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. 2013 Oct 24:7:152.
doi: 10.3389/fnbeh.2013.00152. eCollection 2013.

Cognitive dysfunction, elevated anxiety, and reduced cocaine response in circadian clock-deficient cryptochrome knockout mice

Dimitri De Bundel  1 , Giuseppe Gangarossa, Anne Biever, Xavier Bonnefont, Emmanuel Valjent
Affiliations

Cognitive dysfunction, elevated anxiety, and reduced cocaine response in circadian clock-deficient cryptochrome knockout mice

Dimitri De Bundel et al. Front Behav Neurosci. .

Abstract

The circadian clock comprises a set of genes involved in cell-autonomous transcriptional feedback loops that orchestrate the expression of a range of downstream genes, driving circadian patterns of behavior. Cognitive dysfunction, mood disorders, anxiety disorders, and substance abuse disorders have been associated with disruptions in circadian rhythm and circadian clock genes, but the causal relationship of these associations is still poorly understood. In the present study, we investigate the effect of genetic disruption of the circadian clock, through deletion of both paralogs of the core gene cryptochrome (Cry1 and Cry2). Mice lacking Cry1 and Cry2 (Cry1(-/-)Cry2(-/-) ) displayed attenuated dark phase and novelty-induced locomotor activity. Moreover, they showed impaired recognition memory but intact fear memory. Depression-related behaviors in the forced swim test or sucrose preference tests were unaffected but Cry1(-/-)Cry2(-/-) mice displayed increased anxiety in the open field and elevated plus maze tests. Finally, hyperlocomotion and striatal phosphorylation of extracellular signal-regulated kinase (ERK) induced by a single cocaine administration are strongly reduced in Cry1(-/-)Cry2(-/-) mice. Interestingly, only some behavioral measures were affected in mice lacking either Cry1 or Cry2. Notably, recognition memory was impaired in both Cry1(-/-)Cry2(+/+) and Cry1(+/+)Cry2(-/-) mice. Moreover, we further observed elevated anxiety in Cry1(-/-)Cry2(+/+) and Cry1(+/+)Cry2(-/-) mice. Our data indicate that beyond their role in the control of circadian rhythm, cryptochrome genes have a direct influence in cognitive function, anxiety-related behaviors and sensitivity to psychostimulant drugs.

Keywords: Cry1 and Cry2 knockout mice; learning; mood-related disorder; psychostimulants.

