Lateral hypothalamic area orexin-A influence the firing activity of gastric distension-sensitive neurons and gastric motility in rats

Neuropeptides 57 (2016) 45–52

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Lateral hypothalamic area orexin-A influence the firing activity of gastric distension-sensitive neurons and gastric motility in rats Heling Hao a,1, Xiao Luan a, Feifei Guo a, Xiangrong Sun a, Yanling Gong b, Luo Xu a,⁎,1 a b

Department of Pathophysiology, Medical College of Qingdao University, Qingdao, Shandong 266021, PR China Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, Shandong, PR China

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Article history: Received 21 September 2015 Received in revised form 5 February 2016 Accepted 14 February 2016 Available online 16 February 2016 Keywords: Gastric motility Orexin-A Gastric-distension-sensitive neurons Lateral hypothalamic area Paraventricular nucleus

a b s t r a c t The orexins system consists of two G-protein coupled receptors (the orexin-1 and the orexin-2 receptor) and two neuropeptides, orexin-A and orexin-B. Orexin-A is an excitatory neuropeptide that regulates arousal, wakefulness and appetite. Recent studies have shown that orexin-A may promote gastric motility. We aim to explore the effects of orexin-A on the gastric -distension (GD) sensitive neurons and gastric motility in the lateral hypothalamic area (LHA), and the possible regulation by the paraventricular nucleus (PVN). Extracellular single unit discharges were recorded and the gastric motility was monitored by administration of orexin-A into the LHA and electrical stimulation of the PVN. There were GD neurons in the LHA, and administration of orexin-A to the LHA could increase the firing rate of both GD-excitatory (GD-E) and GD-inhibited (GD-I) neurons. The gastric motility was significantly enhanced by injection of orexin-A into the LHA with a dose dependent manner, which could be completely abolished by pre-treatment with orexin-A receptor antagonist SB334867. Electrical stimulation of the PVN could significantly increase the firing rate of GD neurons responsive to orexin-A in the LHA as well as promote gastric motility of rats. However, those effects could be partly blocked by pre-treatment with SB334867 in the LHA. It is suggested that orexin-A plays an important role in promoting gastric motility via LHA. The PVN may be involved in regulation of LHA on gastric motility. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction The orexins consists of orexin-A (OXA) and orexin-B (OXB), which are both produced from prepro-orexin in the lateral hypothalamus by a cascade of enzymatic reactions (de Lecea et al., 1998). Orexin-A, composed of 33 amino acids including an N-terminal pyroglutamy-l residue and two intramolecular disulfide bridges between cysteine residues (de Lecea et al., 1998). OXA had been identified as the hypothalamic neuropeptides. The orexin neurons were mainly located in the lateral hypothalamic area (LHA) with bilaterally symmetrical distribution (Sakurai et al., 1998). It also existed in the pituitary, pineal gland, adrenal gland, gastrointestinal tract, heart, pancreas and other parts (Sakurai et al., 1998). The fiber of orexin neurons widely projected to the central nervous system, the most important projection area was the paraventricular nuclear (PVN) and central medial nucleus, brainstem and spinal cord. The hypothalamic OXA played a key role in food intake, energy metabolism, arousal/narcolepsy, drug reward responses, and cardiovascular responses (Holmqvist et al., 2005; Stanley et al., 2010). Research revealed that OXA regulate spontaneous phase III-like contractions of the rat stomach (Bülbül et al., 2010b). ⁎ Corresponding author. E-mail address: [email protected] (L. Xu). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.npep.2016.02.005 0143-4179/© 2016 Elsevier Ltd. All rights reserved.

