Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Summary

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Abstract

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Introduction

The mammalian cortex participates in many brain functions, from sensory perception and movement control to abstract information processing and cognition. Various cortical functions build upon different neural circuits, which are made up of different types of neurons communicating and exchanging information at individual synapses. The structure and function of synapses are consistently being modified in response to experiences and pathologies. In the mature brain, synaptic plasticity takes the form of both strength changes and addition/removal of synapses, playing important roles in formation and maintenance of a functional neural circuitry. Dendritic spines are the po....

Protocol

Approval needs to be obtained from home institutions before commencement of the surgery and imaging study. Experiments described in this manuscript were performed in accordance with the guidelines and regulations from the University of California, Santa Cruz Institutional Animal Care and Use Committee.

1. Surgery

1. Autoclave all surgical instruments and sterilize the workspace with 70% alcohol thoroughly before surgery.
2. Anesthetize the mouse by intraperitoneal (IP) injection of KX anesthetic solution (200 mg/kg ketamine and 20 mg/kg xylazine) according to the mouse’s body weight. Note: KX dosage may be ....

Results

In YFP-H line mice²⁵, yellow fluorescent protein expresses in a subset of layer V pyramidal neurons, which project their apical dendrites to the superficial layers in the cortex. Through the thinned-skull preparation, the fluorescently labeled dendritic segments can be repetitively imaged under two-photon microscope over various imaging intervals, ranging from hours to months. Here we show an example of a four-time imaging of the same dendrites over 8 days in the motor cortex of a 1 month old mouse, where indi.......

Discussion

To obtain a successful thinned-skull preparation, several steps in this protocol are crucial. 1) The thickness of the skull. The cranial bone has a sandwich structure, with two layers of high-density compact bone and a middle layer of low-density spongy bone. While the high-speed micro drill is suitable for removing the outer layers of compact bone and spongy bone, the microsurgical blade is ideal for thinning the inner layer of compact bone. As the thickness and stiffness of the skull increases during development, imagi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine	Bioniche Pharma	67457-034-10	Mixed with xylazine for anesthesia
Xylazine	Lloyd laboratories	139-236	Mixed with ketamine for anesthesia
Saline	Hospira	0409-7983-09	0.9% NaCl for injection and imaging
Razor blades	Electron microscopy sciences	72000	Double-edge stainless steel razor blades
Alcohol pads	Fisher Scientific	06-669-62	Sterile alcohol prep pads
Eye ointment	Henry Schein	102-9470	Petrolatum ophthalmic ointment sterile ocular lubricant
High-speed micro drill	Fine Science Tools	18000-17	The high-speed micro drill is suitable for thinning the outer layer of compact bone and targeting a small area
Micro drill steel burrs	Fine Science Tools	19007-14	1.4 mm diameter
Microsurgical blade	Surgistar	6961	The microsurgical blade is suitable for thinning the inner layer of compact bone and middler layer of spongy bone
Cyanoacrylate glue	Fisher Scientific	NC9062131	Fix the head plate onto the skull
Suture	Havard Apparatus	510461	Non-absorbale, sterile silk suture, 6-0 monofilament
Dissecting microscope	Olympus		SZ61
CCD camera	Infinity
Two-photon microscope	Prairie Technologies		Ultima IV
10X objective	Olympus		NA 0.30, air
60X objective	Olympus		NA 1.1, IR permeable, water immersion
Ti-sapphire laser	Spectra-Physics		Mai Tai HP