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Co-localization of hypocretin-1 and hypocretin-2 in the cat hypothalamus and brainstem
Peptides 23 (2002) 1479–1483
Co-localization of hypocretin-1 and hypocretin-2 in the cat hypothalamus and brainstem Jian-Hua Zhang a,∗ , Sharon Sampogna a , Francisco R. Morales b , Michael H. Chase a a
Department of Physiology and the Brain Research Institute, UCLA School of Medicine, University of California, 53-231 CHS, Los Angeles, CA 90095, USA b Departamento de Fisiolog´ıa, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay Received 24 July 2001; accepted 28 February 2002
Abstract Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) are two recently discovered hypothalamic neuropeptides. In the present study, using double immunofluorescent techniques, the co-localization of hcrt-1 and hcrt-2 was examined in neuronal soma and fibers/terminals located, respectively, in the cat hypothalamus and brainstem. In the hypothalamus, all hcrt-1 positive neuronal soma also displayed hcrt-2 immunoreactivity. In the brainstem, both hcrt-1 and hcrt-2 antibodies labeled the same fibers/terminals, indicating that hcrt-1 and hcrt-2 co-localize not only in the neuronal soma (hypothalamus) but also in their fibers/terminals (brainstem). If both peptides are released following neuronal activity, then the distinct effects of these peptides in the brain are likely to depend on the types of postsynaptic receptors that are activated. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Hypocretin; Immunofluorescence; Hypothalamus; Brainstem; Cat
1. Introduction Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) (also known as orexin-A and orexin-B, respectively) are two recently discovered hypothalamic neuropeptides [9,10]. Both hypocretins are derived from the same 130 amino acid residue (rodent) or 131 amino acid residue (human) polypeptide (prepro-hypocretin) by proteolytic processing [3,9,10]. The hcrt-1 is a 33-residue peptide with two intramolecular disulfide bonds in the N-terminal region, whereas hcrt-2 is a linear 28-residue peptide [3,9,10]. Initial studies indicated that hypocretin is involved in the control of food intake [9,10]. However, recent experiments suggest that hypocretins may be involved in the regulation of a sleep and wakefulness [3,5,13,16]. For example, a recent study in our laboratory showed that hypocretinergic processes in the laterodorsal tegmental nucleus (LDT) of cat play an important role in the promotion of wakefulness and the suppression of active sleep [16]. Using in situ hybridization and immunohistochemical techniques, Peyron et al. [8]. studied the distribution of prepro-hypocretin mRNA and peptide in the central nervous system of the rat. They found that prepro-hypocretin-positive ∗
Corresponding author. Tel.: +1-310-825-3417; fax: +1-310-206-3499. E-mail address: [email protected] (J.-H. Zhang).
neurons are located exclusively in the hypothalamus, mostly in the perifornical nucleus and dorsal–lateral hypothalamic area. However, the fibers/terminals of labeled neurons spread widely throughout the brain, including the brainstem [8]. This distribution pattern of hcrts were confirmed later by Date et al. [2] who examined the distribution of hcrt-1 and hcrt-2 in the rat brain using specific antibodies for hcrt-1 and hcrt-2, respectively [2,5]. We observed a similar distribution of hypocretin immunoreactivity in the cat brain [17,18]. In addition, our results show both hcrt-1 and hcrt-2 are located in the same brain regions, suggesting the possibility of co-localization of these two peptides in the same neuron [17,18]. However, until the present investigation, there has been no evidence that hcrt-1 and hcrt-2 are co-expressed in the same neurons. Accordingly, in this study, we examined the possible co-localization of hcrt-1 and hcrt-2 in the neuronal soma and fibers/terminals in the cat hypothalamus and brainstem, respectively.
