Animals and Infection Procedure
transgenic Wistar rats, in which luciferase activity reports transcription of Per1
(originally developed by H. Tei [Yamazaki et al., 2000
]), from the UVa breeding colony were used for bioluminescence recordings. Both male transgenic Per1-luc
and nontransgenic (wild-type) Wistar rats were used for real-time PCR analyses. Rats were placed in a 12:12-hour light:dark (LD) cycle or constant darkness (DD) and had continuous access to food and water. The rats, 5 to 8 weeks old, were infected by intraperitoneal injections with pleomorphic Tb brucei
(AnTat 1/1, derived from stabilate EATRO 1125; Laboratory of Serology, Institute of Tropical Medicine “Prince Leopold,” Antwerp, Belgium), a subspecies not pathogenic to humans, or injected with vehicle (PSG, phosphate buffered glucose saline; 44 mM NaCl, 60 mM NaH2
O, 83 mM D-glucose; pH 8). Infected rats were always matched against vehicle-injected control rats of the same age. A volume of 0.5 mL containing 25,000 to 30,000 parasites was injected in each animal. After 10 days, parasitemia was verified in blood samples, obtained from tail snips, in a light microscope (20× magnification). Body weight was measured once a week from the time of infection in the LD animals. Molecular analyses were performed 42 days postinfection when the animals were euthanized with a halothane overdose. For long-term recording of activity and body temperature, the rats were monitored until they showed severe signs of disease (defined as severe lethargy, inactivity, lack of exploring, and no food or water intake). At this point, they were euthanized with halothane overdose.
Tissue Culture and Bioluminescence Recordings
Forty-two days postinfection, uninfected control and infected rats were euthanized with CO2
. The brain and pituitary glands were quickly removed. The SCN and pituitary tissues were dissected and cultured on membranes (see Supplementary Online Material
; SOM). The cultures were placed within a light-tight incubator holding a constant temperature of 36 °C, and bioluminescence was recorded continuously with Photomultiplier tube (PMT) detector assemblies (Hamamatsu USA, Bridgewater, NJ). The PMT modules were interfaced to a task-dedicated computer for data acquisition (software written by Tom Breeden, University of Virginia). The PMTs were positioned 1 to 2 cm above the culture dishes. Photon counts were integrated over 1-minute intervals. One group of SCN slices was obtained from the infected and control animals that were housed with running wheels and placed into DD as described above. A second group of control and infected animals was maintained under a 12:12 LD cycle in cages without running wheels. Bioluminescence traces were detrended by baseline subtraction of a 24-hour moving average and then smoothed with a 2-hour running average. Detrended and smoothed data were analyzed for period with a χ2
periodogram (Lumicycle Analysis software; Actimetrics Inc., Evanston, IL). Chi-square tests for the period of Per1-luc
expression were considered significant at p
< 0.001. Period values were compared as a function of treatment with an unpaired Student t
-test. All comparisons were considered significant at p
< 0.05. For amplitude analysis, see SOM
Per1-luc transgenic or wild-type Wistar rats were injected with Tb brucei stabilate or vehicle. At day 42 postinfection, total RNA was extracted from tissue samples (SCN, pineal, and spleen) using RNAqueous-4 PCR kit (Ambion, Austin, TX) or the RNeasy Micro kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions, except 100 μL lysis/binding solution was used with the RNAqueous-4 kit (Ambion). Briefly, samples were homogenized, bound to filter, washed, and eluted into H2O. All RNA was treated with DNase I to remove genomic DNA. Spleen total RNA samples were quantified using an Ultrospec 3000 UV/Visible spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). RNA was stored at −80 °C until cDNA synthesis. The iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) was used for cDNA synthesis according to the manufacturer’s directions, using a GeneAmp PCR System 9700 thermocycler (Applied Biosystems Inc., Foster City, CA).
