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Dorsal medullary oxytocin, vasopressin, oxytocin antagonist, and TRH effects on gastric acid secretion and heart rate
Peptides. Vol. 6, pp. 1143-1148, 1985. ~ AnkhoInternationalInc. Printedin the U.S.A.
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Dorsal Medullary Oxytocin, Vasopressin, Oxytocin Antagonist, and TRH Effects on Gastric Acid Secretion and Heart Rate R I C H A R D C. R O G E R S A N D G E R L I N D A E. H E R M A N N
D e p a r t m e n t o f Physiology, University o f N e v a d a School o f Medicine, Reno, N V 89557 R e c e i v e d 3 S e p t e m b e r 1985 ROGERS, R. C. AND G. E. HERMANN. Dorsal medullary oxytocin, vasopressin, oxytocin antagonist, and TRH effects on gastric acM secretion and heart rate. PEPTIDES 6(6) 1143-1148, 1985.--Injections of oxytocin and TRH (11 picomoles), centered on the dorsal motor nucleus of the vagus, substantially increased gastric acid secretion. Additionally, oxytocin, but not TRH, simultaneously produced a consistent reduction in heart rate. Vasopressin injected into the same locus, at doses of 11 and 110 picomoles, had no effect on either function. Both the gastric and cardiac effects of oxytocin were eliminated by the central injections of oxytocin antagonist dEt2Tyr(Et)OrnSVasotocin (ETOV; 6 picomoles) or peripheral administration of atropine (300 p~g/kg, IP). Application of oxytocin or TRH to the area postrema, at double the dosage (22 picomoles) yielded no consistent effects on either gastric secretion or heart rate. These findings indicate that oxytocin in the dorsal motor nucleus of the vagus may act as a regulator of vagally-mediated gastric and cardiovascular functions while TRH effects, in this medullary area, seem limited to the regulation of gastric function. Oxytocin
Vasopressin
TRH
Gastric function
THE hypothalamus and limbic system are responsible for integrating inputs from a vast array of internal and external sensory receptors. The result of this integration is reflected in autonomic efferent activity which regulates the milieu interieur [12]. The paraventricular nucleus of the hypothalamus (PVN) is particularly interesting in this regard. This structure sends axons to both vagal parasympathetic and spinal sympathetic, preganglionic neurons, as well as to vagal sensory neurons in the nucleus of the solitary tract (NST) [20,22]. It seems likely, therefore, that the PVN can control the expression of autonomic functions through direct action on autonomic efferents and on those sensory neurons which form the afferent limb of brainstem autonomic reflexes [17, 18, 19, 25]. Recent immunohistochemical and physiological studies [6, 7, 22, 25, 31] have shown that these direct pathways between the PVN and vagal sensory and motor nuclei contain significant amounts of oxytocin, vasopressin and thyrotropin releasing hormone (TRH), as well as a variety of other peptides. Though it is currently known that TRH produces an increase in gastric secretion via vagal efferent excitation [17,26], it is not known where in the central nervous system TRH exerts this effect. That is, does TRH directly activate vagai neurons in the dorsal medulla or does it act on some other, intermediate locus? The effects of oxytocin and vasopressin on vagally regulated gastrointestinal functions are unknown and are controversial with respect to vagal regulation of cardiac, function [11, 21, 28]. In order to establish whether any of these three PVN neuropeptides have any direct effects on vagal efferent autonomic functions, we decided to inject picomolar quantities of these substances into physiologically identified medullary vagal-motor regions while recording gastric acid output and heart rate.
Heart rate
Oxytocin antagonist
GENERAL METHOD Male, Long-Evans rats (300-400 g) were used for these studies. All were food deprived 24 hr prior to urethane (1.5 g/kg) anesthesia. Dexamethasone (0.2-0.4 mg, SC) was given immediately before surgery to prevent cerebral edema. All animals were prepared for continuous gastric perfusion by placing an infusion catheter (PE50) in the stomach via the cervical esophagus; a drainage catheter (2 mm, o.d.) was secured in the pylorus via a small duodenal incision. After wound closure, cardiac electric activity was continuously monitored from needle electrodes placed subcutaneously in the left thorax and right hind limb. Warmed, unbuffered, saline (37°C, pH 6.8) was infused through the esophageal influx catheter via a Sage T M roller pump (model 371A) at a rate of 2 ml/min. This saline and any gastric secreta were constantly drained through the pyloric efflux catheter and collected in a small beaker. At 10 min intervals, the beaker was changed and the amount of acid secreted was determined by titration with 0.02 M NaOH and a conventional pH meter [17]. Gastric acid secretion was measured continuously throughout the experiment. The animal was placed in a stereotaxic frame with the head held in the nose-down position to allow surgical exposure of the dorsal spinomedullary junction. The obex region of the dorsal medulla was exposed by resecting the dorsal cervical musculature and removing the occipital skull plate. Composite stimulation/injectionelectrodes were constructed from tungsten microelectrodes and muitibarreled micropipette assemblies (Medical Systems). The stimulating microelectrode was electrolytically etched to a tip diameter of approximately 10/~m and then insulated with Epoxylite. Individual, internal tip diameters of the multibarreled micropipettes ranged from 3-8/xm. The metal stimulating elec-
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B
FIG. 1. (A) Combination pipette/electrode array used in these studies. Etched, insulated tungsten wire microelectrode is bent to conform with pipette tip and glued in place with cyanoacrylate, then epoxy. (B) Tip of injection/stimulation array shown in (A). Scale bar= 100/xm.
