Brain Research, 575 (1992) 124-131 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
124 BRES 17537
Morphological and electrophysiological evidence for postsynaptic localization of functional oxytocin receptors in the rat dorsal motor nucleus of the vagus nerve M. Dubois-Dauphin, M. Raggenbass, H. Widmer, E. Tribollet and J.J. Dreifuss Department of Physiology, University Medical Centre, Geneva (Switzerland) (Accepted 29 October 1991)
Key words: Electrophysiology; Neuropeptide; Receptor autoradiography; Solitary-vagal complex; Vagotomy
The vagal complex is innervated by oxytocin immunoreactive axons of hypothalamic origin. The presence of oxytocin binding sites in the dorsal motor nucleus of the vagus nerve of the rat was evidenced by autoradiography with a radioiodinated oxytocin antagonist as ligand. Two weeks following a unilateral vagotomy, distal to the nodose ganglion, binding sites were reduced below the level of detection in the ipsilateral dorsal motor nucleus of the vagus nerve. Choline acetyltransferase immunoreactivity was also markedly reduced in the vagal motoneurons whose axons had been transected. Electrophysiological studies were performed in vitro in brainstem slices from control rats. In antidromically identified vagal motoneurones, oxytocin applied at 0.1-1.0 #M either caused a reversible depolarization or generated, under voltage-clamp conditions, a transient inward current. These responses persisted under the condition of synaptic uncoupling. Taken together these observations favour the notion that oxytocin of hypothalamic origin acts directly on rat vagal motoneurones. INTRODUCTION The dorsal vagal complex, formed of the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve, receives axons originating from cell bodies located in the hypothalamic paraventricular nucleus2°, in particular from vasopressin and oxytocin immunoreactive cell bodies 13'3°'32. The nucleus of the solitary tract
tocin binding sites in the dorsal motor nucleus of the vagus nerve, we investigated the effect of a unilateral vagotomy, distal to the nodose ganglion, on the distribution of oxytocin binding sites in this nucleus using in vitro light microscopic autoradiography. In parallel, the sensitivity to oxytocin of antidromically identified vagal motoneurones was tested by intracellular recordings on brainstem slices from intact rats. The results of both the
receives sensory vagal afferents, but does not send efferent axons into the vagus nerve. The rat dorsal motor nucleus of the vagus nerve contains mainly preganglionic neurones whose axons terminate in the parasympathetic ganglia of thoracic and abdominal viscera 3'12'17'23'27.
morphological and the electrophysiological studies suggest that vagal m o t o n e u r o n e s possess functional oxytocin receptors and, therefore, favours the view that oxytocin acts directly on these neurones.
A physiological role of oxytocin in the vagal complex is well documented. Thus, local infusion of oxytocin affects gastric acid secretion 21'28. This effect was mimicked
MATERIALS AND METHODS
by electrical stimuli applied to the hypothalamic paraventricular nucleus; infusion of a specific oxytocin antagonist into the dorsal motor nucleus of the vagus nerve blocked the effect of the hypothalamic stimulation29. Electrophysiological studies carried out on rat brainstem slices showed that vagal m o t o n e u r o n e s are sensitive to oxytocin 5'2s. In addition, specific binding sites for oxytocin were uncovered in the dorsal motor nucleus of the vagus nerve 7'9'34'35. To assess the pre- or postsynaptic localization of oxy-
Unilateral vagotomy These experiments were conducted on 11 male rats from the Sivz strain, a Sprague-Dawley-derived strain, weighing 220-250 g. Eight animals were anesthetized with sodium pentobarbital (5 mg/100 g). The left or the right vagus nerve was transected distal to the nodose ganglion and approximately 1 cm of the nerve was removed. The proximal cut stump of the vagus nerve was then soaked in a 1-cm-long polythene tubing sealed with wax at one extremity and containing 0.9% NaCI. The tube around the nerve trunk was sealed with Vaseline and secured in the neck cavity with Histoacryl blue tissue adhesive (B. Braun Melsungen, Germany). ChAT immunocytochemistry The effect of the vagotomy on the choline acetyltransferase (CHAT) content of neurones in the dorsal motor nucleus of the va-
Correspondence: M. Dubois-Dauphin, Department of Physiology, University Medical Centre, 9 Avenue de Champel, 1211 Geneva 4, Switzerland. Tel.: 0041.22.22.91.10. Fax: (41) (22) 47.33.34.
125
Fig. 1. ChAT immunoreactivity 2 weeks after unilateral section of the vagus nerve. Note that immunoreactivity disappeared from the ipsilateral dorsal motor nucleus outlined by dotted lines at rostral (A), intermediate (B) and caudal levels (C). Bar: 150/~m. Abbreviations used in figures: AP, area postrema; IO, inferior olive; Sol, nucleus of the solitary tract; 4V, 4th ventricle; 10, dorsal motor nucleus of the vagus nerve; 12, hypoglossal nucleus.
126
Fig. 2. ChAT immunoreactivity in the vagal complex of a rat 2 weeks after unilateral vagotomy. The section was counterstained with cresyl violet to visualize the cell bodies. A: overview: the dorsal motor nucleus contralateral to the vagotomy and both hypoglossal nuclei contain ChAT immunoreactive neurones. The ipsilateral dorsal motor nucleus (on the right) although devoid of ChAT immunoreactivity shows normal number of cell bodies, shown at higher magnification in B (rectangular area outlined in A). Bar: (A) 200 ~m, (B) 85/~m.
gus nerve was assessed on three rats. They were reanesthetized and fixed by intracardiac perfusion with a 4% paraformaldehyde solution in phosphate buffered saline (PBS) pH 7.35. The brain was removed, postfixed for 1 h in the same fixative at 4°C, cryoprotected in a 30% sucrose/PBS solution at 4°C and 40-#m-thick brainstem sections were obtained. Floating sections were incubated for 1 h in normal horse serum, diluted 1/50 in PBS containing 0.1 M L-lysine, then rinsed 3 × 10 min in PBS. Sections were incubated 16 h at 4°C, with mouse monoclonal antibodies raised against CHAT, at the concentration of 7/~g/ml (antibody IE6, a gift from Dr. P. Salvaterra, Beckman Research Institute, Duarte). Following new washes (3 x 10 min), the sections were reacted for 90 min with goat serum containing biotinylated antimouse antibody, diluted 1/100 (Vector Laboratories, Burlingame, CA). After further rinsing, they were incubated for 1 h with avidin-biotin-peroxidase complex. Peroxidase activity was revealed using 0.05% diaminobenzidine in 0.1 M phosphate buffer. Following a 10 min incubation step, 0.013% H20 2 was added and the incubation continued for 10 min. After washing in PBS and rapid rinsing in distilled water, the sections were mounted on chromalum-gelatin coated slides.
