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Functional differences between 4-aminopyridine and tetraethylammonium-sensitive potassium channels in myelinated axons
Neuroscience Letters, 75 (1987) 193--198
193
Elsevier Scientific Publishers Ireland Ltd. NSL 04485
Functional differences between 4-aminopyridine and tetraethylammonium-sensitive potassium channels in myelinated axons J.D. Kocsis*, D.L. Eng, T.R. Gordon
and S.G. Waxman*
Department ~f Neurology, Stanford Universi O' School of Medicine and Palo Alto Veterans Administration Medical Center, Palo Alto, CA 94304 (U.S.A.)
(Received 8 September 1986; Revised version received 24 November t986; Accepted 1 December 1986) Key words:
4-Aminopyridine: Tetraethylammonium; Potassium channel, Peripheral nerve
lntracellular recordings from rat sciatic nerve fibers showed that the potassium channel blocking agents 4-aminopyridine (4-AP) and tetraethylammonium (TEA) had different effects on action potential waveform. When applied alone, TEA did not appreciably alter the waveform of an individual action potential, whereas 4-AP application resulted in action potential broadening and, in some axons, repetitive firing. A prolonged afterhyperpolarization which was blocked by TEA occurred subsequent to repetitive firing. These results indicate the presence of at least two pharmacologically defined potassium channels in mammalian peripheral nerve fibers. The 4-AP-sensitive potassium channels are important for rapid action potential repolarization and the TEA-sensitive potassium channels may serve to modulate axonal excitability during repetitive firing.
T h e H o d g k i n a n d H u x l e y [ l l ] d e s c r i p t i o n o f the a c t i o n p o t e n t i a l in squid giant a x o n emphasizes the i m p o r t a n c e o f s o d i u m a n d p o t a s s i u m c o n d u c t a n c e s : i n w a r d m o v e m e n t o f s o d i u m ions leads to the rising phase o f the action potential, a n d o u t w a r d m o v e m e n t o f p o t a s s i u m ions t o g e t h e r with s o d i u m channel inactivation leads to r e p o l a r i z a t i o n . T h e v o l t a g e - d e p e n d e n t p o t a s s i u m c o n d u c t a n c e in squid a x o n shows a delay in a c t i v a t i o n a n d is t e t r a e t h y l a m m o n i u m (TEA)-sensitive. M o r e recently a variety o f kinetically and p h a r m a c o l o g i c a l l y distinct p o t a s s i u m channels have been described for n e u r o n a l cell bodies, muscle fibers a n d a x o n s (see refs. 1 and 10 for overviews). D u b o i s [8] has d e s c r i b e d 3 types o f p o t a s s i u m c o n d u c t a n c e s in frog m y e l i n a t e d fibers. T w o o f these c o n d u c t a n c e s r a p i d l y activate with one r a p i d l y inactiv a t i n g and the o t h e r slowly inactivating, whereas a third type b o t h activates a n d inactivates slowly. The fast p o t a s s i u m c o n d u c t a n c e s are sensitive to 4 - a m i n o p y r i d i n e (4A P ) whereas the slow p o t a s s i u m c o n d u c t a n c e is not. A l t h o u g h these different p o t a s Corre,sTondence: J.D. Kocsis. *Present address: Department of Neurology (LCI-707), Yale Medical School, P.O. Box 3333, New Haven, CT 06510, U.S.A.
0304-3940/87,'$ 03.50 (~) 1987 Elsevier Scientific Publishers Ireland Ltd.