trode was glued into position such that it aligned with the micropipette array and protruded beyond the pipette array by approximately 40 p~m (Fig. 1). The construction of similar arrays is discussed elsewhere [14,27]. Pipette barrels were filled with Ringer's solution or solutions of 0.011 M arginine vasopressin (Bachem), 0.011 M oxytocin (Bachem), 0.011 M TRH (Bachem), 0.006 M d E_t.,Tyr(Et)OrnSVasotocin (ETOV--oxytocin/vasopressin antagonist; the generous gift of Dr. M. Manning), or 2.5% Pontamine dye, depending on the experimental conditions. Note that Ringer's was used as a vehicle for all injection solutions. The arrays were then mounted in a stereotaxic carrier. The pipettes were connected to a Medical Systems BH-2 micropressure ejector and the stimulating electrode was connected to a conventional physiological stimulator through a constant current stimulus isolation unit. EXPERIMENT
1
Method
Our preliminary studies and the results of others [13,30] indicated that electrical microstimulation (30 p.A, 0.3 msec duration, 10 Hz, 5 sec on/5 sec off, in trains 2-10 rain long) of the left, posteromedial portion of the dorsal motor nucleus of the vagus (DMN) produces a precipitous drop in heart rate as well as an abrupt increase in gastric acid outflow (Fig. 2). Histological analysis verified that these combined effects
were only obtained if the tip of the stimulation electrode was located within the boundaries of the DMN (Fig. 2). Therefore, we used this physiological identification procedure in the present study to guide the placement of our multibarrel arrays. Thus, the pipette/electrode tip was placed on the brainstem surface 0.2 mm lateral and 0.2 mm anterior to obex and directed toward the DMN; approximately 0.5 mm ventral to the brainstem surface. The array was then advanced in 50/~m increments and trains of 30/~A, 0.3 msec, 10 Hz pulses were applied to the stimulating electrode as described above. The pipette/electrode array was left in place when the aforementioned cardiac and gastric effects were obtained. The ejection protocol began one hour after final electrode array placement into this dorsal medullary locus. Ringer's solution (1.0 nl) was always microinjected first as a control measure, followed 60 min later by either oxytocin (0.011 M x 1.0 hi= 11 picomoles), vasopressin (0.011 M x 1.0 nl= 11 picomoles or 0.011 M× 10 hi= 110 picomoles), or TRH (0.011 M x 1.0 nl= 11 picomoles). Resultant changes in heart rate and gastric acid secretion were monitored, as described above. The precise volume of ejection was calibrated by measuring the movement of the meniscus in the pipette with a 200-power monocular microscope while applying trains of pressure pulses (10 and 30 psi, I00-400 msec duration). The details of precision pico-to-nanoliter volume delivery techniques have been described elsewhere [15,24]. At the end of the experiment, a small electrolytic lesion was made by passing cathodal direct current (30 p.A for 30 sec) through the stimulating electrode to mark the location of the array tip for subsequent histological verification of the injection locus. In several animals, an electrolytic lesion was not made, rather, Pontamine dye was injected (1.0 nl) to mark the tip location. This procedure also served as a visual estimate of the degree of spread through the surrounding tissue that such a volume of solution may have. After lesion or dye injection, animals (N=21) received an overdose of pentobarbital and were transcardially perfused with saline and Mirsky's fixative. The brainstems were then removed and stored in a formalin-30% sucrose solution before being sectioned (60 p.m) on a freezing microtome. Sections were then mounted, counterstained with cresyl violet and coverslipped prior to examination with a light microscope. Results
Injection of oxytocin (11 picomoles) into the dorsal posteromedial medulla evoked the same physiological response as elicited by electrical microstimulation (Fig. 2) of the identical locus, i.e., a substantial increase in acid secretion and a signficant decline in heart rate (Figs. 3 and 5). Identical quantities of TRH injected into the same region evoked a similar gastric acid secretory response (Fig. 3) but produced no detectable change in heart rate (Fig. 5). Vasopressin injected in the same identified locus at doses of 11 (N=6) and 110 (N=3) picomoles (0.011 M x l 0 nl) had no effect on either acid secretion (Fig. 3) or heart rate (Fig. 5). Since neither dose of vasopressin evoked changes in these physiological parameters, both dose groups are combined in the figures. In all cases, subsequent histological analysis verified that the array tips were localized within the dorsal motor nucleus of the vagus (DMN) (Fig. 2). Cases in which Pontamine dye was ejected to mark tip location and the degree of dispersion
PEPTIDES AND GASTRIC ACID SECRETION
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FIG. 2. Physiological identification of a DMN injection site. (A) Microstimulation (at vertical hatching; 30/zA, 0.3 msec pulses at 10 Hz, intermittent for 4.5 min) of DMN evokes gastric acid secretion. (B) Effects on heart rate as a consequence of the same microstimulation as in (A) above. (Note different time scale.) (C) Lesion performed through electrode in locus stimulated in (A) and (B) above (posteromedial DMN). DMN =dorsal motor nucleus of the vagus; L=lesion; NST=nucleus of the solitary tract; XII= hypoglossal nucleus. Scale bar= 100/xm.
revealed that injection volumes as small as 1.0 nl could still travel a considerable distance vertically along the cannula track. Of particular concern was the fact that dye injected into the DMN also consistently stained the lateral wall of the area postrema, a significant chemoreceptor structure which contains neurons sensitive to vasopressin and TRH [3,4]. Though this dye "tracking" to the brainstem might have occurred when the electrode was withdrawn, we still wished to control for any peptide contamination of the area postrema, however minor. EXPERIMENT 2
Method In order to verify that the observed physiological responses were attributable to direct effects of these peptides at the site of injection (i.e., dorsal medullary neurons centered on the DMN) and not due to secondary effects of peptide leakage and action at the area postrema, we repeated Experiment ! (N= 12) with the exception that the effective peptide agents (oxytocin and TRH) were applied at double the dosage (0.011 M×2.0 n1=22 picomoles) directly onto the left, dorsolateral area postrema overlying the solitary nucleus and posteromedial DMN. Note that the dura and arachnoid meninges were always removed to fully expose the obex region to the applied peptide solutions. Results Though both oxytocin and TRH produced modest increases in gastric secretion when placed on the area postrema, these effects were not significant (Fig. 3). Furthermore, application of these peptides to the area postrema
resulted in no consistent nor significant alteration in heart rate. EXPERIMENT 3
