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. 2018 Apr 27;13(4):e0196587.
doi: 10.1371/journal.pone.0196587. eCollection 2018.

Comprehensive behavioral analysis of the Cdkl5 knockout mice revealed significant enhancement in anxiety- and fear-related behaviors and impairment in both acquisition and long-term retention of spatial reference memory

Kosuke Okuda  1 , Keizo Takao  2 , Aya Watanabe  1 , Tsuyoshi Miyakawa  2   3 , Masashi Mizuguchi  1 , Teruyuki Tanaka  1
Affiliations

Comprehensive behavioral analysis of the Cdkl5 knockout mice revealed significant enhancement in anxiety- and fear-related behaviors and impairment in both acquisition and long-term retention of spatial reference memory

Kosuke Okuda et al. PLoS One. .

Abstract

Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders. Recently we have generated Cdkl5 KO mice by targeting exon 2 on the C57BL/6N background, and demonstrated postsynaptic overaccumulation of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the hippocampus. In the current study, we subjected the Cdkl5 KO mice to a battery of comprehensive behavioral tests, aiming to reveal the effects of loss of CDKL5 in a whole perspective of motor, emotional, social, and cognition/memory functions, and to identify its undetermined roles. The neurological screen, rotarod, hot plate, prepulse inhibition, light/dark transition, open field, elevated plus maze, Porsolt forced swim, tail suspension, one-chamber and three-chamber social interaction, 24-h home cage monitoring, contextual and cued fear conditioning, Barnes maze, and T-maze tests were applied on adult Cdkl5 -/Y and +/Y mice. Cdkl5 -/Y mice showed a mild alteration in the gait. Analyses of emotional behaviors revealed significantly enhanced anxiety-like behaviors of Cdkl5 -/Y mice. Depressive-like behaviors and social interaction of Cdkl5 -/Y mice were uniquely altered. The contextual and cued fear conditioning of Cdkl5 -/Y mice were comparable to control mice; however, Cdkl5 -/Y mice showed a significantly increased freezing time and a significantly decreased distance traveled during the pretone period in the altered context. Both acquisition and long-term retention of spatial reference memory were significantly impaired. The morphometric analysis of hippocampal CA1 pyramidal neurons revealed impaired dendritic arborization and immature spine development in Cdkl5 -/Y mice. These results indicate that CDKL5 plays significant roles in regulating emotional behaviors especially on anxiety- and fear-related responses, and in both acquisition and long-term retention of spatial reference memory, which suggests that focus and special attention should be paid to the specific mechanisms of these deficits in the CDKL5 deficiency disorder.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. General health and neurological screening.
(A) There is no significant difference in the body weight between Cdkl5 +/Y and -/Y mice. (B) Cdkl5 -/Y mice show a significantly lower body temperature than control mice. (C) There is no significant difference in the grip strength between two genotypes. (D) There is no significant difference in the wire hang latency between two genotypes. (E) There is no significant difference in the hot plate test latency between two genotypes. (F) Acoustic startle response. There is no significant difference between two genotypes either at 110dB or 120 dB sound level. (G) There are no significant differences in the prepulse inhibition (PPI) (%) between genotypes. In Cdkl5 +/Y mice, the PPI induced by 78dB prepulse was larger than that of 74dB prepulse in both startle conditions; however in Cdkl5 -/Y mice, 78dB prepulse does not induce larger PPI over 74dB prepulse in either startle condition, and the genotype difference of PPI induced by 78dB prepulse in 120dB startle condition is markedly large. Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 22. Data indicate means ± SEM.
Fig 2
Fig 2. Cdkl5 -/Y mice exhibit a mild alteration in the gait.
(A) Gait analysis of front paws. Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 19. (i) Percent of stride duration. Cdkl5 KO mice show a significantly lower percentage of brake duration. (ii) Percent of stance duration. There is no significant difference in the duration of brake or propel between two genotypes. (iii) Cdkl5 -/Y mice show a significantly shorter stance width. (iv) There is no significant difference in the stride length between two genotypes. (v) There is no significant difference in the step angle between two genotypes. (vi) There is no significant difference in the paw angle between two genotypes. (B) Gait analysis of hind paws. Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 19. (i) There are no significant differences in the duration of swing, brake, or propel between two genotypes. (ii) There is no significant difference in the duration of brake or propel between two genotypes. (iii) Cdkl5 -/Y mice show a significantly shorter stance width. (iv) There is no significant difference in the stride length between two genotypes. (v) There is no significant difference in the step angle between two genotypes. (vi) Cdkl5 -/Y mice show a significantly small paw angle compared with the control. (C) The rotarod test. There is no significant difference in the latency to fall between two genotypes. Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 22. Data indicate means ± SEM.
