Supplemental Materials
5-choice Apparatus & Behavioral Testing
Sound-insulated 5-hole operant chambers (25×25×25 cm; Med Associates, Inc., St. Albans, VT) were used as described previously. Each chamber had a house light and fan, with an array of 5 square holes (2.5×2.5×2.5 cm, 2.5 cm above the grid floor) arranged horizontally on a curved wall opposite the liquid delivery magazine (Lafayette Instruments, Lafayette, IN). Each hole had a light-emitting diode (LED) at the back and infrared beams, mounted vertically 3 mm from the opening to detect responses. The food delivery magazine contained a well for liquid reinforcement (strawberry Nesquik® plus non-fat milk, 30 μL), delivered by a peristaltic pump (Lafayette Instruments, Lafayette, IN), with an LED at the top. The magazine also contained an infrared beam 5 mm from the floor, recessed 6 mm to detect head entries. The control of stimuli and recording of responses were managed by a SmartCtrl Package 8-In/16-Out with additional interfacing by MED-PC for Windows (Med Associates, Inc.) using custom programming.
Probabilistic Learning Test (PLT)
Mice were trained to respond in apertures for food rewards as previously detailed37, 39-41. Nosepoking in the magazine resulted in a 2 s inter-trial interval (ITI) followed by the illumination of two apertures - one designated the target and the other the non-target for 10 s. Responses in the target stimulus led to reward (strawberry milkshake) or punishment (4 s timeout with house illumination at a 80/20 ratio. The opposite (20/80 reward/punishment) ratio was true for responses to the non-target stimuli. Responses during the ITI were recorded as a premature response, resulting in a 4 s timeout and repeat of the trial. Non-responses within 10 s were recorded as omissions and a 4 s timeout. Performance was assessed over 80 trials or 60 min. Accuracy = target/total choices*100. Reward responsiveness was measured by latency to collect rewards and likelihood of repeating a choice after a reward (win-stay). Punish sensitivity was measured by perseverative responses during timeouts and shifting response choices after punishment (lose-shift).
Progressive Ratio Breakpoint Study (PRBS)
Two days following PLT, mice performed the PRBS in the same testing apparatus, as described in 42, 43. In short, mice were required to poke in a single response port to achieve the reward. The poke requirement to receive the reward increased on a progressive ratio. Breakpoint was defined as the last ratio completed during the course of testing. Mean latencies for response reward retrieval were also calculated.
Elevated Plus Maze (EPM)
The EPM was originally devised to quantify exploratory behavior as a proxy measure of risk-taking. The EPM apparatus consisted of two perpendicularly oriented 64.8 cm long x 6.0 cm wide aluminum runways painted white intersecting in the center. The center region was defined as the 6.0 x 6.0 cm point of overlap between the two runways. Starting from the edge of the center region, one of the runways was enclosed on both sides by a 22.86 cm tall melamine barrier covered by black card stock (closed arm). The other runway was not enclosed by a wall (open arm). During the C57BL/6 study the maze was mounted on four 49.5 cm legs on top of a 86 cm tall table to elevate the maze and prevent the mouse from leaping from the apparatus. During the DAT transgenic study, the EPM was elevated but with only 25 cm above the guillotine doors of a radial arm maze. The maze was isolated from the experimenter using portable partitions. All testing took place under ambient fluorescent room lighting during the animals’ active period. To begin a test, the mouse was always placed into the center region facing the same closed arm. The experimenter then enclosed the maze using the partition and started the test timer. Each animal’s activity was monitored using an overhead camera and was hand scored with ODLog (Macropod Software, Yarraville, Victoria, Australia) by registering and holding a keystroke corresponding to the animal’s location in one of 5 regions of the maze (2 open arms, 2 closed arms, and center). This location was defined by the location of the animal’s forepaws and head in the maze. Open arm time and entries were calculated as the total time or number of entries of the mouse in the open runway. Proportions were calculated by dividing open time/entries by closed time/entries. Time or entries into the center were not included in these calculations. Data was collected over five minutes, and each runway was wiped clean using a Kimwipe (Kimberly-Clark Professionals, Roswell, GA) between each animal.
