INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BOMBESIN (BN), a tetradecapeptide originally isolated in 1971 from the skin of a European frog, Bombina bombina (1), represents a family of peptides with potent biological effects in mammals. On systemic administration, BN robustly suppresses food intake in a variety of species ranging from rodent to human (13, 30, 37). Direct administration of this peptide into the brain also suppresses food intake (17). In addition, central BN evokes other effects, including hypothermia (26), hyperglycemia (4), and compulsive grooming (23). Although BN per se is not present in mammals, several peptides bearing structural homology to this peptide have been detected in mammalian tissue. These BN-like peptides include gastrin-releasing peptide (GRP) and neuromedin B (NMB) (36, 46). Corresponding receptor subtypes have also been characterized (27, 47). The biological effects of BN are likely mediated through the activation of GRP and/or NMB receptors, as it displays high affinity for both these receptor subtypes.

Satiety or meal termination occurs well before food actually gets digested and absorbed. During this preabsorptive phase, the signaling mechanism(s) that participate in meal termination remain partially understood at best. It has been suggested that certain "satiety" peptides are secreted by the gut in response to ingested food, which, through endocrine, neurocrine, paracrine, autocrine, or neural mechanism(s), signal the brain to bring about the termination of a meal (10, 14). Inasmuch as BN-like peptides act to suppress food intake and are present both in the gut and the brain, they could represent one of the physiological mediators of the satiety signal of the "gut-brain" axis (12, 15, 24, 38). However, the mechanism(s) by which BN in the peripheral compartment activates the relevant central circuits remains elusive. Typically, peptides do not easily cross the blood-brain barrier (2, 3). Nonetheless, it is possible that BN-like peptides from the peripheral compartment (whether endogenously released or systemically injected) could still communicate with the brain in an endocrine manner by penetrating weaker area(s) of the blood-brain barrier (such as the area postrema) (3, 18).

Research from our laboratory has shown that immunoneutralization of BN (with BN antiserum) or blockade of its receptors (with receptor antagonists) within the brain attenuates the feeding-suppressant effects of systemically administered BN (33). These observations support the contention that BN-like peptides in the peripheral compartment require BN-responsive receptors in the brain to bring about the suppression of a meal. However, it remains unclear whether BN-like peptide(s) of peripheral origin require visceral afferent neural pathways to relay satiety information to the brain or whether they communicate directly with the brain by penetrating the blood-brain barrier, thus acting in an endocrine manner. The vagus nerve is the most likely potential conduit of such neural communication; for example, it has been shown that subdiaphragmatic vagotomy blocks the effects of systemically administered CCK. However, vagotomy failed to affect the anorectic effects of systemically administered BN. Interestingly, however, it has been demonstrated that a more thorough neural disconnection of the gut from the brain is effective in this regard. Specifically, a combination of subdiaphragmatic vagotomy, higher thoracic section (level T6), and bilateral dorsal rhizotomy from T3 to T6 blocked the feeding-suppressant effects of systemically administered BN (42). This observation suggests that it is unlikely that BN in the peripheral compartment acts directly in an endocrine manner. We therefore propose that peripheral BN suppresses food intake by activating spinal, nonvagal neuronal circuits that eventually provoke the release of BN-like peptides within the central nervous system (CNS). To date, however, the characteristics and/or the identity of the neuronal pathways that convey these effects of systemically administered BN remain unknown.

Capsaicin, a neurotoxin commonly found in chili peppers, has the capacity to excite and subsequently desensitize a subset of primary sensory neurons, including both vagal and spinal fibers (22). This pharmacological deafferentiation technique has the advantage over surgical vagotomy in that the efferent pathways are spared, thus circumventing the debilitating and confounding effects of efferent neural ablation. The capsaicin-lesioned fibers are thought to be peptidergic neurons of small to medium diameter and give rise to unmyelinated (C fiber) or thin-myelinated (A-fiber) axons (22, 43). Thus the objective of the current study was to assess whether neonatal capsaicin exposure alters the adult feeding response to either systemically or centrally administered BN. The results revealed that neonatal capsaicin treatment completely blocked the feeding-suppressant effects of systemically but not centrally administered BN.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals and Apparatus

Capsaicin Treatment

Experiment 1: Impact of Capsaicin Pretreatment on Body Weight Gain and Anorexia Induced by Systemic BN

Experiment 1B: Impact of capsaicin treatment on anorexia induced by systemic BN. Adult male rats (from group 2) neonatally pretreated with capsaicin or vehicle (n = 6-8/group) were systemically injected with BN (0, 4, 8 and 16 µg/kg ip), and their food intake was monitored at 46-50 days of age. BN doses were administered (20-30 min before testing) in a randomized order, according to the Latin Square design. Rats were tested during the light phase (between 0900 and 1200) after having been food deprived for 18 h before testing. Food intake (Kellogg's Froot Loops; to which they were habituated) was monitored at 30-min intervals for 3 h using electronic balances interfaced to a computerized monitoring system (31).

