Title: A Rho-related GTPase Is Involved in Ca2+-dependent Neurotransmitter Exocytosis
Abstract: Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca2+-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA, RhoB, Rac1, and Cdc42 are present in rat brain synaptosomes; however, only Rac1 was associated with highly purified synaptic vesicles. To determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho family, most likely Rac1, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals. Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca2+-dependent neuroexocytosis. Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca2+-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA, RhoB, Rac1, and Cdc42 are present in rat brain synaptosomes; however, only Rac1 was associated with highly purified synaptic vesicles. To determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho family, most likely Rac1, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals. Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca2+-dependent neuroexocytosis. acetylcholine lethal toxin cytotoxic necrotizing factor 1 toxin B paired-pulse facilitation Rho proteins form a subfamily of highly conserved small GTPases belonging to the Ras superfamily. In mammals, Rho GTPases comprise Rho (A to H isoforms), Rac (Rac1 and Rac2 isoforms), Cdc42 (Cdc42Hs and G25K isoforms), and more distant members. Like other monomeric GTPases of the Ras family, Rho proteins act as molecular switches: upon receiving upstream signals, they are converted into an active GTP-bound form that is able to interact with downstream effectors. These comprise protein kinase N, Rho kinase, phosphatidylinositol 3-kinase, phosphatidylinositol 4-kinase, phosphatidylinositol 5-kinase, and the myosin-binding subunit of myosin phosphatase (for review, see Ref. 1.Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (260) Google Scholar).In fine, the activation of Rho-related GTPases leads mainly to a rearrangement of the actin-based cytoskeleton and/or to a regulation of phosphoinositide levels. Rho proteins have been implicated in a large variety of cellular functions, including chemotaxis, cell cycle progression, axonal guidance. and endocytosis (for review, see Refs. 1.Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 2.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar, 3.Ren X.D. Schwartz M.A. Curr. Opin. Genet. Dev. 1998; 8: 63-67Crossref PubMed Scopus (78) Google Scholar). Ca2+-triggered neurotransmitter release and hormone secretion are closely related mechanisms that involve proteins common to both neurons and secretory cells. For example, synaptobrevin, syntaxin, 25-kDa synaptosomal-associated protein, synaptotagmin, soluble N-ethylmaleimide-sensitive factor, and solubleN-ethylmaleimide-sensitive factor attached proteins act in concert to ensure docking and/or fusion in both dense-core granule and synaptic vesicle exocytosis. Consistent with the implication of Rab proteins in vesicle trafficking, the small GTPase Rab3 has been shown to regulate both neurotransmitter and hormone secretion (for review, see Refs. 4.Martin T.F Curr. Opin. Neurobiol. 1994; 4: 626-632Crossref PubMed Scopus (85) Google Scholar, 5.Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1768) Google Scholar, 6.Calakos N. Scheller R.H. Physiol. Rev. 1996; 76: 1-29Crossref PubMed Scopus (311) Google Scholar, 7.Morgan A. Burgoyne R.D. Semin. Cell Dev. Biol. 1997; 8: 141-149Crossref PubMed Scopus (54) Google Scholar, 8.Fernandez-Chacon R. Südhof T.C. Annu. Rev. Physiol. 1999; 61: 753-776Crossref PubMed Scopus (153) Google Scholar, 9.Geppert M. Südhof T.C. Annu. Rev. Neurosci. 1998; 21: 75-95Crossref PubMed Scopus (223) Google Scholar, 10.Bajjalieh S.M. Curr. Opin. Neurobiol. 