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

Nature Methods volume 21, pages 2363–2375 (2024)Cite this article

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Abstract

Advances in optical imaging and fluorescent biosensors enable study of the spatiotemporal and long-term neural dynamics in the brain of awake animals. However, methodological difficulties and fibrosis limit similar advances in the spinal cord. Here, to overcome these obstacles, we combined in vivo application of fluoropolymer membranes that inhibit fibrosis, a redesigned 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 demonstrated a robust ability to monitor axons, identified a spinal cord somatotopic map, performed months-long imaging in freely moving mice, conducted Ca2+ imaging of neural dynamics in behaving mice responding to pain-provoking stimuli and observed persistent microglial changes after nerve injury. The ability to couple in vivo imaging and behavior at the spinal cord level will drive insights not previously possible at a key location for somatosensory transmission to the brain.

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Fig. 1: Surgical design and health validation for long-term spinal cord optical access in awake mice.
Fig. 2: Computational correction of spinal cord motion in awake mice.
Fig. 3: Long-term axonal and neuronal cell body imaging in awake mice.
Fig. 4: Somatotopy and freely moving neural activity identified using large-scale spinal cord imaging.
Fig. 5: Long-term bilateral imaging of spinal cord dorsal horn neural activity in awake mice.
Fig. 6: Long-term imaging of spinal cord microglia before and after injury in awake mice.

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Data availability

The 3D STL and STEP files of the side bars and stabilizing plate along with a 3D model and TIFF stack of the entire mouse body from one of our microCT scans (Fig. 1a) can be found via GitHub at https://github.com/basbaumlab/spinal_cord_imaging and Zenodo at https://doi.org/10.5281/zenodo.11660130 (ref. 86). Any future updates to the design or additional files will be published on those repositories. Due to dataset size, raw imaging data are available from the authors upon request.

Code availability

Code for processing Ca2+ imaging data is available as part of the CIAtah software package under an MIT license (see LICENSE file) via GitHub at https://github.com/bahanonu/ciatah. Code for LD-MCM (feature identification followed by control point motion correction), deformation correction using displacement fields and CS-MCM (cross-session motion correction) is integrated into CIAtah and any future updates will be published on that repository.

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Acknowledgements

We thank the following colleagues for materials and assistance. A. Nimmerjahn and D. Duarte (Salk Institute) for demonstrating their spinal cord setup. A. Gottlieb and Random Technologies for generously gifting Teflon AF material. D. Bernards and T. Desai (UCSF) for helping with the plasma treatment of Teflon AF. Y. Seo and R. Tang (UCSF) for help conducting microCT experiments in the MicroPET/CT, MicroSPECT/CT, MicroCT and Optical Imaging center. MicroCT experiments reported in this publication were supported in part by the Office of the Director, NIH under grant S10OD012301. D. Larson (UCSF) for help optimizing one- and two-photon imaging and microscope maintenance. Data were collected at the Center for Advanced Multiphoton Microscopy with support from the Kavli Institute. K. Herrington and S. Yeon Kim (UCSF) for microscope testing and maintenance help. B. Tiret, P. Schuette and Inscopix, a Bruker company, Mountain View provided the LScape module for nVue 2.0 system. We thank S. Ho (UCSF, Biomaterials and Bioengineering Correlative Microscopy Core) and B. Lee (UCSF) for helping collect scanning electron micrographs of Teflon AF 2400 and PRECLUDE. E. Lam (UCSF) provided help and advice on machining and 3D printing. We thank the following people for reagents and mice. D. McDonald and P. Baluk (UCSF) provided low-magnification objectives. S. Puente and I. Delgado of VICI Metronics helped distribute Teflon AF. W. Xin and J. Chan (UCSF) provided Thy1–YFP-H mice. H. Su and R. Liang (UCSF) provided Thy1–GFP-M mice. B. Roome (McGill University) sent the Phox2a–Cre mouse line. This work was supported by NIH NSR35097306 (A.I.B.), Open Philanthropy (A.I.B.), DARPA 9691 (A.I.B.), HHMI Hanna H. Gray Fellowship (B.A.), NIH R35 NINDS Supplement Funding (B.A.), NIH F32 5F32DE029384 (A.C.), Canadian Institutes of Health Research (PJT-162225, MOP-77556, PJT-153053 and PJT-159839) (A.K.) and NSF Graduate Research Fellowship 2034836 (M.R.C.).

