DOI: 10.5061/dryad.pnvx0k6zr
title: Data from: Multi-dimensional oscillatory activity of mouse GnRH neurons in vivo

description: The gonadotropin-releasing hormone (GnRH) neurons represent the key output
 cells of the neural network controlling mammalian fertility. We used GCaMP
 fiber photometry to record the population activity of the GnRH neuron
 distal projections in the ventral arcuate nucleus where they merge before
 entering the median eminence to release GnRH into the portal vasculature.
 Recordings in freely behaving intact male and female mice revealed abrupt
 ~8-min duration increases in activity that correlated perfectly with the
 appearance of a subsequent pulse of luteinizing hormone (LH). The GnRH
 neuron dendrons also exhibited a low level of unchanging clustered,
 rapidly fluctuating baseline activity in males and throughout the estrous
 cycle in females. In female mice, a gradual increase in basal activity
 that exhibited ~80-min oscillations began in the afternoon of proestrus
 and lasted for 12 h. This was associated with the onset of the LH surge
 that ended several hours before the fall in GCaMP signal. Abrupt 8-min
 duration episodes of GCaMP activity continued to occur on top of the
 rising surge baseline before ceasing in estrus. These observations provide
 the first description of GnRH neuron activity in freely behaving animals.
 They demonstrate that three distinct patterns of oscillatory activity
 occur in GnRH neurons. These are comprised of low-level rapid baseline
 activity, abrupt 8-min duration oscillations that drive pulsatile
 gonadotropin secretion and, in females, a gradual and very prolonged
 oscillating increase in activity responsible for the preovulatory LH
 surge.
description: We used fiber photometry to record GCaMP fluorescence signal in
 freely behaving mice for 6 or 24 h periods using previously described
 methodology (Han, Kane et al. 2019, Han, Morris et al. 2023). This
 included a custom-built photometry system using Doric components (Doric
 Lenses, QC, Canada) and National Instrument data acquisition board (TX,
 USA) based on a previous design (Lerner, Shilyansky et al. 2015). Blue
 (465-490 nm) and violet (405 nm) LED lights were sinusoidally modulated at
 frequencies of 531 and 211 Hz respectively and were focused onto a single
 fiber optic connected to the mouse. The light intensity at the tip of the
 fiber was 30-80 microwatts. Emitted fluorescence signal from the brain was
 collected via the same fiber, passed through a 500-550 nm emission filter
 and focused onto a fluorescence detector (Doric, QC, Canada). The
 emissions were collected at 10 Hz and the two GCaMP6 emissions were
 recovered by demodulating the 465-490 nm signals (calcium-dependent) and
 405 nm (calcium-independent) signals. Signals were either recorded in a
 continuous mode or a scheduled 5s on/10s off mode.
 Analysis was performed in MATLAB with the subtraction of 405
 signal from 465-490 signal to extract the calcium-dependent signal
 followed by an exponential fit algorithm used to correct for baseline
 shift. The signal was converted to DF/F (%) values using the equation
 DF/F=(F<sub>recorded</sub>-F<sub>baseline</sub>)/
 F<sub>baseline</sub>) x 100. The Findpeaks algorithm was used
 to detect dSEs, and the duration and the time of SE to the half width full
 maximum was determined. For deconvolution of signals from 24-h proestrus
 recordings, the movmean algorithm was used to extract 30-min rolling
 average. With this approach, slow oscillatory shifts in
 baseline during the proestrous surge were detected. The onset and offset
 of the surge signals were determined by the points at which there was a
 &gt;5% increment in ΔF relative to all previous timepoints, and when
 the calcium signal returned to 90% of the baseline value, respectively.
