Developmental regulation of the 5-HT7 serotonin receptor and transcription factor NGFI-A in the fetal guinea-pig limbic system: influence of GCs

Abstract

Fetal exposure to excess glucocorticoids (GCs) programs the developing hypothalamo-pituitary-adrenal (HPA) axis, and may predispose offspring to adult-onset disease. During development, serotonin (5-HT) influences transcription of hippocampal GR mRNA via the 5-HT7 receptor. The effect of 5-HT on GR involves the transcription factor NGFI-A. Given the developmental changes which we have previously reported in hippocampal GR mRNA expression, we hypothesized that (1) there are progressive developmental changes in 5-HT7 receptor and NGFI-A mRNA expression in the fetal guinea-pig limbic system, and (2) repeated exposure to synthetic GC treatment will significantly modify developmental expression of these genes. 5-HT7 receptor mRNA was highly expressed in the hippocampus and thalamus at gestational day (gd) 40 (term approximately 70 days), and significantly decreased (P < 0.05) with advancing gestation. Conversely, NGFI-A mRNA expression in the hippocampus and frontal cortex was almost undetectable at gd40, but was dramatically elevated (P < 0.05; 8-fold) near term. Changes in mRNA were refelected by NGFI-A protein levels. These changes were significantly correlated to hippocampal GR expression and fetal plasma cortisol concentrations. Synthetic GC treatment increased NGFI-A mRNA levels in CA1 and the cingulate cortex, but had no effect on 5-HT7 receptor expression. In conclusion our results suggest that (1) limbic 5-HT7 receptor expression is not directly linked to maturation of hippocampal GR in late gestation; (2) the up-regulation of NGFI-A expression near term is driven by glucocorticoid; and (3) premature exposure to synthetic glucocorticoid significantly increases NGFI-A-related transcriptional activity in the fetal limbic system.

Figure 1

A, representative expression of 5-HT7 receptor mRNA in coronal sections of fetal guinea-pig hippocampus at gd40, 50 and 60. Specific 5-HT7 receptor mRNA expression was detected in the hippocampal CA2/3 subfields, dentate gyrus (DG), cingulate cortex (CCx) and paraventricular thalamic nucleus (PVT). Bar: 1.5mm. B, relative levels of 5-HT7 receptor mRNA expression in the hippocampus (CA2/3, CA4), dentate gyrus (DG) and paraventricular nucleus of the thalamus (PVT) in female (open bars) and male (filled bars) fetuses in the second half of gestation. *Significant (P < 0.05) differences compared to gd40 fetuses.

Figure 2

A, representative expression NGFI-A mRNA in coronal sections of fetal guinea-pig hippocampus at gd40, 50 and 60. NGFI-A mRNA expression is shown in hippocampal subfields (CA1, CA2 and CA3), and cingulate cortex (CCx). Bar: 1.5mm. B, relative levels of NGFI-A mRNA expression in the hippocampus (CA1, CA2/3), dentate gyrus (DG) and cingulate cortex in female (open bars) and male (filled bars) fetuses in the second half of gestation. *Significant (P < 0.05) differences compared to previous gestational age.

Figure 3

Upper panel, representative expression NGFI-A mRNA in the CA1 hippocampal subfield in coronal sections of fetal guinea-pig hippocampus at gd40 (left panel), 50 (middle panel) and 60 (right panel), following high resolution silver emulsion autoradiography. Bar: 25μm. Lower panel, immunohistochemical staining of guinea-pig hippocampus (CA1, left panel) and frontal cortex (middle panel) using anti-NGFI-A antibody and DAB visualization. Arrows indicate cells that are darkly stained for NGFI-A protein. Preabsorption of the NGFI-A antibody (right panel) resulted in no detectable signal. Bar: 25μm.

Figure 4

A, representative Western blot for NGFI-A protein in fetal hippocampus during late gestation. Three bands (102, 57 and 45kDa) were detected. Western analysis using preabsorbed antibody completely eliminated all bands. B, relative changes in 45kDa form of NGFI-A protein in female (open bars) and male (filled bars) fetuses in the second half of gestation, expressed as a ratio to tubulin (mean ±s.e.m.). *Significant (P < 0.05) differences compared to previous gestational age. C, relative changes in the 57 and 102kDa bands in female and male fetuses in the second half of gestation (expressed as a ratio to tubulin (mean ±s.e.m.).

Figure 5

Relative levels of GR mRNA expression in the hippocampus (CA1,CA2/3), dentate gyrus (DG) and cingulate cortex in female (open bars) and male (filled bars) fetuses in the second half of gestation. *Significant (P < 0.05) differences compared to gd40 fetuses. These data are presented to validate correlation analysis (see text; Fig. 7).

Figure 6

Plasma cortisol concentrations in female (open bars) and male (filled bars) fetuses in late gestation. *Significant (P < 0.05) differences compared to previous gestational age.

Figure 7

Correlation analysis of NGFI-A mRNA expression (gd40 (•), gd50 (▪) and gd60 (▴)) in CA1 hippocampal subfield to plasma cortisol (left panel) and GR mRNA expression in the CA1 region (right panel) in second half of gestation (data pooled to include both sexes). Both relationships were determined to have statistically significant (P < 0.05) correlations.

Figure 8

A, representative expression of NGFI-A mRNA in the fetal male guinea-pig hippocampus, following treatment with repeated courses of dexamethasone. NGFI-A mRNA expression was detected in the hippocampus (CA1, CA2/3), dentate gyrus (DG), and layers II and IV of the cingulate cortex (Cx(II) and Cx(IV)). Bar: 1.5mm. B, effect of dexamethasone (1mgkg⁻¹ (grey bar) or 10mgkg⁻¹ (filled bar)) or vehicle (open bar) on gd40, 41, 50, 51, 60 and 61 on NGFI-A mRNA expression in the hippocampus (CA1, CA2/3) dentate gyrus (DG), and cingulate cortex (layers II and IV) of female and male fetuses in late gestation. *Significant (P < 0.05) differences compared to vehicle control.