Nociceptin/orphanin FQ modulates energy homeostasis through inhibition of neurotransmission at VMN SF-1/ARC POMC synapses in a sex- and diet-dependent manner
Orphanin FQ (aka nociceptin; N/OFQ) binds to its nociceptin opioid peptide (NOP) receptor expressed in proopiomelanocortin (POMC) neurons within the arcuate nucleus (ARC), a critical anorexigenic component of the hypothalamic energy balance circuitry. It inhibits POMC neurons by modifying neuronal excitability both pre- and postsynaptically. We tested the hypothesis that N/OFQ inhibits neurotransmission at synapses involving steroidogenic factor (SF)-1 neurons in the ventromedial nucleus (VMN) and ARC POMC neurons in a sex- and diet-dependent fashion.
Electrophysiological recordings were done in intact male and in cycling and ovariectomized female NR5A1-Cre and eGFP-POMC mice. Energy homeostasis was assessed in wildtype animals following intra-ARC injections of N/OFQ or its saline vehicle.
N/OFQ (1 μM) decreased light-evoked excitatory postsynaptic current (leEPSC) amplitude more so in males than in diestrus or proestrus females, which was further accentuated in high-fat diet (HFD)-fed males. N/OFQ elicited a more robust outward current and increase in conductance in males than in diestrus, proestrus, and estrus females. These pleiotropic actions of N/OFQ were abrogated by the NOP receptor antagonist BAN ORL-24 (10 μM). In ovariectomized female eGFP-POMC mice, 17β-estradiol (E2; 100 nM) attenuated the N/OFQ-induced postsynaptic response. SF-1 neurons from NR5A1-Cre mice also displayed a robust N/OFQ-induced outward current and increase in conductance that was sexually differentiated and suppressed by E2. Finally, intra-ARC injections of N/OFQ increased energy intake and decreased energy expenditure, which was further potentiated by exposure to HFD and diminished by estradiol benzoate (20 μg/kg; s.c.).
These findings show that males are more responsive to the pleiotropic actions of N/OFQ at anorexigenic VMN SF-1/ARC POMC synapses, and this responsiveness can be further enhanced under conditions of diet-induced obesity/insulin resistance.
KeywordsNociceptin/orphanin FQ Proopiomelanocortin Steroidogenic factor-1 Opioid receptor-like 1 Obesity Insulin resistance
Excitatory postsynaptic currents
Green fluorescent protein
G protein-gated inwardly rectifying K+
G protein coupled receptor
Light-evoked excitatory postsynaptic current
Nociceptin opioid peptide
Yellow fluorescent protein
Nociceptin/orphanin FQ (N/OFQ) is a hepadecapeptide that is similar in structure to the endogenous κ-opioid peptide dynorphin A, yet it does not bind to classical opioid receptors [1, 2]. Its first described physiological role was an increased sensitivity to pain , hence the name nociceptin, but it later proved to be involved in many different physiological processes including cardiovascular and gastrointestinal functions, as well as anxiety [4, 5, 6].
N/OFQ binds with high affinity to its cognate nociceptin opioid peptide (NOP) receptor, which is a G protein coupled receptor (GPCR) that is structured similar to that of classical opioid receptors like the μ-,δ-, and κ-opioid receptors . It has a widespread distribution within the CNS, with higher quantities in the hypothalamus, hippocampus, amygdala, and brainstem [1, 7]. After agonist activation, NOP receptors trigger different intracellular events including a decrease in the activity of adenylyl cyclase . These receptors also couple directly to G protein-gated, inwardly rectifying K+ (GIRK) channels in oocytes , as well as neurons in the dorsal raphe , locus coeruleus , periaqueductal gray , and the hypothalamic arcuate nucleus (ARC) . In addition, they negatively modulate both N-type Ca2+ channels in SH-SY5Y cells  and both N-type as well as P/Q type channels in periaqueductal gray and suprachiasmatic neurons [14, 15].
Bath application of N/OFQ has also been shown to decrease glutamatergic excitatory postsynaptic currents (EPSCs) in rat lateral amygdala, as well as the ARC [16, 17], indicating that N/OFQ acts presynaptically to reduce the amount of glutamate that is released onto its postsynaptic target. Anorexigenic ARC proopiomelanocortin (POMC) neurons have also been shown to be activated from presynaptic glutamatergic inputs emanating from the steroidogenic factor (SF)-1 neurons in the hypothalamic ventromedial nucleus (VMN) [18, 19, 20, 21], which have been shown to synapse directly onto POMC neurons . It has also been shown that chemogenetic stimulation of SF-1 neurons causes a decrease in food intake as well as an increase in energy expenditure and that optogenetic stimulation evokes a robust light-induced EPSC in POMC neurons . It has also been known that a lesioning of the VMN causes rampant hyperphagia and obesity [23, 24]. This indicates that this synaptic connection is important in the homeostatic control of energy balance.
