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Intracellular recordings from crustacean motor axons during presynaptic inhibition
422
Brain Research, 223 (1981) 422-428 Elsevier/North-Holland Biomedical Press
Intracellular recordings from crustacean motor axons during presynaptic inhibition
DOUGLAS A. BAXTER and GEORGE D. BITTNER* Department of Zoology, University of Texas, Austin, TX 78712 (U.S.A.) (Accepted July 9th, 1981) Key words: presynaptic inhibition --crustacean neuromuscular junction - - inhibitory synapses
Action potentials intracellularly recorded from near the nerve terminals of the opener excitor motor axon in crayfish are reduced in amplitude during presynaptic inhibition. The amplitude and sign (hyperpolarizing or depolarizing) of presynaptic inhibitor potentials (PIPs) depends upon the relationship between the resting membrane potential of each excitor axon and its PIP equilibrium potential (which equals the equilibrium potential for 7-aminobutyric acid). Presynaptic inhibition of transmitter release has been known for several decades to be an important means of integration in many vertebrate and invertebrate neurons 1-4,9, 14-16,z3,24,z6,2s. However, an inability to record intracellularly from the affected nerve terminals during presynaptic inhibition has limited our understanding of its cellular mechanism. This paper describes intracellular recordings obtained near the terminal region of the excitor axon to the opener muscle in crayfish while stimulating the inhibitor axon so as to produce presynaptic inhibitory potentials (PIPs). Our data show that presynaptic inhibition is associated with hyperpolarizing and depolarizing PIPs which reduce the amplitude of action potentials in the presynaptic terminal. Experiments were performed on the claw opener muscle of the crayfish, a preparation which has often been used to study the anatomical and physiological basis of presynaptic inhibitionl-SAl-14,17,19,3°,aL The opener muscle in Procarnbarus simulans is innervated by one excitatory and one inhibitory axon which can be independently stimulated in a more proximal limb segment 1,4,14. Both axons branch together on the surface of the opener muscle (Fig. IA). Using 3 M KC1 or 2 M potassium citrate microelectrodes of 20-60 M f~ resistance, intracellular recordings were made from the opener excitor axon (10-20 # m diameter), where it branches in a Y pattern ~7, 0.3-1.0 mm proximal to its nearest synaptic endings on opener muscle fibers as determined from our scanning electron micrographs and from previously published reconstructions of these axons 2,a,19. The inhibitor axon makes synapses upon the excitor axon at these release sites and on axonal constrictions proximal to excitor release sites e,z,19. A few intracellular recordings were also obtained from smaller (4-10/zm) branches (II or * To whom all correspondence should be addressed. 0006-8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
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Fig. 1. A: schematic representation of the excitor (solid line) and inhibitor (dashed line) axons as they branch together to innervate the opener muscle. Intracellular recordings were made from the main trunk of the excitor axon (I), from the Y branch and from more distal secondary (II), tertiary (III), etc., branches of the excitor axon which synapse (open circles) upon muscle fibers. The inhibitor axon makes both presynaptic inhibitory contacts (closed circles) upon the excitor axon and postsynaptic inhibitory terminals upon muscle fibers2,s,la,le. B-D: presynaptic inhibitory potentials (PIPs) intracellularly recorded with DC coupling from the Y branch of the excitor axon. Data for each figure taken from XY plotter output of 100 potentials averaged in a computer of average transients (CAT). B: temporal summation of 8 PIPs produced by 100 Hz stimulation of the inhibitor nerve axon. The membrane potential attained by these summated PIPs (E~P~Ps) was --82.9 mV and is represented by the upper dotted line. The difference between the two dotted lines (A~,I,rPs ~ 1.1 mV, in this case) represents the difference (in mV) between the resting Em (--84.0 mV) and the near maximum effect of the inhibitory transmitter as recorded at the Y branch. In this fiber, EGAaAwas --81.0 mV and thus the ratio of ~ EPIPsto AEGABAwas 0.37. C: depolarizing PIP (DPIP). Resting E m = --78 mV represented by dotted line. The inhibitor axon was stimulated at 2 Hz in both C and D. D: hyperpolarizing PIP (HPIP). Resting E m = --69 mV represented by dotted line. Calibration line equals 0.2 mV, 10 ms for B; and 0.1 mV, 5 ms for C and D. E: plot of single HPIP and DPIP amplitudes ~V) against resting Era (mV) recorded from each of 20 excitor axon terminals. The Em for each fiber was measured at the time of initial penetration. The heavy solid line plots the linear regression (r = 0.87) of PIP amplitude against resting Em. This regression line shows that PIPs reverse in sign at a membrane potential of--70.5 mV. This value (Eih, heavy arrow) represents the apparent equilibrium potential for the inhibitory transmitter recorded at the Y branch. The average EGABA(light arrow) was --68.5 :E 3.5 mV. I I l in Fig. 1A) within 0.1-0.3 m m o f the t e r m i n a l release sites. In some cases, we r e c o r d e d intracellularly f r o m opener muscle fibers n e a r the Y b r a n c h with KCl-filled electrodes 4-s. I n t r a c e l l u l a r recordings were also m a d e from the excitor axon at v a r i o u s points 0.5-4 c m p r o x i m a l to the o p e n e r muscle. A P I P can be r e c o r d e d f r o m the excitor Y b r a n c h during i n h i b i t o r y stimulation (Fig. 1B-D). I n P. simulans, the resting m e m b r a n e p o t e n t i a l (Era) averages - - 7 4 -I- 7 mV S.D. (n ---- 27) a n d P I P s m a y be either h y p e r p o l a r i z i n g ( H P I P , Fig. 1D) or d e p o l a rizing ( D P I P , Fig. 1B, C). T h e sign a n d a m p l i t u d e o f P I P s v a r y with the resting Em for t h a t a x o n (Fig. 1E). P r o x i m a l to the Y, the P I P a m p l i t u d e decreases with a space constant (2) o f 1.7-3.9 ram. This ~. is similar to t h a t r e p o r t e d for o t h e r crustacean m o t o r axonsl8, 20. Three s e p a r a t e results suggest t h a t the P I P s r e c o r d e d at the Y b r a n c h are p r o d u c e d b y ~,-aminobutyric acid ( G A B A ) a n d represent 35-45 % o f their full value at their sites o f origin in opener nerve t e r m i n a l s o f P. simulans.
