Activation of Alpha7 Nicotinic Acetylcholine Receptors Reduce Chronic Pain-Induced Depression-Like Behaviors Via WNT/β-Catenin Pathway

Chronic pain frequently leads to depression and microglia-related inammation is the common mechanism for chronic pain and depression. Activation of alpha 7 nicotinic acetylcholine (α7 nAch) receptor suppresses microglia-related inammation. Thus, we evaluated the activation of α7 nAch receptors on chronic pain-induced depression-like behaviors and explored the potential mechanisms. Methods: Male Wistar rats that received spared nerve injury (SNI) and complete Freund’s adjuvant (CFA) into the unilateral ankle articular cavity received Open eld, sucrose preference, and pain behavior tests. The effect of intracerebroventricular (ICV) injection of α7 nAch receptor agonist PHA and α7 nAch receptor antagonist on depression-like behaviors and cytokines expression was examined. Cytokines were measured in the brain using Enzyme-linked immune absorbent assay. Results: SNI surgery and CFA injection induced depression-like behaviors, and ICV injection of PHA reduced that depression-like behaviors. SNI surgery and CFA injection skewed the medial prefrontal cortex, basolateral amygdala, and ventral hippocampus to a pro-inammatory response and ICV injection of PHA shift brain from pro-inammatory to anti-inammatory response in that sites. The anti-depressive effect and anti-inammatory effect of PHA were reversed by WNT/β-catenin inhibitor. Conclusions: Activation of alpha7 nicotinic acetylcholine receptors reduce chronic pain-induced depression-like behaviors, the mechanisms may be attributed to the activation of WNT/β-catenin pathway and suppression of brain inammation.


Background
Chronic pain is detrimental to human beings and frequently causes psychiatric disorders such as depression [1]. Although effective treatments are available, about one third of patients with depression fail to respond to conventional antidepressant therapies [2], which lead to searching new therapeutic approaches. Recently, depression has been regarded as an in ammatory disease, because suppression of in ammation improved the depressive behaviors. The proin ammatory cytokines affect neuronal activity and results in depression-like behaviors. The in ammation mainly comes from microglia, as minocycline, a microglial inhibitor suppresses brain in ammation and alleviates the depressive behaviors [3]. Thus, targeting microglial-related in ammation is an effective approach to managing psychiatric illness. Alpha 7 nicotinic acetylcholine (α7 nAch) receptor, a subtype of nicotinic acetylcholine receptors, is involved in a variety of biological functions such as synaptic plasticity and neuronal survival; and is considered a prominent therapeutic target in neuropathological diseases [4]. Microglia, an immune cell in the central nervous system, express α7 nAch receptors, and by targeting α7 nAch receptor could change the microglia status and resolve microglial-related neuroin ammation [5]. In recent years, "cholinergic antiin ammatory pathway" has been demonstrated to regulate central nervous in ammation via the α7 nAch receptor of microglia [6]. Activation of microglia α7 nAch receptor also protect the brain from various insults by decreasing the proin ammatory effect of microglia [7]. Thus, activation of α7 nAch receptor may have anti-psychiatric effect.
PHA-543613 (PHA) is an α7 nAch receptor agonist, and we have previously shown that PHA prevent the development and maintenance of neuropathic pain by suppression of microglia-related in ammation [8]. Since chronic pain frequently results in depression and they share a common biological change of in ammation in the central nervous [9], whether targeting α7 nAch receptors can be used for treating chronic pain-induced depression remain to be clari ed. We design this experiment to test the effect of ICV (intracerebroventricular) injection of α7 nAch receptor agonist PHA on the depression-like behaviors induced by SNI surgery and CFA injection and explore the potential signal pathways involved in the antidepressive effect of α7 nAch receptor activation.

