Dopamine and Morphine Tolerance

Dopamine Identified as Key Player

Our Mu Opioid Receptor antibody is used to show that blocking dopamine decreases morphine tolerance: Wen-Ling Dai, Feng Xiong, Bing Yan, Zheng-Yu Cao, Wen-Tao Liu, Ji-Hua Liu1, Bo-Yang Yu. Blockade of neuronal dopamine D2 receptor attenuates morphine tolerance in mice spinal cord. Scientific Reports 6, Article number: 38746 (2016). doi:10.1038/srep38746.

Images: (A) Double immunofluorescence staining showed that MOR (green) and D2DR (red) were co-localized in the mice spinal cord (20X magnification). Chronic morphine treatment increased the co-localization of MOR and D2DR in the spinal cord, and D2DR antagonist sulpiride (8 μg/10 μl, i.t.) reduced the increased co-expression of D2DR with MOR (n = 4). (B) Co-IP experiments showed that D2DR could interact with MOR, and the MOR/D2DR interactions were increased in the spinal dorsal horn after chronic morphine treatment for 7 days while D2DR antagonist sulpiride (8 μg/10 μl, i.t.) disrupted the interactions of the MOR/D2DR (n = 3).

Blockade of D2DR in spinal cord can disrupt the interactions between MOR and D2DR to attenuate morphine tolerance. These findings highlight the possibility of a new clinical strategy to prevent morphine antinociceptive tolerance.

Epigenetics and Pain Research

i-Fect Used to Study Impacts

Our i-Fect siRNA, miRNA and shRNA Trasfection Kit was recently used to study the impact of G9a-specific siRNA (AGUAACGGGCAUCAAUGC) on Mu Opioid Receptors: Yuhao Zhang, Shao-Rui Chen, Geoffroy Laumet, Hong Chen and Hui-Lin Pan. Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-Mediated Transcriptional Repression of µ-Opioid Receptors in Primary Sensory Neurons. First Published on February 25, 2016, doi: 10.1074/jbc.M115.711812... In some SNL rats, G9a-specific siRNA (4 µg) or the negative control siRNA was administered intrathecally. G9a-specific siRNA(AGUAACGGGCAUCAAUGC) or universal negative control siRNA (#SIC001, Sigma-Aldrich) was mixed with i-Fect (Neuromics, Edina, MN) to a final concentration of 400 mg/L for the intrathecal injections...

Figures: G9a knockdown with siRNA reverses the MOR expression in the DRG and the morphine analgesic effect diminished by nerve injury. (A,B) Quantitative PCR (A) and Western blotting (B) analyses show the mRNA and protein levels of MORs in the DRGs of sham and SNL rats treated with control or G9a-specific siRNA (n = 10 rats in each group). The ipsilateral L5 and L6 DRG tissues were removed 24 h after the last siRNA injection. The amount of MOR mRNA and protein was normalized to GAPDH in the same samples, and the mean value of MOR levels in sham control rats was considered to be 1. (C) Time course of the intrathecal morphine effects on the tactile and pressure withdrawal thresholds in sham and SNL rats treated with G9a-specific siRNA or negative control siRNA (n = 9 rats in each group). The withdrawal thresholds after the last siRNA injection were plotted as the baseline control (BL).

Summary: The findings provide new insight into the epigenetic mechanism regulating MOR expression in primary sensory neurons in neuropathic pain. This multidisciplinary approach provides conclusive evidence for G9a as a key chromatin regulator responsible for MOR downregulation in the DRG and the analgesic efficacy of opioids reduced by nerve injury. A better understanding of the epigenetic mechanisms underlying nerve injury-induced downregulation of MORs in primary sensory neurons could help improve the analgesic efficacy of opioids for treating chronic neuropathic pain. G9a inhibitors could be used to enhance the opioid analgesic effect and reduce opioid consumption in patients with chronic neuropathic pain.

Small Molecules-Peptides for Neuroscience Research

Agonists, Antagonists, Inhibitors and Ligands for Studying Neuromodulation

Our friends at R and D Systems/Tocris Bioscience have made available to us select Small Molecules/Peptides. Our focus will be on providing agonists, antagonists, inhibitors and ligands that complement our Neuroscience and Pain Research products and expertise.

