In this study we report that Artn, a growth factor that is elevated in inflamed tissue, increases nAChR subunit transcription in a subunit specific manner. This increase correlated with functional changes in nAChR response properties in DRG neurons and contributed to Artn-induced thermal hypersensitivity. Changes in nAChR subunit expression and nicotine-evoked current primarily affected the peptidergic, GFRα3-positive DRG neuron subpopulation, which is consistent with findings of Flores and colleagues , who showed using in situ hybridization that α3 and β4 are co-expressed in medium-diameter neurons. This observation is also consistent with Spies et al.  who reported small- to medium-sized neurons express the α3 nAChR subunit. In addition, since ~95% of GFRα3 neurons are TRPV1-positive, the overlap in GFRα3/nAChRα3 labeling is consistent with work of Dussor et al. , who showed overlap of TRPV1 and nAChRα3 mRNAs, and studies of Roberts et al., and Rau et al., who used patch clamp analysis of isolated neurons to show functional overlap of nAChR and TRPV1 activity [15, 16].
Functional analysis of cutaneous DRG neurons from wildtype and ART-OE mice revealed three populations of neurons based on their response to nicotine: those with no current (at least to nicotine < 300 μM), and those exhibiting either a fast or slow current. The fast current had biophysical properties consistent with those of α7-subunit containing nAChRs while properties of the slow current were comparable to α3/β4-subunit containing nAChRs . With the exception of the observation that fast currents were blocked by two TRPA1 antagonists, the pharmacological sensitivity of the fast and slow currents were consistent with those of α7- and α3/β4-subunit containing nAChRs, respectively: fast currents were blocked by the α7-selective antagonist MLA and slow currents were activated by the α3/β4 agonist cytisine. Consistent with the changes in subunit expression, the percentage of neurons from ART-OE and WT mice with a fast current was equivalent, whereas slow currents were significantly more prevalent among neurons from ART-OE mice (9 of 89 neurons) compared to WT mice (1 of 74 neurons).
The distribution of fast and slow currents was also consistent with their expression in neurons that express GFRα3: both current types were found only in IB4-negative neurons. This observation raises the possibility that changes in nAChR gene expression are directly regulated by Artn activation of GFRα3 signaling pathways. While another possibility is that this shift reflects increased survival of neurons exhibiting slow current in ART-OE ganglia, our preliminary data from rat suggesting that there is an increase in the proportion of neurons with slow current in the presence of persistent inflammation argues against this possibility .
Changes in nAChR subunit expression also were measured in ganglia of Nrtn-OE mice. Although there was some overlap in the pattern of subunit expression, Nrtn caused a change in nAChRs distinct from that detected in ganglia of ART-OE mice. This suggests that signaling pathways activated downstream of each GFRα receptor can modulate changes in nAChR expression. Furthermore, there is evidence that other growth factors regulate nAChR subunit expression: neuregulin 1 signaling through the ErbB kinase receptor increases nAChRs in developing skeletal muscle  and increases α3 and β4 mRNA in pelvic ganglion neurons . In addition, cultured PC12 cells treated with NGF exhibit increased nicotinic current density and mRNAs that encode nAChRs α3, α5, α7 β2, β4 . With respect to NGF, it is of interest to note that most GFRα3-positive neurons express receptors for NGF, a well-characterized inflammatory mediator. This co-responsiveness is likely to have functional significance; in previous studies we showed a synergistic interaction between Artn and NGF, where a single injection of each factor produced a transient (24 h) heat hyperalgesia but coinjection of Artn and NGF produced hyperalgesia that lasted for more than 6 days . That both NGF and Artn can increase in inflamed tissue and both can modulate nAChR expression suggests the complex interplay between these factors that leads to long-term activation of the GFRα3-positive population could involve changes in cholinergic signaling.
