Presynaptic inhibition by γ-aminobutyric acid in bullfrog sympathetic ganglion cells

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Brain Research, 153 (i978) 398-402 ~¢) Elsevier/North-Holland Biomedical Press

Presynaptic inhibition by ~'-aminobu~rie acid in bullfrog sympathetic ganglion cells E. KATO, K. KUBA and K. KOKETSU Department of Physiology, Kurume University School of Medicine, Kurume 830 (Japan)

(Accepted April 13th, 1978)

In both vertebrate and invertebrate synapses, 7-aminobutyric acid (GABA) is the most probable candidate as a transmitter in the axo-axonal synapses where presynaptic inhibition of excitatory synaptic transmission takes placea, 10,17,20. The mechanism of this type of synaptic inhibition is suggested to be due to depolarization of some part of the presynaptic fiber membrane which would result in reduction of the amplitude of action potentials, and eventually decrease the release of excitatory transmitters. However, there seems to be no direct evidence that GABA, indeed, inhibits release of neurotransmitters from the presynaptic terminal in the vertebrate synapses, although such a case was found in the crayfish neuromuscular junctionS,9, ~,22. It has been reported that GABA inhibits synaptic transmission of mammalian sympathetic ganglion s. Adams and Brown 1,2 concluded that the site of action of GABA in the mammalian ganglion is at the postsynaptic membrane. On the other hand, Koketsu et a1.16 suggested that GABA decreases the release of acetylcholine (ACh) from the preganglionic nerve terminals in the bullfrog sympathetic ganglion. They ascribed this action to the depolarization of the preganglionic nerve terminal which was indeed observed~% In this communication, we report evidence for a presynaptic action of GABA which inhibits release of ACh from the preganglionic nerve terminal in bullfrog sympathetic ganglia. The fact that the bullfrog sympathetic ganglion cell is a spheroidal neurone 23 would eliminate the possibility that the inhibitory action of GABA is caused by the action on remote parts of dendrites of a neurone~ 2. Ninth or tenth lumbar sympathetic ganglia of bullfrogs (Rana catesbeiana) were isolated and conventional intracellular recording techniques were employed is. Intracellular glass electrodes were filled with 3 M KC1 or 3 M K citrate and had tip resistances of 25-100 Mr2. A Wheatstone bridge circuit was used to pass current through a recording electrode. Experiments were carried out at room temperature (20-24 °C). Composition of normal Ringer's solution was the same to that described is. Fast excitatory postsynaptic potentials (fast EPSP) which are generated by the nicotinic action of ACh 15,is were recorded in a low Ca 2+, high Mg 2+ solution (CaCI2 0.74).9 raM; MgCI2 5.4-5.9 raM), the tonicity of which was adjusted by changing the concentration of NaCI. A high K + solution was made by increasing KC1 concentration to

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Fig. 1. A : effects of GABA ( l mM) on the resting membrane potential and resistance in a low Ca 2 high Mg ~+ solution. Constant current pulses (0.2 nA, 250 msec) were applied through a recording intracellular electrode at 0.2 Hz. B: effects of GABA (0.1 mM) on the fast EPS P and the membrane resistance The recordings were made in a low Ca~+-high Mg 2+ solution at 0.2 Hz. The current pulses for electrotonic potentials were 0.15 nA and 250 msec. The records in a, b, c and d are before, 3 min and 7 min after the beginning of application of GABA, and 7 min after removal of GABA, respectively. The small letters correspond to those in Fig. 2A. Only one electrotonic potential is shown in each condition. The per cent changes in the quantal content of fast EPSP and the membrane resistance were plotted in Fig. 2A.

l0 m M and reducing equimolar amount of NaC1. (y-Amino-n-butyric acid was obtained from Wako Pure Chem. Pharm. Co. Japan). As found previously 16, the amplitude of fast EPSP recorded in a low Ca "+, high Mg 2+ solution was markedly decreased in a few minutes after application of GABA (5 # M - I mM; Figs. IB and 2). The depression of the fast EPSP amplitude recovered in 5 to 10 minutes after removal of GABA from the perfusing solution (Figs. 1B and 2). The inhibitory action of GABA seems to be caused by a major presynaptic and minor postsynaptic effect, as described below. When GABA (5 #M-1 mM) was applied to the bullfrog sympathetic ganglion for the first time, the membrane of most cells depolarized only a few mV (Fig. 1A), although there were some cells which were depolarized up to 10 inV. At the same time, the membrane resistance decreased by 20-30 % as seen in Figs. 1 and 2A, which agrees with the findings in mammalian sympathetic ganglion cells1, 2. The decrease in the membrane resistance and slight depolarization explain only a part of the inhibitory action of GABA on the fast EPSP, as will be demonstrated later. Changes in the membrane potential and resistance desensitized quickly and returned to normal values in a few minutes after the commencement of GABA applications (Figs. 1 and 2A). Even after the postsynaptic membrane properties returned to normal, there remained a significant reduction of the fast EPSP amplitude (Fig. 1Be, Fig. 2Ae). After washing out GABA from the ganglion for more than 30 min, readministration of GABA resulted in little or no change in the membrane potential and resistance (Fig. 2B). Furthermore, when the perfusing Ringer solution was slowly changed to the solution

