Delayed satiety-like actions and altered feeding microstructure by a selective type 2 corticotropin-releasing factor agonist in rats: intra-hypothalamic urocortin 3 administration reduces food intake by prolonging the post-meal interval

Abstract

Brain corticotropin-releasing factor/urocortin (CRF/Ucn) systems are hypothesized to control feeding, with central administration of 'type 2' urocortins producing delayed anorexia. The present study sought to identify the receptor subtype, brain site, and behavioral mode of action through which Ucn 3 reduces nocturnal food intake in rats. Non-food-deprived male Wistar rats (n=176) were administered Ucn 3 into the lateral (LV) or fourth ventricle, or into the ventromedial or paraventricular nuclei of the hypothalamus (VMN, PVN) or the medial amygdala (MeA), regions in which Ucn 3 is expressed in proximity to CRF(2) receptors. LV Ucn 3 suppressed ingestion during the third-fourth post-injection hours. LV Ucn 3 anorexia was reversed by cotreatment with astressin(2)-B, a selective CRF(2) antagonist and not observed following equimole subcutaneous or fourth ventricle administration. Bilateral intra-VMN and intra-PVN infusion, more potently than LV infusion, reduced the quantity (57-73%) and duration of ingestion (32-68%) during the third-fourth post-infusion hours. LV, intra-PVN and intra-VMN infusion of Ucn 3 slowed the eating rate and reduced intake by prolonging the post-meal interval. Intra-VMN Ucn 3 reduced feeding bout size, and intra-PVN Ucn 3 reduced the regularity of eating from pellet to pellet. Ucn 3 effects were behaviorally specific, because minimal effective anorectic Ucn 3 doses did not alter drinking rate or promote a conditioned taste aversion, and site-specific, because intra-MeA Ucn 3 produced a nibbling pattern of more, but smaller meals without altering total intake. The results implicate the VMN and PVN of the hypothalamus as sites for Ucn 3-CRF(2) control of food intake.

Figure 1

Effect of lateral ventricle (LV) pretreatment (−20 min) with murine urocortin 3 (Ucn 3) on mean (+ SEM) incremental food (a) and water intake (b) in nondeprived, male Wistar rats (n = 8) housed in automated test cages that detect intake as nosepoke events (Experiment 1). Food intake is normalized for body weight per Kleiber’s mass exponent. *p < 0.05 vs vehicle treatment (within-subject Newman–Keuls test). Panel (c) shows effects of LV injection of Ucn 3 on the formation of a conditioned taste aversion (CTA) (Experiment 2). Data are expressed as the mean (+ SEM) preference ratio for a 7.31mM saccharin solution over water as a function of having received LV Ucn 3 injections immediately after consuming the previously novel saccharin solution, ^§p < 0.05, post-pair preference ratios were significantly higher than pre-pair preference ratios (n = 7/group). Panel (d) shows effects of post-saccharin injections of the positive control 0.15M LiCl (i.p., 20 ml/kg) on the formation of a CTA for the otherwise palatable saccharin solution, as compared to rats receiving isotonic NaCl. LiCl, but not anorectic and hypodipsic doses of Ucn 3, reliably produced a conditioned taste aversion. *p < 0.05 vs NaCl injection, ^#p < 0.05 preconditioning (pre-pair) vs post-pairing (post-pair) preference ratio was significantly lower after LiCl injections, ^§p < 0.05, post-pair preference ratios were significantly higher than pre-pair preference ratios after NaCl injections (n = 7/group) (between-subject Newman–Keuls tests).

Figure 2

Panel (a) shows that adult male, ad libitum fed Wistar rats receiving lateral ventricle (LV) (n = 9), but not subcutaneous (s.c.), injection of 2.5 μg murine Ucn 3 (n = 8) ate less in the subsequent 4 h than vehicle-treated subjects (n = 9) (Experiment 3). This effect was blocked by LV cotreatment with astressin₂B (A₂B), a selective CRF₂ antagonist (n = 6). Each rat received a s.c. injection (0.0, 2.5 μg Ucn 3) followed immediately by an LV injection (0.0, 2.5 μg Ucn 3, or 2.5 μg Ucn 3 + 10 μg A₂B) during the early portion of the dark cycle followed by free access to preweighed chow. Data are expressed as mean (+ SEM) food intake normalized for body weight per Kleiber’s mass exponent. Time course analysis showed an anorectic effect in the second 2 h post-injection bin. *p < 0.05, the s.c. 0.0 μg/LV 2.5 μg (‘central Ucn 3’) group differed significantly from every other group at the total 4 and 3–4 h periods (Dunnett’s test). Panels (b) and (c) show effect of fourth ventricle injection of murine Ucn 3 (b) or human Ucn 1 (c) on nocturnal free feeding in ad libitum fed male Wistar rats (Experiment 4). The 2.9 μg dose of Ucn 1 is equimolar to the 2.5 μg dose of Ucn 3. *p < 0.05, the 2.9 μg Ucn 1 group significantly differs from vehicle group (between-subject Dunnett’s test).

