Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular nucleus stimulation effects on gastric motility

Pepttde~, Vol 8, pp 505-513 ~ Pergamon Journals L t d , 1987 Printed m the U S A

0196-9781/87 $3 00 + 00

Oxytocin, Oxytocin Antagonist, TRH, and Hypothalamic Paraventricular Nucleus Stimulation Effects on Gastric Motility R I C H A R D C. R O G E R S 1 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, The Ohio State University College o f Medic ine, Columbus, O H 43210 R e c e i v e d 28 N o v e m b e r 1986 ROGERS, R. C AND G E HERMANN. Oxytocm. ox)totln antagomst. TRH. and hypothalanm paraventrt~u/ar nu~ leu~ ~nmulation effec t~ on ga~trtc motthty PEPTIDES 8(3) 505-513, 1987 --The roles ofthyrotropin releasing hormone (TRH) and oxytocm as central regulators of gastric motihty were investigated, lhcomolar (4 plcomoles) quantmes of TRH injected into the dorsal motor nucleus of the vagus (DMN) ehclted a slgmficant increase m gastric motlhty while the same quantity of oxytocm ehcited a reductmn m phasic contractile acUwty and tone The actmn of these peptides mimics the excitatory and inhibitory effects of stimulating the paraventncular nucleus of the hypothalamus (PVN); it is hkely that this hypothalamlc structure regulates gastric function through ~ts peptlderglc connections w~th medullary vagal structures Th~s hypothesis is supported by our observations that mjectmns of an oxytocm antagonist into the DMN produced a dlsmhlbltmn of gastric motihty and an increase m the motlhty evoked by subsequent PVN stlmulatmn. Vagotomy ehmmated all subsequent central effects on motility of these peptldes Oxytocm TRH Gastnc motthty

Paraventncular nucleus of the hypothalamus

Dorsal motor nucleus of the vagus

of the present study was to examine whether the site of action of TRH responsible for producing this increase in gastrlc motility might be within the DMN. Additionally, we sought to examine the possible role of oxytocin within this PVN-DMN pathway and its influence on gastric motility. Therefore, the effects of DMN injections of oxytocm or the oxytocin antagonist, dEt2Tyr(Et)Orn ~ Vastocin (ETOV) [1], and PVNmp microstimulation on gastric motility were also examined. It is well known that excitatory vagal influences over gastric motility utilize a muscarinic cholinergic synapse on smooth muscle while inhibitory vagal inputs to gastric smooth muscle are non-cholinergic [6, 7, 13]. We, therefore, used atropine in combination with the above mentioned manipulations to try to establish which (if any) of the effects obtained on gastric motility following the aforementioned manipulations were dependent on a muscarinlc synapse

THE paraventricular nucleus of the hypothalamus (PVN) is emerging as a principal diencephalic controller of autonomic efferent activity. Numerous anatomical studies provide evidence for direct, monosynaptic projections between the PVN and preganglionlc autonomic neurons in the medulla [15, 20, 23, 25, 28]. The existence of these connections have been confirmed with electrophysiological techniques [12]. These PVN-preganglionic vagal efferent connections may be particularly important with regard to forebrain control of gastric function. For example, electrical microstimulation of the medial parvocellular division of the PVN (PVNmp), which projects directly to a medullary area containing the dorsal motor nucleus of the vagus (DMN), evokes large increases in gastric secretion [19, 20, 23, 25]. This PVN-dorsal medullary pathway contains significant amounts of oxytocin and thyrotropm releasing hormone (TRH) [4, 8, 10, 23, 25, 29, 31]. Both of these peptides have been strongly implicated as neurotransmitters released from PVN terminals onto gastric vagal neurons in the DMN which control gastric secretion [3, 5, 8, 10, 20, 23, 25, 29, 31]. Tache reports that intraventricular injections of TRH evoke large increments in gastric motility [26], indicating that TRH might also be a central regulator of gastric motility. Thus, the goal

METHOD

General Pro~ edure~ Male, Long-Evans rats (300-400 g) were food deprived 24

1Requests for repnnts should be addressed to R. C. Rogers, Department of Physiology, The Ohio State Umverslty, College of Medicine, 333 W. 10th Ave , Columbus, OH 43210.

