Neuropeptide S Attenuates the Alarm Pheromone-Evoked Defensive and Risk Assessment Behaviors Through Activation of Cognate Receptor-Expressing Neurons in the Posterior Medial Amygdala

Abstract

Neuropeptide S (NPS) acts by activating its cognate receptor (NPSR). High level expression of NPSR in the posterior medial amygdala suggests that NPS-NPSR system should be involved in regulation of social behaviors induced by social pheromones. The present study was undertaken to investigate the effects of central administration of NPS or with NPSR antagonist on the alarm pheromone (AP)-evoked defensive and risk assessment behaviors in mice. Furthermore, H129-H8, a novel high-brightness anterograde multiple trans-synaptic virus, c-Fos and NPSR immunostaining were employed to reveal the involved neurocircuits and targets of NPS action. The mice exposed to AP displayed an enhancement in defensive and risk assessment behaviors. NPS (0.1-1 nmol) intracerebroventricular (i.c.v.) injection significantly attenuated the AP-evoked defensive and risk assessment behaviors. NPSR antagonist [D-Val⁵]NPS at the dose of 40 nmol completely blocked the effect of 0.5 nmol of NPS which showed the best effective among dose range. The H129-H8-labeled neurons were observed in the bilateral posterodorsal medial amygdala (MePD) and posteroventral medial amygdala (MePV) 72 h after the virus injection into the unilateral olfactory bulb (OB), suggesting that the MePD and MePV receive olfactory information inputs from the OB. The percentage of H129-H8-labeled neurons that also express NPSR were 90.27 ± 3.56% and 91.67 ± 2.46% in the MePD and MePV, respectively. NPS (0.5 nmol, i.c.v.) remarkably increased the number of Fos immunoreactive (-ir) neurons in the MePD and MePV, and the majority of NPS-induced Fos-ir neurons also expressed NPSR. The behavior characteristic of NPS or with [D-Val⁵]NPS can be better replicated in MePD/MePV local injection within lower dose. The present findings demonstrated that NPS, via selective activation of the neurons bearing NPSR in the posterior medial amygdala, attenuates the AP-evoked defensive and risk assessment behaviors in mice.

Keywords: alarm pheromone; antagonist; c-Fos; herpes simplex virus; neural circuit tracing; neuropeptide S; neuropeptide S receptor; posterior medial amygdala.

FIGURE 1

Schematic diagram of the experimental setup. (A) Alarm pheromone collection. An anesthetized donor mouse in the sealed plastic box received electrical stimulation of the perianal region, and the fresh air carried alarm pheromone (orange dots) into the purified water. (B) Control collection. A mouse received electrical stimulation of the subcutaneous part of the napex as vehicle because the donor mouse didn’t release AP in this condition. (C) The test apparatus was used in this study.

FIGURE 2

Behavior responses of mice that were either exposed to AP or vehicle. Saline were i.c.v. injection. Values are means ± SEM (n = 8 in each group). *P < 0.01. Data were analyzed by independent Student’s t-test.

FIGURE 3

Attenuating effects of NPS on the AP-induced defensive and risk assessment behaviors in mice. Saline or NPS were i.c.v. injection. AP + Saline group is same as in Figure 2. Values are means ± SEM (n = 8 in each group). *P < 0.05, **P < 0.01, ***P < 0.001. Data were analyzed by one-way ANOVA and Tukey-Kramer’s multiple comparisons test.

FIGURE 4

Attenuating effects of NPS on the AP-induced defensive and risk assessment behaviors were blocked by [D-Val⁵]NPS. Saline, NPS or NPS + [D-Val⁵]NPS were i.c.v. injection. AP + Saline and AP + 0.5 nmol NPS groups are same as in Figures 2, 3. Values are means ± SEM (n = 8 in each group). *P < 0.05, **P < 0.01, ***P < 0.001. Data were analyzed by one-way ANOVA and Tukey-Kramer’s multiple comparisons test.

FIGURE 5

Anterograde viral tracing and NPS-induced the activated neurons in posterior medial amygdala. (A) Schematic diagram of H129-H8 injection. (B) Schematic drawing shows the section illustrated in (C,D,G,H). (C,D) Ipsilateral coronal section of a H129-H8 (green) treated mouse brain at the MePD (C) and MePV (D) stained with NPSR (red) and DAPI (blue). (E,F) Histograms show quantitative analysis of the number of Fos-ir neurons in the MePD and MePV following NPS (n = 5) and saline (n = 6) i.c.v. injection, respectively. (G,H) Photomicrographs show Fos-ir neurons (black) in the MePD and MePV in NPS- and saline-treated mice, respectively. Values are means ± SEM. *P < 0.001. Data were analyzed by independent Student’s t-test. Arrow (C,D) show the co-expression of H129-H8-labled and NPSR-ir neurons. Bar = 100 μm (C,D), 200 μm (G,H). OB, olfactory bulb; MePD, posterodorsal medial amygdaloid nuclei; MePV, posteroventral medial amygdaloid nuclei; opt, optic tract; sox, supraoptic decussation.

FIGURE 6

NPS-induced Fos-ir neurons bearing NPSR in the MePD and MePV of posterior medial amygdala. Photomicrographs show Fos-ir neurons in the MePD (A) and MePV (D) after NPS i.c.v. administration, NPSR-ir neurons in the MePD (B) and MePV (E), and the co-expression of Fos-ir and NPSR-ir neurons in the MePD (C) and MePV (F), respectively. Yellow (C,F) show the co-expression of Fos-ir and NPSR-ir neurons. Bar = 50 μm.

FIGURE 7

Defensive and risk assessment behavioral test following MePD/MePV local injection of Saline, NPS or NPS + [D-Val⁵]NPS. Values are means ± SEM (n = 8 in each group). *P < 0.05, **P < 0.01, ***P < 0.001. Data were analyzed by one-way ANOVA and Tukey-Kramer’s multiple comparisons test.