Distribution of serotonin 5-hydroxytriptamine 1B (5-HT1B) receptors in the normal rat hypothalamus

Neuroscience Letters 328 (2002) 155–159 www.elsevier.com/locate/neulet

Distribution of serotonin 5-hydroxytriptamine 1B (5-HT1B) receptors in the normal rat hypothalamus I.G. Makarenko a,b, M.M. Meguid a,*, M.V. Ugrumov b a

Department of Surgery, SUNY University Hospital, Neuroscience Program, Surgical Metabolism and Nutrition Laboratory, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA b Laboratory of Hormonal Regulations, Institute of Developmental Biology, Russian Academy of Sciences, 117808 Moscow, Russia Received 8 January 2002; received in revised form 4 March 2002; accepted 5 April 2002

Abstract This is the study first attempting to evaluate distribution of neurons expressing serotonin 5-hydroxytriptamine 1B (5HT1B) receptors in hypothalamus by using immunocytochemistry. The 5-HT1B-immunoreactive neurons were widely distributed in hypothalamus. Accumulations of 5-HT1B neurons occurred in magnocellular nuclei, supraoptic nucleus, paraventricular nucleus (dorsolateral part) and accessory perifornical, circular and retrochiasmatic nuclei. Magnocellular neurons manifested an intense immunostaining suggesting a high level of 5-HT1B receptors. Large and middle-sized neurons with different 5-HT1B staining patterns were scattered throughout lateral hypothalamus, periventricular nucleus and lateral preoptic area. Immunofluorescent double-labeling revealed a great overlapping of the distribution 5-HT1B neurons and dense network of neuropeptide Y-immunoreactive fibers in paraventricular, supraoptic and arcuate nuclei. The potential functional significance of 5-HT1B receptors in the 5-HT control of endocrine functions and feeding are discussed. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: 5-hydroxytryptamine; Brain; Immunohistohemistry; Double-labeling; Magnocellular neurons; Feeding

It has been repeatedly demonstrated that the hypothalamus is intensely innervated by 5-hydroxytriptamine (5-HT) fibers arising from the raphe nucleus [17]. 5-HT afferent fibers innervate synaptically the hypothalamic neurosecretory neurons controlling their functional activity, such as feeding, water-mineral metabolism, thermoregulation, circadian rhythms and reproductive behavior [19]. The discovery of different types of 5-HT receptors over the last decade served to elucidate the uniqueness of the 5-HT action on the target-neurons in different brain regions including the hypothalamus [19]. We focused special attention on 5-HT1B receptors because they contribute to autoregulation of 5-HT neurons, as well as to the control of the targetneurons, which are responsible for the regulation of feeding [14]. 5-HT decreases food intake acting via 5-HT1B receptors [9]. Also, the 5-HT1B receptors are involved in the development of feeding disorders manifested most often in obesity or anorexia [6,9,10,14]. Although numerous physiological and pharmacological studies have evaluated the 5-HT regulatory effects mediated via 5-HT1B receptors, no immunocytochem* Corresponding author. Tel.: 11-315-464-6277; fax: 11-315464-6237. E-mail address: [email protected] (M.M. Meguid).

ical data are currently available concerning their distribution in the hypothalamus (for ref. see refs [1,19]). Therefore, this study aims at mapping 5-HT1B receptors in the hypothalamus of intact adult rats using immunocytochemistry. Ten male Fischer-344 rats (220–250 g) were studied. They were housed in a controlled environment of 26 ^ 18C, 45% humidity, on a 12-h illumination cycle. Food and water were provided ad libitum. Animal experiments were approved by the Committee for the Humane Use of Animals, at SUNY Upstate Medical University, Syracuse, NY. Rats were anaesthetized with a mixture of ketamine, xylazine and acepromazine (150:30:5 mg/ml) via intermuscular injection (0.7 ml/kg body weight) and perfused through the heart first with saline and then with 4% paraformaldehyde (PAF) in 0.1 M phosphate buffer. Brains were removed from their skull, and the forebrains were dissected and post-fixed by immersion in 4% PAF for 4 h at 48C. Then, tissue blocks were cryoprotected in 30% sucrose for 48 h, embedded in Tissue–Tek, frozen in dry ice and stored at 2708C. Coronal 20-mm-thick sections of the forebrain including hypothalamus, were prepared using a cryocut. Every fourth section was collected in 0.02 M phosphate buffer (pH-7.4)

