Gastric motility and food intake in rats after lesions of hypothalamic paraventricular nucleus LORETTA M. FLANAGAN, JANOS AND EDWARD M. STRICKER
DOHANICS,
JOSEPH
G. VERBALIS,
Departments of Behavioral Neuroscience and Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Flanagan, Loretta M., Janos Dohanics, Joseph G. Verbalis, and Edward M. Stricker. Gastric motility and food intake in rats after lesionsof hypothalamic paraventricular nucleus. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R39-R44, 1992.-Systemic administration of cholecystokinin (CCK) or LiCl inhibits gastric motility and food intake in rats. Brain stem-projecting oxytocin (OT) neurons in the hypothalamic paraventricular nucleus (PVN) have been proposed to mediate the inhibitory effects of CCK and LiCl on gastric motility and food intake. In the present studies, we found that basalgastric motility was elevated in rats 12-20 h after knife-cut lesionsof the PVN; however, this effect disappeared3 days later. Furthermore, CCK and LiCl inhibited gastric motility at 12-20 h, 3 days, and 3 wk after PVN lesions, although their effects were blunted. Injection of the local anesthetic lidocaine into the PVN had effects similar to acute PVN lesions.In rats with PVN lesions,the inhibitory effects of CCK and LiCl on food intake were indistinguishable from those in sham-lesionedrats. We concludethat the PVN tonically inhibits gastric motility and that it participates in, but is not essential for, the inhibitory effects of CCK and LiCl on gastric motility and food intake in rats. cholecystokinin; lithium chloride; oxytocin; vagus PARVOCELLULAR OXYTOCIN (OT)-containing
neurons in the hypothalamic paraventricular nucleus (PVN) project to the dorsal motor nucleus of the vagus (DMN), the site of most parasympathetic preganglionic neurons that innervate the stomach (27). The possible role of these PVN oxytocinergic neurons in the control of gastric motility was suggested by findings that gastric motility was inhibited by microinjection of OT into the DMN and that the inhibitory effects on gastric motility of electrical stimulation of the PVN were blocked by microinjection of an OT receptor antagonist into the DMN (23). The PVN has also been implicated in the control of food intake. Bilateral lesions of the PVN have been reported to cause hyperphagia in rats (1,13), as has interruption of fibers descending from PVN (1, 10, 11). Furthermore, systemic treatments with anorexigenic agents such as cholecystokinin (CCK) and LiCl induce c-fos in PVN neurons (20, 31), increase pituitary OT secretion (29, 30), and inhibit gastric motility, gastric emptying, and food intake in rats (6, 17). As a result of these and other findings, it was hypothesized that the PVN integrated autonomic, behavioral, and neuroendocrine responses related to ingestive behavior by way of its pituitary and central projections (26). The present series of experiments had three goals: 1) to determine whether the PVN affects basal gastric motility in rats, 2) to determine whether the PVN plays any role in mediating the inhibitory effects of CCK and LiCl on gastric motility, and 3) to determine whether the PVN is necessary for the anorexigenic effects of CCK 0363-6119/92
and LiCl. In these investigations, the PVN was inactivated either by knife-cut lesions or by direct application of the local anesthetic lidocaine. METHODS Animals. Adult male rats of the Sprague-Dawleystrain (Zivic-Miller, Allison Park, PA) weighing250-350 g wereusedin all experiments.They were housedindividually in wire-meshcages in a temperature-controlled room (21-23°C) with lights on from 8 A.M. to 8 P.M. Purina laboratory Chow pellets and tap water were available ad libitum, except when indicated otherwise. Gastric surgery and measurement of gastric pressure. Rats were anesthetizedwith Equithesin (8.5 g chloral hydrate, 4.25 g MgSO*, and 1.96 g pentobarbital sodium in 200 ml water; 3 ml/kg ip), and after a midline laparotomy PE-90 tubing (Clay Adams, Parsippany, NJ) was inserted 2 cm into the fundus of the stomach via a small puncture made in the antrum. The tubing wassecuredwith a purse-string suture. A collar of Ethicon mesh(RM-54) cementedto the catheter was sutured to the external abdominal musclelayer. The tubing wasthen tunneled subcutaneouslyto the back of the neck, with 2-3 cm protruding through a small incision between the scapulae.The abdomen wasclosedwith sutures,and the catheter was filled with saline and plugged. Rats were given antibiotic (penicillin G benzathine, Bicillin, 0.2 ml im) immediately after surgery,and topical disinfectants were applied to the wound [povidone-iodine (Betadine) and nitrofurazon (Furacin)]. Five to seven days were allowed for recovery. Animals were fasted overnight and then allowedto eat Chow for 1 h. After this feeding period, the rats were placed in polypropylene tubs (27 x 16 X 12 cm) with Sanicel bedding in a quiet room. The catheters were flushed with saline and connected to a pressuretransducer.Recordsof gastric pressurewere obtained with a polygraph (model 79E, Grass Instruments, Quincy, MA), one channel of which was calibrated to detect gastric pressure (O-30 cm water) with a transducer (model P23XL, Spectramed,Oxnard, CA). The output of this amplified signalwent to a polygraph pen and alsoto an integrator (model 7PlOF, Grass Instruments), which signalleda tick on another polygraph pen when gastric pressureincreasedor decreasedby 10 cmH,O. This measure of pressure changes over time (ticks/l0 min) is a function of both the frequency and amplitude of pressurechanges.Becausethese fluctuations in gastric pressureare believed to reflect contractions of gastric smooth muscle, this measureis used as an index of gastric motility (6). Twenty-minute baselinevalues of gastric motility using this index were obtained before each experimental manipulation. Knife-cut lesions. Knife-cut lesionsof the PVN were made using a wire triangle, which produced a conical denervation approximately the size of the PVN (14). Brain surgery was performed on rats anesthetizedwith Equithesin and placed in a stereotaxic instrument (incisor bar positioned 6.5 below the center of the aural bars). After incision of the scalp and craniotomy, the knife waslowered2.0 mm caudal to bregma,on the midline of the sagittal plane, and pointed caudally, until the bottom of the knife rested on the skull. After a few minutes, the
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knife wasrotated 360” three times both clockwiseand counterclockwiseto produce bilateral lesions.Sham lesionsconsistedof a similar procedureexcept that the knife wasloweredto a point just abovethe PVN and wasnot rotated. Wound clips were used to closethe scalp, and rats were returned to their home cages after they recovered from anesthesia. Effects of PVN lesions on gastric pressure. Groups of shamtreated rats and rats with PVN lesions were implanted with gastric catheters at least 1 wk before brain surgery. After the lesions,animalswere deprived of food overnight (approximately 12 h) and then allowedad libitum accessto Chow for 1 h; they ate 7-12 g during this period. Then they were placed in the polypropylene tubs, and gastric catheters were connectedto the polygraph apparatus.After a 20-min baselineperiod, rats were injected with either CCK (1 pg/kg ip) or 0.15 M LiCl (1.5 meq/kg ip), and gastric pressurewas monitored for at least 20 min. Then catheters were plugged and the animals were returned to their home cages. Effects of PVN lesions on food intake. Groups of sham-treated rats and rats with PVN lesionswere maintained on a feeding scheduleof l-h accessto Chow at 10 A.M. and 2-h accessto food at 6 P.M., beginning at least 1 wk after the lesion wasmade.At all other times, food was not present but water was available ad libitum. Stable intakes were establishedafter 7 days of this regimen. Rats were injected with 0.15 M LiCl ip (1.5 or 3.0 meq/kg, given 20 min before the morning feeding session),and its effects on food intake were monitored for 1 h. These doses were given 2 days apart, after which the morning feeding period was reduced to 20 min and stable intakes were reestablished. Rats then were injected with CCK (1 or 5 Mg/kgip, given 5 min before the morning feeding session),and its effects on food intake were monitored for 20 min. These dosesalsowere given 2 days apart. Effects of lidocaine
injection
into the PVN on gastric pressure.
