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Am J Physiol Regul Integr Comp Physiol 284: R1445-R1453, 2003. First published February 20, 2003; doi:10.1152/ajpregu.00554.2002
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Vol. 284, Issue 6, R1445-R1453, June 2003

SPECIAL TOPICS
Peptides that Regulate Food Intake
Effect of peptide histidine isoleucine on consummatory behavior in rats

Pawel K. Olszewski1,2,4, Michelle M. Wirth1, Timothy J. Shaw5, Martha K. Grace1, and Allen S. Levine1,2,3

1 Veterans Affairs Medical Center, Research Service, Minneapolis 55417; Departments of 2 Medicine and of 3 Psychiatry, University of Minnesota, Minneapolis 55455; 4 College of Veterinary Medicine, Minneapolis, Minnesota 55455; and 5 Bethel College, Arden Hills, Minnesota 55112


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peptide histidine isoleucine (PHI) and VIP are derived from the same precursor. While central VIP decreases food intake, potential effects of PHI on feeding have not been studied. In the current study, we found that PHI administered intracerebroventricularly (ICV) or into the hypothalamic paraventricular nucleus (PVN) or central nucleus of the amygdala (CeA) decreased food consumption in overnight-deprived rats. The magnitude of an anorexigenic response to PHI differed depending on the injection route: ICV-infused peptide evoked the most potent effect. We determined that that only PVN- and CeA-injected PHI did not have aversive consequences. In addition, we infused anorexigenic doses of PHI via the same routes and assessed Fos immunoreactivity of PVN oxytocin (OT) and vasopressin (VP) neurons using double immunohistochemistry. OT and VP are thought to promote feeding termination. PHI increased the percentage of Fos-positive OT neurons regardless of the injection route. PVN- and ICV-infused PHI induced activation of VP cells. We conclude that central PHI has an inhibitory influence on food intake in rats. The PVN, with OT and VP neurons, and CeA may be involved in the mediation of anorexigenic effects of PHI.

conditioned taste aversion; oxytocin; vasopressin; paraventricular nucleus of the hypothalamus; amygdala


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PEPTIDE HISTIDINE ISOLEUCINE (PHI) belongs to the pituitary adenylate cyclase-activating polypeptide (PACAP)/ glucagon family derived from a common precursor protein, prepro-VIP. PHI, VIP, peptide histidine valine (PHV), and peptide histidine methionine, among others, are processed from this precursor molecule. Prepro-VIP-related peptides, including PHI, share a structural homology, as well as a certain degree of functional similarities, e.g., by having a stimulatory effect on the release and/or synthesis of glucagon (8), prolactin (20, 28, 40), and melatonin (21, 37, 49), and by affecting circadian rhythmicity (2, 3), heart rate (38), vasomotor functions (15, 19), and activation of the hypothalamic-pituitary-adrenal (HPA) axis (4, 6).

PHI is widespread throughout the central and peripheral nervous systems (12). The presence of this peptide has been demonstrated in numerous central regions, where PHI often colocalizes in neurons with other products of prepro-VIP (13). Interestingly, PHI-containing neuronal elements are encompassed in several brain sites involved in the regulation of consummatory behavior, including the hypothalamic paraventricular (PVN), supraoptic (SON), and arcuate (ARC) nuclei, central nucleus of the amygdala (CeA), and bed nucleus of the stria terminalis (17, 30).

The abundance of receptors for PHI in these feeding-related areas has also been confirmed. There are two well-described subtypes of receptors for PHI, termed VIP1 and VIP2 (18, 24, 45). These receptors do not seem to be selective to PHI alone, but they bind (with different affinities) other products of the prepro-VIP family as well. Recently, Tse et al. (44) reported a discovery of a third subtype of the receptor, which binds PHI and PHV but does not interact with the remaining prepro-VIP-derived molecules. This apparent divergence in the receptor system may serve as an explanation of the fact that physiological and behavioral responses to prepro-VIP-related compounds are not always identical (1, 42).

