Injection Of DAMGO Research Articles

Spinal orexin A attenuates opioid-induced mechanical hypersensitivity in the rat.

BackgroundRepeated administration of opioid analgesics for pain treatment can produce paradoxical hyperalgesia via peripheral and/or central mechanisms. Thus, this study investigated whether spinally (centrally) administered orexin A attenuates opioid-induced hyperalgesia (OIH).Methods[D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), a selective µ-opioid receptor agonist, was used to induce mechanical hypersensitivity and was administered intradermally (4 times, 1-hour intervals) on the rat hind paw dorsum. To determine whether post- or pretreatments with spinal orexin A, dynorphin A, and anti-dynorphin A were effective in OIH, the drugs were injected through an intrathecal catheter whose tip was positioned dorsally at the L3 segment of the spinal cord (5 µg for all). Mechanical hypersensitivity was assessed using von Frey monofilaments.ResultsRepeated intradermal injections of DAMGO resulted in mechanical hypersensitivity in rats, lasting more than 8 days. Although the first intrathecal treatment of orexin A on the 6th day after DAMGO exposure did not show any significant effect on the mechanical threshold, the second (on the 8th day) significantly attenuated the DAMGO-induced mechanical hypersensitivity, which disappeared when the type 1 orexin receptor (OX1R) was blocked. However, intrathecal administration of dynorphin or an anti-dynorphin antibody (dynorphin antagonists) had no effect on DAMGO-induced hypersensitivity. Lastly, pretreatment with orexin A, dynorphin, or anti-dynorphin did not prevent DAMGO-induced mechanical hypersensitivity.ConclusionsSpinal orexin A attenuates mechanical hyperalgesia induced by repetitive intradermal injections of DAMGO through OX1R. These data suggest that OIH can be potentially treated by activating the orexin A-OX1R pathway in the spinal dorsal horn.

Fentanyl induces autism-like behaviours in mice by hypermethylation of the glutamate receptor gene Grin2b

BackgroundEnvironmental factors contribute to autism spectrum disorder. Fentanyl, one of the most widely used opioid analgesics in anaesthesia, can induce neurotoxicity, but its role in autism remains unknown. We determined whether fentanyl induced autism-like behaviours in young mice and the underlying mechanisms. MethodsYoung male and female mice received fentanyl at postnatal days 6, 8, and 10, and performed behavioural tests, including three-chamber social preference, elevated plus maze, grooming behaviour, and open-field test, from postnatal days 30–32. Expression of Grin2b, the gene encoding the GluN2B subunit of the N-methyl-d-aspartate receptor, was assessed in the anterior cingulate cortex of male mice using fluorescence in situ hybridisation histochemistry. We used bisulfite target sequencing to determine Grin2b hypermethylation sites after fentanyl treatment. In the specific activation and rescue experiments, we injected the mu opioid receptor agonist [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin (DAMGO) or Grin2b overexpression lentivirus into the anterior cingulate cortex of male mice. ResultsFentanyl induced autism-like behaviours in both young male and female mice, and downregulated Grin2b expression (0.49-fold [0.08] vs 1.00-fold [0.09]; P<0.01) and GluN2B protein amounts (0.38-fold [0.07] vs 1.00-fold [0.12]; P<0.01) in the anterior cingulate cortex through hypermethylation of Grin2b. The mu-opioid receptor antagonist naloxone and overexpression of Grin2b in anterior cingulate cortex attenuated the fentanyl-induced effects, whereas DAMGO injection into the anterior cingulate cortex induced autism-like behaviours. ConclusionsThese data suggest that fentanyl induces autism-like behaviours in young mice via an epigenetic mechanism. Further research is required to determine possible clinical relevance to autism risk.

Feeding behavior elicited by mu opioid and GABA receptor activation in the lateral septum

The lateral septum (LS), a brain region typically associated with behaviors involving reward, anxiety-like behavior, learning, and memory, has recently received increased interest due to its potential role in eating behavior. Our current results showed that morphine (5 μg) microinjected into the LS produced a stable feeding response. Specifically, across five days of repeated injections, there was no increase or sensitization effect, nor a decrease in feeding or tolerance. Additionally, we found that pretreatment with the broad-spectrum opioid receptor antagonist naloxone blocked morphine-elicited feeding, further supporting a role for LS opioid receptors in the activation of feeding behaviors. We had previously found that the GABAA receptor agonist muscimol produces a similar increase in feeding when injected into the LS. Given the involvement of the LS in multiple behaviors, we next evaluated whether other behaviors might be co-occurring with feeding in response to opioid or GABAA receptor agonist injection into the LS. We assessed eating, drinking, grooming, sleeping, activity levels and resting behavior for 3 h after injection of aCSF, DAMGO, morphine, or muscimol. We found that morphine and muscimol both decreased the latency to eat, and all drugs tested increased food intake. The feeding occurred within 30 min of muscimol injection but was delayed after opioid injections. The absence of increases in other goal-oriented behavior like drinking or grooming or behavioral hyperactivity supports a primary effect of muscimol and the opioids on LS mechanisms of feeding control. Significance statementThe LS is interesting because of its role in a wide range of behaviors including defensive behaviors, social behaviors, learning, memory, and motivation. Although the LS was discovered to have a role in feeding stimulation over 30 years ago, only recently has major progress begun to reveal the underlying mechanisms. The present paper contributes by suggesting that LS GABAA and μ-opioid receptors elicit eating by inhibiting LS neurons that themselves inhibit eating. Importantly, this work informs lateral septal research which may shed light on disordered eating included binge eating and anorexia.

