Because we could visualize sympathetic fibers in live tissue, using the transgenic mouse allowed us to efficiently identify and record from basket cells which are relatively rare. The present results validate the use of pTH-EGFP mice to investigate sympathetic-sensory coupling under pathological conditions. The rapid appearance of sprouting sympathetic fibers (by POD 3) after spinal nerve ligation that we observed in our whole mount preparations is consistent with results obtained with higher resolution, thin section methods in rats . The rapid increase of spontaneous activity (especially, bursting activity) in large diameter cells with high conduction velocities, and their relatively narrow size distribution, is also similar to that previously reported in rats [14, 26, 28, 29]. It is worth noting that cells with high conduction velocities but relatively small diameters have been described as belonging to the small group of nociceptive Aβ-fiber neurons in guinea pig neurons . Cells with similar properties were also observed to express Substance P only after chronic constriction injury in mice  though the N values were too small to be significant. The great reduction in sprouting at POD3 if the dorsal ramus is cut along with the ventral ramus is also similar to observations made in rat . It is of interest that cutting the grey ramus to the L5 DRG, which also reduces sprouting within the DRG, can greatly reduce SNL-induced pain behaviors in the rat [14, 32]. Co-localization of sympathetic sprouts with spontaneously active neurons was demonstrated using the rat sciatic nerve transection model to induce sprouting . These comparisons suggest that studies of sympathetic sprouting give similar results in mice and rats, enhancing the usefulness of transgenic mice in studying sympathetic sprouting.
Sympathetic basket formations around sensory neurons after peripheral nerve injury or inflammation, as first observed by McLachlan et al., initially generated much interest because they provided a possible anatomical basis for sympathetically enhanced or maintained pain states. However, their relevance to pain models has been questioned in part because of conflicting behavioral studies (see discussion in ref ), and in part because basket formations are relatively rare. The data presented in this study provide evidence for important functional roles for these sympathetic sprouts. We find that at a time early after injury (POD 3) sensory neurons with basket formations are much more excitable than neighboring neurons without basket formations; and, in particular, the basket cells have a very high incidence of spontaneous activity (71% showed spontaneous activity, primarily of the bursting pattern). In the data from DRG sections stained for TH, the average incidence of basket formations was 6.8% (Table 1). For comparison, the incidence of spontaneous activity in all DRG cells at this time point was 10% (Figure 3, after allowing for the fact that small cells were excluded from the electrophysiological data). This suggests that over half of the overall spontaneous activity may be attributed to basket cells, despite their overall rarity. In turn, spontaneous activity has been shown to play key roles in pain models, including roles in satellite glia activation, neurotrophin release, spinal cord sensitization, and establishment of pain behaviors[28, 34–37]. Additional evidence for functional relevance of sympathetic sprouting comes from our observations that cells that have nearby sympathetic fibers, but not a full-blown basket or ring formation, have an intermediate electrophysiological phenotype between that of basket cells and cells lacking any nearby sympathetic fibers. Though studies based on microscopy have tended to focus on and quantitate only the basket formations, the cells with the lower density nearby fibers are more numerous (11.5% vs. 6.8%; Table 1) and insofar as the presence of these nearby fibers is also associated with a more excitable phenotype, it seems that these structures may also contribute to a functional role for sympathetic sprouting.
