Intraperitoneal injection of neuropeptide Y (NPY) alters neurotrophin rat hypothalamic levels: Implications for NPY potential role in stress-related disorders

Peptides 32 (2011) 1320–1323

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Intraperitoneal injection of neuropeptide Y (NPY) alters neurotrophin rat hypothalamic levels: Implications for NPY potential role in stress-related disorders Francesca Gelfo a , Paola De Bartolo a,c , Paola Tirassa b , Nicoletta Croce a , Carlo Caltagirone a , Laura Petrosini a,c , Francesco Angelucci a,∗ a

IRCCS Santa Lucia Foundation, Department of Clinical and Behavioral Neurology, 00179 Rome, Italy Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy c Department of Psychology, University of Rome “La Sapienza”, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 3 February 2011 Received in revised form 29 March 2011 Accepted 29 March 2011 Available online 5 April 2011 Keywords: Neuropeptide Y Hypothalamus Neurotrophins BDNF NGF

a b s t r a c t Neuropeptide Y (NPY) is a 36-amino acid peptide which exerts several regulatory actions within peripheral and central nervous systems. Among NPY actions preclinical and clinical data have suggested that the anxiolytic and antidepressant actions of NPY may be related to its antagonist action on the hypothalamic–pituitary–adrenal (HPA) axis. The neurotrophins brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are proteins involved in the growth, survival and function of neurons. In addition to this, a possible role of neurotrophins, particularly BDNF, in HPA axis hyperactivation has been proposed. To characterize the effect of NPY on the production of neurotrophins in the hypothalamus we exposed young adult rats to NPY intraperitoneal administration for three consecutive days and then evaluated BDNF and NGF synthesis in this brain region. We found that NPY treatment decreased BDNF and increased NGF production in the hypothalamus. Given the role of neurotrophins in the hypothalamus, these findings, although preliminary, provide evidence for a role of NPY as inhibitor of HPA axis and support the idea that NPY might be involved in pathologies characterized by HPA axis dysfunctions. © 2011 Elsevier Inc. Published by All rights reserved.

1. Introduction Neuropeptide Y (NPY) is a 36-amino acid peptide belonging to the pancreatic polypeptide family which exerts several regulatory actions (appetite and cardiovascular regulation, cognition modulation, anticonvulsant effects) within peripheral (PNS) and central (CNS) nervous system [5]. Among NPY actions, an involvement in the pathophysiology and treatment of stress-related disorders, such as depression and anxiety, has been proposed [6,16,17,31,32]. More specifically, preclinical and clinical data have suggested that the antidepressant actions of NPY may be related to its antagonist action to the hypothalamic–pituitary–adrenal (HPA) axis [2,8,10,20,40]. In fact, the adaptive function of HPA axis is based on a glucocorticoid feedback mechanism that regulates the duration of the stress response [3,27]. Consequently, stress-related disorders are thought

∗ Corresponding author. Tel.: +39 06 51501550; fax: +39 06 51501552. E-mail addresses: [email protected] (F. Gelfo), [email protected] (P. De Bartolo), [email protected] (P. Tirassa), [email protected] (N. Croce), [email protected] (C. Caltagirone), [email protected] (L. Petrosini), [email protected] (F. Angelucci). 0196-9781/$ – see front matter © 2011 Elsevier Inc. Published by All rights reserved. doi:10.1016/j.peptides.2011.03.023

to involve impairment in this mechanism, resulting in chronic hyperactivation of HPA axis [33,41]. The activity of HPA axis may be also regulated by the neurotrophins brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), proteins involved in the growth, survival and function of neurons in PNS and CNS [42]. Studies performed in rats demonstrated that both intracerebroventricular administration of BDNF [12,29] and intravenous administration of NGF [36] may induce an increase in the activity of HPA axis. At the same time, an involvement of neurotrophins in stress-related disorders has also been evidenced in several studies. Decreased hippocampal BDNF [25] and NGF [24] levels have been described in animal models of depression. Moreover, in humans low BDNF levels in depression [15] and increased NGF levels in anxiety states [1,18] have been found in serum. Furthermore, a recent study in a rat model of depression has evidenced increased BDNF levels in the hypothalamus together with increased systemic levels of adrenocorticotropin hormone (ACTH) and corticotropin-releasing hormone (CRH), suggesting a possible role of BDNF in HPA axis hyperactivation [30]. On the other hand, in rodent models of stress increased hypothalamic NGF levels are hypothesized to be involved in plastic changes underlying stressrelated HPA axis deregulation [1].

