A small molecule ligand for the novel pain target, GPR171, produces minimal reward in mice.

ProSAAS is one of the most abundant proteins in the brain and is processed into several smaller peptides. One of which, BigLEN, is an endogenous ligand for the G protein-coupled receptor, GPR171. Recent work in rodent models has shown that a small-molecule ligand for GPR171, MS15203, increases morphine antinociception and is effective in lessening chronic pain. While these studies provide evidence for GPR171 as a possible pain target, its abuse liability has not yet been assessed and was evaluated in the current study. We first mapped the distribution of GPR171 and ProSAAS throughout the reward circuit of the brain using immunohistochemistry and showed that GPR171 and ProSAAS are localized in the hippocampus, basolateral amygdala, nucleus accumbens, prefrontal cortex. In the major dopaminergic structure, the ventral tegmental area (VTA), GPR171 appeared to be primarily localized in dopamine neurons while ProSAAS is outside of dopamine neurons. Next, MS15203 was administered to mice with or without morphine, and VTA slices were stained for the immediate early gene c-Fos as a marker of neuronal activation. Quantification of c-Fos-positive cells revealed no statistical difference between MS15203 and saline, suggesting that MS15203 does not increase VTA activation and dopamine release. The results of a conditioned place preference experiment showed that treatment with MS15203 produced no place preference indicating a lack of reward-related behavior. Taken together this data provides evidence that the novel pain therapeutic, MS15203, has minimal reward liability. Therefore, GPR171 deserves further exploration as a pain target. SIGNIFICANCE STATEMENT: MS15203, a drug that activates the receptor GPR171, was previously shown to increase morphine analgesia. The authors use in vivo and histological techniques to show that it fails to activate the rodent reward circuitry, providing support for the continued exploration of MS15203 as a novel pain drug, and GPR171 a novel pain target.

GPR171 activation regulates morphine tolerance but not withdrawal in a test-dependent manner in mice.

A newly deorphanized G protein-coupled receptor, GPR171, is found to be highly expressed within the periaqueductal gray, a pain-modulating region in the brain. Our recent research has shown that a GPR171 agonist increases morphine antinociception in male mice and opioid signaling in vitro . The objective of this study was to evaluate the effects of combination treatment in females as well as whether chronic treatment can be used without exacerbating morphine-induced tolerance and withdrawal in female and male mice. Our results demonstrate that activation of GPR171 with an agonist attenuates morphine tolerance in both female and male mice on the tail-flick test, but not the hotplate test. Importantly, the GPR171 agonist in combination with morphine does not exacerbate morphine-induced tolerance and withdrawal during long-term morphine treatment. Taken together, these data suggest that the GPR171 agonist may be combined with morphine to maintain antinociception while reducing the dose of morphine and therefore reducing side effects and abuse liability. The outcome of this study is clearly an important step toward understanding the functional interactions between opioid receptors and GPR171 and developing safer therapeutics for long-term pain management.

Morphine-induced kinase activation and localization in the periaqueductal gray of male and female mice.

Morphine is a potent opioid analgesic with high propensity for the development of antinociceptive tolerance. Morphine antinociception and tolerance are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). However, the majority of research evaluating mu-opioid receptor signaling has focused on males. Here, we investigate kinase activation and localization patterns in the vlPAG following acute and chronic morphine treatment in both sexes. Male and female mice developed rapid antinociceptive tolerance to morphine (10 mg/kg i.p.) on the hot plate assay, but tolerance did not develop in males on the tail flick assay. Quantitative fluorescence immunohistochemistry was used to map and evaluate the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2), protein kinase-C (PKC), and protein kinase-A (PKA). We observed significantly greater phosphorylated ERK 1/2 in the vlPAG of chronic morphine-treated animals which co-localized with the endosomal marker, Eea1. We note that pPKC is significantly elevated in the vlPAG of both sexes following chronic morphine treatment. We also observed that although PKA activity is elevated following chronic morphine treatment in both sexes, there is a significant reduction in the nuclear translocation of its phosphorylated substrate. Taken together, this study demonstrates increased activation of ERK 1/2, PKC, and PKA in response to repeated morphine treatment. The study opens avenues to explore the impact of chronic morphine treatment on G-protein signaling and kinase nuclear transport.