Intravenous administration of OXA stimulated gastric acid secretion and enhanced gastric motility in rats (Bülbül et al., 2010b; Schöne et al., 2011; Yamada et al., 2005). Central and peripheral administration of OXA increased the gastric emptying rate and the frequency of phase III of the interdigestive migrating myoelectric complex (MMC) (Bülbül et al., 2010b). The food intake and gastric motility stimulating effects of OXA were mediated by feeding regulatory hypothalamic centers (Appelbaum et al., 2010). SB334867 is a selective antagonist of orexinA receptors (Rodgers et al., 2001; Yamada et al., 2005). It was the first non-peptide antagonist developed that is selective for the orexin-1 receptor (OXR1), with around 50 × selectivity for OXR1 over OXR2 receptors (Smart et al., 2001). It has been shown to produce sedative and anorectic effects in animals (Haynes et al., 2002), and has been useful in characterizing the orexinergic regulation of brain systems involved with appetite and sleep (Thorpe and Kotz, 2005; Frederick-Duus et al., 2007), as well as other physiological processes (D'anna and Gammie, 2006; Eliassi et al., 2009). Orexin antagonists have multiple potential clinical applications including the treatment of drug addiction, insomnia, obesity and diabetes (Bingham et al., 2006; Borgland et al., 2006; Narita et al., 2006; Lawrence et al., 2006; Sharf et al., 2008). The lateral hypothalamic area (LHA) neurons secreted the neuropeptides melanin-concentrating hormone (MCH) or orexins (OX) and these neurons played important roles in regulating

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ingestion, arousal, locomotor behavior and autonomic function via distinct neuronal circuits (Martin-Fardon and Weiss, 2014; Inutsuka et al., 2014; Tabuchi et al., 2014; Karnani et al., 2011, 2013). Orexin neuronal cell bodies were mainly located in the hypothalamus (Sakurai et al., 1998). The fiber of orexin neurons widely projected throughout the brain suggesting that central OXA action controls a wide array of functions (Petrovich et al., 2012). The paraventricular nucleus (PVN) is an important neuroendocrine and preautonomic output nucleus, and is considered as the important central site for integration of sympathetic nerve activity (Morton et al., 2006; Chaleek et al., 2012). PVN can receive many projections from other parts of the brain (Morrow et al., 1994). Our study is to examine whether exogenous OXA could influence the firing activity of gastric distension (GD) neurons in the LHA, to explore whether administration of OXA might regulate gastric motility in the LHA, and to investigate whether PVN may be involved in the regulation of LHA. 2. Materials and methods

distension by at least 20% from the mean basal firing level. The GD-responsive neurons were further classified into GD-excitatory (GD-E) neurons and GD-inhibitory (GD-I) neurons according to whether the spontaneous discharge increased or decreased with GD, respectively. The change in firing rate of GD-responsive neurons was calculated by 100 × (firing rate of GD-responsive neurons after treatment-firing rate of GD responsive neurons before treatment) / (firing rate of GD-responsive neurons before treatment).

2.2.3. Histochemistry verification After each experiment, pontamine sky blue was injected from the microelectrode to the recorded site by iontophoresis (10 mA, 20 min). The rats were perfused transcardially with 0.9% saline, followed by 10% buffered Formalin solution. The brains were removed; 50 μm thick sections were taken and cannulae and injection tracks were examined with light microscope. Only data obtained from animals whose cannulae and injections were exactly placed in LHA or in PVN were used to analysis. If the recording sites were out of the LHA, the data were excluded from the analysis.

