Immunohistochemistry of pERK

Analysis of the expression of pERK was used for two purposes: first to examine whether intruder stress would activate the serotonergic neurons in the dorsal raphé, median raphé, and rostral medullary raphé; and second to confirm that photo-illumination did in fact inactivate the serotonergic neurons. For both purposes, we examined expression of a cellular activation marker protein, the phosphorylated form of extracellular signal-regulated kinase (pERK), in the serotonergic neurons according to a previously published method (Inui et al., 2016). We selected pERK as it has a more rapid and narrow time window (<15 min) compared to other activation markers including c-Fos (≥30 min) – a commonly used activation marker (Antoine et al., 2014).

For the first purpose, we used Tph2-tTA; TetO-ArchT mice, without the optic fiber implantation, that were exposed to the intruder stress for 3 min. The control mouse in this case was exposed to an empty box for 3 min. Immediately after the intruder, or empty box, was removed, the experimental mouse was deeply anesthetized with urethane (2.0 g/kg, i.p.), and perfused with 0.01 M PBS followed by 4% paraformaldehyde in 0.01 M PBS (pH 7.4). Coronal sections, that included the dorsal raphé, median raphé, and the rostral medullary raphé were made and processed as described above (see section “Confirmation of ArchT (EGFP) Expression in Serotonergic Neurons”), with the exception being that a rabbit anti pERK antibody (1/400, Cell Signaling Technology 4370S) was used instead of an anti GFP antibody. The anti pERK antibody was visualized using biotinylated anti-rabbit IgG antibody (1/200, Jackson Immuno Research Laboratories Inc., 711-065-152) and streptavidin conjugated Alexa 568 (1/200, life technologies).

For the second purpose, we compared four groups of the optic fiber-implanted animals: (1) stressed but without photo-illumination, (2) stressed with photo-illumination, (3) no stress and no photo-illumination, and (4) no stress with photo-illumination. The optic fiber was implanted only into the rostral medullary raphé because pERK was found to have increased only in the medullary raphé, not in the dorsal or median raphé (see section “Results”). The intruder male mouse, confined to a small cage, was introduced to the cage of the resident mouse (male Tph2-tTA; TetO-ArchT bigenic mouse) when the resident’s locomotor activity was at a low, resting level but its eyes were open. The intruder was removed 3 min after introduction. Photo-illumination was given 30 s before intruder introduction and kept on for 180 s during intruder-exposure. Immediately after the intruder was removed, the experimental mouse was deeply anesthetized with urethane (2.0 g/kg, i.p.), and processed as described above. The number of pERK and Tph positive cells were counted in one section per animal – in which the track of the optical fiber was most strongly observed.