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The medial hypothalamus is required for the feeding response to glucoprivation but not to food deprivation
Neuroscience Letters 464 (2009) 6–9
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The medial hypothalamus is required for the feeding response to glucoprivation but not to food deprivation Minemori Watanabe a , Hiroshi Arima a,∗ , Yoshiharu Ozawa a , Motomitsu Goto a , Hiroshi Shimizu a , Ryoichi Banno a , Yoshihisa Sugimura a , Nobuaki Ozaki a , Hiroshi Nagasaki b , Yutaka Oiso a a b
Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan Department of Metabolic Medicine, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan
a r t i c l e
i n f o
Article history: Received 14 May 2009 Received in revised form 14 July 2009 Accepted 2 August 2009 Keywords: Hypothalamus Food intake Food deprivation Glucoprivation
a b s t r a c t While the hypothalamus has been implicated in the regulation of energy balance, the central mechanisms and neural circuit that coordinate the feeding response to energy deficit have not been fully clarified. To better understand the role of the hypothalamus in mediating hyperphagic responses to food deprivation or glucoprivation, we examined the feeding responses in rats in which the medial hypothalamus (MH) was isolated from the rest of the brain. The isolation of the MH was performed with a Halasz’s knife cut, and experiments were performed 7 days after the operation. Food consumption between 9:00 a.m. and 11:00 a.m. in rats which had been fasted overnight was significantly increased compared to that in rats which had access to food ad libitum before the measurement in both the sham and MH-isolated groups, and the absolute values of food consumption in fasted rats were not significantly different between the groups. On the other hand, while an injection of 2-deoxy-d-glucose, which blocks glucose utilization, significantly increased food consumption for 2 h after injection compared to a saline injection in the sham group, it did not increase food intake compared to saline injection in the MH-isolated groups. Thus, it is demonstrated that glucoprivation is not an effective stimulus to induce feeding in MH-isolated rats. © 2009 Elsevier Ireland Ltd. All rights reserved.
Increasing food intake is among the compensatory responses to energy deficit, and serves to restore metabolic fuel. While the central mechanisms underlying the feeding response to energy deficit have not been fully clarified, the hypothalamus and the brainstem are supposed to be critical sites to detect signals related to energy deficit and to coordinate the feeding response [6,7,23]. Previous studies have demonstrated that hyperphagic responses to food deprivation are maintained in decerebrate rats in which the caudal brainstem was surgically isolated from the forebrain with a complete high mesencephalic transection [8]. These data suggest that the brainstem is sufficient for the feeding response. However, as decerebrate rats were not able to eat spontaneously, the experiments were performed in rats in which food was infused directly into the oral cavity. Another approach to clarify the role of the hypothalamus in the feeding responses to food deprivation would be to isolate the hypothalamus from the rest of the brain, but a previous study reported that hypothalamus-isolated rats also did not eat spontaneously [3]. Thus, these studies did not address the issue of whether the hypothalamus is required for the appetitive behavior in response to food deprivation.
