Imaging of Calcium Dynamics in Vasoactive Intestinal Peptide-expressing Neurons of Enteric Nervous System
Marthe Howard, Joseph Margiotta
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.
Before start
Steps
On the day of the experiment prepare solutions of:
- Artificial cerebrospinal fluid (CSF) containing:
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117.9 mmol/L NaCl
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4.7 mmol/L KCL
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25 mmol/L NaHCO3
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1.3 mmol/L NaH2PO4
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1.2 mmol/L MgSO4•7H2O
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2.5 mmol/L CaCl2
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11.1 mmol/L D-glucose
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2 mmol/L sodium butyrate
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20 mmol/L sodium acetate
- 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 7.4.
Detect GCaMP-positive neurons by their basal Ca2+ fluorescence.
Record spontaneous and evoked changes in GCaMP6f-mediated Ca2+ fluorescence intensity.
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.