Rats were implanted with gastric catheters. One week later they were anesthetized and placed in a stereotaxic frame with the incisor bar set to -2.5 mm. An incision wasmadein the scalpto exposethe skull, severalsmallscrewswere fixed into the parietal bone,two smallholeswere drilled in the skull, and two 23-gauge guide cannulaswith 26-gaugeinner stylets were directed bilaterally just above each PVN (0.2 mm behind bregma, 0.4 mm lateral to midline, and 7.5 mm ventral to the surface of the brain). Dental acrylic wasapplied to the skull and allowedto air dry, whereupon the scalp was sutured and prophylactic antibiotics were administered, as describedabove. Three days later, gastric pressurewasrecordedin recently fed rats. After a 20-min baselineperiod, 0.4 ~1 of artificial cerebrospinal fluid (aCSF) was injected through eachguide cannula via 30-gaugetubing that endedjust below the guide cannulas. Twenty minutes later, the sameprocedurewasusedto inject 2% lidocaine bilaterally. Twenty minutes later either CCK (1 pg/ kg) or LiCl (1.5 meq/kg) was administered intraperitoneally, and their effects on gastric pressurewere determined. Histological analysis. After experimentation, all rats with PVN lesionswere deeply anesthetized with Nembutal, given 1,000 U heparin, and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde and 3% picric acid in 0.1 M potassiumphosphate-bufferedsaline.Brains were removed and blocked with the boundaries of the rostra1extent of the optic chiasmand the mammillary bodies.The tissuewaspostfixed in 25% sucrose for an additional 24-48 h and then sectioned (25-40 pm) with a Reichert freezing microtome. Sections were divided sequentially into six dishesand were usedimmediately either for Nissl stains or immunohistochemistry for analysis of residualcells.Lesionswere consideredcompletewhen there was bilateral destruction of all cell bodiesin the area of the PVN. The Nissl staining procedure involved hydrating the tissue mounted on gel-coatedslidesand then staining with cresyl vi-
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olet and rinsing in increasingconcentrations of ethanol. Slides then were treated with a clearing agent (Histoclear), cover slippedwith Permount, and air dried. Processingfor immunocytochemical localization of OT and vasopressin(AVP) neurophysinswasdone usingproceduresdescribed elsewhere(32), whereby avidin-biotin complex (ABC Kit, Vector Laboratories) is usedto increaseoverall sensitivity of the immunocytochemical technique. Primary antibodies to OT- and AVP-neurophysins were raisedin rabbits and donated by Dr. Alan G. Robinson. After the experiments involving rats with intracranial cannulas, placement of the injections was confirmed histologically by injecting 0.4 ~1fast greendye in eachcannula. Rats werethen decapitated, and their brains were quickly dissectedand frozen in isopentane. Sections (40 pm) were cut on a cryostat and counterstained with cresyl violet. Statistical analysis. Previous experiments indicated that intraperitoneal injections of vehicle solution have no significant effect on gastric pressure (6) or on food intake (5). Thus comparisonswere made to baseline values of individual rats. Within-subject comparisonswere madeusinga paired signtest, whereas comparisons between groups were made using the Mann Whitney U test. In studiesof food intake, group comparisonsweremadeusing an unpaired Student’s t test at eachdose. RESULTS Histology. Based on the Konig and Klippel(12) stereotaxic atlas, 26 rats used in the studies of gastric motility had symmetrical lesions destroying the area defined as PVN, and 16 rats used in the studies of food intake had acceptable lesions. In all 42 cases, no Nissl-stained or neurophysin-containing neurons remained within the region of the PVN. However, neurons in the vicinity of the fornix, some of which were oxytocinergic, remained after the lesion. Figure 1 shows coronal hypothalamic sections
Fig. 1. Photomicrograph of coronalhypothalamicsectionfromanintact
rat (A) and from a rat with a PVN knife-cut lesion (B). Tissue was stained both for immunoreactive OT-neurophysin and Nissl substance.
SON,supraopticnucleus.
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from an intact brain and from a representative rat with PVN lesions. Figure 2 presents a composite drawing of the area encompassed by the lesions for all rats used in these studies. Effect of PVN lesions on gastric pressure. Figure 3 illustrates representative tracings of the gastric pressure recordings during the baseline period from a rat with sham lesions and a rat with PVN lesions 12-20 h after surgery. The four rats with PVN lesions had significantly higher baseline gastric motility scores (range 16 to 31) than the six control animals (range 8 to 19; P < 0.01). In contrast, 3 days and 3 wk after PVN lesions, individual gastric motility scores were significantly lower than values obtained 12-20 h after the lesion (both P < 0.01). At these times there were no differences between the gastric motility scores of rats with sham lesions or PVN lesions (n = 5-10 in each group). Both CCK and LiCl continued to reduce gastric motility scores in rats with PVN lesions, but their effects were significantly blunted. More specifically, in rats with sham lesions, gastric motility scores were typically reduced by at least 75% of baseline values when measured 10 min after receiving CCK (1 pg/kg ip; Fig. 4A) and 20 min after receiving LiCl (1.5 meq/l ip; Fig. 5A). In rats studied 3 days and 3 wk after PVN lesions, however, the inhibitory effects of these two treatments on gastric motility scores at those time points were significantly smaller than in rats with sham lesions. Data collected 3 days and 3 wk postlesion were from different sets of animals, and because their baseline gastric motility scores were not significantly different, their data were combined (CCK, P < 0.02, Fig. 4C; LiCl, P < 0.01, Fig. 5C). Both CCK and LiCl treatments also reduced fluctuations in gastric pressure in rats with PVN lesions when measured 12-20 h after surgery (Figs. 4B and 5B), and to a lesser extent than in sham-lesioned rats (combined CCK and LiCl data, 12-20 h postlesion: P < 0.01). It should be noted, however, that the basal gastric motility scores of the rats with PVN lesions were significantly
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Fig. 3. Tracing of gastric pressure recordings of representative 12-20 h after a sham lesion (A) or a complete PVN lesion (B). A
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Fig. 4. Effect of systemic injection of CCK (1 pg/kg ip) on gastric motility in rats (A) 12-20 h after sham lesions or (B) 12-20 h or (C) 3 days or 3 wk after PVN lesions. Shown are motility scores for individual animals expressed as a percent of baseline values. Arrows indicate time of injection. 5.r 120 5 $100 ; -
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Fig. 5. Effect of systemic injection of LiCl (1.5 meq/kg ip) on gastric motility in rats either (A) 12-20 h after sham lesions or (B) 12-20 h or (C) 3 days or 3 wk after PVN lesions. Shown are motility scores for individual animals expressed as a percent of baseline values. Arrows indicate time of injection.
greater than the values observed in control animals, as mentioned. Effect of PVN lesions on food intake. Bilateral lesions of PVN had no significant effect on food intake and did not consistently affect body weight when compared with sham lesions (gains in body weight during 30 days postlesion: PVN lesions, 4.6 t 0.4 g/day; controls, 4.4 t 0.3 g/day). All groups had similar baseline food intakes, and there were no differences between groups in the inhibition of food intake produced by systemic injections of CCK or LiCl at two different doses (Table 1). Fig. 2. Drawings of coronal hypothalamic sections with tracings of the lesions of all rats used in the studies of (A) gastric motility (n = 26) and (B) food intake (n = 16). Tracings were made with a camera lucida, and the drawings were made based on the Konig and Klippel stereotaxic atlas (12, Figs. 29-32). More rostra1 sections are depicted at the top of the figure. F, fornix; OC, optic chiasm.
