Distinct patterns of neuropeptide gene expression in the lateral hypothalamic area and arcuate nucleus are associated with dehydration-induced anorexia

Abstract

We have investigated the hormonal and hypothalamic neuropeptidergic substrates of dehydration-associated anorexia. In situ hybridization and hormone analyses of anorexic and paired food-restricted rats revealed two distinct profiles. First, both groups had the characteristic gene expression and endocrine signatures usually associated with starvation: increased neuropeptide Y and decreased proopiomelanocortin and neurotensin mRNAs in the arcuate nucleus (ARH); increased circulating glucocorticoid but reduced leptin and insulin. Dehydrated animals are strongly anorexic despite these attributes, showing that the output of leptin- and insulin-sensitive ARH neurons that ordinarily stimulate eating must be inhibited. The second pattern occurred only in anorexic animals and had two components: (1) reduced corticotropin-releasing hormone (CRH) mRNA in the neuroendocrine paraventricular nucleus (PVH) and (2) increased CRH and neurotensin mRNAs in the lateral hypothalamic (LHA) and retrochiasmatic areas. However, neither corticosterone nor suppressed PVH CRH gene expression is required for anorexia after dehydration because PVH CRH mRNA in dehydrated adrenalectomized animals is unchanged from euhydrated adrenalectomized controls. We also showed that LHA CRH mRNA was strongly correlated with the intensity of anorexia, increased LHA CRH gene expression preceded the onset of anorexia, and dehydrated adrenalectomized animals (which also develop anorexia) had elevated LHA CRH gene expression with a distribution pattern similar to intact animals. Finally, we identified specific efferents from the CRH-containing region of the LHA to the PVH, thereby providing a neuroanatomical framework for the integration by the PVH of neuropeptidergic signals from the ARH and the LHA. Together, these observations suggest that CRH and neurotensin neurons in the LHA constitute a novel anatomical substrate for their well known anorexic effects.

Fig. 1.

Changes in body weight and food intake during dehydration or food restriction. Mean (±SEM) daily changes in body weight (A) and nocturnal food intake (B) of intact-EU (solid circles,solid lines), ADX-EU (solid squares,solid lines), intact-DE (open circles,dashed lines), ADX-DE (open squares,dashed lines), or intact-food restricted (open circles, solid lines) rats. Dehydration was started on day 0 by replacing drinking water with 2.5% saline.

Fig. 2.

Dehydration and food-restriction both affect arcuate nucleus gene expression. Mean (±SEM) levels of NPY (A), POMC (B), and NT/N (C) mRNA hybridization measured on treatment day 5 in the arcuate nucleus of euhydrated (EU), dehydrated (DE), and paired-food restricted (FR) animals. *p < 0.01; **p < 0.001; ***p < 0.0002 versus EU animals.

Fig. 3.

CRH and neurotensin mRNA hybridization in the lateral hypothalamic area. Dark-field photomicrographs of CRH (A, C, E) and NT/N (B, D, F) mRNA hybridization in the lateral hypothalamic area (LHA) and arcuate nucleus (ARH) at approximately level 27/28 of Swanson (1992) of animals in the three treatment groups killed on treatment day 5. A, B, Serial sections from a control euhydrated animal; C, D, serial sections from a dehydrated animal; E, F, serial sections from a food-restricted animal. 3V, Third ventricle;ME, median eminence.

Fig. 4.

Dehydration and food restriction differentially affect gene expression in the lateral hypothalamic area. Mean (±SEM) levels of CRH (A, C) and NT/N (B, D) mRNA hybridization measured on treatment day 5 in the lateral hypothalamic area of euhydrated (EU), dehydrated (DE), and paired-food restricted (FR) animals. A, B, Total pixel area of the specific hybridization signal; C, D, the mean gray level of the specifically labeled area. See Materials and Methods for further details of the image analysis. *p < 0.0025; **p < 0.0005; ***p < 0.0001 versus EU animals.

Fig. 5.

Dehydration and food restriction differentially affect gene expression in the retrochiasmatic area. Mean (±SEM) levels of CRH (A) and NT/N (B) mRNA hybridization measured on treatment day 5 in the retrochiasmatic area of euhydrated (EU), dehydrated (DE), and paired-food restricted (FR) animals. See Materials and Methods for further details of the image analysis. *p < 0.005; **p < 0.0001 versus EU. ns, Not significant.

Fig. 6.

