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. 2012;7(5):e37058.
doi: 10.1371/journal.pone.0037058. Epub 2012 May 10.

Diacylglycerol kinase β knockout mice exhibit attention-deficit behavior and an abnormal response on methylphenidate-induced hyperactivity

Mitsue Ishisaka  1 , Kenichi Kakefuda, Atsushi Oyagi, Yoko Ono, Kazuhiro Tsuruma, Masamitsu Shimazawa, Kiyoyuki Kitaichi, Hideaki Hara
Affiliations

Diacylglycerol kinase β knockout mice exhibit attention-deficit behavior and an abnormal response on methylphenidate-induced hyperactivity

Mitsue Ishisaka et al. PLoS One. 2012.

Abstract

Background: Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. DGKβ is one of the subtypes of the DGK family and regulates many intracellular signaling pathways in the central nervous system. Previously, we demonstrated that DGKβ knockout (KO) mice showed various dysfunctions of higher brain function, such as cognitive impairment (with lower spine density), hyperactivity, reduced anxiety, and careless behavior. In the present study, we conducted further tests on DGKβ KO mice in order to investigate the function of DGKβ in the central nervous system, especially in the pathophysiology of attention deficit hyperactivity disorder (ADHD).

Methodology/principal findings: DGKβ KO mice showed attention-deficit behavior in the object-based attention test and it was ameliorated by methylphenidate (MPH, 30 mg/kg, i.p.). In the open field test, DGKβ KO mice displayed a decreased response to the locomotor stimulating effects of MPH (30 mg/kg, i.p.), but showed a similar response to an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801 (0.3 mg/kg, i.p.), when compared to WT mice. Examination of the phosphorylation of extracellular signal-regulated kinase (ERK), which is involved in regulation of locomotor activity, indicated that ERK1/2 activation induced by MPH treatment was defective in the striatum of DGKβ KO mice.

