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The normal accumulation of facilitation during presynaptic inhibition
Brain Research, 189 (1980) 535-539 © Elsevier/North-HollandBiomedicalPress
535
The normal accumulation of facilitation during presynaptic inhibition
DOUGLAS A. BAXTER and GEORGE D. BITTNER Department of Zoology, University of Texas, Austin, Texas 78712 (U.S.A.)
(Accepted January 3rd, 1980) Key words: presynapticinhibition-- crayfish-- facilitation
One important feature of chemical transmission by neurons is facilitation which is often defined as the release of increasing amounts of transmitter by successive action potentials. Facilitation is of interest to neurobiologists because the modulation of transmitter release by previous activity may be an important component of learning, memory and other complex behavior 11. Since the level of polarization in the presynaptic terminal has a profound affect on transmitter release, changes in terminal polarization have been suspected to be important in the development of facilitation (see ref. 6 for a review). However, several lines of electrophysiological evidence suggest that facilitation cannot be explained by changes in action potential amplitude because: (i) the development of facilitation can occur in the absence of progressive increases in the action potential amplitude6,14; (ii) artificial depolarization of nerve terminals by current pulses of uniform amplitude can still produce facilitation at frog 12, squid 6 and crayfish synapses15; and (iii) presynaptic membrane depolarizations which vary over a wide range above a minimum amplitude (10-20 mV) produce about the same amount of facilitation, suggesting that the production of facilitation is rather independent of the amplitude of the presynaptic voltage change 6. The implication of these data is that constant frequency trains of conditioning pulses of small presynaptic amplitude should produce about the same amount of facilitation as trains of pulses of large presynaptic amplitude at the same frequency. In the study reported herein, we have used the dactyl opener muscle in the cheliped (claw) of the crayfish Procambarus clarkii to investigate this postulated independence between membrane depolarization and development of facilitation. This muscle is innervated by only two axons, an excitor and an inhibitor which we isolated and independently stimulated in the meropodite segment of the claw s. The inhibitory motor neuron makes both postsynaptic inhibitory junctions on the muscle fibers and presynaptic inhibitory synapses on the excitatory motor axon 1°. The release of GABA (the inhibitory transmitter) onto the excitatory axon reduces the amount of excitatory transmitter released during stimulation7,s and increases the conductance of the axonal membrane 9. The increase in membrane conductance presumably reduces the amplitude of the presynaptic action potential occurring in the excitatory nerve terminals during presynaptic inhibition7.
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Fig. 1. A and B: computer averaged plots of EPSPs intracellularly recorded from a crayfish opener muscle fiber during non-inhibited (A) and inhibited (B) stimulus trains of 100 Hz. Dashed lines indicate the expected decay of the potentials. The amplitude of each EPSP was measured from its peak to the projected 'tail' of the preceding EPSP. The small (less than 5 ~) correction of EPSP size for nonlinear summation of quantal effect was generally ignored4. Calibration mark applies to both A and B. C: growth in EPSP amplitudes calculated for A and B. The facilitation of transmitter release accumulated normally during presynaptic inhibition. Note that the last 3 EPSPs in B and the filled circles in C are not inhibited.
The release of excitatory transmitter was monitored via intracellular recordings of excitatory postsynaptic potentials (EPSPs) from opener muscle fibers a. Once a muscle fiber was penetrated, brief trains of 100 Hz stimuli having anywhere from 8 to 17 equal interval pulses were given to the excitatory and/or inhibitory axons. The trains were repeated every 5 sec and 25 successive trains were averaged with a signalaveraging computer 4. First, the excitatory axon was stimulated alone and the non-