2. Materials and methods 2.1. Animal and tissue preparation Four adult cats (2–3 kg) were employed in the present study. These animals were obtained from and determined
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to be in good health by the UCLA Division of Laboratory Animal Medicine. Animal treatment and handling in these experiments conformed with the policy of the American Physiological Society. The cats were deeply anesthetized with pentobarbital sodium (35 mg/kg, i.v.) and perfused transcardially with 1 l of ice-cold saline (containing 1000 units of heparin) followed by 2.5 l of a fixative containing 4% paraformaldehyde, 15% saturated picric acid and 0.25% glutaraldehyde in 0.1 M phosphate buffer (PBS) (pH 7.4). The whole brain was then removed and postfixed overnight in fresh fixative at 4 ◦ C. Next, the tissue was immersed overnight in 20% sucrose (w/v) in 0.1 M PBS at 4 ◦ C. After freezing with dry ice, it was cut into 15 m coronal sections with a Reichert-Jung cryostat. All sections were collected and stored in a solution of 0.1 M PBS containing 0.3% Triton X-100 and 0.1% sodium azide at 4 ◦ C for later use. 2.2. Double immunofluorescence Immunohistochemical staining was similar to previously published procedures [17]. Briefly, free-floating sections were rinsed several times in ice-cold PBST (0.1 M PBS with 0.3% Triton X-100); they were then incubated simultaneously with goat anti-hcrt-1 (Diagnostic Systems Laboratories Inc., Texas, diluted 1:1500) and rabbit anti-hcrt-2 (Phoenix Pharmaceuticals, Mountain View, CA; diluted
1:1500) antibodies in PBST solution for 3 days at 4 ◦ C. After the sections were rinsed four times in PBST for a total duration of 30 min, they were incubated for 90 min in PBST containing donkey anti-goat IgG conjugated with Rodamine (Vector Laboratories, Burlingame, CA; diluted at 1:300). After rinsing for 20 min with PBST solution, the sections were incubated again with donkey anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) (Vector Laboratories, Burlingame, CA; diluted at 1:200) for 90 min. Finally, the sections were rinsed for 30 min with PBST and coverslipped with aqueous solution; they were then examined under Canon florescent microscopy. Two primary antibodies were used in the present experiment. Antibody to hcrt-1 was a goat polyclonal antibody which was raised against the full-length human hcrt-1, while the antibody to hcrt-2 was a rabbit polyclonal antibody which was raised against the full-length human hcrt-2. The hcrt-2 antibody exhibits 100% cross-reactivity with human, rat and mouse hcrt-2, but no cross-reactivity was detected with hcrt-1 and related peptides from different species (data supplied by Phoenix Pharmaceuticals). In order to confirm the specificity of these antibodies, two types of preadsorption experiments were performed. In the first experiment, hcrt-1 and hcrt-2 antibodies were preadsorbed with the antigens used to generate each antibody, respectively, i.e. hcrt-1 antibody was preadsorbed with
Fig. 1. (A and B) Photomicrographs showing sections of the lateral hypothalamus labeled with hcrt-1 antisera (A) and hcrt-1 antisera preabsorbed with hcrt-2 peptide (B). (C and D) Photomicrographs showing sections of the lateral hypothalamus labeled with hcrt-2 antisera (C) and hcrt-2 antisera preabsorbed with hcrt-1 peptide (D). Note that the immunoreactive labeling is similar in sections stained with antisera (A and C) and sections stained with preadsorbed antisera (B and D). Bars in A–D are 50 m.
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full-length human hcrt-1 antigen (Diagnostic Systems Laboratories Inc., Texas) and hcrt-2 antibody was preadsorbed with full-length human hcrt-2 antigen (Phoenix Pharmaceuticals, Mountain View, CA). In a second experiment, each antiserum was preadsorbed with the other related antigen, i.e. hcrt-1 antibody was preadsorbed with full-length human hcrt-2 antigen and hcrt-2 antibody was preadsorbed with full-length hcrt-2 antigen. The preadsorption of all antibodies was carried out at 4 ◦ C for 24 h with an excess (50 times) amount of antigen, followed by centrifuging for 15 min at 4 ◦ C. Supernatants that contained the preadsorbed antibodies were used to stain brain sections. At the same time, a set of sections was stained with un-preadsorbed hcrt-1 and hcrt-2 antibodies. Both preabsorbed antibodies and un-preabsorbed antibodies were examined simultaneously under the exact same conditions using the immunofluorecent procedures described above. No immunoreactivity was detected in the
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hypothalamus and brainstem in any of these experiments when hcrt-1 and hcrt-2 antibodies were preincubated with hcrt-1 and hcrt-2 antigens, respectively. However, immunostaining was intact and no reduction in the immunoreactivity was observed when hcrt-1 and hcrt-2 antibodies were preincubated with hcrt-2 and hcrt-1 antigens, respectively (Fig. 1).
