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An axonal spike in an identified fast crab motor neuron has sodium and calcium components
Camp. Biochem. Physid. Vol. 89A, No. 3, pp. 45-60, Printed in Great Britain
0300-9629/88
1988
$3.00 + 0.00
Pergamon Press plc
AN AXONAL SPIKE IN AN IDENTIFIED FAST CRAB MOTOR NEURON HAS SODIUM AND CALCIUM COMPONENTS PHILIP J. STEPHENS and PAUL J. CHURCH Villanova University, Department of Biology, Villanova, PA 19085, USA
(Received 7 May 1987) Abstract-l. High intensity intracellular stimulation of the FBE axon produced a spike discharge. The spikes divided into two components, each with a different frequency. 2. Ion replacement and blocking specific ion channels revealed that the FBE spike has calcium and sodium components. 3. The pronounced depolarizing wave that follows the FBE spike is not produced by changes in calcium conductance.
INTRODUCI’ION In a classic series of papers, Hodgkin and Huxley (1952a-d) described the action potential in the squid giant axon in terms of changes in membrane conduc-
tance to sodium and potassium. The rapid depolarization of the membrane during the spike is produced by an increase in sodium conductance. Inactivation of the sodium channels and a delayed increase in potassium conductance hyperpolarize the membrane to a potential below that seen at rest; the membrane then slowly recovers to its resting level. Modifications have been made to the Hodgkin-Huxley model (Meves and Vogel, 1973) and studies of other excitable cells have revealed other mechanisms for spike production. Spikes may be produced by an increase in membrane conductance to sodium and calcium (Koketsu et al., 1959; Niedergerke and Orkand, 1966a, b; Junge, 1967; Kerkut and Gardner, 1967; Niwa and Kawai, 1982), or to calcium alone (Hagiwara and Byerly, 1981). Variability in the properties of potassium channels has also been reported. Other currents, apart from the delayed potassium current described in squid giant axon, include the early potassium (A) current (Connor, 1981), the calcium-activated potassium current and the inward rectifier (Thompson and Aldrich, 1980). Furthermore, in some nerve cells the spike is followed by a depolarizing afterpotential (DAP), which has been explained in terms of an increase in membrane conductance to calcium (Yamigishi and Grundfest, 1971; Lewis, 1984) and an accumulation of potassium in a restricted periaxonal space (Zucker, 1974). The action potential recorded from one of the excitor motor axons to the limb bender muscle of the Pacific shore crab (Pachygrapsus crassipes) is composed of a spike followed by a pronounced DAP (Stephens et al., 1983). The DAP is closely associated with the ability of this axon to produce additional spikes in the periphery under conditions of high temperature or in the presence of ethanol. In the Abbreviations used-DAP, depolarizing after potential; FBE, fast bender excitor; SBE, slow bender excitor.
present study we have performed experiments that have involved the removal of certain ions from the saline or adding specific channel blockers to show that the fast bender excitor (FBE) axon spike has a sodium and a calcium component. A preliminary report of some of the data has been published (Stephens and Church, 1986). MATERIALS AND
METHODS
Pacific shore crabs (P. crassipes) were obtained from the Pacific Biomarine Laboratories (Venice, CA) and were kept individually at room temperature (21 + 1°C). Feeding (Purina rabbit chow) and artificial sea-water changes were performed every 2-3 days and under these conditions the crabs lived well, molted, and frequently regenerated lost limbs. Electrophysiology Preparations were made from autotomixed (not regenerated in the laboratory) second and third walking limbs removed from animals that had been acclimated to laboratory conditions for at least 4 weeks. With its ventral surface uppermost, the autotomixed limb was firmly held to the base of a plastic dish (volume: 10 ml) with dental wax and bathed in a crab saline with the following composition (mM): 470 NaCl; 8 KCl; 2OCaC1,; 10 MgCl,; 5 HEPES buffer, pH 7.2. The cuticle on the ventral surface of the meropodite was removed using a dental drill, and the underlying extensor and flexor muscles were removed. The exposed limb nerve was split along its length with a fine steel pin and most of the nerve was removed; the few remaining bundles contained the excitor axons to the bender muscle (Stephens et al., 1983). The cuticle over the distal portion of the carpopodite~was removed with the aid of a dental drill and the underlying dermal layer was carefully dissected away to expose fibers of the bender muscle. In this way a “window” was created, through which individual bender muscle fibers could be impaled with glass microelectrodes. Brief (0.1 msec duration) electrical shocks were applied to the remaining axon bundles in the center of the meropodite using a pair of silver hook electrodes. A pair of tungsten hooks was placed under the axon bundles (distal to the stimulating electrodes) to apply tension to the axons and to provide support and stability for microelectrode penetration. Light was reflected from below the preparation so that the FBE axon could be visually identified prior to microelectrode penetration. Glass microelectrodes (filled
455
PHILIP
456
J.