0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 34 5- 2

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containing 0.9% NaCl (PBS). The peroxidase anti-peroxidase (PAP) method was used to localize 5-HT1B receptors on free-floating sections with primary polyclonal antibodies AB 5410 raised in guinea pig against 15 amino acid sequence from the third intracellular loop of the 5-HT1Breceptor (Chemicon, Temecula, CA). All solutions were based on PBS containing 0.1% Triton X-100 (Fisher Biotech, Fair Lawn, NJ). Sections were incubated successively with 0.3% H2O2 for 30 min at room temperature (RT), 3% bovine serum albumin (BSA, Sigma, St. Louis, MO) for 1 h RT and primary antibodies (1:2000) with 1% BSA for 48–72 h at 48C. Then, the sections were incubated with a second polyclonal goat anti-guinea pig antibody (Jackson Imm. Res. Lab., West Grove, PA; 1:100) for 2 h at RT and with guinea pig PAP complex (Jackson Imm. Res. Lab., West Grove, PA; 1:600) for 1–2 h at RT. Peroxidase of PAP complex was visualized in Tris-buffered 3,3 0 -diaminobenzidine tetrahydrochloride-H2O2 solution. Finally, sections were mounted on gelatin-coated slides, air-dried, dehydrated in ethanols, xylene, embedded in Mount-Quick (Res. Prod. Int. Corp., Daido Sangyo, IL), and coverslipped. Slides were examined using a conventional light microscope Micromaster (Fisher Scientific, Agawam, MA), and digital images made on Nikon CF160 microscope. The protein database search revealed that there is no crossreactivity between the immunogen sequences of the original peptide for these antibodies and any other proteins. Using the information about immunogen sequence of the protein which was used for developing AB 5410 (Chemicon, Temecula, CA) we compare it with that used Langlois et al. (J. Neurochem. 65 (1995) 2671–2681) and found, that it corresponded to the same 15 amino acid sequence of Langlois’s 25 amino acid sequence from the intracellular loop of the 5-HT1B receptor protein molecule. From this we deduce and conclude that we are using a specific antibody to 5-HT1B receptor protein and hence we are visualizing it on the rat brain sections. The specificity of immunostaining was controlled by omission of primary or secondary antibodies. No immunoreactivity was observed in the control. Moreover, cortical and hippocampal regions from the same levels with the hypothalamus were used as the positive control of the specificity of immunostaining as 5-HT1B messenger RNA (mRNA) has been detected earlier in these brain regions in rats with in situ hybridization [2,3]. In our material neurons with specific granular reaction in the bodies and proximal dendrites were found in the IV–V layers of the anterior and posterior limbic, motor and somatosensory cortex; pyramidal and granular layers of the CA1 hippocampal area. Hypothalamic sections (three rats) were processed for immunofluorescent double labeling for 5-HT1B receptors and neuropeptide Y (NPY). They were incubated in a mixture of primary polyclonal antibodies consisting of guinea-pig anti-5-HT1B receptor (Chemicon, Temecula, CA) at a dilution 1:1000 and rabbit antibodies to NPY

RGG-7180 (Peninsula Lab., San Carlos, CA) at a dilution 1:1500 for 3 days at 48C. Then, after washing in PBS they were successively incubated with Anti-Rabbit IgG TRITC Conjugate No. T6778 (Sigma, Saint Louis, MO) and AntiGuinea pig IgG FITC Conjugate No. F7762 (Sigma, Saint Louis, MO), both at a dilution 1:300 for 2 h at RT. After the final washing, sections were mounted on slides in Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). Slides were examined in Nikon CF160 microscope, using a set of fluorescent filters for simultaneous visualization of rhodamin and fluorescein. Our data showed that 5-HT1B-immunoreactive (IR) neurons are widely distributed in the hypothalamus. Immunocytochemical reaction appear as granular deposits in the neurons and nerve fibers. The density of immunostaining varies significantly in different hypothalamic regions. The most striking accumulations of 5-HT1B-IR neurons were observed in the magnocellular nuclei, the supraoptic nucleus, the paraventricular nucleus (Figs. 1a,b). Some accessory nuclei such as the perifornical, circular and retrochiasmatic nuclei also contained 5-HT1B-IR neurons (Figs. 1c,d). The magnocellular neurons usually have intensive or medium immunostaining, but there are also some lightly labeled cells. The zonality is a characteristic of the distribution of 5-HT1B-IR neurons in the major magnocellular nuclei. As shown in Fig. 1a, they mainly occupy the ventral region of the supraoptic nucleus and the dorsolateral region of the paraventricular nucleus (Fig. 1b). Groups and single magnocellular 5-HT1B-IR neurons are attached to the arch-like blood vessels arising from the meninges at the level of the supraoptic nucleus and extend dorsomedially toward the paraventricular nucleus. Some vessels penetrated the optic chiasma. Besides the 5-HT1B-IR magnocellular neurons, the paraventricular nucleus contain 5-HT1B-IR parvocellular neurons scattered in its ventromedial and periventricular subdivisions. Most of the parvocellular neurons showed weak immunostaining and occasional ones dark labeling (Fig. 1b). In addition to magnocellular nuclei, neurons with different 5-HT1B staining patterns are scattered throughout the lateral hypothalamus (Fig. 1e), periventricular nucleus and lateral preoptic area. The neurons vary in the intensity of immunostaining. The heavily stained neurons are mainly located in the perifornical area and the dorsolateral hypothalamus, whereas the weakly stained neurons are confined to the ventrolateral hypothalamic area. A moderate number of 5-HT1B-IR neurons are detected in other hypothalamic regions, including the arcuate, the dorsomedial, the periventricular and the ventromedial nuclei. Rare weakly stained neurons were observed in the ventral part of the suprachiasmatic nucleus. Immunofluorescent labeling with 5-HT1B receptor antibodies confirmed the data obtained with PAP method concerning the distribution of 5-HT1B receptor expressing neurons in the hypothalamus. Double-labeling of 5-HT1B receptors and NPY revealed a great overlapping of their distribution.