194
sium conductances have been described in voltage-clamp studies of frog myelinated axons, their functional role is not clear. Indeed, voltage-clamp studies of mature mammalian myelinated axons indicate the relative lack of potassium conductances at the node [7]. However, during the course of maturation [12] and following demyelination [4, 15] action potential waveform of mammalian myelinated axons is appreciably affected by potassium channel blocking agents. In order to assess the possible functional significance of different potassium conductances of myelinated fibers, the action potentials (n= 171) of rat sciatic nerves (n=43) were examined using intracellular recording techniques. Action potential waveform and firing characteristics of the axons were studied following application of various combinations of 4-AP and TEA. The nerves of relatively young rats (5 6 weeks old) were examined because the effects of potassium channel blocking agents are decreased in older rats [5, 12]. The results indicate that a 4-AP sensitive potassium conductance has an important role in action potential repolarization. A TEA-sensitive potassium conductance is activated during repetitive firing and leads to a pronounced afterhyperpolarization (AHP). Whereas the 4-AP-sensitive channel may be responsible for rapid action potential repolarization, the TEA-sensitive channel may modulate axonal excitability during repetitive firing. Wistar rats (5 6 weeks old) were deeply anesthetized with sodium pentobarbital (60 mg/kg) and decapitated. The sciatic nerves were removed, desheathed, and placed in a normal electrolyte solution (NS) which was saturated with 95% 02-5% CO2. The NS contained (in mM): NaC1 124, KCI 3.0, NaHzPO4 1.3, MgCI2 2.0, CaC12 2.0, NaHCO3 26.0, dextrose 10. After the nerves stabilized for 30-60 min, they were placed in a nerve recording chamber through which NS continuously flowed. Aluminosilicate glass microelectrodes were pulled on a Brown-Flaming microelectrode puller and filled with 4 M potassium acetate or 2 M KC1. The electrodes were bevelled to a DC resistance of 18(~250 M£2. Axons with resting potentials of 60 mV or greater and action potential amplitudes greater than resting potential were selected for analysis. An action potential recorded from an intra-axonal impalement of a sciatic nerve fiber is shown in Fig. 1A. The action potential had a resting potential of - 7 7 mV and a spike amplitude of 88 mV. With stable intra-axonal impalements single action potentials were never followed by a hyperpolarization in normal electrolyte solution. Indeed, many fibers displayed a depolarizing afterpotential characteristic of normal sciatic nerve fibers [3]. Application of TEA (10 mM) alone had little effect on the action potential waveform (Fig. 1B). However, there were several characteristic changes in action potential waveform following application of 4-AP (1.0 mM). Previous work indicates that 1.0 mM 4-AP will elicit a maximal effect on spike waveform [12]. One effect of 4-AP was to delay repolarization. Note the broadened spike in 4-AP and the delayed return to baseline (Fig. 1C) as compared to the spike recorded in NS (Fig. IA). In another set of sciatic nerve fibers 4-AP led to a delayed depolarization from which a burst of several action potentials arose (Fig. ID). This spike burst response following a single stimulus in the presence of 4-AP is characteristic of sensory (dorsal root) fibers and can be used as a pharmacological means of dis-
195
A 4-AP
20 I mV
25
5.0
6O
B
msec
4-AP+TEA
TEA
G
4-AP 100
Fig. i. Intracellular recordings of action potentials from rat sciatic nerve in normal Ringer (A), 10 m M TEA (B), and 1.0 m M 4-AP (C,D). Note that the action potential recorded in 4-AP (C) is considerably broader than in Ringer (A) or TEA (B). D: in the presence of 4-AP a single whole nerve stimulus leads to a delayed depolarization from which a burst of action potentials arise. Additionally, a prominent A H P follows the burst. E: when T E A is applied in combination with 4-AP (E) the A H P is eliminated and a single stimulus induces increased repetitive firing. F: in the presence of T E A and 4-AP in another axon, slow depolarization shifts and spontaneous burst activity with no A H P s occur. The time calibration in A refers to A C, and the voltage calibration to all traces.