424 (1) W h e n the i n h i b i t o r is stimulated at 100 Hz, the a m p l i t u d e o f the first PIP or the plateau values o f t e m p o r a l l y s u m m a t e d PIPs (AZr~ps in Fig. 1B) vary with resting Em in different excitor axons so that the a p p a r e n t reversal potential (En0 o f single PIPs is - - 7 0 . 5 mV (Fig. 1E) a n d o f AZp~ps is - - 6 9 . 3 mV. W h e n G A B A is a d d e d to the b a t h perfusate in s u p r a - m a x i m a l c o n c e n t r a t i o n (0.5 m M ) , as determined by d o s e - r e sponse curves, the average Em at the Y b r a n c h for these excitor axons is - - 6 8 . 5 ± 3.5 mV (n - - 9). G A B A also p r o d u c e d the same Em value (EGA]~A) at all points, from 0.7 m m distal to 4 cm p r o x i m a l from the Y b r a n c h o f the excitor axon (resting Em values were the same at all these axonal positions); that is, excitor axons a p p e a r to have similar G A B A receptors distributed over their entire length. (2) PIPs recorded distal to the Y b r a n c h (0.1-0.3 m m f r o m the nearest release sites) have a m p l i t u d e s within 5-10 ~ o f those r e c o r d e d at the Y branch. (3) PIPs r e c o r d e d at the Y - b r a n c h decrease in a m p l i t u d e during r a p i d s t i m u l a t i o n (20-100 Hz) o f the i n h i b i t o r a x o n (Fig. 1B), while i n h i b i t o r y p o s t s y n a p t i c potentials (IPSPs) recorded from h y p e r p o l a r i z e d muscle fibers usually show m u c h facilitation o f t r a n s m i t t e r release 1. These d a t a suggest that PIPs decrease in a m p l i t u d e during a t r a i n because they a p p r o a c h the P I P reversal potential. Hence, the m a x i m u m a m p l i t u d e a n d A - - pControl -j . . . . . . . . . . . B. Inhibited . . . . . . . . . . . . . . C. GABA
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Fig. 2. Top traces: action potentials (spikes) intracellularly recorded with DC coupling from the Y branch of the excitor axon. Dotted line at bottom of trace equals resting Era (--79 mV). Dotted line at top of trace equals ÷ 21 mV (the peak voltage). Solid line equals 0 mV. Bottom traces: excitatory junctional potentials (EJPs) intracellularly recorded with DC coupling from a postsynaptic muscle fiber near the Y branch. Dotted line equals resting Em (--70 mV). All traces taken from XY plotter output of 100 potentials averaged using a CAT. A: single excitor action potential and EJP. DAP, depolarizing after-potential. The voltage difference between the foot and peak voltages (arrows) is the total amplitude of the spike (100 mV in this case). B: single excitor action potential and EJP given 2 ms after the last pulse in a train of 10 inhibitor pulses at 100 Hz. The 50% decrease in EJP amplitude (lower trace) resulted from presynaptic inhibition as determined by methods of Baxter and Bittner4. The foot-voltage of the presynaptic action potential was depolarized by 700/~V (AZPIPs) due to stimulation of the inhibitor axon; this voltage is not easily detected at this lower amplification and faster sweep speed. The total spike amplitude was reduced to 93 mV and the peak voltage was reduced by 6.3 mV. C: single excitor action potential and EJP when perfusate contained 0.5 mM GABA. The total spike amplitude was reduced to 81 mV and the peak voltage was reduced by 17 mV. In this fiber the ratio of A ZPtvs to AEaA~A (2 mV in this case) was 0.35. Calibration pulse, upper trace -- 10 mV, 1 ms; calibration pulse, lower trace = 0.25 mV, 5 ms.