Animals and drugs preparation
Our experiments were performed in accordance with the Animal Care and use Committee of Shanghai Jiao Tong University and animal care guidelines of the National Institutes of Health. Male Wistar rats were obtained from the Shanghai Experimental Animal Institute and were kept in a temperature and humidity-controlled environment on a 12-h light/dark cycle (light on between 8:00 AM. to 18:00 P.M.) with ad libitum access to food and water. The α7 nAchR agonist, PHA, and its antagonist, methyllycaconitine citrate (MLA) were purchased from Tocris Biosciences (Minneapolis, USA). PHA was dissolved in 5% dimethylsulfoxide (DMSO) at a dosage of 4 μg in a volume of 4 μL. MLA was dissolved in normal saline and prepared at a dosage of 4 μg in a volume of 4 μL. For the control group, an equal volume of 5% DMSO was used according to our previous study. Minocycline hydrochloride (purchased from Sigma-Aldrich Corp, St Louis, USA) was dissolved in normal saline with a concentration of 40μg/μL. SB431542 (a TGFβ inhibitor; Sigma-Aldrich, St. Louis, USA) were dissolved in 4% DMSO (4 µl, 5 nmol) and used for ICV injection [10]. AZD8055(a mTOR inhibitor; Sigma-Aldrich, St. Louis, USA) was prepared 10 mg/mL with 4% DMSO as vehicle and used 4 µL for ICV injection [11]. IWR-1-endo [12] (IWR, a WNT/β-catenin inhibitor), Numb [13] (a notch inhibitor) and AZD1480 [14] (a JAK inhibitor) were bought from Sigma-Aldrich, St. Louis, USA and were dissolved in 4% DMSO with a concentration of 5 uM and a volume of 4 µL was used for ICV injection.
Animal models establishments Spared nerve injury (SNI) surgery was performed according to our previous study [15] . Brie y, rats received general anesthesia with iso urane 5% induction and 2.5% maintenance using an inhalational anesthesia system (Penlon Sigma Delta, Louisville, KY, USA). The rats were xed on an operation table and the sciatic nerve was exposed at the lateral thigh with an animal surgery microscope (Leica M205FA, Germany); the tibia and common peroneal branches were dissected and tightly ligated with 6-0 absorbable suture and transected, leaving the sural nerve intact. The sciatic nerve of the sham group was only exposed, without ligation and transection. Then the skin was sutured with 5-0 sutures. The rat was returned to their cages for recovery as the surgery was over.
Monoarthritic model was induced by an injection of complete Freund's adjuvant (CFA) into the unilateral ankle articular cavity [16]. The experimental rats were anesthetized with iso urane prior to inducing arthritis. Then the skin around the site of injection was sterilized with 75% alcohol and the right leg of the rat was held, and the fossa of the lateral malleolus of the bula was located. A 28-gauge needle was used vertically to penetrate the skin, and turned distally to insert into the articular cavity from the gap between the tibio bular and tarsus bone until a distinct loss of resistance. Then, a volume of 50 ul of CFA was injected. For the sham group, the same amount of normal saline was injected into the right ankle articular cavity.
For the food intake evaluation, 50g food was provided for each rat in one cage and determined at the indicated day for 24hs.The amount of food remaining after 24hs was individually weighed and subtracted from the original quantity provided. Body weight was recorded before surgery or stimulation, before and after medical intervention. All rats were free access to water.

Pain behavior tests
For mechanical allodynia, the values were measured according to our previous study [17]. Brie y, the rats were placed in a Plexiglas box on a mesh oor 50cm above the table. After 15 min of acclimation, the mechanical withdrawal threshold was measured with an electronic apparatus (IITC Life Science, Woodland Hills, CA, USA). An increasing force was used to stimulate the lateral side of the ipsilateral hind paw. As an immediate paw withdrawal was observed, the value was recorded; the mean value was averaged from 3 repeated measures with an interval of 5 min.
Response rate to cold acetone was done according to our previous study [17]. The positive response was recorded as withdrawal or licking or shaking of the hind paw to a drop of acetone applied to the ventral surface of the paw. The acetone response was repeated for 5 times and the response rate was calculated.

Depression-like behavior tests
The open eld test was done according to our previous study [17]. The rat was placed in a square arena (50 × 50 ×50 cm), which was divided into two areas: a peripheral area and a central (17 × 17 cm) square area. The center distance and total distance were recorded in the experiment, and center/total distance duration were calculated as measures of depression-like behavior, and total running distance was recorded as a measure of general motor behavior. The running track was recorded for 5 min by a camera 2 m above the box. The eld was cleaned with 75% ethanol after each test.