2.1. Animals We used 212 adult male Wistar rats (Qingdao Institute for Drug Control, Shandong, China), weighting 250–300 g. They were housed under a reversed 12 h/12 h light/dark cycle and temperature (22–26 °C) controlled room. Rats were fed with laboratory chow pellets, with free access to water. The study was approved by Animal Care and Use Committee at Qingdao University, and all procedures were performed in accordance with institutional guidelines. 2.2. Electrophysiological experiment 2.2.1. Abdominal and cranial surgery Rats were anesthetized with thiobutabarbitol (100 mg/kg, i.p.; Sigma) and the maintenance anesthetic was given whenever necessary. A balloon was implanted into the stomach, and cranial surgeries were carried out as described previously (Xu et al., 2008). The open part of the brain was covered with warm agar (3% in saline) to improve stability for neuronal recording. A four-barrel glass microelectrode was inserted into the area of LHA (bregma: P: 1.3–2.3 mm, L (R): 1.5–2.5 mm, H: 8.0–9.0 m) (Paxinos and Watson, 2007). 2.2.2. Extracellular electrophysiological recording Four-barrel glass microelectrodes (total tip diameter: 3–10 μm, resistance: 5–15 MΩ) were stereotaxically positioned within the LHA for single-unit recordings and micropressure injection. The recording barrel of the electrode was filled with 0.5 M sodium acetate and 2% pontamine sky blue. The other three barrels connected with a 3-channel pressure injector (PM2000B, Micro Data Instrument, Inc., USA) were filled with a 5 μg solution of orexin-A (Phoenix Pharmaceuticals, Burlingame, CA, USA), a 6 μg solution of SB334867 (Sigma, St Louis, MO, USA), a selective antagonist of orexin-A receptors, and normal saline (NS), respectively. Drugs were ejected onto the surface of firing cells with short-pulse gas pressure (1500 ms, 5.0–15.0 psi). Once the microelectrode was advanced into the area of the LHA, the extracellular action potentials of single unit were recorded. The recorded signals were amplified using MEZ8201 amplifier (Nihon Kohden, Tokyo, Japan) and displayed on an oscilloscope (VC-II, Nihon Kohden, Tokyo, Japan). Electrical signals from the amplifier were input into SUMP-PC bioelectric signal processing system and all data were stored in a computer for subsequent analysis. The frequency of basal firing was determined by the average frequency of 120 s baseline data before drug administration. After the firing pattern had become stable, the unit was tested with a GD stimulus to determine whether there was input from gastric mechanoreceptors. A neuron was identified as a GD-responsive neuron if its mean firing frequency changed via gastric

2.3. Measurement of gastric motility in conscious rats 2.3.1. Group of experiments This study consisted of three parts: Part A (n = 48), to study the effects and potential mechanisms of orexin-A in the LHA on gastric motility. The rats were randomly divided into six groups (n = 8, each group): (a) 0.5 μL NS group; (b) 0.05 μg/0.5 μL orexin-A group; (c) 0.5 μg/0.5 μL orexin-A group; (d) 5.0 μg/0.5 μL orexin-A group; (e) 5 μg/0.5 μL SB334867 group; (f) 5 μg/0.5 μL SB334867 + 0.5 μg/0.5 μL orexin-A group; Part B (n = 48), to explore the effects of electrical stimulation of the PVN on gastric motility. The rats were divided into six groups (n = 8, each group): (a) PVN sham stimulation group (SS); (b) PVN electrical stimulation group (ES); (c) 0.5 μL normal saline (NS) + sham stimulation group (NS + SS); (d) 0.5 μL normal saline + electrical stimulation group (NS + ES); (e) 6 μg/0.5 μL SB334867(SB) + sham stimulation group (SB + SS); (f) 6 μg/0.5 μL SB334867 + electrical stimulation group (SB + ES); Part C (n = 36), to study the effects of orexin-A in the LHA on gastric secretion. The rats were randomly divided into six groups (n = 6, each group): (a) 0.5 μL NS group; (b) 0.05 μg/0.5 μL orexin-A group; (c) 0.5 μg/0.5 μL orexin-A group; (d) 5.0 μg/0.5 μL orexin-A group; (e) 5 μg/0.5 μL SB334867 group; (f) 5 μg/0.5 μL SB334867+ 0.5 μg/0.5 μL orexin-A group.

2.3.2. Animal surgery After fasting for 18 h, rats were anesthetised with thiobutabarbitol (100 mg/kg, i.p.; Sigma) and placed in a stereotaxic frame (Narashige SN-3, Tokyo, Japan). A stainless steel guide cannula (24-gauge) was implanted into the LHA (administration site) or the PVN (stimulation site, from bregma: P, 1.6–1.9 mm, L (R), 0.1–0.7 mm, H, 7.7–8.4 mm) respectively. The injection cannula (29-gauge) was connected to a syringe by a 10-cm piece of polyethylene tubing. A monopolar electrode was put into the cannula placed focally in the PVN for electrical stimulation when necessary. After the cannula was implanted, a midline laparotomy was performed. Briefly, the abdominal cavity was exposed and contractile force transducers were sutured onto the serosa of the gastric antrum, 0.5 cm caudal to the pyloric ring, to measure circular muscle motility (Gong et al., 2013). The lead wires of the force transducers were tunneled subcutaneously and exteriorised at the nape of the neck, protruding 2–3 cm through a small incision between the scapulae. Following this, the abdomen was closed with suture and the animals were allowed to recover for 5 days before administrating chemicals.

