Baclofen has a presynaptic action at the crayfish neuromuscular junction

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Brain Rew,arch. 31 111984) 152-15h Elbevlc)-

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Baciofen has a presynaptic action at the crayfish neuromuscular j u ~ i o n SUSAN R. BARRY

Marine Biological Laboratory, Woods Hole. MA 02543 (U.S.A.) (Accepted May 22nd, 1984)

Key words: baclofen

crayfish neuromuscular junction - - gamma-aminobutyric acid - - presynaptic inhibition G A B A receptors transmitter release presynaptic receptors

The action of baclofen, a G A B A analog, was studied at the crayfish neuromuscular junction (NMJ) Baclofen depressed the amplitude of excitatory junction potentials (ejps) without affecting muscle input resistance and reduced the frequency of spontaneous miniature ejps without affecting their size. Thus. baclofen may mediate presynaptic inhibition by depressing transmitter release from the excitatory nerve. The site of baclofen's effect at the crayfish NMJ may parallel its site of action in the vertebrate nervous system.

The action of baclofen, a G A B A analog, was studied at the neuromuscular junction (NMJ) of the crayfish opener muscle. Gamma-aminobutyric acid ( G A B A ) mediates both presynaptic and postsynaptic inhibition at the crayfish NMJ. Experiments were directed toward determining whether baclofen mimicked presynaptic or postsynaptic actions of G A B A . Baclofen (Lioresal) is used clinically to treat spasticity. The drug may act as a G A B A agonist on a pharmacologically distinct class of G A B A receptors in vertebrate sympathetic and dorsal root ganglion neuronsn-6,t3, 21. G A B A depolarizes the cell body of sympathetic neurons by causing an increase m chloride conductance. This effect is blocked by bicuculline. a G A B A antagonist I. G A B A also inhibits transmitter release from sympathetic nerve terminals tTan action which is not blocked by bicuculline but is mimicked by baclofen 4-6. Thus, the G A B A receptors on the cell bodies and nerve terminals of sympathetic neurons may be pharmacologically different. The effects of baclofen may result in part by binding to G A B A receptors on the nerve terminal and mimicking presynaptic inhibition. The crayfish NMJ is an excellent preparation for contrasting possible presynaptic and postsynaptic actions of baclofen. The crayfish opener muscle is in-

nervated by both an excitatorv and an inhibitory axon. Stimulation of the excitatory nerve produces depolarizing excitatory junction potentials (ejps) on the muscle. The inhibitory axon synapses not only on the muscle but also on the excitatory nerve terminalSu.23. Inhibitory nerve stimulation produces a postjunctional increase in chloride conductance and also depresses transmitter release from the excitatory nerve terminal. The inhibitory transmitter is GABA20. Thus. application of G A B A to the crayfish NMJ reduces ejp amplitude by two mechanisms. G A B A acts postsynaptically by decreasing the muscle input resistance and presynapticalty by depressing transmitter release from the excitatory nerve. The G A B A receptors on the muscle and nerve terminal are pharmacologically different. Dudel 7 showed that certain G A B A analogs, such as fl-guanidino-propionic acid. mimicked presynaptic, but not postsynaptic, inhibition. Other G A B A agonists, such as guanidino-acetic acid. acted only postsynapticatly. In the present study, baclofen was tested at the crayfish NMJ to determine whether it was selective for one class of invertebrate G A B A receptors as it is for one class of vertebrate G A B A receptors. A preliminary report of this investigation has been published elsewhere 3.

Correspondence and present address: S. R Barry, Department of Physiology, University of Michigan. Medical Science Building I1. Room 7773. Ann Arbor. M148109. U.S.A. ntwu~_uoo':t/fla/¢o~fvi (~ 10R~.l::l~e~vierScience Publishers B.V.

153 Baclofen's effects on both evoked ejps and spontaneous miniature ejps (mejps) at the crayfish NMJ were investigated. Experiments were performed on the opener muscle (abductor of the dactyl) of the isolated first or second walking leg of the crayfish Procambarus clarkii (Carolina Biological). Opener muscle fibers were impaled with one or two 10 mr2 microelectrodes filled with 2 M KAc. The resting potential of the muscle cells averaged -90 to -95 inV. To evoke ejps, the nerve bundle, which contains the excitatory but not the inhibitory axon, was exposed in the meropodite segment of the leg and stimulated with a platinum hook electrode. Stimulus trains of 0.1 ms pulses at a frequency of 75 pps for 75-200 ms were used to elicit trains of facilitating ejps 7.H. Ejps and mejps were photographed from the oscilloscope using a Grass Kymograph camera, and the size of these potentials was measured from the photographic records. Mejp amplitudes were at least twice as great as the background noise level of the recording system. Experiments were performed at 20-25 °C. The preparation was continually perfused with van Harreveld's solution 10 to which 11 mm glucose was added 16. A stock solution of 10 mM (+)-baclofen (Ciba-Geigy) was prepared in 100 mM HC1 and diluted to a final concentration with van Harreveld's solution 13. G A B A (Sigma) was dissolved directly into van Harreveld's solution. Drugs were applied by perfusion: Baclofen depressed excitatory transmission at the crayfish NMJ. In 3 experiments, 10 -4 M baclofen reversibly decreased ejp amplitude by 16-26% (Fig. 1, for 3 experiments, P < 0.005, P < 0.025, P < 0.10, Student's t-test). The reduction in ejp size was not ac-