Effect of lidocaine injection into the PVN on gastric pressure. Injection of aCSF into the PVN had no signif-
icant effect on gastric pressure. Figure 6 illustrates gastric pressure recordings from two representative rats during the baseline period and after bilateral microinjection of lidocaine (0.4 pi/side) into the PVN. After this treatment,
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Table 1. Effect of systemic injection of CCK or LiCl on food intake in rats with PVN lesions and sham-lesioned controls
CCK 1 Pdk 5 Pdk
n
Food intake,
66.9dl.O 56.4t5.3
12 8
49.9t5.0 49.1t14.3
60.6t8.3 41.5t9.7
16 5
44.92 15.4 23.8dl.l
Food intake,
8 8 7 8
%
%
LiCl 1.5 meq/kg 3 mea/kg:
,q 1
PVN Lesions
Sham Lesions n
LiCl PVN Lidocaine
Values are means t SE expressed as a percent of baseline food intakes. There were no significant differences between groups in the effects of each dose of CCK or LiCl on food intake.
5 min
Fig. 6. Tracings of gastric pressure recordings of 2 representative rats before and after microinjection of lidocaine into the PVN bilaterally.
gastric motility scores increased within minutes and remained elevated for at least 30-40 min. On the other hand, injection of lidocaine into the PVN did not eliminate the inhibition of gastric motility produced by systemic injection of CCK or LiCl. As shown in Fig. 7, gastric motility was inhibited by systemic injection of CCK (n = 5, P < 0.01) or LiCl (n = 4, P < 0.05) given 20 min after microinjection of lidocaine into the PVN, a time when gastric motility scores were still elevated by lidocaine. The distribution of dye provided histological verification of the microinjection site just dorsal to the PVN in all five animals for which data were presented. However, when the injections occurred lateral, dorsal, or ventral to the PVN (n = 7), or into the third cerebral ventricle (n = 5), the lidocaine injection produced no change in gastric pressure. DISCUSSION
Gastric afferents stimulate oxytocinergic neurons in the hypothalamic PVN (28). Moreover, electrical stimulation of the PVN decreases gastric motility in rats (24). The present experiments sought to provide additional information regarding the influence of the PVN on gastric function in rats. Our results suggest that the PVN exerts a tonic inhibitory effect on gastric motility, because bilateral lesions of PVN caused an acute increase in gastric motility, which was still evident even 12-20 h after the lesion. The same effect was seen within minutes after acute anesthetization of the PVN, but neither effect was seen when lesions or anesthetization occurred at sites adjacent to PVN. Together with our recent findings that intracerebroventricular (icv) injection of an OT receptor
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Fig. 7. Effect of (A) CCK or (B) LiCl on gastric motility after microinjection of lidocaine into the PVN. Shown are motility scores for 4 animals, expressed as a percent of baseline values. Arrows indicate times of injection.
antagonist similarly increased gastric motility acutely in conscious rats (7), and the present observations that the inhibitory effects of CCK and LiCl on gastric motility were significantly blunted in rats with PVN lesions, these results strongly support previous proposals that parvocellular oxytocinergic neurons play an important role in the central control of gastric function (15, 23). On the other hand, increases in gastric motility were no longer evident 3 days or 3 wk after PVN lesions. Since gastric function is likely to have multiple redundant central control mechanisms, these findings suggest that after damage to the PVN other elements controlling gastric function assume greater significance than they have normally. However, despite this effect, it is noteworthy that CCK and LiCl both remain able to inhibit gastric motility even by 12-20 h after PVN lesions while basal motility still is elevated. More acute disruption of PVN activity by lidocaine anesthetization similarly did not eliminate the inhibitory effects of CCK and LiCl on gastric motility, which would be expected if the PVN played a critical role in these processes, since presumably the tests conducted during PVN anesthetization did not allow sufficient time for compensatory mechanisms to be established. Thus these results demonstrate that the PVN is not essential for producing the inhhbitory effects of CCK and LiCl on gastric motility in rats. Given that systemic CCK appears to affect gastric motility by stimulating receptors located on afferent fibers of the gastric vagus that project to the nucleus tractus solitarius (NTS) in the brain stem (16,21,25), whereas LiCl is thought to stimulate chemoreceptors in the area postrema (2, 22), the present results allow the possibility that the inhibition of gastric motility induced by these two agents is mediated in part by local circuits in the brain stem, from the NTS and area postrema to the dorsal motor nucleus of the vagus. Aside from its putative role in the central control of gastric function, the hypothalamic PVN also has been suspected of playing an important role in the control of food intake. This possibility has been suggested by observations that hyperphagia in rats is caused by bilateral lesions of the PVN or of fibers projecting caudally from the PVN (1, 10, 11, 13). The most effective lesions in producing hyperphagia in rats appear to destroy the cauda1 PVN and the adjacent perifornical region, which contains both cells and fiber tracts projecting caudally, including some containing OT. The knife used in the
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present series of studies avoided damage to this perifornical area, and perhaps for that reason the animals did not become hyperphagic despite complete destruction of all PVN tissue as classically defined. In contrast, it is noteworthy that PVN lesions produced by a similar but slightly larger knife, which damaged the fornices and dorsomedial hypothalamus in addition to the PVN, have been reported to produce significant increases in body weight in rats (4). Thus it would appear from these studies that laterally situated neurons, perhaps including parvocellular OT-containing fibers in this region, are critical to the phenomenon of hyperphagia induced by lesions of the PVN area. Presumably, these perifornical neurons in the caudal PVN provide a component of inhibition in the central neural control of food intake that, when damaged, disinhibits the system and leads to hyperphagia. This hypothesis is consistent with substantial recent evidence suggesting a role of brain OT-containing neurons in the inhibition of food intake (6, 11, 17-19, 29, 30). Although it is not yet clearly established what the role of the PVN is in the central control of food intake and gastric motility, the present observations that CCK and LiCl had undiminished inhibitory effects on food intake in rats with PVN lesions allow the possibility that the laterally situated parvocellular oxytocinergic neurons, projecting caudally from the PVN, participate in the inhibition of food intake induced by vagal (e.g., CCK) or toxic (e.g., LiCl) stimuli into the NTS. More medially situated parvocellular OT-containing neurons may be involved in modulating the inhibition of gastric motility caused by those same stimuli. The present results may be contrasted with the results of parallel experiments in which an OT-receptor antagonist given icv blunted the inhibition of food intake by CCK and LiCl, by 28-G% (19). Perhaps the present PVN lesions were ineffective