High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics.

Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the 'opioid epidemic.' We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.

GPR171 Agonist Reduces Chronic Neuropathic and Inflammatory Pain in Male, But Not Female Mice.

Chronic pain is a growing public health crisis that requires exigent and efficacious therapeutics. GPR171 is a promising therapeutic target that is widely expressed through the brain, including within the descending pain modulatory regions. Here, we explore the therapeutic potential of the GPR171 agonist, MS15203, in its ability to alleviate chronic pain in male and female mice using a once-daily systemic dose (10 mg/kg, i.p.) of MS15203 over the course of 5 days. We found that in our models of Complete Freund's Adjuvant (CFA)-induced inflammatory pain and chemotherapy-induced peripheral neuropathy (CIPN), MS15203 did not alleviate thermal hypersensitivity and allodynia, respectively, in female mice. On the other hand, MS15203 treatment decreased the duration of thermal hypersensitivity in CFA-treated male mice following 3 days of once-daily administration. MS15203 treatment also produced an improvement in allodynia in male mice, but not female mice, in neuropathic pain after 5 days of treatment. Gene expression of GPR171 and that of its endogenous ligand BigLEN, encoded by the gene PCSK1N, were unaltered within the periaqueductal gray (PAG) in both male and female mice following inflammatory and neuropathic pain. However, following neuropathic pain in male mice, the protein levels of GPR171 were decreased in the PAG. Treatment with MS15203 then rescued the protein levels of GPR171 in the PAG of these mice. Taken together, our results identify GPR171 as a GPCR that displays sexual dimorphism in alleviation of chronic pain. Further, our results suggest that GPR171 and MS15203 have demonstrable therapeutic potential in the treatment of chronic pain.

Differences in antinociceptive signalling mechanisms following morphine and fentanyl microinjections into the rat periaqueductal gray.

BACKGROUND: Morphine and fentanyl are two of the most commonly used opioids to treat pain. Although both opioids produce antinociception by binding to mu-opioid receptors (MOR), they appear to act via distinct signalling pathways. OBJECTIVE: This study will reveal whether differences in morphine and fentanyl antinociception are the result of selective activation of G-protein signalling and/or selective activation of pre- or postsynaptic MORs. METHODS: The contribution of each mechanism to morphine and fentanyl antinociception was assessed by microinjecting drugs to alter G-protein signalling or block potassium channels linked to pre- and postsynaptic MORs in the ventrolateral periaqueductal gray (PAG) of male Sprague-Dawley rats. RESULTS: Both morphine and fentanyl produced a dose-dependent antinociception when microinjected into the PAG. Enhancement of intracellular G-protein signalling by microinjection of the Regulator of G-protein Signalling 4 antagonist CCG-63802 into the PAG enhanced the antinociceptive potency of morphine, but not fentanyl. Microinjection of α-dendrotoxin into the PAG to block MOR activation of presynaptic Kv + channels caused a significant rightward shift in the dose-response curve of both morphine and fentanyl. Microinjection of tertiapin-Q to block MOR activation of postsynaptic GIRK channels caused a larger shift in the dose-response curve for fentanyl than morphine antinociception. CONCLUSIONS: These findings reveal different PAG signalling mechanisms for morphine and fentanyl antinociception. In contrast with fentanyl, the antinociceptive effects of morphine are mediated by G-protein signalling primarily activated by presynaptic MORs. SIGNIFICANCE: Microinjection of the opioids morphine and fentanyl into the periaqueductal gray (PAG) produce antinociception via mu-opioid receptor signalling. This study reveals differences in the signalling mechanisms underlying morphine and fentanyl antinociception in the PAG. In contrast with fentanyl, morphine antinociception is primarily mediated by presynaptic opioid receptors and is enhanced by blocking RGS proteins.

