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4 Veterans Affairs Medical Center and 3 Minnesota Obesity Center, Minneapolis 55417; and Departments of 1 Neuroscience, 5 Food Science and Nutrition, and 2 Medicine, University of Minnesota, St. Paul, Minnesota
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Orexin A (OX-A) administered in the lateral hypothalamus (LH) increases feeding in a dose-dependent manner. The LH is a relatively large neural structure with a heterogeneous profile of neural inputs, efferent projections, and orexin receptor distribution. We sought to determine the LH region most sensitive to the feeding stimulatory effect of OX-A injection. Fifty-six male Sprague-Dawley rats were fitted with cannulas 1 mm above four separate LH regions ~1 mm apart in the rostral-caudal direction. There were 14-16 animals/LH region. After recovery, animals received either artificial cerebrospinal fluid or OX-A (250, 500, or 1,000 pmol). To determine whether there is a circadian effect of LH OX-A on the feeding response, we performed injections at 0200, 0900, 1400, and 2100. Food intake was measured at 1, 2, and 4 h after injection. The most rostral extent of the LH was the only region in which injection of OX-A significantly stimulated feeding. Within this region, feeding was increased at all times of the day, although the most robust and only significant feeding response occurred after the afternoon injection (1400) of OX-A. To determine the extent to which the metabolic status of the rat contributed to the circadian specificity of orexin-induced feeding, animals were placed on a restricted diet and injected with OX-A in the most rostral region of the LH. Under these conditions, OX-A significantly increased feeding and more robustly when compared with animals on a nonrestricted diet. These data suggest that the rostral LH is the only region of the LH sensitive to the injection of OX-A, and the metabolic status of the animal at the time of injection may influence the feeding response to OX-A.
melanin-concentrating hormone; hypothalamus; hypocretin; restricted diet
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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OREXIN A (OX-A, also known as hypocretin 1) is a recently identified neurotransmitter that may play an important role as a neuroregulator of homeostasis (15, 44). OX-A induces food intake when administered in the ventricular system, the hypothalamic paraventricular nucleus (PVN), the lateral hypothalamic (LH) area, the dorsomedial nucleus, and the perifornical area of the rat brain (16, 44, 48). This increase in feeding behavior after OX-A administration is transient, since 24-h food intake and body weight are unaffected after chronic ventricular infusion of OX-A (21). A role for OX-A in feeding behavior is not surprising since it is localized within the LH, a classic feeding center (8, 15, 44). Electrical stimulation of the LH increases feeding behavior, whereas chemical lesions in this region result in anorexia and death (5).
Defining the areas of the LH most relevant to OX-A-induced feeding is important because the LH is a large and complex neural center containing a diverse population of neurotransmitters and receptors. Within the LH, there are numerous intralateral hypothalamic connections in addition to connectivity between the LH and several other brain regions (5). Orexin and melanin-concentrating hormone (MCH)-containing neurons are present in the LH in separate neuronal populations. Orexin-containing neurons coexpress dynorphin, an orexigenic opioid peptide (12), whereas MCH neurons coexpress cocaine amphetamine-related transcript, an anorectic peptide (7, 52). The two identified orexin receptors, OX1R and OX2R, are expressed by orexin neurons within the LH, and MCH neurons express OX1R (3, 13). Although OX1R has a much greater affinity for OX-A than orexin B, OX2R has a similar affinity for both OX-A and orexin-B (44). Orexin neurons also express ANG II, dynorphin, and leptin receptors (4, 12). The functional significance of the receptor signaling system for these neuron populations is not yet clearly understood.