537 inhibited train of EPSPs was plotted (Fig. 1A). Then the excitatory train was repeated while the inhibitory axon was stimulated at a relative latency (phase angle, 0, in Fig. 1B) which produced maximum presynaptic inhibitionT,8; that is, each inhibitory action potential preceded each excitatory action potential by 1-3 msec. After 4-14 paired excitatory and inhibitory stimuli, the inhibitory axon was no longer stimulated and the remaining excitatory action potentials were released from inhibition (Fig. 1B). The same qualitative results were observed using single presentations of the two trains and displaying the amplified EPSPs on a storage oscilloscope. Furthermore, these results were stable over a period of several hours in any given muscle fiber. EPSP amplitudes facilitated rapidly during the non-inhibited train (Fig. 1C). The amplitudes of the presynaptically inhibited EPSPs (inh-EPSPs) did not grow as rapidly and the last inh-EPSP of the inhibited train was reduced by 50-70 ~o compared to the corresponding EPSP in the non-inhibited train (Fig. 1C). Two lines of evidence indicated that this reduction in EPSP amplitude was of presynaptic rather than postsynaptic origin. Firstly, when the stimulation of the excitatory axon preceded the stimulation of the inhibitory axon by 1 msec, inhibition of the EPSP was much less effective. This reversal of the phase angle for optimum presynaptic inhibition typically resulted in reduction of the last inh-EPSP in an inhibited train by an average of 8 ~o (range 1-17 ~) rather than 50-70 ~. Secondly, we performed a direct test of the effect of the inhibitor on postsynaptic membrane conductance by injecting a 2-3 msec depolarizing current pulse into the impaled muscle fiber to displace its membrane potential to about the same exctent as that produced by the excitatory stimulation alone. The inhibitory axon was stimulated at the same frequency as before and the depolarizing pulse was injected at the point in time corresponding to the last inhEPSP. The per cent reduction of the artificial depolarization was used as an index of postsynaptic inhibition. Postsynaptic inhibition was found to have contributed only 2-9 ~o (mean --~ 3.5 ~o) of the observed 50-70 ~ reduction of the last inh-EPSP in an inhibited train. The effect of presynaptic inhibition alone upon facilitation was calculated in 27 muscle fibers by subtracting the amount of postsynaptic inhibition from the total amount of inhibition. In 18 of these fibers, presynaptic inhibition reduced the last inhEPSP amplitude by 40-72 ~o (mean ---- 55 ~o) below the corresponding EPSP amplitude in the non-inhibited train. In these fibers when stimulation of the inhibitor was suddenly stopped, the amplitude for the EPSP which followed presynaptic inhibition abruptly increased to within 10~o above or below the amplitude calculated for the corresponding EPSP in the non-inhibited train (mean ----- 4~o below the EPSP amplitude observed in the non-inhibited train). The amplitudes for the 2-6 noninhibited EPSPs following inhibition remained within 10~ above or below the amplitudes calculated for the corresponding EPSPs in the non-inhibited train. For example, in Fig. 1C the amplitude for the twelfth inh-EPSP in the inhibited train was reduced 43 ~ below the amplitude for the twelfth EPSP in the non-inhibited train. However, after the presynaptic inhibition was terminated, the amplitude for the noninhibited thirteenth EPSP in the previously inhibited train increased to within 5 ~ of the amplitude for the thirteenth EPSP in the non-inhibited train. Furthermore, the
538 amount of postsynaptic inhibition remaining at this synapse was 3 ~i at the thirteenth pulse of the previously inhibited train. Hence, the EPSP amplitude following inhibition was within 2)o~ of the amplitude at the thirteenth pulse of a non-inhibited train. In other words, although the inh-EPSP amplitudes were reduced by presynaptic inhibition, the facilitation process accumulated normally and upon removal of the inhibition, this accumulated facilitation was expressed as a dramatic increase in the EPSP amplitude. Not all of the fibers clearly demonstrated as large an increase in EPSP amplitude following release from inhibition. In 5 of the 27 preparations, the presynaptic inhibition reduced the amplitude of the last inh-EPSP by 50-78 o~ (mean ~- 69 'J'~'i)but the EPSP amplitude immediately following inhibition remained below the EPSP amplitude observed in the non-inhibited train (mean =: 35 o/,,, below the non-inhibited EPSP amplitudes; range 25-45~). Lastly, in 4 of the 27 preparations, very little reduction in EPSP amplitude was seen during the presynaptically inhibited train. This study shows that the facilitation process can accumulate normally during presynaptic inhibition of transmitter release at crustacean neuromuscular junctions. The functional implication of this result for the crayfish or other organisms is that the suppression of a muscle movement or more complex behavior by presynaptic inhibition can be followed by a fully facilitated movement or behavior upon removal of presynaptic inhibitione,5,13. These results from crustacean synapses agree with the data from squid synapses 6 that artificial, uniform depolarizations of greatly different amplitudes produce about the same amount of facilitation. Hence, basic cellular mechanisms for facilitation may well be similar in most organisms and may have undergone a rather conservative evolution, as have other cellular mechanisms for the oroduction of action potentials and the release of transmitter. This work was supported in part by R C D A Grant NS00070 to G.D.B.