∗ Corresponding author. Tel.: +81 52 744 2142; fax: +81 52 744 2206. E-mail address: [email protected] (H. Arima). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.08.005
The feeding responses to glucoprivation, induced by injection of either insulin or 2-deoxy-d-glucose (2DG), which blocks glucose utilization [2], have also been studied extensively [4,5,16,17,21,27,28]. High-dose insulin injection is reported to induce a hyperphagic response in decerebrate rats [4], suggesting that the brainstem is sufficient to detect the glucoprivic signals and increase food intake. On the other hand, glucoprivation reportedly did not induce hyperphagic responses in rats in which the medial hypothalamus (MH) was lesioned [12,13,17]. It is also demonstrated that the delivery of anti-dopamine -hydroxylase conjugated to saporin (DSAP) to the paraventricular nuclei (PVN) in the hypothalamus, which causes retrograde degeneration of brainstem catecholamine neurons [1,21], abolished the feeding response to 2DG [21], suggesting that catecholaminergic projection from the brainstem to the PVN is required for the feeding response to glucoprivation. Thus, feeding responses to glucoprivation closely depend on the experimental conditions. To clarify whether the neural connections between the hypothalamus and the rest of the brain are required in hyperphagic responses to deprivation or glucoprivation, the current study examined the feeding responses in rats in which the MH is isolated from the rest of the brain. Our previous paper showed that the MH-isolated rats were obese and could eat spontaneously [30]. Male Sprague–Dawley rats [250–300 g body weight (BW); Chubu Science Materials, Nagoya, Japan] were housed individually
M. Watanabe et al. / Neuroscience Letters 464 (2009) 6–9
in plastic cages under controlled conditions (23.0 ± 0.5 ◦ C, lights on from 9:00 a.m. to 9:00 p.m.), handled every day and had access to standard chow and water ad libitum until the experiments. All procedures were performed in accordance with the institutional guidelines for animal care at Nagoya University Graduate School of Medicine and approved by the Animal Experimentation Committee. Isolation of the MH was performed according to Halasz’s method [9] with minor modifications as reported previously [30]. In brief, rats were anesthetized with an intraperitoneal (ip) injection of pentobarbital (50 mg/kg) for stereotaxic surgery. The coordinates of the MH isolation were 2.8 mm posterior to the bregma and 9.4 mm ventral to the skull surface. A knife 3.5 mm in height with a 2 mm radius was lowered at the midline with the blade pointing laterally, and rotated. With this operation, the arcuate, dorsomedial, paraventricular, suprachiasmatic and ventromedial nuclei, but not the lateral hypothalamic area, were isolated. In sham-operated rats, the knife was lowered to the same coordinates but was not rotated. Daily food intake and BW were monitored after the operation, and experiments were performed 7 days after the operation. The food consumption between 9:00 a.m. and 11:00 a.m. was measured in both the MH-isolated and sham-operated rats which had been fasted overnight. The food consumption was also measured in both the MH-isolated and sham-operated rats which had access to food ad libitum before the measurement. Either 750 mg/kg 2DG (Sigma, St. Louis, MO) dissolved in isotonic saline or vehicle was injected (2% BW) ip between 9:00 a.m. and 10:00 a.m. in both the MH-isolated and sham-operated rats which had access to food ad libitum before the injection, and the subsequent food consumption over a 2 h period was measured. Statistical significance of the differences between the groups was calculated by one-way ANOVA followed by Fisher’s protected least significant difference test. Results are expressed as means ± SE, and differences were considered significant at p < 0.05. The group size was eight in all experiments. Consistent with our previous paper [30], BW (sham: 261 ± 2 g; MH-isolation: 307 ± 4 g; n = 32 for each group; p < 0.01) and daily food intake (sham: 23 ± 1 g; MH-isolation: 33 ± 2 g; n = 32 for each group; p < 0.01) on day 6 were significantly increased in the MHisolated rats compared to the sham-operated rats. Rats in both the sham and MH-isolated groups were divided into four groups for the following experiments. The food consumption between 9:00 a.m. and 11:00 a.m. in rats which had access to food ad libitum before the measurement was significantly increased in the MH-isolated group compared to the sham group (Fig. 1A), demonstrating that MH-isolated rats tend to eat food even after the lights are turned on. The food consumption in fasted rats was significantly increased compared to that in rats which had access to food ad libitum before the measurement in both sham and MH-isolated groups, and the absolute values of food consumption in fasted rats were not significantly different between the sham and MH-isolated groups (Fig. 1A). When fasted and ad libitum rats were compared, however, the differences were significantly less in MH-isolated groups (4.0 ± 0.6 g) than in sham groups (6.3 ± 0.3 g). The food consumption in rats injected with saline was significantly increased in the MH-isolated group compared to the sham group (Fig. 1B). The food consumption was significantly increased after 2DG injection in the sham, but not in the MH-isolated group, and the absolute values in the MH-isolated rats injected with 2DG were significantly lower compared to those in the sham group (Fig. 1B). Isolation of MH was confirmed at the end of experiments. A representative photograph of a brain section −2.80 mm of bregma stained with cresyl violet is shown in Fig. 2.