3. Results Similar to our previous study [17], in the hypothalamus, hcrt-1 immunoreactive (hcrt-1-ir) neuronal somata were found mainly in the lateral hypothalamic area (LHA) at the level of tuberal cinereum (Fig. 2A and B) and in the dorsal and posterior hypothalamic areas, although a few positive neuronal somata were scattered in other hypothalamic regions. In the LHA, most of hcrt-1-ir neuronal somata
Fig. 2. Immunofluorescent photomicrographs of the same sections of the lateral hypothalamic area (LHA) (A and B), dorsal hypothalamus (DH) (C and D) and laterodorsal tegmental nucleus (LDT) (E and F) after double staining with antisera to hypocretin-1 (hcrt-1) (A, C, and E) and hypocretin-2 (hcrt-2) (B, D, and F). Note that both antibodies label the same neuronal soma and fibers in these regions. Bars in A–D are 40 m, bars in E–F are 20 m.
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were located dorsal and lateral to the fornix. In addition to the soma, hcrt-1-ir fibers with varicosities were observed in almost all hypothalamic regions (Fig. 2A–D) with a high density of fibers in the suprachismatic, infundibular, tuberomamillary, supramamillary, and premamillary nuclei. When compared with hcrt-1-ir somata and fibers in the hypothalamus, hcrt-2-ir neuronal somata and fibers exhibited a similar localization. In fact, in all regions, hcrt-1 and hcrt-2 labeled the same neuronal somata and fibers (Fig. 2A–D).In the brainstem, a high density of hcrt-1-ir fibers with varicosities were present in the nucleus raphe dorsalis (DRN), the LDT, and the locus coeruleus (LC). Under high magnification, it was found that all hcrt-1 labeled fibers and varicosities were found to contain hcrt-2 immunoreactivity, indicating that both hcrt-1 and hcrt-2 are co-localized in fibers and varicosities in these regions (Fig. 2E and F).
4. Discussion In the present experiment, using double immunoflorescent techniques, we demonstrated that both hcrt-1 and hcrt-2 immunoreactivity are present in the same neuronal somata in the hypothalamus and in the same fibers/terminals in the brainstem, indicating that hcrt-1 and hcrt-2 are co-localized not only in neuronal somata but also in their fibers/terminals. It is likely that co-localization occurs in other brain regions [2,5], because hcrt-1 and hcrt-2 are similarly expressed in the same regions throughout the brain. In rat brain, two types of receptors are found for hcrt-1 and hcrt-2, e.g. hcrt receptor 1 (hcrtr1) and hcrt receptor 2 (hcrtr2) [9,10]. Both receptors belong to the class of G protein-coupled cell surface receptors. The hcrtr2 binds hcrt-1 and hcrt-2 with similar affinities, whereas hcrtr1 is 30–100 times more potent for hcrt-1 then for hcrt-2. Therefore, hcrtr1 is considered to be a selective receptor for hcrt-1, while hcrtr2 is a non-selective receptor for both hcrt-1 and hcrt-2. Recent studies using in situ hybridization techniques show a marked difference, almost complementary, in the distribution of hcrtr1 and hcrtr2 in the rat brain [1,7,15]. For example, Marcus et al. found the strongest hybridization signal for hcrtr1 in the rat brain is located in the LC [7]. However, this nucleus contained little hcrtr2 mRNA, indicating that hcrtr1 is the predominant type of hcrt receptor in the LC. On the other hand, following local administration of hcrt-1 and hcrt-2 into the LC, Bourgin et al. found that only hcrt-1 suppressed REM sleep in a dose-dependent manner and increased wakefulness at the expense of deep, slow-wave sleep [1]. No effects of hcrt-2 were detected. Thus, although both hcrt-1 and hcrt-2 are co-expressed in the terminals of axons located in the LC, only hcrt-1 is effective in the regulation of wakefulness in this nucleus and this effect is mediated by hcrtr1 receptors. Therefore, different functions of hcrt-1 and hcrt-2 in these brain regions appear to depend on the type of postsynaptic receptor.