Inspiratory and Phase‐Spanning Dorsolateral Pontine Neurons Are Silent During Fentanyl‐Induced Apnea

Overdoses caused by the opioid agonist fentanyl have increased exponentially in recent years. These overdose deaths are the result of inadequate ventilation. While much research has focused on opioid modulation of medullary respiratory nuclei, pontine mediated effects of opioids have received less attention. Given the interconnectivity of the pontine Kölliker-Fuse/Parabrachial complex (KF/PB) with rhythmogenic nuclei in the medulla, their involvement in controlling respiratory rate and pattern, and their sensitivity to opioids, we investigate the role they play in opioid mediated respiratory depression and hypothesize that dorsolateral pontine inspiratory, phase-spanning, and post-inspiratory neurons are silenced by opioids. To address this, we have done single unit recordings in the working heart brainstem in situ rat preparation. Dorsolateral pontine neural units (n=48) were recorded extracellularly and systemically exposed to fentanyl and then the opioid antagonist naloxone. A phase-fraction 2-D histogram (heatmap) was generated to compare all unit's action potential spiking over respiratory phases and during periods following fentanyl administration. Each neural unit was categorized at baseline into 4 main groups of discharge identity: Inspiratory, Expiratory, Phase-spanning, and Tonic. Systemic application of fentanyl (300nM) resulted in a consistent decline in fictive respiratory output, transitioned to fast shallowing breathing, and eventually resulted in cessation of respiratory output (apnea). Out of 48 extracellular single unit recordings in the dorsolateral pons, 25 (52%) were completely silent (firing frequency of 0 Hz) suggesting post-synaptic opioid receptor activation during fentanyl-induced apnea; 17/20 inspiratory, 6/12 phase spanning (4/9 IE & 2/3 EI), 1/13 expiratory (1/5 Post I Dec) and 1/3 tonic neurons. To note, many neurons that were not completely silenced by opioids did fire at a much lower firing frequency, suggesting pre-synaptic opioid receptors also contribute to opioid-induced loss of pontine neural activity. Our hypothesis was partially supported, as the majority of inspiratory neurons (17/20) and a subpopulation of phase-spanning neurons (6/12) were completely silenced by fentanyl. Surprisingly post-inspiratory neurons were mainly insensitive to opioids (4/5). These results suggest that loss of post-inspiratory activity with DAMGO injection into the KF may depend critically on post-synaptic inhibition of inspiratory and a subset of IE phase-spanning neurons, which in turn, could decrease local pre-synaptic excitation of post-inspiratory neurons. The majority of pontine expiratory neurons were not silenced by fentanyl (12/13). Due to the KF/PB role in expiratory duration, sustained activity of pontine expiratory neurons may facilitate sustained apnea during opioid overdose. These results warrant further investigation into the contribution of the dorsolateral pons in respiratory pattern formation and opioid-induced respiratory depression.

Positive allosteric modulation of the cannabinoid type-1 receptor (CB1R) in periaqueductal gray (PAG) antagonizes anti-nociceptive and cellular effects of a mu-opioid receptor agonist in morphine-withdrawn rats.

Opioid drugs are a first-line treatment for severe acute pain and other chronic pain conditions, but long-term opioid drug use produces opioid-induced hyperalgesia (OIH). Co-administration of cannabinoids with opioid receptor agonists produce anti-nociceptive synergy, but cannabinoid receptor agonists may also produce undesirable side effects. Therefore, positive allosteric modulators (PAM) of cannabinoid type-1 receptors (CB1R) may provide an option reducing pain and/or enhancing the anti-hyperalgesic effects of opioids without the side effects, tolerance, and dependence observed with the use of ligands that target the orthosteric binding sites. This study tested GAT211, a PAM of cannabinoid type-1 receptors (CB1R), for its ability to enhance the anti-hyperalgesic effects of the mu-opioid receptor (MOR) agonist DAMGO in rats treated chronically with morphine (or saline) and tested during withdrawal. We tested the effects of intra-periaqueductal gray (PAG) injections of (1) DAMGO, (2) GAT211, or (3) DAMGO + GAT211 on thermal nociception in chronic morphine-treated rats that were hyperalgesic and also in saline-treated control rats. We used slice electrophysiology to test the effects of DAMGO/GAT211 bath application on synaptic transmission in the vlPAG. Intra-PAG DAMGO infusions dose-dependently reversed chronic morphine-induced hyperalgesia, but intra-PAG GAT211 did not alter nociception at the doses we tested. When co-administered into the PAG, GAT211 antagonized the anti-nociceptive effects of DAMGO in morphine-withdrawn rats. DAMGO suppressed synaptic inhibition in the vlPAG of brain slices taken from saline- and morphine-treated rats, and GAT211 attenuated DAMGO-induced suppression of synaptic inhibition in vlPAG neurons via actions at CB1R. These findings show that positive allosteric modulation of CB1R antagonizes the behavioral and cellular effects of a MOR agonist in the PAG of rats.