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Author notes

  1. These authors contributed equally: Biafra Ahanonu, Andrew Crowther.

Authors and Affiliations

  1. Department of Anatomy, University of California, San Francisco, CA, USA

    Biafra Ahanonu, Andrew Crowther & Allan I. Basbaum

  2. Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada

    Artur Kania

  3. Department of Cell Biology and Anatomy, and Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada

    Artur Kania

  4. Neuroscience Graduate Program, University of California, San Francisco, CA, USA

    Mariela Rosa-Casillas

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  1. Biafra Ahanonu
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Contributions

B.A., A.C. and A.I.B. designed the project and wrote the manuscript. A.C. and B.A built the instrumentation for surgery and imaging and developed the surgical and imaging protocols. A.C. and B.A. performed surgeries, histology, imaging, image processing and data analysis. B.A. developed and tested the motion correction algorithms and performed animal behavior. M.R.C. assisted with experiments and manuscript preparation and created the supplementary surgery videos. A.K. provided the Phox2a–Cre mouse line.

Corresponding author

Correspondence to Allan I. Basbaum.

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Extended data

Extended Data Fig. 1 Awake spinal imaging experimental overview and designs.

a, Spinal cord imaging workflow. Several steps, such as microCT validation, are optional. b, Spinal implant chamber components: 3D printed and laser cut stainless or mild steel side bars, stabilizing plates, and the protective snap-on cover. Scale bar, 1 cm. c, Spinal cord surgery setup made from commercially available components and 3D printed parts, see Supplementary Table 4 for a parts list. d, Side bars technical diagram; units in mm. e, Stabilizing plate technical diagram; units in mm. f, Several (#1–6) iterative designs (top row, CAD; bottom row, real image) of the stabilizing plate with different positioning of the clamping/handling tabs. Side bars are included for size comparison. Scale bar, 1 cm. g, Horizontal view of the spinal cord implant chamber and optional screws (3D model). h, Spinal implant chamber (see f) with miniature screws. i, Protective cover for the spinal window (3D model); colors as in Fig. 1a. i”, magnified view of the cover (semi-transparent for visualization) on the spinal implant. j, Technical diagram of side bar cover; units in mm. k, Coronal view of an implant. Screws are optional. Note the dorsal-oriented attachment of the metal chamber components (red and blue pieces) to the T12-L1 vertebrae, compared to prior strategies (green pieces). Side clamps are used to manipulate the chamber during surgery and imaging. Colors for items are the same as in Fig. 1a. l, Survival curves (Kaplan-Meier estimator), as in Fig. 1g, illustrate the fibrosis onset probability PRECLUDE + Teflon AF (n = 36) or only Kwik-Sil (n = 10) surgeries; Kwik-Sil curve is not at zero (blue arrow) as n = 2 mice were fibrosis free or deceased at time of analysis. Censored data points indicate mice that died (X) or are still alive (circle) at the time of analysis. The purple arrow indicates time points with multiple alive mice.

Extended Data Fig. 2 Blueprint of chamber implantation and fluoropolymer characterization.

a, Vertebral anatomy, using actual mouse vertebrae, critical to the chamber implantation procedure, including the lamina (blue shading), dorsal spinous process (DSP, red circle), and facet joint (green circle). b, Horizontal view of the T12-L1 vertebrae of the spinal column (3D microCT reconstruction). Note: only the two circled facet joints are surgically exposed and rest above the side bars after correct placement. c, Side bar edges are manually tapered by a grinding wheel before implantation. Scale bar, 1 mm. d, Side view showing an implant. Note the dorsal-oriented attachment of the side bar. e, Spinal process needles are superglued to the side bars (red dots) and dental cement covers the implant (yellow) with the T13 lamina kept cement free for laminectomy. f, Spinal process needles bore through the DSP of T12 and L1 (solid blue circles). g, Spinal column dissection of a chamber-implanted mouse showing chamber components placement at T13. h, The lateral offset (solid red line) of the laminectomy is critical for dorsal horn imaging. i, Cross-section of the T13 vertebra (microCT micrograph). Red lines: lateral extent to which the T13 lamina is transected during laminectomy, to access the dorsal horn. j, Scanning electron micrographs (SEM) of PRECLUDE. Magnifications (top-left to bottom-right): 136X, 281X, 1180X, 3400X, 8850X, 30830X. k, SEM of Teflon AF 2400. Yellow line: edge of Teflon. Magnifications (top-left to bottom-right): 42X, 387X, 1490 K X, 4460X. Each micrograph (j-k) is from a single piece of PRECLUDE (j) or Teflon AF 2400 (k). We observed a similar Teflon AF 2400 texture across 5 other independent samples. l, PRECLUDE and Teflon AF confocal micrographs demonstrate transparency and minimal autofluorescence of Teflon AF. Brightness and contrast matched across images in l”. Scale bar, 2 mm. m, Mean projection image from one-photon imaging of 1-µm yellow-green microspheres with or without Teflon AF; brightness and contrast matched. Scale bar, 20 µm. n, Two-photon imaging of the same microsphere slide as in m. Arrows indicate the beads used for the measurements in o. Scale bar, 20 µm. o, Profile through 10 beads matched in two-photon imaging (as in n) with and without Teflon AF.

