Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5335-5339, July 1986 Neurobiology Biochemical and electrophysiological evidence of functional receptors in the rat superior cervical ganglion (autoradiography/inositol phosphollpids/neurohypophyseal peptides/receptor characterization/sympathetic system) MARIA KIRALY*, SYLVIE AUDIGIERt, ELIANE TRIBOLLETt, C. BARBERISt, M. DoLIvo*, AND J. J. DREIFUSS4§ *Institute of Physiology, Faculty of Medicine, 1005 Lausanne, Switzerland; tCentre National de la Recherche Scientifique-Institut National de la Santd et de la Recherche Mddicale Center of Pharmacology-Endocrinology, 34033 Montpellier, France; and lDepartment of Physiology, University Medical Center, 1211 Geneva, Switzerland Communicated by Ewald R. Weibel, March 31, 1986

ABSTRACT Binding of radioactive vasopressin-but not vasopressin receptors, whose intracellular signal is depen- of -was detected by autoradiography and by labeling dent upon the hydrolysis ofmembrane inositol phospholipids ofmembranes obtained from the rat superior cervical ganglion. and which are distinct from the -type (V2) receptors In both instances binding could be displaced by V1 (smooth associated with adenylate cyclase (15, 16). Moreover, we muscle-type) but not by V2 (kidney-type) , indicating show in electrophysiological studies that AVP applied at low that the ganglionic vasopressin receptors are similar to those concentrations causes a reduction in the amplitude ofthe fast present on hepatocytes and vascular smooth muscle. In ac- excitatory postsynaptic potential (f-EPSP) elicited by stim- cordance with the V1 character of the receptors, vasopressin ulation of the preganglionic nerve. activated the turnover of membrane inositol lipids, and this effect was abolished by a structural analogue known to act as a vasopressor antagonist. A possible physiological role of MATERIALS AND METHODS vasopressin was suggested by intracellular recordings obtained Binding of [3H]AVP to Crude Membrane Preparations. from ganglion cells in vitro. Vasopressin induced a reduction in Superior cervical ganglia were dissected from stunned male the amplitude of the fast excitatory postsynaptic potential Wistar rats (200-250 g, body weight). The afferent and evoked by electrical stimulation of the preganglionic nerve. efferent nerves were cut off, and the connective tissue This reduction in ganglionic transmission was antagonized by sheaths were removed. For each experiment, the ganglia the same synthetic structural analogue that blocked the effect from 15 rats were dissected and immersed in ice-cold 0.32 M of vasopressin on inositol lipids. This study provides evidence sucrose, 2 mM EDTA. The ganglia were then homogenized for the presence of functional vasopressin receptors in a rat in 1.0 ml of 50 mM Tris HCl (pH 7.4), 5 mM MgCl2. The sympathetic ganglion and thus suggests that vasopressin may membranes were separated by centrifugation for 10 min at play a role in peripheral autonomic function. 10,000 x g (Eppendorf5414). The sediment was resuspended in 1.2 ml of the same medium. To eliminate the residual The neuropeptides arginine vasopressin (AVP) and oxytocin connective tissue the suspension was filtered through a (OT) are present not only in the hypothalamoneuro-hypo- stainless steel grid (200 Am, side length). This final suspen- physial system but also in neural pathways that project to sion, containing about 1 mg of protein per ml, was used for various areas of the central nervous system (1-3). Vasopres- the binding studies. All experiments were performed using sin- and oxytocin-containing axon terminals are found in freshly prepared membranes. particular in the proximity of cell clusters believed to par- Binding of [3H]AVP was measured at 30'C for 20 min ticipate in autonomic functions, such as the nucleus of the according to Audigier and Barberis (17). solitary tract-dorsal motor nucleus of the vagus complex, as Autoradiography. Superior cervical ganglion and brain well as the cell bodies of sympathetic preganglionic neurones were dissected from adult male rats (300-350 g) of Sprague- in the intermediolateral