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

Author(s)

Cartarozzi, Luciana Politti ^{[1, 2]} ; Rieder, Phillip ^[1] ; Bai, Xianshu ^[1] ; Scheller, Anja ^[1] ; Rodrigues de Oliveira, Alexandre Leite ^[2] ; Kirchhoff, Frank ^[1]

Affiliation

^[1] Univ Saarland, CIPMM, Mol Physiol, Bldg 48, D-66421 Homburg - Germany

^[2] Univ Estadual Campinas, UNICAMP, Lab Nerve Regenerat, Cidade Univ Zeferino Vaz, Rua Monteiro Lobato 255, BR-13083970 Campinas, SP - Brazil

• Document type: Journal article
• Source: JOURNAL OF NEUROSCIENCE METHODS; v. 299, p. 8-15, APR 1 2018.
• Web of Science Citations: 6

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 mu m in the grey matter ventral horn of the mouse spinal cord. (C) 2018 Elsevier B.V. All rights reserved. (AU)

• FAPESP's process: 15/08297-8 - Analysis of MHC class I-dependent reactivity of glial cell after nervous system lesions by in vivo two-photon imaging
• Grantee: Luciana Politti Cartarozzi
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• FAPESP's process: 13/16134-6 - Immunomodulatory mechanisms of mesenchymal stem cells in glial reactivity and correlation with nervous system regenerative capacity after lumbar root axotomy
• Grantee: Luciana Politti Cartarozzi
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 14/06892-3 - Use of mesenchymal stem cells in the CNS/PNS interface: repair of proximal lesions
• Grantee: Alexandre Leite Rodrigues de Oliveira
• Support Opportunities: Research Projects - Thematic Grants