425 sign of the voltage deflection produced by temporally summated PIPs (AY~pIps in Fig. IB) provides an estimate of the maximum amplitude and sign of the deflection produced by the inhibitory transmitter upon the axon (AEIh). Since GABA is almost certainly the inhibitory neurotransmitter (see refs. 11-13 and 17, and section (1) above), AEIh should approximate AEGABA(Fig.2C) at the sites of presynaptic inhibition. The ratio of A~pips recorded at the Y branch to AEGABAWaS0.38 i 0.07 (n = 7). Since AEpIps should decrement along the excitor nerve axon, whereas AEGABAis the same at all points along the axon, these data suggest that AEpips recorded at the Y branch are decremented by about 60 ~ with regard to AEIh generated at the nearest presynaptic release sites in P. simulans. In other words, the recording site at the Y branch is within a single space constant of the presynaptic inhibitory terminals. This conclusion is in agreement with calculations based on cable theory made for P. clarkii 7. Having provided evidence that membrane voltage at the Y branch is coupled to sites of presynaptic inhibitor action in the excitor axon ofP. simulans, we examined the effects of inhibitor stimulation on excitor action potentials intracellularly recorded at the Y branch (terminal action potential; records taken from more distal branches gave similar results). We found (Fig. 2A) that terminal action potentials have a peak voltage of +20 to +30 mV, a foot-to-peak total amplitude of 93 4- 4 mV (n = 25), a duration of 1 ms at one-half peak amplitude, a depolarizing after-potential (DAP) of 10 4- 2 mV at 2 ms after the rising phase of the spike, and a DAP duration of 30-50 ms. We then stimulated the inhibitor 10 times at 10 ms intervals so as to facilitate the release of inhibitory neurotransmitter. The excitor axon was stimulated 1-2 ms after the 10th inhibitory pulse so that the excitor axon potential arrived at its terminal 0.5-1.0 ms after the last inhibitor action potential reached its terminal1, TM. This stimulus paradigm produces maximal presynaptic inhibitory effects1,4,14as evidenced by a maximum reduction of excitory junctional potentials (EJPs) recorded from adjacent muscle fibers (Fig. 2B; postsynaptic inhibition was usually rather small as determined by conventional techniques1,4). This stimulus paradigm reduces the peak voltage and total amplitude of the terminal action potential by 4-8 mV (mean ---- 6.1 41.2 mV, n = 11). This observed 4-8 mV reduction in the amplitude of the excitatory action potential during presynaptic inhibition is probably an underestimate of the maximum spike reduction which occurs at the sites of presynaptic inhibition because the ;t calculated above applies to PIPs which passively spread toward the recording electrode. During an action potential actively propagating toward the nerve terminals, the ;t is probably much less, perhaps one-quarter of the value for a steady-state ~22. We find that the addition of GABA to the bath perfusate in high concentration (0.5 mM) as determined by dose-response curves reduces the total amplitude of excitor axon potentials by 10-20 mV at all points along the excitor axon (Fig. 2C). Hence, compared to inhibitor stimulation at the optimum interval for presynaptic inhibition, high GABA concentrations have greater effects on total spike amplitude. The magnitude difference on total spike amplitude between high bath GABA and rapid inhibitory stimulation may be explained by the fact that the axon is sensitive to GABA along its entire length whereas the effect of the inhibitory stimulation is restric-
426 ted to specific release sites some distance away from the Y branch. Thus, the reduction in excitor spike amplitude during bath application of GABA may represent the maximum effect of presynaptic inhibition at the excitor terminals. Several observations suggest that the reduction in excitor spike amplitude during presynaptic inhibition results from a membrane conductance increase at the terminals rather than a presynaptic voltage change per se. First, small alterations (I-3 ms) of the stimulus paradigm to increase or decrease the optimum delay between the last inhibitory spike and the single excitor spike produce much less presynaptic inhibition t,:~,15 and much less reduction of excitor spike amplitude compared to the optimum timing interval (Baxter and Bittner, in preparation). Second, the time course (2-6 ms) for the reduction in the excitor spike amplitudes is much shorter than the time course (20-50 ms) of a PIP voltage change, but this 2-6 ms effect is similar to the rise and decay times of the postsynaptic conductance changes produced by GABA released from this same inhibitor axon*. Third, large or small, depolarizing or hyperpolarizing PIPs reduce spike amplitude by the same extent (Baxter and Bittner, in preparation). Thus, the reduction in excitor spike amplitude during presynaptic inhibition is independent of the sign, amplitude and duration of a PIP, but is correlated with the expected conductance change associated with a PIP. In summary, it would appear that intracellular potentials recorded from the Y branch of the excitor nerve axon to the crayfish opener muscle reflect electrical activity occurring in the excitor nerve terminals. The effect of presynaptic inhibition is to reduce the amplitude of action potentials which invade the excitor terminals. The time course and amplitude of this reduction is consistent with a brief presynaptic action