Postpartum scarcity-adversity inflicts sex-specific cerebellar adaptations and reward behaviors in adolescence

Abstract

Early life adversity in the form of poor postnatal care is a major developmental stressor impacting behavior later in life. Previous studies have shown the impact of early life stress on neurobehavioral abnormalities. Specifically, research has demonstrated how limited bedding and nesting (LBN) materials can cause behavioral deficits in adulthood. There is, however, a limited understanding of how LBN influences sex-specific neurobehavioral adaptation in adolescence, a developmental stage susceptible to psychiatric diseases including substance use disorder. LBN and stress-naive c57BL/6 adolescent male and female mouse offspring were tested for a battery of behaviors including open field, novel object recognition, elevated plus maze, social preference, and morphine-induced conditioned place preference. There was a significant sex-specific deficit in social preference in male mice exposed to LBN compared to stress-naïve counterparts and both LBN males and females had a higher preference towards the drug-paired chamber in the morphine-induced conditioned place preference test. These behavioral deficits were concomitant with sex-specific increases in the transcription factor, Klf9 in the deep cerebellar nuclei (DCN) of males. Further, mRNA levels of the circadian gene Bmal1, which is known to be transcriptionally regulated by Klf9, were decreased in the DCN. Since Bmal1 has recently been implicated in extracellular matrix modulation, we examined perineuronal nets (PNN) and observed depleted PNN in the DCN of males but not female LBN mice. Overall, we provide a novel understanding of how postpartum adversity impinges on the cerebellar extracellular matrix homeostasis, likely, through disruption of the circadian axis by Klf9 that might underlie sex-specific behavioral adaptations in adolescence.

Keywords: Adolescence; Cerebellum; Circadian; Perineuronal nets; Postpartum adversity.

Figure 1:. Timeline of behavioral and biomolecular experiments.

Dams from naïve and LBN groups were subjected to pup-directed behaviors. This included kicking/stomping frequency (PND2–9), time spent in the rearing hut (PND2–9), and pup retrieval on PND7. Adolescent (PND28–37) male and female offspring from naive and LBN-exposed dams were exposed to a behavioral battery of open field, novel object recognition, elevated plus maze, social preference, and conditioned place preference for morphine. A separate cohort of adolescent males and females (PND30) that underwent LBN or control rearing conditions (but not tested for behaviors) was used for qPCR, immunoblotting, and neuroimaging experiments.

Figure 2:. LBN females exhibit abusive behaviors towards the pups.

a. Time spent in the rearing paper hut during PND2–9 between naive and LBN dams (n=5/group). b. Frequency of kicking/stomping the pups during PND2–9 between naïve and LBN dams (n=5/group). c. Pup retrieval between naïve and LBN dams (n=5/group) on PND7 when abusive behaviors are at their peak. All data are represented as mean ± SEM. **p<0.01 between LBN and naïve dams for pup retrieval behavior.

Figure 3:. LBN induces behavioral dysregulation pertaining to social and opioid rewards.

a. Total distance in meters in the open field test. b. number of central entries in the open field test. c. time spent in the central zone (in seconds) in the open field behavior. d. time spent in the open arm in the elevated plus maze test. e. total number of open arm entries in the elevated plus maze test. f. recognition index in the novel object recognition behavior. g. discrimination index in the social preference test. h. time spent in morphine paired chamber in the conditioned place preference test. n=9–12/group for all behaviors. All data are represented as mean ± SEM. *p<0.05 between naïve and LBN females in the number of central entries (open field), time spent in the central zone (open field), and time spent in the morphine chamber (CPP); **p<0.01 between naïve and LBN males in the discrimination index of the social preference test; ***p<0.001 between naïve and LBN males in the time spent in the morphine chamber of the CPP test.

Figure 4:. LBN induces cerebellar adaptations in adolescence.

Gene expression changes of a. Klf1 b. Trx1, c. Kdm5c, d. Klf9, e. Cry2, f. Bmal1, g. Nr1d2, h. Per1, i. Per2 and j. Dec2 in the DCN of male and female mice exposed to LBN and naïve conditions early in life; n=8/group. k. Klf9 protein expression between naïve and LBN males in the DCN region; n=5–6/group. All data are represented as mean ± SEM. *p<0.05 between naïve and LBN males for Klf9 protein expression; ***p<0.001 between naïve and LBN males for Klf9 and Bmal1 gene and expression.

Figure 5.. LBN disrupts PNN homeostasis in the DCN of males.

a. PNN intensity in the DCN region of LBN and naïve males and females, represented as arbitrary units. b. representative images of PNN intensity (average of all the somas from 3 sections of each animal) between naïve and LBN males and females; n=3 animals/sex/condition. White arrows indicate PNN fluorescence around the soma. * p<0.05 between naïve and LBN males.