Postnatal handling increases the expression of cAMP-inducible transcription factors in the rat hippocampus: the effects of thyroid hormones and serotonin

Abstract

Postnatal handling increases glucocorticoid receptor expression in the rat hippocampus, thus altering the regulation of hypothalamic synthesis of corticotropin-releasing hormone and the hypothalamic-pituitary-adrenal response to stress. The effect on glucocorticoid receptor gene expression represents one mechanism by which the early environment can exert a long-term effect on neural development. The handling effect on hippocampal glucocorticoid receptor expression is dependent on peripheral thyroid hormone release and the activation of ascending serotonergic pathways. In primary hippocampal cell cultures, serotonin (5-HT) increases glucocorticoid receptor expression, and this effect appears to be mediated by increased cAMP levels. In the current studies we examined the in vivo effects of handling on hippocampal cAMP-protein kinase A (PKA) activity. In 7-d-old rat pups, we found that (1) postnatal handling increased adenylyl cyclase activity and hippocampal cAMP levels, (2) the effect of handling on cAMP levels was completely blocked by treatment with either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or the 5-HT receptor antagonist, ketanserin, and (3) handling also increased hippocampal PKA activity. We then examined the effects of handling on cAMP-inducible transcription factors. Handling rapidly increased levels of the mRNAs for nerve growth factor-inducible factor A (NGFI-A) (zif268, krox24) and activator protein-2 (AP-2) as well as for NGFI-A and AP-2 immunoreactivity throughout the hippocampus. Finally, we found that the effects of handling on NGFI-A and AP-2 expression were significantly reduced by concurrent treatment with either PTU or ketanserin, effects that paralleled those on cAMP formation. NGFI-A and AP-2 have been implicated in the regulation of glucocorticoid receptor expression during development. Thus, these findings suggest that postnatal handling might alter glucocorticoid receptor gene expression via cAMP-PKA pathways involving the activation of NGFI-A and AP-2.

Fig. 1.

Mean (±SEM) level of cAMP in hippocampi from acutely handled (AcH), chronically handled (ChH), and nonhandled (NH)animals. Acutely handled rats were handled only on day 7, whereas chronically handled animals were handled once per day from days 1 to 7; animals were killed immediately after handling on day 7. NH animals were killed immediately after removal from the home cage. *p < 0.001, n = 7–9 per group.

Fig. 2.

A, Mean (±SEM) level of cAMP in hippocampi from chronically handled (H) and nonhandled (NH) animals treated with either propylthiouracil (PTU) or saline (Sal) vehicle. *p < 0.01,n = 7–8 per group. B, Mean (±SEM) level of cAMP in hippocampi from chronically handled (H) and nonhandled (NH) animals treated with either ketanserin (Ket) or saline vehicle. *p < 0.01,n = 7–8 per group. For both studies, animals were killed immediately after handling on day 7 (H) or after removal from the home cage (NH).

Fig. 3.

Mean (±SEM) levels of specific [³H]forskolin binding (femtomol/milligram) in various hippocampal cell fields in chronically handled (H) and nonhandled (NH) animals killed immediately after handling on day 7 (H) or after removal from the home cage (NH). All H versusNH comparisons are significant at p< 0.001, n = 10–12 per group.

Fig. 4.

Mean (±SEM) level of hippocampal protein kinase A activity in nonhandled (NH), acutely handled (AH), and chronically handled (H) animals killed at various times after handling on day 7. AH animals were killed immediately after handling on day 7. **p < 0.01, *p < 0.05,n = 5–7 per group.

Fig. 5.

Mean (±SEM) grains per cell as a function of the area of the cell for NGFI-A mRNA in situ hybridization in various hippocampal regions from nonhandled (NH) and chronically handled (H) animals killed at various times (in minutes) after handling on day 7. For all regions, each handling time point is significantly different from the NH group atp < 0.01 (n = 3–4 per group). The inset provides a representative photomicrograph of grains over Nissl-stained neurons in the CA1 region of Ammon's horn.

Fig. 6.