1 Atwood, H. L. and Bittner, G. D., Matching of excitatory and inhibitory inputs to crustacean muscle fibers, J. NeurophysioL, 34 (1971) 157-170. 2 Atwood, H. L. and Wolcott, B., Recording of electrical activity and movement from legs of walking crabs, Canad. J. ZooL, 43 (1968) 657-665. 3 Bittner, G. D., Differentiation of nerve terminals in the crayfish opener muscle and its functional significance, J. gen. PhysioL, 51 (1968) 731-758. 4 Bittner, G. D. and Sewell, V. L., Facilitation at crayfish neuromuscular junctions, J. comp. PhysioL, 109 (1976) 287-308. 5 Carew, T. J., Pinsker, H., Rubinson, K. and Kandel, E. R., Physiological and biochemical properties of neuromuscular transmission between identified motorneurons and gilt muscle in Aplysia, J. Neurophysiol., 37 (1974) 1020-1040. 6 Charlton, M. P. and Bittner, G. D., Presynaptic potentials and facilitation of transmitter release in the squid giant synapse, J. gen. PhysioL, 72 (1978) 487-511. 7 Dudel, J., Presynaptic inhibition of the excitatory nerve terminals in the neuromuscular junction of the crayfish, Pfliigers Arch. ges. PhysioL, 277 (1963) 537-557. 8 Dudel, J. and Kuffler, S. W., Presynaptic inhibition at the crayfish neuromuscular junction, J. PhysioL (Lond.), 155 (1961) 534-562. 9 Fuchs, P. A., Conduction and Inhibition in a Crayfish Motor Axon, Ph.D. Thesis, Stanford University, 1979.
539 10 Jahromi, S. S. and Atwood, H. L., Three-dimensionalultrastructure of the crayfish neuromuscular apparatus, J. Cell BioL, 63 (1974) 599-613. 11 Kandel, E. R., Behavioral Biology ofAplysia, W. H. Freeman, San Francisco, 1979. 12 Katz, B. and Miledi, R., Tetrodotoxin and neuromuscular transmission, Proc. Roy Soc. B, 167 (1967) 8-22. 13 Kennedy, D., Identified Neurons and Behavior of Arthropods, Plenum Press, New York, 1977, 111 PP. 14 Martin, K. L. and Pilar, G., Pre-synaptic and post-synaptic events during post-tetanic potentiation and facilitation in the avian ciliary ganglion, J. Physiol. (Lond.), 175 (1964) 17-30. 15 Zucker, R. S., Crayfish neuromuscular facilitation activated by constant pre-synaptic action potentials and depolarizing pulses, J. Physiol. (Lond.), 241 (1974) 69-90.
535
The normal accumulation of facilitation during presynaptic inhibition
DOUGLAS A. BAXTER and GEORGE D. BITTNER Department of Zoology, University of Texas, Austin, Texas 78712 (U.S.A.)
(Accepted January 3rd, 1980) Key words: presynapticinhibition-- crayfish-- facilitation
One important feature of chemical transmission by neurons is facilitation which is often defined as the release of increasing amounts of transmitter by successive action potentials. Facilitation is of interest to neurobiologists because the modulation of transmitter release by previous activity may be an important component of learning, memory and other complex behavior 11. Since the level of polarization in the presynaptic terminal has a profound affect on transmitter release, changes in terminal polarization have been suspected to be important in the development of facilitation (see ref. 6 for a review). However, several lines of electrophysiological evidence suggest that facilitation cannot be explained by changes in action potential amplitude because: (i) the development of facilitation can occur in the absence of progressive increases in the action potential amplitude6,14; (ii) artificial depolarization of nerve terminals by current pulses of uniform amplitude can still produce facilitation at frog 12, squid 6 and crayfish synapses15; and (iii) presynaptic membrane depolarizations which vary over a wide range above a minimum amplitude (10-20 mV) produce about the same amount of facilitation, suggesting that the production of facilitation is rather independent of the amplitude of the presynaptic voltage change 6. The implication of these data is that constant frequency trains of conditioning pulses of small presynaptic amplitude should produce about the same amount of facilitation as trains of pulses of large presynaptic amplitude at the same frequency. In the study reported herein, we have used the dactyl opener muscle in the cheliped (claw) of the crayfish Procambarus clarkii to investigate this postulated independence between membrane depolarization and development of facilitation. This muscle is innervated by only two axons, an excitor and an inhibitor which we isolated and independently stimulated in the meropodite segment of the claw s. The inhibitory motor neuron makes both postsynaptic inhibitory junctions on the muscle fibers and presynaptic inhibitory synapses on the excitatory motor axon 1°. The release of GABA (the inhibitory transmitter) onto the excitatory axon reduces the amount of excitatory transmitter released during stimulation7,s and increases the conductance of the axonal membrane 9. The increase in membrane conductance presumably reduces the amplitude of the presynaptic action potential occurring in the excitatory nerve terminals during presynaptic inhibition7.
