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Interactive report

Loss of 24 h rhythm and light-induced c-fos mRNA expression in the

suprachiasmatic nucleus of the transgenic hypertensive TGR(mRen2)27

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rat and effects on cardiovascular rhythms

*

¨

Bjorn Lemmer , Stefan Hauptfleisch, Klaus Witte

Institute of Pharmacology and Toxicology, University of Heidelberg, Maybachstr. 14-16, 68169 Mannheim, Germany Accepted 27 September 2000

Abstract

Immediate early genes, especially c-fos, are thought to play an essential role in photic entrainment of circadian rhythms. A special characteristic of the transgenic hypertensive TGR(mRen2)27 rat strain, expressing an additional mouse renin2 gene, is the inverse blood pressure rhythm in relation to those in heart rate and activity resulting in internal desynchronisation of these physiological rhythms. Assessment of c-fos mRNA expression by microdissection and RT-PCR in the suprachiasmatic nucleus showed, that in contrast to normotensive Sprague–Dawley rats the 24 h and circadian rhythm of c-fos mRNA expression in TGR(mRen2)27 rats is abolished. Moreover, light-induced c-fos expression within the nucleus could be found in the normotensive controls, but was absent in transgenic hypertensive rats. The light pulse applied during the subjective night, at CT 14, significantly phase delayed rhythms in blood pressure, heart rate and activity in the normotensive rats by about 2 h, whereas in the transgenic hypertensive animals rhythms in blood pressure and heart rate were unaffected, only activity showed a slight phase shift. In conclusion, these data suggest that the transgene in TGR leads not only to a disturbance of the cardiovascular system but also influences the light entrainment response, which is accompanied by a suppressed c-fos mRNA expression in the suprachiasmatic nucleus.  2000 Elsevier Science B.V. All rights reserved.

Theme: Endocrine and autonomic regulation

Topic: Cardiovascular regulation: central control

Keywords: TGR(mRen2)27; Suprachiasmatic nucleus; c-fos mRNA; Heart rate; Blood pressure; Entrainment

1. Introduction brought into synchronisation with the external environment [1,22]. In rodents light-induced phase shifts of behavioural Most behavioural and physiological parameters in mam- rhythms are known to be positively correlated with the mals display at least some evidence of a 24 h temporal induction of the transcription factor Fos in the SCN and it structure, reflecting an innate temporal programme pro- has been suggested that Fos itself mediates these light-vided by biological clocks. The suprachiasmatic nucleus induced phase shifts [9,17,24]. Alterations of endogenous (SCN) of the hypothalamus serves as the main Zeitgeber rhythms, including severe changes in the rhythmic pattern for such circadian rhythms. Light induction of clock genes of blood pressure (BP) have been detected in transgenic might be a general factor through which the body clock is hypertensive TGR(mRen2)27 rats [11,26,27]. This rat strain was introduced to determine the role of a key component, the renin gene, in the overall control of blood pressure. Harbouring a 24 kb transgene spanning the

Published on the World Wide Web on 12 October 2000. mouse Ren2 gene, the transgenic TGR(mRen2)27 rat is

All authors are employees of the Ruprecht–Karls-University of _{characterised by an overexpressed renin–angiotensin} sys-Heidelberg, Faculty of Clinical Medicine Mannheim, working at the _{tem [13] and develops a fulminant hypertension [15]. An} Institute of Pharmacology and Toxicology.

interesting and special characteristic of this rat strain is the

*Corresponding author. Tel.: 149-621-330030; fax: 1

49-621-inverse blood pressure rhythm in relation to heart rate and

3300333.

E-mail address: [email protected] (B. Lemmer). activity leading to internal desynchronisation [11]. Thus,

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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 (200mm 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 TGR(mRen2)27 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 TGR(mRen2)27 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

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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 P/ACE 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 (J&W Scientific), 100mm 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 0.2mm. 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 sepa-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 embluX06769uRNCFOSR (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

gbuX17163uRSJUNAP1 (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-39antisense 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-gbuM19533uRATCYCA (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

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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 (n}₅_5).

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

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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 TGR(mRen2)27 (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

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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 values}₆_{SEM (n}₅_5). 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

(P,0.05) in TGR than SDR.