7
Fig. 1. Feeding responses to food deprivation and glucoprivation in medial hypothalamus (MH)-isolated and sham-operated rats. (A) Food consumption between 9:00 a.m. and 11:00 a.m. was measured in both MH-isolated (isolation) and shamoperated (sham) rats which had been fasted overnight (fast) or had access to food ad libitum (ad lib) before the measurement. (B) Either 750 mg/kg 2DG dissolved in isotonic saline or vehicle (saline) was injected (2% BW) ip in both the sham-operated (sham) and MH-isolated rats (isolation) which had access to food ad libitum before injection, and the subsequent food consumption for 2 h was measured. Results are expressed as mean ± SE (n = 8). *p < 0.05 vs. values in ad libitum groups (A) and in saline groups (B). † p < 0.05 vs. values in ad libitum (A) and in saline groups (B) in the sham. # p < 0.05 vs. values in the 2DG group in the sham.
In the present study, we examined feeding responses to deprivation and glucoprivation in rats in which the MH was isolated from the rest of the brain. Successful isolation of the MH was confirmed not only histologically but also via the hyperphagic and obese phenotypes, as reported previously [30]. Using this model, we demonstrated that the hyperphagic response to 2DG was abolished, whereas the response to food deprivation was maintained in the MH-isolated rats. As the feeding response to food deprivation was more robust than that induced by 2DG injection in sham rats, it is possible that the effects of MH-isolation on the feeding response were somehow masked in fasting. Nevertheless, the clear difference in the responses to fasting and glucoprivation in MH-isolated rats suggests that neural circuits to induce feeding responses are different between fasting and glucoprivation. The hyperphagic response to deprivation in the MH-isolated rats shown in the present study is consistent with a previous paper showing that decerebrate rats ingested more after food deprivation than under control conditions [8]. Feeding behavior consists of appetitive behavior and consummatory components [6,7,23], and the hypothalamus has been implicated in appetitive behavior [11,25,29]. By using MH-isolated rats which could eat spontaneously, we demonstrated that not only consummatory responses but also appetitive behavior were maintained in these rats. It is important, however, to keep in mind that not only metabolic signals related to energy deficit but also the gastrointestinal status after deprivation affects the feeding response. Seeley et al. demonstrated
8
M. Watanabe et al. / Neuroscience Letters 464 (2009) 6–9
The feeding response to glucoprivation was reportedly abolished in the MH-lesioned rats [12,13,17], in which neural as well as humoral regulation in the MH was lost. On the other hand, we previously demonstrated that the neurons in the MH remained intact after its isolation [30]. Thus, the present study not only confirmed previous studies using MH-lesioned rats [12,13,17], but also demonstrated that neural connections between MH and the rest of the brain are required for the feeding response. Several lines of evidence suggest that signals related to glucopenia are detected at the brainstem [22,24]. Ritter et al. demonstrated that the hyperphagic response to glucoprivation was abolished in rats in which the adrenergic neurons projecting to the PVN were destroyed by DSAP injection [21]. While DSAP injection at the PVN may have affected the adrenergic projections in other areas, in light of our data, it seems possible that projections from the brainstem to the hypothalamus are required for the hyperphagic response to glucoprivation. Alternatively, the isolation of glucose-inhibited neurons in the hypothamamus such as arcuate and ventromedial nuclei, which could directly sense decreases in glucose concentrations [14,15,18], might be responsible for the changes in feeding response to glucoprivation. On the other hand, our findings are in contrast to previous studies showing that glucoprivation elicited a feeding response in decerebrate rats [4]. As these rats could not eat spontaneously, one possible explanation for the discrepancy between the studies could be that appetitive behavior was exclusively abolished by the isolation of the MH in the current study while the consummatory response was left intact, although a previous study demonstrated that the consummatory response to glucoprivation was also abolished in rats injected with DSAP at the PVN [10]. In conclusion, our data demonstrated that the MH-isolated rats displayed a hyperphagic response to food deprivation but not to glucoprivation. Whether this is mediated by pathways that are also involved in the hyperphagic response of intact rats remains to be investigated. References Fig. 2. Isolation of medial hypothalamus. (A) Isolation was confirmed at the end of the experiments, and arrows indicate the Halasz’s knife cut in a brain section −2.80 mm of bregma stained with cresyl violet. (B) The drawing of a brain section was modified from Ref. [20]. The dotted line corresponds to the knife cut in (A). Arc: arcuate nucleus; VMH: ventromedial hypothalamic nucleus; LH: lateral hypothalamic area; DMD: dorsomedial hypothalamic nucleus; 3V: 3rd ventricle.