Fig 3
Fig 3. Cdkl5 -/Y mice show significantly enhanced anxiety-like behaviors.
(A-D) The light/dark transition test. (A) Cdkl5 -/Y mice show a significantly decreased distance traveled in the light chamber and an increased distance in the dark chamber. (B) Cdkl5 -/Y mice show a significantly decreased stay time in the light chamber. (C) Cdkl5 -/Y mice show a significantly decreased number of transitions between chambers. (D) Cdkl5 -/Y mice show a tendency to increase in the latency to light chamber, but without statistical significance. (E-H) The open field test. (E) (i) Total distance traveled (cm) in 120 minutes. Each bin represents a mean value in 5 minutes. There is no significant difference between two genotypes (p = 0.8038). (ii) Total distance traveled during the first 5 minutes. Each bin represents a mean value in one minute. Marked reduction in the distance traveled of Cdkl5 -/Y mice is observed. (F) The vertical activity. Cdkl5 -/Y mice show a significantly decreased vertical activity in the whole period, compared with the control mice (p = 0.0001). There is a significant time x genotype effect (p = 0.0004). (G) Center time (sec). In the whole period, there is no significant difference of the center time between two genotypes (p = 0.1719). However, in the first 5 minutes (first bin), Cdkl5 -/Y mice show a marked decrease in the center time. (H) Stereotypic counts. In the whole period, there is no significant difference between two genotypes (p = 0.1719), but time x genotype effect is significant (p < 0.0001). (I-L) The elevated plus maze test. (I) Cdkl5 -/Y mice show a significantly decreased number of entries into all arms. (J) Cdkl5 -/Y mice show a significantly decreased distance traveled. (K) There is no difference in the number of entries into open arms between two genotypes. (L) Cdkl5 -/Y mice show a significantly decreased time spent on the open arms. Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 22. Data indicate means ± SEM.
Fig 4
Fig 4. Cdkl5 -/Y mice show altered depressive-like behaviors.
(A, B) The Porsolt forced swim test. (A) Cdkl5 -/Y mice show a significant decrease of immobility time in the first day (p < 0.0001), and a tendency to decrease in the second day (p = 0.0672). (B) There is no significant difference in the distance traveled between two genotypes either in day 1 or day 2. (C) The tail suspension test. Cdkl5 -/Y mice show a significant increase of the immobility time (p = 0.0105). Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 22. Data indicate means ± SEM.
Fig 5
Fig 5. Social interaction of Cdkl5 -/Y mice is uniquely altered.
(A-E) The social interaction test in a novel environment (one-chamber test). Cdkl5 -/Y mice show a significant increase in the total duration of contacts (A), a significant increase in the mean duration per contact (B), a significant decrease in the number of contacts (C), a significant decrease in the total duration of active contacts (D), and a significant decrease in the distance traveled (E). Cdkl5 +/Y, n = 11 pairs; Cdkl5 -/Y, n = 11 pairs. Data indicate means ± SEM. (F-K) The three-chamber social interaction test. Cdkl5 -/Y mice show a significant decrease in the total distance traveled (F), and a tendency toward decrease in the average speed (G). (H) Time spent in each chamber (sec). Cdkl5 -/Y mice show a significant preference for the center chamber, whereas Cdkl5 +/Y mice spend significantly more time in other chambers. (I) Time spent around cage (sec). Cdkl5 +/Y mice spend significantly more time around the mouse cage compared to the empty cage. Cdkl5 -/Y mice showed a tendency toward preference for the mouse cage without statistical significance. (J) Number of entries in each chamber. Both genotypes similarly show a significant difference between the center and stranger chamber, a significant difference between the center and empty chamber, and no significant difference between the stranger and empty chamber. (K) Number of entries around cage. Both genotypes show no significant difference in the number of entries around cage between the stranger side and the empty side. Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 22. Data indicate means ± SEM.
Fig 6
Fig 6. Twenty-four hour monitoring revealed increased interaction and decreased activity during daytime in Cdkl5 -/Y mice.
(A) (i) Mean number of particles. Cdkl5 -/Y mice spend significantly more time staying together compared to Cdkl5 +/Y mice during daytime (p < 0.001), but not in the nighttime (p = 0.2436). The genotype difference during the whole period was significant (p = 0.0012). (ii) Activity level (A.U.). The activity level of Cdkl5 -/Y mice is significantly decreased compared to Cdkl5 +/Y mice during daytime (p = 0.0004), but not in the nighttime (p = 0.1535). The genotype difference during the whole period was not significant (p = 0.791). (B) Mean values in the middle three days. Cdkl5 -/Y mice show significant decreases in the number of particles (i) and activity level (ii) during daytime. Cdkl5 +/Y, n = 11 pairs; Cdkl5 -/Y, n = 11 pairs. Data indicate means ± SEM.
Fig 7
Fig 7. Cdkl5 -/Y mice show an increased freezing time and a decreased distance traveled during the pretone period in the altered context.