As mentioned above, the NOP receptor is expressed within the hypothalamus, where it regulates many homeostatic properties. Within the ARC, it works through GIRK channels to inhibit POMC neurons [12, 25]. When N/OFQ has been administered centrally, it has been shown to increase both food intake and body weight in mice, effects which are more pronounced in high-fat diet (HFD)-fed animals . It also decreases energy expenditure and ultimately leads to hyperleptinemia and hyperinsulinemia . Administration into the ARC has been shown to be particularly efficacious, leading to greater increases in food intake in comparison to other nuclei . Intracerebroventricular (I.C.V) injections of N/OFQ also produce a hypothermic effect in adult rats due to the activation of NOP receptors . Within the VMN, N/OFQ has been shown to hyperpolarize leptin receptor expressing neurons by NOP receptor activation, which can be reversed by the GIRK channel blocker SCH23390 . Injections into the VMN and the nucleus accumbens have also been shown to increase food intake in rats . In addition, co-administration of the selective NOP receptor antagonist [Nphe1]NC(1-13)NH2 into the third ventricle (3V) reverses the potent orexigenic effects of N/OFQ  in male rats, which suggests that N/OFQ regulates energy intake and expenditure, at least in part, by inhibiting neurotransmission at VMN SF-1/ARC POMC synapses.
There is considerable precedence for sex differences in the hypothalamic regulation of energy homeostasis . While we do not know if there are sex differences in the NOP receptor-mediated regulation of energy homeostasis, we do know that there are sex differences in its regulation of nociception. In in vivo electrophysiological recordings taken from the medullary dorsal horn, N/OFQ reduced the N-methyl-d-aspartate (NMDA)-evoked responses in males and ovariectomized (OVX) females, but not in proestrus or OVX, estradiol benzoate (EB)-treated females . It has also been shown that an intrathecal injection of N/OFQ attenuated the NMDA receptor-mediated nociceptive response in males and OVX females but not in OVX, EB-treated females . Injection of N/OFQ administered into the same region also produced an increased tail flick latency (TFL) to a thermal stimulus in males as well as OVX females, which was reversed with pretreatment of the NOP antagonist UFP-101. In OVX, EB-treated females, however, there was no significant increase in the TFL upon injection of N/OFQ . It has also been shown that activation of GPR30, estrogen receptor (ER)α, the Gq-coupled membrane ER (Gq-mER), but not ERβ abolishes the NOP-mediated antinociception in males and OVX females .
We also know that gonadal steroid hormones exert activational effects upon the NOP receptor-mediated regulation of the hypothalamic energy balance circuitry. For example, in ovariectomized, estradiol-primed female rats, the ability of N/OFQ to activate GIRK channels in POMC neurons and decrease miniature EPSC frequency are significantly diminished . Estradiol attenuates these pleiotropic actions of N/OFQ on POMC neurons by binding to either ERα or the Gq-mER, leading to a signaling cascade that includes PKC, protein kinase A (PKA), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), and PLC . Moreover, progesterone administered to OVX, estrogen-primed females restores the sensitivity of POMC neurons to these pre- and postsynaptic actions of N/OFQ .
Thus, there is sufficient precedence to suggest that the NOP receptor-mediated regulation of energy homeostasis and POMC neuronal excitability is sexually differentiated and subject to modulatory influences from diet and gonadal steroid hormones. Moreover, there is compelling evidence that N/OFQ-sensitive glutamatergic input onto POMC neurons arises, in large part, from SF-1 neurons in the dorsomedial VMN [18, 19, 20, 22]. We therefore hypothesized that N/OFQ modulates energy homeostasis through an inhibition of excitatory neurotransmission at VMN SF-1/ARC POMC and by modulating postsynaptic conductances in both cell types in a sex- and diet-dependent manner.
Materials and methods
Adult male and female Topeka guinea pigs (580–879 g; 40–79 days of age) were bred in-house or purchased on demand from Elm Hill Breeding Labs (Clemsford, MA, USA). Male and female NR5A1-Cre mice (18–43 g; 52–144 days of age) were purchased from Jackson Laboratories (Stock #012462) and bred in house. Male and female eGFP-POMC mice (20–43 g; 55–93 days of age) were also purchased from Jackson Laboratories (Stock #009593) and bred in house as well. Animals were housed under a 12:12-h light/dark cycle (light on at 6 a.m. and off at 6 p.m.), with food and water available ad libitum. Intact female NR5A1-Cre and eGFP-POMC mice were checked the day of experimentation by vaginal lavage to evaluate cell cytology and thus determine the stage of the estrous cycle. On the day of experimentation, MRIs were performed using EchoMRI™-100H and EchoMRI™-130 Body Composition Analyzers for Live Small Animals (Mice) and Organs (EchoMRI LLC, Houston, TX, USA) in order to determine the total fat mass as well as lean mass. Fat dissections were also taken from the perirenal, gonadal, and abdominal regions during terminal harvest along with photographic documentation. All procedures were approved by the Western University of Health Sciences’ IACUC and IBC in accordance with institutional guidelines based on NIH standards.