STEPIBNS
with 2M-notassium acetate; resistance 15-25MCI) were used to impale individual axons and single bender muscle fibers: identification of an imnaled axon as that of the FBE is g&n in the Results sect&. In the present study two ~cr~l~tr~s were usuaIiy placed close to one another (within about 30 am) in the FBE axon. One electrode was used to record evoked activity, while the second electrode was used to pass current into the axon, either to produce action potentials or to correct for changes in the membrane potential produced by bathing the preparation in modified saline solutions. Intracellularly recorded nerve and muscle activity was conventionally displayed on the screen of an oscilloscope (Nicolet 4094) and stored on discs for later analysis. No attempts were made to measure the space constant since the axon was stretched to provide tension and stability for microelectrode penetration. All experiments were performed at 21°C. The temperature of the preparation was controlled by heat exchange between the saline and a glass coil, which was fitted to the margin of the dish and connected to a ~e~ostatic~iy controlled,
constantly circulating water bath. The temperature of the saline in the dish was monitored using a thermal probe placed close to the meropodite. Fresh crab saline was constantly perfused into the dish at a rate of about lOml/min; suction was used to provide circulation and to maintain a constant depth of saline. RESULTS
Identification of the FBE axon A microelectrode was placed in a bender muscle fiber known to be innervated by only the FBE axon; the slow bender excitor (SBE) innervates fibers only
Fig. 1. Identitication of the FBE axon. (A) An extracelltiar shock applied to the limb nerve caused the synchronous appearance of an epsp in a bender muscle fiber (upper trace) and an action potential in the FBI3 axon (lower trace). (B) A stimulus shock applied to the FBE axon through a second microelectrode depolarized the axon and produced an action potential and an epsp. Calibration pulse. (at the beginning of traces) 5 mV and 1 msec.
and PAUL J.CHURCH in the central and medial portion of the window, while the FBE axon supplies all of the accessible fibers (Lazarus et al., 1982). Individual axons in the limb nerve were penetrated with a second microelectrode. Iden~~tion of the impaled axon as that of the FBE was based upon three criteria: (1) careful grading of the electrical shocks applied at either polarity through the extracellular hook electrodes caused the synchronous appearance of an action potential in the axon and an excitatory postsynaptic potential (epsp) in the bender muscle fiber (Fig. IA). (2) The axon spike was followed by a pronoun~d DAP (Fig. IA); the SBE has a spike followed by a hyperpolarizing afterpotential (Stephens et al., 1983). (3) Injection of current through the microelectrode depolarized the membrane and produced one or more action potentials in the FBE axon and concomitant epsps in the bender muscle fibers (Fig. 1B). The FBE axons had a mean resting potential of -78.9 mV (SE f 3.0; R = 36) and a mean spike amplitude of 12.6 mV above zero (SE + 4.8; n = 29). The ion components of the spike (a) Current injection into the FBE axon. injection of current through a microelectrode in the FBE axon revealed that the threshold for the production of action potentials is about 20 mV above resting (Fig. 1B). An increase in the amount of injected current produced further membrane depolarization and a spike discharge (Fig. 2A). Further increases in the amount of current injected into the FBE axon produced action potentials with a decreased amplitude and resulted in a division or splitting of each action potential into two components. This is illustrated in Fig. 2B-E, which shows records (ED) and a graph (E) of the inter-spike intervals for the two components during the discharge. In most preparations the two components were easily differentiated by size (Fig. 2D). During the evoked discharge, the delay between the first and second components increased progressively so that periodically the second component fused with the first component of the succeeding action potential (Fig. 2C, D-open arrow). (b) ion replacement studies. Brief electrical shocks applied to the limb nerve through extracellular hook electrodes evoked an action potential in the FBE axon and an epsp in the bender muscle fibers (Fig. 1A). Bathing preparations in a saline in which all sodium ions had been replaced with choline caused the evoked FBE action potential to decrease in amplitude and split into two components (Fig. 3). During a short (about 30 set) time period, the smaller second component was recorded at progressively increasing intervals after the stimulus shock until it failed and only the first component remained (Fig. 3B-D). In normal saline, injection of current through a microelectrode in the FBE axon depolarized the membrane and produced a train of action potentials (Figs 2A and 4A). Bath applications of 2-4 piM tetr~otox~ (TTX) progressively (and reversibly) abolished the spikes at the end of the train until only a single spike was recorded at the onset of depolarization (Fig. 4B-D). At this stage, increasing the amount of current injected into the axon did not increase the number of evoked spikes. The single
Fast crab axon action potential
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(A)
(El
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IO
Spike
15
20
25
number
Fig. 2. Stimulation of the FBE axon through the microelectrode. Injection of current through a microelectrode depolarized the FBE axon above threshold and produced a spike discharge, which was recorded through a second microelectrode (A). Increasing the amount of applied current caused the evoked spikes to split into two components (B), which at higher current intensity occurred at different frequencies (C). This can be seen clearly at a higher sweep speed (D), where the different frequencies caused the second (smaller) component to fuse with the first component of the succeeding spike to produce a larger amplitude response (open arrow). The interspike intervals for the evoked spikes in this trace are shown graphically in (E): circles, smaller component; squares, larger component. In all traces the solid arrow indicates zero membrane potential. Calibration 20 mV (A-C), 10 mV (D) and 10 msec (A-C), 5 msec (D).
small-amplitude
spike persisted
for more than 30 min
and was considered to be a m-resistant spike. Application of TTX had a minimal effect on the FBE membrane potential (f l-2 mV) but increased the threshold for FBE axon spike production by 15-20 mV, when compared with normal saline (n = 5). When two microelectrodes were placed in the FBE axon at either end of the meropodite, it was found that the TTX-resistant spike propogated down the axon, but did not produce an epsp in the bender muscle. Bathing preparations in a saline containing 5 mM calcium (15 mM magnesium was added to maintain osmolarity) depolarized the FBE axon by 10-l 5 mV.