The present study provides multiple lines of evidence demonstrating that the additional expression of the mouse renin gene (mRen2) in transgenic hypertensive rats not only results in the development of a severe hypertension but also alters the light induced expression of c-fos mRNA and the phase-shifting effect of light on physiological circadian rhythms.

expres-Fig. 5. Twenty-four hour expression of c-Jun mRNA in the SCN of _{sion of a mouse renin gene in the transgenic rat not only} normotensive (SDR) and transgenic hypertensive (TGR) rats kept under a _{results in severe hypertension but also affects clock} 12 h:12 h LD schedule. Analysis of the mRNA by RT-PCR with capillary

mechanisms responsible for regulating cardiovascular

electrophoresis and laser-induced fluorescence detection with respect to

rhythms. In this respect the data extend our previous

Cyclophilin (n56, mean6SEM). Horizontal black bars represent the dark

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pressure rhythms are internally dissociated from those in There is now indirect evidence that the overexpression HR and MA [11] but also less efficiently coupled to of the renin–angiotensin system by introduction of the regulation by light. mouse renin2 gene into TGR may be the main factor The role of c-fos mRNA in circadian rhythm regulation responsible for the disturbed blood pressure rhythm. within the SCN is discussed controversially. There are Recently, we could give evidence that the plasma renin indications that light induced c-fos mRNA is not directly activity and the corticosterone concentration are not only correlated with a phase response to a light pulse [6,20]. elevated in TGR but peak predominantly during the light Honrado et al. [8] observed that c-fos knockout mice were span (at which blood pressure is highest in TGR), plasma still capable to respond to light pulses, but they showed a aldosterone being about seven times higher in TGR than in dampened phase response curve. There is also evidence SDR [13]. Moreover, blood pressure reductions in TGR by that c-fos mRNA is not only temporally but also spatially angiotensin converting enzyme (ACE) inhibitors [12] and differently expressed in the SCN. Schwartz et al. [21] and angiotensin AT -receptor antagonists [19] are greater1

Sumova et al. [23] demonstrated that the spontaneous Fos during daytime than during the night, again indicating that rhythm in darkness is limited to the dorsomedial part of the the disturbed renin–angiotensin system is involved in the SCN, and the light-induced Fos protein occurred only in inverse circadian blood pressure pattern. However, no data the ventrolateral part of the SCN. Since we measured the are available at present concerning the outcome of the c-fos expression in a whole SCN extract, we cannot extra renin gene onto the local renin–angiotensin and distinguish between c-fos expression in different parts of vasopressin system in the central nervous system of TGR the SCN. However, the light-induced c-fos mRNA exceed- including the SCN.

ing the basal level in DD may reflect the induction in the In summary, we have demonstrated that the additional retinorecipent zone of the SCN. expression of the mouse renin gene not only results in a Describing the light-induced phase shift and expression disruption of normal physiological rhythmicity of blood of the clock genes Per1 and Per2 in the SCN of rats Yan et pressure but also influences the circadian clock itself. The al. [31] found that the induction only occurs in the transgenic hypertensive TGR(mRen2)27 showed an ventrolateral neurones of the SCN (VLSCN). Therefore, abolished 24 h and circadian rhythm in c-fos mRNA they concluded that the phase shift of overall rhythmicity expression in the suprachiasmatic nucleus and a blunted would only occur through the oscillatory coupling of the induction of c-fos mRNA after application of light pulses. VLSCN and the dorsomedial part of the SCN (DMSCN). Although c-fos is an ubiquitous transcription factor our The requirement of co-ordinated gene expression could results indicate that it may contribute to a reduced neuronal also help to explain the predicament that light induction of responsiveness in the SCN of TGR rats as a consequence c-fos mRNA at CT 2 had no phase shifting effects in SDR. of the overexpressed renin–angiotensin system.

In the transgenic hypertensive rat the small light-induced increase in c-fos expression was not significant. Whether

this blunted increase may be responsible for the lack of a _{Acknowledgements} phase-shifting effect at CT 14 on HR and BP is an open

question. Recently, Amy et al. [2] showed that the _{This work was supported by a grant of the Deutsche} magnitude of the phase delay response after a given light _{Forschungsgemeinschaft (Le 318 / 10-1 and 2). We thank} pulse is proportional to the number of cells in the SCN that _{Zenon Baraniak for excellent technical assistance with the} exhibit Fos induction. On the other hand, at CT 14, the _{CE / LIF system.}

basal c-fos mRNA level in the SCN of TGR was sig-nificantly higher than in the normotensive control (P,

0.05) suggesting that the reduced c-fos induction might be _References caused by the negative feedback of Fos on its own gene

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Pharmacol. 23 (1994) 311–314. [25] B.G.M. Vandeginste, D.L. Massart, L.M.C. Buydens, S. De Jong, P.J. ¨

[13] B. Lemmer, K. Witte, A. Schanzer, A. Findeisen, Circadian rhythms Lewi, J. Smeyers-Verbeke, Handbook of Chemometrics and Qual-in the renQual-in–angiotensQual-in-system and adrenal steroids may contribute imetrics: Part B, Elsevier, Amsterdam, 1998.

to the inverse blood pressure rhythm in hypertensive [26] K. Witte, B. Lemmer, Free-running rhythms in blood pressure and TGR(mREN2)27 rats, Chronobiol. Int. 17 (2000) 645–658. heart rate in normotensive and transgenic hypertensive rats, [14] D.L. Massart, B.G.M. Vandeginste, L.M.C. Buydens, S. De Jong, P.J. Chronobiol. Int. 12 (1995) 237–247.

