Work Description

Date:
18 February 2025

Dataset title:
Sex and time of day alter the interactions between hypothalamic glia and the neural circuits controlling reproduction

Dataset creators:
C.D. Phillips, R.A. DeFazio, S.M. Moenter

Dataset contacts:
C.D. Phillips (chrystip@umich.edu) and S.M. Moenter (smoenter@umich.edu)

Funding:
Supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health (NIH) under award number R37HD034860 (to S.M. Moenter). C.D. Phillips was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the NIH under award number F31HD110102

Key points:
- We investigated the extent to which hypothalamic glia regulate gonadotropin-releasing hormone (GnRH) neuron and arcuate kisspeptin (KNDy) neuron activity, and if sex, reproductive cycle stage, and the time of day modulate this regulation.

- Activating preoptic glia stimulates proximal GnRH neurons and induces luteinizing hormone (LH) release regardless of sex, cycle stage, or time of day, although LH is released more rapidly in males

- Activating arcuate glia doesn't stimulate proximal KNDy neurons nor induce LH release regardless of time of day, but LH is released in males, but not females, in a KNDy-neuron-independent manner

- The gliotransmitter prostaglandin E2 (PGE2) stimulates GnRH neurons, but not KNDy neurons. This effect is stronger in males compared to females and is stronger during the morning

Research overview:
An upstream network, including glia and arcuate nucleus (ARC) kisspeptin neurons, controls hormone secretion from preoptic area (POA) gonadotropin-releasing hormone (GnRH) neurons, which form the final common pathway for the central control of fertility. In males, chemogenetic activation of Gq-mediated signaling in POA glia activated GnRH neurons and downstream luteinizing hormone (LH) release, while chemogenetic activation of ARC glia had no effect on ARC kisspeptin neurons. We characterized sex differences and time-of-day effects in these critical circuits to understand their effects on reproduction. Chemogenetic activation of glial fibrillary acidic protein (GFAP)-expressing cells increased intracellular calcium concentrations regardless of sex, brain region, or time of day. Activation of POA glia or treatment with the gliotransmitter analog dimethyl prostaglandin E2 (dmPGE2) increased GnRH neuron firing rate, and these responses were dependent upon sex and time of day. In contrast, ARC kisspeptin neuron firing rate was unresponsive to ARC glia activation or dmPGE2 regardless of sex or time of day. POA glial activation increased LH levels in males and females but the response in males was more rapid. ARC glia activation had no effect on LH in females and the response in males was delayed compared to POA glia activation. A similar LH response persisted after ARC kisspeptin neuron ablation, suggesting it is not mediated by those neurons. GnRH neurons rather than arcuate kisspeptin neurons are thus the main target of glial regulation of reproductive neuroendocrine output and this regulation is dependent on sex and time of day.

Methodology:
The data analyzed are gathered from a variety of experiments which include extracellular recordings, serial blood sample collection, calcium imaging, and histological colocalization. Analysis was conducted using Prism 10 (GraphPad, Version 10.4.1 [627]) and SPSS (IBM, Version 29.0.0.0). See manuscript for further details.

Date coverage:
From 2021-06-01 to 2024-12-01

Instruments and/or Software specifications:
Prism 10 (GraphPad, Version 10.4.1, SPSS (IBM, Version 29.0.0.0); Olympus Cellsense software (Evident), ImageJ (Version 1.53q, Igor Pro 8 (Sutter Instruments); PatchMaster (HEKA Elektronic)

Files contained here:
Each Excel document contains raw and processed data gathered from one or more sets of experiments, which are further described below. The Word document contains the Supplemental Tables summarizing the statistical analysis (see Methodology) of the data gathered in the aforementioned Excel documents.

- "CDP_Supplemental_Tables.docx" contains 30 Supplemental Tables describing statistical outcomes (see the manuscript for further context to each table).

- "Calcium_imaging_with_chemogenetics_.xlsx" contains data from the experiments assessing if chemogenetic activation of POA and ARC glia induces increases in glial intracellular calcium levels (see Figures 2 and 3 in the manuscript). Factors that were addressed in these experiments included sex, reproductive cycle stage (diestrus and proestrus), and time of day (morning [AM] and afternoon [PM]). Noteworthy spreadsheets are described below:

- "Male summary" contains key biometric information and processed data from males across both brains regions, both times of day, and using either a control or a designer receptor exclusively activated by designer drug (DREADD) virus. The processed data is measured as area under the curve (AUC), which is a proxy for deltaF/F0 calculations of fluorescence induced by intracellular calcium changes.

- "Female summary" contains key biometric information and processed data from females across both brain regions, both cycle stages, both times of day, and using either a control or DREADD virus. Data presentation is similar to that in "Male summary".