When the membrane potential was restored to its resting level by current injection through a second microelectrode, the action potential produced by nerve stimulation had a smaller spike amplitude than in normal saline (Fig. SA). When calcium ions were replaced with barium, an ion that can pass through calcium channels (Hagiwara and Byerly, 1981), the spike amplitude was the same as in normal saline (Fig. 5B). Preparations were bathed in a saline in which 10mM sodium chloride was replaced with 1OmM cadmium chloride, so that cadmium blocked the calcium channels (Hagiwara and Byerly, 1981). When compared with the response recorded in normal
PHILIP J. STEPHENS
458
and PAUL J. CHURCH
(A)
(01 *
Fig. 3. The effects of choline on the FBE axon spike. Perfusion with saline in which sodium ions had been replaced with choline caused the FBE spike (evoked by brief extracellular shocks) to split into two components (A-C). The stimulus-response time delay for the smaller second component (open arrow) increased with time until it failed (D). In all traces the solid arrow indicates zero membrane potential. Calibration 20 mV, 10 ms.
(8)
(A) *
(D)
1 L
-I
’
i-
Fig. 4. The effects of TTX on the FBE axon spike. Injection of current through a microelectrode in the FBE axon caused membrane depolarization and a spike discharge, which was recorded through a second microelectrode (A). Application of ‘TTX (2-4 pM) decreased the number of spikes (B, C) until only a single ‘ITX-resistant spike was recorded (D). This ‘FIX-resistant spike remained for at least 30 min. Recordings in (C) and (D) were made with two microelectrodes located about 5 mm apart in the FBE axon. In all traces the solid arrow indicates zero membrane potential. Calibration 20 mV (A, B), 40 mV (C, D) and 10 msec (A-C), 13 msec (D).
Fast crab axon action potential
459
fD)
Fig. 5. The effect of low-calcium, barium and cadmium on the FBE spike. In saline containing 5mM calcium and 25 mM magnesium (A), the evoked spike decreased in amplitude and increased its time of decay when compared with an action potential recorded in normal saline (superimposed-open arrow). Replacing calcium with barium did not effect the spike but increased DAP amplitude (B), while 10 mM cadmium (replacing 10 mM sodium) blocked the calcium channels and resulted in an action potential which was smaller and slower than that evoked in normal saline (superimposed-open arrow) (C). In cadmium saline, high intensity current applied to the FBE axon through a second microelectrode evoked a train of spikes (D). The spikes in this train did not split into two components. In all traces the solid arrow indicates zero membrane potential. Calibration 20 mV, (A, C), 40 mV (B), 4 mV (C) and 10 msec (AC), 13 msec (D).
saline, the action potential recorded in cadmiumcontaining saline had a decreased amplitude and an increased spike rise and decay time (Fig. 5C). At this stage, application of 24pM TTX abolished the evoked spike, even when the stimulus intensity was increased. When preparations were perfused with a salinecontaining cadmium or low-calcium and highmagnesium levels, injection of high levels of current into the FBE axon through a microelectrode provoked a spike discharge. In these preparations the evoked spikes did not split into two components (Fig. SD). Bathing these preparations in normal saline or in a saline in which the calcium ions had been replaced with barium, resulted in high intensity stimulation producing spikes that split into two components (Fig. 2B-D). DISCUSSION
In the present study, the FBE axon spike split into two components when the axon was perfused with sodium-free (choline) saline (Fig. 3) or injected with
high levels of current (Fig. 2B-D). The splitting of the spike into two components may be explained by the presence of two populations of channels, or by a damaged area of membrane around the electrode whose response is delayed relative to the nearby, undamaged membrane. Our ion replacement experiments make this latter possibility less likely, since one component always remained when preparations were perfused with a saline in which calcium or sodium was replaced with another ion or when the channels were blocked (Figs 3-5). The decline in spike amplitude in the presence of TTX (Fig. 4) or when sodium was replaced with choline (Fig. 3) demonstrates that there is a population of voltage-sensitive sodium channels. The presence of a TTX-resistant spike, however, indicates the existence of a second population of channels and our results suggest that these are calcium channels. The evidence for this is: (1) bathing preparations in a saline containing cadmium ions (to block calcium channels) or in low-calcium, high-magnesium saline reduced the amplitude of the spike (Fig. 5); (2) application of TTX to cadmium-containing saline
PHILIP J.