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Fig. 1. A: 10-4 M baclofenreversiblydecreased ejp amplitude by 25%. Ejps were evoked by a train of stimuli to the excitatory motor nerve at twice threshold (0.1 ms pulses, 75 pps, 75 ms train duration). B: 10-4 M baclofen had no effect on the muscle input resistance. The muscle fiber was impaled with both a stimulating and recording electrode and 160 ms hyperpolarizing current pulses (2.5 × 10-8 A) were delivered through the stimulating electrode.

companied by a change in membrane potential or input resistance of the muscle (Fig. 1). To measure input resistance, the muscle fiber was impaled with both a stimulating and recording electrode and hyperpolarizing current pulses were delivered through the stimulating electrode. Baclofen, up to 10 -3 M, did not affect the muscle's response to the hyperpolarizing current pulses. Similarly, Nistri and Constanti 19 have reported that baclofen does not alter the input resistance of the lobster claw opener muscle. 10-4 M G A B A reversibly reduced ejp amplitude at the crayfish NMJ by 100%. Thus, G A B A was a more potent inhibitor of ejps than baclofen. However, 10-4 M G A B A also reduced muscle input resistance by up to 50%. Bicuculline has been reported to antagonize GABA's action on crustacean muscle 22. Thus, baclofen's lack of effect on muscle input resistance is consistent with its proposed selective effect on 'bicuculline-insensitive' receptors. Since baclofen depressed excitatory transmission without affecting the muscle input resistance, the drug may alter the response of the muscle to the excitatory transmitter or may decrease transmitter release from the excitatory nerve terminal. To distinguish between these two possibilities, the effects of baclofen on the frequency and size of spontaneous mejps were determined. The results of these experiments indicate that baclofen's action at the crayfish NMJ is presynaptic. Mejps at the crayfish neuromuscular junction result from the spontaneous release of transmitter quanta from the excitatory motor nervel0j2. They occur randomly at an average frequency of about one per second in normal saline (van Harreveld's solution with 11 mM glucose). The average mejp frequency was increased by elevating the osmolarity of the saline from 430 to 470 mosm with 40 mM glucose. Mejps vary in size from about 25 to 200/~V. This size variation results in part because the muscle is multipie-innervated. Thus, an intracellular microelectrode records mejps arising from many synaptic junctions. Changes in the frequency and amplitude of spontaneous minature potentials have been used as a measure of fluctuations in transmitter release from crustacean and vertebrate neuromuscular junctions2.U, 14js. Changes in the amplitude distribution of mejps may indicate fluctuations in the size of the quanta released or changes in the postsynaptic response. In

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Fig. 2.10 -4 M baclofen reduced the frequency of spontaneous mejps (AI but had no effect on their size (B). In (A), mejp frequencies calculated for 30-s time intervals were plotted against time. 10 -4 M baclofen reduced mejp frequency by an average of 30%. In (B) the amplitudes of mejps were grouped into 10 p V bins and then plotted against the frequency of their occurrence. 10-4 M baclofen did not affect the amplitude distribution of the mejps. The amplitude histograms in (B) were plotted after measuring the size of 449 mejps recorded in saline and 316 melPS recorded in l0 -4 M baclofen. All data were obtained from one cell.

contrast, variations in the frequency of melps without corresponding changes in amplitude may indicate fluctuations in the quantal content or number of quanta released from the nerve terminal. Baclofen (10 -4 M) reduced the frequency of spontaneous mejps by 30 to 45% but had no effect on the amplitude distribution of the mejps (6 experiments. Figs. 2 and 3). To determine this, the amplitude of mejps was plotted against the frequency of their occurrence. Baclofen reduced the frequency of each size category of mejps to the same extent. The Kolmogorov-Simirnov test was used to show that the distribution of mejp amplitude did not differ significantly in the presence and absence of baclofen. In 2 experiments, 10-5 M baclofen also depressed mejp frequency by 22 and 40% while 10 -6 M baclofen had no effect. Thus, the threshold concentration for baclofen's action is probably between 10 -6 and 10-5 M. Since baclofen depressed ejp amplitude and mejp f r e q u e n c y w i t h o u t a f f e c t i n g m e j p a m p l i t u d e or m u s cle i n p u t r e s i s t a n c e , the d r u g m a y act b y d e p r e s s i n g

transmitter release from the excitatory nerve terminal. Since the size of mejps was not changed, baclo-