DAMGO-induced μ opioid receptor internalization and recycling restore morphine sensitivity in tolerant rat

This study investigated the effect of DAMGO-induced μ opioid receptor (MOR) internalization on morphine tolerance. Male Sprague-Dawley rats (200–250 g) aged 6–8 weeks were administered morphine via intrathecal (i.t.) injection (15 μg/10 μl twice daily for 6 days) to induce antinociceptive tolerance, which was evaluated using the tail-flick and paw-withdrawal tests. Response latency was calculated as the percentage of maximum possible effect (%MPE). A bolus of DAMGO was administered by i.t. injection on day 6, and the tail-flick and paw-withdrawal tests were carried out 24, 48, and 72 h later. Membrane and cytosolic MOR expression was assessed by western blotting. HEK293 cells were transfected with MOR-FLAG plasmid and after 6 days of morphine treatment (10 μM), the cells were treated with 1 μM DAMGO, and MOR localization was examined by immunofluorescence analysis 30 and 60 min later. Repeated morphine treatment induced tolerance after 5 days; however, i.t. DAMGO administration restored morphine sensitivity and enhanced acute morphine-induced antinociception after 24, 48, and 72 h. In HEK293 cells, DAMGO treatment stimulated MOR internalization after 30 min and MOR recycling to the membrane after 1 h. Membrane and cytoplasmic MOR expression in vivo was unchanged 24, 48, and 72 h after i.t. DAMGO injection. Morphine does not cause significant MOR internalization or downregulation, and can readily induce tolerance. DAMGO counters this effect by enhancing receptor endocytosis, thereby reversing morphine-induced antinociceptive tolerance and restoring its analgesic efficacy.

Interaction Between Oxytocin and Opioidergic System on Food Intake Regulation in Neonatal Layer Type Chicken

The aim of the current study was to determine possible interaction of central oxytocin and opioidergic system on food intake regulation in neonatal layer-type chicken. In experiment 1, FD3 chicken ICV injected with control solution, oxytocin (10 µg), β-FNA (µ receptor antagonist, 5 µg) and oxytocin (10 µg) + β-FNA were injected. Experiments 2–6 were similar to experiments 1, except chicken injected with nor-BNI (κ receptor antagonist, 5 µg), NTI (δ receptor antagonist, 5 µg), DAMGO (µ receptor agonist, 62.25 pmol), U-50488H (κ receptor agonist, 10 nmol), DPDPE (δ receptor agonist, 20 pmol) instead of β-FNA. In experiment 7, control solution, DAMGO (125 pmol), d(CH2)5Tyr(Me)-[Orn8]-vasotocin (oxytocin antagonist, 5 µg) and DAMGO + d(CH2)5Tyr(Me)-[Orn8]-vasotocin were ICV injected to FD3 chicken. Experiments 8 and 9 were similar to experiments 7, except chicken injected with U-50488H (30 nmol) and DPDPE (40 pmol) instead of DAMGO. Then, cumulative food intake was recorded at 30, 60 and 120 min after injection. According to the results, ICV injection of the oxytocin (10 µg) significantly decreased food intake compared to control group (P < 0.05). Co-injection of the oxytocin + β-FNA and oxytocin + U-50488H significantly decreased hypophagic effect of the oxytocin (P < 0.05). While, co-injection of the oxytocin + nor-BNI or oxytocin + DAMGO significantly amplified hypophagic effect of the oxytocin in chicken (P < 0.05). In addition, ICV injection of DAMGO (125 pmol) significantly decreased cumulative food intake compared to control group (P < 0.05). However, co-addministration of the DAMGO + (CH2)5Tyr(Me)-[Orn8]-vasotocin significantly decreased hypophagic effect of the DAMGO (P < 0.05) in chicken. These results suggested there are interconnection between oxytocin and opioidergic system on central food intake regulation, which mediates via µ and κ opioidergic receptors in neonatal layer-type chicken.

Activation of MORs in the VTA induces changes on cFos expression in different projecting regions: Effect of inflammatory pain

Chronic pain is a worldwide major health problem and many pain-suffering patients are under opioid based therapy. Epidemiological data show that pain intensity correlates with the risk of misuse of prescription opioids, and other drugs of abuse including alcohol. This increased vulnerability to suffer Substance Use Disorders could be, in part, caused by functional changes that occur over the mesocorticolimbic system, a brain pathway involved in reward processing and addiction. Previous data in rats revealed that inflammatory pain desensitizes mu opioid receptors (MORs) in the ventral tegmental area (VTA). As a consequence, pain alters dopamine release in the nucleus accumbens (NAc) derived from MOR activation in the VTA and also increases intake of high doses of heroine. Given that the VTA neurons target different brain regions, in the present study we first analyzed changes induced by inflammatory pain in the MOR dependent activation pattern of the main VTA projecting areas. To do that, we administered two doses (7 or 14 ng) of DAMGO (MORs agonist) or artificial cerebrospinal fluid (aCSF) focally into the VTA of rats and measured the activation in projection areas by cFos immunohistochemistry. Our results show that focal injections of DAMGO in the VTA increases cFos expression in the majority of its projecting areas, namely NAc, basolateral amygdala (BLA), cingulate cortex (ACC) and bed nucleus of the stria terminalis (BNST), as compared to aCSF. Second, we analyzed whether inflammatory pain would affect to cFos expression using a group of rats injected with CFA in the hind paw. In this case, we found that cFos expression was not significantly different between DAMGO and aCSF administered rats in BLA, ACC and BNST. Our results confirm that inflammatory pain induces desensitization of VTA MORs in a region dependent manner which can be very relevant for addictive behaviours.