in blunting the inhibition of food intake induced by CCK and LiCl because critical oxytocinergic fibers, whose function was blocked by the OT-receptor antagonist, were not damaged by our PVN lesions. Alternatively, because evaluation of food intake in the present study was not done until 2-3 wk after the lesions, perhaps adaptive adjustments in the central systems that control food intake occurred after the lesions, which could not occur after acute injection of the OT antagonist. If so, then a comparable blunting of their inhibitory effects on eating would be expected if CCK and LiCl were given to rats shortly after anesthetization of PVN. These possibilities await further investigation. An important role of PVN in the mediation of the anorexia induced by CCK in rats has been suggested by Crawley and Kiss (3). In their studies, rats with PVN knife-cut lesions were deprived of food overnight and then placed in a novel open field with palatable mash available. Food consumption was not suppressed in animals with PVN lesions after systemic injection of 5 or 10 in rats Pdk cw wh ereas it was markedly inhibited with sham lesions. Because of the many differences in protocol between that study and the present experiments, including the environment in which the feeding test was conducted, the feeding schedule, the doses of CCK used,
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and the food being consumed, it remains possible that the PVN has an important role in the control of food intake but one that can be seen only in certain experimental paradigms. Alternatively, because the knife used to produce the lesions in the previous study (3) was larger than the one used in the present study, it is again possible that its damage to critical perifornical fibers in the PVN accounted for the observed failure of CCK to inhibit food intake. Finally, it seems possible that the brain stem plays a prominent role in the control of food intake (5,8, 9, 20). These possibilities are not mutually exclusive. In summary, the present findings that acute PVN lesions and anesthetization of the PVN increase basal gastric motility in rats and that PVN lesions blunt the inhibition of gastric motility induced by CCK and LiCl administered systemically suggest that the PVN has an important role in the central control of both tonic and phasic gastric function. Although the present findings that PVN lesions did not alter the CCK- or LiCl-induced inhibition of food intake do not support a role for the PVN in mediating these effects, these lesions purposely avoided damage to more lateral perifornical areas of the hypothalamus. In light of numerous other reports suggesting a role of the PVN in the control of food intake, it seems clear that more detailed investigations of the hypothalamic structures involved in these effects are warranted. We gratefully acknowledge the expert contributions of Dr. Gloria Hoffman in the histological assessment of the lesions. This work was supported in part by National Institutes of Health (NIH) Grant MH-25140 (MERIT Award to E. M. Stricker and J. G. Verbalis), NIH National Research Service Award MH-09770 (to L. M. Flanagan), and NIH Research Career Development Award DK-02014 (to J. Dohanics). Portions of this work were submitted by L. M. Flanagan in partial fulfillment of the requirements for the PhD degree. Some of these findings were presented in preliminary form at the annual meeting of the Society for Neuroscience, St. Louis, MO, November 1990. Current address and address for reprint requests: L. M. Flanagan, Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Ave., New York, NY 10021-6399. Received 29 August 1991; accepted in final form 21 January 1992. REFERENCES 1. Aravich, P. F., and A. Sclafani. Paraventricular hypothalamic lesions and medial hypothalamic knife cuts produce similar hyperphagic syndromes. Behau. Neurosci. 97: 970-983, 1983. 2. Borison, H. L., and S. C. Wang. Physiology and pharmacology of vomiting. Pharmacol. Rev. 5: 193-230, 1953. 3. Crawley, J. N., and J. 2. Kiss. Paraventricular nucleus lesions abolish the inhibition of feeding induced by systemic cholecystokinin. Peptides 6: 927-235, 1985. 4. Darlington, D. N., J. Shinsako, and M. F. Dallman. Paraventricular lesions: hormonal and cardiovascular responses to hemorrhage. Brain Res. 439: 289-301, 1988. 5. Flanagan, L. M., R. E. Blackburn, J. G. Verbalis, and E. M. Stricker. Hypertonic NaCl inhibits gastric motility and food intake in rats with lesions in the rostra1 AV3V region. Am. J. Physiol. 263 (Regulatory Integrative Cornp. Physiol. 32): R9-R14, 1992. 6. Flanagan, L. M., J. G. Verbalis, and E. M. Stricker. Effects of anorexigenic treatments on gastric motility in rats. Am. J. Physiol. 256 (Regulatory Integrative Cornp. Physiol. 25): R955R961, 1989. 7. Flanagan, L. M., B. R. Olson, A. F. Sved, J. G. Verbalis, and E. M. Stricker. Gastric motility in conscious rats given
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oxytocin and an oxytocin antagonist centrally. Bruin Res. In press. 8. Grill, H. J., and J. M. Kaplan. Caudal brainstem participates in the distributed neural control of feeding. In: Handbook of Behavioral Neurobiology, edited by E. M. Stricker. New York: Plenum Press, 1990, vol. 10, p. 125-149. 9. Grill, H. J., and G. P. Smith. Cholecystokinin decreases sucrose intake in chronic decerebrate rats. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R853-R856, 1988. 10. Kirchgessner, A. L., and A. Sclafani. PVN-hindbrain pathway involved in the hypothalamic hyperphagic-obesity syndrome. Physiol. Behav. 42: 517-528, 1988. 11. Kirchgessner, A. L., A. Sclafani, and G. Nilaver. Histochemical identification of a PVN-hindbrain feeding pathway. Physiol. Behav. 42: 529-543, 1988. 12. Konig, J. F. R., and R., A. Klippel. The Rat Brain: A Stereotaxic Atlas. Baltimore: Williams & Wilkins, 1963. 13. Leibowitz, S. F., N. J. Hammer, and K. Chang. Hypothalamic paraventricular nucleus lesions produce overeating and obesity in the rat. PharmacoZ. Biochem. Behau. 27: 1031-1040, 1981. 14. Makara, G. B., E. Stark, M. Karteszi, M. Palkovits, and Gy. Rappay. Effects of paraventricular lesions on stimulated ACTH release and CRF in stalk-median eminence of the rat. Am. J. Physiol. 240 (Endocrinol. Metab. 3): E441-E446, 1981. 15. McCann, M. J., and R. C. Rogers. Oxytocin excites gastricrelated neurones in rat dorsal vagal complex. J. Physiol. Land. 428: 95-108, 1990. 16. McCann, M. J., J. G. Verbalis, and E. M. Stricker. Capsaicin pretreatment attenuates multiple responses to cholecystokinin in rats. J. Auton. Nerv. Syst. 23: 265-272, 1988. 17. McCann, M. J., J. G. Verbalis, and E. M. Stricker. LiCl and CCK inhibit gastric emptying and feeding and stimulate OT secretion in rats. Am. J. Physiol. 256 (Regulatory Integrative Comp. Physiol. 25): R463-R468, 1989. 18. Olson, B. R., M. D. Drutarosky, M.-S. Chow, V. J. Hruby, E. M. Stricker, and J. G. Verbalis. Oxytocin and an oxytocin agonist administered centrally decrease food intake in rats. Peptides 12: 113-118, 1991. 19. Olson, B. R., M. D. Drutarosky, E. M. Stricker, and J. G. Verbalis. Brain oxytocin receptor antagonism blunts the effects of anorexigenic treatments in rats: Evidence for central oxytocin inhibition of food intake. Endocrinology 129: 785-791, 1991. 20. Olson, B. R., G. E. Hoffman, A. F. Sved, E. M. Stricker, and J. G. Verbalis. c-fos is expressed in anatomically distinct brain nuclei following inhibition of or potentiation of food intake and gastric motility in rats. Sot. Neurosci. Abstr. 17: 1366, 1991.