For Artn, identifying which of the numerous GFR signaling cascades such as src, PKA, PLC and MAPKs  that could potentially influence nAChR gene transcription will require further study. Nevertheless, the fact that heterogeneity in nAChR subunit composition is largely responsible for heterogeneity in nAChR signaling suggests the subpopulation-specific expression of nAChRs could contribute to the unique functional properties exhibited by GFR- and trk-positive neuronal subtypes [10, 30, 41].
That mecamylamine and hexamethonium reduce thermal sensitivity in ART-OE mice and WT mice injected with Artn, suggest that nAChR activity contributes to thermal sensitivity. Preliminary data suggest ACh levels are elevated in CFA inflamed skin (at 3 d post CFA injection a 1.46-fold increase in ACh was measured in inflamed skin relative to skin of naïve mice (CFA 8.9 μg/ml vs. naive 4.7 μg/ml supernatant; n = 3 CFA mice and 2 naïve mice)). ACh is also elevated in human skin conditions such as atopic dermatitis . The increase in ACh raises the possibility that endogenous ACh could contribute to persistent depolarization of nociceptive afferents. Persistent depolarization would require the slowly inactivating α3β4-type channels increased by Artn and could account for the increased sensitivity to thermal stimuli . While the fast currents should also contribute to activation of nociceptive afferents, the persistent α3β4-type channels are also likely to contribute to the aversive responses elicited by ACh, nicotine and other cholinergic agonists applied to the skin, arterial, tongue, nasal, or ocular surfaces [21–24]. Consistent with the suggestion that nicotine can generate a persistent depolarizing drive in nociceptive afferents, in a rat skin-saphenous nerve preparation, nicotine not only induced a dose-dependent activation of C-fibers, but sensitized them to heat stimulation . Such a mechanism could also account for the well established link between smoking and the severity of chronic pain conditions, such as lower back pain , diabetic neuropathy [45, 46], fibromyalgia and pancreatitis . The presence of nAChRs on peptidergic afferents also suggests that these channels contribute to the regulation of the efferent function of these afferents, e.g., transdermal iontophoresis of ACh in humans causes flare by neuropeptide release which can be blocked by hexamethonium .
Previous studies of ART-OE mice have shown elevation of TRPV1 and TRPA1 mRNAs in GFRα3-neurons and behavioral sensitivity to their respective agonists, capsaicin and mustard oil. Because functional interactions between TRPV1, TRPA1 and nAChR channels are reported in DRG neurons [31, 49, 50], we explored a possible interaction between the increase in nAChR subunits and TRP gene expression using ART-OE x TRPV1−/−/TRPA1−/− hybrid mice. Results show that even in the absence of functional TRPV1 and TRPA1, Artn overexpression in skin increased transcription of mRNAs encoding α3 and β4 to levels equivalent to those in ART-OE mice. Therefore, the elevation in nAChRs is not a compensatory response to the increase in TRPV1 and TRPA1 in GFRα3 neurons and suggests a more direct role for Artn in nAChR regulation.
While we have focused on the role of peripheral nAChRs in the present study, changes in receptor density or function at central afferent terminals may also occur. In contrast to the pronociceptive role for nAChR signaling in peripheral terminals, the majority of studies using systemic or intrathecal delivery of nAChR agonists show spinal nAChR activation is antinociceptive [11, 51–53]. Antinociception may result from changes in neurotransmitter release at presynaptic terminals and/or activation of descending noradrenergic and serotonergic systems [54, 55]. Our behavioral and pharmacological data does not rule out the possibility of altered spinal presynaptic or descending signaling in response to long-term changes in Artn level in the periphery. Indeed, repeated injection of Artn was reported to block hyperalgesia and normalize neurochemical changes related to nerve injury in a rat model . In addition, Artn (neublastin) is currently in Phase 2 clinical trial for treatment of sciatica-related pain . In this preliminary study, some subjects reported a higher reduction in pain compared to placebo treated subjects. However, several subjects also reported mild to moderate adverse events that included headache, feeling hot, generalized pruritus and burning sensations. These events may result from the known effects of Artn on TRPV1 and TRPA1 expression and perhaps, as indicated by results of this study, on nAChR signaling as well.