Abstract
Mouse embryonic spinal cord neurons in culture exhibit spontaneous calcium oscillations from day in vitro (DIV) 6 through DIV 10. Such spontaneous activity in developing spinal cord contributes to maturation of synapses and development of pattern-generating circuits. Here we demonstrate that these calcium oscillations are regulated by κ opioid receptors (KORs). The κ opioid agonist dynorphin (Dyn)-A (1–13) suppressed calcium oscillations in a concentration-dependent manner, and both the nonselective opioid antagonist naloxone and the κ-selective blocker norbinaltorphimine eliminated this effect. The KOR-selective agonist (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) mimicked the effect of Dyn-A (1–13) on calcium oscillations. A κ-specific peptide antagonist, zyklophin, was also able to prevent the suppression of calcium oscillations caused by Dyn-A (1–13). These spontaneous calcium oscillations were blocked by 1 μM tetrodotoxin, indicating that they are action potential-dependent. Although the L-type voltage-gated calcium channel blocker nifedipine did not suppress calcium oscillations, the N-type calcium channel blocker ω-conotoxin inhibited this spontaneous response. Blockers of ionotropic glutamate receptors, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline and dizocilpine maleate (MK-801), also suppressed calcium oscillations, revealing a dependence on glutamate-mediated signaling. Finally, we have demonstrated expression of KORs in glutamatergic spinal neurons and localization in a presynaptic compartment, consistent with previous reports of KOR-mediated inhibition of glutamate release. The KOR-mediated inhibition of spontaneous calcium oscillations may therefore be a consequence of presynaptic inhibition of glutamate release.
Footnotes
This work was supported by National Institutes of Health National Institute on Drug Abuse [Grant R01-DA023924].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.071456.
-
ABBREVIATIONS:
- CNS
- central nervous system
- KOR
- κ opioid receptors
- DIV
- day(s) in vitro
- nor-BNI
- norbinaltorphimine
- DAMGO
- [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin
- DPDPE
- [d-Pen2,d-Pen5]-enkephalin
- NMDA
- N-methyl d-aspartate
- AM
- acetoxymethyl ester
- PSD
- postsynaptic density
- NBQX
- 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione
- MK-801
- (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d] cyclohepten-5,10-imine hydrogen (dizocilpine) maleate
- Dyn
- dynorphin
- U69593
- (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide
- TBST
- Tris-buffered saline-Tween 20
- PBS
- phosphate-buffered saline
- Cy-3
- cyanine 3
- TTX
- tetrodotoxin
- CI
- confidence interval.
- Received January 26, 2011.
- Accepted March 21, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|