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
pressure BP for TGR and SDR controls under LD 12:12 conditions collected for 3 days. Time is referenced to zeitgeber time ZT where ZT
3.1. Telemetric measurement of heart rate, blood
12 is defined as the beginning of darkness n55.
pressure and motor activity In normotensive SDR telemetric measurements of heart
different lighting conditions, i.e. under LD 12:12, under rate HR and systolic blood pressure sBP in L and in D
constant darkness DD or as response to the 1 h light were 313.9621.6 354.1619.5 beat min and 111.464.3
pulse at two circadian times. The 5 min measurements of 118.064.7 mmHg. In TGR both HR 370.6641.1
MA, HR and sBP are displayed using the daily mean of 437.2637.7
beats min and
systolic blood
pressure each function see Fig. 1 as cut-off value. It can be seen
228.7610.4 218.6610.5 mmHg were higher in both that patterns in HR and sBP in TGR are more fragmented
phases P,0.001 compared to SDR. Fig. 1 displays the than those in the normotensive controls. However, both
group-averaged hourly mean waveforms of motor activi- strains are well adapted to the 12:12 light dark regime.
ty MA, heart rate and systolic blood pressure obtained for both strains under 12:12 h light–dark conditions. Heart
3.2. Phase response of motor activity, heart rate and rate and blood pressure are plotted as percent difference
blood pressure to a 1 h light pulse
from the 24 h mean. Whereas the absolute values in HR and sBP significantly differed between SDR and TGR both
Apparent shifts induced by the light pulse were calcu- in L and D see above, the relative variation around the
lated as the difference between acrophase in the reference 24-mean was similar, but the rhythm in BP in TGR was
DD period and the DD period in which the light pulse was inverse to that in SDR Fig. 1. In Fig. 2 the data on MA,
given. The results were also confirmed by the commonly HR and sBP are plotted as actograms obtained under
used ‘‘eye-fit’’ method. One h light pulses differently
254 B
Fig. 3. Phase-shifting effects of the light pulses on motor activity MA, heart rate HR, diastolic dBP and systolic sBP blood pressure.
Animals maintained under a 12:12 lighting cycle were released to DD for 48 h and exposed to a 1 h light pulse at CT2 and CT 14 and then kept in
DD for another 5 days. Light-induced phase-shifts were calculated as the difference between acrophase of a reference DD period and the DD
period in which the light pulses was given n55; mean h6SEM.
hypertensive rats nor in the normotensive controls Fig. 3; see also Fig. 2 for the actograms.
3.3. Twenty-four hour rhythmicity in c-fos and c-Jun mRNA expression
In Sprague–Dawley rats maintained under a 12:12 h light dark cycle a stable and robust rhythm in c-fos mRNA
expression in the SCN was found by RT-PCR with the highest value at ZT 2 Fig. 4. Thereafter, c-fos mRNA
decreased reaching lowest values during the late day and early night ZT 10–14. When fitting a 24 h cosine curve
to the data Fig. 4 a highly significant rhythm was
Fig. 2. Here 5 min data collections are plotted as actograms of motor
confirmed P,0.006 with an acrophase at ZT 2:2361:11
activity MA, heart rate HR and systolic blood pressure sBP of a representative normotensive Sprague–Dawley SDR and transgenic
h. In contrast, there was no rhythmic expression in c-fos
hypertensive TGRmRen227 TGR rat throughout the experiment. The
mRNA in the SCN of the TGR Fig. 4. This was mainly
daily mean of each parameter was used as the cutoff value. Bars on the
due to the fact that in TGR expression of c-fos mRNA in
right side represent the light regime with open bars for the 12 h:12 h
the SCN was significantly suppressed at ZT 2 and ZT 6
light dark cycle LD and black bars demonstrating days under constant
P,0.05. The expression of c-Jun mRNA in the SCN of
darkness DD. Arrows and open circles mark administration of the light pulses. The actogram nicely demonstrated the inverse blood pressure
both strains was only weak and showed neither a signifi-
rhythm in TGR. The reduced responsiveness of all three parameters in
cant rhythmicity nor a response to light pulses Fig. 5.
TGR to the given light pulses is also clearly visible.
3.4. Light-induced c-fos mRNA expression affected the physiological functions of the two rat strains.
The light pulses at CT 14 significantly induced c-fos In SDR the 1 h light pulse given during the early
mRNA expression in the SCN of SDR Fig. 6. Interest- subjective night at CT 14 caused a phase delay P,0.05
ingly, we found also a significant c-fos mRNA induction at in all four parameters, averaging 2 h Fig. 3; see also Fig.
CT 2 P,0.05, during the early subjective day. The 2 for the actograms. In contrast, in TGR the light pulse at
absolute amount of increase in c-fos induced by light was CT 14 did affect neither HR nor sBP, a minor delay of 0.5
about the same at both time points. In contrast, light pulses h was observed in motor activity Fig. 3. The light pulse
given to TGR either during the subjective night CT 14 or given during the early subjective day, at CT 2, had no
the subjective day CT 2 did not result in an induction of effect on MA, HR, and BP, neither in the transgenic
c-fos mRNA. Moreover, in the normotensive SDR a
B . Lemmer et al. Brain Research 883 2000 250 –257
255
Fig. 4. Twenty-four hour expression of c-fos mRNA in the SCN of Fig. 6. Spontaneous and light-induced c-fos mRNA expression in the
normotensive SDR and transgenic hypertensive TGR rats kept under a SCN of normotensive SDR and transgenic hypertensive TGR rats
12 h:12 h LD schedule. Analysis of the mRNA by RT-PCR with capillary during the subjective day and subjective night. Animals maintained under
electrophoresis and laser-induced fluorescence detection with respect to a 12:12 lighting cycle were released to DD for 48 h, exposed to a 1 h
Cyclophilin n56, mean6SEM. Horizontal black bars represent the dark light pulse at CT2 and CT 14 and sacrificed thereafter. DD controls were
phase of the cycle. decapitated under dim red light. Shown are mean values6SEM n55.
Analysis of mRNA by RT-PCR with capillary electrophoresis and laser- induced fluorescence detection with respect to Cyclophilin. There is also a
significant difference between the basal level of c-fos mRNA at CT 14 in
significant P,0.0005 spontaneous variation in c-fos
the DD controls of SDR and TGR P,0.05.
mRNA CT 2 and CT 14 can be noticed in DD, which was absent in the transgenic hypertensive TGR. Interest-
ingly, c-fos mRNA at CT 14 was significantly higher
4. Discussion