column of the spinal cord (4, 5). Dawley-derived stock (SIVZ) under sodium pentobarbital Electrophysiological studies have demonstrated that AVP anesthesia (5 mg/100 g of body weight, intraperitoneally), and OT affect neuronal activity in these regions (3, 6, 7). In and autoradiographs were obtained according to a method addition, the peptides are releasable by a calcium-dependent adapted from Van Leeuwen and Wolters (18). Organs were mechanism (8, 9). These findings have prompted an interest immediately frozen in isopentane (2-methyl butane) at - 30'C in the possible role of AVP and OT as centrally acting and cryostat sections (15 Aum thick) were cut, collected on modulators of autonomic functions. chrome/alum/gelatin-coated slides and dried overnight in a Hanley et al. (10) detected the presence of AVP-like dessicator at +40C. They were preincubated at room tem- immunoreactivity in the rat superior cervical ganglion. In perature for 20 min by dipping the slides in 50 mM Tris HCl view of previous reports showing that substance P (11, 12), (pH 7.4) containing 100 mM NaCl and 50 'M guanosine enkephalin (13), and other peptides (14) may act directly on 5'-triphosphate, then rinsed twice for 5 min in 50 mM the excitability of the ganglionic neurones and/or exert Tris HCl alone. Each section was covered with 200 A.l of the presynaptic actions in sympathetic ganglia by reducing the incubation medium [50 mM Tris HCl 0.1 mM amount of acetylcholine released from axon terminals (13), it (pH 7.4), was of importance to assess whether AVP and OT might affect neurotransmission in the Abbreviations: AVP, arginine vasopressin; f-EPSP, fast excitatory superior cervical ganglion. postsynaptic potential; OT, oxytocin; [Phe2,Orn8]VT, [2-phenylal- In the present study we first explore the presence of anine,8-ornithine]; [Thr?,Gly7]OT, [4-threonine,7-glycine]- binding sites for AVP in the ganglion. We then characterize oxytocin; HO-[Thr4,Gly7]OT, [1-(L-2-hydroxy-3-mercaptopropionic these sites as belonging to the smooth muscle-type (V1) acid,4-threonine,7-glycine]oxytocin; dDAVP,1-deamino[8-D-argi- nine]vasopressin; d(CH2)5Tyr(Et)VAVP, [1-(.3-mercapto-,4,,8-cyclo- pentamethylene propionic acid,2-0-ethyltyrosine,4-valine]vas- The publication costs of this article were defrayed in part by page charge opressin; dTyr(Me)VDAVP, [1-(/3-mercaptopropionic acid),2-0- payment. This article must therefore be hereby marked "advertisement" methyltyrosine,4-valine,8-D-arginine]vasopressin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom all correspondence should be addressed. 5335 Downloaded by guest on September 23, 2021 5336 Neurobiology: Kiraly et al. Proc. Natl. Acad. Sci. USA 83 (1986)

bacitracin, 3 mM MgCl2, and 0.1% bovine serum albumin] [3H][Phe3]arginine vasopressin (40-60 Ci/mmol; 1 Ci = 37 containing [3H]AVP (1.5 nM) or [3H]OT (3.5 nM) either alone GBq; New England Nuclear) and [3H][Tyr2]oxytocin (34.4 or with 10 ,M of the same unlabeled hormone to determine Ci/mmol; New England Nuclear) were purified by HPLC and the nonspecific binding. Structural analogues were also used affinity chromatography on a neurophysin-Sepharose col- at various concentrations as competing agents. umn as described (17). myo-2-[3H]Inositol (16.5 Ci/mmol) Incubation was carried out for 1 hr at room temperature in a was obtained from New England Nuclear. humid chamber followed by two 5-min washes in ice-cold incubation medium. The slides were dried with cold air, placed RESULTS in a vacuum dessicator with paraformaldehyde at 800C for 2 hr, then placed in an x-ray cassette in contact with tritium-sensitive [3H]AVP Binding. Concentration-dependent specific bind- LKB Ultrofilm for 4 months at 40C. The film was developed in ing at equilibrium was determined on membranes obtained D-19 for S min, and the sections were stained with cresyl violet. from rat superior cervical ganglia. The results of four inde- The autoradiographic images were analyzed semi-quanti- pendent experiments-one of which is illustrated in Fig. 1- tatively using computerized densitometry. The results are indicate that [3H]AVP bound to an apparently