F. Gelfo et al. / Peptides 32 (2011) 1320–1323

Altogether these findings suggest that NPY may affect neurotrophin synthesis at hypothalamic levels, as a mechanism of HPA axis regulation. With this in mind, we exposed young adult rats to NPY intraperitoneal administration for three consecutive days and then evaluated BDNF and NGF synthesis in the hypothalamus.

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Table 1 Body weight of NPY- and saline-treated rats measured at each time point of treatment. NPY treatment did not affect body weight, and no interaction between treatment and time was shown. Time

Treatment Saline

2. Materials and methods 2.1. Animals Adult male Wistar rats (250–300 g; Harlan, Italy) were used in the study. Rats were kept under standard conditions on a 12/12 h dark/light cycle and were allowed access to water and food during acclimation ad libitum. Animals were maintained according to the guidelines for ethical conduct developed by the European Communities Council Directive of November 24, 1986 (86/609/EEC). All efforts were made to minimize pain or discomfort of the animals. 2.2. NPY treatment Neuropeptide Y (NPY) (human, rat–cat. n. H-6375) was bought from Bachem AG – Switzerland. It was reconstituted in sterile distilled H2 O at a concentration of 0.1 mM, filtered/sterilized and stored at −20 ◦ C. At 15 p.m., rats were intraperitoneally injected with either vehicle (saline) or NPY (60 ␮g/kg) for three consecutive days (n = 8 animals/group). The present NPY dose was extrapolated on the basis ofprevious studieswhere NPY was given either intraperitoneally [26,28] or intravenously [19] in adult rodents. NPY at this dose was able to pass the blood brain barrier [21,22] as demonstrated by effects on brain-controlled functions such as breathing and cardiovascular functions. 2.3. Tissue dissection 24 hours after the last injection, rats from saline- and NPYtreated groups were decapitated and the brains were quickly removed and dissected on ice using a binocular dissection microscope. The whole hypothalamus was collected according to Glowinski and Iversen’s method [13]. For measurement of neurotrophins in hypothalamus they were extracted in 1 ml extraction buffer/100 mg tissue and homogenized in an ice-cold lysis buffer containing 137 mM NaCl, 20 mM Tris–HCl (pH 8.0), 1% NP40, 10% glycerol, 1 mM PMSF 10 ␮g/ml aprotinin, 1 ␮g/ml leupetin and 0.5 mM sodium vanadate. The tissue homogenate solutions were centrifuged with 14,000 × g for 25 min at 4 ◦ C. The supernatants were collected and used for quantification of NGF and BDNF. 2.4. BDNF and NGF determination by enzyme-linked immunosorbent assay (ELISA) Concentrations of BDNF and NGF proteins were assessed using a two-site enzyme immunoassay kit (Promega, USA). Briefly, 96well immunoplates (NUNC) were coated with 50 ␮l/well with the corresponding captured antibody which binds the neurotrophin of interest overnight at 4 ◦ C. The next day serial dilutions of known amounts of BDNF and NGF ranging from 0 to 500 pg/ml were performed in duplicate for generating the standard curve. Then the plates were washed three times with wash buffer and the standard curves and supernatants of brain tissue homogenates were incubated in the coated wells (100 ␮l each) for 2 h at room temperature (RT) with shaking. After additional washes, the antigen was incubated with second specific antibody for 2 h at RT (BDNF)or overnight at 4 ◦ C (NGF), as specified in the protocol. The plates were washed again with wash buffer and then incubated with an

Day 1 (1st injection) Day 2 (2nd injection) Day 3 (3rd injection) Day 4 (sacrifice)

290 294 298 302

± ± ± ±

NPY 9.89 9.48 11.10 10.83

298 303 304 307

± ± ± ±

4.54 5.31 4.44 4.99

N = 8 animals/group. Values are expressed in grams. Data represent means ± SEM.

