Figure 3.

Pcdhγ siRNA knock-down in vitro alters axon outgrowth in naive but not injured adult sensory neurons. Confirmation of Pcdhγ mRNA knock-down in vitro by siRNA compared with Scrambled siRNA [A, mRNA Pcdhγ siRNA, *p = 0.04, Pcdhγ KD vs scrambled control siRNA (Scrambled siRNA); one-tailed Mann–Whitney n = 3/group (alternately tested as p = 0.02 two-tailed unpaired Student's t test)]. Representative images of uninjured Pcdhγ siRNA-treated neurons (top row) and Scrambled siRNA siRNA-treated neurons (bottom row) following a 72-h incubation (B). The neurons depicted have an outgrowth average close to the mean of the respective groups. Analysis of neurite outgrowth (C–F) in naive uninjured DRG sensory neurons indicated longer average neurite outgrowth per neuron in the Pcdhγ siRNA group and greater numbers of branch points [(C) length of neurite outgrowth, **p = 0.0083, Pcdhγ KD vs Scrambled siRNA; and (F) branchpoint numbers, **p = 0.0041, Pcdhγ KD vs Scrambled siRNA; two-tailed unpaired Student's t tests; n = 4/group]. Neurite outgrowth was also examined in DRG neurons following a prior axotomy injury (72 h before culture) and their contralateral controls examined 24 h after plating (G–I). In contrast to the naive uninjured neurons above, increased neurite outgrowth was not identified either ipsilateral or contralateral to injury in response to Pcdhγ siRNA (G; overall neurite outgrowth). However, axotomy injury induced neurons to grow longer branches (H; individual branch length) and in the case of Pcdhγ KD neurons, fewer branching points [I; (H) for longest branch length, p = 0.0009 ANOVA, post hoc, **p = 0.0007 Pcdhg KD Inj vs Pcdhg KD Con (contralateral),*p = 0.03 Scrambled siRNA Inj vs Scrambled siRNA Con, two-tailed unpaired Student's t tests; n = 4/group; (I) branching points, p = NS ANOVA; within culture two-way comparison *p = 0.036 Pcdhg KD Inj vs Pcdhg KD Con, two-tailed unpaired Student's t test; n = 4/group]. Sholl analysis indicated that intact contralateral DRG sensory neurons treated with Pcdhγ siRNA, but not neurons with a prior axotomy injury, had greater arborization. An example is shown of an Pcdhγ Con contralateral neuron with Sholl shells placed every 10 µm from the soma center (J). The number of shell crossings from 20 randomly selected cells from each experiment at each 10-µm shell and averaged following a 24-h incubation is shown (K). Areas under the curve are quantified in L, averaged per experiment [(L) areas p = 0.003 ANOVA, post hoc, **p = 0.0038 Pcdhg KD Con vs Pcdhg KD Inj, *p = 0.046 Scrambled siRNA Con vs Scrambled siRNA Inj, two-tailed unpaired Student's t test, n = 4 for Pcdhγ and Scrambled siRNA con (contralateral) and 5 for Pcdhγ and Scrambled siRNA Inj].