homogeneous expressed in fmol of [3H]AVP/mg of protein (19). population of specific binding sites. The estimated equilibri- AnaIysis of Labeled Inositol Phospholipids. Desheathed um dissociation constant was 1.5 nM, a value that is similar ganglia from 25 rats were incubated for 2 hr at 370C in a to that determined for the AVP binding sites characterized in shaking incubator, in 6 ml of Krebs solution (NaCl, 125 mM; the rat hippocampus (17, 24). A mean maximal binding KCl, 3.5 mM; KH2PO4, 1.25 mM; MgSO4, 1.2 mM; CaCl2, capacity of 196 fmol/mg of protein was found, a value much 0.75 mM; NaHCO3, 25 mM; glucose, 10 mM) containing 2 higher than the 39 fmol/mg ofprotein previously observed for liM myo-2-[3H]inositol, gassed with 95% 02/5% CO2. The hippocampal membranes. ganglia were then rinsed with the same medium containing no The detected vasopressin binding sites exhibited a high inositol. Each ganglion was transferred to a tube containing degree of specificity. Unlabeled structural analogues com- 230 Al of prewarmed, oxygenated Krebs solution. After 15 peted for [ H]AVP binding to almost the same maximal min, 10 mM LiCl was added to inhibit the hydrolysis of extent but exhibited marked differences in efficiency (Fig. 1). inositol phosphates (20); 20 min later a peptide was added, Thus the selective oxytocic [Thr4,Gly7]OT and the and incubation usually was continued for another 20 min. selective agonist dDAVP inhibited [3HJATP Incubations were terminated by adding 12.5 ml of 70% binding with low affinities (Ki = 3.7 ,uM and 1.0 8vM, (wt/vol) HClO4. Inositol phosphates were separated using a respectively), while the vasopressor agonist [Phe2,0m ]VT small column of Dowex 1-xlO (100-200 mesh, formate form) had a high affinity (Ki = 12.3 nM). The antagonist as described (21). Total lipids were extracted from the d(CH2)5Tyr(Et)VAVP inhibited [3H]AVP binding with an acid-treated ganglia with chloroform/methanol/12 M HCl even higher affinity (Ki = 1.7 nM). (100:200:1, vol/vol). The solvent was evaporated; [3H]ino- Autoradiography. AVP binding was found throughout the sitol cpm were determined by scintillation counting, and the superior cervical ganglion (Fig. 2), but its distribution ap- percentage incorporation of [3H]inositol into total lipids was peared patchy. Comparison ofenlarged autoradiographs with calculated. the corresponding cresyl violet-stained sections showed that Electrophysiological Recordings. The superior cervical gan- the most intensely labeled areas corresponded to densely glion along with its preganglionic and postganglionic nerves packed neuronal cell groups, while little or no specific was isolated from male Wistar rats (100-200 g) under ure- labeling was observed over fiber tracts. In the most intensely thane anesthesia. The ganglion was mounted in a recording labeled areas a mean binding capacity of 530 fmol/mg of chamber and was continuously superfused with prewarmed protein and 655 fmol/mg ofprotein was found in two separate (33TC) Krebs solution (NaCl, 117.0 mM; KCl, 4.7 mM; experiments. These values are higher than the maximal MgCl2, 1.2 mM; NaH2PO4, 1.2 mM; CaCl2, 25.0 mM; binding capacity of crude membrane preparations obtained NaHCO3, 25.0 mM, glucose; 11.5 mM) equilibrated with 95% from the total ganglion (see above). This difference may be 02/5% C02. The volume of the chamber was adjusted to due to a difference in the strains used in Geneva and 0.5-0.8 ml and the solution flow rate to 2 ml/min. Montpellier, respectively, or more likely, to the fact that, Ganglion cells were impaled with glass microelectrodes following autoradiography, quantitative measurements were filled with 2 M potassium acetate. Tip resistance of the performed only over markedly labeled areas; in contrast, electrodes was between 30 and 80 MQl. Membrane potentials crude membrane preparations probably included membranes were recorded intracellularly by using a high-input-imped- ance amplifier (WPI M701), displayed on a digital storage Table 1. Biological activities of AVP, OT, and analogues oscilloscope (Gould 2040), and traced with a pen recorder Relative biological