Animals and general housing

All animals were maintained in a 12:12 h LD cycle [lights on at 07.00 h, Zeitgeber time (ZT0)], constant temperature (22 ± 1 °C) and free access to food (Rodent Laboratory Chow 5001) and water, unless otherwise stated. Experimental procedures used in this study were in strict accordance with the Mexican norms for animal handling, Norma Oficial Mexicana NOM-062-ZOO-1999, which conforms to international guidelines for animal handling, and were approved by the Ethics Committee (063/2016) in the Faculty of Medicine UNAM. Experiments conform to international guidelines on the ethical use of animals; procedures were aimed at minimizing the number of animals used and their suffering.

Experimental designs

Re-entrainment with chocolate after acute jet-lag

To determine whether a piece of chocolate can aid to re-entrain in jet-lag conditions, rats were randomly assigned to one of three groups that were exposed to a sudden 6 hour phase advance (6PA): 1. The control group (n = 8), exposed to the 6PA, with ad libitum regular food access and NO chocolate; 2. the chocolate-previous rats (CH-P; n = 8); starting with the day of 6PA they received daily a piece (5 g) of milk chocolate (Kinder chocolate Maxi, Ferrero) coinciding with the onset of the previous night; 3. the chocolate–new rats (CH-N group n = 8), starting with the day of 6PA they received daily a piece (5 g) of milk chocolate coinciding with the start of the new night. after 10 days of baseline in an LD cycle (lights on at 1900 h, = ZT0), rats were exposed to the 6PA by turning off the light 6 h earlier, at 1300 h external time. The chocolate administration continued for the 12 days following the 6PA shift. After the 6PA, the following 12 days were monitored and the number of transitory cycles for achieving the new expected acrophase were determined for general activity and core temperature using a cosinor analysis (see cosinor analysis). The new expected acrophase for each group was estimated by subtracting 6 hours from the values obtained in the baseline. The daily new acrophases were statistically compared with the expected acrophase using a one-way ANOVA for repeated measures followed by a Dunnett´s post hoc test. Data that resulted statistically different from the expected acrophase were classified as transitory cycles, whereas when data were statistically similar to the expected acrophase for 3 consecutive days, the re-entrainment to the new phase was classified as reached. A one-way ANOVA was used to compare the number of days required by each group to achieve re-entrainment to the new acrophase for general activity and core temperature.

Re-entrainment with food after acute jet-lag

Rats exposed to a sudden 6 hour phase advance (6PA) were randomly assigned to 1 of 2 experimental groups: Food access in previous night (Food-P) or Food access in the new night (Food-N). After 10 days of baseline in an LD cycle with lights-on at 0700 h and lights-off at 1900 h, all rats were exposed to a 6 hours phase advance (6PA) as previously described. The shift was accomplished by ending the light period 6 h earlier, resulting in lights-off at 1300 h new ZT12 and lights-on at 0100 h external time (new ZT0). After the 6PA both groups were exposed daily to a restricted food access of 12 h food and 12 h fasting during 10 days. The Food-P group (n = 12) were fasted for 12 h and received food during 12 hours in the time of their previous night, while the Food-N group (n = 12) were fasted for 12 h previously to 6PA, received food during 12 hours in the new night (geographic time: 1300 h to 0100 h) for 10 days after the 6PA.

The number of transitory cycles necessary for re-entrainment was determined for general activity and temperature using a cosinor analysis as described before.

Re-entrainment with the chocolate components after acute jet-lag

Rats were randomly assigned to one of six groups exposed to a 6PA: the control group receiving ad libitum food and was not exposed to other feeding conditions, experimental rats after the 6PA received daily either 5 g of chocolate or pellets containing the separate elements of a chocolate bar; gelatin was used as the base to prepare all the pellets, therefore, one group received only gelatin, other group received the smell and fat of chocolate provided by cocoa butter, the calories of chocolate were mimicked with sugar and the chocolate flavor was accomplished by adding cocoa flavor with Splenda to gelatin. After 5 days in this protocol rats were left in constant darkness for 24 hours without chocolate access to determine the endogenous circadian phase. The daily phase after the 6PA was compared with the expected acrophase by sustracting 6 h form the baseline acrophase. A one-way ANOVA for repeated measure was used followed by a Dunnett´s pos hoc test to determine significant differences, as described for experiment 1. On day 6 under DD the achieved acrophase was compared with the expected acrphase and the difference was used as indicator of the re-entrainment effect of the element using a cosinor analysis as previously described.