Methocinnamox (MCAM) is an Effective Long‐Term Antagonist of Peripheral Mu, but not Kappa or Delta Opioid Receptors In Vivo

The United States is currently in the midst of an opioid abuse epidemic; deaths from synthetic opioids like fentanyl and its derivatives are increasing. Currently naloxone (Narcan), a competitive mu opioid receptor antagonist is the only pharmacological treatment for opioid overdose; reversing both respiratory depression and profound sedation. However, naloxone has a relatively short duration of action and thus its effects are limited. A long lasting mu opioid receptor antagonist called MCAM has been shown to attenuate the antinociceptive effects of morphine in mice for more than 48 hours (Broadbear et al., J Pharmacol Exp Ther, 294:933–940, 2000) and due to this long duration of action, may make a better treatment for opioid overdose and perhaps for opioid abuse. Because MCAM reduces mu receptor function for a longer period relative to kappa or delta opioid receptors, we have begun to examine if pain relief from either kappa or delta receptor agonists will be active while mu receptors remain occupied by MCAM. Using the Hargreaves method for thermal heat nociception, we determined the effect of intraplantar (i.pl.) pretreatment of MCAM or vehicle on peripherally mediated reduction in PGE2‐evoked thermal allodynia by DAMGO (mu agonist), U50488 (kappa agonist) or DPDPE (delta agonist). Injection of DAMGO (20μg/50 μl i.pl.), U50488 (0.1μg/50μl i.pl.) or DPDPE (20μg/50 μl i.pl.) produced a significant reduction in PGE2‐evoked thermal allodynia. Pretreatment with MCAM 15 min prior to PGE2 injection at 10nM (0.25μg/50μl i.pl), 50nM (1.25μg/50μl i.pl) or 20nM (0.5μg/50μl i.pl), had no effect on PGE2‐induced thermal hypersensitivity; however, it significantly inhibited the antinociceptive response to DAMGO, U50488 and DPDPE. The DAMGO‐mediated antinociception remained blocked 24 and 48h after a single administration of MCAM. By contrast, when tested 24h after administration, MCAM had no effect on U50488‐ or DPDPE‐mediated antinociceptive responses. Thus, MCAM displayed long‐term antagonism at mu opioid receptors but only a short antagonist effect at either kappa or delta opioid receptors. These data suggest that MCAM's long duration of antagonist action is selective for mu opioid receptors and that peripherally restricted delta and kappa receptor agonists may be a viable option for pain relief.Support or Funding InformationThe Addiction Research, Treatment &amp; Training (ARTT) Center of Excellence at UT Health San Antonio.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Central Opioidergic and Adrenergic systems Mediates Food Intake via α1, α2 and β2 Receptors in Neonatal Layer-Type Chicken

The aim of the current study was to determine possible interaction of central Opioidergic and Adrenergic systems on food intake regulation in neonatal layer-type chicken. In experiment 1, chicken ICV injected with control solution, DAMGO (µopioid receptors agonist, 125 pmol), parazosin (α1 receptor antagonist, 10 nmol) and DAMGO + parazosin. In experiment 2, control solution, DAMGO (125 pmol), yohimbine (α2 receptor antagonist, 13 nmol) and DAMGO + yohimbine were ICV injected. In experiment 3, FD3 birds ICV injected with control solution, DAMGO (125 pmol), metoprolol (β1 receptor antagonist, 24 nmol) and DAMGO + metoprolol. In experiment 4, FD3 chicks received ICV injection of control solution, DAMGO (125 pmol), ICI 118,551 (β2 receptor antagonist, 5 nmol) and DAMGO + ICI 118,551. Experiments 5–8 were similar to experiments 1–4, except chicken injected with DPDPE (δ opioid receptors agonist, 40 nmol) instead of DAMGO. Experiments 9–12 were similar to experiments 1–4, except chicken injected with U-50488H (κ opioid receptors agonist, 30 nmol) instead of DAMGO. Then, cumulative food intake was recorded at 30, 60 and 120 min after injection. According to the results, ICV injection of the DAMGO significantly decreased food intake while DPDPE and U-50488H significantly increased food intake in neonatal layer type chicken (P < 0.05). Co-injection of the DAMGO + ICI 118,551 decreased DAMGO-induced hypophagia (P < 0.05). Also, co-injection of the DPDPE + parazosin diminished hyperphagic effect of the DPDPE (P < 0.05). In addition, co-injection of the U-50488H + yohimbine diminished U-50488H-induced hyperphagia (P < 0.05). These results suggested there are interconnection between adrenergic and opioidergic systems on central food intake regulation which mediates via α1, α2 and β2 receptors in neonatal layer-type chicken.