that, although decerebrate and intact rats showed a similar intake response to gastric preload, food intake after deprivation was not higher than that following stomach evacuation in decerebrate rats [26]. Thus, it is possible that the hyperphagic responses of the MHisolated rats shown in the current study were due to the absence of a gastrointestinal inhibitory influence and not to metabolic signals related to energy deficit. Actually, while MH-lesioned rats still displayed a hyperphagic response to food deprivation, the relative increase was significantly less compared to sham rats. Previous studies showed that 2DG increased food intake when injected during daytime, as in this study, but it did not affect food intake when injected in the evening [19], suggesting that 2DG was not powerful enough to induce hyperphagic responses when the intrinsic requirement for food intake is great. In this study, the suprachiasmatic nucleus, the center of circadian rhythm, was isolated by the knife cut and the MH-isolated rats ate more than sham rats in the 2 h after the light was turned on. However, the absolute values of food consumption in sham rats injected 2DG was much higher than those in MH-isolated groups, indicating that negative effects of 2DG on food intake in MH-isolated groups cannot be explained by the possible disturbance of circadian rhythm.
[1] W.W. Blessing, D.A. Lappi, R.G. Wiley, Destruction of locus coeruleus neuronal perikarya after injection of anti-dopamine-B-hydroxylase immunotoxin into the olfactory bulb of the rat, Neurosci. Lett. 243 (1998) 85–88. [2] J. Brown, Effects of 2-deoxyglucose on carbohydrate metablism: review of the literature and studies in the rat, Metabolism 11 (1962) 1098–1112. [3] G.D. Ellison, Appetitive behavior in rats after circumsection of the hypothalamus, Physiol. Behav. 3 (1968) 221–226. [4] F.W. Flynn, H.J. Grill, Insulin elicits ingestion in decerebrate rats, Science 221 (1983) 188–190. [5] G.S. Fraley, S. Ritter, Immunolesion of norepinephrine and epinephrine afferents to medial hypothalamus alters basal and 2-deoxy-D-glucose-induced neuropeptide Y and agouti gene-related protein messenger ribonucleic acid expression in the arcuate nucleus, Endocrinology 144 (2003) 75–83. [6] H.J. Grill, Distributed neural control of energy balance: contributions from hindbrain and hypothalamus, Obesity (Silver Spring) 14 (Suppl 5) (2006) 216S–221S. [7] H.J. Grill, J.M. Kaplan, The neuroanatomical axis for control of energy balance, Front Neuroendocrinol. 23 (2002) 2–40. [8] H.J. Grill, R. Norgren, Chronically decerebrate rats demonstrate satiation but not bait shyness, Science 201 (1978) 267–269. [9] B. Halasz, L. Pupp, Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotrophic area, Endocrinology 77 (1965) 553–562. [10] B. Hudson, S. Ritter, Hindbrain catecholamine neurons mediate consummatory responses to glucoprivation, Physiol. Behav. 82 (2004) 241–250. [11] S.P. Kalra, P.S. Kalra, NPY and cohorts in regulating appetite, obesity and metabolic syndrome: beneficial effects of gene therapy, Neuropeptides 38 (2004) 201–211. [12] B.M. King, S.P. Grossman, Response to glucoprivic and hydrational challenges by normal and hypothalamic hyperphagic rats, Physiol. Behav. 18 (1977) 463–473. [13] B.M. King, B.A. Stamoutsos, S.P. Grossman, Delayed response to 2-deoxy-Dglucose in hypothalamic obese rats, Pharmacol. Biochem. Behav. 8 (1978) 259–262. [14] B.E. Levin, A.A. Dunn-Meynell, V.H. Routh, Brain glucose sensing and body energy homeostasis: role in obesity and diabetes, Am. J. Physiol. 276 (1999) R1223–R1231. [15] B.E. Levin, V.H. Routh, L. Kang, N.M. Sanders, A.A. Dunn-Meynell, Neuronal glucosensing: what do we know after 50 years? Diabetes 53 (2004) 2521–2528.
