The Journal of Neuroscience, June 1991, 1 f(6): 1609-l 616 Vasopressin Generates a Persistent Voltage-dependent Sodium Current in a Mammalian Motoneuron Mario Raggenbass, Michel Goumaz, Edoardo Sermasi, Eliane Tribollet, and Jean Jacques Dreifuss Department of Physiology, University Medical Center, CH-1211 Geneva, Switzerland During the period of life that precedes weaning, the facial diographical, and biochemical studies have suggestedthat nucleus of the newborn rat is rich in 3H-vasopressin binding vasopressinprobably also plays a role as a neurotransmitteri sites, and exogenous arginine vasopressin (AVP) can excite neuromodulator (Audigierand Barberis, 1985; Buijs, 1987; Du- facial motoneurons by interacting with V, (vasopressor-type) bois-Dauphin andzakarian, 1987;Van Leeuwen, 1987; Freund- receptors. We have investigated the mode of action of this Mercier et al., 1988; Poulin et al., 1988; Tribollet et al., 1988), peptide by carrying out single-electrode voltage-clamp re- and on the basisof behavioral studies, it has been claimed that cordings in coronal brainstem slices from the neonate. Facial this peptide may influence memory storage and retrieval (De motoneurons were identified by antidromic invasion follow- Wied, 1980). Electrophysiological studies have indicated that ing electrical stimulation of the genu of the facial nerve. vasopressincan directly excite selected populations of central When the membrane potential was held at or near its resting neurons (Suzue et al., 1981; Miihlethaler et al., 1982; Ma and level, vasopressin generated an inward current whose mag- Dun, 1985; Peters and Kreulen, 1985; Raggenbasset al., 1987, nitude was concentration related; the lowest peptide con- 1988, 1989; Carette and Poulain, 1989; Liou and Albers, 1989; centration still effective in eliciting this effect was 10 nM. Sun and Guyenet, 1989). Although some information is avail- The vasopressin-induced current, I,,,, was resistant to te- able concerning the cellular mode of action of vasopressinas a trodotoxin (TTX) and was insensitive to a reduction in ex- hormone (Mollard et al., 1988; Van Renterghem et al., 1988; tracellular calcium concentration. It was sustained, was in- Korbmacher et al., 1989; Martin et al., 1989), the membrane ward at all potentials tested (- 120 to -25 mV), and increased mechanismby which vasopressinaffects neuronal bioelectrical in magnitude during depolarization. lAvp was not generated activity is not yet known. We have approachedthis problem by by the blockade of a potassium current, because it did not recording from vasopressin-sensitivefacial motoneurons from reverse at hyperpolarized potentials, was not affected by a the newborn rat (Tribollet et al., 199 l), using the in vitro brain two-fold increase in the transmembrane potassium gradient, slice technique and the single-electrodevoltage-clamp method. and was not modified by the potassium channel blockers tetraethylammonium bromide (TEA), 4-aminopyridin (4-AP), Materials and Methods barium, cesium, quinine, glibenclamide, and apamin. Also, Zntracellular recordings. The animalsused were 2-lo-d-old malerats lAvp was not affected by changes in the transmembrane chlo- from a Sprague-Dawley-derived strain. Coronal brainstem slices, 300- ride gradient. In contrast, it could be reduced by partially 400 pm thick, were cut using a vibratome, transferred to a recording chamber, and perfused, at 34-35”C, with a solution containing (in mM) substituting extracellular sodium with equimolar N-methyl- 135 NaCl. 15 NaHCO,. 1 MaSO,. 1.25 KH,PO,. 2 CaCl,. 10 alucose. o-glucamine or Tris. Our results suggest that vasopressin and, unlessotherwise stated,-5 I&l. This soluti& wassaturated with increases the excitability of facial motoneurons by gener- 95%0,/5% CO, (pH, 7.35-7.45).A low-calcium,high-magnesium so- ating a persistent sodium-dependent membrane current that lution, containing 0.1 mM CaCl, and 16 mM MgSO,, was used in some experiments to block synaptic transmission and to reduce membrane is voltage gated and TTX resistant. calcium currents. When barium was added to the solution, phosphates were omitted, and MgSO, was replaced by MgCl,. N-methyl-D-gluca- The nonapeptide arginine vasopressin,which is synthesized in mine and Tris, which were used in sodium ion substitution experiments, the hypothalamus and secretedinto the circulation at the level were titrated to pH 7.4 with concentratedHCl. Microelectrodescon- tainedeither 3 M KC1or 3 M K-acetate(pH, 7.4; 30-80 MQ); in some of the neurohypophysis, exerts a variety of peripheral effects. experiments,facial neuronswere cesium-loadedby impalementwith Thus, by acting on V,-type, phospholipaseC-coupled receptors, microelectrodes filled with 2 M CsCl or Cs-acetate (pH, 7.4). Recordings it causesvasoconstriction and stimulates glycogenolysisin he- were done usingan Axoclamp-2A amplifier. In the single-electrode patocytes; by binding to V,-type, adenylate cyclase-coupledre- voltage-clampmode, the switchingfrequency was 2-3 kHz, and the ceptors, it enhanceswater reabsorption in the kidney (Jard, headstage was continuously monitored. Membrane potential and cur- rent wereplotted on paperon line. Somedata weredigitized usinga 1983; Manning et al., 1987). Immunocytochemical, autora- homemade A/D converter and stored on the hard disk of an IBM-AT- compatible computer for further analysis. Antidromic activation of fa- cial neurons was achieved by delivering constant current pulses (1 O- Received Oct. I, 1990; revised Jan. 2, 199 1; accepted Jan. 4, 199 1. 