Diacylglycerol kinase β knockout mice exhibit lithium-sensitive behavioral abnormalities

Abstract

Background: Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). DGKβ is widely distributed in the central nervous system, such as the olfactory bulb, cerebral cortex, striatum, and hippocampus. Recent studies reported that the splice variant at the COOH-terminal of DGKβ was related to bipolar disorder, but its detailed mechanism is still unknown.

Methodology/principal findings: In the present study, we performed behavioral tests using DGKβ knockout (KO) mice to investigate the effects of DGKβ deficits on psychomotor behavior. DGKβ KO mice exhibited some behavioral abnormalities, such as hyperactivity, reduced anxiety, and reduced depression. Additionally, hyperactivity and reduced anxiety were attenuated by the administration of the mood stabilizer, lithium, but not haloperidol, diazepam, or imipramine. Moreover, DGKβ KO mice showed impairment in Akt-glycogen synthesis kinase (GSK) 3β signaling and cortical spine formation.

Conclusions/significance: These findings suggest that DGKβ KO mice exhibit lithium-sensitive behavioral abnormalities that are, at least in part, due to the impairment of Akt-GSK3β signaling and cortical spine formation.

Figure 1. Locomotor activity test of WT and DGKβ KO mice.

WT (n = 10) and DGKβ KO (n = 9) mice were placed in individual home cages, and their locomotion was assessed every hour for 24 hr. (a) Locomotor activity throughout the 24-hr period and (b) locomotor activity was analyzed separately during the day and night. ^*; p<0.05, ^**; p<0.01 vs. WT mice.

Figure 2. Open field test of WT and DGKβ KO mice.

(a–e) Assessment of the single-dose lithium effect on DGKβ KO mice in the open field test. Representative images show typical examples of WT (a) and DGKβ KO (b) mice exploring behavior in the open field test. After drug treatment, each group of mice was placed in the open field apparatus and their distance traveled (c), number of scratching behaviors (d) and time spent in the center area (e) were measured. (n = 5 to 9) ^*; p<0.05, ^**; p<0.01 vs. WT mice. (f–h) Assessment of the chronic lithium effect on DGKβ KO mice in the open field test. After 10-days of drug treatment, mice were subjected to the open field test, and distance traveled (f), time spent in the center area (g) and the frequency of entering the center area (h) were measured for one hour. (n = 6 or 7) ^#; p<0.05 vs. control mice.

Figure 3. Elevated plus maze test of WT and DGKβ KO mice.

Representative images show typical examples of WT (a) and DGKβ KO (b) mice exploring in the elevated plus maze apparatus. After drug treatment, each group of mice was placed in the elevated plus maze apparatus for 10 min, and the number of entries into (c) and the time spent in open arms (d) were assessed. (e) After 10-days of drug treatment, mice were subjected to the elevated plus maze test again, and their time spent in open arms as a ratio to the pre test was assessed. (n = 7 to 9) ^*; p<0.05, ^**; p<0.01 vs. WT mice. ^#; p<0.05 vs. control mice.

Figure 4. Antidepressant-like behaviors of DGKβ KO mice.

(a) Immobile time of forced swim test. Thirty-minutes after drug treatment, mice were placed in water for a period of 6 min; only the last 5 min immobility time was measured. (b) Immobile time of tail suspension test. Thirty-minutes after drug treatment, mice were tail suspended with an adhesive tape 50 cm above the floor over a period of 7 min; immobility time was measured in only the last 6 min. (n = 7 to 9) ^*; p<0.05, ^**; p<0.01 vs. WT mice.

Figure 5. Western blot analysis of Akt-GSK3β signaling.

(a, b) Phosphorylated Akt (Ser473) and GSK3β were decreased in the cortex of control group of DGKβ KO mice. (c, d) Quantitative analysis of Western blotting showed that phosphorylated Akt (Ser473) and GSK3β were decreased in the cortex of DGKβ KO mice, and that lithium treatment attenuated this effect. (n = 7) ^*; p<0.05, ^**; p<0.01 vs. WT mice. ^#; p<0.05, ^##; p<0.01 vs. control mice.

Figure 6. Histological analysis of the cortex in DGKβ KO mice.

(a–d) Cresyl violet staining. (a, b) Representative photomicrographs show coronal sections stained with cresyl violet. (c, d) DGKβ KO mice showed no defects in the layered structure of the cerebral cortex. Scale bar  = 100 µm (e–i) Golgi staining. (e, f) Representative photomicrographs show pyramidal neurons in the cortex stained by Golgi. Scale bar  = 100 µm (g, h) Representative photomicrographs show high-magnification images of apical dendritic segments. Scale bar  = 5 µm (i) Quantitative analysis of spine density in WT mice and DGKβ KO mice. (n = 11 or 15) ^**; p<0.01 vs.WT mice.