Method If the intramedullary effects of oxytocin on gastric secretion and heart rate regulation are due to specific activation of oxytocin receptors, then these effects should be blocked by a structural antagonist of oxytocin action [dEt~Tyr(Et)OrnSVasotocin (ETOV)] [1]. Animals (N=3) were prepared as described previously. Here, however, the pipette/electrode array was filled with 0.006 M ETOV, 0.011 M oxytocin, and Ringer's solution. The tip of the pipette/electrode array was placed in a site which yielded cardiac slowing and an increment in gastric acid secretion following electrical microstimulation (as described previously). After a one hour stabilization period, 1.0 nl of Ringer's solution was injected as a vehicle control. One hour later, a 1.0 nl volume of 0.006 M ETOV (6 picomoles) was injected. This antagonist pretreatment was then followed 10 min later by a 1.0 nl injection of 0.011 M oxytocin (11 picomoles) into the same site. Lastly, if intramedullary oxytocin effects on gastric and cardiac function are mediated via vagal efferents, then one would predict that these central effects could be antagonized by the vagal post-ganglionic blocker atropine. Therefore, additional animals (N=3) were prepared for the collection of gastric secretions and monitoring ECG activity as described before. Pipette/electrode arrays containing Ringer's and 0.011 M oxytocin were placed in the medullary locus which elicited gastric acid secretion and cardiac slowing in re-
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FIG. 3. Histogram illustrating the effects on gastric acid secretion in response to injection of various peptides into a physiologically identified pool of DMN neurons. Note that all effects are compared to the averaged (l hr) gastric acid secretion following a control injection of Ringer's (1.0 nl) into the DMN. Data plotted as means_+SEM. RC--is the averaged (l hr) gastric acid secretion following a control injection of Ringer's (1.0 nl) into the DMN. All such control values of secretion are equalized at 100% for purposes of comparison. VA--peak gastric acid secretion following injection of vasopressin (ll picomoles; N=6 and l l0 picomoles; N=3) into a pool of DMN neurons. OX--peak gastric acid secretion following injection of oxytocin (l 1 picomoles) into a pool of DMN neurons. TRH--peak gastric acid secretion following injection of TRH (11 picomoles) into a pool of DMN neurons. ETOV OX--peak gastric acid secretion following the oxytocin receptor antagonist ETOV (6 picomoles) followed by oxytocin (11 picomoles) injected into the same physiologically identified DMN locus. ATRO OX--peak gastric acid secretion following atropine (300 /zg/kg, IP) followed by oxytocin (ll picomoles) injected into a pool of DMN neurons. OX AP--peak gastric acid secretion following oxytocin (22 picomoles) applied onto the area postrema. TRH AP--peak gastric acid secretion following TRH (22 picomoles) applied onto the area postrema. *=p<0.05, sign test.
sponse to microstimulation. After a one hour stabilization period, a 1.0 nl Ringer's vehicle control was injected into the dorsal medulla. One hour later, a 300 ~g/kg dose of atropine was peripherally administered (IP). This atropine pretreatment was then followed 10 min later by an intramedullary injection of oxytocin (11 picomoles). The effects of atropine on central TRH-evoked acid secretion were not studied here. It is clear from other work [26] that atropine antagonizes the gastrointestinal effects of large doses of TRH administered intraventricularly. Results
Pretreatment with ETOV completely antagonized the effects of a subsequent intramedullary injection of oxytocin on gastric acid secretion (Fig. 3) and heart rate (Fig. 5). Both parameters remained at baseline levels following oxytocin injection in all animals tested that had been pretreated with ETOV. Additionally, pretreatment with peripherally administered atropine also completely antagonized the gastric and cardiac rate effects of centrally injected oxytocin. Indeed, as Figs. 3 and 5 illustrate, this dose of atropine produced a decline in acid output and a small elevation in heart rate, characteristic of the effects of atropine [29].
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FIG. 4. Typical time course of the gastric secretory responses following central application of peptides observed in this study. Top---effects of TRH (I 1 picomoles) injected into DMN (at "0") of preparation DMG19. Middle--effects of oxytocin (11 picomoles) injected into DMN (at "0") of preparation DMG3. Bottom--effects of vasopressin (11 picomoles) injected into DMN (at "0") of preparation DMG22.
DISCUSSION The results presented herein establish that pico-molar quantities of oxytocin and TRH injected into the dorsal motor nucleus of the vagus (DMN) produce significant increases in gastric acid secretion. Oxytocin also produces a concomitant, modest, though consistent, decline in heart rate, while TRH is without effect in this regard. Vasopressin is without effect on either measure. Selected dye injections revealed the possibility of minor injection leakage along the pipette track from the DMN into the area postrema. However, applications of oxytocin and TRH (at double the dosage) directly onto the area postrema did not yield significant gastric acid secretion or cardiac deceleration. Though it is clear that our central peptide injections invaded both the DMN and some of the overlying medial NST, this fact does not detract from the main result of this report that oxytocin and TRH significantly affect parasympathetic functions when injected in very small amounts into the "dorsal vagal complex." This may be attributed to the fact that several oxytocin- and TRHcontaining pathways originating in the forebrain appear to terminate in both the DMN and NST [6, 7, 20, 22]. Furthermore, activation of one such path (PVN to dorsal medulla) may affect vagal efferent activity by acting directly on the DMN or indirectly by altering the gain or setpoint of vagovagal reflexes [ 17,18]. Our results support the conclusions of Charpak et al. [5], who determined that oxytocin, or an oxytocin agonist, excited DMN neurons in vitro and that this excitation was
P E F F I D E S A N D GASTRIC ACID SECRETION completely antagonized by a synthetic structural analog of oxytocin. Vasopressin was ineffective in activating DMN neurons in this preparation. On the basis of these results, one would predict that oxytocin injected into the DMN should evoke parasympathetic effects, such as elevated gastric secretion and bradycardia similar to those observed following peripheral vagal stimulation or central DMN stimulation [13,30]. Further, a specific oxytocin antagonist should eliminate these effects of centrally injected oxytocin [1]. Finally, atropine pretreatment should eliminate the subsequent visceral effects of central oxytocin injections if these visceral effects are mediated by parasympathetic efferents. Our results verify these predictions. Though our observations on heart rate following intramedullary administration of oxytocin and vasopressin are in contrast with those of Vallejo et al. [28] and Schmid et al. [21], it should be noted that their central injections were centered on the nucleus of the solitary tract (NST), while ours were centered on the DMN. It should also be noted that the division of the NST involved in baroreflex