In vivo two-photon imaging of motoneurons and adjacent glia in the ventral spinal cord

https://doi.org/10.1016/j.jneumeth.2018.01.005Get rights and content

Highlights

  • In vivo imaging of spinal motoneurons and adjacent glia by two-photon laser-scanning microscopy (2P-LSM).

  • Cell-type identification by using transgenic mice with cell-specific expression of fluorescent proteins.

  • Study of immediate glial responses to motoneuron axotomy.

Abstract

Background

Interactions between motoneurons and glial cells are pivotal to regulate and maintain functional states and synaptic connectivity in the spinal cord. In vivo two-photon imaging of the nervous system provided novel and unexpected knowledge about structural and physiological changes in the grey matter of the forebrain and in the dorsal white matter of the spinal cord.

New method

Here, we describe a novel experimental strategy to investigate the spinal grey matter, i.e. the ventral horn motoneurons and their adjacent glial cells by employing in vivo two-photon laser-scanning microscopy (2P-LSM) in anesthetized transgenic mice.

Results

After retrograde tracer labelling in transgenic mice with cell-specific expression of fluorescent proteins and surgical exposure of the lumbar intumescence groups of motoneurons could be visualized deeply localized in the ventral horn. In this region, morphological responses of microglial cells to ATP could be recorded for an hour. In addition, using in mice with expression of GCaMP3 in astrocytes, physiological Ca2+ signals could be recorded after local noradrenalin application.

Comparison with existing methods

Previous in vivo imaging protocols were restricted to the superficial dorsal white matter or upper layers of the dorsal horn. Here, we modified a multi-step procedure originally established for a root-crush injury. We adapted it to simultaneously visualize motoneurons and adjacent glial cells in living animals.

Conclusion

A modified surgery approach is presented to visualize fluorescently labelled motoneurons and glial cells at a depth of more than 200 μm in the grey matter ventral horn of the mouse spinal cord.

Graphical abstract

Summary of a novel in vivo 2P-LSM imaging approach to study motoneurons of the ventral spinal cord. For accurate cell localization, tracer was injected in the hindlimb muscles of transgenic mice with selective expression of fluorescent proteins in microglia, astrocytes or neurons. Subsequently, motoneurons and their adjacent glial cells at the lumbar intumescence could be detected in the living mouse using a combination of careful surgery and 2P-LSM imaging.

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Introduction

Lesions to the spinal cord result in severe loss of motor and sensory function that involves neuronal degeneration as well as acute reactions of the adjacent glial cells, microglia and astrocytes. To date, the knowledge about in vivo immediate responses to injury occurring at the minute time scale such as shape changes of microglial processes or transients of intracellular Ca2+ changes in astrocytes remained largely elusive. So far, most of the morphological information on acute, trauma-associated spinal cord events is based on two-photon laser-scanning microscopy (2P-LSM) of superficial layers in the dorsal white matter. Electrophysiological approaches have been used to quantify the impairment and putative regeneration of axon function without direct information of the underlying structural alterations. However, parameters such as the acute activation of microglial cells or the visualization of intracellular signaling cascades could provide important mechanistic information on scar formation and subsequent degenerative or healing processes.

2P-LSM has turned out as an important methodology for cellular investigations in vivo taking advantage of genetically modified mice with cell-specific expression of various fluorescent reporter proteins. In this way, novel and often unexpected findings about the dynamic behavior of cells have been described under physiological or pathological conditions (Davalos and Akassoglou, 2012). 2P-SLM is an optical imaging approach that relies on fluorescence emission detection (Denk et al., 1990). Initially it was restricted to cultured cells or acutely isolated brain slices. However, it was very quickly adapted to anesthetized rodents which represented an “intravital” preparation. The first observations of the brain revolutionized the field of neuroscience. Suddenly, researchers could visualize the dynamics of cellular processes in living animals (Nayak et al., 2012).

Several approaches have been introduced for imaging the spinal cord (Cupido et al., 2014; Davalos and Akassoglou, 2012; Dibaj et al., 2010; Dray et al., 2009; Fenrich et al., 2012; Johannssen and Helmchen, 2010, Johannssen and Helmchen, 2013), e.g. allowing observation of dorsal funiculus axons and glial cells. The use of Texas Red-dextran by injection into the tail vein was employed for the evaluation of the vasculature and its close relationship with astrocytes and microglia. Micro-lesioning neuronal fibers allowed real-time investigation of acute microglial changes, as well as chronic changes up to 6 months (Laskowski and Bradke, 2013). Imaging of the spinal cord, so far, has been largely restricted to the superficial white matter of the dorsal columns. The surgical complexity to reach ventral positions of the spinal cord prevented an adequate analysis. However, for a better understanding of the mechanisms that re-establish muscle innervation after a peripheral nerve injury, direct visualization of fast Ca2+ changes or structural alterations could be a significant advancement.

Therefore, the present work was designed to establish a novel approach to image spinal motoneurons (located at lamina IX of Rexed) and surrounding glial cells in vivo employing 2P-LSM.

Section snippets

Transgenic mice and animal licenses

To visualize neurons and glial cells we used transgenic mice in which neurons were labelled by transgenic expression of the yellow fluorescent protein EYFP under the control of Thy1 promoter (TgN(Thy1-EYFP)) (Winter et al., 2007), astrocytes by the cyan fluorescent protein ECFP under control of the GFAP promoter (TgN(GFAP-ECFP)) (Hirrlinger et al., 2005) and microglia by the green fluorescent protein EGFP from the CX3CR1 gene locus (TgH(CX3CR1-EGFP)) (Jung et al., 2000). Double as well as

Results

Here, we demonstrate a novel methodological approach that allows the in vivo visualization of lumbar spinal cord motoneurons and their adjacent glial cells by 2P-LSM taking advantage of transgenic mice with cell-specific fluorescent protein expression. For this purpose, we developed a surgical protocol that can be carried out in less than one hour with subsequent image acquisition. The protocol consists of the following main steps depicted in Fig. 1, Fig. 2:

  • skin incision along the sagittal

Discussion

So far, imaging of the spinal cord had been restricted to the dorsal white matter, the dorsal horn superficial layers (Fenrich et al., 2012; Cupido et al., 2014) or to ex vivo preparations (Greenberg et al., 2014). With this work, we demonstrate the feasibility of 2P-imaging in deep layers of the ventral spinal cord gray matter employing anesthetized transgenic mice. This novel approach combining skilled surgery, 2P-LSM and living mice with transgenic labelling of selected cell populations will

Conclusion

Combining transgenic animal models with 2P-LSM and surgical techniques, it is possible to image in vivo, and in a reproducible way, morphological changes as well as physiological signals of motoneurons and their adjacent glial cells localized in the ventral horn of the lumbar spinal cord.

Acknowledgements

The authors are grateful to Frank Rhode for technical assistance and to Daniel Rhode for animal husbandry. We thank Prof. Dr. Elaine Del Bel (University of São Paulo, Brazil) for continuous support as the Brazilian coordinator of the PROBRAL-I CAPES/DAAD exchange programme. This work was supported by grants from Deutsche Forschungsgemeinschaft SPP1757, SFB894 and FOR2289; Fondation pour l’Aide a la Recherche sur la Sclerose En Plaques and Association Française contre les Myopathies (ARSEP-AFM),

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