Autoradiography Two weeks after unilateral vagotomy, five animals were reanesthetized, decapitated, their brain removed and frozen at -25°C in isopentan. Sixteen-/xm-thick coronal sections of the brainstem were cut in a cryostat, laid on chromalum-gelatin coated slides, air-dried and processed for light microscopic autoradiography. Three unoperated rats were used as controls. Oxytocin binding sites were detected using 125I-labelled d(CH2) 5[Tyr(Me)2,Thr4,Tyr(NH2)9]ornithine vasotocin, ([125I]OTA), as ligands. The compound was synthesized and kindly provided by Dr. M. Manning and was monoiodinated with 125I-labelled Na to a specific activity of about 2000 Ci/mmol. Slides were dipped in 0.2% paraformaldehyde in PBS (pH 7.4), then rinsed twice in 50 mM Tris-HCl (pH 7.4). Incubation was carried out for 1 h at room temperature in a humid chamber by covering each slide with 400 /tl of the incubation medium (50 mM Tris-HC1, 0.1 mM bacitracine, 5 mM MgC12, 0.1% bovine serum albumin) containing 0.05 nM [125I]OTA. Non-specific binding was evaluated by incubating adjacent sections with medium containing in addition 1 ,aM nonradioactive oxytocin. Incubation was followed by 2 × 5 min washes in ice-cold incubation medium and a quick rinse in distilled water.
127 The slides were rapidly dried and apposed to fl-Amersham film for 3-5 days. Alternate sections were processed similarly to detect vasopressin binding sites using 1.5 nM [3H]vasopressin as ligand (Du Pont New England Nuclear, Boston, MA) in the presence of 5 nM of nonradioactive OH[Thr4,Gly7]oxytocin. The latter compound is a selective oxytocin agonist22 and was added to prevent binding of [3H]vasopressin to oxytocin receptors. Non-specific labelling was assessed in adjacent sections treated in the same conditions, except that the incubation medium contained in addition 10 t~M non-radioactive arginin-vasopressin. Sections were then placed in contact with a tritium sensitive film (LKB Ultrofilm) in an X-ray cassette for 3 months at 4°C. Films were developed in Kodak D-19, and the sections were stained with cresyl violet.
In all c o n t r o l animals,
[I25I]OTA b i n d i n g was ob-
s e r v e d bilaterally in the dorsal m o t o r nucleus of the vagus n e r v e and in the i n f e r i o r olive (Fig. 3 A ) . A u t o r a d i o g r a p h i c labelling was d e t e c t e d in the a n t e r i o r and m i d d l e p a r t of the dorsal m o t o r nucleus b u t n o t in its caudal, cervical, aspect.
[3H]Vasopressin b i n d i n g sites
w e r e d e t e c t e d in a r e a p o s t r e m a , in the nucleus of the solitary tract and in the i n f e r i o r olive.
Electrophysiology The animals used were 32 adult male rats of the same strain as used for the morphological studies, weighing 200-300 g. Following decapitation, the brain was gently removed and a block of brainstem containing the dorsal medulla was prepared. Coronal brainstem slices, 300 to 400-~m-thick, were cut using a vibratome (Campden Instruments, London, UK) and incubated in a thermoregulated (33.5-34.5°C) recording chamber at the interface between a perfusion solution and an oxygenated, humidified atmosphere. Unless otherwise stated, the composition of the perfusion solution was (in mM): NaC1 138, KCl 2, NaHCO 3 15, KH2PO 4 1.25, MgSO 4 1, CaC12 2, and glucose 10. This solution was saturated with 95% 02/5% CO 2 (pH 7.35-7.45). Intracellular recordings were obtained using glass micropipettes filled with 3 M potassium-acetate (pH 7.4; 50-100 MQ). Voltage and current signals were amplified using an Axoclamp-2A (Axon Instruments Inc., Foster City, CA), digitized and stored on the hard disk of an AT-386-type personal computer. In the single-electrode voltage-clamp mode, the switching frequency was 2-3 kHz; the headstage was continuously monitored. In order to activate antidromically vagal motoneurones, bipolar stimulation electrodes, made of twisted nichrome wires (100/~m in diameter, isolated except for their tips), were positioned ventrolaterally to the dorsal motor nucleus of the vagus nerve, along the course followed by the axons arising from this nucleus 12. Stimuli were constant current pulses (10-200/~A), delivered at 0.5-1 Hz. Antidromic invasion of the soma was considered to be successful if the following criteria were obeyed: (a) the evoked action potentials arose at fixed latency with respect to the stimulus artefact; (b) they followed the stimulation at high frequency; (c) they could dissociate into an axon initial segment component and a somatodendritic component. Synaptic uncoupling was achieved either by perfusing the preparation with a modified solution, containing reduced (0.1 mM) calcium and increased (16 mM) magnesium, or by adding tetrodotoxin (TI'X) at 1-2 ktM to the normal perfusion solution. Oxytocin was obtained from Bachem (Bubendorf, Switzerland) and TI'X was from Sigma (St. Louis, MO).
RESULTS
ChAT immunoreactivity and receptor autoradiography T w o w e e k s after u n i l a t e r a l v a g o t o m y , C h A T i m m u n o r e a c t i v e cell b o d i e s w e r e p r e s e n t in t h e facial and perihypoglossal nuclei, a n d in the dorsal m o t o r n u c l e u s cont r a l a t e r a l to the v a g o t o m y in all t h r e e animals studied. In c o n t r a s t , t h e ipsilateral n u c l e u s was d e v o i d of C h A T i m m u n o r e a c t i v i t y a l o n g its e n t i r e r o s t r o c a u d a l e x t e n s i o n (Fig. 1). H o w e v e r , cresyl v i o l e t stained cell b o d i e s w e r e clearly visible in t h e ipsilateral as well as in t h e c o n t r a lateral dorsal m o t o r nucleus (Fig. 2).
Fig. 3. Autoradiograms of oxytocin and vasopressin binding sites in an intact rat (A) and in a rat 2 weeks after unilateral vagotomy (B,C). A: [a25I]OTA binding sites are detected bilaterally in the dorsal motor nucleus (10) and in the inferior olive (IO) of a normal rat. B: in a vagotomized animal [lzSI]OTA binding disappeared from the dorsal motor nucleus ipsilateral to the vagotomy but is present in the contralateral dorsal motor nucleus and in both inferior olives. C: [3H]vasopressin binding in area postrema (AP), in the nucleus of the solitary tract (Sol) and in the inferior olive of the same vagotomized rat. This autoradiogram was obtained from a section 150 #m caudal to the section which generated the autoradiogram shown in B. The specificity of the binding was ascertained by its displacement with an excess of non-radioactive oxytocin or non-radioactive vasopressin (not shown). Bar: 1 mm.
128
Fig. 4. Cresyl violet staining of sections from which autoradiograms (A) and (B) in Fig. 3 were obtained. A is from the control and B from the vagotomized animal. Bar: 300 ~m.