There is a well-established connection between orexin and states of arousal (32, 47) such that orexin increases arousal (20). Most illustrative of this is the finding that the canine condition of narcolepsy is because of an inherited mutation in the canarc-1 gene, which encodes OX2R (30). Lack of orexin signaling may also play a role in the human condition of narcolepsy, since human narcoleptic patients have extremely low cerebrospinal fluid (CSF) orexin levels (37). Moreover, analysis of human narcoleptic brain tissue revealed a significant loss of orexin-producing neurons within the hypothalamus (37, 39, 51). Consistent with the role of orexin in arousal states, orexin signaling varies across the light-dark cycle. Rodent studies indicate that orexin peptide levels within the CSF and within the hypothalamus exhibit diurnal variation, with high levels of OX-A during the dark phase and lower levels of OX-A in the light phase (18, 55). It is possible that this diurnal variation results from connections with the suprachiasmatic nucleus (SCN), since there are direct projections between the SCN and LH (1). However, the relationship between the SCN and orexin-containing neurons remains unclear.
In the present study, we sought to define the LH region most important for OX-A-induced feeding by targeting four different regions within the LH separated ~1 mm apart in the rostral-caudal direction. Furthermore, we have previously observed that OX-A appears to stimulate feeding more robustly when injected in the afternoon vs. the morning. However, this circadian feeding response to discrete OX-A administration has never been tested rigorously. Thus we also tested the effect of OX-A on feeding in four different LH regions at four different times of day. We hypothesized that OX-A-induced feeding is location and time sensitive. Finally, to evaluate whether sensitivity to OX-A signaling is influenced by metabolic status, we tested the effect of OX-A administration on food intake in rats maintained on a food-restricted diet. We hypothesized that, because OX-A-induced feeding is relatively mild compared with other orexigenic peptides, exogenous administration of OX-A would not stimulate intake because of masking by the strong feeding stimulus induced by chronic restriction.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The treatment of animals in these studies fully conforms with the Guiding Principles for Research Involving Animals and Human Beings of the American Physiological Society (2), and these studies received local institutional animal care and use committee approval.
Animals
Male Sprague-Dawley rats (Harlan, Madison, WI) weighing 250-350 g were housed individually in conventional hanging cages with a 12:12-h light-dark photoperiod (lights on at 0700) in a temperature-controlled room (21-22°C). Teklad Lab Chow and water were allowed ad libitum, except where noted. For the night-time injections (0200 and 2100), animals were habituated to a reverse light-cycle room for 2 wk before injections.Cannulation
Rats were anesthetized with Nembutal (40 mg/kg) and were fitted with a 27-gauge stainless steel guide cannula (Plastics One, Austin, TX) placed just above the LH in four different LH regions. The lateral hypothalamus (LH) regions targeted were ~1 mm apart in the rostral-to-caudal direction. Stereotaxic coordinates were determined from the rat brain atlas by Paxinos and Watson (38) and are listed in Table 1. To maintain the cannula within the boundary of the LH as anterior/posterior coordinates changed, the dorsal/ventral and medial/lateral coordinates were amended as well. The injector extended 1 mm beyond the end of the guide cannula. For all cannulations, the incisor bar was set at 3.3 mm below the ear bars. Regions targeted and representative schematics of correctly placed LH cannulas are shown in Fig. 1. At least 7 days elapsed after surgery before experimental trials.