Lewi, J. Smeyers-Verbeke, in: Handbook of Chemometrics and [27] K. Witte, B. Lemmer, Development of inverse circadian blood Qualimetrics: Part A, Elsevier, Amsterdam, 1997. pressure pattern in transgenic hypertensive TGR(mRen2)27 rats, [15] J.J. Mullins, J. Peters, D. Ganten, Fulminant hypertension in Chronobiol. Int. 16 (1999) 293–303.

transgenic rats harbouring the mouse Ren-2 gene, Nature 344 (1990) [28] K. Witte, A. Schnecko, R.M. Buijs, J. van der Vliet, E. Scalbert, P.

541–544. Delagrange, B. Guardiola-Lemaitre, B. Lemmer, Effects of

SCN-[16] C.S. Pittendrigh, On the mechanism of the entainment of a circadian lesions on circadian blood pressure rhythm in normotensive and rhythm by light cycles, in: J. Aschoff (Ed.), Circadian Clocks, transgenic hypertensive rats, Chronobiol. Int. 15 (1998) 135–145. North-Holland Publishing Company, Amsterdam, 1965, pp. 277– [29] K. Witte, P. Zuther, B. Lemmer, Analysis of telemetric time series

297. data for periodic components using DQ-Fit, Chronobiol. Int. 14

[17] B. Rusak, L. McNaughton, H.A. Robertson, S.P. Hunt, Circadian (1997) 561–574.

variation in photic regulation of immediate-early gene mRNAs in rat [30] C.T. Wittwer, K.M. Ririe, R.V. Andrew, D.A. David, R.A. Gundry, suprachiasmatic nucleus cells, Brain Res. Mol. Brain Res. 14 (1992) U.J. Balis, The LightCycler: a microvolume multisample fluorimeter 124–130. with rapid temperature control, BioTechniques 22 (1997) 176–181. [18] H. Sano, H. Hayashi, M. Makino, H. Takezawa, M. Hirai, H. Saito, [31] L. Yan, S. Takekida, Y. Shigeyoshi, H. Okamura, Per1 and Per2 gene S. Ebihara, Effects of suprachiasmatic lesions on circadian rhythms expression in the rat suprachiasmatic nucleus: circadian profile and of blood pressure, heart rate and locomotor activity in the rat, Jpn. the compartment-specific response to light, Neuroscience 94 (1999)

Circ. J. 59 (1995) 565–573. 141–150.

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‘‘control’’ week under DD was added.

2.6. Capillary electrophoresis (CE) with laser-induced

2.5. RT-PCR fluorescence detection(LIF)

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

gbuX17163uRSJUNAP1 (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-39antisense 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-gbuM19533uRATCYCA (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

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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

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 (n}₅_5).

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

from the 24 h mean. Whereas the absolute values in HR

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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

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 TGR(mRen2)27 (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

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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 (P,0.05) in TGR than SDR.

Here we provide first evidence that the 24 h rhythmicity of the transcription factor c-fos in the SCN, already described by histochemical methods [4,6,21] in normoten-sive rats, is abolished in the transgenic hypertennormoten-sive TGR at the level of the mRNA. Moreover, in contrast to the normotensive SDR, a light pulse failed to induce an expression of c-fos mRNA in the SCN of transgenic TGR. In this study we also were able to demonstrate that in normotensive rats a light pulse at CT 14 can shift the onset of the blood pressure and heart rate rhythms as earlier evidenced for other physiological functions such as motili-ty [7,16]. Again, TGR did not respond to the light pulse at CT 14. These findings indicate that the additional expres-Fig. 5. Twenty-four hour expression of c-Jun mRNA in the SCN of _{sion of a mouse renin gene in the transgenic rat not only} normotensive (SDR) and transgenic hypertensive (TGR) rats kept under a _{results in severe hypertension but also affects clock} 12 h:12 h LD schedule. Analysis of the mRNA by RT-PCR with capillary

mechanisms responsible for regulating cardiovascular electrophoresis and laser-induced fluorescence detection with respect to

rhythms. In this respect the data extend our previous Cyclophilin (n56, mean6SEM). Horizontal black bars represent the dark

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regulation by light. mouse renin2 gene into TGR may be the main factor

