Comprehensive behavioral phenotyping of a new Semaphorin 3 F mutant mouse

Abstract

Background: Semaphorin 3 F (Sema3F) is a secreted type of the Semaphorin family of axon guidance molecules. Sema3F and its receptor neuropilin-2 (Npn-2) are expressed in a mutually exclusive manner in the embryonic mouse brain regions including olfactory bulb, hippocampus, and cerebral cortex. Sema3F is thought to have physiological functions in the formation of neuronal circuitry and its refinement. However, functional roles of Sema3F in the brain remain to be clarified. Here, we examined behavioral effects of Sema3F deficiency through a comprehensive behavioral test battery in Sema3F knockout (KO) male mice to understand the possible functions of Sema3F in the brain.

Results: Male Sema3F KO and wild-type (WT) control mice were subjected to a battery of behavioral tests, including neurological screen, rotarod, hot plate, prepulse inhibition, light/dark transition, open field, elevated plus maze, social interaction, Porsolt forced swim, tail suspension, Barnes maze, and fear conditioning tests. In the open field test, Sema3F KO mice traveled shorter distance and spent less time in the center of the field than WT controls during the early testing period. In the light/dark transition test, Sema3F KO mice also exhibited decreased distance traveled, fewer number of transitions, and longer latency to enter the light chamber compared with WT mice. In addition, Sema3F KO mice traveled shorter distance than WT mice in the elevated plus maze test, although there were no differences between genotypes in open arm entries and time spent in open arms. Similarly, Sema3F KO mice showed decreased distance traveled in the social interaction test. Sema3F KO mice displayed reduced immobility in the Porsolt forced swim test whereas there was no difference in immobility between genotypes in the tail suspension test. In the fear conditioning test, Sema3F KO mice exhibited increased freezing behavior when exposed to a conditioning context and an altered context in absence of a conditioned stimulus. In the tests for assessing motor function, pain sensitivity, startle response to an acoustic stimulus, sensorimotor gating, or spatial reference memory, there were no significant behavioral differences between Sema3F KO and WT mice.

Conclusions: These results suggest that Sema3F deficiency induces decreased locomotor activity and possibly abnormal anxiety-related behaviors and also enhances contextual memory and generalized fear in mice. Thus, our findings suggest that Sema3F plays important roles in the development of neuronal circuitry underlying the regulation of some aspects of anxiety and fear responses.

Fig. 1

General health and neurological screen. a body weight, (b) body temperature, (c) grip strength, and (d) wire hang latency. Data indicate means ± SEM (n = 19 for Sema3F KO mice; n = 20 for WT controls)

Fig. 2

Normal motor coordination, pain sensitivity and sensorimotor function in Sema3F KO mice. a The latency to fall from an accelerating rotarod was measured by three trials per day for two consecutive days in the rotarod test. There was no significant difference in the latency to fall between Sema3F KO mice and WT controls. b The hot plate test was used to evaluate sensitivity to a painful stimulus. Mice were placed on a hot plate and latency to the first hindpaw response was recorded. There was no significant difference in the latency between the genotypes. c, d The startle response/prepulse inhibition tests were performed to examine startle responses to loud stimuli (110 or 120 dB) and inhibition of the startle response by prepulse stimulus (74 or 78 dB). c The startle response and (d) the prepulse inhibition were not significantly different between Sema3F KO mice and WT controls. Data indicate means ± SEM (n = 19 for Sema3F KO mice; n = 20 for WT controls)

Fig. 3

Decreased locomotor activity and increased anxiety-related behavior in Sema3F KO mice. a-d Light/dark transition test. a Distance traveled, (b) number of transitions between the light and dark chambers, (c) latency to enter the light chamber, and (d) stay time in the light chamber (n = 17 for Sema3F KO mice; n = 18 for WT controls). e-h Open field test. e Distance traveled, (f) vertical activity, (g) time spent in center area, and (h) stereotypic behavior counts (n = 19 for Sema3F KO mice; n = 20 for WT controls). i-m Elevated plus maze test. i Number of total entries into arms, (j) distance traveled, (k) percentage of entries into open arms, (l) percentage of time spent in open arms, and (m) time spent in open arms, center area, and closed arms (n = 19 for Sema3F KO mice; n = 20 for WT controls). n-r Social interaction test in a novel environment. n Number of contacts, (o) total duration of contacts, (p) total duration of active contacts, (q) mean duration per contact, and (r) distance traveled (n = 9 for each genotype)

Fig. 4

Hyperactivity in forced swim test in Sema3F KO mice. a, b Porsolt forced swim test. Mice were placed in a water-filled cylinder, and immobility time was measured every 1 min for a 10-min period on the first and second days. Sema3F KO mice spent significantly less time in immobility (a) and traveled significantly longer distances (b) than their WT controls. N = 19 for Sema3F KO mice; N = 20 for WT controls. c Tail suspension test. There was no significant difference in immobility between Sema3F KO mice and WT controls. All data indicate means ± SEM (n = 18 for Sema3F KO mice; n = 15 for WT controls)

Fig. 5

Increased fear memory in Sema3F KO mice. Freezing behavior was measured in the contextual and cued fear conditioning test to assess fear memory in Sema3F KO mice. a There were no significant differences in freezing between Sema3F KO mice and WT controls during the conditioning session. b, c Sema3F KO mice exhibited more freezing than WT controls in the re-exposure to the same context approximately 24 hours after the conditioning session and in the altered context with absence or presence of the auditory cue. Data indicate means ± SEM (n = 18 for Sema3F KO mice; n = 15 for WT controls)

Fig. 6

Normal spatial reference memory in Sema3F KO mice. Spatial reference memory was tested in the Barnes circular maze. a, b During the training session, there were no significant differences in the number of errors and latency to reach the correct target hole between Sema3F KO mice and WT controls. c, d In the retention probe tests, 1 day and 10 days after the last training session, Sema3F KO mice and WT controls spent similar time in the correct target hole. Data indicate means ± SEM (n = 18 for Sema3F KO mice; n = 15 for WT controls)