M. Watanabe et al. / Neuroscience Letters 464 (2009) 6–9 [16] S. Luquet, C.T. Phillips, R.D. Palmiter, NPY/AgRP neurons are not essential for feeding responses to glucoprivation, Peptides 28 (2007) 214–225. [17] E.E. Muller, A. Pecile, D. Cocchi, V.R. Olgiati, Hyperglycemic or feeding response to glucoprivation and hypothalamic glucoreceptors, Am. J. Physiol. 226 (1974) 1100–1109. [18] S. Muroya, T. Yada, S. Shioda, M. Takigawa, Glucose-sensitive neurons in the rat arcuate nucleus contain neuropeptide Y, Neurosci. Lett. 264 (1999) 113–116. [19] C. Naito, Y. Yoshitoshi, K. Higo, H. Ookawa, Effects of long-term administration of 2-deoxy-D-glucose on food intake and weight gain in rats, J. Nutr. 103 (1973) 730–737. [20] G. Paxinos, C. Watson, The Rat Brain in Sterotaxic Coordinates, second ed., Academic Press, San Diego, 1986. [21] S. Ritter, K. Bugarith, T.T. Dinh, Immunotoxic destruction of distinct catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation, J. Compd. Neurol. 432 (2001) 197–216. [22] S. Ritter, T.T. Dinh, 2-Mercaptoacetate and 2-deoxy-D-glucose induce Fos-like immunoreactivity in rat brain, Brain Res. 641 (1994) 111–120. [23] S. Ritter, T.T. Dinh, A.J. Li, Hindbrain catecholamine neurons control multiple glucoregulatory responses, Physiol. Behav. 89 (2006) 490–500.
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[24] S. Ritter, T.T. Dinh, Y. Zhang, Localization of hindbrain glucoreceptive sites controlling food intake and blood glucose, Brain Res. 856 (2000) 37–47. [25] M.W. Schwartz, S.C. Woods, D. Porte Jr., R.J. Seeley, D.G. Baskin, Central nervous system control of food intake, Nature 404 (2000) 661–671. [26] R.J. Seeley, H.J. Grill, J.M. Kaplan, Neurological dissociation of gastrointestinal and metabolic contributions to meal size control, Behav Neurosci 108 (1994) 347–352. [27] D.K. Sindelar, L. Ste Marie, G.I. Miura, R.D. Palmiter, J.E. McMinn, G.J. Morton, M.W. Schwartz, Neuropeptide Y is required for hyperphagic feeding in response to neuroglucopenia, Endocrinology 145 (2004) 3363–3368. [28] G.P. Smith, A.N. Epstein, Increased feeding in response to decreased glucose utilization in the rat and monkey, Am. J. Physiol. 217 (1969) 1083–1087. [29] B.G. Stanley, S.E. Kyrkouli, S. Lampert, S.F. Leibowitz, Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity, Peptides 7 (1986) 1189–1192. [30] M. Watanabe, H. Arima, K. Fukushima, M. Goto, H. Shimizu, M. Hayashi, R. Banno, I. Sato, N. Ozaki, H. Nagasaki, Y. Oiso, Direct and indirect modulation of neuropeptide Y gene expression in response to hypoglycemia in rat arcuate nucleus, FEBS Lett. 582 (2008) 3632–3638.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The medial hypothalamus is required for the feeding response to glucoprivation but not to food deprivation Minemori Watanabe a , Hiroshi Arima a,∗ , Yoshiharu Ozawa a , Motomitsu Goto a , Hiroshi Shimizu a , Ryoichi Banno a , Yoshihisa Sugimura a , Nobuaki Ozaki a , Hiroshi Nagasaki b , Yutaka Oiso a a b
Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan Department of Metabolic Medicine, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan
a r t i c l e
i n f o
Article history: Received 14 May 2009 Received in revised form 14 July 2009 Accepted 2 August 2009 Keywords: Hypothalamus Food intake Food deprivation Glucoprivation
a b s t r a c t While the hypothalamus has been implicated in the regulation of energy balance, the central mechanisms and neural circuit that coordinate the feeding response to energy deficit have not been fully clarified. To better understand the role of the hypothalamus in mediating hyperphagic responses to food deprivation or glucoprivation, we examined the feeding responses in rats in which the medial hypothalamus (MH) was isolated from the rest of the brain. The isolation of the MH was performed with a Halasz’s knife cut, and experiments were performed 7 days after the operation. Food consumption between 9:00 a.m. and 11:00 a.m. in rats which had been fasted overnight was significantly increased compared to that in rats which had access to food ad libitum before the measurement in both the sham and MH-isolated groups, and the absolute values of food consumption in fasted rats were not significantly different between the groups. On the other hand, while an injection of 2-deoxy-d-glucose, which blocks glucose utilization, significantly increased food consumption for 2 h after injection compared to a saline injection in the sham group, it did not increase food intake compared to saline injection in the MH-isolated groups. Thus, it is demonstrated that glucoprivation is not an effective stimulus to induce feeding in MH-isolated rats. © 2009 Elsevier Ireland Ltd. All rights reserved.
