Contribution of caudal brainstem to d -fenfluramine anorexia

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Contribution of caudal brainstem to dfenfluramine anorexia Article in Psychopharmacology · May 1997 DOI: 10.1007/s002130050253 · Source: PubMed

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University of Illinois at Chicago

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Psychopharmacology (1997) 130:375–381

© Springer-Verlag 1997

O R I G I N A L I N V E S T I G AT I O N

&roles:Harvey J. Grill · Jamie C.K. Donahey · Lynne King Joel M. Kaplan

Contribution of caudal brainstem to d-fenfluramine anorexia

&misc:Received: 30 July 1996 / Final version: 23 November 1996

&p.1:Abstract Of the central 5-HT substrates that may mediate the anorexic actions of systemically administered dfenfluramine (d-FEN), those in the forebrain have received the most attention. As a counterpoint to this forebrain focus, we evaluated the contribution of caudal brainstem substrates to the anorexic action of d-FEN. Two experimental protocols were employed. In one we compared the feeding response (intra-oral intake of 12.5% glucose) of intact and chronic supracollicular decerebrate (CD) rats to systemic administration of d-FEN. In the other, d-FEN was administered via fourth intracerebroventricular (ICV) injection to determine whether a dose-related suppression of intra-oral intake could be obtained. A dose-dependent suppression of intra-oral intake was obtained in the CD rat treated with d-FEN (0–8 mg/kg, delivered IP 20 min before testing). The threshold dose was two to three times higher in CD rats than in their intact controls, but the dynamic range of the doseresponse curves of the two groups were overlapping with similar slopes of decline and with comparable maximal intake suppression. Fourth ICV administration of d-FEN in the intact rat yielded a dose-related suppression of intra-oral intake. Intake was also suppressed by fourth ICV d-FEN (30 mg) when rats drank 12.5% glucose solution from a spout. The reduced intra-oral intake following fourth ICV d-FEN treatment was partially attenuated by the systemic administration of the serotonin antagonist metergoline (0.4 mg/kg; IP). The CD results demonstrate the sufficiency of caudal brainstem receptors in mediating intake suppressive responses to systemic d-FEN. The fourth ICV results suggest further that 5-HT receptors in the caudal brainstem play a significant role in normal meal size control in the neurologically intact rat. &kwd:Key words Serotonin · Food intake · Chronic decerebrate · Fourth ventricle · Intra-oral intake&bdy: H.J. Grill (✉) · J.C.K. Donahey · L. King · J.M. Kaplan Department of Psychology, University of Pennsylvania, 3815 Walnut Street, Philadelphia, PA 19104, USA&/fn-block:

Introduction Of the central serotonergic systems that may mediate the anorexic actions of systemically administered fenfluramine (Copp et al. 1967; Neill et al. 1990; Samanin and Grignaschi 1996), those in the forebrain, particularly in the hypothalamus, have received the greatest attention (e.g., Weiss et al. 1986; Curzon 1990). A hypothalamic orientation is reinforced, for example, by reliable intake suppression following d-fenfluramine (d-FEN) microinjection into the paraventricular nucleus (e.g., Weiss et al. 1986; Leibowitz et al. 1987; Shor-Posner et al. 1986). While the paraventricular nucleus may be sufficient for an intake response to d-FEN, it does not appear to be necessary. Thus, d-FEN and other systemically administered serotonergic agonists remain effective after bilateral radio frequency lesions of paraventricular nucleus (Fletcher et al. 1993). Such results indicate the need to expand the anatomical perspective on the system that underlies serotonergic effects on ingestive behavior. It is surprising, on a priori grounds, that caudal brainstem (CBS) substrates have not yet been studied extensively in this regard. Contained within its boundaries are all of the serotonin producing cell bodies and a prominent distribution of 5-HT receptors that encompass structures whose relevance to intake control has been amply demonstrated (e.g., Lanca and Van der Kooy 1985; Thor and Helke 1987; Hoffman and Mezey 1989; Palacios et al. 1991; Wright et al. 1995). A CBS orientation is supported by c-FOS induction studies in which CBS regions are activated by peripheral d-FEN injection (e.g., Li and Rowland 1993). Li et al. (1994) went further to demonstrate that lesions of one caudal brainstem site highlighted in the c-FOS profile, the lateral parabrachial nucleus, significantly attenuated the response to systemic d-FEN. This demonstration suggests that CBS mediation of dFEN anorexia should be explored further. In the present study, we provide two complementary approaches to the CBS contribution to d-FEN anorexia in the rat. In one, the feeding response to systemically administered d-FEN in neurologically intact subjects is

