Mouse anti th

To test if the CDt-projecting neurons were dopaminergic, we double-labeled 500 μm-interval SN slices with CTB and TH antibodies. After 1 h permeabilization with 0.05% Triton X-100 in TBS, the slices were blocked for 1 h in a solution containing 3% normal goat serum, 2% bovine serum albumin, and 0.05% Triton X-100 in TBS and then incubated with rabbit anti-CTB (1:1500; GenWay) and mouse anti-TH (1:3000; Immunostar) antibody overnight at room temperature. After three washes with PBS, the slices were incubated for 2 h at room temperature with goat anti-mouse IgG antibody conjugated with Alexa 488 for monkey SM or Alexa 647 for monkey ZO (1:200; Invitrogen) and goat anti-rabbit IgG conjugated with Alexa 594 for both monkeys (1:200; Invitrogen). The slices were air dried overnight at room temperature, and then mounted with VECTASHIELD (Vector). The cell images were scanned using fluorescence microscope (Zeiss AXIO imager M2). To test if the CDh-projecting neurons were dopaminergic, we examined co-localization of the TH signal and the remaining DY signal after the double labeling procedure.

Mice were deeply anaesthetised using 3.5 $\text{\%}$ isoflurane, injected with a lethal dose of pentobarbital (Euthatal, Boehringer Ingelheim) intraperitoneally, and transcardially perfused with 1X PBS followed by 10 $\text{\%}$ formalin solution. Following perfusion, the brain was extracted and placed in 10 $\text{\%}$ formalin for short-term storage. Prior to sectioning, the brain was placed into a 30 $\text{\%}$ sucrose solution until it sank, for cryoprotection. The brain was then mounted upright in OCT (Sakura FineTek) and 40 μm slices were made using a cryostat (Leica CM1850 UV). Slices were washed five times in 1X PBS before overnight incubation on a rotating platform at room temperature in primary solution: 1:5000 mouse anti-TH (ImmunoStar, Cat #22941) in PBS-T (0.4 $\text{\%}$ Triton in 1X PBS), to label TH-positive (including dopamine) cells. The following day, slices were washed five time in 1X PBS before a 2-hour secondary incubation, in 1:1000 Alexa Fluor 594-goat anti-mouse (Biolegend, Cat #405326) in PBS-T. Slices were then washed five times in 1X PBS before being mounted and allowed to dry. Mounting medium with DAPI (Vectashield, Vector Laboratories) was added to stain cell bodies, before adding the coverslip and sealing with nail polish. Slices were then imaged at 10x magnification using a confocal microscope (Leica DMi8) and LAS X software (Leica).

Animals were sacrificed at ZT2, immediately following a 2‐hr physical restraint stressor. Four percent Paraformaldehyde fixed and frozen brain tissue was sectioned in 30 µm increments through the LC on a Leica (Buffalo Grove, IL) cryostat, stored in 1x PBS with sodium azide, and later processed for immunohistochemistry. Briefly, all sections were washed 3x for 10 min in 1x PBS, permeabilized in PBS–Triton (0.5%), blocked in 5% normal goat serum (in PBS–Triton 0.5%), and incubated in primary antibody overnight (24 hr) at 4°C. Sections were then washed in PBS and incubated in secondary antibody (24 hr) at 4°C. Tissue was mounted on poly‐lysine coated microscope glass and coverslipped under Prolong Gold (Molecular Probes). The following antibodies were used: mouse anti‐TH (ImmunoStar Hudson, WI), rabbit anti‐GR (Santa Cruz, Dallas, TX), and Alexa 488 conjugated anti‐mouse and Alexa 594 conjugated anti‐rabbit IgG (Molecular Probes) at 1:1000. Fluorescent images were collected on an Olympus fluorescent microscope provided by the University of Michigan Microscopy and Imaging core facility (University of Michigan). Immuno‐positive GR cells were counted using the ImageJ binary thresholding algorithm (San Diego Plugin). The average number of cells was compared between the LRT and HRT animals. The average values for each group were then evaluated using an independent sample t‐test.

Brain tissue sections were incubated overnight at 4°C with rabbit anti-TH antibody (1:100), then treated with the appropriate biotinylated secondary antibody for 2 h at room temperature followed by a third antibody for 30 min (MXB, Fuzhou, China). The sections were developed in diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA) for 3–5 min, then immersed in distilled water to halt the reaction. The sections were dehydrated and sealed with neutral gum and images were acquired on a light microscope (ECHO, San Diego, CA, USA). For immunofluorescence labeling, mouse brain sections were blocked with sheep serum (Beyotime Institute of Biotechnology) at room temperature for 1 h, then incubated overnight at 4°C with a mixture of primary antibodies consisting of mouse anti-GFAP (1:100) and rabbit anti-TH (1:100), or mouse anti-TH (1:100; ImmunoStar, Hudson, WI, USA) and rabbit anti-Iba1 (1:400; Wako Pure Chemical Industries). After rinsing, the sections were incubated with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) cross-adsorbed secondary antibodies (both 1:500, Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at room temperature. Images were acquired with a confocal laser scanning microscope (A1; Nikon, Tokyo, Japan).

The left hemispheres of the dissected brains were sectioned coronally on a freezing microtome (Leica SM2010R) at 40 μm. Immunohistochemical stainings were performed on free-floating sections. The SNpc sections, were given an initial antigen-retrieval incubation in Tris/EDTA (pH 9.0) at 80 °C for 45 min. All the sections were quenched with 3% H₂O₂/10% MetOH for 30 min then blocked with 5% Normal Horse Serum (NHS) for SNpc and Normal Goat Serum29 (link) for Striatum before overnight RT incubation with primary antibody (mouse anti-TH 1:10000 for SNpc, Immunostar, Wisconsin for SNpc; rabbit anti-TH 1:4000, Millipore, California for Striatum). On the second day, sections were incubated with the corresponding biotinylated secondary antibody (horse anti-mouse 1:200; goat anti-mouse 1:200, Vector Laboratories) for 1 h at RT. This was followed by a 30-min incubation with an avidin-biotin peroxidase solution (ABC Elite, Vector Laboratories), and the antigen was visualized using 3,3-diaminobenzidine (DAB) as a chromogen. Sections were mounted on glass slides, dehydrated with increasing concentrations of ethanol and pure xylene, and finally coverslipped using DPX mounting medium (Sigma-Aldrich, Gillingham). Sections with uneven, blurry, not penetrating staining were excluded from analyses.