Multiplex translaminar imaging in the spinal cord of behaving mice

Abstract

While the spinal cord is known to play critical roles in sensorimotor processing, including pain-related signaling, corresponding activity patterns in genetically defined cell types across spinal laminae have remained challenging to investigate. Calcium imaging has enabled cellular activity measurements in behaving rodents but is currently limited to superficial regions. Here, using chronically implanted microprisms, we imaged sensory and motor-evoked activity in regions and at speeds inaccessible by other high-resolution imaging techniques. To enable translaminar imaging in freely behaving animals through implanted microprisms, we additionally developed wearable microscopes with custom-compound microlenses. This system addresses multiple challenges of previous wearable microscopes, including their limited working distance, resolution, contrast, and achromatic range. Using this system, we show that dorsal horn astrocytes in behaving mice show sensorimotor program-dependent and lamina-specific calcium excitation. Additionally, we show that tachykinin precursor 1 (Tac1)-expressing neurons exhibit translaminar activity to acute mechanical pain but not locomotion.

Fast, Accurate, and Versatile Data Analysis Platform for the Quantification of Molecular Spatiotemporal Signals

“…We employed AQuA2 to analyze translaminar astrocyte calcium activity in the spinal dorsal horn of Tg(GFAP:GCaMP6f) mice. The experimental setup, depicted in Figure 5A, involved securing the mouse on a spherical treadmill and optically recording GCaMP6f activity through a microprism implanted near the lateral edge of the spinal gray matter 8 . During the recordings, a pressure stimulus was applied to the mouse’s proximal tail.…”

“…Within the spinal cord, individual laminae contain unique neuronal populations, exhibit distinct connectivity patterns, and serve specialized functional roles 41,42 . A recently introduced translaminar imaging approach offers the capability for rapid measurements across spinal laminae, providing an opportunity to explore and comprehend the interactions and dynamics inherent to these distinct laminae 8 . Yet, the absence of a mature analysis tool for this novel imaging technique requires researchers to manually partition the field of view (FOV) into equally sized ROIs for analysis.…”

“…Imaging cellular and molecular activity across space and time has emerged as a crucial approach in many fields such as neuroscience 1 , cell biology 2 , pathology 3 , and developmental biology 4 . Recent developments in modern genetically encoded fluorescent probes 5–7 and advanced imaging techniques 8–10 have enabled observation of a wide range of signals including calcium ions, ATP, neurotransmitters, neuromodulators, and other molecules, expanding greatly the breadth and depth of scientific studies. However, with the rapid growth of data generation and the revealing of complex spatiotemporal activity patterns such as heterogeneous spatial footprints and various propagations (see top row of Figure 3), quantifying and understanding the data has become a limiting factor.…”

Selective modification of ascending spinal outputs in acute and neuropathic pain states

“…It is therefore not known how the tuning of individual ALT neurons is altered or preserved during the induction and maintenance of persistent pain states, or whether nociceptive and neuropathic pain signals are transmitted from the spinal cord to the brain by the same or different ensembles of neurons. In vivo and ex vivo cellular imaging techniques can now be applied to the superficial dorsal horn of the spinal cord, allowing direct visualization of population sensory responses in spinal projection neurons and interneurons 26,[30][31][32][33][34][35][36] . We have adapted this approach to allow repeated imaging cellular function in vivo over prolonged time scales, and applied it to track how sensory coding in spinal output circuitry is transformed during the development and maintenance of persistent neuropathic pain.…”

Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cordin vivo

“…This limits studies to only a few superficial regions of the spinal cord, such as the dorsal axonal tracts and restricted grey matter regions outlining the dorsal horn for acute or long-term imaging through window preparations (Farrar & Schaffer, 2014; Lorenzana et al, 2015; Ruschel et al, 2015; Tang et al, 2015; Tedeschi et al, 2019; Wu et al, 2022; Yang et al, 2017). Efforts to visualize cellular functions beyond superficial laminar regions require invasive procedures such as imaging through a chronically implanted microprism (Shekhtmeyster, Carey, et al, 2023) or surgically exposing the spinal cord and angling of the mice to image parts of the ventral horn in an acute preparation (Cartarozzi et al, 2018).…”