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Tetrodotoxin resistant electrically excitable responses of receptor cells
Brain Research, 62 (1973) 253-259
253
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
Tetrodotoxin resistant electrically ,excitable responses of receptor cells
BIRGIT ZIPSER AND M. V. L. BENNETT Department of Anatomy, Rose F. Kennedy Center Jbr Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461 (U.S.A.)
(Accepted August 6th, 1973)
Electroreceptors are receptors specialized for the detection of electric fields2,a. They are found in electric fish and in some non-electric fish as well. They are 'secondary receptors' in that the initial stimulus transduction or transformation is carried out by neuroepithelial cells which then transmit synaptically to the afferent nerve fibers. They are modified mechanoreceptors of the lateral line and as such are innervated by cranial nerves of the acoustico-lateralis complex. In the weakly electric teleosts, the South American gymnotids and African mormyrids, the receptors can be divided into tonic and phasic classes. The receptor cells of phasic receptors in general respond to appropriate electrical stimuli with a graded regenerative response or even an all-or-none spike. In contrast the tonic receptor cells behave linearly to applied stimuli; there is no obvious electrical response although the stimuli alter the release of transmitter and thereby the nerve discharge. It is of interest to know the ionic basis of the electrical responses of the phasic receptors. The present study utilizes tetrodotoxin (TTX), which has proven in many tissues to be a specific blocker of Na channels. This drug failed to affect the receptor cell activity, although it blocked nerve impulses in the same animals. These findings suggest that some ion such as Ca 2+ is involved in the responses of the receptor cells. A preliminary report of these findings has been presented is. The fish used were 10-20 cm long mormyrids (Gnathonemuspetersii) and 20-25 cm long gymnotids (Gymnotus carapo). In most of the experiments the fish were curarized (10 mg/kg) and respired by perfusing aerated aquarium water through the mouth. Receptor cell and nerve responses were monopolarly recorded at the receptor openings with Ringer agar filled electrodes 50-100 #m in diameter. A similar electrode was used to pass current pulses. A chlorided silver wire in the aquarium water bath served as an indifferent electrode. To reduce shunting and increase response amplitude, the water level often was lowered so that the receptor was either barely covered with water or completely out in the air. In the latter case, the skin was kept moist by occasional rinsings. TTX (Sigma Chemical Co.; St. Louis, Mo.) was given intraperitoneally. The doses used for mormyrids were 0.15 mg/kg (which is 15 times the LDs0 for mice10).
254
SHORT COMMUNICATIONS
GYMNOTID TONIC RECEPTOR CONTROL A '. 'r '. '. ', ',
B.
O."
c .
TTX
I
!i lOmse¢
Fig. I. The effect of TTX on a tonic receptor of Gymnotus. Upper trace, recording of nerve responses at the opening of the receptor on the skin. Lower trace, current applied through a second electrode. A - C before and D after the injection of TTX. A: the nerve innervating the tonic receptor is firing spontaneously. B: the rate is increased by an anodal stimulus. C: the rate is decreased by a cathodal stimulus. D: the nerve impulses are blocked by TTX and are absent even when a well suprathreshold stimulus is given. (The change in time constant in the record is due to increased shunting following rinsing, but similar rinsing prior to TTX injection did not prevent recording of the responses.)
This dose quickly paralyzed a mormyrid in one experiment in which the fish was not injected with curare. In another single experiment a dose of TTX of 25 mg/kg had no effect on mormyrid phasic receptors. The absence of nerve impulses in the lateral line nerve after the injection of TTX was established by bipolar recording and stimulation at the termination of each experiment. Gymnotids were injected with doses of 1 mg/kg (100 times LDs0 for mice). Block of impulses in the lateral line nerve was confirmed in several experiments. The effect of TTX on a tonic receptor of Gymnotus is shown in Fig. 1. External to the receptor, spike activity can be recorded which was shown by recording and stimulating in the afferent nerve to be the afferent nerve impulse 1,3. As is typical of these tonic receptors the nerve impulse frequency is increased by anodal stimuli and decreased by cathodal stimuli. However, the potential changes at the receptor opening show no indication of a response in addition to that of the nerve fiber. TTX blocks the nerve response within a few minutes of intraperitoneal injection (Fig. 1D). The effect of TTX on a phasic receptor of Gymnotus is illustrated in Fig. 2. The responses to anodal stimuli are graded oscillations at both onset and termination of the stimulus (A,B). Later oscillations may be preceded by a small positivity (arrows) which has been shown to be the nerve impulse, and under these recording conditions
255
SHORT COMMUNICATIONS
GYMNOTID PHASIC RECEPTOR CONTROL
TTX
A
C
B
D
TTX E
l~ - ' ' ' -
F
/VVV%'~
.
