Brain Research, 575 (1992) 155-158

~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

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BRES 25099

G proteins are involved in the regulation of transmitter release at an Aplysia cholinergic synapse G. Baux, P. Fossier and L. Tauc Laboratoire de Neurobiologie Cellulaire et Mol~culaire, C.N.R.S. F-91198 Gif-sur-Yvette (France)

(Accepted 10 December 1991) Key words: G protein; Acetylcholine release; Calcium current; Synapse; Aplysia; Electrophysiology

At an identified cholinergic synapse of the Aplysia buccal ganglion, presynaptic injections of guanosine 5"-O-3-thiotriphosphate(GTP-7-S) depressed the amplitude of evoked postsynaptic responses. This reduction of acetylcholine (ACh) release by GTP-),-S, prevented by preinjection of guanosine 5"-O-2-thiodiphosphate(GDP-fl-S) in the presynaptic neuron, was due to a reduction of the number of ACh quanta released. The mean amplitude of the evoked miniature postsynaptic current (MPSC) was unchanged. The presynaptic Ca2+ influx was lowered. Voltage-dependent Ca 2+ and K + channels are currently reported as being the target of G protein regulation 5a°'a2'33. At synapses, influx of Ca 2+ is the trigger of transmitter release 3'25'43 whereas a change in K + conductance, by modifying the action potential of the presynaptic neuron, alters the activation of presynaptic voltagedependent Ca 2+ channels and the release of transmitter ~' 21,22. A direct involvement of specific G proteins in the mechanism of transmitter release itself is also under consideration 2,lmS'17,26-2s. Such possible regulations of transmitter release by G proteins could be tested at an identified cholinergic synapse in the buccal ganglion of Aplysia californica. We focused our attention on the effects of activation of presynaptic G proteins on the presynaptic Ca 2+ current, on the number of evoked quanta and on the size of ACh quantum. At this synapse, the short (500/~m) and thick (tens of microns) presynaptic axon permits the induction of transmitter release directly by applying depolarizing steps from the soma of the voltage clamped presynaptic neuron 4'16'35. The calculations of the mean amplitude (imin) and of the decay time (T) of the evoked miniature postsynaptic currents (elementary postsynaptic events due to the release of individual quanta of acetylcholine (ACh)) forming the postsynaptic response (Fig. 1) were done as described previously4a6'35. The number of quanta (Q) released by a given presynaptic depolarization can be determined by dividing the total quantity of current of the postsynaptic response (mean amplitude of the response, I, multiplied by its duration, 3 s) by that

of a miniature (mean calculated amplitude, imin, multiplied by its calculated decay time, 7); Q = 1.3/imin.T (Fig. 1). Experiments were performed at room temperature in artificial sea water (in mM: NaC1 460, KC1 10, CaCI 2 11, MgC12 25, MgSO4 28, Tris-HC1 10, pH 7.8) in the presence of tetrodotoxin (100/~M) to block sodium spike generation. The role of G proteins on transmitter release was investigated by the introduction, into the presynaptic soma, of the non-hydrolysable guanosine 5-triphosphate (GTP) analogue, guanosine 5"-O-3-thiotriphosphate, GTP-y-S2° for the activation, or of the non hydrolysable guanosine-5-diphosphate (GDP) analogue, guanosine 5"-O-2-thiodiphosphate, GDP-fl-S a4, for the inactivation of G protein activity. These two compounds (obtained from Sigma and Boehringer, respectively) were ionophoretically injected into the presynaptic soma by an appropriate constant current. Ionophoretic injection of GTP-y-S into the presynaptic neuron led to a decrease of the postsynaptic response (Fig. 1,I) evoked by a 3 s rectangular depolarization of the voltage clamped presynaptic neuron. The decrease in amplitude of the postsynaptic response started about 10 min after the beginning of the injection (Fig. 1,I). This delay probably corresponds to the migration (transport and/or diffusion) of GTP-y-S from the soma to the terminal and is similar to the delay observed following injection of other molecules of comparable molecular weight such as choline or A f h 23'29. The final concentration of GTP-~,-S obtained in the neuron (a constant current of 10 nA for 90 s was used) was estimated to be

Correspondence: G. Baux, Laboratoire de Neurobiologie Cellulaire et Mol6culaire, C.N.R.S., F-91198 Gif-sur-Yvette cedex, France. Fax: (33) (1) 69-82-35-80.