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2.3.3. Gastric motility recordings Following an overnight fasting, the rats were acclimatized for 30 min before recording to avoid influence from the new environment at recording area. The rats were moving freely during the recording, and provided with water but deprived of food. Gastric motility was recorded on a polygraph (3066–23; Chengdu Precision Instruments, Sichuan, China). After recording baseline motility for 30 min, orexin-A or SB334867 was slowly injected via the cranial cannula. For the control group, equal volumes of 0.9% saline were injected. The amplitude and frequency of gastric motility were measured. For each animal, recordings were made for 1–2 h per day, and lasted several days with at least 2 day interval. 2.3.4. Measurement of gastric secretion Gastric acid secretion was collected by the pylorus-ligation method as described previously (Nobuhiko et al., 1999). Rats underwent pylorus ligation and were anesthetised with isoflurane, then received LHA injection of orexin-A. One hours later, we collected the gastric juice and determined gastric acid output. The volume of gastric secretion was measured and the amount of gastric acid determined by titration with 0.01 N NaOH to a pH of 7.0. 2.4. Electrical stimulation A monopolar stimulation electrode (RH NE-100 01 × 50 mm; David Kopf Instruments, Tujunga, CA, USA), insulated with epoxy to within 200 μm of the tip, was inserted into the PVN. Stimulation was from a stimulator with a radiofrequency output of square-wave current

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impulses, 20 μA in intensity and 0.5 ms in duration, delivered for 10 s at 50 Hz. 2.5. Statistical analysis All data were expressed as mean ± SD and were processed with SPSS 17.0 statistics software. Paired Student's t-test was used to study the firing rate difference of the same unit neuron before and after treatment. Non-Paired Student's t-test and one-way analysis of variance followed by post hoc Bonferroni's tests were applied to study the comparison among two or multi-set experimental data. P b 0.05 was considered statistically significant. 3. Results 3.1. Electrophysiological experiment 3.1.1. Effects of orexin-A and SB-334867 on discharges of GD-responsive neurons in the LHA 80 rats were used to observe the effects of orexin-A and SB334867 on discharges of GD-responsive neurons in the LHA. 154 neurons were identified as GD neurons. 94 neurons (61.0%) were excited by GD (GD-E), the firing rate increased from 4.15 ± 0.56 to 7.62 ± 0.88 Hz (P b 0.05, Fig. 1A). Orexin-A significantly increased the firing rate of 58 GD-E neurons (61.7%) to 9.34 ± 1.06 Hz (P b 0.05, Fig. 1A,C), with a change rate of 44.6 ± 9.8%. The firing rate of 19 GD-E (20.2%) neurons was decreased (P b 0.05) and 17 GD-E (18.1%) neurons had no change (P N 0.05, Fig. 1A,C).

Fig. 1. Effects of orexin-A and SB334867 administration to the LHA on the discharges of GD-responsive neurons. Orexin-A administration to the LHA increased the firing frequency of GD-E neurons (A) and GD-I neurons (B). (C) The change of firing rate (%) of GD-responsive neurons in the LHA induced by orexin-A. Normal saline or SB334867 alone had no effect on the firing frequency of neurons. After SB334867 pretreatment the effects induced by orexin-A were abolished. Scale bar, 60 s. **P b 0.01 vs. NS and ##P b 0.01 vs. 1orexin-A. Data are shown as mean ± SD. The change of firing rate of GD-responsive neurons was calculated by 100 × (firing rate of GD-responsive neurons after treatment-firing rate of GD-responsive neurons before treatment) / (firing rate of GD-responsive neurons before treatment).