Central Opioidergic System Interplay with Histamine on Food Intake in Neonatal Chicks: Role of µ-Opioid and H1/H3 Receptors

ABSTRACT The present study was designed to examine the role of Opioidergic and Histaminergic systems on feeding behavior in 3-hour food deprived neonatal meat- type chicks. In experiment 1, chicks received intracerebroventricular (ICV) injection of (A) control solution, (B) α-FMH (alpha fluoromethyl histidine; 250 nmol), (C) DAMGO (µ-opioid receptor agonist, 125 pmol) and (D) α-FMH + DAMGO. Experiments 2-4 were similar to experiment 1, except chicken ICV injected with Chlorpheniramine (histamine H1 receptors antagonist; 300 nmol), famotidine (histamine H2 receptors antagonist; 82 nmol) and Thioperamide (histamine H3 receptors antagonist; 300 nmol) instead of the α-FMH. In experiments 5-8, birds ICV injected with the same procedure as experiments 1-4, except they were injected with DPDPE (δ-opioid receptor agonist, 40 nmol) instead of DAMGO. Experiments 9-12 were similar to the experiments 1-4, except neonatal broilers ICV were injected with U-50488H (κ-opioid receptor agonist, 30 nmol) instead of DAMGO. Then the cumulative food intake was measured until 120 min post injection. According to the results, ICV injection of DAMGO, significantly decreased food intake (p 0.05). Also, the hyperphagic effect of DPDPE and U-50488 had no affect by α-FMH, Chlorpheniramine, famotidine and Thioperamide (p>0.05). These results suggested that an interconnection between central opioidergic and histaminergic systems on feeding behavior is mediated via µ-opioid and H1/H3 receptors in neonatal broilers.

The effect of DAMGO injections on the respiratory pattern varies between subareas of the ventral respiratory column in adult rabbits

We have previously described how sequential microinjections of high concentrations of the mu‐opioid agonist [D‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) into the ventral respiratory column (VRC) depressed ventilation with rather variable changes in respiratory pattern in the adult, in vivo, decerebrate rabbit model (1). This study aimed to distinguish whether DAMGO injections into subareas of the VRC that were functionally identified by their differential respiratory pattern response to local microinjection of α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) resulted in different effects on the respiratory pattern. The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3–4 kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Nerve activity was recorded from the c5 rootlet of the phrenic nerve, time averaged and used to calculate inspiratory and expiratory durations. Gridwise localized pressure injections of AMPA (50 μM, 70 nl) utilizing a multibarrel glass pipette with a tip diameter of 20μm into the VRC rostral of obex identified two areas with distinctly different pattern changes: An area (on average 2mm rostral from obex, 2.5mm lateral from midline, 4.5mm ventral to the dorsal surface) where AMPA injection caused significant tachycardia with decreased peak phrenic activity (PPA) and a second area (on average 1mm rostral, 2 mm lateral, 3.5mm ventral) where AMPA caused less tachycardia and an increase in PPA. In twelve animals, DAMGO injection (100μM, 700nl) into the more rostral area caused a decrease in inspiratory duration (from 1.2±0.1sec to 0.9±0.2sec, mean±SE) while injection into the more caudal area caused a small increase (from 1.3±0.1sec to 1.4±0.1sec). These effects were significantly different (p&lt;0.001, 2‐way ANOVA, factors: location, drug). DAMGO injection increased expiratory duration similarly in both areas (from 1.1±0.2 sec to 1.9±0.4sec and from 1.5±0.3sec to 2.2±0.3sec, difference between areas not significant). This resulted in no change in respiratory rate in the rostral area but a decrease in rate in the caudal area (from 25±3 bpm to 20±3bpm, difference between areas p&lt;0.001). PPA was similarly decreased in both areas (from 100% to 81±7% and from 100% to 95±2%, difference not significant). While the rostral area in location and AMPA response may match the preBötzinger area as described, e.g., by Marchenko (2), respiratory pattern can also be affected in more caudal subareas of the VRC. We suggest that glutamate agonists can elicit tachypnea in various subareas of the VRC and that injection of DAMGO into these individual subareas can have different effects on the respiratory pattern. This may be an explanation for diverging results of DAMGO injection between different studies using glutamate agonists for functional identification of their target.Support or Funding InformationNIH R01GM112960‐01A1 (Dr. Stucke) and VA merit grant I01BX000721 (Dr. Zuperku).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Peripheral DAMGO Decreases Group III and IV Afferents' Responses to Contraction in Rats Whose Femoral Arteries are Occluded