1999; 9: 321-328Crossref PubMed Scopus (50) Google Scholar). On the other hand, despite the similarities between the release of the content of synaptic vesicles and large dense-core granules, several recent studies have revealed mechanistic differences between these two exocytotic processes (for review, see Refs. 7.Morgan A. Burgoyne R.D. Semin. Cell Dev. Biol. 1997; 8: 141-149Crossref PubMed Scopus (54) Google Scholar and 11.Edwards R.H. Curr. Biol. 1998; 8: 883-885Abstract Full Text Full Text PDF PubMed Google Scholar). In endocrine cells, members of the Rho family have been proposed to regulate exocytosis. Indeed, Cdc42 and Rac control regulated secretion in pancreatic beta cells (12.Kowluru A. Li G. Rabaglia M.E. Segu V.B. Hofmann F. Aktories K. Metz S.A. Biochem. Pharmacol. 1997; 54: 1097-1108Crossref PubMed Scopus (81) Google Scholar), basophilic leukemia cells (13.Prepens U. Just I. von Eichel-Streiber C. Aktories K. J. Biol. Chem. 1996; 271: 7324-7329Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and mast cells (14.Brown A.M. O'Sullivan A.J. Gomperts B.D. Mol. Biol. Cell. 1998; 9: 1053-1063Crossref PubMed Scopus (79) Google Scholar). In chromaffin cells, RhoA localized on secretory granules controls subplasmalemmal actin and exocytosis by regulating a granule-associated phosphatidylinositol 4-kinase (15.Gasman S. Chasserot-Golaz S. Hubert P. Aunis D. Bader M.-F. J. Biol. Chem. 1998; 273: 16913-16920Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 16.Gasman S. Chasserot-Golaz S. Popoff M.R. Aunis D. Bader M.-F. J. Biol. Chem. 1997; 272: 20564-20571Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Rho-like GTPases have been implicated in actin filament dynamics and organelle movement in growth cones (for review, see Ref. 17.Luo L. Jan L.Y. Jan Y.N. Curr. Opin. Neurobiol. 1997; 7: 81-86Crossref PubMed Scopus (172) Google Scholar). The aim of our study was to probe the presence of Rho proteins in nerve terminals and to determine their possible implication in neurotransmitter release. We found that Rac1, RhoA, RhoB, and Cdc42 are present in nerve terminals, with Rac1 selectively associated with purified synaptic vesicles. The role played by these small GTPases in neurotransmission was addressed by monitoring acetylcholine (ACh)1 release from identified cholinergic neurons in the Aplysia buccal ganglion. The function of Rho-related GTPases was impaired by presynaptic microinjection of bacterial toxins known to selectively activate or inactivate subgroups of Rho proteins (for review, see Ref.18.Aktories K. Trends Microbiol. 1997; 5: 282-288Abstract Full Text PDF PubMed Scopus (136) Google Scholar). Our results suggest that a Rho-related protein, most likely Rac1, plays a major role in neurotransmission by controlling a yet undefined Ca2+-dependent late step of synaptic vesicle exocytosis. Lethal toxin (LT) from Clostridium sordellii strain IP82 was purified as described previously (19.Popoff M.R. Infect. Immun. 1987; 55: 35-43Crossref PubMed Google Scholar). Recombinant C3 exoenzyme was expressed in Escherichia colistrain Sure (Stratagene) from pMRP143, consisting of the DNA fragment coding for the mature C3 protein (20.Popoff M.R. Hauser D. Boquet P. Eklund M.W. Gill D.M. Infect. Immun. 1991; 59: 3673-3679Crossref PubMed Google Scholar) under the control of the iota toxin gene promoter in vector pJIR750 (21.Marvaud J.C. Gibert M. Inoue K. Fujinaga Y. Oguma K. Popoff M.R. Mol. Microbiol. 1998; 29: 1009-1018Crossref PubMed Scopus (67) Google Scholar). After sonicating bacterial cells in 10 mm Tris-HCl (pH 8.5), the extract was clarified by centrifugation, treated with protamine sulfate (2 mg/ml; Merck) for 30 min at 40 °C, and centrifuged again. The supernatant was loaded onto a QAE-Sepharose A50 column (Amersham Pharmacia Biotech) equilibrated in 10 mm Tris-HCl (pH 8.5). The flow-through containing the purified C3 enzyme showed a single band of 25 kDa on SDS-polyacrylamide gel electrophoresis. Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic E. colistrains, was purified as described previously (22.Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 23.Fiorentini C. Fabbri A. Flatau G. Donelli G. Matarrese P. Lemichez E. Falzano L. Boquet P. J. Biol. Chem. 1997; 272: 19532-19537Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Stock solutions (20 μm) were prepared in 50 μm Tris-HCl (pH 7.4) containing 100 μm NaCl. A recombinant C-terminal 14.8–31.5 kDa peptide corresponding to the catalytic region of CNF1 was produced as a glutathione S-transferase fusion protein. It was purified according to a previously described procedure (24.Lemichez E. Flatau G. Bruzzone M. Boquet P. Gauthier M. Mol. Microbiol. 1997; 24: 1061-1070Crossref PubMed Scopus (119) Google Scholar). A stock solution (20 μm) was prepared in sodium phosphate buffer (pH 7.4) containing reduced glutathione (20 mm). All toxins were stored at −80 °C in 3–5-μl aliquots. The various buffers used for storage of the toxins had no effect on evoked ACh release. The biological activity of each batch of toxin was verified by the ability to induce morphological changes and cytoskeletal modifications (C3, ToxB, and LT: cell rounding and disruption of stress fibers; and CNF1: cell spreading and increase in stress fibers), visualized by staining of HeLa or Vero cells with fluorescein-conjugated phalloidin (22.Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 23.Fiorentini C. Fabbri A. Flatau G. Donelli G. Matarrese P. Lemichez E. Falzano L. Boquet P. J. Biol. Chem. 1997; 272: 19532-19537Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 25.Popoff M.R. Chaves-Orlate E. Lemichez E. von Eichel-Streiber C. Thelestam M. Chardin P. Cussac D. Antonny B. Chavrier P. Flatau G. Giry M. de Gunzburg J. Boquet P J. Biol. Chem. 1996; 271: 10217-10224Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The activity of C3 was further determined by its ability to induce [32P]ADP-ribosylation of membrane-bound RhoA present in purified chromaffin granule preparations (15.Gasman S. Chasserot-Golaz S. Hubert P. Aunis D. Bader M.-F. J. Biol. Chem. 1998; 273: 16913-16920Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The enzymatic activity of CNF1 on recombinant RhoA (22.Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar) or chromaffin granule-associated RhoA (data not shown) was demonstrated by an increase in the apparent molecular mass of the molecule on SDS gels. Synaptic vesicles were prepared from rat brains according to Huttner et al. (26.Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (890) Google Scholar). Briefly, 14 rat brains were placed in ice-cold buffered sucrose (320 mm sucrose and 4 mm Hepes-NaOH (pH 7.4)). From this point on, the material was kept at 4 °C. Cerebral cortices were dissected free of cerebellum, brainstem, and most of the midbrain and homogenized in the same buffer containing 1 μm phenylmethylsulfonyl fluoride and 2 μg/ml pepstatin in a Teflon/glass homogenizer. The homogenate was then centrifuged for 10 min at 1100 × g. The resulting supernatant was centrifuged for 15 min at 9200 ×g, and the pellet was resuspended in 10 ml of buffered sucrose/brain and centrifuged for 15 min at 10,500 ×g. The resulting pellet (synaptosomes) was resuspended in buffered sucrose, diluted with 9 volumes of ice-cold H2O (hypotonic lysis of synaptosomes), and immediately homogenized. Protease inhibitors (pepstatin and phenylmethylsulfonyl fluoride) and 1m Hepes (pH 7.4) at a final concentration of 7.5 mm were added, and the homogenate was incubated on ice for 30 min and then centrifuged for 20 min at 25,500 × g. The pellet containing synaptosomal membranes (i.e. plasma membrane, mitochondria, and granules) was saved. The supernatant was