Figure 3

Illustration of reconstructed injection sites from Experiment 5. Correct bilateral injection placements (‘hits’) are indicated as closed circles in the ventromedial (VMN; n = 7), paraventricular nuclei of the hypothalamus (PVN; n = 8), and medial amygdala (MeA; n = 6) (panels a, b and c, respectively). Incorrect injection placements (‘misses’) are indicated in panels (d), (e) and (f) for the VMN (n = 5), PVN (n = 4), and MeA (n = 2), respectively. Brain structure diagrams of coronal sections are adapted from Paxinos and Watson (Paxinos and Watson, 1998), the numbers refer to anterior–posterior distance from bregma in mm, L: left, R: right side of the brain.

Figure 4

Light photomicrograph of coronal brain sections showing example of injector placements (black arrowheads) within (panel a) the dorsomedial subdivision of the ventromedial hypothalamic nucleus (VMN), (b) the dorsal aspect of the paraventricular nucleus of the hypothalamus (PVN), and (c) the posterodorsal medial amygdala (MeA). Numbers (top right of each panel) indicate approximate anterior–posterior distance from bregma in mm. Ic, internal capsule; f, fornix; LH, lateral hypothalamus; mt, medial tubercle; opt, optic tract; st, stria terminalis; III, third ventricle.

Figure 5

Effect of brain site-specific injections of murine urocortin 3 (Ucn 3) on mean (+ SEM) incremental food and water intake in nondeprived, male Wistar rats housed in automated test cages that detect intake as nosepoke events (Experiment 5). Food intake is normalized for body weight per Kleiber’s mass exponent. Intake is shown in 2-h bins following bilateral injection into the ventromedial (VMN; n = 7) (panels a: food and d: water), paraventricular nuclei of the hypothalamus (PVN; n = 8) (b: food, e: water), or medial amygdala (MeA; n = 6) (c: food, f: water). ^#p < 0.01, *p < 0.05 compared to vehicle (within-subjects Newman–Keuls test).

Figure 6

Effect of bilateral injection of murine Ucn 3 into the ventromedial (VMN; n = 7), paraventricular nuclei of the hypothalamus (PVN; n = 8), or medial amygdala (MeA; n = 6) on the mean (+ SEM) average post-meal interval during the third–fourth post-injection hours (a) and on aspects of feeding bout microstructure (b–e) during the first 4 post-injection hours. Intra-VMH and PVN treatment prolonged the post-meal interval during the period of anorexia (a). No treatment altered the number of feeding bouts (b), but intra-VMH Ucn 3 infusion reduced the average bout size (c) and slowed the within-bout eating rate (d). Intra-PVN Ucn 3 infusion also slowed the within-bout eating rate and increased the serial irregularity of feeding within bouts, calculated as the entropy of the probability joint return map for feeding (e). Based on log-transformed frequency histogram analysis of inter-feeding and inter-drinking intervals, a feeding bout was defined as three or more feeding events in which consecutive feeding events were < 35 s apart. *p < 0.05 vs within-subjects vehicle treatment.

Figure 7

Illustration of the serial regularity within feeding bouts after bilateral injection of murine Ucn 3 into the paraventricular nucleus of the hypothalamus. Panels reflect doses of 0.0 (a, d), 0.1 (b, e), or 0.5 μg murine Ucn 3/side (c, f). Return maps (panels a–c) are constructed from ‘within-bout’ inter-feeding intervals (IFIs) from one representative rat (# PVN-1) during the first 4 post-injection hours. Under vehicle treatment, rats showed serial regularity from pellet to pellet reflected in a ‘clustered’ return map. Ucn 3 increased the dispersion/irregularity of successive feeding events, reflected as an increased spread and quantified as an increased return map entropy. Panels (d), (e), and (f) show density plots of the IFI return maps (joint IFI probability distribution) in a color-coded form from the representative rat (# PVN-1). The spreading and decreased ‘peakedness’ of the pattern indicates the irregularity of feeding within bouts that resulted from Ucn 3 treatment. Warmer colors (toward red) indicate higher local probabilities.