Neurotrace 530 615

At the end of the last TD test session under Stress condition, 6 mice in each genotype were randomly selected for cFos immunostaining, which was performed using standard procedures (M. Buhusi et al., 2016 (link)), using a rabbit anti cFos primary antibody (Cell Signaling Technologies, Danvers, CA, Antibody Registry AB_2247211, 1:300 dilution), Alexa488-conjugated goat anti rabbit secondary antibody and NeuroTrace 530/615 (Life Technologies, Carlsbad, CA). NeuroTrace neuronal labeling was used to visualize the regions of interest. A Zeiss LSM710 laser scanning confocal microscope was used for image acquisition. Neuronal activation was estimated by counting cFos-positive nuclei in corresponding areas in 2 sections / region of interest / mouse (OFC: bregma 2.10/2.34, PrL: bregma 1.78/2.10, Acb-shell and core: bregma 1.10/1.34, DS-med and lat: bregma 0.98/1.34) (Franklin & Paxinos, 2008 ), averaged over two independent observers unaware of genotype (inter-observer reliability r=0.36, p<0.01).

Mice were perfused and geniculate ganglia were exposed by removing the brain and bone dorsal to the ganglia as described previously^{24 (link)}. The greater superficial petrosal and facial nerves were cut, and the ganglia were extracted by blunt dissection. In order to quantify the number of Thy1-GCaMP3-positive neurons per ganglia, fluorescent Nissl staining (NeuroTrace 530/615, Molecular Probes) was used to label neurons, and the ratio of Nissl-positive to GCaMP3-positive neurons determined; greater than 90% of all neurons are labeled by Thy1-GCaMP3 (n=6 ganglia). Ganglia were mounted in Vecta-shield mounting medium and imaged with a Zeiss 510 confocal microscope (Zeiss ×10, 0.45 NA microscope objective). For control KCl stimulations, dissected ganglia were submersed in imaging buffer (Hank’s Buffered Salt Solution with Ca²⁺ and 10 mM HEPES). Suture thread was used to mount the ganglia onto a coverslip within a custom imaging chamber. A depolarizing solution (potassium chloride, 500 mM) was applied to cells between washes with imaging buffer.

Dissected wholemount retinas or sections were incubated in 5% normal donkey serum in phosphate-buffered saline (PBS) with 1% Triton-X for three hours. They were then rinsed in PBS and incubated in primary antibodies diluted in PBS with 1% Triton-X for 72 hrs. Primary antibodies used in this study are listed in Table 1. In addition, Hoechst (Invitrogen, Eugene, OR; 1:1000), NeuroTrace 530/615 (ThermoFisher Scientific, Waltham, MA; #N21482, 1:500) and PNA lectin conjugated to Alexa Fluor 647 (ThermoFisher Scientific, Waltham, MA; #L32460, 1:500) were used and added to the solution of primary antibodies. Retinas were subsequently rinsed in PBS and incubated overnight in the secondary antibodies. All secondary antibodies were raised in donkey and conjugated to AlexaFluor dyes (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:200). All steps were conducted under agitation at 4°C.