Considering the importance of PHI in the regulation of various neuroendocrine processes and the presence of this peptide and the appropriate receptors in feeding-related brain areas, surprisingly little attention has been drawn to a possible involvement of central PHI in the control of consummatory behavior. Thus far, investigators have tried to link feeding only to circulating PHI, whereas the potential influence of this peptide acting within the brain has not been the main focus of research. At the peripheral level, it has been shown that meal ingestion causes a short-lived increase in the plasma PHI profile in humans (14). Also, central and peripheral vagal stimulation leads to a release of PHI in the stomach, where this peptide appears to affect gastric motility (29). It is noteworthy that indirect evidence points also to the likely role of the central pool of PHI in feeding control. It has been reported that central injection of this peptide activates hypothalamic oxytocin (OT) and vasopressin (VP) systems (5, 10, 34); importantly, OT and VP are thought to inhibit food intake through satiety- and aversion-related mechanisms (7, 23).

Evidence that seems more complete has been obtained regarding the role in feeding regulation of the PHI's counterpart VIP. Similarly to PHI, VIP acting in the peripheral tissues appears to affect some aspects of ingestive behavior. Kulkosky and colleagues (22) found that peripheral VIP decreased food intake associated with drinking in water-deprived rats. Some authors have observed an increased release of VIP to the general circulation due to food ingestion (16, 36). Importantly, VIP seems to influence consummatory behavior also through independent central mechanisms. Woods et al. (47) reported that ICV-injected VIP reduced meal size by 25% in rats. PVN and ICV injections of VIP as well as PHI generate dose-dependent increases in plasma ACTH and corticosterone levels in rats (5, 6). Another study linked this VIP-induced ACTH and corticosterone response to feeding (4).

On the basis of the above-mentioned observations, in the current project we sought to investigate potential anorexigenic effects of PHI acting within the central circuitry. First, we examined whether central PHI decreases food consumption in overnight-deprived rats. We studied the presumed anorexigenic effects of PHI administered ICV or directly into the PVN and CeA (2 feeding-related sites that contain receptors for PHI) (45). In conditioned taste aversion (CTA) experiments, we examined whether PHI injected at minimal anorexigenic doses and administered via the same routes has adverse consequences; the purpose of such approach is to test whether a PHI-generated decrease in feeding depends on sickness-/malaise-inducing properties of this peptide. In addition, we infused consumption-altering doses of PHI into the lateral ventricle, in the PVN, or in the CeA, and assessed activation of OT and VP neurons in two hypothalamic sites where these cells are amassed: in the PVN and SON. We also determined c-Fos immunoreactivity (IR; a marker of neuronal activation) of chemically unidentified PVN, SON, and CeA neurons after PHI treatment. Standard double immunohistochemistry for Fos and OT/VP allowed us to study activation of OT and VP neurons in the PVN and SON.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Adult male Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA) weighing ~300 g at the beginning of the experiment were used in the studies. Animals were housed individually in wire-mesh cages with a 12:12-h light/dark schedule (lights on at 0700) in a temperature- and humidity-controlled room. Water and food (Rodent Chow; Teklad, Indianapolis, IN) were available ad libitum, except when noted otherwise.

All behavioral manipulations as well as surgical and injection procedures presented in the current project adhere to the Guiding Principles in the Care and Use of Animals of the American Physiological Society and were approved by the Institutional Animal Care and Use Committee of the Minneapolis Veterans Affairs Medical Center.

Surgical Procedures

All rats were equipped with an indwelling stainless steel cannula in either the right lateral ventricle (20 gauge), hypothalamic PVN (26 gauge), or CeA (26 gauge). The stereotaxic coordinates, assessed according to the atlas of Paxinos and Watson (35), were as follows: 1) ICV: 1.5 mm lateral to the midline, 1.0 mm caudal to bregma, and 3.5 mm below the surface of the skull; 2) PVN: 0.5 mm lateral to the midline, 1.9 mm caudal to bregma, and 7.3 mm below the surface of the skull; and 3) CeA: 3.9 mm lateral, 2.5 mm caudal to bregma, and 7.0 mm below the surface of the skull. The injector needle extended 1 mm below the tip of the guide cannula. Dental cement was used to secure the cannula to two screws inserted in the skull. Surgeries were performed under Nembutal anesthesia (50 mg/kg body wt ip). Seven days of postoperative recovery were allowed before the injection trials began.

Water intake measurement after administration of angiotensin II (100 ng; Sigma Diagnostics, St. Louis, MO) provided verification of cannula placement in the lateral ventricle: those rats that drank <5 ml of water within 20 min after the injection of the peptide were excluded from the study.