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60 neurons (39.0%) were inhibited by GD (GD-I), exhibiting a lower firing rate from 4.02 ± 0.48 to 2.38 ± 0.36 Hz (P b 0.05, Fig. 1B,C). Orexin-A remarkably increased 43 of GD-I neurons (71.7%) discharging rate to 7.25 ± 0.96 Hz (P b 0.05, Fig. 1B,C), with a change rate of 163.7 ± 5.7%. 11 GD-I neurons (18.3%) were increased (P b 0.05) and 6 GD-I neurons (10.0%) had no change (P N 0.05). After the pre-treatment with SB334867 in the LHA, the orexin-A-induced responses were abolished. Administration of SB334867 alone had no effect on the activity of the GD-responsive neurons in the LHA. Control injection of 0.9% normal saline was always carried out to confirm the specificity of the responses to orexin-A (Fig. 1A,B,C). 3.1.2. Effects of electrical stimulation of the PVN on the discharges of orexin-A responsive GD neurons in the LHA In this experiment, electrical stimulation the PVN was performed to observe the discharge activity of GD neurons in LHA. 46 out of 58 (79.3%) GD-E neurons responsive to orexin-A in the LHA were excited by electrical stimulating the PVN, with firing rate increased from 4.16 ± 0.68 Hz to 9.23 ± 1.24 Hz (P b 0.01, Fig. 2A,C), with a change rate of 43.8 ± 9.6%.7 GD-E neurons were inhibited (P b 0.05) and 5 had no response (P N 0.05, Fig. 2A,C). Out of 43 GD-I neurons responsive to orexin-A, 29 neurons (67.4%) were also excited by electrical stimulating the PVN, and firing rate increased from 4.22 ± 0.55 Hz to 9.76 ± 1.03 Hz(P b 0.01, Fig. 2B,C), with a change rate of 38.2 ± 9.6%,while 8 were inhibited (P b 0.05) and 6 had no change (P N 0.05, Fig. 2B,C). Meanwhile, the electrical stimulating PVN induced responses were diminished by pre-treatment with orexin-A receptor antagonist

SB334867 in the LHA for both orexin-A responsive GD-E (Fig. 2A) and GD-I neurons (Fig. 2B,C). 3.2. Gastric motility recordings 3.2.1. Effects of orexin-A on gastric motility in the LHA Orexin-A administered to the LHA dose-dependently increased the amplitude and frequency of gastric motility recordings compared with the saline group (Figs. 3, 4). The increase in amplitude and frequency after injection of 0.05 μg, 0.5 μg, 5.0 μg orexin-A had a latency of 5 min and lasted for around 5–10, 10–15, 10–20 min respectively (Figs. 3, 4). However, there was no effect after administration of 1.0 μL mixture of 0.5 μg orexin-A and 5.0 μg SB334867 (Figs. 3, 4). Meanwhile, when given SB334867 or saline alone, the amplitude and frequency showed no significant change. 3.2.2. Effects of electrical stimulation of the PVN on gastric motility In this experiment, monopolar concentric electrodes were advanced into the PVN to elicit electrical stimulation and the stimulus parameters had been described above. Sham stimulation was conducted as the same procedure with electrical stimulation but no current was passed through the electrode. NS or SB334867 was injected to the LHA singly or before sham or electrical stimulation whenever necessary. Gastric motility in conscious rats was recorded for 60 min. Results showed that the amplitude and frequency increased with a latency of 3 min after electrical stimulation of the PVN and reached the peak at around 13 min (Figs. 5, 6A,B). The promotional effect induced by electrical

Fig. 2. Effects of electrical stimulation of the PVN on the discharges of orexin-A-responsive GD neurons in the LHA. Both orexin-A-responsive GD-E neurons (A) and GD-I neurons (B) were mostly excited by electrical stimulation of the PVN. However, pretreatment with SB334867 in the LHA could partially diminish the responses induced by electrically stimulating the PVN. Scale bar, 60 s. The change of firing rate (%) of orexin-A-responsive GD neurons in the LHA induced by electrical stimulation of the PVN was shown in (C). #P b 0.05 vs. 1stimulation. Data are shown as mean ± SD.

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Fig. 3. Effects of different dose of orexin-A on gastric motility in the LHA. There was a latency of ~5 min after injection of orexin-A to the LHA. When orexin-A was injected to the LHA, the amplitude of the gastric contractions increased(B). Increasing the dose of injected orexin-A led to a further increase in gastric motility(C and D). Compared with the NS group(A), there was no significant difference after injection of SB334867 alone or a mixture of orexin-A and SB334867(E and F).

stimulation of the PVN was partly blocked (P b 0.05) by administration of SB334867 in the LHA (Figs. 5, 6A,B). However, administration of NS or SB334867 to the LHA alone had no significant effect on the gastric motility.

