CRH gene expression in hypothalamic PVN is down-regulated by daily handling as early as the 9th postnatal day
As shown in , CRH-mRNA levels in PVN of the 9-day-old, daily-handled rats were significantly lower (233.3 ± 8.8 nCi/g) than those of undisturbed pups (545.0 ± 25.0 nCi/g, P < 0.01). Decreased CRH-mRNA signal over PVN is evident in a dark-field photomicrograph derived from a handled rat, compared with a matched section from an undisturbed animal ().
FIG. 1 CRH-mRNA expression in paraventricular nucleus (PVN) of 9-day-old rats experiencing daily handling on postnatal days 2–8 is reduced compared with that of undisturbed immature rats. A, Semiquantitative analysis of signal over PVN was achieved after (more ...)
GR gene expression in hippocampal CA1 is not yet altered in handled 9-day-old rats
shows that, on postnatal day 9, GR-mRNA levels in hippocampal CA1 did not differ between rats that were handled daily on postnatal days 2–8 and those raised undisturbed (65.0 ± 2.9 nCi/g vs. 67.3 ± 8.96 nCi/g). demonstrates robust GR-mRNA signal in CA1 at this age, but (in contrast to older animals) little GR expression in the dentate gyrus granule cell layer.
FIG. 2 Glucocorticoid receptor (GR)-mRNA levels in hippocampal CA1 of 9-day-old rats experiencing daily handling on postnatal days 2–8 do not differ from those of undisturbed pups. A, Semiquantitative analysis of signal over hippocampal CA1 was achieved (more ...)
Modulation of CRH expression in PVN upon handling is sustained beyond the first three weeks of life and is not accompanied by changes in CRH-mRNA levels in amygdala
To further clarify the evolution of CRH-mRNA expression as a consequence of early-life experience, two additional ages were examined. As shown in , CRH-mRNA levels in PVN were persistently reduced in handled rats, when measured on post-natal day 23 (475.8 ± 35.4 nCi/g handled vs. 622.5 ± 24.8 nCi/g in undisturbed animals) and on day 45 (446.0 ± 92.0 nCi/g vs. 860.0 ± 140.0 nCi/g). In contrast, analysis of CRH-mRNA levels in ACe did not reveal significant effects of early-life handling. Thus, CRH-mRNA levels in ACe in handled and undisturbed rats averaged 69.0 ± 5.2 nCi/g and 57.1 ± 9.1 nCi/g, respectively on postnatal day 23. By day 45, CRH-mRNA levels averaged 82.5 ± 7.5 nCi/g in handled pups, and 76.7 ± 3.3 nCi/g in the undisturbed cohort.
GR gene expression in hippocampal CA1 of rats subjected to early-life handling is up-regulated on the 45th, but not on the 23rd postnatal day
To better resolve the timing of GR up-regulation, two additional ages were examined. shows that GR-mRNA levels in hippocampal CA1 did not differ between daily-handled and undisturbed rats immediately following the handling paradigm (day 9) or 2 weeks later (for day 23: 72.1 ± 5.3 nCi/g in handled vs. 71.8 ± 8.9 nCi/g in undisturbed rats). However, by the 45th postnatal day, following puberty, GR-mRNA levels were higher in rats experiencing early-life handling (102.3 ± 7.4 nCi/g vs. 54.7 ± 2.8 nCi/g, P < 0.01). In the 9-day-old rat, little GR-gene expression was found over the DG granule cell layer (). Therefore, the early effects of the experimental manipulations described here on this transcript's expression could not be determined. Comparison of GR-mRNA signal over DG in 23- and 45-day old rats revealed an up-regulation of GR expression on the 45th postnatal day in the handled group (, 106.7 ± 9.5 nCi/g vs. 70.7 ± 9.4 nCi/g), as shown also in . This figure depicts coronal brain sections at the level of the dorsal hippocampus, following in situ hybridization for GR-mRNA. Similar expression magnitude and pattern in handled (A) and undisturbed (B) rats are evident on postnatal day 23. By postnatal day 45, GR signal is enhanced in a rat subjected to early-life handling (C) compared with one raised undisturbed (D).