Following the discovery of hcrt-1 and hcrt-2, their functions in the brain have been extensively studied [5,13]. It has been shown that hcrt-1 is involved in regulating a variety of brain functions such as feeding and drinking behaviors [6,14], sympathetic and cardiovascular activity [11,12], energy homeostasis, arousal, locomotor activity and regulation of the sleep–wake cycle [2,13]. On the other hand, although hcrt-2 is also involved in some of these regulatory functions [6,11,12,14], there are a number of behavioral, neuroendocrine and neurochemical effects of hcrt-2 which are clearly different from those of hcrt-1 [4]. For example, Sweet at al. [14] found that although both hcrt-1 and hcrt-2 stimulated feeding behavior following intraventricular injections, only hcrt-1 stimulated feeding following microinjection into hypothalamic regions such as the perifornical region and the lateral hypothalamus. In addition, following the central administration of hcrt-1 and hcrt-2 into rat brain ventricles, Jones et al. [4] found that while hcrt-1 caused robust whole body grooming, hcrt-2 only increased head grooming. On the other hand, hcrt-1 stimulated corticosterone levels, while hct-2 failed to alter the level of corticosterone [4]. Finally, hcrt-2 increased plasma TSH levels, while hcrt-1 did not. These data indicate widespread but different roles for hcrt-1 and hcrt-2 within the CNS [4]. Because we have shown that hcrt-1 and hcrt-2 are co-expressed in the same neurons, differences between the functions of these two peptides is likely to be due to distinct types of postsynaptic receptors that are present on the target cells of hypocretinergic terminals.
Acknowledgments We wish to thank Dr. J.K. Engelhardt for critically editing this manuscript. This study was funded by Grants MH 43362, NS 23426, NS 09999, HL 60296 and AG 04307.
References [1] Bourgin P, Huitron-Resendiz S, Spier AD, Fabre V, Morte B, Criado JR, et al. Hypocretin-1 modulates rapid eye movement sleep through activation of locus coeruleus neurons. J Neurosci 2000;20:7760–5. [2] Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, et al. Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. PNAS USA 1999;96:748–53. [3] de Lecea L, Kilduff TS, Peyron C, Gao X-B, Foye PE, Danielson PE, et al. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. PNAS USA 1998;95:322–7. [4] Jones DNC, Gartlon J, Parker F, Taylor SG, Routledge C, Hemmati P, et al. Effects of centrally administered orexin-B and orexin-A: a role for orexin-1 receptors in orexin-B-induced hyperactivity. Psychopharmacology 2000;153:210–8. [5] Kliduff TS, Peyron C. The hypocretin/orexin ligand-receptor system: implications for sleep and sleep disorders. TINS 2000;23:359–65. [6] Kunii K, Yamanaka A, Nambu T, Matsuzaki I, Goto K, Sakurai T. Orexins/hypocretins regulate drinking behavior. Brain Res 1999;842:256–61.
J.-H. Zhang et al. / Peptides 23 (2002) 1479–1483 [7] Marcus JN, Aschkenasi CJ, Lee CE, Chemelli RM, Saper CB, Yanagisawa M, et al. Differential expression of orexin receptors 1 and 2 in the rat brain. J Comp Neurol 2001;435:6–25. [8] Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, et al. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 1998;18:9996–10015. [9] Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptor that regulate feeding behavior. Cell 1998;92:573–85. [10] Sakurai T. Orexins and orexin receptors: implication in feeding behavior. Regulatory Peptides 1999;85:25–30. [11] Samson WK, Gosnell B, Chang J-K, Resch ZT, Murphy TC. Cardiovascular regulatory actions of the hypocretins in brain. Brain Res 1999;83:248–53. [12] Shirasaka T, Nakazato M, Matsukura S, Takasaki M, Kannan H. Sympathetic and cardivascular actions of orexins in conscious rats. Am J Physiol 1999;277:R1780–5.