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Injections
Injections in the LH were given in a 0.5-µl volume over 30 s by the use of a 33-gauge internal cannula (Plastics One). The injector was left in place for an additional 30 s to ensure diffusion from the tip of the injector and to minimize possible backflow in the guide cannula. All injections took place within a 1-h range for each time of day tested as follows: 0200-0300, 0900-1000, 1400-1500, and 2100-2200. A stylet was kept in place in the guide cannula at all times excluding injections.Verification of Cannula Placement
After all experiments, brains were dissected out and stored in a 10% formaldehyde solution for later placement verification by histological examination. Schematics of coronal sections demonstrating the LH regions targeted are shown in Fig. 1. For study 1, initial animal numbers were n = 12/region. Data from animals with incorrectly placed cannulas were excluded from the final analysis. Cannula placement was deemed incorrect when the injection occurred outside the LH and beyond a 0.5-mm radius from the targeted site. For study 1, four animals were removed based on either cannula extrusion (n = 2) or incorrect cannula placement (n = 2). Additionally, based on the histology of the actual injection sites, several rats were placed in a different region than originally intended. The final number of animals left in each analysis for each LH region is as follows: region 1: n = 15; region 2: n = 13; region 3: n = 6; region 4: n = 10. For study 2, the initial number of animals was 20. Based on cannula placement verification, three animals were removed, leaving n = 17 for the final analysis.Drugs
OX-A was purchased from RBI (Natick, MA) for study 1 and from Phoenix Pharmaceuticals (Belmont, CA) for study 2 and was dissolved in artificial cerebral spinal fluid (aCSF) before use.Food Intake Measurements
Standard laboratory rat chow (Teklad) was allowed ad libitum until the start of each experimental trial. Just before injection, chow was removed, and, immediately after injection, preweighed pellets of the chow were placed inside the rat cage. At 1, 2, and 4 h after injection, pellets and spillage were weighed and subtracted from the initial weight to quantify the amount of food eaten. The same laboratory chow was used for all of the experimental trials.Experimental Design
Study 1: regional and circadian specificity of LH OX-A-induced feeding. Male Sprague-Dawley rats were fitted with cannulas 1 mm above four separate LH regions ~1 mm apart in the rostral-caudal direction. Correct injection sites are shown in Fig. 1. There were 6-15 animals/LH region. After recovery, animals received either aCSF or OX-A (250, 500, or 1,000 pmol) with an injector that extended 1 mm beyond the end of the implanted guide cannula so that the injection occurred in the center of the LH region under study. To determine whether there is a circadian effect of LH OX-A injections, we performed four separate studies in which injections were performed at 0200, 0900, 1400, and 2100. Food intake was measured at 1, 2, and 4 h after injection. Each rat received each treatment one time on separate days, with at least 48 h between treatments to allow drug clearance from the central nervous system and for normal feeding patterns to be reestablished. Treatments were randomly assigned and given in a counterbalanced design, such that each treatment was represented equally (to the greatest extent possible with uneven animal numbers) on each injection day.
After these studies were completed, and we determined that injections in region 1 at 1400 produced the most robust feeding response to OX-A (see Fig. 3), we performed one additional study of nine new animals to verify that this response was repeatable in animals that had not received any prior injections. These newly cannulated animals received either injections of aCSF or a high dose of OX-A (1,000 pmol) at either 0200 or 1400, and food intake was measured at 2 h after injection.Study 2: effect of food restriction on LH OX-A-induced feeding. Male Sprague-Dawley rats were fitted with cannulas placed in region 1 (Table 1). Animals were fed a restricted diet (<19 g) until they reached 90% of their baseline body weight. This body weight was then maintained via a restricted diet (~19 g chow/day) for 3 wk. Chow was administered at 1300. On the day of the experiment, 1,000 pmol OX-A or aCSF was administered between 1200 and 1300 before the presentation of chow ad libitum. A crossover design was used, such that all animals received both treatments. Food intake 2 h postinjection was measured by weighing food before its placement in the cage and then at 2 h postinjection. Animals were then allowed ad libitum access to chow for 1 mo. After this time, OX-A or aCSF was administered between 1200 and 1300 in a crossover design with 48 h between injections. Food intake at 2 h postinjection was measured.
Statistical Analysis
Study 1: regional and circadian specificity of LH OX-A-induced feeding. All data were first analyzed by repeated-measures three-factor ANOVA (factors: LH region, time of day, and OX-A dose) to determine regional, circadian, and treatment effects. Where main effects were observed, data were further analyzed separately by one-factor ANOVA within each LH region targeted at each time point. Post hoc testing for significant ANOVAs was performed using paired t-tests.
