Long-term optical imaging of the spinal cord in awake, behaving animals

3D model of the recording chamber implanted at the T12-L1 vertebrae. Bone is from our microCT data. Created using PTC Creo with Render Studio.

How does the spinal cord process sensory stimuli in awake animals? We released a preprint on bioRxiv demonstrating new experimental and computational approaches to enable easier, more consistent, and cost-effective long-term access to the spinal cord.

This should allow researchers to investigate a variety of questions about the spinal cord and its interaction with the brain and other systems without the confounds of anesthesia. Further, it allows us to start addressing the many questions of pain percepts and their modification by internal state and external stimuli that can only be answered in awake animals.

With the variety of techniques for manipulating cellular activity or signal transduction along with the plethora of imaging tools and sensors, this is an exciting time! More to come.

The manuscript bioRxiv preprint can be found at:

• https://www.biorxiv.org/content/10.1101/2023.05.22.541477v1.full
• https://doi.org/10.1101/2023.05.22.541477

Abstract of the preprint:

Advances in optical imaging approaches and fluorescent biosensors have enabled an understanding of the spatiotemporal and long-term neural dynamics in the brain of awake animals. However, methodological difficulties and the persistence of post-laminectomy fibrosis have greatly limited similar advances in the spinal cord. To overcome these technical obstacles, we combined in vivo application of fluoropolymer membranes that inhibit fibrosis; a redesigned, cost-effective implantable spinal imaging chamber; and improved motion correction methods that together permit imaging of the spinal cord in awake, behaving mice, for months to over a year. We also demonstrate a robust ability to monitor axons, identify a spinal cord somatotopic map, conduct Ca²⁺ imaging of neural dynamics in behaving animals responding to pain-provoking stimuli, and observe persistent microglial changes after nerve injury. The ability to couple neural activity and behavior at the spinal cord level will drive insights not previously possible at a key location for somatosensory transmission to the brain.

Several key takeaways are as follows:

• We developed an improved surgical preparation and implant chamber that is both cost-effective and improves animal health and well-being.
  • The final preparation does not affect animal behavior or sensory processing.
  • The text describes the method in detail along with a full list of supplies to help others get started.
• We identified two materials (PRECLUDE and Teflon AF 2400) that when used in sequence allow for long-term optical access to the spinal cord by inhibiting fibrosis.
  • Teflon AF 2400 can be purchased at https://www.vicimetronics.com/products/teflon-af-films-1.
• Created a computational work flow to handle the different types of motion that researchers encounter with large-scale spinal cord imaging.
  • e.g. rapid motion of hundreds of microns in the presence of occluding objects (e.g. overlying vasculature).
  • This include handling large motion using deep learning (e.g. DeepLabCut) to track spinal cord vasculature then conduct control point-based and rigid motion correction.
• Conducted bilateral spinal cord imaging in awake, behaving mice for months to over a year across a variety of preparations and imaging modalities (one- and two-photons):
  • Axon imaging in Thy1-GFP animals.
  • Axon and cell body imaging of increasing protein expression with AAV-PHP.eB-GFP (spinal cord, cell bodies) and AAV-PHP.S-tdTomato (DRG, axons).
  • Calcium imaging of spinal cord projection neurons in awake, behaving animals responding to noxious stimuli.
    • We demonstrate tracking over months of the same spinal cord projection neurons.
    • Used Phox2a-Cre (https://doi.org/10.1016/j.celrep.2020.108425) mice crossed with GCaMP6 lines (Ai148 or Ai162, see https://portal.brain-map.org/explore/toolkit/mice).
  • Microglia using CX3CR1-GFP mice.
    • Show that we can track changes in microglial dynamics for months after injury.
• Demonstrate that you can observe somatotopy using our large-scale preparation, e.g. more rostral spinal cord activation by hindpaw stimulation compared to tail stimulation.
  • This has implications for changes in somatotopy after injury that may drive aberrant behavior.
• Used 3D printed materials to create a microCT compatible spinal implant chamber that allows us to obtain high-resolution in vivo images and 3D reconstruction of the laminectomy region along with the entire mouse without the issues associated with metal devices.

Videos

We released a subset of the supplementary videos with the preprint that highlight several of the advances and key results. There will be additional videos forthcoming.