Nociceptin/orphanin FQ neurons in the Arcuate Nucleus and Ventral Tegmental Area Act via Nociceptin Opioid Peptide Receptor Signaling to Inhibit Proopiomelanocortin and A10 Dopamine Neurons and Thereby Modulate Ingestion of Palatable Food

Abstract

The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits neuronal activity via its cognate nociceptin opioid peptide (NOP) receptor throughout the peripheral and central nervous systems, including those areas involved in the homeostatic and hedonic regulation of energy homeostasis. We thus tested the hypothesis that N/OFQ neurons in the hypothalamic arcuate nucleus (ARC) and ventral tegmental area (VTA) act via NOP receptor signaling to inhibit nearby anorexigenic proopiomelanocortin (POMC) and A₁₀ dopamine neuronal excitability, respectively, and thereby modulate ingestion of palatable food. Electrophysiologic recordings were performed in slices prepared from transgenic male and ovariectomized (OVX) female N/OFQ-cre/enhanced green fluorescent protein-POMC, N/OFQ-cre and tyrosine hydroxylase-cre animals to see if optogenetically-stimulated peptide release from N/OFQ neurons could directly inhibit these neuronal populations. Binge-feeding behavioral experiments were also conducted where animals were exposed to a high-fat-diet (HFD) for one hour each day for five days and monitored for energy intake. Photostimulation of ARC and VTA N/OFQ neurons produces an outward current in POMC and A₁₀ dopamine neurons receiving input from these cells. This is associated with a hyperpolarization and decreased firing. These features are also sex hormone- and diet-dependent; with estradiol-treated slices from OVX females being less sensitive, and obese males being more sensitive, to N/OFQ. Limited access to HFD causes a dramatic escalation in consumption, such that animals eat 25-45% of their daily intake during that one-hour exposure. Moreover, the NOP receptor-mediated regulation of these energy balance circuits are engaged, as N/OFQ injected directly into the VTA or ARC respectively diminishes or potentiates this binge-like increase in a manner heightened by diet-induced obesity or dampened by estradiol in females. Collectively, these findings provide key support for the idea that N/OFQ regulates appetitive behavior in sex-, site- and diet-specific ways, along with important insights into aberrant patterns of feeding behavior pertinent to the pathogenesis of food addiction.

Keywords: A(10) dopamine neurons; ARC; Nociceptin; POMC; VTA; energy balance; food addiction; sex differences.

Fig. 1.

Photostimulation (photostim) of ARC N/OFQ neurons inhibits POMC neurons in male N/OFQ-cre/eGFP-POMC mice. A, N/OFQ immunostaining in the ARC visualized with AF546. B, eYFP ChR2 reporter signal in the cells in A. C, Composite overlay (20X). D & E, Low power images of the eYFP ChR2 reporter signal in ARC N/OFQ neurons juxtaposed with the eGFP reporter signal in ARC POMC neurons. F, DIC image (40X) of a recorded ARC neuron. G, eYFP/ChR2-labelled N/OFQ fibers and perikaryal in the immediate vicinity of the cell in F. H, eGFP POMC reporter signal in the cell in F. I, Prominent N/OFQ fiber labelling and a conspicuous absence of N/OFQ somas in the VMN. J, Optogenetic stimulation of ARC N/OFQ neurons elicits a robust and reversible outward current (n = 9) in the POMC neuron shown in H that is blocked by the NOP receptor antagonist BAN ORL 24 (10 μM; K; n = 5). L, I/V relationships generated prior to and following photostimulation show the increased slope conductance that again is antagonized by BAN ORL 24 (M). N & O, Composite bar graphs illustrating the NOP receptor-mediated outward current and increase in slope conductance. Bars represent means and lies 1 SEM. Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *, p < 0.05, relative to Photostimulation alone, Student’s t test

Fig. 2.

The photostimulation (photostim)-induced outward current in POMC neurons is associated with a hyperpolarization and a decrease in firing, which is abrogated by BAN ORL 24. (A; n=8) Representative trace of a current clamp recording in a slice from an intact male N/OFQ-cre/eGFP-POMC mouse showing the reversible hyperpolarization and decrease in firing (n = 8) that is not apparent in slices pre-treated with BAN ORL 24 (C; n=7) or injected (inj.) with an eYFP blank-containing AAV (B; n=5). D-F, the photo-stimulation-induced increase in slope conductance (or lack thereof) faithfully mirrors the extent of the hyperpolarization and decrease in firing. These effects are summarized in the composite bar graphs seen in G and H. Bars represent means, and lines 1 SEM of the hyperpolarization magnitude (mV; G) and the change in firing (H). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05, relative to photostimulation alone, one-way ANOVA/LSD (G); relative to baseline, Kruskal-Wallis/median-notched box-and-whisker analysis (H).