Peripheral artery disease (PAD) is characterized by restricted blood flow to the legs. Patients with PAD show an exaggerated pressor response to mild exercise, an effect attributable to the exercise pressor reflex, whose afferent arm is comprised of the thinly myelinated Group III and unmyelinated Group IV afferents. We simulated the blood flow patterns to muscle in PAD by ligating the femoral artery of rats 72 hours prior to an experiment. Previously, we found that DAMGO, a mu‐opioid agonist injected into the femoral artery, attenuated the exaggerated exercise pressor reflex in rats with ligated femoral arteries. To follow on these findings, we recorded the responses of Group III and IV afferents to thirty seconds of static contraction before and after injecting DAMGO (1 μg) into the superficial epigastric artery in rats with patent femoral arteries and in rats with ligated femoral arteries.In rats with patent femoral arteries, contraction increased afferent discharge in both Group III afferents (0.5 ± 0.2 to 2.0 ± 0.3 impulses/s, n=9, p&lt;0.001) and Group IV afferents (0.4 ± 0.1 to 1.0 ± 0.2 imp/s, n=8, p&lt;0.001). DAMGO did not change the responses to contraction of either Group III (0.5 ± 0.2 to 1.6 ± 0.4 imp/s; p=0.83) or Group IV (0.3 ± 0.1 to 0.7 ± 0.2 imp/s; p=0.34) afferents. In contrast, in rats with “ligated” femoral arteries, DAMGO injection (1 μg) significantly decreased the responses to contraction of both Group III afferents (n=9, p&lt;0.01) and Group IV afferents (n=9; p&lt;0.01).In addition to contraction, we tested the effect of DAMGO on afferents' response to injecting the TRPV1 receptor agonist, capsaicin (0.2 μg), into the superficial epigastric artery. In rats with patent femoral arteries, approximately half (7/13) of Group III afferents responded to capsaicin (0.4 ± 0.2 to 2.4 ± 0.6 imp/s, p&lt;0.001). DAMGO seemed to slightly, though not significantly, reduce the responses of these seven Group III afferents to capsaicin injection (p=0.27). Likewise, most (9/10) of the Group IV afferents tested responded to capsaicin (0.3 ± 0.9 to 2.1 ± 0.4 imp/s, p&lt;0.001). DAMGO seemed to reduce the responses of these nine Group IV afferents, but the effect was not statistically significant (p=0.09).DAMGO had no significant effect on the responses of Group III and IV afferents to capsaicin whose impulse activity was recorded from “ligated” rats. Only half (6/12) of the Group III afferents responded to capsaicin (0.5 ± 0.1 to 2.4 ± 0.4 imp/s, p&lt;0.01); DAMGO reduced the response of these six afferents slightly, but not significantly (p=0.11). Again, most (9/11) Group IV afferents from “ligated” rats responded to capsaicin (0.4 ± 0.1 to 1.9 ± 0.3 imp/s, p&lt;0.001), but DAMGO injection had no effect on the responses of these nine afferents (p=0.94).These findings are in agreement with our previous studies which showed that peripheral DAMGO injection attenuated the exercise pressor reflex in rats with ligated femoral arteries. In contrast, DAMGO injection had only modest effect on the exercise pressor reflex in rats with patent femoral arteries. Together, these studies suggest potential therapeutic value in using opioids peripherally to treat patients with PAD, with less risk of adverse side effects and dependence.Support or Funding InformationFunded by NIH grants R01 AR059397 and P01 HL134609‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Peripheral µ-opioid receptors attenuate the responses of group III and IV afferents to contraction in rats with simulated peripheral artery disease.

Patients with peripheral artery disease show an exaggerated pressor response to mild exercise, an effect attributable to the exercise pressor reflex, whose afferent arm comprises the thinly myelinated group III and unmyelinated group IV afferents. Previously, we found that DAMGO, a µ-opioid agonist injected into the femoral artery, attenuated the exaggerated exercise pressor reflex in rats with ligated femoral arteries, a preparation that simulates the blood flow patterns to muscle that is seen in patients with peripheral artery disease. Continuing this line of investigation, we recorded the responses of group III and IV afferents to static contraction before and after injecting DAMGO (1 µg) into the superficial epigastric artery in rats with patent femoral arteries and in rats with ligated femoral arteries. In rats with patent arteries, DAMGO did not change the responses to contraction of either group III ( n = 9; P = 0.83) or group IV ( n = 8; P = 0.34) afferents. In contrast, in rats with ligated femoral arteries, DAMGO injection (1 µg) significantly decreased the responses to contraction of both group III afferents ( n = 9, P < 0.01) and group IV afferents ( n = 9; P < 0.01). DAMGO did not significantly attenuate the responses of either group III or IV afferents to capsaicin in rats with either patent or ligated femoral arteries. These findings are in agreement with our previous studies that showed that peripheral DAMGO injection attenuated the exercise pressor reflex in rats with ligated femoral arteries but had only a modest effect on the exercise pressor reflex in rats with patent femoral arteries. NEW & NOTEWORTHY In an animal model of peripheral artery disease, we show that the µ-opioid agonist, DAMGO reduces the afferent response rate resulting from stimulated static contraction. These results suggest that peripherally active opioid agonists that do not cross the blood-brain barrier may be therapeutic for treatment of peripheral artery disease without the negative and addictive side effects associated with opioids in the central nervous system.

µ-Opioid receptors inhibit the exercise pressor reflex by closing N-type calcium channels but not by opening GIRK channels in rats.