The sucrose preference experiment followed a previous study with minor modi cations [17]. Brie y, all of the rats were habituated with 1% sucrose for 48 h before the experiments. On the day of the experiment, each rat received an 8-h uid and food deprivation and then was exposed to two bottles for 1 h, one lled with 1% sucrose and the other tap water. The sucrose intake percentage was calculated with a formula: (sucrose/(sucrose + water)) × 100%.

Intracerebroventricular catheter placement and injection
Intracerebroventricular (ICV) catheter placement and injection followed the procedures used in our previous study [17]. Brie y, rats were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg). Then, the rat was xed on a stereotaxic apparatus. The surgery was done and a catheter was placed with stereotaxic coordinates: 0.8 mm posterior, 1.5 mm left lateral, and 4.5 mm ventral from the bregma. The guide cannula was xed with dental cement and the rat was returned to individual cage for recovery. The ICV cannulation was veri ed by injection of methylene blue through the cannula after experiment. For each daily injection, rats were anesthetized with iso urane as above; rats received bilateral microinjection of PHA and inhibitors or minocycline (160 μg) or saline (control) into the cerebral ventricle. A total volume of 4.0 μl was infused into each side over 10 min, and the injection syringe was left in place for an additional 5 min to allow for diffusion. The microinjections were performed daily for six days, followed by behavior tests.
In ammatory cytokines measurements with enzyme-linked immune-absorbent assay After the last behavior tests, rats were deeply anesthetized with pentobarbital sodium (100 mg/kg) followed by decapitation according to our previous study [17]. Tissues were collected for ELISA followed our previously study. Brie y, the brains were placed in a chilled matrix and micro-dissected on a chilled glass plate. The medial prefrontal cortex (mPFC) was obtained from a 2-mm-thick slice ranging from approximately 5 to 3 mm anterior of Bregma. The ventral hippocampus (VH) was isolated from a 4-mmthick section ranging from approximately 3.2 to 7.2 mm posterior of Bregma and was separated from cortex and underlying brain structures. The basolateral amygdala (BA) was dissected out ranging from approximately 1.2 to 3.2 mm posterior of Bregma just lateral to the optic tracks. After the samples were collected, these tissues were homogenized with normal saline and centrifuged at 4000 rpm, 4 °C for 10 min. The supernatants were used for IL-10, IL-1β, TNF-α and IL-18 assay using ELISA kits (Nanjingjiancheng Biocompany, Nanjing, China) and the procedures followed the manufacturer's instructions

Statistical analysis
The data are shown as mean ± SD and were analyzed with one-way or two-way analysis of variance (ANOVA) with repeated measures. Bonferroni post hoc analysis was conducted if a statistically signi cant difference was found. Nonparametric Wilcoxon or unpaired "t" test was performed for comparison of non-repeated measures. P < 0.05 was considered statistically signi cant. The statistical analysis was performed using GraphPad Prism software (GraphPad Prism 5.0, GraphPad Software Inc., San Diego, CA, USA).

SNI surgery induced depression-like behaviors after surgery
The depression-like behaviors were measured from POD14 onward, and the results showed that SNI surgery decreased center distance and center/total distance compared to sham group in the open eld test from POD7 to POD 21 after surgery ( Fig 1A and C respectively). SNI surgery did not affect total running distance in the open eld test after surgery ( Fig 1B). In the sucrose preference test, SNI decreased sucrose intake percentage from POD7 to POD21 (Fig 1D). In addition, SNI decreased food intake and weight gain compared to sham after surgery from POD7 to POD21 (Fig 1 E and F respectively). The paw withdrawal threshold and response to acetone were recorded before and after SNI surgery. The results showed that SNI surgery decreased paw withdrawal threshold and increased response to acetone after surgery from POD7 to POD21 (Fig 1 G and H respectively).
