Two-photon imaging of neural activity and structural plasticity in the rodent spinal cord

[摘要] In my PhD thesis, I used two‐photon imaging to investigate neuronal circuits andglia cells in the spinal cord of living mice. To achieve this, a major effort first was toestablish a mouse spinal cord preparation suitable for stable and long‐lastingimaging experiments. Without adequate stabilisation, the spinal cord was prone tolarge‐scale movement artefacts clearly hampering high‐resolution imaging in vivo.To overcome these limitations, I employed strategies to optimise the animalsposture, namely rigid clamping of the vertebral column to isolate the spinal cordfrom breathing‐related movements. In addition, I developed strategies to dampentissue movements remaining after posture optimisation. These improvementsmade it possible to image the structural plasticity of genetically labelled microgliacells with subcellular resolution for many hours in anesthetized mice. In aparadigm of focal spinal cord injury, microglia became rapidly activated anddisplayed high levels of filopodial motility clearly directed towards the injury site.In addition, I adapted Ca2+ indicator loading techniques to stain neuronal networksin the mouse superficial dorsal horn to visualize activity patterns of painprocessingneurons. Despite the heavily myelinated surrounding tissue, neuronalpopulations within the first two laminae could be visualized after Ca2+ indicatorloading. The preparation was sufficiently stable to for the first time resolve fast,individual Ca2+ transients in the spinal cord of living rodents. In agreement withthe role of dorsal horn circuits in nociceptive processing I found low rates ofspontaneous activity but could reliably evoke increased activity levels by electricalstimulation of primary afferent fibres in situ. Specifically, also natural sensorystimulation applied to the paw elicited Ca2+ transients in a subset of dorsal hornneurons.In a parallel project, I collaborated with Klas Kullander’s group to investigateactivity patterns of identified Renshaw cells during an in vitro model oflocomotion. Using two‐photon Ca2+ imaging in the isolated neonatal mouse SC, wefound that several subclasses of Renshaw cells are differentially engaged inongoing locomotion. In addition, the activities of the different Renshaw cell populations were correlated with subgroups of simultaneously recordedmotoneurons. Afferent inputs delivered during ongoing locomotion perturbed thelocomotor rhythm and the nature of perturbations depended on stimulus timingduring either the flexor‐ or extensor‐related cycle phase. On the local circuit level,we observed that correlations between specific Renshaw cells and motoneuronsubpopulations were boosted by sensory input and that this effect also dependedon stimulus timing. In a broader context, these results can be interpreted asreflections of synaptic strengthening of developing locomotor modules by sensoryinputs.

[发布机构] ETH Zurich

[效力级别] 570 Life sciences

[关键词] Brain Research Institute;570 Life sciences;biology;610 Medicine & health