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The precise neural mechanisms underlying the pathogenesis of depression are largely unknown, though stress-induced brain inflammation and serotonergic plasticity are thought to be centrally involved. Moreover, we previously demonstrated that neuropeptide FF receptor 2 (NPFFR2) overexpression provokes depressive-like behaviors in mice. Here, we assess whether NPFFR2 is involved in priming of depressive-like behaviors and downregulation of serotonergic 1A receptor (5HT1AR) after lipopolysaccharide (LPS) treatment. The forced swimming test (FST) and sucrose preference test (SPT) were used to quantify depressive-like phenotypes in wild-type (WT) and NPFFR2-knockout (KO) mice. A single dose of LPS (i.p. 1 mg/kg) readily caused increases in toll-like receptor 4 and tumor necrosis factor-α along with decreases in 5-HT1AR mRNA in the ventral hippocampus of WT mice. Furthermore, LPS treatment of WT mice increased immobility time in FST and decreased sucrose preference in SPT. In contrast, none of these effects were observed in NPFFR2-KO mice. While WT mice injected with lentiviral 5-HT1AR shRNA in the ventral hippocampus displayed an unaltered response after LPS challenge, LPS-challenged NPFFR2-KO mice displayed a profound decrease in sucrose preference when pretreated with 5-HT1AR shRNA. Taken together, these results suggest that NPFFR2 modulates LPS-induced depressive-like behavioral phenotypes by downregulating 5HT1AR in the ventral hippocampus.

Mental stress is highly related to many clinical symptoms and disorders, as it activates the hypothalamic-pituitary-adrenocortical (HPA) axis to affect a wide variety of physiological functions. Furthermore, stress leads to the aberrations in HPA axis activity and disruptions of body homeostasis. It was previously shown that neuropeptide FF (NPFF) regulates the HPA axis through the activation of hypothalamus paraventricular nucleus (PVN), and genetic overexpression or pharmacological stimulation of NPFF receptor 2 (NPFFR2) triggers hyperactivity of HPA axis and suppresses behavioral correlates of emotion in mice. In this study, we further examined the role of NPFFR2 in stress response in mice by utilizing a single prolonged stress (SPS). SPS is considered a model of post-traumatic stress disorder (PTSD), and mice undergo physical restraint, forced swimming, and ether anesthesia within a day followed by social isolation for one week. NPFFR2 knockout B6 mice were generated by CRISPR/Cas9 technology and exposed to SPS. The NPFFR2 knockouts showed resistance to stress exposure-induced anxiety-like behaviors and HPA axis hyperactivity. Additionally, the hippocampal mRNA levels of glucocorticoid receptor and mineralocorticoid receptor were reduced in wild-type (WT) mice but not in NPFFR2 knockouts after stress exposure. Our data also suggested that NPFFR2 knockout mice have stronger negative feedback on the HPA axis after exposure to SPS. Mice with intra-PVN Npffr2 shRNA injection displayed trends toward resistance to SPS exposure in both behavioral and molecular assays. Together, our findings suggest that NPFFR2 may be a potential therapeutic target for disorders relating to stress/anxiety and HPA dysregulation.

Background: Stimulation of trigeminovascular pathway is widely used to establish the headache animal model. Headache is a common neurological disorder, in which symptomatic attacks are mediated by calcitonin-gene-related peptide (CGRP). CGRP is synthesized and released from the trigeminal ganglion to transmit pain signals under stimulation. On the other hand, Neuropeptide FF (NPFF) is a candidate transmitter/modulator for migraine, and stimulation of its receptor, NPFFR2, increases the expression and release of CGRP in mice sensory neurons. Here, we investigate the impact of NPFFR2 on trigeminal CGRP level in a capsaicin-induced headache mouse model., Methods: Mice were intracisternally injected with capsaicin into the cisterna magna to activate the trigeminovascular pathway and induce headache symptoms. Mice pretreated with Npffr2-shRNA or NPFFR2 knockouts were adopted to test the impact of NPFFR2 on capsaicin-induced CGRP upregulation in trigeminal ganglion. The gene silencing effect of Npffr2-shRNA in trigeminal ganglion was confirmed by real-time PCR. Trigeminal CGRP level was determined by immunofluorescence staining, and the percentage of CGRP-positive cell was calculated after setting the signal intensity threshold by Image J software. Amount of trigeminal CGRP in NPFFR2 overexpressed mice was also measured by CGRP ELISA., Findings: Infusion of capsaicin into the cisterna magna upregulated the CGRP in trigeminal ganglion and induced spontaneous pain behaviors including the reduction of locomotor activity and the increase of freezing behavior. Intracisternal injection of Npffr2-shRNA reduced the mRNA of Npffr2 in trigeminal ganglion. Mice pretreatment with Npffr2-shRNA prevented capsaicin-induced CGRP upregulation in trigeminal ganglion. Similarly, CGRP upregulation was also reduced in NPFFR2 knockout mice. On the contrary, trigeminal CGRP was increased in NPFFR2 overexpressed mice., Conclusions: Reducing the level of NPFFR2 leads to the downregulation of capsaicin-induced CGRP in trigeminal ganglion, which would consequently attenuate the activation of trigeminovascular pathway. Thus, NPFFR2 could serve as a potential target for neuromodulation of cephalic pain.