For all surgeries, animals were administered carprofen (Vetranal by Sigma Aldrich, 5 mg/mL; give 5 mg/kg; s.c.; both preemptively and 1 day post-surgery) to mitigate against surgical and postoperative pain, as well as sulfamethoxazole/trimethoprim suspended in their drinking water (0.48 g/L) in order to minimize the potential for postoperative infections. For some experiments, both NR5A1-Cre and eGFP-POMC female mice were OVX while they were under 2% isoflurane anesthesia. In order to focally inject adeno-associated viral vector (AAV) constructs, NR5A1-Cre mice were anesthetized with 2% isoflurane and placed in a stereotaxic frame. An incision was made to expose the skull, and a single hole was drilled on one side of the mid-sagittal suture so that an injection needle could be slowly lowered into the dorsomedial subdivision of the VMN (coordinates from bregma: AV − 0.6 mm, ML ± 0.3 mm, and DV − 5.6 mm from dura). A unilateral injection of a Cre-recombinase-dependent AAV vector containing cation channel rhodopsin-2 (ChR2; AAV1.EF1a.DIO.ChR2 (E123A).YFP.WPRE.jGH; 7.2 × 1012 genomic copies/mL; 300 nL total volume; University of Pennsylvania Vector Core; Addgene plasmid #35507) was given over 2 min. The injection needle remained in place for 10 min after infusion to allow for diffusion from the tip and was then slowly removed from the brain to reduce potential spread of the virus from the desired anatomical location. Animals were used for experimentation 2–3 weeks after viral injection and 1–2 weeks after OVX. Only those animals that (a) showed clear evidence of accurate AAV injection in the VMN (as indicated by the fluorescence of the YFP reporter) and (b) maintained their bright, alert, and responsive status and regained a positive growth trajectory post-surgery were included in the present study.
The stereotaxic implantation of a guide cannula into the ARC of the mice was performed similar to that described above. Briefly, once anesthetized, an animal was secured in a stereotaxic frame (Stoelting, Wood Dale, IL, USA), and a midline incision was made through the scalp. A hole was then drilled in the skull, through which a 26-gauge guide cannula (Plastics One, Roanoke, VA, USA) was lowered 1 mm above the ARC using the following coordinates: AP − 0.6 mm, ML − 0.3 mm, DV − 4.9 mm. The guide cannula was fastened in place with C&B Metabond dental cement (Parkell, Edgewood, NY, USA) applied to the surgical field. Finally, a stylet was inserted into the guide cannula to keep the lumen patent. The animals were allowed to recover for 1 week prior to the start of experimentation. Only those animals in which we could verify accurate guide cannula placement within the ARC were included in this study.
All drugs were purchased from Tocris Bioscience/R&D Systems (Minneapolis, MN, USA) unless otherwise stated. For electrophysiological experiments, the GABAA receptor antagonist 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (SR 95531) was dissolved in Ultrapure H20 to a stock concentration of 10 mM, and the stock solution was diluted further with artificial cerebrospinal fluid (aCSF) to the working concentration of 10 μM. N/OFQ was prepared as a 1 mM stock solution in UltraPure H20 and diluted further with aCSF to the working concentration of 1 μM. The NOP receptor antagonist (2R)-1-(phenylmethyl)-N-[3-(spiro[isobenzofuran-1(3H),4′-piperidin]-1-yl)propyl-2-pyrrolidinecarboxamide (BAN ORL 24 (BAN)) was prepared as a 10 mM stock solution in UltraPure H2O and diluted further with aCSF to the working concentration of 10 μM. The Na+ channel blocker octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethano-10aH-[1,3]dioxocino[6,5-d]pyrimidine-4,7,10,11,12-pentol (tetrodotoxin, TTX) was prepared as a 1 mM stock solution in UltraPure H20 and diluted further with aCSF to the working concentration of 500 nM. 1, 3, 5(10)-Estratrien-3, 17β-diol (17β-estradiol (E2); Steraloids, RI, USA) was dissolved in punctilious ethanol to a stock concentration of 1 mM, which was further diluted to a working concentration of 100 nM. All aliquots of the stock solutions were stored at either four or − 20 °C until needed for experimentation.
For all behavioral experiments, N/OFQ was prepared as a 1.5 mM stock solution by dissolving it in filtered saline and injected directly into the ARC at a 0.3 nmol dose. Estradiol benzoate (EB; Steraloids, Newport, RI, USA) was initially prepared as a 1 mg/mL stock solution in punctilious ethanol. A known quantity of this stock solution was added to a volume of sesame oil sufficient to produce a final concentration of 100 μg/mL following evaporation of the ethanol.
