In Vivo Imaging of Geniculate Ganglion Neuron Responses Using Epifluorescent Microscopy

Samenvatting

Source: Fowler, B. E., et al. In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli. J. Vis. Exp. (2021)

This video demonstrates a protocol for delivering tastants to the surgically exposed geniculate ganglion in an anesthetized mouse and using GCaMP fluorescence with epifluorescent microscopy to visualize real-time neuronal responses, aiding studies on peripheral taste signal processing.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Run tastant panel
   1. Use suction to remove liquid from over the geniculate. Optionally place an absorbent point to help mitigate seepage and aid in microscope navigation.
   2. Place the animal on an absorbent pad under the microscope. Locate the geniculate ganglion: useful landmarks include the hole left in the bulla, the hole in the temporal bone, and the seventh nerve. Using the FITC/GFP (Fluorescein Isothiocyanate/ green fluorescent protein) filter on the epifluorescence scope, check for individual GCaMP-expressing geniculate ganglion neurons. A 10x objective (working distance of 10 mm) will provide sufficient resolution to track the activity of individual cells, but a 20x objective (working distance of 12 mm) can also be used.
   3. Place dispensing needle for tastant line firmly in animal's mouth. Place a Petri dish below the animal's mouth to catch fluid.
   4. Ensure that the camera is viewing the microscope's field of view. Synchronize the start of the video recording with the start of the tastant presentation.
   5. During recording, watch the live feed for responses, drift, and seepage.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
1ml Syringe with luer lock	Fisher Scientific	14-823-30
27 Gauge Blunt Dispensing Needle	Fisher Scientific	NC1372532
Absorbent Points	Fisher Scientific	50-930-668
Acesulfame K	Fisher Scientific	A149025G
BD Allergist Trays with Permanently Attached Needle	Fisher Scientific	14-829-6D
Blunt Retractors	FST	18200-09
Breadboard	Thor Labs	MB8
Custom Head Post Holder	eMachineShop		See attached file 202410.ems
Custom Metal Head Post	eMachineShop		See attached file 202406.ems
DC Temperature Controller	FHC	40-90-8D
Digital Camera, sCMOS OrcaFlash4 Microscope Mounted	Hamamatsu	C13440
Disection Scope	Leica	M80
LED Cold Light Source	Leica Mcrosystems	KL300LED
Luer Lock 1/16" Tubing Adapters	Fisher	01-000-116
Microscope	Olympus	BX51WI
Mini-series Optical Posts	Thorlabs	MS2R
Petri dishes	Fisher Scientific	FB0875713A
Pressurized air	Airgas	AI Z300
Self Sticking Labeling Tape	Fisher Scientific	159015R
Silicone Pinch Valve Tubing 1/32" x 1/16" o.d. (per foot)	Automate Scientific	05-14
Snap25-2A-GCaMP6s-D	JAX	25111
Student Fine Scissors	Fine Science Tools	91460-11
Surgical Probe	Roboz Surgical Store	RS-6067
Surgical Probe Holder	Roboz Surgical Store	RS-6061
BD Intramedic Tubing	Fisher Scientific	22-046941
Two Stage Gas Regulator	Airgas	Y12FM244B580-AG
Tygon vinyl tubing - 1/16"	Automate Scientific	05-11
Valvelink8.2 digital/manual controller	Automate Scientific	01-18
Valvelink8.2 Pinch Valve Perfusion System	Automate Scientific	17-pp-54
Xylazine	Anased	NADA# 139-236