tinguishing sensory and motor fibers [13]. Fibers that show spike broadening with the absence of both a delayed depolarization and spike burst activity in the presence of 4-AP are characteristic of motor (ventral root) fibers [12]. Over half of immature
196 sciatic nerve fibers respond to a single stimulus with spike burst activity in the presence of 4-AP [12]. In the presence of 4-AP a prominent A H P follows the burst of action potentials elicited by a single stimulus (Fig. 1D). The peak amplitude of the A H P shown in Fig. I E is 9 mV, and the duration is about 100 ms. The 4-AP-induced A H P was blocked by TEA (Fig. I E). Note the increased spike discharge after TEA application. The AHP was eliminated by TEA thereby indicating that 10 mM TEA was sufficient to elicit a maximal response with respect to the AHP. The combination of 4-AP and TEA led to increased spontaneous action potential activity and occasional shifts in membrane potential. Note the slow depolarization shift and the burst of action potentials with no A H P in the presence o f TEA and 4-AP (Fig. 1F). In motor fibers, i.e. fibers that did not display a delayed depolarization and action potential burst activity in 4-AP [5, 13], an AHP was not present following a single stimulus. However, a hyperpolarization could be induced even in the absence of 4-AP during and following repetitive stimulation. This type of stimulus-evoked hyperpolarization in NS was present in all fibers from which recordings were obtained indicating that both sensory and motor fibers could display the AHP in the absence of 4-AP, given the proper stimulus. This A H P was sensitive to TEA. Fig. 2A shows a hyperpolarization induced in a fiber following a stimulus train (200 Hz for 100 ms). The hyperpolarization was similar to that seen in sensory fibers after a single stimulus in 4-AP, and was blocked by TEA (Fig. 2B). Dubois [8] has demonstrated that kinetically fast potassium channels of frog myelinated fibers are sensitive to I mM 4-AP application whereas a third slow type of potassium channel is not. The slow channels are sensitive to TEA. The existence of several types of potassium channels in myelinated fibers has prompted speculation about their functional roles [2, 8, 9, 14]. However, voltage-clamp studies of mature mammalian peripheral nerve fibers indicate a relative absence of voltage-gated potassium conductances at the node [6, 7]. Potassium channels of these fibers are located at the internodal axon membrane [7]. The functional role of the internodal potassium channels is uncertain. Differences in the effects of 4-AP and TEA on myelinated axons have been previously noted in the peripheral [2, 8, 19] and central [14] nervous systems.
I
B
I
75
mV
6o
Fig. 2. In the presence of normal drug-free Ringer repetitivestimulation leads to a prominent AHP (A) that is eliminated by TEA (B). The action potentialsare 'clipped' to show the AHP at a higher magnification.
197
Blockade of the 4-AP-sensitive potassium conductance in the present study led to a delayed repolarization and to a number of hyperexcitability phenomena such as action potential burst activity and increased spontaneous impulse activity. Spike burst activity following a single stimulus in the presence of 4-AP is characteristic of sensory axons [5, 13]. A prominent AHP in both sensory and motor fibers was elicited during repetitive stimulation in normal Ringer, and following a single stimulus to sensory fibers in the presence of 4-AP. Taken together these results suggest functional differences between the 4-AP- and TEA-sensitive channels. A single action potential could activate the 4-AP-sensitive channels which would quickly lead to repolarization and prevent appreciable activation of the slower TEA-sensitive potassium channel. After 4-AP blockade of the fast channels, the greater depolarization associated with broadened action potentials and action potential burst activity may lead to increased activation of the slower TEA-sensitive channel as shown by the appearance of a prominent TEA-sensitive AHP. In the presence of 4-AP, sensory fibers may display an AHP following a single stimulus because of the increased depolarization associated with the burst activity. The effects of 4-AP on sciatic nerve are attenuated during the course of development [5, 12]. The results of the present study were obtained from relatively young animals where pronounced 4-AP effects are present. Demyelinated axons, like immature fibers, are sensitive to 4-AP, thus suggesting the internodal localization of the 4-AP-sensitive channel [4, 15]. It may be that mature fibers have a tighter seal between myelin and axon membranes in the paranodal region which impedes 4-AP from reaching the internodal axon membrane. The