One concern with studies involving multiple injections in specific sites is the potential for decreased effectiveness of injections over time. Thus we also performed a one-factor ANOVA (day = independent variable; 2-h food intake = dependent variable) of the feeding data resulting from OX-A (1,000 pmol) treatment only at each injection time and within each LH region. These analyses revealed no significant differences in the 2-h feeding response to OX-A over time (P > 0.05). However, because this analysis is limited in power to detect differences, for region 1 (the only region showing sensitivity to OX-A) we performed a similar analysis but included all doses of orexin collapsed across the time of day (correcting for differences in basal intake), which resulted in an n of 7-9 animals/day and a power of 0.78. Like the first analysis, this analysis with enhanced power revealed no significant difference in response to orexin (0- to 2-h food intake above baseline) due to day [P = 0.2301, F(15,154) = 1.266]. Animals may also become conditioned to either eat or not eat after injection of various compounds, and thus the effect of day on food intake for all saline-treated animals was analyzed by one-factor ANOVA at each injection time (day = independent variable; 2-h food intake = dependent variable). This analysis revealed no significant differences in 2-h food intake in the control animals resulting from treatment day (P > 0.05). Separation of the data into LH "regions" is somewhat arbitrary in that the regions selected were based on distance apart in the rostral-to-caudal direction. To obtain a better understanding of the relationship between the LH site of injection along the rostral-to-caudal axis and feeding response, 0- to 4-h food intake from the groups treated with 1,000 pmol OX-A at the 1400 injection time was regressed to distance from the bregma using simple linear regression analysis.Study 2: effect of food restriction on LH OX-A-induced feeding. Data were analyzed by a repeated-measures two-factor ANOVA [factors: energy status (restricted or ad libitum) and treatment (OX-A or aCSF)]. Food intake at 2 h after OX-A or aCSF injection in animals on either a restricted or ad libitum diet was analyzed by paired t-test (independent factor = aCSF or OX-A; dependent variable = food intake). To determine the effect that energy status (restricted or ad libitum) had on the feeding response elicited by OX-A, the individual difference between food intake 2 h post-aCSF injection and food intake 2 h post-OX-A injection was calculated for each animal within each energy status condition. Thus a "grams above baseline" value was obtained for each animal for both of the energy status conditions. The effect of energy status on the OX-A-induced increase in food intake above baseline levels was analyzed by a paired t-test (independent factor = energy status; dependant variable = 2-h intake).
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Study 1: Regional and Circadian Specificity of LH OX-A-Induced Feeding
Three-factor ANOVA indicated that, at 1 and 2 h after injection, there were significant main effects of the LH region (1 h: P = 0.0008, 2 h: P = 0.0050), orexin dose (1 h: P = 0.0101, 2 h: P = 0.0235), and time of day (1 and 2 h: P < 0.0001). By 4 h after injection, the only significant main effect was time of day (P < 0.0001), which was the result of the normal daily variation in food intake. Post hoc analysis indicated that the data from region 1 were significantly different from regions 2, 3, and 4 (P < 0.05) and that data from regions 2, 3, and 4 were not different from one another (P > 0.05). Thus the 0- to 1-h and 0- to 2-h data from region 1 at each time of day were further analyzed using one-factor ANOVA to determine the effect of OX-A treatment. ANOVA indicated that OX-A was effective at stimulating feeding by 2 h after the 1400 injection (P = 0.0394). For comparison with the other regions, the 0- to 2-h intake periods at this injection time (1400) are shown in Fig. 2. Post hoc analysis of orexin effects in region 1 at 1400 at 2 h after injection indicated that the 500- and 1,000-pmol doses of OX-A significantly stimulated feeding above that observed after treatment with aCSF (P = 0.0329 and P = 0.0078, respectively; Fig. 2). The lack of ability of OX-A injection to stimulate feeding (P > 0.05) after injection in region 1 at any other time of day is shown in Fig. 3. In an additional experiment, in which OX-A (1,000 pmol) was injected in region 1 of newly cannulated animals (n = 9) that had received no prior injections, we found that OX-A did not significantly increase feeding at 0200 (vehicle: 2.2 ± 0.7 g; OX-A: 3.1 ± 0.5 g, P = 0.2638), whereas, in these same animals, OX-A at 1400 significantly stimulated feeding (vehicle: 0.3 ± 0.1 g; OX-A: 2.9 ± 0.5 g, P = 0.0006). These data are very similar to that observed in rats that had received multiple injections in region 1 (Fig. 3), indicating that the number of injections received in these studies did not influence the results observed. Simple regression analysis indicated that the increase in the 0- to 4-h food intake observed at the 1400 injection time after 1,000 pmol OX-A was significantly correlated with the distance of the injection site from bregma [F(1,40) = 5.888, P = 0.0198, R = 0.358; Fig. 4].