The role of c-fos mRNA in circadian rhythm regulation responsible for the disturbed blood pressure rhythm. within the SCN is discussed controversially. There are Recently, we could give evidence that the plasma renin indications that light induced c-fos mRNA is not directly activity and the corticosterone concentration are not only correlated with a phase response to a light pulse [6,20]. elevated in TGR but peak predominantly during the light Honrado et al. [8] observed that c-fos knockout mice were span (at which blood pressure is highest in TGR), plasma still capable to respond to light pulses, but they showed a aldosterone being about seven times higher in TGR than in dampened phase response curve. There is also evidence SDR [13]. Moreover, blood pressure reductions in TGR by that c-fos mRNA is not only temporally but also spatially angiotensin converting enzyme (ACE) inhibitors [12] and differently expressed in the SCN. Schwartz et al. [21] and angiotensin AT -receptor antagonists [19] are greater1

In the transgenic hypertensive rat the small light-induced increase in c-fos expression was not significant. Whether

this blunted increase may be responsible for the lack of a _{Acknowledgements} phase-shifting effect at CT 14 on HR and BP is an open

basal c-fos mRNA level in the SCN of TGR was sig-nificantly higher than in the normotensive control (P,

0.05) suggesting that the reduced c-fos induction might be _References caused by the negative feedback of Fos on its own gene

[2] S.P. Amy, R. Chari, A. Bult, Fos in the suprachiasmatic nucleus of comparison to their normotensive controls. The higher

house mouse lines that reveal a different phase-delay response to the amount of spontaneous c-fos mRNA in the SCN of TGR

same light pulse, J. Biol. Rhythms 15 (2000) 95–102.

activation of angiotensin II in neurones [10]. Since no data [4] C. Beaule, S. Amir, Photic entrainment and induction of immediate-early genes within the rat circadian system, Brain Res. 821 (1999) were available on a 24-h rhythmicity of c-Jun expression

95–100. we also investigated the mRNA of this transcription factor

[5] P. Chomczynski, A reagent for the single-step simultaneous isolation both in TGR and SDR. However, there was only a weak _{of RNA, DNA and Proteins from cell and tissue samples,} BioTech-expression of c-Jun mRNA in the SCN of both rat strains niques 5 (1993) 532–536.

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893. 1043–1053.

Pharmacol. 23 (1994) 311–314. [25] B.G.M. Vandeginste, D.L. Massart, L.M.C. Buydens, S. De Jong, P.J. ¨

transgenic rats harbouring the mouse Ren-2 gene, Nature 344 (1990) [28] K. Witte, A. Schnecko, R.M. Buijs, J. van der Vliet, E. Scalbert, P.

541–544. Delagrange, B. Guardiola-Lemaitre, B. Lemmer, Effects of

297. data for periodic components using DQ-Fit, Chronobiol. Int. 14

[17] B. Rusak, L. McNaughton, H.A. Robertson, S.P. Hunt, Circadian (1997) 561–574.

Circ. J. 59 (1995) 565–573. 141–150.

Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol883.Issue2.2000:

Interactive report

Loss of 24 h rhythm and light-induced c-fos mRNA expression in the

suprachiasmatic nucleus of the transgenic hypertensive TGR(mRen2)27

rat and effects on cardiovascular rhythms

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Bjorn Lemmer , Stefan Hauptfleisch, Klaus Witte

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Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol883.Issue2.2000:

Interactive report

Loss of 24 h rhythm and light-induced c-fos mRNA expression in the

suprachiasmatic nucleus of the transgenic hypertensive TGR(mRen2)27

rat and effects on cardiovascular rhythms

*

¨

Bjorn Lemmer , Stefan Hauptfleisch, Klaus Witte

Parts

Dokumen yang terkait

PERENCANAAN PORTAL KELUAR KAMPUS III UMM

PERENCANAAN ULANG (REDESIGN) STRUKTUR ATAS PROYEK RUMAH SUSUN SEDERHANA 2 (RUSUNAWA) UMM MALANG DENGAN MENGGUNAKAN BALOK PRATEGANG PARSIAL

PERSEPSI MAHASISWA TENTANG MATERI TAYANGAN SANG PEMBURU DI LATIVI ( Studi Pada Mahasiswa Jurusan Ilmu Komunikasi UMM Angkatan 2002)

APRESIASI MAHASISWA TERHADAP TAYANGAN Â“OPERA VAN JAVAÂ” DI TRANS7 (Studi Pada Mahasiswa Jurusan Ilmu Komunikasi UMM Angkatan 2008)

FAKTOR-FAKTOR PENYEBAB KESULITAN BELAJAR BAHASA ARAB PADA MAHASISWA MA’HAD ABDURRAHMAN BIN AUF UMM

38 Mahasiswa UMM Pilih KKN di Padang

FPP UMM Seleksi Sarjana Membangun Desa

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Pengajian Muhammadiyah di UMM Akan Hadirkan BJ Habibie

Robert John Pope: UMM Miliki Intellectual Honesty

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