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21. Raybould, H. E., R. J. Gayton, and G. J. Dockray. Mechanisms of action of peripherally administered cholecystokinin octapeptide on brain stem neurons in the rat. J. Neurosci. 8: 30183024, 1988. 22. Ritter, S., J. J., McClone, and K. W. Kelley. Absence of lithium-induced taste aversion after area postrema lesion. Bruin Res. 201: 501-506, 1980. 23. Rogers, R. C., and G. E. Hermann. Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular stimulation: effects on gastric motility. Peptides 8: 505-513, 1987. 24. Sakaguchi, T., and M. Ohtake. Inhibition of gastric motility induced by activation of the hypothalamic paraventricular nucleus. Bruin Res. 335: 365-367, 1985. 25. Smith, G. P., C. Jerome, and R. Norgren. Afferent axones in abdominal vagus mediate satiety effect of cholecystokinin in rats. Am. J. Physiol. 249 (Regulatory Integrative Comp. Physiol. 18): R638-R641, 1985. 26. Stricker, E. M., M. J. McCann, L. M. Flanagan, and J. G. Verbalis. Neurohypophyseal secretion and gastric function: biological correlates of nausea. In: Biowarning System in the Brain, edited by H. Takagi, Y. Oomura, M. Ito, and M. Otsuka. Tokyo: Univ. of Tokyo Press, 1988, p. 295-307. 27. Swanson, L. W., and P. E. Sawchenko. 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. Neural. 205: 260-272, 1982. 28. Ueta, Y., H. Kannan, and H. Yamashita. Gastric afferents to the paraventricular nucleus in the rat. Exp. Bruin Res. 84: 487494, 1991. 29. Verbalis, J. G., M. J. McCann, C. M. McHale, and E. M. Stricker. Oxytocin secretion in response to cholecystokinin and food intake: Differentiation of nausea from satiety. Science Wash. DC 232: 1417-1419, 1986. 30. Verbalis, J. G., C. M. McHale, T. W. Gardiner, and E. M. Stricker. Oxytocin and vasopressin secretion in response to stimuli producing learned taste aversions in rats. Behav. Neurosci. 100: 466-475, 1986. 31. Verbalis, J. G., E. M. Stricker, A. G. Robinson, and G. E. Hoffman. Cholecystokinin activates c-fos expression in hypothalamic oxytocin and corticotropin-releasing hormone neurons. J. NeuroendocrinoZ. 3: 205-213, 1991. 32. Watson, R. E., S. J. Weigand, R. W. Clough, and G. E. Hoffman. Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 7: 155-159, 1986.
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DOHANICS,
JOSEPH
G. VERBALIS,
Departments of Behavioral Neuroscience and Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Flanagan, Loretta M., Janos Dohanics, Joseph G. Verbalis, and Edward M. Stricker. Gastric motility and food intake in rats after lesionsof hypothalamic paraventricular nucleus. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R39-R44, 1992.-Systemic administration of cholecystokinin (CCK) or LiCl inhibits gastric motility and food intake in rats. Brain stem-projecting oxytocin (OT) neurons in the hypothalamic paraventricular nucleus (PVN) have been proposed to mediate the inhibitory effects of CCK and LiCl on gastric motility and food intake. In the present studies, we found that basalgastric motility was elevated in rats 12-20 h after knife-cut lesionsof the PVN; however, this effect disappeared3 days later. Furthermore, CCK and LiCl inhibited gastric motility at 12-20 h, 3 days, and 3 wk after PVN lesions, although their effects were blunted. Injection of the local anesthetic lidocaine into the PVN had effects similar to acute PVN lesions.In rats with PVN lesions,the inhibitory effects of CCK and LiCl on food intake were indistinguishable from those in sham-lesionedrats. We concludethat the PVN tonically inhibits gastric motility and that it participates in, but is not essential for, the inhibitory effects of CCK and LiCl on gastric motility and food intake in rats. cholecystokinin; lithium chloride; oxytocin; vagus PARVOCELLULAR OXYTOCIN (OT)-containing
neurons in the hypothalamic paraventricular nucleus (PVN) project to the dorsal motor nucleus of the vagus (DMN), the site of most parasympathetic preganglionic neurons that innervate the stomach (27). The possible role of these PVN oxytocinergic neurons in the control of gastric motility was suggested by findings that gastric motility was inhibited by microinjection of OT into the DMN and that the inhibitory effects on gastric motility of electrical stimulation of the PVN were blocked by microinjection of an OT receptor antagonist into the DMN (23). The PVN has also been implicated in the control of food intake. Bilateral lesions of the PVN have been reported to cause hyperphagia in rats (1,13), as has interruption of fibers descending from PVN (1, 10, 11). Furthermore, systemic treatments with anorexigenic agents such as cholecystokinin (CCK) and LiCl induce c-fos in PVN neurons (20, 31), increase pituitary OT secretion (29, 30), and inhibit gastric motility, gastric emptying, and food intake in rats (6, 17). As a result of these and other findings, it was hypothesized that the PVN integrated autonomic, behavioral, and neuroendocrine responses related to ingestive behavior by way of its pituitary and central projections (26). The present series of experiments had three goals: 1) to determine whether the PVN affects basal gastric motility in rats, 2) to determine whether the PVN plays any role in mediating the inhibitory effects of CCK and LiCl on gastric motility, and 3) to determine whether the PVN is necessary for the anorexigenic effects of CCK 0363-6119/92
and LiCl. In these investigations, the PVN was inactivated either by knife-cut lesions or by direct application of the local anesthetic lidocaine. METHODS Animals. Adult male rats of the Sprague-Dawleystrain (Zivic-Miller, Allison Park, PA) weighing250-350 g wereusedin all experiments.They were housedindividually in wire-meshcages in a temperature-controlled room (21-23°C) with lights on from 8 A.M. to 8 P.M. Purina laboratory Chow pellets and tap water were available ad libitum, except when indicated otherwise. Gastric surgery and measurement of gastric pressure. Rats were anesthetizedwith Equithesin (8.5 g chloral hydrate, 4.25 g MgSO*, and 1.96 g pentobarbital sodium in 200 ml water; 3 ml/kg ip), and after a midline laparotomy PE-90 tubing (Clay Adams, Parsippany, NJ) was inserted 2 cm into the fundus of the stomach via a small puncture made in the antrum. The tubing wassecuredwith a purse-string suture. A collar of Ethicon mesh(RM-54) cementedto the catheter was sutured to the external abdominal musclelayer. The tubing wasthen tunneled subcutaneouslyto the back of the neck, with 2-3 cm protruding through a small incision between the scapulae.The abdomen wasclosedwith sutures,and the catheter was filled with saline and plugged. Rats were given antibiotic (penicillin G benzathine, Bicillin, 0.2 ml im) immediately after surgery,and topical disinfectants were applied to the wound [povidone-iodine (Betadine) and nitrofurazon (Furacin)]. Five to seven days were allowed for recovery. Animals were fasted overnight and then allowedto eat Chow for 1 h. After this feeding period, the rats were placed in polypropylene tubs (27 x 16 X 12 cm) with Sanicel bedding in a quiet room. The catheters were flushed with saline and connected to a pressuretransducer.Recordsof gastric pressurewere obtained with a polygraph (model 79E, Grass Instruments, Quincy, MA), one channel of which was calibrated to detect gastric pressure (O-30 cm water) with a transducer (model P23XL, Spectramed,Oxnard, CA). The