460
STEPHENS and
abolished all spikes; (3) replacing calcium with barium did not change the amplitude of the FBE spike (Fig. 5B); (4) when calcium channels were blocked with cadmium, high intensity stimulation of the axon produced spikes with only a single component (Fig. SD), not two, as seen in normal saline (Fig. 2C, 0). Thus, our data suggest that the FBE axon spike is produced by an increase in the conductance of the membrane to calcium and sodium. Furthermore, the firing of the two components at different frequencies (Fig. 2B-E), indicates that the two populations of voltage-sensitive channels have different opening and closing kinetics. A TTX-resistant spike has been reported in the distal regions of the stretcher inhibitory axon in lobsters (Kawai and Niwa, 1980; Niwa and Kawai, 1982). In this axon, the ‘FIX-resistant spike is also considered to be produced by an increase in membrane conductance to calcium. Moreover, it has been suggested that the density of the voltage-sensitive calcium channels increases towards the periphery. In the present study we have found that the TTXresistant spike propagates along the FBE axon in the meropodite but does not produce an epsp. This may be explained by the increase in the threshold for producing TTX-resistant spikes and the presence of areas along the axon with a low safety factor for conduction, for example, branchpoints and bottlenecks (Jahromi and Atwood, 1974). The FBE axon spike is followed by a pronounced depolarizing afterpotential (DAP) (Fig. 1A). The DAP has been explained by a slow increase in membrane conductance to calcium (Yamagishi and Grundfest, 1971; Lewis, 1984). This explanation seems unlikely for the FBE axon since reducing calcium levels in the saline or blocking calcium channels did not abolish the DAP, in fact it increased the DAP amplitude (Fig. 5A, C). These results may be accounted for if some of the potassium channels are ~lcium-activate. This would produce an overall decrease in membrane potassium permeability, a decrease in the decay time of the spike and a decrease in the amount of membrane hyperpolarization after the spike (or an increase in the amount of membrane depolarization produced by a conductance increase in another ion after the spike). Acknowledgements-This
hall Foundation
work was funded by the Whiteand the Research Corporation.
REFERENCES Connor
J. A. (1981) The fast K+ channel firing. In Moliuscax Nerve Cells (Edited by Byrne J. H.), pp. 125-133. Cold Spring New York. Hagiwara S., and Byerly L. (1981) Calcium Rev. Neurosci. 4, 69-125.
and repetitive Koester J. and Harbor Press, channel. Ann.
PAUL
J. CHURCH
Hodgkin A. L. and Huxley A. F. (1945) Resting and action potentials in single nerve fibers. J. Physiot., Lond. 104, 176-195. Hodgkin A. L. and Huxley A. F. (19%) Currents carried bysodimn and potassium ions through the membrane of the giant axon of Lo&o. J. Fhvsiol.. Land. 116.449472. Hodgkin A. L. and Ht&ey A. F: (19j2b) The components of membrane conductance in the giant axon of L&go. J. Physiol., Land. 116, 474496.
Hodgkin A. L. and Huxley A. F. (1952~) The dual effect of membrane potential on sodium conductance in the giant axon of Lojigo. J. Physioi., Land. 116,497-X%. Hodakin A. L. and Huxley A. F. 11952d) A a~nti~tive of mgmbrane current and its application to conduction and excitation in nerve. J. Physiof., Land. 117, 500-544. Jahromi S. S. and Atwood H. L. (1974) Three dimensional ultra-structure of the crayfish neuromuscular junction. J. Cell Biol. 63, 599-613.
Junge D. (1967) Multi-ionic action potentials in molluscan giant neurons. Nature 215, 546-548. Kawai N. and Niwa A. (1980) Neuromuscular transmission without sodium activation of the presynaptic terminal in the lobster. J. Physiol., Land. 305, 73085. Kerkut G. A. and Gardner D. R. (1967)The role of calcium ions in the action ootential of Helix asversa neurons. Cotnp. Biockem. Phisiol. 20, 147-162.
_
Koketsu K.. Cerf J. A. and Nishi S. (1959) Further observations oh the activity of frog spihal ganglion cells in sodium-free solutions. J. N~rophysiol. 22, 693-703. Lazarus R. E.. Steuhens P. J. and Mind&o H. (1983) The peripheral generation of action potentials in excitatory motor neurons of a crab. J. exv. Zoo/. 229, 129-136. Lewis D. V. (1984) Spike aRer&ents in R15 of Aplysia: Their relationship to slow inward current and calcium influx. J. Neurovhysioi. 51. 387-403. Meves H. and VogeIW. (1973) Calcium inward currents in internally perfused giant axons. J. Physiol., Land. 235, 225-265.
Niedergerke R. and Orkand R. K. (1966a) The dual effect of calcium on the action potential of the frog’s heart. J. Physiol., Laud. 1%
291L31 1.