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Study 2: Effect of Food Restriction on LH OX A-Induced Feeding
A two-factor repeated-measures ANOVA indicated that there was a main effect of treatment (P < 0.0001) and energy status (P < 0.0001) on food intake. Thus OX-A significantly increased feeding regardless of energy status conditions, and energy status significantly affected food intake. Additionally, a significant interaction between treatment and energy status was found (P < 0.004), indicating that energy status significantly influences the feeding response elicited by OX-A. Paired t-tests indicated that OX-A injected in region 1 of the LH at 1300 significantly increased feeding compared with vehicle treatment 2 h postinjection when animals were food restricted (P < 0.05, Fig. 5A) or on a freely fed diet (P < 0.05, Fig. 5A). Animals receiving OX-A consumed significantly more grams above baseline levels when diet was restricted (5.4 ± 1.0 g) compared with those with free access to chow (1.7 ± 0.5 g; P < 0.005, Fig. 5B), as revealed by paired t-test.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The results of this study demonstrate that there is a distinct region within the LH that is sensitive to OX-A and that this sensitivity is dependent on the time of day the injection is given. Additionally, these data show that a restricted diet may enhance sensitivity to exogenously administered OX-A. OX-A increased feeding only when injected in the most rostral portion of the LH, and no significant effect of OX-A on feeding was observed when OX-A was injected in any other region of the LH (Fig. 2). Furthermore, OX-A was found to augment feeding only when administered during the light phase (normal resting) of the light-dark cycle (Fig. 3). On a restricted diet, rats ate 5.4 g above baseline (control levels) after OX-A administration (Fig. 5). This is a twofold greater increase in food intake resulting from OX-A injected in the LH during nonrestricted conditions (48) and may indicate an increased sensitivity to OX-A under these conditions. Although we hypothesized that the strong feeding stimulus induced by chronic restriction would mask OX-A feeding effects, these findings are consistent with other reports of OX-A efficacy in food intake stimulation after 24 h of food deprivation (49). Furthermore, orexin neurons in the LH show increased Fos-LI under restricted diet conditions (33).
The LH has complex circuitry that enables it to act as a site of integrative processing to maintain homeostasis. With multiple neurotransmitters and several distinct populations of neural phenotypes, the LH is likely to be functionally diverse. The finding that OX-A elicits behavioral responses when administered to the LH is somewhat surprising because the LH is the primary site of orexin production (44). In addition, it is unclear which cell type(s) respond to exogenous administration of OX-A, because orexin and MCH neurons occupy similar regions of the LH and express OXR1 receptors (3, 13). Thus defining which population(s) is/are responsible for the behavioral responses seen with exogenous OX-A administration in the LH region is precluded in the current study.
MCH has been shown to increase feeding (41, 42), and thus OX-A may exert an orexigenic effect via stimulation of MCH signaling. However, this finding does not rule out the possibility of an autoreceptor mechanism for OX-A action. Orexin and MCH perikarya have been found in close proximity to each other, and synaptic contacts have been observed between them (4). It is likely that these two neuronal populations communicate within the LH in an interrelated fashion. With the use of c-fos-ir, it has previously been demonstrated that several key feeding regulatory sites are activated with injection of OX-A in this region of the LH, namely the arcuate nucleus, the nucleus of the solitary tract (NTS), and the PVN of the hypothalamus (34). Furthermore, it has been shown that there is reciprocal connectivity between orexin neurons in the LH and neuropeptide Y (NPY)/AGRP containing neurons in the arcuate nucleus (22). When substimulatory doses of NPY and OX-A were coinjected in the ventricles, a feeding response was observed (43). Additionally, the feeding response elicited by ventricular OX-A was attenuated by prior administration of NPY receptor (Y1) antagonists (23, 24, 53). These studies suggest that NPY signaling may play an important role in the feeding regulatory properties of OX-A.