regulation is considerably removed (i.e., 0.5 mm anterior and lateral) from that overlying our injection sites [9]. The NST region potentially involved in our study (i.e., directly above the posteromedial DMN) is related to subdiaphragmatic visceral sensory processing [2, 8, 10, 16, 18]. Thus, these conflicting reports may reflect real differences in oxytocin and vasopressin action in the anterior NST and the DMN, respectively. Detailed electrophysiological studies on physiologically identified NST and DMN neurons will be required to determine whether this is so. The differential effects of TRH and oxytocin on the same pool of dorsal vagal neurons is interesting in light of current knowledge of the organization of the DMN. That is, several anatomical and physiological studies directed at determining organ-specific relationships within the DMN have found, at best, rudimentary "viscero-topic" relationships, i.e., vagal " m o t o r neurons" innervating the gut, heart, liver, and pancreas are scattered throughout the DMN in no particular discernible order ([2, 8, 9, 10, 16, 18], W. Laughton, personal communication). It is possible that vagal motor function may be controlled by a set of highly specific, descending, point-to-point connections, i.e., randomly scattered hypothalamic neurons of the paraventricular nucleus, which are responsible for controlling gastric function, may make specific connections with equally randomly scattered vagal " m o t o r " neurons which innervate the stomach. However, it is also possible that the specificity of descending visceral motor control is n o t coded in specific connections between limbic/hypothalamic neurons and their vagal-motor counterparts. Rather, descending viscero-topic control may be encoded on the cell surface of DMN neurons in the form of the peptide receptors which they may or may not possess. Such an arrangement would not require either specific inputs from specific forebrain sites or, obviously, any specific organization of vagal motor neurons in the brainstem. Instead, it simply requires that neuropeptides involved in descending viscero-motor control be released widely throughout the DMN and that DMN neurons which make connections with one class of target organ have a different population of peptide receptors than DMN neurons serving a different tissue type. With such a scheme, all specific information concerning the type of visceral response " o r d e r e d " by descending influences is contained in the mix of neuropeptides released and the receptors contacted rather than the specific anatomical con-
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FIG. 5. Cardiovascular effects of oxytocin injected into the DMN (filled triangles). Oxytocin (11 picomoles) injected at "'0" produced a modest but consistent bradycardia. Injection of Ringer's, vasopressin (either dose), or TRH into the same locus had no detectable effect on heart rate. Likewise, when central injections of ETOV preceded microinjection of oxytocin into the DMN, the cardiovascular effects normally elicited by oxytocin were completely antagonized. These null results are not plotted as they all lie together on thel00% axis. Atropine pretreatment (300/xg/kg, IP; filled circles) yielded a characteristic, mild tachycardia; subsequent central injection of oxytocin into the DMN had no effect on the heart rate.
nections (i.e., labeled lines). This may explain how a relatively small pool of apparently randomly ordered vagal " m o t o r " neurons, which produce a variety of effects when stimulated electrically, can be induced to act differentially by two different peptides applied to the identical locus. Such an effect was clearly demonstrated in this study; TRH was a potent promoter of gastric acid secretion while oxytocin evoked both bradycardia and gastric secretion when applied to the DMN. Similar reports of " s p e c t r a " of peptide effects on visceral nerve function have appeared recently [23] which support this control scheme. Though suggestive, substantial supportive electrophysiological, immunocytochemical, and neuropharmacological evidence will be required before such a neural control hypothesis can be substantiated. ACKNOWLEDGEMENTS
This work was supported by an NIH grant (AM35346) and an American Heart Association, Nevada Affiliate grant to R.C.R. The oxytocin antagonist was the generous gift of Dr. M. Manning of the Department of Biochemistry, Medical College of Ohio, Toledo, OH 43699.
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ROGERS AND HERMANN
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18. Rogers, R. C. and G. E. Hermann. Gastric-vagal solitary neurons excited by paraventricular nucleus microstimulation. J Auton Nerv Syst, in press. 19. Rogers, R. C. and D. O. Nelson. Neurons of the vagal division of the solitary nucleus activated by the paraventricular nucleus of the hypothalamus. J Auton Nerv Syst 10: 193-197, 1984. 20. Sawchenko, P. E. and L. W. Swanson. 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: 260-272, 1982. 21. Schmid, P. G., F. M. Sharabi, G. Guo, F. M. Abboud and M. D. Thames. Vasopressin and oxytocin in the neural control of the circulation. Fed Proe 43: 97-102, 1984. 22. Sofroniew, M. V. and U. Schrell. Evidence for a direct projection from oxytocin and vasopressin neurons in hypothalamic paraventricular nucleus to the medulla oblongata: immunohistochemical visualization of both the horseradish peroxidase transported and the peptide produced by the same neurons. Neurosei Lett 22: 211-217, 1981. 23. Somiya, H. and T. Tonoue. Neuropeptides as central integrators of autonomic nerve activity: effects ofTRH, SRIF, VIP, and bombesin on gastric and adrenal nerves. Regu! Pept 9: 47-52, 1984. 24. Stone, T. W. Mieroiontophoresis and Pressure I~'l'ection, IBRO Handbook Series: Methods ofNeurosciences, vol 8. New York: John Wiley and Sons, 1985. 25. Swanson, L. W. and P. E. Sawchenko. Hypothalamic integration: organization of the paraventricular and supraoptic nuclei. Annu Rev Neurosei 6: 269-324, 1983. 26. Tache, Y., W. Vale and M. Brown. Thyrotropin-releasing hormone--CNS action to stimulate gastric acid secretion. Nature 287: 14%151, 1980. 27. Tamura, Y. and S. Maruyama. An apparatus for the assembly of a combined single barrel recording electrode and a multibarreled micropipette. J Neurosei Methods 1: 24%252, 1979. 28. Vallejo, M., D. A. Carter and S. L. Lightman. Haemodynamic efforts of arginine vasopressin microinjections into the nucleus tractus solitarius: a comparative study of vasopressin, a selective receptor agonist and antagonist and oxytocin. Neurosei Left 52: 247-252, 1984. 29. Weiner, N. Atropine, scopolamine, and related muscarinic drugs. In: The Pharmacological Basis of Therapeutics. 6th editions, Chapter 7, edited by A. G. Gilman, L. S. Goodman and A. Gilman. New York: Macmillan, Inc., 1980. 30. Wyrwricka, W. and R. Garcia. Effect of electrical stimulation of the dorsal nucleus of the vagus nerve on gastric secretion in cats. Exp Neurol 65: 315-325, 1979. 31. Zimmerman, E. A., G. Nilaver, A. Hou-Yu and A. J. Silverman. Vasopressinergic and oxytocinergic pathways in the central nervous system. Fed Proc 43: 91-96, 1984.