Two weeks after unilateral vagotomy, [125I]OTAbinding was symmetrical in the inferior olive (Fig. 3B), as was [3H]vasopressin binding in the nucleus of the solitary tract (Fig. 3C). In contrast, in all 5 animals studied, [12SI]OTA binding was no longer detectable in the dorsal motor nucleus ipsilateral to the vagus nerve transection (Fig. 3B). Cell bodies in the dorsal motor nucleus were as intensely stained with cresyl violet in the nucleus ipsilateral to the vagotomy as in the control animals (Fig. 4).
Electrophysiological study Intracellular recordings were obtained from 36 antidromically identified vagal motoneurones. They had resting membrane potentials ranging from -50 to -70 mV and were either silent or discharged spontaneously, at frequencies < 5 action potentials/s. Their average membrane resistance was 145 _ 15 Mf~ (mean _ S.E.M.; n = 21). Typically, these neurones were endowed with a transient, voltage-dependent potassium conductance of the A-type 37. Twenty-eight neurones (i.e. 78%) re-
129
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50 s
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B
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60 s
Fig. 5. Effect of oxytocin on two antidromically identified vagal motoneurones, A and B. A: left panel, three superimposed voltage traces, recorded under current-clamp conditions, showing the response of the neurone to pairs of shocks, delivered at 20, 30 and 40 ms intervals ventromedially to the dorsal motor nucleus of the vagus nerve. Stimulus artifacts are marked by asterisks. Note that the first action potential in each pair arose at fixed latency; note also that at 50 Hz, the action potential generated by the second stimulus invaded the axon initial segment but failed to propagate into the neuronal soma. Right panel: ratemeter records showing the oxytocin-induced increase in firing rate in the normal solution (top trace) and in a low-calcium, high-magnesium solution (bottom trace). The peptide was added to the perfusion solution at 1 #M for the time indicated by the horizontal line above each record. In the modified solution the neurone lost its spontaneous activity but still responded to oxytocin. B, left panel: three superimposed voltage traces recorded under current-clamp conditions and representing the neuronal response to a single shock. The action potential was due to antidromic invasion, since it tended to dissociate spontaneously into a low amplitude initial segment component and a high amplitude somatodendritic component. Note that in one case, the initial segment spike failed to invade the soma. Right panel: voltage-clamp records of the membrane current generated in the same neurone by oxytocin at 1 #M in the normal solution (top trace) and in a solution containing 1 #M T r x (bottom trace). Holding potential, -50 inV.
sponded to oxytocin dissolved in the perfusion solution at 0.1 or 1 pM. Under current-clamp conditions, oxytocin induced a reversible membrane depolarization, accompanied by an increase in firing frequency (n = 16; Fig. 5B, right panel, top trace). Under voltage-clamp conditions -- the cell membrane potential being held at or near its resting level -- oxytocin generated an inward current (n = 12; Fig. 5B, right panel, top trace). In order to determine whether the action of oxytocin was direct, the response to the peptide recorded in the normal solution was compared with that recorded in conditions of synaptic uncoupling. In four out of four current-clamped neurones, the oxytocin-induced depolarization and the increase in firing rate persisted in a lowcalcium, high-magnesium solution, although in the latter solution the neuronal excitability was reduced (Fig. 5A, right panel, bottom trace). In seven out of seven voltageclamped neurones, the inward current generated by oxytocin in the presence of TTX or in a low-calcium, highmagnesium solution was superimposable on that generated in control solutions (Fig. 5B, right panel, bot-
tom trace).
DISCUSSION
A pathway of oxytocin containing axons arises in the rat hypothalamic paraventricular nucleus 13'2°'a° and innervates the dorsal vagal complex, where synapses containing oxytocin have been detected by immunoelectron microscopy32'36. Oxytocin can be released in vitro from rat brainstem slices containing the dorsal vagal complex by a membrane depolarization in the presence of extracellular calcium4. Since oxytocin has been shown to excite vagal motoneurones in vitro, it has been conjectured that oxytocin may exert a direct, stimulatory effect on vagal motoneurones 7'25. By contrast, Siaud et al. 32 have postulated an indirect route, where oxytocin-containing hypothalamic axons project to and contact catecholaminergic interneurones in the rat medulla which, in turn, form synapses with vagal preganglionic motoneurones. In confirmation of earlier results, binding sites for oxytocin were detected in the dorsal motor nucleus of the vagus nerve (and in the inferior olive) using [125I]OTA, a selective oxytocin receptor ligand. Its high affinity and specific activity permit to visualize small numbers of oxytocin binding sites, which escape detection with tritiated o x y t o c i n 7'9'31'34'35.
130 Two weeks after transection of one vagus nerve, distally to the nodose ganglion, the ipsilateral dorsal motor nucleus showed signs of altered metabolic activity: a marked reduction of ChAT activity and of the density of oxytocin binding sites. The fact that these effects were due to the unilateral vagotomy is supported by the following arguments: (a) at two weeks, unilateral vagotomy had little effect on the number of cells present in the ipsilateral dorsal motor nucleus of the vagus nerve, based on cresyl violet staining of cells (see also refs. 1,14,15). (b) Unilateral vagotomy produced a reduction in ChAT activity which affected only the ipsilateral dorsal motor nucleus of the vagus nerve; the dorsal motor nucleus contralateral to the transected side showed normal ChAT immunoreactivity, as did the perihypoglossal nuclei on both sides (see also refs, 1,2,19). (c) Unilateral vagotomy reduced [12511OTA binding in the ipsilateral dorsal motor nucleus of the vagus nerve; the dorsal motor nucleus contralateral to the transected side showed normal radioligand binding, as did the inferior olives on both sides. This latter observation suggests strongly that binding sites for oxytocin are associated with vagal motoneurones and are down-regulated in chromatolytic neurones. Another control is provided by the fact that the density of vasopressin binding sites in area postrema and in the nucleus of the solitary tract was the same on left and right side in unilaterally vagotomized rats, as in intact rats. Since we performed the vagotomy distal to the nodose ganglion, we were able to achieve axotomy without causing afferent nerve degeneration lz. Nodose ganglionectomy, by lesioning the cell bodies of afferent vagal fibers would have resulted in the degeneration of axon terminals in the nucleus of the solitary tract and in the dorsal motor nucleus of the vagus nerve ~°. Actually, Phillips et al. 24 reported that nodose ganglionectomy reduced the binding of vasopressin in the medial subdivision of the ipsilateral nucleus of the solitary tract, suggesting that some vasopressin receptors are located on viscerosensory afferent axons coursing in the vagus nerve. However, other subdivisions of the nucleus of the solitary tract showed no loss of vasopressin binding after ganglionectomy and are therefore possibly located postsynaptically on solitary tract neurones 6'11'1s. The electrophysiological data show that oxytocin can directly excite neurones located in the region of the dorsal motor nucleus of the vagus nerve. This excitation is due to a peptide-induced inward membrane current, which is resistant to TTX and which is not synaptically mediated, since it persisted in a low-calcium, high-magnesium solution. The ionic basis of this current is presently under investigation in our laboratory.