Extended Data Fig. 3 Validation of spinal implant with microCT, animal health, and behavior.

a, 3D printed phantom of skull and spinal column. To evaluate impact on microCT scans, a 3D printed spinal chamber (Surgical Guide) is implanted with different cements and metallic screws. b, Horizontal view from microCT scan of phantom in a. Yellow bars: acquisition planes with reconstruction artifacts due to metallic screws; cyan arrows highlight reduced reconstruction of spinal chamber and column. Scale bars, 2 mm. c, Coronal view of scan as in b shows metal screw details and artifact scan lines. Scale bars, 2 mm. d, Coronal sections of the phantom without (left) and with (right) metal screws in the acquisition plane. Scale bar, 2 mm. e, Coronal section from microCT scan (resolution: 20 μm) of a dissected mouse spinal column, placed inside a 3D printed test piece, using the same material (BioMED Clear) as for the 3D printed spinal chamber. Scale bar, 2 mm. f, Off-axis and sagittal views of 3D reconstructed microCT scan as in e. g, Pipeline for 3D reconstruction of microCT scans. h, Coronal view of mouse with 3D printed spinal chamber showing an acquisition plane at the T13 laminectomy location. Scale bar, 2 mm. i, 3D reconstruction of the mouse in Fig. 1h-j and h with bone (gray), spinal chamber (blue), and glass coverslip (red). Inset: magnified view highlights the T13 laminectomy and spinal chamber. j, Change in weight of an additional cohort of individual animals after chamber implant. Two mice, ‘2’ and ‘3’, are replotted from Fig. 1l. k, Model error (sum of score map cross-entropy and body part location L1-distance losses) as a function of DeepLabCut iterations for model trained (600,000 iterations) using data from 3 mice in an open field. l, Mean (per animal) latency to fall in all three trials on an accelerating rotarod, comparing naïve (n = 14) and different post-surgery times (n = 12/8/8, 2, 10, 5, 5, 5, 5). Error bars are mean ± SD. Two-way ANOVA including all trials followed by one-way ANOVA with Dunnett post-hoc per trial (one star, P < 0.05).

Extended Data Fig. 4 Histological analysis post chamber implant and laminectomy.

a, Examples of EGFP+ fluorescence in naïve (a) and post-surgical (a', a'', and a''') CX3CR1-EGFP mouse spinal cord whole mounts and immunohistochemistry (coronal sections) with Neurotrace and anti-GFAP. Scale bars, 500 µm (whole-mount) and 200 µm (coronal slices). b, Spinal cord dissection and histology of a CX3CR1-EGFP mouse 1 week after chamber implantation. The whole-mount image (left) shows dorsal root ganglia in relation to the implant and the associated spinal segments. b’, cross-sectional views of EGFP (green) and GFAP staining (red) show minimal gliosis near the implant. Scale bars, 1 mm (whole-mount) and 200 µm (coronal slices). c, Quantification of microgliosis in naïve mice (n = 2) along with those after spinal chamber implant (1 week, n = 1) and laminectomy (1 week, n = 1 and 1 month, n = 2).