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compared to that of the chronically maintained decerebrate rat, surviving a complete transection of the neuraxis at the meso-diencephalic juncture. In the other, CBS serotonin substrates are targeted by fourth ICV injection of d-FEN. The decerebration strategy has shown the CBS to be sufficient for various aspects of feeding control (Grill and Kaplan 1990). Because chronic decerebrate (CD) rats do not approach food, their ingestive competence must be evaluated with a specialized intake test involving direct oral infusion of nutritive fluids. Intra-oral infusions are actively ingested by CD rats (and their intact controls) via rhythmic, coupled movements of the jaw and tongue and coordinated swallowing actions (Kaplan and Grill 1989). As is the case with intact rats, the amount consumed when the CD meets the satiety criterion of the “intra-oral intake test” is sensitive to stimulus concentration (Flynn and Grill 1988) and to post-ingestive inhibitory feedback arising from the gastrointestinal tract (Grill and Kaplan 1992; Seeley et al. 1994). In addition, CD and intact rats respond similarly to a range of pharmacological treatments, including cholecystokinin (Grill and Smith 1988; Kaplan and Sodersten 1994), bombesin (Flynn and Robillard 1992), insulin (Flynn and Grill 1983) and apomorphine (Kaplan and Sodersten 1994). The CD rat, however, fails to respond to certain treatments that dramatically affect their neurologically intact controls. It does not increase its water or sodium chloride intake in response to depletion treatments (Grill and Miselis 1981; Grill et al. 1986), nor is its sucrose intake sensitive to the systemic/metabolic aspect of food deprivation (Seeley et al. 1993). The CD preparation, therefore, can be a discriminating tool for evaluating whether or not the CBS contributes to the ingestive effects of treatments of interest. One element of a case for the sufficiency of CBS substrates in mediating d-FEN anorexia would be provided if intra-oral intake of the CD is reduced, as it is in intact rats (Wolgin et al. 1988; Kaplan et al. 1996), following systemic injection of d-FEN. Such a result would be compelling, but would not by itself speak to the role of CBS in normal intake control in the neurologically intact rat. The CBS may play a more prominent role after, than it does before the transection. Thus, the relevant substrates may be reorganized (e.g., by receptor upregulation; Frankfurt et al. 1993; Manrique et al. 1994) in a manner that amplifies its sensitivity to d-FEN. Alternatively, the CBS contribution to d-FEN’s intake effect may reflect the unmasking of function that is otherwise served predominantly by forebrain substrates. As an approach to this issue, we deliver fourth ICV injections of d-FEN to target serotonin substrates in the caudal brainstem of intact rats. The effect of fourth ICV d-FEN delivery is evaluated using the intra-oral intake test and, in order to provide a bridge between intake methodologies, in a more standard paradigm, the spout-licking test. Our results, taken together, argue for increased attention to CBS serotonin systems in ingestion control.