Hypothalamic slice preparation
On the day of experimentation, the animal was briefly anesthetized with 32% isoflurane and rapidly decapitated. The brain was removed from the skull, and the hypothalamic area was dissected. We then mounted the hypothalamic block on a cutting platform that, for the guinea pig, was secured in a vibratome well filled with ice-cold, oxygenated (95% O2, 5%CO2) aCSF (NaCl, 124; NaHCO3 26; dextrose 10, HEPES 10; KCl 5; NaH2PO4 2.6; MgSO4 2; CaCl2 1; in mM). For the mice, we used a sucrose-based cutting solution (NaHCO3 26; dextrose 10, HEPES 10; Sucrose 208; KCl 2; NaH2PO4 1.25; MgSO4 2; CaCl2 1; in mM). Four to five coronal slices (300 μm) through the rostrocaudal extent of the ARC were then cut. The slices were transferred to an auxiliary chamber containing oxygenated aCSF at room temperature and maintained there until the electrophysiological recording.
Whole-cell patch clamp electrophysiological recordings from ARC neurons using biocytin-filled electrodes were performed in hypothalamic slices prepared from gonadally intact male/female NR5A1-Cre and eGFP-POMC mice, as well as OVX female NR5A1-Cre and eGFP-POMC mice. To ascertain whether the hypothesized pleiotropic actions of N/OFQ occurred in other species, recordings in slices from male and periovulatory female guinea pigs were also conducted. During recordings, the slices were maintained in a chamber perfused with warmed (35 °C), oxygenated aCSF in which the CaCl2 concentration raised to 2 mM. Artificial CSF and all drugs (diluted with aCSF) were perfused via peristaltic pump at a rate of 1.5 mL/min. Patch electrodes were prepared from borosilicate glass (World Precision Instruments, Sarasota, FL, USA; 1.5 mm OD) pulled on a P-97 Flaming Brown puller (Sutter Instrument Co., Novato, CA, USA), and filled with an internal solution containing the following (in mM): potassium gluconate 128, NaCl 10, MgCl2 1, EGTA 11, HEPES 10, ATP 1, GTP 0.25, 0.5% biocytin, adjusted to a pH of 7.3 with KOH and osmolality 286–320 mOsm. Electrode resistances varied from 3 to 8 MΩ.
For guinea pig experiments, whole-cell patch clamp recordings were performed using a Multiclamp 700A preamplifier (Axon Instruments, Foster City, CA, USA) that amplified potentials and passed current through the electrode. Membrane currents were recorded in voltage clamp with access resistances ranging from 8 to 20 MΩ. The signals underwent analog-digital conversion via a Digidata 1322A interface coupled to pClamp 10.5 software (Axon instruments). For the transgenic mouse experiments, recordings were made on an Olympus BX51 W1 fixed stage microscope outfitted with infrared differential interference contrast video imaging. A Multiclamp 700B preamplifier (Molecular Devices) amplified potentials and passed current through the electrode. Membrane currents underwent analog-digital conversion with a Digidata 1550A interface (Molecular Devices) coupled to pClamp 10.5 software. The access resistance, resting membrane potential (RMP), and input resistance were monitored throughout the course of all recordings. If the access resistance deviated greater than 10% of the original value, the recording was ended. Low-pass filtering of the currents was conducted at a frequency of 2 kHz. The liquid junction potential was calculated to be − 10 mV and corrected for during data analysis using pClamp software. All recordings for the presynaptic studies were performed under a holding potential of − 75 mV, while those for the postsynaptic studies were performed at a holding potential of − 60 mV.
For the optogenetic studies described in experiments 1–3 that were designed primarily to test the hypothesis that N/OFQ decreases glutamate release at VMN SF-1/ARC POMC synapses in a sex- and diet-dependent manner, recordings were performed in slices from NR5A1-Cre mice that were injected with a ChR2-containing viral vector into the VMN 2–3 weeks prior to experimentation. Once glutamatergic SF-1-expressing fibers (visualized with YFP) impinging on ARC neurons were encountered, functional synaptic connectivity was ascertained by applying a photo-stimulus (25–100-ms pulses delivered every 2 s) from a light-emitting diode (LED) blue light source (470 nm) controlled by a variable 2A driver (ThorLabs, Newton, NJ, USA) that directly delivered the light path through the Olympus 40X water-immersion lens to generate a fast excitatory postsynaptic current (EPSC) as described previously . We then determined whether the neuron under consideration was a likely POMC neuron by prescreening for intrinsic membrane currents like the A-type K+ current and the hyperpolarization-activated mixed cation current [37, 39, 40] via the current-voltage (I/V) relationships generated as described in the next paragraph. Baseline light-evoked excitatory postsynaptic currents (leEPSCs) were generated in the presence of the GABAA receptor antagonist SR95531 (10 μM), either alone or with the NOP receptor antagonist BAN (10 μM), by photostimulating the SF-1 neurons from a holding potential of − 75 mV. After generating baseline leEPSCs over three to five 10-sweep trials, we would then co-apply N/OFQ (1 μM) amidst SR95531 with or without BAN for an additional 4–6 min, after which we would then generate leEPSCs in the presence of N/OFQ over another three to five 10-sweep trials. The N/OFQ was then allowed to clear the slice for 10 min before generating washout leEPSCs over another three to five 10-sweep trials. To analyze the leEPSCs collected prior to and in the presence of N/OFQ, alone and in conjunction with BAN, we would measure the amplitude of each leEPSC for each sweep in every trial and generate an average.