_
_
_ d i lmsec
Fig. 2. The effect of TTX on phasic receptors of Gymnotus. Display, stimulation and recording as in Fig. I. A-B: graded oscillatory responses of a receptor are elicited by anodal stimuli. The brief positive potentials indicated by arrows in B have been shown to be nerve impulses. C-D: the oscillations persist after the application of TTX. Nerve impulses are blocked. E: oscillations recorded from a phasic receptor after administration of TTX. F: an anodal stimulus briefly increases the amplitude of the oscillations, perhaps by synchronizing a number of different receptors.
the nerve impulse stimulates the receptor cells to generate additional oscillations 1. Following TTX administration the oscillations persist, but they are reduced in amplitude and more rapidly damped. Nerve impulses are no longer observed. Much of the reduction in oscillatory behavior is ascribable to loss of the nerve impulse, but the possibility remains of some slight depressive effect of TTX itself. When the skin resistance becomes sufficiently high as a result of drying, the receptors begin to oscillate continually 1. That oscillations occur in the absence of stimulation unequivocally demonstrates their active, regenerative nature, for no energy is applied from an external source. These oscillations were also observed after TTX administration (Fig. 2E-F). Responses of receptor cells in the two kinds of phasic receptors in mormyrids are unaffected by TTX. This result is illustrated for a large receptor in Fig. 3. Responses to just threshold and well suprathreshold stimuli are compared before and after TTX. The threshold response to an anodal stimulus is a brief all-or-none spike (Fig. 3A) and neither threshold nor amplitude are affected by TTX (Fig, 3C). Even a strong anodal stimuli evoke only a single spike at the onset of the stimuli (Fig. 3B, note reduced gain). However, a delayed spike appears irregularly after termination of the stimuli (Fig. 3B, arrow). These responses are also unaffected by TTX (Fig. 3D). (There is evidence that the delayed spike is an anode break responsel, 3. Because the outer face of the receptor cells acts as a differentiating capacity, the inner face of the receptor cells is hyperpolarized transiently at the end of the stimulus.) The medium receptors of mormyrids respond with a graded biphasic potential at the onset of anodal stimuli (Fig. 4A). This response appears to be somewhat re-
256
SHORT COMMUNICATIONS
MORMYRID LARGE REC EPTOR CONTROL
A
i
TTX
!
c
i
I
i
, s
/
O
I
F t
./
.._d
m
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Fig. 3. Lack of effect of TTX on a mormyrid large receptor. Display, stimulation and recording as in
Fig. 1. A: a threshold anodal stimulus evokes at its onset either a subthreshold response or a spike. Two superimposed sweeps. B: suprathreshold anodai stimuli evoke a single spike at the onset of each stimulus and a delayed spike (arrow) after the termination of some fraction of the stimuli. Recorded at lower gains, two superimposed sweeps. C and D: after TTX the responses are unchanged.