Chocolate effects in the experimental shift-work condition

Rats were randomly assigned to a Control (CNT) or a Shift-work (WRK) condition. These groups were further subdivided in: the control group (CNT) receiving regular chow ad libitum during the entire experiment, the control-chocolate for dinner group (CH-D) receiving daily 5 g of chocolate immediately after lights on (ZT0) and the control-chocolate for breakfast group (CH-B) receiving daily 5 g of chocolate immediately after lights off (ZT12). In a similar way the WRK rats were subdivided in three groups: The WRK group received chow ad libitum during the entire experiment, the work rats receiving chocolate for dinner (WCH-D) received daily 5 g of chocolate 10 minutes before entering to the slow rotating wheel for (ZT2); the work rats receiving chocolate for breakfast (WCH-B) received daily 5 g of chocolate at ZT 12. Rats assigned to the shift-work protocol were placed in slow rotating drums (1 revolution / 3 min) that are normally used for sleep deprivation (33 cm diameter, 633 cm wide) from Monday to Friday for 8 h (from ZT2 to ZT10) during 4 weeks, as previously reported54. The scheduled chocolate was only provided from Monday to Friday associated with the shift-work protocol. During weekends, all rats remained undisturbed in their home cages. Rats were weighed before starting the baseline and once / week during the first 3 week of the experimental manipulations. Body weight gain was calculated for this interval and for each group. At the end of the 3rd working week rats underwent surgery to implant a jugular cannula (n = 4–6 per group) in order to obtain blood samples for a 24 h cycle.

Rats were allowed to recover during the weekend and continued their work protocol during week 4. One series of rats was used to evaluate postprandial thermogenesis. At the end of week 4 the change in core temperature was compared between WRK rats and WRK rats receiving chocolate either for breakfast or dinner.

Monitoring of general activity and core temperature

All rats were housed in individual cages (45 × 30 × 35 cm) placed on plates with tilt sensors, in soundproof lockers with controlled lighting conditions. General activity in the home cage was continuously monitored with the tilt sensors. Behavioral events were collected with a digitized system and automatically stored every minute in a PC for further analysis SPAD9 (Data processing system, 1.1.1 version; Omnialva SA. De CV. Mexico City, Mexico) based on MATLAB. Double plotted actograms were obtained for each animal by collecting the sum of activity for 15 min intervals. Daily activity counts were fitted to a nonlinear cosinor analysis for estimating daily individual acrophases (see cosinor analysis below). For each rat, normalized activity counts for the last day of the base line, and for the following days after the 6PA were analyzed individually with the cosinor analysis, to obtain the daily acrophases. In order to evaluate core body temperature, rats underwent a brief surgery to receive intra-abdominal temperature sensors (iButton Sensor- Temperature Logger; Maxim integrated products, USA) as previously described55. Briefly, one week before starting baseline, rats were anaesthetized with an intramuscular dose of xylazine (Procin 8 mg/kg) and ketamine (Inoketam 40 mg/kg). Under anesthesia a small incision was performed in the abdominal cavity and the temperature sensor, previously sterilized, was introduced in the peritoneum. Abdominal muscles were sutured with absorbable catgut (000) and skin was sutured with surgical suture (Atramat, International Farmaceutica, SA. de CV. Mexico). Rats were left for 1 week to recover before starting the baseline. Temperature sensors were programmed to store samples every 30 min. Data for the baseline and for the 12 following days after the 6PA were analyzed with the cosinor test to obtain acrophases as described for general activity. The acrophase for the last day of the base line was compared with the acrophases following the 6PA a one way ANOVA for repeated mesurements followed by a Dunnett’s pos thoc test for general activity. For daily temperature profiles data in the shift-work model data are presented as average per hour. Postprandial thermogenesis in response to chocolate was evaluated at the end of week 4, in a normal work day. The postprandial response was compared between WRK rats and rats receiving chocolate (WCH-B and WCH-D). The temperature change was determined every 30 min. by comparing temperature prior chocolate presentation with the temperature of the 5 h after chocolate intake for WCH-B and WCH-D rats.

Metabolic and hormonal rhythms

At the end of week 3 of the work protocol, animals in the shift-work protocol and their controls were anesthetized with an intramuscular dose of xylazine (Procin 8 mg/kg) and ketamine (Inoketam 40 mg/kg) and cannulated in the internal jugular vein with a polyethylene silicon tube (0.025 in. i.d. and 0.047 in. o.d.; Silastic Laboratory tubing; Dow Corning Corp., Midland, MI, USA) filled with heparin (500 U/ml) as anti-coagulant as previously reported54. The outer end of the cannula was fixed in the back between both shoulder blades and clotted with a small needle. Rats were allowed to recover during the weekend and on Monday the work protocol was reinitiated. At the end of the 4th working week blood samples were obtained distributed in 2 days (Thursday and Friday) to cover a 24 h cycle with 3 h intervals; 4 samples were obtained one day at ZT0, ZT6, ZT12 and ZT18 and the other 4 samples were obtained the next day (ZT3, ZT9, ZT15, AND ZT21) Blood samples (500 ul) were collected in Eppendorf tubes (1.8 ml) containing a clot-activator gel and were centrifuged at 2500 r.p.m. during 10 min, serum was stored in 100 µl aliquots at −45 °C until assay. Aliquots were processed with colorimetric methods for determination of glucose, triglycerides (TG) and melatonin.

