Motivated behaviors and levels of 3α,5α-THP in the midbrain are attenuated by knocking down expression of pregnane xenobiotic receptor in the midbrain ventral tegmental area of proestrous rats

Abstract

Introduction: Progesterone (P4 ) and its product, 5α-pregnan-3α-ol-20-one (3α,5α-THP), act in the midbrain ventral tegmental area (VTA) to alter motivated behaviors, such as mating, and motor and anxiety behavior. Of interest is whether 3α,5α-THP formation requires the pregnane xenobiotic receptor (PXR), which is expressed in the midbrain of rats.

Aim: The role of PXR in the midbrain for 3α,5α-THP formation, which precedes modulation of motivated behaviors, was investigated.

Methods: Rats had estrous cycle phase determined and were assessed when they were in diestrus or proestrus. Diestrous and proestrous rats were infused with control or antisense oligodeoxyribonucleotides (AS-ODNs) targeted against PXR to the VTA.

Main outcome measures: In pilot studies, PXR gene and protein expression in the midbrain were determined with quantitative reverse transcriptase polymerase chain reaction and Western blotting, respectively. Diestrous and proestrous rats infused with control or AS-ODNs to the VTA were tested for anxiety (open field and plus maze), social (social interaction), and sexual (paced mating) behavior. Expression of PXR in the midbrain was verified with Western blotting. Plasma estradiol, P4 , dihydroprogesterone (DHP), and 3α,5α-THP levels, and brain P4 , DHP, and 3α,5α-THP levels were measured. We predicted that proestrous rats infused with PXR AS-ODNs would have decreased anti-anxiety, social, and sexual behavior, lower midbrain expression of PXR, and lower midbrain levels of 3α,5α-THP compared with controls.

Results: Results supported the hypothesis that formation of 3α,5α-THP requires PXR and may be important for motivated behaviors. PXR AS-ODN, compared with control, infusions to the VTA reduced PXR expression and 3α,5α-THP levels in the midbrain and attenuated sexual receptivity of proestrous rats.

Conclusions: Knockdown of PXR in the midbrain reduces 3α,5α-THP levels and sexual receptivity of proestrous rats. Thus, PXR in the midbrain may be required for the observed increase in 3α-5α-THP during proestrus, which has subsequent effects on motivated, reproductive behaviors.

Keywords: Allopregnanolone; Lordosis; Mating; Neurosteroids; Progesterone; Receptivity.

Figure 1

Brains were visually inspected during dissection to verify that infusions were to the midbrain (shown on left, indicated by arrowhead) or outside of the midbrain (shown on right, indicated by arrowhead). Data from 20 rats that had infusions outside of the midbrain were not included in statistical analyses.

Figure 2

Depicts mean (+SEM) relative optical density of midbrain PXR bands to actin bands on the same blots of cycling rats administered vehicle control (diestrous n=11, proestrous n=13) or PXR AS-ODNs (diestrous n=13, proestrous n=9) infusions that were behaviorally-tested and had plasma and brain levels of steroids measured. Inset depicts representative picture of blot run for this experiment. In each blot, lanes were run with positive controls (liver and heart) and experimental conditions. * indicates a reduction due to PXR AS-ODN, compared to control, infusions of proestrous rats (P<0.05).

Figure 3

Depicts mean (+ SEM) entries to inner 8 squares in the open field of cycling rats administered vehicle control (diestrous n=11, proestrous n=13) or PXR AS-ODNs (diestrous n=13, proestrous n=9) infusions. * indicates a reduction due to PXR AS-ODN, compared to control, infusions (p<0.05).

Figure 4

Depicts mean (+ SEM) open arm time in the elevated plus maze of cycling rats administered vehicle control (diestrous n=11, proestrous n=13) or PXR AS-ODNs (diestrous n=13, proestrous n=9) infusions.

Figure 5

Depicts the mean (+S.E.M.) time spent in social interaction of cycling rats administered vehicle control (diestrous n=11, proestrous n=13) or PXR AS-ODNs (diestrous n=13, proestrous n=9) infusions.

Figure 6

Depicts the mean (+S.E.M.) lordosis quotients of cycling rats administered vehicle control (diestrous n=11, proestrous n=13) or PXR AS-ODNs (diestrous n=13, proestrous n=9) infusions. * indicates a reduction due to PXR AS-ODN, compared to control, infusions among proestrous rats (P<0.05).

Figure 7

Depicts mean (+SEM) 3α,5α-THP levels of cycling rats administered vehicle control (diestrous n=11, proestrous n=13) or PXR AS-ODNs (diestrous n=13, proestrous n=9) infusions. * indicates a reduction due to PXR AS-ODN, compared to control, infusions among proestrous rats (P<0.05).