Targeting Cannabinoid 1 and Delta Opioid Receptor Heteromers Alleviates Chemotherapy-Induced Neuropathic Pain.

Cannabinoid 1 (CB1R) and delta opioid receptors (DOR) associate to form heteromers that exhibit distinct pharmacological properties. Not much is known about CB1R-DOR heteromer location or signaling along the pain circuit in either animal models or patients with chemotherapy-induced peripheral neuropathy (CIPN). Here, we use paclitaxel to induce CIPN in mice and confirm the development of mechanical allodynia. Under these conditions, we find significant increases in CB1R-DOR heteromers in the dorsal spinal cord of mice with CIPN as well as in postmortem spinal cords from human subjects with CIPN compared to controls. Next, we investigated receptor signaling in spinal cords of mice with CIPN and found that treatment with a combination of low signaling doses of CB1R and DOR ligands leads to significant enhancement in G-protein activity that could be selectively blocked by the CB1R-DOR antibody. Consistent with this, administration of subthreshold doses of a combination of ligands (CB1R agonist, Hu-210, and DOR agonist, SNC80) leads to significant attenuation of allodynia in mice with CIPN that is not seen with the administration of individual ligands, and this could be blocked by the CB1R-DOR antibody. Together, these results imply that CB1R-DOR heteromers upregulated during CIPN-associated mechanical allodynia could serve as a potential target for treatment of neuropathic pain including CIPN.

Opioid-Induced Signaling and Antinociception Are Modulated by the Recently Deorphanized Receptor, GPR171.

ProSAAS is one of the most widely expressed proteins throughout the brain and was recently found to be upregulated in chronic fibromyalgia patients. BigLEN is a neuropeptide that is derived from ProSAAS and was recently discovered to be the endogenous ligand for the orphan G protein-coupled receptor GPR171. Although BigLEN-GPR171 has been found to play a role in feeding and anxiety behaviors, it has not yet been explored in pain and opioid modulation. The purpose of this study was to evaluate this novel neuropeptide-receptor system in opioid-induced antinociception. We found that GPR171 is expressed in GABAergic neurons within the periaqueductal gray, which is a key brain area involved in pain modulation and opioid functions. We also found that, although the GPR171 agonist and antagonist do not have nociceptive effects on their own, they oppositely regulate morphine-induced antinociception with the agonist enhancing and antagonist reducing antinociception. Lastly, we showed that the GPR171 antagonist or receptor knockdown decreases signaling by the mu-opioid receptor, but not the delta-opioid receptor. Taken together, these results suggest that antagonism of the GPR171 receptor reduces mu opioid receptor signaling and morphine-induced antinociception, whereas the GPR171 agonist enhances morphine antinociception, suggesting that GPR171 may be a novel target toward the development of pain therapeutics. SIGNIFICANCE STATEMENT: GPR171 is a recently deorphanized receptor that is expressed within the periaqueductal gray and can regulate mu opioid receptor signaling and antinociception. This research may contribute to the development of new therapeutics to treat pain.

Lack of Antinociceptive Cross-Tolerance With Co-Administration of Morphine and Fentanyl Into the Periaqueductal Gray of Male Sprague-Dawley Rats.

Tolerance to the antinociceptive effect of mu-opioid receptor agonists, such as morphine and fentanyl, greatly limits their effectiveness for long-term use to treat pain. Clinical studies have shown that combination therapy and opioid rotation can be used to enhance opioid-induced antinociception once tolerance has developed. The mechanism and brain regions involved in these processes are unknown. The purpose of this study was to evaluate the contribution of the ventrolateral periaqueductal gray (vlPAG) to antinociceptive tolerance and cross-tolerance between administration and co-administration of morphine and fentanyl. Tolerance was induced by pretreating rats with morphine or fentanyl or low-dose combination of morphine and fentanyl into the vlPAG followed by an assessment of the cross-tolerance to the other opioid. In addition, tolerance to the combined treatment was assessed. Cross-tolerance did not develop between repeated vlPAG microinjections of morphine and fentanyl. Likewise, there was no evidence of cross-tolerance from morphine or fentanyl to the co-administration of morphine and fentanyl. Co-administration did not cause cross-tolerance to fentanyl. Cross-tolerance was only evident to morphine or morphine and fentanyl combined in rats pretreated with co-administration of low doses of morphine and fentanyl. This finding is consistent with the functionally selective signaling that has been reported for antinociception and tolerance after morphine and fentanyl binding to the mu-opioid receptor. This research supports the notion that combination therapy and opioid rotation may be useful clinical practices to decrease opioid tolerance and other side effects. PERSPECTIVE: This preclinical study shows that there is a decrease in cross-tolerance between morphine and fentanyl within the periaqueductal gray, which is a key brain region in opioid antinociception and tolerance.