µ-Opioid G protein-coupled receptors (MOR) interact with ion channels to decrease neuronal excitability. In humans, intrathecal administration of the MOR agonist fentanyl inhibits the exercise pressor reflex, an effect that can be attributed to either the opening of inward rectifying potassium channels (GIRK) or the closing of N-type calcium channels. The purpose of this study was to determine if the highly selective MOR agonist [d-Ala2, N-MePhe4,Gly-ol]-enkephalin (DAMGO) attenuates the exercise pressor reflex and which of these two channels are responsible for this effect. In decerebrate rats, we determined the effect of intrathecal injection of either tertiapin-LQ, which blocks the GIRK channel or ω-conotoxin-GVIA, which blocks the N-type calcium channel on the exercise pressor reflex, which was evoked by contracting the triceps surae muscles. Initially, we established that intrathecal injection of DAMGO inhibited the exercise pressor reflex relative to no intrathecal injection or intrathecal saline injection ( P < 0.001, n = 5). We then found that intrathecal injection of two doses of tertiapin-LQ (1 and 10 µg) had no effect on the exercise pressor reflex ( n = 6 and n = 7, respectively; P > 0.05). Importantly, neither dose of tertiapin-LQ prevented the DAMGO-induced inhibition of the exercise pressor reflex. Last, we found that intrathecal injection of ω-conotoxin-GVIA markedly attenuated the exercise pressor reflex ( P < 0.001, n = 7). The cardioaccelerator response to contraction did not appear to be effected in any of the experiments. We conclude that N-type voltage-gated calcium channel inhibition appears to be the mechanism by which MOR activation inhibits the exercise pressor reflex in decerebrate rats.

Interaction Between Central Opioidergic and Glutamatergic Systems on Food Intake in Neonatal Chicks: Role of NMDA, AMPA and mGLU1 Receptors

The present study was designed to examine the role of opioidergic and glutamatergic systems on feeding behavior in neonatal meat-type chicken. In experiment 1, FD3 neonatal broilers ICV injected with (A) saline, (B) DAMGO (µ-opioid receptor agonist, 125 pmol), (C) MK-801 (NMDA glutamate receptors antagonist, 15 nmol) and (D) combination of DAMGO plus MK-801. Experiments 2–5 were similar to experiment 1, except FD3 chicks ICV injected with CNQX (AMPA glutamate receptors antagonist, 390 nmol), AIDA (mGLU1 receptors antagonist, 2 nmol), LY341495 (mGLU2 receptors antagonist, 150 nmol) and UBP1112 (mGLU3 receptors antagonist, 2 nmol) instead of MK-801, respectively. In experiments 6–10, FD3 chicks ICV injected as the same as procedure to the experiments 1–5, except to inject with DPDPE (δ-opioid receptor agonist, 40 nmol) instead of the DAMGO. The experiments 11–15 were similar to the experiments 1–5, except neonatal broilers ICV injected with U-50488H (κ-opioid receptor agonist, 30 nmol) instead of DAMGO. Then the cumulative food intake measured until 120 min post injection. According to the results, ICV injection of DAMGO, significantly decreased food intake (P < 0.05) while DPDPE and U-50488H increased feeding behavior compared to the control group (P < 0.05). Co-injection of the DAMGO + MK-801 and DAMGO + AIDA, significantly decreased DAMGO-induced hypophagia in neonatal chicks (P < 0.05). Also, co-injection of the DPDPE + CNQX significantly amplified DPDPE induced feeding behavior (P < 0.05). These results suggested interconnection between central opioidergic and glutamatergic systems on feeding behavior mediates via µ- and δ-opioid receptor with NMDA, AMPA and mGLU1 receptors in FD3 neonatal broilers. These findings may shed light on the circuitry underlying interconnection between central opioidergic and glutamatergic systems on feeding behavior.

Role of Opioid Receptors on Food Choice and Macronutrient Selection in Meat-Type Chick

The present study was designed to examine the role of opioid receptors on food choice and macronutrient selection in neonatal chicks. In this study, 13 experiments designed, experiments 1–3 for effect of specific opioid receptors on appetite and experiments 4–13 on effect of opioid receptors on food choice and macronutrient selection in meat-type chick. In experiment 1, chicken intracerebroventricular (ICV) injected with 125, 250 and 500 pmol of DAMGO (µ-opioid receptor agonist). Experiment 2 was conducted to investigate the effect of DPDPE (δ-opioid receptor agonist) at doses of 20, 40 and 80 nmol. In experiment 3 ICV injection of the U-50488H (κ-opioid receptor agonist, of 10, 20 and 40 nmol) was done. In experiment 4, birds injected with saline and different diets: standard diet without fat, diet containing nutrient energy 20 % higher than standard, diet containing nutrient energy 20 % lower than standard and standard diet containing fat were offered to them to investigate desire of chicken to diets. Experiments 5–7 were similar to experiment 4, except, birds ICV injected with 125, 250 and 500 pmol of DAMGO. In experiments 8–10 chicken received ICV injection of DPDPE (20, 40 and 80 nmol). The experiments 11–13 was similar to previous experiments which birds injected with different doses of U-50488H (10, 20 and 40 nmol), respectively. Then the cumulative food intake measured until 180-min post injection. According to the results, ICV injection of DAMGO diminished food intake while DPDPE and U-50488H increased appetite (P < 0.05). Despite anorexigenic effect, ICV injection of DAMGO increased birds desire to eat fat containing standard diet compared to the standard diet without fat (P < 0.05). These findings suggest endogenous opioids governing preferences for fat rich foods.