Experiment 2: Response to Central or Systemic BN Injection After Capsaicin Exposure

Experiment 2B: Impact of capsaicin treatment on anorexic effects of centrally injected BN. SURGERY: Adult male rats (42-47 days of age; controls, n = 6; capsaicin treated, n = 6) were anesthetized with pentobarbital sodium (50 mg/kg ip) and placed in a stereotaxic instrument with the skull leveled. Stainless steel guide cannulas (22 gauge) aimed at the third ventricle were implanted (on the midline, 4.5 mm posterior to bregma and 4.3 mm ventral to the skull surface), according to coordinates from Paxinos and Watson (39). The cannulas were anchored by three skull screws and acrylic dental cement. The cannulas were plugged by a removable stainless steel wire stylet.

INTRACEREBROVENTRICULAR BN INJECTIONS. Rats were entrained to a regimen of daily 4-h food (Purina Rat Chow) access for 2 wk before the experiment. Injection of BN into the third ventricle [0 (saline), 0.1, 0.25, and 0.5 µg icv] began after a 7-day postsurgical recovery period. The peptide doses, given in a randomized order according to the Latin Square design, were infused at a rate of 3 µl/min, and testing commenced immediately thereafter. Food intake was monitored electronically as described earlier.

Data Analysis

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experiment 1A: Impact of Capsaicin Pretreatment on Body Weight Gain

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of capsaicin treatment on body weight gain. Data points represent means ± SE of body weight of control and capsaicin-treated female (A) and male (B) rats during first 6 wk of life. ** Significant differences between capsiacin-treated and control groups (P < 0.01).

Experiment 1B: Impact of Capsaicin Pretreatment on Anorexic Effects of Systemic BN

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of capsaicin treatment on suppression of food intake induced by systemically (A) or centrally (B) administered bombesin (BN). Data points represent means ± SE of food consumed over 30 min (g). ** Significant within-group differences from control (saline) condition (P < 0.01);  significant differences between dose-matched capsaicin-treated and vehicle controls (P < 0.01).

As depicted in Fig. 2A, the lowest dose of BN (4 µg/kg ip) failed to significantly affect food intake in either group. At the 8 µg/kg dose, BN markedly suppressed food intake in the controls during the initial 30-min period (P < 0.01) but failed to significantly affect food intake in the capsaicin-pretreated animals. At the highest dose tested, BN (16 µg/kg) suppressed food intake in the control animals at the 30-min interval (see Fig. 2B), and this suppression was long lasting (evident up to 2-h postinjection; data not shown). However, even at this supramaximal dose, BN failed to suppress food intake in the capsaicin-pretreated rats.

Experiment 2A: Confirmation of the Loss of Response to Systemically Administered BN in Capsaicin-Exposed Rats

Experiment 2B: Impact of Capsaicin Treatment on Anorexic Effects of Centrally Injected BN

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Peptides of the bombesin family are thought to be one of the physiological mediators of satiety (meal termination). When administered systemically, BN (and related peptides) dose dependently suppresses food intake in a variety of species (14, 16, 24, 26). The present results clearly demonstrate that the anorexic effect of systemic BN is completely abolished in adult rats that were exposed to a single dose of capsaicin on postnatal day 2. This suggests that systemic BN requires intact capsaicin-sensitive fibers to communicate with the brain and thus argues against the notion that systemically administered BN directly affects brain functioning in an endocrine manner. From previous research, it appears that, although capsaicin treatment lesions the vagal afferent projections, this conduit of communication may not be essential for the elicitation of the BN effects. Indeed, surgical subdiaphragmatic vagotomy does not block the feeding-suppressant effects of systemically administered BN. The suggestion that systemic BN may deploy an alternate neural pathway (such as spinal afferents) for mediation of its effects is supported by the present findings and by those of Stuckey et al. (42), demonstrating that a more complete surgical neural disconnection of the gut from the brain abrogates the feeding-suppressant effects of systemically administered BN.

The present results from neonatally capsaicin-lesioned rats are not fully concordant with some earlier studies where capsaicin was administered in adulthood (28, 41). Whereas capsaicin treatment failed to affect BN-induced anorexia in one study (41), it attenuated the effects of systemically administered BN in another (28). Some of the discrepancies between these studies may be attributable to the differences in testing conditions (such as extent of food deprivation at testing, doses of BN used, etc.). However, we believe that the major contributing factor must have been the age at which the capsaicin lesion was inflicted (day 2 in our study vs. adulthood in others). Indeed, the unmyelinated primary afferent neurons are particularly sensitive to capsaicin during the initial 12 days of life but relatively resistant to such degeneration beyond the age of 14 days (21). Furthermore, it has been established that capsaicin exposure in adulthood, particularly if injected in a single dose, fails to permanently destroy all of the C fibers (20). This contrasts with the actions of capsaicin administered early in life, where the neurotoxic effects are more extensive and permanent (11, 20). Thus the subset of fibers that escape (or recover) from damage induced by adult capsaicin exposure must be important in the transmission of BN-mediated afferent signals to the brain.

