Additional file 3: of Relationships between the acoustic startle response and prepulse inhibition in C57BL/6J mice: a large-scale meta-analytic study

Figure S2. Scatter plots of the amplitudes of acoustic startle response and percentages of prepulse inhibition of the startle response in different ages of male C57BL/6J mice. Relationships between acoustic startle responses to 110- and 120-dB pulse stimuli and percentages of prepulse inhibition were assessed by by Spearman’s rank correlation coefficients (Rho) and p values in 1363 mice in total (2–3-month old, n = 757; 4–5-month old, n = 389; 6–7-month old, n = 167; 8–12-month old, n = 50). Scatter plot of the amplitudes of startle response to pulse stimulus and percentages of prepulse inhibition in each age groups of mice at 74–110 dB (A–E), 78–110 dB (F–J), 74–120 dB (K–O), and 78–120 dB (P–T) trials. (PDF 1237 kb

Additional file 4: of Relationships between the acoustic startle response and prepulse inhibition in C57BL/6J mice: a large-scale meta-analytic study

Figure S3. Examples of relationships between the acoustic startle response and prepulse inhibition in mutant and wild-type mice. (A–C) Scatter plots of acoustic startle amplitudes and percentages of prepulse inhibition in mutant strains of mice with a C57BL/6 background (for Apc1638T/1638T mice, Onouchi et al., 2014; for Gria4−/− mice, Sagata et al., 2010; for Zfhx2−/− mice, Komine et al., 2012) that were obtained from the Mouse Phenotype Database ( http://www.mouse-phenotype.org ) show that the interpretation of differences in PPI levels between mutant and wild-type mice may be confounded by differences in basal startle reactivity (refer to Discussion and Fig. 4). The scatter plots suggest that differences in PPI levels between Apc1638T/1638T and Apc+/+ mice and between Gria4−/− and Gria4+/+ mice may result from low startle reactivity in mutants (A, B), and the lower levels of PPI in Zfhx2−/− may be a result of their higher startle reactivity (C). (PDF 472 kb

Additional file 2: of Relationships between the acoustic startle response and prepulse inhibition in C57BL/6J mice: a large-scale meta-analytic study

Figure S1. Scatter plots of the latency to peak of acoustic startle response and percentages of prepulse inhibition of the startle response in male C57BL/6J mice. Relationships between behavioral measures were assessed by Spearman’s rank correlation coefficients (Rho) and p values in 721 mice (2–3-month old, n = 423; 4–5-month old, n = 198; 6–7-month old, n = 64; 8–12-month old, n = 36). Scatter plots of the latency to peak of startle response and amplitude of startle response at 110-dB (A) and 120-dB (D) stimuli are shown, and scatter plots of the latency to peak of startle response and percentage of prepulse inhibition at 74–110 dB (B), 78–110 dB (C), 74–120 dB (E), and 78–120 dB (F) trials are presented. (PDF 530 kb

Enhanced depression-like behavior of <i>Zfhx2</i>-deficient mice.

<p>(A, B) Porsalt forced swim test: the percentage of time in immobile posture of the mutant mice was significantly higher (A) and the distance traveled by the mutant mice was significantly shorter (B) than those of the controls. (C) Tail suspension test: the percentage of time in immobile posture of the mutant mice was significantly higher than that of the control mice.</p

List of behavioral tests.

*<p>; Age (weeks old) of the youngest animals of the group at the start of the test. The oldest animals are 3 weeks (1st group) or 4 weeks (2nd group) older than the age indicated.</p>**<p>; Sections in the text where the results are described. Subsections of Results are; 2, General characteristics of <i>Zfhx2</i>-deficient mice; 3, Locomotor activity and anxiety-like behavior; 4, Depression-like behavior; 5, Sensorimotor gating; 6, Spatial reference memory; 7, Other behavioral tests.</p

Structure and expression of mouse <i>Zfhx2</i>.

