Peripheral Nerve Injury Increases Glutamate-Evoked Calcium Mobilization in Adult Spinal Cord Neurons

Abstract

BackgroundCentral 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.ResultsBath-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.ConclusionsBulk-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.

Constitutive μ-Opioid Receptor Activity Leads to Long-Term Endogenous Analgesia and Dependence

“…Using live-cell Fura-2 ratiometric analysis in adult spinal cord slices (25), we found that glutamate-evoked [Ca 2+ ] i in lamina II neurons was potentiated at 3 d after CFA and then resolved by day 21 (F 3,17 = 15, P < 0.0001; Fig. 2A); this coincides with the temporal onset and resolution of inflammatory hyperalgesia.…”

Neuroplasticity of ascending and descending pathways after somatosensory system injury: reviewing knowledge to identify neuropathic pain therapeutic targets

“…26,27 The aforementioned neurotransmitters interact with N-methyl-D-aspartate receptor (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), metabotropic receptors of glutamate (mGluR), neurokinin-1 receptor (NK1R) and purinergic receptors (P2X) of spinal nociceptive projection neurons [28][29][30][31][32][33][34][35] causing their depolarization and the generation of painful signals to be scattered throughout the nociceptive spinothalamic pathway. In turn, this chemical neurotransmission causes an influx of calcium ions in the spinal nociceptive neurons of second order, 36 a process that activates calcium-dependent intracellular cascades, inducing phosphorylation sensitization 37 and ionic channel and membrane receptor overexpression (Table 3). Moreover, the neurotransmitters and Voltage-gated ion channels (increase of their expression)…”

Endogenous Analgesia, Dependence, and Latent Pain Sensitization

“…pERK is typically observed following high-threshold (Aδ- or C-fiber) or noxious stimulation (Ji et al, 1999), however, during the maintenance phase of chronic pain (> 2 days post CFA) a low-threshold (Aβ-fiber) stimulation (Matsumoto et al, 2008) or light-touch (Gao and Ji, 2010a) can induce spinal pERK expression, suggestive of injury-induced central sensitization (Ji et al, 2003). We found that after pain resolution (21d), light-touch was unable to effectively activate superficial DH neurons unless LS was disinhibited by NTX (Corder et al, 2013); 2) Within spinal cord slices, we visualized intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in populations of DH neurons using Fura-2 labeling and wide-field fluorescence microscopy (Doolen et al, 2012). NTX administration to slices taken from post-hyperalgesia animals precipitated ↑ [Ca 2+ ] I , indicating that LS survives in spinal nociceptive neurons within our slice preparation, and that opioid receptor activity tonically suppresses it (Corder et al, 2013); 3) NTX produced cAMP superactivation in the DH of post-hyperalgesia mice (Corder et al, 2013).…”