 The highest value observed during the slow oscillatory phase of the surge
 was determined to be the peak, and FWHM values were determined from the
 onset, peak and offset values. The time taken between one trough to
 another in individual oscillations was calculated as the duration of each
 oscillation. The second phase of the deconvolution detected all dSE
 signals using ‘findpeaks’ algorithm and signals between -60 to 360 s
 around each peak were separated to visualize the remaining low amplitude
 ‘residual’ signal. A threshold of 5% above baseline was used to extract
 the residual signal. Residual signals with peaks occurring &gt; 420s
 from the preceding peak were considered to represent separate
 clusters. To examine the relationship between calcium
 episodes with LH pulses, freely behaving mice were attached to the fiber
 photometry system, and 4-mL blood samples were obtained every 5 to 10
 minutes from the tail tip over a period of 120 to 240 minutes. To assess
 the relationship between the long calcium increment during proestrus
 evening and the LH surge, female mice were attached to the fiber
 photometry system in the morning of proestrus and blood samples (3-mL)
 collected every 3 hours for 18 hours until the morning of estrus. Levels
 of LH were measured by in-house LH ELISA (Steyn, Wan et al. 2013) with an
 assay sensitivity of 0.04 ng/mL and intra-assay coefficient of variation
 of 8.2%.
 <em>Immunohistochemistry</em>
 Adult GnRH-Cre,GCaMP6s mice were given a lethal overdose of
 pentobarbital (3mg/100mL, i.p.) and perfused transcardially with 4%
 paraformaldehyde. Brains were processed for dual GFP and GnRH
 immunofluorescence. For GFP immunostaining,  anti-chicken GFP (1:5000,
 Aves Lab) was used followed by AlexaFluor 488-conjugated goat-anti-chicken
 (1:1000). For GnRH cell body immunostaining, GA2 guinea pig anti-GnRH
 antisera (1:3000, gift from G.Anderson, New Zealand) was used in
 combination with AlexaFluor 647-conjugated goat anti-guinea pig
 immunoglobulin (1:500, Thermo Fisher Scientific, USA). For GnRH dendron
 immunostaining, rabbit anti-GnRH (1:20,000, LR1, gift of R.Benoit,
 Montreal) antisera was used followed by biotinylated goat anti-rabbit
 immunoglobulin (1:1000, Jackson Immunoresearch) and AlexaFluor
 568-conjugated Streptavidin (1:400, Thermo Fisher Scientific, USA).
 Imaging was performed using a Leica SP8 Laser Scanning Confocal Microscope
 (Leica Microsystems) at the Cambridge Advanced Imaging Center and analyzed
 using ImageJ. <em>Statistical
 Analysis</em> All statistical analyses were
 performed in Prism 10 (GraphPad software Inc.). All values given in this
 study are mean ± SEM, and significance is defined as P &lt;0.05* or
 P&lt;0.01**. For inter-peak interval analysis in females across the
 estrous cycle and the residual cluster activity analysis, Kruskal Wallis
 ANOVA followed by Dunn’s post-hoc tests was used.   
description: # Multi-dimensional oscillatory activity of mouse GnRH neurons in vivo
 [https://doi.org/10.5061/dryad.pnvx0k6zr](https://doi.org/10.5061/dryad.pnvx0k6zr) ## Description of the data and file structure Description of the data and file structure. We have submitted 5 Excel files in total corresponding to figures 2-6  in the paper. Each figure has one Excel sheet with panels corresponding to each tab. For example, file Elife_GnRH_Fig2 Tab Fig2C corresponds to Fig 2C in the original article. More details of data collection and analysis can be found in the figure legends and Methods section of the original article. Applicable for all Species: mice Missing values are listed as blank; All data are plotted and analyzed using Graphpad Prism 10 as described in Methods section. ### Files and variables #### File: Elife\_GnRH\_Fig2.xlsx Tab A: Figure 2A. A representative example of 24-h GCaMP photometry recording. Column A:Time in minutes, Column B: Clock time (24h), Column C: deltaF/F in percentage. Tab B: Figure 2B. Example of 8-h GCaMP6 photometry recording in a male mouse with a misplaced fiber optic. Column A:Time in minutes, Column B: Clock time (24h), Column C: deltaF/F in percentage. Tab C: Figure 2C. Average high-resolution profile of an dSE in male mice showing a rapid onset followed by a gradual decrease back to baseline. A:Time in seconds, Column B: Normalized amplitude (peak value = 1.0), Column C:  standard error of mean Tab D: Figure 2D. (D) Inter-peak intervals combined from all recordings displayed as a percentage of all intervals occurring in 10-min bins. Column A: Bin centre in minutes, Column B: frequency displayed as percentage Tab E: Figure 2E. Representative example showing the relationship of dSEs to pulsatile LH secretion. Column A: Time in minutes, Column B: deltaF/F in percentage, Column E: Time of blood sampling (relative to recording time), Column F: LH in ng/ml  Tab F: Figure 2F. Normalized increase in LH plotted against the dSEs, with the time 0 being the peak of dSE. A:Time in minutes, Column B: Normalized amplitude (peak value = 1.0). Column C = standard error of mean, Column F = Time of blood sampling (relative to recording time), Column G: Normalized LH value (peak = 1.0), Column H: Standard error of the mean #### File: Elife\_GnRH\_Fig3.xlsx Tab A-D: Figure 3Ai (A), 3Aii (B), 3Aiii (C), 3Aiv(D). Representative example of 6-h GCaMP6 photometry recording from female mice in (A) metestrus, (B) diestrus, (C) proestrus, and (D) estrus. Tab E: Panels to the right of each Figure 3Ai-iv. Frequency distribution of of Inter-peak intervals displayed as a percentage of all intervals occurring in 10-min bins. Column A: Bin center in minutes, Column B: metestrus, Column C: diestrus, Column D: proestrus, Column E: Estrus Tab F: Figure 3B.  Histograms showing inter-peak intervals of dSEs across the estrous cycle. Column A: Animal ID, Column B: metestrus , Column C: diestrus, Column D: proestrus, Column E = estrus.  Tab G: Figure 3C. Average high-resolution profile of dSEs in female mice showing a rapid onset followed by a gradual decrease in the signal.  A:Time in seconds, Column B: Normalized amplitude (peak value = 1.0), Column C: standard error of mean Tab H: Figure 3D. Representative female example showing the relationship of dSEs to pulsatile LH secretion. Column A: Time in minutes, Column B: deltaF/F in percentage, Column E: Time of blood sampling (relative to recording time), Column F: LH in ng/ml Tab I: Figure 3E. Normalized increase in LH plotted against the dSEs, with the time 0 being the peak of dSE. A:Time in minutes, Column B: Normalized amplitude (peak value = 1.0). Column C = standard error of mean, Column F = Time of blood sampling (relative to recording time), Column G: Normalized LH value (peak = 1.0), Column H: Standard error of the mean #### File: Elife\_GnRH\_Fig4.xlsx Tab A: Figure 4C. The duration of the baseline activity in male (Column A)  and female mice.  M= metestrus (Column B), D = diestrus (Column C) , P = proestrus (Column D) , E= estrus (Column E) Tab B: Fig 4D. The intra-cluster frequency in male (Column A)  and female mice.  M= metestrus (Column B), D = diestrus (Column C) , P = proestrus (Column D) , E= estrus (Column E). #### File: Elife\_GnRH\_Fig5.xlsx Tab A: Figure 5A. 21-h GCaMP recordings from four proestrous female mice. Column A: Time in minutes. Column B: Clock time (24h), Column C: Fig5A-#1, Column D: Fig5A-#2, Column E: Fig 5A-#3, Column F: Fig5A-#4.   Tab B: Figure 5B. A representative example showing the relationship of the slow increase in baseline calcium activity with LH surge. Column A: Time in minutes, Column B: Clock time (24h), Column E: Time of blood sampling (relative to recording time), Column F: LH in ng/ml #### File: Elife\_GnRH\_Fig6.xlsx Tab A: Fig 6A, Tab B: Fig 6B. Representative examples of 24-h photometry recordings from two female mice starting at proestrus showing (Column B) the original recording, (Column C) 30-min rolling average highlighting the LH surge signal, (Column D) the LH surge signal subtracted from the original recording, displaying the dendron synchronization episodes, and (Column E)  the residual baseline signal after subtracting both the surge and pulse profiles. Column A: Time in minutes.