Early Handling Exerts Anxiolytic Effects and Alters Brain Mitochondrial Dynamics in Adult High Anxiety Mice

Abstract

Early handling (EH), the brief separation of pups from their mother during early life, has been shown to exert beneficial effects. However, the impact of EH in a high anxiety background as well as the role of brain mitochondria in shaping EH-driven responses remain elusive.Here, we used a high (HAB) vs. normal (NAB) anxiety-related behavior mouse model to study how EH affects pup and dam behavior in divergent anxiety backgrounds. We also investigated EH-induced effects at the protein and mRNA levels in adult male HAB mice in the hypothalamus, the prefrontal cortex, and the hippocampus by examining the same mitochondrial/energy pathways and mitochondrial dynamics mechanisms (fission, fusion, biogenesis, and mitophagy) in all three brain regions.EH exerts anxiolytic effects in adult HAB but not NAB male mice and does not affect HAB or NAB maternal behavior, although basal HAB vs. NAB maternal behaviors differ. In adult HAB male mice, EH does not impact oxidative phosphorylation (OXPHOS) and oxidative stress in any of the brain regions studied but leads to increased protein expression of glycolysis enzymes and a correlation of anxiety-related behavior with Krebs cycle enzymes in HAB mice in the hypothalamus. Intriguingly, EH alters mitochondrial dynamics by increasing hypothalamic DRP1, OPA1, and PGC1a protein levels. At the mRNA level, we observe altered, EH-driven mitochondrial dynamics mRNA signatures which predominantly affect the prefrontal cortex.Taken together, our results show that EH exerts anxiolytic effects in adulthood in high anxiety and modulates mitochondrial dynamics pathways in a brain region-specific manner.

Keywords: Early life; HAB; Maternal behavior; Mitochondria; Neonatal handling; Post-natal handling.

Fig. 1

Early handling (EH) induces anxiolytic effects in HAB EH male mice. a EH paradigm experimental setup. EH was applied to the pups from PND 1 to PND 14. SPAT was performed on PND 31, followed by DaLi (PND 64), OFT (PND 65), FST (PND 66), and sampling on PND 70. b HAB EH male mice enter more times in the DaLi light compartment (*p = 0.0240; HAB EH n = 20, HAB NH n = 17, NAB EH n = 14, and NAB NH n = 7) and show decreased latency to the first entry in the DaLi light compartment (*p = 0.0366; HAB EH n = 19, HAB NH n = 17, NAB EH n = 14, and NAB NH n = 7) compared to HAB NH mice. No EH-induced anxiolytic effects are observed in NAB male mice. c EH does not affect body weight on PND 70 in male HAB and NAB mice (HAB EH n = 20, HAB NH n = 17, NAB EH n = 15, and NAB NH n = 7). EH, early handling; NH, no handling; HAB EH, early handling HAB; HAB NH, no handling HAB; NAB EH, early handing NAB; NAB NH, no handling NAB; PND, post-natal day; HYP, hypothalamus; PFC, prefrontal cortex; HIP, hippocampus

Fig. 2

Early handling (EH) does not affect maternal care behavior in HAB and NAB dams. a Experimental setup for maternal observations. Maternal behaviors are presented from more (licking-grooming, arched-back nursing, nursing, nest building) towards less (self-maintenance, exploration, inactivity) active care of the pups. Maternal observations were collected from PND 2 to PND 7 on predetermined time points per day. b EH does not affect maternal behavior in HAB EH (n = 5) vs. HAB NH (n = 6) and in NAB EH (n = 4) vs. NAB NH (n = 4) dams, as shown by the time course analysis of daily assessment of maternal observations. Here, we only discuss differences between HAB EH vs. HAB NH and NAB EH vs. NAB NH dams. EH, early handling; HAB EH, early handling HAB; HAB NH, no handling HAB; NAB EH, early handing NAB; NAB NH, no handling NAB; PND, post-natal day

Fig. 3

Divergent basal maternal behavior in HAB and NAB dams. a Pie charts for the maternal observations for HAB NH and NAB NH in %. Maternal behaviors are presented from more active (licking-grooming, arched-back nursing, nursing, nest building) towards less active (self-maintenance, exploration, inactivity) care of the pups. Data are presented as the average % per behavior for the 6-day observation period. b Maternal behavior summary for the 6-day observation period and time course analysis of behaviors for HAB NH (n = 6) vs. NAB NH (n = 4) dams. Increased arched-back nursing on PND 2 (**p = 0.0098), PND 3 (p = 0.0818 (T), PND 4 (*p = 0.0200), and PND 6 (**p = 0.0077) and licking-grooming on PND 3 (**p = 0.0093) were observed in HAB NH compared to NAB NH dams. HAB NH dams show decreased nest building behavior on PND 3 (p = 0.0544 (T)) and PND 6 (*p = 0.0439) and increased inactivity on PND 7 (*p = 0.0378) compared to NAB NH dams. Significant line effects for the 6-day observation period are noted on the bar graphs. EH, early handling; HAB NH, no handling HAB; NAB NH, no handling NAB; PND, post-natal day; LG, licking-grooming; ABN, arched-back nursing; NU, nursing; NE, nest building; SM, self-maintenance; EXPLR, exploration; IN, inactivity; T, trend

