Mania- and anxiety-like behavior and impaired maternal care in female diacylglycerol kinase eta and iota double knockout mice

Abstract

Genome-wide association studies linked diacylglycerol kinase eta and iota to mood disorders, including bipolar disorder and schizophrenia, and both genes are expressed throughout the brain. Here, we generated and behaviorally characterized female mice lacking Dgkh alone, Dgki alone, and double Dgkh/Dgki-knockout (dKO) mice. We found that fewer than 30% of newborn pups raised by dKO females survived to weaning, while over 85% of pups survived to weaning when raised by wild-type (WT) females. Poor survival under the care of dKO mothers was unrelated to pup genotype. Moreover, pups from dKO dams survived when fostered by WT dams, suggesting the poor survival rate of dKO-raised litters was related to impaired maternal care by dKO dams. Nest building was similar between WT and dKO dams; however, some dKO females failed to retrieve any pups in a retrieval assay. Pups raised by dKO dams had smaller or absent milk spots and reduced weight, indicative of impaired nursing. Unlike WT females, postpartum dKO females showed erratic, panicked responses to cage disturbances. Virgin dKO females showed behavioral signs of anxiety and mania, which were not seen in mice lacking either Dgkh or Dgki alone. Our research indicates that combined deletion of Dgkh and Dgki impairs maternal behavior in the early postpartum period, and suggests female dKO mice model symptoms of mania and anxiety.

Keywords: Dgkh; Dgki; anxiety; bipolar disorder; diacylglycerol kinase; female mice; knockout mice; mania; maternal behavior; mouse behavior; postpartum.

Figure 1.. Generation of Dgkh-knockout mice using CRISPR/Cas9.

A, Integration of a STOP cassette arrests translation at the start of the first catalytic domain of DGKH. PH = pleckstrin homology. C1 = cysteine-rich (diacylglycerol-binding). SAM = sterile alpha motif. B, The 22-base-pair cassette (red), containing a STOP codon and KpnI restriction site, was inserted into Exon 9 of the Dgkh gene. C, PCR amplification of tail DNA and digestion with KpnI. Without KpnI digestion, the amplified fragment from the Dgkh^−/− allele is 628 bp. D, Western blot using 30 μg of protein isolated from cerebral cortices of WT, Dgkh^−/−, Dgki^−/−, and dKO female mice.

Figure 2.. Poor survival of offspring raised by dKO females.

A, Survival rate of pups born from dKO dams was significantly reduced after birth relative to those born from WT dams. B, The average proportion of each litter that survived to weaning was dependent on genotype of the mother. dKO fostered = litters born from dKO dams fostered with recently postpartum WT dams. C, Litter size on the day of birth based on the genotype of the mother. D, Percentage of slitter with a milk spot present, shown by genotype of the mother. E, Weight of pups raised by WT or dKO mothers. F, Time taken to retrieve the first pup and all three pups in the pup retrieval assay, based on the genotype of the mother. G, Percentage of mothers, tested in (F), that retrieved all three pups to the nest. Number of pups indicated on graphs in (A) and (E). Number of litters indicated on graphs in (B-D). Bars in (B-F) represent mean ± SEM. P < *0.05, **0.01, ***0.001, ****0.0001.

Figure 3.. Deletion of Dgkh and/or Dgki does not alter responses or habituation to acoustic startle tone in female mice.

A, Startle responses to a 120-decibel (dB) tone. B, The % decrease in startle response from (A) when the 120-dB tone was preceded by a non-startling tone of 74, 78, 82, 86, or 90 dB. Same mice were used in (A) and (B); number of mice indicated on graph in (A). Bars represent mean ± SEM.

Figure 4.. dKO females show mood-disorder-like phenotypes.

A, In the forced swim test, mice were placed in a large cylinder of water for six minutes. Time spent immobile (i.e. not swimming) was measured for the final four minutes. B, Time in closed arm versus aversive open arms of an elevated plus maze was measured in a five-minute period. Number of mice indicated on graphs. Bars represent mean ± SEM. P < **0.01, ***0.001, ****0.0001.

Figure 5.. Locomotion and center behavior in an open field were unaffected by Dgkh and/or Dgki loss in female mice.

In the 60-minute test, the total distance covered (A-C), vertical rearing activity (D-F), and the distance covered (G-I) and time spent (J-L) in the center of an open field were tracked. Number of mice indicated in graphs. Data represent mean ± SEM. P < *0.05.

Figure 6.. Sucrose preference and sleep patterns were disrupted in dKO female mice.

A, The preference of a 1% sucrose solution over water was measured over 24 h. B-F, Wake and sleep behaviors were measured continuously for eight days, and the following measurements for each animal were calculated by averaging across days. The average percentage of time each mouse spent asleep was calculated in 1-h (B) and 6-h (C) bins over the 12-h light and 12-h dark phases. The average length of sleep bouts in the light and dark phases (D) and the average length of wake bouts in light (E) and dark (F) phases were calculated in 6-h bins. Number of mice indicated on graphs in (A) and (B). Same mice from (B) are represented in (C-F). Bars represent mean ± SEM. P < *0.05, **0.01.