The Journal of Neuroscience, January 1, 1998, 18(1):559–572 Leptin Receptor Immunoreactivity in Chemically Defined Target Neurons of the Hypothalamus Marie-Louise Håkansson,1 Hilary Brown,1 Nico Ghilardi,2 Radek C. Skoda,2 and Bjo¨ rn Meister1 1Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden, and 2Department of Pharmacology, Biozentrum, University of Basel, 4056 Basel, Switzerland The adipose tissue-derived hormone leptin regulates body showed LR-LI in the periphery of individual cells. In magnocel- weight homeostasis by decreasing food intake and increasing lular neurons of the SON and PVN, LR-LI was demonstrated in energy expenditure. The weight-reducing action of leptin is vasopressin- and oxytocin-containing neurons. In parvocellular thought to be mediated primarily by signal transduction through neurons of the PVN, LR-LI was demonstrated in many the leptin receptor (LR) in the hypothalamus. We have used corticotropin-releasing hormone-containing neurons. LR-IR immunohistochemistry to localize LR-immunoreactive (LR-IR) neurons were mainly seen in the ventromedial aspect of the cells in the rat brain using an antiserum against a portion of the arcuate nucleus, where LR-LI co-localized with neuropeptide Y. intracellular domain of LR that is common to all LR isoforms. In the ventrolateral part of the arcuate nucleus, LR-LI was The antiserum recognized the short and long isoforms of LR in present in many large adrenocorticotropic hormone-IR transfected hematopoietic BaF3 cells. To examine the chemical proopiomelanocortin-containing neurons and in a few galanin-, nature of target cells for leptin, direct double-labeling immuno- neurotensin-, and growth hormone-releasing hormone- fluorescence histochemistry was applied. The results show containing neurons. In the dorsomedial arcuate nucleus, few extensive distribution of LR-like immunoreactivity (LR-LI) in the tyrosine hydroxylase (dopamine)-containing neurons were seen brain with positively stained cells present, e.g., in the choroid to have LR-LI. Melanin-concentrating hormone-containing neu- plexus, cerebral cortex, hippocampus, thalamus, and hypothal- rons in the lateral hypothalamus had LR-LI. Based on the amus. In the hypothalamus, strongly LR-IR neurons were immunohistochemical results, possible interactions of leptin present in the supraoptic nucleus (SON) and paraventricular with brain mechanisms are discussed. nucleus (PVN), periventricular nucleus, arcuate nucleus, and lateral hypothalamus. Weaker LR-IR neurons were also dem- Key words: arcuate nucleus; CRH; dopamine; galanin; onstrated in the lateral and medial preoptic nuclei, suprachias- GHRH; immunohistochemistry; lateral hypothalamic area; matic nucleus, ventromedial and dorsomedial nuclei, and tube- LHRH; MCH; neurotensin; NPY; oxytocin; paraventricular nu- romammillary nucleus. Confocal laser scanning microscopy cleus; POMC; somatostatin; supraoptic nucleus; vasopressin The recently cloned obese (ob) gene encodes a circulating signal- Results from parabiosis studies using ob/ob and db/db mice ing factor called leptin produced by adipocytes that regulates (Coleman, 1973), together with the demonstration of failure of body weight homeostasis (Zhang et al., 1994b). The crystal struc- db/db mice to respond to injection of leptin and the high levels of ture of leptin has revealed a four-helix bundle similar to that of leptin in adipose tissue combined with high serum levels of leptin the long-chain helical cytokine family (Zhang et al., 1997). Ho- in db/db mice (Campfield et al., 1995; Halaas et al., 1995), mozygous ob/ob mice that have a mutation in the ob gene exhibit indicate that db/db mice may be defective in reception of the ob obesity, diabetes, and infertility. Exogenous administration of gene product signal. Recently, the gene for mouse leptin recep- recombinant leptin corrects the defects in ob/ob mice and induces tors (LR) was cloned and shown to be mutated in db/db mice weight reduction in mice with diet-induced obesity as well as in (Tartaglia et al., 1995; Chen et al., 1996; Lee et al., 1996). LR is normal mice by decreasing food intake and increasing energy a single transmembrane-spanning receptor and a member of the expenditure (Campfield et al., 1995; Halaas et al., 1995; Pelley- cytokine receptor superfamily that includes the gp130 signal- mounter et al., 1995; Stephens et al., 1995). Injection of leptin transducing component of the receptors for interleukin-6, gran- directly into the third ventricle reduces body weight at low doses ulocyte colony-stimulating factor, and leukemia-inhibitory factor (Campfield et al., 1995), suggesting that leptin acts primarily via (Tartaglia et al., 1995). Four different LR (originally designated the CNS. Ob-Ra–d) variants arise from the db gene via alternate splicing Mutations in the mouse diabetes (db) gene results in a pheno- (Lee et al., 1996). A fifth isoform represents a soluble form of the type indistinguishable from ob/ob mice (Hummel et al., 1966). receptor (Lee et al., 1996). One of the splice variants (LR isoform b), which