activity* (Gould 2200). Electrical stimuli were applied to the pregan- glionic nerve at a rate of 0.5 Hz using a suction electrode. Compound Antidiuretict Vasopressort Oxytocict Substances were applied by superfusion. AVP 100.0 100.0 5.3 Peptides and Other Substances Used. AVP and OT were [Phe2,Orn]VT 0.5 33.0 1.0 purchased from Bachem Fine Chemicals (Bubendorf, Swit- dDAVP 371.5 0.001 0.1 zerland). The vasopressin structural analogues: [2-phenylal- dTyr(Me)VDAVP 619.0 Antagonist§ Antagonist anine,8-ornithine]vasotocin, [Phe2,0m8]VT; [4-threonine,7- d(CH2)5Tyr(Et)VAVP Antagonistll Antagonistl Antagonist glycine]oxytocin, [Thr4,Gly7]OT; [1-(L-2-hydroxy-3-mercap- OT 1.2 1.1 100.0 topropionic acid,4-threonine,7-glycine)]-oxytocin, HO- [Thr4,GIyI'OT 0.002 <0.001 176.3 [Thr4,Gly7]OT; 1-deamino[8-D-arginine]vasopressin (d- HO-[Thr4,Gly7IOT 0.001 <0.001 206.2 DAVP); [1-(I3-mercapto-3,B3-cyclopentamethylene propionic *From Sawyer et al. (22) and Manning et al. (23). acid), 2-O-ethyltyrosine,4-valine]vasopressin, d(CH2)5Tyr- tPercent of AVP taken as standard. (Et)VAVP; and [1-(,fmercaptopropionic acid), 2-0-methyl- tPercent of OT taken as standard. tyrosine,4-valine,8-D-arginine]vasopressin, dTyr(Me)- §pA2 = 7.01 (pA2 is the negative logarithm of A2, the concentration of antagonist reducing the response to vasopressin by 50%6 of that VDAVP, were a gift of M. Manning, Medical College ofOhio obtained in the absence of the antagonist). (Toledo, OH). The relative biological activities of these IpA2 = 7.57. compounds are given in Table 1. IpA2 = 8.16. Downloaded by guest on September 23, 2021 Neurobiology: Kiraly et al. Proc. Natl. Acad. Sci. USA 83 (1986) 5337

a) - CX c0E0, :3 02

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50 100 150 200 -10 -9 -8 -7 -6 -5 [3H]AVP bound, fmol/mg of protein log [unlabeled peptide] FIG. 1. Specific ['H]AVP binding to crude membranes from superior cervical ganglia. (Left) Scatchard plot of concentration-dependent binding. Each point represents the mean of triplicate determinations of one typical experiment. Linear regression analysis was performed to estimate the dissociation constant and maximal binding capacity. (Right) Displacement of ['H]AVP binding by unlabeled structural analogues. Membranes were incubated in the presence ofa constant amount of [3H]AVP (1 nM) and various amounts ofunlabeled peptides. Specific binding was measured in the presence (B) as well as in the absence (BO) of the unlabeled peptide. Ordinate shows the ratio B/Bo for different peptides at the concentrations indicated. Each point represents the mean of triplicate determinations. x, d(CH2)5Tyr(Et)VAVP; *, [Phe2,Orn8]VT; A, dDAVP; m, [Thr?,Gly']OT. from regions devoid of AVP binding sites. The values found posed to 100 nM AVP, and it was suppressed by the AVP in the ganglion were also higher than those seen in the brain. antagonist d(CH2)5Tyr(Et)VAVP. By contrast the specific In these same animals, the septal area was the most intensely oxytocic agonist [Thr4,Gly7]OT produced no significant labeled brain structure by autoradiography and had a mean change in inositol phosphate accumulation. binding capacity of 229 fmol/mg of protein, whereas in the Electrophysiological Effect ofAVP on Ganglion Cells. When hippocampus binding amounted to 94 fmol/mg of protein. superfused for 1 min at 10-5 or 10-6 M, AVP produced a Using [3H]OT, no binding sites were detected in the ganglion. gradual membrane depolarization in 50% of the 60 cells The [3H]AVP binding was inhibited by the vasopressor tested, averaging 4.2 ± 0.9 mV (n = 22) and 2.2 ± 0.8 mV agonist [Phe2,0M8]VT, while neither the potent antidiuretic (n = 8), respectively. In the remaining cells AVP did not agonist nor the selective oxytocic agonist displaced AVP affect the membrane potential. In seven out of nine cells a binding (Fig. 2). With 100 nM [Phe2,0M8]VT, the displace- moderate increase in membrane resistance was induced by ment was almost 60%. In contrast, neither 100 nM dTyr(Me)- AVP 10-5 M; no change in input resistance could be detected VDAVP nor 150 nM HO-[Thr4,Gly7]OT inhibited [3H]AVP in the remaining two cells. Neither