The purpose of the present study was to test the prediction that the unique manifestation of chemotherapeutic-induced peripheral neuropathy (CIPN) would be reflected in a specific pattern of changes in the regulation of the intracellular Ca2+ concentration ([Ca2+]i) in subpopulations of cutaneous neurons. To test this prediction, we characterized the pattern of changes in mechanical nociceptive threshold associated with paclitaxel administration (2 mg/kg, iv, every other day for four days), as well as the impact of target of innervation and paclitaxel treatment on the regulation of [Ca2+]i in subpopulations of putative nociceptive and non-nociceptive neurons. Neurons innervating the glabrous and hairy skin of the hindpaw as well as the thigh were identified with retrograde tracers, and fura-2 was used to assess changes in [Ca2+]i. Paclitaxel was associated with a persistent decrease in mechanical nociceptive threshold in response to stimuli applied to the glabrous skin of the hindpaw, but not the hairy skin of the hindpaw or the thigh. However, in both putative nociceptive and non-nociceptive neurons, resting [Ca2+]i was significantly lower in neurons innervating the thigh after treatment. The magnitude of the depolarization-evoked Ca2+ transient was also lower in putative non-nociceptive thigh neurons. More interestingly, while paclitaxel had no detectable influence on either resting or depolarization-evoked Ca2+ transients in putative non-nociceptive neurons, in putative nociceptive neurons there was a subpopulation- specific decrease in the duration of the evoked Ca2+ transient that was largely restricted to neurons innervating the glabrous skin. These results suggest that peripheral nerve length alone, does not account for the selective distribution of CIPN symptoms. Rather, they suggest the symptoms of CIPN reflect an interaction between the toxic actions of the therapeutic and unique properties of the neurons deleteriously impacted.

Key pointsr There has been little if any systematic analysis of the Na + -Ca 2+ exchanger (NCX) in sensory neurons despite conflicting results in the literature regarding the extent to which it contributes to the regulation of intracellular Ca 2+ in these neurons.r While the differential distribution and/or biophysical properties of NCX isoforms may contribute to these conflicting results, only indirect evidence points to the consequences of NCX activity on afferent function.r NCX activity is restricted to a subpopulation of putative nociceptive neurons. r All three NCX isoforms are expressed in putative nociceptive afferents and appear to be differentially distributed along major neuron compartments: central axon, cell body and peripheral axon.r NCX 3 plays a dominant role in the regulation of the duration of the evoked Ca 2+ transient in the cell body, regulation of the action potential conduction velocity, and establishment of nociceptive threshold. Abstract The Na+ -Ca 2+ exchanger (NCX) appears to play an important role in the regulation of the high K + -evoked Ca 2+ transient in putative nociceptive dorsal root ganglion (DRG) neurons. The purpose of the present study was to (1) characterize the properties of NCX activity in subpopulations of DRG neurons, (2) identify the isoform(s) underlying NCX activity, and (3) begin to assess the function of the isoform(s) in vivo. In retrogradely labelled neurons from the glabrous skin of adult male Sprague-Dawley rats, NCX activity, as assessed with fura-2-based microfluorimetry, was only detected in putative nociceptive IB4+ neurons. There were two modes of NCX activity: one was evoked in response to relatively large and long lasting (ß325 nM for >12 s) increases in the concentration of intracellular Ca 2+ ([Ca 2+ ] i ), and a second was active at resting [Ca 2+ ] i > ß150 nM. There also were two modes of evoked activity: one that decayed relatively rapidly (<5 min) and a second that persisted (>10 min). Whereas mRNA encoding all three NCX isoforms (NCX1-3) was detected in putative nociceptive cutaneous neurons with single cell PCR, pharmacological analysis and small interfering RNA (siRNA) knockdown of each isoform in vivo suggested that NCX2 and 3 were responsible for NCX activity. Western blot analyses suggested that NCX isoforms were differentially distributed within sensory neurons. Functional assays of excitability, action potential propagation, and nociceptive behaviour suggest NCX activity has little influence on excitability per se, but instead influences axonal conduction velocity, resting membrane potential, and nociceptive threshold. Together these results indicate that the function of NCX in the regulation of [Ca 2+ ] i in putative nociceptive neurons may be unique relative to other cells in which these exchanger isoforms have been characterized and it has the potential to influence sensory neuron properties at multiple levels.

We have recently demonstrated that in a rat model of chemotherapy-induced peripheral neuropathy (CIPN), there is a significant decrease in the duration of the depolarization-evoked Ca2+ transient in small diameter, IB4+, and capsaicin-responsive neurons innervating the glabrous skin of the hindpaw. This change was specific to the transient duration and significantly smaller if not undetectable in neurons innervating the dorsal skin of the hindpaw or the skin of the inner thigh. Given the importance of mitochondria in intracellular Ca2+ regulation and the findings of chemotherapy-associated increase in mitotoxicity along the sensory neuron axons, we hypothesized that CIPN is due to both increases and decreases in mitochondria function, with changes manifest in distinct subpopulations of afferents. To begin to test this hypothesis, we used confocal microscopy and Ca2+ imaging in combination with pharmacological manipulations to study paclitaxel-induced changes in retrograde tracer-labeled neurons from naïve, vehicle-treated, and paclitaxel-treated rats. Paclitaxel treatment was not associated with decreased mitochondrial membrane potential or increased superoxide levels in the somata of putative nociceptive glabrous skin neurons. However, it was associated with significant increases in the relative contribution of mitochondria to the control of the evoked Ca2+ transient duration in putative nociceptive glabrous skin neurons, as well as increases in mitotracker and Tom20 staining which reflected an increase in mitochondrial volume. Furthermore, the relative contribution of the sarco-endoplasmic reticulum Ca2+ ATPase to the regulation of the duration of the depolarization evoked Ca2+ transient was also increased in this subpopulation of neurons from paclitaxel treated rats. Our results indicate that the paclitaxel-induced decrease in the duration of the evoked Ca2+ transient is due to both direct and indirect influences of mitochondria. It remains to be determined if and how these changes contribute to the manifestation of CIPN.