156

I

between 50 and 500 #M. This range of concentrations has previously been used in other neurons to activate G proteins 6,18,19,34,38,42 postsynaptic

response(nS) 3O0

The irreversible depression of the postsynaptic response by GTP-7-S was characterized by a reduction in

GTP-)'-S

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a

400 nS ""-,I . ~

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TIME (minJ I I 60 70

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after GTP- r - S injection (B)

Injection of GDP-fl-S into the presynaptic n e u r o n led to an increase (5-30%) in the size of the postsynaptic response and of the n u m b e r of quanta released (Fig. 2). This could be the consequence of the blockade of G proteins involved in a pre-existent active process induc-

m I(vA)

100%

the n u m b e r of quanta (Q) released by the presynaptic depolarization (Fig. 1,II). As the amplitude imin of the evoked MPSC was not significantly changed (Fig. 1,II), it can be concluded that the activation of G proteins did not modify the size of the presynaptic A C h quantum.

o6 t-

Fig. 1, Depression of the postsynaptic response after ionophoretic injection of GTP-7-S into the presynaptic neuron. I: time course of the decrease of the postsynaptic response after GTP-7-S ionophoretic injection (10 nA for 90 s) into the presynaptic neuron. Inserts represent postsynaptic responses before (A) and after (B) injection of GTP-7-S (10 nA for 90 s). Acetylcholine release was induced by long-lasting depolarizations (3 s to +10 mV) of the voltage clamped presynaptic neuron in the presence of tetrodotoxin. Using this technique, the presynaptic stimulus remained constant whatever the changes in conductances of the presynaptic membrane, a: postsynaptic current in the cell voltage clamped at -80 mV; b: ac trace of the same postsynaptic current with higher amplification; c: current and d: potential measured in the presynaptic neuron voltage clamped at -50 mV with a + 10 mV step. A: control postsynaptic response; B: postsynaptic response 35 min after GTP-7-S injection into the presynaptic neuron. II: for the same experiment, statistical analysis of the postsynaptic response allows the calculation of the amplitude, imin and of the decay time, T, of the evoked postsynaptic currents constituting the long duration postsynaptic response• From these values, the number of evoked miniature postsynaptic currents can be deduced and thus the number of released quanta, Q. The histograms show the values of I (mean postsynaptic current amplitude expressed as a conductance), imin (expressed as a conductance), T and Q in control situation (A) and after injection of GTP-7-S (B). 100% represent 285 nS for I, 1.35 nS for imin(n = 8), 11 ms for T and 57575 quanta for Q. III: decrease of presynaptic Ca2+ current after intracellular injection of GTP-7-S. I-V curves represent the Ca2+ current of the presynaptic neuron recorded in presence of tetrodotoxin (I00/~M), tetraethylammonium (50 mM) and 4-aminopyridine (10 mM) in artificial seawater containing 55 mM of CaCI2. The neuron was voltage-clamped at -30 mV. (0) control; (©) after ionophoretic injection of GTP-7-S (constant injecting current: 10 nA for 90 s); (11) after ionophoretic injection of GTP-7-S (constant injecting current: 10 nA for 4 min); (&) current after bath application of lanthanum ions to block the Ca2+ current.

ing a depression of transmitter release. The replacement of endogenous G D P by the non-hydrolysable analogue GDP-fl-S prevented the depressing effect on transmitter release induced by GTP-7-S injections. In the experiment illustrated in Fig. 2, we took advantage of the presence of two equivalent presynaptic neurons which synapse on the same postsynaptic neuron: the first presynaptic n e u r o n (Pre 1) was injected with GTP-7-S wh.:'ch induced a rapid decrease of the postsynaptic response; in the second presynaptic n e u r o n (Pre 2) which was preloaded with GDP-fl-S, the effect of the GTP-7-S injection was markedly reduced. Most likely, in Pre 2, a great n u m b e r of G protein alpha subunits were occupied by GDP-fl-S and thus made 'inactivatable' by

