The Journal of Neuroscience, July 15, 1999, 19(14):6111–6121 Distinct Patterns of Neuropeptide Gene Expression in the Lateral Hypothalamic Area and Arcuate Nucleus Are Associated with Dehydration-Induced Anorexia Alan G. Watts, Graciela Sanchez-Watts, and Andrea B. Kelly The Neuroscience Program and the Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-2520 We have investigated the hormonal and hypothalamic neu- required for anorexia after dehydration because PVH CRH ropeptidergic substrates of dehydration-associated anorexia. mRNA in dehydrated adrenalectomized animals is unchanged In situ hybridization and hormone analyses of anorexic and from euhydrated adrenalectomized controls. We also showed paired food-restricted rats revealed two distinct profiles. First, that LHA CRH mRNA was strongly correlated with the intensity both groups had the characteristic gene expression and endo- of anorexia, increased LHA CRH gene expression preceded the crine signatures usually associated with starvation: increased onset of anorexia, and dehydrated adrenalectomized animals neuropeptide Y and decreased proopiomelanocortin and neu- (which also develop anorexia) had elevated LHA CRH gene rotensin mRNAs in the arcuate nucleus (ARH); increased circu- expression with a distribution pattern similar to intact animals. lating glucocorticoid but reduced leptin and insulin. Dehydrated Finally, we identified specific efferents from the CRH-containing animals are strongly anorexic despite these attributes, showing region of the LHA to the PVH, thereby providing a neuroana- that the output of leptin- and insulin-sensitive ARH neurons that tomical framework for the integration by the PVH of neuropep- ordinarily stimulate eating must be inhibited. The second pat- tidergic signals from the ARH and the LHA. Together, these tern occurred only in anorexic animals and had two compo- observations suggest that CRH and neurotensin neurons in the nents: (1) reduced corticotropin-releasing hormone (CRH) LHA constitute a novel anatomical substrate for their well mRNA in the neuroendocrine paraventricular nucleus (PVH) and known anorexic effects. (2) increased CRH and neurotensin mRNAs in the lateral hypo- Key words: feeding behavior; anorexia; neuropeptides; thalamic (LHA) and retrochiasmatic areas. However, neither corticotropin-releasing hormone; arcuate nucleus; lateral hypo- corticosterone nor suppressed PVH CRH gene expression is thalamus; paraventricular nucleus; leptin; glucocorticoid Feeding in rats is regulated, like other motivated behaviors, by medial (DMH) nuclei, and retrochiasmatic area (RCH), with interactive inhibitory and stimulatory networks (Stricker, 1990). further contributions from the lateral hypothalamic area (LHA) Rat ingestive behaviors consistently and predictably occur during and paraventricular nucleus (PVH) (Elmquist et al., 1998a,c). In the dark period with unlimited food and water. Eating and drink- this context, neuropeptide Y (NPY) stimulates food intake when ing are alternately initiated and terminated by integrative mech- injected into the brain, whereas altered NPY and proopiomela- anisms involving peptidergic neural circuits, visceral sensory nocortin (POMC) mRNA levels in the ARH occur during depri- feedback, and circulating nutrient and hormone levels (Woods et vation (for review, see Woods et al., 1998). Regarding behavioral al., 1998). Dysfunction in this physiological network disturbs inhibition, deprivation-induced eating is suppressed by intraven- eating behaviors with serious consequences. For example, disrup- tricular injection of corticotropin-releasing hormone (CRH) or tion of leptin and associated neuropeptide signaling results in neurotensin (Morley, 1987; Woods et al., 1998). Agonists of the obesity (Elmquist et al., 1999), whereas an inability to restrain melanocortin (MC) 3/4 receptor reduce food intake (Fan et al., inhibitory mechanisms leads to anorexia, with consequent nega- 1997; Thiele et al., 1998), whereas MC3/4R antagonist adminis- tive energy balance and body wasting (Schwartz et al., 1995). tration or MC4R knockout mice provoke hyperphagia (Hruby et Several neuropeptides are strongly implicated in activating al., 1995; Huszar et al., 1997). Although the PVH is implicated in eating, and the detailed organization of their cognate neural mediating some of these anorectic effects (Heinrichs et al., 1993), circuits is being clarified. For example, to regulate eating, leptin the details of the circuits responsible for generating anorexia targets neuropeptide-containing neurons in a primary network remain less clearly defined than those responsible for stimulating consisting of the arcuate (ARH), ventromedial (VMH), dorso- eating. To examine this question we have used in situ hybridization to Received Feb. 25, 1999; revised April 27, 1999; accepted April 27, 1999. compare the dynamics of four key neuropeptide mRNAs (CRH, This study was supported by Grant NS 29728 (A.G.W.) from the National Institute neurotensin, NPY, POMC) in the ARH, PVH, LHA, and RCH of Neurological Diseases and Stroke, National Institutes of Health. We thank Drs. P. during dehydration-associated anorexia and pair-fed food depri- Dobner (NT/N), D. Larhammar (NPY), J. Majzoub (CRH), and J. Roberts (POMC) for gifts of cDNAs for probe preparation. We also thank Cynthia Kay- vation. Although thirst is the obvious behavioral correlate of Nishiyama and Dr. JunQi Zheng for technical assistance. dehydration, when prolonged it also generates an intense an- Correspondence should be addressed to Dr. Alan G. Watts, Program in Neural, Informational and Behavioral Sciences, Hedco Neuroscience Building, MC 2520, orexia (Watts, 1999). Because digestion requires significant water University of Southern California, Los Angeles, CA 90089-2520. investment, some of which is subsequently lost by colonic excre- Copyright © 1999 Society for Neuroscience 0270-6474/99/196111-11$05.00/0 tion, dehydration-associated anorexia is a physiologically gener- 6112 J. Neurosci., July 15, 1999, 19(14):6111–6121 Watts et al. • Neuropeptide Gene Expression Associated with Dehydration-Induced Anorexia ated behavioral reflex critical for protecting the fluid compart- Radioimmunoassays. Concentrations of serum leptin, plasma ACTH, ment. We have recently shown that when animals are dehydrated insulin, and corticosterone were all measured by double-antibody radio- immunoassays (RIAs) using commercially available 125I-labeled antigen by replacing drinking water with 2.5% saline, anorexia develops kits. Plasma insulin and leptin were measured using kits purchased from relatively slowly but is rapidly reversed once access to water is Linco Research (St. Charles, MI), with the lower sensitivity limits being restored, a behavioral structure consistent with the presence of 100 pg/ml and 0.5 ng/ml, respectively, whereas the intra-assay coefficients simultaneous activated sets of neural circuits that independently of variation were ,9.4% and ,6.8%, respectively. Plasma ACTH con- stimulate or suppress eating (Watts, 1999). We hypothesize that centrations were determined using an RIA kit purchased from ICN Biochemicals (Costa Mesa, CA). The lower sensitivity limit was 30 pg/ml, modified patterns of appetite-regulating neuropeptide gene ex- and the intra-assay coefficient of variation was ,10.5%. Plasma cortico- pression apparent during dehydration but not food restriction are sterone concentrations were determined using an RIA kit purchased associated with anorexia, whereas changes seen during both from ICN Biochemicals. The lower sensitivity limit was 12 ng/ml, and the dehydration and food restriction are likely associated with hunger intra-assay coefficient of variation was ,8.5%. For all hormones, all and the ensuing bout of eating that occurs once water is returned samples were run in single assays. In situ hybridization. Eight series of one in eight 15-mm-thick frontal (Watts, 1999). Because these data identified the LHA as a sections were cut through the hypothalamus and saved in ice-cold potas- potential mediator of anorexia, we also used injections of Phaseo- sium PBS (KPBS) containing 0.25% paraformaldehyde, pH 7.4. Sections lus vulgaris leucoagglutinin (PHA-L) to determine whether the were mounted the same day onto poly-L-lysine-coated gelatin-subbed region containing CRH neurons provides afferent projections to slides, vacuum-desiccated overnight, post-fixed in KPBS/4% paraformal- the PVH. dehyde for 1 hr at room temperature, rinsed five times for 5 min in clean KPBS, air-dried, and then stored at 270°C in air-tight containers con- Some of these data have been reported previously in abstract taining silica-gel desiccant for hybridization at a later date. Serial sec- form (Watts and Sanchez-Watts, 1998; Watts et al., 1998) tions were saved for thionin staining. Sections were hybridized using 35S-labeled cRNA probes for ppCRH MATERIALS AND METHODS [a 700 bp fragment encoding part of exon 1 and all of exon 2 (Frim et al., Animals. Adult male Sprague Dawley rats [250–260 gm body weight 1990)], ppNT/N [a 336 bp fragment encoding part of exon 4 (Kislauskis (BW) at the start of the experiment] were individually caged and main- and Dobner, 1990)], POMC [a 538 bp fragment encoding part of exon 3 tained on a 12 hr light/dark schedule with lights on at 6 A.M. with (Drouin and Goodman, 1980)], or NPY [a 287 bp fragment encoding part unlimited access to food (Teklad rodent diet 8604) and water. Food of exon 2 (Larhammar et al., 1987)] mRNAs synthesized