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. 2024 Mar 7;14(1):138.
doi: 10.1038/s41398-023-02679-w.

Phenotypes for general behavior, activity, and body temperature in 3q29 deletion model mice

Daisuke Mori  1   2   3 , Ryosuke Ikeda  4 , Masahito Sawahata  5   6 , Sho Yamaguchi  7 , Akiko Kodama  4 , Takashi Hirao  4 , Yuko Arioka  8   9 , Hiroki Okumura  8   5 , Chihiro Inami  8   5   7 , Toshiaki Suzuki  4 , Yu Hayashi  4 , Hidekazu Kato  4 , Yoshihiro Nawa  4 , Seiko Miyata  4 , Hiroki Kimura  4 , Itaru Kushima  4   10 , Branko Aleksic  4 , Hiroyuki Mizoguchi  5 , Taku Nagai  5   11 , Takanobu Nakazawa  12 , Ryota Hashimoto  13 , Kozo Kaibuchi  14 , Kazuhiko Kume  7 , Kiyofumi Yamada  5 , Norio Ozaki  8   15
Affiliations

Phenotypes for general behavior, activity, and body temperature in 3q29 deletion model mice

Daisuke Mori et al. Transl Psychiatry. .

Abstract

Whole genome analysis has identified rare copy number variations (CNV) that are strongly involved in the pathogenesis of psychiatric disorders, and 3q29 deletion has been found to have the largest effect size. The 3q29 deletion mice model (3q29-del mice) has been established as a good pathological model for schizophrenia based on phenotypic analysis; however, circadian rhythm and sleep, which are also closely related to neuropsychiatric disorders, have not been investigated. In this study, our aims were to reevaluate the pathogenesis of 3q29-del by recreating model mice and analyzing their behavior and to identify novel new insights into the temporal activity and temperature fluctuations of the mouse model using a recently developed small implantable accelerometer chip, Nano-tag. We generated 3q29-del mice using genome editing technology and reevaluated common behavioral phenotypes. We next implanted Nano-tag in the abdominal cavity of mice for continuous measurements of long-time activity and body temperature. Our model mice exhibited weight loss similar to that of other mice reported previously. A general behavioral battery test in the model mice revealed phenotypes similar to those observed in mouse models of schizophrenia, including increased rearing frequency. Intraperitoneal implantation of Nano-tag, a miniature acceleration sensor, resulted in hypersensitive and rapid increases in the activity and body temperature of 3q29-del mice upon switching to lights-off condition. Similar to the 3q29-del mice reported previously, these mice are a promising model animals for schizophrenia. Successive quantitative analysis may provide results that could help in treating sleep disorders closely associated with neuropsychiatric disorders.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Generation of 3q29-del mice by CRISPR/Cas9.
a Structure of the human 3q29 microchromosomal region and its homologous mouse 16B2-3 region and strategies for generating mouse models of 3q29 deletion. b, c Mixture of two guide RNAs, Cas9 protein, and donor DNA were injected into the pronucleus of mouse fertilized eggs, and genotyping PCR was performed on the two F0 embryos. d Genomic DNA was prepared from the tail of No. 2 mouse in (b), and deletion of a region corresponding to the human 3q29 microchromosomal region was confirmed using array CGH for mouse. No. 2 mouse in (b) was a male, and the litter obtained by mating with female mice of the C57BL/6J strain (e); deletion of the Tfrc region from Bdh1 was also confirmed in the F1 pups by PCR (f). g Whole brain lysates were prepared from whole brains of 3q29-del and WT mice immediately after birth and immunoblotted with antibodies against DLG and PAK2, genes within the 3q29 region. The relative protein levels of both DLG1 and PAK2 were reduced by about half in 3q29-del mouse brains. P0 mouse brains were obtained from WT and 3q29-del, respectively, and DLG1 or PAK2 signals were calculated relative to GAPDH as an internal standard, and both were significantly downregulated in 3q29-del (two-tailed t-test).
Fig. 2
Fig. 2. Physical characteristics and histopathological analysis of 3q29-del mice.