In the present study, OX-A only elicited a positive effect on feeding
when administered to the most rostral region of the LH (
1.8 mm to
bregma). On the basis of previous localization studies (4, 40,
44), it appears that MCH and orexin neurons are distributed from
about 2.1 to 3.7 mm to bregma. MCH neurons are distributed largely
within the zona incerta region, whereas orexin neurons are localized in
the dorsal perifornical area (40), although the exact
rostral-caudal distribution of the receptor expression is not clear. On
the basis of diffusion coefficients and characteristics
(36) and empirical data (14), the spread of
OX-A from the actual injection site would fill the LH region but would
be no more than 1 mm. For region 1, this 1-mm radius includes the perifornical area, the PVN, and some of the zona incerta,
and thus some OX-A likely reached these regions. However, the
concentration of OX-A at these areas would be minimal, since most would
be taken up near the site of injection (36), and thus the
receptors affected by the injections in region 1 are likely
those occupying the rostral LH. Orexin B does not have a significant
effect on feeding when administered to this region (48),
and thus the feeding effects observed are likely mediated through OX1R
expressed by either MCH or orexin neurons. However, Dube et al.
(16) found that injection of OX-A in the more caudal LH
(3.6 to bregma) significantly stimulated feeding, which is inconsistent with the present data, and the reason for this discrepancy is unclear.
When administered to the ventricles, OX-A can induce feeding in the middark cycle and the early light cycle, but not at the beginning of the dark cycle (21, 54). Consistent with this finding, our data indicate that OX-A administered to the LH induces feeding only during the middle of the light period. Recently, a diurnal variation in OX-A signaling has been reported. Prepro-orexin mRNA was shown to be elevated during the light cycle and was lower during the dark cycle (50). Extracellualar orexin peptide levels (via microdialysis; see Ref. 55) and c-fos (17) were found to be low at 1300 and high at night. This variation suggests that orexin signaling may be coupled to different states of behavioral arousal. The circadian specificity of OX-A-induced feeding coincides with decreased feeding and low arousal signals from other sources, such as NPY (11, 46). Thus the condition for which the internal signaling milieu proves favorable for orexin signaling appears to be when other stimulators of appetite are low. Under conditions of heightened "hunger" signaling, one might not expect exogenous administration of OX-A to yield increases in food intake but, rather, that feeding effects of orexin signaling would be masked by other proappetitive signals. However, we found the opposite result. In rats on a restricted diet, baseline hunger level at 1300 (before injection) is presumably high. However, food intake (above baseline levels) after OX-A stimulation of the LH was >5 g, a two- to threefold increase in food intake (above baseline) compared with that observed in OX-A-treated nonrestricted animals (Fig. 5).
It is possible that sensitivity to the effects of OX-A-induced feeding may coincide with low glucose levels. Within the hypothalamus reside glucosensing neurons (GSN), which are divided into glucose-excited (GE) and glucose-inhibited (GI) neurons. These neurons exhibit connectivity with metabolic regulation centers (for review, see Ref. 28). OX-A-containing neurons may be GIs, since hypoglycemia activates orexin-containing neurons (35), increases prepro-orexin mRNA levels (19), and induces c-fos-like activity in orexin neurons (6, 33). Furthermore, orexin-containing neurons have excitatory processes that terminate on GSN (31). The NTS, which also contains GSNs, projects to the LH. Under hypoglycemic conditions, there is an increase in c-Fos immunolabeling of neurons in the NTS and increases in prepro-orexin mRNA in the LH, suggesting a role for the NTS in mediating glucose-related orexin signaling (9). Direct injection of OX-A in the NTS does not result in increased food intake (16). Implications for the relation between orexin signaling and metabolic status stem from the finding that hypoglycemia induced by 48-h food deprivation is associated with an increase in orexin mRNA in the LH. Although no increase in orexin mRNA is observed after a restricted diet paradigm (10), c-fos immunostaining in orexin neurons increases when rats are placed on a restricted diet (27). Taken together, these data suggest a link between OX-A-containing neurons and regulation of short-term energy homeostasis, possibly via sensitivity to blood glucose levels.
