Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

Summary

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Abstract

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

Protocol

1. To visualize neurons in the intact brain using two-photon microscopy, a preparation where neurons are labeled with fluorescent markers is used. In the experiments presented here we use the GFP-M transgenic mouse line which labels layer 5 pyramidal cells¹. Layer 5 pyramidal cells project dendritic processes into superficial layers, allowing the visualization of dendrites and dendritic spines up to a depth of 300 μm below the level of the pia. An alternate approach is to label cells using viral markers (see Lowery et al., submitted to JOVE).
2. Sterilize the workspace using 70% ethanol and autoclave tools for aseptic surgery. Cover operating surfa....

Discussion

Chronic imaging using two-photon microscopy is becoming an increasingly popular technique to study morphological changes triggered during plasticity^2-5. Here we demonstrate a thinned-skull preparation to follow identified dendritic spines in the intact mouse brain on different imaging days. In this protocol, the skull is left intact, causing minimal damage to the cortex and resulting in very low levels of neuroinflammation which may alter brain function⁶. This allows the animal to be imaged immedia.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fentanyl Citrate		In House		Fentanyl Cocktail: Anesthesic Cocktail; IP Dose: 0.0125 ml/g Final cocktail conc.: 0.05 mg/kg
Medetomindine Hydrochloride		In House		Fentanyl Cocktail: Anesthesic Cocktail; IP Dose: 0.0125 ml/g Final cocktail conc.: 0.05 mg/kg
Midazolam HCL		In House		Fentanyl Cocktail: Anesthesic Cocktail; IP Dose: 0.0125 ml/g Final cocktail conc.: 0.05 mg/kg
2,2,2-tribrom–thanol				Avertin Cocktail: Anesthetic; IP Dose: 0.0075 cc/ g Final cocktail conc.: 1%
2-methyl-2-butanol (Final concentration: 0.775%)				Avertin Cocktail: Anesthetic; IP Dose: 0.0075 cc/ g Final cocktail conc.: .775%
TC-1000 Temp. Control System for Mice		CWE, Inc.	TC-1000 Mouse	Body Temp. Regulation
Toberadex		In House		Eye Ointment
10% Ferric Chloride		Ricca Chemical Company	3120-32	Thin-skull preparation
Microsurgical Blade		Sable Industries	S-6400	Thin-skull preparation
Cyanoacrylate 404,401		Loctite	P/N 46551	Thin-skull preparation
Cyanoacrylate 401		Loctite	P/N 40140	Thin-skull preparation
Zip Kicker Glue Accelerator		Pacer Technology	PT-29	Thin-skull preparation
Micro Drill Steel Burrs 0.7mm tip diameter		Fine Science Tools	19008-07	Thin-skull preparation
Microtorque Control Box and Tech2000 Handpiece		Ram Products, Inc.	TECH2000ON/OFF	Thin-skull preparation
#6-0 (0.7 metric ) silk suture		Ethicon Inc.	K8894H	Taper C-1
Eye Dressing Forceps, 10cm, tip width 0.5mm, curved	Surgical Tools	Fine Science Tools	11152-10
Extra Fine Bonn Scissors, 8.5cm, straight tip, cutting edge 13mm	Surgical Tools	Fine Science Tools	14084-08
Standard Pattern Forceps, straight, 2.5mmx1.35mmtip, 12cm	Surgical Tools	Fine Science Tools	11000-12