(A) Surgical and behavioral timeline for animals that underwent optogenetic stimulation in the OFC or mPFC. Examples of testing and illumination protocols on days 3 and 4 are shown, where yellow indicates continuous photostimulation with 620-nm light.
(B) In slice preparation from PV-Cre NpHR+/− mice, we demonstrate optogenetic suppression of stimulus current-induced spiking in a NpHR+/−-expressing PV interneuron. For slice experiments, a 545 ± band-pass excitation filter on white light emitting diode (LED) light was used. The light power at the plane of focus was 28.2 μW/mm2. A more in-depth analysis of the effects of optogenetic stimulation is presented in .
(C) The effects of continuous illumination (yellow bars) over the OFC during various test phases of the attentional set-shifting task were assessed in NpHR−/− mice (shown in black, n = 4) and NpHR+/− mice (shown in gray, n = 8). There is a significant interaction among light stimulation, genotype, and task phase (three-way ANOVA; F(2,69) = 7.963, p = 0.001). Specifically, NpHR+/− mice take significantly more trials to reach criterion (t(3,19) = 5.053, p = 0.0002) than NpHR−/− littermates under light stimulation during RR learning.
(D) Similarly, under continuous illumination over the OFC, NpHR+/− mice commit significantly more errors during RR learning than NpHR−/− animals (t(3,19) = 3.541, p = 0.0065). A significant three-way interaction among light stimulation, genotype, and task phase on errors commit during the task was also reported (F(2,69) = 4.208, p = 0.019).
(E) Postmortem c-Fos analysis revealed continuous optical inhibition of PV interneurons in the OFC leads to an increase in c-Fos expression in this region t(1,10) = 3.108, p = 0.011). Data shown for lateral orbitofrontal (LO) cortex; ventral orbitofrontal (VO) expression was also significant t(1,10) = 2.841, p = 0.018) and can be found in .
(F) Effects of continuous photostimulation with 570-nm light over the mPFC. A significant interaction among light stimulation, genotype, and trial phase was reported (three-way ANOVA; F(2,69) = 3.232, p = 0.045). Specifically, NpHR+/− animals take significantly more trials to meet criterion during the RS phase than NpHR−/− mice t(8,31) = 5.686, p < 0.0001). These effects were not present in any other task phase or in the absence of light.
(G) Optogenetic inhibition of mPFC interneurons also affected errors committed during the task, with a three-way interaction found among light stimulation, genotype, and task phase (F(2,69) = 4.590, p = 0.013). NpHR−/+ mice (n = 6) committed significantly more errors than NpHR−/− mice (n = 5) (t(8,31) = 5.374, p < 0.0001) committed selectively during the RS task phase. Again, effects were only observed in the presence of photostimulation.
(H) Postmortem c-Fos analysis revealed that optically silencing PV interneurons in the mPFC led to an increase in c-Fos expression (t(1,11) = 3.216, p = 0.008). Data shown for the infralimbic cortex (IL); expression in the prelimbic cortex (PL) was also significant (t(1,11) = 2.883, p = 0.015) and can be found in .
For all plots, data show means ± SEM. Significant main effects and main interactions are reported, but only significant Bonferroni comparisons are shown. For c-Fos expression, data are analyzed using two-tailed Student’s t tests. Significance is denoted as follows: *p < 0.05, ***p < 0.001