As for the ZW-Per crosses, the MGE transplants were also among the spinal neurons that had taken up the WGA (arrow, Figure 4K). We estimate that 32.5% ±
3.8% of MGE cells contained the WGA tracer. We also observed WGA+, but not GFP+, neurons that were enveloped by terminals that derive from the MGE cells (arrowhead, Figure 4L). These studies demonstrate that myelinated afferents, the great majority of which respond to innocuous peripheral stimulation, make connections with transplanted MGE cells and that MGE cells also target circuits engaged by primary afferents. If the connections established between DRG neurons and MGE grafts are, in fact, functional, then PD0332991 mouse a peripheral stimulus should “activate” the MGE transplants. Because the transneuronal WGA studies revealed a myelinated afferent fiber connection with MGE transplants, we asked whether innocuous peripheral inputs could activate grafted neurons. One month after transplantation, MGE-transplanted mice walked on a rotarod for 90 min, a condition that predominantly engages low threshold mechanoreceptive (myelinated)
afferents (Neumann et al., 2008; Figure S3). Consistent with the results from the tracing studies, we found that this nonnoxious LBH589 molecular weight peripheral input induced expression of Fos in 18.6% ± 6.3% MGE cells (Figures 5A–5C). The majority of Fos+ MGE cells predominated in deeper laminae (III-V) and around the central canal, a region that receives input from myelinated muscle spindle and joint afferents. We next asked whether a noxious chemical stimulus (hindpaw injection of 1% formalin), which engages both unmyelinated and myelinated axons (Bráz and Basbaum, 2010 and Shields et al., 2010), can activate the MGE cells. Figure 5B illustrates that the injection of formalin
evoked Fos in both host and GFP+ transplanted neurons in the dorsal horn, ipsilateral to the noxious stimulus (1 month posttransplantation; Figures 5D–5I and S3). In the superficial dorsal horn, where Fos+ neurons predominate, we found that 34.6% ± 13.4% of MGE neurons expressed Fos. On the other hand, injection Org 27569 of formalin in the hindpaw of 1 week-transplanted mice, despite inducing significant Fos in host neurons, did not evoke Fos in the transplanted neurons (Figures 5J–5L). In fact, by following the grafted cells from 1 to 4 weeks after transplantation, we conclude that it takes at least 2 weeks for the MGE-derived cells to respond to a peripheral noxious input, which coincides with the time when they acquire a neuronal phenotype. Taken together, these functional assays indicate that 1 month after transplantation, presumptive GABAergic MGE neurons are “activated” by nonnoxious, as well as noxious, inputs and confirm the existence of functional connections between primary afferent neurons and transplants. We next asked whether grafted cells made connections with host spinal cord neurons.