Reduction of lipocalin-type prostaglandin D synthase in the preoptic area of female mice mimics estradiol effects on arousal and sex behavior

Abstract

In female rodents, sleep and activity levels fluctuate over the estrous cycle. When estradiol (E2) levels are highest, sleep is reduced whereas locomotion is increased. The preoptic area (POA) is a key site for estrogenic regulation of these functions. However, molecular mechanisms by which E2 acts to reduce sleep and increase activity are unclear. Recently, we demonstrated a twofold reduction in lipocalin-type prostaglandin D synthase (L-PGDS) transcript levels, after E2 treatment, in the ventrolateral POA (VLPO), a putative sleep-active nucleus. Catalytic activity of L-PGDS produces PGD2, an endogenous somnogen. Thus, we hypothesized that decreases in PGD2 in the VLPO may contribute to the generalized arousal mediated by estrogens. To test this, we infused (i) antisense oligonucleotides (oligos), containing locked nucleic acid moieties (an improved technology), targeted to L-PGDS mRNA, (ii) scrambled sequence control oligos, or (iii) saline vehicle into the VLPO of ovariectomized female mice treated with E2 or oil. Arousal states and activity levels were assessed in response to a series of sensory stimuli (vestibular, olfactory, and somatosensory). The vestibular stimulus, which was administered first, resulted in the strongest responses and elicited significantly different responses among the groups: all groups in the E2 cohort demonstrated increases in overall home cage activity and duration of that activity compared with the oil-treated control groups. As predicted from E2 suppression of L-PGDS transcript levels, the responses of the locked nucleic acid antisense oligo-treated animals from the oil cohort did not differ from the E2-treated groups, such that they also demonstrated increases in activity and duration of activity compared with their controls. Thus, reducing L-PGDS in the VLPO of oil-treated females mimicked the effect of E2 on activity and arousal and represents a unique molecular pathway through which E2 may modulate these functions.

Fig. 1.

AS LNA-oligos, specific for L-PGDS mRNA reduce the target protein in the VLPO. (A) Representative electrophoretic lanes from an immunoblot of VLPO tissue from ovariectomized adult female Swiss–Webster mice treated with saline vehicle (Sal), LNA-scrambled (Scrm), or AS LNA to L-PGDS (AS). A total of 2 μg of total protein was loaded onto each lane. The blot was probed with a rabbit polyclonal Ab to L-PGDS, which recognized the appropriatesized bands and with a mouse monoclonal to GAPDH. (B) Quantification of L-PGDS immunoreactive bands after treatment with saline vehicle, scrambled LNA, or LNA-AS. Data are represented as a ratio of the optical density (O.D.) of L-PGDS-immunoreactive band to the GAPDH-immunoreactive band. The mean optical density of L-PGDS protein-immunoreactive bands was reduced in the LNA-AS-treated animals compared with the saline vehicle and scrambled LNA control-treated animals. (^**, P < 0.01.)

Fig. 2.

Histology of the injection site. Representative photomicrographs of Nissl-stained tissue from an untreated brain (A) and a brain microinjected with AS LNA-oligos to L-PGDS (B and C). (A) The VLPO is a triangle-shaped cluster of neurons along the base of the brain, just lateral to the optic chiasm. (B) A representative needle track from a successful microinjection. The arrowheads represent the tract, and the arrow demarcates the injection site. (Scale bars represent 50 μm.) (C) A high-power view of the injection site shown in B. Typically, the microinjection did little if any damage to the VLPO and surrounding structures. (Scale bar represents 10 μm.)

Fig. 3.

Analysis of generalized arousal. The vestibular stimulus elicited the strongest overall response and resulted in a significantly increased duration of general activity for both E₂-treated mice (ANOVA; ^**, P < 0.01) and the oil-treated AS group (ANOVA; ^*, P < 0.05) compared with the oil-treated control groups (saline and scrambled). Also, E₂-treated mice (^**, P < 0.01) and the oil-treated AS group (^*, P < 0.05) exhibited more general activity (as measured by HACTV) compared with the oil-treated control groups (saline and scrambled). Finally, AS administration to both oil- and E₂-treated animals resulted in an increase in total distance traveled compared with the oil-treated saline and scrambled oligo controls. Neither the olfactory nor somatosensory stimulus elicited significantly different behaviors among the treatment groups.

Fig. 4.

Activity levels after the vestibular stimulus. The activity levels during the first 5 min after the vestibular stimulus administration were analyzed. During this time, E₂-treated animals, regardless of AS treatment and the oil-treated AS group (ANOVA; ^*, P < 0.05) exhibited more general activity movements (as measured by HACTV) compared with the oil-treated control groups (saline and scrambled). There were no significant differences in the total distanced traveled (P = 0.47).

Fig. 5.

Female sex behavior. Females were tested during the dark phase of the cycle under red light. Quantification of lordosis behavior revealed a significant effect of AS treatment (^*, P < 0.05). Post hoc analysis demonstrated that the display of lordosis in response to the mounting of a stud male was significantly increased in E₂-treated, saline-infused animals compared with the oil-treated saline and scrambled control animals. Moreover, infusion of the AS oligos to oil-treated animals significantly increased the display of lordosis compared to the oil-treated saline and scrambled controls. This level of behavior was not significantly different from the E₂-treated saline controls.