the amplitude nor the time binding significantly (Fig. 3). course of the action potential elicited by direct intracellular Accumulation of Inositol Phosphates. Incubation of rat current injection were significantly altered in the presence of superior cervical ganglia for 20 min with 10 nM AVP caused vasopressin. an appreciable accumulation oflabeled inositol phosphates in Effects of AVP on Evoked Synaptic Potentials. AVP was the tissue (Table 2), confirming previous results (21). This applied to neurons in which action potential initiation was accumulation was even prevented either by hyperpolarizing the membrane potential greater when the ganglia were ex- or by adjusting the stimulus intensity such that the f-EPSP A D was subthreshold and fairly uniform in amplitude. AVP superfused for 1 min at concentrations ranging from 5 x 10-8 to 10-5 M reduced the amplitude of the f-EPSP in 63 out of 130 cells tested; this effect was fully reversible within 5-30

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z 10 100 nM 10 100 nM 20 150 nM FIG. 2. [3H]AVP binding in sections of superior cervical ganglia. [Phe2,Orn8]VT dTyr(Me)VDAVP [Thr4,Gly7]OT A-C are autoradiograms obtained from one ganglion; D-F were obtained from a different animal. A and D show the labeling in FIG. 3. Displacement of [3H]AVP bound in superior cervical sections incubated with 1.5 nM [3H]AVP; most of this labeling was ganglion by structural AVP analogues. The results obtained by inhibited by 10 AuM unlabeled AVP (B). The oxytocin agonist autoradiography (see Fig. 2) were measured by densitometry and are HO-[Thr4,Gly']OT (150 nM, C) and the antidiuretic agonist expressed in percent of the maximal specific binding. Note that only dTyr(Me)VDAVP (100 nM, E) failed to displace [3H]AVP while the the vasopressor agonist [Phe2,Orn8]VT significantly displaced the vasopressor agonist [Phe2,Orn8]VT (100 nM, F) did. (Bar = 0.6 mm.) bound [3H]AVP. Downloaded by guest on September 23, 2021 5338 Neurobiology: Kiraly et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 2. Accumulation of inositol phosphates in ganglia exposed and by labeling ofcrude membrane preparations. In addition, to AVP and structural analogues it was observed that the ganglion possesses functional AVP [3H]Inositol receptors-i.e., capable of activating inositol lipid turnover phosphates and of affecting ganglionic transmission of impulses. In Compound Concentration (dpm/102 dpm) n contrast, OT neither bound nor exerted any effect at com- concentrations. Control 4.3 ± 0.9 10 parable AVP 10 nM 9.0 ± 1.9 3 The binding experiments and the results of competition AVP 100 nM 19.8 ± 2.6 3 studies using AVP structural analogues revealed a single AVP + l0 class of binding sites characterized by high affinity for AVP nM40t04 3 d(CH2)5Tyr(Et)VAVP 1 MM 4.0 ± 0.4 3 and low affinity for OT. AVP binding was displaced with low d(CH2)5Tyr(Et)VAVP 1 MM 3.8 ± 0.1 3 concentrations of a vasopressor agonist and a vasopressor [Thr4,Gly7]OT 10 UM 4.3 ± 0.1 3 antagonist, but not of an antidiuretic agonist. It was thus concluded that the AVP receptors present in the cervical Isolated ganglia were labeled with [3H]inositol. After a 20-min incubation with 10 mM LiCl ganglia were incubated in the presence superior ganglion are ofthe V1 type characteristic ofvascular ofthe peptides for 20 min. Values represent means ± SD (n = number smooth muscle cells and of hepatocytes (15, 16). The maxi- of ganglia analyzed) of the combined inositol phosphates expressed mal binding capacity of ganglionic membranes was much relative to an incorporation of 102 dpm into total ganglionic lipids. higher than that which we had found for membranes obtained from the central nervous system (17). min after washout (Fig. 4A). The concentration-response The presence of high-affinity binding sites is consistent relationship is shown in Fig. 4B. Under these conditions, the with the view that AVP affects cells located in the superior minimum effective concentration of AVP was 5 x 10-8 M. cervical ganglion. This hypothesis is supported by the fact When applied for longer periods (5-10 min), AVP