generative, for an inflection can be observed on the rising phase of larger responses (arrows). T T X had no noticeable effects on these responses (Fig. 4B). The failure of T T X to block the electrical responses of electroreceptor cells suggests to us that these responses may be mediated by Ca currents. The insensitivity of Ca channels to T T X is well knownT, 11. The sensitivity of the Na channels of nerve and muscle is also well established, and T T X blocked excitability of the nerve of the fishes studied here. To be sure there are a few instances of T T X resistant N a currents in molluscs, and the vertebrates that make the toxin are also insensitive to it 10. Also it has been reported recently that the spike of muscle becomes insensitive to T T X following denervation, although it remains N a dependent 14. The polarity of the externally recorded receptor responses, and the polarity of stimuli required to excite, both indicate that the responses are generated by the inner, presynaptic faces of the receptor cells. This location is such that T T X ought to reach the active membrane. No obvious barrier other than a basement membrane is seen electron microscopically and T T X blocks the nerve impulses at a point which must be very close to the receptor cells. Glutamate, which is about half the molecular weight of TTX, excites the afferent fibers when applied to the inner surface of the skin. Presumably glutamate acts on the nerve 17. While it remains desirable to demonstrate directly the accessibility of the inner faces of the receptor cells to blood born molecules of a comparable size, it seems very likely that T T X reaches the active membrane. There is some additional indirect evidence that the receptor cells have Ca 2+ channels. Synaptic transmission at medium receptors of mormyrids is depressed by
257
SHORT COMMUNICATIONS
MORMYRID MEDIUM RECEPTOR CONTROL
TTX
_
Ill
2 mime Fig. 4. Lack of effect of TTX on the response of a mormyrid medium receptor. Display, stimulation and recording as in Fig. 1. A: the onset of an anodal pulse elicitsa graded and relatively slow biphasic response of the receptor cells. B: this response is unchanged after TTX administration. Several superimposed sweeps in each record. high Mg and low Ca solutions, and similar results have been obtained with the ampulla of Lorenzini 16,17. By analogy with neuronal synapses these results suggest that depolarization increases Ca permeability and causes Ca influx that leads to transmitter release. At the squid giant synapse presynaptic Ca spikes can be obtained by blocking Na channels with TTX and K channels with TEA and increasing the Ca gradient across the membranetL The explanation is that a small number of Ca channels become able to generate a spike when shunting by other ions is sufficiently reduced. Apparently similar responses occur at the frog neuromuscular junctionlL Comparable treatments have now been found to lead to TTX insensitive spike activity in the presynaptic membrane of receptor cells of the ampulla of Lorenzini 4. This membrane is otherwise not obviously excitable ~3. Presumably some non-linearity is present in the normal membrane but it is too shunted by other conductances to be seen under the recording conditions. The preceding discussion suggests a simple basis for the difference in responsiveness of phasic and tonic electroreceptors. Phasic receptors have a relatively large number o f Ca channels compared to K channels. Small depolarizations open sufficient Ca channels that net inward current results and oscillations or all-or-none responses are developed. In tonic receptors K channels are more numerous and although Ca channels are opened by depolarization, they are sufficiently short circuited by the K channels that net inward current is not produced. Probably the primitive lateral line receptor corresponds to the tonic receptor and is not obviously excitable 9. Evolution of the phasic receptor may then have involved a change in the proportion of K and Ca channels, a mechanism that might be simpler developmentally than making Na channels which were not there previously (although it would be expected that D N A for Na channels would be present in the receptor cells). Known Ca spikes in other tissues are quite slow in time course. In contrast the
258
SHORT COMMUNICATIONS
spikes of mormyrid large receptors are very brief and responses of gymnotid phasic receptors are comparable in duration to ordinary nerve action potentials. The significance of this difference is obscure. Although no other receptor cells have been shown to generate electrical responses, there are interesting similarities between mammalian vestibular receptors and electroreceptors. One class of afferent vestibular fiber, apparently innervating type 11 hair cells, is tonically and fairly regularly activeS, 6. The morphology of the synapses suggests chemically mediated transmission and the resting frequency of discharge is modulated up and down by appropriate direction of stimulation. These receptors appear analogous to tonic receptors. A second class of fibers, presumably coming from type I hair cells, are active irregularlyS, 6. A type ! hair cell is almost completely enveloped by the large calyx terminal of its afferent fiber which would greatly impede the currents required for conventional chemical transmission. Furthermore, there are close appositions between hair cell and nerve terminal as at other electrical synapsesS, 15. These two features suggest strongly that transmission is electrical at this synapse. Both irregular spontaneous activity and electrical transmission occur in the large (phasic) receptor of mormyrids. It may well be that depolarization generated at the kinocilium of the type 1 hair cells is amplified by electrical responsiveness of the cell in order to generate adequate presynaptic depolarization to excite the afferent fiber. While further direct evidence is required, the results presented here are suggestive of similarities between interneuronal synapses and those of receptor cells, which are also of ectodermal origin. Because of experimental convenience the study of electroreceptors may provide results of interest with respect to the acoustic and vestibular receptors of higher animals and perhaps of relevance to interneuronal synapses as well. This work was supported in part by grants from the National Institute of Health (NB-07512, HD-04248 and 2T01 GM00102) and the Alfred P. Sloan Foundation. Michael V. L. Bennett was a Kennedy Scholar.