Mean (±SEM) grains per cell as a function of the area of the cell for the AP-2 mRNA in situ hybridization in various hippocampal regions from nonhandled (NH) and chronically handled (H) animals killed at various times points (in minutes) after handling on day 7. For all regions, each handling time point is significantly different from the NH group atp < 0.05 (n = 3–4 per group). The inset provides a representative photomicrograph of grains over Nissl-stained neurons in the CA1 region of Ammon's horn.

Fig. 7.

Increased expression of AP-2- and NGFI-A-like immunoreactivity in dorsal hippocampus of chronically handled (H) and nonhandled (NH) animals on day 7 of life. Hanimals were killed 120 min after handling. Note the rather uniform increase throughout the hippocampus.

Fig. 8.

Western immunoblot analysis of NGFI-A- and AP-2-like immunoreactivity (measures are derived from 3–4 blots per group, each from tissue from separate animals) with antibodies to either NGFI-A or AP-2, which detects the 88 kDa NGFI-A band or the 49.5 kDa AP-2 band. Twenty-five micrograms of protein were loaded from hippocampal homogenates from chronically handled (H) and nonhandled (NH) animals killed immediately after handling on day 7 of life. Representative blots are shown for each study.A, Animals were treated with PTU, a thyroid synthesis inhibitor, or saline for days 1–7 of life. Handling produced a significant (p < 0.001) increase in both NGFI-A and AP-2 expression. Treatment with PTU significantly reduced NGFI-A and AP-2 expression in chronically handled rats (H+PTU vs H+Sal, p < 0.01 for both transcription factors). B, Animals were treated with ketanserin (2 μg/g body weight), a 5-HT receptor blocker, or saline for days 1–7 of life. Handling produced a significant (p < 0.001) increase in both NGFI-A and AP-2 expression in hippocampus. Treatment with ketanserin significantly reduced NGFI-A and AP-2 expression in chronically handled rats (H+PTU vs H+Sal,p < 0.01 for both transcription factors).C, To correct for potential loading errors, all blots were stripped and reprobed for α-tubulin staining. Shown here are representative blots for the AP-2 and NGFI-A studies. For each study, the data are derived from four to five independent experiments.

Fig. 9.

Left, Representative Western immunoblot analysis of CREB-like immunoreactivity in hippocampal samples from chronically handled (H) and nonhandled (NH) animals on day 7 of life.Middle, Representative results of immunocytohistochemical studies of phospho-CREB-like immunoreactivity in dorsal hippocampus of chronically handled and nonhandled animals on day 7 of life. Handled animals were killed 60 min after handling. Note the rather high and uniform levels of staining throughout the hippocampus in both groups of animals. Right, Representative Western blot analysis of phospho-CREB-like immunoreactivity with an antibody that detects the 44 kDa phospho-CREB band in hippocampal samples from chronically handled (H) and nonhandled (NH) animals on day 7 of life.

Fig. 10.

Handling provides for multisensoral stimulation of pups, which leads to various changes in pup physiology and thus in the sensory characteristics of the pups. This, in turn, leads to changes in maternal behavior (Liu et al., 1997; Francis et al., 1999) that appear to mediate the handling effect on hippocampal glucocorticoid receptor gene expression. In response to the handling manipulation, there occurs an increase in circulating levels of triiodothyronine that stimulates 5-HT activity at the level of the hippocampus (see introductory remarks). The increase in both thyroid hormones and 5-HT are obligatory for the handling effect on hippocampal glucocorticoid receptor expression. In vitrostudies with cultured hippocampal neurons reveal that 5-HT can directly modulate glucocorticoid receptor expression in hippocampal neurons and that this effect is mediated by a 5-HT₇-like receptor, which is positively coupled to cAMP. The results of the current studies show that handling increases hippocampal cAMP formation, PKA activity, and mRNA levels for AP-2 and NGFI-A. Recent studies (Diorio et al., 1997) show that handling also increases AP-2 and NGFI-A binding to their respective consensus sequences, such as those found on a promoter for the human glucocorticoid receptor gene. We propose that these steps are involved in the molecular basis of the handling effect on hippocampal glucocorticoid receptor gene expression.