produced that AVP activated the inositol lipid turnover in the ganglion. a complete blockade ofthe f-EPSPs even at concentrations as The results shown in Table 2 furthermore identify these low as 10-8 M. OT depressed the f-EPSP amplitude only in receptors as being of the V1 type. Even a most potent and 5% of the cells tested and less powerfully than AVP. selective OT agonist had no effect on the turnover ofinositol To examine the specificity of AVP action, we applied the phosphates (Table 2). same vasopressin antagonist, d(CH2)5Tyr(Et)VAVP, as was The idea that functional and selective vasopressin recep- used in the binding study. Superfused at 1 4M, this com- tors are present in the ganglion is also supported by the pound did not by itself affect the amplitude of f-EPSPs, but observation that AVP affected ganglionic transmission. At effectively antagonized the depressing effect ofAVP (Fig. 5). high concentration it caused membrane depolarization, as AVP decreased neither the amplitude nor the time course of described (25, 26); at lower concentrations, AVP depressed the postsynaptic depolarization produced by superfusion of synaptic It did acetylcholine, thus suggesting a presynaptic site for the AVP transmission. so by reducing the amplitude of action. the f-EPSPs without producing a noticeable change in either resting membrane potential or in membrane resistance. The fact that OT was much less potent than AVP, and the DISCUSSION observation that the depressing effect of AVP was antago- In the present study binding of [3H]AVP in the superior nized by d(CH2)5Tyr(Et)-VAVP (Fig. 5), are in full agreement cervical ganglion of the rat was shown by autoradiography with the results obtained by [3H]AVP binding (Fig. 1). Control AVP 1 uM Wash A i_+

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AVP concentration, M FIG. 4. (A) Effect of 1 uM vasopressin (1 min application) on the amplitude off-EPSPs elicited in response to stimulation ofthe preganglionic nerve (arrows indicate stimulation artifacts). Four traces of consecutives f-EPSPs recorded from a rat superior cervical cell are shown. There was full recovery 10 min after washout with AVP-free Krebs solution. (B) Concentration-response curve showing the reduction of f-EPSPs amplitude in response to AVP. The reduction was calculated by averaging f-EPSP amplitudes during a 1-min control period and a 1-min period at the height ofthe peptide effect. Each point represents the mean, the vertical bars the standard errors ofthe mean, and the values in parentheses the number of cells tested. Downloaded by guest on September 23, 2021 Neurobiology: Kiraly et al. Proc. Natl. Acad. Sci. USA 83 (1986) 5339

Control AVP 10 AM Wash

4 1 FIG. 5. Effect of AVP and of an AVP antagonist on f-EPSPs in response to pre- ANT 10-6M AVP 6M +ANT 10-M Wash recorded ganglionic stimulation on a single ganglionic neurone. Three consec- utive traces are shown. Arrows indicate stimulation artifacts. (Up- per) Reversible decrease in f- EPSP amplitude in response to the application of vasopressin, 1 ,AM. (Lower) The antagonist d(CH2)5- Tyr(Et)VAVP had no effect per se I5 mV but effectively antagonized the ac- 5 ms tion of AVP. The question then arises how endogenous vasopressin 3. Muhlethaler, M., Raggenbass, M. & Dreifuss, J. J. (1985) in could reach the receptors present in the ganglion. At least Neurotransmitter Actions in the Vertebrate Nervous System, three possibilities might be considered: (i) release from axon eds. Rogawski, M. A. & Barker, J. L. (Plenum, New York), terminals of extraganglionic origin, (ii) diffusion from the pp. 439-458. is 4. Swanson, L. W. & McKellar, S. (1979) J. Comp. Neurol. 