functional properties attributed to the 4-AP- and TEA-sensitive channels may be present in the mature rat, but may not be observed because of the inaccessibility of the internodal potassium channels to exogenous 4-AP application. These results indicate that unlike squid giant axon, mammalian myelinated fibers primarily utilize a 4-AP-sensitive potassium channel, not a TEA-sensitive channel, for repolarization of individual action potentials. The TEA-sensitive channel, activated by prolonged depolarization during multiple spike discharge, results in a prominent AHP and may be important in the regulation of repetitive firing. This work was supported in part by the NIH and the Medical Research Service of the Veterans Administration. 1 Adams, P.R. and Galvan, M., Voltage-dependent currents of vertebrate neurons and their role in membrane excitability, In A.V. Delgado-Escueta, A.A. Ward, Jr., D.M. Woodbury and R.J. Porter (Eds.)~ Advances in Neurology, Vol. 44, Raven, New York, 1986, pp. 137 170. 2 Baker, M., Bostock H. and Grafe, P., Accommodation in rat myelinated axons depends on two pharmacologically distinct types of potassium channels, J. Physiol. (London), 369 (1985) 102P. 3 Barrett, E.F. and Barrett, J.N., Intracellular recording from vertebrate myelinated axons: mechanism of the depolarizing afterpotentials, J. Physiol. (London), 323 (1982) 117 144. 4 Bostock, H., Sears, T.A. and Sherratt, R.M., The effects of 4-aminopyridine and tetraethylammonium ions on normal and demyelinated m a m m a l i a n nerve fibers, J. Physiol. (London), 313 (1980) 30t 315. 5 Bowe, C.M., Kocsis, J.D. and W a x m a n , S.G., Differences between ventral and dorsal spinal roots in
198
6 7 8 9 I0 11 12
13 14 15
response to blockade of potassium channels during maturation, Proc. Roy. Soc. London Ser. B, 224 (1985) 355-356. Brismar, T., Potential clamp analysis of membrane currents in rat myelinated nerve fibres, J. Physiol. (London), 298 (1980) 171- 184. Chiu, S.Y. and Ritchie, J.M., Evidence for the presence of potassium channels in the paranodal region of acutely demyelinated mammalian single nerve fibres, J. Physiol. (London) 313 (1981) 415-431. Dubois, J.M., Evidence for the existence of three types of potassium channels in the frog Ranvier node membrane, J. Physiol. (London) 318 ( 1981 ) 297 316. Grafe, P., Martins, P. and Bostock, H., Three types of potassium channels in rat spinal root axons. Pfliiger's Arch., 405 (1985) R53. Hille, B., Ionic Channels of Excitable Membranes, Sinauer, Sunderland, MA, 1984, pp. 99 114. Hodgkin, A.L. and Huxley, A.F., A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. (London), 117 (1952) 500-544. Kocsis, J.D., Ruiz, J.A. and Waxman, S.G., Maturation of mammalian myelinated fibers: changes in action-potential characteristics following 4-aminopyridine application, J. Neurophysiot., 50 (1983) 449-463. Kocsis, J.D., Bowe, C.M. and Waxman, S.G., Different effects of 4-aminopyridine on sensory and motor fibers: pathogenesis ofparesthesias, Neurology, 36 (1986) 117 120. Kocsis, J.D., Gordon, T.R. and Waxman, S.G., Mammalian optic nerve fibers display two pharmacologically distinct potassium channels, Brain Res. 383 (1986) 357 361. Targ, E.G. and Kocsis, J.D., 4-aminopyridine leads to restoration of conduction in demyelinated rat sciatic nerve, Brain Res., 328 (1984) 358 361.
193
Elsevier Scientific Publishers Ireland Ltd. NSL 04485
Functional differences between 4-aminopyridine and tetraethylammonium-sensitive potassium channels in myelinated axons J.D. Kocsis*, D.L. Eng, T.R. Gordon
and S.G. Waxman*
Department ~f Neurology, Stanford Universi O' School of Medicine and Palo Alto Veterans Administration Medical Center, Palo Alto, CA 94304 (U.S.A.)
(Received 8 September 1986; Revised version received 24 November t986; Accepted 1 December 1986) Key words:
4-Aminopyridine: Tetraethylammonium; Potassium channel, Peripheral nerve
lntracellular recordings from rat sciatic nerve fibers showed that the potassium channel blocking agents 4-aminopyridine (4-AP) and tetraethylammonium (TEA) had different effects on action potential waveform. When applied alone, TEA did not appreciably alter the waveform of an individual action potential, whereas 4-AP application resulted in action potential broadening and, in some axons, repetitive firing. A prolonged afterhyperpolarization which was blocked by TEA occurred subsequent to repetitive firing. These results indicate the presence of at least two pharmacologically defined potassium channels in mammalian peripheral nerve fibers. The 4-AP-sensitive potassium channels are important for rapid action potential repolarization and the TEA-sensitive potassium channels may serve to modulate axonal excitability during repetitive firing.