output of this amplified signalwent to a polygraph pen and alsoto an integrator (model 7PlOF, Grass Instruments), which signalleda tick on another polygraph pen when gastric pressureincreasedor decreasedby 10 cmH,O. This measure of pressure changes over time (ticks/l0 min) is a function of both the frequency and amplitude of pressurechanges.Becausethese fluctuations in gastric pressureare believed to reflect contractions of gastric smooth muscle, this measureis used as an index of gastric motility (6). Twenty-minute baselinevalues of gastric motility using this index were obtained before each experimental manipulation. Knife-cut lesions. Knife-cut lesionsof the PVN were made using a wire triangle, which produced a conical denervation approximately the size of the PVN (14). Brain surgery was performed on rats anesthetizedwith Equithesin and placed in a stereotaxic instrument (incisor bar positioned 6.5 below the center of the aural bars). After incision of the scalp and craniotomy, the knife waslowered2.0 mm caudal to bregma,on the midline of the sagittal plane, and pointed caudally, until the bottom of the knife rested on the skull. After a few minutes, the
$2.00 Copyright 0 1992 the American Physiological
Society
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knife wasrotated 360” three times both clockwiseand counterclockwiseto produce bilateral lesions.Sham lesionsconsistedof a similar procedureexcept that the knife wasloweredto a point just abovethe PVN and wasnot rotated. Wound clips were used to closethe scalp, and rats were returned to their home cages after they recovered from anesthesia. Effects of PVN lesions on gastric pressure. Groups of shamtreated rats and rats with PVN lesions were implanted with gastric catheters at least 1 wk before brain surgery. After the lesions,animalswere deprived of food overnight (approximately 12 h) and then allowedad libitum accessto Chow for 1 h; they ate 7-12 g during this period. Then they were placed in the polypropylene tubs, and gastric catheters were connectedto the polygraph apparatus.After a 20-min baselineperiod, rats were injected with either CCK (1 pg/kg ip) or 0.15 M LiCl (1.5 meq/kg ip), and gastric pressurewas monitored for at least 20 min. Then catheters were plugged and the animals were returned to their home cages. Effects of PVN lesions on food intake. Groups of sham-treated rats and rats with PVN lesionswere maintained on a feeding scheduleof l-h accessto Chow at 10 A.M. and 2-h accessto food at 6 P.M., beginning at least 1 wk after the lesion wasmade.At all other times, food was not present but water was available ad libitum. Stable intakes were establishedafter 7 days of this regimen. Rats were injected with 0.15 M LiCl ip (1.5 or 3.0 meq/kg, given 20 min before the morning feeding session),and its effects on food intake were monitored for 1 h. These doses were given 2 days apart, after which the morning feeding period was reduced to 20 min and stable intakes were reestablished. Rats then were injected with CCK (1 or 5 Mg/kgip, given 5 min before the morning feeding session),and its effects on food intake were monitored for 20 min. These dosesalsowere given 2 days apart. Effects of lidocaine
injection
into the PVN on gastric pressure.
Rats were implanted with gastric catheters. One week later they were anesthetized and placed in a stereotaxic frame with the incisor bar set to -2.5 mm. An incision wasmadein the scalpto exposethe skull, severalsmallscrewswere fixed into the parietal bone,two smallholeswere drilled in the skull, and two 23-gauge guide cannulaswith 26-gaugeinner stylets were directed bilaterally just above each PVN (0.2 mm behind bregma, 0.4 mm lateral to midline, and 7.5 mm ventral to the surface of the brain). Dental acrylic wasapplied to the skull and allowedto air dry, whereupon the scalp was sutured and prophylactic antibiotics were administered, as describedabove. Three days later, gastric pressurewasrecordedin recently fed rats. After a 20-min baselineperiod, 0.4 ~1 of artificial cerebrospinal fluid (aCSF) was injected through eachguide cannula via 30-gaugetubing that endedjust below the guide cannulas. Twenty minutes later, the sameprocedurewasusedto inject 2% lidocaine bilaterally. Twenty minutes later either CCK (1 pg/ kg) or LiCl (1.5 meq/kg) was administered intraperitoneally, and their effects on gastric pressurewere determined. Histological analysis. After experimentation, all rats with PVN lesionswere deeply anesthetized with Nembutal, given 1,000 U heparin, and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde and 3% picric acid in 0.1 M potassiumphosphate-bufferedsaline.Brains were removed and blocked with the boundaries of the rostra1extent of the optic chiasmand the mammillary bodies.The tissuewaspostfixed in 25% sucrose for an additional 24-48 h and then sectioned (25-40 pm) with a Reichert freezing microtome. Sections were divided sequentially into six dishesand were usedimmediately either for Nissl stains or immunohistochemistry for analysis of residualcells.Lesionswere consideredcompletewhen there was bilateral destruction of all cell bodiesin the area of the PVN. The Nissl staining procedure involved hydrating the tissue mounted on gel-coatedslidesand then staining with cresyl vi-
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olet and rinsing in increasingconcentrations of ethanol. Slides then were treated with a clearing agent (Histoclear), cover slippedwith Permount, and air dried. Processingfor immunocytochemical localization of OT and vasopressin(AVP) neurophysinswasdone usingproceduresdescribed elsewhere(32), whereby avidin-biotin complex (ABC Kit, Vector Laboratories) is usedto increaseoverall sensitivity of the immunocytochemical technique. Primary antibodies to OT- and AVP-neurophysins were raisedin rabbits and donated by Dr. Alan G. Robinson. After the experiments involving rats with intracranial cannulas, placement of the injections was confirmed histologically by injecting 0.4 ~1fast greendye in eachcannula. Rats werethen decapitated, and their brains were quickly dissectedand frozen in isopentane. Sections (40 pm) were cut on a cryostat and counterstained with cresyl violet. Statistical analysis. Previous experiments indicated that intraperitoneal injections of vehicle solution have no significant effect on gastric pressure (6) or on food intake (5). Thus comparisonswere made to baseline values of individual rats. Within-subject comparisonswere madeusinga paired signtest, whereas comparisons between groups were made using the Mann Whitney U test. In studiesof food intake, group comparisonsweremadeusing an unpaired Student’s t test at eachdose. RESULTS Histology. Based on the Konig and Klippel(12) stereotaxic atlas, 26 rats used in the studies of gastric motility had symmetrical lesions destroying the area defined as PVN, and 16 rats used in the studies of food intake had acceptable lesions. In all 42 cases, no Nissl-stained or neurophysin-containing neurons remained within the region of the PVN. However, neurons in the vicinity of the fornix, some of which were oxytocinergic, remained after the lesion. Figure 1 shows coronal hypothalamic sections
Fig. 1. Photomicrograph of coronalhypothalamicsectionfromanintact
rat (A) and from a rat with a PVN knife-cut lesion (B). Tissue was stained both for immunoreactive OT-neurophysin and Nissl substance.
