Activating mu-opioid receptors in the lateral parabrachial nucleus increases c-Fos expression in forebrain areas associated with caloric regulation, reward and cognition

Abstract

The pontine parabrachial nucleus (PBN) has been implicated in the modulation of ingestion and contains high levels of mu-opioid receptors (MOPRs). In previous work, stimulating MOPRs by infusing the highly selective MOPR agonist [d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) into the lateral parabrachial region (LPBN) increased food intake. The highly selective MOPR antagonist d-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) prevented the hyperphagic action of DAMGO. The present experiments aimed to analyze both the pattern of neural activation and the underlying cellular processes associated with MOPR activation in the LPBN. Male Sprague-Dawley rats received a unilateral microinfusion of a nearly maximal hyperphagic dose of DAMGO into the LPBN. We then determined the level of c-Fos immunoreactivity in regions throughout the brain. MOPR activation in the LPBN increased c-Fos in the LPBN and in the nucleus accumbens, hypothalamic arcuate nucleus, paraventricular nucleus of the thalamus and hippocampus. Pretreatment with CTAP prevented the increase in c-Fos translation in each of these areas. CTAP also prevented the coupling of MOPRs to their G-proteins which was measured by [(35)S] guanosine 5'-O-[gamma-thio]triphosphate ([(35)S]GTPgammaS) autoradiography. Together, these data strongly suggest that increasing the coupling of MOPRs to their G-proteins in the LPBN disinhibits parabrachial neurons which subsequently leads to excitation of neurons in regions associated with caloric regulation, ingestive reward and cognitive processes in feeding.

Figure 1

Coronal section through the parabrachial nucleus. Regions of interest quantified are the ventral lateral (LPBV), central lateral (LPBC1 and LPBC2, respectively), external lateral (LPBE), central medial (MPBC) and external medial (MPBE) PBN.

Figure 2

Unilateral infusion of DAMGO into the LPBN (N=7) increased the expression of c-Fos in the LPBN on the side ipsilateral to the infusion site. Data are represented as the mean number of c-Fos positive cells identified (mean ± SEM) in each subregion of the LPBN/mm² on the ipsilateral and contralateral sides of the brain. Asterisks indicate significant difference from corresponding value for vehicle: *p<0.05, **p<0.01, Student-Newman-Keuls test after two-factor repeated measures ANOVA.

Figure 3

Coronal sections through the PBN after immunohistochemical staining for c-Fos. Groups include those infused: twice with vehicle (VEH/VEH); vehicle followed by DAMGO (VEH/DAMGO); the MOPR antagonist CTAP followed by vehicle (CTAP/VEH); and CTAP followed by DAMGO (CTAP/DAMGO). Bar in lower right corner of panels indicates scale (0.5mm).

Figure 4

Infusion of the MOPR agonist DAMGO into the LPBN robustly increased the number of c-Fos positive nuclei found in the LPBV, LPBC1, LPBC2, and LPBE subregions. Data are represented as the mean number of c-Fos positive cells (means ± SEM) identified in each subregion of the LPBN/mm². Groups include VEH/VEH (N=7), VEH/DAMGO (N=7; same rats as in Fig. 2 above), CTAP/VEH (N=6) and CTAP/DAMGO (N=5). Asterisks indicate significant difference from corresponding value for vehicle: *p<0.05, **p<0.01, Student-Newman-Keuls test after two-factor repeated measures ANOVA.

Figure 5

Autoradiograms of [³⁵S]GTPγS incorporation in coronal sections from the PBN of a male Sprague–Dawley rat after incubation in vitro with no drug (Basal), 1 µM DAMGO, both 1 µM DAMGO and 100nM CTAP or unlabeled GTPγS (Non-Specific). Arrows indicate [³⁵S]GTPγS incorporation in the external lateral subregion of the LPBN in one set of autoradiograms from one rat.