Niederaerke R. and Grkand R. K. (1966b) The dependence of thpeaction potential of the frog’s hea’rt on the external and intra~llu~r sodium concentration. J. Physiol., Land. l&1, 312-334. Niwa A. and Kawai N. (1982) Tetrodotoxin-resistant propagating action potentials .in presynaptic axon of the lobster. J. Neurovhysiol. 47, 353-361. Stephens P. J. and *Church P. J. (1986) The spike of a slow crab axon has sod&n and calcium components. A&r. Sot. Neurosci. 12, 1348. Stephens P. J., Frascella P. A. and Mindrebo N. (1983) Effects of ethanol and temperature on a crab motor axon action potential: A possible mechanism for peripheral spike generation. J. exp. Biol. 103, 289-301. Thomnson S. H. and Aldrich R. W. (1980) Membrane pot&sium channels. In The Ceil Surface and Neuronal Function (Edited bv Cotmar C. W., Poste G. and Nicolson, G. L.), pp. -49-85. El~vier;No~h Holland Biomedical Press, Amsterdam. Yamagishi S. and Grundfest H. (1971) Contributions of various ions to the resting and action potentials of the crayfish medial giant axons. J. Membr. Biol. 5, 345-365. Zucker R. S. (1974) Excitability changes in crayfish motor neurone terminals. J. Physiok, Land. 241, 111-126.
0300-9629/88
1988
$3.00 + 0.00
Pergamon Press plc
AN AXONAL SPIKE IN AN IDENTIFIED FAST CRAB MOTOR NEURON HAS SODIUM AND CALCIUM COMPONENTS PHILIP J. STEPHENS and PAUL J. CHURCH Villanova University, Department of Biology, Villanova, PA 19085, USA
(Received 7 May 1987) Abstract-l. High intensity intracellular stimulation of the FBE axon produced a spike discharge. The spikes divided into two components, each with a different frequency. 2. Ion replacement and blocking specific ion channels revealed that the FBE spike has calcium and sodium components. 3. The pronounced depolarizing wave that follows the FBE spike is not produced by changes in calcium conductance.
INTRODUCI’ION In a classic series of papers, Hodgkin and Huxley (1952a-d) described the action potential in the squid giant axon in terms of changes in membrane conduc-
tance to sodium and potassium. The rapid depolarization of the membrane during the spike is produced by an increase in sodium conductance. Inactivation of the sodium channels and a delayed increase in potassium conductance hyperpolarize the membrane to a potential below that seen at rest; the membrane then slowly recovers to its resting level. Modifications have been made to the Hodgkin-Huxley model (Meves and Vogel, 1973) and studies of other excitable cells have revealed other mechanisms for spike production. Spikes may be produced by an increase in membrane conductance to sodium and calcium (Koketsu et al., 1959; Niedergerke and Orkand, 1966a, b; Junge, 1967; Kerkut and Gardner, 1967; Niwa and Kawai, 1982), or to calcium alone (Hagiwara and Byerly, 1981). Variability in the properties of potassium channels has also been reported. Other currents, apart from the delayed potassium current described in squid giant axon, include the early potassium (A) current (Connor, 1981), the calcium-activated potassium current and the inward rectifier (Thompson and Aldrich, 1980). Furthermore, in some nerve cells the spike is followed by a depolarizing afterpotential (DAP), which has been explained in terms of an increase in membrane conductance to calcium (Yamigishi and Grundfest, 1971; Lewis, 1984) and an accumulation of potassium in a restricted periaxonal space (Zucker, 1974). The action potential recorded from one of the excitor motor axons to the limb bender muscle of the Pacific shore crab (Pachygrapsus crassipes) is composed of a spike followed by a pronounced DAP (Stephens et al., 1983). The DAP is closely associated with the ability of this axon to produce additional spikes in the periphery under conditions of high temperature or in the presence of ethanol. In the Abbreviations used-DAP, depolarizing after potential; FBE, fast bender excitor; SBE, slow bender excitor.