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Fig. 1. A and B: computer averaged plots of EPSPs intracellularly recorded from a crayfish opener muscle fiber during non-inhibited (A) and inhibited (B) stimulus trains of 100 Hz. Dashed lines indicate the expected decay of the potentials. The amplitude of each EPSP was measured from its peak to the projected 'tail' of the preceding EPSP. The small (less than 5 ~) correction of EPSP size for nonlinear summation of quantal effect was generally ignored4. Calibration mark applies to both A and B. C: growth in EPSP amplitudes calculated for A and B. The facilitation of transmitter release accumulated normally during presynaptic inhibition. Note that the last 3 EPSPs in B and the filled circles in C are not inhibited.
The release of excitatory transmitter was monitored via intracellular recordings of excitatory postsynaptic potentials (EPSPs) from opener muscle fibers a. Once a muscle fiber was penetrated, brief trains of 100 Hz stimuli having anywhere from 8 to 17 equal interval pulses were given to the excitatory and/or inhibitory axons. The trains were repeated every 5 sec and 25 successive trains were averaged with a signalaveraging computer 4. First, the excitatory axon was stimulated alone and the non-
537 inhibited train of EPSPs was plotted (Fig. 1A). Then the excitatory train was repeated while the inhibitory axon was stimulated at a relative latency (phase angle, 0, in Fig. 1B) which produced maximum presynaptic inhibitionT,8; that is, each inhibitory action potential preceded each excitatory action potential by 1-3 msec. After 4-14 paired excitatory and inhibitory stimuli, the inhibitory axon was no longer stimulated and the remaining excitatory action potentials were released from inhibition (Fig. 1B). The same qualitative results were observed using single presentations of the two trains and displaying the amplified EPSPs on a storage oscilloscope. Furthermore, these results were stable over a period of several hours in any given muscle fiber. EPSP amplitudes facilitated rapidly during the non-inhibited train (Fig. 1C). The amplitudes of the presynaptically inhibited EPSPs (inh-EPSPs) did not grow as rapidly and the last inh-EPSP of the inhibited train was reduced by 50-70 ~o compared to the corresponding EPSP in the non-inhibited train (Fig. 1C). Two lines of evidence indicated that this reduction in EPSP amplitude was of presynaptic rather than postsynaptic origin. Firstly, when the stimulation of the excitatory axon preceded the stimulation of the inhibitory axon by 1 msec, inhibition of the EPSP was much less effective. This reversal of the phase angle for optimum presynaptic inhibition typically resulted in reduction of the last inh-EPSP in an inhibited train by an average of 8 ~o (range 1-17 ~) rather than 50-70 ~. Secondly, we performed a direct test of the effect of the inhibitor on postsynaptic membrane conductance by injecting a 2-3 msec depolarizing current pulse into the impaled muscle fiber to displace its membrane potential to about the same exctent as that produced by the excitatory stimulation alone. The inhibitory axon was stimulated at the same frequency as before and the depolarizing pulse was injected at the point in time corresponding to the last inhEPSP. The per cent reduction of the artificial depolarization was used as an index of postsynaptic inhibition. Postsynaptic inhibition was found to have contributed only 2-9 ~o (mean --~ 3.5 ~o) of the observed 50-70 ~ reduction of the last inh-EPSP in an inhibited train. The effect of presynaptic inhibition alone upon facilitation was calculated in 27 muscle fibers by subtracting the amount of postsynaptic inhibition from the total amount of inhibition. In 18 of these fibers, presynaptic inhibition reduced the last inhEPSP amplitude by 40-72 ~o (mean ---- 55 ~o) below the corresponding EPSP amplitude in the non-inhibited train. In these fibers when stimulation of the inhibitor was suddenly stopped, the amplitude for the EPSP which followed presynaptic inhibition abruptly increased to within 10~o above or below the amplitude calculated for the corresponding EPSP in the non-inhibited train (mean ----- 4~o below the EPSP amplitude observed in the non-inhibited train). The amplitudes for the 2-6 noninhibited EPSPs following inhibition remained within 10~ above or below the amplitudes calculated for the corresponding EPSPs in the non-inhibited train. For example, in Fig. 1C the amplitude for the twelfth inh-EPSP in the inhibited train was reduced 43 ~ below the amplitude for the twelfth EPSP in the non-inhibited train. However, after the presynaptic inhibition was terminated, the amplitude for the noninhibited thirteenth EPSP in the previously inhibited train increased to within 5 ~ of the amplitude for the thirteenth EPSP in the non-inhibited train. Furthermore, the