T h e H o d g k i n a n d H u x l e y [ l l ] d e s c r i p t i o n o f the a c t i o n p o t e n t i a l in squid giant a x o n emphasizes the i m p o r t a n c e o f s o d i u m a n d p o t a s s i u m c o n d u c t a n c e s : i n w a r d m o v e m e n t o f s o d i u m ions leads to the rising phase o f the action potential, a n d o u t w a r d m o v e m e n t o f p o t a s s i u m ions t o g e t h e r with s o d i u m channel inactivation leads to r e p o l a r i z a t i o n . T h e v o l t a g e - d e p e n d e n t p o t a s s i u m c o n d u c t a n c e in squid a x o n shows a delay in a c t i v a t i o n a n d is t e t r a e t h y l a m m o n i u m (TEA)-sensitive. M o r e recently a variety o f kinetically and p h a r m a c o l o g i c a l l y distinct p o t a s s i u m channels have been described for n e u r o n a l cell bodies, muscle fibers a n d a x o n s (see refs. 1 and 10 for overviews). D u b o i s [8] has d e s c r i b e d 3 types o f p o t a s s i u m c o n d u c t a n c e s in frog m y e l i n a t e d fibers. T w o o f these c o n d u c t a n c e s r a p i d l y activate with one r a p i d l y inactiv a t i n g and the o t h e r slowly inactivating, whereas a third type b o t h activates a n d inactivates slowly. The fast p o t a s s i u m c o n d u c t a n c e s are sensitive to 4 - a m i n o p y r i d i n e (4A P ) whereas the slow p o t a s s i u m c o n d u c t a n c e is not. A l t h o u g h these different p o t a s Corre,sTondence: J.D. Kocsis. *Present address: Department of Neurology (LCI-707), Yale Medical School, P.O. Box 3333, New Haven, CT 06510, U.S.A.
0304-3940/87,'$ 03.50 (~) 1987 Elsevier Scientific Publishers Ireland Ltd.
194
sium conductances have been described in voltage-clamp studies of frog myelinated axons, their functional role is not clear. Indeed, voltage-clamp studies of mature mammalian myelinated axons indicate the relative lack of potassium conductances at the node [7]. However, during the course of maturation [12] and following demyelination [4, 15] action potential waveform of mammalian myelinated axons is appreciably affected by potassium channel blocking agents. In order to assess the possible functional significance of different potassium conductances of myelinated fibers, the action potentials (n= 171) of rat sciatic nerves (n=43) were examined using intracellular recording techniques. Action potential waveform and firing characteristics of the axons were studied following application of various combinations of 4-AP and TEA. The nerves of relatively young rats (5 6 weeks old) were examined because the effects of potassium channel blocking agents are decreased in older rats [5, 12]. The results indicate that a 4-AP sensitive potassium conductance has an important role in action potential repolarization. A TEA-sensitive potassium conductance is activated during repetitive firing and leads to a pronounced afterhyperpolarization (AHP). Whereas the 4-AP-sensitive channel may be responsible for rapid action potential repolarization, the TEA-sensitive channel may modulate axonal excitability during repetitive firing. Wistar rats (5 6 weeks old) were deeply anesthetized with sodium pentobarbital (60 mg/kg) and decapitated. The sciatic nerves were removed, desheathed, and placed in a normal electrolyte solution (NS) which was saturated with 95% 02-5% CO2. The NS contained (in mM): NaC1 124, KCI 3.0, NaHzPO4 1.3, MgCI2 2.0, CaC12 2.0, NaHCO3 26.0, dextrose 10. After the nerves stabilized for 30-60 min, they were placed in a nerve recording chamber through which NS continuously flowed. Aluminosilicate glass microelectrodes were pulled on a Brown-Flaming microelectrode puller and filled with 4 M potassium acetate or 2 M KC1. The electrodes were bevelled to a DC resistance of 18(~250 M£2. Axons with resting potentials of 60 mV or greater and action potential amplitudes greater than resting potential were selected for analysis. An action potential recorded from an intra-axonal impalement of a sciatic nerve fiber is shown in Fig. 1A. The action potential had a resting potential of - 7 7 mV and a spike amplitude of 88 mV. With stable intra-axonal impalements single action potentials were never followed by a hyperpolarization in normal electrolyte solution. Indeed, many fibers displayed a depolarizing afterpotential characteristic of normal sciatic nerve fibers [3]. Application of TEA (10 mM) alone had little effect on the action potential waveform (Fig. 1B). However, there were several characteristic changes in action potential waveform following application of 4-AP (1.0 mM). Previous work indicates that 1.0 mM 4-AP will elicit a maximal effect on spike waveform [12]. One effect of 4-AP was to delay repolarization. Note the broadened spike in 4-AP and the delayed return to baseline (Fig. 1C) as compared to the spike recorded in NS (Fig. IA). In another set of sciatic nerve fibers 4-AP led to a delayed depolarization from which a burst of several action potentials arose (Fig. ID). This spike burst response following a single stimulus in the presence of 4-AP is characteristic of sensory (dorsal root) fibers and can be used as a pharmacological means of dis-
195
A 4-AP
20 I mV
25
5.0
6O
B
msec
4-AP+TEA
TEA
G
4-AP 100
Fig. i. Intracellular recordings of action potentials from rat sciatic nerve in normal Ringer (A), 10 m M TEA (B), and 1.0 m M 4-AP (C,D). Note that the action potential recorded in 4-AP (C) is considerably broader than in Ringer (A) or TEA (B). D: in the presence of 4-AP a single whole nerve stimulus leads to a delayed depolarization from which a burst of action potentials arise. Additionally, a prominent A H P follows the burst. E: when T E A is applied in combination with 4-AP (E) the A H P is eliminated and a single stimulus induces increased repetitive firing. F: in the presence of T E A and 4-AP in another axon, slow depolarization shifts and spontaneous burst activity with no A H P s occur. The time calibration in A refers to A C, and the voltage calibration to all traces.