The antidromically activated oxytocin-responsive neurones were vagal preganglionic motoneurones. Although some recordings could have been obtained from neurones located in the adjacent nucleus of the solitary tract, electrical stimulation of sensory axons in the vagus nerve would have lead, at best, to orthodromic activation of these neurones. Recordings could also have been obtained, occasionally, from hypoglossal motoneurones. However, the latter can be readily distinguished from vagal motoneurones since they are devoid of voltage-dependent A-type potassium channels16.Our electrophysiological results suggest that at least part of the [~zSI]OTA binding sites detected by autoradiography in the dorsal motor nucleus of the vagus nerve are functional receptors located on vagal motoneurones. This is in accordance with previous results showing that oxytocin excited in vitro brainstem neurones which, following intracelluiar staining with Lucifer yellow, could be identified as being vagal motoneurones on the basis of their location and morphology25. We have previously provided evidence that the vasopressin binding sites detected in the nucleus of the solitary tract are functional receptors affecting neuronal excitability 26. The question of the significance of the binding sites for neurohypophysial peptides present in the inferior olive has not been addressed so far. Since all raphe nuclei, including the raphe pallidus, which is located in the immediate vicinity of the inferior olive, contain vasopressin- and oxytocin-immunoreactive axons 33, the inferior olive may be another target for neurohypophysial peptides. In conclusion, the presence of an oxytocin immunoreactive innervation of the dorsal motor nucleus of the vagus nerve of hypothalamic origin, the presence of oxytocin binding sites in this nucleus, their disappearance from the nucleus ipsilateral to a unilateral vagotomy, the sensitivity to oxytocin of antidromically identified vagal motoneurones, all point to a direct effect of hypothalamic oxytocin on motoneurones of the dorsal motor nucleus of the vagus nerve. On the other hand, our results do not preclude an additional, indirect action via interneurones located in the nucleus of the solitary tract.
Acknowledgements. This work was supported in part by the Swiss National Foundation (Grant 31-28624.90). We thank Drs. M. Manning (Toledo, OH) for the gift of oxytocin structural analogs, S. Jard and C. Barberis (Montpellier, France) for radioiodinating OTA, P. Salvaterra for the gift of antiserum and Ms. A. Marguerat, M. Berti and D. Machard for technical assistance. Michel DuboisDauphin gratefully acknowledges receipt of a Career Development Award of the Prof. Dr. Max Clo~tta Foundation.
131 REFERENCES 1 Aldskogius, H., Barron, K.D. and Regal, R., Axon reaction in dorsal motor vagal and hypoglossal neurons of the adult rat. Light microscopy and RNA-cytochemistry, J. Comp. Neurol., 193 (1980) 165-177. 2 Aldskogius, H., Barron, K.D. and Regal, R., Axon reaction in dorsal motor vagal and hypoglossal neurons of the adult rat. Incorporation of [3H]-leucine, Exp. Neurol., 85 (1984) 139-151. 3 Altschuler, S.M., Ferenci, D.A., Lynn, R.B. and Miselis, R.R., Representation of the cecum in the lateral dorsal motor nucleus of the vagus nerve and commissural subnucleus of the nucleus tractus solitarii in rat, J. Comp. Neurol., 304 (1991) 261-274. 4 Buijs, R.M. and van Heerikhuize, J.J., Vasopressin and oxytocin release in the brain -- a synaptic event, Brain Res., 252 (1982) 71-76. 5 Charpak, S., Armstrong, W.E., M0hlethaler, M. and Dreifuss, J.J., Stimulatory action of oxytocin on neurones of the dorsal motor nucleus of the vagus nerve, Brain Res., 300 (1984) 8389. 6 Dashwood, M.R., Muddle, J.R. and Spyer, K.M., Opiate receptor subtypes in the nucleus tractus solitarii of the cat: the effect of vagal section, Eur. J. Pharmacol., 155 (1988) 85-92. 7 Dreifuss, J.J., Raggenbass, M., Charpak, S., Dubois-Dauphin, M. and Tribollet, E., A role of central oxytocin in autonomic functions: its action in the motor nucleus of the vagus nerve, Brain Res. Bull., 20 (1988) 765-770. 8 Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.J., Bankowski, K., Manning, M. and Sawyer, W.H., 12SI-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr(NH2)9]OVT: a selective oxytocin receptor ligand, Eur. J. Pharmacol., 147 (1988) 197-207. 9 Elands, J., Beetsma, A., Barberis, C. and de Kloet, R., Topography of the oxytocin receptor system in rat brain: an autoradiographical study with a selective radioiodinated oxytocin antagonist, J. Chem. Neuroanat., 1 (1988) 293-302. 10 Helke, C.J., Handelmann, G.E. and Jacobowitz, D.M., Choline acetyltransferase activity in the nucleus tractus solitarius: regulation by the afferent vagus nerve, Brain Res. Bull., 10 (1983) 433-436. 11 Helke, C.J., Shults, C.V., Chase, T.N. and O'Donohue, T.L., Autoradiographic localization of substance P receptors in rat medulla: effect of vagotomy and nodose ganglionectomy, Neuroscience, 12 (1984) 215-223. 12 Kalia, M. and Sullivan, J.M., Brainstem projections of sensory and motor components of the vagus nerve in the rat, J. Comp. Neurol., 211 (1982) 248-264. 13 Kalia, M., Fuxe, K., H6kfelt, T., Harfstrand, A., Lang, R.E. and Ganten, D., Distribution of neurophysin II immunoreactive nerve fibers within the subnuclei of the nucleus of the tractus solitarius of the rat, Brain Res., 321 (1984) 71-82. 14 Laiwand, R., Werman, R. and Yarom, Y., Time course and distribution of motoneuronal loss in the dorsal motor vagal nucleus of guinea pig after cervical vagotomy, J. Comp. Neurol., 256 (1987) 527-537. 15 Lams, B.E., Isacson, O. and Sofroniew, M.V., Loss of transmitter-associated enzyme staining following axotomy does not indicate death of brainstem cholinergic neurons, Brain Res., 475 (1988) 401-406. 16 Laursen, A.M. and Reckling, J.C., Electrophysiological properties of hypoglossal motoneurons of guinea pig studied in vitro, Neuroscience, 30 (1989) 619-637. 17 Leslie, R.A., Gwyn, D.G. and Hopkins, D.A., The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat, Brain Res. Bull., 8 (1982) 3743. 18 Leslie, R.A., Murphy, K.M. and Robertson, H.A., Nodose ganglionectomy selectively reduces muscarinic cholinergic and delta opioid binding sites in the dorsal vagal complex of the cat, Neuroscience, 32 (1989) 481-492. 19 Lewis, ER., Scott, J.A. and Navaratnam, V., Localization in the dorsal motor nucleus of the vagus in the rat, J. Anat., 107
