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

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.