Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons
<p>Abstract</p> <p>Background</p> <p>Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca<sup>2+</sup> mobilization. We used Fura-2 AM bulk loading of mouse slices together with wide-field Ca<sup>2+</s...
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SAGE Publishing,
2012-07-01T00:00:00Z.
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Summary: | <p>Abstract</p> <p>Background</p> <p>Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca<sup>2+</sup> mobilization. We used Fura-2 AM bulk loading of mouse slices together with wide-field Ca<sup>2+</sup> imaging to measure glutamate-evoked increases in extracellular Ca<sup>2+</sup> to test the hypotheses that: 1. Exogenous application of glutamate causes Ca<sup>2+</sup> mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca<sup>2+</sup> mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca<sup>2+</sup> transients; and 4. The magnitude of glutamate-evoked Ca<sup>2+</sup> responses increases in the setting of peripheral neuropathic pain.</p> <p>Results</p> <p>Bath-applied glutamate robustly increased [Ca<sup>2+</sup>]<sub>i</sub> 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. Ca<sup>2+</sup> transients were glutamate concentration-dependent with a K<sub>d</sub> = 0.64 mM. Ca<sup>2+</sup> 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 [Ca<sup>2+</sup>]<sub>i</sub> also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca<sup>2+</sup> imaging, glutamate elicited a Ca<sup>2+</sup> response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca<sup>2+</sup>]<sub>i</sub>, 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 Ca<sup>2+</sup> signals.</p> <p>Conclusions</p> <p>Bulk-loading fura-2 AM into spinal cord slices is a successful means for determining glutamate-evoked Ca<sup>2+</sup> mobilization in naïve adult dorsal horn neurons. AMPA receptors mediate the majority of these responses. Peripheral neuropathic injury potentiates Ca<sup>2+</sup> signaling in dorsal horn.</p> |
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Item Description: | 10.1186/1744-8069-8-56 1744-8069 |