(1970) 197-208. 20 Luiten, P.G.M., ter Horst, G.J., Karst, H. and Steffens, A.B., The course of paraventricular hypothalamic efferents to autonomic structures in medulla and spinal cord, Brain Res., 329 (1985) 374-378. 21 McCann, M.J. and Rogers, R.C., Oxytocin excites gastric-related neurones in the rat dorsal vagal complex, J.Physiol., 428 (1990) 95-108. 22 Manning, M. and Sawyer, W.H., Development of selective agonists and antagonists of vasopressin and oxytocin. In R.W. Schrier (Ed.), Vasopressin, New York, Raven, 1985, pp. 131144. 23 Norgren, R. and Smith, G.P., Central distribution of subdiaphragmatic vagal branches in the rat, J. Comp. Neurol., 273 (1988) 207-223. 24 Phillips, P.A., Widdop, R., Chai, S.Y., Kelly, J., Mooser, J., Trinder, D. and Johnston, C.I., Reduced V 1 vasopressin binding in the rat nucleus solitarii after nodose ganglionectomy, Clin. Exp. Pharmacol. Physiol., 17 (1990) 321-325. 25 Raggenbass, M., Dubois-Dauphin, M., Charpak, S. and Dreifuss, J.J., Neurons in the dorsal motor nucleus of the vagus nerve are excited by oxytocin in the rat but not in the guinea pig, Proc. Natl. Acad. Sci., U.S.A., 84 (1987) 3926-3930. 26 Raggenbass, M., Tribollet, E., Dubois-Dauphin, M. and Dreifuss, J.J., Vasopressin receptors of the vasopressor (V1) type in the nucleus of the solitary tract of the rat mediate direct neuronal excitation, J. Neurosci., 9 (1989) 3929-3936. 27 Rinaman, L., Card, J.E, Schwaber, J.S. and Miselis, R.R., U1trastructural demonstration of a gastric monosynaptic vagal circuit in the nucleus of the solitary tract in rat, J. Neurosci., 9 (1989) 1985-1996. 28 Rogers, R.C. and Herman, G.E., Dorsal medullary oxytocin, vasopressin, oxytocin antagonist, and TRH effects on gastric acid secretion and heart rate, Peptides, 6 (1985) 1143-1148. 29 Rogers, R.C. and Herman, G.E., Hypothalamic paraventricular nucleus stimulation-induced gastric acid secretion and bradycardia suppressed by oxytocin antagonist, Peptides, 7 (1986) 695-700. 30 Sawchenko, P.E. and Swanson, L.W., Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat, J. Comp. Neurol., 205 (1982) 260-272. 31 Shapiro, L.E. and Insel, T.R., Ontogeny of oxytocin receptors in rat forebrain: a quantitative study, Synapse, 4 (1989) 259266. 32 Siaud, P., Denoroy, L., Assenmacher, I. and Alonso, G., Comparative immunocytochemical study of the catecholaminergic and peptidergic afferent innervation to the dorsal vagal complex in rat and guinea pig, J. Comp. Neurol., 290 (1990) 323-335. 33 Sofroniew, M.V., Vasopressin, oxytocin and their related neurophysins. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 4: GABA and Neuropeptides in the CNS, Part L Elsevier, Amsterdam, 1985, pp. 93-95. 34 Tribollet, E., Barberis, C., Jard, S., Elands, J., Dubois-Dauphin, M., Marguerat, A. and Dreifuss, J.J., Mapping and analysis of receptors for neurohypophysial peptides present in the brain. In B.T. Pickering, J.B. Wakerley and A.J.S. Summerlee (Eds.), Neurosecretion: Cellular Aspects of the Production and Release of Neuropeptides, Plenum, New York, 1988, pp. 81-87. 35 Tribollet, E., Charpak, S., Schmidt, A., Dubois-Dauphin, M. and Dreifuss, J.J., Appearance and transient expression of oxytocin receptors in fetal, infant, and peripubertal rat brain studied by autoradiography and electrophysiology, J. Neurosci., 9 (1989) 1764-1773. 36 Voorn, E and Buijs, R.M., An immunoelectromicroscopical study comparing vasopressin, oxytocin, substance P and enkephalin containing nerve terminals in the nucleus of the solitary tract in the rat, Brain Res., 270 (1983) 169-173. 37 Yarom, Y., Sugimori, M. and Llinas, R., Ionic currents and firing patterns of mammalian vagal motoneurons in vitro, Neuroscience, 16 (1985) 719-737.
124 BRES 17537
Morphological and electrophysiological evidence for postsynaptic localization of functional oxytocin receptors in the rat dorsal motor nucleus of the vagus nerve M. Dubois-Dauphin, M. Raggenbass, H. Widmer, E. Tribollet and J.J. Dreifuss Department of Physiology, University Medical Centre, Geneva (Switzerland) (Accepted 29 October 1991)
Key words: Electrophysiology; Neuropeptide; Receptor autoradiography; Solitary-vagal complex; Vagotomy
The vagal complex is innervated by oxytocin immunoreactive axons of hypothalamic origin. The presence of oxytocin binding sites in the dorsal motor nucleus of the vagus nerve of the rat was evidenced by autoradiography with a radioiodinated oxytocin antagonist as ligand. Two weeks following a unilateral vagotomy, distal to the nodose ganglion, binding sites were reduced below the level of detection in the ipsilateral dorsal motor nucleus of the vagus nerve. Choline acetyltransferase immunoreactivity was also markedly reduced in the vagal motoneurons whose axons had been transected. Electrophysiological studies were performed in vitro in brainstem slices from control rats. In antidromically identified vagal motoneurones, oxytocin applied at 0.1-1.0 #M either caused a reversible depolarization or generated, under voltage-clamp conditions, a transient inward current. These responses persisted under the condition of synaptic uncoupling. Taken together these observations favour the notion that oxytocin of hypothalamic origin acts directly on rat vagal motoneurones. INTRODUCTION The dorsal vagal complex, formed of the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve, receives axons originating from cell bodies located in the hypothalamic paraventricular nucleus2°, in particular from vasopressin and oxytocin immunoreactive cell bodies 13'3°'32. The nucleus of the solitary tract
tocin binding sites in the dorsal motor nucleus of the vagus nerve, we investigated the effect of a unilateral vagotomy, distal to the nodose ganglion, on the distribution of oxytocin binding sites in this nucleus using in vitro light microscopic autoradiography. In parallel, the sensitivity to oxytocin of antidromically identified vagal motoneurones was tested by intracellular recordings on brainstem slices from intact rats. The results of both the
receives sensory vagal afferents, but does not send efferent axons into the vagus nerve. The rat dorsal motor nucleus of the vagus nerve contains mainly preganglionic neurones whose axons terminate in the parasympathetic ganglia of thoracic and abdominal viscera 3'12'17'23'27.
morphological and the electrophysiological studies suggest that vagal m o t o n e u r o n e s possess functional oxytocin receptors and, therefore, favours the view that oxytocin acts directly on these neurones.