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NPY was visualized in the fibers heavy innervating the supraoptic (Fig. 2), paraventricular, and arcuate nuclei. Neurons with bright 5-HT1B receptor labeling were tightly surrounded with NPY-IR fibers. Perifornical and lateral hypothalamus contain less dense network of NPY-IR fibers and some close appositions between fibers and receptor immunoreactive neurons were visible. This is the first study that we are aware of in which the

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neurons synthesizing 5-HT1B receptors in the hypothalamus are mapped using immunocytochemistry. In contrast to earlier immunocytochemical studies [13], we detected both 5-HT1B-IR neuronal cell bodies in addition to nerve fibers. Bonaventure et al.’s study [2] focused primarily on the distribution of the neurons expressing 5-HT1B receptors in different brain regions, in which the hypothalamus was only mentioned in passing. Their study, done in guinea pig,

Fig. 1. Distribution of the neurons with 5-HT1B receptor immunoreactivity in the supraoptic nucleus (a), magnocellular (PVm) and parvocellular (PVp) paraventricular nucleus (b); circular nucleus (c); retrochiasmatic (d); dorsolateral hypothalamus (e); and arcuate nucleus (f). ARC, arcuate nucleus; C, circular nucleus; F, fornix; LH, lateral hypothalamus; OC, optic chiasma; RC, retrochiasmatic nucleus; SO, supraoptic nucleus; and V, 3rd ventricle. Bar ¼ 100 mm.

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using an indirect approach to detect of 5-HT1B receptors via, in situ hybridization of 5-HT1B mRNA, or an autoradiographic detection of the radioligand recognizing both the 5-HT1B receptor and the 5-HT1D receptor. Despite our direct immunocytochemical approach, our control data in general are in agreement with the in situ hybridization data, which show a widespread distribution of 5-HT1B receptors in the brain with particularly high concentration in the cortex, hippocampus, and the hypothalamus. In this immunocytochemical study, the highest concentration of 5-HT1B-IR neurons was observed in the magnocellular nuclei [12]. The neurons of this location showed a particularly intense immunostaining suggesting a high level of the expression of 5-HT1B receptors. The magnocellular nuclei receive quite low innervation by 5-HT fibers [16]. This discrepancy between the high receptor expression and the low innervation by 5-HT fibers can be explained according to the literature analysis by Barnes and Sharp [1]. They stressed that 5-HT1B receptors may be located on non5-HT nerve terminals and may has a role not only as autoreceptors but heteroreceptors, thus controlling the transmitter release from the target neuron. According to our data, the 5-HT1B-IR neurons are concentrated in the ventral region of the supraoptic nucleus, which is occupied predominantly by vasopressinergic neurons [12]. The latter are innervated by 5-HT fibers [6]. Without doublelabeling, it is difficult to interpret whether or not oxitocinergic neurons also expressed 5-HT1B receptors, because other parts of the nucleus contain some immunoreactive neurons [15]. Physiological studies failed to show 5-HT influence on the secretory activity of oxytocinergic neurons in the supraoptic nucleus [18]. Interactions between serotonin and vasopressin in the anterior hypothalamus was revealed in physiological and pharmacological studies induces aggres-

