B . Lemmer et al. Brain Research 883 2000 250 –257
251
the highest levels in blood pressure are detectable during 2.2. Tissue preparation and RNA isolation
the resting phase of the animal. Interestingly, these rats are normotensive at birth and develop hypertension simul-
Rats were sacrificed under bright light during the taneously with the inverse blood pressure rhythm at the
daytime or under dim red light during the nighttime and in age of 5–11 weeks [27]. The exact mechanism involved in
experiments under free-run conditions. Brains were rapidly the onset of hypertension during maturation and the
dissected and the block of tissue containing the hypo- development of the inverse blood pressure rhythm in this
thalamus was cut into coronal slices 200 mm in thickness transgenic rat strain is still unclear [27]. Earlier studies
and transferred on to slides. The whole SCN was cut out have shown that the blood pressure rhythms in the
under a microscope using a NIH-micro punch 26 gauge normotensive Sprague–Dawley rats SDR as well as the
and was fresh-frozen in liquid nitrogen prior to analysis. inverse pattern in TGR are preserved under free-run
Extraction of total RNA from single SCN was done by a conditions in constant darkness [26]. Both in normotensive
modified Chomczynski method [5], using 100 ml per mg rats [18,28] and in TGR [28], ablation of the suprach-
tissue TriPure reagents Roche Molecular Biochemicals, iasmatic nucleus abolished the rhythm in motility and also
Mannheim, Germany. Extracted total RNA was then those in heart rate and blood pressure. These data indicate
diluted in 10 ml of a buffer containing 1 mM sodium that — at least in the rat — cardiovascular rhythms must
citrate, pH 6.5 to reduce decomposition of mRNA. The also be under the control of the central clock located in the
concentration of total RNA was determined with an UV SCN.
spectrophotometer GeneQuant II, Amersham-Pharmacia In order to get more insight into the disturbed circadian
Biotech, Freiburg, Germany. Samples were kept frozen at rhythm regulation within the cardiovascular system we
2808C until analysed. investigated the expression pattern of mRNAs in the SCN
of normotensive Sprague–Dawley and transgenic hyper- 2.3. Measurement of activity, heart rate and blood
tensive TGR rats. Assuming that an independent over- pressure
expressed brain renin–angiotensin system could alter the regulation of gene expression in this region we studied the
The Dataquest IV system Data Sciences Inc., St. Paul, expression of the immediate early genes IEG c-fos and
MN was used to measure systolic blood pressure sBP, c-Jun. The IEGs c-fos and c-Jun were selected because
diastolic blood pressure dBP, heart rate HR and motili- they can be induced by angiotensin II and at least c-fos
ty MA by telemetry. Transmitters were implanted into plays a role in the light induced entrainment of the
six Sprague–Dawley and six transgenic hypertensive circadian clock. Furthermore, in order to study coupling
TGRmRen227 rats 8 weeks of age as described in processes of the clock, the effect of light pulses at two
detail [11]. Briefly, animals were anaesthetised with en- Zeitgeber times CT 2, CT 14 were investigated both on
flurane Abbott, Wiesbaden, Germany and the transmitter c-fos mRNA in the SCN as well as on the onset of the
body was implanted into the peritoneal cavity with the rhythms in BP, HR and MA in both rat strains.
sensing catheter placed into the abdominal aorta. Each animal was housed in a single cage and three cages were
placed on one shelf inside a ventilated, light- and sound- tight enclosure. Measurement was taken every 5 min