a Sperm from mature 3q29-del males were artificially inseminated with oocytes from the C57BL/6J strain and implanted into sham-pregnant ICR strains. The pups were weighed every other week from 3 to 7 weeks of age, respectively. The Mann–Whitney U test showed significant differences in the weights of pups of both sexes and at all ages. b HE staining in the sagittal section of the brain of 16-week-old 3q29-del. c The immunoblot analysis for the quantification of 3q29-del brain apoptosis. Several areas of the adult brains were isolated under a stereomicroscope and prepared as total tissue lysate. Subsequently, 20 μg of protein was applied into each well. Cleaved PARP were detected using the apoptosis western blot cocktail. β-Actin was co-detected as an internal control. d For each brain region, the ratio of the signal intensity of cleaved PARP corrected for 3q29-del β-actin to WT was calculated and tested using the Mann–Whitney t-test. e, f TUNEL staining of the sagittal sections of the adult 3q29-del and WT mice. TUNEL-positive signals in the hippocampus (e) and cortex (mainly primary somatosensory cortex) (f) were measured and compared in terms of the number of signals per millimeter square. g The sagittal sections of the brains of the 3q29-del and WT mice immediately after birth were stained with TUNEL. Moreover, the TUNEL signals in the hippocampal and cortical regions were measured, and the number of signals per millimeter square was compared between the 3q29-del and WT mice. The bar indicates 200 μm. All TUNEL assays were tested for significance using the Mann–Whitney t-test.
Fig. 3
Fig. 3. Pathological analysis of PV neurons constituting the 3q29-del brain via immunostaining.
The immunohistochemistry of the brains of adult 3q29-del and WT mice. The whole brains of the mice were thinly sliced (coronal) and immunostained with anti-Parvalbumin (PV) antibodies. The fluorescence of the whole sections was observed, and the granular signals were measured in four regions with the help of Fiji (Image J2): cortex, hippocampus, thalamus, and hypothalamus. Furthermore, the number of signals per mm2 was compared between the WT (a) and 3q29-del (b) mice, and statistical analysis was performed using Student’s t-test (c). Bars = 1 mm.
Fig. 4
Fig. 4. General behavioral analysis of 3q29-del model mice.
a Result of locomotor activity test. Total activity over a 2 h measurement period (top left), activity every 5 min (top right), activity divided into the first half hour and the second half hour (center left). b Comparison of the number of rearing observed under free movement of mice in the open field test. c Result of novel object recognition test. Data represent the mean ± SEM (N = 11 for WT male mice, N = 12 for WT female mice, N = 11 for 3q29-del male mice, N = 12 for 3q29-del female mice). Refer also Table 1 and Supplementary Fig. 2.
Fig. 5
Fig. 5. 24-h locomotor activity test.
a Total activity in 24 h (WT mice: N = 10; 3q29-del mice: N = 10). b Graph of activity plotted hourly for the results in (a). Indicated as mean ± SE of 10 animals. Multiple t-tests were performed at each time period. c Graph of activity every 15 min for the 6 h after the switch from light-on to light-off in the measurement in (b).
Fig. 6
Fig. 6. Long-term activity and body temperature measurement with a miniature accelerometer: Nano-tag.
a, b The data are from the L-D cycle of a mouse model in which the Nano-Tag was surgically implanted intraperitoneally and displayed by the Nano-Tag Viewer software. Data for the first 20 days are shown. The horizontal axis is the time axis from ZT15 (AM0) to ZT15 (PM12) of the next day, the histograms on the vertical axis show the amount of activity for each 5 min, and the line graphs show the body temperature; (a) WT, (b) 3q29-del mice. Overall data are shown in Supplementary Fig. 4. Mean ± standard error of 6-week data from five animals each of (c) WT (d) 3q29-del measured in the L-D cycle plotted by time. The vertical axis shows activity in 5 min and the horizontal axis shows time in twenty-four hrs. e, f Enlarged views from the data in (c) and (d) for the 3 h from ZT11 to ZT14. g Activity phase analysis under L-D cycle conditions [53]. The time at which the 24 h moving average activity level exceeded the 3 h moving average activity level was defined as the onset of activity (onset) and the time at which it fell below (offset). The middle point was defined as the point at which the cumulative amount of activity from the start of the activity was half of the total amount of activity for the entire period (16 h). The time of the maximum value of the 1-h moving average was set as (peak). Statistical analysis was performed with a two-tailed t-test and was significant for onset (p = 0.001). h Circadian cycle determined by FFT analysis from each individual’s data in the D-D cycle for 13 days. According to t-test, 3q29-del was 5.3 min longer than WT, and the difference from WT was significant (p = 0.009).
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