of G A B A to increase the conductance of the terminal membrane of the excitor axon. One might expect that reduction of 6-20 mV in total spike amplitude at the nerve terminals would greatly reduce the amount of transmitter released 1°,2t. This mechanism is similar to that originally hypothesized to account for presynaptic inhibition in this preparation 14. Presynaptic inhibition via a conductance increase differs from that suggested for various CNS synapses in which depolarization per se of nerve terminals is presumed to produce a decrease in spike amplitude and hence a decrease in transmitter release 15,23,24,z6. Finally, presynaptic inhibition in Aplysia CNS synapses may occur by a direct effect of the inhibitory transmitter to block calcium channels 29. This latter mechanism would not be expected to reduce excitor spike amplitude to the extent that we have observed in crayfish motor terminals. Our data show that the sign of a PIP in crayfish excitor terminals is determined by the relationship between the resting Em and EaA13a. Hence, in some synapses presynaptic inhibition could just as easily be associated with primary afferent hyperpo* The rise time of the conductance change produced by this inhibitor axon on postsynaptic muscle fibers is very brief (1-3 ms) and decays rapidly from its peak value25. The decay time of the IPSP voltage change is determined more by the muscle membrane time constant than the conductance change produced by the inhibitory transmitter 25. The time course of GABA release and conductance change in the presynaptic excitor axon might be rather similar to the time course in the postsynaptic muscle. A brief conductance change would account for the observation that alterations of 1-3 ms in the optimum interval between the last inhibitor pulse and the single excitor pulse are associated with a dramatic decrease in the amount of presynaptic inhibition.
427 larization a n d a threshold increase for spike generation as well as p r i m a r y afferent depolarization a n d a threshold decrease as generally reported in crayfish a n d vertebrate C N S synapseslS,2a,24,~s. I n fact, G A B A has recently been reported to produce a threshold increase or a threshold decrease in different afferent fibers in cat spinal cord 27. This work was supported in part by a n N I H Career development A w a r d 00070 a n d N S F Research G r a n t BNS-80-22248 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 Jones, A., Presynaptic inhibition in crustacean muscle: axo-axonal synapses, Experientia, 23 (1967) 1036-1038. 3 Atwood, H. L. and Morin, W. A., Neuromuscular and axo-axonal synapses of crayfish opener muscle, J. Ultrastruct. Res., 32 (1970) 351-369. 4 Baxter, D. A. and Bittner, G. D., The normal accumulation of facilitation during presynaptic inhibition, Brain Research, 189 (1980) 535-539. 5 Bittner, G. D., Differentiation of nerve terminals in the crayfish opener muscle and its functional significance, J. gen. Physiol., 51 (1968) 731-758. 6 Bittner, G. D., The differentiation of crayfish muscle fibers during development, J. exp. 2ool., 167 (1968) 439-456. 7 Bittner, G. D. and Harrison, J., A reconsideration of the Poisson hypothesis for transmitter release at the crayfish neuromuscular junction, J. Physiol. (Lond.), 206 (1970) 1-23. 8 Bittner, G. D. and Kennedy, D., Quantitive aspects of transmitter release, J. Cell Biol., 47 (1970) 585-592. 9 Bryan, J. S. and Krasne, F. B., Presynaptic inhibition: the mechanism of protection from habituation of the crayfish lateral giant fibre escape response, J. PhysioL (Lond.), 271 (1977) 369-390. 10 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. 11 Dudel, J., Presynaptic inhibition of the excitatory nerve terminals in the neuromuscularjunction of the crayfish, Pfliigers Arch., 277 (1963) 537-557. 12 Dudel, J., The mechanism of presynaptic inhibition at the crayfish neuromuscularjunction, Plfiigets Arch., 284 (1965) 66-80. 13 Dudel, J. and Hatt, H., Four types of GABA receptors in crayfish leg muscles characterized by desensitization and specific antagonist, Pfliigers Arch., 364 (1976) 217-222. 14 Dudel, J. and Kuffier, S. W., Presynaptic inhibition at the crayfish neuromuscular junction, J. Physiol. (Lond.), 155 (1961) 534-562. 15 Eccles, J. C., 7he Physiology of Synapses, Academic Press, New York, 1964, 220 pp. 16 Frank, K. and Fuortes, M. G. F., Presynaptic and post-synaptic inhibition of monosynaptic reflexes, Fed. Proc., 16 (1957) 39-40. 17 Fuchs, P. A. and Getting, P. A., Ionic basis of presynaptic inhibitory potentials at crayfish claw opener, J. Neurophysiol., 43 (1980) 1547-1557. 18 Hodgkin, A. L. and Rushton, W. A. H., The electrical constants of a crustacean nerve fibre, Proc. roy. Soc. B, 133 (1946) 4a.~ A.79. 19 Jahromi, S. S. and Atwood, H. L., Three-dimensional ultrastructure of the crayfish neuromuscular apparatus, J. Cell Biol., 63 (1974) 599-613. 20 Kawai, N. and Niwa, A., Neuromuscular transmission without sodium activation of the presynaptic nerve terminal in the lobster, J. Physiol. (Lond.), 305 (1980) 73-85. 21 Katz, B. and Miledi, R., The study of synaptic transmission in the absence of nerve impulses, J. Physiol. (Lond.), 192 (1967) 407-436. 22 Katz, B. and Miledi, R., The effect of local blockage of motor nerve terminals, J. Physiol. (Lond.), 199 (1968) 729-741. 23 Kennedy, D., Calabrese, R. L. and Wine, J. J., Presynaptic inhibition: primary afferent depolarization in crayfish neurons, Science, 186 (1974) 451-454.