Placement of the PVN cannula was initially verified based on the increase in food intake after the administration of neuropeptide Y (NPY; 117 pmol; Peninsula Laboratories, Belmont, CA). Animals that did not consume at least 3 g of chow within 1 h postinjection were considered to have an incorrectly placed cannula.

To avoid the possibility of confounding consumption-related side effects of NPY and angiotensin treatments, rats tested with either of these substances for the proper placement of an ICV and PVN cannula were not used in feeding or CTA studies for 4 days after the behavioral assessment of the cannula positioning. After the completion of experiments, rats were euthanized and brains were dissected out to determine cannula positioning in the PVN and CeA by histologic examination. Data from animals with an incorrect cannula placement were discarded.

Effect of ICV-, PVN-, and CeA-Injected PHI on Deprivation-Induced Feeding

After 18 h of food deprivation, animals were injected either 1) ICV with 0, 10, 20, or 30 nmol PHI-27 (Bachem, Torrance, CA) in a volume of 5 µl saline (n = 10) or 2) in the PVN with 0, 1, 3, or 10 nmol PHI in a volume of 0.5 µl saline (n = 9); or 3) in the CeA with 0, 1, 3, or 10 nmol PHI in 0.5 µl saline (n = 10). Each rat used in the study received each dose of the peptide; 3-4 days without any treatment preceded each experimental trial. Injections were performed in a counterbalanced fashion. We did not observe any differential effects associated with the order of injections or day when a given injection was administered (data not shown). All injections took place between 1000 and 1100. Immediately after treatment, preweighed chow pellets were placed in the hopper. These pellets and collected spillage were weighed and subtracted from the initial weight to quantify the amount of food eaten 1, 2, 4, and 24 h postinjection.

Data are presented as means ± SE and were analyzed using a one-factor ANOVA with repeated measures. For treatments showing a main effect by ANOVA, means were compared by post hoc Fisher's protected least-significant difference test (PLSD), and values were considered significantly different when P < 0.05.

Effect of ICV-, PVN-, and CeA-Injected PHI on Acquisition of CTA

Rats were accustomed to having access to water for 30 min (1030-1100) per day, for 4 days. On the fifth day, rats were given a novel 0.1% saccharin solution instead of water. After 30 min of drinking, they were injected with 1) 20 nmol PHI ICV (specifications as above), 2) 3 nmol PHI in the PVN (specifications as above), 3) 10 nmol PHI in the CeA (specifications as above), or 4) LiCl (127 mg/kg body wt ip). Isotonic saline was administered via the same routes in control animals. PHI was injected at the lowest doses found to reduce food intake. Rats treated with LiCl, a substance known to cause a powerful aversive effect, served as a positive control for CTA development (32, 46). During the next 2 days after injections, rats were presented only with water. On the eighth day, a standard two-bottle preference test was used to assess acquisition of CTA to the saccharin solution.

Control groups that underwent unconditioned stimulation (treatment not paired with the presentation of the saccharin solution) were also included.

Six animals per group were used in the taste aversion experiments.

The amount of ingested saccharin solution expressed as the percentage of total fluid intake was assessed during a two-bottle test per animal. This parameter was used to determine the acquisition of CTA. The results were averaged per experimental group and were analyzed using ANOVA followed by Fisher's PLSD test (significant when P < 0.05).

Effect of ICV-, PVN-, and CeA-Injected PHI on Fos-IR of OT/VP Neurons and on Fos-IR of Unidentified Neurons in the PVN, SON, and CeA

Rats placed on ad libitum access to food and water received a single injection of PHI 1) ICV at 20 nmol, 2) in the PVN at 3 nmol, or 3) in the CeA at 10 nmol. These doses were chosen based on the results of behavioral experiments as the lowest effective doses of PHI that affect consummatory behavior. Saline injected via the same routes served as a control solution (n = 4 or 5/group). All infusions were performed between 1030 and 1200. To prevent the induction of c-fos expression due to feeding or drinking, chow and water were immediately removed from the cages of injected rats. In addition to the fact that, in rats, consummatory activity is minimal during the light phase of the light/dark cycle, we measured food intake during 1 h preceding injections. As expected, animals ingested 0.0-0.3 g of chow during that period.