present study we have performed experiments that have involved the removal of certain ions from the saline or adding specific channel blockers to show that the fast bender excitor (FBE) axon spike has a sodium and a calcium component. A preliminary report of some of the data has been published (Stephens and Church, 1986). MATERIALS AND
METHODS
Pacific shore crabs (P. crassipes) were obtained from the Pacific Biomarine Laboratories (Venice, CA) and were kept individually at room temperature (21 + 1°C). Feeding (Purina rabbit chow) and artificial sea-water changes were performed every 2-3 days and under these conditions the crabs lived well, molted, and frequently regenerated lost limbs. Electrophysiology Preparations were made from autotomixed (not regenerated in the laboratory) second and third walking limbs removed from animals that had been acclimated to laboratory conditions for at least 4 weeks. With its ventral surface uppermost, the autotomixed limb was firmly held to the base of a plastic dish (volume: 10 ml) with dental wax and bathed in a crab saline with the following composition (mM): 470 NaCl; 8 KCl; 2OCaC1,; 10 MgCl,; 5 HEPES buffer, pH 7.2. The cuticle on the ventral surface of the meropodite was removed using a dental drill, and the underlying extensor and flexor muscles were removed. The exposed limb nerve was split along its length with a fine steel pin and most of the nerve was removed; the few remaining bundles contained the excitor axons to the bender muscle (Stephens et al., 1983). The cuticle over the distal portion of the carpopodite~was removed with the aid of a dental drill and the underlying dermal layer was carefully dissected away to expose fibers of the bender muscle. In this way a “window” was created, through which individual bender muscle fibers could be impaled with glass microelectrodes. Brief (0.1 msec duration) electrical shocks were applied to the remaining axon bundles in the center of the meropodite using a pair of silver hook electrodes. A pair of tungsten hooks was placed under the axon bundles (distal to the stimulating electrodes) to apply tension to the axons and to provide support and stability for microelectrode penetration. Light was reflected from below the preparation so that the FBE axon could be visually identified prior to microelectrode penetration. Glass microelectrodes (filled
455
PHILIP
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STEPIBNS
with 2M-notassium acetate; resistance 15-25MCI) were used to impale individual axons and single bender muscle fibers: identification of an imnaled axon as that of the FBE is g&n in the Results sect&. In the present study two ~cr~l~tr~s were usuaIiy placed close to one another (within about 30 am) in the FBE axon. One electrode was used to record evoked activity, while the second electrode was used to pass current into the axon, either to produce action potentials or to correct for changes in the membrane potential produced by bathing the preparation in modified saline solutions. Intracellularly recorded nerve and muscle activity was conventionally displayed on the screen of an oscilloscope (Nicolet 4094) and stored on discs for later analysis. No attempts were made to measure the space constant since the axon was stretched to provide tension and stability for microelectrode penetration. All experiments were performed at 21°C. The temperature of the preparation was controlled by heat exchange between the saline and a glass coil, which was fitted to the margin of the dish and connected to a ~e~ostatic~iy controlled,
constantly circulating water bath. The temperature of the saline in the dish was monitored using a thermal probe placed close to the meropodite. Fresh crab saline was constantly perfused into the dish at a rate of about lOml/min; suction was used to provide circulation and to maintain a constant depth of saline. RESULTS
Identification of the FBE axon A microelectrode was placed in a bender muscle fiber known to be innervated by only the FBE axon; the slow bender excitor (SBE) innervates fibers only
Fig. 1. Identitication of the FBE axon. (A) An extracelltiar shock applied to the limb nerve caused the synchronous appearance of an epsp in a bender muscle fiber (upper trace) and an action potential in the FBI3 axon (lower trace). (B) A stimulus shock applied to the FBE axon through a second microelectrode depolarized the axon and produced an action potential and an epsp. Calibration pulse. (at the beginning of traces) 5 mV and 1 msec.
and PAUL J.CHURCH in the central and medial portion of the window, while the FBE axon supplies all of the accessible fibers (Lazarus et al., 1982). Individual axons in the limb nerve were penetrated with a second microelectrode. Iden~~tion of the impaled axon as that of the FBE was based upon three criteria: (1) careful grading of the electrical shocks applied at either polarity through the extracellular hook electrodes caused the synchronous appearance of an action potential in the axon and an excitatory postsynaptic potential (epsp) in the bender muscle fiber (Fig. IA). (2) The axon spike was followed by a pronoun~d DAP (Fig. IA); the SBE has a spike followed by a hyperpolarizing afterpotential (Stephens et al., 1983). (3) Injection of current through the microelectrode depolarized the membrane and produced one or more action potentials in the FBE axon and concomitant epsps in the bender muscle fibers (Fig. 1B). The FBE axons had a mean resting potential of -78.9 mV (SE f 3.0; R = 36) and a mean spike amplitude of 12.6 mV above zero (SE + 4.8; n = 29). The ion components of the spike (a) Current injection into the FBE axon. injection of current through a microelectrode in the FBE axon revealed that the threshold for the production of action potentials is about 20 mV above resting (Fig. 1B). An increase in the amount of injected current produced further membrane depolarization and a spike discharge (Fig. 2A). Further increases in the amount of current injected into the FBE axon produced action potentials with a decreased amplitude and resulted in a division or splitting of each action potential into two components. This is illustrated in Fig. 2B-E, which shows records (ED) and a graph (E) of the inter-spike intervals for the two components during the discharge. In most preparations the two components were easily differentiated by size (Fig. 2D). During the evoked discharge, the delay between the first and second components increased progressively so that periodically the second component fused with the first component of the succeeding action potential (Fig. 2C, D-open arrow). (b) ion replacement studies. Brief electrical shocks applied to the limb nerve through extracellular hook electrodes evoked an action potential in the FBE axon and an epsp in the bender muscle fibers (Fig. 1A). Bathing preparations in a saline in which all sodium ions had been replaced with choline caused the evoked FBE action potential to decrease in amplitude and split into two components (Fig. 3). During a short (about 30 set) time period, the smaller second component was recorded at progressively increasing intervals after the stimulus shock until it failed and only the first component remained (Fig. 3B-D). In normal saline, injection of current through a microelectrode in the FBE axon depolarized the membrane and produced a train of action potentials (Figs 2A and 4A). Bath applications of 2-4 piM tetr~otox~ (TTX) progressively (and reversibly) abolished the spikes at the end of the train until only a single spike was recorded at the onset of depolarization (Fig. 4B-D). At this stage, increasing the amount of current injected into the axon did not increase the number of evoked spikes. The single
Fast crab axon action potential
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(A)
(El
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5
IO
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15
20
25
number
Fig. 2. Stimulation of the FBE axon through the microelectrode. Injection of current through a microelectrode depolarized the FBE axon above threshold and produced a spike discharge, which was recorded through a second microelectrode (A). Increasing the amount of applied current caused the evoked spikes to split into two components (B), which at higher current intensity occurred at different frequencies (C). This can be seen clearly at a higher sweep speed (D), where the different frequencies caused the second (smaller) component to fuse with the first component of the succeeding spike to produce a larger amplitude response (open arrow). The interspike intervals for the evoked spikes in this trace are shown graphically in (E): circles, smaller component; squares, larger component. In all traces the solid arrow indicates zero membrane potential. Calibration 20 mV (A-C), 10 mV (D) and 10 msec (A-C), 5 msec (D).
