9-11. The soma of the giant neuron R2 of Aplysia californica is capable of producing all-or-none Ca spikes in the absence of Na (see ref. 2). The R2 axon spike is abolished by Na-free or TTX-containing mediumS, a, suggesting that it may be a pure Na spike. This report will describe the ability of R2 axon to produce conducted, TTX-insensitive, Ca spikes in the presence of intracellular tetraethylammonium (TEAl) and Ba spikes in the absence of TEA. The preparation of the visceral ganglion together with the right connective containing the axon of R2 has been described previously 6,s, and involves pretreatment with Pronase. Microelectrodes (2-15 Mg)) were used for somatic and axonal impalement. Extracellular recordings indicate that regenerative activity in the axon is abolished by Na-free medium as close as 250 #m from the axo-somatic junction s. Accordingly, all axonal impalements were made at least 1.5 mm from the soma. The axonal electrode was filled with 3 M KC1. Iontophoretic application of TEA was accomplished by passing current pulses (800 msec, 150-400 hA, 1/sec) between a 2.5 M TEA Br and a 3 M potassium acetate (KAc) electrode in the soma. Somatic membrane potential was recorded with the KAc electrode, or with a KC1 electrode in the studies of Ba spikes. Experiments were conducted at 21-23 °C. Solution changes were made by perfusing at least 20 times the chamber volume of each solution. Normal saline (NS) had the composition: NaC1 494 mM, KC1 11 mM, CaC12 11 mM, MgCl2 19 mM, MgSO4 30 mM, Tris.HC1 (pH = 7.7) 10 raM. Variations in concentrations of ions were achieved by substituting osmotically equivalent amounts of Tris.HCl (pK = 8.3).
178
I
v,
I
NS
L
2
1
NS
...................
B I
Q
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NS
,
C
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Ns
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Fig. 1. Effect of TEAl on somatic and axonal action potentials. Somatic and axonal membrane potentials are depicted. Dashed lines indicate bath potentials. The axon was impaled 2.0 mm from the soma. Row A shows spikes in NS. Row B was recorded two hours after iontophoresis of TEA into the soma. B1 in is NS. B2 and 3 show that the axon spike is reduced 3 and 10 rain after exposure to Na-free medium. Row C was recorded 90 min after row B. C2 shows an axon spike after 15 min in Na-free medium.
Fig. 1, row A, shows somatic a n d a x o n a l action potentials in N S generated by an i n t r a s o m a t i c c u r r e n t pulse. The b a t h potentials for the axon a n d s o m a electrodes are i n d i c a t e d by d a s h e d lines. The axon was i m p a l e d 2.0 m m f r o m the soma. Subsequent to the records in row A, 3.6 × 10 -4 C o f charge was passed over a 50 min p e r i o d between the T E A a n d K A c electrodes in the soma. The records in row B were filmed two hours after the i o n t o p h o r e t i c current was discontinued. D u r i n g this p e r i o d the d u r a t i o n o f the axon spike increased, indicating t h a t T E A diffuses d o w n the axon, as r e p o r t e d previously 1. B1 was r e c o r d e d in n o r m a l saline. B2 a n d 3 show action potentials rec o r d e d 3 a n d 10 min after the N a was replaced by Tris buffer. The s o m a c o n t i n u e d to fire, b u t the a x o n spike was greatly reduced. Because o f the electrotonic coupling between the s o m a a n d this p a r t o f the axon, it is n o t clear if the a x o n a l response h a d a regenerative c o m p o n e n t . C1 shows action potentials in N S r e c o r d e d 90 min after o b t a i n i n g record B3. The increased d u r a t i o n o f somatic a n d axonal a c t i o n potentials indicates t h a t at this time o u t w a r d current h a d further been decreased by the TEAI. C2 shows that the a x o n c o n t i n u e d to fire 15 min after N a was r e m o v e d f r o m the bath. The rate o f rise a n d c o n d u c t i o n velocity were b o t h decreased by the r e m o v a l o f Na, indicating t h a t N a
179 current contributes to the rising phase of the axon spike. C3 shows action potentials 10 min after immersion in NS. The axon spike in C2 was an active, not an electrotonic, event because (1) it had a greater amplitude and overshoot potential than the soma spike, and (2) an extracellular electrode at this location on the axon showed a negative potential during the rising phase o f the intracellular potential, indicating net inward current at this location. In the absence o f TEAl the negative extracellular potential of the axon is abolished by Na-free medium 6. The presence o f an action potential in Na-free medium at a given location on the axon apparently requires time for a sufficient concentration o f TEAi to diffuse to that location. The diffusion is slow enough in the course of an experiment that the spikes never c o n d u c t more than a few m m distal to the soma. In the absence o f TEAi, I was unable to elicit an axon spike in Na-free medium enriched with 100 m M T E A Br. At distances < 3 m m from the soma this concentration o f extracellular T E A causes prolonged axon spikes in the presence o f Na. A possible effect o f the anion, acetate, was dismissed after injecting 10-3 C between two K A c electrodes in the soma over a 2-h period. N o prolongation o f spikes was observed, n o r was the axon capable o f firing in the absence o f Na, as close as 250 # m from the soma. Fig. 2 shows that the axon spike in Na-free medium has several features characteristic of a Ca spike. I n this preparation, impaled at 1.6 m m f r o m the soma, an axon spike could be elicited in Na-free medium one h o u r after passing 2.5 × 10-4 C o f charge between the iontophoretic electrodes in the soma. Somatic and axonal potentials are indicated together with intrasomatic stimulating current. Fig. 2A is in NS one h o u r after injection o f TEA, and shows two axon spikes in response to a single somatic spike. Fig. 2B shows that the axon continued to fire in Na-free medium. Fig. 2C shows that the addition o f 3 0 / z M T T X failed to abolish the action potentials in b o t h the soma and axon. This concentration of T T X abolishes b o t h the axon spike and the N a c o m p o n e n t of the somatic spike in the absence o f TEAi (see refs. 2 and 8). In Na-free m e d i u m
A
c
Txomn 50 r~
D
oNo3ocomin E
ONQOCaSmnFONoOCoEGTA2mn
Fig. 2. Study of action potentials in Na-free medium. Membrane potentials and intrasomatic stimulating currents are shown. The axon was impaled 1.6 mm from the soma. All records were filmed one hour after iontophoretic application of TEA. A is in NS. B and C show that 30 pM TTX does not abolish the spikes in O Na. D and E show that neither the addition of 30 mM Co z+ nor the removal of Ca from the medium completely abolishes spikes. Addition of 1 mM EGTA to the Na- and Ca-free medium rapidly abolishes regenerative activity. G is in NS.