One hour after the treatment, animals were deeply anesthetized (Nembutal; 100 mg/kg body wt ip) and perfused with 50 ml of saline followed by 500 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and postfixed overnight in the same fixative at 4°C. Coronal Vibratome sections (40 µm thick) were cut through the regions of the SON, PVN, and CeA. They were processed as free-floating sections for standard single (Fos) and double (Fos-OT and Fos-VP) immunostaining.

Sections were pretreated for 10 min in 3% H2O2 and 10% methanol [in Tris buffered saline (TBS); pH 7.4] and incubated for 36 h at 4°C in the goat-anti-Fos antibody (1:9,000; Santa Cruz Biotechnology). Subsequently, tissue was incubated for 60 min in the rabbit-anti-goat antibody (1:400; room temperature; Vector Laboratories, Burlingame, CA). After a 60-min incubation in the avidin-biotin complex (room temperature), peroxidase in the sections was visualized with 0.05% diaminobenzidine (DAB), 0.01% H2O2, and 0.3% nickel sulfate. The vehicle for all incubations in antibodies was a mixture of 0.25% gelatin and 0.5% Triton X-100 in TBS. Rinsing steps were done in TBS alone. After the completion of c-Fos staining, some sections containing the PVN and SON were further processed to visualize OT and VP. Procedure was similar as in the staining for the first antigen; however, rabbit-anti-OT (1:11,000; Phoenix Pharmaceuticals, Belmont, CA) and rabbit-anti-VP (1:5,000; generously provided by Dr. R. M. Buijs of the Netherlands Institute for Brain Research, Amsterdam, The Netherlands) were used as primary antibodies; sections were incubated for 1 h in goat-anti-rabbit antibody (1:400; Vector Laboratories). Nickel sulfate was not added to the DAB solution to obtain the brown instead of black staining.

Sections were mounted on gelatin-coated slides, air-dried, dehydrated in alcohols, soaked in xylene, and embedded in Entellan (Merck).

Staining Analysis

Single staining for Fos. The number of c-Fos-positive nuclear profiles was counted bilaterally (in ICV-injected animals) and unilaterally (ipsilaterally to the cannula; in site specifically injected animals) for each neuroanatomical region of interest (the PVN, SON, and CeA) with boundaries defined according to the atlas of Paxinos and Watson (35) on six sections per animal. Images provided by Dage-MTI DC 3CCD camera attached to a Nikon Eclipse 400 microscope were analyzed using Scion Image software. Densities of Fos-IR nuclei (per 1 mm2) were averaged per animal and then per experimental group.

Double staining for Fos and OT or VP. Activation of OT and VP cells was studied by the analysis of c-Fos-IR in the immunohistochemically characterized neurons in the PVN and SON. Twelve sections per region that contained neurons expressing OT or VP were used for the analysis in each animal. The following estimates per section and then, by adding up the numbers, per region were assessed: 1) the total number of OT/VP neurons and 2) the total number of Fos-IR nuclear profiles colocalizing with OT/VP. The percentage of Fos-positive OT and VP neurons in the PVN and SON was calculated per animal. The percentages were averaged for each experimental group.

Data are expressed as means ± SE. Results of immunohistochemical studies were analyzed using a t-test, and values were considered significantly different when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of PHI on Feeding and CTA

ICV administration of PHI at 20 and 30 nmol resulted in a decrease of deprivation-induced feeding by ~40% during the 0- to 1 (P = 0.0024 and P = 0.0067, respectively)-, 0- to 2 (P = 0.0001 and P = 0.0006, respectively)-, and 0- to 4-h periods (P = 0.0053 and P = 0.0085, respectively) postinjection. No dose of the peptide affected 24-h food intake (Fig. 1; Table 1).


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  		Fig. 1.   Effect of peptide histidine isoleucine (PHI) injected ICV (A) into the paraventricular nucleus (PVN; B) or into the central nucleus of the amygdala (CeA; C) on feeding induced by overnight deprivation. Animals were presented with chow immediately after treatment; food intake was measured 1, 2, and 4 h postinjection. * Significantly different from controls: P < 0.05 (repeated measures).


