Laser Nanosurgery of Cerebellar Axons In Vivo

Summary

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Abstract

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals^1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo^3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex^5-9.

This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Introduction

Axonal transection resulting from mechanical injury, toxic insult or neurodegenerative diseases is usually followed by degeneration of the distal part of the axon that is detached from the cell body^10-13. With a few exceptions^2,7,14,15, severed axons in the CNS of adult animals are usually unable to activate a regrowth program¹⁶.

Little is known about the real-time dynamics of degenerative events at the cellular and subcellular level. The development of new strategies for limiting neuronal damage and promoting neuronal regrowth requires, as a first step, clarifying the mechanism by which singularly injured n....

Protocol

1. Axonal Labeling

1. Climbing fibers can be labeled by injecting either organic dyes conjugated to high molecular weight dextrans or plasmid/viruses that induce the expression of fluorescent proteins^26-29. In this protocol, the organic dye Alexa Fluor Dextran 488 is injected into the inferior olive to label climbing fibers and visualize them in the cerebellar cortex (Figure 1). All the procedures described here have been approved by the Italian Ministry of Health.
2. Prepare the glass capillary tube by pulling it on a micropipette puller. Trim the tip of the glass capillary tube with scissors till the external diameter is a....

Results

This protocol described how to perform axonal labeling, in vivo imaging and laser axotomy on single neurons. The timeline of the experiment is shown in Figure 1.

An example of CFs labeled with Alexa Fluor 488 Dextran and visualized under the cranial window by in vivo two-photon microscopy is reported in Figure 2. As previously reported^6,27, the ascending branches display a high stability throughout the observation period of several.......

Discussion

This protocol shows how to label neurons of the inferior olive with a fluorescent dye. Subsequently, the method to perform a cranial window on the cerebellar cortex is described. This technique provides optical access to the terminal portion of olivocerebellar neurons, the climbing fibers. Unfortunately, the outcome of both labeling and craniotomy surgery is quite low even in the hands of skilled operators (usually 1 out of 3 mice is labeled, and 1 out of 3 cranial windows remains clear after 1-2 weeks).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lab standard stereotaxic, rat and mouse	Stoelting	51670
Borosilicate glass with filament	Sutter Instrument Inc	BF100-50-10
Germinator 500 (Glass bead sterilizer)	Roboz
Microinjection dispense system	Picospritzer
Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R)	Warner Instruments	64-0720
Ti:Sapphire laser, 120 fsec width pulses, 90 MHz repetition rate	Coherent	Chameleon
Spongostan, hemostatic sponge	Ferrosan	MS0005
Galvanometric mirrors	GSI Lumonics	VM500+
Objective	Olympus	XLUMPLFLN 20XW
Piezoelectric stage	Physik Instrumente	P-721
Photomultiplier modules	Hamamatsu Photonics	H7710-13
LabVIEW System Design Software	National Instruments
Voren, 1 mg/ml (dexamethasone-21- isonicotinate)	Boheringer Ingelheim
Rymadil (carprofen)	Pfizer
Lidocaine clorohydrate 2%	ATI
Alexa488 dextran	Life Technologies	D22910