Apr 14, 2022

Public workspaceImaging of Calcium Dynamics in Vasoactive Intestinal Peptide-expressing Neurons of Enteric Nervous System

DOI
dx.doi.org/10.17504/protocols.io.14egn76xpv5d/v1
  • 1Department of Neurosciences, University of Toledo College of Medicine and Life Sciences, Toledo, Ohio
Marlena S Pela
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DOI: dx.doi.org/10.17504/protocols.io.14egn76xpv5d/v1
Protocol Citation: Joseph Margiotta, Marthe Howard 2022. Imaging of Calcium Dynamics in Vasoactive Intestinal Peptide-expressing Neurons of Enteric Nervous System. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn76xpv5d/v1
Manuscript citation:
https://doi.org/10.1016/j.jcmgh.2021.08.016
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 11, 2022
Last Modified: April 14, 2022
Protocol Integer ID: 59375
Abstract
The protocol was published on behalf of the investigators by the SPARC project team.

The University of Toledo Health Science Campus animal care and use committee approved animal care, breeding procedures, and experimental protocols. Animals were housed in an Association of Laboratory animal Care-approved facility with a 12-hour light cycle with food (standard chow) and water ad libitum.

One male VIP-GCAmP reporter mouse, age 4 months, was used in this study. The mouse was purchased from the Jackson Laboratories, and all genotyping was performed using primers recommended by the Jackson Laboratories according to their protocols.
Materials
Mouse strains:
ABCDEF
Formal strain nameCommonly used nameMouse genome informatics IDJax Lab stock numberRRIDStrain background
Vip-tm1(cre)Zjh-JVip-IRES-cre4431361031628IMSR_JAX:031628C57BL/6J
B6J.Cg-Gt(ROSA)26Sortm95.1(CAGGCaMP6f)Hze/MwarJAi95(RCL-GCaMP6f)-D (C57BL/6J) or Ai95D (C57BL/6J)5558090 028865IMSR_JAX:028865C57BL/6J
Mice that contained a floxed GCaMP6f construct within the Gt(ROSA)26 locus were bred with mice that expressed Cre recombinase under the control of the Vip promoter.

Artificial cerebrospinal fluid:
NaCl - catalogue #: S9888, Sigma, St. Louis, MO
KCL - catalogue #: P3911, Sigma, St. Louis, MO
NaHCO3 - catalogue #: S6014, Sigma, St. Louis, MO
NaH2PO4 - catalogue #: S8282, Sigma, St. Louis, MO
MgSO4•7H2O - catalogue #: 230391, Sigma, St. Louis, MO
CaCl2 - catalogue #: C1016, Sigma, St. Louis, MO
D-glucose - catalogue #: G5767, Sigma, St. Louis, MO
sodium butyrate - catalogue #: B5887, Sigma, St. Louis, MO
sodium acetate - catalogue #: S2889, Sigma, St. Louis, MO

Equipment:
Picospritzer II, Parker Instrumentation Corp, Barnstaple, UK
DM6000 FS Leica fluorescence microscope - Leica, Buffalo Grove, IN
Prime 95B CMOS camera - Teledyne Photometrics, Tuscon, AZ
Metamorph software - Molecular Devices, Downington, PA


On the day of the experiment prepare solutions of:

  1. Artificial cerebrospinal fluid (CSF) containing:

  • 117.9 mmol/L NaCl
  • 4.7 mmol/L KCL
  • 25 mmol/L NaHCO3
  • 1.3 mmol/L NaH2PO4
  • 1.2 mmol/L MgSO4•7H2O
  • 2.5 mmol/L CaCl2
  • 11.1 mmol/L D-glucose
  • 2 mmol/L sodium butyrate
  • 20 mmol/L sodium acetate

2. Artificial CSF with test reagents, e.g., agonists.
Euthanize the mouse.
Remove the colon.
Open the excised proximal colon segment along the mesenteric border.
Pinn the colon segment mucosal side down onto a Sylgard surface lining a glass coverslip attached to the bottom of a plastic imaging chamber containing artificial CSF perfused and bubbled with Carbogen (95% O2/5% CO2) to oxygenate and achieve Ph7.4 .
Note
In experiments that involve recording neural activity leading up to spontaneous Colonic Motor Complexes (CMCs) heat the artificial CSF to Temperature35 °C .

Detect GCaMP-positive neurons by their basal Ca2+ fluorescence.

Note
To do this we used an upright DM6000 FS Leica fluorescence microscope (Leica, Buffalo Grove, IL) and Prime 95B Scientific CMOS camera (Photometrics, Tucson, AZ).

Images were collected with Metamorph software (Molecular Devices, Downington, PA) at a 40-Hz sampling rate and 25-ms exposure time.

Record spontaneous and evoked changes in GCaMP6f-mediated Ca2+ fluorescence intensity.

Note
We acquired 12-bit images using a 1.44-megapixel CMOS camera capable of capturing at 40 frames/sec (Prime 95B; Teledyne Photometrics, Tuscon, AZ) controlled by Meta-Morph software (version 7.10.1.161; Molecular Devices, Silicon Valley, CA).

At second 20 focally deliver an agonist by pressure microperfusion (10psi,10 sec; via Picospritzer II; Parker Instrumentation Corp, Barnstaple, UK) from blunt glass micropipettes (diameter, 5–10 mm) delivered within 50 µm of an adjacent myenteric ganglion.

Note
Here we used dimethylphenyl-piperazinum (DMPP) Concentration10 micromolar (µM) .