Fig 1.
Fluorescent photomicrographs of coronal sections from a female OXTR-Venus mouse and a female wild type mouse.
Immunocytochemistry using an anti-GFP antibody was conducted to enhance and preserve the fluorescent signal from Venus. A. Numerous immunoreactive OXTR-Venus cells were observed in various regions in the section from an OXTR-Venus mouse. B. No immunoreactive-cells were observed in the brain section from a wild type mouse. Scale bar = 1 mm.
Fig 2.
Fluorescent photomicrographs showing OXTR-Venus immunoreactivity in coronal sections from a female (left panels) and a male (right panels) OXTR-Venus mouse. A. At the level of OVLT, OXTR-Venus cells were found in the layers of the cortex (Cg1, Cg2, M1, M2, S1, and S2). Especially dense populations of OXTR-Venus cells were observed in the piriform cortex (Pir, DEn and IEn), insular cortex (AIP), lateral and medial septum (LS and MS), and anterior part of medial division of the bed nucleus of the stria terminalis (STMA). A moderate population of OXTR-Venus cells was found in the medial preoptic area (MPOA) surrounding the OVLT. B. At the level of the anterior commissure (AC), a dense population of OXTR-Venus cells was additionally found in the intermediate and ventral parts of the LS (LSI and LSV), and divisions of the BNST (STMA, STLP, and STLV). In the preoptic areas, magnocellular preoptic nucleus (MCPO) and Septohypothalamic nucleus (Shy) had a cluster of OXTR-Venus cells, whereas not so dense a population of OXTR-Venus cells was observed in the medial preoptic area (MPOA). A sexually dimorphic distribution of OXTR-Venus cells occurred in the AVPV where it appeared only in the female; however, the difference is not very clear due to low magnification of images. C. At the level of the supraoptic nucleus (SON), a prominent cluster of OXTR-Venus cells was additionally found in several areas in the amygdala (ACo, CxA, BMA, CeC, CeL, CeM, MeAD) and hypothalamus (AH, SCN, PVN). A sparse population of OXTR-Venus cells were also found in the thalamus (PVA). D. At the level of the posterior hypothalamus, a dense population of OXTR-Venus cells was observed in the VMH, while a sparse population was found in the CA1, CA2, and CA3 layers of the hippocampus. Overall, there is no obvious sex difference in the distribution of OXTR-Venus between sexes.
Fig 3.
The sexually dimorphic distribution of OXTR-Venus cells in the AVPV.
A. Fluorescent photomicrographs showing OXTR-Venus immunoreactivity in (a) the preoptic-hypothalamic regions at the level of the AC, (b) 5 sections (200 μm) posterior to the AC, and (c) 15 sections (600 μm) posterior to the AC from a female (top panels) and a male (bottom panels) OXTR-Venus mouse. B. The mean number of OXTR-Venus neurons in the AVPV (i-a), in the medial preoptic area (MPOA) including the AVPV (ii-a), and the MPOA without the AVPV (iii-a) on each brain section from 8 female and 6 male OXTR-Venus mice are plotted from the 10th section anterior from the AC to the 25th section posterior from the AC. The mean total number of OXTR-Venus cells in females was greater than in males in the AVPV (i-b) and in the MPOA including the AVPV (ii-b). There were no significant differences between sexes in the number of OXTR-Venus cells in the MPOA excluding the AVPV (iii-b). All numerical data are expressed as the mean ± SEM. AC: anterior commissure; POA: preoptic area. Scale bars = 200 μm.
Fig 4.
OXTR-Venus cells in the AVPV express functional OXTR.
A. Single-cell RT-PCR analysis of OXTR expression in OXTR-Venus cells. Expression of OXTR mRNA was detected in 8 of 10 Venus cells harvested from OXTR-Venus heterozygous (+/-) females, but not detected from any of 4 OXTR-Venus cells obtained from homozygous (+/+) females. B. Examples of the effect of oxytocin on membrane potential and the firing pattern of OXTR-Venus (+/-) cells. a. Bath applications of oxytocin (100 nM) repeatedly caused membrane depolarization. b. The oxytocin-mediated depolarization caused repetitive firing. c. Application of oxytocin had no effect on OXTR-Venus (+/+) cells. C. In OXTR-Venus (+/-) cells, the mean membrane potentials before, during, and after the application of oxytocin (100 nM) were -60.36 ± 5.97 mV, -52.76 ± 3.47 mV, and -60 ± 3.74 mV, respectively. The application of oxytocin resulted in significant depolarization in OXTR-Venus (+/-) cells. In OXTR-Venus (+/+) cells, the mean membrane potentials before, during, and after the application of oxytocin (100 nM) were -67.57 ± 2.39 mV, -68.73 ± 2.35 mV, and -68.02 ± 2.24 mV, respectively. The application of oxytocin did not cause significant change in membrane potentials of OXTR-Venus (+/+) cells (F(2,4) = 1.33, p = 0.19). D. The mean amplitude of depolarization in response to application of oxytocin was significantly (t(14) = -5.35; p<0.0001) greater among OXTR-Venus (+/-) cells (7.6 ± 1.004 mV) than in OXTR-Venus (+/+) cells (-1.17 ± 1.3 mV).
Fig 5.
OXTR-Venus cells in the AVPV express ERα.
A. Double confocal fluorescence photomicrographs of ERα immunoreactivity and OXTR-Venus from a female and a male OXTR-Venus (+/-) mouse. Many OXTR-Venus cells were seen in the AVPV from the female, whereas virtually no detectable OXTR-Venus cell was found in the AVPV from the male. The merged image shows that all OXTR-Venus cells were immunoreactive to ERα. B. Single-cell RT-PCR analysis of ERα expression in OXTR-Venus cells. The same set of cDNA derived from individually harvested OXTR-Venus cells used in Fig 2A was used. ERα mRNA was detected from all cells except cell #4.
Fig 6.
Expression of OXTR in the sexually dimorphic OXTR-neurons is supported by estrogen.
A. Plot of the average number of OXTR-Venus neurons in the AVPV/section in the serial sections from the OVLT to SCN in intact, OVX, OVX+E2, and OVX+Vehicle female mice. B. The mean total number of OXTR-Venus cells in the AVPV from intact female and OVX. The mean total number of OXTR-Venus cells in OVX was significantly lower than that of females. C. Fluorescence microscopic images of OXTR-Venus cells in the AVPV from an OVX+E2 and an OVX+Vehicle. The group of OXTR-Venus cells observed in the AVPV in the intact females was not observed in OVX+Vehicle; however, the group of OXTR-Venus cells was observed in the OVX+ E2 mouse. D. The mean total number of OXTR-Venus cells in the AVPV of OVX+E2 was significantly higher than that of OVX+Vehicle. All numerical data are expressed as the mean ± SEM.