(A) Distance traveled (cm) at footshock 1 (FS1) (i), footshock 2 (FS2) (ii), and footshock 3 (FS3) (iii) during conditioning. Cdkl5 -/Y mice show a significantly decreased distance traveled in response to the first footshock (p = 0.0158) compared with control mice. There are no significant differences upon the second and third footshock between two genotypes. (B) Cdkl5 -/Y mice show a significantly decreased freezing time during conditioning. (C) (i,ii) Contextual testing one day after conditioning. There are no significant differences in the freezing time (i) and distance traveled (ii) between two genotypes. (iii,iv) Cued testing with altered context one day after conditioning. Cdkl5 -/Y mice show a significantly increased freezing time (p = 0.0002) (iii) and a significantly decreased distance traveled (p = 0.0038) (iv) during the pretone period. There are no significant differences in the freezing time (iii) and distance traveled (iv) in response to the conditioned tone. (D) (i,ii) Contextual testing seven days after conditioning. There are no significant differences in the freezing time (i) and distance traveled (ii) between two genotypes. (iii,iv) Cued testing with altered context seven days after conditioning. Cdkl5 -/Y mice show a significantly increased freezing time (p = 0.0006) (iii) and a significantly decreased distance traveled (p = 0.0448) (iv) during the pretone period. There are no significant differences in the freezing time (iii) and distance traveled (iv) in response to the conditioned tone. Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 22. Data indicate means ± SEM.
Fig 8
Fig 8. Acquisition and long-term retention of reference memory, and working memory are significantly impaired in Cdkl5 -/Y mice.
(A-F) Barnes maze test. Number of errors (A), latency (B), distance traveled (C) to reach the target during training. Cdkl5 -/Y mice show a significant increase in the number of errors (p = 0.0002) (A), latency (p < 0.0001) (B) and distance traveled (p < 0.0001) (C) to reach the target hole, compared to the control mice. (D) In the first probe test (PT1) performed 24 hours after the 12th training, Cdkl5 -/Y mice spend significantly less time at the target hole than control mice (p = 0.0009), and spend similar time at the adjacent hole to the target hole (p = 0.1869). (E) In the second probe test (PT2) performed 24 hours after the 21st training, Cdkl5 -/Y mice spend similar time at the target hole to the control mice (p = 0.1615), and spend significantly more time at the target hole compared to the adjacent hole (p = 0.0031). (F) In the third probe test after 30 days, Cdkl5 -/Y mice spend significantly less time at the target hole than control mice (p = 0.0167), and spend similar time at the adjacent hole to the target hole (p = 0.3926). (G-J) T-maze test. (G) Cdkl5 -/Y mice show a significantly decreased percentage of correct responses than control mice in all 3 trials. (H) Cdkl5 -/Y mice spend significantly longer time to finish 10 trials than control mice. (I) There is no significant difference in the distance traveled between two genotypes. (J) Scheme of the T-maze. Cdkl5 +/Y, n = 22; Cdkl5 -/Y, n = 22. Data indicate means ± SEM.
Fig 9
Fig 9. Dendritic arborization and spine geometry of hippocampal CA1 pyramidal neurons are impaired in Cdkl5 -/Y mice.
Results for basal dendrites (A-H) and apical dendrites (I-P). Cdkl5 +/Y, n = 34 neurons; Cdkl5 -/Y, n = 32 neurons. Bar graphs (A-D, I-L) show overall mean values, and line graphs (E-H, M-P) show mean values on 10-μm increment concentric circles. There are no significant differences in the mean numbers of basal and apical dendrites between two genotypes (A,I). The number of nodes (B,F), endings (C,G), intersections (E), and length (D,H) of basal dendrites are significantly decreased in Cdkl5 -/Y mice. The numbers of intersections (M) and length (P) of apical dendrites are significantly decreased in Cdkl5 -/Y mice. (Q,R) Morphometric analysis of spines on apical dendrites. Cdkl5 +/Y, 8,150 spines, n = 29 neurons; -/Y, 6,831 spines, n = 32 neurons. The mean length (Q) and density (R) are increased in Cdkl5 -/Y mice. (S) Spine-type classification analysis. Cdkl5 +/Y, 3,442 spines, n = 15 neurons; -/Y, 4,294 spines, n = 15 neurons. Cdkl5 -/Y mice show a significantly lower percentage of stubby spines and a significantly higher percentage of thin spines. (A-D, I-L) are analyzed by ANOVA, (E-H, M-P) are analyzed by repeated measure ANOVA, and (Q-S) are analyzed by Student’s t-test. Data indicate mean ± SEM.
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This work was supported by Grants-in-Aid for Scientific Research 21659252, 23500381, 15K09614 (TT, MM) and Research Fellowship for Young Scientists 12J04298 (KO) from Japan Society for the Promotion of Science (http://www.jsps.go.jp/english/), Grants-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) (TT, KO, KT, TM) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.mext.go.jp/en/), Comprehensive Research on Disability Health and Welfare (Neurological and Muscle diseases) Grant 24-007 of the Ministry of Health, Labor and Welfare of Japan (http://www.mhlw.go.jp/english/) (TT), research grants from The Japan Epilepsy Research Foundation (http://www.epi-fj.jp) (TT), The Mother and Child Health Foundation (http://www.glico.co.jp/boshi/) (TT), and Japan Rett Syndrome Support Organization (http://www.npo-rett.jp) (TT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.