180 30 m M COC12 blocks the Ca spike in R2 somaL Fig. 2D shows that neither the soma spike nor the axon spike were completely blocked by 30 m M Co ~÷, although both were markedly reduced. Even the removal o f Ca f r o m the Na-free medium failed to prevent regenerative events in the soma and axon (Fig. 2E). However, the addition o f 1 m M ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), a Ca chelating agent, to the Na- and Ca-free medium rapidly abolished all regenerative activity (Fig. 2F). This result suggests that in a nominally Ca-free medium some Ca may be retained in extracellular sites near the membrane. Fig. 2G shows recovery in NS. The rising phase of the axon spike in NS precedes that of the soma, in agreement with earlier work showing a spike triggering zone on the proximal axon ~0. Subsequent experiments (in preparation) show that the overshoot potential of the axon spike in Na-free medium responds to changes in extracellular Ca concentration approximately in the manner predicted by the Nernst equation for a Ca electrode. These results strongly indicate that the axon spike in Na-free medium is a Ca spike and that, unlike the Ca spike in squid axon 14, inward current is passing t h r o u g h a Ca channel separate from the N a channel. Fig. 3 shows that, in the absence of TEA, Ba spikes can be elicited in the R2 axon. Ba is more permeable than Ca in Ca channels, and in some preparations blocks K efflux ~. In Fig. 3A, B and C spikes were generated by stimulating the cut end of the right connective. The axon was impaled 5.8 m m from the soma. The stimulus artifact is most obvious in Fig. 3A. The dashed line is the zero potential for both the somatic and axonal traces. N o t e the variation in sweep speeds. Fig. 3A is in NS. Fig. 3B shows that
A
B NS
.
.
.
L .
.
.
.
.
.
!
50 ms
Is
OJs
50 ms
Fig. 3. Ba spikes in the soma and axon. Somatic and axonal potentials are indicated. The dashed line is bath potential for both the somatic and the axonal electrode. Note changes in sweep speed. Axon impaled 5.8 mm from the soma. In A, B and C spikes were generated by stimulating the end of the connective with a hook electrode 2.5 cm distal (rostral) to the axon microelectrode. A is in NS. B and C are in 60 mMBa 2+ (see text), and show a single antidromic axon spike followed by a somatic action potential and a train of orthodromic axon spikes. D shows that 6 mM Ca 2+ inhibits the Ba spike.
181 a x o n spikes can be g e n e r a t e d in a Na-free m e d i u m in which all o f the C a a n d M g are replaced by a n e q u i m o l a r c o n c e n t r a t i o n o f Ba (60 m M ) . A single a n t i d r o m i c axon spike was generated at the distal end o f the right connective a n d i n v a d e d the soma, causing a 3.5-sec somatic spike (Fig. 3C), which in turn caused repetitive o r t h o d r o m i c spikes in the axon. The a d d i t i o n o f 6 m M Ca e+ effectively p r e v e n t e d the c o n d u c t i o n o f the a n t i d r o m i c a x o n spike ( n o t shown). Fig. 3D shows t h a t in this solution the d u r a t i o n a n d a m p l i t u d e o f the somatic a c t i o n potential, generated by i n t r a s o m a t i c stimulation, decreased. The decreased rate o f rise o f the a x o n spike suggests t h a t it decremented. C a m a y have two a c t i o n s : (1) it m a y c o m p e t i t i v e l y inhibit Ba c u r r e n t 3, a n d / o r (2) it m a y activate p o t a s s i u m c o n d u c t a n c O , 12A3, which shunts the i n w a r d current. A s r e p o r t e d a b o v e for the Ca spike, 30 # M T T X has little or no influence on a c t i o n p o t e n t i a l s in 60 m M Ba. A divalent channel, distinct f r o m the N a channel a n d c a p a b l e o f passing a net i n w a r d current, exists in the a x o n o f R2. This channel m a y be c o m p a r a b l e with the voltage-sensitive Ca channel o f the s o m a o f this cell 2, a n d with that o f p r e s y n a p t i c terminals 9-11. T E A i m a y b l o c k the early o u t w a r d K efflux 15 which usually counteracts i n w a r d Ca current. I t h a n k D. Junge for the facilities a n d his help, a n d M. Brodwick, R. Eckert, a n d S. H a g i w a r a for c o m m e n t s o n the manuscript.
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