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155 fen probably did not affect the response of the muscle to the excitatory transmitter. Thus, baclofen may mediate presynaptic but not postsynaptic inhibition at the crayfish NMJ. Experiments were then directed toward examining how baclofen may depress transmitter release at the crayfish NMJ. Takeuchi and Takeuchi 24 showed that G A B A reduced transmitter release at the opener muscle junction by increasing the chloride conductance of the excitatory nerve terminal. To determine whether baclofen also altered nerve terminal chloride conductance, the opener muscle was bathed in saline in which the chloride was completely replaced with acetate. When 10-4 M baclofen was added to the chloride-deficient saline, a 4 to 20-fold increase in the frequency of spontaneous mejps was seen. This effect was transient lasting only 12-80 s (4 experiments, Fig. 3). Baclofen's dramatic but brief enhancement of mejp frequency is identical to that reported for G A B A by Takeuchi and Takeuchi 24. The effects can be explained by assuming that G A B A and baclofen open chloride channels in the excitatory nerve terminal. In chloride-free saline, chloride ions would flow out and depolarize the axonal terminal, resulting in a large increase in transmitter release. This effect is transient because chloride ions rapidly redistribute inside and outside the nerve terminal. In normal saline, the increase in chloride conductance produced by baclofen or G A B A may depress transmitter release perhaps by short-circuiting the action potential invading the nerve terminal. Baclofen acts differently on the somata of chick dorsal root ganglion cells13,21. On these cells, both baclofen-sensitive and baclofen-insensitive G A B A receptors are found. G A B A , when bound to the baclofen-insensitive receptors, causes an increase in chloride conductance. In contrast, G A B A or baclofen bound to the second class of receptors decreases the calcium conductance during an action potential. Baclofen's primary action at the crayfish NMJ, may not involve inhibition of a calcium conductance. Baclofen depressed spontaneous transmitter release to a greater extent than evoked release. While calcium influx is necessary to initiate transmitter release 1 Adams, P. R. and Brown, D. A., Actions of GABA on sympathetic ganglion cells, J. Physiol. (Lond.), 250 (1975) 85-120. 2 Augustine, G. J. and Levitan, H., Neurotransmitter re-

during a nerve impulse, spontaneous release does not necessarily involve calcium14,15. Since baclofen primarily affected spontaneous resting release, it is unlikely that baclofen's principle inhibitory action is to depress the calcium current that occurs during an action potential. Instead, baclofen probably acts by increasing the nerve terminal chloride conductance. The difference in the mechanism by which baclofen decreases transmitter release from crayfish and vertebrate nerve terminals may reflect a difference in GABA's actions in the various species. At the crayfish NMJ, G A B A ' s presynaptic action is reduced or abolished in chloride-deficient saline 23. In contrast, GABA's inhibitory effect on transmitter release from sympathetic nerve terminals is not affected by removal of extracellular chlorideS. At the crayfish NMJ, G A B A ' s inhibitory action is not affected by increasing the external calcium concentration 23. Yet, G A B A depresses calcium conductance in chick sensory neurons13, 21. Thus, G A B A , and therefore baclofen, receptors may be coupled to chloride channels in crayfish motor axons and coupled to calcium channels in vertebrate sensory and perhaps sympathetic nerve terminals. In conclusion, baclofen may mediate presynaptic, but not postsynaptic, inhibition at the crayfish NMJ. The drug may bind to G A B A receptors on the excitatory nerve terminal but not to receptors on the muscle. When bound to presynaptic receptors, baclofen probably depresses transmitter release at least in part by increasing the chloride conductance of the nerve terminal. Finally, the presynaptic site of action of baclofen at the crayfish NMJ may parallel its site of action in the vertebrate nervous system. This work was supported by a Grass Foundation Fellowship for research at the Marine Biological Laboratory in Woods Hole, MA. I thank Ciba-Geigy for their generous gift of (+)-baclofen and Dr. Alan Gelperin for his generous loan of equipment. I also appreciate the advice and support of Drs. M. Goy, C. K. Govind, G. Augustine, J. E. Brown, D. McCulloh, E. McGlade-McCulloh, and R. L. Macdonald. 1 thank Michaele Weeks for secretarial assistance. lease and nerve terminal morphology at the frog neuromuscular junction affected by the dye erythrosin B., J. Physiol. (Lond.), 334 (1983) 47-63. 3 Barry, S. R., Presynaptic action of baclofen, a GABA ana-

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