When necessary, slices were then processed after recording for immunohistochemistry using various phenotypic markers of ARC POMC neurons. Slices were initially fixed with 4% paraformaldehyde in Sorenson’s phosphate buffer (pH 7.4) for 120–180 min. They were then immersed overnight in 20% sucrose dissolved in Sorensen’s buffer and frozen in Tissue-Tek embedding medium (Miles, Inc., Elk-hart, IN, USA) the next day. Coronal sections (20 μm) were cut on a cryostat and mounted on chilled slides. These sections were then washed with 0.1 M sodium phosphate buffer (pH 7.4) and then processed with streptavidin-Alexa Flour (AF) 546 (Molecular Probes, Inc., Eugene, OR, PA, USA) at a dilution of 1:600. After localizing the biocytin-filled neuron via fluorescence microscopy, the appropriate sections were processed further with polyclonal antibodies directed against α-melanocyte-stimulating hormone (α-MSH, Immunostar, Inc., Hudson, WI, USA; 1:200 dilution), β-endorphin (Immunostar, Inc.; 1:400 dilution), cocaine and amphetamine-regulated transcript (CART; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA; 1:200 dilution), or SF-1 (Abcam, Cambridge, MA, USA; 1:300), and again evaluated using fluorescence immunohistochemistry.
Feeding and metabolic studies
The feeding and metabolic studies were performed using a four-station Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, Ohio, USA) from which we monitored cumulative food intake, meal size, meal frequency, rate of consumption and several measures of energy expenditure (O2 consumption, CO2 production, and respiratory exchange ratio (RER) and metabolic heat production as described and validated previously ). These studies were conducted under conditions in which food and water were available ad libitum. The animals were allowed to acclimate in their CLAMS chamber over a 3-day period. Each day they were weighed, handled, and returned to their respective chambers. After the 3-day acclimation session, we initiated the 5-day monitoring phase during which the animals were weighed, injected each day at 4:00 pm (2–3 h in advance of the nocturnal peak in energy consumption) with either N/OFQ (0.3 nmol) or its 0.9% saline vehicle (0.2 μL) administered directly into the ARC. OVX females were also injected with either EB (20 μg/kg; s.c.) or its sesame oil vehicle (1 mL/kg; s.c.) every other day at the same time beginning on acclimation day 2. The mice were immediately placed back in their feeding chambers and monitoring took place continuously around the clock for the next 5 days.
Cumulative food intake was taken as the total amount of food consumed at 1, 2, and 4 h after either N/OFQ or vehicle administration. Meal size is the amount of food eaten in a given hour divided by the number of meals in that same hour. The parameters of energy intake, meal pattern, and energy expenditure were continuously written to computer via an A/D converter.
Data were analyzed using Statgraphics software (Statgraphics Centurion XVI Version 16.1 17, Starpoint Technologies, INC.) and checked for normality using Bartlett’s test. Comparisons between two groups were made with either the Student’s t test (for parametric data) or the Mann-Whitney U test (for non-parametric data). Comparisons made between more than two groups were performed using either the one-way, multi-factorial, repeated-measures multi-factorial, or rank-transformed multi-factorial analysis of variance (ANOVA; the first three for parametric data, the last one for non-parametric data) followed by the least significant difference (LSD) test, or alternatively via the Kruskal-Wallis test followed by the median-notched box-and-whisker analysis (for non-parametric data). If a significant interaction was encountered among the experimental variables following multi-factorial analyses, we then performed a one-way ANOVA to elucidate significant differences among the various treatment groups. Differences were considered statistically significant if the alpha probability was 0.05 or less.