Interaction Between Opioidergic and Dopaminergic Systems on Food Intake in Neonatal Layer Type Chicken

Central regulatory mechanisms for food intake regulation vary among animals. Evidence from animal studies suggests central opioids and dopamine have prominent role on appetite regulation but their interaction(s) have not been studied in layer-type chicken. Thus, in this study six experiments designed to investigate intracerebroventricular (ICV) administration of SCH23390 (D1 like receptors antagonist), Sulpride (D2 like receptors antagonist), DAMGO (μ-opioid receptors agonist), DPDPE (δ-opioid receptors agonist), U-50488H (κ-opioid receptors agonist) on feeding behavior in 3 h food deprived neonatal layer-type chickens. In experiment 1, chicks ICV injected with control solution, SCH23390 (2.5 nmol), DAMGO (125 pmol) and their combination (SCH23390 + DAMGO). In experiment 2: control solution, SCH23390 (2.5 nmol), DPDPE (δ-opioid receptors agonist, 40 pmol) and SCH23390 + DPDPE were applied to the birds. In experiment 3, injections were control solution, SCH23390 (2.5 nmol), U-50488H (30 nmol) and SCH23390 + U-50488H. In experiments 4–6 were similar to experiments 1–3 except Sulpride (2.5 nmol) applied instead of SCH23390. Then, cumulative food intake was recorded until 120 min after injection. According to the results, ICV injection of DAMGO (125 pmol) significantly decreased food intake but co-injection of DAMGO + SCH23390 diminished DAMGO-induced hypophagia (P 0.05). Furthermore, Sulpride had no role on DAMGO, DPDPE and U-50488H-induced food intake (P > 0.05). These results suggest there is an interaction between opioidergic and dopaminergic systems via μ and D1 receptors in appetite regulation in chicken.

Opioid-induced Respiratory Depression Is Only Partially Mediated by the preBötzinger Complex in Young and Adult Rabbits In Vivo.

The preBötzinger Complex (preBC) plays an important role in respiratory rhythm generation. This study was designed to determine whether the preBC mediated opioid-induced respiratory rate depression at clinically relevant opioid concentrations in vivo and whether this role was age dependent. Studies were performed in 22 young and 32 adult New Zealand White rabbits. Animals were anesthetized, mechanically ventilated, and decerebrated. The preBC was identified by the tachypneic response to injection of D,L-homocysteic acid. (1) The μ-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO, 100 μM) was microinjected into the bilateral preBC and reversed with naloxone (1 mM) injection into the preBC. (2) Respiratory depression was achieved with intravenous remifentanil (0.08 to 0.5 μg kg(-1) min(-1)). Naloxone (1 mM) was microinjected into the preBC in an attempt to reverse the respiratory depression. (1) DAMGO injection depressed respiratory rate by 6 ± 8 breaths/min in young and adult rabbits (mean ± SD, P < 0.001). DAMGO shortened the inspiratory and lengthened the expiratory fraction of the respiratory cycle by 0.24 ± 0.2 in adult and young animals (P < 0.001). (2) During intravenous remifentanil infusion, local injection of naloxone into the preBC partially reversed the decrease in inspiratory fraction/increase in expiratory fraction in young and adult animals (0.14 ± 0.14, P < 0.001), but not the depression of respiratory rate (P = 0.19). PreBC injections did not affect respiratory drive. In adult rabbits, the contribution of non-preBC inputs to expiratory phase duration was larger than preBC inputs (3.5 [-5.2 to 1.1], median [25 to 75%], P = 0.04). Systemic opioid effects on respiratory phase timing can be partially reversed in the preBC without reversing the depression of respiratory rate.

Neuronal cytochrome P450 activity and opioid analgesia: relevant sites and mechanisms

Recent studies suggest a functional role for neuronal cytochrome P450 monooxygenase (P450) activity in opioid analgesia. To characterize the relevant receptors, brain areas, and circuits, detailed in vitro and in vivo studies were performed with the highly selective μ opioid receptor agonist DAMGO in neuronal P450-deficient mutant (Null) and control mice. Homogenates of brain regions and spinal cord showed no differences in DAMGO-induced activation of [35S]- GTPγS binding between Null and control mice, indicating no genotype differences in µ opioid receptor signaling, receptor affinities or receptor densities. Intracerebroventricular (icv) DAMGO produced robust, near-maximal, analgesic responses in control mice which were attenuated by 50% in Null mice, confirming a role for µ opioid receptors in activating P450-associated responses. Intra-periaqueductal gray (PAG) and intra-rostral ventromedial medulla (RVM) injections of DAMGO revealed deficits in Null (vs. control) analgesic responses, yet no such genotype differences were observed after intrathecal DAMGO administration. Taken with earlier published findings, the present results suggest that activation of µ opioid receptors in both the PAG and in the RVM relieves pain by mechanisms which include nerve-terminal P450 enzymes within inhibitory PAG-RVM projections. Spinal opioid analgesia, however, does not seem to require such P450 enzyme activity.