The Contribution of the Descending Pain Modulatory Pathway in Opioid Tolerance.

Opioids remain among the most effective pain-relieving therapeutics. However, their long-term use is limited due to the development of tolerance and potential for addiction. For many years, researchers have explored the underlying mechanisms that lead to this decreased effectiveness of opioids after repeated use, and numerous theories have been proposed to explain these changes. The most widely studied theories involve alterations in receptor trafficking and intracellular signaling. Other possible mechanisms include the recruitment of new structural neuronal and microglia networks. While many of these theories have been developed using molecular and cellular techniques, more recent behavioral data also supports these findings. In this review, we focus on the mechanisms that underlie tolerance within the descending pain modulatory pathway, including alterations in intracellular signaling, neural-glial interactions, and neurotransmission following opioid exposure. Developing a better understanding of the relationship between these various mechanisms, within different parts of this pathway, is vital for the identification of more efficacious, novel therapeutics to treat chronic pain.

Enhanced antinociception with repeated microinjections of apomorphine into the periaqueductal gray of male and female rats.

Dopamine neurons in the ventrolateral periaqueductal gray (PAG) have been reported to contribute to antinociception. The objective of this study was to determine how this dopamine-mediated antinociception differs from what is known about morphine-induced antinociception. Microinjection of the dopamine receptor agonist apomorphine into the PAG produced a dose-dependent increase in hot plate latency and a decrease in open field activity that was greater in male than in female rats. The peak antinociceptive effect occurred 5 min after apomorphine administration. Surprisingly, the antinociceptive potency of apomorphine was enhanced following systemic administration of the opioid receptor antagonist naloxone in male, but not in female rats. The antinociceptive potency of microinjecting apomorphine into the ventrolateral PAG in male and female rats was also enhanced following twice-daily injections for 2 days. The characteristics of apomorphine-induced antinociception differ from previous reports of morphine antinociception following PAG microinjections in that morphine antinociception peaks at 15 min, is blocked by naloxone, and is susceptible to tolerance with repeated administration. These results indicate that apomorphine-induced antinociception is distinct from opioid-induced antinociception, and that dopamine receptor agonists may provide a novel approach to pain modulation.

The BigLEN-GPR171 Peptide Receptor System Within the Basolateral Amygdala Regulates Anxiety-Like Behavior and Contextual Fear Conditioning.

Studies show that neuropeptide-receptor systems in the basolateral amygdala (BLA) play an important role in the pathology of anxiety and other mood disorders. Since GPR171, a recently deorphanized receptor for the abundant neuropeptide BigLEN, is expressed in the BLA, we investigated its role in fear and anxiety-like behaviors. To carry out these studies we identified small molecule ligands using a homology model of GPR171 to virtually screen a library of compounds. One of the hits, MS0021570_1, was identified as a GPR171 antagonist based on its ability to block (i) BigLEN-mediated activation of GPR171 in heterologous cells, (ii) BigLEN-mediated hyperpolarization of BLA pyramidal neurons, and (iii) feeding induced by DREADD-mediated activation of BigLEN containing AgRP neurons in the arcuate nucleus. The role of GPR171 in anxiety-like behavior or fear conditioning was evaluated following systemic or intra-BLA administration of MS0021570_1, as well as following lentiviral-mediated knockdown of GPR171 in the BLA. We find that systemic administration of MS0021570_1 attenuates anxiety-like behavior while intra-BLA administration or knockdown of GPR171 in the BLA reduces anxiety-like behavior and fear conditioning. These results indicate that the BigLEN-GPR171 system plays an important role in these behaviors and could be a novel target to develop therapeutics to treat psychiatric disorders.