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FIG 1 Mmmture extralummal strata gauge measurements of gastric motdzty following brief electrical stimulation (2 sec, 10/~A, 10 Hz, 0 3 msec duration) of the DMN. (A) Control sUmulatlon before atropine (B) Same ammal and stlmulauon parameters as above but 30 minutes after atropme (400 p.g/kg, IP)

hr prior to urethane (1 5 g/kg, 1P) anesthesm Dexamethasone (0.2-0.4 mg, SC) was given immedmtely before surgery to prevent cerebral edema at the time of craniotomy In all subjects (n=36), a minmture strain gauge [2] was sutured to the corpus of the stomach such that contracUons of gastric circular smooth muscle (i.e., gastric motdity) could be monitored; EKG actwity was recorded using two transdermal electrodes Animals were divided into two groups. The first group (n=24) was prepared for bralnstem (Le., DMN) mampulat~ons. In th~s group, the ammal was placed m a stereotaxic frame with the head held in the nose-down position (10-12 mm below ear bar zero) to allow surglcal exposure of the dorsal spinomedullary junction The obex region of the dorsal medulla was exposed by resectmg the dorsal cervical musculature and removing the occipital skull plate. Composite stimulation/injection electrodes were constructed from tungsten microelectodes and multibarreled glass micropipettes (individual internal tip diameter=7-10 /zm) as described m earher reports [16, 18, 19, 27]. Pipette barrels were filled with artificial cerebrospinal flmd (ACSF; glucose- and calcmm-free), 0.002 M dE-t_,Tyr(Et)Orns Vasotocm ( E T O V - - o x y t o c m antagomst, the generous gift of Dr. M. Manning), 0.002 M oxytocm, and 0 002 M TRH dissolved m ACSF. The mlcroplpettes were connected to a mlcropressure ejecUon system [16,18], the stimulating electrode was connected to a conventional physiologtcal sumulator through a constant current stimulus ~solaUon umt The composite electrode was then mounted m a stereotax~c carner and directed toward the left dorsal motor nucleus of the vagus (DMN) under physiological guidance. Briefly, it has been shown in the rat that electrical sUmulation of the DMN evokes an increase in phasic gastric contractions which are superamposed upon a decline m gastric tone; such stimulation also evokes a sharp drop in heart rate [14,22]. Furthermore, a dose of atropine (400/zg/kg, IP) delivered prior to stimulation of vagal efferents eliminates the increase in gastric contractions but preserves the reduction m tone. These data have been used to support a mechanism of dual vagal control over gastric motility. A chohnerglc,

muscanmc mechamsm is responsible for the increase in gastnc contractions while a non-muscanmc mechanism is responsible for the simultaneous reducuon in tone [6,7]. Therefore, we used these physiological indices as a guide to directmg the electrode/pipette tip to the DMN. The p~petteelectrode Up was placed on the dorsal surface of the bramstem 0.2 mm lateral and 0 2 mm anterior to the obex Trams of electrical pulses (2 sec, 25 /zA, 10 Hz, 0.3 msec duration) were applied to the electrode as it was advanced slowly into the brainstem until pronounced bradycardla, increase m phasic gastric contractions, and decrease m tone were observed (Fig. 1) Subsequent histological analysis vemfied that these results were obtained only when the stimulating electrode was placed m the DMN (Fig. 2) The other group (n= 12) was prepared for sUmulatlon of the PVN. Due to the nose-down position of the head m the stereotaxic frame, the vertical axis of the m~croelectrode carrier was tilted at approximately a 12 degree angle rostrally such that lambda and bregma were co-planar and perpendicular to the electrode. A trephination in the frontal skull plate was made directly over the left PVN (1.1 mm posterior to bregma, 0.5 mm lateral to midline, 6.0--7.5 mm ventral to surface of brain). A tungsten microelectrode, electrolytlcally etched to a tip dmmeter of approximately 10/zm, insulated with Epoxyhte, and having an impedance range of 100 kohm at 1 kHz, was directed toward the PVN. Our preliminary studies (Fig. 9) demonstrated that trams of electrical stimulation (50 tzA, 20 Hz, 0.3 msec duration) of the medial parvocellular portion of the PVN (PVNmp) ehcits a modest bradycardla [19] and a change m gastric motihty which is characterized by an increase m motility followed closely by a reduction. Therefore, we used this Identification procedure m the present study to guide the placement of the PVNm,, stimulating electrode. Th~s mlcroelectrode was lowered to 6.0 mm below the surface of the brain. Thirty to sixty second trams of stimulation, as described above, were applied to the PVNmp electrode as ~t was advanced through the brain at 250/zm increments. A transient bradycardia and change m gastric motdity coincident with stimulation were taken as positive ewdence that the electrode was placed in