100 /IA, 0.1 msec) to the genu of the facial nerve. The elicited action This work was supported in part by Grant 3 1.28624.90 from the Swiss National potentials were considered to be due to antidromic invasion if (1) they Science Foundation. We thank Dr. M. M. Manning for the gift of the selective oxytocin agonist, Dr. D. Bertrand for helpful discussions, and Mrs. D. Machard aroseat fixed latency following stimulation,(2) they followedhigh- for excellent technical assistance. frequency stimulation, and (3) they could dissociate, in double-shock Correspondence should be addressed to Mario Raggenbass,Ph.D., DCpartement studies, into initial segment and somatodendritic components. de Physiolrgie, Centre M6clical Universitaire, 9 Avenue de Champel, CH- 12 11 Light microscopic autoradiography. Following dissection, brain tissue Geneve, Switzerland. was frozen in 2-methylbutane at -25°C. Series of 15-pm-thick sections Copyright 0 1991 Society for Neuroscience 0270-6474/91/l 11609-08$03.00/O were cut in a crvostat. mounted on gelatin-coated slides. and. after 1610 Raggenbass et al. l Vasopressin-induced Sodium Current in a Rat Motoneuron Figure I. Vasopressin binding sites in the facial nucleus of a newborn rat. Au- toradiograms in A and B were obtained by incubating coronal brainstem sec- tions with )H-vasopressin (A) or with -‘H-vasopressin supplemented with an excess of nonradioactive vasopressin (B). C and D show the ventrolateral re- gion on the right side of A at a higher magnification. D is the cresyl violet- stained section used to generate auto- radiograms A and C. Note that labeling is most intense in the area containing large facial-motoneuron perikarya. Scale bars: A and B, 1 mm; C and D, 0.5 mm. drying, stored at -80°C. Prior to the binding procedure, the sections These cells had resting membrane potentials negative to -50 were slightly fixed for 5 min in 0.2% paraformaldehyde in phosphate- mV and fired no action potentials at rest. Their average input buffered saline (PBS; pH, 7.4). Each slide was incubated for 1 hr at room temperature in 400 ~1 of a solution containing 50 mM Tris-HCl resistancewas 25 f 12 MQ (mean + SD, n = 180). Eighty- (DH.~&. SUDDk- seven neuronscould be activated antidromically following elec- 0.025% bacitracin. 5 mM M&l,.1 _I and 0.1% BSA and -_ mented’&itb 3 nM 3H-vasopressin and 5 nM HO[Thr4,Gly7]oxytocin. trical stimulation of the genu of the facial nerve and were thus The presence of this last compound, a selective oxytocin agonist, pre- identified as facial motoneurons (Fig. 2B). The average input vented the labeling by 3H-vasopressin of oxytocin binding sites. In order to determine non-specific binding, adjacent sections were incubated in resistance of these identified motoneurons was 25 f 11 MB (mean + SD; iz = 84). Vasopressin,dissolved in the perfusion the same solution as above but supplemented with 1 PM nonradioactive vasopressin. The slides were washed in ice-cold medium, rinsed in solution, affected 82% of the recorded neurons. Of the identified distilled water, dried, and placed in contact with tritium-sensitive films motoneurons, 84% responded to the peptide. When the mem- in an x-ray cassette for 3-4 months. Films were developed for 5 min brane potential was voltage clamped at or near its resting level, in Kodak D19, and sections were stained with cresyl violet. Chemical compounds. Vasopressin (8-L-arginine vasopressin) was vasopressingenerated a sustainedinward current (Fig. 2A); un- purchased from Bachem (Bubendorf, Switzerland). 3H-vasopressin (spe- der current-clamp conditions, the peptide causeda membrane cific activity, 67 Wmmol) was from Du Pont-New England Nuclear depolarization (seeFig. 5B). These effects were fully reversible. (Boston, MA) and was purified by HPLC and affinity chromatography The peak current evoked by vasopressin,added to the perfusion (Pradelles et al., 1972). The oxytocic-selective agonist HO[Thr4,Gly7]- solution at 1 PM for 30 set, ranged from 0.1 to 1.4 nA, and the oxytocin ([ 1 -(L-2-hydroxy-3-mercaptopropionic acid),4-threonine,7gly- cineloxytocin; Lowbridge et al., 1977) was kindly provided by Dr. M. peak depolarization ranged from 3 to 20 mV. The action of M. Manning: (Denartment of Biochemistrv. Medical College of Ohio, vasopressinwas concentration dependent, the lowest peptide Toledo, OHj. ‘Tetrodotoxin (TTX), glibenclamide, apamine, and qui: concentration still effective in eliciting an effect being 10 nM nine hydrochloride were from Sigma (St. Louis, MO). Tetraethylam- (except in one neuron, where a measurableeffect was evoked monium bromide (TEA), 4-aminopyridin (4-AP), Na-isethionate, and N-methyl-D-ghtcamine were from Fh&a (Buchs, Switzerland).