<p>(A) Structure of ZFHX2 and related proteins. ZFHX2 is a protein of 2562 amino acids containing 18 zinc fingers (green ovals) and three homeodomains (red squares). (B) <i>Zfhx2</i>, <i>Zfhx3</i>, and <i>Zfhx4</i> mRNA detected by semi-quantitative RT-PCR in various RNA sources. Note that cDNAs were amplified for 30 cycles for brains of different developmental stages, whereas for 35 cycles for various adult tissues. (C–E) Expression of <i>Zfhx2</i> (C), <i>Zfhx3</i> (D), and <i>Zfhx4</i> (E) mRNA in the parasagittal sections of an E15.5 mouse brain. These three genes were expressed in substantially similar patterns with the highest expression level of <i>Zfhx3</i>. (F, G) Expression of <i>Zfhx2</i> mRNA (F) and the ZFHX2 protein (G) was compared on adjacent coronal sections of an E15.5 brain. mRNA expressed in the thalamic region (Th) was translated, whereas mRNA expressed in the cerebral cortex (Cx) was not translated: this situation made the expression patterns of ZFHX2 and ZFHX3 more alike in the protein level than in the mRNA level. (H–J) Expression of <i>Zfhx2</i> (H), <i>Zfhx3</i> (I), and <i>Zfhx4</i> (J) mRNA in the coronal sections of an adult brain. Cerebral cortex (Cx), hippocampus (Hp), thalamus (Th), caudate putamen (CP). Expression levels of all three genes were decreased compared with those in the embryonic brain, but <i>Zfhx2</i> maintained higher level of expression than the others. (K–P) Expression of ZFHX2 protein in the adult brain. The pyramidal layer of hippocampus (K, Py), the suprachiasmatic nucleus (L, SCN), laterodorsal thalamic nucleus (M, LD), lateral geniculate nucleus (N, LGN), substantia nigra pars compacta (O, SNc), and magnocellular part of the red nucleus suprachiasmic (P, RMC). (Q–Y) Double-color in situ hybridization with <i>Zfhx2</i> and tyrosine hydroxylase (<i>Th</i>) probes. The <i>Zfhx2</i> mRNA (red) was highly expressed in the <i>Th</i> mRNA (green)-positive cells in the substantia nigra pars compacta (SNc). <i>Zfhx2</i> mRNA was coexpressed with <i>Th</i> mRNA also in the ventral tegmental area (VTA) at a slightly lower level.</p

Additional file 2: Figure S1. of Age-related changes in behavior in C57BL/6J mice from young adulthood to middle age

Correlation between rotarod latency and body weight. Correlations between rotarod latency (s) and body weight (g) in (A) all age groups, (B) 2–3-month-old group, (C) 4–5-months-old group, (D) 6–7-months-old group, and (E) 8–12-months-old group. (F) Correlation between rotarod latency (s) and body weight (g) in each age group with body weights ranging from 27.5 to 32.5 g and (G) the mean rotarod latency. (EPS 2206 kb

Locomotor activity measured in open field test.

<p>Distance traveled (A, B), stereotypic behavior (C), vertical activity (D), and time spent in the center area (E). Measurements are blocked in 5 min (A, C, D, and E) or in 1 min (B, shown only for the first 10 min). The <i>Zfhx2</i>-deficient mice were generally hyperactive but were hypoactive in the initial period in the open field.</p

Spatial reference memory.

<p>Barnes maze test was performed to assess spatial reference memory. There was no significant difference in latency (A) and number of errors (B) to reach the target hole during the training period. (C) Probe trial conducted 1 day after the last training session. The results were analyzed using two-way ANOVA followed by Fisher’s PLSD post hoc test. A significant difference was found in the effect of (genotype) × (hole position ( = distance from target)) interaction (<i>F</i>_11,418 = 2.989, <i>p</i> = 0.0008), although the difference of the time spent around the target did not reach a statistically significant level (genotype effect, <i>F</i>_1,38 = 4.003, <i>p</i> = 0.0526).</p

Comprehensive Behavioral Analysis of Cluster of Differentiation 47 Knockout Mice

<div><p>Cluster of differentiation 47 (CD47) is a member of the immunoglobulin superfamily which functions as a ligand for the extracellular region of signal regulatory protein α (SIRPα), a protein which is abundantly expressed in the brain. Previous studies, including ours, have demonstrated that both CD47 and SIRPα fulfill various functions in the central nervous system (CNS), such as the modulation of synaptic transmission and neuronal cell survival. We previously reported that CD47 is involved in the regulation of depression-like behavior of mice in the forced swim test through its modulation of tyrosine phosphorylation of SIRPα. However, other potential behavioral functions of CD47 remain largely unknown. In this study, in an effort to further investigate functional roles of CD47 in the CNS, CD47 knockout (KO) mice and their wild-type littermates were subjected to a battery of behavioral tests. CD47 KO mice displayed decreased prepulse inhibition, while the startle response did not differ between genotypes. The mutants exhibited slightly but significantly decreased sociability and social novelty preference in Crawley’s three-chamber social approach test, whereas in social interaction tests in which experimental and stimulus mice have direct contact with each other in a freely moving setting in a novel environment or home cage, there were no significant differences between the genotypes. While previous studies suggested that CD47 regulates fear memory in the inhibitory avoidance test in rodents, our CD47 KO mice exhibited normal fear and spatial memory in the fear conditioning and the Barnes maze tests, respectively. These findings suggest that CD47 is potentially involved in the regulation of sensorimotor gating and social behavior in mice.</p></div