Forced Swim Test (FST)
The FST was devised to screen for compounds with putative antidepressant efficacy, based on observing so-called “behavioral despair”. Mice were placed in a clear glass beaker with a diameter of 15 cm, a height of 24 cm, and 15 cm of tap water at 25 °C. Each test lasted 6 min and immobility duration scored for all 6 min by trained investigators. The primary outcome measure immobility was defined as no movement except minor corrections required to keep afloat. After the test, the animal was dried and returned to an unoccupied cage with a heating pad before returning to its home cage. The water was replaced between every 5-6 animals in order to retain correct temperature and cleanliness.
Quantification of TH+ and SST+ neurons in the Paraventricular nucleus
Mice were deeply anesthetized, then intracardially perfused with 50 mL phosphate buffer solution (PBS) followed by 50 mL 4% ice-cold PFA (10 mL/min). Brains were harvested and post-fixed overnight in 4% PFA at 4°C, then cryo-protected in 30% sucrose for 48 hours. Brains were snap frozen on dry ice and 30μm-thick sections were collected with a standard Leica Microtome (SM 2010R). Horizontal brain sections were collected through the hypothalamus for each mouse and stored in PBS for immediate use or in cryoprotectant solution for long-term storage at -20°C. Hypothalamic dopaminergic and somatostatin neurons were identified with standard 3,3′-diaminobenzidine (DAB) immunohistochemistry (IHC) for tyrosine hydroxylase (TH) and somatostatin (SST). Sections were washed 3 times for 10 min in PBS, then blocked in 5% horse normal serum / 0.3% Triton in PBS for 1 hour before overnight incubation with primary antibody (mouse monoclonal anti-TH, 1:500, Millipore; rabbit anti-SST, Chemicon 1:300) solution at 4°C. Sections were then washed 3 times for 10’ in PBS, then incubated with secondary antibody (biotinylated anti-mouse, 1:100, Vector) solution for 1 hour at room temperature. Following 3 PBS washes, sections were incubated in ABC solution (Vector) for 1 hour, washed again 3 times and incubated in fresh DAB solution for 4 minutes. After 3 final PBS washes, sections were counterstained with Giemsa solution (Harleco), then mounted in gelatin (Millipore) on glass slides, and coverslipped with Cytoseal mounting media (Thermo Scientific). Stained tissue was imaged with a slide scanner (Leica AperioNanozoomer). Stereological quantification of TH+ and SST+ neurons in the PVN of the hypothalamus was performed blind with the Stereologer software (Stereology Resource Center, Inc.).
Quantification of CRH neurons
For quantification of hypothalamic CRH+ neurons, transgenic mice expressing Cre-recombinase under the CRH promoter (CRH-ires-Cre mice, B6(Cg)-Crh tm1(cre)Zjh/J, Jackson Laboratory) were crossed with a floxed-tdTomato reporter line (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, Jackson Laboratory). Resulting offspring (selectively expressing tdTomato under the CRH-promoter) were exposed for 14 days to different photoperiods, then perfused and processed for IHC as described above. CRH/tdTomato fluorescence is easily detectable with confocal microscope and do not require staining. For TH fluorescent IHC, sections were washed 3 times for 10’ in PBS, then blocked in 5% horse normal serum/0.3% Triton in PBS for 1 hour before overnightincubation with the primary antibody (mouse monoclonal anti-TH, 1:500, Millipore) solution at 4°C. Sections were then washed 3 times for 10’ in PBS, then incubated in secondary antibody (anti-mouse 647, 1:500, AlexaFluor) solution for 1 hour at room temperature. After 3 more PBS washes sections were mounted in gelatin on glass slides and coverslipped with FluorMount mounting media. Tissue was imaged with a Leica SPe Confocal Microscope. Hypothalamic CRH+ and TH+ cells were quantified with Photoshop CS counting tool.