SON,supraopticnucleus.
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from an intact brain and from a representative rat with PVN lesions. Figure 2 presents a composite drawing of the area encompassed by the lesions for all rats used in these studies. Effect of PVN lesions on gastric pressure. Figure 3 illustrates representative tracings of the gastric pressure recordings during the baseline period from a rat with sham lesions and a rat with PVN lesions 12-20 h after surgery. The four rats with PVN lesions had significantly higher baseline gastric motility scores (range 16 to 31) than the six control animals (range 8 to 19; P < 0.01). In contrast, 3 days and 3 wk after PVN lesions, individual gastric motility scores were significantly lower than values obtained 12-20 h after the lesion (both P < 0.01). At these times there were no differences between the gastric motility scores of rats with sham lesions or PVN lesions (n = 5-10 in each group). Both CCK and LiCl continued to reduce gastric motility scores in rats with PVN lesions, but their effects were significantly blunted. More specifically, in rats with sham lesions, gastric motility scores were typically reduced by at least 75% of baseline values when measured 10 min after receiving CCK (1 pg/kg ip; Fig. 4A) and 20 min after receiving LiCl (1.5 meq/l ip; Fig. 5A). In rats studied 3 days and 3 wk after PVN lesions, however, the inhibitory effects of these two treatments on gastric motility scores at those time points were significantly smaller than in rats with sham lesions. Data collected 3 days and 3 wk postlesion were from different sets of animals, and because their baseline gastric motility scores were not significantly different, their data were combined (CCK, P < 0.02, Fig. 4C; LiCl, P < 0.01, Fig. 5C). Both CCK and LiCl treatments also reduced fluctuations in gastric pressure in rats with PVN lesions when measured 12-20 h after surgery (Figs. 4B and 5B), and to a lesser extent than in sham-lesioned rats (combined CCK and LiCl data, 12-20 h postlesion: P < 0.01). It should be noted, however, that the basal gastric motility scores of the rats with PVN lesions were significantly
A
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Fig. 3. Tracing of gastric pressure recordings of representative 12-20 h after a sham lesion (A) or a complete PVN lesion (B). A
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Fig. 4. Effect of systemic injection of CCK (1 pg/kg ip) on gastric motility in rats (A) 12-20 h after sham lesions or (B) 12-20 h or (C) 3 days or 3 wk after PVN lesions. Shown are motility scores for individual animals expressed as a percent of baseline values. Arrows indicate time of injection. 5.r 120 5 $100 ; -
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Fig. 5. Effect of systemic injection of LiCl (1.5 meq/kg ip) on gastric motility in rats either (A) 12-20 h after sham lesions or (B) 12-20 h or (C) 3 days or 3 wk after PVN lesions. Shown are motility scores for individual animals expressed as a percent of baseline values. Arrows indicate time of injection.
greater than the values observed in control animals, as mentioned. Effect of PVN lesions on food intake. Bilateral lesions of PVN had no significant effect on food intake and did not consistently affect body weight when compared with sham lesions (gains in body weight during 30 days postlesion: PVN lesions, 4.6 t 0.4 g/day; controls, 4.4 t 0.3 g/day). All groups had similar baseline food intakes, and there were no differences between groups in the inhibition of food intake produced by systemic injections of CCK or LiCl at two different doses (Table 1). Fig. 2. Drawings of coronal hypothalamic sections with tracings of the lesions of all rats used in the studies of (A) gastric motility (n = 26) and (B) food intake (n = 16). Tracings were made with a camera lucida, and the drawings were made based on the Konig and Klippel stereotaxic atlas (12, Figs. 29-32). More rostra1 sections are depicted at the top of the figure. F, fornix; OC, optic chiasm.
Effect of lidocaine injection into the PVN on gastric pressure. Injection of aCSF into the PVN had no signif-
icant effect on gastric pressure. Figure 6 illustrates gastric pressure recordings from two representative rats during the baseline period and after bilateral microinjection of lidocaine (0.4 pi/side) into the PVN. After this treatment,
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B
CCK ,
A
Table 1. Effect of systemic injection of CCK or LiCl on food intake in rats with PVN lesions and sham-lesioned controls
CCK 1 Pdk 5 Pdk
n
Food intake,
66.9dl.O 56.4t5.3
12 8
49.9t5.0 49.1t14.3
60.6t8.3 41.5t9.7
16 5
44.92 15.4 23.8dl.l
Food intake,
8 8 7 8
%
%
LiCl 1.5 meq/kg 3 mea/kg:
,q 1
PVN Lesions
Sham Lesions n
LiCl PVN Lidocaine
Values are means t SE expressed as a percent of baseline food intakes. There were no significant differences between groups in the effects of each dose of CCK or LiCl on food intake.
5 min
Fig. 6. Tracings of gastric pressure recordings of 2 representative rats before and after microinjection of lidocaine into the PVN bilaterally.
gastric motility scores increased within minutes and remained elevated for at least 30-40 min. On the other hand, injection of lidocaine into the PVN did not eliminate the inhibition of gastric motility produced by systemic injection of CCK or LiCl. As shown in Fig. 7, gastric motility was inhibited by systemic injection of CCK (n = 5, P < 0.01) or LiCl (n = 4, P < 0.05) given 20 min after microinjection of lidocaine into the PVN, a time when gastric motility scores were still elevated by lidocaine. The distribution of dye provided histological verification of the microinjection site just dorsal to the PVN in all five animals for which data were presented. However, when the injections occurred lateral, dorsal, or ventral to the PVN (n = 7), or into the third cerebral ventricle (n = 5), the lidocaine injection produced no change in gastric pressure. DISCUSSION
Gastric afferents stimulate oxytocinergic neurons in the hypothalamic PVN (28). Moreover, electrical stimulation of the PVN decreases gastric motility in rats (24). The present experiments sought to provide additional information regarding the influence of the PVN on gastric function in rats. Our results suggest that the PVN exerts a tonic inhibitory effect on gastric motility, because bilateral lesions of PVN caused an acute increase in gastric motility, which was still evident even 12-20 h after the lesion. The same effect was seen within minutes after acute anesthetization of the PVN, but neither effect was seen when lesions or anesthetization occurred at sites adjacent to PVN. Together with our recent findings that intracerebroventricular (icv) injection of an OT receptor
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Fig. 7. Effect of (A) CCK or (B) LiCl on gastric motility after microinjection of lidocaine into the PVN. Shown are motility scores for 4 animals, expressed as a percent of baseline values. Arrows indicate times of injection.