Increasing food intake is among the compensatory responses to energy deficit, and serves to restore metabolic fuel. While the central mechanisms underlying the feeding response to energy deficit have not been fully clarified, the hypothalamus and the brainstem are supposed to be critical sites to detect signals related to energy deficit and to coordinate the feeding response [6,7,23]. Previous studies have demonstrated that hyperphagic responses to food deprivation are maintained in decerebrate rats in which the caudal brainstem was surgically isolated from the forebrain with a complete high mesencephalic transection [8]. These data suggest that the brainstem is sufficient for the feeding response. However, as decerebrate rats were not able to eat spontaneously, the experiments were performed in rats in which food was infused directly into the oral cavity. Another approach to clarify the role of the hypothalamus in the feeding responses to food deprivation would be to isolate the hypothalamus from the rest of the brain, but a previous study reported that hypothalamus-isolated rats also did not eat spontaneously [3]. Thus, these studies did not address the issue of whether the hypothalamus is required for the appetitive behavior in response to food deprivation.
∗ Corresponding author. Tel.: +81 52 744 2142; fax: +81 52 744 2206. E-mail address: [email protected] (H. Arima). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.08.005
The feeding responses to glucoprivation, induced by injection of either insulin or 2-deoxy-d-glucose (2DG), which blocks glucose utilization [2], have also been studied extensively [4,5,16,17,21,27,28]. High-dose insulin injection is reported to induce a hyperphagic response in decerebrate rats [4], suggesting that the brainstem is sufficient to detect the glucoprivic signals and increase food intake. On the other hand, glucoprivation reportedly did not induce hyperphagic responses in rats in which the medial hypothalamus (MH) was lesioned [12,13,17]. It is also demonstrated that the delivery of anti-dopamine -hydroxylase conjugated to saporin (DSAP) to the paraventricular nuclei (PVN) in the hypothalamus, which causes retrograde degeneration of brainstem catecholamine neurons [1,21], abolished the feeding response to 2DG [21], suggesting that catecholaminergic projection from the brainstem to the PVN is required for the feeding response to glucoprivation. Thus, feeding responses to glucoprivation closely depend on the experimental conditions. To clarify whether the neural connections between the hypothalamus and the rest of the brain are required in hyperphagic responses to deprivation or glucoprivation, the current study examined the feeding responses in rats in which the MH is isolated from the rest of the brain. Our previous paper showed that the MH-isolated rats were obese and could eat spontaneously [30]. Male Sprague–Dawley rats [250–300 g body weight (BW); Chubu Science Materials, Nagoya, Japan] were housed individually
M. Watanabe et al. / Neuroscience Letters 464 (2009) 6–9
in plastic cages under controlled conditions (23.0 ± 0.5 ◦ C, lights on from 9:00 a.m. to 9:00 p.m.), handled every day and had access to standard chow and water ad libitum until the experiments. All procedures were performed in accordance with the institutional guidelines for animal care at Nagoya University Graduate School of Medicine and approved by the Animal Experimentation Committee. Isolation of the MH was performed according to Halasz’s method [9] with minor modifications as reported previously [30]. In brief, rats were anesthetized with an intraperitoneal (ip) injection of pentobarbital (50 mg/kg) for stereotaxic surgery. The coordinates of the MH isolation were 2.8 mm posterior to the bregma and 9.4 mm ventral to the skull surface. A knife 3.5 mm in height with a 2 mm radius was lowered at the midline with the blade pointing laterally, and rotated. With