Figure 6

The MOPR antagonist CTAP blocks G-protein coupling stimulated by DAMGO in the external LPBN. Data are shown as percentage of basal level of GTPγS incorporation (means ± SEM). Data (N=3) were calculated from individual means of multiple sections from three rats (total of 42 sections for all rats). Asterisks indicate significant difference from corresponding value for vehicle: **p<0.01, Student-Newman-Keuls test after two-factor repeated measures ANOVA.

Figure 7

Coronal section through the nucleus accumbens (NAcc). Regions of interest quantified include the dorsomedial shell (DM shell), ventromedial shell (VM shell), dorsal core (D core), ventral core (V core), and lateral core (L core) ac = anterior commissure.

Figure 8

Coronal sections through the NAcc. Sections are from VEH/VEH and VEH/DAMGO conditions. Bar in lower right corner of panels indicates scale (0.5mm).

Figure 9

Infusion of the MOPR agonist DAMGO into the central region of the LPBN increases the number of c-Fos-IR nuclei in the dorsomedial shell (DM shell), ventromedial shell (VM shell) and dorsal core (D core) of the NAcc (Veh/Veh N=9; Veh/DAMGO N=7). No difference was found in the number of c-Fos-IR nuclei between the contralateral and ipsilateral sides of the NAcc (see figure insert). Data are represented as the average number of c-Fos positive cells (means ± SEM) identified in each subregion of the NAcc/mm². Asterisks indicate significant difference from corresponding value for vehicle: *p<0.05, **p<0.01, Student-Newman-Keuls test after two-factor repeated measures ANOVA.

Figure 10

Infusion of the MOPR agonist DAMGO into the central subregion of the LPBN increased c-Fos-IR in the arcuate nucleus of the hypothalamus but not in the ventromedial nucleus of the hypothalamus (VMH), paraventricular nucleus (PVH) or lateral hypothalamus (LH) (Veh/Veh N=4; Veh/DAMGO N=4) (see Figure 11). Similarly, there was no change in c-Fos-IR nuclei in the VTA or the central nucleus or basolateral nucleus of the amygdala. Increased c-Fos-IR was also seen in the CA1, CA3 and dentate regions of the hippocampal formation (see Figure 12). Data are represented as the average number of c-Fos positive cells (means ± SEM)/ mm². Groups include VEH/VEH and VEH/DAMGO. Asterisks indicate significant difference from corresponding value for vehicle: *p<0.05, Student-Newman-Keuls test after two-factor repeated measures ANOVA.

Figure 11

Coronal sections through the hypothalamus. Sections are from VEH/VEH and VEH/DAMGO conditions. Bar in lower right corner of panels indicates scale (0.5mm) f = fornix.

Figure 12

Coronal sections through the hippocampal formation. Sections are from VEH/VEH and VEH/DAMGO conditions. Bar in lower right corner of panels indicates scale (0.5mm).

Figure 13

Coronal sections through the paraventricular nucleus of the thalamus. Sections are from VEH/VEH and VEH/DAMGO conditions. Bar in lower right corner of panels indicates scale (0.5mm) 3V=third ventricle.

Figure 14

A. Immunolabeling of the parabrachial nucleus primarily in the external lateral subnucleus (scp, superior cerebellar peduncle), by antibodies directed against MOPRs and microtubule-associated protein 2 (MAP2). The postage stamp insert in the upper right corner shows staining only for MAP2. The larger image shows double labeling for MOPRs (FITC, green) and MAP2 (linked to rhodamine). Note the extensive overlap of MOPR and MAP2 staining (yellow), thereby suggesting that MOPRs are localized to dendrites within this region, as reported by others. B. Immunolabeling of a section adjacent to that shown for MOPR/MAP2 (A), showing staining for the microtubular protein tau (TRITC) (upper right inset). Note the much smaller degree of apparent overlap of tau with the MOPRs (larger image) when compared to MAP2/MOPR double labeling in A. These non-quantitative data are for descriptive puposes , indicating the possibility of either dendritic or axonal/terminal localization of MOPRs in mediating the cellular and behavioral actions of DAMGO.