Region-, age-, and sex-specific effects of fetal diazepam exposure on the postnatal development of neurosteroids

Abstract

Fetal exposure to diazepam (DZ), a positive modulator of GABA(A) receptors and an agonist at mitochondrial benzodiazine receptors, induces long-term neural and behavioral effects. This study evaluated whether the early manipulation influenced the normal development of brain levels of neurosteroids or altered steroid action at GABA(A) receptors. Pregnant dams were injected over gestation days 14 through 20 with DZ (2.5 mg/kg) or the vehicle. Male and female offspring were analyzed at five postnatal ages. The levels of progesterone (P), dihydroprogesterone (DHP), 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP), testosterone (T), dihydrotestosterone, and 5alpha-androstan-3alpha,17beta diol were measured in the cerebral cortex and diencephalon. The results indicated that development of brain steroid levels and the impact of fetal DZ exposure were region- and sex-specific. Age-related changes in brain steroids did not mirror associated changes in circulating P and T. Age regulated the levels of all 3 progestins in the cerebral cortex, and fetal DZ exposure interacted with the development of P and DHP. The development of 3alpha,5alpha-THP in the cortex was markedly influenced by sex, with levels in males decreasing over postnatal development whereas they increased over postpubertal development in females. An adolescent surge in T levels was observed in male cortex and fetal DZ exposure prevented that surge. Steroid levels in the diencephalon were altered by age mainly in females, and DZ exposure had little effect in this region. The data support region-specific regulation of brain steroid synthesis. Only in the cerebral cortex are relevant mechanisms readily modifiable by fetal DZ exposure. However, neither sex nor fetal DZ exposure altered the response of GABA(A) receptors in adult cortex to neurosteroid.

Fig. 1

Levels (ng/g) of progesterone, DHP, and 3α,5α-THP (THP) measured in the cerebral cortex of male and female rats at different postnatal ages from PD 7 to PD 60. Steroids were measured in Long–Evans rats exposed in utero to DZ (2.5 mg/kg) over gestational days 14–20 and in controls (VEH). The control group consisted of animals whose dams had either not been injected in utero or were injected with the vehicle. Data are expressed as mean ± SEM. Significance is designated as follows: *significant difference between treatment groups in respective sex at indicated age; #significantly different from control group, respective sex (collapsed across age); ¶significantly different from unmarked ages, respective sex (collapsed over treatment) and significantly different from other sex, same age. See text for other significant results.

Fig. 2

Levels (ng/g) of testosterone, DHT, and 3α-diols (Diols) measured in the cerebral cortex of male and female rats at different postnatal ages from PD 7 to PD 60. Steroids were measured in Long–Evans rats exposed in utero to DZ (2.5 mg/kg) over gestational days 14–20 and in controls (VEH). The control group consisted of animals whose dams had either not been injected in utero or were injected with the vehicle. Data are expressed as mean ± SEM. Significance is designated as follows: *significant difference between treatment groups in respective sex at indicated age, #significantly different from control group (collapsed over age), respective sex, ^significantly different from the other sex, collapsed over age and treatment group. See text for other significant results.

Fig. 3

Levels (ng/g) of progesterone, DHP, and 3α,5α-THP (THP) measured in the diencephalon of male and female rats at different postnatal ages from PD 7 to PD 60. Steroids were measured in Long–Evans rats exposed in utero to DZ (2.5 mg/kg) over gestational days 14–20 and in controls (VEH). The control group consisted of animals whose dams had either not been injected in utero or were injected with the vehicle. Data are expressed as mean ± SEM. Significance is designated as follows: *significant difference between treatment groups in respective sex at indicated age, ¶significantly different from unmarked ages, respective sex (collapsed over treatment) and significantly different from other sex, same age.

Fig. 4

Levels (ng/g) of testosterone, DHT, and 3α-diols (Diols) measured in the diencephalon of male and female rats at different postnatal ages from PD 7 to PD 60. Steroids were measured in Long–Evans rats exposed in utero to DZ (2.5 mg/kg) over gestational days 14–20 and in controls (VEH). The control group consisted of animals whose dams had either not been injected in utero or were injected with the vehicle. Data are expressed as mean ± SEM. Significance is designated as follows: #significantly different from control group, respective sex (collapsed across age).