Erin N. Bobeck

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.

Extracellular Signal-Regulated Kinase 1/2 Activation Counteracts Morphine Tolerance in the Periaqueductal Gray of the Rat

Repeated administration of opioids produces long-lasting changes in μ-opioid receptor (MOR) signaling that underlie behavioral changes such as tolerance. Mitogen-activated protein kinase (MAPK) pathways, including MAPK extracellular signal-regulated kinases (ERK1/2), are modulated by opioids and are known to produce long-lasting changes in cell signaling. Thus, we tested the hypothesis that ERK1/2 activation contributes to the development and/or expression of morphine tolerance mediated by the periaqueductal gray (PAG). Changes in phosphorylated ERK1/2 expression were assessed with confocal microscopy and compared to behavioral measures of tolerance to the antinociceptive effects of chronic morphine administration. Repeated microinjection of morphine into the PAG produced tolerance and caused a significant increase in ERK1/2 phosphorylation, an effect not evident with acute morphine microinjection. Microinjection of the MAPK/ERK kinase inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene ethanolate (U0126), into the PAG had no effect on antinociception produced by acute morphine administration. However, repeated coadministration of U0126 and morphine into the PAG blocked ERK1/2 phosphorylation and enhanced the development of morphine tolerance. Coadministration of U0126 with morphine only on the test day also enhanced the expression of morphine tolerance. Administration of the irreversible opioid receptor antagonist β-chlornaltrexamine blocked the activation of ERK1/2 caused by repeated morphine microinjections, demonstrating that ERK1/2 activation was a MOR-mediated event. In summary, these studies show that chronic morphine administration alters ERK1/2 signaling and that disruption of ERK1/2 signaling enhances both the development and expression of morphine tolerance. Contrary to expectations, these data indicate that ERK1/2 activation opposes the development of morphine tolerance.

Drug Dependent Sex Differences in Periaqueducatal Gray Mediated Antinociception in the Rat

ABSTRACT 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 30 min) and long duration (60 min) compared to the antinociception produced following microinjection of fentanyl, bicuculline, or kainic acid (peak effect at 3 min; duration less than 30 min). 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 GABAA receptor antagonist bicuculline) produces greater antinociception in males.

Glutamate modulation of antinociception, but not tolerance, produced by morphine microinjection into the periaqueductal gray of the rat

The periaqueductal gray (PAG) plays an important role in morphine antinociception and tolerance. Co-localization of mu-opioid and NMDA receptors on dendrites in the PAG suggests that glutamate may modulate morphine antinociception. Moreover, the involvement of glutamate in spinally mediated tolerance to morphine suggests that glutamate receptors may contribute to PAG mediated tolerance. These hypotheses were tested by microinjecting glutamate receptor antagonists and morphine into the ventrolateral PAG (vPAG) of the rat. Microinjection of the non-specific glutamate receptor antagonist kynurenic acid or the NMDA receptor antagonist MK-801 into the vPAG did not affect nociception. However, co-administration of these antagonists with morphine into the vPAG enhanced the acute antinociceptive effects of morphine as measured by a leftward shift in the morphine dose-response curves. Repeated microinjections of morphine into the vPAG caused a rightward shift in the dose-response curve for antinociception whether the glutamate receptor antagonists kynurenic acid or MK-801 were co-administered or not. The lack of effect of microinjecting glutamate receptor antagonists into the vPAG indicates that tonic glutamate release in the PAG does not contribute to nociceptive tone. That these antagonists enhance morphine antinociception indicates that endogenous glutamate counteracts the antinociceptive effect of morphine in the vPAG. However, this compensatory glutamate release does not contribute to tolerance to the antinociceptive effects of microinjecting morphine into the vPAG. Previous research showing that glutamate contributes to spinal mechanisms of tolerance indicate that different tolerance mechanisms are engaged in the vPAG and spinal cord.

Identification of GPR83 as the receptor for the neuroendocrine peptide PEN

Functional coupling occurs between PEN-GPR83 and bigLEN-GPR171, ligand-receptor pairs implicated in feeding. PEN adopts GPR83 Neuropeptides produced by proteolytic processing of the protein proSAAS include PEN and bigLEN and are implicated in the regulation of appetite. The receptor for bigLEN is GPR171, which was an orphan G protein–coupled receptor (GPCR) until this ligand was identified. Here, Gomes et al. identify the receptor for PEN as the orphan GPCR GPR83, a receptor implicated in behavior, learning, and metabolic regulation. These two ligand-receptor pairs altered each other’s pharmacological properties. Proximity ligation assays and immunofluorescence colocalization assays in mouse brain indicated that the two receptors were present in some of the same cells in the region of the brain that controls feeding. Because PEN and bigLEN are produced from the same precursor, they are sometimes packaged in the same vesicles and are released together. GPR83 also colocalizes and functionally interacts with the receptor for ghrelin, another peptide involved in regulation of organismal metabolism by the central nervous system. Knowing the ligand for GPR83 will enable the investigation of how GPR83, GPR171, and other receptors interact to control body weight, which has implications for treating metabolic disorders associated with being underweight or overweight. 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.

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 β-funaltrexamine hydrochloride (β-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 β-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.

Change in functional selectivity of morphine with the development of antinociceptive tolerance.

Opioids, such as morphine, are the most effective treatment for pain but their efficacy is diminished with the development of tolerance following repeated administration. Recently, we found that morphine activated ERK in opioid‐tolerant but not in naïve rats, suggesting that morphine activation of μ‐opioid receptors is altered following repeated morphine administration. Here, we have tested the hypothesis that μ‐opioid receptor activation of ERK in the ventrolateral periaqueductal gray (vlPAG) is dependent on dynamin, a protein implicated in receptor endocytosis.

Endothelin‐converting enzyme 2 differentially regulates opioid receptor activity

Opioid receptor function is modulated by post‐activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co‐endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin‐converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization.

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.

OPIOID RECEPTOR INTERNALIZATION CONTRIBUTES TO DERMORPHIN-MEDIATED ANTINOCICEPTION

Microinjection of opioids into the ventrolateral periaqueductal gray (vlPAG) produces antinociception in part by binding to mu-opioid receptors (MOPrs). Although both high and low efficacy agonists produce antinociception, low efficacy agonists such as morphine produce limited MOPr internalization suggesting that MOPr internalization and signaling leading to antinociception are independent. This hypothesis was tested in awake, behaving rats using DERM-A594, a fluorescently labeled dermorphin analog, and internalization blockers. Microinjection of DERM-A594 into the vlPAG produced both antinociception and internalization of DERM-A594. Administration of the irreversible opioid receptor antagonist beta-chlornaltrexamine (beta-CNA) prior to DERM-A594 microinjection reduced both the antinociceptive effect and the number of DERM-A594 labeled cells demonstrating that both effects are opioid receptor-mediated. Pretreatment with the internalization blockers dynamin dominant-negative inhibitory peptide (dynamin-DN) and concanavalinA (ConA) attenuated both DERM-A594 internalization and antinociception. Microinjection of dynamin-DN and ConA also decreased the antinociceptive potency of the unlabeled opioid agonist dermorphin when microinjected into the vlPAG as demonstrated by rightward shifts in the dose-response curves. In contrast, administration of dynamin-DN had no effect on the antinociceptive effect of microinjecting the GABA(A) receptor antagonist bicuculline into the vlPAG. The finding that dermorphin-induced antinociception is attenuated by blocking receptor internalization indicates that key parts of opioid receptor-mediated signaling depend on internalization.