Presenting Author University of Minnesota Minneapolis, Minnesota
NMDA receptor (NMDAr) plays an important role in synaptic plasticity and one of the targets for chronic pain management is antagonism of the NMDA receptors (NMDAr). Agmatine is an endogenous aminoguanidine that preferentially antagonizes GluN2B-containing NMDArs. Our previous studies showed that the intrathecal administration of agmatine, an endogenous aminoguanidine that selectively antagonizes GluN2B-containing NMDArs, reverses the mechanical hypersensitivity without motor side effects. In this study, we demonstrated the spinal modulatory effect of agmatine and other NMDAr antagonists utilizing calcium imaging.
The aim of this study is to use a newly developed NMDAr-mediated calcium transient assay (Figure 1.) in ex vivo mouse spinal cord slices and examine the effect of agmatine and other NMDAr antagonists in this assay. We hypothesize that agmatine and other NMDAr antagonists can dose-dependently inhibit NMDAr-mediated calcium transient in mouse spinal cord dorsal horn. Female and male ICR mice (4-6 weeks) were perfused before spinal cord extraction and ex vivo spinal slices were incubated with the calcium indicator dye Fluo-4. Intracellular Ca2+ was visualized by single plane two-photon microscopy. Time-lapse of images were acquired and the peak amplitude of fluorescence intensity was analyzed by Student’s t-test.
We found that NMDAr-mediated calcium transients were elicited by an NMDA drug mixture containing NMDAr agonists (NMDA, glycine) and AMPA receptor antagonists (NBQX). 5mM agmatine incubation significantly attenuated the NMDAr-mediated calcium transients compared to the control. Increasing agmatine concentration dose-dependently attenuates NMDAr-mediated calcium transients. Other NMDAr antagonists including AP5 and ifenprodil, showed dose-dependent inhibition of NMDAr-mediated calcium transients, supporting that the newly developed calcium imaging assay is NMDAr specific. These data suggest that agmatine attenuates NMDAr-mediated calcium transients in the spinal cord dorsal horn, which is consistent with our previous research demonstrating that agmatine is an effective inhibitor of NMDAr in the spinal cord.
Support or Funding Information
These studies were supported by NIDA Grant R01DA035931 to C.A.F. as well as the College of Pharmacy of the University of Minnesota.
Figure 1. The fluorescent intensity of calcium indicator is measured from a single cell at mouse spinal cord dorsal horn. NMDAr agonists mixture application at time point 1 increases the fluorescent intensity, but after the incubation in 5mM agmatine, the response is attenuated at time point 2 despite adding the same NMDAr agonists mixture. 1mM glutamate application results in robust response at time point 3, ensuring the attenuated response at time point 2 is not due to the cellular viability.