ICV injection of α7 nAch receptor agonist PHA and microglia inhibitor MIN decreased depression-like behaviors after SNI surgery The behavior results showed that SNI surgery decreased center distance and center/total distance from POD10 to POD18, and ICV PHA or MIN improved that decrement of center distance and center/total distance on POD18 (Fig 2A and C respectively). SNI decreased sucrose intake percentage from POD10 to POD18 and ICV PHA or MIN decreased that effect on POD18 (Fig 2D). SNI surgery decreased food intake and weight gain from POD10 to POD18 compared to sham group, and ICV PHA or MIN decreased that effect on POD18 (Fig 2E and F respectively). Although SNI decreased paw withdrawal threshold and increased response to acetone, ICV PHA or MIN did not affect paw withdrawal threshold and response to acetone (Fig 2G and H respectively). The time course for surgery and experiment was shown in Fig 2I. PHA decreased depression-like behaviors after SNI surgery by the activation of α7 nAch receptors and suppression of brain in ammation The results showed that ICV co-administration of α7 nAch receptor antagonist MLA decreased antidepressive effect of PHA in center distance and center/total distance in the open eld test (Fig 3A and C respectively) and the total distance running was not different between groups after SNI surgery on POD18 ( Fig 3B). PHA increased sucrose intake percentage and food intake and weight gain after SNI, while coadministration MLA decreased that effect (Fig 3D, E and F respectively). Both MLA did not affect the effect of PHA on paw withdrawal threshold and response to acetone (Fig 3G and H respectively).
In addition, ICV PHA increased IL-10 in the mPFC, BA and VH compared to SNI+Vehicle group, and MLA decreased that effect (Fig 4A, E and I respectively). However, SNI increased IL-1β in the mPFC, BA and VH compared to Naive group, and ICV PHA decreased IL-1β in the mPFC, BA and VH compared to SNI+Vehicle group, while MLA decreased the effect of PHA (Fig 4B, F  and VH compared to Naive group, and ICV PHA decreased their content in the mPFC, BA and VH compared to SNI+Vehicle group, while MLA decreased the effects of PHA. PHA decreased depressive-like behaviors after CFA injection by the activation of α7 nAch receptors and suppression of brain in ammation In the CFA experiment, the CFA injection and ICV cannulation were performed on the same day after anesthesia. The results showed that CFA decreased center distance and center/total distance in the open eld test, and did affect total running distance moved, compared to naïve group, and PHA improved the behaviors in the open led tests, whereas MLA decreased the effect of PHA (Fig 5A, B and C respectively). CFA decreased sucrose intake percentage, food intake and weight gain, and PHA reversed that effect, and the effects was abrogated by MLA (Fig 5D, E and I respectively). CFA decreased mechanical anodynia and increased response to cold, whereas ICV PHA and MLA did not affect that response (Fig 5G and H  respectively). The research protocol was shown in Fig 5I. CFA did not affect IL-10 content in the mPFC BA and VH, and ICV PHA increased IL-10 content in the mPFC and BA, and the effect was decreased by MLA (Fig 6A). CFA increased IL-1β content in the mPFC BA and VH, and ICV PHA decreased IL-1β increment in that sites, and the effect was decreased by MLA ( Fig 6B). CFA increased TNF-α content in the mPFC BA and VH, and ICV PHA decreased TNF-α content in the mPFC, BA and VH, the effect was abolished by MLA ( Fig 6C). CFA increased IL-18 content in the mPFC BA and VH, and ICV PHA decreased IL-18 content in the mPFC and VH, while the effect of PHA was decreased by MLA (Fig 6D).
PHA decreased depressive-like behaviors via WNT/β-catenin signal pathway PHA increased center distance, center/total distance ratio, sucrose intake percentage, food intake and weight gain after SNI surgery and signal pathway inhibitors of TGFβ, mTOR, WNT/β, notch, JAK were coadministered to explore the potential anti-depressive mechanism of PHA. The results found WNT/βcatenin inhibitor IWR reversed the anti-depressive effect of PHA on center distance, center/total distance ratio, sucrose intake percentage, food intake percentage and weight gain (Fig 7A, B, C, D, E and F respectively). Surprisingly, mTOR inhibitor AZD8055 also reversed the effect of PHA on food intake percentage and weight gain (Fig 7E and F respectively). And TGFβ, notch and JAK pathway inhibitors did not affect anti-depressive behaviors of PHA.