centrifuged for 2 h at 48,000 rpm in a Ti-60 rotor (Beckman Instruments). The resulting supernatant was cleared by centrifugation at 100,000 × g and saved (cytosol). The pellet (crude synaptic vesicles) was resuspended in 4 ml of 30 mm sucrose and 4 mm Hepes (pH 7.4), homogenized, passed five times back and forth through a 25-gauge needle, loaded on a continuous gradient of 50–800 mm sucrose in 4 mm Hepes (pH 7.4), and centrifuged for 5 h at 26,000 rpm in an SW 28 rotor (Beckman Instruments). After centrifugation, 2-ml fractions were collected. Fractions in the 200–400 mm sucrose region were pooled and chromatographed on a glyceryl-coated controlled-pore glass bead column (GLY 03000B, Electro-Nucleonics Inc.) to obtain highly purified synaptic vesicles (26.Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (890) Google Scholar). Protein content of the various fractions was analyzed by the Bradford procedure (Bio-Rad). SDS-polyacrylamide gel electrophoresis was performed on 12% acrylamide gels in Tris/glycine buffer (27.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). Proteins were transferred to nitrocellulose sheets, and blots were developed with secondary antibodies coupled to alkaline phosphatase (Sigma); immunoreactive bands were detected with 5-bromo-4-chloro-3-indolyl phosphate (0.15 mg/ml) and nitro blue tetrazolium (0.3 mg/ml) in 40 mm sodium carbonate and 5 mm MgCl2 (pH 9.8). In some experiments, blots were developed with secondary antibodies coupled to horseradish peroxidase (Amersham Pharmacia Biotech), and immunoreactive bands were revealed with the ECL system (Amersham Pharmacia Biotech). Mouse monoclonal antibodies against Rac1 (Transduction Laboratories) were used at 1:500 dilution. Mouse monoclonal antibodies against RhoA (Santa Cruz Biotechnology) were used at 1:50 dilution. Rabbit polyclonal antibodies against RhoB and Cdc42 (Santa Cruz Biotechnology) were used at 1:50 and 1:100 dilutions, respectively. Mouse monoclonal anti-synaptotagmin antibodies against the C2A domain (clone 1D12), a generous gift from Dr. Masami Takahashi (Mitsubishi Kasei Institute, Tokyo, Japan), were diluted 1:1000. Rabbit antibodies against the cytosolic epitope EQEGYQPNYGQ of synaptophysin (28.Taubenblatt P. Dedieu J.C. Gulik-Krzywicki T. Morel N. J. Cell Sci. 1999; 112: 3559-3567Crossref PubMed Google Scholar) were kindly provided by Dr. Nicolas Morel (Laboratoire de Neurobiologie Cellulaire, CNRS, Gif-sur-Yvette, France) and utilized at 1:1000 and 1:2000 dilutions. Experiments were performed at identified cholinergic synapses (29.Gardner D. Science. 1971; 173: 550-553Crossref PubMed Scopus (144) Google Scholar) in buccal ganglia of Aplysia californica(70–120 g of body weight; Marinus Inc., CA) according to previously published procedures (30.Johannes L. Doussau F. Clabecq A. Henry J.-P. Darchen F. Poulain B. J. Cell Sci. 1996; 109: 2875-2884Crossref PubMed Google Scholar, 31.Doussau F. Clabecq A. Henry J.-P. Darchen F. Poulain B. J. Neurosci. 1998; 18: 3147-3157Crossref PubMed Google Scholar). Briefly, two presynaptic cholinergic interneurons (100–150 μm in diameter) and one postsynaptic neuron (150–200 μm in diameter) in the buccal ganglion were impaled with two glass microelectrodes (3 m KCl and Ag/AgCl2, 2–10 megaohms). ACh release from a presynaptic neuron was monitored by evoking an action potential at 40-s intervals. In several experiments, 1- or 2-s trains of stimuli were generated by using supraliminar depolarizing pulses of 5 ms separated by a repolarizing phase of adequate duration (SMP-311 pulse generator, Bio-Logic S. A., Grenoble, France). ACh release was estimated by measuring the amplitude of the evoked postsynaptic current (at these synapses, it is a Cl−current) using a conventional two-electrode voltage-clamp technique and subsequently converting it to an