Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Summary

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

Abstract

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Introduction

The ability to perform in vivo multiphoton fluorescence microscopy in the cortex of mice is paramount to the study of cellular signaling and structure^{1,2,3,4,5,6,7,8,9}, disease pathology^10,11, and cellular development^12,13. With the implantation....

Protocol

All animal experiments were performed in accordance with guidelines approved by the IACUC at the University of Nebraska Medical Center.

1. Before surgery

1. Prepare pipettes for viral injections. Pull borosilicate glass capillaries using a pipette puller and bevel the pipette at a 20° angle. Sterilize the pipettes overnight.
2. Prepare fresh pieces of sterile gel foam by cutting them into small squares. Submerge the gel foam in a sterile microfuge tube containing 0.5 mL of.......

Discussion

Here, we have presented a protocol for the implantation of chronic cranial windows for in vivo imaging of cortical astrocytes and neurons in awake, head-restrained mice on an air-lifted home cage. Specific examples have been provided of the cranial window application for imaging astrocytes that express GECIs and neuronal synaptic structures. With the use of multiphoton microscopy, astrocytic calcium signaling dynamics and structural synaptic dynamics can be recorded repeatedly over days.

Materials

Name	Company	Catalog Number	Comments
15o Pointed Blade	Surgistar	6500	Surgery Tools
19 G Needles	BD	305186	Surgery Supply
AAV1-CAG-FLEX-tdTomato	Addgene	28306-AAV1	Viral Vector
AAV1-CaMKII-0-4-Cre	Addgene	105558-AAV1	Viral Vector
Acteone	Fisher Scientific	A16P4	Reagent
Alcohol Prep Pads	Fisher Scientific	Covidien 5750	Surgery Supply
Beveler	Narishige		Equipment
Borosilicate Glass	World Precision Instruments	TW100F-4	Surgery Supply
Carbide Burs	SS White Dental	14717	Surgery Tools
Carprofen (Rimadyl), 50 mg/mL	Zoetis Mylan Institutional, LLC.		Drug
Compressed Air	Fisher Scientific	23-022-523	Surgery Supply
Cotton Tip Applicators	Puritan	836-WC NO BINDER	Surgery Supply
Cover Glass, No. 1 thickness, 3 mm/5 mm	Warner Instruments	64-0720, 64-0700	Surgery Supply
Dental Drill	Aseptico		Equipment
Dexamethasone, 4 mg/mL	Mylan Institutional, LLC.		Drug
Dissecting Microscope	Nikon		Equipment
Duralay Liquid  (dental cement liquid)	Patterson Dental	602-8518	Reagent
Duralay Powder  (dental cement powder)	Patterson Dental	602-7932	Reagent
Enrofloxacin, 2.27%	Bayer		Drug
Eye Ointment	Dechra	17033-211-38	Surgery Supply
Fiber Lite High Intensity Illuminator	Dolan-Jenner Industries		Equipment
Forceps (Large)	World Precision Instruments	14099	Surgery Tools
Forceps (Small)	World Precision Instruments	501764	Surgery Tools
GCaMP6f B6; 129S-Gt(ROSA)26Sortm95.1(CAGGCaMP6f)Hze/J	The Jackson Laboratory	Stock No: 024105	Mouse line
Germinator	Fisher Scientific		Equipment
GLAST-CreER Tg(Slc1a3-cre/ERT) 1Nat/J	The Jackson Laboratory	Stock No: 012586	Mouse Line
Headplate	Neurotar	Model 1, Model 3	Surgery Supply
Hemostatic forceps	World Precision Instruments	501705	Surgery Tools
Holder for 15o Pointed Blade	World Precision Instruments	501247	Surgery Tools
Holder for Scalpel Blades	World Precision Instruments	500236	Surgery Tools
Iodine Prep Pads	Avantor	15648-926	Surgery Supply
Isoflurane	Piramal		Surgery Supply
Isoflurane table top system with Induction Box	Harvard Apparatus		Equipment
Isoflurane Vaporizer	SurgiVet		Equipment
Krazy Glue	Office Depot	KG517	Reagent
Loctite 401	Henkel	40140	fast-curing instant adhesive
Loctite 454	Fisher Scientific	NC9194415	cyanoacrylate adhesive gel
Micropipette Puller	Sutter Instruments		Equipment
Multiphoton Microscope			Equipment
Nitrogen	Matheson	NI M200	Gas
Oxygen	Matheson	OX M250	Gas
Picospritzer	Parker		intracellular microinjection dispense system
Pipette Tips	Rainin	17014340	Surgery Supply
Rodent Hair Trimmer	Wahl		Equipment
Saline (0.9% Sodium Chloride)	Med Vet International	RX0.9NACL-30BAC	Surgery Supply
Scalpel Blades, Size 11	Integra	4-111	Surgery Tools
Scissors	World Precision Instruments	503667	Surgery Tools
Stereotaxic Instrument	Stoelting		Equipment
Sugi Sponge Strips (sponge strips)	Kettenbach Dental	31002	Surgery Supply
SURGIFOAM (gel foam)	Ethicon	1972	Surgery Supply
Syringe with 26 G Needle	BD	309625	Surgery Supply
Tamoxifen	Sigma Aldrich	T5648-1G	Reagent
Ti:Sapphire Laser	Coherent		Equipment
Transfer Pipettes	Fisher Scientific	13-711-9AM	Surgery Supply
Water Blanket	Fisher Scientific		Equipment
Xylocaine MPF with Epinephrine (1:200,000), 10 mg/mL	Fresenius Kabi USA		Drug