Neuropeptide FF (NPFF) belongs to the RFamide family and is known as a morphine-modulating peptide. NPFF regulates various hypothalamic functions through two receptors, NPFFR1 and NPFFR2. The hypothalamic-pituitary-adrenal (HPA) axis participates in physiological stress response by increasing circulating glucocorticoid levels and modulating emotional responses. Other RFamide peptides, including neuropeptide AF, neuropeptide SF and RFamide related peptide also target NPFFR1 or NPFFR2, and have been reported to activate the HPA axis and induce anxiety- or depression-like behaviors. However, little is known about the action of NPFF on HPA axis activity and anxiety-like behaviors, and the role of the individual receptors remains unclear. In this study, NPFFR2 agonists were used to examine the role of NPFFR2 in activating the HPA axis in rodents. Administration of NPFFR2 agonists, dNPA (intracerebroventricular, ICV) and AC-263093 (intraperitoneal, IP), time-dependently (in rats) and dose-dependently (in mice) increased serum corticosteroid levels and the effects were counteracted by the NPFF receptor antagonist, RF9 (ICV), as well as corticotropin-releasing factor (CRF) antagonist, α-helical CRF(9-41) (intravenous, IV). Treatment with NPFFR2 agonist (AC-263093, IP) increased c-Fos protein expression in the hypothalamic paraventricular nucleus and induced an anxiogenic effect, which was evaluated in mice using an elevated plus maze. These findings reveal, for the first time, that the direct action of hypothalamic NPFFR2 stimulates the HPA axis and triggers anxiety-like behaviors., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Neuropeptide FF (NPFF) is a morphine-modulating peptide that regulates the analgesic effect of opioids, and also controls food consumption and cardiovascular function through its interaction with two cognate receptors, NPFFR1 and NPFFR2. In the present study, we explore a novel modulatory role for NPFF-NPFFR2 in stress-related depressive behaviors. In a mouse model of chronic mild stress (CMS)-induced depression, the expression of NPFF significantly increased in the hypothalamus, hippocampus, medial prefrontal cortex (mPFC) and amygdala. In addition, transgenic (Tg) mice over-expressing NPFFR2 displayed clear depression and anxiety-like behaviors with hyperactivity in the hypothalamic-pituitary-adrenal (HPA) axis, reduced expression of glucocorticoid receptor (GR) and neurogenesis in the hippocampus. Furthermore, acute treatment of NPFFR2 agonists in wild-type (WT) mice enhanced the activity of the HPA axis, and chronic administration resulted in depressive and anxiety-like behaviors. Chronic stimulation of NPFFR2 also decreased the expression of hippocampal GR and led to persistent activation of the HPA axis. Strikingly, bilateral intra-paraventricular nucleus (PVN) injection of NPFFR2 shRNA predominately inhibits the depressive-like behavior in CMS-exposed mice. Antidepressants, fluoxetine and ketamine, effectively relieved the depressive behaviors of NPFFR2-Tg mice. We speculate that persistent NPFFR2 activation, in particular in the hypothalamus, up-regulates the HPA axis and results in long-lasting increases in circulating corticosterone (CORT), consequently damaging hippocampal function. This novel role of NPFFR2 in regulating the HPA axis and hippocampal function provides a new avenue for combating depression and anxiety-like disorder., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The present study investigates the modulatory effects of neuropeptide FF (NPFF) receptors on the mesolimbic dopaminergic pathway controlled by opioid receptors. A stable NPFF(2) receptor agonist, dNPA, was injected into the ventral tegmental area (VTA) and the release of dopamine and serotonin within the nucleus accumbens (NAc), induced by intraperitoneal injection of morphine, was monitored using the brain microdialysis, in non-constrained rat. dNPA decreased systemic morphine-induced elevation of dopamine and serotonin metabolites within the NAc. Furthermore, co-injected with morphine into the VTA, NPFF inhibited morphine-induced stereotypy 60-120min after the injection. This neurochemical and behavioural anti-opioid effect mediated by NPFF(2) receptors at the level of VTA suggests the involvement of NPFF in the rewarding effects of opiates on the mesolimbic dopamine system., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Neuropeptide FF (NPFF) participates in many physiological functions associated with opioids in the mammalian CNS. We established a pheochromocytoma PC-12 cell line clone stably expressing rat NPFF1 receptors. Both NPFF and FMRFamide activated NPFF1 receptors to couple with Gi/o protein and inhibited adenylyl cyclase activity in PC-12/rNPFF1 cells, but there were no effects on MAPKs (ERK1/2 and p38 MAPK) or PI3K/Akt pathway. FMRFamide also inhibited DARPP-32/Thr34 phosphorylation in the presence of forskolin. Similarly, PFR(Tic)amide, a 'super-agonist' of NPFF receptors, inhibited the production of cAMP and slightly decreased DARPP-32/Thr34 phosphorylation in PC-12/rNPFF1 cells. Intracerebroventricular injections of PFR(Tic)amide blocked behavioral sensitization of locomotor activity to amphetamine, and intrathecal injection of PFR(Tic)amide caused a dose-dependent antinociception in vivo in rats. Thus, "over-activation" of NPFF receptors by PFR(Tic)amide induced different bio-effects from those induced by NPFF in vivo.