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Lesioning mu opioid receptor-containing neurons in the ventrolateral periaqueductal gray attenuates morphine analgesia in male but not female rats
Loyd DR, Murphy AZ (2007) Lesioning mu opioid receptor-containing neurons in the ventrolateral periaqueductal gray attenuates morphine analgesia in male but not female rats. Neuroscience 2007 Abstracts 921.4/NN15. Society for Neuroscience, San Diego, CA.
Summary: Chronic pain will affect four out of five persons at some point across the lifespan. While the opioid-based narcotic morphine is the most prevalent treatment for chronic pain in clinical settings, it is becoming increasingly clear that morphine produces a significantly greater degree of analgesia in males compared to females. In both somatic and visceral pain models, the ED50 for morphine is generally two-fold higher for females than for males. The midbrain periaqueductal gray (PAG) and its descending projections to the rostral ventromedial medulla (RVM) is the primary circuit for opioid-based analgesia. We have recently shown that the PAG-RVM pathway is sexually dimorphic both in its anatomical organization and in its activation during persistent pain. Interestingly, while female rats have a greater number of PAG neurons that project to the RVM, inflammatory pain activates these cells to a greater degree in males. Additionally, systemic morphine inhibits the pain-induced activation of PAG neurons in males, but not females. Sex differences in neuronal activity during pain and morphine analgesia are prominent in the ventrolateral PAG, a region containing a large population of mu opioid receptor-containing neurons. We have recently shown that females have significantly lower levels of mu opioid receptors (MOR) in this region, however it is not known whether sex differences in MOR expression contribute to our observed sex differences in morphine analgesia. To test the role of ventrolateral PAG MOR in morphine analgesia, the cytotoxin saporin conjugated to the MOR agonist dermorphin (Der-Sap) was injected into the ventrolateral PAG to site-specifically lesion MOR-containing neurons. Twenty-eight days later, rats received an intraplantar injection of CFA to induce persistent pain and twenty-four hours later morphine was administered systemically using a cumulative dosing paradigm (1.8 -18mg/kg). Lesions of PAG MOR-containing neurons resulted in a two-fold rightward shift in morphine ED50 values in male rats compared to controls. Interestingly, in females no difference was noted in morphine ED50 for Der-Sap treated females versus controls suggesting that the PAG is not a critical site for morphine analgesia in females. Der-Sap treatment had no significant impact on baseline paw withdrawal latencies or CFA-induced hyperalgesia. These results indicate that the PAG is a primary locus for systemic morphine analgesia in males only and suggests the necessity for the development of sex-specific treatments for persistent pain in females.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Role of spinal cord µ-opioid receptor expressing dorsal horn neurons in morphine analgesia
Kline IV RH, Wiley RG (2006) Role of spinal cord µ-opioid receptor expressing dorsal horn neurons in morphine analgesia. Neuroscience 2006 Abstracts 643.19. Society for Neuroscience, Atlanta, GA.
