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 

δ- and μ-opioid receptors co-expression and Nociceptive Pain

Dr. Tomas Hokfelt and his team at Karolinska Institute recently published use of our Opioid Receptor Antibodies and Substance P Antibody.They show the interplay of DOR and MOR in modulation of nociceptive afferent transmission and opioid analgesia.

Hai-Bo Wanga, Bo Zhaoa, Yan-Qing Zhonga, Kai-Cheng Li, Zi-Yan Li, Qiong Wang, Yin-Jing Lua, Zhen-Ning Zhang, Shao-Qiu He, Han-Cheng Zheng, Sheng-Xi Wu, Tomas G. M. Hökfelt, Lan Baob, and Xu Zhanga. Coexpression of δ- and μ-opioid receptors in nociceptive sensory neurons. PNAS July 20, 2010 vol. 107 no. 29 13117-13122.

Immunostaining. Adult rats, mice, and Oprd1 exon 1-deleted mice were fixed. Cryostat sections of L4 and L5 DRGs and spinal cord segments were processed for immunofluorescence staining (13) with Rb anti-DOR13–17 (1:2,000–1:60,000; DiaSorin and 1:4,000–1:60,000; Neuromics), Rb anti-DOR12–18 (1:30,000–1:120,000; Alomone), Rb anti-DOR1358–372 (1:1,000–1:2,000; Lifespan Biosciences), Rb anti-MOR (1:1,000; Neuromics); guinea pig anti-SP (1:500; Neuromics), and mouse anti-CGRP (1:1,000; Biogenesis) antibodies. IB4-labeling was carried out with fluorescein-labeled GSL I-IB4 (1:200). The Myc-DOR1–transfected HEK293 cells and neurons were fixed and processed with mouse anti-Myc antibodies (1:500; DSHB). Nuclear DAPI staining was used to indicate HEK293 cells in control experiments.

Images: Distinct distribution patterns of DORs in subsets of DRG neurons of mice. Immunostaining with antibodies against DOR13–17 [A: 1:30,000, antibody 1 (ab #1); DiaSorin and C: antibody 2 (ab #2); Neuromics] shows DORs in small DRG neurons and afferent fibers in spinal laminae I–II. This immunostaining pattern is abolished by the antiserum preabsorption or the deletion of Oprd1 exon 1. Reduction in immunostaining is quantitatively assayed by determining the percentage of positive DRG neurons (B; n = 6) and fluorescence intensity (Ifluo.) in the laminae I–II (D; n = 5). **P < 0.01; ***P < 0.001. (Scale bars: A and C, 40 μm.). DOR labeling (anti-DOR13–17, 1:30,000; DiaSorin) associated with vesicles in peptidergic small DRG neurons (E and F) is absent in Oprd1 exon 1-deleted mice (G). Colocalization of DORs and neuropeptides is shown by correlated peaks of Ifluo. measured along lines. (Scale bar: 8 μm.) (H) Immunostaining with antibodies against DOR12–18 (1:60,000; Alomone) shows the presence of DORs on the cell surface of large DRG neurons of mice. (Scale bar: 25 μm.) This staining pattern is abolished by preabsorption and is absent in Oprd1 exon 1-deleted mice. (Scale bar: 80 μm.) (I) Triple-immunostaining shows that DOR+ large DRG neurons contain neither SP nor CGRP. (Scale bar: 80 μm.)

Immunoblotting.The samples were processed for SDS/PAGE, transferred, probed with Rb antibodies against MOR (1:500; Neuromics), phospho-DOR1 (1:1,000; Neuromics), phospho-MOR (1:1,000; Neuromics), Myc (1:500; DSHB), Flag (1:1,000; Sigma), or actin (1:50,000; Chemicon) and visualized with enhanced chemiluminescence (19).

Featured and Related Reagents: Mu Opioid Receptor-Rabbit Mu Opioid Receptor-Guinea Pig Delta Opioid Receptor 3-17 Delta Opioid Receptor 358-372 Delta Opioid Receptor 358-372 Kappa Opioid Receptor phospho-Mu Opioid Receptor (Ser375) MOR-1C  ORL 1-Pure

ORL1-Whole Serum 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

Staining Neuron-Glial Cultures-Related Markers

I have been receiving a growing number of requests for best techniques related to staining cultures of primary neurons and glia. I wanted to share this short, step by step protocol.

These requests are often catalyzed by a search of our growing Neuron/Glial Markers catalog. The objective being to find the right markers for a particular assay. I wanted to share examples of the potency of several Neurofilament or NF markers for labeling neurons:

1. Neurofilament NF-L-Mouse Monoclonal Antibody (Clone: DA2) and Neurofilament alpha-internexin/NF66-Whole Serum-Rabbit Antibody

Images: Cells grown from adult rat brainLarge cell in middle is stained with mouse monoclonal to NF-L clone DA2 (green). Another type of neuronal lineage cell was stained with rabbit polyclonal to alpha-internexin (red). These cells were mitotic but had several characteristics of neurons. Rat spinal cord homogenate showing the major intermediate filament proteins of the nervous system (lane 1). The remaining lanes show blots of this material stainted with various antibodies including NF-L. Protocols on data-sheet.

2. Neurofilament NF-H, phosphylated-Mouse Monoclonal and Neurofilament NF-L-Purified Chicken Polyclonal.

Image: View of mixed neuron/glial cultures stained with chicken polyclonal NF-L (red) and phosphorylated NF-H The NF-L protein is assembled into neurofilaments which are found throughout the axons, dendrites and perikarya of these cells. In contrast the phosphorylated NF-H has a much rmore restricted expression pattern, being found only in developed axonal neurofilaments. Since both proteins are found in neurofilaments, the red and green patterns overlap, so that neurofilaments containing NF-L and phosphorylated NF-H appear yellowish. In contrast neurofilaments containing only NF-L appear red. Protocol on datasheet.

Neurofilament Markers

TRPV1 & P2X3-Daily Double

Low pH and Chronic Muscle Pain

Our Pain and Inflammation Antibodies are routinely used for chronic pain. I would like to highlight a recent publication referencing use of our  Guinea Pig TRPV1 and Pig 2X3 Antibodies and Blocking Peptides:

James P. Lund,Somayeh Sadeghi,Tuija Athanassiadis, Nadia Caram Salas, François Auclair, Benoît Thivierge, Isabel Arsenault, Pierre Rompré, Karl-Gunnar Westberg, and Arlette Kolta.Assessment of the Potential Role of Muscle Spindle Mechanoreceptor Afferents in Chronic Muscle Pain in the Rat Masseter Muscle. PLoS One. 2010; 5(6): e11131. Published online 2010 June 15 doi:10.1371/journal.pone.0011131.

Conclusions/Significance

Low pH leads to changes in several electrical properties of MSA, including initiation of ectopic action potentials which could propagate centrally but could also invade the peripheral endings causing glutamate release and activation of nearby nociceptors within the spindle capsule. This peripheral drive could contribute both to the transition to, and maintenance of, persistent muscle pain as seen in some “functional” pain syndromes.

Images: Photomicrographs of trigeminal ganglion neurons stained with TRPV1 and P2X3.

Related Reagents:

Pain and Inflammation Research Antibodies

Neurotransmission

Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes