DOI: 10.5061/dryad.zpc866tm1
title: Data from: GCaMP6f-expressing chromaffin cells in murine adrenal slices exhibit dynamic spontaneous calcium responses that do not require nerve input

description: Chromaffin cells of the adrenal medulla exhibit spontaneous action
 potentials and intracellular Ca2+ responses that, when dissociated in
 vitro, result in catecholamine secretion. However, spontaneous activity in
 these cells is nearly abolished when splanchnic nerve input is blocked in
 vivo. To address this discrepancy, we examined spontaneous Ca2+ responses
 in chromaffin cells using an intermediate preparation: adrenal slices from
 transgenic mice expressing the genetically encoded Ca2+ indicator GCaMP6f
 selectively in these cells. Within this preparation, the three-dimensional
 structure and extracellular environment surrounding chromaffin cells are
 retained, as is input from distal splanchnic nerve terminals, which
 spontaneously release the neurotransmitter acetylcholine. Chromaffin cells
 within these slices displayed spontaneous Ca2+ responses with frequencies
 and amplitudes that varied greatly within and between individual cells.
 However, population averages remained stable, providing a tool to measure
 the cellular and molecular mechanisms underlying these responses. While
 overall average frequencies and amplitudes of spontaneous Ca2+ responses
 depended on the influx of extracellular Ca2+ through voltage-gated Ca2+
 channels, they did not require Ca2+ release from intracellular stores,
 splanchnic nerve input, or activation of voltage-gated sodium channels.
 Together, these results suggest that spontaneous activity in chromaffin
 cells in adrenal slices is generated autonomously, similar to that in
 dissociated chromaffin cells. By contrast, spontaneous activity in
 chromaffin cells in intact animals likely represents a distinct form that
 depends on basal input from the intact splanchnic nerve. This study
 provides a foundation to further explore the diverse mechanisms mediating
 chromaffin cell activation.
description: # Data from: GCaMP6f-expressing chromaffin cells in murine adrenal slices
 exhibit dynamic spontaneous calcium responses that do not require nerve
 input Dataset DOI: [10.5061/dryad.zpc866tm1](10.5061/dryad.zpc866tm1) ##
 Description of the data and file structure We collected spontaneous
 calcium fluorescence transients from chromaffin cells within adrenal
 slices from mice expressing the calcium indicator GCaMP6f. To determine
 which mechanisms contributed to this spontaneous activity, we conducted
 several experiments in which we blocked various ion channels and receptors
 while recording calcium activity in six one-minute videos spaced five
 minutes apart. The first three videos were recorded prior to (pre-), and
 the last three videos after (post-), perfusing the blocker of choice. The
 drugs we used included EGTA to deplete extracellular calcium,
 cyclopiazonic acid (CPA) to deplete intracellular calcium stores,
 nifedipine (NIF) to selectively block L-type voltage-gated calcium
 channels, tetrodotoxin (TTX) to block voltage-gated sodium channels,
 hexamethonium (HEX) to block nicotinic acetylcholine receptors, and
 atropine (ATR) to block muscarinic acetylcholine receptors. We also
 included control experiments in which we demonstrated the lack of effect
 of ethanol (ETH) and DMSO, the solvents in which NIF and CPA were
 dissolved, respectively. For each drug experiment, we have provided 6
 Excel .xlsx data files, including 3 from male and 3 from female mouse
 subjects/slices, to which we added the drug indicated in the file title.
 In turn, each Excel .xlsx file contains 6 tabs, the data on each tab
 representing that from each of 6 videos that were acquired at 5 minute
 intervals. Tabs 1-3 contain fluorescence transients from chromaffin cells
 (1 cell per column) captured prior to, and tabs 4-6 contain fluorescence
 transients captured after, drug perfusion. The transients were obtained
 after background subtraction was performed in Image J. Only transients
 that met the criteria described in the Methods Section of the manuscript
 were considered active and included in analyses. ### Files and variables
 #### File: NO_DRUG **Description:** spontaneous calcium transients from 3
 male and 3 female subjects (6 excel files), 6 videos each (tabs), in the
 absence of drug #### File: EGTA **Description:** spontaneous calcium
 transients from 3 male and 3 female subjects (6 excel files), 6 videos
 each (tabs), before (tabs 1-3) and after (tabs 4-6) EGTA perfusion to
 deplete extracellular calcium #### File: CPA **Description:** spontaneous
 calcium transients from 3 male and 3 female subjects (6 excel files), 6
 videos each (tabs), before (tabs 1-3) and after (tabs 4-6) CPA perfusion
 to deplete intracellular calcium stores from the ER #### File: NIF
 **Description:** spontaneous calcium transients from 3 male and 3 female
 subjects (6 excel files), 6 videos each (tabs), before (tabs 1-3) and
 after (tabs 4-6) nifedipine perfusion to block L-type voltage-gated
 calcium channels #### File: TTX **Description:** spontaneous calcium
 transients from 3 male and 3 female subjects (6 excel files), 6 videos
 each (tabs), before (tabs 1-3) and after (tabs 4-6) tetrodotoxin perfusion
 to block voltage-gated sodium channels #### File: HEX
 **Description:** spontaneous calcium transients from 3 male and 3 female
 subjects (6 excel files), 6 videos each (tabs), before (tabs 1-3) and
 after (tabs 4-6) hexamethonium perfusion to block nicotinic acetylcholine
 receptors #### File: ATROPINE **Description:** spontaneous calcium
 transients from 3 male and 3 female subjects (6 excel files), 6 videos
 each (tabs), before (tabs 1-3) and after (tabs 4-6) atropine perfusion to
 block muscarinic acetylcholine receptors #### File: ETH
 **Description:** spontaneous calcium transients from 3 male and 3 female
 subjects (6 excel files), 6 videos each (tabs), before (tabs 1-3) and
 after (tabs 4-6) ethanol perfusion to demonstrate the lack of effect of
 this solvent #### File: DMSO **Description:** spontaneous calcium
 transients from 3 male and 3 female subjects (6 excel files), 6 videos
 each (tabs), before (tabs 1-3) and after (tabs 4-6) DMSO perfusion to
 demonstrate the lack of effect of this solvent \[ATR-Atropine,
 CPA- Cyclopentyladenosine, DMSO- Dimethyl Sulfoxide, EGTA- Ethylene Glycol
 Tetraacetic Acid, ETH- Ethanol, HEX- Hexamethonium, NIF- Nifedipine,
 TTX- Tetrodotoxin, NoDrug- Control] ### **Code/software** Image J was used
 to obtain fluorescence transients from chromaffin cells recorded in videos
 of adrenal slices from GCaMP6f mice. Transients were transferred into
 Excel files, 1 column per chromaffin cell transient. Excel files were read
 into Matlab, which was used to identify calcium events (troughs and peaks)
 and to calculate frequencies and amplitudes.