tinguishing sensory and motor fibers [13]. Fibers that show spike broadening with the absence of both a delayed depolarization and spike burst activity in the presence of 4-AP are characteristic of motor (ventral root) fibers [12]. Over half of immature
196 sciatic nerve fibers respond to a single stimulus with spike burst activity in the presence of 4-AP [12]. In the presence of 4-AP a prominent A H P follows the burst of action potentials elicited by a single stimulus (Fig. 1D). The peak amplitude of the A H P shown in Fig. I E is 9 mV, and the duration is about 100 ms. The 4-AP-induced A H P was blocked by TEA (Fig. I E). Note the increased spike discharge after TEA application. The AHP was eliminated by TEA thereby indicating that 10 mM TEA was sufficient to elicit a maximal response with respect to the AHP. The combination of 4-AP and TEA led to increased spontaneous action potential activity and occasional shifts in membrane potential. Note the slow depolarization shift and the burst of action potentials with no A H P in the presence o f TEA and 4-AP (Fig. 1F). In motor fibers, i.e. fibers that did not display a delayed depolarization and action potential burst activity in 4-AP [5, 13], an AHP was not present following a single stimulus. However, a hyperpolarization could be induced even in the absence of 4-AP during and following repetitive stimulation. This type of stimulus-evoked hyperpolarization in NS was present in all fibers from which recordings were obtained indicating that both sensory and motor fibers could display the AHP in the absence of 4-AP, given the proper stimulus. This A H P was sensitive to TEA. Fig. 2A shows a hyperpolarization induced in a fiber following a stimulus train (200 Hz for 100 ms). The hyperpolarization was similar to that seen in sensory fibers after a single stimulus in 4-AP, and was blocked by TEA (Fig. 2B). Dubois [8] has demonstrated that kinetically fast potassium channels of frog myelinated fibers are sensitive to I mM 4-AP application whereas a third slow type of potassium channel is not. The slow channels are sensitive to TEA. The existence of several types of potassium channels in myelinated fibers has prompted speculation about their functional roles [2, 8, 9, 14]. However, voltage-clamp studies of mature mammalian peripheral nerve fibers indicate a relative absence of voltage-gated potassium conductances at the node [6, 7]. Potassium channels of these fibers are located at the internodal axon membrane [7]. The functional role of the internodal potassium channels is uncertain. Differences in the effects of 4-AP and TEA on myelinated axons have been previously noted in the peripheral [2, 8, 19] and central [14] nervous systems.
I
B
I
75
mV
6o
Fig. 2. In the presence of normal drug-free Ringer repetitivestimulation leads to a prominent AHP (A) that is eliminated by TEA (B). The action potentialsare 'clipped' to show the AHP at a higher magnification.
