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

Abstract

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 combinedin vivoapplication 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.

Towards a Brighter Constellation: Multi-Organ Neuroimaging of Neural and Vascular Dynamics in the Spinal Cord and Brain

“…2 ); (iii) surgical procedures are more complex and hence require more training than brain procedures; (iv) the spinal cord has a smaller tool builder community than the brain, and advancements happen at slower pace; (v) in general the longevity of spinal cord window implants is lower than for the brain; (vi) the highly-mobile structure of the spinal cord makes it challenging to design implants cable of withstanding traditional restraint paradigms used for brain imaging, limiting the complexity and number of experimental trials that can be performed under awake spinal restraint. 58…”

“…66 Also, treating an implant with gradually releasing anti-inflammatory agents might increase the duration of single-cell resolved imaging by suppressing fibrosis. 58 Besides surgical preparation, habituation of the animal remains an equally important factor in maintaining spinal cord window quality when it comes to awake behavior. We found that high mobility of the spinal cord makes it more prone to injury.…”

“…60 Their work laid the foundations for several recent studies building on this original design that have in turn made important, unique contributions. 15,58,60 While recent progress has been substantial, prior spinal implant designs have limitations we sought to mitigate in our work. First, we found the process of restraining an awake animal using custom-fabricated fine screw-based holders time consuming and stressful for the animal when compared to the streamlined restraint process using our standard headpost implants.…”

“…2); (iii) surgical procedures are more complex and hence require more training than brain procedures; (iv) the spinal cord has a smaller tool builder community than the brain, and advancements happen at slower pace; (v) in general the longevity of spinal cord window implants is lower than for the brain; (vi) the highly-mobile structure of the spinal cord makes it challenging to design implants cable of withstanding traditional restraint paradigms used for brain imaging, limiting the complexity and number of experimental trials that can be performed under awake spinal restraint. 58 Several crucial breakthroughs in overcoming these challenges have been led by Schaffer and colleagues. 17,59,60 Their spinal chamber implant design and procedure revolutionized the field by allowing chronic 2-photon imaging of blood flow in spinal stroke 59 and axonal morphology chronic changes following spinal cord injury.…”

The Bayliss–Starling Prize Lecture: The developmental physiology of spinal cord and cortical nociceptive circuits

“…mechanical pinch) (NS) or wide dynamic range (WDR) cells, which encode input from both low‐ and high‐intensity modalities (see William & Coggeshall, 2003). Importantly, adult cutaneous receptive fields are not fixed but are subject to rapid changes in response to contextual factors, such as stimulus history, supraspinal connections and anaesthesia (Ahanonu et al., 2023; Dubner & Ruda, 1992; Sandkühler, 2009). Such flexibility is made possible by the presence of subliminal inputs surrounding a receptive field, which might or might not be recruited over minutes, depending on nearby events (Woolf & King, 1990), controlled by inhibitory circuits within the dorsal horn (Sivilotti & Woolf, 1994; Zeilhofer et al., 2012).…”