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[13] Sutcliffe JG, de Lecea L. The hypocretins: excitatory neuromodulatory peptides for multiple homeostatic systems, including sleep and feeding. J Neurosci Res 2000;62:161–8. [14] Sweet DC, Levine AS, Billington CJ, Kotz CM. Feeding response to central orexins. Brain Res 1999;821:535–8. [15] Trivedi P, Yu H, MacNeil DJ, van der Ploeg LHT, Guan XM. Distribution of orexin receptor mRNA in the rat brain. FEBS Lett 1998;438:71–5. [16] Xi M-C, Morales FR, Chase MH. Effects on sleep and wakefulness of the injection of hypocretin-1 (orexin-A) into the laterodorsal tegmental nucleus of the cat. Brain Res 2001;901:259–64. [17] Zhang J-H, Sampogna S, Morales FR, Chase MH. Orexin (hypocretin)-like immunoreactivity in the cat hypothlamus: a light and electron microscopic study. Sleep 2001;24:67–76. [18] Zhang J-H, Sampogna S, Morales FR, Chase MH. Hypocretin (orexin)-like immunoreactivity in the cat brainstem. Soc Neurosci Abstr, vol. 30, Program No. 908.7.
Co-localization of hypocretin-1 and hypocretin-2 in the cat hypothalamus and brainstem Jian-Hua Zhang a,∗ , Sharon Sampogna a , Francisco R. Morales b , Michael H. Chase a a
Department of Physiology and the Brain Research Institute, UCLA School of Medicine, University of California, 53-231 CHS, Los Angeles, CA 90095, USA b Departamento de Fisiolog´ıa, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay Received 24 July 2001; accepted 28 February 2002
Abstract Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) are two recently discovered hypothalamic neuropeptides. In the present study, using double immunofluorescent techniques, the co-localization of hcrt-1 and hcrt-2 was examined in neuronal soma and fibers/terminals located, respectively, in the cat hypothalamus and brainstem. In the hypothalamus, all hcrt-1 positive neuronal soma also displayed hcrt-2 immunoreactivity. In the brainstem, both hcrt-1 and hcrt-2 antibodies labeled the same fibers/terminals, indicating that hcrt-1 and hcrt-2 co-localize not only in the neuronal soma (hypothalamus) but also in their fibers/terminals (brainstem). If both peptides are released following neuronal activity, then the distinct effects of these peptides in the brain are likely to depend on the types of postsynaptic receptors that are activated. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Hypocretin; Immunofluorescence; Hypothalamus; Brainstem; Cat
1. Introduction Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) (also known as orexin-A and orexin-B, respectively) are two recently discovered hypothalamic neuropeptides [9,10]. Both hypocretins are derived from the same 130 amino acid residue (rodent) or 131 amino acid residue (human) polypeptide (prepro-hypocretin) by proteolytic processing [3,9,10]. The hcrt-1 is a 33-residue peptide with two intramolecular disulfide bonds in the N-terminal region, whereas hcrt-2 is a linear 28-residue peptide [3,9,10]. Initial studies indicated that hypocretin is involved in the control of food intake [9,10]. However, recent experiments suggest that hypocretins may be involved in the regulation of a sleep and wakefulness [3,5,13,16]. For example, a recent study in our laboratory showed that hypocretinergic processes in the laterodorsal tegmental nucleus (LDT) of cat play an important role in the promotion of wakefulness and the suppression of active sleep [16]. Using in situ hybridization and immunohistochemical techniques, Peyron et al. [8]. studied the distribution of prepro-hypocretin mRNA and peptide in the central nervous system of the rat. They found that prepro-hypocretin-positive ∗
Corresponding author. Tel.: +1-310-825-3417; fax: +1-310-206-3499. E-mail address: [email protected] (J.-H. Zhang).
