However, both peak firing rate and duration of the discharges in response to either capsaicin or histamine were significantly reduced in the cKO mice (Figures 6I–6N). Based on these findings, we hypothesized that the activity in the cKO mice of the lamina I projection neurons in response to algogenic and pruritogenic stimuli is not sufficient to drive the supraspinal sites that are required for the full expression of supraspinally mediated pain behaviors. To test this hypothesis, we next evaluated noxious stimulus-evoked Fos induction in a major supraspinal target of NK1 receptor-expressing lamina I projection neurons, namely the lateral parabrachial nucleus of the dorsolateral pons (Al-Khater

and Todd, 2009). Figure 7 illustrates that the number of formalin-induced Fos-immunoreactive neurons in the parabrachial nucleus Enzalutamide is indeed significantly reduced in the cKO compared to WT mice. Taken together, we conclude that loss of a population of excitatory

interneurons in the superficial dorsal horn underlies the reduced activity of supraspinal loci critical to the full expression of pain behaviors in response to noxious stimulation. Significant deficits in learning, memory, and emotional processes unquestionably contribute to the experience of pain or itch. Thus, even though our findings indicate that a deficit in the transmission of pain and itch messages from the spinal cord to the brain is the critical contributor to the behavioral phenotype in the cKO mice, it was important to address Cytoskeletal Signaling inhibitor a possible contribution of diminished higher cortical function in these mice. To this end, we assessed the mice in traditional tests of learning, memory and anxiety. Figure S6A shows that the TR4 cKO perform as well as PDK4 their WT littermates in the Morris Water maze. The TR4 cKO and WT mice also performed comparably in the open field test (Figure S6B); however, we did observe a small, but significant increase in the time spent in the

open arms of the zero maze (Figure S6C), which suggests that these mice are somewhat less anxious than the WT mice. It is unlikely, however, that this contributes significantly to the dramatically reduced pain and itch phenotypes observed in the cKO mice. Consistent with this conclusion, when we crossed the floxed TR4 mice with an αCaMKII-Cre line, which restricted TR4 deletion to the forebrain (Silva et al., 1992; Tsien et al., 1996), or when we used a Cre-line that selectively targets the hypothalamus (SF1Cre) (Dhillon et al., 2006), we found that pain and itch behaviors were completely normal. Furthermore, and not surprisingly, we found no anatomical reorganization at the spinal cord level (data not shown). On the other hand, when we used a Pax3-Cre line, which is heavily expressed in the dorsal horn spinal cord (Tsai et al.