A short half-life GFP mouse model for analysis of suprachiasmatic nucleus organization - PubMed (original) (raw)

A short half-life GFP mouse model for analysis of suprachiasmatic nucleus organization

Joseph LeSauter et al. Brain Res. 2003.

Abstract

Period1 (Per1) is one of several clock genes driving the oscillatory mechanisms that mediate circadian rhythmicity. Per1 mRNA and protein are highly expressed in the suprachiasmatic nuclei, which contain oscillator cells that drive circadian rhythmicity in physiological and behavioral responses. We examined a transgenic mouse in which degradable green fluorescent protein (GFP) is driven by the mPer1 gene promoter. This mouse expresses precise free-running rhythms and characteristic light induced phase shifts. GFP protein (reporting Per1 mRNA) is expressed rhythmically as measured by either fluorescence or immunocytochemistry. In addition the animals show predicted rhythms of Per1 mRNA, PER1 and PER2 proteins. The localization of GFP overlaps with that of Per1 mRNA, PER1 and PER2 proteins. Together, these results suggest that GFP reports rhythmic Per1 expression. A surprising finding is that, at their peak expression time GFP, Per1 mRNA, PER1 and PER2 proteins are absent or not detectable in a subpopulation of SCN cells located in the core region of the nucleus.

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Figures

Fig. 1

(A) Locomotor activity of a Per1::GFP transgenic mouse in DD shows a very strong and precise rhythm. To facilitate inspection of the rhythms, the daily activity is plotted twice on a 48-h horizontal time scale (labeled 0–48 h at the top). In this animal, light pulses (white stars) presented at the end and at the beginning of the subjective night induced a phase advance of 1.95 circadian hours and a phase delay of 0.93 circadian hour. (B) The periodogram taken from day 1 to day 15 (11–14 to 11–28 included) depicts a strong amplitude rhythm with a period of 22.9 h, the line near the bottom of the plot indicates the amplitude’ significance level, _P_=0.01. (C) The phase response curve (PRC) to a 30-min light pulse given at specific time points (_x_-axis) shows phase delays and advances at the beginning and end of night, and very little effect during the middle of the day. Seven animals represented by different symbols, each received several light pulses at different times. (D) Mean wheel running activity profile for 10 Per1::GFP mice. The mice are not active during subjective day. Most of the wheel running occurs at the beginning of the subjective night, with a secondary peak at the end of subjective night.

Fig. 2

(A) _Per1_-driven GFP fluorescence cycles in the in vitro SCN. The heavy line shows the average of normalized fluorescence from four individual recordings, depicted by the differing symbols (○ ▲ ☆ –), as it changes during the course of one cycle. The gray line indicates the background staining of the outside of the SCN (right _y_-axis). (B) An image captured at ZT1.5 shows the low level of fluorescence intensity in the SCN at the beginning of the subjective day. By ZT7.5, the fluorescence intensity peaks and then begins to decline to a lower fluorescence intensity level in the SCN while the autofluorescence outside of the SCN begins to rise as demonstrated in the image captured at ZT15.5. OC: optic chiasm; 3V: third ventricle. Scale bar: 100 μm.

Fig. 3

Per1 mRNA, GFP, PER1 and PER2 are rhythmic in the SCN. Left: bar graph depicting the rhythmic circadian pattern of Per1 mRNA, GFP, PER1 (in the same animals) and PER2 (in a different group) immunoreactivity in the SCN in DD. Right: photomicrographs showing the difference in levels of GFP immunoreactivity at two times in DD.

Fig. 4

Photomicrographs showing the regional distribution of Per1 mRNA (CT6), GFP, PER1 (CT10) and PER2-ir (CT14) at five levels of the SCN from rostral to caudal. In the first column, the sections were stained with digoxigenin. The next three columns show the same double-labeled sections. In the second column, the sections were photographed with a GFP filter (Chroma, # 41020 HQ). In the third column they were photographed with a Texas red filter (Chroma, # 96109 C). The fourth column is an overlay of the first two columns showing the double-labeled cells. The fourth section shows PER2 labeled with Cy3. The distribution of all proteins and mRNA overlaps. Note the paucity of immunoreactivity in the central and lateral SCN at the 3rd and 4th levels (arrows). The bottom row is a high power view of GFP and PER1 in the medial SCN (white box on column 4, level 3). Most cells are double labeled, although with different intensities for each protein. A few cells seem to contain either GFP or PER1 alone (white arrows). This may be due to a difference in the timing of accumulation of both proteins (see Section 4). * Indicates a staining artifact (level 2).

Fig. 5

Photomicrographs showing the regional distribution of GFP in the mid to caudal SCN (levels 3 and 4 in Fig. 4) at the time of peak expression, CT 10. Double-staining for bis-benzimide shows that the regions devoid of GFP is a cell-rich zone of the SCN.

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A short half-life GFP mouse model for analysis of suprachiasmatic nucleus organization - PubMed (original) (raw)