Experiment 1: N/OFQ significantly decreases optogenetically stimulated leESPC amplitude due to the activation of the NOP receptor
Experiment 2: The N/OFQ-induced decrease in glutamatergic input onto POMC neurons due to NOP activation is sexually differentiated
Now that we know that the N/OFQ-induced decrease in glutamatergic input onto ARC POMC neurons emanating from SF-1 neurons in the dorsomedial VMN is due to the activation of the NOP receptor, we next wanted to examine if this action is sexually differentiated. Figure 4 shows the representative basal leEPSCs for NR5A1-Cre females during diestrus and proestrus. The degree of the N/OFQ-induced reduction in leEPSC amplitude seen in proestrus (Fig. 4a) and diestrus (Fig. 4b) is significantly smaller than that seen in the previous figure with the intact male recording (Figs. 3a, 4c; Kruskal-Wallis/median-notched box- and- whisker plot, test statistic = 11.1162, p < 0.005). Data from estrus and metestrus females are not included here due to the lack of functional synapses capable of generating a leEPSC even under baseline conditions. This indicates that the N/OFQ-induced decrease in excitatory neurotransmission at VMN SF-1/ARC POMC synapses is sexually differentiated, with males being more sensitive than females during certain stages of the estrous cycle.
Experiment 3: Long-term exposure to HFD further accentuated the N/OFQ-induced decrease in leEPSC amplitude
Experiment 4: N/OFQ postsynaptically inhibits POMC and SF-1 neurons by inducing an outward current due to the activation of the NOP receptor
Experiment 5: The N/OFQ-induced outward current and increase in conductance is sexually differentiated
We then ventured to see if the observed sex differences in the postsynaptic effects of N/OFQ extended to other species. Here, we again performed electrophysiological recordings from ARC POMC neurons using the same whole-cell patch clamp techniques in intact male and periovulatory female guinea pigs. As expected, N/OFQ caused a pronounced hyperpolarization and a complete cessation of firing in identified POMC neurons (Additional file 3: Figure S3B: Student’s t test, t = 3.082, p < 0.030). More importantly, the disparity in the N/OFQ-induced outward current between male and periovulatory female guinea pigs was very similar to that of the male and female mice, in that there was a robust response and increase in conductance for the male (Additional file 4: Figure S4A), while in the female this effect was attenuated (Additional file 4: Figure S4B). The inequities in the N/OFQ-induced increase in slope conductance seen in Additional file 4: Figure S4C further illustrate the conserved nature of this sex difference in postsynaptic activation of GIRK channels in POMC neurons (multi-factorial ANOVA/LSD: Fvoltage = 0.39 (df = 1, p < 0.60),;Fsex = 5.27 (df = 1, p < 0.040), Finteraction = 0.35 (df = 1, p < 0.60)).
Experiment 6: Long term exposure to HFD accentuates the N/OFQ-induced outward current and increase in conductance in a sex-dependent manner
Experiment 7: N/OFQ also produces an outward current and increases conductance in SF-1 neurons located in the dorsomedial VMN
Experiment 8: Direct injection of N/OFQ into the ARC significantly increases food intake and modulates energy expenditure in a sex- and diet- dependent
The results generated from this project demonstrate that N/OFQ modulates energy homeostasis via pleiotropic actions at VMN SF-1/ARC POMC synapses in a sex- and diet-dependent manner. These findings are based on the following observations: (1) N/OFQ decreases leEPSC amplitude in POMC neurons upon photostimulation of SF-1 neurons via activation of NOP receptors; (2) this effect is significantly more pronounced in males than in proestrus and diestrus females; (3) HFD further accentuates the N/OFQ-induced presynaptic inhibition of excitatory neurotransmission at VMN SF-1/ARC POMC synapses; (4) N/OFQ induces a prominent, reversible outward current in both POMC and SF-1 neurons associated with an increase in conductance that robustly hyperpolarizes and decreases firing in these cells, again via activation of NOP receptors; (5) these N/OFQ-induced postsynaptic effects are sexually differentiated, fluctuate during the estrous cycle, enhanced in POMC neurons from HFD-fed females, and markedly attenuated by E2; and (6) N/OFQ delivered directly into the ARC increases energy intake and decreases energy expenditure, which is further potentiated by long-term exposure to HFD in males and, to a lesser extent, in OVX females, and diminished by E2. Collectively, these findings validate our working hypothesis that males are more responsive than females to the multi-faceted effects of N/OFQ within the hypothalamic energy balance circuitry, which can be further accentuated under conditions of diet-induced obesity/insulin resistance in sex-specific ways.
N/OFQ pleotropically modulates neurotransmission at VMN SF-1/ARC POMC synapses in males due to the activation of the NOP receptor
We have demonstrated that N/OFQ decreases glutamatergic input from VMN SF-1 neurons onto ARC POMC neurons due to the activation of the NOP receptor. This finding aligns with other examples of N/OFQ-induced presynaptic inhibition of glutamatergic input onto neurons located in other brain regions like the suprachiasmatic nucleus (SCN), where Gompf et al. found that bath application of N/OFQ produced a concentration-dependent inhibition of glutamatergic EPSCs , or in the rat lateral amygdala, where extracellular application of N/OFQ was found to again dose dependently decrease EPSC amplitude . It also coincides with previous reports that N/OFQ decreases EPSC amplitude in unidentified ARC neurons  and reduces mEPSC frequency in ARC POMC neurons, in a manner sensitive to NOP receptor antagonism or genetic ablation [25, 41]. Most importantly, this study is the first to clearly delineate the anatomical origin of the N/OFQ-sensitive glutamatergic input that impinges on POMC neurons.
