JOURNAL OF NEUROBIOLOGY, VOL. 6, NO. 4, PP. 429-433
Inhibitory Transmission at a Molluscan Neuromuscular Junction Nervous inhibition of the heart of molluscs has been known since the last century (Ransom, 1883; Krijgsman and Divaris, 1955). However, no other peripheral neuromuscular inhibition has been reported in molluscs despite its widespread occurrance in other invertebrates (Gerschenfeld, 1973). In the lamellibranch mollusc, Mytilus, nervous stimulation can cause relaxation of tension in smooth muscle displaying a catch. These neurons act not by inhibiting excitation, but by selective relaxation of the catch mechanism (Twarog, 1967a). Excitatory junctional potentials produced by axon stimulation have been recorded in a variety of molluscan muscles (Twarog, 196713; Kater, Heyer, and Hegman, 1971; Kupferman, Pinsker, Castelucci, and Kandel, 1971) and recently depolarizing and hyperpolarizing junctional potentials were recorded in gill muscle of Aplysia (Carew, Pinsker, Rubinson, Schwartz, and Kandel, personal communication). In this communication evidence is provided for peripheral inhibition at a neuromuscular junction in the buccal mass of Aplysia. The fine structure and electrophysiology of excitatory junctional transmission are reported elsewhere (Orkand and Orkand, 1975). The experimental preparation consisted of the buccal ganglion and lower extrinsic protractor muscle of Aplysia californica pinned to a Sylgard floor of a Perspex chamber and bathed in “Instant Ocean” sea water. The solution was kept at 10°C to reduce spontaneous activity. Figure 1A is a diagram of the experimental arrangement for intracellular stimulation and recording with a single microelectrode from neurons in the buccal ganglion (Martin and Pilar, 1963). Intracellular recordings were obtained from muscle fibers and extracellular stimulation was applied to the R-4 root connecting the ganglion with the muscle. The experimental apparatus consisted of conventional high impedance preamps and a bridge circuit. To penetrate neurons through the thick connective tissue, which encapsulates the ganglion, the electrode holder was mounted on the diaphragm of a 2 in. speaker and caused to vibrate a t 100 Hz. Neurons which might innervate the muscle were first located by soaking the R-4 root in a 0.4 M cobalt chloride solution. This solution will diffuse up the innervating axon and fill the cell soma in about three days when kept a t 4OC. The preparation is then rinsed in a 10% ammonium sulfide solution in sea water at room temperature. The precipitating reaction takes place rapidly, leaving a cobalt sulfide precipitate which clearly identifies the innervating cell bodies (Pearson and Fourtner, 1973). Three cells were located in the right ganglion with this technique. Figure 1B shows simultaneous records from the most medial neuron located with this technique on the rostra1 surface (abutting the buccal mass) of the lower medial quadrant of the buccal ganglion and an innervated muscle fiber. The neuron was depolarized to give a train of action potentials by current passed through the intracellular elec429 01975 by John Wiley & Sons, Inc.
BANKS
430
C.R.O.
(A)
MUSCLE
Fig. 1 (continued).
trode. Each action potential in the neuron was followed by a hyperpolarizing junctional potential in the muscle. These potentials ranged from 0.5 to 1 mV in amplitude and occurred with a “fixed” latency of about 100 msec. With increased depolarization the frequency of action potentials increased, eventually leading to a fusion of the hyperpolarizing junctional potentials (Fig. 2D). To test the possibility that these potentials could inhibit excitatory processes, excitatory junctional potentials (e.j.p.’s) were elicited in the muscle by root stimulation in the presence and absence of hyperpolarizing junctional potentials. The experiment was feasible because the spike threshold of the excitatory axons in the root was much below that of the axon of the neuron producing hyperpolarizing junctional potentials. The results of one such experiment is shown in Figure 2. The e.j.p. observed during stimulation of the cell body (2B) was reduced in amplitude from that with no inhibitory stimulation (2A and 2C). It is apparent that the hyperpolarizing potentials also increased the rate of decay of the e.j.p.’s. In three experiments the average e.j.p. amplitude was reduced by 17% and the average time for half decay of the e.j.p. reduced by 79% by comparable trains of impulses. To test physiologically if the axon of the inhibitory cell body ran in the R-4 root, the strength of the stimulus to the root was progressively increased. Figure 3 shows that as the stimulus strength was increased, a graded excitatory synaptic potential was first seen in the cell body, then an orthodromic spike arose from the synaptic potential and finally an antidromic spike with zero latency and no synaptic prepotential. The appearance of the antidromic spike confirmed that the inhibitory cell axon does run in the R-4 root. It is likely that the inhibitory connection to the muscle is monosynaptic because, as shown in Figure 1,the inhibitory junctional potentials follow spikes
MOLL USCA N I N H I R 17’10N
431
Fig. l(A). Diagram of the experimental arrangement for intracellular stimulation and recording with 3 M KCl electrode from neurons of the buccal ganglion, recording from muscle fibers and extracellular stimulation of R-4 root, which connects the ganglion to the muscle. (B) Record of spikes (top trace) in neuron produced by current through intracellular electrode. These are followed one to one, and with a “fixed” latency by hyperpolarizing postsynaptic potentials in the lower extrinsic protractor muscle (bottom trace).