small-amplitude
spike persisted
for more than 30 min
and was considered to be a m-resistant spike. Application of TTX had a minimal effect on the FBE membrane potential (f l-2 mV) but increased the threshold for FBE axon spike production by 15-20 mV, when compared with normal saline (n = 5). When two microelectrodes were placed in the FBE axon at either end of the meropodite, it was found that the TTX-resistant spike propogated down the axon, but did not produce an epsp in the bender muscle. Bathing preparations in a saline containing 5 mM calcium (15 mM magnesium was added to maintain osmolarity) depolarized the FBE axon by 10-l 5 mV.
When the membrane potential was restored to its resting level by current injection through a second microelectrode, the action potential produced by nerve stimulation had a smaller spike amplitude than in normal saline (Fig. SA). When calcium ions were replaced with barium, an ion that can pass through calcium channels (Hagiwara and Byerly, 1981), the spike amplitude was the same as in normal saline (Fig. 5B). Preparations were bathed in a saline in which 10mM sodium chloride was replaced with 1OmM cadmium chloride, so that cadmium blocked the calcium channels (Hagiwara and Byerly, 1981). When compared with the response recorded in normal
PHILIP J. STEPHENS
458
and PAUL J. CHURCH
(A)
(01 *
Fig. 3. The effects of choline on the FBE axon spike. Perfusion with saline in which sodium ions had been replaced with choline caused the FBE spike (evoked by brief extracellular shocks) to split into two components (A-C). The stimulus-response time delay for the smaller second component (open arrow) increased with time until it failed (D). In all traces the solid arrow indicates zero membrane potential. Calibration 20 mV, 10 ms.
(8)
(A) *
(D)
1 L
-I
’
i-
Fig. 4. The effects of TTX on the FBE axon spike. Injection of current through a microelectrode in the FBE axon caused membrane depolarization and a spike discharge, which was recorded through a second microelectrode (A). Application of ‘TTX (2-4 pM) decreased the number of spikes (B, C) until only a single ‘ITX-resistant spike was recorded (D). This ‘FIX-resistant spike remained for at least 30 min. Recordings in (C) and (D) were made with two microelectrodes located about 5 mm apart in the FBE axon. In all traces the solid arrow indicates zero membrane potential. Calibration 20 mV (A, B), 40 mV (C, D) and 10 msec (A-C), 13 msec (D).
Fast crab axon action potential
459
fD)
Fig. 5. The effect of low-calcium, barium and cadmium on the FBE spike. In saline containing 5mM calcium and 25 mM magnesium (A), the evoked spike decreased in amplitude and increased its time of decay when compared with an action potential recorded in normal saline (superimposed-open arrow). Replacing calcium with barium did not effect the spike but increased DAP amplitude (B), while 10 mM cadmium (replacing 10 mM sodium) blocked the calcium channels and resulted in an action potential which was smaller and slower than that evoked in normal saline (superimposed-open arrow) (C). In cadmium saline, high intensity current applied to the FBE axon through a second microelectrode evoked a train of spikes (D). The spikes in this train did not split into two components. In all traces the solid arrow indicates zero membrane potential. Calibration 20 mV, (A, C), 40 mV (B), 4 mV (C) and 10 msec (AC), 13 msec (D).
saline, the action potential recorded in cadmiumcontaining saline had a decreased amplitude and an increased spike rise and decay time (Fig. 5C). At this stage, application of 24pM TTX abolished the evoked spike, even when the stimulus intensity was increased. When preparations were perfused with a salinecontaining cadmium or low-calcium and highmagnesium levels, injection of high levels of current into the FBE axon through a microelectrode provoked a spike discharge. In these preparations the evoked spikes did not split into two components (Fig. SD). Bathing these preparations in normal saline or in a saline in which the calcium ions had been replaced with barium, resulted in high intensity stimulation producing spikes that split into two components (Fig. 2B-D). DISCUSSION
In the present study, the FBE axon spike split into two components when the axon was perfused with sodium-free (choline) saline (Fig. 3) or injected with
high levels of current (Fig. 2B-D). The splitting of the spike into two components may be explained by the presence of two populations of channels, or by a damaged area of membrane around the electrode whose response is delayed relative to the nearby, undamaged membrane. Our ion replacement experiments make this latter possibility less likely, since one component always remained when preparations were perfused with a saline in which calcium or sodium was replaced with another ion or when the channels were blocked (Figs 3-5). The decline in spike amplitude in the presence of TTX (Fig. 4) or when sodium was replaced with choline (Fig. 3) demonstrates that there is a population of voltage-sensitive sodium channels. The presence of a TTX-resistant spike, however, indicates the existence of a second population of channels and our results suggest that these are calcium channels. The evidence for this is: (1) bathing preparations in a saline containing cadmium ions (to block calcium channels) or in low-calcium, high-magnesium saline reduced the amplitude of the spike (Fig. 5); (2) application of TTX to cadmium-containing saline
PHILIP J.
