Pancreatic polypeptide and its central Y4 receptors are essential for cued fear extinction and permanent suppression of fear

Abstract

Background and purpose: Avoiding danger and finding food are closely related behaviours that are essential for surviving in a natural environment. Growing evidence supports an important role of gut-brain peptides in modulating energy homeostasis and emotional-affective behaviour. For instance, postprandial release of pancreatic polypeptide (PP) reduced food intake and altered stress-induced motor activity and anxiety by activating central Y4 receptors.

Experimental approach: We characterized [K(30) (PEG2)]hPP2-36 as long-acting Y4 receptor agonist and injected it peripherally into wildtype and Y4 receptor knockout (Y4KO) C57Bl/6NCrl mice to investigate the role of Y4 receptors in fear conditioning. Extinction and relapse after extinction was measured by spontaneous recovery and renewal.

Key results: The Y4KO mice showed impaired cued and context fear extinction without affecting acquisition, consolidation or recall of fear. Correspondingly, peripheral injection of [K(30) (PEG2)]hPP2-36 facilitated extinction learning upon fasting, an effect that was long-lasting and generalized. Furthermore, peripherally applied [K(30) (PEG2)]hPP2-36 before extinction inhibited the activation of orexin-expressing neurons in the lateral hypothalamus in WT, but not in Y4KO mice.

Conclusions and implications: Our findings suggests suppression of excessive arousal as a possible mechanism for the extinction-promoting effect of central Y4 receptors and provide strong evidence that fear extinction requires integration of vegetative stimuli with cortical and subcortical information, a process crucially depending on Y4 receptors. Importantly, in the lateral hypothalamus two peptide systems, PP and orexin, interact to generate an emotional response adapted to the current homeostatic state. Detailed investigations of feeding-relevant genes may thus deliver multiple intervention points for treating anxiety-related disorders.

Figure 1

Context and cued fear extinction is impaired in Y4KO mice. (A) Experimental timescale of fear conditioning and extinction experiments; (B) compared with WT mice, Y4KO mice exhibit unchanged acquisition and (C) expression of context fear, but (D) delayed extinction of context fear and (E) impaired extinction of cued fear. (F) While WT mice display an increase in fear expression upon reinstatement (RI), Y4KO mice display equal freezing levels during extinction recall (ER) and reinstatement, suggesting complete absence of fear extinction memory (WT: n = 9, Y4KO: n = 8; data are expressed as mean ± SEM, *P < 0.05, significantly different from WT; # P<0.05, significantly different from ER).

Figure 2

Cued fear extinction is facilitated by peripheral injection of the long‐acting Y₄ receptor agonist [K³⁰(PEG2)]hPP_2‐36. (A) Experimental procedure of fear conditioning and extinction experiments. (B) Following fear acquisition, male WT mice were divided into two equal groups; (C) peripheral injection of [K³⁰(PEG2)]hPP_2‐36 in fasted mice (16 h, before and during fear extinction) resulted in facilitated extinction of CS‐induced fear compared with saline‐injected controls. (D) Freezing to the CS was reduced in the [K³⁰(PEG2)]hPP_2‐36 group during extinction recall tested under drug‐free conditions with food available ad libitum. (E) An additional fear extinction session of the saline and [K³⁰(PEG2)]hPP_2‐36 groups under drug‐free and fed conditions resulted in (F) equal freezing levels during extinction recall 2, demonstrating similar composition of the groups (saline: n = 8, [K³⁰(PEG2)]hPP_2‐36: n = 8) Data are expressed as mean ± SEM, *P < 0.05, significantly different from saline.

Figure 3

Peripheral injection of [K³⁰(PEG2)]hPP_2‐36 facilitates fear extinction by activation of Y₄ receptors. (A) Experimental timeline of fear conditioning experiments. (B) Facilitated fear extinction in male WT mice but not in (C) Y4KO mice after peripheral injections of [K³⁰(PEG2)]hPP_2‐36 60 min before fear extinction. (D) Equal freezing levels of all groups during the first CS, demonstrating equal fear expression, (E) reduced freezing level of [K³⁰(PEG2)]hPP_2‐36‐injected WT mice compared with those of all other experimental groups (CS13). (F) While both WT groups displayed extinction of conditioned fear, Y4KO mice exhibited still high freezing levels (CS20) and (G) extinction of conditioned fear in all four treatment groups at CS 25 (WT: saline: n = 10, [K³⁰(PEG2)]hPP_2‐36: n = 10; Y4KO: saline: n = 10, [K³⁰(PEG2)]hPP_2‐36: n = 10; data are expressed as mean ± SEM. *P < 0.05, PP significantly different from saline. #P < 0.05, WT significantly different from Y4KO.

Figure 4

Treatment with [K³⁰(PEG2)]hPP_2‐36 before fear extinction results in long‐term suppression of fear. (A) Experimental timeline of fear conditioning experiments. (B) Male WT mice were divided into two groups with equal fear acquisition, (C) facilitated extinction during three consecutive extinction sessions (E1–E3) in [K³⁰(PEG2)]hPP_2‐36‐treated mice compared with saline‐injected controls and equal freezing levels during extinction recall (ER), but decreased freezing of [K³⁰(PEG2)]hPP_2‐36 group during (D) spontaneous recovery and (E) renewal on days 12 and 13 respectively (saline: n = 7, [K³⁰(PEG2)]hPP_2‐36: n = 7; data are expressed as mean ± SEM. *P < 0.05, significantly different from saline.

Figure 5

Peripheral injection of [K³⁰(PEG2)]hPP_2‐36 before fear extinction specifically reduces the activation of orexin neurons in the lateral hypothalamus. Male WT and Y4KO mice were fasted before and during fear extinction (16 h) and injected with [K³⁰(PEG2)]hPP_2‐36 60 min before fear extinction; all mice were perfused 90 min after the end of extinction training. Representative images of immunohistochemistry for c‐Fos, orexin and overlay for (A–C) saline‐injected and (D–F) [K³⁰(PEG2)]hPP_2‐36‐injected WT mice. Arrows point to examples of dual‐labelled neurons, higher magnification in A and F with Hoechst staining for labelling nuclei; scale bar 100 μm. Histograms depicting quantification of (G) percentage of cFos/orexin double‐labelled neurons and (H) number of orexin neurons in the lateral hypothalamus of WT and Y4KO mice (WT: saline: n = 7, [K³⁰(PEG2)]hPP_2‐36: n = 7; Y4KO: saline: n = 7, [K³⁰(PEG2)]hPP_2‐36: n = 7; data are expressed as mean ± SEM. *P < 0.05, significantly different from saline. #P < 0.05, WT significantly different from Y4KO.