anti-IgY HRP for 1 h at RT. After another wash the plates were incubated with a TMB/Peroxidase substrate solution for 15 min and phosphoric acid 1 M (100 ␮l/well) was added to the wells. The colorimetric reaction product was measured at 450 nm using a microplate reader (Dynatech MR 5000, Germany). Neurotrophin concentrations were determined from the regression line for the neurotrophin standard (ranging from 7.8 to 500 pg/ml-purified mouse BDNF or NGF) incubated under similar conditions in each assay. For each assay kit, the cross-reactivity with other related neurotrophic factors, e.g. NT-3 and NT-4 was less than 3%. Neurotrophin concentration was expressed as pg/g wet weight and all assays were performed in triplicate. 2.5. Statistical analysis Data on BDNF and NGF levels were analyzed by one-way analysis of variance (ANOVA) considering NPY and saline treatments as variables. Differences in body weight were evaluated with ANOVA for repeated measures. p-Values ≤0.05 were considered statistically significant. 3. Results 3.1. Effects of NPY treatment on body weight Since NPY is involved in the control of food intake we measured body weight in NPY- and saline-treated rats during all days of treatment. Data on body weights are shown in Table 1. NPY treatment did not affect body weight (f-value = 0.412; p-value = 0.531), and no interaction between treatment and time was evidenced (f-value = 1.660; p-value = 0.190). An effect of time was present (f-value = 38.24; p-value < 0.0001) as rats of both groups equally gained weight during the course of the treatment. 3.2. Effects of NPY treatment on neurotrophin synthesis in the hypothalamus BDNF and NGF levels in the hypothalamus are shown in Fig. 1. ANOVA showed that NPY treatment significantly affected hypothalamic BDNF levels (f-value = 14.38; p < 0.01). NPY-treated rats had significantly lower BNDF levels than saline-treated rats. A significant effect of NPY on hypothalamic NGF levels was also evidenced. NPY-treated rats had significantly higher NGF levels as compared to saline-treated rats (f-value = 5.88; p-value < 0.05). 4. Discussion This study was performed to characterize the effect of intraperitoneal injection of NPY on the production of neurotrophins in the hypothalamus. We found that NPY treatment had an opposite effects on neurotrophins in the rat hypothalamus. BDNF protein level was decreased while NGF was increased by NPY treatment.

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BDNF

pg/gr WW

250 200 150

B

NGF 1000

** pg/gr WW

A

*

800 600

vehicle NPY

100

400

50

200

0

0

Fig. 1. Effect of NPY peripheral administration on neurotrophin production in the rat hypothalamus. Rats were intraperitoneally injected with either vehicle (saline) or NPY (60 ␮g/kg) for three consecutive days (n = 8 animals/group). Figure shows BDNF (A) and NGF (B) levels in the hypothalamus of NPY- (NPY) and saline- (vehicle) treated rats. Data represent means ± SEM. WW: wet weight. Asterisks indicate significant differences between groups (*p < 0.05; **p < 0.01).

To the best of our knowledge, this is the first study that analyses the effects of NPY peripheral administration on rat hypothalamic neurotrophin levels. In fact, in most of the previous studies a central administration of the peptide was used [7,8,10,40], although NPY has been shown to cross the blood–brain barrier [21,22]. BDNF is highly localized in the ventromedial hypothalamic nucleus (VMN), but is also found in the lateral hypothalamic area, dorsomedial nucleus (DMN), and paraventricular nucleus (PVN) [38]. The VMN is also highly sensitive to the orexigenic actions of NPY and NPY is reported to robustly inhibit VMN neurons [9]. Thus, one possibility is that NPY reduces BDNF synthesis in the hypothalamus by inhibiting activity of VMN neurons. Another option is that the effects of neuropeptide Y on BDNF may be mediated by a peripheral action of the peptide. For example, an increased release of the peptide vasopressin, which can be induced by NPY itself [23,35], may also affect central levels of neurotrophins in the rat brain [43]. Interestingly, several studies have shown that central or peripheral NPY administration is able to antagonize HPA axis hyperactivation [33,41]. Studies performed in rats documented that central administration of NPY reverses the anxiogenic effects of increased CRH [8,10]. Moreover, in humans it has been reported that intravenous injections of NPY decrease CRH and ACTH secretion, an effect associated with inhibition of HPA axis activity [2]. In line with these studies there is some evidence that neurotrophins, particularly BDNF, in the hypothalamus are involved in the regulation of HPA axis. In fact, it has been shown that a stressful situation induces an increase in BDNF level in the rat hypothalamus [34,39] while administration of BDNF into the rat brain is able to induce HPA axis stimulation [12,29]. The significance of these findings is still uncertain. They could be of relevance when elucidating the role of NPY in depression. Depression is characterized by hyperactivation of the HPA axis [23] and, accordingly, a decrease of NPY in depressed patients has been reported [32]. Moreover, antidepressant drugs increase NPY in animal models and humans [6,17,31,32]. These and our present findings suggest that the so-called “BDNF hypothesis” of depression, based on the observation that BDNF is reduced in depression and increased by antidepressant drugs, must be integrated with new findings on the interaction between BDNF itself and other biologically active peptides, such as NPY. Supporting this idea, recent data obtained in rats have shown that depression and/or antidepressant-like drug activity are not always associated with BDNF changes in the hippocampus [11,14]. The decrease of BDNF hypothalamic levels reported in the present study after NPY peripheral administration fits well with the published literature. One can speculate that NPY inhibitory effects on HPA axis might be mediated by the reduction in hypothalamic BDNF levels and that BDNF may act as an intercellular messenger that mediates the stress