2-APB=TRP Channel Modulator

We will be adding about 10 new molecules/peptides per month. Here's a sampling our our most recent additions:

Name	Type	Bioactivity
(±)-trans-ACPD	Agonist	Potent NMDA agonist. Also group II mGluR agonist
(S)-(-)-5-Fluorowillardiine	Agonist	Very potent AMPA agonist
(S)-4-Carboxyphenylglycine	Antagonist	Competitive group I mGluR antagonist/weak group II agonist
2-APB	Modulator	TRP channel modulator. Also IP3 receptor antagonist
2-Methylthioadenosine triphosphate tetrasodium salt	Agonist	P2 purinergic agonist
AM 404	Modulator	Vanilloid receptor agonist. Also anandamide transport inhibitor
BRL 52537 hydrochloride	Ligand	Potent and selective κ opioid receptor agonist
CNQX	Antagonist	Potent AMPA/kainate antagonist
Clocinnamox mesylate	Antagonist	Irreversible μ-opioid receptor antagonist
Endomorphin-1	Agonist	Potent and selective μ opioid receptor agonist
Endomorphin-2	Agonist	Potent and selective μ opioid receptor agonist
FIT	Agonist	Irreversible δ opioid receptor agonist
GBR 13069 dihydrochloride	Agonist	Potent dopamine uptake inhibitor
L-NIO dihydrochloride	Inhibitor	Potent eNOS inhibitor
L-Quisqualic acid	Agonist	Very potent group I mGluR agonist
N-Benzylnaltrindole hydrochloride	Agonist	Opioid receptor selective non-peptide antagonist
Nociceptin	Inhibitor	Endogenous NOP agonist
O-Phospho-L-serine	Antagonist	Group III mGluR agonist; enhances neuronal differentiation
Ro 51	Antagonist	Potent P2X3, P2X2/3 antagonist
cis-ACPD	Agonist	Potent NMDA agonist. Also group II mGluR agonist

We will be posting new additions and related data and publications

Mu Opioid Receptor, Aging and Immune Response

Mu Opioid Receptors (MOR) play an integral role in modulating perception of pain. Our Opioid Receptor and Opioid Neuropeptide Antibodies have been widely used and extensively published by Pain Researchers.

Here researchers determine a pivotal role of synaptic IGF-1R/Fyn signaling controlled by MOR downstream signaling cascades were crucial for the age-dependent neuroimmune modulation following traumatic stress: Hui Zhao, Xiaocong Zhao, Xiaoding Cao and Gencheng Wu. Age-Dependent Neuroimmune Modulation of IGF-1R in the Traumatic Mice. Immunity & Ageing 2012, 9:12 doi:10.1186/1742-4933-9-12.

Highlights: Fyn activity, as well as its molecular connection with IGF-1R was dependent on MOR, on account of that their coupling was obviously not able to be observed when lack of MOR, accordingly, there was not improvement from the immuno-suppression mediated by traumatic stress in MOR (−/−) mice. Likewise, there was not remarkable change in MOR expression, as well as the association of MOR with IGF-1R or Fyn in the synaptic zone. Then, it is plausible that a characteristic feature of IGF-1R was probably due to age-dependent Fyn activation that was triggered by MOR signaling cascades, this specialized process was mainly concentrated within synaptic zone and might contribute to the recovery from traumatic stress mediated immuno-suppression.

Images: MOR expression during traumatic stress. 2-month and 1-year mice were killed 1 and 3 days after traumatic stress (n = 5 for each group), synaptoneurosome from frontal cortex was prepared, Western blot analysis was used to detect MOR expression (A). Immunoprecipitation was used to analyze alterations of MOR and IGF-1R/Fyn interaction. The immunoprecipitation antibody was anti-Fyn (B) or anti-IGF-1R (D) and the immunoblotting antibody was anti-MOR. Panel C and E depict quantitative analysis of B and D respectively. Data are presented as percentage of control, values represent mean ± SD for 3 independent experiments. Con: control; T3: 3 days after trauma.

I will continue to post new applications for our MOR antibodies.

