The melanocortinergic pathway is rapidly recruited by emotional stress and contributes to stress-induced anorexia and anxiety-like behavior

Abstract

Neurons producing melanocortin receptor agonist, alpha-MSH derived from proopiomelanocortin, and antagonist, agouti-related protein, are known to be sensitive to metabolic stress such as food deprivation and glucoprivation. However, how these neurons respond to emotional/psychological stress remained to be elucidated. We report here that acute emotional stressors, i.e. restraint and forced swim, evoked mRNA expression of c-fos, a neuronal activation marker, in a high percentage of proopiomelanocortin neurons (up to 53% for restraint stress and 62% for forced swim), with marked variations along the rostro-caudal axis of the arcuate nucleus. In contrast, only a small population of agouti-related protein neurons in this brain region was activated. These neuronal activation patterns were correlated with behavioral reactions. Both stressors suppressed feeding and induced anxiety-like behavior in the elevated plus-maze test, as reflected by a reduction in the percentage of entries and time spent in the open arms. Central pretreatment with SHU9119, a melanocortin receptor antagonist, dose dependently attenuated the anorectic and anxiogenic effects elicited by acute restraint or forced swim. These results indicate that the melancortinergic pathway can be rapidly recruited by acute emotional stress, and that activation of melanocortin signaling is involved in mediating stress-induced anorexia and anxiety.

Fig. 1

Images of film autoradiograms (×2) showing c-fos mRNA expression in the brain under nonstressful condition (A), 30 min after the onset of restraint (B), or forced swim stress (C). Arc, Arcuate nucleus; BLA, basolateral amygdala; CeA, central amygdala; CTX, cortex; DMH, dorsomedial hypothalamus; Hippo, hippocampus; MeA, medial amygdala; VMH, ventromedial hypothalamus.

Fig. 2

Microscopic images showing induction of c-fos mRNA expression in POMC neurons in the arcuate nucleus under the basal condition (control, A and A’) or after acute restraint (B and B’) or forced swim (C and C’) stress. A–C, Bright-field microscopic images (×50) showing the distribution of POMC-expressing neurons (detected by digoxigenin-labeled riboprobe, dark purple cells). A’–C’, High magnification of dark-field microscopic images (×100) showing that POMC neurons (dark purple cells) coexpress c-fos mRNA (detected by ³⁵S-labeled riboprobe, clusters of white grains). Black arrowheads indicate cells not double labeled for c-fos mRNA and POMC mRNA. Yellow arrowheads indicate cells double labeled for c-fos mRNA and POMC mRNA.

Fig. 3

Number of POMC neurons and POMC neurons double labeled for c-fos mRNA along the rostro-caudal axis of the arcuate nucleus under basal condition (control), or after restraint or forced swim stress. Cell counts were made on nine sections per animal corresponding to nine coronal planes from Bregma −2.1 to Bregma −4.2 mm. Sections for cell counting were anatomically matched between animals within the same treatment group as well as between different treatments. A, Control. B, Restraint stress. C, Forced swim. D, Distribution of the percentages of POMC neurons positive for c-fos mRNA along the rostral-caudal axis of the arcuate nucleus under basal condition (naive) or after restraint and forced swim. Data are expressed as mean ± SEM (n = 5 per group). *, P < 0.05, **, P < 0.01, restraint vs. forced swim stress.

Fig. 4

Microscopic images showing induction of c-fos mRNA expression in AgRP neurons in the arcuate nucleus under basal condition (control A and A’) or after acute restraint (B and B’) and forced swim (C and C’) stress. A–C, Bright-field microscopic images (×50) showing the distribution of AgRP-expressing neurons (detected by digoxigenin-labeled riboprobe, dark purple cells). A’–C’, High magnification of dark-field microscopic images showing AgRP neurons (dark purple cells) double labeled for c-fos mRNA (detected by ³⁵S-labeled riboprobe, clusters of white grains) (×100). Black arrowheads indicate cells not double labeled for c-fos mRNA and AgRP mRNA. Yellow arrowheads indicate cells double labeled for c-fos mRNA and AgRP mRNA.

Fig. 5

Number of AgRP neurons and AgRP neurons double labeled for c-fos mRNA along the rostro-caudal axis of the arcuate nucleus under basal condition (control) or after restraint and forced swim stress. Cell counts were made on sections corresponding to nine coronal planes from Bregma −2.1 to Bregma −4.2 mm. Brain sections for cell counting were adjacent to those used for POMC cell counting in Fig. 3. A, Control. B, Restraint stress. C, Forced swim. D, Distribution of the percentages of AgRP neurons positive for c-fos mRNA along the rostro-caudal axis of the arcuate nucleus under basal condition (control) or after restraint and forced swim stress. Data are expressed as mean ± SEM (n = 5 per group).

Fig. 6

Effect of acute restraint and forced swim stress on feeding behavior. Rats were injected icv with 0, 0.05, or 0.5 nmol SHU9119 30 min before exposure to either no stress (control), restraint for 30 min, or forced swim stress for 10 min. icv injections were performed at 1 h before the dark cycle, with stressors applied 30 min after icv injection. The treatment groups were as follows: vehicle followed by no stress (n = 12), 0.05 nmol SHU9119 followed by no stress (n = 5), 0.5 nmol SHU9119 followed by no stress (n = 6), vehicle followed by restraint stress (n = 9), 0.05 nmol SHU9119 followed by restraint stress (n = 6), 0.5 nmol SHU9119 followed by restraint stress (n = 6), vehicle followed by forced swim stress (n = 7), 0.05 nmol SHU9119 followed by forced swim stress (n = 6), and 0.5 nmol SHU9119 followed by forced swim stress (n = 6). Food was provided 30 min after the onset of either stressor. Food intake was measured over 30 min (A) and 120 min (B) and adjusted by body weight. Data are expressed as mean ± SEM. **, P < 0.01, compared with the nonstressed group. ^#, P < 0.05, ^##, P < 0.01, compared with the vehicle-treated condition combined with the same stress exposure.

Fig. 7

Effect of acute restraint and forced swim stress on anxiety-like behavior in the elevated plus-maze test. Rats were injected icv with 0, 0.05, or 0.5 nmol SHU9119 30 min before exposure to either no stress (control), restraint for 30 min, or forced swim stress for 10 min. The treatment groups were as follows: vehicle followed by no stress (n = 10), 0.5 nmol SHU9119 followed by no stress (n = 5), vehicle followed by restraint stress (n = 13), 0.05 nmol SHU9119 followed by restraint stress (n = 5), 0.5 nmol SHU9119 followed by restraint stress (n = 5), vehicle followed by forced swim stress (n = 8), 0.05 nmol SHU9119 followed by forced swim stress (n = 5), and 0.5 nmol SHU9119 followed by forced swim stress (n = 5). Elevated plus-maze tests were performed for 5 min after acute stress. The number of entries into the open arms and closed arms and time spent in the open arms and closed arms was measured. Entries into the open arms/total entries into all arms [OTR(e)] (A) and time spent in the open arms/total time spent [OTR(t)] in all arms (B) were calculated. Data are expressed as mean ± SEM. **, P < 0.01; ***, P < 0.001, compared with the nonstressed control group. ^#, P < 0.05; ^##, P < 0.01 compared with the vehicle-treated condition combined with the same stress exposure. ⁺, P < 0.05; ⁺⁺, P < 0.01 compared with 0.05 nmol SHU9119.