Orexin prevents depressive-like behavior by promoting stress resilience

Hypothalamic neuropeptide orexin has been implicated in the pathophysiology of psychiatric disorders and accumulating clinical evidence indicates a potential link between orexin and depression. However, the exact role of orexin in depression, particularly the underlying neural substrates and mechanisms, remains unknown. In this study, we reveal a direct projection from the hypothalamic orexinergic neurons to the ventral pallidum (VP), a structure that receives an increasing attention for its critical position in rewarding processing, stress responses, and depression. We find that orexin directly excites GABAergic VP neurons and prevents depressive-like behaviors in rats. Two orexin receptors, OX1R and OX2R, and their downstream Na+–Ca2+ exchanger and L-type Ca2+ channel co-mediate the effect of orexin. Furthermore, pharmacological blockade or genetic knockdown of orexin receptors in VP increases depressive-like behaviors in forced swim test and sucrose preference test. Intriguingly, blockage of orexinergic inputs in VP has no impact on social proximity in social interaction test between novel partners, but remarkably strengthens social avoidance under an acute psychosocial stress triggered by social rank. Notably, a significantly increased orexin level in VP is accompanied by an increase in serum corticosterone in animals exposed to acute stresses, including forced swimming, food/water deprivation and social rank stress, rather than non-stress situations. These results suggest that endogenous orexinergic modulation on VP is especially critical for protecting against depressive reactions to stressful events. The findings define an indispensable role for the central orexinergic system in preventing depression by promoting stress resilience.


Stereotaxic surgery.
Adult male rats were anesthetized using sodium pentobarbital (40 mg/kg, i.p.) and mounted on a stereotaxic frame (1404, David Kopf Instruments, Tujunga, CA) for stereotactic brain surgery under aseptic conditions. Throughout the surgery, body temperature was monitored by a rectal thermistor probe and maintained at 36-38°C on a heating pad. The scalp was incised to expose the skull. Small holes were drilled in the skull above the targeted coordinates according to the rat brain atlas of Paxinos and Watson 1 , and the dura was gently broken to allow uninterrupted passage of Hamilton syringes or microinjection cannulae.

Intra-VP lentivirus microinjection. The concentrated lentivirus was delivered into
the VP bilaterally (A 0.0 --0.3, L 2.5, and H 7.6) using a 1 µl Hamilton syringe with a thin 25-gauge metal needle, and the injection was driven by an infusion pump (KDS100, KD Scientific, Holliston, MA; the injection volume and flow rate 1 μl at 0.1 μl/min).
After the injection, the needle was left in place for 10 additional minutes and then slowly withdrawn. The rats treated with lentivirus were caged individually and allowed to recover for 14 days before further behavioral studies. eGFP positivity in the VP was After the implantation, animals were caged individually and allowed to recover for at least 3 days. During the behavioral testing sessions, two stainless-steel injection tubes (length 13mm, o.d. 0.5 mm, i.d. 0.3 mm) were inserted to protrude 2 mm beyond the tip of the guide tubes and just above the VP (to minimize lesioning the nuclei) for microinjection of orexin A (1 μM, Tocris, Bristol, UK), TCS1102 (20 nM, Tocris), and saline (0.9 % NaCl) using Hamilton syringes (0.5 μl each side, lasting 2 min). The effective extent of the drug diffusion in the present study was estimated by using extracellular electrophysiological recording units 0.5-2.0 mm away from the injection site according to our previous reports 2, 3 and restricted in the VPs.

Behavioral assessments
Forced swim test. The forced swim test took place in a vertical glass cylinder (50 cm in height × 20 cm in diameter) containing 35 cm of freshwater at 25 ± 2 °C. In the 5 first day, the rats were forced to swim for 10 min and thereafter dried with heater. After 24 h, rats were re-exposed to forced swimming for 10 min and behavior was videotaped.
The total time spent immobile were measured by video analysis (TopView Animal Behavior Analyzing System; CleverSys Inc, Reston, VA).

Sucrose preference test.
Rats were habituated to one bottle of water and one bottle of 1% sucrose in home cages for 1 week. Sucrose consumption was then measured for one-hour preceded by a 20 hours food and water deprivation. The amount of fluid intake was measured by weighing the bottles before and after the one-hour test. The preference score was calculated as sucrose intake relative to total fluid intake. The sucrose preference baseline was averaged from two baseline fluid intake tests performed before rat grouped and separated by at least 5 days. Sucrose preference tests were conducted immediately after drug microinjection or weekly after lentivirus infusion.
Open field test. Rats were tested in a sound-isolated, dimly illuminated room (20 lux) in an open-field box (50 cm × 50 cm). Rats were allowed to explore the box freely and their behavior was recorded for 10 min. The number of times rats rearing and the total distance they travelled were calculated (TopView Animal Behavior Analyzing System).
Novel social proximity test. In each test, two rats that met for the first time were randomly selected from separate cages. The tested rat was placed in the center of a 6 plastic open field apparatus (50 × 50 cm) simultaneously with the other novel partner or a cloud of cotton which was put in an iron cage (16 × 22 cm) in one corner of the arena. Time of head approaching and staying close to the cage (≤ 5 cm) and the times of forepaws climbing the cage in 10 minutes were recorded and calculated (TopView Animal Behavior Analyzing System).