1 BENNETT, M. V. L., Mechanisms of electroreception. In P. CAHN(Ed.), Lateral Line Detectors,
Indiana Univ. Press, Bloomington, Ind., 1967, pp. 313-393. 2 BENNETT,M. V. L., Electrolocation in fish. In Orientation: Sensory Basis, Ann. N. Y. Acad. ScL, 188 (1971) 242-269. 3 BENNETT, M. V. L., Electroreception. In W. S. HOARAND D. J. RANDALL(Eds.), Fish Physiology, Vol. 5, Academic Press, New York, 1971, pp. 493-573. 4 CLUSIN, B., AND BENNETT, M. V. L., Calcium electrogenesis in skate electroreceptors, Biol. Bull., in press. 5 FERNANDEZ,C., GOLDBERG,J. M., ANDABEND,W. K., Response to static tilts of peripheral neurons innervating otolith organs of the squirrel monkey, J. Neurophysiol., 35 (1972) 978-997. 6 GOLDBERG, J. M., AND FERNANDEZ, C., Physiology of peripheral neurons innervating semicircular canals of the squirrel monkey. III. Variation among units in their discharge properties, J. Neurophysiol., 34 (1971) 676-684. 7 HAGIWARA, S., AND NAKAJIMA, S., Differences in Na and Ca spikes as examined by application of tetrodotoxin, procaine and manganese ions, J. gen. Physiol., 49 (1966) 793-806. 8 HAMILTON,D. W., The calyceal synapse of type I vestibular hair cells, J. Ultrastruct. Res., 23 (1968) 98-114. 9 HARRIS,G. G., FRISHKOPF,L. S., ANDFLOCK,/~.,Receptor potentials from hair cells of the lateral line, Science, 167 (1970) 76-79.
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259
10 KAO, C. Y., Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena, Pharmacol. Rev., 18 (1966) 997-1099. 11 KATZ, B., AND MILEDI, R., Tetrodotoxin resistant electric activity in presynaptic terminals, J. Physiol. (Lond.), 203 (1969) 459--487. 12 "KATZ, B., ANOMILEDI, R., Spontaneous and evoked activity of motor nerve endings in calcium Ringer, J. Physiol. (Lond.), 203 (1969) 689-706. 13 OBARA, S., AND BENNETT, M. V. L., Mode of operation of ampullae of Lorenzini of the skate, Raja, J. gen. Physiol., 60 (1972) 534-557. 14 REDFERN,P., LUNDH, H., AND THESLEFF,S., Tetrodotoxin resistant action potentials in denervated rat skeletal muscle, Europ. J. Pharmacol., 11 (1970) 263-265. 15 SPOENDLIN,H., Some morphofunctional and pathological aspects of the vestibular sensory epithelia. In 2nd Symposium on the Role of Vestibular Organs in Space Exploration, NASA, Washington, D.C., 1966, pp. 99-115. 16 STEINBACH,A. B., Transmission from receptor cells to afferent nerve fibers. In M. V. L. BENNETT (Ed.), Synaptic Transmission and lnterneuronal Communication. Raven Press, New York, in press. 17 STEINBACH,A. B., AND BENNETT, M. V. L., Effect of divalent ions and drugs on synaptic transmission in phasic electroreceptors in a mormyrid fish, J. gen. Physiol., 58 (1971) 580-598. 18 ZIPSER, B., Tetrodotoxin resistant electrically excitable responses of receptor cells, Biophys. Soc. Abstr. 15th Ann. Meeting, (1971) 44a.
