GCaMP expression in GnRH neurons.

(A) Photomicrographs at the level of organum vasculosm of the laminae terminalis (OVLT) showing GFP immunofluorescence representing GCaMP6 (green), GnRH (purple) and merged channels showing double labelled cells (white). (B) Photomicrographs of four mice at the level of arcuate nucleus showing the location of the end of the optic fiber (dotted line) in relation to GnRH neuron dendrons labeled with GnRH (red), GFP representing GCaMP6 (green) and merged channels showing double labelled (yellow). 3V, third ventricle.

GnRH neuron dendron activity in male mice.

(A) A representative example of 24-h GCaMP6 photometry recording showing abrupt dendron synchronized episodes (dSEs) occurring on lower amplitude baseline activity in a male mouse. Below, expanded view of the trace indicated by the horizontal line. (B) Example of 8-h GCaMP6 photometry recording in a male mouse with a misplaced fiber optic. All recorded signals are under 3% of baseline. Below, expanded view of the trace indicated by the horizontal line. (C) Average high-resolution profile of an dSE in male mice (N=7 mice) showing a rapid onset followed by a gradual decrease back to baseline. ‘a’ and ‘b’ give values at full-width half maximum (FWHM). (D) Inter-peak intervals combined from all recordings (n= 96 dSEs) displayed as a percentage of all intervals occurring in 10-min bins. W=0.87, P<0.0001****, Shapiro Wilk normality test. Skewness = 1.39, Kurtosis=1.81. (E) Representative example showing the relationship of dSEs (black) to pulsatile LH secretion (red). (F) Normalized increase in LH plotted against the dSEs, with the time 0 being the peak of dSE.

GnRH neuron dendron activity in female mice.

(A) Representative example of 6-h GCaMP6 photometry recording from female mice in (i) metestrus, (ii) diestrus, (iii) proestrus, and (iv) estrus. To the right of each trace are histograms showing the mean inter-peak intervals for dSEs from all recordings across metestrus (n=53 dSEs), diestrus (n=88 dSEs), proestrus (n=62 dSEs) and estrus (n=42 dSEs) displayed as a percentage of all intervals occurring in 10-min bins. In all cycles, the data failed normality test (metestrus [W=0.92, P=0.0007***], diestrus [W=0.88, P<0.0001****], proestrus [W=0.92, P=0.0007***], estrus [W=0.94, P=0.03], Shapiro-Wilk test). (B) Histograms showing inter-peak intervals of dSEs across the estrous cycle. M = metestrus (N=8), D = diestrus (N=8), P = proestrus (N=6), E = estrus (N=5). *P<0.05, **P<0.01, Kruskal Wallis followed by Dunn’s post-hoc test. (C) Average high-resolution profile of dSEs in female mice showing a rapid onset followed by a gradual decrease in the signal. ‘a’ and ‘b’ show the values at full-width half maximum (FWHM). (D) A representative example showing the relationship of dSEs (black) to pulsatile LH secretion (red). (E) Normalized increase in LH plotted against all dSEs, with the time 0 being the peak of the dSE.

Baseline high-frequency cluster activity of GnRH neuron dendrons in male and female mice.

Representative 4-h GCaMP recordings in (A) two male mice and (B) a female mouse across the different stages of the estrous cycle showing high frequency baseline activity and larger increases in calcium activity representing dendron synchronization episodes (dSEs) indicated by red asterisks. The peaks of dSEs reaching >30% dFF were cut off to optimize the display of the low amplitude activities. Histograms showing the duration (C) and the intra-cluster frequency (D) of the baseline activity in male and female mice. No significance was found across the groups. M= metestrus, D = diestrus, P = proestrus, E= estrus.

Slow oscillating increases in calcium activity on the afternoon of proestrus in female mice.

(A) Examples of 21-h GCaMP recordings from four proestrous female mice. Note the prolonged (>10h) gradual oscillating increase in calcium signal beginning on the afternoon of proestrus. The sharp dSEs continue until approximately the plateau phase of the increased baseline at which time they stop or slow. (B) A representative example showing the relationship of the slow increase in baseline calcium activity (black) with LH surge (red). Note that the rise in LH occurs in alongside the initial rise in calcium activity but returns to baseline several hours before the calcium signal.

Deconvolution of the GCaMP signal across the proestrous surge reveals multimodal activity patterns of GnRH neuron dendron.

(A-B) Representative examples of 24-h photometry recordings from two female mice starting at proestrus showing (i) the original recording, (ii) 30-min rolling average highlighting the LH surge signal (red), (iii) the LH surge signal subtracted from the original recording, displaying the dSEs (blue), and (iv) the residual baseline signal (pink) after subtracting both the surge and pulse profiles.