- "Charts & graphs" contains calculations of sample sizes per experimental group and graphical summaries of the male and female data.

- "Data calculations" contains behind-the-scene calculations for "Charts & graphs"

- The remaining spreasheets (e.g., Male AM | POA) contain the raw data gathered using Olympus Cellsense software (Evident) and extracted using both ImageJ (Version 1.53q) and Igor Pro 8 (Sutter Instruments).

- "LH_measurements___extracellular_recordings_with_chemogenetics.xlsx" contains data from the experiments assessing if chemogenetic activations of POA and ARC glia 1) increase LH levels (see Figures 6, 7, and 8 in the manuscript) and 2) increases GnRH neuron or KNDy neuron firing rates (see Figures 4 and 5 in the manuscript). Factors that were addressed in these experiments included sex, reproductive cycle stage, and time of day. Noteworthy spreadsheets are described below:

- "Male summary" contains key biometric information and processed data from males across both brains regions, both times of day, and using either a control or DREADD virus. The processed data is divided into two sections: LH measurements are entered to the
right of the biometric information, and the extracellular recordings are futher to the right of the LH data. The data for LH levels (ng/mL) are collected at 10 min intervals (t1 - t17), and extracellular recordings are measured as firing rate (Hz).

- "Female summary" contains key biometric information and processed data from females across both brain regions, both cycle stages, both times of day, and using either a control or DREADD virus. Data presentation is similar to that in "Male summary".

- "Charts & graphs" contains calculations of sample sizes per experimental group and graphical summaries of the male and female data.

- "Data calculations" contains behind-the-scene calculations for "Charts & graphs"

- The remaining spreasheets (e.g., Male AM | POA) contain the raw data gathered using Olympus Cellsense software (Evident) and PatchMaster (HEKA Elektronic), and the data was extracted using Igor Pro 8 (Sutter Instruments).

- "KNDy_neuron_ablation_cell_count.xlsx" contains data from the experiment assessing if KNDy neuron ablation alters the LH level increase seen when ARC glia are chemogenetically activated in males (see Figure 8 in the manuscript).

- "dmPGE2_extracellular_recordings.xlsx" contains data from the experiments assessing if dmPGE2 is sufficient to activate GnRH or KNDy neurons (see Figure 9 and 10 in the manuscript). Factors that were addressed in these experiments included sex and time of day. Noteworthy spreadsheets are described below:

- "Male summary" contains key biometric information and processed data from males across both brains regions and both times of day. The processed data for extracellular recordings are measured as firing rate (Hz).

- "Female summary" contains key biometric information and processed data from females across both brain regions, both cycle stages, and both times of day. Data presentation is similar to that in "Male summary".

- "Charts & graphs" contains calculations of sample sizes per experimental group and graphical summaries of the male and female data. NOTE: The 200 nM dmPGE2 data, but not the 1 uM dmPGE2 data, is charted and graphed.

- "Data calculations" contains behind-the-scene calculations for "Charts & graphs"

- The remaining spreadsheets (e.g., Male AM | POA) contain the raw data gathered using Olympus Cellsense software (Evident) and PatchMaster (HEKA Elektronic), and the data was extracted using Igor Pro 8 (Sutter Instruments).

- "Extracellular_recordings_with_chemogenetics_and_EP1_2_antags.xlsx" contain data from the experiment assessing if PGE2 signaling is necessary to increase GnRH neuron firing rates. Factors that were addressed in these experiments included sex and time of day. Noteworthy spreadsheets are described below:

- "Male summary" contains key biometric information and processed data from males across both brains regions and both times of day using the DREADD virus. The processed data for extracellular recordings are measured as firing rate (Hz).
- "Female summary" contains key biometric information and processed data from females across both brain regions and both times of day using the DREADD virus. Data presentation is similar to that in "Male summary".
- "Charts & graphs" contains calculations of sample sizes per experimental group and graphical summaries of the male and female data.
- "Data calculations" contains behind-the-scene calculations for "Charts & graphs"
- The remaining spreasheets (e.g., Male AM | POA) contain the raw data gathered using Olympus Cellsense software (Evident) and PatchMaster (HEKA Elektronic), and the data was extracted using Igor Pro 8 (Sutter Instruments).

Related publication:
Phillips, C.D., et al. (2025). Sex and time of day alter the interactions between hypothalamic glia and the neural circuits controlling reproduction. Endocrinology. Forthcoming.

Use and access:
This data set is made available under a Creative Commons Attribution 4.0 International (CC BY 4.0).

To cite data:
Phillips, C. D., DeFazio, R. A., Moenter, S. M. Sex and time of day alter the interactions between hypothalamic glia and the neural circuits controlling reproduction [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/qtnn-sk87