in a one to one fashion at “fixed” latency and in addition there is no histological evidence of neuronal cell bodies which could act as interneurons in the muscle (Orkand and Orkand, 1975). These results provide the first evidence for peripheral inhibition in a molluscan neuromuscular system. It is possible that some of the inhibition is prejunctional (Dude1 and Kuffler, 1964). However, the appearance of a hyperpolarizing potential which leads to a small reduction in amplitude of the e.j.p. and an accelerated rate of decay of the e.j.p. is consistent with the suggestion that the inhibitory transmitter increases the postjunctional membrane conductance to an ion or ions whose equilibrium potential is more negative than the resting potential (Fatt and Katz, 1953).
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Fig. 2. Intracellular records from neuron cell body (top trace) and muscle fiber (bottom trace). (A) Neuron is hyperpolarized by intracellular current (Wheatstone bridge not completely balanced) while root is stimulated once to give an e.j.p. in muscle. (B) Neuron is depolarized by same current to give a train of spikes during e.j.p. in muscle. Note hyperpolarization in muscle, decreased amplitude of e.j.p. and increased rate of decay of e.j.p. (C) Repeat of control e.j.p. 2 sec after B. (D) Fused hyperpolarizing potentials in muscle produced by depolarization and spikes in neuron. No root stimulation.
Fig. 3. Intracellular recordings from the neuron cell body during root stimulation. Stimulus increased left to right. Initial response is excitatory synaptic potential which upon increase in stimulus intensity gives rise to a spike in cell soma. Further increase of stimulus elicits an antidromic spike. Line beneath trace indicates time of stimulus.
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433
Contraction of this muscle results from summation and facilitation of e.j.p.’s (Orkand and Orkand, 1975), therefore, such an increase in conductance seems well suited to inhibit contraction. This investigation was supported by U.S. Public Health Service No. 08346 and training grant No. GM-448-13. The author thanks Dr. R. Orkand and Dr. D. Junge for their suggestions and criticisms and also Don Finegold for his technical assistance in these experiments. REFERENCES DUDEL,J. and KUFFLER,S. W. (1961). Presynaptic inhibition at the crayfish neuromuscular junction. J . Physiol. 155: 543-562. FATT, P. and KATZ,B. (1953). The effect of inhibitory nerve impulses on a crustacean muscle fiber. J.Physiol. 121: 374-389. GERSCHENFELD, H. M. (1973). Chemical transmission in invertebrate central nervous systems and neuromuscular junction. Physiol. Reu. 53: 1-119. KATER, S. B., HEYER,C. and HEGMAN,J. P. (1971). Neuromuscular transmission in the gastropod mollusc Heliosoma triuoluis: Identification of motor neurons. 2. Vergl. Physiol. 74: 127-139. KRIJGSMAN,B. J. and DIVARIS,0. A. (1955). Contractile and pacemaker mechanisms of the heart of molluscs. Biol. Reu. 30: 1-39. I., PINSKER,H., CASTELLUCCI, V. and KANDEL,E. R. (1971). Central and periKUPFERMAN, pherial control of gill movements in Aplysia. Science 174: 1252-1256. MARTIN,A. R. and PILAR,G. (1963). An analysis of electrical coupling at synapses in the avian ciliary ganglion. J . Physiol. 130 114-122. ORKAND,P. M. and ORKAND,R. K. (1975). Neuromuscular junction in the buccal mass of A p lysia: Fine structure and electrophysiology of excitatory transmission. J. Neurobiol. 6. PEARSON,K. G. and FOURTNER,C. R. (1973). Identification of the somata of common inhibitory motoneurons in the metathoracic ganglion of the cockroach. Canadian J . 2001.51: 859866. RANSOM,W. B. (1884). On the cardiac rhythm of invertebrate. J. Physiol. 5: 261-341. TWAROG,B. M. (1967a). The regulation of catch in molluscan muscle. J . Gen. Physiol. 5 0 157-161. TWAROG, B. M. (1967b). Excitation of Mytilus smooth muscle. J. Physiol. 192: 857-808.