PHA decreased brain in ammation via WNT/β-catenin and mTOR pathway The cytokines were measured after behavior tests. The results showed that PHA increased IL-10 and decreased IL-18 in mPFC, BA and VH induced after SNI surgery, and WNT/β-catenin inhibitor IWR reversed those effect (Fig 8A and D respectively). In addition, PHA decreased IL-1β content in mPFC and VH after SNI surgery, however, the change was reversed by mTOR inhibitor AZD8055 and WNT/β-catenin inhibitor IWR (Fig 8B). Similarly, PHA decreased the content of TNF in mPFC, BA and VH after SNI surgery, and the content change of TNFαin mPFC and BA was reversed by mTOR inhibitor AZD8055 and WNT/β-catenin inhibitor IWR (Fig 8C).

Discussion
Our results showed that ICV α7 nAch receptor agonists PHA decreased depression-like behaviors induced by SNI and CFA, the mechanisms could be due to the activation of WNT/β-catenin signal pathway and suppression of brain in ammation.
Pain and depression comorbidity have been observed in clinics and investigated in animal models [18]. In the study, we found that SNI-induced depression-like behaviors after surgery. The results were con rmed by decreased center and center/total distance in the open eld tests, and decreased sucrose and food intake, and weight gain compared to naïve rats. SNI surgery did not affect the total distance moved in the open eld test, which excluded the possibility that the effect was attributed to motor impairment. Pain behaviors of paw withdrawal threshold and response to acetone suggest the successful establishment of pain animal models. In addition, animals received CFA injection, which is proin ammatory and induces pain, also present depressive behaviors, which con rm chronic pain induces depressive conditions. Clinical study has demonstrated that chronic pain frequently results in depression [1], and chronic pain contributed to the recurrence, longer duration and increased sadness of depressive episodes [19]. In addition, pain and depression were intertwined and co-exacerbate physical and psychological symptoms. The mechanism may be explained by some overlap biological process [20]. For example, injuries to sensory pathways have been demonstrated to affect the same brain regions that are involved in mood disorders [9].
Recently, depression has been shown to be an in ammatory disease, and inhibition of in ammation reduces depression-like behaviors [3]. In clinical study, neuroin ammation of cytokines has been analyzed in CSF of post-mortem brain tissue, and results found immune cells are involved in the immune response using both postmortem tissue and positron emission tomography (PET) imaging [21].The specialized immune cells in the brain are called microglia that comprise 5%-10% of total brain cells and their main functions are to maintain CNS homeostasis and to cope with damage or infection [22]. In many neurodegenerative and neuropsychiatric diseases, microglia are activated and contribute to pathology by promoting neuroin ammation [23]. In depressive conditions, microglia are activated and induce cascade proin ammatory response in the brain and contribute to the neuropathology and depression-like behaviors [24]. Evidence in rodents found that after insult, microglial indoleamine 2, 3-dioxygenase and kynurenine pathway are activated and generate excessive neurotoxic quinolinic acid, microglia shift to proin ammatory pro le, and generates a host of cytokines, including interleukin IL-1β, TNF-α and so on [25]. The proin ammatory quinolinic acid and cytokines from microglia inhibit hippocampal neurogenesis and induce depression-like behaviors, whereas treatment with minocycline, a microglia inhibitor, counteracted this effect. Thus, suppression of the brain microglia-related in ammation is effective approach to treating depression.
