Article Figures & Data
Figures
- Figure 1.
Hypothalamic lordosis circuit. Estradiol acts initially on NPY neurons in the ARH, which project to and activate POMC/β-End neurons. These neurons project to the MPN where the release of β-End activates and internalizes MORs. When these receptors are internalized, lordosis is attenuated. Neurons from the MPN project further to the VMH, where signals from other circuits are integrated before projecting to lower brain regions and ultimately to the spinal motor neurons responsible for the manifestation of lordosis behavior. 3V, 3rd ventricle; OC, optic chiasm; ME, median eminence. Figure adapted from Micevych and Christensen (2012), with permission.
- Figure 2.
POMC expression in tdTomato-labeled cell bodies. IHC was run to confirm that tdTomato was expressed in POMC neurons in the ARH. A, tdTomato in magenta. B, Rabbit anti-POMC in green. C, Merged image showing tdTomato and POMC-ir. 3V, 3rd ventricle. Scale bar = 50 µm.
- Figure 3.
ChR2 expression in ARH and MPN. A representative image showing (A) soma and fiber expression of ChR2-tdTomato in the ARH, and (B) fiber expression in the MPN in the same animal. The MPN is shown at the level containing the medial (m) and lateral (l) subdivisions. 3V, 3rd ventricle. Scale bars = 100 µm.
- Figure 4.
Photostimulation of ChR2 in the MPN significantly attenuates the LQ. A two-way ANOVA followed by Tukey’s multiple comparisons test indicated a significant interaction between virus type and behavior test on the LQ (F(2,28) = 3.96, p = 0.03). Photostimulation of ChR2-expressing fibers in the MPN significantly reduced the LQ as compared to all other conditions (denoted by *). There was no difference in initial sexual receptivity between any of the three groups. In Pomc-cre mice expressing ChR2 in β-End terminals, photostimulation significantly attenuated lordosis (photostimulation, white bar) as compared to the pre-test without photostimulation (pre-test, white bar; simple main effect p = 0.01). Photostimulation of ChR2 (white bar) also significantly reduced the LQ as compared to both mice with the control virus (gray bar, simple main effect p = 0.001) and mice with incorrectly placed fiber optic cannulae (black bar, simple main effect p = 0.03). Photostimulation of ChR2 (photostimulation, white bar) furthermore significantly reduced the LQ as compared to the pre-test LQ of both mice receiving the control virus (pre-test, gray bar, simple main effect p = 0.002) and those with incorrectly placed fiber optic cannulae (pre-test, black bar, simple main effect p = 0.03). Mice that received the control AAV exhibited no difference (p > 0.99) in LQ between the pre-test and during photostimulation, nor did mice expressing ChR2 but with incorrect placement of the fiber optic cannula (p > 0.99). There was no difference in LQ between mice that received the control virus and those with ChR2 but incorrect placement of fiber optic cannulae in any condition. Values are expressed as mean ± SEM.
- Figure 5.
Photostimulation of ChR2 in β-End terminals in the MPN internalizes the MOR. Photostimulation induced the internalization of MOR within cells in the MPN. A, A one-way ANOVA indicated that there was a significant difference in receptor internalization between groups (F(2,11) = 5.15, p = 0.03, r 2 = 0.48). Tukey’s multiple comparisons test indicated that mice with ChR2 in β-End terminals in the MPN showed a greater percentage of the area of the image covered by MOR-ir (mean, 8.7 ± 1.8%) as compared to both mice that received the control AAV (mean, 4.3 ± 0.4%; *p = 0.05) and those with ChR2 but incorrect fiber optic placement (mean, 3.7 ± 0.4%, **p = 0.04). Images show DAPI (blue) and MOR (green) in a representative cell from group 1 (B) and group 2 (C). Scale bar = 2 µm. All values expressed as mean ± SEM.
- Figure 6.
cFOS expression in the ARH is increased following photostimulation. IHC for cFOS was run to confirm activity in POMC neurons in the ARH. A, Following photostimulation, cFOS trended toward greater expression in neurons in mice with ChR2 than those that had the control virus (ChR2 mean, 41.3 ± 9.6%; control AAV mean, 15.7 ± 6.2%; p = 0.09), though this effect was not statistically significant, likely due to a lack of statistical power. tdTomato in magenta (B), cFOS in green (C), and merged image with both tdTomato and cFOS (D) in a representative mouse expressing ChR2 in the ARH. Double-labeled neurons appear yellow. Scale bar = 50 µm. 3V, 3rd ventricle. All values expressed as mean ± SEM.
Tables
Group Virus Hormone replacement n Group 1 ChR2 EB+P4 7 Group 2 Control EB+P4 6 Group 3 ChR2 Oil 4 Group 4 Control Oil 4 Group 5 ChR2 EB+P4 4 Presented are the groups of mice used in this study, including AAV, hormone treatment, and number of mice per group. All hormones were dissolved in safflower oil and delivered subcutaneously 2 h before lights off. Mice that did not receive hormone replacement instead received an equal volume of safflower oil only.
n Group JAX #005965 JAX #010714 Group 1 6 1 Group 2 4 2 Group 3 2 2 Group 4 2 2 Group 5 4 0 Two strains of Pomc-cre mice were used in this study; provided are the number of each strain of mouse used in each experimental group (described in Table 1).
Antibody Dilution Source Rabbit anti-MOR 1:4000 Neuromics, #RA10104 Rabbit anti-cFOS 1:5000 Abcam, #ab190289 Rabbit anti-POMC 1:8000 Phoenix Pharmaceuticals Inc., #H-029-30 Provided is the species in which the antibody was raised, dilution of antibody in microliters, and commercial source of primary antibodies used.
Antibody Dilution Source Biotinylated goat anti-rabbit IgG 1:300 Vector Laboratories, #BA-1000 Streptavidin Alex Fluor 488 1:2000 Molecular Probes, #S-11223 Alexa Fluor 488 donkey anti-rabbit IgG 1:2000 Jackson ImmunoResearch, #711-545-152 Provided is the type of secondary antibody including species in which it was raised, dilution of antibody in microliters, and commercial source.
Mean SEM Group 1 79.9 3.3 Group 2 86.8 4.5 Group 5 82.0 5.2 Presented is the mean and the SEM of the pre-test LQ score for each of the groups that received hormone replacement.
Mean SEM Group 1 52.6 9.0 Group 2 88.0 4.8 Group 5 81.4 3.0 Presented is the mean and the SEM of the LQ score during photostimulation for each of the groups that received hormone replacement.
In this issue