Our data show that OX-A administered to the LH stimulates feeding only when injected during the normal resting time for rats, and, because OX-A induces arousal (32, 45), it is possible that the feeding response seen at this time is secondary to OX-A-induced arousal. However, if increased arousal were the primary cause for the elevation in feeding after OX-A, then OX-A-induced arousal would always be coincident with increased feeding. We recently found that, although OX-A increases activity during both light and dark cycles, indicating enhanced arousal in both situations, increases in feeding are only observed during the light cycle, demonstrating that feeding is not always coincident with increased arousal (26). Furthermore, as previously noted, studies that link hypoglycemia and orexin-containing neurons suggest a strong link between orexin signaling and metabolic status. These hypoglycemic conditions are somewhat parallel to the metabolic status of food-restricted animals, and, in these animals, we found that administration of exogenous OX-A was potently orexigenic, which suggests that these metabolic conditions sensitize orexin signaling. In contrast, the feeding response (above baseline levels) in food-deprived rats after central administration of the well-established orexigenic neuropeptide NPY remains the same relative to that response observed during ad libitum-fed conditions (29). The mechanism by which food restriction increases sensitivity of orexin signaling is unclear. It is possible that a change in orexin receptor density or the relative proportion of OX1R to OX2R mediates the sensitization. Recently, Kurose et al. (27) demonstrated a decrease in OX2R mRNA within the PVN when rats were placed on a restricted diet. Exactly what aspect of the restricted diet and what function sensitization to orexin may serve endogenously are not clear. It is clear, however, that some component of orexin signaling may be influenced by metabolic status.
There is a possibility that the behavioral effects noted with discrete injections are due to diffusion of injectate outside the intended site and action at unintended sites. For example, the region within which OX-A was found to elicit a feeding response is at the same rostrocaudal level as the PVN. The PVN is a site of NPY action and an important component of the hypothalamic feeding network (5). Both orexin receptors are expressed within the PVN (3, 13), and microinjection of OX-A in this site elicits feeding (16). However, the possibility that the feeding behavior observed with microinjection of OX-A in the LH results from action in the PVN is diminished by the current data demonstrating that injections in sites in close proximity did not elicit the same behavioral response. This finding of localized effects is similar to previous studies of hindbrain injections of opioid antagonist (25). If injections do in fact diffuse over a larger region than intended, one would expect no difference in the behavioral response resulting from injection in adjacent sites. However, we found that injection in sites very close in proximity yields different behavioral outcomes. Other groups have also demonstrated specificity with discrete injections of OX-A (16).
In summary, we demonstrated that the effect of OX-A-stimulated feeding is localized in one region of the LH and that OX-A-induced feeding occurs only after injection during the light phase, a time at which rats are resting and not engaging in a high level of feeding activity. Finally, we demonstrated that food-restricted rats exhibit an increased sensitivity to OX-A injections in the LH. These results suggest that sensitivity to OX-A signaling may be influenced by the metabolic status of the animal, which likely plays a role in the circadian specificity of OX-A feeding stimulation.
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ACKNOWLEDGEMENTS |
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We thank Jennifer Lockie for expert technical assistance with the food intake measurements.
This work was supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57573.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Catherine Kotz, Veterans Affairs Medical Center, Geriatric, Research, Education and Clinical Center (11G), One Veterans Dr., Minneapolis, MN 55417 (E-mail: kotzx004{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.
10.1152/ajpregu.00344.2002
Received 5 June 2002; accepted in final form 9 December 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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