253
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
Tetrodotoxin resistant electrically ,excitable responses of receptor cells
BIRGIT ZIPSER AND M. V. L. BENNETT Department of Anatomy, Rose F. Kennedy Center Jbr Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461 (U.S.A.)
(Accepted August 6th, 1973)
Electroreceptors are receptors specialized for the detection of electric fields2,a. They are found in electric fish and in some non-electric fish as well. They are 'secondary receptors' in that the initial stimulus transduction or transformation is carried out by neuroepithelial cells which then transmit synaptically to the afferent nerve fibers. They are modified mechanoreceptors of the lateral line and as such are innervated by cranial nerves of the acoustico-lateralis complex. In the weakly electric teleosts, the South American gymnotids and African mormyrids, the receptors can be divided into tonic and phasic classes. The receptor cells of phasic receptors in general respond to appropriate electrical stimuli with a graded regenerative response or even an all-or-none spike. In contrast the tonic receptor cells behave linearly to applied stimuli; there is no obvious electrical response although the stimuli alter the release of transmitter and thereby the nerve discharge. It is of interest to know the ionic basis of the electrical responses of the phasic receptors. The present study utilizes tetrodotoxin (TTX), which has proven in many tissues to be a specific blocker of Na channels. This drug failed to affect the receptor cell activity, although it blocked nerve impulses in the same animals. These findings suggest that some ion such as Ca 2+ is involved in the responses of the receptor cells. A preliminary report of these findings has been presented is. The fish used were 10-20 cm long mormyrids (Gnathonemuspetersii) and 20-25 cm long gymnotids (Gymnotus carapo). In most of the experiments the fish were curarized (10 mg/kg) and respired by perfusing aerated aquarium water through the mouth. Receptor cell and nerve responses were monopolarly recorded at the receptor openings with Ringer agar filled electrodes 50-100 #m in diameter. A similar electrode was used to pass current pulses. A chlorided silver wire in the aquarium water bath served as an indifferent electrode. To reduce shunting and increase response amplitude, the water level often was lowered so that the receptor was either barely covered with water or completely out in the air. In the latter case, the skin was kept moist by occasional rinsings. TTX (Sigma Chemical Co.; St. Louis, Mo.) was given intraperitoneally. The doses used for mormyrids were 0.15 mg/kg (which is 15 times the LDs0 for mice10).
254
SHORT COMMUNICATIONS
GYMNOTID TONIC RECEPTOR CONTROL A '. 'r '. '. ', ',
B.
O."
c .
TTX
I
!i lOmse¢
Fig. I. The effect of TTX on a tonic receptor of Gymnotus. Upper trace, recording of nerve responses at the opening of the receptor on the skin. Lower trace, current applied through a second electrode. A - C before and D after the injection of TTX. A: the nerve innervating the tonic receptor is firing spontaneously. B: the rate is increased by an anodal stimulus. C: the rate is decreased by a cathodal stimulus. D: the nerve impulses are blocked by TTX and are absent even when a well suprathreshold stimulus is given. (The change in time constant in the record is due to increased shunting following rinsing, but similar rinsing prior to TTX injection did not prevent recording of the responses.)
This dose quickly paralyzed a mormyrid in one experiment in which the fish was not injected with curare. In another single experiment a dose of TTX of 25 mg/kg had no effect on mormyrid phasic receptors. The absence of nerve impulses in the lateral line nerve after the injection of TTX was established by bipolar recording and stimulation at the termination of each experiment. Gymnotids were injected with doses of 1 mg/kg (100 times LDs0 for mice). Block of impulses in the lateral line nerve was confirmed in several experiments. The effect of TTX on a tonic receptor of Gymnotus is shown in Fig. 1. External to the receptor, spike activity can be recorded which was shown by recording and stimulating in the afferent nerve to be the afferent nerve impulse 1,3. As is typical of these tonic receptors the nerve impulse frequency is increased by anodal stimuli and decreased by cathodal stimuli. However, the potential changes at the receptor opening show no indication of a response in addition to that of the nerve fiber. TTX blocks the nerve response within a few minutes of intraperitoneal injection (Fig. 1D). The effect of TTX on a phasic receptor of Gymnotus is illustrated in Fig. 2. The responses to anodal stimuli are graded oscillations at both onset and termination of the stimulus (A,B). Later oscillations may be preceded by a small positivity (arrows) which has been shown to be the nerve impulse, and under these recording conditions
255
SHORT COMMUNICATIONS
GYMNOTID PHASIC RECEPTOR CONTROL
TTX
A
C
B
D
TTX E
l~ - ' ' ' -
F
/VVV%'~
.
