Referee 3's Review:

**********
N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.
**********

The authors of this manuscript have tested the hypothesis that mTOR and its downstream effectors (S6kinase and 4E-BP) are localized to sensory axons in the skin and tested the effects of the mTOR inhibitor rapamycin on pain thresholds. Their data suggest that sustained activity in the mTOR pathway is necessary for the maintenance of A- fiber nociceptive thresholds and that mTOR is an important mediator of secondary hyperalgesia. The experiments are well thought out and their reasoning is clearly explained. The authors have used a number of techniques to reach their conclusions. Overall this is an excellent manuscript with high quality, well controlled experiments.
I have, however, several concerns that the authors should address:

1) The phospho 4E-BP antibody used here recognizes both 4E-BP1 and 4E- BP2. 4E-BP2 is the primary neuronal 4E-BP (see, for instance Banko et al., (2007) Neurobiology of Learning and Memory). The authors routinely refer to phospho 4E-BP immunoreactivity as 4E-BP1 in their manuscript. I do not think such an exclusion of 4E-BP2 is warranted.
2) The authors have localized mTOR, phospho-mTOR and downstream effectors to sensory afferents that terminate in the dermis. There was little immunoreactivity in the epidermis. The authors seem to exclude the possibility that C-fibers contain mTOR and its effector molecules. Are they sure that: 1) mTOR and its effectors are not present in C-fibers of the viscera, 2) that mTOR and its effectors are not present in C-fibers but the proteins just do not localize to endings within the epidermis and 3) that protein levels are not below detection limits and 4) that other modes of translation, such as IRES-dependent translation, are not occurring in C-fibers? Some language in the discussion seems very certain that this pathway does not function in C-fibers; however, the authors did record some changes in C-fiber sensitivity with rapamycin. This needs some clarification between the strength of the positive findings of the manuscript and the lack of exhaustive efforts to support what appear to be negative findings.
3) It is not particularly useful to use Molar concentrations for injections into the paw. I believe the injections of rapamycin (250uM) would be equivalent to 12.5 ug (roughly). This would be a more informative dose for the reader.
4) Several important manuscripts are not cited. For instance, in the introduction the authors mention the presence of FMRP in sensory axons. This work has now been expanded to show that FMRP-KO mice have deficits in mGluR-mediated peripheral nociception (Price et al., (2007) J Neuroscience). This paper also shows that a peripheral injection of rapamycin (nearly the same dose -- 10ug) inhibits the second phase of the formalin test and that SNI-induced mechanical hyperalgesia is strongly reduced in FMRP-KO mice. These findings are highly relevant to the present manuscript. Additionally, the authors cite work of the Kandel group on local translation in aplysia. A series of papers from the group of Terry Walters (Weragoda and Walters (2007) J Physiology and Weragoda, Ferrer and Walters (2004) J Neuroscience) are also very relevant to the present discussion. These papers show that the mTOR pathway plays an important role in axonal hyperexcitability (in aplysia) after axonal injury. These citations should be included.
5) The sentence on page 6 line 16 about mTOR-mediated translation in the absence of an external stimuli is directly contradicted by some areas of the discussion where possible roles of BDNF and insulin signaling are discussed. This passage should be revised.
6) The last two sentences of page 14 are very hard to understand. I believe the authors are referring to protein degradation or protein half-life and not "loss" of proteins from sensory axons. How is replenishing occurring if mTOR is inhibited? I am not sure that I understand what the authors wish to convey here.
In conclusion, the authors of this manuscript have provided a highly detailed and exciting body of work that represents an important advance in our understanding of peripheral nociceptive sensitivity and hyperalgesia.