188, bloodstream, and (iii) local synthesis. A central origin 87-106. unlikely, since injection of large amounts of True Blue into 5. Sawchenko, P. E. & Swanson, L. W. (1982) J. Comp. Neurol. the ganglion did not retrogradely label central vasopres- 205, 260-272. sinergic neurones (E.T., unpublished observations). An al- 6. Gilbey, M. P., Coote, J. H., Fleetwood-Walker, S. & ternative source of vasopressin in the ganglion might be Peterson, D. F. (1982) Brain Res. 251, 283-296. provided by axons ofprimary sensory neurones (27), similar 7. Charpak, S., Armstrong, W. E., Muhlethaler, M. & Dreifuss, to the substance P innervation of the inferior mesenteric J. J. (1984) Brain Res. 300, 83-89. ganglion of the guinea pig (11, 28). Regarding the second 8. Buijs, R. M. & van Heerikhuize, J. J. (1982) Brain Res. 252, possibility, the existence of a blood/ganglion barrier (29) 71-76. its 9. Buijs, R. M. (1983) Pharmacol. Ther. 22, 127-141. does not support the view that vasopressin could reach 10. Hanley, M. R., Benton, H. P., Lightman, S. L., Todd, K., ganglionic receptors by diffusion from the circulation. In Bone, E., Fretten, P., Palmer, S., Kirk, C. J. & Michell, R. H. support of the third possibility is the report showing by (1984) Nature (London) 309, 258-261. immunocytochemistry that a vasopressin-like compound is 11. Tsunoo, A., Konishi, S. & Otsuka, M. (1982) Neuroscience 7, localized with noradrenaline in neurones of the rat superior 2025-2037. cervical ganglion (10). However, this compound apparently 12. Dun, N. J. & Kiraly, M. (1983) J. Physiol. (London) 340, possesses a larger molecular weight than vasopressin. 107-120. Regardless of the origin and nature of the endogenous 13. Konishi, S., Tsunoo, A. & Otsuka, M. (1979) Nature (London) compound, our results clearly show the presence in a 282, 515-516. sympathetic ganglion of authentic and functionally active 14. Dun, N. J. (1983) in Autonomic Ganglia, ed. Elfin, L. G. (Wiley, Chichester), pp. 345-366. receptors. Previous observations using immuno- vasopressin 15. Michell, R. H., Kirk, C. J. & Billah, M. M. (1979) Biochem. cytochemical and electrophysiological approaches suggest Soc. Trans. 7, 86-89. that AVP and OT could modulate the activity of the auto- 16. Jard, S. (1983) in Current Topics in Membranes and Transport, nomic nervous system by acting onto its centrally located Vol. 18: Membrane Receptors, ed. Kleinzeller, A. (Academic, neurones (4-7). The present study extends these findings by New York), pp. 255-285. showing that neurones in a sympathetic ganglion probably 17. Audigier, S. & Barberis, C. (1985) EMBO J. 4, 1407-1412. represent an additional target for AVP action. AVP may, 18. Van Leeuwen, F. M. & Wolters, P. (1983) Neurosci. Lett. 41, therefore, participate in the control ofautonomic functions at 61-66. 19. Rostene, W. H., Fischette, C. T., Rainbow, T. C. & McEwen, both central and peripheral levels. B. S. (1983) Neurosci. Lett. 37, 143-148. 20. Berridge, M. J., Downes, C. P. & Hanley, M. R. (1982) This work was supported in part by Grants 3.318.082 and Biochem. J. 206, 587-595. 3.560.0.83 from the Swiss National Science Foundation, ATP 21. Bone, E. A., Fretten, P., Palmer, S., Kirk, C. J. & Michel, 953.134 from the Centre National de la Recherche Scientifique, U264 R. H. (1984) Biochem. J. 221, 803-811. from the Institut National de la Sante et de la Recherche Mddicale, 22. Sawyer, W. H., Grzonka, Z. & Manning, M. (1981) Mol. Cell. and TW 84.308 from the European Training Programme in Brain and Endocrinol. 22, 117-134. Behaviour Research. We thank Ms. M. Maillard and A. Marguerat 23. Manning, M., Lammek, B., Kolodziejczyk, A. M., Seto, J. & for excellent technical assistance, Dr. W. E. Rostene for access to Sawyer, W. H. (1981) J. Med. Biol. 24, 701-706. his densitometer, and Dr. M. Manning for the gift of peptides. 24. Barberis, C. (1983) FEBS Lett. 162, 400-405. 25. Kiraly, M., Maillard, M., Dreifuss, J. J. & Dolivo, M. (1985) Neurosci. Lett. 62, 89-95. 1. Sofroniew, M. V. (1985) in Handbook ofChemical Neuroanat- 26. Peters, S. & Kreulen, D. L. (1985) Brain Res. 339, 126-129. omy, Vol. 4: GABA and Neuropeptides in the CNS, eds. 27. Kai-Kai, M. A., Swann, R. W. & Keen, P. (1985) Neurosci. Bjorklund, A. & Hbkfelt, T. (Elsevier, Amsterdam), pp. Lett. 55, 83-88. 93-165. 28. Jiang, Z., Dun, N. J. & Karczmar, A. G. (1982) Science 217, 2. De Vries, G. J., Buijs, R. M., van Leeuwen, F. W., Caffe, 739-741. A. R. & Swaab, D. F. (1985) J. Comp. Neurol. 233, 236-254. 29. Depace, D. N. (1982) Anat. Rec. 204, 357-363. Downloaded by guest on September 23, 2021