Brain Research 883 2000 250–257 www.elsevier.com locate bres
Interactive report
Loss of 24 h rhythm and light-induced c-fos mRNA expression in the suprachiasmatic nucleus of the transgenic hypertensive TGRmRen227
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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 TGRmRen227 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 TGRmRen227 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 : TGRmRen227; 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 TGRmRen227 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
1
Published on the World Wide Web on 12 October 2000.
mouse Ren2 gene, the transgenic TGRmRen227 rat is
q
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: 149-621-
inverse blood pressure rhythm in relation to heart rate and
3300333. E-mail address
: bjoern.lemmerurz.uni-heidelberg.de B. Lemmer.
activity leading to internal desynchronisation [11]. Thus,
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 8 9 - 9
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.