The data presented here show an association between spontaneous activity in vitro and the presence of sympathetic fibers around the neuron. This suggests a highly localized interaction between the sympathetic fibers and nearby sensory neurons - neighboring cells lacking nearby fibers have different properties. This may reflect long-lasting, pro-excitatory interactions in vivo that are preserved in our acutely isolated in vitro preparation, and/or may indicate ongoing release of excitatory sympathetic transmitters in our in vitro preparation. Some evidence for the latter possibility was obtained in rat DRG, in medium but not large diameter cells . Do spontaneously active neurons attract sympathetic fibers, or do nearby sympathetic fibers enhance excitability and help drive spontaneous activity? Or could both processes occur in a positive-feedback cycle? This study provides evidence for interactions going in both directions. On the one hand, spontaneous activity is highest on day 1, before much sympathetic sprouting has occurred, suggesting spontaneous activity cannot be initiated by sympathetic inputs. However, we also found that simply cutting the small dorsal ramus in addition to the larger ventral ramus that is usually ligated in the SNL model, reduced sprouting on POD3 and also dramatically reduced spontaneous activity to levels seen in control animals. Insofar as spontaneous activity is initiated by axonal ligation, cutting both dorsal and ventral ramus would have been expected to increase spontaneous activity, not decrease it, though such an effect would have been quite small given the very small number of lumbar DRG neurons with projections in the dorsal ramus (less than 3% in rat ). This suggests that it is more likely the large reduction in sympathetic sprouting that explains the observation that cutting the dorsal ramus greatly reduces spontaneous activity.
The literature describes a number of possible mechanisms for the postulated two-way interaction between spontaneous activity and sympathetic sprouting. Spontaneously active cells may release neurotrophic factors that attract sympathetic fibers, either directly or via activation of surrounding satellite glia cells that then synthesize neurotrophic factors (reviewed in ). In the rat sciatic nerve transection model, blocking nerve activity early after injury reduces sympathetic sprouting, and increasing activity increases sprouting. Conversely, sensory neurons (and their associated satellite glia) also express receptors for sympathetic neurotransmitters norepinephrine and ATP that have excitatory effects on neurons, particularly after injury or inflammation [38–40], and sympathectomy reduces SNL-induced spontaneous activity recorded in rat dorsal root fibers .
The membrane capacitance distributions suggested that spontaneously active cells as well as basket cells were in the size range near the boundary between medium and large diameter cells. The very largest cells were not generally spontaneously active and did not have basket formations. However, in experiments in which conduction velocity was measured, basket cells always had conduction velocities above 9 m/s; though the higher velocities were not accurately measured due to the shortness of the stimulated dorsal root and the physiological temperature used. These velocities were clearly distinct from the much slower, accurately measured values seen in a subset of cells including small cells. The conduction velocities and size distribution suggested that spontaneously active cells and cells with basket formations might overlap with myelinated nociceptors. Though most nociceptors are small cells with nonmyelinated axons, a significant minority of cells conducting in the Aαβ range show nociceptive (high) thresholds and project into lamina I/II of the spinal cord [42, 43]. In rat it is estimated that 20% of Aαβ cells are myelinated nociceptors . Consistent with this possibility, we observed that several commonly used nociceptive markers - TrkA, CGRP, and substance P - were markedly enriched in basket cells compared to cells lacking nearby sympathetic fibers. TrkA in particular has been proposed as a marker for the myelinated nociceptors , and an association of TrkA with sprouting induced by NGF overexpression in mice has been reported . However, there are also numerous mechanisms by which Aαβ neurons that are not normally nociceptive can contribute to abnormal pain states . Further studies are needed to more precisely define the physiological roles and projections of the basket cells and spontaneously active cells.
We found that substance P was largely confined to small and medium diameter cells (Additional file1, Figure S1), while CGRP and TrkA had a somewhat broader distribution, similar to previous studies in both rats [46–49] and mice [50, 51]. Some studies, primarily in rat, have shown upregulation of substance P and/or CGRP specifically in medium or large diameter neurons in various pain models including SNL [50–54]. Our immunohistochemical data did not provide clear evidence for this phenomenon, possibly because we studied a much earlier time point than most of the above studies or because the time course may differ in mice. Comparison of cells with upregulated substance P or CGRP in previous studies, with the basket cells described in this study, is confounded by the fact that the basket cells described here (especially the spontaneously active ones) would have been defined as having Aβ fibers in functional studies but probably would be classified as "medium" diameter cells in many microscopy studies; the categorization of cells as small, medium, or large diameter does not correlate very precisely with C, Aδ, and Aβ conduction velocities respectively . The present study was not designed to specifically address the question of selective upregulation of substance P or CGRP in medium and large diameter cells, but does show that these nociceptive markers are enriched in cells with nearby sprouting sympathetic fibers.