3.2.3. Effects of orexin-A on gastric secretion in the LHA Representative responses of gastric acid secretion are shown in Fig. 7. LHA injection of orexin-A induced dose-dependent increase of gastric acid secretion compared with the saline group. The gastric juice volume was

Fig. 4. Effects of orexin-A on the amplitude and frequency of gastric motility in the LHA. After administration of orexin-A in the LHA, the amplitude and frequency of the gastric contraction significantly increased. The amplitude (A) and frequency (B) of gastric motility induced by orexin-A increased dose-dependently compared to the saline control group. n = 8, *P b 0.05, **P b 0.01 versus NS group; ΔP b 0.05, ΔΔP b 0.01 versus 0.05 μg orexin-A group, and #P b 0.05, ##P b 0.01 versus 0.5 μg orexin-A group. Data are expressed as mean ± SD. Percentage of the frequency and amplitude change was derived from the equation: frequency or amplitude change = (frequency or amplitude after microinjection-frequency or amplitude before microinjection) / frequency or amplitude before microinjection × 100%.

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Fig. 5. Effects of electrical stimulation of the PVN on the gastric motility. The amplitude of gastric motility increased after electrical stimulation of the PVN(B). The excitatory effect induced by electrical stimulation (D) was reduced by injecting SB334867 to the LHA (F). Gastric motility showed no change in the SS, NS + SS or SB334867 + SS groups (A, C and E).

observed after orexin-A injections into the LHA (Fig. 7A). Fig. 7(B) shows the gastric acid output pattern over time following microinjection of orexin-A into the LHA. However, there was no significant change after injection of 1.0 μL mixture of 0.5 μg orexin-A and 5 μg SB334867 (Fig. 7). When given SB334867 alone, the gastric secretion showed no remarkably change compared with the saline group. 4. Discussion We have previously demonstrated that neurons in the LHA are sensitive to gastric distension (Xu et al., 2008). In our present study,

the data have shown that orexin-A administered to the LHA could excite most of these neurons, including GD-E and GDI neurons and promote gastric motility, which could be eliminated by SB334867 pretreatment. These data indicate that LHA could receive signals from the gastrointestinal tract and that exogenous orexin-A could promote gastric motility via its receptor in the LHA. Krowicki et al. (2002) have demonstrated that orexin acts in the dorsal motor nucleus in the medulla to increase gastric contractility in rats. About 15% orexin-A-positive neurons in the lateral hypothalamic area project to the dorsal vagal complex. Therefore, we speculate gastric-distension responsive neurons in LHA may affect neuronal activity of other brain areas and finally regulates

Fig. 6. Effects of electrical stimulation of the PVN on the amplitudes and frequencies of gastric motility. After electrical stimulating the PVN, the amplitude (A) and frequency (B) of the gastric contraction increased compared with the SS group. The excited effect was partly blocked by administration of SB334867 in LHA before electrical stimulating PVN. n = 8, *P b 0.05, **P b 0.01 versus SS group; #P b 0.05, ##P b 0.01 versus SB + SS group; ΔP b 0.05, ΔΔP b 0.01 versus NS + SS group. Data are represented as mean ± SD.

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Fig. 7. Stimulatory effects of LHA injection of orexin-A on (A) juice volume, (B) gastric acid secretion. LHA injection of orexin-A induced dose-dependent increase of gastric acid secretion compared with the saline group. However, there was no remarkably difference after injection SB334867 alone or yya mixture of 0.5 μg orexin-A and 5 μg SB334867 (Fig. 7). *P b 0.05, **P b 0.01 versus NS group.