                              
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  		Table 1.   Feeding response in overnight-deprived rats observed 24 h after administration of saline or PHI into the lateral ventricle, PVN, or CeA

When ICV-injected PHI at the lowest anorexigenic dose (20 nmol) was associated with an ingestion of a novel saccharin solution, it supported the development of a taste aversion to saccharin. The magnitude of the PHI-induced aversive response was similar to that observed due to treatment with LiCl (Fig. 2).


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  		Fig. 2.   Effect of PHI (A) and LiCl (B) on acquisition of a conditioned taste aversion (CTA) to the 0.1% saccharin solution. On the conditioning day, after their first exposure to saccharin, rats were injected with LiCl (127 mg/kg body wt ip) or with PHI ICV (20 nmol), into the PVN (3 nmol), or into the CeA (10 nmol); saline served as a vehicle. Three days later, a 2-bottle preference test (saccharin vs. water) was used to assess acquisition of CTA. Amount of ingested saccharin solution is expressed as the percentage of total fluid intake. * Significantly different from controls: P < 0.05.

When infused directly into the PVN, 3 and 10 nmol PHI significantly reduced feeding in deprived animals in the 0- to 2 (P = 0.0121 and P = 0.0256, respectively)- and 0- to 4-h periods (P = 0.0048 and P = 0.0307, respectively) by 10-20%, but no dose of PVN-injected PHI affected food intake 1 or 24 h after the treatment (Fig. 1; Table 1). Similarly, CeA administration of PHI caused a 10-20% decrease in feeding 2 and 4 h postinjection (P = 0.0453 and P = 0.0394, respectively). However, only the highest 10-nmol dose of this peptide produced an anorexigenic response (Fig. 1; Table 1).

In contrast to ICV PHI, neither PVN nor CeA administration of this peptide (at 3 and 10 nmol, respectively) after the ingestion of 0.1% saccharin by rats affected their later preference for the saccharin solution compared with controls (Fig. 2).

Total fluid intake in a two-bottle preference test did not differ significantly between groups in CTA experiments; unconditioned stimulation did not produce taste aversion (data not shown).

Effect of PHI on Fos-IR in Selected Sites and on Fos-IR of OT and VP Neurons

Immunohistochemical studies using single staining for c-Fos revealed that PHI administered into the lateral ventricle or into specific sites led to an increase in the density of Fos-IR nuclear profiles in the PVN and CeA. Elevated numbers of Fos-positive nuclei in the SON could be observed only after an ICV infusion of this peptide (Table 2).

                              
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  		Table 2.   Densities of Fos-IR nuclear profiles in the PVN, SON, and CeA in rats injected ICV, into the PVN, or into the CeA with saline or anorexigenic doses of PHI

Double immunostaining allowed us to observe a significant increase in activation of OT neurons in the PVN as a result of administration of PHI via each of the three routes. The percentage of Fos-positive OT cells was highest (almost 40%) due to ICV-infused PHI. VP-containing PVN neurons exhibited elevated c-Fos-IR after ICV and PVN, but not CeA injection of PHI. Only ICV PHI treatment generated a significant increase in activation of OT and VP cells in the SON (Figs. 3 and 4).


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  		Fig. 3.   Effect of PHI injected ICV (20 nmol) into the PVN (3 nmol) or into the CeA (10 nmol) on Fos immunoreactivity of oxytocin (A and C) and vasopressin (B and D) neurons localized in the PVN (A and B) and supraoptic nucleus (SON; C and D). Saline served as a control vehicle. Animals were perfused 1 h after treatment. * Significantly different from controls: P < 0.05.



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  		Fig. 4.   Photomicrographs depicting coronal sections through the hypothalamic PVN of rats injected ICV with saline (top) or 20 nmol PHI (bottom). Sections were double stained for c-Fos and oxytocin (OT; left) or c-Fos and vasopressin (VP; right). Large arrows, Fos-positive OT/VP neurons; small arrows, OT/VP neurons devoid of Fos. Scale bar = 0.05 mm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A member of the prepro-VIP-derived family, PHI, has been implicated in the regulation of a variety of physiological and behavioral mechanisms (28, 38, 39, 43). Limited data available thus far have pointed to feeding-related processes as being potentially modulated by PHI (14, 29). The results of the current project present a possibility that central PHI is involved in the control of consummatory behavior.

