Results

for the C7 segment Ganetespib purchase in each experiment are shown in Table 2, while those for the L4 segment are in Table 3. In each case, the analysis was carried out on 10 randomly selected 60 μm thick Vibratome sections, and retrogradely labelled cells on the right side (contralateral to the injection site) were counted. Examples of retrogradely labelled lamina I neurons are shown in Fig. 4. In experiments 1–3, in which Fluorogold was injected into the PAG and CTb into the LPb, between 40 and 60 cells that contained one or both tracers were identified in lamina I on the contralateral side in the 10 sections selected from C7. The great majority of these (85–100%) were labelled with CTb, while between 50% and 77% contained Fluorogold (Table 2). In experiments 4–6 (Fluorogold injected into LPb, CTb into CVLM) the numbers of labelled lamina I cells in the 10 sections from C7 ranged from 54 to 79. Virtually all of these (98–100%) were labelled with Fluorogold, while between 83% and 89% were CTb-positive (Table 2). In experiments 7–10 (Fluorogold injected into LPb, CTb into dorsal medulla) AG-014699 solubility dmso 40–60 labelled lamina I cells were present in the 10 sections from C7. Most of these (97–100%) were labelled with Fluorogold and 15–33% were labelled with CTb. In the L4 segment from these experiments between 70 and 113 lamina I neurons were labelled in the 10 selected

sections. Most of these (96–98%) contained Fluorogold, while 22–27% were labelled with CTb. The main findings Dimethyl sulfoxide of this study were: (1) that in the C7 segment the great majority of lamina I neurons retrogradely labelled from PAG or CVLM were labelled from LPb, as we have previously reported for the mid-lumbar cord (Spike et al., 2003); and (2) that in both C7 and L4 segments most of the cells in this lamina that were labelled following injection of tracer into the dorsal medulla were also labelled from LPb. Although it is possible that we underestimated the numbers of retrogradely labelled cells in these experiments due to lack of sensitivity for the detection of one or both tracers, we feel

that this is unlikely for two reasons. Firstly, there was a clear distinction between cells that were positive and negative for each of the tracers, which suggests that none of the retrogradely labelled cells contained such low levels of tracer that these were close to the limits of detection. Secondly Al Ghamdi et al. (2009) have recently shown that following injection of CTb into the CVLM and Fluorogold into the LPb, immunostaining for the two tracers with a method identical to that used in the present study resulted in the detection of retrograde label in 99% of the large and medium-sized lamina I neurons (those with soma areas > 200 μm2 when viewed in the horizontal plane) that expressed the neurokinin 1 receptor, which is found on ∼ 80% of projection neurons in this lamina (Todd, 2002).