PHA is a α7 nAch receptor agonist, and our results showed that ICV injection of PHA decreased depression-like behaviors, including open eld test, sucrose preference test and food intake induced by SNI, the reason could be attributed to the activation of α7 nAch receptors, as co-administration of α7 nAch receptor antagonist MLA abolished that effect. Consistently, our previous study showed that IT injection of PHA prevents the development and maintenance of chronic pain. The reason is attributed to suppression of proin ammatory response in the spinal cord. In addition, PHA decreased in ammatory cytokines release from microglia in vitro by activation of α7 nAch receptors in primary microglia cultures [8]. Study have demonstrated that the in ammation in the brain majorly comes from microglia, as microglia suppression prevents the depression-like behaviors in animals [3]. In our study, we included a group of rats received MIN, a microglia inhibitor, and results showed that ICV MIN decreased SNI-induced depression-like behaviors and brain proin ammatory response. This con rmed depression is a microglial in ammatory disease and suppression of microglia-related in ammation reversed depression-like behaviors induced by SNI, which is consistent with our previous study.
The cholinergic anti-in ammatory pathway controls cytokine production and in ammation in the body [26]. Experimental evidence suggests that cholinergic anti-in ammatory signaling requires a7 nAch receptors expressed on non-neuronal cytokine-producing microglia in the central nervous system. a7 nAch receptor agonists inhibit cytokine release and shows protective effect in a variety of experimental lethal in ammatory models and has been exploited to counteract abnormal chronic and hyper-activated pro-in ammatory conditions [6]. Our study con rmed that suppression of in ammation in the brain is a promising therapeutic target for treating depression by activation of a7 nAch receptors. In order to con rm that the effect of α7 nAch receptor activation is not limited to SNI-induced depression, we included CFA model to prove the general effect of α7 nAch receptor agonist in chronic pain-induced depression. Our results showed that CFA-induced depression-like behaviors, including decreased center distance, center/total distance, sucrose intake percentage and food intake, and ICV PHA provided antidepressive effect induced by CFA. In addition, the anti-depressive effect of PHA was reversed by MLA, which con rmed that PHA's anti-depressive effect by activation of α7 nAch receptors. Further, PHA decreased CFA-induced brain in ammation, including increased IL-10, decreased IL-1β, TNF-α and IL-18 in the brain.
To further explore the mechanisms of the anti-depressive effect of PHA, signal pathway inhibitors were administered at the same time. The results showed that WNT/β-catenin inhibitor IWR reversed the antidepressive effect of PHA in the open eld test and sucrose preference test, and both WNT/β-catenin inhibitor IWR and mTOR inhibitor suppressed food intake and weight gain, whereas TGF-β, Notch, and JAK pathway inhibitors did not affect that effect. This suggests the anti-depressive effect of PHA through WNT/β-catenin, while feeding behavior of food and weight gain are affected by both WNT/β-catenin and mTOR signal pathway. WNT/β-catenin are secreted glycoproteins that signals through the frizzled receptors and are essential for normal embryonic development [27,28]. Study have demonstrated WNT/βcatenin signaling is associated in depression and antidepressant response. The WNT/β-catenin pathway in nucleus accumbens is down-regulated in the mouse model of social defeat model of depression, and knock down of the signaling renders mice more susceptible to social defeat stress and promotes depression-like behaviors [29]. In addition, different classes of antidepressant treatments, including SSRIs, SNRI, and chronic electroconvulsive shock, showed anti-depressive effect by activation of WNT/β-catenin pathway [30]. However, our study suggests activation of a7 nAch receptors showed anti-depressive effect via the WNT/β-catenin pathway.
Our result showed that the mTOR inhibitor decreased weight gain of PHA. the reason may be attributed to that mTOR controls the body metabolism and increases food intake and consequently result in weight gain [31]. Thus, food intake and weight gain are also affected by mTOR inhibitors in our experiments. The in ammatory cytokine concentration was measured and the results showed WNT/β-catenin inhibitor IWR reversed the IL-10, IL-1β, TNF-α and IL-18 content change induced by PHA, which con rmed PHA present its anti-in ammation and anti-depressive effect via WNT/β-catenin signal pathway. Surprisingly, IL-1β, TNF-α content change were reversed also by mTOR pathway inhibitors. This indicates that mTOR pathway is also anti-in ammatory in chronic pain-induced depressive animal models. Although previous study has demonstrated that the activation of mTOR reversed depression-like behaviors, the mechanisms may be explained by suppression of in ammation [32,33]. However, mTOR pathway inhibitor did not affect anti-depressive effect of PHA, and this suggested mTOR maybe a downstream signal of WNT/βcatenin, or at least not a major contributor of the anti-depressive effect of PHA [33]. PHA activates WNT/βcatenin and results in the increment of IL-10 and the decrement of IL-1β, TNF-α, IL18 release, and at the same time, activates mTOR which result in enhanced decrement of IL-1β and TNF-α release. As previous study has demonstrated IL-10 can resolve the proin ammatory response, the anti-depressive effect of PHA should be attributed to the activation of WNT/β-catenin and increased IL-10 release [34].