_
_
_ d i lmsec
Fig. 2. The effect of TTX on phasic receptors of Gymnotus. Display, stimulation and recording as in Fig. I. A-B: graded oscillatory responses of a receptor are elicited by anodal stimuli. The brief positive potentials indicated by arrows in B have been shown to be nerve impulses. C-D: the oscillations persist after the application of TTX. Nerve impulses are blocked. E: oscillations recorded from a phasic receptor after administration of TTX. F: an anodal stimulus briefly increases the amplitude of the oscillations, perhaps by synchronizing a number of different receptors.
the nerve impulse stimulates the receptor cells to generate additional oscillations 1. Following TTX administration the oscillations persist, but they are reduced in amplitude and more rapidly damped. Nerve impulses are no longer observed. Much of the reduction in oscillatory behavior is ascribable to loss of the nerve impulse, but the possibility remains of some slight depressive effect of TTX itself. When the skin resistance becomes sufficiently high as a result of drying, the receptors begin to oscillate continually 1. That oscillations occur in the absence of stimulation unequivocally demonstrates their active, regenerative nature, for no energy is applied from an external source. These oscillations were also observed after TTX administration (Fig. 2E-F). Responses of receptor cells in the two kinds of phasic receptors in mormyrids are unaffected by TTX. This result is illustrated for a large receptor in Fig. 3. Responses to just threshold and well suprathreshold stimuli are compared before and after TTX. The threshold response to an anodal stimulus is a brief all-or-none spike (Fig. 3A) and neither threshold nor amplitude are affected by TTX (Fig, 3C). Even a strong anodal stimuli evoke only a single spike at the onset of the stimuli (Fig. 3B, note reduced gain). However, a delayed spike appears irregularly after termination of the stimuli (Fig. 3B, arrow). These responses are also unaffected by TTX (Fig. 3D). (There is evidence that the delayed spike is an anode break responsel, 3. Because the outer face of the receptor cells acts as a differentiating capacity, the inner face of the receptor cells is hyperpolarized transiently at the end of the stimulus.) The medium receptors of mormyrids respond with a graded biphasic potential at the onset of anodal stimuli (Fig. 4A). This response appears to be somewhat re-
256
SHORT COMMUNICATIONS
MORMYRID LARGE REC EPTOR CONTROL
A
i
TTX
!
c
i
I
i
, s
/
O
I
F t
./
.._d
m
I ImlDe¢
Fig. 3. Lack of effect of TTX on a mormyrid large receptor. Display, stimulation and recording as in
Fig. 1. A: a threshold anodal stimulus evokes at its onset either a subthreshold response or a spike. Two superimposed sweeps. B: suprathreshold anodai stimuli evoke a single spike at the onset of each stimulus and a delayed spike (arrow) after the termination of some fraction of the stimuli. Recorded at lower gains, two superimposed sweeps. C and D: after TTX the responses are unchanged.