2. Material and methods throughout the study period.
2.1. Animals 2.4. Photic entrainment experiment
All animal experimentation were approved by the Radiotelemetric devices allow to measure motor activity
German federal regulations regarding experiments in ani- MA, heart rate HR and blood pressure BP in freely
mals. These regulations are in accordance with the Euro- moving unrestrained animals without affecting their be-
pean Communities Council Directive 86 609 EEC. Male haviour. Thus, this method was used to study the effect of
Sprague–Dawley SDR n562 and heterozygote male a light pulse on these physiological functions in both rat
transgenic hypertensive TGRmRen227 rats n562, both strains. Prior to the experiments animals were kept under a
from M1B A S Ry, Denmark were used for all experi- 12:12 h light dark schedule. After the hypertension in the
ments. In detail, six rats from either strain were used for TGR was fully established, at an age of 14 weeks, lighting
telemetry, 36 for the determination of c-fos at six circadian conditions were changed to constant darkness DD for 48
times six per time point and 20 rats in the experiments on h. First, all animals received a 1 h light pulse 100 Lux
light entrainment ten without with light induction. If not during the subjective night at circadian time 14 CT 14;
especially mentioned animals were maintained under a with the activity onset used as a reference point set to CT
standard 12:12 h light 100 Lux dark schedule LD; lights 12. After additional 5 days under DD the animals were
on is set to ‘‘zeitgeber’’ time ZT 0, and lights off to ZT 12 re-entrained to a 12:12 h light dark cycle for one week.
with food and water ad libitum. Following 48 h under DD, the second light pulse was
252 B
applied during the subjective day at CT 2. Then the rats amplicon of 335 bp in size. For test-to-test variation and
were again placed in DD for 5 days and entrained experiment consistency a positive control was amplified
thereafter to LD 12:12 h for 1 week after which a with each RT-PCR.
‘‘control’’ week under DD was added. 2.6. Capillary electrophoresis CE with laser-induced
2.5. RT-PCR fluorescence detection
LIF Single tube RT-PCR was performed with the Titan
Determination of PCR products was carried out by CE One-Tube RT-PCR kit Roche Molecular Biochemicals,
in a PACE System 2100 Beckmann Instruments Mannheim, Germany as described by the manufacturer.
equipped with a laser-induced fluorescence detector using The determination of amplification efficiency for a number
an argon ion laser source. A 488 nm excitation was used of consecutive cycles was done for each mRNA tested. For
and fluorescence emission was collected through a band these experiments a mixture of total RNA over all time
pass filter at 520 nm. Separation was performed with a points and from both rat strains were used to assure that
DB-17 coated capillary JW Scientific, 100 mm I.D. and afterwards all amplification reactions were performed in
37 cm in length effective length is 30 cm. The coating the linear range of the enzymatic reaction [30]. In addition,
thickness was 0.2 mm. The electrophoresis buffer consisted Cyclophilin was used as an endogenous internal standard,
of 0.7 w w hydroxypropylmethyl cellulose Sigma to correct tube-to-tube variations. To minimise the prob-
Deisenhofen, Germany
in NF-TBE
buffer Roth,
lems associated with DNA contamination primers were Karlsruhe, Germany with 7 M urea and 0.1 M NaCl, pH
selected to span at least one intron boundary of the 8.5. The buffer was replaced after each run. Without a prior
genomic sequence. This will result in a PCR product from desalting step the samples were applied to the capillary by
genomic contamination that will be larger in size than the pressure injection for 60 s. The subsequent separations
product generated from the cDNA. The exact match and were performed with a voltage ramp for the first 2 min
location of the primers were controlled by a BLAST search followed by 20 min at a constant voltage of 180 V cm.
http: www.ncbi.nlm.nih.gov BLAST . The sense and The mRNA expression was quantified by integrating the
antisense primers were all synthesised and 59-end labelled peak areas of the primers and the PCR products Gold TM
with fluorescein by MWG-Biotech Ebersberg, Germany. Software, Beckmann Instruments and calculating the
RT-PCR was carried out with a PTC 200 Thermocycler relation between PCR product and the total measured
MJ Research. The samples were placed into the pre- fluorescence area5100. In order to control the sepa-
heated block with heated lid and cDNA was synthesised at ration efficiency and size of the RT-PCR products a
508C for 30 min. The RT-enzyme was than deactivated at molecular weight marker of fluorescein-labelled DNA-
948C for 2 min. A 3-step cycling programme was used for fragments from 50 to 500 bp, in 50 bp steps with a known
amplification consisting of a denaturation step at 948C for concentration of 5 fmol ml fragment Sizer 50–500,
30 s, annealing at the calculated annealing temperature Amersham-Pharmacia Biotech, Freiburg, Germany, was
Tm for each case for 30 s and an elongation step at 688C injected prior and after each 12th run of analysis.