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Dorsal Medullary Oxytocin, Vasopressin, Oxytocin Antagonist, and TRH Effects on Gastric Acid Secretion and Heart Rate R I C H A R D C. R O G E R S A N D G E R L I N D A E. H E R M A N N
D e p a r t m e n t o f Physiology, University o f N e v a d a School o f Medicine, Reno, N V 89557 R e c e i v e d 3 S e p t e m b e r 1985 ROGERS, R. C. AND G. E. HERMANN. Dorsal medullary oxytocin, vasopressin, oxytocin antagonist, and TRH effects on gastric acM secretion and heart rate. PEPTIDES 6(6) 1143-1148, 1985.--Injections of oxytocin and TRH (11 picomoles), centered on the dorsal motor nucleus of the vagus, substantially increased gastric acid secretion. Additionally, oxytocin, but not TRH, simultaneously produced a consistent reduction in heart rate. Vasopressin injected into the same locus, at doses of 11 and 110 picomoles, had no effect on either function. Both the gastric and cardiac effects of oxytocin were eliminated by the central injections of oxytocin antagonist dEt2Tyr(Et)OrnSVasotocin (ETOV; 6 picomoles) or peripheral administration of atropine (300 p~g/kg, IP). Application of oxytocin or TRH to the area postrema, at double the dosage (22 picomoles) yielded no consistent effects on either gastric secretion or heart rate. These findings indicate that oxytocin in the dorsal motor nucleus of the vagus may act as a regulator of vagally-mediated gastric and cardiovascular functions while TRH effects, in this medullary area, seem limited to the regulation of gastric function. Oxytocin
Vasopressin
TRH
Gastric function
THE hypothalamus and limbic system are responsible for integrating inputs from a vast array of internal and external sensory receptors. The result of this integration is reflected in autonomic efferent activity which regulates the milieu interieur [12]. The paraventricular nucleus of the hypothalamus (PVN) is particularly interesting in this regard. This structure sends axons to both vagal parasympathetic and spinal sympathetic, preganglionic neurons, as well as to vagal sensory neurons in the nucleus of the solitary tract (NST) [20,22]. It seems likely, therefore, that the PVN can control the expression of autonomic functions through direct action on autonomic efferents and on those sensory neurons which form the afferent limb of brainstem autonomic reflexes [17, 18, 19, 25]. Recent immunohistochemical and physiological studies [6, 7, 22, 25, 31] have shown that these direct pathways between the PVN and vagal sensory and motor nuclei contain significant amounts of oxytocin, vasopressin and thyrotropin releasing hormone (TRH), as well as a variety of other peptides. Though it is currently known that TRH produces an increase in gastric secretion via vagal efferent excitation [17,26], it is not known where in the central nervous system TRH exerts this effect. That is, does TRH directly activate vagai neurons in the dorsal medulla or does it act on some other, intermediate locus? The effects of oxytocin and vasopressin on vagally regulated gastrointestinal functions are unknown and are controversial with respect to vagal regulation of cardiac, function [11, 21, 28]. In order to establish whether any of these three PVN neuropeptides have any direct effects on vagal efferent autonomic functions, we decided to inject picomolar quantities of these substances into physiologically identified medullary vagal-motor regions while recording gastric acid output and heart rate.
Heart rate
Oxytocin antagonist
GENERAL METHOD Male, Long-Evans rats (300-400 g) were used for these studies. All were food deprived 24 hr prior to urethane (1.5 g/kg) anesthesia. Dexamethasone (0.2-0.4 mg, SC) was given immediately before surgery to prevent cerebral edema. All animals were prepared for continuous gastric perfusion by placing an infusion catheter (PE50) in the stomach via the cervical esophagus; a drainage catheter (2 mm, o.d.) was secured in the pylorus via a small duodenal incision. After wound closure, cardiac electric activity was continuously monitored from needle electrodes placed subcutaneously in the left thorax and right hind limb. Warmed, unbuffered, saline (37°C, pH 6.8) was infused through the esophageal influx catheter via a Sage T M roller pump (model 371A) at a rate of 2 ml/min. This saline and any gastric secreta were constantly drained through the pyloric efflux catheter and collected in a small beaker. At 10 min intervals, the beaker was changed and the amount of acid secreted was determined by titration with 0.02 M NaOH and a conventional pH meter [17]. Gastric acid secretion was measured continuously throughout the experiment. The animal was placed in a stereotaxic frame with the head held in the nose-down position to allow surgical exposure of the dorsal spinomedullary junction. The obex region of the dorsal medulla was exposed by resecting the dorsal cervical musculature and removing the occipital skull plate. Composite stimulation/injectionelectrodes were constructed from tungsten microelectrodes and muitibarreled micropipette assemblies (Medical Systems). The stimulating microelectrode was electrolytically etched to a tip diameter of approximately 10/~m and then insulated with Epoxylite. Individual, internal tip diameters of the multibarreled micropipettes ranged from 3-8/xm. The metal stimulating elec-
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FIG. 1. (A) Combination pipette/electrode array used in these studies. Etched, insulated tungsten wire microelectrode is bent to conform with pipette tip and glued in place with cyanoacrylate, then epoxy. (B) Tip of injection/stimulation array shown in (A). Scale bar= 100/xm.