Identification of a small-molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake.

Several neuropeptide systems in the hypothalamus, including neuropeptide Y and agouti-related protein (AgRP), control food intake. Peptides derived from proSAAS, a precursor implicated in the regulation of body weight, also control food intake. GPR171 is a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) for BigLEN (b-LEN), a peptide derived from proSAAS. To facilitate studies exploring the physiological role of GPR171, we sought to identify small-molecule ligands for this receptor by performing a virtual screen of a compound library for interaction with a homology model of GPR171. We identified MS0015203 as an agonist of GPR171 and demonstrated the selectivity of MS0015203 for GPR171 by testing the binding of this compound to 80 other membrane proteins, including family A GPCRs. Reducing the expression of GPR171 by shRNA (short hairpin RNA)-mediated knockdown blunted the cellular and tissue response to MS0015203. Peripheral injection of MS0015203 into mice increased food intake and body weight, and these responses were significantly attenuated in mice with decreased expression of GPR171 in the hypothalamus. Together, these results suggest that MS0015203 is a useful tool to probe the pharmacological and functional properties of GPR171 and that ligands targeting GPR171 may eventually lead to therapeutics for food-related disorders.

Collybolide is a novel biased agonist of κ-opioid receptors with potent antipruritic activity.

Among the opioid receptors, the κ-opioid receptor (κOR) has been gaining considerable attention as a potential therapeutic target for the treatment of complex CNS disorders including depression, visceral pain, and cocaine addiction. With an interest in discovering novel ligands targeting κOR, we searched natural products for unusual scaffolds and identified collybolide (Colly), a nonnitrogenous sesquiterpene from the mushroom Collybia maculata. This compound has a furyl-δ-lactone core similar to that of Salvinorin A (Sal A), another natural product from the plant Salvia divinorum Characterization of the molecular pharmacological properties reveals that Colly, like Sal A, is a highly potent and selective κOR agonist. However, the two compounds differ in certain signaling and behavioral properties. Colly exhibits 10- to 50-fold higher potency in activating the mitogen-activated protein kinase pathway compared with Sal A. Taken with the fact that the two compounds are equipotent for inhibiting adenylyl cyclase activity, these results suggest that Colly behaves as a biased agonist of κOR. Behavioral studies also support the biased agonistic activity of Colly in that it exhibits ∼10-fold higher potency in blocking non-histamine-mediated itch compared with Sal A, and this difference is not seen in pain attenuation by these two compounds. These results represent a rare example of functional selectivity by two natural products that act on the same receptor. The biased agonistic activity, along with an easily modifiable structure compared with Sal A, makes Colly an ideal candidate for the development of novel therapeutics targeting κOR with reduced side effects.

Identification of GPR83 as the receptor for the neuroendocrine peptide PEN.

PEN is an abundant peptide in the brain that has been implicated in the regulation of feeding. We identified a receptor for PEN in mouse hypothalamus and Neuro2A cells. PEN bound to and activated GPR83, a G protein (heterotrimeric guanine nucleotide)-binding protein)-coupled receptor (GPCR). Reduction of GPR83 expression in mouse brain and Neuro2A cells reduced PEN binding and signaling, consistent with GPR83 functioning as the major receptor for PEN. In some brain regions, GPR83 colocalized with GPR171, a GPCR that binds the neuropeptide bigLEN, another neuropeptide that is involved in feeding and is generated from the same precursor protein as is PEN. Coexpression of these two receptors in cell lines altered the signaling properties of each receptor, suggesting a functional interaction. Our data established PEN as a neuropeptide that binds GPR83 and suggested that these two ligand-receptor systems-PEN-GPR83 and bigLEN-GPR171-may be functionally coupled in the regulation of feeding.