for 45 s. After 10 cycles, further cycles were carried out with an additional 5 s elongation per cycle. Subsequent to
2.7. Rhythmic c-fos and c-Jun mRNA expression the cycling program a prolonged elongation of 7 min at
688C was performed. For c-fos mRNA the following The 24 h expression pattern of c-fos and c-Jun mRNA in
primers were constructed: antisense: 59-ACA GTA CGT the SCN of SDR and TGR was examined using six time
GGA TAT AGC GA-39 and sense: 59-CCT CGA GGG points throughout the 24 h cycle ZT 2, ZT 6, ZT 10, ZT
GTT CCC GTA GA-39 using the gene sequence: 14, ZT 18 and ZT 22. Six rats of each strain were used for
embl uX06769uRNCFOSR Genbank EMBL. PCR reaction
each time point. Rats were kept in a 12:12 h light dark was performed at Tm: 528C and 30 cycles, the amplicon is
schedule and sample preparation was performed as de- 497 bp in size. The 59-AGG GGA GTT CAT CCG CAA
scribed above. Data derived from RT-PCR CE-LIF quanti- TC-39 antisense and 59-AAA CTT GAG AAC TTG ACT
fication were submitted to a rhythm analysis [29]. GG-39 sense primer, constructed after the gene sequence
gb uX17163uRSJUNAP1 Genbank EMBL, were used to
2.8. Light-induced c-fos mRNA expression detect the c-Jun mRNA using an annealing temperature of
Tm: 548C and 32 additional cycles. The amplicon was 417 According to the protocol described above for the
bp in size. The housekeeping gene Cyclophilin was measurement of physiological functions, five animals of
amplified using the 59-GAT GGG TAA AAT GCC CGC each strain SDR, TGR were exposed to an 1 h light pulse
AA-39 antisense and 59-GGT GAC TTC ACA CGC CAT 100 Lux either during the subjective day CT 2 or
AA-39 sense primer, constructed after the gene sequence during the subjective night CT 14 and sacrificed there-
gb uM19533uRATCYCA Genbank EMBL. PCR was car-
after. Control animals n55 per strain at each time point ried out at Tm: 608C and 24 cycles. The reaction gave an
remained in darkness and were decapitated under dim red
B . Lemmer et al. Brain Research 883 2000 250 –257
253
light. Preparation of tissue and extraction of mRNA was done as described above.
2.9. Data presentation, rhythm analysis, analysis of phase shifts and statistical analysis
The telemetric data of BP, HR and MA were collected every 5 min for each single rat and plotted as actograms
using the daily mean of each parameter as the cutoff value Circadia software; distributed by R. Mistlberger, Simon
Fraser University, Burnaby, BC, Canada. Since the pattern in dBP and sBP were about the same only sBP data are
presented here. Phase shifts in the rhythms of HR, dBP, sBP and MA were calculated by three independent meth-
ods. The time series analysis tool CUSUM [14,25] pro- grammed in Excel Microsoft Corp., was used to calculate
the activity onset as well as onset of heart rate and blood pressure prior to and following each pulse. The small
circadian amplitude in BP made it difficult to clearly define its onset. Therefore, in a second analysis the 24 h
acrophases were calculated for all functions from data covering four subsequent days from each of the three
experiments in DD reference DD, DD with light pulse at CT 2 and CT 14, resp.. Briefly, light-induced phase shifts
were calculated as the difference between acrophases of the reference DD period and the DD period in which the
light pulse was given. These results were additionally confirmed by the commonly used ‘‘eye-fitting’’ method as
described [7]. Statistical analyses were done by ANOVA
´ with a post-hoc Scheffe’s F-test or a Student’s t-test where
appropriate StatView, Abacus Concepts Inc., Berkely, CA.
Fig. 1. Group-averaged hourly mean6SE waveforms of motor activity MA and relative percent of 24 h mean heart rate HR and blood
3. Results