Although we have built the case for capsaicin-sensitive neural pathway(s) of systemic origin in the transmission of BN effects, it could also be argued that capsaicin may damage and/or downregulate BN-sensitive circuits within the brain. This point is particularly relevant with the use of neonatal capsaicin, inasmuch as the blood-brain barrier is weaker in neonates, thus increasing the likelihood of capsaicin-elicited damage to the brain. Indeed, there have been reports of capsaicin-induced neural damage within the brains of neonatal rats exposed to capsaicin (40). There is also considerable evidence pointing to the importance of brain BN/GRP receptors in the mediation of the effects of systemically administered (32, 35) as well as endogenously released BN-like peptides (34). Thus the loss of the feeding-suppressant effects of systemic BN in neonatally capsaicin-treated rats may not be due to the loss of peripheral afferent fibers, but to the loss of central BN-sensitive neurons. If capsaicin exposure did lesion or downregulate BN-sensitive neural circuits, then systemic BN may be expected to lose its potency and/or efficacy irrespective of whether the effects were mediated neurally or by direct penetration into the brain. Accordingly, we assessed the effects of BN injected directly into the brain (third ventricle) of animals neonatally exposed to capsaicin. In sharp contrast to the loss of response to peripherally administered BN, centrally administered BN maintained full potency and efficacy, despite capsaicin exposure. This suggests that capsaicin exposure did not adversely affect the brain system(s) using BN-like peptides and further supports our contention that peripheral BN must communicate with the brain through capsaicin-sensitive neural pathways. The peripheral site(s) where BN actions are initiated remain to be identified. In this regard, Kirkham et al. (25) suggested that the satiety-like effect of peripheral BN may be mediated by receptors perfused by the celiac artery because the levels of BN required to suppress food intake are much lower when infused here than when administered either intraperitoneally or by superior mesenteric infusion. Thus it remains possible that the afferent arm of the BN-responsive and capsaicin-sensitive circuit may originate from the vicinity of sites perfused by the celiac artery.

A palatable diet was selected in experiment 1, along with overnight deprivation, to ensure a strong feeding response and to maximize any differences due to the feeding-suppressive effects of BN. We previously showed that BN is effective at attenuating intake of both chow and snack food (31). On the other hand, because we used a highly palatable food, it could be argued that the capsaicin-treated rats failed to suppress intake after central BN administration due to an independent alteration of hedonic responsiveness to the Froot Loops rather than an interruption of BN activity. Altered responsiveness to palatable foods by capsaicin-treated rats was observed previously (7). However, in our hands, the saline-treated capsaicin-exposed rats do not demonstrate an increase in intake of palatable food, suggesting that the reward system was unaltered.

Although the major objective of the present investigation was to assess the role of capsaicin-sensitive neurons in the mediation of the anorexic or satiety effects of BN, we were also interested in evaluating the ontogenic behavioral and growth changes induced by neonatal capsaicin exposure. Thus animals were monitored for both weight and various behaviors on a weekly basis, starting from the first week of life. Consistent with earlier studies (6, 45), we noted that the capsaicin-exposed rats showed a small but significant reduction in body weight starting ~4 wk after capsaicin administration. As we did not monitor daily food intake, it is unclear whether the weight gain differences were due to altered energy intake or expenditure. However, some insights can be gleaned from activity levels, which were recorded in the present study. Sniffing (a form of exploratory activity) decreased transiently at weeks 4 and 5 (data not shown), which may be related to the decreased olfactory sensitivity (8). In addition, capsaicin treatment failed to significantly alter locomotor activity (assessed over a 24-h period) but transiently increased grooming and scratching behavior (peaking at week 5; data not shown). The latter observation is concordant with an earlier report (44). Thus the time course of the behavioral and weight gain changes do not support the idea that capsaicin-elicited weight gain changes are due to altered behavioral activity.

In summary, the present findings suggest that peripherally injected BN employs capsaicin-sensitive afferent neurons to exert its feeding-suppressant effect on the CNS. This study shows, for the first time, that in contrast to the loss of systemic effects of BN, capsaicin exposure left the central mechanisms mediating the feeding-suppressant effects of BN fully functional. Thus, whereas the afferent arm of one of the satiety circuits that is activated by BN-like peptide(s) is capsaicin sensitive, the efferent arm of that system is completely resistant to actions of this neurotoxin. Capsaicin exposure also caused a reduction in the rate of body weight gain, which appeared to be unrelated to the activity profile of the animals. Future challenges include a more precise identification of specific subpopulation of the capsaicin-sensitive afferent neurons activated by systemically injected and/or endogenously released BN-like peptides involved in meal termination.

Perspectives