Fig. 3.

Optogenetic stimulation of ARC N/OFQ neurons produces an outward current in POMC neurons from OVX female N/OFQ-cre/eGFP-POMC mice, which is markedly attenuated by E₂. (A) Membrane current trace showing the robust and reversible outward current (n = 12) during a recording from an ethanol (EtOH; 0.01% v:v) vehicle pre-treated slice. The magnitude of this current is dramatically dampened by E₂ (100 nM; B; n = 11). C & D, Parallel changes were observed with the photostimulation (photostim)-induced increase in slope conductance. These effects are captured in composite form in E and F. Bars represent means, and lines 1 SEM of the change in membrane current (pA; E) and conductance (nS; F). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05, relative to EtOH vehicle alone, Student’s t-test.

Fig. 4.

Optogenetic activation of VTA N/OFQ neurons in male mice induces NOP receptor-mediated outward currents in downstream neurons receiving synaptic input. A & B, eYFP ChR2 reporter staining in the VTA at 4X and 40X. C, DIC image of a recorded neuron surrounded by the eYFP-filled N/OFQ fibers seen in B. D-G, Photostimulation (photostim) of N/OFQ neurons elicits a reversible outward current in the cell in C due to activation of a K⁺ conductance via a NOP receptor-mediated mechanism (D: n = 8; E: n = 6). These effects are compositely expressed in H and I. Bars represent means, and lines 1 SEM of the change in membrane current (ΔI; H) and slope conductance (Δg; I). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05, relative to photostimulation alone, Student’s t-test.

Fig. 5.

The outward current caused by photostimulation (photostim) of VTA N/OFQ neurons corresponds with a NOP receptor-mediated membrane hyperpolarization and reduction in firing. A & B, Representative voltage traces depicting the photostimulation-induced hyperpolarization (n = 7) and abolition of firing that are antagonized by BAN ORL 24 (n = 5). The composite bar graphs in C and D summarize these effects. Bars represent means, and lines 1 SEM of the change in membrane voltage (C; mV) and the change in firing relative to baseline (D). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05, relative to photostimulation alone, Student’s t test (C); Kruskal-Wallis/median-notched box-and-whisker analysis (D).

Fig. 6.

N/OFQ elicits an outward current in A₁₀ dopamine neurons during voltage clamp recordings in slices from male TH-cre mice via a NOP receptor-mediated mechanism. A, TH immunostaining in the VTA. B, eYFP signal seen in these A₁₀ dopamine neurons. C, composite overlay. D & E, eYFP signal from A₁₀ dopamine neurons in VTA slices seen at 4X and 40X. F, DIC image of the recorded A₁₀ dopamine neuron seen in E. G & H, Bath applied N/OFQ (1 μM) produces a robust and reversible outward current in A₁₀ dopamine neurons (n = 11) that is abrogated by BAN ORL 24 (n = 8). I & J, This NOP receptor-mediated response is associated with an increased K⁺ conductance. These effects are summarized compositely in K and L. Bars represent means and lines 1 SEM of the change in membrane current (K; pA) and slope conductance (L; nS). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05 relative to N/OFQ alone, Student’s t-test.

Fig. 7.

The NOP receptor-mediated outward current in A₁₀ dopamine neurons in male mice is associated with a membrane hyperpolarization and cessation of firing. A & B, Representative voltage traces obtained during current clamp recordings in slices from male TH-cre mice illustrating the N/OFQ-induced hyperpolarization (n = 14) and suppression of firing that is negated by BAN ORL 24 (n = 10). C & D, These effects are mirrored by the increase in K⁺ conductance or lack thereof, and summarized compositely in E and F. Bars represent means and lines 1 SEM of the hyperpolarization (E; mV) and change in firing relative to baseline (F). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05, relative to photostimulation alone, Student’s t test (E); Kruskal-Wallis/median-notched box-and-whisker analysis (F).

Fig. 8.