response. Supporting this hypothesis, in a recent study using a rat model of depressive-like state induced by chronic restraint stress it was shown that hypothalamic BDNF levels were increased, in association with systemic levels of ACTH and CRH [30]. However, since in the present study we did not use a rat model of depression speculations on BDNF-NPY interaction in such disorder are at present limited. In contrast to BDNF results, we found that NPY peripheral administration significantly increased hypothalamic NGF levels. NGF and its receptor are located in the hypothalamic regions with a high concentration in PVN [4] and, like BDNF, NGF is also able to activate HPA axis by mediating the release of HPA axis mediators [36]. NPY also regulates the activity of PVN where it appears to play an important role in body weight and feeding regulation [5,37]. Thus it is possible that NGF changes are related to the effect of NPY on PVN where NGF is produced or is active through its receptor. Another possibility is that NGF increases as a compensatory effect to the inhibitory action of NPY on HPA axis. Furthermore, increased serum NGF levels have been reported as an acute stress reaction [1] suggesting that the treatment per se may contribute to alter NGF peripheral levels in our rats. Nevertheless, it is worth to note that, in case of a chronic stress condition such as that observed in generalized anxiety disorder (GAD), an increase peripheral levels of NGF may indicate a good response to cognitive-behavioral therapy [18]. If this is true, then we can suppose that an NPY-mediated increase of NGF may be associated with NPY anxiolytic properties. However, as for BDNF, this hypothesis needs to be validated in animal models of chronic stress condition. 5. Conclusions In conclusion, this study demonstrated that systemic administration on NPY (at a dosage that did not induce orexigenic effects) reduces BDNF and increases NGF production in the hypothalamus. Given the role of neurotrophins in the hypothalamus, these findings, although preliminary, provide evidence for a role of NPY as an inhibitor of HPA axis further supporting the idea that NPY might be involved in pathologies characterized by HPA axis dysfunctions. Contributors Authors Francesco Angelucci and Francesca Gelfo designed the study and wrote the protocol. Authors Laura Petrosini, Nicoletta Croce, and Carlo Caltagirone managed the literature searches and analyses. Authors Francesco Angelucci and Francesca Gelfo undertook the statistical analysis. Authors Francesco Angelucci, Francesca