In the study, several cytokines were chosen to evaluate the brain in ammatory response. Previous studies have demonstrated that IL-10, IL-1β, TNF-α and IL-18 were related to depression, and modulation of in ammation can induce or decrease depression-like behaviors. In our study, both SNI and CFA increased IL-1β, TNF-α and IL-18 in the brain, and ICV PHA increased IL-10, and decreased IL-1β, TNF-α and IL-18 content. This con rmed increased anti-in ammatory IL-10 suppressed pro-in ammatory response after chronic pain insult [35]. In addition, followed our previous study, tissue of mPFC BA and VH were acquired to testify the in ammatory pro le after SNI or CFA, because those sites have been proved to be involved in depression and showed increased cytokines expression after insult [36]. Our research veri ed that these sites are proper and effective for the study of depression and anti-depressive treatment response.

Conclusions
Our study suggests that the activation of α7 nAch receptor presents anti-depressive-like behaviors, and the reason could be attributed to the activation of WNT/β-catenin pathway and suppression of in ammation in the brain.   The effect of PHA on brain cytokines production. ICV PHA increased IL-10 in the mPFC (A), BA (E) and VH (I) compared to SNI+Vehicle group, and MLA decreased that effect. SNI increased IL-1β in the mPFC (B), BA (F) and VH (J) compared to Naive group, and ICV PHA decreased IL-1β in the mPFC, BA and VH compared to SNI+Vehicle group, while MLA decreased the effect of PHA. SNI increased TNF-α (C, G and K) and IL-18 (D, H and L) in the mPFC, BA and VH compared to Naive group, and ICV PHA decreased their content in the mPFC, BA and VH compared to SNI+Vehicle group, while MLA decreased the effects of PHA. "a" denotes the statistically signi cant difference compared to the SNI + vehicle group. "b" denotes the statistically signi cant difference compared to the SNI + PHA group. ELISA enzymelinked immuneabsorbent assay. "a" denotes the statistically signi cant difference compared to the CFA + vehicle group. "b" denotes the statistically signi cant difference compared to the CFA + PHA group.  PHA decreased depressive-like behaviors via WNT/β-catenin signal pathway. IWR (WNT/β-catenin inhibitor) reversed the anti-depressive effect of PHA on center distance (A), center/total distance ratio (C), sucrose intake percentage D, food intake percentage (E) and weight gain (F), while did not affect total running distance(B) in the open eld test after SNI surgery. However, AZD8055 (mTOR inhibitor) also reversed the effect of PHA on food intake percentage (E) and weight gain (F), Whereas SB431542(TGFβ inhibitor), Numb (notch inhibitor) and AZD1480 (JAK inhibitor) did not affect anti-depressive behaviors of PHA. "a" denotes the statistically signi cant difference compared to the SNI group. "b" denotes the statistically signi cant difference compared to the PHA group. PHA decreased brain in ammation via WNT/β-catenin and mTOR pathway. PHA increased IL-10 (A) and decreased IL-18 (D) in mPFC, BA and VH after SNI surgery, and IWR decreased those effect. (B) PHA decreased IL-1β content in mPFC and VH after SNI surgery, the effect was reversed by AZD8055 and IWR.
(C) PHA decreased the content of TNF in mPFC, BA and VH after SNI surgery, and the content change of TNFα in mPFC and BA was reversed by AZD8055 and IWR. "a" denotes the statistically signi cant difference compared to the SNI group. "b" denotes the statistically signi cant difference compared to the PHA group.