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Fig. 2. Effects of G A B A (A, 0. l mM: B, I raM) on fast EPSP amplitude (E)), quantal content of fast

EPSP (©) and membrane resistance (L~). The ordinates are percent changes of each parameter compared to those in the absence of GABA. Each square with vertical bars denotes the mean amplitude and S.E. of 24 fast EPSP's recorded for 2 min. Quantal contents were calculated from 43 fast EPSP's recorded for the time interval shown by the horizontal bars by the variance method7. Vertical bars for open circles are the S.E. of quantal content calculated using the equation . For the first time, GABA (0.1 raM) was applied to the ganglion cell in the experiment shown in A. Thirty minutes after the experinaent of A, the same ganglion cell was treated with GABA (1 raM) in B. Note lack of changes in the membrane resistance and marked reductions of quantal content• •

11

containing G A B A (0.1-1 mM), taking a few minutes for the complete exchange of the solutions, the postsynaptic effects of GABA were negligible in most cells even during the first application. Most of the experiments in the present study were carried out under this condition. The mean changes in the membrane potential and resistance after the slow application of G A B A (0.1-1 mM) were depolarization of 1.7 5_ 0.5 mV (S.E., n = 47) and 99.0 i 4.0% (n = 10) of the control input resistance. The mean decrease of the amplitude of the fast EPSP under the same condition was 51.4 :j-: 8.7 oj~ (n -- 14) of the control. Quantal content of the fast EPSP which was measured from the variance of the fast EPSP amplitudes 7 was significantly decreased by the action of G A B A at the concentrations of 5 btM-1 m M as shown in Fig. 2. The mean decrease in quantal content 5 min after application of GABA (0. l-1 raM) was 42.1 ~ 3.7 ~ (n = 14) of that in the normal solution where quantal content varied from l to 5. These results clearly indicate that GABA decreased the release of transmitter (ACh) from the preganglionic nerve terminal induced by its stimulation. This conclusion is further supported by the fact that G A B A did not affect the sensitivity of the postsynaptic membrane to ACh 16. To test the possibility that G A B A may inhibit transmitter release by the depolarizing action at the presynaptic terminal membrane 16, the effect o f GABA on spontaneous miniature excitatory postsynaptic potentials (MEPSP) was examined. The frequency of MEPSP was not affected by G A B A (0.1 mM). Under the same condition, the amplitude of MEPSP remained almost unchanged, although a slight de-

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401 crease was seen in some cells. The mean changes in the frequency and amplitude of MEPSP in the presence of GABA (0.1 raM) were 103.9 ~ 7 . 7 ~ (n = 7) and 109.5 ± 7.9 °/0 (n 7), respectively. On the other hand, depolarization of the preganglionic membrane by increasing the external K + concentration to 10 m M markedly increased the MEPSP frequency from 2.6 i 0.5/rain (n -- 5) in normal Ringer to 32.8 ± 6/rain (n -- 9) as found by Blackman et al. 4. Even under this condition, GABA (0.1-1 mM) did not change the M EPSP frequency (95.7 ± 5.9 ~;, n -- 5) nor its amplitude (105.7 ~L 9.0 ~ , n 5), although quantal content of the fast EPSP was decreased to about 60 /o/o of the control (61.2 ± 6.8)~, n 6). Thus, lack of effects of GABA on MEPSP frequency would indicate that the marked action of GABA on the evoked release of ACh does not seem to be directly caused by the membrane depolarization of the strategic site for ACh release in the presynaptic terminal since such depolarization would be expected to increase the frequency of MEPSP as shown in the high K + solution. Furthermore, the absence of effect of GABA on the amplitude of MEPSP supports the conclusion that the inhibitory action of GABA on the fast EPSP is mainly presynaptic under the present experimental conditions. The preganglionic axon in the bullfrog sympathetic ganglion divides into many unmyelinated arborizations which form many synaptic boutons on the cell soma z3. Therefore, depolarization recorded by the sucrose gap method from the preganglionic terminal, but not from the axon t6, may occur at some portion of the unmyelinated segments distant from the synaptic knob. This depolarization, whatever the mechanism is, may impair invasion of the action potential to the strategic site for release, causing an overall decrease in the amount of ACh released 14,17. Another possibility would be that there may be a large increase by GABA in the conductance of the terminal membrane participating in release, say, enhancement of Cl- conductance (Gcl), as in case of the somata of primary afferent neurones13,19, whether or not it causes a small depolarization which does not affect MEPSP frequency. The shunting effect produced by the increase in the membrane conductance would reduce the size of the terminal spike resulting in depression of transmitter release, as has been suggested in the crayfish neuromuscular junction where GABA increases the Gcl of the excitatory nerve terminal21,22. The third possibility would be that the depolarizing action of GABA and its inhibitory action on ACh release are independent, and the latter action may be due to a direct action on the mechanism of excitation-secretion coupling, say, the Ca 2~ gate mechanism. However, there is no evidence for the possibility. Since there seems to be no evidence for a neuron containing GABA and axoaxonal synapses in the bullfrog sympathetic ganglion, the physiological significance of the present study would lie elsewhere. Brown and Galvan 5 have shown that glial cells in mammalian sympathetic ganglion can accumulate GABA at a relatively low concentration up to 1 #M, precluding untoward effects on neuronal excitability1, '~. In the case of bullfrog sympathetic ganglion cell, the action of GABA seems to be both presynaptic and postsynaptic, although the former appears to predominate. If one assumes that glial cells in the bullfrog ganglion have a similar uptake mechanism of GABA, hormonal control, by GABA, of ganglionic synaptic transmission at both the presynaptic and postsynaptic sites seems possible. This matter is now under investiga-

