title: Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons.
abstract: BACKGROUND
  Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca2+ mobilization. We used Fura-2 AM bulk loading of mouse slices together with wide-field Ca2+ imaging to measure glutamate-evoked increases in extracellular Ca2+ to test the hypotheses that: 1. Exogenous application of glutamate causes Ca2+ mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca2+ mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca2+ transients; and 4. The magnitude of glutamate-evoked Ca2+ responses increases in the setting of peripheral neuropathic pain.
  
  
  RESULTS
  Bath-applied glutamate robustly increased [Ca2+]i in 14.4 ± 2.6 cells per dorsal horn within a 440 x 330 um field-of-view, with an average time-to-peak of 27 s and decay of 112 s. Repeated application produced sequential responses of similar magnitude, indicating the absence of sensitization, desensitization or tachyphylaxis. Ca2+ transients were glutamate concentration-dependent with a Kd = 0.64 mM. Ca2+ responses predominantly occurred on neurons since: 1) Over 95% of glutamate-responsive cells did not label with the astrocyte marker, SR-101; 2) 62% of fura-2 AM loaded cells exhibited spontaneous action potentials; 3) 75% of cells that responded to locally-applied glutamate with a rise in [Ca2+]i also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca2+ imaging, glutamate elicited a Ca2+ response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca2+]i, while NMDA (AP-5), kainate (UBP-301) and class I mGluRs (AIDA) did not. Compared to sham controls, peripheral nerve injury significantly decreased mechanical paw withdrawal threshold and increased glutamate-evoked Ca2+ signals.
  
  
  CONCLUSIONS
  Bulk-loading fura-2 AM into spinal cord slices is a successful means for determining glutamate-evoked Ca2+ mobilization in naïve adult dorsal horn neurons. AMPA receptors mediate the majority of these responses. Peripheral neuropathic injury potentiates Ca2+ signaling in dorsal horn.
authors: Department of Physiology, School of Medicine, University of Kentucky Medical Center, Lexington, KY 40536, USA.
authors: Suzanne
authors: S
authors: Doolen
authors: Camille B
authors: CB
authors: Blake
authors: Bret N
authors: BN
authors: Smith
authors: Bradley K
authors: BK
authors: Taylor
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000375:Aging / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D059348:Peripheral Nerve Injuries / Q000378:metabolism* / Q000473:pathology* / Q000503:physiopathology; D020671:Posterior Horn Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D017470:Receptors, Glutamate / Q000378:metabolism; D013116:Spinal Cord / Q000473:pathology* / Q000503:physiopathology
chemical_list: D018691:Excitatory Amino Acid Antagonists; D005971:Glutamates; D017470:Receptors, Glutamate; D002118:Calcium