428 24 Kennedy, D., McVittie, J., Calabrese, R. L., Fricke, R. A., Craelius, W. and Chiapella, P., Inhibition of mechanosensory interneurons in crayfish. I. Presynaptic inhibition from giant fibers, J. Neurophysiol., 43 (1980) 1495-1509. 25 Onodera, K. and Takeuchi, A., Inhibitory postsynaptic current in voltage-clamped crayfish muscle, Nature (Lond.), 263 (1976) 153 154. 26 Pearson, K. G. and Goodman, C. S., Presynaptic inhibition of transmission from identified interneurons in locust central nervous system, J. Neurophysiol., 45 (1981) 501 515. 27 Randic, M., Semba, K. and Murase, K., Presynaptic effects of gamma-aminobutyric acid on intraspinal single cutaneous afferent C- and A-fibers, Neurosei. Abstr., l0 (1980) 571. 28 Schmidt, R. F., Presynaptic inhibition in the vertebrate central nervous system, Ergebn. Physiol., 63 (1971) 20-101. 29 Shapiro, E., Castellucei, V. F. and Kandel, E. R., Presynaptic inhibition in Aplysia involves a decrease in the Ca +~- current of presynaptic neuron, Proe. nat. Aead. Sci. USA, 77 (1980) 11851189. 30 Takeuchi, A. and Takeuchi, N., A study of the inhibitory action of 7-amino-butyric acid on the neuromuscular transmission in the crayfish, J. Physiol. (Lond.), 183 (1966) 418-432. 31 Takeuchi, A. and Takeuchi, N., On the permeability of the presynaptic terminal of the crayfish neuromuscular junction during synaptic inhibition and the action of 7-amino-butyric acid, J. Physiol. (Lond.), 183 (1966) 433~149.
Brain Research, 223 (1981) 422-428 Elsevier/North-Holland Biomedical Press
Intracellular recordings from crustacean motor axons during presynaptic inhibition
DOUGLAS A. BAXTER and GEORGE D. BITTNER* Department of Zoology, University of Texas, Austin, TX 78712 (U.S.A.) (Accepted July 9th, 1981) Key words: presynaptic inhibition --crustacean neuromuscular junction - - inhibitory synapses
Action potentials intracellularly recorded from near the nerve terminals of the opener excitor motor axon in crayfish are reduced in amplitude during presynaptic inhibition. The amplitude and sign (hyperpolarizing or depolarizing) of presynaptic inhibitor potentials (PIPs) depends upon the relationship between the resting membrane potential of each excitor axon and its PIP equilibrium potential (which equals the equilibrium potential for 7-aminobutyric acid). Presynaptic inhibition of transmitter release has been known for several decades to be an important means of integration in many vertebrate and invertebrate neurons 1-4,9, 14-16,z3,24,z6,2s. However, an inability to record intracellularly from the affected nerve terminals during presynaptic inhibition has limited our understanding of its cellular mechanism. This paper describes intracellular recordings obtained near the terminal region of the excitor axon to the opener muscle in crayfish while stimulating the inhibitor axon so as to produce presynaptic inhibitory potentials (PIPs). Our data show that presynaptic inhibition is associated with hyperpolarizing and depolarizing PIPs which reduce the amplitude of action potentials in the presynaptic terminal. Experiments were performed on the claw opener muscle of the crayfish, a preparation which has often been used to study the anatomical and physiological basis of presynaptic inhibitionl-SAl-14,17,19,3°,aL The opener muscle in Procarnbarus simulans is innervated by one excitatory and one inhibitory axon which can be independently stimulated in a more proximal limb segment 1,4,14. Both axons branch together on the surface of the opener muscle (Fig. IA). Using 3 M KC1 or 2 M potassium citrate microelectrodes of 20-60 M f~ resistance, intracellular recordings were made from the opener excitor axon (10-20 # m diameter), where it branches in a Y pattern ~7, 0.3-1.0 mm proximal to its nearest synaptic endings on opener muscle fibers as determined from our scanning electron micrographs and from previously published reconstructions of these axons 2,a,19. The inhibitor axon makes synapses upon the excitor axon at these release sites and on axonal constrictions proximal to excitor release sites e,z,19. A few intracellular recordings were also obtained from smaller (4-10/zm) branches (II or * To whom all correspondence should be addressed. 0006-8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
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Membrane Potenliol (my)
Fig. 1. A: schematic representation of the excitor (solid line) and inhibitor (dashed line) axons as they branch together to innervate the opener muscle. Intracellular recordings were made from the main trunk of the excitor axon (I), from the Y branch and from more distal secondary (II), tertiary (III), etc., branches of the excitor axon which synapse (open circles) upon muscle fibers. The inhibitor axon makes both presynaptic inhibitory contacts (closed circles) upon the excitor axon and postsynaptic inhibitory terminals upon muscle fibers2,s,la,le. B-D: presynaptic inhibitory potentials (PIPs) intracellularly recorded with DC coupling from the Y branch of the excitor axon. Data for each figure taken from XY plotter output of 100 potentials averaged in a computer of average transients (CAT). B: temporal summation of 8 PIPs produced by 100 Hz stimulation of the inhibitor nerve axon. The membrane potential attained by these summated PIPs (E~P~Ps) was --82.9 mV and is represented by the upper dotted line. The difference between the two dotted lines (A~,I,rPs ~ 1.1 mV, in this case) represents the difference (in mV) between the resting Em (--84.0 mV) and the near maximum effect of the inhibitory transmitter as recorded at the Y branch. In this fiber, EGAaAwas --81.0 mV and thus the ratio of ~ EPIPsto AEGABAwas 0.37. C: depolarizing PIP (DPIP). Resting E m = --78 mV represented by dotted line. The inhibitor axon was stimulated at 2 Hz in both C and D. D: hyperpolarizing PIP (HPIP). Resting E m = --69 mV represented by dotted line. Calibration line equals 0.2 mV, 10 ms for B; and 0.1 mV, 5 ms for C and D. E: plot of single HPIP and DPIP amplitudes ~V) against resting Era (mV) recorded from each of 20 excitor axon terminals. The Em for each fiber was measured at the time of initial penetration. The heavy solid line plots the linear regression (r = 0.87) of PIP amplitude against resting Em. This regression line shows that PIPs reverse in sign at a membrane potential of--70.5 mV. This value (Eih, heavy arrow) represents the apparent equilibrium potential for the inhibitory transmitter recorded at the Y branch. The average EGABA(light arrow) was --68.5 :E 3.5 mV. I I l in Fig. 1A) within 0.1-0.3 m m o f the t e r m i n a l release sites. In some cases, we r e c o r d e d intracellularly f r o m opener muscle fibers n e a r the Y b r a n c h with KCl-filled electrodes 4-s. I n t r a c e l l u l a r recordings were also m a d e from the excitor axon at v a r i o u s points 0.5-4 c m p r o x i m a l to the o p e n e r muscle. A P I P can be r e c o r d e d f r o m the excitor Y b r a n c h during i n h i b i t o r y stimulation (Fig. 1B-D). I n P. simulans, the resting m e m b r a n e p o t e n t i a l (Era) averages - - 7 4 -I- 7 mV S.D. (n ---- 27) a n d P I P s m a y be either h y p e r p o l a r i z i n g ( H P I P , Fig. 1D) or d e p o l a rizing ( D P I P , Fig. 1B, C). T h e sign a n d a m p l i t u d e o f P I P s v a r y with the resting Em for t h a t a x o n (Fig. 1E). P r o x i m a l to the Y, the P I P a m p l i t u d e decreases with a space constant (2) o f 1.7-3.9 ram. This ~. is similar to t h a t r e p o r t e d for o t h e r crustacean m o t o r axonsl8, 20. Three s e p a r a t e results suggest t h a t the P I P s r e c o r d e d at the Y b r a n c h are p r o d u c e d b y ~,-aminobutyric acid ( G A B A ) a n d represent 35-45 % o f their full value at their sites o f origin in opener nerve t e r m i n a l s o f P. simulans.