OXYTOCIN, TRH AND PVN STIMULATION

FIG 2 Histological verification of electrode placements Upper panel coronal section through the dorsal medulla showing the marker lesion in the left dorsal motor nucleus of the vagus coinciding with the location of the tip of the combined stimulating electrode-reJection mlcroplpette. Scale bar= 1 mm Lower panel coronal section through the dorsal hypothalamus showing the posmon of a marker lesion m the PVNmp coincident with the location of the hypothalamlc stimulation electrode Scale bar=l mm. C=central canal, DMN=dorsal motor nucleus of the vagus, H=hypoglossal nucleus, PVNm~=magnocellular paraventrlcular nucleus, PVNm~=medlal parvocellular division of the paraventncular nucleus

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the PVNmn. Subsequent histological analysis (Fig. 2) always verified this assumption

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Experimental Procedures DMN-pepnde effi'~ ts on gastric motthty Once the electrode/pipette array was positioned in the DMN, a minimum period of 30 minutes was allowed for the collection of baseline motility records. At the end of this period, artificial CSF (ACSF; 2 nanohters) was injected into the DMN as a vehicle control, followed 30 minutes later by an injection of either TRH (2 nanohters × 0.002 M = 4 picomoles) or oxytocm (2 nanoliters × 0.002 M = 4 plcomoles). All injections were made under direct visual control according to methods described elsewhere [16,18]. A total of 24 animals were used for this section of the project. Data collected m this section of the study were converted to 5 minute motility indices [15] for statistical and graphical purposes. Briefly, the motility index assigns a numerical value to an epoch of strain gauge data according to the formula' MI=(N ×A~ 2)+(N ×A2 0 × 2 + ( N × A 4 ~ ) × 4 + ( N × A ~ ) × 8 where MI=mo]lhty per unit time (in this case, 5 minutes), (N × A~ 2) = number of contractions m the amphtude range from "'just-detectable"* to twice justdetectable contraction (N × A., ~) = number of contrachons in the amphtude range from 2 to 4 times the just-detectable contraction (N × A~4 s) = number ofcontrachons m the amplitude range from 4 to 8 hmes the just-detectable contraction (N × A>s) = all those contractions that are at least 8 hmes greater than the just-detectable contraction (*For these studies, "just-detectable'" gastric contractions that could be discriminated from respiratory movements were 0.5 g in amplitude.) To determine whether these effects on motility produced by DMN peptide injections were attributable to vagal mnervation, six of the 24 animals included In this study were prepared as described previously except that they also had suture loops placed around the cervical vagal trunks such that vagotomy could be performed during the course of the experiment. In these cases, ACSF and TRH (n=3) or oxytocm (n=3) were injected as described above. Following the initial peptlde injection, the animal was vagotomized by pulling the vagal loops. Once a stable basehne was re-established, TRH or oxytocin was again injected into the DMN. Another six animals of this group of 24 were used to determine if atropine interfered with the gastric motility effects caused by the DMN peptlde injections. Thus, basic preparations and protocols in this section of the study proceded exactly as in the vagotomy study above except that the animal received a 400 /zg/kg dose of atropine sulfate, IP (rather than vagotomy) 30 minutes after the first DMNpeptide injection. P V N Mtcroanmulatton The basic protocol for examining PVN microstimulation effects on gastric motility was the same as described above except that the PVN was microstimulated (50 tzA, 20 Hz, 0 3 msec, 2 min) in place of peptide injections into the DMN A total of 12 animals were used to establish the effects of PVN microstimulation on gastric motility. Of this group of twelve,