Q (%) G D P - !3-S

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Pre 1 (*) GTP- y -S Pre 2 ('J) G D P - I~-S + G T P - y - S I

I 40

I

I 60

Ie

I

I 80

"'*....4, ......... 4 . 1 I I 100

I 120

TIME (min.) I I 140

Fig. 2. GDP-fl-S prevents the action of GTP-7-S. Taking advantage of the presence of two functionally equivalent presynaptic neurons (insert), we injected GTP-7-S (10 nA, 120 s) in one of them (Pre 1, O) whereas the second (Pre 2, C)) was loaded (10 nA, 3 rain) with GDP-fl-S before injection with GTP-7-S (10 nA, 120 s). The loading with GDP-fl-S prevented the depressive action of GTP-7-S since the number of quanta released (Q) by the depolarization of the presynaptic neuron 2 (O) was significantly less decreased than that evoked by the presynaptic neuron 1 (0). Note the small increase of the number of quanta released by the presynaptic neuron 2 after intracellular injection of GDP-fl-S. 100% represents 65000 quanta (0) and 67500 quanta (©).

157 GTP-7-S. This e x p e r i m e n t further illustrates the specificity of action of GTP-~-S. Similar results were o b t a i n e d when, instead of long lasting depolarizations of the presynaptic neuron, action igotentials were used to release A C h (not shown). In this study, transmitter release was e v o k e d by depolarization of the presynaptic n e u r o n under voltageclamp excluding that any change in the presynaptic K + current could modify the polarization of the terminal and t h e r e b y affect transmitter release. It may be suggested that either a modification of the transmitter release mechanism itself or of the presynaptic Ca 2÷ influx could account for the depression of transmitter release following GTP-),-S injection. The latter hypothesis could be tested in o u r p r e p a r a t i o n . A m o d u l a t o r y action of G proteins on Ca 2+ currents cannot be directly examined from the analysis of the long-lasting presynaptic currents in Fig. 1 (Ic) since any change in the inward Ca 2+ current would be m a s k e d by the large o u t w a r d K + current. To isolate the presynaptic Ca 2+ current, we c l a m p e d the presynaptic n e u r o n at - 3 0 m V to inactivate the early transient K + current 9, while o t h e r v o l t a g e - d e p e n d e n t ionic currents were b l o c k e d pharmacologically4'16. Intracellular ionophoretic injection of G T P w - S into the presynaptic cell, p e r f o r m e d u n d e r the same conditions as in Fig. 1 (injection current of 10 n A for 90 s), led to a 47% decrease of the p e a k amplitude of the Ca 2÷ current (Fig. 1, III). It also a p p e a r e d that v o l t a g e - d e p e n d e n t outward

K + currents (Fig. 1, Ic) were unchanged by the GTP7-S injection, contrary to results r e p o r t e d for o t h e r Aplysia neurons where G proteins are thought to regulate the opening of different K + channels coupled to acetylcholine, d o p a m i n e , histamine and F M R F a m i d e receptors 7'3t'32'4°'41. Presynaptic Ca 2÷ channels can thus be

