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 elusive. Calcium imaging has enabled cellular activity measurements in behaving rodents but is currently limited to superficial regions. 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 new integrated system addresses multiple challenges of previous wearable microscopes, including their limited working distance, resolution, contrast, and achromatic range. The combination of these innovations allowed us to uncover that dorsal horn astrocytes in behaving mice show somatosensory program-dependent and lamina-specific calcium excitation. Additionally, we show that tachykinin precursor 1 (Tac1)-expressing neurons exhibit upper laminae-restricted activity to acute mechanical pain but not locomotion.

A perspective on astrocyte regulation of neural circuit function and animal behavior

“…Recent studies have also indicated that spinal cord astrocytes are regionally heterogeneous (Kronschläger et al, 2021; Molofsky et al, 2014; Shekhtmeyster, Carey, et al, 2021), that this heterogeneity is functionally relevant (Kelley et al, 2018; Kohro et al, 2020) and associated with distinct spatiotemporal excitation patterns. For example, using translaminar imaging in behaving mice with chronically implanted microprisms, astrocytes in sensory and premotor areas of the spinal dorsal horn were found to respond to noxious mechanical stimuli and locomotion in a lamina‐specific manner (Shekhtmeyster, Carey, et al, 2021). In contrast, Tac1‐expressing neurons, involved in pain processing, responded to the same noxious stimuli with calcium spiking in sensory regions but did not respond to locomotion, suggesting that lamina‐specific astrocyte activity depends on the neuronal cell type.…”

“…To date, most spinal cord studies involving astrocytes have been performed in reduced preparations or anesthetized animals, mainly due to the technical difficulty of obtaining stable recordings from this CNS region during animal behavior, which typically results in large amplitude and nonlinear tissue displacements ( Nelson et al., 2019 ). The few studies that have been conducted in awake mice demonstrate that anesthesia powerfully suppresses spinal neuron and astrocyte activity and that sensorimotor-driven astrocyte excitation is complex and regionally heterogeneous ( Sekiguchi et al, 2016 ; Shekhtmeyster, Carey, et al, 2021 ; Shekhtmeyster, Duarte, et al, 2021 ). For example, using quantitative sensory and motor assays together with in vivo calcium imaging, superficial (i.e., lamina I–II) dorsal horn astrocytes were found to respond to innocuous sensory stimuli (tail pinch) with asynchronous increases in the frequency but not amplitude or duration of their microdomain transients.…”

“…In contrast, noxious stimuli evoked concerted calcium transients throughout the superficial astrocyte syncytium within and across spinal segments, both in the presence and absence of a flight response ( Sekiguchi et al, 2016 ; Shekhtmeyster, Duarte, et al., 2021 ). Using the same standardized behavioral assays, local neural activity was measured and compared to astrocyte excitation using separate groups of mice ( Sekiguchi et al, 2016 ; Shekhtmeyster, Carey, et al., 2021 ). As expected, innocuous sensory stimuli of increasing intensity elevated the number and amplitude of responding excitatory neurons, suggesting that astrocytes’ microdomain transients are driven primarily by synaptic activity.…”

Neurophotonic Tools for Microscopic Measurements and Manipulation: Status Report

“…More recently, miniscopes have also enabled real-time measurement of cellular activity in the spinal cord of behaving mice. 372,[377][378][379] In the future, we can expect these imaging systems to become even more powerful. Improved electronics and optics (e.g., new CMOS sensors, custom micro-optics, electronic focusing systems) will enhance 1P miniscopes' ability to record at higher resolution, sensitivity, contrast, and speed, in multiple colors and across larger FOVs or tissue volumes.…”

“…Improved electronics and optics (e.g., new CMOS sensors, custom micro-optics, electronic focusing systems) will enhance 1P miniscopes' ability to record at higher resolution, sensitivity, contrast, and speed, in multiple colors and across larger FOVs or tissue volumes. 352,372,[378][379][380] While initial tether-free designs exist [Fig. 19(a)], further improvements in onboard or wireless power and data transmission technology are needed to reduce overall device weight and boost recording duration at desired image resolutions and frame rates.…”