We also found that N/OFQ not only modulates the presynaptic inputs onto ARC POMC neurons emanating from VMN SF-1 neurons, but that it also activates somatodendritic NOP receptors on both SF-1 and POMC neurons to hyperpolarize and thereby inhibit these cells. Again, these data are in agreement with prior reports of similar postsynaptic responses in other brain regions such as the SCN . They are also congruent with previous findings in the ARC, where N/OFQ produces a robust outward current and hyperpolarization that is associated with an increase in conductance, and abrogated by NOP receptor antagonists, genetic ablation of the NOP receptor, and GIRK channel blockers [12, 25, 41].
The pleiotropic actions of N/OFQ at VMN SF-1/ARC POMC synapses are sexually differentiated
Our present study also demonstrates that both the pre- and postsynaptic effects of N/OFQ are sexually differentiated; with males being more sensitive than females during certain stages of their estrous cycle. This is consistent with other sex differences we have seen previously at this particular synaptic connection, with males being more sensitive to retrograde, EC-mediated inhibition of glutamatergic input emanating from SF-1 neurons onto POMC neurons . N/OFQ has also proven to have gender-specific modulations in other regions. Flores et al.  also found that N/OFQ inhibits NMDA-evoked excitatory responses in trigeminal nociceptive neurons from male and OVX female rats, but not in proestrus or OVX, estradiol-treated female rats. In addition, Claiborne et al. reported that N/OFQ failed to produce antinociceptin in proestrus rats, and estradiol dose dependently diminished the antinociception seen in OVX females . Conversely, testosterone facilitated the antinociceptive effect of N/OFQ . Moreover, activation of ERα and Gq-mER attenuates NOP-mediated antinociceptin in males and OVX females , as well as the activation of GIRK channels in ARC POMC neurons , via signal transduction pathways that include extracellular signal-regulated kinase, PKC, PKA, PI3K, and neuronal nitric oxide synthase (nNOS).
In the present study, we saw that the pleiotropic actions of N/OFQ at SF-1/POMC synapses varied across the estrous cycle. For example, during metestrus, the NOP receptor-mediated activation of GIRK channels in POMC neurons is similar to that observed in males, whereas in diestrus, proestrus, and estrus females it is markedly attenuated. Moreover, the NOP receptor-mediated presynaptic inhibition of glutamatergic input from SF-1 neurons onto POMC neurons is significantly diminished during diestrus and proestrus as compared to males, whereas in estrus and metestrus females the number of functional inputs is greatly reduced. The fluctuations in the N/OFQ-induced responses are similar to what we have demonstrated previously in OVX, estradiol-primed females—with and without progesterone treatment. Indeed, Borgquist et al. found that progesterone treatment following estradiol priming restores the responsiveness of POMC neurons to the postsynaptic, N/OFQ-induced activation of GIRK channels that was blunted by estradiol treatment alone . Progesterone also reinstates the ability of N/OFQ to presynaptically inhibit glutamatergic input, and markedly dampens the ability of N/OFQ to presynaptically inhibit GABAergic input, onto POMC neurons in estradiol-primed females . This would explain why, during diestrus and proestrus, when estradiol levels are on the rise [45, 46], the pleiotropic actions of N/OFQ at SF-1/POMC synapses are appreciably diminished. Indeed, hypothalamic circuits are most sensitive to the feedback actions of estradiol during this period [47, 48]. Conversely, while progesterone has been reported to peak during estrus [45, 49], it has also been documented to be elevated during metestrus as well [46, 50], and it is clear that the progesterone-induced increase in the responsiveness of SF-1/POMC synapses to N/OFQ manifests during this stage of the cycle.