424 (1) W h e n the i n h i b i t o r is stimulated at 100 Hz, the a m p l i t u d e o f the first PIP or the plateau values o f t e m p o r a l l y s u m m a t e d PIPs (AZr~ps in Fig. 1B) vary with resting Em in different excitor axons so that the a p p a r e n t reversal potential (En0 o f single PIPs is - - 7 0 . 5 mV (Fig. 1E) a n d o f AZp~ps is - - 6 9 . 3 mV. W h e n G A B A is a d d e d to the b a t h perfusate in s u p r a - m a x i m a l c o n c e n t r a t i o n (0.5 m M ) , as determined by d o s e - r e sponse curves, the average Em at the Y b r a n c h for these excitor axons is - - 6 8 . 5 ± 3.5 mV (n - - 9). G A B A also p r o d u c e d the same Em value (EGA]~A) at all points, from 0.7 m m distal to 4 cm p r o x i m a l from the Y b r a n c h o f the excitor axon (resting Em values were the same at all these axonal positions); that is, excitor axons a p p e a r to have similar G A B A receptors distributed over their entire length. (2) PIPs recorded distal to the Y b r a n c h (0.1-0.3 m m f r o m the nearest release sites) have a m p l i t u d e s within 5-10 ~ o f those r e c o r d e d at the Y branch. (3) PIPs r e c o r d e d at the Y - b r a n c h decrease in a m p l i t u d e during r a p i d s t i m u l a t i o n (20-100 Hz) o f the i n h i b i t o r a x o n (Fig. 1B), while i n h i b i t o r y p o s t s y n a p t i c potentials (IPSPs) recorded from h y p e r p o l a r i z e d muscle fibers usually show m u c h facilitation o f t r a n s m i t t e r release 1. These d a t a suggest that PIPs decrease in a m p l i t u d e during a t r a i n because they a p p r o a c h the P I P reversal potential. Hence, the m a x i m u m a m p l i t u d e a n d A - - pControl -j . . . . . . . . . . . B. Inhibited . . . . . . . . . . . . . . C. GABA
~°°~..,
EG~BA
Fig. 2. Top traces: action potentials (spikes) intracellularly recorded with DC coupling from the Y branch of the excitor axon. Dotted line at bottom of trace equals resting Era (--79 mV). Dotted line at top of trace equals ÷ 21 mV (the peak voltage). Solid line equals 0 mV. Bottom traces: excitatory junctional potentials (EJPs) intracellularly recorded with DC coupling from a postsynaptic muscle fiber near the Y branch. Dotted line equals resting Em (--70 mV). All traces taken from XY plotter output of 100 potentials averaged using a CAT. A: single excitor action potential and EJP. DAP, depolarizing after-potential. The voltage difference between the foot and peak voltages (arrows) is the total amplitude of the spike (100 mV in this case). B: single excitor action potential and EJP given 2 ms after the last pulse in a train of 10 inhibitor pulses at 100 Hz. The 50% decrease in EJP amplitude (lower trace) resulted from presynaptic inhibition as determined by methods of Baxter and Bittner4. The foot-voltage of the presynaptic action potential was depolarized by 700/~V (AZPIPs) due to stimulation of the inhibitor axon; this voltage is not easily detected at this lower amplification and faster sweep speed. The total spike amplitude was reduced to 93 mV and the peak voltage was reduced by 6.3 mV. C: single excitor action potential and EJP when perfusate contained 0.5 mM GABA. The total spike amplitude was reduced to 81 mV and the peak voltage was reduced by 17 mV. In this fiber the ratio of A ZPtvs to AEaA~A (2 mV in this case) was 0.35. Calibration pulse, upper trace -- 10 mV, 1 ms; calibration pulse, lower trace = 0.25 mV, 5 ms.
425 sign of the voltage deflection produced by temporally summated PIPs (AY~pIps in Fig. IB) provides an estimate of the maximum amplitude and sign of the deflection produced by the inhibitory transmitter upon the axon (AEIh). Since GABA is almost certainly the inhibitory neurotransmitter (see refs. 11-13 and 17, and section (1) above), AEIh should approximate AEGABA(Fig.2C) at the sites of presynaptic inhibition. The ratio of A~pips recorded at the Y branch to AEGABAWaS0.38 i 0.07 (n = 7). Since AEpIps should decrement along the excitor nerve axon, whereas AEGABAis the same at all points along the axon, these data suggest that AEpips recorded at the Y branch are decremented by about 60 ~ with regard to AEIh generated at the nearest presynaptic release sites in P. simulans. In other words, the recording site at the Y branch is within a single space constant of the presynaptic inhibitory terminals. This conclusion is in agreement with calculations based on cable theory made for P. clarkii 7. Having provided evidence that membrane voltage at the Y branch is coupled to sites of presynaptic inhibitor action in the excitor axon ofP. simulans, we examined the effects of inhibitor stimulation on excitor action potentials intracellularly recorded at the Y branch (terminal action potential; records taken from more distal branches gave similar results). We found (Fig. 2A) that terminal action potentials have a peak voltage of +20 to +30 mV, a foot-to-peak total amplitude of 93 4- 4 mV (n = 25), a duration of 1 ms at one-half peak amplitude, a depolarizing after-potential (DAP) of 10 4- 2 mV at 2 ms after the rising phase of the spike, and a DAP duration of 30-50 ms. We then stimulated the inhibitor 10 times at 10 ms intervals so as to facilitate the release of inhibitory neurotransmitter. The excitor axon was stimulated 1-2 ms after the 10th inhibitory pulse so that the excitor axon potential arrived at its terminal 0.5-1.0 ms after the last inhibitor action potential reached its terminal1, TM. This stimulus paradigm produces maximal presynaptic inhibitory effects1,4,14as evidenced by a maximum reduction of excitory junctional potentials (EJPs) recorded from adjacent muscle fibers (Fig. 2B; postsynaptic inhibition was usually