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Gelfo, and Paola Tirassa wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript. Role of funding source This work was supported by grant nEUROsyn Italian Ministry of Health (RC2008) in the frame of ERA-Net Neuron. The funding source had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Conflict of interest The authors declare no conflicting financial or other competing interests. Acknowledgements Supported by grant nEUROsyn Italian Ministry of Health (RC2008) in the frame of ERA-Net Neuron. References [1] Alleva E, Santucci D. Psychosocial vs. “physical” stress situations in rodents and humans: role of neurotrophins. Physiol Behav 2001;73:313–20. [2] Antonijevic IA, Murck H, Bohlhalter S, Frieboes RM, Holsboer F, Steiger A. Neuropeptide Y promotes sleep and inhibits ACTH and cortisol release in young men. Neuropharmacology 2000;39:1474–81. [3] Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB. The role of corticotropinreleasing factor in depression and anxiety disorders. J Endocrinol 1999;160:1–12. [4] Badowska-Szalewska E, Klejbor I, Ludkiewicz B, Domaradzka-Pytel B, Dziewiatkowski J, Spodnik JH, et al. Immunoreactivity of c-Fos NGF and its receptor TrkA in the periventricular zone of the rat hypothalamus after open field exposure. Pol J Vet Sci 2006;9:171–80. [5] Beck B. Neuropeptide Y in normal eating and in genetic and dietary-induced obesity. Philos Trans R Soc Lond B Biol Sci 2006;361:1159–85. [6] Bjørnebekk A, Mathé AA, Brené S. The antidepressant effects of running and escitalopram are associated with levels of hippocampal NPY and Y1 receptor but not cell proliferation in a rat model of depression. Hippocampus 2010;20:820–8. [7] Britton KT, Southerland S, Van Uden E, Kirby D, Rivier J, Koob G. Anxiolytic activity of NPY receptor agonists in the conflict test. Psychopharmacology (Berl) 1997;132:6–13. [8] Britton KT, Akwa Y, Spina MG, Koob GF. Neuropeptide Y blocks anxiogenic-like behavioral action of corticotropin-releasing factor in an operant conflict test and elevated plus maze. Peptides 2000;21:37–44. [9] Chee MJ, Myers Jr MG, Price CJ, Colmers WF. Neuropeptide Y suppresses anorexigenic output from the ventromedial nucleus of the hypothalamus. J Neurosci 2010;30:3380–90. [10] Ehlers CL, Somes C, Seifritz E, Rivier JE. CRF/NPY interactions: a potential role in sleep dysregulation in depression and anxiety. Depress Anxiety 1997;6:1–9. [11] Elfving B, Plougmann PH, Müller HK, Mathé AA, Rosenberg R, Wegener G. Inverse correlation of brain and blood BDNF levels in a genetic rat model of depression. Int J Neuropsychopharmacol 2010;13:563–72. [12] Givalois L, Naert G, Rage F, Ixart G, Arancibia S, Tapia-Arancibia L. A single brain-derived neurotrophic factor injection modifies hypothalamo– pituitary–adrenocortical axis activity in adult male rats. Mol Cell Neurosci 2004;27:280–95. [13] Glowinski J, Iversen LL. Regional studies of catecholamines in the rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]dopa in various regions of the brain. J Neurochem 1966;13:655–69. [14] Hansson AC, Rimondini R, Heilig M, Mathé AA, Sommer WH. Dissociation of antidepressant-like activity of escitalopram and nortriptyline on behaviour and hippocampal BDNF expression in female rats. J Psychopharmacol 2011 [Epub ahead of print]. [15] Hashimoto K. Brain-derived neurotrophic factor as a biomarker for mood disorders: an historical overview and future directions. Psychiatry Clin Neurosci 2010;64:341–57. [16] Heilig M. The NPY system in stress, anxiety and depression. Neuropeptides 2004;38:213–24.