FIG. 4 Time-course of the effects of early-life experience on glucocorticoid receptor (GR)-mRNA expression in the hippocampal formation. A, Significant (*) up-regulation of GR-mRNA levels in hippocampal CA1 of daily handled rats emerges starting at the 45-day (more ...)
Early-life handling experience does not influence GR-mRNA levels in hypothalamic PVN and frontal cortex
Analysis of GR-mRNA levels in PVN and in frontal cortex failed to demonstrate effects of early-life handling. Thus, GR-mRNA levels in PVN of handled and undisturbed rats averaged 72.3 ± 7.9 nCi/g and 63.0 ± 13.0 nCi/g, respectively, on postnatal day 9; corresponding values were 71.7 ± 4.4 nCi/g and 75.3 ± 4.5 nCi/g on postnatal day 23. Finally, on postnatal day 45, GR-mRNA levels in PVN averaged 70.0 ± 20.0 and 54.3 ± 6.7 nCi/g, respectively. In frontal cortex, levels of GR-mRNA were 46.0 ± 5.6 in handled rats and 46.7 ± 2.4 nCi/g in undisturbed rats on postnatal day 9. In 23-day-old rats GR-mRNA levels averaged 25.1 ± 2.2 and 27.7 ± 1.7 nCi/g in handled and undisturbed rats, respectively, and no differences were observed also on postnatal day 45 (52.0 ± 10.5 nCi/g and 49.0 ± 2.1 nCi/g, respectively in handled and undisturbed groups).
Early-life experiences influence the hormonal stress response by the 23rd and 45th postnatal days, but not on postnatal day 9
Analyses of the hormonal stress responses of rats handled early in life compared with the undisturbed groups were carried out on postnatal days 9, 23, and 45. On day 9, robust elevations of both plasma ACTH and CORT in response to age-appropriate stress were observed. shows significant increases of plasma ACTH at both time points after stress onset. Whereas a robust effect of stress was noted (F2,15 = 11; P = 0.0016), no effect of the early life experience was revealed by two-way ANOVA (P = 0.59). CORT levels were in line with ACTH values: basal AM levels were 0.99 ± 0.12 and 1.14 ± 0.15 mg/dl in the handled and undisturbed groups, respectively. In response to stress, CORT levels of both groups rose, to 2.67 ± 0.41 and 2.59 ± 0.35 mg/dl at the 20 min time-point and 4.49 ± 0.40 and 3.51 ± 0.26 μg/dl at 60 min. Two-way ANOVA revealed a robust effect of stress (F2,30 547.68; P < 0.0001), but not of the early-life experience (F1,30 = 1.54; P = 0.22). By the 23rd day of life, (i.e. before alteration of hippocampal GR-mRNA) significant effects of the early-life handling experience on plasma ACTH and CORT responses to stress were evident. demonstrates a diminished induction of plasma ACTH by stress in the handled group: two-way ANOVA revealed both an effect of stress (F2,35 = 20.30 P < 0.001) and a robust effect of the early life treatment (F = 6.32; P = 0.017). Plasma CORT response to stress and early-life treatment was similar, showing significant effects of stress (F2,16 = 29.55) and of handling (F1,16 = 12.77). As shown in , this effect of early life treatment on the hormonal stress response persisted at least to postnatal day 45 when, in addition to robust effects of stress (P = 0.0046), two-way ANOVA showed a strong handling effect (F = 7.7; P = 0.018). As evident from the figure, basal AM levels of ACTH were not significantly different in handled and undisturbed groups at all three ages. Similarly, for CORT (see 9 day levels, above) basal values were 2.39 ± 0.44 and 4.42 ± 0.44 μg/dl on postnatal day 23, and 3.496±0.86 vs. 4.81 ± 0.41 μg/dl on postnatal day 45.
FIG. 5 Basal and stress-induced plasma ACTH as a function of age and early-life experience. A, On P9, plasma ACTH levels increase significantly (*) after age-appropriate stress, but do not differ in handled vs. undisturbed animals (P = 0.59, effect of handling, (more ...)