trode was glued into position such that it aligned with the micropipette array and protruded beyond the pipette array by approximately 40 p~m (Fig. 1). The construction of similar arrays is discussed elsewhere [14,27]. Pipette barrels were filled with Ringer's solution or solutions of 0.011 M arginine vasopressin (Bachem), 0.011 M oxytocin (Bachem), 0.011 M TRH (Bachem), 0.006 M d E_t.,Tyr(Et)OrnSVasotocin (ETOV--oxytocin/vasopressin antagonist; the generous gift of Dr. M. Manning), or 2.5% Pontamine dye, depending on the experimental conditions. Note that Ringer's was used as a vehicle for all injection solutions. The arrays were then mounted in a stereotaxic carrier. The pipettes were connected to a Medical Systems BH-2 micropressure ejector and the stimulating electrode was connected to a conventional physiological stimulator through a constant current stimulus isolation unit. EXPERIMENT
1
Method
Our preliminary studies and the results of others [13,30] indicated that electrical microstimulation (30 p.A, 0.3 msec duration, 10 Hz, 5 sec on/5 sec off, in trains 2-10 rain long) of the left, posteromedial portion of the dorsal motor nucleus of the vagus (DMN) produces a precipitous drop in heart rate as well as an abrupt increase in gastric acid outflow (Fig. 2). Histological analysis verified that these combined effects
were only obtained if the tip of the stimulation electrode was located within the boundaries of the DMN (Fig. 2). Therefore, we used this physiological identification procedure in the present study to guide the placement of our multibarrel arrays. Thus, the pipette/electrode tip was placed on the brainstem surface 0.2 mm lateral and 0.2 mm anterior to obex and directed toward the DMN; approximately 0.5 mm ventral to the brainstem surface. The array was then advanced in 50/~m increments and trains of 30/~A, 0.3 msec, 10 Hz pulses were applied to the stimulating electrode as described above. The pipette/electrode array was left in place when the aforementioned cardiac and gastric effects were obtained. The ejection protocol began one hour after final electrode array placement into this dorsal medullary locus. Ringer's solution (1.0 nl) was always microinjected first as a control measure, followed 60 min later by either oxytocin (0.011 M x 1.0 hi= 11 picomoles), vasopressin (0.011 M x 1.0 nl= 11 picomoles or 0.011 M× 10 hi= 110 picomoles), or TRH (0.011 M x 1.0 nl= 11 picomoles). Resultant changes in heart rate and gastric acid secretion were monitored, as described above. The precise volume of ejection was calibrated by measuring the movement of the meniscus in the pipette with a 200-power monocular microscope while applying trains of pressure pulses (10 and 30 psi, I00-400 msec duration). The details of precision pico-to-nanoliter volume delivery techniques have been described elsewhere [15,24]. At the end of the experiment, a small electrolytic lesion was made by passing cathodal direct current (30 p.A for 30 sec) through the stimulating electrode to mark the location of the array tip for subsequent histological verification of the injection locus. In several animals, an electrolytic lesion was not made, rather, Pontamine dye was injected (1.0 nl) to mark the tip location. This procedure also served as a visual estimate of the degree of spread through the surrounding tissue that such a volume of solution may have. After lesion or dye injection, animals (N=21) received an overdose of pentobarbital and were transcardially perfused with saline and Mirsky's fixative. The brainstems were then removed and stored in a formalin-30% sucrose solution before being sectioned (60 p.m) on a freezing microtome. Sections were then mounted, counterstained with cresyl violet and coverslipped prior to examination with a light microscope. Results
Injection of oxytocin (11 picomoles) into the dorsal posteromedial medulla evoked the same physiological response as elicited by electrical microstimulation (Fig. 2) of the identical locus, i.e., a substantial increase in acid secretion and a signficant decline in heart rate (Figs. 3 and 5). Identical quantities of TRH injected into the same region evoked a similar gastric acid secretory response (Fig. 3) but produced no detectable change in heart rate (Fig. 5). Vasopressin injected in the same identified locus at doses of 11 (N=6) and 110 (N=3) picomoles (0.011 M x l 0 nl) had no effect on either acid secretion (Fig. 3) or heart rate (Fig. 5). Since neither dose of vasopressin evoked changes in these physiological parameters, both dose groups are combined in the figures. In all cases, subsequent histological analysis verified that the array tips were localized within the dorsal motor nucleus of the vagus (DMN) (Fig. 2). Cases in which Pontamine dye was ejected to mark tip location and the degree of dispersion
PEPTIDES AND GASTRIC ACID SECRETION
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FIG. 2. Physiological identification of a DMN injection site. (A) Microstimulation (at vertical hatching; 30/zA, 0.3 msec pulses at 10 Hz, intermittent for 4.5 min) of DMN evokes gastric acid secretion. (B) Effects on heart rate as a consequence of the same microstimulation as in (A) above. (Note different time scale.) (C) Lesion performed through electrode in locus stimulated in (A) and (B) above (posteromedial DMN). DMN =dorsal motor nucleus of the vagus; L=lesion; NST=nucleus of the solitary tract; XII= hypoglossal nucleus. Scale bar= 100/xm.
revealed that injection volumes as small as 1.0 nl could still travel a considerable distance vertically along the cannula track. Of particular concern was the fact that dye injected into the DMN also consistently stained the lateral wall of the area postrema, a significant chemoreceptor structure which contains neurons sensitive to vasopressin and TRH [3,4]. Though this dye "tracking" to the brainstem might have occurred when the electrode was withdrawn, we still wished to control for any peptide contamination of the area postrema, however minor. EXPERIMENT 2
Method In order to verify that the observed physiological responses were attributable to direct effects of these peptides at the site of injection (i.e., dorsal medullary neurons centered on the DMN) and not due to secondary effects of peptide leakage and action at the area postrema, we repeated Experiment ! (N= 12) with the exception that the effective peptide agents (oxytocin and TRH) were applied at double the dosage (0.011 M×2.0 n1=22 picomoles) directly onto the left, dorsolateral area postrema overlying the solitary nucleus and posteromedial DMN. Note that the dura and arachnoid meninges were always removed to fully expose the obex region to the applied peptide solutions. Results Though both oxytocin and TRH produced modest increases in gastric secretion when placed on the area postrema, these effects were not significant (Fig. 3). Furthermore, application of these peptides to the area postrema
resulted in no consistent nor significant alteration in heart rate. EXPERIMENT 3