rather small as determined by conventional techniques1,4). This stimulus paradigm reduces the peak voltage and total amplitude of the terminal action potential by 4-8 mV (mean ---- 6.1 41.2 mV, n = 11). This observed 4-8 mV reduction in the amplitude of the excitatory action potential during presynaptic inhibition is probably an underestimate of the maximum spike reduction which occurs at the sites of presynaptic inhibition because the ;t calculated above applies to PIPs which passively spread toward the recording electrode. During an action potential actively propagating toward the nerve terminals, the ;t is probably much less, perhaps one-quarter of the value for a steady-state ~22. We find that the addition of GABA to the bath perfusate in high concentration (0.5 mM) as determined by dose-response curves reduces the total amplitude of excitor axon potentials by 10-20 mV at all points along the excitor axon (Fig. 2C). Hence, compared to inhibitor stimulation at the optimum interval for presynaptic inhibition, high GABA concentrations have greater effects on total spike amplitude. The magnitude difference on total spike amplitude between high bath GABA and rapid inhibitory stimulation may be explained by the fact that the axon is sensitive to GABA along its entire length whereas the effect of the inhibitory stimulation is restric-
426 ted to specific release sites some distance away from the Y branch. Thus, the reduction in excitor spike amplitude during bath application of GABA may represent the maximum effect of presynaptic inhibition at the excitor terminals. Several observations suggest that the reduction in excitor spike amplitude during presynaptic inhibition results from a membrane conductance increase at the terminals rather than a presynaptic voltage change per se. First, small alterations (I-3 ms) of the stimulus paradigm to increase or decrease the optimum delay between the last inhibitory spike and the single excitor spike produce much less presynaptic inhibition t,:~,15 and much less reduction of excitor spike amplitude compared to the optimum timing interval (Baxter and Bittner, in preparation). Second, the time course (2-6 ms) for the reduction in the excitor spike amplitudes is much shorter than the time course (20-50 ms) of a PIP voltage change, but this 2-6 ms effect is similar to the rise and decay times of the postsynaptic conductance changes produced by GABA released from this same inhibitor axon*. Third, large or small, depolarizing or hyperpolarizing PIPs reduce spike amplitude by the same extent (Baxter and Bittner, in preparation). Thus, the reduction in excitor spike amplitude during presynaptic inhibition is independent of the sign, amplitude and duration of a PIP, but is correlated with the expected conductance change associated with a PIP. In summary, it would appear that intracellular potentials recorded from the Y branch of the excitor nerve axon to the crayfish opener muscle reflect electrical activity occurring in the excitor nerve terminals. The effect of presynaptic inhibition is to reduce the amplitude of action potentials which invade the excitor terminals. The time course and amplitude of this reduction is consistent with a brief presynaptic action of G A B A to increase the conductance of the terminal membrane of the excitor axon. One might expect that reduction of 6-20 mV in total spike amplitude at the nerve terminals would greatly reduce the amount of transmitter released 1°,2t. This mechanism is similar to that originally hypothesized to account for presynaptic inhibition in this preparation 14. Presynaptic inhibition via a conductance increase differs from that suggested for various CNS synapses in which depolarization per se of nerve terminals is presumed to produce a decrease in spike amplitude and hence a decrease in transmitter release 15,23,24,z6. Finally, presynaptic inhibition in Aplysia CNS synapses may occur by a direct effect of the inhibitory transmitter to block calcium channels 29. This latter mechanism would not be expected to reduce excitor spike amplitude to the extent that we have observed in crayfish motor terminals. Our data show that the sign of a PIP in crayfish excitor terminals is determined by the relationship between the resting Em and EaA13a. Hence, in some synapses presynaptic inhibition could just as easily be associated with primary afferent hyperpo* The rise time of the conductance change produced by this inhibitor axon on postsynaptic muscle fibers is very brief (1-3 ms) and decays rapidly from its peak value25. The decay time of the IPSP voltage change is determined more by the muscle membrane time constant than the conductance change produced by the inhibitory transmitter 25. The time course of GABA release and conductance change in the presynaptic excitor axon might be rather similar to the time course in the postsynaptic muscle. A brief conductance change would account for the observation that alterations of 1-3 ms in the optimum interval between the last inhibitor pulse and the single excitor pulse are associated with a dramatic decrease in the amount of presynaptic inhibition.
427 larization a n d a threshold increase for spike generation as well as p r i m a r y afferent depolarization a n d a threshold decrease as generally reported in crayfish a n d vertebrate C N S synapseslS,2a,24,~s. I n fact, G A B A has recently been reported to produce a threshold increase or a threshold decrease in different afferent fibers in cat spinal cord 27. This work was supported in part by a n N I H Career development A w a r d 00070 a n d N S F Research G r a n t BNS-80-22248 to G.D.B.
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