neurons are located exclusively in the hypothalamus, mostly in the perifornical nucleus and dorsal–lateral hypothalamic area. However, the fibers/terminals of labeled neurons spread widely throughout the brain, including the brainstem [8]. This distribution pattern of hcrts were confirmed later by Date et al. [2] who examined the distribution of hcrt-1 and hcrt-2 in the rat brain using specific antibodies for hcrt-1 and hcrt-2, respectively [2,5]. We observed a similar distribution of hypocretin immunoreactivity in the cat brain [17,18]. In addition, our results show both hcrt-1 and hcrt-2 are located in the same brain regions, suggesting the possibility of co-localization of these two peptides in the same neuron [17,18]. However, until the present investigation, there has been no evidence that hcrt-1 and hcrt-2 are co-expressed in the same neurons. Accordingly, in this study, we examined the possible co-localization of hcrt-1 and hcrt-2 in the neuronal soma and fibers/terminals in the cat hypothalamus and brainstem, respectively.
2. Materials and methods 2.1. Animal and tissue preparation Four adult cats (2–3 kg) were employed in the present study. These animals were obtained from and determined
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to be in good health by the UCLA Division of Laboratory Animal Medicine. Animal treatment and handling in these experiments conformed with the policy of the American Physiological Society. The cats were deeply anesthetized with pentobarbital sodium (35 mg/kg, i.v.) and perfused transcardially with 1 l of ice-cold saline (containing 1000 units of heparin) followed by 2.5 l of a fixative containing 4% paraformaldehyde, 15% saturated picric acid and 0.25% glutaraldehyde in 0.1 M phosphate buffer (PBS) (pH 7.4). The whole brain was then removed and postfixed overnight in fresh fixative at 4 ◦ C. Next, the tissue was immersed overnight in 20% sucrose (w/v) in 0.1 M PBS at 4 ◦ C. After freezing with dry ice, it was cut into 15 m coronal sections with a Reichert-Jung cryostat. All sections were collected and stored in a solution of 0.1 M PBS containing 0.3% Triton X-100 and 0.1% sodium azide at 4 ◦ C for later use. 2.2. Double immunofluorescence Immunohistochemical staining was similar to previously published procedures [17]. Briefly, free-floating sections were rinsed several times in ice-cold PBST (0.1 M PBS with 0.3% Triton X-100); they were then incubated simultaneously with goat anti-hcrt-1 (Diagnostic Systems Laboratories Inc., Texas, diluted 1:1500) and rabbit anti-hcrt-2 (Phoenix Pharmaceuticals, Mountain View, CA; diluted
1:1500) antibodies in PBST solution for 3 days at 4 ◦ C. After the sections were rinsed four times in PBST for a total duration of 30 min, they were incubated for 90 min in PBST containing donkey anti-goat IgG conjugated with Rodamine (Vector Laboratories, Burlingame, CA; diluted at 1:300). After rinsing for 20 min with PBST solution, the sections were incubated again with donkey anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) (Vector Laboratories, Burlingame, CA; diluted at 1:200) for 90 min. Finally, the sections were rinsed for 30 min with PBST and coverslipped with aqueous solution; they were then examined under Canon florescent microscopy. Two primary antibodies were used in the present experiment. Antibody to hcrt-1 was a goat polyclonal antibody which was raised against the full-length human hcrt-1, while the antibody to hcrt-2 was a rabbit polyclonal antibody which was raised against the full-length human hcrt-2. The hcrt-2 antibody exhibits 100% cross-reactivity with human, rat and mouse hcrt-2, but no cross-reactivity was detected with hcrt-1 and related peptides from different species (data supplied by Phoenix Pharmaceuticals). In order to confirm the specificity of these antibodies, two types of preadsorption experiments were performed. In the first experiment, hcrt-1 and hcrt-2 antibodies were preadsorbed with the antigens used to generate each antibody, respectively, i.e. hcrt-1 antibody was preadsorbed with
Fig. 1. (A and B) Photomicrographs showing sections of the lateral hypothalamus labeled with hcrt-1 antisera (A) and hcrt-1 antisera preabsorbed with hcrt-2 peptide (B). (C and D) Photomicrographs showing sections of the lateral hypothalamus labeled with hcrt-2 antisera (C) and hcrt-2 antisera preabsorbed with hcrt-1 peptide (D). Note that the immunoreactive labeling is similar in sections stained with antisera (A and C) and sections stained with preadsorbed antisera (B and D). Bars in A–D are 50 m.