apparent membrane conductance by taking into account the null potential for Cl−(i.e. the reversal potential of the postsynaptic response). The holding potential of the postsynaptic neuron was maintained at 30 mV above E Cl−. Dissected buccal ganglia were maintained at 22 °C using a Peltier plate system and superfused continuously (50 ml/h) with a physiological control medium containing NaCl (460 mm), KCl (10 mm), CaCl2 (33 mm), MgCl2 (50 mm), and MgSO4 (28 mm) in 10 mm Tris-HCl (pH 7.5). This dication-rich medium has a high [Ca2+]/[Mg2+] ratio (0.42) to minimize fluctuations in evoked ACh release due to spontaneous neuron firing activity. To modify the extracellular CaCl2 concentration, the respective concentrations of CaCl2 and MgCl2 were calculated according to the following equations: [CaCl2] (mm) = Q(83 + [MgSO4])/(Q + 1) and [MgCl2] (mm) = 83 − [CaCl2], whereQ is the [Ca2+]/[Mg2+] ratio. When CdCl2 (Sigma) was used, it was added directly to the control medium. To reduce the intracellular concentration of Ca2+ ions, EGTA was applied intraneuronally by pressure injection (see below). Possible intracellular pH changes were avoided by preparing EGTA in Tris-HCl (pH 7.4) with a 2.2-fold excess of Tris base. To limit the toxin action to a given neuron without modifying ACh release by a change in the ACh receptor efficiency, toxins were microinjected into presynaptic neurons. The sample to be injected was mixed with a vital dye (10% (v/v) fast green; Sigma). The samples were air pressure-injected under visual and electrophysiological monitoring. The injected volume was in the range of 1% of the cell body volume. Following intracellular injection, only neurons with membrane potentials of −60 to −45 mV and with no alterations in the action potentials were utilized. Unless indicated, data are presented as mean ± S.D. Statistical significance of the data was calculated by paired or unpaired t tests. The intracellular distribution of Rho proteins in presynaptic terminals was investigated in synaptosomes prepared from rat brains since the amount of neuronal tissue that can be collected from Aplysia does not allow subcellular fractionation. Fig.1 shows a Western blot analysis of the soluble and membrane-bound fractions obtained from purified synaptosomes by hypotonic lysis. Using specific antibodies raised against various members of the Rho family, we found that RhoA, RhoB, Rac1, and Cdc42 were present in the presynaptic terminals. In contrast to RhoA, which was largely cytosolic, and Cdc42, which was present in both cytosolic and membrane-bound fractions, RhoB and Rac1 were mostly detected in the particulate fraction containing synaptosomal plasma membrane, mitochondria, large dense-core particles, and a huge amount of synaptic vesicles. To probe the direct association of Rho proteins with the membrane of synaptic vesicles, crude synaptic vesicles obtained by high speed centrifugation (see “Experimental Procedures”) were further fractionated on a 50–800 mmsucrose velocity gradient. Fig. 2 shows the distribution of two synaptic vesicle marker proteins estimated by immunoreplica analysis in the fractions collected from the sucrose gradient. Synaptic vesicles were distributed in fractions 7–18 as revealed by the immunosignal for synaptophysin and synaptotagmin. Fractions 7–18 were also labeled with anti-RhoB and anti-Rac1 antibodies (Fig. 2 B), suggesting that RhoB and Rac1 may associate with the membrane of synaptic vesicles. Accordingly, previous reports suggested the presence of several low molecular mass GTP-binding proteins in brain synaptic vesicles (32.Matsuoka I. Dolly J.O. Biochim. Biophys. Acta. 1990; 1026: 99-104Crossref PubMed Scopus (10) Google Scholar) and cholinergic vesicles from the electric organ of marine ray (33.Ngsee J.K. Elferink L.A. Scheller R.H. J. Biol. Chem. 1991; 266: 2675-2680Abstract Full Text PDF PubMed Google Scholar). Note that RhoB and, to a certain extent, Rac1 were present in larger amounts in fractions 15–18 (Fig. 2 B). These higher density fractions also displayed some immunoreactivity with antibodies raised against RhoA and Cdc42, thereby revealing some heterogeneity of the vesicle population with respect to the presence of Rho GTPases. Therefore, the association of monomeric GTPases on synaptic vesicles was further probed in vesicle preparations obtained by controlled-pore glass chromatography (26.Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (890) Google Scholar). As illustrated in Fig. 2 C, we detected an immunosignal only for Rac1 in highly purified synaptic vesicles, indicating that Rac1 is the sole member of the Rho family associated with the membrane of synaptic vesicles.Figure 2Rac1 is associated with purified synaptic vesicles. A, total protein profile of fractions collected from a sucrose velocity gradient layered with the crude synaptic vesicle fraction; B, immunoblot analysis of synaptic vesicle markers and Rho proteins in each fraction of the gradient (10 μg of protein/lane); C, immunoblot analysis of synaptic vesicles purified by permeation chromatography on a controlled-pore glass column (CPG).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the buccal ganglion ofAplysia, two identified presynaptic neurons make cholinergic synapses with the same postsynaptic neuron (29.Gardner D. Science. 1971; 173: 550-553Crossref PubMed Scopus (144) Google Scholar, 31.Doussau F. Clabecq A. Henry J.-P. Darchen F. Poulain B. J. Neurosci. 1998; 18: 3147-3157Crossref PubMed Google Scholar). Thus, ACh release from presynaptic neurons can be monitored by measuring the amplitude of the evoked transmembrane current in the postsynaptic neuron. To probe the role of Rho-related GTPases in neurotransmitter release, we used ToxB, which specifically glucosylates Rac, Cdc42, and Rho (34.Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (883) Google Scholar); LT, which glucosylates Rac, but has no effect on Rho and Cdc42 (25.Popoff M.R. Chaves-Orlate E. Lemichez E. von Eichel-Streiber C. Thelestam M. Chardin P. Cussac D. Antonny B. Chavrier P. Flatau G. Giry M. de Gunzburg J. Boquet P J. Biol. Chem. 1996; 271: 10217-10224Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar); and C3, which ADP-ribosylates RhoA, RhoB, RhoC, andAplysia Rho (35.Quilliam L.A. Lacal J.C. Bokoch G.M. FEBS Lett. 1989; 247: 221-226Crossref PubMed Scopus (29) Google Scholar, 36.Sekine A. Fujiwara M. Narumiya S. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar) and, under certain circumstances, Rac (35.Quilliam L.A. Lacal J.C. Bokoch G.M. FEBS Lett. 1989; 247: 221-226Crossref PubMed Scopus (29) Google Scholar, 37.Didsbury J. Weber R.F. Bokoch G.M. Evans T. Snyderman R. J. Biol. Chem. 1989; 264: 16378-16382Abstract Full Text PDF PubMed Google Scholar, 38.Just I. Mohr C. Schallehn G. Menard L. Didsbury J.R. Vandekerckhove J. van Damme J. Aktories K. J. Biol. Chem. 1992; 267: 10274-10280Abstract Full Text PDF PubMed Google Scholar, 39.Menard L. Tomhave E. Casey P.J. Uhing R.J. Snyderman R. Didsbury J.R. Eur. J. Biochem. 1992; 206: 537-546Crossref PubMed Scopus (43) Google Scholar). Glucosylation or ADP-ribosylation in the effector domain of the various Rho, Rac, and Cdc42 isoforms disrupts their interaction with downstream effectors and thereby inactivates the intracellular pathways controlled by these GTPases. ToxB, LT, or C3 was pressure-injected into one presynaptic neuron, and the second presynaptic neuron was injected with the buffer used for toxin injection. In this way, we had an internal control for the stability of evoked neuroexocytosis for the duration of the experiments (up to 24 h). As shown in Fig. 3, all three toxins inhibited