Injections of this peptide into the lateral ventricle, in the PVN, and in the CeA reduced the amount of consumed food in overnight-deprived rats compared with saline-treated controls. This anorexigenic effect was relatively short lasting regardless of the route of administration, and it could not be detected 24 h postinjection. The finding of short-lived hypophagia induced by centrally delivered PHI appears somewhat similar to (although, due to the lack of sufficient data, cannot be linked to) peripherally related results obtained in human studies in which meal ingestion caused only a transient elevation of plasma PHI levels and starvation did not impact the profile of the circulating peptide (14). It remains to be elucidated whether activity of the PHI system in the brain leads to inhibition of consumption and whether this potential activity is also reflected by the rise of the concentration of circulating PHI. Importantly, the current set of food intake data allows us to propose that PHI delivered to, thus acting within, the central nervous system reduces ingestive behavior in rats and that the PVN and CeA appear to play a role in integrating anorexigenic properties of centrally active PHI.

It has been shown that ICV infusion of VIP, a peptide closely related to PHI, decreases food consumption in rats (47). Our present findings are in agreement with this earlier report, as we observed a particularly dramatic (~40%) reduction in feeding response in animals that received PHI injection into the lateral cerebral ventricle. Therefore, it seems that the two members of the PACAP/glucagon family affect food intake in a similar manner when administered ICV in rats. Unfortunately, to our knowledge, no data on the influence of intra-PVN and intra-CeA VIP have been published to date.

Another interesting issue related to anorexigenic properties of PHI and VIP, as members of the same peptide group, is that according to the initial evidence, both peripheral and central pools of these peptides appear to modulate consumption (7, 14, 16, 22, 23, 29, 36, 47). It has been shown that some peptides belonging to the PACAP/glucagon family are capable of crossing the blood-brain barrier (see, e.g., Ref. 9). Unfortunately, to our knowledge, this issue has not been investigated in relationship to PHI or VIP. Studies on a potential ability of these two peptides to penetrate through the blood-brain barrier could shed more light on whether there is a relationship between the peripheral status of PHI and VIP and some aspects of their centrally mediated feeding effects.

It should be emphasized that, regardless of similar anorexigenic properties of ICV-infused VIP and PHI, the role of these two peptides in feeding control does not necessarily have to be identical. In fact, while studying the role of peripheral PHI in feeding control, the same group of investigators that observed a meal consumption-induced increase in PHI plasma levels in human subjects could not detect simultaneous changes in the VIP plasma concentration (14). It is likely, then, that also within the central nervous system, various members of the PACAP/glucagon family affect distinct mechanisms related to ingestive behavior. The hypothesis of differential effects of VIP and PHI can be supported by a recent discovery of the third receptor subtype for prepro-VIP-derived peptides that does not bind VIP, but does interact with PHI (44). Thus far this receptor has been identified and cloned in various organs, including the brain, only in the goldfish. Therefore, future studies are needed to verify this receptor's presence and localization in other vertebrate species.

It should be noted that, although our data suggest the possibility that PHI plays a role in the regulation of food intake through central mechanisms, additional experiments involving antagonist injections, lesions, or knife-cut techniques, among others, are necessary to properly assess whether central PHI indeed functions as an endogenous regulator of food intake. In this respect, there is also a need of testing whether centrally delivered PHI exhibits anorexigenic properties when animals are subjected to various feeding conditions. In the current project, we showed that ICV and intraparenchymal administration of this peptide decrease consumption in rats stimulated to eat by overnight deprivation. This is one of several standard paradigms used to evaluate a potential inhibitory action of a particular compound on food intake. Follow-up studies should provide detailed characterization of this peptide's effect on feeding under conditions that differ in food availability (e.g., chronic deprivation, scheduled feeding, nocturnal vs. diurnal, free feeding) and palatability (such as macronutrient and flavor preference).