J.-H. Zhang et al. / Peptides 23 (2002) 1479–1483
full-length human hcrt-1 antigen (Diagnostic Systems Laboratories Inc., Texas) and hcrt-2 antibody was preadsorbed with full-length human hcrt-2 antigen (Phoenix Pharmaceuticals, Mountain View, CA). In a second experiment, each antiserum was preadsorbed with the other related antigen, i.e. hcrt-1 antibody was preadsorbed with full-length human hcrt-2 antigen and hcrt-2 antibody was preadsorbed with full-length hcrt-2 antigen. The preadsorption of all antibodies was carried out at 4 ◦ C for 24 h with an excess (50 times) amount of antigen, followed by centrifuging for 15 min at 4 ◦ C. Supernatants that contained the preadsorbed antibodies were used to stain brain sections. At the same time, a set of sections was stained with un-preadsorbed hcrt-1 and hcrt-2 antibodies. Both preabsorbed antibodies and un-preabsorbed antibodies were examined simultaneously under the exact same conditions using the immunofluorecent procedures described above. No immunoreactivity was detected in the
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hypothalamus and brainstem in any of these experiments when hcrt-1 and hcrt-2 antibodies were preincubated with hcrt-1 and hcrt-2 antigens, respectively. However, immunostaining was intact and no reduction in the immunoreactivity was observed when hcrt-1 and hcrt-2 antibodies were preincubated with hcrt-2 and hcrt-1 antigens, respectively (Fig. 1).
3. Results Similar to our previous study [17], in the hypothalamus, hcrt-1 immunoreactive (hcrt-1-ir) neuronal somata were found mainly in the lateral hypothalamic area (LHA) at the level of tuberal cinereum (Fig. 2A and B) and in the dorsal and posterior hypothalamic areas, although a few positive neuronal somata were scattered in other hypothalamic regions. In the LHA, most of hcrt-1-ir neuronal somata
Fig. 2. Immunofluorescent photomicrographs of the same sections of the lateral hypothalamic area (LHA) (A and B), dorsal hypothalamus (DH) (C and D) and laterodorsal tegmental nucleus (LDT) (E and F) after double staining with antisera to hypocretin-1 (hcrt-1) (A, C, and E) and hypocretin-2 (hcrt-2) (B, D, and F). Note that both antibodies label the same neuronal soma and fibers in these regions. Bars in A–D are 40 m, bars in E–F are 20 m.
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were located dorsal and lateral to the fornix. In addition to the soma, hcrt-1-ir fibers with varicosities were observed in almost all hypothalamic regions (Fig. 2A–D) with a high density of fibers in the suprachismatic, infundibular, tuberomamillary, supramamillary, and premamillary nuclei. When compared with hcrt-1-ir somata and fibers in the hypothalamus, hcrt-2-ir neuronal somata and fibers exhibited a similar localization. In fact, in all regions, hcrt-1 and hcrt-2 labeled the same neuronal somata and fibers (Fig. 2A–D).In the brainstem, a high density of hcrt-1-ir fibers with varicosities were present in the nucleus raphe dorsalis (DRN), the LDT, and the locus coeruleus (LC). Under high magnification, it was found that all hcrt-1 labeled fibers and varicosities were found to contain hcrt-2 immunoreactivity, indicating that both hcrt-1 and hcrt-2 are co-localized in fibers and varicosities in these regions (Fig. 2E and F).
