Figure 2. Activation of descending LC:SC but not ascending LC^:PFC is anti-nociceptive.

(A) Retrograde transduction strategy with canine adenoviral vectors (CAV2) to target noradrenergic LC neurons with projections to the spinal cord (LC^:SC) or prefrontal cortex (LC^:PFC). Similar numbers of LC neurons were transduced by injections in lumbar spinal cord (L3-4, 380) and prefrontal cortex (361). In both cases, >99% of neurons were DBH+. (scale bar 100 µm). (B) The Hargreaves’ test (radiant heat) was used to measure hind- paw withdrawal latency. PSEM308 activation of LC^:SC but not LC^:PFC caused a robust anti-nociception (increase in withdrawal latency). The LC^:SC anti-nociceptive effect was only seen in the ipsilateral hind paw (same side as spinal CAV2 injection). (C) Representative images of double immunofluorescence showing DBH-positive fibres in the spinal cord that were anterogradely filled with EGFP after transduction of LC^:SC neurons. Quantification of the percentage of DBH+ fibres that were EGFP+ showed a three-fold higher density of EGFP-labelled fibres ipsilateral to vector injection (Mann-Whitney test, *p<0.05) which is consistent with the lateralized analgesic effect seen in (B). (D) Representative images and comparison of the percentage of EGFP labelled fibres in cortical areas and the spinal cord after LC^:SC or LC^:PFC transduction (N = 3 in each group). Data analysed with two-way repeated measures ANOVA with Bonferroni’s post hoc tests (**p<0.01, ***p<0.001, ****p<0.0001).