CRH mRNA in the LHA is correlated with the degree of anorexia, and CRH mRNA levels in the LHA return to control values 24 hr after anorexia is reversed. A, Correlation between food intake (expressed as the percentage reduction relative to that measured during the dark period immediately preceding treatment day 0) and total pixel area of the specific CRH mRNA hybridization signal measured on treatment day 5 in individual euhydrated and dehydrated rats. See Results for levels of significance. B, Mean (±SEM) CRH mRNA hybridization signal in the lateral hypothalamic area of animals before (0 hr), 5 hr, or 24 hr after the return of drinking water. Values were measured as total pixel area and expressed as a percentage relative to those in euhydrated (EU) animals on treatment day 5. ***p < 0.00025 versus EU animals. ns, Not significant.

Fig. 7.

Dehydration and food-restriction effects on CRH gene expression in the paraventricular nucleus and lateral hypothalamic area of adrenalectomized rats. Mean (±SEM) levels of CRH mRNA hybridization measured on treatment day 5 in the dorsal aspect of the medial parvicellular part of the hypothalamic paraventricular nucleus from euhydrated (EU), dehydrated (DE), paired-food restricted (FR) intact animals (A), or from EU or DE adrenalectomized (ADX) (B) animals.C, Mean (±SEM) levels of CRH mRNA hybridization in the lateral hypothalamic area of EU or DE-ADX animals. See Materials and Methods for further details of the image analysis. ***p < 0.0005 versus EU animals.ns, Not significant.

Fig. 8.

CRH and neurotensin mRNA hybridization in the paraventricular nucleus retrochiasmatic area. Dark-field photomicrographs of CRH (A, C, E) and NT/N (B, D, F) mRNA hybridization in the paraventricular nucleus (PVH) and retrochiasmatic area (RCH) at approximately level 25/26 of Swanson (1992) of animals in the three treatment groups killed on treatment day 5. A, B, Serial sections from a control euhydrated animal; C, D, sections from a dehydrated animal;E, F, sections from a food-restricted animal.3V, Third ventricle; PVHmpd, dorsal aspect of the medial parvicellular part of the PVH.

Fig. 9.

Maps of PHAL-labeled projections from the lateral hypothalamic area to the paraventricular nucleus. PHAL-immunoreactive processes from four representative injections plotted onto maps at levels 26 and 27 of Swanson (1992). PHAL immunoreactively labeled neuronal cell bodies at injection sites (black dots) are also shown where visible. Maps are arranged depending on the rostrocaudal position of the injection site, with the most rostral case (LP 15) first. Cases LP 15, LP 18, and LP 3 (located at the same dorsoventral position as the LHA-crh, but further caudal at level 29) were all control injections placed outside the region of the LHA-crh; case LP 6 was in the center of the LHA-crh [Level 27A (Kelly and Watts, 1998)]. Note the large number of PHAL-labeled fibers in the parvicellular regions of the PVH in case LP 6, but not in any of the control injections. See Kelly and Watts (1998) for complete descriptions of the injection sites. 3V, Third ventricle; AHN, anterior hypothalamic nucleus;ARH, arcuate nucleus; dp, dorsal parvicellular part of the PVH; fx, fornix;lp, lateral parvicellular part of the PVH;mpd, dorsal aspect of the medial parvicellular part of the PVH; mpv, ventral aspect of the medial parvicellular part of the PVH; pv, periventricular part of the PVH;RE, nucleus reuniens; SBPV, subparaventricular zone; VMH, ventromedial nucleus;ZI, zona incerta.

Fig. 10.

PHAL-labeled projections from the lateral hypothalamic area to the paraventricular nucleus. Dark-field photomicrographs of PHAL-immunoreactive processes in the region of the hypothalamic paraventricular nucleus (PVH) at approximately levels 26 and 27 of Swanson (1992). Injection LP 6 (A) was centered in the LHA-crh (level 27A/28); case LP 18 (B) was a control injection centered in the region immediately medial and ventral to the fornix at level 26/27 ofSwanson (1992). Note the large number of PHAL-labeled fibers in the parvicellular regions of the PVH in the animal injected in the LHA-crh (A) but not the control injection (B). See Kelly and Watts (1998) for further detailed descriptions of the injection sites. 3V, Third ventricle; AHN, anterior hypothalamic nucleus;dp, dorsal parvicellular part of the PVH;fx, fornix; lp, lateral parvicellular part of the PVH; mpd, dorsal aspect of the medial parvicellular part of the PVH; mpv, ventral aspect of the medial parvicellular part of the PVH; SBPV, subparaventricular zone.

Fig. 11.

Summary of changes in gene expression. Schematic diagram summarizing those patterns of gene expression that are exclusive to dehydrated (anorexic) animals (black bars) and those that are common to both dehydrated and food-restricted (hungry) animals (gray bars). The height of each symbol represents relative levels of gene expression.