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three animals were also prepared with vagal loops to determine if vagotomy eliminated the gastric motility effects of PVN stimulaUon. Simdarly, three other ammals received an injection of atropine (400 p.g/kg, IP) to determine if muscannlc blockade interrupted the effects of PVN stimulation on gastric motility. The fourth subgroup of these animals (n=3) was prepared for both PVN mlcrostlmulation and for injection of ACSF and ETOV into the DMN as described above. Here, ACSF (2 nanoliters) was injected into the DMN, as a volume/vehicle control measure, 5 minutes prior to the first PVN sumulation ETOV (4 picomoles m 2 nanoliters) was injected into the DMN 30 minutes later 0.e., 5 minutes prior to the second PVN stimulation) to determine whether any of the gastric motility effects of PVN stimulation are interrupted by this oxytocin antagonist. At the end of each experiment, small electrolytic lesions were made by passing cathodal direct current (30/zA for 30 sec) through the stimulating electrodes in both the bramstem and hypothalamus to mark their locations for subsequent histological verification of the injection and stimulation loci.

RESULTS

Effects on Gastric Monhty o[" TRH and Oxyto( in InJections Into the D M N As Fig. 3 illustrates, control injections of ACSF (2 nanohters) into the DMN had no effect on the baseline contractde state of the stomach. However, TRH (4 picomoles in 2 nanoliters) evokes a large increase m phasic contraction amplitude and a moderate increase m contraction frequency Oxytocin (4 picomoles in 2 nanoliters) has the opposite effect--a reduction in contractile activity. These effects are graphically depicted in Fig. 4.

OXYTOCIN, TRH AND PVN S T I M U L A T I O N

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FIG 4 Gastric motdlty as measured by the 5-minute motility index for periods 5 minutes before DMN rejection (-5-0), 5 minutes after DMN rejection (0-5), and 10 minutes after rejection (5-10). Upper panel, effects of DMN rejections of ACSF (2 nanohters), TRH (2 nanohters × 2 mM) and oxytocm (2 nanohters × 2 mM) on gastric motility At this dosage, TRH produces a slgmficant elevation m gastric motihty for both post-rejection periods (* =p<0.05 S~gnTest; standard error bars added as a measure of within-group variance) Oxytocm, at the same dosage under identical conditions, produces a s~gmficant reduction m gastnc motd~ty for both post-treatment periods (*=p<0 05, Sign Test) Lower panel, effects ofACSF, TRH and oxytocm on thefrequenc y of gastric contractions for the periods mentioned above TRH produces a slgmficant increase m the frequency of contractions (*=p<0 05, Sign Test), whde oxytocm produces a transRory reduction m the frequency of contractions (*=p<0.05, Sign Test). For both upper and lower panels, ACSF group N= 12, TRH group N=6, OX group N=6

FIG. 5 Effect of vagotomy on gastric contractions produced by rejections of TRH into the DMN. (A) Pre-vagotomy control rejection of TRH (2 nanohters × 2 mM). (B) Vagotomy (at VX) 30 mln followmg TRH rejection m (A) above under basal motdlty condmons (C) Effects of injections of TRH into the DMN 30 mm after vagotomy m (B) above Note that TRH doses 10x higher than the original effective dose now have no effect on motlhty These results are typical of the 3 preparations done under these conditions

Figures 5 and 6 dlustrate that vagotomy completely eliminates these DMN-oxytocin and -TRH effects on gastric motility. This was true for every case. Similarly, atropine completely ehminates any effect that DMN-TRH rejections have on gastric motility (Fig. 7). Though atropine reduces the inhibitory effect of DMN oxytocm injections, it does not completely eliminate this inhibition. As Fig. 8 demonstrates, it is possible to detect a slight reduction in gastric motility in an atropinized preparation following oxytocin injections into the DMN.

