Comprehensive behavioral test battery of CD47 KO mice.

<p>Age (w): age in weeks at the beginning of each test.</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

Physical characteristics, neuromuscular strength, motor coordination, nociception, and gait in CD47 KO and wild-type mice.

<p>No significant differences between genotypes were detected in body weight (A), body temperature (B), grip strength (C), or latency to fall in the wire hang test (D). (E) Likewise, no significant differences between the genotypes were observed in latency to fall from the rotarod (F) or latency to the first paw response in the hot plate test. CD47 KO mice, n = 20; wild-type mice, n = 20. (G–H) CD47 KO mice were also subjected to gait analysis. (G) The mutants showed significant decreases in stance width for front and hind paws compared to wild-type mice. (H) Paw angles for hind paws, but not for front paws, were significantly narrower in the mutants than in wild-type mice. CD47 KO mice, n = 19; wild-type mice, n = 20.</p

Normal startle response and decreased PPI in CD47 KO mice.

<p>(A) CD47 KO mice demonstrated normal acoustic startle responses to the 110 dB and 120 dB startle stimuli. (B) CD47 KO mice showed a significant reduction in PPI for the 74 and 78 dB prepulse sound level compared to wild-type mice. PPI in the mutants was significantly lower than that in wild-type mice when the startle stimulus was 120 dB, whereas only a trend for reduced PPI was observed in the mutants when startle stimulus was 110 dB. CD47 KO mice, n = 20; wild-type mice, n = 20.</p

Normal social behaviors of CD47 KO mice in a novel environment and the home cage.

<p>(A–E) In the social interaction test performed in a novel environment, there were no significant differences between the genotypes in total duration of contacts (A), number of contacts (B), total duration of active contacts (C), mean duration per contact (D), or distance traveled (E). CD47 KO mice, n = 10 pairs; wild-type mice, n = 10 pairs. (F, G) Social interaction in the home cage was monitored over a 9-day period. There were no significant differences between the genotypes in the mean numbers of particles, i.e., whether 1 (mice together) or 2 (mice apart) subjects were detected (F) or activity level (G). CD47 KO mice, n = 8 pairs; wild-type mice, n = 8 pairs.</p

Normal locomotor activity in CD47 KO mice.

<p>In the open field test, (A) CD47 KO mice stayed significantly longer in the center of area of the apparatus than wild-type mice during the earlier part of the experimental period, but not over the entire experimental period. No significant differences were detected in total distance traveled (B), vertical activity (C), or stereotypic counts (D). CD47 KO mice, n = 19; wild-type mice, n = 20. CD47 KO mice and wild-type mice were also subjected to the light/dark transition test (E–F). Time spent in the light box (E) and number of transitions (F) did not significantly differ between genotypes. CD47 KO mice, n = 20; wild-type mice, n = 20. The elevated plus maze test was also performed (G–H). There were no significant differences between the genotypes in the number of entries to the open arms (G) or time spent on the open arms (H). CD47 KO mice, n = 20; wild-type mice, n = 20.</p

Normal depression-like behavior of CD47 KO mice in the tail suspension test.

<p>There was no significant genotype effect for the percentage of immobility time in the tail suspension test. CD47 KO mice, n = 19; wild-type mice, n = 20.</p

Normal fear and spatial memory in CD47 KO mice.

<p>(A) In the fear conditioning test, no significant differences between genotypes were observed in freezing during conditioning, context testing, or cued testing in an altered context. CD47 KO mice, n = 19; wild-type mice, n = 20. (B–E) In the training session of the Barnes maze test, no significant differences between the genotypes were detected in the latency to the 1^st hole (the escape box) (B), or error to the 1^st hole in the probe test (C). In a retention test performed either 24 h (D) or 2 weeks (E) after the last training session, there were no significant differences between the genotypes in time spent around the hole where the escape box had been placed (target). CD47 KO mice, n = 19; wild-type mice, n = 20.</p

Survival of TH-positive neurons and outgrowth from SIRPα mutant cultures.

<p>TH-immunohistochemistry over the tissue slice area demonstrates that the TH-positive neurons appeared healthy in control culture (a), and cultures from CD47^−/− (b), SIRPα mutant (c), and control culture treated with antibodies against TSP-1 (d). Measurements of TH-positive nerve fiber density (e), length (f), and astrocytic migration revealed similar values for SIRPα wildtypes and mutants. Scale bar = 25 µm.</p

TH-positive nerve fiber outgrowth and astrocytic migration in CD47^−/− cultures.

<p>TH-positive nerve fiber outgrowth and vimentin–positive astrocytic migration from CD47^+/+ (a, c) and CD47^−/− (b, d) VM cultures after 7 (a, b) and 14 (c, d) DIV. The astrocytic migration was shorter at 7 DIV as compared to 14 DIV. Short TH-positive nerve fiber outgrowth was observed in CD47^+/+ (a) and CD47^−/− (b) cultures at 7 DIV, while at 14 DIV the TH-positive nerve fibers had reached over longer distances and with higher density in CD47^−/− cultures (d) compared to control cultures (c). The dotted line delineates the border of the tissue slice. Scale bar = 100 µm.</p