Method If the intramedullary effects of oxytocin on gastric secretion and heart rate regulation are due to specific activation of oxytocin receptors, then these effects should be blocked by a structural antagonist of oxytocin action [dEt~Tyr(Et)OrnSVasotocin (ETOV)] [1]. Animals (N=3) were prepared as described previously. Here, however, the pipette/electrode array was filled with 0.006 M ETOV, 0.011 M oxytocin, and Ringer's solution. The tip of the pipette/electrode array was placed in a site which yielded cardiac slowing and an increment in gastric acid secretion following electrical microstimulation (as described previously). After a one hour stabilization period, 1.0 nl of Ringer's solution was injected as a vehicle control. One hour later, a 1.0 nl volume of 0.006 M ETOV (6 picomoles) was injected. This antagonist pretreatment was then followed 10 min later by a 1.0 nl injection of 0.011 M oxytocin (11 picomoles) into the same site. Lastly, if intramedullary oxytocin effects on gastric and cardiac function are mediated via vagal efferents, then one would predict that these central effects could be antagonized by the vagal post-ganglionic blocker atropine. Therefore, additional animals (N=3) were prepared for the collection of gastric secretions and monitoring ECG activity as described before. Pipette/electrode arrays containing Ringer's and 0.011 M oxytocin were placed in the medullary locus which elicited gastric acid secretion and cardiac slowing in re-
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FIG. 3. Histogram illustrating the effects on gastric acid secretion in response to injection of various peptides into a physiologically identified pool of DMN neurons. Note that all effects are compared to the averaged (l hr) gastric acid secretion following a control injection of Ringer's (1.0 nl) into the DMN. Data plotted as means_+SEM. RC--is the averaged (l hr) gastric acid secretion following a control injection of Ringer's (1.0 nl) into the DMN. All such control values of secretion are equalized at 100% for purposes of comparison. VA--peak gastric acid secretion following injection of vasopressin (ll picomoles; N=6 and l l0 picomoles; N=3) into a pool of DMN neurons. OX--peak gastric acid secretion following injection of oxytocin (l 1 picomoles) into a pool of DMN neurons. TRH--peak gastric acid secretion following injection of TRH (11 picomoles) into a pool of DMN neurons. ETOV OX--peak gastric acid secretion following the oxytocin receptor antagonist ETOV (6 picomoles) followed by oxytocin (11 picomoles) injected into the same physiologically identified DMN locus. ATRO OX--peak gastric acid secretion following atropine (300 /zg/kg, IP) followed by oxytocin (ll picomoles) injected into a pool of DMN neurons. OX AP--peak gastric acid secretion following oxytocin (22 picomoles) applied onto the area postrema. TRH AP--peak gastric acid secretion following TRH (22 picomoles) applied onto the area postrema. *=p<0.05, sign test.
sponse to microstimulation. After a one hour stabilization period, a 1.0 nl Ringer's vehicle control was injected into the dorsal medulla. One hour later, a 300 ~g/kg dose of atropine was peripherally administered (IP). This atropine pretreatment was then followed 10 min later by an intramedullary injection of oxytocin (11 picomoles). The effects of atropine on central TRH-evoked acid secretion were not studied here. It is clear from other work [26] that atropine antagonizes the gastrointestinal effects of large doses of TRH administered intraventricularly. Results
Pretreatment with ETOV completely antagonized the effects of a subsequent intramedullary injection of oxytocin on gastric acid secretion (Fig. 3) and heart rate (Fig. 5). Both parameters remained at baseline levels following oxytocin injection in all animals tested that had been pretreated with ETOV. Additionally, pretreatment with peripherally administered atropine also completely antagonized the gastric and cardiac rate effects of centrally injected oxytocin. Indeed, as Figs. 3 and 5 illustrate, this dose of atropine produced a decline in acid output and a small elevation in heart rate, characteristic of the effects of atropine [29].
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FIG. 4. Typical time course of the gastric secretory responses following central application of peptides observed in this study. Top---effects of TRH (I 1 picomoles) injected into DMN (at "0") of preparation DMG19. Middle--effects of oxytocin (11 picomoles) injected into DMN (at "0") of preparation DMG3. Bottom--effects of vasopressin (11 picomoles) injected into DMN (at "0") of preparation DMG22.
DISCUSSION The results presented herein establish that pico-molar quantities of oxytocin and TRH injected into the dorsal motor nucleus of the vagus (DMN) produce significant increases in gastric acid secretion. Oxytocin also produces a concomitant, modest, though consistent, decline in heart rate, while TRH is without effect in this regard. Vasopressin is without effect on either measure. Selected dye injections revealed the possibility of minor injection leakage along the pipette track from the DMN into the area postrema. However, applications of oxytocin and TRH (at double the dosage) directly onto the area postrema did not yield significant gastric acid secretion or cardiac deceleration. Though it is clear that our central peptide injections invaded both the DMN and some of the overlying medial NST, this fact does not detract from the main result of this report that oxytocin and TRH significantly affect parasympathetic functions when injected in very small amounts into the "dorsal vagal complex." This may be attributed to the fact that several oxytocin- and TRHcontaining pathways originating in the forebrain appear to terminate in both the DMN and NST [6, 7, 20, 22]. Furthermore, activation of one such path (PVN to dorsal medulla) may affect vagal efferent activity by acting directly on the DMN or indirectly by altering the gain or setpoint of vagovagal reflexes [ 17,18]. Our results support the conclusions of Charpak et al. [5], who determined that oxytocin, or an oxytocin agonist, excited DMN neurons in vitro and that this excitation was
P E F F I D E S A N D GASTRIC ACID SECRETION completely antagonized by a synthetic structural analog of oxytocin. Vasopressin was ineffective in activating DMN neurons in this preparation. On the basis of these results, one would predict that oxytocin injected into the DMN should evoke parasympathetic effects, such as elevated gastric secretion and bradycardia similar to those observed following peripheral vagal stimulation or central DMN stimulation [13,30]. Further, a specific oxytocin antagonist should eliminate these effects of centrally injected oxytocin [1]. Finally, atropine pretreatment should eliminate the subsequent visceral effects of central oxytocin injections if these visceral effects are mediated by parasympathetic efferents. Our results verify these predictions. Though our observations on heart rate following intramedullary administration of oxytocin and vasopressin are in contrast with those of Vallejo et al. [28] and Schmid et al. [21], it should be noted that their central injections were centered on the nucleus of the solitary tract (NST), while ours were centered on the DMN. It should also be noted that the division of the NST involved in baroreflex