Materials and methods Subjects Male Sprague-Dawley rats (Charles River) weighing 350–450 g were housed individually in hanging cages and maintained on a 12:12-h light:dark schedule. Individual rats were tested at the same time each day, between 5 and 8 h after lights on. Pelleted food and tap water were available ad libitum, except for rats in expt 1, as noted below. Experiment 1: d-FEN dose-response analysis in intact and CD rats Surgery Rats received a complete transection of the neuraxis at the mesodiencephalic juncture according to the procedure described in Grill and Norgren (1978). Briefly, the decerebration was accomplished in two hemitransection procedures separated by at least 7 days. For each, rats were anesthetized with ketamine (9 mg/kg) and xylazine (1.5 mg/kg) IM. Each hemitransection was made with a hand-held blunted spatula. During the second hemitransection surgery, rats received two intra-oral cannula. A separate group (n=9) of neurologically intact rats received intra-oral cannulae (PE-100) under ketamine/xylazine anesthesia. Each cannula was led from just lateral to the first maxillary molar to emerge at the top of the head. Stainless steel tubing (19 G) was press fitted into the distal end of the tube and secured to the skull with screws and dental acrylic (see Grill et al. 1987 for details.) At least 1 week was allowed for recovery before habituation training and testing began. Tube-feeding schedule Because CD rats do not spontaneously feed (see Introduction), CD (n=10) and intact control (n=6) rats were maintained with four daily 9-ml tube feedings of milk diet (1:1 Bordens sweetened condensed milk:water, supplemented with 1 ml Polyvisol with iron per 600 ml). Tube feedings were separated by a minimum of 2 h. In addition, because of labile body temperature, rectal temperature was taken after each feeding. CD rats were placed on heating pads when hypothermic and under a cooling fan when hyperthermic. Apparatus Rats were run in groups of four to six in individual hanging cages (18×25×36 cm). Intra-oral infusions were driven by an infusion pump (Harvard Apparatus, Pump 44). Each infusion line was led through a miniature three-way solenoid valve (Lee) either to a waste dish or to the rat. A PC-AT personal computer with custom software and interface, controlled infusion delivery to the rat by solenoid activation, and tracked cumulative intake. Procedure Stimulus selection. &p.2:d-FEN has been shown to reduce intake of a broad range of solid and liquid foods (for review see Cooper 1992) including sweetened milk diet (Wolgin et al. 1988), sucrose (Asin et al. 1992) and glucose (Kaplan et al. 1996) solutions. Because CD rats do not approach food, intra-oral delivery of a liquid stimulus is required for ingestive analysis. A 12.5% glucose solution was therefore chosen as the model food for all experiments presented here. Intra-oral intake test. &p.2:The intra-oral glucose infusion was initiated 5 min after the rat was placed into the test cage. Satiety criterion. &p.2:When the solution was first seen to drip from the rat’s mouth, the infusion was halted for 30 s. The test was termi-

377 nated if the rat rejected glucose a second time within 60 s after the infusion was resumed. Otherwise, the test continued until this criterion (two rejections within a 90-s period) was met.

Experiment 3: effect of systemic metergoline injection on the intake response to fourth ICV-administered d-FEN Subjects

Habituation training. &p.2:Beginning at least 1 week after surgery, rats received an intra-oral intake test with 12.5% glucose during each of ten daily habituation training sessions. The intra-oral infusion was delivered at 0.75 ml/min.

Eight naive rats received fourth ICV and intra-oral cannula surgery, 5TG verification tests, and habituation training, as described above.

Experimental design

Experimental design

After habituation training, each rat received five injections of dFEN (1, 2, 4, 6, 8 mg/kg) and one vehicle injection. An IP injection was delivered every second or third day, 30 min before the beginning of the intra-oral intake test. An intake test was run on the alternate days with no injection to assure stability of baseline intake. Drug presentation order was counterbalanced across rats using a modified Latin square design.

Each rat received two injections, one IP and one ICV, prior to each of four intra-oral intake tests run on consecutive days. The IP injection was either 0.4 mg/kg metergoline dissolved in acetic acid and neutralized to pH 6.5 with NaOH, or the acetic acid/NaOH vehicle (pH 6.5), delivered in each case in a volume of 1.0 ml/kg. IP injections were delivered 3 h before intake testing (see Grignaschi and Samanin 1993). The fourth ICV injection, delivered 10 min prior to intake testing, was either 120 µg d-FEN in 3 µl of distilled water or 3 µl water vehicle. Each of the four combinations of IP and ICV injections was tested once with presentation order counterbalanced across the four test sessions.