has a long cytoplasmic domain of 302 amino acids, is Received June 6, 1997; revised Oct. 27, 1997; accepted Nov. 3, 1997. expressed primarily in the hypothalamus and is spliced abnor- This research was supported by Swedish Medical Research Council Grant 04X- 10358, grants from Stiftelsen Tornspiran, Åhle´n-Stiftelsen, Magnus Bergvalls Stif- mally in C57BL/Ks db/db mice (Chen et al., 1996; Ghilardi et al., telse, Åke Wibergs Stiftelse, Stiftelsen Sigurd, and Elsa Goljes Minne, and funds 1996; Lee et al., 1996). The C57BL/Ks db/db mutation generates from the Karolinska Institute. a new splice donor that interferes with the correct splicing of LR Correspondence should be addressed to Dr. Bjo¨rn Meister, Department of Neu- roscience, Karolinska Institute, S-171 77 Stockholm, Sweden. isoform b mRNA and is predicted to cause absence of the LR Copyright © 1997 Society for Neuroscience 0270-6474/97/180559-14$05.00/0 isoform b protein in db/db mice (Lee et al., 1996). The intracel- 560 J. Neurosci., January 1, 1998, 18(1):559–572 Håkansson et al. • Leptin Receptor in Hypothalamic Neurons Figure 1. Immunofluorescence photomicrographs of hematopoietic BaF3 cells transfected with short (A) or long (B) LR isoforms or control BaF3 cells (C). There is strong LR-LI in transfected cells (A, B) but absence of LR-LI in control cells (C). Weak fluorescence in control cells is represented by autofluorescence (C). Scale bar, 50 mm. lular domain of short LR isoform a, which is present in db/db chemical nature of LR-immunoreactive (LR-IR) hypothalamic mice, contains the box 1 motif but lacks the box 2 motif, required neurons with emphasis on chemical messengers that have been for activation of cytoplasmic tyrosine kinases of the janus kinase implicated previously in the regulation of ingestive behavior. (JAK) family (Chen et al., 1996; Lee et al., 1996). JAKs phos- phorylate cytoplasmic target proteins such as signal transducers MATERIALS AND METHODS and activators of transcription (STAT). Phosphorylation of STAT Male Sprague Dawley rats (150–200 g body weight;B&KUniversal, proteins induces dimerization and translocation into the nucleus Stockholm, Sweden) were anesthetized with sodium pentobarbital (Me- and results in specific activation of gene transcription (Ihle, 1996). bumal; 40 mg/kg, i.p.) and perfused via the ascending aorta with 50 ml 21 The short form of LR present in db/db mice is therefore unable to of Ca -free Tyrode’s solution (37°C) followed by 50 ml of formalin– picric acid fixative (37°C) (4% paraformaldehyde and 0.4% picric acid in activate the JAK–STAT pathway (Baumann et al., 1996; Ghilardi 0.16 M phosphate buffer, pH 6.9). Perfusions were continued thereafter et al., 1996; Ghilardi and Skoda, 1997). for 6 min with ice-cold fixative as above. Some rats received an injection The weight-reducing effects of leptin are likely to be mediated of colchicine (120 mgin20ml of 0.9% NaCl; Sigma, St. Louis, MO) into by JAK–STAT signal transduction via LR in the hypothalamus. the lateral ventricle 24 hr before sacrifice. Colchicine is known to arrest Specific 125I-leptin binding sites have been demonstrated in the axonal transport, thereby increasing levels of transmitters, enzymes, and peptides and proteins in the cell soma. hypothalamic arcuate nucleus–median eminence complex and in Tissues were dissected out rapidly, post-fixed in the same fixative for 90 the choroid plexus (Banks et al., 1996). LR mRNA is present in min, and rinsed for at least 24 hr in 0.1 M phosphate buffer, pH 7.4, several hypothalamic nuclei, including the arcuate, paraventricu- containing 10% sucrose, 0.02% Bacitracin (Sigma), and 0.01% sodium lar, supraoptic, ventromedial, and dorsomedial nuclei and lateral azide (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer, pH 7.4. Sections were cut (14 mm) in a cryostat and incubated at 4°C overnight hypothalamic area, as revealed by in situ hybridization (Håkans- with goat polyclonal antiserum to LR (antiserum sc-1834, lot G116, son et al., 1996; Huang et al., 1996; Mercer et al., 1996a,b; Fei et diluted 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Antiserum al., 1997). Because chemical lesioning of the hypothalamic arcu- sc-1834 has been raised to an epitope corresponding to amino acids ate nucleus results in severe obesity (see Meister, 1991), this 877–894 (KNCSWAQGLNFQKRTDTL) mapping at the C terminus of nucleus has been assumed to play a major role for the action of the short form of mouse LR. The mouse immunogen peptide sequence shows 94% homology with the rat sequence (one amino acid mismatch). leptin on the CNS. Within the arcuate nucleus, LR mRNA- BaF3 cells stably transfected with LR expression constructs were expressing neurons are primarily present in the ventromedial grown as described previously (Ghilardi and Skoda, 1997), fixed in 4% part, and they have been shown to contain neuropeptide Y (NPY) paraformaldehyde and 0.4% picric acid in 0.16 M phosphate buffer, pH mRNA (Håkansson et al., 1996; Huang et al., 1996; Schwartz et 6.9, and incubated with primary antiserum to LR.