regulation is considerably removed (i.e., 0.5 mm anterior and lateral) from that overlying our injection sites [9]. The NST region potentially involved in our study (i.e., directly above the posteromedial DMN) is related to subdiaphragmatic visceral sensory processing [2, 8, 10, 16, 18]. Thus, these conflicting reports may reflect real differences in oxytocin and vasopressin action in the anterior NST and the DMN, respectively. Detailed electrophysiological studies on physiologically identified NST and DMN neurons will be required to determine whether this is so. The differential effects of TRH and oxytocin on the same pool of dorsal vagal neurons is interesting in light of current knowledge of the organization of the DMN. That is, several anatomical and physiological studies directed at determining organ-specific relationships within the DMN have found, at best, rudimentary "viscero-topic" relationships, i.e., vagal " m o t o r neurons" innervating the gut, heart, liver, and pancreas are scattered throughout the DMN in no particular discernible order ([2, 8, 9, 10, 16, 18], W. Laughton, personal communication). It is possible that vagal motor function may be controlled by a set of highly specific, descending, point-to-point connections, i.e., randomly scattered hypothalamic neurons of the paraventricular nucleus, which are responsible for controlling gastric function, may make specific connections with equally randomly scattered vagal " m o t o r " neurons which innervate the stomach. However, it is also possible that the specificity of descending visceral motor control is n o t coded in specific connections between limbic/hypothalamic neurons and their vagal-motor counterparts. Rather, descending viscero-topic control may be encoded on the cell surface of DMN neurons in the form of the peptide receptors which they may or may not possess. Such an arrangement would not require either specific inputs from specific forebrain sites or, obviously, any specific organization of vagal motor neurons in the brainstem. Instead, it simply requires that neuropeptides involved in descending viscero-motor control be released widely throughout the DMN and that DMN neurons which make connections with one class of target organ have a different population of peptide receptors than DMN neurons serving a different tissue type. With such a scheme, all specific information concerning the type of visceral response " o r d e r e d " by descending influences is contained in the mix of neuropeptides released and the receptors contacted rather than the specific anatomical con-
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FIG. 5. Cardiovascular effects of oxytocin injected into the DMN (filled triangles). Oxytocin (11 picomoles) injected at "'0" produced a modest but consistent bradycardia. Injection of Ringer's, vasopressin (either dose), or TRH into the same locus had no detectable effect on heart rate. Likewise, when central injections of ETOV preceded microinjection of oxytocin into the DMN, the cardiovascular effects normally elicited by oxytocin were completely antagonized. These null results are not plotted as they all lie together on thel00% axis. Atropine pretreatment (300/xg/kg, IP; filled circles) yielded a characteristic, mild tachycardia; subsequent central injection of oxytocin into the DMN had no effect on the heart rate.
nections (i.e., labeled lines). This may explain how a relatively small pool of apparently randomly ordered vagal " m o t o r " neurons, which produce a variety of effects when stimulated electrically, can be induced to act differentially by two different peptides applied to the identical locus. Such an effect was clearly demonstrated in this study; TRH was a potent promoter of gastric acid secretion while oxytocin evoked both bradycardia and gastric secretion when applied to the DMN. Similar reports of " s p e c t r a " of peptide effects on visceral nerve function have appeared recently [23] which support this control scheme. Though suggestive, substantial supportive electrophysiological, immunocytochemical, and neuropharmacological evidence will be required before such a neural control hypothesis can be substantiated. ACKNOWLEDGEMENTS
This work was supported by an NIH grant (AM35346) and an American Heart Association, Nevada Affiliate grant to R.C.R. The oxytocin antagonist was the generous gift of Dr. M. Manning of the Department of Biochemistry, Medical College of Ohio, Toledo, OH 43699.
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ROGERS AND HERMANN
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18. Rogers, R. C. and G. E. Hermann. Gastric-vagal solitary neurons excited by paraventricular nucleus microstimulation. J Auton Nerv Syst, in press. 19. Rogers, R. C. and D. O. Nelson. Neurons of the vagal division of the solitary nucleus activated by the paraventricular nucleus of the hypothalamus. J Auton Nerv Syst 10: 193-197, 1984. 20. Sawchenko, P. E. and L. W. Swanson. 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: 260-272, 1982. 21. Schmid, P. G., F. M. Sharabi, G. Guo, F. M. Abboud and M. D. Thames. Vasopressin and oxytocin in the neural control of the circulation. Fed Proe 43: 97-102, 1984. 22. Sofroniew, M. V. and U. Schrell. Evidence for a direct projection from oxytocin and vasopressin neurons in hypothalamic paraventricular nucleus to the medulla oblongata: immunohistochemical visualization of both the horseradish peroxidase transported and the peptide produced by the same neurons. Neurosei Lett 22: 211-217, 1981. 23. Somiya, H. and T. Tonoue. Neuropeptides as central integrators of autonomic nerve activity: effects ofTRH, SRIF, VIP, and bombesin on gastric and adrenal nerves. Regu! Pept 9: 47-52, 1984. 24. Stone, T. W. Mieroiontophoresis and Pressure I~'l'ection, IBRO Handbook Series: Methods ofNeurosciences, vol 8. New York: John Wiley and Sons, 1985. 25. Swanson, L. W. and P. E. Sawchenko. Hypothalamic integration: organization of the paraventricular and supraoptic nuclei. Annu Rev Neurosei 6: 269-324, 1983. 26. Tache, Y., W. Vale and M. Brown. Thyrotropin-releasing hormone--CNS action to stimulate gastric acid secretion. Nature 287: 14%151, 1980. 27. Tamura, Y. and S. Maruyama. An apparatus for the assembly of a combined single barrel recording electrode and a multibarreled micropipette. J Neurosei Methods 1: 24%252, 1979. 28. Vallejo, M., D. A. Carter and S. L. Lightman. Haemodynamic efforts of arginine vasopressin microinjections into the nucleus tractus solitarius: a comparative study of vasopressin, a selective receptor agonist and antagonist and oxytocin. Neurosei Left 52: 247-252, 1984. 29. Weiner, N. Atropine, scopolamine, and related muscarinic drugs. In: The Pharmacological Basis of Therapeutics. 6th editions, Chapter 7, edited by A. G. Gilman, L. S. Goodman and A. Gilman. New York: Macmillan, Inc., 1980. 30. Wyrwricka, W. and R. Garcia. Effect of electrical stimulation of the dorsal nucleus of the vagus nerve on gastric secretion in cats. Exp Neurol 65: 315-325, 1979. 31. Zimmerman, E. A., G. Nilaver, A. Hou-Yu and A. J. Silverman. Vasopressinergic and oxytocinergic pathways in the central nervous system. Fed Proc 43: 91-96, 1984.