DAT RT-PCR
DAT RNA expression levels across photoperiods were measured by quantitative RT-PCR. Mice were deeply anesthetized before decapitation. Brains were dissected on an ice pad in RNA-se free condition. Isolation of the hypothalamus was achieved byfine dissection under the scope.Tissue was stored in 0.5 mL RNA-Later (Quiagen) at -40°C. RNA extraction was performed with a QuiagenRNAeasy mini-kit according to manufacturer’s instructions and tested for purity and integrity. Total RNA was reverse transcribed by random decamer primer protocol (RETROscript kit, Ambion) according to manufacturer’s instructions. TAQman Real-Time PCR analysis was conducted on a Light Cycler 480 system (Roche), and the data was processed and analyzed using the comparative ΔCT method 44. DAT primers (Bio-Rad Laboratories), designed using the Roche online tool (Assay Design Center), were validated before use.
Experiment 1: Photoperiod-induced behavioral and immunohistochemical switching in C57BL/6 mice
C57BL/6 mice were housed in the 3 photoperiod conditions (SA, NA, or LA) for 14 days. At the end of each time period, mice were tested in the EPM then the FST over two days, and on the third day the mice were sacrificed for immunohistochemical analysis.
Experiment 2: Lack of photoperiod-induced behavioral and immunohistochemical switching in CLOCK ∆19 mice
Male and female CLOCK ∆19 mutant (Mt; n=32) and WT (n=46) mice were housed in the 3 photoperiod conditions (SA, NA, LA) for 14 days, after which they were tested in the EPM then the FST over two days.
Experiment 3: Susceptibility of photoperiod-induced behavioral and immunohistochemical switching in DAT HY mice
Cohort 1 of DAT HY and WT mice were housed in the 3 photoperiod conditions (SA, NA, LA) for 14 days, after which they were tested in the EPM then the FST over two consecutive days.
Cohort 2 of DAT HY and WT mice were trained to respond for single food rewards in the 5-choice chambers (FR1), Mice were then tested in the PLT and PRBS paradigms over 3 days, with HAB2 training interspersed between tests. The performance of each mice from were counterbalanced into 3 groups with WT and HY mice equally represented such that no difference in performance was observed between the three groups. After counterbalancing, each group was housed in the 3 photoperiod conditions (SA, NA, LA) for 14 days, after which they were retested in the PLT or PRBS paradigms. Afterwards, mice were sacrificed for immunohistochemical analysis (see Supplemental Table for sample sizes).
c57Bl/6 / DAT-WT / DAT-HTTH / SST / TH / SST / TH / SST
SA / 6 / 6 / 7 / 6 / 9 / 9
NA / 6 / 6 / 7 / 6 / 6 / 6
LA / 6 / 6 / 6 / 6 / 6 / 6
Supplemental Table: Summary of the number of animals per genotype per photoperiod used in the quantification of TH- and SST-immunoreactive neurons
Supplementary Figure 1: Photoperiod doesn’t affect the number of CRH+ neurons of C57Bl/6. 2-week exposure to short active (SA) or long active (LA) photoperiod has no significative effect on the number of CRH+ neurons (One-way between subject ANOVA [F(2,9) = 0.98, p = 0.43).
Supplementary Figure 2: Effects of photoperiod condition on depression-relevant behavior in ClockΔ19 (Mut)and their wildtype (WT) littermate mice. Consistent with C57BL/6 mice, short active (SA, 19L:5D) photoperiods increased depression-relevant behaviors in the forced swim test (FST), compared with mice in a normal active (NA; 12L:12D) photoperiod, irrespective of genotype. Additionally, Clock Mut mice exhibited less depression relevant behavior irrespective of photoperiod compared with WT littermates. Long active (LA; 5L; 19D) photoperiod conditions did not affect behavior. Data presented as individual data points plus mean ±S.E.M. * denotes p<0.05 compared with indicated population.