antagonist similarly increased gastric motility acutely in conscious rats (7), and the present observations that the inhibitory effects of CCK and LiCl on gastric motility were significantly blunted in rats with PVN lesions, these results strongly support previous proposals that parvocellular oxytocinergic neurons play an important role in the central control of gastric function (15, 23). On the other hand, increases in gastric motility were no longer evident 3 days or 3 wk after PVN lesions. Since gastric function is likely to have multiple redundant central control mechanisms, these findings suggest that after damage to the PVN other elements controlling gastric function assume greater significance than they have normally. However, despite this effect, it is noteworthy that CCK and LiCl both remain able to inhibit gastric motility even by 12-20 h after PVN lesions while basal motility still is elevated. More acute disruption of PVN activity by lidocaine anesthetization similarly did not eliminate the inhibitory effects of CCK and LiCl on gastric motility, which would be expected if the PVN played a critical role in these processes, since presumably the tests conducted during PVN anesthetization did not allow sufficient time for compensatory mechanisms to be established. Thus these results demonstrate that the PVN is not essential for producing the inhhbitory effects of CCK and LiCl on gastric motility in rats. Given that systemic CCK appears to affect gastric motility by stimulating receptors located on afferent fibers of the gastric vagus that project to the nucleus tractus solitarius (NTS) in the brain stem (16,21,25), whereas LiCl is thought to stimulate chemoreceptors in the area postrema (2, 22), the present results allow the possibility that the inhibition of gastric motility induced by these two agents is mediated in part by local circuits in the brain stem, from the NTS and area postrema to the dorsal motor nucleus of the vagus. Aside from its putative role in the central control of gastric function, the hypothalamic PVN also has been suspected of playing an important role in the control of food intake. This possibility has been suggested by observations that hyperphagia in rats is caused by bilateral lesions of the PVN or of fibers projecting caudally from the PVN (1, 10, 11, 13). The most effective lesions in producing hyperphagia in rats appear to destroy the cauda1 PVN and the adjacent perifornical region, which contains both cells and fiber tracts projecting caudally, including some containing OT. The knife used in the
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present series of studies avoided damage to this perifornical area, and perhaps for that reason the animals did not become hyperphagic despite complete destruction of all PVN tissue as classically defined. In contrast, it is noteworthy that PVN lesions produced by a similar but slightly larger knife, which damaged the fornices and dorsomedial hypothalamus in addition to the PVN, have been reported to produce significant increases in body weight in rats (4). Thus it would appear from these studies that laterally situated neurons, perhaps including parvocellular OT-containing fibers in this region, are critical to the phenomenon of hyperphagia induced by lesions of the PVN area. Presumably, these perifornical neurons in the caudal PVN provide a component of inhibition in the central neural control of food intake that, when damaged, disinhibits the system and leads to hyperphagia. This hypothesis is consistent with substantial recent evidence suggesting a role of brain OT-containing neurons in the inhibition of food intake (6, 11, 17-19, 29, 30). Although it is not yet clearly established what the role of the PVN is in the central control of food intake and gastric motility, the present observations that CCK and LiCl had undiminished inhibitory effects on food intake in rats with PVN lesions allow the possibility that the laterally situated parvocellular oxytocinergic neurons, projecting caudally from the PVN, participate in the inhibition of food intake induced by vagal (e.g., CCK) or toxic (e.g., LiCl) stimuli into the NTS. More medially situated parvocellular OT-containing neurons may be involved in modulating the inhibition of gastric motility caused by those same stimuli. The present results may be contrasted with the results of parallel experiments in which an OT-receptor antagonist given icv blunted the inhibition of food intake by CCK and LiCl, by 28-G% (19). Perhaps the present PVN lesions were ineffective in blunting the inhibition of food intake induced by CCK and LiCl because critical oxytocinergic fibers, whose function was blocked by the OT-receptor antagonist, were not damaged by our PVN lesions. Alternatively, because evaluation of food intake in the present study was not done until 2-3 wk after the lesions, perhaps adaptive adjustments in the central systems that control food intake occurred after the lesions, which could not occur after acute injection of the OT antagonist. If so, then a comparable blunting of their inhibitory effects on eating would be expected if CCK and LiCl were given to rats shortly after anesthetization of PVN. These possibilities await further investigation. An important role of PVN in the mediation of the anorexia induced by CCK in rats has been suggested by Crawley and Kiss (3). In their studies, rats with PVN knife-cut lesions were deprived of food overnight and then placed in a novel open field with palatable mash available. Food consumption was not suppressed in animals with PVN lesions after systemic injection of 5 or 10 in rats Pdk cw wh ereas it was markedly inhibited with sham lesions. Because of the many differences in protocol between that study and the present experiments, including the environment in which the feeding test was conducted, the feeding schedule, the doses of CCK used,
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and the food being consumed, it remains possible that the PVN has an important role in the control of food intake but one that can be seen only in certain experimental paradigms. Alternatively, because the knife used to produce the lesions in the previous study (3) was larger than the one used in the present study, it is again possible that its damage to critical perifornical fibers in the PVN accounted for the observed failure of CCK to inhibit food intake. Finally, it seems possible that the brain stem plays a prominent role in the control of food intake (5,8, 9, 20). These possibilities are not mutually exclusive. In summary, the present findings that acute PVN lesions and anesthetization of the PVN increase basal gastric motility in rats and that PVN lesions blunt the inhibition of gastric motility induced by CCK and LiCl administered systemically suggest that the PVN has an important role in the central control of both tonic and phasic gastric function. Although the present findings that PVN lesions did not alter the CCK- or LiCl-induced inhibition of food intake do not support a role for the PVN in mediating these effects, these lesions purposely avoided damage to more lateral perifornical areas of the hypothalamus. In light of numerous other reports suggesting a role of the PVN in the control of food intake, it seems clear that more detailed investigations of the hypothalamic structures involved in these effects are warranted. We gratefully acknowledge the expert contributions of Dr. Gloria Hoffman in the histological assessment of the lesions. This work was supported in part by National Institutes of Health (NIH) Grant MH-25140 (MERIT Award to E. M. Stricker and J. G. Verbalis), NIH National Research Service Award MH-09770 (to L. M. Flanagan), and NIH Research Career Development Award DK-02014 (to J. Dohanics). Portions of this work were submitted by L. M. Flanagan in partial fulfillment of the requirements for the PhD degree. Some of these findings were presented in preliminary form at the annual meeting of the Society for Neuroscience, St. Louis, MO, November 1990. Current address and address for reprint requests: L. M. Flanagan, Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Ave., New York, NY 10021-6399. Received 29 August 1991; accepted in final form 21 January 1992. REFERENCES 1. Aravich, P. F., and A. Sclafani. Paraventricular hypothalamic lesions and medial hypothalamic knife cuts produce similar hyperphagic syndromes. Behau. Neurosci. 97: 970-983, 1983. 2. Borison, H. L., and S. C. Wang. Physiology and pharmacology of vomiting. Pharmacol. Rev. 5: 193-230, 1953. 3. Crawley, J. N., and J. 2. Kiss. Paraventricular nucleus lesions abolish the inhibition of feeding induced by systemic cholecystokinin. Peptides 6: 927-235, 1985. 4. Darlington, D. N., J. Shinsako, and M. F. Dallman. Paraventricular lesions: hormonal and cardiovascular responses to hemorrhage. Brain Res. 439: 289-301, 1988. 5. Flanagan, L. M., R. E. Blackburn, J. G. Verbalis, and E. M. Stricker. Hypertonic NaCl inhibits gastric motility and food intake in rats with lesions in the rostra1 AV3V region. Am. J. Physiol. 263 (Regulatory Integrative Cornp. Physiol. 32): R9-R14, 1992. 6. Flanagan, L. M., J. G. Verbalis, and E. M. Stricker. Effects of anorexigenic treatments on gastric motility in rats. Am. J. Physiol. 256 (Regulatory Integrative Cornp. Physiol. 25): R955R961, 1989. 7. Flanagan, L. M., B. R. Olson, A. F. Sved, J. G. Verbalis, and E. M. Stricker. Gastric motility in conscious rats given
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