Behavioral Deficits in Mice with Postnatal Disruption of Ndel1 in Forebrain Excitatory Neurons: Implications for Epilepsy and Neuropsychiatric Disorders

Abstract

Dysfunction of nuclear distribution element-like 1 (Ndel1) is associated with schizophrenia, a neuropsychiatric disorder characterized by cognitive impairment and with seizures as comorbidity. The levels of Ndel1 are also altered in human and models with epilepsy, a chronic condition whose hallmark feature is the occurrence of spontaneous recurrent seizures and is typically associated with comorbid conditions including learning and memory deficits, anxiety, and depression. In this study, we analyzed the behaviors of mice postnatally deficient for Ndel1 in forebrain excitatory neurons (Ndel1 CKO) that exhibit spatial learning and memory deficits, seizures, and shortened lifespan. Ndel1 CKO mice underperformed in species-specific tasks, that is, the nest building, open field, Y maze, forced swim, and dry cylinder tasks. We surveyed the expression and/or activity of a dozen molecules related to Ndel1 functions and found changes that may contribute to the abnormal behaviors. Finally, we tested the impact of Reelin glycoprotein that shows protective effects in the hippocampus of Ndel1 CKO, on the performance of the mutant animals in the nest building task. Our study highlights the importance of Ndel1 in the manifestation of species-specific animal behaviors that may be relevant to our understanding of the clinical conditions shared between neuropsychiatric disorders and epilepsy.

Keywords: Lis1 Reelin; Nde1; Ndel1; epilepsy; hippocampus; mouse behavior; schizophrenia.

Figure 1

Behaviors of Ndel1 CKO mice in species-specific tasks. (A) Nest building: Ndel1 CKO mice, heterozygous CKO, and WT littermates were monitored for their ability to build nest around 5–8 weeks of age, after cage change. Representative photographs of nests built by mice with the respective genotypes 24–48 h after cage change. The Ndel1 CKO failed to build their nest. This phenotype was highly penetrant and observed in more than 95% of the Ndel1 CKO analyzed. A total of 120 mice were monitored for this phenotype over a period of ~5 years. The bar graph depicts the relative area with bedding material in the cages of WT, Het and CKO mice. Mean ± SD, ^***P<0.005, NS: nonsignificant. N = 3–8 mice, average age: ~7 weeks. (B) In-cage behavioral ethograms for the 2 WT, 2 Het, and 2 CKO shown in (A) and Supplementary Videos 1–6. The ethograms illustrate the 4 mutually exclusive behaviors (sitting, walking, rearing, hiding under nesting material) displayed by the mice at each time point over 300 s. The graphs show the cumulative time each mouse engaged in 1 of the 4 behaviors. (C) Y maze: During the 600 s acquisition of the Y maze mice of all three genotypes (WT, Het, and CKO) spent equivalent amounts of time in zones one and two. During the 300 s retention trial Ndel1 CKO mice spent significantly less time in the novel zone. Mean ± SD, ^*P<0.05. (D) Open Field: Representative examples of paths taken by a WT and a CKO mouse during 300 s in the open field and quantification of the total time spent as well as the time spent in the outer, middle, and center zones. Ndel1 CKO mice spent significantly longer in the center zone compared to the other two genotypes. Quantification of the total distance traveled as well as the distance traveled in the outer, middle and center zones. Ndel1 CKO mice traveled significantly less distance than WT and Het mice. Mean ± SD, ^*P < 0.05, ^**P < 0.01. (E) Forced swim: Ndel1 CKO mice spent significantly more time climbing and significantly less time floating than the other two genotypes (WT and Het). Mean ± SD, ^**P < 0.01. (F) Dry cylinder: Ndel1 CKO mice spent less time touching the walls (more time not touching the walls) compared with the other two genotypes. Mean ± SD, ^*P < 0.05. 7 mice of each genotype were subjected to the tests in (B-E) at 7 weeks of age.

Figure 2

Gross anatomy and molecular changes in the hippocampus and neocortex of Ndel1 CKO mice. (A) Bilayered CA1 of Ndel1 CKO mice at ~7 weeks of age is associated with intense astrogliosis in the CA1 and stratum lacunosum moleculare. GFAP detects astrocytes while WFS1 labels preferentially CA1 pyramidal neurons. (B) Neocortical lamination of Ndel1 CKO mice appears intact at 12 weeks of age (the average lifespan of the mutant mice is 10 ± 4 weeks, see Jiang et al. 2016; Kiroski et al. 2020), as indicated by immunofluorescent staining with Cux1 (that label neurons of layers 2 and 3) and TBR1 antibodies (that label neurons of layer 6). (C) Levels of proteins potentially linked to Ndel1 biology, Ndel1 CKO phenotypes, epilepsy and/or schizophrenia in the neocortex and hippocampus of Ndel1 CKO mice. (D) Bar graph quantifications for the levels of proteins (in arbitrary units - A.U.) and ratios of phospho-kinase/total kinase illustrated in (C) in the hippocampus, n = 3–8 mice. (E) Bar graph quantifications for the levels of proteins (in arbitrary units - A.U.) and ratios of phospho-kinase/total kinase illustrated in (C) in the neocortex, n = 3–8 mice. For (D-E): mean ± SD; Student’s t-test; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; n = 3–8 mice, average age: ~9 weeks. (F) Crude synaptosome fractionation determines the amounts of GluR1 found in the preclear lysate (S1), cytosolic fraction (S2), pellet 1 (P1), or crude synaptic compartment (P2). The amount of protein in each fraction is estimated in percentage. Representative blots from hippocampal lysates of 2 Ndel1 CKO and 2 WT out of 6 animals of each genotype depict a subtle decrease of GluR1 (3.2 ± 3.1%; mean ± SD, n = 6; Student’s t-test; P = 0.0468) in the synaptic compartment. PSD-95, a postsynaptic marker, was used a control for the procedure. Average age of the animals: 9.5 weeks.

Figure 3

Reelin improves the performance of the Ndel1 CKO mice in the nest building task. (A-C) Representative photographs depicting the performance of Ndel1 CKO mice injected at 5, 8, or 9 weeks of age with (A) the purified full length protein (n = 3), (B) a commercially available central active fragment of Reelin (n = 2) or (C) a control solution (n = 3). All mice were monitored for their performance after injection for up to 8 days. Red traces delimit the area with bedding material. (D) Bar graph showing the relative area with bedding material in the cage before and after control or Reelin injection in Ndel1 CKO mice. Mean ± SD, Student’s t-test, ^***P < 0.001, NS: nonsignificant; n = 8 mice in total, average age: ~8 weeks; A.U.: arbitrary units. (E) Levels of Ndel1, Lis1, and GFAP in WT, Ndel1 CKO mice, and Ndel1 CKO mice injected with Reelin. Data were analyzed and compared using One-way ANOVA and Tukey’s multiple comparison test. ^*P < 0.05, ^**P < 0.01; n = 3/group.