An observed decrease in food intake after injection of a given peptide does not allow one to define mechanisms through which this compound induces hypophagia, as animals terminate food consumption due to a variety of reasons ranging from satiation to sickness/malaise. A CTA test is a standard model used to determine whether anorexia induced by a substance of interest is related to sickness (11, 27). When consumption of a novel flavor is paired with exposure to a toxic agent that causes an unpleasant gastrointestinal sensation, animals will avoid this particular ingestant in the future; thus they will exhibit a CTA toward a presented flavor. This acquired behavioral response is accompanied by a wide array of neural and endocrine changes. For example, administration of a powerful aversive substance, LiCl, enhances c-Fos-IR in a variety of brain sites, including the nucleus of the solitary tract, area postrema, CeA, PVN, and SON (31). In addition, an increase in the plasma OT and (to a lesser degree) VP levels and, thus, activation of OT and VP neurons in the PVN and SON, has been observed as a result of LiCl treatment (32, 46).

In the current studies, we found that ICV-injected PHI at a minimal anorexigenic dose (20 nmol) did evoke a strong aversive response, as measured by subsequent intake of a treatment-associated saccharin solution. As expected, this CTA-inducing treatment led to a significant increase in Fos-IR of OT and VP neurons in the PVN and SON. In addition, elevated numbers of Fos-positive nuclei could be seen in the CeA, PVN, and SON of rats injected ICV with PHI. Our finding suggests that, at least to some degree, anorexigenic response to ICV PHI may stem from aversive consequences of this treatment. To shed more light on this issue, it would be of particular importance to determine whether PHI-containing neurons respond to treatments that cause a CTA, which would allow us to classify this peptide as a mediator of aversive responsiveness. In this context, PHI should be also studied in relationship to its general involvement in feeding-related homeostatic regulation, because, for example, an increased number of PHI-IR perikarya have been found in the PVN of salt-loaded rats (25).

In contrast to ICV PHI, neither PVN (3 nmol) nor CeA (10 nmol) injection of this peptide after the ingestion of saccharin, affected animals' later preference for the saccharin solution compared with controls. Importantly, doses of site specifically injected PHI that did not cause a CTA, did result in an inhibition of feeding. These results indicate that there is no aversive component to PHI-induced anorexia mediated by the PVN and CeA, thus inhibition of ingestive behavior evoked by stimulation of the PVN and CeA with this peptide cannot be attributed to sickness or malaise. However, one should not exclude a possibility that the receptors for PHI present in either of the two regions, aside from contributing to the termination of food intake, do mediate certain aspects of a CTA. In the taste aversion experiments of the current project, we used only the lowest anorexigenic doses of PHI. It remains to be elucidated whether administration of PHI at doses higher than these required to decrease feeding leads to aversive responses.

The results of CTA studies bring about an interesting question as to why, within the range of anorexigenic doses, ICV administration of PHI causes a relatively powerful aversive response, whereas PHI infused into the CeA or PVN fails to induce a CTA. In the preceding paragraph, we mentioned that one of the possible explanations of this phenomenon is the fact that PHI doses higher than these used in the current project may be necessary to generate a CTA in a site-specific injection paradigm. We also emphasize an alternative explication of this puzzling issue. We hypothesize that, as a result of generalized ICV infusion, binding of PHI to its receptors in various areas surrounding the ventricle occurs, affecting activity of numerous central pathways via direct and indirect input. Simultaneous activation by PHI of various types of circuitry involved in different physiological processes (in addition to those related to food intake) may potentially cause nonspecific behavioral responses, including a CTA. It is likely that intrasite, i.e., intra-PVN and -CeA, injections of this peptide may limit the affected populations of receptors for PHI and, more precisely, target those that are engaged in feeding control. Therefore, we propose that a CTA observed due to ICV administration of PHI may stem from the binding of this peptide occurring simultaneously in multiple sites, thus engaging various actions of PHI. Our immunohistochemical data provide indirect evidence supporting this notion: single staining revealed that c-Fos-IR levels in the PVN and SON are higher due to the action of ICV- vs. PVN- or CeA-administered PHI. Also, more OT and VP neurons contain Fos-positive nuclei after ICV injection of PHI rather than site-specific infusion of this substance. Importantly, other anorexigenic peptides follow a similar pattern to the one observed with PHI, e.g., alpha -melanocyte stimulating hormone generates aversive effects when injected ICV but not in the PVN (33).