A physiological role of oxytocin in the vagal complex is well documented. Thus, local infusion of oxytocin affects gastric acid secretion 21'28. This effect was mimicked
MATERIALS AND METHODS
by electrical stimuli applied to the hypothalamic paraventricular nucleus; infusion of a specific oxytocin antagonist into the dorsal motor nucleus of the vagus nerve blocked the effect of the hypothalamic stimulation29. Electrophysiological studies carried out on rat brainstem slices showed that vagal m o t o n e u r o n e s are sensitive to oxytocin 5'2s. In addition, specific binding sites for oxytocin were uncovered in the dorsal motor nucleus of the vagus nerve 7'9'34'35. To assess the pre- or postsynaptic localization of oxy-
Unilateral vagotomy These experiments were conducted on 11 male rats from the Sivz strain, a Sprague-Dawley-derived strain, weighing 220-250 g. Eight animals were anesthetized with sodium pentobarbital (5 mg/100 g). The left or the right vagus nerve was transected distal to the nodose ganglion and approximately 1 cm of the nerve was removed. The proximal cut stump of the vagus nerve was then soaked in a 1-cm-long polythene tubing sealed with wax at one extremity and containing 0.9% NaCI. The tube around the nerve trunk was sealed with Vaseline and secured in the neck cavity with Histoacryl blue tissue adhesive (B. Braun Melsungen, Germany). ChAT immunocytochemistry The effect of the vagotomy on the choline acetyltransferase (CHAT) content of neurones in the dorsal motor nucleus of the va-
Correspondence: M. Dubois-Dauphin, Department of Physiology, University Medical Centre, 9 Avenue de Champel, 1211 Geneva 4, Switzerland. Tel.: 0041.22.22.91.10. Fax: (41) (22) 47.33.34.
125
Fig. 1. ChAT immunoreactivity 2 weeks after unilateral section of the vagus nerve. Note that immunoreactivity disappeared from the ipsilateral dorsal motor nucleus outlined by dotted lines at rostral (A), intermediate (B) and caudal levels (C). Bar: 150/~m. Abbreviations used in figures: AP, area postrema; IO, inferior olive; Sol, nucleus of the solitary tract; 4V, 4th ventricle; 10, dorsal motor nucleus of the vagus nerve; 12, hypoglossal nucleus.
126
Fig. 2. ChAT immunoreactivity in the vagal complex of a rat 2 weeks after unilateral vagotomy. The section was counterstained with cresyl violet to visualize the cell bodies. A: overview: the dorsal motor nucleus contralateral to the vagotomy and both hypoglossal nuclei contain ChAT immunoreactive neurones. The ipsilateral dorsal motor nucleus (on the right) although devoid of ChAT immunoreactivity shows normal number of cell bodies, shown at higher magnification in B (rectangular area outlined in A). Bar: (A) 200 ~m, (B) 85/~m.
gus nerve was assessed on three rats. They were reanesthetized and fixed by intracardiac perfusion with a 4% paraformaldehyde solution in phosphate buffered saline (PBS) pH 7.35. The brain was removed, postfixed for 1 h in the same fixative at 4°C, cryoprotected in a 30% sucrose/PBS solution at 4°C and 40-#m-thick brainstem sections were obtained. Floating sections were incubated for 1 h in normal horse serum, diluted 1/50 in PBS containing 0.1 M L-lysine, then rinsed 3 × 10 min in PBS. Sections were incubated 16 h at 4°C, with mouse monoclonal antibodies raised against CHAT, at the concentration of 7/~g/ml (antibody IE6, a gift from Dr. P. Salvaterra, Beckman Research Institute, Duarte). Following new washes (3 x 10 min), the sections were reacted for 90 min with goat serum containing biotinylated antimouse antibody, diluted 1/100 (Vector Laboratories, Burlingame, CA). After further rinsing, they were incubated for 1 h with avidin-biotin-peroxidase complex. Peroxidase activity was revealed using 0.05% diaminobenzidine in 0.1 M phosphate buffer. Following a 10 min incubation step, 0.013% H20 2 was added and the incubation continued for 10 min. After washing in PBS and rapid rinsing in distilled water, the sections were mounted on chromalum-gelatin coated slides.
Autoradiography Two weeks after unilateral vagotomy, five animals were reanesthetized, decapitated, their brain removed and frozen at -25°C in isopentan. Sixteen-/xm-thick coronal sections of the brainstem were cut in a cryostat, laid on chromalum-gelatin coated slides, air-dried and processed for light microscopic autoradiography. Three unoperated rats were used as controls. Oxytocin binding sites were detected using 125I-labelled d(CH2) 5[Tyr(Me)2,Thr4,Tyr(NH2)9]ornithine vasotocin, ([125I]OTA), as ligands. The compound was synthesized and kindly provided by Dr. M. Manning and was monoiodinated with 125I-labelled Na to a specific activity of about 2000 Ci/mmol. Slides were dipped in 0.2% paraformaldehyde in PBS (pH 7.4), then rinsed twice in 50 mM Tris-HCl (pH 7.4). Incubation was carried out for 1 h at room temperature in a humid chamber by covering each slide with 400 /tl of the incubation medium (50 mM Tris-HC1, 0.1 mM bacitracine, 5 mM MgC12, 0.1% bovine serum albumin) containing 0.05 nM [125I]OTA. Non-specific binding was evaluated by incubating adjacent sections with medium containing in addition 1 ,aM nonradioactive oxytocin. Incubation was followed by 2 × 5 min washes in ice-cold incubation medium and a quick rinse in distilled water.
127 The slides were rapidly dried and apposed to fl-Amersham film for 3-5 days. Alternate sections were processed similarly to detect vasopressin binding sites using 1.5 nM [3H]vasopressin as ligand (Du Pont New England Nuclear, Boston, MA) in the presence of 5 nM of nonradioactive OH[Thr4,Gly7]oxytocin. The latter compound is a selective oxytocin agonist22 and was added to prevent binding of [3H]vasopressin to oxytocin receptors. Non-specific labelling was assessed in adjacent sections treated in the same conditions, except that the incubation medium contained in addition 10 t~M non-radioactive arginin-vasopressin. Sections were then placed in contact with a tritium sensitive film (LKB Ultrofilm) in an X-ray cassette for 3 months at 4°C. Films were developed in Kodak D-19, and the sections were stained with cresyl violet.
In all c o n t r o l animals,
[I25I]OTA b i n d i n g was ob-
s e r v e d bilaterally in the dorsal m o t o r nucleus of the vagus n e r v e and in the i n f e r i o r olive (Fig. 3 A ) . A u t o r a d i o g r a p h i c labelling was d e t e c t e d in the a n t e r i o r and m i d d l e p a r t of the dorsal m o t o r nucleus b u t n o t in its caudal, cervical, aspect.