4. Discussion In the present experiment, using double immunoflorescent techniques, we demonstrated that both hcrt-1 and hcrt-2 immunoreactivity are present in the same neuronal somata in the hypothalamus and in the same fibers/terminals in the brainstem, indicating that hcrt-1 and hcrt-2 are co-localized not only in neuronal somata but also in their fibers/terminals. It is likely that co-localization occurs in other brain regions [2,5], because hcrt-1 and hcrt-2 are similarly expressed in the same regions throughout the brain. In rat brain, two types of receptors are found for hcrt-1 and hcrt-2, e.g. hcrt receptor 1 (hcrtr1) and hcrt receptor 2 (hcrtr2) [9,10]. Both receptors belong to the class of G protein-coupled cell surface receptors. The hcrtr2 binds hcrt-1 and hcrt-2 with similar affinities, whereas hcrtr1 is 30–100 times more potent for hcrt-1 then for hcrt-2. Therefore, hcrtr1 is considered to be a selective receptor for hcrt-1, while hcrtr2 is a non-selective receptor for both hcrt-1 and hcrt-2. Recent studies using in situ hybridization techniques show a marked difference, almost complementary, in the distribution of hcrtr1 and hcrtr2 in the rat brain [1,7,15]. For example, Marcus et al. found the strongest hybridization signal for hcrtr1 in the rat brain is located in the LC [7]. However, this nucleus contained little hcrtr2 mRNA, indicating that hcrtr1 is the predominant type of hcrt receptor in the LC. On the other hand, following local administration of hcrt-1 and hcrt-2 into the LC, Bourgin et al. found that only hcrt-1 suppressed REM sleep in a dose-dependent manner and increased wakefulness at the expense of deep, slow-wave sleep [1]. No effects of hcrt-2 were detected. Thus, although both hcrt-1 and hcrt-2 are co-expressed in the terminals of axons located in the LC, only hcrt-1 is effective in the regulation of wakefulness in this nucleus and this effect is mediated by hcrtr1 receptors. Therefore, different functions of hcrt-1 and hcrt-2 in these brain regions appear to depend on the type of postsynaptic receptor.
Following the discovery of hcrt-1 and hcrt-2, their functions in the brain have been extensively studied [5,13]. It has been shown that hcrt-1 is involved in regulating a variety of brain functions such as feeding and drinking behaviors [6,14], sympathetic and cardiovascular activity [11,12], energy homeostasis, arousal, locomotor activity and regulation of the sleep–wake cycle [2,13]. On the other hand, although hcrt-2 is also involved in some of these regulatory functions [6,11,12,14], there are a number of behavioral, neuroendocrine and neurochemical effects of hcrt-2 which are clearly different from those of hcrt-1 [4]. For example, Sweet at al. [14] found that although both hcrt-1 and hcrt-2 stimulated feeding behavior following intraventricular injections, only hcrt-1 stimulated feeding following microinjection into hypothalamic regions such as the perifornical region and the lateral hypothalamus. In addition, following the central administration of hcrt-1 and hcrt-2 into rat brain ventricles, Jones et al. [4] found that while hcrt-1 caused robust whole body grooming, hcrt-2 only increased head grooming. On the other hand, hcrt-1 stimulated corticosterone levels, while hct-2 failed to alter the level of corticosterone [4]. Finally, hcrt-2 increased plasma TSH levels, while hcrt-1 did not. These data indicate widespread but different roles for hcrt-1 and hcrt-2 within the CNS [4]. Because we have shown that hcrt-1 and hcrt-2 are co-expressed in the same neurons, differences between the functions of these two peptides is likely to be due to distinct types of postsynaptic receptors that are present on the target cells of hypocretinergic terminals.
Acknowledgments We wish to thank Dr. J.K. Engelhardt for critically editing this manuscript. This study was funded by Grants MH 43362, NS 23426, NS 09999, HL 60296 and AG 04307.
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