Serotonin and Pain

Brainstem facilitations and descending serotonergic controls contribute to visceral nociception but not pregabalin analgesia in rats.

Pregabalin is used to treat Neuropathic Pain. Here the authors show state-dependent pregabalin analgesia in neuropathy does not apply to visceral pain. Our rabbit polyclonal Mu Opioid Receptor Antibody is used to demonstrate intra-RVM Derm-SAP locally ablates a substantial proportion of MOR and serotonergic cells: Shafaq Sikandar, Kirsty Bannister, Anthony H. Dickenson. Brainstem facilitations and descending serotonergic controls contribute to visceral nociception but not pregabalin analgesia in rats. Neuroscience Letters. Volume 519, Issue 1, 21 June 2012, Pages 31–36.... For MOR staining, a rabbit polycolonal μ-opioid receptor primary antibody (1:10,000 TTBS; Neuromics, MN, USA, RA10104)...

These findings are important for understanding the limitations of Preglabin as an analgesic for viceral pain. Check out all our Opioid Publications.

Opioid Addiction During Pregnancy-Implications for Neuro-development

I would like to thank Dr. Carmen Sato-Bigbee, Virginia Commonwealth University School of Medicine, for kindly sharing this important study. The publication also references use of our Mu Opioid and Nociceptin/Orphanin FQ Receptor Antibodies: Andrew C. Eschenroeder, Allison A. Vestal-Laborde, Emilse S. Sanchez, Susan E. Robinson, Carmen Sato-Bigbee. Oligodendrocyte responses to buprenorphine uncover novel and opposing roles of μ-opioid- and nociceptin/orphanin FQ receptors in cell development: Implications for drug addiction treatment during pregnancy. Glia Volume 60, Issue 1, pages 125–136, January 2012.

Highlights: Oligodendrocytes are responsible for making myelin in the CNS. The authors have shown We have shown previously that rat brain myelination is significantly altered by buprenorphine, an opioid analogue currently used in clinical trials for managing pregnant opioid addicts. In this study, perinatal exposure to low levels of this drug induced accelerated and increased expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic drug doses delayed MBP brain expression and resulted in a decreased number of myelinated axons. We have now found that this biphasic-dose response to buprenorphine can be attributed to the participation of both the l-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOP receptor) in the oligodendrocytes. This is the first study showing the potential role of the NOP receptor in myelination.

High levels of opiate exposure could negatively disrupt the normal interplay between these two systems altering the developmental pattern of brain myelination. Understanding this pathway, could help researchers find ways to favorably modulate myelination and protect neuro-development of fetuses exposed to high levels of opiates during pregnancy.

Related Data:

Direct treatment of immature oligodendrocytes with buprenorphine alters MBP expression in a dose-specific manner. Cells isolated from 9-day-old rat brains were incubated for 4 days in CDM with or without 0.25, 0.5, 1.0, and 3.0 lM buprenorphine. MBP levels were determined by western blotting using b-actin levels as loading controls. Figures correspond to representative experiments. Results in the bar graph are expressed as percentage of controls (0 lM buprenorphine) 6 SEM from five experiments and correspond to the combined scanning of the four major MBP isoforms. **P <0.005 and ***P<0.0001.

Pre-oligodendrocytes express both MOR and the NOP receptor. Cells isolated from 9-day-old rat brain were allowed to fully attachon the culture plates by overnight incubation and stained by double
immunocytochemistry with O4 (green) together with anti-MOR or anti-NOP receptor antibodies (red). Scale bar: 20 lm. The western blot shows MOR and NOP receptor expression in two different samples of developing oligodendrocytes directly isolated from 9-day-old rat brains.

I will be posting future studies investigating the molecular mechanisms by which buprenorphine and methadone affect myelination and neuron-glial interactions. These should provide deeper understanding into these developmental processes and new and better strategies for the managing of both pregnant addicts and drug addiction in adolescence.

Opioid Induced Itch

Our widely used and frequently published Opioid Receptors Antibodies are used for a spectrum of pain research. Here is an interesting study on the root causes of opioid induced itch.