Interestingly, both intra-PVN and intra-CeA infusions of PHI at the lowest hypophagia-inducing dose evoked activation of OT neurons in the PVN. Also, elevated percentage of Fos-positive VP cells in the PVN was observed after administration of PHI into this region. These data suggest that OT and VP neurons in the PVN may play a role of integrators of PHI-derived information. On the basis of the outcome of our experiments and previous reports that have focused on infusions of PHI in the PVN (5), we speculate that OT and VP cells in the PVN may be directly targeted by PHI. In addition, our results indicate that the PVN OT system receives either single- or multisynaptic pathway input that originates from CeA neuronal populations sensitive to PHI, as the increase in activation of OT cells in the PVN occurred due to injection of PHI into the amygdala.

Aside from their involvement in the development of taste aversion, OT and VP have been proposed as part of central satiety mechanisms (7, 23). Thus an observed increase in activation of PVN OT/VP neurons after anorexia- but not aversion-inducing PHI treatments allows us to propose a hypothesis that the OT/VP system may mediate the inhibitory effect of PHI on food consumption. We stress the fact that more studies are needed to further substantiate this concept. In particular, experiments assessing a consummatory response to centrally administered PHI in rats pretreated with an antagonist of the receptor for OT or VP would shed more light on the issue whether OT and VP are a necessary component of mechanisms that promote PHI-induced termination of food intake.

Considering the fact that PHI administered ICV or into specific sites promoted an increase in Fos-IR of biochemically unidentified neurons in the PVN and CeA, it is highly probable that PHI modifies food intake by interacting with populations of cells that contain peptides other than OT and VP. Hence, it is imperative that future studies delineate relationships of PHI with a wider network of neuropeptidergic systems. In this context, there appears to be a need of conducting additional studies that would involve retrograde tracing and knife-cut techniques, among others, to address the issue of functional neuroanatomy of those feeding-related central networks that contain PHI.

In summary, we found that centrally delivered PHI had a short-term (i.e., lasting <24 h) inhibitory influence on food intake in rats. The magnitude of anorexigenic responses evoked by central PHI depends on the injection route/site: ICV administration of this peptide causes a more powerful effect than area-specific infusions. However, ICV-infused PHI supports the development of CTA, whereas there is no aversive component to hypophagia induced by this peptide injected in the CeA or PVN. Finally, the PVN (with OT and VP neurons) and CeA, neuronal populations that exhibit elevated Fos-IR due to PHI treatment, may be considered as potential mediators of anorexigenic effects of PHI.

Perspectives

A concomitant occurrence of aversion and inhibition of food intake induced by an injection of a neuroregulatory peptide makes it difficult to define the nature of this compound's influence on feeding. In the current study, we demonstrate that injection of PHI may result in both aversion and early termination of food intake. However, the aversive consequence of PHI could be observed only when this peptide was infused into the lateral ventricle; direct administration of minimal anorexigenic doses of this substance into the PVN or CeA decreased feeding without causing a CTA. The PVN and CeA are involved in both aversion as well as aversion-independent inhibition of ingestive behavior, including satiety (26, 32, 33, 48). Thus the fact that intra-PVN or -CeA injection of PHI decreased food intake without any aversive consequences leads us to propose a hypothesis that PHI may act as a potential endogenous inhibitor of consummatory behavior whose anorexigenic action is independent from aversion-related mechanisms. It appears that the main challenge of the future studies will be to provide an accurate characterization of PHI's function in the process of feeding regulation, i.e., whether this peptide contributes to a decrease in consumption by being part of, for example, satiety- or motivation-governing central mechanisms. As has been the case for other peptidergic regulators of ingestive behavior, a wide array of behavioral and metabolic/molecular studies will have to be employed.


    ACKNOWLEDGEMENTS

We thank Dr. R. M. Buijs of the Netherlands Institute for Brain Research, Amsterdam, The Netherlands, for a generous gift of the rabbit-anti-VP antibody.

This project was supported by the National Institutes of Health, National Research Service Award T32DA-07097 from the National Institute of Drug Abuse, and by the Department of Veterans Affairs, the National Institute of Drug Abuse (DA-03999), and the Minnesota Obesity Center (DK-50456).


    FOOTNOTES

Address for reprint requests and other correspondence: P. K. Olszewski, Research Service 151, Veterans Affairs Medical Center, 1 Veterans Dr., Minneapolis, MN 55417 (E-mail: olsze005{at}umn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 20, 2003;10.1152/ajpregu.00554.2002

Received 9 September 2002; accepted in final form 6 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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