For spleen samples, quantitative real-time PCR was performed on the MyiQ real-time PCR detection system (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad), and data were analyzed using the MyiQ software (Bio-Rad). For SCN and pineal samples, an ABI Prism 7000 sequence detection system (Applied Biosystems) was used with 1× QuantiTect SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA). There was 0.5 to 1 μL cDNA that was used in a total volume of 25 μL per reaction, and all analyses were performed in triplicate. For primers, see SOM
. For the spleen, 2 samples were used from each animal that were averaged to obtain a single value per animal. Data were normalized to the levels of RNA encoding cyclophilin
, and relative differences were calculated using the 2×ΔΔCT
method (Livak and Schmittgen, 2001
). All values of expression levels were calculated relative to values of control mice at ZT 7. Two-way analysis of variance (ANOVA) was performed to determine the main effects of time of day and infection on mRNA expression levels. In all analyses, a significance level of p
< 0.05 was considered statistically significant.
Wheel-Running Activity Recordings
Transgenic rats were placed in individual cages with running wheels in a 12:12-hour LD cycle. Wheel activity was monitored with ClockLab (Actimetrics Inc.). After 3 to 4 days, rats were infected with Tb brucei or injected with vehicle. One group of rats was released into constant darkness (DD). Continuous activity recordings were performed until the rats showed severe signs of disease and were euthanized with halothane overdose.
Cage Activity and Body Temperature Recordings
Transgenic rats were anesthetized with halothane, and a transmitter (Mini Mitter, Bend, OR) was quickly implanted into the peritoneal cavity. The rats were placed in individual cages in a 12:12-hour LD cycle. General locomotor activity and body temperature were monitored using the Dataquest system (Data Sciences International, St. Paul, MN). Two weeks later, the rats were injected with Tb brucei or vehicle and released into DD. Continuous recordings were performed until the rats showed severe signs of disease and were euthanized with halothane overdose.
Statistical Analyses of Activity and Temperature
Two statistical methods were used to analyze wheel running, cage activity, and body temperature data (Pincus, 1991
). The first method employed is the fast Fourier transform-nonlinear least squares (FFT-NLLS) as previously described. Briefly, time series were linear-regression detrended by FFT-NLLS to produce zero-mean, zero-slope data. An FFT power spectrum was first calculated. The period, phase, and amplitude of the most powerful spectral peak were used to initialize a 1-component cosine function, which was NLLS minimized to the detrended time series. The statistical significance of each derived rhythmic component was assessed by way of the relative amplitude error (RAE; ratio of amplitude error to most probable amplitude). Theoretically, this metric ranges from 0.0 to 1.0, with 0.0 indicating a rhythm known to infinite precision (zero error) and 1.0 indicating a rhythm that was not statistically significant (error exceeds most probable value). Intermediate values were indicative of varying degrees of rhythmic determination.
The second method used for analysis of rhythmic parameters was the COSOPT algorithm. COSOPT imports data and calculates the mean value and its corresponding standard deviation (SD). Arithmetic linear regressions detrend of the original time series was not performed. Variable weighing of individual time points (by SEMs from replicate measures) was accommodated during analysis for the presence of rhythms at periods between 18 and 30 hours.
Circadian period and phasemax (time of maximal activity/temperature) were assessed. An additional number of parameters were analyzed as detailed below: Significant circadian spectral power describes the relative amount of circadian versus noncircadian rhythmic patterning. Calculations were performed using the algorithm MC-FFT. A Fourier spectral assessment was performed on data that were performed on data zero padded to at least 2 times the original data series length. Empirical resampling was employed to evaluate statistical significance at 95% probability. One thousand temporally shuffled, randomized surrogates of each original time series were analyzed to produce 1000 corresponding surrogate Fourier spectra from which the mean power and SD were calculated. The fractions of significant spectral power in the 18- to 30-hour period range were then determined. Alpha is a measure of the relative duration active (0 = no activity, and 1 = no rest). For temperature data, it is the relative duration during which the temperature remained above the local mean temperature. ApEn (approximate entropy) is a measure of temporal patterning irregularity, the larger the value the greater irregularity. ApEn calculation is based on quantification of regularity to statistically discriminate time series. ApEn ratio is a relative measure of “maximally” random temporal patterning (0 = completely ordered condition, 1 = maximal randomness). ApEn Z scores represent the number of standard deviations that the observed temporal patterning differs from the maximally random condition. Statistical comparisons between experimental groups were made using the Student t-test with Welsh correction.