538 amount of postsynaptic inhibition remaining at this synapse was 3 ~i at the thirteenth pulse of the previously inhibited train. Hence, the EPSP amplitude following inhibition was within 2)o~ of the amplitude at the thirteenth pulse of a non-inhibited train. In other words, although the inh-EPSP amplitudes were reduced by presynaptic inhibition, the facilitation process accumulated normally and upon removal of the inhibition, this accumulated facilitation was expressed as a dramatic increase in the EPSP amplitude. Not all of the fibers clearly demonstrated as large an increase in EPSP amplitude following release from inhibition. In 5 of the 27 preparations, the presynaptic inhibition reduced the amplitude of the last inh-EPSP by 50-78 o~ (mean ~- 69 'J'~'i)but the EPSP amplitude immediately following inhibition remained below the EPSP amplitude observed in the non-inhibited train (mean =: 35 o/,,, below the non-inhibited EPSP amplitudes; range 25-45~). Lastly, in 4 of the 27 preparations, very little reduction in EPSP amplitude was seen during the presynaptically inhibited train. This study shows that the facilitation process can accumulate normally during presynaptic inhibition of transmitter release at crustacean neuromuscular junctions. The functional implication of this result for the crayfish or other organisms is that the suppression of a muscle movement or more complex behavior by presynaptic inhibition can be followed by a fully facilitated movement or behavior upon removal of presynaptic inhibitione,5,13. These results from crustacean synapses agree with the data from squid synapses 6 that artificial, uniform depolarizations of greatly different amplitudes produce about the same amount of facilitation. Hence, basic cellular mechanisms for facilitation may well be similar in most organisms and may have undergone a rather conservative evolution, as have other cellular mechanisms for the oroduction of action potentials and the release of transmitter. This work was supported in part by R C D A Grant NS00070 to G.D.B.
1 Atwood, H. L. and Bittner, G. D., Matching of excitatory and inhibitory inputs to crustacean muscle fibers, J. NeurophysioL, 34 (1971) 157-170. 2 Atwood, H. L. and Wolcott, B., Recording of electrical activity and movement from legs of walking crabs, Canad. J. ZooL, 43 (1968) 657-665. 3 Bittner, G. D., Differentiation of nerve terminals in the crayfish opener muscle and its functional significance, J. gen. PhysioL, 51 (1968) 731-758. 4 Bittner, G. D. and Sewell, V. L., Facilitation at crayfish neuromuscular junctions, J. comp. PhysioL, 109 (1976) 287-308. 5 Carew, T. J., Pinsker, H., Rubinson, K. and Kandel, E. R., Physiological and biochemical properties of neuromuscular transmission between identified motorneurons and gilt muscle in Aplysia, J. Neurophysiol., 37 (1974) 1020-1040. 6 Charlton, M. P. and Bittner, G. D., Presynaptic potentials and facilitation of transmitter release in the squid giant synapse, J. gen. PhysioL, 72 (1978) 487-511. 7 Dudel, J., Presynaptic inhibition of the excitatory nerve terminals in the neuromuscular junction of the crayfish, Pfliigers Arch. ges. PhysioL, 277 (1963) 537-557. 8 Dudel, J. and Kuffler, S. W., Presynaptic inhibition at the crayfish neuromuscular junction, J. PhysioL (Lond.), 155 (1961) 534-562. 9 Fuchs, P. A., Conduction and Inhibition in a Crayfish Motor Axon, Ph.D. Thesis, Stanford University, 1979.
539 10 Jahromi, S. S. and Atwood, H. L., Three-dimensionalultrastructure of the crayfish neuromuscular apparatus, J. Cell BioL, 63 (1974) 599-613. 11 Kandel, E. R., Behavioral Biology ofAplysia, W. H. Freeman, San Francisco, 1979. 12 Katz, B. and Miledi, R., Tetrodotoxin and neuromuscular transmission, Proc. Roy Soc. B, 167 (1967) 8-22. 13 Kennedy, D., Identified Neurons and Behavior of Arthropods, Plenum Press, New York, 1977, 111 PP. 14 Martin, K. L. and Pilar, G., Pre-synaptic and post-synaptic events during post-tetanic potentiation and facilitation in the avian ciliary ganglion, J. Physiol. (Lond.), 175 (1964) 17-30. 15 Zucker, R. S., Crayfish neuromuscular facilitation activated by constant pre-synaptic action potentials and depolarizing pulses, J. Physiol. (Lond.), 241 (1974) 69-90.