Brain 5alpha-dihydroprogesterone and allopregnanolone synthesis in a mouse model of protracted social isolation

Abstract

Allopregnanolone (ALLO), is a brain endogenous neurosteroid that binds with high affinity to gamma-aminobutyric acid type A (GABA(A)) receptors and positively modulates the action of GABA at these receptors. Unlike ALLO, 5alpha-dihydroprogesterone (5alpha-DHP) binds with high affinity to intracellular progesterone receptors that regulate DNA transcription. To investigate the physiological roles of ALLO and 5alpha-DHP synthesized in brain, we have adopted a mouse model involving protracted social isolation. In the frontal cortex of mice, socially isolated for 6 weeks, both neurosteroids were decreased by approximately 50%. After administration of (17beta)-17-(bis-1-methyl amino carbonyl) androstane-3,5-diene-3-carboxylic acid (SKF105,111), an inhibitor of the enzyme (5alpha-reductase Type I and II) that converts progesterone into 5alpha-DHP, the ALLO and 5alpha-DHP content of frontal cortex of both group-housed and socially isolated mice decreased exponentially to 10%-20% of control values in about 30 min. The fractional rate constants (k h(-1)) of ALLO and 5alpha-DHP decline multiplied by the ALLO and 5alpha-DHP concentrations at any given steady-state estimate the rate of synthesis required to maintain that steady state. After 6 weeks of social isolation, ALLO and 5alpha-DHP biosynthesis rates were decreased to 30% of the values calculated in group-housed mice. Moreover, in socially isolated mice, the expression of 5alpha-reductase Type I mRNA and protein was approximately 50% lower than in group-housed mice whereas 3alpha-hydroxysteroid oxidoreductase mRNA expression was equal in the two groups. Protracted social isolation in mice may provide a model to investigate whether 5alpha-DHP by a genomic action, and ALLO by a nongenomic mechanism down-regulate the action of drugs acting as agonists, partial agonists, or positive allosteric modulators of the benzodiazepine recognition sites expressed by GABA(A) receptors.

Figure 1

Social isolation decreases ALLO and 5α-DHP but not pregnenolone (PREG) or progesterone (PROG) content in the frontal cortex of mice. Each bar represents the mean ± SEM of five to six mice either group-housed (GH, open bars) or socially isolated (SI, filled bars) for 6 weeks. *, P < 0.01 when SI mice were compared with GH mice.

Figure 2

Semilogarithimic plot of the decline of ALLO (A) and 5α-DHP (B) from mouse frontal cortex after i.p. administration of SKF105,111 (48 μmol/kg). (A) The regression equation for ALLO is as follows: log [ALLO]pmol/g = 0.80 − 0.035time in group-housed (GH, ●) mice and log[ALLO]pmol/g = 0.51 − 0.028time in socially isolated (SI, ○) mice. The fractional rate constant (k) of ALLO decline in GH and SI mice is 4.8 h⁻¹ and 3.9 h⁻¹, respectively. (B) The regression equation for 5α-DHP is log[5α-DHP]pmol/g = 0.99 − 0.02time in GH (●) mice and log[5α-DHP]pmol/g = 0.71 − 0.012time in SI (○) mice. The fractional rate constant (k) of 5α-DHP decline in GH and SI mice is 2.8 h⁻¹ and 1.7 h⁻¹, respectively. The neurosteroids were measured at 0, 15, and 30 min after SKF105,111 treatment. Each point is the mean ± SEM of five to six mice. The turnover rate (TR) (pmol⋅g⁻¹⋅h⁻¹) was calculated by multiplying k/h by the ALLO or 5α-DHP concentration at steady state. **, Differences between SI_TR and GH_TR for ALLO or 5α-DHP were statistically significant (P < 0.01, Student's t test).

Figure 3

Brain distribution of 5α-reductase Type I and 3α-HSOR mRNAs. The mRNAs were determined by competitive RT-PCR with mutant internal standards (16). Each value is the mean ± SE of three to five animals. OB, olfactory bulb; FC, frontal cortex; ST, striatum; HIP, hippocampus; HYP, hypothalamus; BS, brainstem; and CB, cerebellum.

Figure 4

5α-reductase Type I mRNA (A), 5α-reductase Type I protein (B), and 3α-HSOR (C) mRNA content in frontal cortex of group-housed (GH, open bars) and socially isolated (SI, filled bars) mice. Each value is the mean ± SE of at least six animals. *, P < 0.01 with Student's t test comparison. mRNAs were quantified by competitive RT-PCR with mutant internal standards (16). (Inset in B) Relative content of 5α-reductase Type I protein (5α-R, molecular mass ≈30 kDa) was estimated by Western blot using OD ratio with β-actin (47 kDa) as a control for overall protein content and blotting efficiency.