We used behavioral and imaging methods to study the role of Pirt, a recently identified regulator of TRPV1 
, in itch sensation. Large reductions in the response to histamine and HTMT in mutant mice show a clear role for Pirt in histaminergic itch. Nonhistaminergic itch as induced by CQ, α-Me-5HT, and ET-1 is also markedly decreased. Unexpectedly, although we observed a cellular change in the response to SLIGRL, the behavioral deficits in the KO were not significant. This may be explained by the high variability of behavioral responses obtained with this pruritogen.
Notably, these results allude to a mechanism that is independent of TRPV1, which does not participate in itch mediated by several compounds that produce scratching deficits in Pirt KO mice. This opens up a number of possibilities as to how Pirt functions mechanistically in itch sensation. Other TRP channels such as TRPV3 and TRPV4 have been implicated in itch 
and Pirt may interact with these as it does with TRPV1. Beyond the TRP family, other ion channels found in DRG neurons may contribute to itch sensation, and furthermore, PIP2
has been linked to ion channel function in many contexts 
. This provides a possible explanation if Pirt does not directly modify channel activity (see below). PIP2
and phosphoinositide signaling as a whole are also involved in other means of ion channel regulation such as membrane targeting and vesicle trafficking 
. A recent study 
suggests direct binding of TRPV1 to PIP2
may not be Pirt-dependent. However, the lack of a Pirt effect on TRPV1 is likely due to the concentration of capsaicin used in the study, i.e. 0.8–1.0 µM. We found a positive effect of Pirt only when a higher capsaicin concentration, i.e. 5 µM, was used to activate TRPV1 
. Moreover, as the deficits in Pirt−/−
mice are not limited to TRPV1-dependent forms of itch, we do not expect PIP2
modulation of TRPV1 by Pirt to completely account for the various Pirt−/−
Considering many compounds are known to exert their pruritogenic effects by acting as ligands for GPCRs, including endothelin receptors, Mrgprs, and the histamine H1 receptor, Pirt may modulate some aspect(s) of the GPCR signaling cascade. The extensive network of the GPCR pathway presents various opportunities for regulation. For example, the enzyme phospholipase C β3 (PLCβ3) has been shown to be critical for several varieties of itch and likely acts downstream of particular GPCRs that serve as itch receptors 
. Thus, one possible mechanism is that Pirt modulates hydrolysis of PIP2
by PLCβ3. A recent study 
has shown that chronic changes in PIP2
levels affect TRPV1 and TRPA1 activity in a concentration- and cell type-dependent manner, suggesting other sensory neuron factors like Pirt are important for PIP2
-mediated TRP regulation. Specific G proteins, including both α and βγ subunits, may also have distinct roles in itch transduction 
Another study 
involving our laboratory found that TRPA1 is required for itch induced by CQ but not histamine. While seemingly contradictory, these results do not preclude roles for both Pirt and TRPA1 in detecting CQ itch. Pirt does not contribute to direct activation of TRPA1 by mustard oil or formalin, but because CQ generates itch by first directly activating MrgprA3 followed by downstream TRPA1 activation, the overall process can accommodate functions for both Pirt and TRPA1. Although it does not directly modify TRPA1 activity, Pirt may modulate PIP2
and/or other aspects of the G protein signaling pathway that constitutes CQ itch transduction.
neurons show diminished Ca2+
responses to several compounds we tested. While these results are consistent with impaired signaling of these pruritogens, it remains to be demonstrated whether the deficits are directly linked to itch. For example, histamine elicits responses in a relatively broad population in DRG that is probably not limited to the neurons required for detecting itch. Work from our laboratory and others 
has indicated the existence of itch-sensing neurons in the DRG that are a subset of nociceptive neurons and would produce itch sensation upon activation. It would be worthwhile to examine if the reduction in histamine responsiveness found in Pirt−/−
neurons is restricted to itch-sensing neurons, e.g. the MrgprA3-expressing population described recently 
. This can also be addressed by asking whether the targeted removal of Pirt from these purported itch-sensing DRG neurons produces cellular and behavioral deficits akin to those in the Pirt global KO mouse.
Our results provide further evidence that multiple molecular (TRPV1-dependent and -independent) and cellular (direct and indirect activation of sensory fibers) pathways are involved in itch signaling and demonstrate that Pirt plays a role in many of them. We have shown the presence of Pirt is indispensable for proper itch sensation. How it fits into this process mechanistically remains to be determined and may elucidate the different signaling pathways required to produce all varieties of itch. As Pirt is found in over 80% of DRG neurons 
, it is probable that it serves functions beyond thermal pain and now itch. Its mechanism of action in these contexts may shed light on other somatosensory processes to which Pirt contributes.
At the same time, the specific expression pattern of Pirt, which is limited to sensory neurons, indicates its potential use as a target for itch therapeutics. There are a number of mouse models to investigate various aspects of medical itch 
that can be utilized to see if Pirt plays a role in any facet of itch in its various pathological manifestations such as inflammatory, dermatitis, etc. Our results establish a critical role for Pirt in sensing itch and generate new questions to advance knowledge of itch from both basic science and clinical perspectives.