Dysregulation of diurnal rhythms of serotonin 5-HT2C and corticosteroid receptor gene expression in the hippocampus with food restriction and glucocorticoids

Abstract

Both serotonergic dysfunction and glucocorticoid hypersecretion are implicated in affective and eating disorders. The adverse effects of serotonergic (5-HT)2C receptor activation on mood and food intake, the antidepressant efficacy of 5-HT2 receptor antagonists, and the hyperphagia observed in 5-HT2C receptor knockout mice all suggest a key role for increased 5-HT2C receptor-mediated neurotransmission. Glucocorticoids, however, downregulate 5-HT2C receptor mRNA in the hippocampus, and it is unclear how increased 5-HT2C receptor sensitivity is achieved in the presence of elevated glucocorticoid levels in depression. Here we show a monophasic diurnal rhythm of 5-HT2C receptor mRNA expression in the rat hippocampus that parallels time-dependent variations in 5-HT2C receptor agonist-induced behaviors in open field tests. Rats entrained to chronic food restriction show marked but intermittent corticosterone hypersecretion and maintain an unaltered 5-HT2C receptor mRNA rhythm. The 5-HT2C receptor mRNA rhythm, however, is suppressed by even modest constant elevations of corticosterone (adrenalectomy + pellet) or with elevated corticosterone during the daytime (8 A.M.), whereas a normal rhythm exists in animals that have the same dose of corticosterone in the evening (6 P.M.). Thus, animals showing even a transient daytime corticosterone nadir exhibit normal hippocampal 5-HT2C receptor mRNA rhythms, even in the presence of overt corticosterone hypersecretion. Chronic food restriction also abolishes the normal diurnal variation in hippocampal glucocorticoid receptor (GR) and mineralocorticoid receptor mRNAs and produces, unusually, both elevated corticosterone and increased GR. The mismatch between elevated glucocorticoids and maintained 5-HT2C receptor and increased GR gene expression in the hippocampus provides a new model to dissect mechanisms that may underlie affective and eating disorders.

Fig. 1.

Diurnal variation of 5-HT_2C receptor mRNA expression and mCPP-induced behaviors. A, 5-HT_2C receptor mRNA expression in the CA1 subregion of the hippocampus over a 24 hr period. Receptor mRNA levels were determined by in situ hybridization histochemistry. The mean number of grains/subregion/rat was standardized to the expression observed in the control rats at 8 P.M. (20) (100%). Values represent mean ± SEM; n = 4. *p < 0.05 compared with 8 A.M. (8).B–D, Sensitivity to mCPP-induced inhibition;B, locomotor behavior; C, rearings in an open field. The % number of crossings of open field zones and % number of rearings in a 5 min period after mCPP (1 mg/kg, i.p.) 30 min before testing, compared with controls (saline injected) tested at the same time of day. Numbers per group = 6–8. D,The % inhibition of food intake 30 min after mCPP (1 mg/kg, i.p.) compared with control animals tested at the same time of day. Animals were fasted overnight before testing, and the test period was for 1 hr. Number of animals per group = 8. All columns represent mean results per group ±SEM. *p < 0.05 compared with controls tested at the same time of day. Time of day: 9 = 9 A.M.; 13 = 1 P.M.; 17 = 5 P.M.; 21 = 9 P.M.

Fig. 2.

Twenty-four hour profile of plasma corticosterone (Plasma B) levels in control (•) and in animals after 3 week food restriction (□). Values represent mean ± SEM;n = 4–8. ▨ represents period of food availability for food-restricted animals, and black barsrepresent period of darkness. Time notation as in Figure 1legend.

Fig. 3.

Twenty-four hour profile of (A) 5-HT_2C and (B) 5-HT_1A receptor mRNA expression in control (▪) and food-restricted animals (▵) in various hippocampal subregions. Receptor mRNA levels were determined byin situ hybridization histochemistry. The mean number of grains/subregion/rat was standardized to the expression observed in the control rats at 8 P.M. (20) (100%). Values represent mean ± SEM; n = 4. *p < 0.05 compared with peak value. Time notation as in Figure 1legend.

Fig. 4.

Twenty-four hour profile of (A) MR and (B) GR mRNA expression in control (▪) and food-restricted (▵) animals in various hippocampal subregions. Receptor mRNA levels were determined by in situhybridization histochemistry. The mean number of grains/subregion/rat was standardized to the expression observed in the control rats at 8 P.M. (20) (100%). Values represent mean ± SEM;n = 4. *p < 0.05 compared with peak value. Time notation as in Figure 1 legend.

Fig. 5.

Plasma corticosterone levels obtained at 8 A.M. (08.00) and 8 P.M. (20.00) in sham-adrenalectomized (black and open columns) rats or rats adrenalectomized with a 100 mg corticosterone slow-release pellet subcutaneously (striped and stipled columns). *p < 0.05 compared with 8 A.M.

Fig. 6.

The effect of constant corticosterone levels on the diurnal rhythm of (a) 5-HT_2C receptor (5-HT_2C R mRNA), (b) 5-HT_1A receptor (5-HT_1AR mRNA), (c) MR (MR mRNA), and (d) GR (GR mRNA) mRNA expression in hippocampal subfields. Expression was measured in sham-operated animals at 8 A.M. (black columns) and 8 P.M. (open columns) and compared with adrenalectomized rats with a corticosterone pellet at 8 A.M. (striped column) and 8 P.M. (stipled column). Receptor mRNA levels were determined by in situhybridization histochemistry. The mean number of grains/subregion/rat was standardized to the expression observed in the control rats at 8 P.M. (100%). Values represent mean ± SEM; n= 5–6. *p < 0.05 compared with value at 8 A.M. in sham-operated rats.

Fig. 7.

The effect of pulsatile corticosterone replacement on the diurnal rhythm of 5-HT_2C receptor mRNA expression in ventral CA1 of the hippocampus and plasma corticosterone levels. Sham-operated controls (□) compared with adrenalectomized rats with corticosterone replacement (•). A, Sham-operated controls; B, corticosterone injection (20 mg/kg, s.c.) at 8 A.M. (8); C, corticosterone injection (20 mg/kg, s.c.) at 6 P.M. The arrowrepresents time of corticosterone injection. Receptor mRNA levels were determined by in situ hybridization histochemistry. The mean number of grains/subregion/rat was standardized to the expression observed in the control rats at 8 P.M. (20) (100%). Values represent mean ± SEM; n = 4. *p < 0.05 compared with 8–10 A.M. control value. ⋆ p < 0.05 compared with 8–10 A.M. corticosterone replacement value. Time notation as in Figure 1legend.

Fig. 8.

5-HT_2C receptor mRNA expression in other brain regions at 8 A.M. (open columns) and 8 P.M. (striped columns). Values represent mean ± SEM;n = 4–6. *p < 0.05 compared with alternate time point. LH, Lateral hypothalamus;DMN, dorsomedial nucleus; VMN, ventromedial nucleus; PVN, paraventricular nucleus;RSGC, retrosplenic granular cortex; BNST, bed nucleus of the stria terminalis; POA, preoptic area;dSCN, dorsal suprachiasmatic nucleus;SCN, suprachiasmatic nucleus.

Fig. 9.

Correlation of 24 hr profiles of plasma corticosterone with hippocampal 5-HT_2C receptor mRNA diurnal rhythmicity. Dotted line represents normal nadir levels of corticosterone.