402 tion. F u r t h e r m o r e , the presynaptic effect of G A B A on the presynaptic terminals in bullfrog sympathetic ganglia can be used as a model for the action of G A B A in the central nervous system. We t h a n k Dr. R. A. Nicoll for reading the m a n u s c r i p t a n d valuable suggestions. This work was supported by a g r a n t (257037) from the Ministry of Education of Japan.

1 Adams, P. R. and Brown, D. A., Action of 7'-aminobutyric acid (GABA) on ral sympathetic ganglion cells, Brit. J. Pharmacol., 47 (1973) 639P-640P. 2 Adams, P. R. and Brown, D. A., Actions of y-aminobutyric acid on sympathetic ganglion cells, J. Physiol. (Lond.), 250 (1975) 85-120. 3 Barker, J. L. and Nicoll, R. A., The pharmacology and ionic dependency of amino acid responses in the frog spinal cord, J. Physiol. (Lond.), 228 (1973) 259-277. 4 Blackman, J. G., Ginsborg, B. L. and Ray, C., Spontaneous synaptic activity in sympathetic ganglion cells of the frog, J. Physiol. (Lond.), 167 (1963) 389-401. 5 Brown, D. A. and Galvan, M., Influence of neuroglial transport on the action of ~,-aminobutyric acid on mammalian ganglion ceils, Brit. J. Pharmacol., 59 (1977) 373-378. 6 De Groat, W. C., The actions of 7-aminobutyric acid and related amino acids on mammalian autonomic ganglia, J. Pharmaeol. exp. Ther., 172 (1970) 384-396. 7 Del Castillo, J. and Katz, B., Quantal components of the end-plate potential, J. Physiol. (Lond.), 124 (1954) 560-573. 8 Dudel, J., The action of inhibitory drugs on nerve terminals in crayfish muscle, Pfliigers Archly. ges. Physiol., 284 (1965) 81-94. 9 Dudel, J. and Kuffler, S. W., Presynaptic inhibition at the crayfish neuromuscular junction, J. Physiol. (Lond.), 155 (1961)543-562. 10 Eccles, J. C., The Physiology of Synapses, Springer, Berlin, 1964, pp. 220-238. 1l Edwards, C. and lkeda, K., Effects of 2-PAM and succinylcholine on neuromuscular transmission in the frog, J. Pharmacol. exp. Ther., 138 (1962) 322-328. 12 Frank, K.,Basic mechanisms ofsynaptic transmission in the central nervous system, LR.E. Trans. Med. Electronics, ME-6 (1959) 85-88. 13 Gallagher, J. P., Higashi, H. and Nishi, S., Characterization and ionic basis of GABA-induced depolarizations recorded in vitro from cat primary afferent neurones, J. PhysioL (Lond.), 275 (1978) 263-282. 14 Hatt, H. and Smith, D. O., Synaptic depression related to presynaptic axon conduction block, J. Physiol. (Lond.), 259 (1976) 367-393. 15 Koketsu, K., Cholinergic synaptic potentials and the underlying ionic mechanisms, Fed. Proc., 28 (1969) 101-112. 16 Koketsu, K., Shoji, T. and Yamamoto, K., Effects of GABA on presynaptic nerve terminals in bullfrog (Rana catesbiana) sympathetic ganglia, Experientia (Basel), 30 (1974) 382-383. 17 Krnjevi6, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418-540. 18 Nishi, S. and Koketsu, K., Electrical properties and activities of single sympathetic neurons in frogs, J. cell. cornp. Physiol., 55 (1960) 15-30. 19 Nishi, S., Minota, S. and Karczmar, A. G., Primary afferent neurones: The ionic mechanism of GABA-mediated depolarization, Neuropharmacology, 13 (1974) 215-219. 20 Schmidt, R. F., Presynaptic inhibition in the vertebrate central nervous system, Ergebn. Physiol., 63 (1971) 20-101. 21 Takeuchi, A. and Takeuchi, N.,A study of the inhibitory action ofy-aminobutyric acid on neuromuscular transmission in the crayfish, J. Physiol. (Lond.), 183 (1966) 418-432. 22 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,-aminobutyric acid, J: Physiol. (Lond.), 183 (1966) 433~1-49. 23 Taxi, M. J., Etude l'ultrastruture des zones synaptiques dans les ganglions sympathiques de la grenouille, C.R. Acad. Sci. (Paris), 252 (1961) 174-176.