this operation, the arcuate, dorsomedial, paraventricular, suprachiasmatic and ventromedial nuclei, but not the lateral hypothalamic area, were isolated. In sham-operated rats, the knife was lowered to the same coordinates but was not rotated. Daily food intake and BW were monitored after the operation, and experiments were performed 7 days after the operation. The food consumption between 9:00 a.m. and 11:00 a.m. was measured in both the MH-isolated and sham-operated rats which had been fasted overnight. The food consumption was also measured in both the MH-isolated and sham-operated rats which had access to food ad libitum before the measurement. Either 750 mg/kg 2DG (Sigma, St. Louis, MO) dissolved in isotonic saline or vehicle was injected (2% BW) ip between 9:00 a.m. and 10:00 a.m. in both the MH-isolated and sham-operated rats which had access to food ad libitum before the injection, and the subsequent food consumption over a 2 h period was measured. Statistical significance of the differences between the groups was calculated by one-way ANOVA followed by Fisher’s protected least significant difference test. Results are expressed as means ± SE, and differences were considered significant at p < 0.05. The group size was eight in all experiments. Consistent with our previous paper [30], BW (sham: 261 ± 2 g; MH-isolation: 307 ± 4 g; n = 32 for each group; p < 0.01) and daily food intake (sham: 23 ± 1 g; MH-isolation: 33 ± 2 g; n = 32 for each group; p < 0.01) on day 6 were significantly increased in the MHisolated rats compared to the sham-operated rats. Rats in both the sham and MH-isolated groups were divided into four groups for the following experiments. The food consumption between 9:00 a.m. and 11:00 a.m. in rats which had access to food ad libitum before the measurement was significantly increased in the MH-isolated group compared to the sham group (Fig. 1A), demonstrating that MH-isolated rats tend to eat food even after the lights are turned on. The food consumption in fasted rats was significantly increased compared to that in rats which had access to food ad libitum before the measurement in both sham and MH-isolated groups, and the absolute values of food consumption in fasted rats were not significantly different between the sham and MH-isolated groups (Fig. 1A). When fasted and ad libitum rats were compared, however, the differences were significantly less in MH-isolated groups (4.0 ± 0.6 g) than in sham groups (6.3 ± 0.3 g). The food consumption in rats injected with saline was significantly increased in the MH-isolated group compared to the sham group (Fig. 1B). The food consumption was significantly increased after 2DG injection in the sham, but not in the MH-isolated group, and the absolute values in the MH-isolated rats injected with 2DG were significantly lower compared to those in the sham group (Fig. 1B). Isolation of MH was confirmed at the end of experiments. A representative photograph of a brain section −2.80 mm of bregma stained with cresyl violet is shown in Fig. 2.
7
Fig. 1. Feeding responses to food deprivation and glucoprivation in medial hypothalamus (MH)-isolated and sham-operated rats. (A) Food consumption between 9:00 a.m. and 11:00 a.m. was measured in both MH-isolated (isolation) and shamoperated (sham) rats which had been fasted overnight (fast) or had access to food ad libitum (ad lib) before the measurement. (B) Either 750 mg/kg 2DG dissolved in isotonic saline or vehicle (saline) was injected (2% BW) ip in both the sham-operated (sham) and MH-isolated rats (isolation) which had access to food ad libitum before injection, and the subsequent food consumption for 2 h was measured. Results are expressed as mean ± SE (n = 8). *p < 0.05 vs. values in ad libitum groups (A) and in saline groups (B). † p < 0.05 vs. values in ad libitum (A) and in saline groups (B) in the sham. # p < 0.05 vs. values in the 2DG group in the sham.