FLOYD W. BANKS Department of Biology University of California Los Angeles, California Accepted for publication January 16,1974
Inhibitory Transmission at a Molluscan Neuromuscular Junction Nervous inhibition of the heart of molluscs has been known since the last century (Ransom, 1883; Krijgsman and Divaris, 1955). However, no other peripheral neuromuscular inhibition has been reported in molluscs despite its widespread occurrance in other invertebrates (Gerschenfeld, 1973). In the lamellibranch mollusc, Mytilus, nervous stimulation can cause relaxation of tension in smooth muscle displaying a catch. These neurons act not by inhibiting excitation, but by selective relaxation of the catch mechanism (Twarog, 1967a). Excitatory junctional potentials produced by axon stimulation have been recorded in a variety of molluscan muscles (Twarog, 196713; Kater, Heyer, and Hegman, 1971; Kupferman, Pinsker, Castelucci, and Kandel, 1971) and recently depolarizing and hyperpolarizing junctional potentials were recorded in gill muscle of Aplysia (Carew, Pinsker, Rubinson, Schwartz, and Kandel, personal communication). In this communication evidence is provided for peripheral inhibition at a neuromuscular junction in the buccal mass of Aplysia. The fine structure and electrophysiology of excitatory junctional transmission are reported elsewhere (Orkand and Orkand, 1975). The experimental preparation consisted of the buccal ganglion and lower extrinsic protractor muscle of Aplysia californica pinned to a Sylgard floor of a Perspex chamber and bathed in “Instant Ocean” sea water. The solution was kept at 10°C to reduce spontaneous activity. Figure 1A is a diagram of the experimental arrangement for intracellular stimulation and recording with a single microelectrode from neurons in the buccal ganglion (Martin and Pilar, 1963). Intracellular recordings were obtained from muscle fibers and extracellular stimulation was applied to the R-4 root connecting the ganglion with the muscle. The experimental apparatus consisted of conventional high impedance preamps and a bridge circuit. To penetrate neurons through the thick connective tissue, which encapsulates the ganglion, the electrode holder was mounted on the diaphragm of a 2 in. speaker and caused to vibrate a t 100 Hz. Neurons which might innervate the muscle were first located by soaking the R-4 root in a 0.4 M cobalt chloride solution. This solution will diffuse up the innervating axon and fill the cell soma in about three days when kept a t 4OC. The preparation is then rinsed in a 10% ammonium sulfide solution in sea water at room temperature. The precipitating reaction takes place rapidly, leaving a cobalt sulfide precipitate which clearly identifies the innervating cell bodies (Pearson and Fourtner, 1973). Three cells were located in the right ganglion with this technique. Figure 1B shows simultaneous records from the most medial neuron located with this technique on the rostra1 surface (abutting the buccal mass) of the lower medial quadrant of the buccal ganglion and an innervated muscle fiber. The neuron was depolarized to give a train of action potentials by current passed through the intracellular elec429 01975 by John Wiley & Sons, Inc.
BANKS
430
C.R.O.
(A)
MUSCLE
Fig. 1 (continued).
trode. Each action potential in the neuron was followed by a hyperpolarizing junctional potential in the muscle. These potentials ranged from 0.5 to 1 mV in amplitude and occurred with a “fixed” latency of about 100 msec. With increased depolarization the frequency of action potentials increased, eventually leading to a fusion of the hyperpolarizing junctional potentials (Fig. 2D). To test the possibility that these potentials could inhibit excitatory processes, excitatory junctional potentials (e.j.p.’s) were elicited in the muscle by root stimulation in the presence and absence of hyperpolarizing junctional potentials. The experiment was feasible because the spike threshold of the excitatory axons in the root was much below that of the axon of the neuron producing hyperpolarizing junctional potentials. The results of one such experiment is shown in Figure 2. The e.j.p. observed during stimulation of the cell body (2B) was reduced in amplitude from that with no inhibitory stimulation (2A and 2C). It is apparent that the hyperpolarizing potentials also increased the rate of decay of the e.j.p.’s. In three experiments the average e.j.p. amplitude was reduced by 17% and the average time for half decay of the e.j.p. reduced by 79% by comparable trains of impulses. To test physiologically if the axon of the inhibitory cell body ran in the R-4 root, the strength of the stimulus to the root was progressively increased. Figure 3 shows that as the stimulus strength was increased, a graded excitatory synaptic potential was first seen in the cell body, then an orthodromic spike arose from the synaptic potential and finally an antidromic spike with zero latency and no synaptic prepotential. The appearance of the antidromic spike confirmed that the inhibitory cell axon does run in the R-4 root. It is likely that the inhibitory connection to the muscle is monosynaptic because, as shown in Figure 1,the inhibitory junctional potentials follow spikes
MOLL USCA N I N H I R 17’10N
431
Fig. l(A). Diagram of the experimental arrangement for intracellular stimulation and recording with 3 M KCl electrode from neurons of the buccal ganglion, recording from muscle fibers and extracellular stimulation of R-4 root, which connects the ganglion to the muscle. (B) Record of spikes (top trace) in neuron produced by current through intracellular electrode. These are followed one to one, and with a “fixed” latency by hyperpolarizing postsynaptic potentials in the lower extrinsic protractor muscle (bottom trace).