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Figures

Figure 1
Figure 1
Spontaneous locomotor activity in Cry1−/−Cry2−/− mice. (A) When exposed to an enclosed environment, horizontal activity (top panel) declined more rapidly [Genotype × Time: F(3, 111) = 3.29, P = 0.023; Genotype: F(1, 37) = 3.70, P = 0.062; Time: F(3, 111) = 69.21, P < 0.0001] and vertical activity (bottom panel) remained low [Genotype × Time: F(3, 111) = 0.27, P = 0.84; Genotype: F(1, 37) = 14.43, P = 0.0005; Time: F(3, 111) = 8.66, P < 0.0001] during the initial habituation period in Cry1−/−Cry2−/− mice (n = 18) compared to wild type (WT) mice (n = 21). (B) Over a circadian period, the increase in horizontal activity during the dark phase (top panel) was lower [Genotype × Time: F(95, 1520) = 3.48, P < 0.0001; Genotype: F(1, 16) = 49.60, P = 0.015; Time: F(95, 1520) = 18.54, P < 0.0001] in Cry1−/−Cry2−/− mice compared to WT mice. Similarly, the increase in vertical activity during the dark phase (bottom panel) was less pronounced [Genotype × Time: F(95, 1520) = 1.59, P = 0.0003; Genotype: F(1, 16) = 1.79, P = 0.20; Time: F(95, 1520) = 4.89, P < 0.0001] in Cry1−/−Cry2−/− mice (n = 10) compared to WT mice (n = 8). *P < 0.05 Cry1−/−Cry2−/− mice vs. WT mice.
Figure 2
Figure 2
Cognitive processing, depression-related behavior, and anxiety-related behavior in Cry1−/−Cry2−/− mice. (A) During the training phase of the object recognition test (left panel), Cry1−/−Cry2−/− mice (n = 11) required more time [t(26) = 5.971, P < 0.0001] to reach the criterion of 40 s total object exploration compared to wild type (WT) mice (n = 17). During the test phase, 24 h later, Cry1−/−Cry2−/− mice did not show a preference for the novel object as opposed [t(26) = 3.895, P = 0.0006] to WT mice, which had a retention index above 0 [t(16) = 7.389, P < 0.0001]. (B) In the auditory fear conditioning test, Cry1−/−Cry2−/− mice (n = 8) readily associate the tone with the ensuing foot shock, but display higher [Genotype × Time: F(3, 45) = 2.52, P = 0.07; Genotype: F(1, 45) = 10.81, P = 0.005; Time: F(3, 45) = 58.98, P < 0.0001] freezing levels compared to WT mice (n = 10). During the test, 24 h later, both genotypes showed an elevated freezing response to tone presentation [Genotype × Time: F(17, 204) = 0.58, P = 0.9; Genotype: F(1, 204) = 0.03, P = 0.86; Time: F(17, 204) = 30.32, P < 0.0001]. (C) In the forced swim test, immobility times did not differ [Genotype × Time: F(4, 96) = 1.52, P = 0.20; Genotype: F(1, 96) = 0.03, P = 0.87; Time: F(4, 96) = 47.61, P < 0.0001] between Cry1−/−Cry2−/− mice (n = 8) and WT mice (n = 13). (D) In the sucrose preference test, both Cry1−/−Cry2−/− mice (n = 8) and WT mice (n = 12) expressed a clear preference (left panel) for 1% sucrose over water [Genotype × Treatment: F(5, 90) = 1.05, P = 0.39; Genotype: F(1, 90) = 0.12, P = 0.73; Treatment: F(5, 90) = 5.91, P < 0.0001] and total fluid intake (right panel) was significantly higher for 1% sucrose over water in both genotypes [Genotype × Treatment: F(5, 90) = 1.05, P = 0.39; Genotype: F(1, 90) = 0.12, P = 0.73; Treatment: F(2, 26) = 10.75, P < 0.0004]. (E) In the open field test, overall activity (left panel) was lower [t(36) = 3.498, P = 0.0013] in Cry1−/−Cry2−/− mice compared to WT mice. Moreover, the time spent in the center of the open field (right panel) was lower [t(36) = 7.225, P < 0.0001] in Cry1−/−Cry2−/− mice (n = 18) compared to WT mice (n = 20). (F) In the elevated plus maze test, Cry1−/−Cry2−/− mice (n = 18) spent less [t(47) = 5.75, P < 0.0001] time in the open arms compared to WT mice (n = 20). Moreover, the number of arm entries was lower for the open arms but not for the closed arms of the plus maze in Cry1−/−Cry2−/− mice compared to WT mice [Genotype × Arm: F(1, 51) = 11.80, P = 0.0012; Genotype: F(1, 51) = 11.43, P = 0.0014; Arm: F(17, 204) = 44.67, P < 0.0001]. (***P < 0.001 Cry1−/−Cry2−/− mice vs. WT mice. +P < 0.05 vs. chance level. +++P < 0.001 vs. chance level. °°°P < 0.001 for treatment or time effect.
Figure 3
Figure 3
Cocaine-induced acute hyperlocomotion and locomotor sensitization in Cry1−/−Cry2−/− mice. (A and C) Locomotor activity induced by repeated cocaine administration (7.5 and 15 mg/kg) in wild type (WT) and Cry1−/−Cry2−/− mice (n = 10–12 per experimental group). Data (means ± sem) were analyzed using Two-Way ANOVA repeated measures: A [Time × Genotype: F(4, 76) = 2.783, P = 0.0325; Time: F(4, 76) = 17.95, P < 0.0001; Genotype: F(1, 19) = 3.883, P = 0.0635], C [Time × Genotype: F(4, 76) = 3.302, P = 0.015; Time: F(4, 76) = 53.45, P < 0.0001; Genotype: F(1, 19) = 17.98, P = 0.0004]. (B and D) Cumulative locomotor activity of WT and Cry1−/−Cry2−/− mice over a 30 min period post cocaine administration. Data (means ± sem) were analyzed using Two-Way ANOVA repeated measures: (B) [Time × Genotype: F(2, 38) = 3.907, P = 0.0288; Time: F(2, 38) = 41.05, P < 0.0001; Genotype: F(1, 19) = 8.998, P = 0.0074], (D) [Time × Genotype: F(2, 38) = 0.8807, P = 0.4228; Time: F(2, 38) = 48.61, P < 0.0001; Genotype: F(1, 19) = 14.73, P = 0.0012]. (E) Locomotor activity induced by repeated cocaine administration (15 mg/kg) in WT (n = 12), Cry1−/−/Cry2+/+ (n = 12) and Cry1+/+/Cry2−/− (n = 5) mice. Data (means ± sem) were analyzed using Two-Way ANOVA repeated measures: [Time × Genotype: F(8, 104) = 0.3432, P = 0.9470; Time: F(4, 104) = 9.940, P < 0.0001; Genotype: F(2, 26) = 0.4848, P = 0.6213]. (F) Cumulative locomotor activity of WT, Cry1−/−/Cry2+/+ and Cry1+/+/Cry2−/− mice over a 30 min period post cocaine Data (means ± sem) were analyzed using Two-Way ANOVA repeated measures: [Time × Genotype: F(4, 52) = 0.9167, P = 0.4613; Time: F(2, 52) = 16.52, P < 0.0001; Genotype: F(2, 26) = 0.07071, P = 0.9319]. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
Cocaine-induced ERK activation in Cry1−/−Cry2−/− mice. (A) Immunofluorescence pictures showing P-ERK in the NAcc Core and Shell of wild type (WT) and Cry1−/−Cry2−/− mice treated with vehicle (veh) (WT, n = 3; Cry1−/−Cry2−/−, n = 3), cocaine 7.5 mg/kg (Coc7.5) (WT, n = 3; Cry1−/−Cry2−/−, n = 3) and cocaine 15 mg/kg (Coc15) (WT, n = 4; Cry1−/−Cry2−/−, n = 4). (B) Quantification of P-ERK positive neurons in NAcc core (a), NAcc medial shell (b), NAcc ventral shell (c), dorso-medial striatum (d), and ventro-medial striatum (e). Data (means ± sem) were analyzed using an unpaired Student's t-test: *p < 0.05, **p < 0.01. (C) Quantification of ERK1 and ERK2 expression levels in wild type (WT) (n = 6) and Cry1−/−Cry2−/− mice (n = 6) (means ± sem).
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