In the present study, we examined feeding responses to deprivation and glucoprivation in rats in which the MH was isolated from the rest of the brain. Successful isolation of the MH was confirmed not only histologically but also via the hyperphagic and obese phenotypes, as reported previously [30]. Using this model, we demonstrated that the hyperphagic response to 2DG was abolished, whereas the response to food deprivation was maintained in the MH-isolated rats. As the feeding response to food deprivation was more robust than that induced by 2DG injection in sham rats, it is possible that the effects of MH-isolation on the feeding response were somehow masked in fasting. Nevertheless, the clear difference in the responses to fasting and glucoprivation in MH-isolated rats suggests that neural circuits to induce feeding responses are different between fasting and glucoprivation. The hyperphagic response to deprivation in the MH-isolated rats shown in the present study is consistent with a previous paper showing that decerebrate rats ingested more after food deprivation than under control conditions [8]. Feeding behavior consists of appetitive behavior and consummatory components [6,7,23], and the hypothalamus has been implicated in appetitive behavior [11,25,29]. By using MH-isolated rats which could eat spontaneously, we demonstrated that not only consummatory responses but also appetitive behavior were maintained in these rats. It is important, however, to keep in mind that not only metabolic signals related to energy deficit but also the gastrointestinal status after deprivation affects the feeding response. Seeley et al. demonstrated
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M. Watanabe et al. / Neuroscience Letters 464 (2009) 6–9
The feeding response to glucoprivation was reportedly abolished in the MH-lesioned rats [12,13,17], in which neural as well as humoral regulation in the MH was lost. On the other hand, we previously demonstrated that the neurons in the MH remained intact after its isolation [30]. Thus, the present study not only confirmed previous studies using MH-lesioned rats [12,13,17], but also demonstrated that neural connections between MH and the rest of the brain are required for the feeding response. Several lines of evidence suggest that signals related to glucopenia are detected at the brainstem [22,24]. Ritter et al. demonstrated that the hyperphagic response to glucoprivation was abolished in rats in which the adrenergic neurons projecting to the PVN were destroyed by DSAP injection [21]. While DSAP injection at the PVN may have affected the adrenergic projections in other areas, in light of our data, it seems possible that projections from the brainstem to the hypothalamus are required for the hyperphagic response to glucoprivation. Alternatively, the isolation of glucose-inhibited neurons in the hypothamamus such as arcuate and ventromedial nuclei, which could directly sense decreases in glucose concentrations [14,15,18], might be responsible for the changes in feeding response to glucoprivation. On the other hand, our findings are in contrast to previous studies showing that glucoprivation elicited a feeding response in decerebrate rats [4]. As these rats could not eat spontaneously, one possible explanation for the discrepancy between the studies could be that appetitive behavior was exclusively abolished by the isolation of the MH in the current study while the consummatory response was left intact, although a previous study demonstrated that the consummatory response to glucoprivation was also abolished in rats injected with DSAP at the PVN [10]. In conclusion, our data demonstrated that the MH-isolated rats displayed a hyperphagic response to food deprivation but not to glucoprivation. Whether this is mediated by pathways that are also involved in the hyperphagic response of intact rats remains to be investigated. References Fig. 2. Isolation of medial hypothalamus. (A) Isolation was confirmed at the end of the experiments, and arrows indicate the Halasz’s knife cut in a brain section −2.80 mm of bregma stained with cresyl violet. (B) The drawing of a brain section was modified from Ref. [20]. The dotted line corresponds to the knife cut in (A). Arc: arcuate nucleus; VMH: ventromedial hypothalamic nucleus; LH: lateral hypothalamic area; DMD: dorsomedial hypothalamic nucleus; 3V: 3rd ventricle.
that, although decerebrate and intact rats showed a similar intake response to gastric preload, food intake after deprivation was not higher than that following stomach evacuation in decerebrate rats [26]. Thus, it is possible that the hyperphagic responses of the MHisolated rats shown in the current study were due to the absence of a gastrointestinal inhibitory influence and not to metabolic signals related to energy deficit. Actually, while MH-lesioned rats still displayed a hyperphagic response to food deprivation, the relative increase was significantly less compared to sham rats. Previous studies showed that 2DG increased food intake when injected during daytime, as in this study, but it did not affect food intake when injected in the evening [19], suggesting that 2DG was not powerful enough to induce hyperphagic responses when the intrinsic requirement for food intake is great. In this study, the suprachiasmatic nucleus, the center of circadian rhythm, was isolated by the knife cut and the MH-isolated rats ate more than sham rats in the 2 h after the light was turned on. However, the absolute values of food consumption in sham rats injected 2DG was much higher than those in MH-isolated groups, indicating that negative effects of 2DG on food intake in MH-isolated groups cannot be explained by the possible disturbance of circadian rhythm.
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