Conclusions/significance: These findings suggest that DGKβ KO mice showed attention-deficit and hyperactive phenotype, similar to ADHD. Furthermore, the hyporesponsiveness of DGKβ KO mice to MPH was due to dysregulation of ERK phosphorylation, and that DGKβ has a pivotal involvement in ERK regulation in the striatum.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. DGKβ KO mice showed an attention-deficit behavior in object-based attention test.
(A) Mice were exposed to five objects for 3 min (training session), then, after an interval of 10 s, they were exposed to two objects that include a familiar and a novel objects for 3 min (retention session). (B) Object exploration time during the 3-min training session. (C) The novel-object discriminating abilities of mice were expressed as a recognition index. Values are expressed as the mean ± S.E.M. (KO: n = 8, WT: n = 9) *; p<0.01 vs. WT mice (t-test). (D) Mice were exposed to five objects for 6 min (training session), then, after an interval of 10 sec, they were exposed to two objects that include a familiar and a novel objects for 3 min (retention session). (E) Object exploration time during the 6-min training session. (F) The novel-object discriminating abilities of mice were expressed as a recognition index. Values are expressed as the mean ± S.E.M. (KO: n = 6, WT: n = 7). (G) The effect of MPH on the recognition index in retention phase after 3-min training phase. Values are expressed as the mean ± S.E.M. (n = 6, 7) *; p<0.05, **; p<0.01 vs. vehicle-treated WT mice, #; p<0.05 vs. vehicle-treated KO mice (Tukey's test).
Figure 2
Figure 2. DGKβ KO mice showed an abnormal response to methylphenidate.
(A) The locomotor activity after various doses of MPH. Each mouse was placed in a locomotor activity monitor for an initial period of 30 min and then injected with vehicle or MPH (0.3, 3, 30 mg/kg, i.p.). Horizontal activities of 90 min after drug treatment were recorded. Values are expressed as the mean ± S.E.M. (n = 5) *; p<0.05 vs. vehicle-treated WT mice (t-test), #; p<0.05, ##; p<0.01 vs. vehicle-treated WT mice (Dunnett's test). Each mouse was placed in a locomotor activity monitor for an initial period of 30 min (shown as arrow) and then injected with vehicle or MPH (30 mg/kg). Horizontal activity was recorded every 5 min for a 2-h period. Locomotor activity throughout the 2-h period of WT (B) and DGKβ KO (C) mice. Values are expressed as the mean ± S.E.M. (n = 4 to 10) *; p<0.05, **; p<0.01 vs. vehicle-treated group (t-test). (D) Rearing behavior of WT and DGKβ KO mice in the first 5 min of open field test. Values are expressed as the mean± S.E.M. (n = 5, 7) *; p<0.05 vs. WT mice (t-test). (E) Rearing behavior of MPH/vehicle treated WT and KO mice for 5 min after drug treatment. Values are expressed as the mean ± S.E.M. (n = 4 to 10) *; p<0.05, **; p<0.01 vs. vehicle-treated KO mice group (t-test). (F) The duration of the stereotyped behaviors for 5 min after drug treatment. Values are expressed as the mean ± S.E.M. (n = 4 to 10) *; p<0.05, **; p<0.01 vs. vehicle-treated WT mice group (t-test).
Figure 3
Figure 3. DGKβ KO mice showed normal responses to MK-801-induced hyperactivity.
Each mouse was placed in a locomotor activity monitor for an initial period of 30 min (shown as arrow) and then injected with vehicle or MK-801 (0.3 mg/kg). Horizontal activity was recorded every 5 min for a 2-h period. Locomotor activity throughout the 2-h period of WT (A) and DGKβ KO (B) mice. Values are expressed as the mean ± S.E.M. (n = 4 or 5) *; p<0.05, **; p<0.01. (C) Total horizontal activities of 90 min after drug treatment were recorded. Values are expressed as the mean ± S.E.M. (n = 4 or 5) *; p<0.05, **; p<0.01 vs. vehicle-treated WT mice (t-test), ##; p<0.01 vs. vehicle-treated KO mice (t-test).
Figure 4
Figure 4. Dopaminergic systems in the striatum of the DGKβ KO mice.
(A) Representative photograph of the coronal sections of the striatum immunostained for tyrosine hydroxylase (TH) of WT and DGKβ KO mice. Scale bar shows 300 µm. (B) Optical density of TH-positive fibers in the striatum of WT and DGKβ KO mice. Values are expressed as the mean ± S.E.M. (n = 3). (C) Representative immunoblots showing the expression levels of D1 dopamine receptor (D1DR) and D2 dopamine receptor (D2DR) in the striatum of WT and DGKβ KO mice. (D) Protein levels of D1DR and D2DR are quantified relative to the GAPDH levels. Values are expressed as the mean ± S.E.M. (n = 6). (E) The retention time of haloperidol-induced catalepsy of WT and DGKβ KO mice. Values are expressed as the mean ± S.E.M. (n = 6 to 12).
Figure 5
Figure 5. Western blot analysis of the phosphorylation levels of ERK1/2 and GluR1 in the striatum.
Phosphorylated and total ERK1/2 levels in the striatum were measured by Western blot analysis. (A) Representative immunoblots showing the expression levels of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) in the striatum of WT and DGKβ KO mice 5 min after drug treatment. (B) Phosphorylation levels of ERK1/2 are quantified relative to the t-ERK1/2 levels. Values are expressed as the mean ± S.E.M. (n = 5 to 8) **; p<0.01 v.s. vehicle-treated WT mice group (t-test). (C) Representative immunoblots showing the expression levels of phosphorylated GluR1 (p-GluR1) in the striatum of WT and DGKβ KO mice 40 min after drug treatment. (D) Phosphorylation levels of GluR1 are quantified relative to the β-actin levels. Values are expressed as the mean ± S.E.M. (n = 6 to 8) *; p<0.01 vs. vehicle-treated WT mice group, ##; p<0.01 vs. vehicle-treated KO mice group (t-test).
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