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.
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 animals1,2. 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 vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
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.
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 body10-13. With a few exceptions2,7,14,15, 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 neuronal cells degenerate and regenerate. This study is most directly addressed by monitoring the dynamics of a single neuron in vivo. While one-photon fluorescence imaging techniques are limited by intense scattering of visible light, two-photon excitation reaches deep cortical layers in live mice with subcellular resolution3,4,17. Taking advantage of transgenic mice in which fluorescent proteins are selectively expressed in subpopulations of neurons18-20, TPF microscopy has been applied to the exploration of synaptic plasticity and axonal elongation during development in vivo21,22.The capability of singularly damaged neurons to regrow after injury can be investigated by coupling in vivo monitoring by two-photon imaging with a model of injury specifically targeted to the axon of interest. Multi-photon absorption of femtosecond pulses has been used to disrupt single dendrites or even single spines5,23. Moreover, this injury paradigm allows cutting single axonal branches without disrupting the contacting dendrite6. Within the context of dissecting the features that allow specific neuronal population to regenerate their axons once damaged, cerebellar climbing fibers (CFs) are a useful model since they retain remarkable plastic properties after injury even in adult animals24,25. Recently, long-term imaging of CFs showed that these axons are capable of regrowing in the days that follow laser axotomy6.
This protocol describes how to label olivocerebellar neurons and their axonal elongation through anterograde tracing. Once the neurons of interest are fluorescently labeled, they can be monitored repeatedly at arbitrary time points for weeks or months under a cranial window. The procedure to dissect single axonal branches by laser axotomy in vivo will then be illustrated.
The techniques presented here provide new insights into the mechanism of axonal remodeling in vivo and may help the development of therapeutic strategies to limit neuronal degeneration and promote axonal regrowth.
1. Axonal Labeling
2. Optical Window on the Cerebellar Cortex
NOTE: The dye is transported in 2 weeks from the inferior olivary nucleus up to the cerebellar cortex to the CFs terminals (see Figure 1). The sparsely labeled CFs can be visualized under the permanent cranial window that can be performed as follows:
3. In Vivo Multi-photon Laser Axotomy
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 reported6,27, the ascending branches display a high stability throughout the observation period of several days.
Figure 3 shows an example of axonal severing by laser axotomy of a single branch of a CF. As previously described, the damage was performed by irradiating a distal branch (~30 μm below the pia) of a CF with a high energy dose of infrared light. As shown in the second panel of Figure 3, 15 min after laser irradiation the distal portion of the axon started swelling and degenerating. The day after, the portion of the axon distal to the lesion site completely disappeared (Figure 3, d17). The degenerated portion is highlighted by the red arrowheads in the time course. The last two panels also show the progressive formation of a new transverse branch (green arrowhead) from the injured axon (more details on the sprouting of new branches after laser axotomy in vivo can be found in6).
Figure 1. Timeline of the laser nanosurgery experiment. After the injection into the inferior olivary nucleus, two weeks of recovery will allow the anterograde dextran-conjugated dye to label the climbing fibers. The cerebellar cortex is then exposed by performing a permanent cranial window. From 2-7 later on, the labeled CFs can be imaged under the two-photon microscope in vivo at arbitrary time intervals. The laser nanosurgery on the labeled axons can be performed at any time after the cranial window has been implanted.
Figure 2. Climbing fibers labeling. The left panel shows an example of a CF labeled with Alexa Fluor 488 Dextran and visualized in vivo by TPF microscopy. Right panels display time-lapse images of the same CF from day 16 (d16) to d18. Scale bar, 10 µm.
Figure 3. Laser axotomy on a single branch of a climbing fiber. The neuron was irradiated where the red arrow points on d16. Red arrowheads highlight the disappearance of the distal portion of the axon after laser axotomy (d18). A newly formed transverse branch is pointed out by the green arrowhead. Scale bar, 10 µm.
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).
The procedure to perform laser axotomy in living mice is then presented. This method allows dissecting fluorescent neurons in targeted sites. While commonly used injury paradigms, either by mechanical severing or the use of chemicals, cause massive disruption of brain tissue, the key strength of this model is the high spatial confinement and specificity. Indeed, previous studies showed that single spines can be ablated while sparing the structural integrity of the parent dendrite5; moreover, single axonal branches can be disrupted avoiding the formation of a persistent glial scar6. The degeneration process can be then followed in real-time, as well as eventual remodeling of the injured neuron. This method is therefore a powerful tool to study the basic mechanism of axonal structural plasticity in vivo.
The possibility of dissecting selected structures depends to a first approximation on the depth of the structure and on its fluorescence emission intensity. Although two-photon microscopy allows imaging neurons up to 600-800 μm deep, we found that the nanosurgery is limited to the first layers of the cortex (up to 300 mm).
In this protocol laser axotomy was targeted to cerebellar climbing fibers, but it can be used to dissect any fluorescent structure in live brain. Laser irradiation has been used to dissect single neuronal cell bodies, dendritic branches, axons or single spines of GFP-labeled pyramidal neurons in the cortex of transgenic mice5,8,9,23.
The authors have nothing to disclose.
We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission’s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the “Stem Cells Call for Proposals.” This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by “Ente Cassa di Risparmio di Firenze”.
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 fs width pulses, 90 MHz repetition rate | Coherent | Chameleon | |
Spongostan, haemostatic 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 |