460
STEPHENS and
abolished all spikes; (3) replacing calcium with barium did not change the amplitude of the FBE spike (Fig. 5B); (4) when calcium channels were blocked with cadmium, high intensity stimulation of the axon produced spikes with only a single component (Fig. SD), not two, as seen in normal saline (Fig. 2C, 0). Thus, our data suggest that the FBE axon spike is produced by an increase in the conductance of the membrane to calcium and sodium. Furthermore, the firing of the two components at different frequencies (Fig. 2B-E), indicates that the two populations of voltage-sensitive channels have different opening and closing kinetics. A TTX-resistant spike has been reported in the distal regions of the stretcher inhibitory axon in lobsters (Kawai and Niwa, 1980; Niwa and Kawai, 1982). In this axon, the ‘FIX-resistant spike is also considered to be produced by an increase in membrane conductance to calcium. Moreover, it has been suggested that the density of the voltage-sensitive calcium channels increases towards the periphery. In the present study we have found that the TTXresistant spike propagates along the FBE axon in the meropodite but does not produce an epsp. This may be explained by the increase in the threshold for producing TTX-resistant spikes and the presence of areas along the axon with a low safety factor for conduction, for example, branchpoints and bottlenecks (Jahromi and Atwood, 1974). The FBE axon spike is followed by a pronounced depolarizing afterpotential (DAP) (Fig. 1A). The DAP has been explained by a slow increase in membrane conductance to calcium (Yamagishi and Grundfest, 1971; Lewis, 1984). This explanation seems unlikely for the FBE axon since reducing calcium levels in the saline or blocking calcium channels did not abolish the DAP, in fact it increased the DAP amplitude (Fig. 5A, C). These results may be accounted for if some of the potassium channels are ~lcium-activate. This would produce an overall decrease in membrane potassium permeability, a decrease in the decay time of the spike and a decrease in the amount of membrane hyperpolarization after the spike (or an increase in the amount of membrane depolarization produced by a conductance increase in another ion after the spike). Acknowledgements-This
hall Foundation
work was funded by the Whiteand the Research Corporation.
REFERENCES Connor
J. A. (1981) The fast K+ channel firing. In Moliuscax Nerve Cells (Edited by Byrne J. H.), pp. 125-133. Cold Spring New York. Hagiwara S., and Byerly L. (1981) Calcium Rev. Neurosci. 4, 69-125.
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Koketsu K.. Cerf J. A. and Nishi S. (1959) Further observations oh the activity of frog spihal ganglion cells in sodium-free solutions. J. N~rophysiol. 22, 693-703. Lazarus R. E.. Steuhens P. J. and Mind&o H. (1983) The peripheral generation of action potentials in excitatory motor neurons of a crab. J. exv. Zoo/. 229, 129-136. Lewis D. V. (1984) Spike aRer&ents in R15 of Aplysia: Their relationship to slow inward current and calcium influx. J. Neurovhysioi. 51. 387-403. Meves H. and VogeIW. (1973) Calcium inward currents in internally perfused giant axons. J. Physiol., Land. 235, 225-265.
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Niederaerke R. and Grkand R. K. (1966b) The dependence of thpeaction potential of the frog’s hea’rt on the external and intra~llu~r sodium concentration. J. Physiol., Land. l&1, 312-334. Niwa A. and Kawai N. (1982) Tetrodotoxin-resistant propagating action potentials .in presynaptic axon of the lobster. J. Neurovhysiol. 47, 353-361. Stephens P. J. and *Church P. J. (1986) The spike of a slow crab axon has sod&n and calcium components. A&r. Sot. Neurosci. 12, 1348. Stephens P. J., Frascella P. A. and Mindrebo N. (1983) Effects of ethanol and temperature on a crab motor axon action potential: A possible mechanism for peripheral spike generation. J. exp. Biol. 103, 289-301. Thomnson S. H. and Aldrich R. W. (1980) Membrane pot&sium channels. In The Ceil Surface and Neuronal Function (Edited bv Cotmar C. W., Poste G. and Nicolson, G. L.), pp. -49-85. El~vier;No~h Holland Biomedical Press, Amsterdam. Yamagishi S. and Grundfest H. (1971) Contributions of various ions to the resting and action potentials of the crayfish medial giant axons. J. Membr. Biol. 5, 345-365. Zucker R. S. (1974) Excitability changes in crayfish motor neurone terminals. J. Physiok, Land. 241, 111-126.