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[17] Jiménez-Vasquez PA, Diaz-Cabiale Z, Caberlotto L, Bellido I, Overstreet D, Fuxe K, et al. Electroconvulsive stimuli selectively affect behavior and neuropeptide Y (NPY) and NPY Y(1) receptor gene expressions in hippocampus and hypothalamus of Flinders Sensitive Line rat model of depression. Eur Neuropsychopharmacol 2007;17:298–308. [18] Jockers-Scherübl MC, Zubraegel D, Baer T, Linden M, Danker-Hopfe H, SchulteHerbrüggen O, et al. Nerve growth factor serum concentrations rise after successful cognitive-behavioural therapy of generalized anxiety disorder. Prog Neuropsychopharmacol Biol Psychiatry 2007;31:200–4. ´ [19] Kaczynska K, Szereda-Przestaszewska M. NPY Y(1) receptors are involved in cardio-respiratory responses to intravenous injection of neuropeptide Y in anaesthetized rats. Pharmacol Res 2010;62:444–9. [20] Kask A, Rägo L, Harro J. Alpha-helical CRF(9-41) prevents anxiogenic-like effect of NPY Y1 receptor antagonist BIBP3226 in rats. Neuroreport 1997;8:3645–7. [21] Kastin AJ, Akerstrom V. Nonsaturable entry of neuropeptide Y into brain. Am J Physiol 1999;276:E479–82. [22] Kastin AJ, Pan W. Blood–brain barrier and feeding: regulatory roles of saturable transport systems for ingestive peptides. Curr Pharm Des 2008;14:1615–9. [23] Larsen PJ, Jukes KE, Chowdrey HS, Lightman SL, Jessop DS. Neuropeptide-Y potentiates the secretion of vasopressin from the neurointermediate lobe of the rat pituitary gland. Endocrinology 1994;134:1635–9. [24] Marais L, van Rensburg SJ, van Zyl JM, Stein DJ, Daniels WM. Maternal separation of rat pups increases the risk of developing depressive-like behavior after subsequent chronic stress by altering corticosterone and neurotrophin levels in the hippocampus. Neurosci Res 2008;61:106–12. [25] Martinowich K, Manji H, Lu B. New insights into BDNF function in depression and anxiety. Nat Neurosci 2007;10:1089–93. [26] Mazzocchi G, Malendowicz LK, Macchi C, Gottardo G, Nussdorfer GG. Further investigations on the effects of neuropeptide Y on the secretion and growth of rat adrenal zona glomerulosa. Neuropeptides 1996;30:19–27. [27] Mello AA, Mello MF, Carpenter LL, Price LH. Update on stress and depression:the role of the hypothalamic–pituitary–adrenal (HPA) axis. Rev Bras Psiquiatr 2003;25:231–8. [28] Moriya R, Shirakura T, Hirose H, Kanno T, Suzuki J, Kanatani A. NPY Y2 receptor agonist PYY(3-36) inhibits diarrhea by reducing intestinal fluid secretion and slowing colonic transit in mice. Peptides 2010;31:671–5. [29] Naert G, Ixart G, Tapia-Arancibia L, Givalois L. Continuous i.c.v. infusion of brain-derived neurotrophic factor modifies hypothalamic–pituitary–adrenal axis activity, locomotor activity and body temperature rhythms in adult male rats. Neuroscience 2006;139:779–89. [30] Naert G, Ixart G, Maurice T, Tapia-Arancibia L, Givalois L. Brain-derived neurotrophic factor and hypothalamic–pituitary–adrenal axis adaptation processes in a depressive-like state induced by chronic restraint stress. Mol Cell Neurosci 2011;46:55–66. [31] Nikisch G, Agren H, Eap CB, Czernik A, Baumann P, Mathé AA. Neuropeptide Y and corticotropin-releasing hormone in CSF mark response to antidepressive treatment with citalopram. Int J Neuropsychopharmacol 2005;8:403–10. [32] Nikisch G, Mathé AA. CSF monoamine metabolites and neuropeptides in depressed patients before and after electroconvulsive therapy. Eur Psychiatry 2008;23:356–9. [33] Paschos KA, Veletza S, Chatzaki E. Neuropeptide and sigma receptors as novel therapeutic targets for the pharmacotherapy of depression. CNS Drugs 2009;23:755–72. [34] Rage F, Givalois L, Marmigère F, Tapia-Arancibia L, Arancibia S. Immobilization stress rapidly modulates BDNF mRNA expression in the hypothalamus of adult male rats. Neuroscience 2002;112:309–18. [35] Sato K, Crofton JT, Wang YX, Share L. Effects of gender on the central actions of neuropeptide Y and norepinephrine on vasopressin and blood pressure in the rat. Brain Res 1995;689:71–8. [36] Scaccianoce S, Lombardo K, Nicolai R, Affricano D, Angelucci L. Studies on the involvement of histamine in the hypothalamic–pituitary–adrenal axis activation induced by nerve growth factor. Life Sci 2000;67:3143–52. [37] Simpson KA, Martin NM, Bloom SR. Hypothalamic regulation of food intake and clinical therapeutic applications. Arq Bras Endocrinol Metabol 2009;53:120–8. [38] Smith MA, Makino S, Kim SY, Kvetnansky R. Stress increases brain-derived neurotrophic factor messenger ribonucleic acid in the hypothalamus and pituitary. Endocrinology 1995;136:3743–50. [39] Tapia-Arancibia L, Rage F, Givalois L, Arancibia S. Physiology of BDNF: focus on hypothalamic function. Front Neuroendocrinol 2004;25:77–107. [40] Thorsell A, Slawecki CJ, Ehlers CL. Effects of neuropeptide Y and corticotropinreleasing factor on ethanol intake in Wistar rats: interaction with chronic ethanol exposure. Behav Brain Res 2005;161:133–40. [41] Tsigos C, Chrousos GP. Hypothalamic–pituitary–adrenal axis, neuroendocrine factors and stress. J Psychosom Res 2002;53:865–71. [42] Weisenhorn DM, Roback J, Young AN, Wainer BH. Cellular aspects of trophic actions in the nervous system. Int Rev Cytol 1999;189:177–265. [43] Zhou AW, Li WX, Guo J, Du YC. Facilitation of AVP(4-8) on gene expression of BDNF and NGF in rat brain. Peptides 1997;18(8):1179–87.