[3H]Vasopressin b i n d i n g sites
w e r e d e t e c t e d in a r e a p o s t r e m a , in the nucleus of the solitary tract and in the i n f e r i o r olive.
Electrophysiology The animals used were 32 adult male rats of the same strain as used for the morphological studies, weighing 200-300 g. Following decapitation, the brain was gently removed and a block of brainstem containing the dorsal medulla was prepared. Coronal brainstem slices, 300 to 400-~m-thick, were cut using a vibratome (Campden Instruments, London, UK) and incubated in a thermoregulated (33.5-34.5°C) recording chamber at the interface between a perfusion solution and an oxygenated, humidified atmosphere. Unless otherwise stated, the composition of the perfusion solution was (in mM): NaC1 138, KCl 2, NaHCO 3 15, KH2PO 4 1.25, MgSO 4 1, CaC12 2, and glucose 10. This solution was saturated with 95% 02/5% CO 2 (pH 7.35-7.45). Intracellular recordings were obtained using glass micropipettes filled with 3 M potassium-acetate (pH 7.4; 50-100 MQ). Voltage and current signals were amplified using an Axoclamp-2A (Axon Instruments Inc., Foster City, CA), digitized and stored on the hard disk of an AT-386-type personal computer. In the single-electrode voltage-clamp mode, the switching frequency was 2-3 kHz; the headstage was continuously monitored. In order to activate antidromically vagal motoneurones, bipolar stimulation electrodes, made of twisted nichrome wires (100/~m in diameter, isolated except for their tips), were positioned ventrolaterally to the dorsal motor nucleus of the vagus nerve, along the course followed by the axons arising from this nucleus 12. Stimuli were constant current pulses (10-200/~A), delivered at 0.5-1 Hz. Antidromic invasion of the soma was considered to be successful if the following criteria were obeyed: (a) the evoked action potentials arose at fixed latency with respect to the stimulus artefact; (b) they followed the stimulation at high frequency; (c) they could dissociate into an axon initial segment component and a somatodendritic component. Synaptic uncoupling was achieved either by perfusing the preparation with a modified solution, containing reduced (0.1 mM) calcium and increased (16 mM) magnesium, or by adding tetrodotoxin (TI'X) at 1-2 ktM to the normal perfusion solution. Oxytocin was obtained from Bachem (Bubendorf, Switzerland) and TI'X was from Sigma (St. Louis, MO).
RESULTS
ChAT immunoreactivity and receptor autoradiography T w o w e e k s after u n i l a t e r a l v a g o t o m y , C h A T i m m u n o r e a c t i v e cell b o d i e s w e r e p r e s e n t in t h e facial and perihypoglossal nuclei, a n d in the dorsal m o t o r n u c l e u s cont r a l a t e r a l to the v a g o t o m y in all t h r e e animals studied. In c o n t r a s t , t h e ipsilateral n u c l e u s was d e v o i d of C h A T i m m u n o r e a c t i v i t y a l o n g its e n t i r e r o s t r o c a u d a l e x t e n s i o n (Fig. 1). H o w e v e r , cresyl v i o l e t stained cell b o d i e s w e r e clearly visible in t h e ipsilateral as well as in t h e c o n t r a lateral dorsal m o t o r nucleus (Fig. 2).
Fig. 3. Autoradiograms of oxytocin and vasopressin binding sites in an intact rat (A) and in a rat 2 weeks after unilateral vagotomy (B,C). A: [a25I]OTA binding sites are detected bilaterally in the dorsal motor nucleus (10) and in the inferior olive (IO) of a normal rat. B: in a vagotomized animal [lzSI]OTA binding disappeared from the dorsal motor nucleus ipsilateral to the vagotomy but is present in the contralateral dorsal motor nucleus and in both inferior olives. C: [3H]vasopressin binding in area postrema (AP), in the nucleus of the solitary tract (Sol) and in the inferior olive of the same vagotomized rat. This autoradiogram was obtained from a section 150 #m caudal to the section which generated the autoradiogram shown in B. The specificity of the binding was ascertained by its displacement with an excess of non-radioactive oxytocin or non-radioactive vasopressin (not shown). Bar: 1 mm.
128
Fig. 4. Cresyl violet staining of sections from which autoradiograms (A) and (B) in Fig. 3 were obtained. A is from the control and B from the vagotomized animal. Bar: 300 ~m.
Two weeks after unilateral vagotomy, [125I]OTAbinding was symmetrical in the inferior olive (Fig. 3B), as was [3H]vasopressin binding in the nucleus of the solitary tract (Fig. 3C). In contrast, in all 5 animals studied, [12SI]OTA binding was no longer detectable in the dorsal motor nucleus ipsilateral to the vagus nerve transection (Fig. 3B). Cell bodies in the dorsal motor nucleus were as intensely stained with cresyl violet in the nucleus ipsilateral to the vagotomy as in the control animals (Fig. 4).
Electrophysiological study Intracellular recordings were obtained from 36 antidromically identified vagal motoneurones. They had resting membrane potentials ranging from -50 to -70 mV and were either silent or discharged spontaneously, at frequencies < 5 action potentials/s. Their average membrane resistance was 145 _ 15 Mf~ (mean _ S.E.M.; n = 21). Typically, these neurones were endowed with a transient, voltage-dependent potassium conductance of the A-type 37. Twenty-eight neurones (i.e. 78%) re-
129
A
rnV *
*
*
*
20
n
~
spikes/s
50 s
ms
B
]3o
mV
*
0.1 nA
I0 ms
60 s
Fig. 5. Effect of oxytocin on two antidromically identified vagal motoneurones, A and B. A: left panel, three superimposed voltage traces, recorded under current-clamp conditions, showing the response of the neurone to pairs of shocks, delivered at 20, 30 and 40 ms intervals ventromedially to the dorsal motor nucleus of the vagus nerve. Stimulus artifacts are marked by asterisks. Note that the first action potential in each pair arose at fixed latency; note also that at 50 Hz, the action potential generated by the second stimulus invaded the axon initial segment but failed to propagate into the neuronal soma. Right panel: ratemeter records showing the oxytocin-induced increase in firing rate in the normal solution (top trace) and in a low-calcium, high-magnesium solution (bottom trace). The peptide was added to the perfusion solution at 1 #M for the time indicated by the horizontal line above each record. In the modified solution the neurone lost its spontaneous activity but still responded to oxytocin. B, left panel: three superimposed voltage traces recorded under current-clamp conditions and representing the neuronal response to a single shock. The action potential was due to antidromic invasion, since it tended to dissociate spontaneously into a low amplitude initial segment component and a high amplitude somatodendritic component. Note that in one case, the initial segment spike failed to invade the soma. Right panel: voltage-clamp records of the membrane current generated in the same neurone by oxytocin at 1 #M in the normal solution (top trace) and in a solution containing 1 #M T r x (bottom trace). Holding potential, -50 inV.
sponded to oxytocin dissolved in the perfusion solution at 0.1 or 1 pM. Under current-clamp conditions, oxytocin induced a reversible membrane depolarization, accompanied by an increase in firing frequency (n = 16; Fig. 5B, right panel, top trace). Under voltage-clamp conditions -- the cell membrane potential being held at or near its resting level -- oxytocin generated an inward current (n = 12; Fig. 5B, right panel, top trace). In order to determine whether the action of oxytocin was direct, the response to the peptide recorded in the normal solution was compared with that recorded in conditions of synaptic uncoupling. In four out of four current-clamped neurones, the oxytocin-induced depolarization and the increase in firing rate persisted in a lowcalcium, high-magnesium solution, although in the latter solution the neuronal excitability was reduced (Fig. 5A, right panel, bottom trace). In seven out of seven voltageclamped neurones, the inward current generated by oxytocin in the presence of TTX or in a low-calcium, highmagnesium solution was superimposable on that generated in control solutions (Fig. 5B, right panel, bot-
tom trace).