197
Blockade of the 4-AP-sensitive potassium conductance in the present study led to a delayed repolarization and to a number of hyperexcitability phenomena such as action potential burst activity and increased spontaneous impulse activity. Spike burst activity following a single stimulus in the presence of 4-AP is characteristic of sensory axons [5, 13]. A prominent AHP in both sensory and motor fibers was elicited during repetitive stimulation in normal Ringer, and following a single stimulus to sensory fibers in the presence of 4-AP. Taken together these results suggest functional differences between the 4-AP- and TEA-sensitive channels. A single action potential could activate the 4-AP-sensitive channels which would quickly lead to repolarization and prevent appreciable activation of the slower TEA-sensitive potassium channel. After 4-AP blockade of the fast channels, the greater depolarization associated with broadened action potentials and action potential burst activity may lead to increased activation of the slower TEA-sensitive channel as shown by the appearance of a prominent TEA-sensitive AHP. In the presence of 4-AP, sensory fibers may display an AHP following a single stimulus because of the increased depolarization associated with the burst activity. The effects of 4-AP on sciatic nerve are attenuated during the course of development [5, 12]. The results of the present study were obtained from relatively young animals where pronounced 4-AP effects are present. Demyelinated axons, like immature fibers, are sensitive to 4-AP, thus suggesting the internodal localization of the 4-AP-sensitive channel [4, 15]. It may be that mature fibers have a tighter seal between myelin and axon membranes in the paranodal region which impedes 4-AP from reaching the internodal axon membrane. The functional properties attributed to the 4-AP- and TEA-sensitive channels may be present in the mature rat, but may not be observed because of the inaccessibility of the internodal potassium channels to exogenous 4-AP application. These results indicate that unlike squid giant axon, mammalian myelinated fibers primarily utilize a 4-AP-sensitive potassium channel, not a TEA-sensitive channel, for repolarization of individual action potentials. The TEA-sensitive channel, activated by prolonged depolarization during multiple spike discharge, results in a prominent AHP and may be important in the regulation of repetitive firing. This work was supported in part by the NIH and the Medical Research Service of the Veterans Administration. 1 Adams, P.R. and Galvan, M., Voltage-dependent currents of vertebrate neurons and their role in membrane excitability, In A.V. Delgado-Escueta, A.A. Ward, Jr., D.M. Woodbury and R.J. Porter (Eds.)~ Advances in Neurology, Vol. 44, Raven, New York, 1986, pp. 137 170. 2 Baker, M., Bostock H. and Grafe, P., Accommodation in rat myelinated axons depends on two pharmacologically distinct types of potassium channels, J. Physiol. (London), 369 (1985) 102P. 3 Barrett, E.F. and Barrett, J.N., Intracellular recording from vertebrate myelinated axons: mechanism of the depolarizing afterpotentials, J. Physiol. (London), 323 (1982) 117 144. 4 Bostock, H., Sears, T.A. and Sherratt, R.M., The effects of 4-aminopyridine and tetraethylammonium ions on normal and demyelinated m a m m a l i a n nerve fibers, J. Physiol. (London), 313 (1980) 30t 315. 5 Bowe, C.M., Kocsis, J.D. and W a x m a n , S.G., Differences between ventral and dorsal spinal roots in
198
6 7 8 9 I0 11 12
13 14 15
response to blockade of potassium channels during maturation, Proc. Roy. Soc. London Ser. B, 224 (1985) 355-356. Brismar, T., Potential clamp analysis of membrane currents in rat myelinated nerve fibres, J. Physiol. (London), 298 (1980) 171- 184. Chiu, S.Y. and Ritchie, J.M., Evidence for the presence of potassium channels in the paranodal region of acutely demyelinated mammalian single nerve fibres, J. Physiol. (London) 313 (1981) 415-431. Dubois, J.M., Evidence for the existence of three types of potassium channels in the frog Ranvier node membrane, J. Physiol. (London) 318 ( 1981 ) 297 316. Grafe, P., Martins, P. and Bostock, H., Three types of potassium channels in rat spinal root axons. Pfliiger's Arch., 405 (1985) R53. Hille, B., Ionic Channels of Excitable Membranes, Sinauer, Sunderland, MA, 1984, pp. 99 114. Hodgkin, A.L. and Huxley, A.F., A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. (London), 117 (1952) 500-544. Kocsis, J.D., Ruiz, J.A. and Waxman, S.G., Maturation of mammalian myelinated fibers: changes in action-potential characteristics following 4-aminopyridine application, J. Neurophysiot., 50 (1983) 449-463. Kocsis, J.D., Bowe, C.M. and Waxman, S.G., Different effects of 4-aminopyridine on sensory and motor fibers: pathogenesis ofparesthesias, Neurology, 36 (1986) 117 120. Kocsis, J.D., Gordon, T.R. and Waxman, S.G., Mammalian optic nerve fibers display two pharmacologically distinct potassium channels, Brain Res. 383 (1986) 357 361. Targ, E.G. and Kocsis, J.D., 4-aminopyridine leads to restoration of conduction in demyelinated rat sciatic nerve, Brain Res., 328 (1984) 358 361.