Summary: The role of spinal cord μ-opioid receptor expressing dorsal horn neurons in morphine analgesia is not clearly understood. Using lumbar intrathecal (i.t.) injections of the targeted toxin dermorphin-saporin to selectively destroy these cells, we sought to determine the effect of this lesion on the antinociceptive activity of systemic and i.t. morphine on the hotplate test. We examined the antinociceptive effects of morphine across a range of stimulus intensities (44, 47 & 52oC) in order to assess responses mediated by C or Aδ thermal nociceptors. Experiment 1 (systemic morphine): Sixteen Sprague Dawley male rats were injected with 500ng dermorphin-saporin i.t. or PBS and hotplate testing resumed one week after injections. Baseline hotplate responses were monitored for three weeks after which systemic morphine dose response curves (0, 2.5, 5, &10 mg/kg s.c.) were performed. Experiment 2 (spinal intrathecal morphine): Twelve Long Evans female rats were surgically implanted with indwelling lumbar i.t. catheters (8.5cm), underwent baseline hotplate testing for 7 days, had i.t. morphine dose response curves (0, 0.01, 0.1, & 1 μg) performed at 44 & 52oC seven days before and eight days after dermorphin-saporin injections. The dependent measures for the hotplate test were: 1) latencies to the first lick or guard response (all temperatures) and 2) the cumulative durations and amounts of licking and guarding events (44 and 47oC). Loss of lamina II MOR-expressing dorsal horn neurons after dermorphin-saporin was confirmed in spinal cord sections from each rat stained for MOR1 and MOR1C using standard immunoperoxidase techniques on adjacent 40 μm sections from the L4 spinal segment. Baseline responses to noxious heat did not decrease after i.t. dermorphin-saporin. The antinociceptive activity of systemic morphine was attenuated in dermorphin-saporin treated rats at 44 & 47oC; this effect was least striking on the 52oC hotplate and greatest on the 44oC hotplate. The dermorphin-saporin-induced lesion reduced the antinociceptive effects of intrathecal morphine more than systemic morphine. Based on the above findings are others not included here, we conclude that dorsal horn MOR expressing neurons are necessary for morphine to exert its maximum antinociceptive and analgesic effects.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
A selective lesioning method to probe the role of intercalated (ITC) amygdala neurons in the extinction of classically conditioned fear responses
Likhtik E, Apergis-Schoute J, Pare D (2006) A selective lesioning method to probe the role of intercalated (ITC) amygdala neurons in the extinction of classically conditioned fear responses. Neuroscience 2006 Abstracts 370.19. Society for Neuroscience, Atlanta, GA.
Summary: The acquisition of conditioned fear responses (CRs) is thought to involve the potentiation of synapses conveying information about the conditioned stimulus (CS) to the basolateral (BLA) amygdala. Expression of CRs would depend on transfer of potentiated CS inputs by the BLA to the central amygdala (CE). In contrast, the mechanisms of extinction remain controversial. It was proposed that ITC neurons, which receive BLA inputs and generate feedforward inhibition in CE, are in a key position to mediate extinction. In this view, potentiation of BLA inputs to ITC cells during extinction training, would dampen the impact of CS-related BLA activity on CE neurons, inhibiting CRs. However, this idea is difficult to test because ITC cells occur in small, lateromedially dispersed clusters, making conventional lesioning methods inadequate. The present study aimed to find an effective way of eliminating ITC cells, taking advantage of the fact that, compared to the rest of the amygdala, they exhibit strong immunoreactivity for mu opioid receptors (muORs). First, we performed electron microscopic observations to determine whether muORs are expressed by ITC cells vs. afferents to ITC cells. This test revealed that muORs are concentrated in the postsynaptic membrane density of asymmetric synapses found on ITC cells. Next, we tested whether it is possible to obtain selective ITC lesions by injecting the toxin saporin conjugated to the mu opioid agonist dermorphin (DER-SAP) in the proximity of ITC cells. Thus, rats received intra-amygdaloid pressure injections of DER-SAP in one hemisphere and of vehicle on the contralateral side. Seven days later, the animals were perfused and the tissue processed to reveal muOR. DER-SAP injections produced a marked reduction in muOR immunoreactivity at the BLA-CE border, where ITC cells are usually located. Thus, selective lesioning of ITC cells can be achieved using this method. We are currently testing the impact of such ITC lesions on extinction learning.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization.
Vera-Portocarrero LP, Zhang ET, Ossipov MH, Xie JY, King T, Lai J, Porreca F (2006) Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization. Neuroscience 140(4):1311-1320. doi: 10.1016/j.neuroscience.2006.03.016
Summary: Rats were treated with 1.5 pmol of dermorphin-SAP (Cat. #IT-12) or saporin (Cat. #PR-01) into each side of the rostral ventromedila medulla, followed by spinal nerve ligation. The data indicate that mu opioid-expresing neurons are necessary to maintain nerve injury-induced central sensitization.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Saporin (Cat. #PR-01)
Descending facilitation from the rostral ventromedial medulla maintains visceral pain in rats with experimental pancreatitis.