It has long been known that electrical stimulation of mixed vagal efferents produces changes in gastric motility patterns which are characterized by an increase in phasic contractions superimposed on a reduction in gastric muscular tone [13,22]. It seems clear that this mixed pattern of excitation and inhibition results from stimulation of two kinds of vagal neurons; one corresponding to the classical cholinergic excitatory pathway which terminates on gastric smooth muscle. The other, an inhibitory pathway, probably uses a nicotinic synapse between pre- and post-ganglionic neurons while the contact with gastric smooth muscle may be purinergic or peptldergic [6]. Though little IS presently known about how the central nervous system regulates these excitatory and inhibitory vagal paths to the stomach, there is ample evidence that central neural influences can powerfully modulate gastric motility by acting on these vagal outputs [7]. In this report, we have presented preliminary evidence which suggests that these vagal excitatory and inhibitory pathways controlling gastric smooth muscle may be controlled to some degree by the release of TRH and oxytocin from the terminal endings

tion m ammals pretreated with ETOV (Fig. 10). Vagotomy (n=3) eliminated the effects of PVNmp stimulation on gastric motlhty (Fig. 11). Intraperitoneai atropine (400/zg/kg, n=3) eliminated the increase in gastric motility produced by PVNmp stimulation but did not completely ehminate the PVN stimulation-reduced reduction in gastric tone (Fig. ll). DISCUSSION

Effects o f PVNm~ Sttmulanon and ETOV on Gastric Monhty Preliminary and subsequent experimental results revealed that electrical microstimulation of the PVNmpproduced an elevation in phasic gastric contractions often followed by a modest reduction in gastric motility (Fig. 9). The injection of the oxytocin antagonist, ETOV (4 picomoles in 2 nanoliters), into the DMN (n=3) prior to PVN stimulation caused an immediate increase in gastric motility and augmented the increase in motility to subsequent PVNmp stimulation. No decline in motility was observed following PVNmp stimula-

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FIG 7. (A) Effect on gastric moUhty of an mjecUon of TRH into the DMN prior to atropmlzahon (B) Effect of an identical TRH mjectton 30 minutes following an IP mjecuon of atropine (400 ~g/kg) These results are typical of the 3 preparatmns done under these condmons.

o f c e r t a i n h y p o t h a l a m i c p a r a v e n t n c u l a r n e u r o n s , ~ e., the PVNmw Specifically, p i c o m o l a r a m o u n t s o f T R H i n j e c t e d into physiologically identified p o o l s o f D M N n e u r o n s p r o d u c e s u b s t a n t i a l m ( r e a r e ~ in m o t d i t y . T h e s e T R H effects are e l i m i n a t e d b y v a g o t o m y a n d a t r o p i n e , facts w h i c h confirm t h a t T R H acts o n a n e x c i t a t o r y vagal e f f e r e n t p a t h w a y utilizing a m u s c a r i n i c s y n a p s e o n gastric s m o o t h m u s c l e [26]. T h l s c o n c l u s i o n is also s u p p o r t e d b y (a) o u r o b s e r v a U o n t h a t s t i m u l a t i o n o f the PVNmo p r o v o k e s a n initml i n c r e a s e m m o t d l t y as well as (b) the e x i s t e n c e of a s u b s t a n t m l T R H c o n t a i n i n g p a t h w a y f r o m the P V N to the d o r s a l m e d u l l a [10, 20, 23, 25]. H o w e v e r , the d e v e l o p m e n t o f a specific T R H a n t a g o m s t will still be n e c e s s a r y to p r o v e the h y p o t h e s i s t h a t a T R H - c o n t a i n m g P V N - D M N path is r e s p o n s i b l e for augm e n t m g gastric motility. I n j e c t i o n s o f o x y t o c m into t h e D M N m p i c o m o l a r a m o u n t s ehc~t a s u b s t a n t i a l r e d u c n o n in gastric motd~ty,

