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Role of M 2 , M 3 , and M 4 muscarinic receptor subtypes in the spinal cholinergic control of nociception revealed using siRNA in rats
Author(s) -
Cai YouQing,
Chen ShaoRui,
Han HeeDong,
Sood Anil K.,
LopezBerestein Gabriel,
Pan HuiLin
Publication year - 2009
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2009.06396.x
Subject(s) - muscarinic acetylcholine receptor , muscarine , nociception , spinal cord , dorsal root ganglion , chemistry , muscarinic acetylcholine receptor m2 , gene knockdown , receptor , muscarinic acetylcholine receptor m3 , pharmacology , small interfering rna , neuropathic pain , microbiology and biotechnology , neuroscience , medicine , biology , biochemistry , rna , gene
Muscarinic acetylcholine receptors (mAChRs) are involved in the control of nociception in the spinal cord. The M 2 , M 3 , and M 4 mAChR subtypes are present in the spinal dorsal horn. However, the role of the individual subtypes in the anti‐nociceptive effect produced by mAChR agonists is uncertain. Here, we determined the contribution of M 2 , M 3 , and M 4 subtypes to spinal muscarinic analgesia by using small‐interference RNA (siRNA) targeting specific mAChR subtypes in rats. The neuronal uptake and distribution of a chitosan‐siRNA conjugated fluorescent dye in the spinal cord and dorsal root ganglion were confirmed after intrathecal injection. The control and gene‐specific siRNA‐chitosan complexes were injected intrathecally for three consecutive days. Quantitative reverse‐transcription polymerase chain reaction analysis showed that treatment with siRNA targeting M 2 , M 3 , or M 4 subtype produced a large reduction in the corresponding mRNA levels in the dorsal root ganglion and dorsal spinal cord. Also, the protein levels of the mAChR subtypes in the spinal cord were significantly down‐regulated by siRNA treatment, as determined by the immunoprecipitation and receptor‐binding assay. Treatment with the M 2 ‐siRNA caused a large reduction in the inhibitory effect of muscarine on the nociceptive withdrawal threshold. Furthermore, M 4 knockdown at the spinal level significantly reduced the anti‐nociceptive effect of muscarine. However, the anti‐nociceptive effect of muscarine was not significantly changed by the M 3 ‐specific siRNA. Our study suggests that chitosan nanoparticles can be used for efficient delivery of siRNA into the neuronal tissues in vivo . Our findings also provide important functional evidence that M 2 and M 4 , but not M 3 , contribute to nociceptive regulation by mAChRs at the spinal level.