Vera-Portocarrero LP, Yie JX, Kowal J, Ossipov MH, King T, Porreca F (2006) Descending facilitation from the rostral ventromedial medulla maintains visceral pain in rats with experimental pancreatitis. Gastroenterology 130(7):2155-2164. doi: 10.1053/j.gastro.2006.03.025
Summary: Here the authors investigated the role of ascending or descending pathways in the mediation of pain caused by pancreatitis. Rats received 1.5 pmol injections of dermorphin-SAP (Cat. #IT-12) into each side of the rostral ventromedial medulla. Abdominal hypersensitivity was tested using von Frey filaments. Although the ablation of mu-opioid receptor-expressing neurons by dermorphin-SAP did not prevent the initial expression of pancreatitis pain, maintenance of this pain was absent. The data link maintenance of pancreatitis pain to descending pathways.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Featured Article: Targeted toxins in pain
Wiley RG (2006) Featured Article: Targeted toxins in pain. Targeting Trends 7(2)
Related Products: 192-IgG-SAP (Cat. #IT-01), Anti-DBH-SAP (Cat. #IT-03), Anti-SERT-SAP (Cat. #IT-23), SP-SAP (Cat. #IT-07), SSP-SAP (Cat. #IT-11), Dermorphin-SAP / MOR-SAP (Cat. #IT-12),
Facilitatory influences from the rostral ventromedial medulla (RVM) are required for pancreatic nociception
Vera-Portocarrero LP, Xie Y, King T, Lai J, Porreca F (2005) Facilitatory influences from the rostral ventromedial medulla (RVM) are required for pancreatic nociception. Neuroscience 2005 Abstracts 623.18. Society for Neuroscience, Washington, DC.
Summary: Pain is a frequent complaint of patients with pancreatitis or pancreatic cancer. An animal model of pancreatitis induced by dibutyltin dichloride (DBTC) is characterized by abdominal hypersensitivity to mechanical stimuli that appears by day 3 after induction of pancreatitis and persists for at least 10 days. We have used this model to evaluate the role of descending pain modulatory pathways from the RVM in the processing of visceral pain. Pancreatitis was induced in rats by a single tail vein injection of DBTC. Animals were monitored for mechanical sensitivity of the abdominal area as an index of pancreatic nociception using von Frey hairs applied to the surface of the abdomen and recording the frequency of withdrawals from stimulation. Six days after DBTC injection, when mechanical hypersensitivity was fully developed, lidocaine, or saline, was microinjected into the RVM. Lidocaine, but not saline, given into the RVM produced a time-related reversal of mechanical hypersensitivity which peaked by 20 min after injection in animals with pancreatitis. RVM lidocaine had no effect on rats without pancreatitis. A second group of rats received a single microinjection of the cytotoxin dermorphin-saporin into the RVM in order to ablate mu opioid receptor expressing cells that have been proposed to drive descending pain facilitation. 28 days later, the rats received DBTC and their response to mechanical stimulation was monitored daily. These rats showed mechanical hypersensitivity on day 3 after DBTC, but the sensory threshold reverted to normal level by day 6, while rats that had been pretreated with dermorphin, saporin, or water exhibited persistent mechanical hypersensitivity after DBTC out to day 10. These data suggest that a blockade of the descending input from the RVM by lidocaine is sufficient to block the pancreatitis-induced visceral pain, and that the mu opioid receptor expressing cells in the RVM are critical for the persistent pain state.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Increased formalin behavior after selective destruction of μ opiate receptor-expressing dorsal horn neurons: impaired descending analgesic control?
Datta S, Kline IV RH, Wiley RG (2005) Increased formalin behavior after selective destruction of μ opiate receptor-expressing dorsal horn neurons: impaired descending analgesic control?. Neuroscience 2005 Abstracts 623.15. Society for Neuroscience, Washington, DC.