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FIG 8 Effects of oxytocm, ACSF and TRH on moUhty patterns following atropmlzaUon Upper panel mh~bmon of gastric motility following an rejection of oxytocm into the DMN (2 nanohters × 2 raM) prior to atropine mjechon Second panel effect on gastric motdRy of an ~dentlcal oxytocm mJecUon into the DMN 30 minutes after an IP rejection of atropme (400/xg/kg). Note the reduction m basehne strata, indicating a considerable relaxation of the stomach Note further that though basal motd~ty ~s considerably reduced, ~t ~s stdl possible to detect a period of relatively reduced motdlty followmg oxytocm (OX2). Third panel: 30 mmutes following the second oxytocm reJection (OX2), note that a control ACSF mjecuon (2 nanohters) has no effect on motd~ty Bottom panel 30 minutes following the ACSF reJection above, note that (as m Fig. 7) TRH has no effect on motlhty following atropmlzatlon These results are typical of those obtained in 6 s~mllar preparaUons

t h e s e effects are also e l i m i n a t e d b y v a g o t o m y . T h o u g h atr o p i n i z a t l o n r e d u c e s the a p p a r e n t m a g m t u d e o f the c e n t r a l o x y t o c i n effect, a small decline in motility u n d e r t h e s e condltions is still o b s e r v e d . T h e s e effects are difficult to Interpret, since a t r o p m i z a t i o n p r o d u c e s a d r a m a t i c r e d u c t i o n in gastric motility b y itself [30]. P e r h a p s o x y t o c i n m a y b e act i v a t i n g a n i n h i b i t o r y v a g a l p a t h w a y to gastric s m o o t h muscle w h o s e final e f f e r e n t is n o t d e p e n d e n t on a m u s c a r i n i c

OXYTOCIN, TRH AND PVN S T I M U L A T I O N

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FIG. 9. Effect of stimulating the PVNm, (50/xA, 20 Hz, 0.3 msec for 5 minutes; stlmulatmn on at "PVN STIM" bar) on gastric motihty Note modest reduction in post-stimulus motility These results are typical of 12 similar preparations

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FIG 10. Upper panel, (control) effect of PVNm, stimulation (50 #A, 20 Hz, 0.3 msec for 2 minutes) on gastric motility following a (control) injection of artificial

CSF (CSF--2 nanohters) into the DMN Lower panel effect of an ETOV inJection into the DMN (2 nanohters x 2 mM) followed by PVNmo stimulation as above Note the ~mmedlate increase in motility following the central injection of the oxytocm antagomst, as well as the augmented effect PVNmostimulation has on motlllty These results are typical of those obtained in 3 such preparatmns

synapse. Thus, atropine only masks the effect by producing a parallel reduction in motlhty due to the removal of the excitatory cholinergic influence on gastric smooth muscle. The concept that oxytocm is used as a neurotransmitter between the PVN and gastric vagal neurons has been strengthened considerably by the existence of the oxytocm antagonist, ETOV [1,19]. In the present study, we found that an injection of 4 picomoles of the antagonist ETOV into the DMN eliminates the inhibitory component of PVN stimulation effects on gastric motility. The observation that ETOV injections into the DMN provoke an immediate i n c r e a s e in motility suggests that the PVN may exert a tonic oxytocmergic inhibition on gastric motility through actlvatmn of the inhibitory vagal path to the stomach. (Though ETOV is also a vasopressin antagonist, this fact does not confound the result since vasopressin has no effect on the activity of DMN neurons [4] and vasopressin has none of the cardiovascular or gastric effects that oxytocin has when injected into the dorsal medulla [18].) As with the case of TRH, there is considerable anatomical evidence for a PVNmoDMN pathway containing significant amounts of oxytocin [20, 23, 25, 29].

An examination of the effects of PVNrno stimulation or DMN stimulation on gastric motility yields very similar results. In both instances, one observes an increase in phasic gastric contractions superimposed on a reduction in gastric " t o n e . " Vagotomy eliminates both stimulation effects and atropine eliminates the excitatory aspects but preserves the inhibitory components of b o t h PVNmp and DMN stimulation. These biphaslc effects of DMN stimulation have been explained by the existence of two intermixed populations of excitatory and inhibitory vagal efferent neurons [6,13]. Given that a monosynaptic connection between the PVN and the DMN exists, it also seems likely that an equivalent mixture of excitatory and mhibitory "premotor" neurons exist in the PVN which act on their counterparts in the DMN Based on the previously cited anatomical and physiological studies and the results presented herein, we hypothesize the existence of the following hypothalamic circuit which modulates gastric motility (see Fig. 12). A pool of oxytocmergic neurons in the PVN project directly to the DMN. This pathway specifically activiates DMN cells responsible for producing a reduction in gastric motility. A second pool of TRHergic neurons intermixed with the