Fig. 4

No effects of early handling (EH) on OXPHOS, oxidative stress, and metabolism in adult HAB male mice. a Representative data of OXPHOS complex I NDUFB8 and complex III UQCRC2 proteins in hypothalamus, prefrontal cortex, and hippocampus indicating no expression differences in HAB EH (n = 11) vs. HAB NH (n = 10) mice. b Representative Western blots of GSR, PRX, CAT, and SOD2 in hypothalamus, prefrontal cortex, and hippocampus, indicating no expression differences between HAB EH and HAB NH male mice (for quantification data see Fig. S2). c No differences in TAC between HAB EH (n = 20) and HAB NH (n = 17) plasma. d No differences in TAC between HAB EH and HAB NH male mice in hypothalamus (HAB EH n = 10, HAB NH n = 8), prefrontal cortex (HAB EH n = 9, HAB NH n = 10), and hippocampus (HAB EH n = 11, HAB NH n = 10). e No differences in the amount of carbonylated proteins per mg protein content between HAB EH (n = 7) and HAB NH (n = 6) mice in the prefrontal cortex. f The amount of carbonylated proteins negatively correlates with the latency to the first entry in the DaLi light compartment for HAB EH mice (Pearson r =  − 0.7724, *p = 0.0418) and shows a trend towards positive correlation with the number of entries to the light compartment (Pearson r = 0.7502, p = 0.0521 (T)) in HAB EH mice. Full Western blot data and loading control quantifications are provided in Figs. S3 and S4, respectively. HAB EH, early handling HAB; HAB NH, no handling HAB; HYP, hypothalamus; PFC, prefrontal cortex; HIP, hippocampus; CI, OXPHOS complex I; CIII, OXPHOS complex III; T, trend

Fig. 5

Early handling (EH) affects metabolism pathways in the hypothalamus. a Increased protein expression of PKLR (*p = 0.0150, Welch’s test) and a trend towards increased expression of ENO1 (p = 0.0661 (T), Welch’s test) in the hypothalamus of HAB EH (n = 11) vs. HAB NH (n = 10) mice. b No differences in protein expression of CS and ISOD and SDHA in the hypothalamus of HAB EH (n = 11) vs. HAB NH (n = 10) mice. c Hypothalamic CS expression strongly correlates with the number of entries in the DaLi in HAB EH mice (Pearson r =  − 0.8695, ***p = 0.0005). d Hypothalamic ISOD expression in HAB EH hypothalamus positively correlates with the number of entries (Pearson r = 0.6172, *p = 0.0431) and negatively correlates with the latency to the first entry in the light compartment (Pearson r =  − 0.6092, *p = 0.0467) in DaLi. e No differences in LDHB or in GSK-3b protein expression in the hypothalamus of HAB EH (n = 11) vs. HAB NH (n = 10) mice. Full Western blot data and loading control quantifications are provided in Figs. S3 and S4, respectively. HAB EH, early handling HAB; HAB NH, no handling HAB; T, trend

Fig. 6

Early handling (EH) induces alterations in mitochondrial dynamics in adult HAB brain in a region-specific manner. a Increased protein expression of DRP1 (*p = 0.0221, Welch’s t-test), PGC1a (*p = 0.0102, Welch’s t-test) and OPA1 (**p = 0.0030, Welch’s t-test) in the hypothalamus of HAB EH (n = 11) vs. HAB NH (n = 10) male mice. b A trend towards increased protein expression of PRKN (p = 0.0832 (T), Welch’s t-test) in the hippocampus of HAB EH (n = 10) vs. HAB NH (n = 10) male mice. c Altered mRNA expression in the prefrontal cortex of HAB EH male mice. Increased Fis1 (*p = 0.0121, Welch’s test), decreased Opa1 (*p = 0.0246, Mann–Whitney test), increased Prkn (*p = 0.0289, Mann–Whitney test), increased Pgc1a (*p = 0.0224, Welch’s test), and decreased Tfam (**p = 0.0093, Welch’s test) mRNA levels in the prefrontal cortex of HAB EH vs. HAB NH male mice (HAB EH n = 9, HAB NH n = 7, for Fis1 in hippocampus EH n = 8, HAB NH n = 6). d Decreased Tfam mRNA expression (*p = 0.0240, Welch’s test) in the hypothalamus of HAB EH (n = 9) vs. HAB NH (n = 7) male mice. e Increased Slc25a46 mRNA expression (*p = 0.0362, Welch’s test) in the hippocampus of HAB EH (n = 9) vs. HAB NH (n = 7) male mice. f Schematic representation of different brain mitochondrial dynamics gene/protein regulatory networks affected by EH (green arrow: higher expression in HAB EH mice; red arrow: lower expression in HAB EH mice). Full Western blot data and loading control quantifications are provided in Figs. S3 and S4, respectively. HAB EH, early handling HAB; HAB NH, no handling HAB; HYP, hypothalamus; PFC, prefrontal cortex; HIP, hippocampus; T, trend