in a one to one fashion at “fixed” latency and in addition there is no histological evidence of neuronal cell bodies which could act as interneurons in the muscle (Orkand and Orkand, 1975). These results provide the first evidence for peripheral inhibition in a molluscan neuromuscular system. It is possible that some of the inhibition is prejunctional (Dude1 and Kuffler, 1964). However, the appearance of a hyperpolarizing potential which leads to a small reduction in amplitude of the e.j.p. and an accelerated rate of decay of the e.j.p. is consistent with the suggestion that the inhibitory transmitter increases the postjunctional membrane conductance to an ion or ions whose equilibrium potential is more negative than the resting potential (Fatt and Katz, 1953).
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RANKS
Fig. 2. Intracellular records from neuron cell body (top trace) and muscle fiber (bottom trace). (A) Neuron is hyperpolarized by intracellular current (Wheatstone bridge not completely balanced) while root is stimulated once to give an e.j.p. in muscle. (B) Neuron is depolarized by same current to give a train of spikes during e.j.p. in muscle. Note hyperpolarization in muscle, decreased amplitude of e.j.p. and increased rate of decay of e.j.p. (C) Repeat of control e.j.p. 2 sec after B. (D) Fused hyperpolarizing potentials in muscle produced by depolarization and spikes in neuron. No root stimulation.
Fig. 3. Intracellular recordings from the neuron cell body during root stimulation. Stimulus increased left to right. Initial response is excitatory synaptic potential which upon increase in stimulus intensity gives rise to a spike in cell soma. Further increase of stimulus elicits an antidromic spike. Line beneath trace indicates time of stimulus.
MOLLUSCAN INHIBITION
433
Contraction of this muscle results from summation and facilitation of e.j.p.’s (Orkand and Orkand, 1975), therefore, such an increase in conductance seems well suited to inhibit contraction. This investigation was supported by U.S. Public Health Service No. 08346 and training grant No. GM-448-13. The author thanks Dr. R. Orkand and Dr. D. Junge for their suggestions and criticisms and also Don Finegold for his technical assistance in these experiments. REFERENCES DUDEL,J. and KUFFLER,S. W. (1961). Presynaptic inhibition at the crayfish neuromuscular junction. J . Physiol. 155: 543-562. FATT, P. and KATZ,B. (1953). The effect of inhibitory nerve impulses on a crustacean muscle fiber. J.Physiol. 121: 374-389. GERSCHENFELD, H. M. (1973). Chemical transmission in invertebrate central nervous systems and neuromuscular junction. Physiol. Reu. 53: 1-119. KATER, S. B., HEYER,C. and HEGMAN,J. P. (1971). Neuromuscular transmission in the gastropod mollusc Heliosoma triuoluis: Identification of motor neurons. 2. Vergl. Physiol. 74: 127-139. KRIJGSMAN,B. J. and DIVARIS,0. A. (1955). Contractile and pacemaker mechanisms of the heart of molluscs. Biol. Reu. 30: 1-39. I., PINSKER,H., CASTELLUCCI, V. and KANDEL,E. R. (1971). Central and periKUPFERMAN, pherial control of gill movements in Aplysia. Science 174: 1252-1256. MARTIN,A. R. and PILAR,G. (1963). An analysis of electrical coupling at synapses in the avian ciliary ganglion. J . Physiol. 130 114-122. ORKAND,P. M. and ORKAND,R. K. (1975). Neuromuscular junction in the buccal mass of A p lysia: Fine structure and electrophysiology of excitatory transmission. J. Neurobiol. 6. PEARSON,K. G. and FOURTNER,C. R. (1973). Identification of the somata of common inhibitory motoneurons in the metathoracic ganglion of the cockroach. Canadian J . 2001.51: 859866. RANSOM,W. B. (1884). On the cardiac rhythm of invertebrate. J. Physiol. 5: 261-341. TWAROG,B. M. (1967a). The regulation of catch in molluscan muscle. J . Gen. Physiol. 5 0 157-161. TWAROG, B. M. (1967b). Excitation of Mytilus smooth muscle. J. Physiol. 192: 857-808.
FLOYD W. BANKS Department of Biology University of California Los Angeles, California Accepted for publication January 16,1974
