Direct inhibition of hypocretin/orexin neurons in the lateral hypothalamus by nociceptin/orphanin FQ blocks stress-induced analgesia in rats

Abstract

We recently demonstrated that hypocretin/orexin (Hcrt) and nociceptin/orphanin FQ (N/OFQ) systems coordinately regulate nociception in a mouse model of stress-induced analgesia (SIA). However, the site of N/OFQ action on modulation of SIA was elusive, since N/OFQ was administered via intracerebroventricular (i.c.v.) injection acting on widely distributed N/OFQ receptors (NOP) in the brain. In the present study, we tested the hypothesis that N/OFQ modulates the SIA directly via the inhibition of the Hcrt neurons in the lateral hypothalamus. Using both fluorescent and electron microscopy, we found that N/OFQ-containing neurons are located in the lateral hypothalamus and the N/OFQ-containing fibers make direct contacts with the Hcrt neurons. Paw thermal nociceptive test revealed that the immobilization restraint of the rat increased the thermal pain threshold by 20.5 ± 7.6%. Bilateral microinjection of N/OFQ (9 μg/side) into the rat perifornical area of the lateral hypothalamus, the brain area in which the Hcrt neurons are exclusively located, abolished the SIA. Activity of Hcrt neurons in the same animals was assessed using Fos immunohistochemistry. Percentage of Fos(+)/Hcrt neurons was lower in rats injected with N/OFQ than rats injected with saline, with the difference between groups stronger in the Hcrt neurons located medially to the fornix than in Hcrt neurons located laterally to the fornix. These results suggest that N/OFQ modulation of SIA is mediated by direct inhibition of Hcrt neuronal activity in the perifornical area. The uncovered peptidergic interaction circuitry may have broad implication in coordinated modulation by Hcrt and N/OFQ on other stress adaptive responses.

Fig. 1

Hcrt-immunoreactive cells (green color) and N/OFQ-immunoreactive cells (red color) are located in the perifornical area (A and A’) and in the area dorsal to perifornical area (B and B’). A” shows merged images of A and A’, and B” shows merged images of B and B’.

Fig. 2

Electron micrograph showing an asymmetrical synaptic membrane specialization (black arrow) between the N/OFQ bouton-like structure and the Hcrt immunolabeled dendrite in the lateral hypothalamus. Annotations to the photographs indicate the different cellular elements: A to indicate axon terminals, arrowheads to indicate Hcrt IHC postsynaptically, rER to point to rough endoplasmaic reticulum, and m to indicate mitochondria. Scale bar represents 1 µm.

Fig. 3

Microinjection of N/OFQ into the rat lateral hypothalamus abolishes stress-induced analgesia. Distribution of Hcrt- and Fos-immunoreactive cells in a representative brain section of a rat injected with N/OFQ (A) or with saline (B). Location of the injection site in the same brain section as A indicated by Fluorogold fluorescence (C). Note that the location of the injection site is dorsal to the distribution of Hcrt neurons. Location of the injection site in the same brain section as B indicated by Fluorogold fluorescence (D). The insert in (E) showing high magnification of the brain area marked by a rectangle in A and C. The insert in (F) showing high magnification of the brain area marked by a rectangle in B and D. Hcrt cells are labeled in red, and Fos nuclei are labeled in black. f, fornix. (G) Plantar test in rats before and after restraint stress. The plantar test was repeated in four measurements. The first baseline measurement was performed 30 min before the restraint, the second measure was done immediately after the release from the restraint (0 min), the third and fourth measures were made at 30 min and 60 min after the release from the restraint, respectively. *P<0.05 between N/OFQ and saline groups at the same timepoint.

Fig. 4

Local microinjection of N/OFQ depresses Fos expression in Hcrt neurons in the rats. Representative images show Hcrt-immunoreactive cells (red color) expressing Fos (black color) in the rats injected with N/OFQ (A) or with saline (B). Percentage of Fos⁺Hcrt neurons is shown in rats injected with N/OFQ or saline (C). The counts were analyzed separately for the Hcrt neurons located medially and laterally to the fornix (f), as schematically shown in the insert. Total counts include Hcrt neurons located on both sides of the fornix. Paired comparisons versus corresponding saline group, Mann Whitney U test: *p < 0.05; **p <. 0.02; ***p < 0.01. Scale bar represents 100 µm.