1 Abrams, T.W., Castellucci, V.E, Camardo, J.S., Kandel, E.R. and Lloyd, P.E., Two endogenous neuropeptides modulate the gill and siphon withdrawal reflex in Aplysia by presynaptic facilitation involving cAMP-dependent closure of a serotonin-sensitive potassium channel, Proc, Natl. Acad. Sci. USA, 81 (1984) 7956-7960. 2 Ahnert-Hilger, G., Brautigam, M. and Gratzl, M., Ca2÷ stimulated catecholamine release from toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G proteins, Biochemistry, 26 (1987) 78427848. 3 Augustine, G.J. and Charlton, M.P., Calcium dependence of presynaptic calcium current and post-synaptic response at the squid giant synapse, J. Physiol., 381 (1986) 619-640. 4 Baux, G., Fossier, P. and Tauc, L., Histamine and FLRFamide regulate in opposite ways acetylcholine release at an identified neuro-neuronal synapse in Aplysia, J. Physiol., 429 (1990) 147168. 5 Birnbaumer, L., Abramowitz, J. and Brown, A.M., Receptoreffector coupling by G proteins, Biochim. Biophys. Acta, 1031 (1990) 163-224. 6 Bley, K.R. and Tsien, R.W., Inhibition of Ca2÷ and K ÷ channels in sympathetic neurons by neuropeptides and other ganglionic transmitters, Neuron, 2 (1990) 379-391. 7 Brezina, V., Guanosine 5"-triphosphate analogue activates potassium current modulated by neurotransmitters in Aplysia neurones, J. Physiol., 407 (1988) 15-40. 8 Brezina, V., Eckert, R. and Erxleben, C., Suppression of calcium current by an endogenous neuropeptide in neurones of Aplysia californica, J. Physiol., 388 (1987) 565-595. 9 Brezina, V., Eckert, R. and Erxleben, C., Modulation of po-

tassium conductances by an endogenous neuropeptide in neutones of Aplysia californica, J. Physiol., 382 (1987) 267-290. Brown, A.M., Ion channels as G protein effectors, N.LP.S., 6 (1991) 158-161. Chin, G.J., Vogel, S.S., Elste, A.M. and Schwartz, J.H., Characterization of synaptophysin and G proteins in synaptic vesicles and plasma membrane of Aplysia californica, Brain Research, 508 (1990) 265-272. Dolphin, A.C., Regulation of calcium channel activity by GTP binding proteins and second messengers, Biochim. Biophys. Acta, 1091 (1991) 68-80. Dolphin, A.C. and Scott, R.H., Calcium channel currents and their inhibition by (-)baclophen in rat sensory neurones: modulation by guanine nucleotides, J. Physiol., 386 (1987) 1-17. Eckstein, F., Cassel, D., Levkovitz, H., Lowe, M. and Selinger, Z., Guanosine 5"-0-2-thiodiphosphate: an inhibitor of adenylate cyclase stimulation by guanine nucleotides and fluorides ions, J. Biol. Chem., 254 (1979) 9829-9834. Fisher von Mollard, G., Sudhof, T.C. and Jahn, R., A small GTP-binding protein dissociates from synaptic vesicles during exocytosis, Nature, 349 (1991) 79-81. Fossier, P., Baux, G. and Tauc, L., Activation of protein kinase C by presynaptic FLRFamide receptors facilitates transmitter release at an Aplysia cholinergic synapse, Neuron, 5 (1990) 479-486. Gomperts, B.D., Ge: a GTP-binding protein mediating exocytosis, Annu. Rev. Physiol., 52 (1990) 591-606. Hescheler, J., Rosenthal, W., Trautwein, W. and Schultz, G., The GTP-binding protein Go regulates neuronal calcium channels, Nature, 325 (1987) 445-447. Holz, G.G., Rane, S.G. and Dunlap, K., GTP-binding proteins

considered to be one of the effectors by which G proteins could m o d u l a t e transmitter release at the identified buccal ganglion synapse. A t this cholinergic synapse known to be involved in the control of feeding behavior 36'37, the next step will be to d e t e r m i n e if G proteins could be transducers between Ca 2÷ channels and presynaptic receptors 4. It will be first tested for histamine presynaptic receptors, the activation of which decreases the a m o u n t of A C h released by reducing Ca 2÷ influx 4 as does GTP-y-S. Activation of G proteins was r e p o r t e d to mimic the decrease in voltage d e p e n d e n t Ca 2+ currents obtained through the activation of o p i a t e receptors TM, a2 receptors 3°, d o p a m i n e receptors 39, G A B A B receptors 13, somatostatin receptors 24 and F M R F a m i d e receptors in Aplysia s. F u r t h e r experiments using specific antibodies or cholera and pertussis toxins are n e e d e d to further characterize the G proteins involved.

We are grateful to L.E. Trudeau for comments on the manuscript. The work was supported by Grant 89/145 from DRET to L.T.

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