Glucose was measured using a commercial colorimetric kit (GPSL-0507, ELI Tech Clinical Systems). TG were assessed with a commercial kit (TGML-0427, ELI Tech Clinical Systems), melatonin was determined by the ELISA method, with a commercial kit (Enzyme immunoassay for the direct, quantitative determination of melatonin; of IBL International, Germany). A cosinor analysis was used to obtain daily acrophases of the metabolic variables/ individual (see cosinor analysis).

Immunohistochemical staining

One series of the groups employed for the jet-lag study was used to obtain the brains in two time points. Brains were obtained 1 day after the 6PA either at their new ZT1 (one hour after the lights onset) or new ZT13 (one hour after the light offset). ZT13 also corresponded to one hour after chocolate intake for the CH-N group. For the CH-P group, night sampling was performed at ZT19, one hour after chocolate intake.

One series of rats for the shift-work protocol was used for brain collection and immunohistochemistry. On week 4 of the shift-work protocol, rats were perfused one hour after chocolate intake (ZT1) or 12 h later (ZT13) to obtain a day-night pattern.

Rats were anaesthetized with an overdose of sodium pentobarbital (Sedalphorte 65 mg/ml, Pisa, Mexico) and were perfused transcardially with 250 ml of 0.9% saline followed by 250 ml of fixative 4% paraformaldehyde in phosphate buffer saline (PBS, 0.1 M, pH 7.2).

For the night-time points rats were anesthetized under dim red light and their eyes covered. The time between anesthesia and the start of fixation was of 5–6 min. Brains were removed and post fixed for 24 h in paraformaldehyde 4%. After post fixation they were cryoprotected in 30% sucrose for 3–4 days. Brains were frozen and cut in sections of 40 μm at −18 °C. Sections were collected in four series; one series was processed for c-Fos as previously described36. Free floating sections were incubated in c-Fos antibody raised in rabbit (1:2500; Santa Cruz biotechnology, Santa Cruz, CA, USA) in phosphate buffer 0.1 M, pH 7.2 with 0.9% saline 1% (PBS) 0.25% gelatin (G), and 0.3% Triton X-100 (PBSGT) for 72 h in 4 °C. This was followed by incubation in secondary antibody, goat anti-rabbit (Vector Laboratories, California USA), 1:200 in PBSGT for 2 h at room temperature, followed by incubation in avidin–biotin complex (0.9% avidin and 0.9% biotin solutions; Vector Laboratories, California, USA) in PBSGT for 2 h at room temperature. Between incubations sections were rinsed three times for 10 min in PBS. Tissues were reacted with diaminobenzidine (0.01%), nickel (0.05%) and hydrogen peroxide (35 μl,/ 100 mL 30% H2O2) to obtain a blue color. Tissues were mounted, dehydrated and cover slipped with microscopy Entellan (Merck, Darmstadt, Germany).

SCN c-Fos quantification in Jet-Lag and Shift-work animals

One medial section of the SCN per animal was selected, images were digitalized with Leica camera ICC50 HD attached to a Leica DM500. Picture were taken with an objective 20X and analyzed with ImageJ software by determining SCN ventral and dorsal regions bilaterally of the SCN. For counting c-Fos positive cells background was subtracted (50%); threshold was determined, and particle analysis was set for particles of 1.0–2.0 circularity and 20–150 pixels. The counts of the dorsal and the ventral regions were added to determine the total amount of c-Fos in the SCN. The results were graphed as total, dorsal and ventral cFos expressing cells and were compared with a two-way ANOVA for the factors groups and time (day-night).

Cosinor analysis

The cosinor analysis was used to obtain daily acrophases for each individual, mean and standard error of the mean (SEM) were obtained / group. The cosinor analysis was performed with MATLAB version 5.3 using the least square method to fit a sine wave to a time series (in this case to 24 h). The formula used was: Y(t)=M + Acophase (2πt/τ + φ) + e(t); Y = collected data; M = mesor; A = amplitude; φ = acrophase; T = period; e = error at each time. The obtained acrophase/day was compared with the expected acrophase using the Student “t” test (significant values set at p < 0.05). Days when the acrophase was statistically different (indicated with asterisks) from the new expected acrophase were considered transitory cycles, while re-entrainment was achieved when no statistical difference between the mean daily acrophase and the expected new acrophase was indicated.

Statistical analysis

Graphics and statistical analysis were elaborated using the GraphPad Prism version 6.00 for macOS, GraphPad Software, La Jolla California USA, www.graphpad.com.