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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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SAGE Publishing
2012-07-01
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| Series: | Molecular Pain |
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| Online Access: | http://www.molecularpain.com/content/8/1/56 |
| _version_ | 1811279682965864448 |
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| author | Doolen Suzanne Blake Camille B Smith Bret N Taylor Bradley K |
| author_facet | Doolen Suzanne Blake Camille B Smith Bret N Taylor Bradley K |
| author_sort | Doolen Suzanne |
| collection | DOAJ |
| description | <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> |
| first_indexed | 2024-04-13T01:00:05Z |
| format | Article |
| id | doaj.art-a84c99e1a5b249c9a6dd5c2ca810e53d |
| institution | Directory Open Access Journal |
| issn | 1744-8069 |
| language | English |
| last_indexed | 2024-04-13T01:00:05Z |
| publishDate | 2012-07-01 |
| publisher | SAGE Publishing |
| record_format | Article |
| series | Molecular Pain |
| spelling | doaj.art-a84c99e1a5b249c9a6dd5c2ca810e53d2022-12-22T03:09:31ZengSAGE PublishingMolecular Pain1744-80692012-07-01815610.1186/1744-8069-8-56Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neuronsDoolen SuzanneBlake Camille BSmith Bret NTaylor Bradley K<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>http://www.molecularpain.com/content/8/1/56PainAMPA receptorCentral sensitizationCalcium imaging |
| spellingShingle | Doolen Suzanne Blake Camille B Smith Bret N Taylor Bradley K Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons Molecular Pain Pain AMPA receptor Central sensitization Calcium imaging |
| title | Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons |
| title_full | Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons |
| title_fullStr | Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons |
| title_full_unstemmed | Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons |
| title_short | Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons |
| title_sort | peripheral nerve injury increases glutamate evoked calcium mobilization in adult spinal cord neurons |
| topic | Pain AMPA receptor Central sensitization Calcium imaging |
| url | http://www.molecularpain.com/content/8/1/56 |
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