the function of neurons in brain stem area such as dorsal motor nucleus to directly innervate gut. When orexin-A injection into the LHA could excite the dorsal motor nucleus, and then orexin-A may enhance the gastric contractility via the vagal pathways. In addition, the firing frequency of GD neurons was increased dramatically by injection of orexin-A in the LHA. It indicated that orexin-A might modulate the gastric motility perhaps via changing the excitability of the LHA neurons. Our study showed that electrical stimulation of the PVN excited most of the orexin-A-responsive GD neurons in the LHA. Nonetheless, pretreatment with SB334867 in the LHA decreased the firing rate of orexin-A-responsive GD-E neurons and GD-I neurons following by electrical stimulation of the PVN. These results indicate that neurons in the PVN may regulate the activity of GD-responsive neurons in the LHA. LHA is an important center for regulating appetite, food intake, and gastric functions (Sheng et al., 2014; Goforth et al., 2014). The orexigenic orexin-A was expressed in the feeding related hypothalamus nuclei, such as the LHA, the PVN, and the ARC (Louis et al., 2010; Anderson et al., 2014). The function of orexin-A in the LHA has been studied by many research (Tabuchi et al., 2014), while little work has focused on the effect of orexin-A on the excitability of GD neurons in the LHA. Our results showed that administration of orexin-A in the LHA could excite the GD-E neurons and GD-I neurons. It suggested that LHA could receive the afferent information from stomach, and orexin-A may regulate the excitability of GD neurons in the LHA. Bülbül et al. reported that i.c.v. injection of orexin-A promoted gastric motility (Bülbül et al., 2010b). Several experiments also had shown that i.c.v. injection of orexin-A accelerates stomach emptying in rodents (Ehrström et al., 2005b; Bülbül et al., 2010c) and suppressed gastroduodenal motility in mice (Ehrström et al., 2005a). Bülbül M reported central orexin-A modulated postprandial gastric contractions of rats in non-stressed conditions (Bülbül et al., 2010a). Nozu et al. discovered that endogenous orexin-A in the brain was involved in the vagal-dependent stimulation of gastric contractions (Nozu et al., 2012). Some research evidence showed that brain orexin-A played a role in the pathophysiology of functional gastrointestinal disorders (Okumura and Nozu, 2011).

Orexin-A also regulated the feeding behavior of rats (Choi et al., 2010; Inutsuka et al., 2014; Thorpe and Kotz, 2005). PVN is an important integrated nucleus, which receives afferent inputs from the other sites including the LHA, ARC, subfornical organ, organum vasculosum of the lamina terminalis, medial preoptic area, and suprachiasmatic nucleus (Morton et al., 2006; Chaleek et al., 2012; Morrow et al., 1994). The LHA is considered as one of the most extensively interconnected areas of the hypothalamus (Tabuchi et al., 2014). The LHA can receive and consolidate a vast array of interoceptive and exteroceptive information and widely project to several areas containing the amygdala, hippocampal formation, arcuate, dorsomedial as well as the paraventricular (Gong et al., 2013; Sheng et al., 2014). It has been observed that microinjection of orexin-A into the LHA increased feeding and wakefulness (Inutsuka et al., 2014) as well as induced c-fos immunoreactivity in the LHA and in other regions involved in feeding control (Anderson et al., 2014; Inutsuka et al., 2014). Eliass reported that endogenous orexin-A acts on the ventromedial hypothalamus to stimulate acid secretion (Eliassi et al., 2009). These findings revealed that LHA may be a vital center for the mediation of gastric motility and gastric secretion. In the current study, the retrograde tracing combining and immunofluorescent trials showed orexin-A and FG double-labeled neurons in the PVN after intra-LHA injection of the FG, implying the orexin-A neural projections from the PVN to the LHA. Subsequently, the function of projection from the PVN to the LHA was investigated with electrical physiological techniques (Larsen et al., 1994). As expected, electrical stimulation of the PVN considerably increased the discharges of GD-responsive neurons in the LHA and accelerated gastric motility. Nonetheless, pretreatment with the SB334867 in the LHA diminished either the discharges of some GD-E neurons and GD-I neurons or gastric motility induced by electrical stimulation of the PVN. These results revealed that the PVN played a role in the regulation of LHA neurons on gastric motility. Our data also suggested that orexin-A was one of the orexigenic peptides involved in the mediation on the gastric motility and gastric secretion. In our study, the results indicated that orexin-A administered in the LHA played a vital role in the regulation of gastric motility and the PVN may participate in the regulatory process. Orexin-A as a neuropeptide,

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