ACh release. More important, neither the action potential that triggers ACh release at nerve terminals nor the transmembrane resting potential and the membrane resistance of the injected neurons were significantly modified after injection of the toxins (data not shown). Therefore, the inhibition of ACh release induced by LT, ToxB, or C3 is not due to a modification of membrane excitability. The mean inhibition induced by the three toxins, calculated 3 and 20 h after injection, is shown in Fig.3 B. The three toxins induced an almost complete inhibition of neurotransmitter release 20 h after injection (Fig.3 B). Note, however, that high doses of C3 (2 μm final intraneuronal concentration) were required to abolish neurotransmission (Fig. 3 B). Collectively, these results suggest that Rac is the most likely candidate to regulate a rate-limiting step of exocytosis in neurons since it is the sole GTPase inhibited by LT, ToxB, and high concentrations of C3. Rho isoforms can be constitutively activated by CNF1, which catalyzes the deamidation of glutamine 63. This residue is conserved in RhoA, RhoB, RhoC, andAplysia Rho. Transformation of glutamine 63 to glutamic acid leads to inhibition of both intrinsic and Rho GTPase-activating protein-stimulated GTPase activity of Rho proteins (22.Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 40.Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar). Thus, the CNF1 action on Rho is equivalent to the amino acid substitution used to generate dominant-positive Rho mutants. Cdc42 can also be activated by a high concentration of this enzyme (22.Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 23.Fiorentini C. Fabbri A. Flatau G. Donelli G. Matarrese P. Lemichez E. Falzano L. Boquet P. J. Biol. Chem. 1997; 272: 19532-19537Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 40.Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar). To further evaluate the implication of Rho and Cdc42 in neuroexocytosis, dichainal CNF1 (200 nm final intraneuronal concentration) or a CNF1 recombinant catalytic moiety (data not shown) was microinjected into presynaptic neurons. As illustrated in Fig.4 A, no significant modification of ACh release was observed for at least 150 min. In longer experiments, CNF1 induced an alteration of membrane excitability. We also examined whether Rho activation by CNF1 would be able to restore ACh release previously inhibited by LT. LT (50 nm final concentration) was first injected into a presynaptic neuron, and after ACh release had stabilized, CNF1 (200 nm final concentration) was injected into the same neuron. As illustrated in Fig. 4 B, CNF1 was unable to rescue the ACh release inhibited by LT treatment. These experiments suggest that neither Rho nor Cdc42 plays a crucial role in neurotransmitter release. To further define the step in neurotransmitter release that is controlled by Rac, we examined the effect of high frequency stimulation on LT-induced blockage of ACh release. Trains of stimuli were elicited at 50 Hz for 1 or 2 s. Under control conditions, after a brief facilitation (31.Doussau F. Clabecq A. Henry J.-P. Darchen F. Poulain B. J. Neurosci. 1998; 18: 3147-3157Crossref PubMed Google Scholar), ACh release declined slightly during a train stimulus (Fig.5 A, open circles), probably due to an imbalance between the replenishment of the readily releasable pool of vesicles and the number of vesicles that undergo exocytosis. In LT-injected neurons, the time course of ACh release evoked by a 50-Hz stimulation train was greatly modified (Fig.5 A, closed circles): ACh release stabilized for ∼400 ms (404 ± 70 ms, n = 46) and then increased to reach within 1 s a level that was similar to that observed before LT injection (Fig. 5 A, compareopen and closed circles). This indicates that LT-induced inhibition of AC