Diet-induced obesity renders A₁₀ dopamine neurons more quiescent and prolongs the inhibitory effect of N/OFQ. Composite bar graphs depicting the markedly hyperpolarized RMP (A) and reduced basal firing rate (B) seen in A₁₀ dopamine neurons from obese TH-cre males. C & D, Representative voltage traces from chow-(n = 22) and HFD-fed (n = 12) males illustrating the more prolonged inhibitory response of A₁₀ dopamine neurons to N/OFQ under conditions of diet-induced obesity. The last two bar graphs show the lack of effect of diet-induced obesity on the magnitude of the N/OFQ-induced hyperpolarization (E) or change in conductance (F). Bars represent means and line 1 SEM. Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05, relative to chow-fed controls, Student’s t-test.

Fig. 9.

E₂ attenuates the outward current caused by optogenetic stimulation of VTA N/OFQ neurons during voltage clamp recordings in slices from OVX female N/OFQ-cre mice. A & B, Representative traces show the estrogenic diminution in the magnitude of the photostimulation (photostim)-induced outward current (A: n = 14; B: n = 9) as well as the accompanying increase in slope conductance shown in C and D. These effects are compositely represented in the bar graphs shown in E and F. Bars represent means and lines 1 SEM of the change in membrane current current (E; pA) and slope conductance (F; nS). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05 relative to EtOH vehicle, Student’s t-test.

Fig. 10.

E₂ diminishes the outward current in A₁₀ dopamine neurons caused by exogenously administered N/OFQ during voltage clamp recordings in slices from OVX female TH-cre mice. A & B, Representative traces illustrating how E₂ reduces the amplitude of the outward current caused by bath application of N/OFQ (A: n = 8; B: n = 7) along with the increase in slope conductance shown in C and D. These effects are compositely expressed in the bar graphs shown in E and F. Bars represent means and lines 1 SEM of the change in membrane current current (E; pA) and slope conductance (F; nS). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05 relative to EtOH vehicle, Student’s t-test (E).

Fig. 11.

Likewise, E₂ dampens the N/OFQ-induced membrane hyperpolarization and abolition of firing seen in A₁₀ dopamine neurons during current clamp recordings in slices from OVX female TH-cre mice. A & B, Representative traces depicting how E2 physiologically antagonizes the hyperpolarization and decrease in firing caused by N/OFQ (A: n = 11; B: n = 8), as well as the increase in K⁺ conductance shown in C and D. These changes are reflected in the composite bar graphs of E and F. Bars represent means and lines 1 SEM of the hyperpolarization (E; mV) and change in firing relative to baseline (F). Numbers above parenthesis in bar graphs are indicative of the number of cells recorded for that treatment, while numbers in parenthesis are indicative of the number of animals used for that treatment. *p < 0.05, relative to EtOH alone, Student’s t test (E); Kruskal-Wallis/median-notched box-and-whisker analysis (F).

Fig. 12.

Intermittent access to HFD dramatically escalates consumption in wildtype mice, which is accentuated by diet-induced obesity and hypoestrogenic conditions. A, Time course illustrating the rapidity of the escalation in binge eating over the five days of the monitoring period, which is especially prominent in obese males and OVX females. B, Bar graph illustrating the disproportionate energy consumption during the one-hour exposure to HFD; particularly in obese males. C, Bar graph depicting cumulative energy intake observed during the other 23 hours of the day. D, Bar graph demonstrating that the extensive binge-like hyperphagia occurs irrespective of time of day. Bars represent means and vertical lines 1 SEM. Numbers above bars indicate number of animals used per treatment group. *, p < .05 relative to binge day 1; #, p < .05 relative to non-binging controls; ^, p < .05 relative to males; %, p < .05 relative to chow-fed controls, repeated measures multi-factorial ANOVA/LSD, n = 6 – 9.

Fig. 13.

NOP receptors regulate binge-eating behavior in a site- and sex-specific manner. A, Bar graph depicting how intra-VTA N/OFQ (0.3 nmole/0.2 μL) dampens the escalation in the consumption of palatable food seen in males exposed to HFD for five weeks prior to commencing the binge-feeding protocol; an effect attenuated by the NOP receptor antagonist UFP-101 (10 nmole/0.2 μL). B, Bar graph illustrating that intra-ARC N/OFQ potentiates binge feeding in male mice. C, Bar graph demonstrating that the N/OFQ-induced decrease in the binge-like intake seen in sesame oil-treated (1 mL/kg; s.c.) OVX female mice is reversed by EB treatment (20 μg.kg; s.c.); particularly in those rendered obese following five weeks of HFD feeding. Numbers above bars indicate number of animals used per treatment group. *, p < .05 relative to saline-treated controls; #, p < .05 relative to lean controls; ^, p < .05 relative to sesame oil-treated controls, repeated measures multi-factorial ANOVA/LSD, n = 6 – 9.