Ligand-biased activation of extracellular signal-regulated kinase 1/2 leads to differences in opioid induced antinociception and tolerance.

Opioids produce antinociception by activation of G protein signaling linked to the mu-opioid receptor (MOPr). However, opioid binding to the MOPr also activates ß-arrestin signaling. Opioids such as DAMGO and fentanyl differ in their relative efficacy for activation of these signaling cascades, but the behavioral consequences of this differential signaling are not known. The purpose of this study was to evaluate the behavioral significance of G protein and internalization dependent signaling within ventrolateral periaqueductal gray (vlPAG). Antinociception induced by microinjecting DAMGO into the vlPAG was attenuated by blocking Gαi/o protein signaling with administration of pertussis toxin (PTX), preventing internalization with administration of dynamin dominant-negative inhibitory peptide (dyn-DN) or direct inhibition of ERK1/2 with administration of the MEK inhibitor, U0126. In contrast, the antinociceptive effect of microinjecting fentanyl into the vlPAG was not altered by administration of PTX or U0126, and was enhanced by administration of dyn-DN. Microinjection of DAMGO, but not fentanyl, into the vlPAG induced phosphorylation of ERK1/2, which was blocked by inhibiting receptor internalization with administration of dyn-DN, but not by inhibition of Gαi/o proteins. ERK1/2 inhibition also prevented the development and expression of tolerance to repeated DAMGO microinjections, but had no effect on fentanyl tolerance. These data reveal that ERK1/2 activation following MOPr internalization contributes to the antinociceptive effect of some (e.g., DAMGO), but not all opioids (e.g., fentanyl) despite the known similarities for these agonists to induce ß-arrestin recruitment and internalization.

Contribution of adenylyl cyclase modulation of pre- and postsynaptic GABA neurotransmission to morphine antinociception and tolerance.

Opioid inhibition of presynaptic GABA release in the ventrolateral periaqueductal gray (vlPAG) activates the descending antinociception pathway. Tolerance to repeated opioid administration is associated with upregulation of adenylyl cyclase activity. The objective of these studies was to test the hypothesis that adenylyl cyclase contributes to opioid tolerance by modulating GABA neurotransmission. Repeated microinjections of morphine or the adenylyl cyclase activator NKH477 into the vlPAG decreased morphine antinociception as would be expected with the development of tolerance. Conversely, microinjection of the adenylyl cyclase inhibitor SQ22536 reversed both the development and expression of morphine tolerance. These behavioral results indicate that morphine tolerance is dependent on adenylyl cyclase activation. Electrophysiological experiments revealed that acute activation of adenylyl cyclase with forskolin increased the frequency of presynaptic GABA release. However, recordings from rats treated with repeated morphine administration did not exhibit increased basal miniature inhibitory postsynaptic current (mIPSC) frequency but showed a decrease in mean amplitude of mIPSCs indicating that repeated morphine administration modulates postsynaptic GABAA receptors without affecting the probability of presynaptic GABA release. SQ22536 reversed this change in mIPSC amplitude and inhibited mIPSC frequency selectively in morphine tolerant rats. Repeated morphine or NKH477 administration also decreased antinociception induced by microinjection of the GABAA receptor antagonist bicuculline, further demonstrating changes in GABA neurotransmission with morphine tolerance. These results show that the upregulation of adenylyl cyclase caused by repeated vlPAG morphine administration produces antinociceptive tolerance by modulating both pre- and postsynaptic GABA neurotransmission.

Differential development of antinociceptive tolerance to morphine and fentanyl is not linked to efficacy in the ventrolateral periaqueductal gray of the rat.