Summary: Spinal intrathecal injection of dermorphin-saporin (derm-sap) selectively destroys dorsal horn neurons expressing the mu-opiate receptor (MOR). In the present study, we sought to determine the effect of derm-sap (500 ng, i.t.) on responses to intraplantar formalin injection (25 ul of 5%). After formalin injection, rats were immediately placed into a clear observation chamber with a video camera beneath the floor. Rats were videotaped for 90 minutes and their behavior scored offline for one minute out of every 5 minutes. 120 minutes after formalin injection rats were anesthetized with pentobarbital and perfused with formalin. Spinal cord sections were stained for MOR and cholecystokinin (CCK) using standard immunoperoxidase techniques on adjacent 40 um sections from L4 spinal segment. Coded sections were used to assess MOR staining intensity by quantitative densitometry. Derm-sap treated rats showed no separation between phase I and II and spent more time than vehicle controls licking/guarding/biting the injected hindpaw during both phase I and II. Derm-sap significantly decreased dorsal horn MOR. Staining for CCK showed time dependant changes after derm-sap which was not present in PBS controls. These same derm-sap treated rats performed normally on hotplate at 44, 47 and 52 C and had normal analgesic responses to systemic morphine on 44, 47 and 52 C hotplates. We interpret these data to indicate that loss of the dorsal horn MOR-expressing neurons reduces the effect of descending analgesic mechanisms. Supported by NIH R21-DA14380 and Department of Veterans Affairs.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Pain facilitatory cells in the rostral ventromedial medulla coexpress opioid-μ receptors and cholecystokinin type 2 receptors
Zhang W, Gardell SE, Xie Y, Luo M, Rance NE, Vanderah TW, Porreca F, Lai J (2005) Pain facilitatory cells in the rostral ventromedial medulla coexpress opioid-μ receptors and cholecystokinin type 2 receptors. Neuroscience 2005 Abstracts 394.17. Society for Neuroscience, Washington, DC.
Summary: Pain transmission can be modulated by descending input to the spinal dorsal horn from the rostral ventromedial medulla (RVM). RVM neurons that facilitate nociception are termed “ON-cells”, which are inhibited by mu-opioids, suggesting that they express opioid mu receptors (MOR). Focal application of cholecystokinin (CCK8(s)) into the RVM elicits acute thermal and tactile hypersensitivity and induces ON-cell activity. In situ hybridization using riboprobes for either rat MOR or rat cholecystokinin type-2 receptor (CCK-2) confirms the expression of these receptors in the RVM. Pretreatment with a toxin conjugate, CCK8(s)-saporin results in a significant loss of CCK-2 positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The pretreatment also significantly reduces the number of neurons labeled for MOR in the RVM, suggesting that MOR and CCK-2 may be co-localized in some RVM cells. Consistent with these data, similar pretreatment with the toxin conjugate, dermorphin-saporin, which selectively targets MOR expressing neurons, significantly reduces the number of MOR labeled cells in the RVM, blocks RVM CCK8(s) induced hyperalgesia and reduces the number of CCK-2 positive cells in the RVM. In situ hybridization using 35S-labeled CCK-2 riboprobes and Digoxigenin-labeled MOR riboprobes shows that over 80% of labeled RVM neurons co-express both MOR and CCK-2, ~15% express only CCK-2, and very few cells express only MOR. These findings represent the first direct demonstration of the phenotype of pain facilitatory neurons in the RVM. Together with previous studies showing that RVM CCK-2 antagonists reverse nerve injury-induced pain, this phenotype provides strong support for the view that endogenous CCK is a critical mediator of the descending pain facilitation, particularly in the maintenance of experimental neuropathic pain. Support Contributed By: NIDCR R01 DE016458
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), CCK-SAP (Cat. #IT-31)
Molecular neurosurgery with targeted toxins
Wiley RG, Lappi DA (2005) Molecular neurosurgery with targeted toxins. Humana Press, Totowa, New Jersey.
Summary: The idea behind the book was to provide a road map for the users of Molecular Neurosurgery to see how experienced scientists used these exceptional reagents in their work. Experiments with several targeted toxins are described, and readers can get an idea either specifically about a targeted toxin that they’re using, or about how a type of molecule is used and at what dosage, in a paradigm similar to theirs.
Related Products: 192-IgG-SAP (Cat. #IT-01), ME20.4-SAP (Cat. #IT-15), Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11), Dermorphin-SAP / MOR-SAP (Cat. #IT-12), IB4-SAP (Cat. #IT-10), CTB-SAP (Cat. #IT-14)