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FIG 11 Effects of atropine and vagotomy on gasmc motility following PVNmp stimulation Upper panel, effect of PVNmo stlmulatmn (50/xA, 20 Hz, 0 3 msec for 2 minutes) on gastric motility (N=12). Middle panel: effect of PVNmp stimulation on gastric motihty following atropine (400/zg/kg IP) (N =3). Note modest reduction in motility produced by the identical PVNmo stimulation as above Lower panel, effect of PVNmo stimulation on gastric motihty following vagotomy (N=3)

FIG 12 Summary of proposed mechanism for dual PVN peptiderglc control of gastric motlhty. A mixed pool of TRHergic and oxytocmerglc neurons in the PVN,,,, project to the DMN TRHerglc neurons activate the chohnergic excitatory path whde oxytocmergic neurons activate the non-chohnergic mhibitory vagal pathway This model should not be interpreted so as to exclude other descending mfluences.

o x y t o c i n e r g i c n e u r o n s also p r o j e c t s d i r e c t l y to t h e D M N . H o w e v e r , this T R H e r g i c p a t h w a y is r e s p o n s i b l e for m c r e a s mg gastric mottlity via t h e " c l a s s i c a l " c h o l i n e r g l c p a t h w a y to t h e s t o m a c h . T h u s , a c c o r d i n g to this s c h e m e , the P V N h a s

the m e a n s to b o t h i n c r e a s e a n d d e c r e a s e gastric a c t w i t y levels via t h e s e t w o different p e p t i d e r g l c p a t h w a y s to the brainstem.

ACKNOWLEDGEMENTS This work was supported by grants from the National Institutes of Health (AM 32980, NS 24530) and the Nevada Affiliate of the American Heart Assocmtlon

REFERENCES

1 Bankowskl, K., M Manning, J Seto, J. Halder and W H Sawyer Design and synthesis of potent in vivo antagonists of oxytocln Int J Pept Protein Res 16: 382-391, 1980. 2 Bass, P. and J. N. Wiley Contractile force transducer for recording muscle activity in unanesthetized animals J Appl Phystol 32: 567-570, 1972. 3. Buijs, R. and J. Van Heerikulze. Vasopressin and oxytocln release in the bram: a synaptlc event Brain Res 252: 71-76, 1982. 4 Charpak, S., W Armstrong, M. Muhlethaler and J Dreifuss StImulatory action of oxytocin on neurons of the dorsal nucleus of the vagus nerve. Brain Res 300: 83-89, 1984. 5 Elde, R and T Hokfelt. Localization of hypophysiotrophlc peptides and other biologically active peptldes within the brain Anna Rev Nearosci 41: 587-602, 1979

6 Gillesple, J. S. Non-adrenergic non-chohnerglc inhibitory control of gastrointestinal motility In Motdtty m the Dzgesttve Tract, edited by M Weinbeck New York" Raven Press, 1982, pp. 51-66 7. Gllhs, R. A., J. A. Quest, F D. Pagam and W P Norman. Control centers in the central nervous system for regulating gastrointestinal motility. In Handbook o f Physiology, Section on the Ahmentary Tract, edtted by J. D Wood. Bethesda, MD American Physiological Society, 1987, in press 8 Hokfelt, T., K. Fuxe, O. Johansson, S. Jeffcoate and N White. Thyrotropin-releaszng hormone (TRH) containing nerve terminals m certain brainstem nuclei and in the spinal cord Neurosc Lett 1: 133-139, 1975