Social interaction test under psychosocial stress. A pair of rats in similar weight
were simultaneously loaded into the opposite ends of a plexiglass pipe tube (length 60 cm, o.d. 72 mm, i.d. 68 mm) and held until both rats strived to move forward. A trial will last until one of the rats is pushed out, or for a maximum of 5 minutes. The winner for 3 repeated trials was considered as a dominator and the losing rat as subordinate.
The pair of rats was conducted to test social proximity 15 min after the tube test, in which the caged dominator was introduced as a psychosocial stressor for subordinate.
After behavioral tests, the animals were deeply anaesthetized and the microinjection sites were histologically identified as we previously reports. 3,4 Data from rats in which the injection sites were deviated from the VP were excluded from further analysis.

Quantitative real time RT-PCR
In this experiment, three independent groups of RNA pools each from five adult 7 animals were used as biological replicates. VP tissue punches were collected from coronal brain slices according to the rat brain atlas of Paxinos and Watson 1

Retrograde tracing with Fluoro-Gold
For retrograde tracing experiments, stereotaxic surgery was conducted on adult rats as described above. 4  Experimental conditions were repeated three times to account for technical and biological variation.

Immunohistochemistry
Adult rats were given an overdose of sodium pentobarbital and perfused transcardially with 100 ml of saline, followed by 450-500 ml of 4% sodium phosphate-

Whole-cell patch clamp recordings on brain slices
Thirty-seven adult rats of either sex were decapitated under sodium pentobarbital anaesthesia. After decapitation under sodium pentobarbital (40 mg/kg) anesthesia, the coronal slices (300 μM in thickness) containing VP were obtained according to the rat brain atlas 1  used to discriminate the specific type of VGCCs coupled to orexin receptors. Besides, the selective Na + channel blocker tetrodotoxin (TTX, 0.3 μM), the relatively selective A-type K + channel blocker 4-aminopyridine (4-AP, Sigma) at a low concentration (500 μM), as well as the K + channel blocker tetraethylammonium chloride (TEA, 20 mM; Sigma) and Cs + (5 mM, Sigma), were employed to eliminate Na + and K + component in isolating Ca 2+ current. Moreover, Cs + -based internal pipette solution was used to block K + channels from the inside of membrane. Cs 2+ -based pipette solution was composed of the following (in mM): 100 CsCl, 20 TEACl, 5 MgCl2, 2 BAPTA, 10 HEPES, 5 Na2ATP, 0.5 Na3GTP, and CsOH/HCl for pH 7.3. Sucrose was added to bring the 14 osmolality to 305 mOsm.

Sample processing for ELISA
Fifteen minutes after behavioral test, the rats were anesthetized with isoflurane then drawn blood from eyeball and decapitated. Blood was collected in serum tubes and brain tissue sample was dissected similar to those described in our previous publication. 5, 6 For the blood sample, blood was allowed to coagulate at room temperature in serum tubes for 1 hour then centrifuged 3000 rpm for 10 min. Serum was aliquoted and stored at -80 °C. For the brain tissue sample collected from the VP, tissue was weighed and immersed in 0.5 M acetic acid and then boiled for 10 min. The VP samples were centrifuged for 30 s at 5000 rpm and the supernatants were air dried under a hood overnight, and the dried samples were subsequently stored at -80 °C.
Corticosterone kit (KGE009; R&D Systems Inc., Minneapolis, MN, USA) and orexin-A kit (FEK-003-30; Phoenix Pharmaceuticals, Dayton, OH, USA) were used for the ELISA testing. Upon completion of the assays, the fluorescence intensities of the 96 well microplates were read by an assay reader (Tecan, Männedorf, Switzerland).

Experimental design and statistical analysis
All experiments and data analysis were performed blind to the conditions of the experiments. In behavioral experiments, the animals were randomly grouped by 15 different treatments. Sample sizes were based on previous experiments. 3,4,7,8 All data were analyzed with SPSS 17.0 and presented as median (horizontal bar) with 25th-75th (box) and 10th-90th (whiskers) percentiles. Data were tested for normal distribution and homogeneity of variance. Two-tailed unpaired and paired Student's t test, one-way, two-way and repeated measures two-way analysis of variance (ANOVA), and post hoc Bonferroni-corrected t test was employed for statistical analysis. P-values of < 0.05 were considered to be significant. significantly decreased ox1r mRNA in VP (n = 5), so did LV-shOX2R-eGFP (n = 5).