Xian-Yu Liu, Zhong-Chun Liu, Yan-Gang Sun, Michael Ross1, Seungil Kim, Feng-Fang Tsai, Qi-Fang Li, Joseph Jeffry, Ji-Young Kim, Horace H. Loh, Zhou-Feng Chen. Unidirectional Cross-Activation of GRPR by MOR1D Uncouples Itch and Analgesia Induced by Opioids. Cell, Volume 147, Issue 2, 14 October 2011, Pages 261-262.

Root Causes: Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the μ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCβ3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.

Let-7 microRNAs and Nociceptive Pain

Our Opioid Receptor Antibodies have set a potent standard for studying Nociceptive and Neuropathic Pain. Related Publications.

We want to recognize Dr. Zaijie Jim Wang and his team for being the first to use our Mu Opioid Receptor for studying the potential role of microRNAs in Nociception.

Ying He, Cheng Yang, Chelsea M. Kirkmire, and Zaijie Jim Wang. Regulation of Opioid Tolerance by let-7 Family MicroRNA Targeting the µ Opioid Receptor. The Journal of Neuroscience, July 28, 2010, 30(30):10251-10258; doi:10.1523/JNEUROSCI.2419-10.2010
Abstract: MicroRNA has emerged as a critical regulator of neuronal functions. This study aimed to test whether let-7 microRNAs can regulate the µ opioid receptor (MOR) and opioid tolerance. Employing bioinformatics, we identified a let-7 binding site in the 3'-untranslated region (UTR) of MOR mRNA, which was experimentally confirmed as a direct target of let-7. The repressive regulation of MOR by let-7 was revealed using a LNA-let-7 inhibitor to knockdown let-7 in SH-SY5Y cells. Conversely, morphine significantly upregulated let-7 expression in SH-SY5Y cells and in a mouse model of opioid tolerance. The LNA-let-7 inhibitor decreased brain let-7 levels and partially attenuated opioid antinociceptive tolerance in mice. Although chronic morphine treatment did not change overall MOR transcript, polysome-associated mRNA declined in a let-7-dependent manner. let-7 was identified as a mediator translocating and sequestering MOR mRNA to P-bodies, leading to translation repression. These results suggest that let-7 plays an integral role in opioid tolerance.

• Western blot analysis. Western blot analysis was performed as previously described (Tang et al., 2006) using the anti-µ opioid receptor antibody (1:1000; Neuromics). The expression of β-actin was similarly determined from the same blots using a monoclonal antibody (1:10,000; Sigma).
• For immunofluorescence analysis, the antibody for hDcp1a (Santa Cruz Biotechnology) and MOR were used at 1:500 and 1:5000 dilutions, respectively. Secondary anti-goat and anti-mouse antibodies labeled with Alexa 488 and Alexa 594 fluorochromes (Invitrogen), respectively, were used at 1:500 dilutions.

Related Reagent Links:
All Opioid Receptor Antibodies
Pain and Inflammation       
Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion Channels,
Biogenic Amines and more
i-Fect Transfection Kit -gene silencing of DOR,
NaV1.8 tetrodotoxin-resistant sodium channel, NTS2 and more in-vitro and in vivo
Primary Neurons and Astrocytes-Primary human,
rat and mouse neurons and astrocytes 

No Pain; No Gain

We work hard to make sure our Pain and Inflammation antibodies continue to be a gold standard for researchers. We follow up with virtually all researchers to make sure they work to expecations in each unique application.

We also look for references in current publication. Although published in 2006, this one just crossed our radar scope.

It contains multiple images of 3 of our top sellers: Mu Opioid Receptor, VR1 C-terminus (TRPV1) and VR1 N-Terminus (TRPV1).

Shao-Rui Chen and Hui-Lin Pan. Loss of TRPV1-expressing Sensory Neurons Reduces Spinal : Opioid Receptors but Paradoxically Potentiates Opioid Analgesia. J Neurophysiol (February 8, 2006). doi:10.1152/jn.01343.2005

Amped Up ORL-1

We have been collaborating with a customer to amp up our ORL-1. Here's an example of a resulting IHC images.

Image: ORL 1 staining in rat brain (singulate cortex). ORL1 Detection was done using anti-rabbit Cy3 conjugated antibodies (red color). DAPI was used to counterstain cell nuclei (blue color). Working dilution is 1:40-1:100.