OXYTOCIN, TRH AND PVN STIMULATION

9. Korner, P I Central nervous control of autonomic cardxovascular function. In: Handbook of Phystology, The Cardiovascular System, section 2, vol 1, chapter 20. Bethesda, MD American Physiological Society, 1979. 10. Kubek, M. J , M. A. Rea, Z. I. Modes and M. H. Aprison. Quantltatlon and charactenzatlon of thyrotropin releasing hormone in vagal nuclei and other regions of the medulla oblongata of the rat. J Neurochem 40: 1307-1313, 1983 11. Lawrence, D., J Clnello, Q J. Pittman and K Lederis. The effect of the vasopressin antagomst d(CH2)sdTyrVAVP on the cardiovascular responses to stanulatlon of the PVN Proc West Pharma~ol Soc 27: 15-17, 1984 12. Lawrence, D and O J. lhttman InteracUons between descendmg paraventrlcular neurons and vagal motor neurons. Bram Res 332: 158-160, 1985 13 Martmson, J. Stu&es on the efferent vagal control of the stomach. Atta PhyJtol Scand 65: Suppl 255, 1-24, 1965. 14. Nosaka, S , T Yamamoto and K Yasunaga. Locahzation of vagal car&omhlbltory preganghomc neurons within the rat bramstem J Comp Neurol 186: 7%82, 1979 15 Ormsbee, H S. and P. Bass Gastroduodenal motor gradients m the dog after pyloroplasty Am J Phy~tol 230: 38%397, 1976 16. Rogers, R. C. An inexpensive plcoliter-volume pressure ejectlon system Bram Res Bull 15: 66%671, 1985. 17. Rogers, R C., H. Klta, L. L. Butcher and D. Nown Afferent project|ons to the dorsal motor nucleus of the vagus. Bram Res Bull 5: 365-373, 1980. 18. Rogers, R. C. and G. E. Hermann. Dorsal medullary oxytocm, vasopressm and TRH effects on gastric acid secretmn and heart rate Pepttde.~ 6: 1143-1148, 1985 19 Rogers, R C and G. E. Hermann Hypothalamlc paraventncular nucleus stimulation-reduced gastric acid secretion and bradycardm suppressed by oxytocm antagomst. Pepttdes 7: 695-700, 1986 20. Sawchenko, P E. and L W Swanson Immunohlstochemlcal ~dentlficatlon of neurons m the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord m the rat. J Comp Neurol 205: 260-272, 1982.

513

21 Schmid, P. G , F M. Sharabi, G. Guo, F M Abboud and M. D. Thames. Vasopressln and oxytocm m the neural control of the circulation. Fed Pro~ 43: 97-102, 1984. 22 Semba, T , N Klmura and K Fugii Bulbar influence on gastric motdlty. Jpn J Phy~tol 19: 521-533, 1969. 23 Sofromew, M. V and U. Schrell Evidence for a direct projection from oxytocln and vasopressln neurons m hypothalam~c paraventricular nucleus to the medulla oblongata' lmmunohlstochemlcal wsualizatlon of both the horseradish peroxidase transported and the peptide produced by the same neurons Neuros~l Lett 22: 211-217, 1981 24 Stone, T W Mt~rotontophorests and Pressure Eje~ tton. IBRO Handbook Series Methods ofNeurosclences, vol 8 New York John Wiley and Sons, 1985. 25 Swanson, L. W and P E Sawchenko. Hypothalamlc integration organization of the paraventncular and supraoptic nuclex Annu Rev Neurosct 6: 26%324, 1983. 26. Tache, Y The role of TRH m the central regulation of gastric function. Winter Peptlde Conference, 1987, m press. 27 Tamura, Y. and S. Maruyama An apparatus for the assembly of a combined single barrel recording electrode and a multlbarreled mlcropipette J Neuros~t Method~ 1: 24%252, 1979 28 ter Horst, G J., P. G M Lulten and F Kmpers. Descending pathways from hypothalamus to dorsal motor vagus and amblguus nuclei m the rat J Auton Nerv Syst 11: 5%75, 1984 29. Voorn, P and R M. Buijs. An ~mmunomlcroscopical study comparing vasopressln, oxytocm, substance P, and enkephahn-contammg nerve terminals m the nucleus of the solitary tract. Brain Res 270: 16%173, 1983 30 Wemer, N Atropine, scopolamine and related muscarinlc drugs In: The Pharma~ologt¢ Bast~ o f Therapeutics, 6th edlt~on, e&ted by A. G. Gdman, L S Goodman and A. Gilman. New York' Macmdlan Inc., 1980 31 Zlmmerman, E A , G Ndaver, A. Hou-Yu and A J Silverman. Vasopressmerglc and oxytocmerglc pathways in the central nervous system. Fed Pro~ 43: 91-96, 1984