DISCUSSION
A pathway of oxytocin containing axons arises in the rat hypothalamic paraventricular nucleus 13'2°'a° and innervates the dorsal vagal complex, where synapses containing oxytocin have been detected by immunoelectron microscopy32'36. Oxytocin can be released in vitro from rat brainstem slices containing the dorsal vagal complex by a membrane depolarization in the presence of extracellular calcium4. Since oxytocin has been shown to excite vagal motoneurones in vitro, it has been conjectured that oxytocin may exert a direct, stimulatory effect on vagal motoneurones 7'25. By contrast, Siaud et al. 32 have postulated an indirect route, where oxytocin-containing hypothalamic axons project to and contact catecholaminergic interneurones in the rat medulla which, in turn, form synapses with vagal preganglionic motoneurones. In confirmation of earlier results, binding sites for oxytocin were detected in the dorsal motor nucleus of the vagus nerve (and in the inferior olive) using [125I]OTA, a selective oxytocin receptor ligand. Its high affinity and specific activity permit to visualize small numbers of oxytocin binding sites, which escape detection with tritiated o x y t o c i n 7'9'31'34'35.
130 Two weeks after transection of one vagus nerve, distally to the nodose ganglion, the ipsilateral dorsal motor nucleus showed signs of altered metabolic activity: a marked reduction of ChAT activity and of the density of oxytocin binding sites. The fact that these effects were due to the unilateral vagotomy is supported by the following arguments: (a) at two weeks, unilateral vagotomy had little effect on the number of cells present in the ipsilateral dorsal motor nucleus of the vagus nerve, based on cresyl violet staining of cells (see also refs. 1,14,15). (b) Unilateral vagotomy produced a reduction in ChAT activity which affected only the ipsilateral dorsal motor nucleus of the vagus nerve; the dorsal motor nucleus contralateral to the transected side showed normal ChAT immunoreactivity, as did the perihypoglossal nuclei on both sides (see also refs, 1,2,19). (c) Unilateral vagotomy reduced [12511OTA binding in the ipsilateral dorsal motor nucleus of the vagus nerve; the dorsal motor nucleus contralateral to the transected side showed normal radioligand binding, as did the inferior olives on both sides. This latter observation suggests strongly that binding sites for oxytocin are associated with vagal motoneurones and are down-regulated in chromatolytic neurones. Another control is provided by the fact that the density of vasopressin binding sites in area postrema and in the nucleus of the solitary tract was the same on left and right side in unilaterally vagotomized rats, as in intact rats. Since we performed the vagotomy distal to the nodose ganglion, we were able to achieve axotomy without causing afferent nerve degeneration lz. Nodose ganglionectomy, by lesioning the cell bodies of afferent vagal fibers would have resulted in the degeneration of axon terminals in the nucleus of the solitary tract and in the dorsal motor nucleus of the vagus nerve ~°. Actually, Phillips et al. 24 reported that nodose ganglionectomy reduced the binding of vasopressin in the medial subdivision of the ipsilateral nucleus of the solitary tract, suggesting that some vasopressin receptors are located on viscerosensory afferent axons coursing in the vagus nerve. However, other subdivisions of the nucleus of the solitary tract showed no loss of vasopressin binding after ganglionectomy and are therefore possibly located postsynaptically on solitary tract neurones 6'11'1s. The electrophysiological data show that oxytocin can directly excite neurones located in the region of the dorsal motor nucleus of the vagus nerve. This excitation is due to a peptide-induced inward membrane current, which is resistant to TTX and which is not synaptically mediated, since it persisted in a low-calcium, high-magnesium solution. The ionic basis of this current is presently under investigation in our laboratory.
The antidromically activated oxytocin-responsive neurones were vagal preganglionic motoneurones. Although some recordings could have been obtained from neurones located in the adjacent nucleus of the solitary tract, electrical stimulation of sensory axons in the vagus nerve would have lead, at best, to orthodromic activation of these neurones. Recordings could also have been obtained, occasionally, from hypoglossal motoneurones. However, the latter can be readily distinguished from vagal motoneurones since they are devoid of voltage-dependent A-type potassium channels16.Our electrophysiological results suggest that at least part of the [~zSI]OTA binding sites detected by autoradiography in the dorsal motor nucleus of the vagus nerve are functional receptors located on vagal motoneurones. This is in accordance with previous results showing that oxytocin excited in vitro brainstem neurones which, following intracelluiar staining with Lucifer yellow, could be identified as being vagal motoneurones on the basis of their location and morphology25. We have previously provided evidence that the vasopressin binding sites detected in the nucleus of the solitary tract are functional receptors affecting neuronal excitability 26. The question of the significance of the binding sites for neurohypophysial peptides present in the inferior olive has not been addressed so far. Since all raphe nuclei, including the raphe pallidus, which is located in the immediate vicinity of the inferior olive, contain vasopressin- and oxytocin-immunoreactive axons 33, the inferior olive may be another target for neurohypophysial peptides. In conclusion, the presence of an oxytocin immunoreactive innervation of the dorsal motor nucleus of the vagus nerve of hypothalamic origin, the presence of oxytocin binding sites in this nucleus, their disappearance from the nucleus ipsilateral to a unilateral vagotomy, the sensitivity to oxytocin of antidromically identified vagal motoneurones, all point to a direct effect of hypothalamic oxytocin on motoneurones of the dorsal motor nucleus of the vagus nerve. On the other hand, our results do not preclude an additional, indirect action via interneurones located in the nucleus of the solitary tract.
Acknowledgements. This work was supported in part by the Swiss National Foundation (Grant 31-28624.90). We thank Drs. M. Manning (Toledo, OH) for the gift of oxytocin structural analogs, S. Jard and C. Barberis (Montpellier, France) for radioiodinating OTA, P. Salvaterra for the gift of antiserum and Ms. A. Marguerat, M. Berti and D. Machard for technical assistance. Michel DuboisDauphin gratefully acknowledges receipt of a Career Development Award of the Prof. Dr. Max Clo~tta Foundation.
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