UNLABELLED: Systemic administration of morphine typically produces greater tolerance than higher efficacy mu-opioid receptor (MOPr) agonists such as fentanyl. The objective of the present study was to test this relationship by measuring antinociceptive efficacy and tolerance to morphine and fentanyl microinjected into the ventrolateral periaqueductal gray (vlPAG). MOPr agonist efficacy was evaluated by microinjecting the irreversible opioid receptor antagonist ß-funaltrexamine hydrochloride (ß-FNA) into the vlPAG prior to a dose-response analysis of morphine and fentanyl antinociception. In contrast to systemic administration of morphine and fentanyl, microinjection of these drugs into the vlPAG had similar efficacy as measured by similar reductions in maximal antinociception following ß-FNA administration. Analysis of tolerance revealed a rightward shift in the dose-response curve to a single pretreatment with morphine, but not fentanyl. The magnitude of tolerance to morphine was comparable following 1, 4, or 8 pretreatments. Tolerance to fentanyl also was evident following 4 or 8 microinjections. These data are surprising in that antinociceptive efficacy appears to vary depending on the site of administration. Moreover, the similar efficacy following microinjection of morphine and fentanyl into the vlPAG was associated with comparable tolerance, with the 1 exception of no tolerance to acute administration of fentanyl. PERSPECTIVE: These data reveal that antinociceptive tolerance following vlPAG administration of opioids develops rapidly and is evident with both morphine and fentanyl, and the magnitude is relatively consistent regardless of the number of pretreatments.

The influence of non-nociceptive factors on hot-plate latency in rats.

UNLABELLED: The hot plate is a widely used test to assess nociception. The effect of non-nociceptive factors (weight, sex, activity, habituation, and repeated testing) on hot-plate latency was examined. Comparison of body weight and hot-plate latency revealed a small but significant inverse correlation (light rats had longer latencies). Habituating rats to the test room for 1 hour prior to testing did not decrease hot-plate latency except for female rats tested on days 2 to 4. Hot-plate latency decreased with repeated daily testing, but this was not caused by a decrease in locomotor activity or learning to respond. Activity on the hot plate was consistent across all 4 trials, and prior exposure to a room-temperature plate caused a similar decrease in latency as rats tested repeatedly on the hot plate. Despite this decrease in baseline hot-plate latency, there was no difference in morphine antinociceptive potency. The present study shows that weight, habituation to the test room, and repeated testing can alter baseline hot-plate latency, but these effects are small and have relatively little impact on morphine antinociception. PERSPECTIVE: This manuscript shows that non-nociceptive factors such as body weight, habituation, and repeated testing can alter hot-plate latency, but these factors do not alter morphine potency. In sum, the hot-plate test is an easy to use and reliable method to assess supraspinally organized nociceptive responses.

Drug dependent sex-differences in periaqueducatal gray mediated antinociception in the rat.

Mu-opioid receptor (MOPr) agonists, such as morphine, produce greater antinociception in male compared to female rats. The ventolateral periaqueductal gray (vlPAG) appears to contribute to this sex-difference despite fewer vlPAG output neurons projecting to the rostral ventromedial medulla in male compared to female rats. This greater projection in female rats suggests that non-opioid activation of vlPAG output neurons should produce greater antinociception in female compared to male rats. This hypothesis was tested by comparing the time course and antinociceptive potency of microinjecting MOPr agonists (morphine, DAMGO, fentanyl) and non-opioid compounds (bicuculline, kainic acid) into the vlPAG of female and male rats. Microinjection of morphine or DAMGO produced antinociception that had a slow onset (peak from 15 to 30min) and long duration (60min) compared to the antinociception produced following microinjection of fentanyl, bicuculline, or kainic acid (peak effect at 3min; duration less than 30min). No sex-differences in the time courses were evident. All five compounds caused a dose-dependent antinociception when microinjected into the vlPAG. Antinociceptive potency was significantly greater in male compared to female rats following microinjection of morphine, DAMGO, and bicuculline, but not following microinjection of fentanyl or kainic acid. In no case did activation of the vlPAG produce greater antinocicepiton in female compared to male rats. These findings demonstrate that the vlPAG can produce comparable antinociception in female and male rats, but antinociception produced by inhibition of GABAergic neurons (whether by morphine or the GABA(A) receptor antagonist bicuculline) produces greater antinociception in males.