Experiment 2: fourth ICV delivery of d-FEN: dose-response analysis Surgery Nine naive rats received, during the same surgical procedure, intra-oral cannulae (see above) and a stereotaxically implanted fourth ICV cannula. The ICV infusion assembly (Plastic One) consists of a guide cannula (22 G) and injector (28 G). The cannula was advanced into the brain through a small hole drilled in the skull on the midline, 2.5 mm anterior to the occipital crest, and positioned 5.5 mm below the dura (1 mm above the injection site). The ICV cannula hub and the protruding portion of the intra-oral cannulae were secured to the skull with screws and dental acrylic. An obturator was then inserted into the guide cannula. Procedure Fourth ICV injection. &p.2:d-FEN was prepared for fourth ICV injection in sterile distilled water vehicle. The solution to be infused was loaded into a Hamilton syringe connected to the injector that when inserted, extended 1 mm beyond the tip of the guide cannula. For each injection, 3 µl of vehicle or drug+vehicle was infused over a 1-min period. The injector was removed from the guide cannula 30 s later and the obturator was replaced. Verification of cannula placement. &p.2:We verified cannula placement prior to behavioral testing by determining whether a rise in plasma glucose followed fourth ICV injection of 210 µg 5-thio-D-glucose in 3 µl distilled water (Flynn and Grill 1985). A rise in plasma glucose, mediated by the sympathoadrenal hyperglycemic response, was obtained in all rats included in this analysis (mean increase in plasma glucose of 150 mg/dl plasma from preinjection values). To verify cannula placement after testing, a functional [5TG] probe, and in most cases a post-mortem inspection of the location of 3 µl of injected India ink, was conducted.

Experiment 4: fourth ICV d-FEN effect on ingestion of glucose obtained from a drinking spout Four naive rats were maintained as described above. Rats received fourth ICV cannula surgery and 5TG verification tests, described above, prior to the experiment. Apparatus Rats were tested in individual hanging wire test chambers. A drinking spout (Girton Inc.) was mounted on the front panel of each chamber, 4 cm above the chamber floor. Fluid was delivered by a PE-100 tube with its flared end fitted tightly in the opening at the tip of the drinking spout. Fluid (12.5% glucose), maintained under constant pressure (4.0 psi) was delivered to each chamber from individual reservoirs. The fluid line from reservoir to spout was interrupted by a two-way solenoid valve. The duration of solenoid activation was calibrated to deliver a preset volume of 8 µl of glucose to the drinking spout. The system registered (and stored) the time of occurrence of each lick event via a lickometer circuit that passed less than 50 µA through the rat. With each lick registration, the corresponding solenoid was activated for the appropriate precalibrated duration. Predicted intake (number of licks×lick volume) was confirmed by pre- and post-test weight measurements of the fluid reservoir’s content. Intake test Rats were placed into the testing chamber. The test meal began with the first spout-lick and ended 60 min later. Procedure

Experimental design After habituation training (see Materials and methods, expt 1), each rat received four injections of d-FEN (60, 120, 240, 480 µg) and one vehicle injection. An injection was delivered every second day, 10 min before the beginning of the intra-oral intake test (see Materials and methods, expt 1). An intake test was run on the alternate days with no ICV injection to assess any carry-over effect from the previous day’s treatment and to assure stability in baseline intake. Drug presentation order was counterbalanced across rats using a modified Latin square design.

Habituation training. &p.2:Rats received a series of 14 daily habituation training sessions prior to testing. During each 1-h session, rats had access to the drinking spout which delivered 12.5% glucose. Experimental design d-FEN (30 µg) in water vehicle (3 µl) or vehicle alone was delivered 30 min before the licking test. Injection conditions were counterbalanced.

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Results Experiment 1 CD and intact dose-response profiles were compared via two separate two-way ANOVAs (CD/intact×dose), with percent of vehicle baseline intake (Fig. 1) and absolute intake (Fig. 1, inset) as respective dependent variables. The ANOVA on percent of baseline intake revealed a significant effect of dose [F(5,70)=9.30, P