Figure 1
RFRP-3 immunoreactivity and neuronal activity in the dorsomedial hypothalamus of female C57BL/6J mice sampled at different time points of the day of diestrus and proestrus. (A) LH blood concentrations; (B) number of RFRP-3-ir neurons in the DMH; (C) percentage of c-Fos–expressing RFRP-3-ir neurons; (D) Photograph where arrows show c-Fos–positive (brown) RFRP3-ir (blue) neurons at ZT0 (08:00 am) on the day of proestrus; scale bar = 100 µm. Data are presented as mean ± SEM of n = 5 mice for each experimental point. Two-way ANOVA was used to assess significant variations among different time points and estrous stages, followed by Sidak’s multiple comparisons test. Daily differences with a value of P < 0.05 were considered as significant and are indicated by *. Cycle stage differences were insignificant (P > 0.05).
Figure 2
Distribution of arginine vasopressin (AVP)-immunoreactive (ir) fibers in the dorsomedial hypothalamus of female C57BL/6J mice sampled at different time points of the day of diestrus and proestrus. (A) AVP-ir fiber density (arbitrary unit) in the DMH; (B) Percentage of RFRP-3-ir neurons with close AVP-ir fiber appositions; (C) Photograph shows RFRP-3-ir neurons and AVP-containing fibers at ZT8 (16:00) on the day of proestrus; scale bar = 200 µm. Data are presented as mean ± SEM of n = 5 mice for each experimental point. Two-way ANOVA was used to assess significant variations among different time points and estrous stages, followed by Sidak’s multiple comparisons test. Daily differences with a value of P < 0.05 were considered as significant and are indicated by *. Cycle stage differences were insignificant (P > 0.05).
Figure 3
Electrical firing characteristics of RFRP-3 neurons in the dorsomedial hypothalamus at two time points and stages of the estrous cycle in female C57BL/6J mice. (A) Representative traces of RFRP-3 neurons firing in the DMH at two time points (ZT6-10 and ZT11-14) during diestrus and proestrus; (B) Mean spike frequency of RFRP-3 neurons in the DMH at two time points during the day of diestrus (ZT6-10; n=22 neurons out of 7 mice and ZT11-14; n=42 neurons out of 10 mice) and proestrus (ZT6-10; n=30 neurons out of 7 mice and ZT11-14; n=30 neurons out of 9 mice); (C) InterSpike Interval CV (ISI-CV) of RFRP-3 neurons in the DMH at two time points during the day of diestrus and proestrus. Data are presented as mean ± SEM. Two-way ANOVA was used to assess significant variations in the firing properties among different time points and estrous stages. Differences among groups were considered significant for P < 0.05 and are indicated by *.
Figure 4
Effect of AVP on RFRP-3 electrical activity in the dorsomedial hypothalamus at different time points and stages of the estrous cycle in female C57BL/6J mice. (A) Sample trace illustrating the effect of AVP on RFRP-3 neuron firing in Diestrus at ZT6-10. (B) Average time course of relative InterSpikeInterval (ISI) in response to AVP (1µM) application of RFRP-3 neurons in the DMH at ZT 6-10 (n=6 neurons out of 5 mice) and ZT11-14 (n=7 neurons out of 3 mice) during the day of diestrus. (C) Summary bar graph of the AVP effect on mean spike frequency of RFRP-3 neurons in the DMH during the day of diestrus. (D) Average time course of relative InterSpikeInterval (ISI) in response to AVP (1µM) application of RFRP-3 neurons in the DMH at ZT 6-10 (n=7 neurons out of 5 mice) and ZT11-14 (n=4 neurons out of 3 mice) in the day of proestrus. (E) Summary bar graph of the AVP effect on mean spike frequency of RFRP-3 neurons in the DMH the day of proestrus. Data are presented as mean ± SEM. Repeated measures Two-way ANOVA was used to assess significant variations in the relative ISI among different time points in diestrus and proestrus. A paired Student t-test was used to compare RFRP mean firing frequency between baseline and after AVP application. Differences among groups were considered significant for P < 0.05 and are indicated by *.