Diet-induced obesity further modifies N/OFQ’s pleotropic effects at VMN SF-1/ARC POMC synapses in a sexually disparate manner
This present study also demonstrates that long-term exposure to HFD, in males, further accentuates the N/OFQ-induced decrease in excitatory neurotransmission at this specific synapse. Diet-induced obesity has long been associated with dysregulated neuroendocrine function within the hypothalamic energy balance circuitry. For instance, Fabelo et al. found previously that males exposed long-term to HFD exhibit a more pronounced reduction in leEPSC amplitude in POMC neurons upon optogenetic stimulation of SF-1 neurons that occurs via enhanced retrograde EC-mediated signaling, whereas in females this is due to a loss of functional excitatory synapses altogether . The alterations caused by diet-induced obesity/insulin resistance are associated with disordered PI3K/Akt signaling in the ARC and VMN. For example, the sexually differentiated increase in inhibitory EC tone at SF-1/POMC synapses seen with diet-induced obesity/insulin resistance is linked to reduced PI3K/Akt signaling in male but not female animals [21, 38]. On the other hand, diet-induced obesity promotes an insulin-dependent increase in PI3K signaling in the VMN . PI3K and the energy sensor AMPK are counter-regulatory signaling molecules involved in the hypothalamic control of energy balance [52, 53]. Thus, the reduction in ARC PI3K/Akt signaling seen with obese/insulin resistance could pave the way for testosterone-induced AMPK activation in males that increases EC and N/OFQ tone at SF-1/POMC synapses [20, 54]. Interestingly, diet-induced obesity/insulin resistance enhances NOP receptor-mediated activation of GIRK channels in POMC neurons from female but not male animals. Coupled with the accentuated EC- and N/OFQ-induced presynaptic inhibition of glutamatergic input from SF-1 neurons onto POMC neurons, it stands to reason that the enhancement of this Gi/o-coupled metabotropic receptor-mediated response may represent a more global form of adaptive plasticity occurring with diet-induced obesity/insulin resistance at these synapses (see Fig. 15).
We also found that E2 decouples NOP receptors from their GIRK channels in both POMC and SF-1 neurons from both chow-fed and obese females. This is entirely consistent with prior reports demonstrating that in POMC neurons, or in excitatory inputs impinging upon POMC neurons, E2 rapidly uncouples metabotropic Gi/o-coupled receptors like μ-opioid, GABAB, and CB1 receptors from their effector systems [32, 55, 56, 57]. This occurs via activation of ERα and the Gq-coupled mER via signaling pathways that include PLC, PI3K, nNOS, PKC, and PKA [32, 37, 57, 58]. In addition, the decoupling of inhibitory ORL1 receptors from GIRK channels in SF-1 neurons lies in agreement with the ERα/PI3K-mediated increase in the excitability of these cells . Moreover, knockout of ERα in VMN SF-1 neurons leads to reduced energy expenditure, whereas in POMC neurons it leads to hyperphagia . Given that both SF-1 and POMC neurons are anatomical substrates for the actions of insulin [38, 51, 61], this ability of E2 to markedly attenuate inhibitory NOP receptor-mediated neurotransmission at every node comprising these anorexigenic VMN SF-1/ARC POMC synapses may represent a novel means of protection against the development of central insulin resistance.
Direct administration of N/OFQ into the ARC causes a time-dependent increase food intake and decrease in energy expenditure
Consistent with the ability of N/OFQ to inhibit both SF-1 and POMC neurons, as well glutamate release at the synapses formed between them, our study found that direct administration of N/OFQ into the ARC causes an increase in energy intake corresponding with parallel changes in meal pattern, as well as a decrease in energy expenditure. These effects were prominent in male and OVX female animals and markedly diminished by E2. We also found that the N/OFQ-induced increases in energy intake and various indices of meal pattern are more pronounced in HFD-fed males and OVX females, but not in OVX, EB-treated females. The N/OFQ-induced changes in energy intake that we observed presently are congruent with the findings of Matushita et al., who reported that the N/OFQ-induced hyperphagia and increased adiposity were further exaggerated in animals exposed to a HFD . The ability of E2 to antagonize these effects is in keeping with numerous prior demonstrations that it negatively modulates the signal transduction elicited by orexigenic, Gi/o-coupled receptors (for review see [32, 62]). Polidori et al. examined the site-specific effect of N/OFQ in a number of different limbic and hypothalamic structures and found that the ARC is by far the region most sensitive to the hyperphagic effects of the neuropeptide . While Matsushita et al. found no change in rectal temperature or spontaneous locomotor activity with N/OFQ , NOP receptor knockout mice and NOP antisense-treated rats are reported to exhibit higher core body temperatures than their respective controls [5, 63]. This latter finding is indicative of a decrease in energy expenditure, which aligns with our findings that N/OFQ decreased O2 consumption and CO2 production, as well as metabolic heat production in the OVX female. These actions are appreciably attenuated by E2 in both chow- and HFD-fed OVX females and further potentiated in obese males.
This study was supported by PHS Grant DA024314 and intramural funding from Western University of Health Sciences.
Availability of data and materials
The dataset is available from the corresponding author on reasonable request.
JH and CF performed all stereotaxic and survival surgeries. JH and CF performed all electrophysiological recordings. JH, CF, RC, LP, and CM performed all metabolic studies. JH, CF, and EJW performed data analysis for all electrophysiology and metabolic studies, while RC analyzed data for metabolic studies. JH and EJW created all figures and performed all statistical analyses. JH and EJW generated the manuscript, while JH, CF, RC, LP, CM, and EJW edited the final manuscript. EJW, JH, and CF designed the experiments. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All procedures were approved by the Western University of Health Sciences’ IACUC in accordance with institutional guidelines based on NIH standards.
Consent for publication
The authors declare that they have no competing interests.
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