sive behavior. This occurred, via V1A receptors, while 5-HT diminishes this effect acting via 5-HT1B receptors [9]. We demonstrated in this study that numerous large neurons immunostained intensely in the dorsolateral magnocellular subdivision of the paraventricular nucleus. These data point to a high level of the expression of 5-HT1B receptors. In contrast to the supraoptic nucleus, in the paraventricular nucleus oxytocinergic neurons receive strong 5-HT innervation, providing the stimulating control of the oxytocin secretion [18]. Ongoing studies should elucidate, whether this regulation is mediated via 5-HT1B receptors. From the physiological view point, the most intriguing observation in this study related to 5-HT1B-IR neurons in the arcuate nucleus. Indeed, the 5-HT1B receptors mediate the 5-HT inhibiting influence on the NPY secretion in the arcuate-paraventricular system [4,9], suggesting that 5-HT’s food intake inhibitory effects modulate NPY orexinogenic function. According to the earlier morphological studies, the 5-HT fibers innervate the NPY neurons at the level of cell bodies in the arcuate nucleus and distal axons in the paraventricular nucleus [8,16]. Bearing in mind that 5-HT is a potent anorexic factor [14] and NPY stimulates feeding [4], the interactions between both these systems contribute to response for the acute regulation of feeding and energy homeostasis [4] on a minute to minute basis. Our double labeling material revealed a great degree of overlapping of the distribution 5HT1B-IR neurons and NPY-IR fibers with high concentration in paraventricular, supraoptic and arcuate nuclei and thus provide additional evidence pointing to the hypotalamic nuclei which are involved in food intake regulation. In addition to our observations in the magnocellular nuclei, numerous 5-HT1B-IR neurons were observed in the lateral hypothalamus which is known as a site of interaction of different neuronal systems, involved in food intake regula-

Fig. 2. Immunofluorescent doublelabeling: 5-HT1B receptor immunoreactive neurons (green) in the supraoptic nucleus are surrounded with NPY-imunoreactive (yellow) fibers. Bar ¼ 100 mm.

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tion [7,20]. The receptors are expressed most probably in the neurons, which were earlier demonstrated to be involved in food intake and body weight regulation [5,11]. Noteworthy, these neurons are under the control not only by inhibitory 5HT effects but also by the inhibitory effects of cholecystokinin [21] in response to different nutrients. It should be determined in the future whether these neurons produce melaninconcentrating hormone or orexin, which contribute to the regulation of feeding in the lateral hypothalamus [5]. Thus, the neurons expressing 5-HT1B receptors are widely distributed throughout the hypothalamus with particularly high concentrations occurring in magnocellular nuclei and dorsolateral hypothalamus on both sites known for their contribution in the activity of regulation of food intake among other functions. This work was supported by DK/NCI 70239 awarded to M.M. Meguid and by Scientist Exchange Program of the Office of International Affairs, National Cancer Institute awarded to I.G. Makarenko (2001–2002). The authors thank Tomoko Tada for her technical assistance and George Reynolds and Rich Whelsky for their photographic skills. [1] Barnes, N.M. and Sharp, T., A review of central 5-HT receptors and their function, Neuropharmacology, 38 (1999) 1083–1152. [2] Bonaventure, P., Voorn, P., Luyten, W.H., Jurzak, M., Schotte, A. and Leysen, J.E., Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function, Neuroscience, 82 (1998) 469–484. [3] Bruinvels, A.T., Landwehrmeyer, B., Gustafson, E.L., Durkin, M.M., Mengod, G., Branchek, T.A., Hoyer, D. and Palacios, J.M., Localization of 5-HT1B, 5-HT1D alpha, 5HT1E and 5-HT1F receptor messenger RNA in rodent and primate brain, Neuropharmacology, 33 (1994) 367–386. [4] Dryden, S., Frankish, H.M., Wang, Q. and Williams, G., Increased feeding and neuropeptide Y (NPY) but not NPY mRNA levels in the hypothalamus of the rat following central administration of the serotonin synthesis inhibitor p-chlorophenylalanine, Brain Res., 724 (1996) 232–237. [5] Elias, C.F., Saper, C.F., Maratos-Flier, E., Tritos, N.A., Lee, C., Kelly, J., Tatro, J.B., Hoffman, G.E., Ollmann, M.M., Barsh, G.S., Sakurai, T., Yanagisawa, M. and Elmquist, J.K., Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area, J. Comp. Neurol., 402 (1998) 442–459. [6] Ferris, C.F., Melloni, R.H., Koppel, G., Perry, K.W., Fuller, R.W. and Delville, Y., Vasopressin/serotonin interactions in the anterior hypothalamus control aggressive behavior in golden hamsters, J. Neurosci., 11 (1997) 4331–4340.

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