Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons

Molecular Pain

Bradley K Taylor¹  Bret N Smith¹  Camille B Blake¹  Suzanne Doolen¹ 

[1] Department of Physiology, School of Medicine, University of Kentucky Medical Center, Lexington, KY, 40536, USA

关键词: Calcium imaging;    Central sensitization;    AMPA receptor;    Pain;   

Others  :  863800
DOI  :  10.1186/1744-8069-8-56

 received in 2012-05-30, accepted in 2012-07-05,  发布年份 2012

【 摘 要 】

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 K_d = 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.

【 授权许可】

   
2012 Doolen et al.; licensee BioMed Central Ltd.

【 图 表 】

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