generative, for an inflection can be observed on the rising phase of larger responses (arrows). T T X had no noticeable effects on these responses (Fig. 4B). The failure of T T X to block the electrical responses of electroreceptor cells suggests to us that these responses may be mediated by Ca currents. The insensitivity of Ca channels to T T X is well knownT, 11. The sensitivity of the Na channels of nerve and muscle is also well established, and T T X blocked excitability of the nerve of the fishes studied here. To be sure there are a few instances of T T X resistant N a currents in molluscs, and the vertebrates that make the toxin are also insensitive to it 10. Also it has been reported recently that the spike of muscle becomes insensitive to T T X following denervation, although it remains N a dependent 14. The polarity of the externally recorded receptor responses, and the polarity of stimuli required to excite, both indicate that the responses are generated by the inner, presynaptic faces of the receptor cells. This location is such that T T X ought to reach the active membrane. No obvious barrier other than a basement membrane is seen electron microscopically and T T X blocks the nerve impulses at a point which must be very close to the receptor cells. Glutamate, which is about half the molecular weight of TTX, excites the afferent fibers when applied to the inner surface of the skin. Presumably glutamate acts on the nerve 17. While it remains desirable to demonstrate directly the accessibility of the inner faces of the receptor cells to blood born molecules of a comparable size, it seems very likely that T T X reaches the active membrane. There is some additional indirect evidence that the receptor cells have Ca 2+ channels. Synaptic transmission at medium receptors of mormyrids is depressed by
257
SHORT COMMUNICATIONS
MORMYRID MEDIUM RECEPTOR CONTROL
TTX
_
Ill
2 mime Fig. 4. Lack of effect of TTX on the response of a mormyrid medium receptor. Display, stimulation and recording as in Fig. 1. A: the onset of an anodal pulse elicitsa graded and relatively slow biphasic response of the receptor cells. B: this response is unchanged after TTX administration. Several superimposed sweeps in each record. high Mg and low Ca solutions, and similar results have been obtained with the ampulla of Lorenzini 16,17. By analogy with neuronal synapses these results suggest that depolarization increases Ca permeability and causes Ca influx that leads to transmitter release. At the squid giant synapse presynaptic Ca spikes can be obtained by blocking Na channels with TTX and K channels with TEA and increasing the Ca gradient across the membranetL The explanation is that a small number of Ca channels become able to generate a spike when shunting by other ions is sufficiently reduced. Apparently similar responses occur at the frog neuromuscular junctionlL Comparable treatments have now been found to lead to TTX insensitive spike activity in the presynaptic membrane of receptor cells of the ampulla of Lorenzini 4. This membrane is otherwise not obviously excitable ~3. Presumably some non-linearity is present in the normal membrane but it is too shunted by other conductances to be seen under the recording conditions. The preceding discussion suggests a simple basis for the difference in responsiveness of phasic and tonic electroreceptors. Phasic receptors have a relatively large number o f Ca channels compared to K channels. Small depolarizations open sufficient Ca channels that net inward current results and oscillations or all-or-none responses are developed. In tonic receptors K channels are more numerous and although Ca channels are opened by depolarization, they are sufficiently short circuited by the K channels that net inward current is not produced. Probably the primitive lateral line receptor corresponds to the tonic receptor and is not obviously excitable 9. Evolution of the phasic receptor may then have involved a change in the proportion of K and Ca channels, a mechanism that might be simpler developmentally than making Na channels which were not there previously (although it would be expected that D N A for Na channels would be present in the receptor cells). Known Ca spikes in other tissues are quite slow in time course. In contrast the
258
SHORT COMMUNICATIONS
spikes of mormyrid large receptors are very brief and responses of gymnotid phasic receptors are comparable in duration to ordinary nerve action potentials. The significance of this difference is obscure. Although no other receptor cells have been shown to generate electrical responses, there are interesting similarities between mammalian vestibular receptors and electroreceptors. One class of afferent vestibular fiber, apparently innervating type 11 hair cells, is tonically and fairly regularly activeS, 6. The morphology of the synapses suggests chemically mediated transmission and the resting frequency of discharge is modulated up and down by appropriate direction of stimulation. These receptors appear analogous to tonic receptors. A second class of fibers, presumably coming from type I hair cells, are active irregularlyS, 6. A type ! hair cell is almost completely enveloped by the large calyx terminal of its afferent fiber which would greatly impede the currents required for conventional chemical transmission. Furthermore, there are close appositions between hair cell and nerve terminal as at other electrical synapsesS, 15. These two features suggest strongly that transmission is electrical at this synapse. Both irregular spontaneous activity and electrical transmission occur in the large (phasic) receptor of mormyrids. It may well be that depolarization generated at the kinocilium of the type 1 hair cells is amplified by electrical responsiveness of the cell in order to generate adequate presynaptic depolarization to excite the afferent fiber. While further direct evidence is required, the results presented here are suggestive of similarities between interneuronal synapses and those of receptor cells, which are also of ectodermal origin. Because of experimental convenience the study of electroreceptors may provide results of interest with respect to the acoustic and vestibular receptors of higher animals and perhaps of relevance to interneuronal synapses as well. This work was supported in part by grants from the National Institute of Health (NB-07512, HD-04248 and 2T01 GM00102) and the Alfred P. Sloan Foundation. Michael V. L. Bennett was a Kennedy Scholar.
1 BENNETT, M. V. L., Mechanisms of electroreception. In P. CAHN(Ed.), Lateral Line Detectors,
Indiana Univ. Press, Bloomington, Ind., 1967, pp. 313-393. 2 BENNETT,M. V. L., Electrolocation in fish. In Orientation: Sensory Basis, Ann. N. Y. Acad. ScL, 188 (1971) 242-269. 3 BENNETT, M. V. L., Electroreception. In W. S. HOARAND D. J. RANDALL(Eds.), Fish Physiology, Vol. 5, Academic Press, New York, 1971, pp. 493-573. 4 CLUSIN, B., AND BENNETT, M. V. L., Calcium electrogenesis in skate electroreceptors, Biol. Bull., in press. 5 FERNANDEZ,C., GOLDBERG,J. M., ANDABEND,W. K., Response to static tilts of peripheral neurons innervating otolith organs of the squirrel monkey, J. Neurophysiol., 35 (1972) 978-997. 6 GOLDBERG, J. M., AND FERNANDEZ, C., Physiology of peripheral neurons innervating semicircular canals of the squirrel monkey. III. Variation among units in their discharge properties, J. Neurophysiol., 34 (1971) 676-684. 7 HAGIWARA, S., AND NAKAJIMA, S., Differences in Na and Ca spikes as examined by application of tetrodotoxin, procaine and manganese ions, J. gen. Physiol., 49 (1966) 793-806. 8 HAMILTON,D. W., The calyceal synapse of type I vestibular hair cells, J. Ultrastruct. Res., 23 (1968) 98-114. 9 HARRIS,G. G., FRISHKOPF,L. S., ANDFLOCK,/~.,Receptor potentials from hair cells of the lateral line, Science, 167 (1970) 76-79.
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10 KAO, C. Y., Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena, Pharmacol. Rev., 18 (1966) 997-1099. 11 KATZ, B., AND MILEDI, R., Tetrodotoxin resistant electric activity in presynaptic terminals, J. Physiol. (Lond.), 203 (1969) 459--487. 12 "KATZ, B., ANOMILEDI, R., Spontaneous and evoked activity of motor nerve endings in calcium Ringer, J. Physiol. (Lond.), 203 (1969) 689-706. 13 OBARA, S., AND BENNETT, M. V. L., Mode of operation of ampullae of Lorenzini of the skate, Raja, J. gen. Physiol., 60 (1972) 534-557. 14 REDFERN,P., LUNDH, H., AND THESLEFF,S., Tetrodotoxin resistant action potentials in denervated rat skeletal muscle, Europ. J. Pharmacol., 11 (1970) 263-265. 15 SPOENDLIN,H., Some morphofunctional and pathological aspects of the vestibular sensory epithelia. In 2nd Symposium on the Role of Vestibular Organs in Space Exploration, NASA, Washington, D.C., 1966, pp. 99-115. 16 STEINBACH,A. B., Transmission from receptor cells to afferent nerve fibers. In M. V. L. BENNETT (Ed.), Synaptic Transmission and lnterneuronal Communication. Raven Press, New York, in press. 17 STEINBACH,A. B., AND BENNETT, M. V. L., Effect of divalent ions and drugs on synaptic transmission in phasic electroreceptors in a mormyrid fish, J. gen. Physiol., 58 (1971) 580-598. 18 ZIPSER, B., Tetrodotoxin resistant electrically excitable responses of receptor cells, Biophys. Soc. Abstr. 15th Ann. Meeting, (1971) 44a.