Extended Data Fig. 5 Deep-learning feature detection and control point motion correction.

a, Comparison of reference frame 42 (cyan) to movement frame 804 (red, overlaid on cyan image) before and after LD-MCM motion correction. Scale bar, 300 µm. b, Example of DLC-identified vascular features used for cross-session registration (DLC model trained using day 41). Scale bar, 300 µm. c, Model error as a function of DLC iterations (500,000 iterations, n = 4 mice). d, Spearman’s correlation of each feature to other features in a movie from a Phox2a-Cre; Ai162 mouse. Green arrow, a feature that has reduced correlation with all other features and can thus be removed to improve motion correction. e, Point clouds with each dot (2001 frames) represents the rostrocaudal and mediolateral location of that feature on an individual frame during an imaging session (~6 min, 13.9 Hz, mouse from a). f, DLC tracks (1) large mediolateral shifts in the field of view (yellow arrow) and (2) camera errors that result in a split of the field of view (yellow line). Only showing features with confidence >0.1. Scale bar, 300 µm. g, Labeling (DeepLabCut, 20 frames from day 75) of vascular features in a Phox2a-Cre; Ai162 (GCaMP6s) mouse across 52 neural activity imaging sessions, spanning nearly 5 months. Scale bar, 300 µm. h, Feature locations (normalized to the session mean location) across 13 features tracked in raw and LD-MCM motion corrected movies.Green lines, frames shown in i. i, Frames before and after LD-MCM motion correction. Yellow dots: tracked features with the line showing connected features indicating improvement with LD-MCM. Scale bar, 300 µm. j, Performance of LD-MCM as a function of the number of features used for control point registration (n = 10 movies, n = 2 mice). Mean, median, and standard deviation calculated per movie for each combination of imaging session, parameter value, trial, and feature. Then the mean is taken across all features for the final displayed values (each data point). Boxplots in all figures display the 1st, 2nd (median), and 3rd quartiles with whiskers indicating 1.5*IQR; outliers are omitted.

Extended Data Fig. 6 Deformation-based motion correction using displacement fields.

a, Each motion correction method run on the movie (5,000 frames, 13.9 Hz) from a Phox2a-Cre; Ai162 (GCaMP6s) mouse displays 1: the mean of all movie frames, 2: combined numerical gradient in both lateral directions on the mean frame, 3: the standard deviation over all movie frames (hence visibility of neurons on left and right side of the spinal cord), and 4: ΔF/F frames. Arrows indicate areas of interest where differences between methods are most evident. b, 2D correlation coefficient of all frames to the mean frame of the movie (as in a) for displacement field motion correction compared to raw, TurboReg, and NoRMCorre. All movies (except raw) were spatially filtered to remove large magnitude, low-frequency changes in fluorescence, which artificially enhances correlations. c, Histogram of 2D correlation coefficients over all frames from b. d, Spearman’s rho of all frames to the mean frame of the movie (as in a) for displacement field motion correction compared to raw, TurboReg, and NoRMCorre. All movies (except raw) were spatially filtered to remove large magnitude, low-frequency changes in fluorescence, which artificially enhances correlations. e, Histogram of Spearman’s rho values over all frames from d.

Extended Data Fig. 7 Long-term imaging of cell bodies and axons in the spinal cord of awake mice.

a, Clarity of GFP+ axons (Thy1-GFP mouse) with increasing sCMOS camera exposure times (LED power held constant). Yellow box: magnified section on the right. Yellow arrows: features with increased signal and minimal blur at 10-ms exposure. As a trade-off between SNR and clarity, we used 5–20-ms exposure times. Scale bars, 300 and 50 µm. b, Frames cropped to highlight cross-session matched areas from individual imaging sessions from a Thy1-GFP animal. Scale bar, 200 µm. c, Spearman correlation coefficient to the mean frame of a raw movie from a Thy1-GFP mouse (as in b). d, Increase in tdTomato expression in the dorsal columns after retro-orbital injection of AAV-PHP.S-tdTomato. Day 56, shows 10- and 100-ms exposure. Scale bar, 300 µm. e, Near daily imaging of GFP and tdTomato fluorescence normalized to baseline (pre retro-orbital injection). Magnified view of Fig. 3m highlights tdTomato signal increase from baseline.

Extended Data Fig. 8 Transient angiogenesis and vascular dynamics in awake and anesthetized states.

a, Individual frames across imaging sessions show onset and reversal of angiogenesis in the spinal cord of a CX3CR1-EGFP mouse. Scale bar, 300 µm. b, Change in spinal cord vessel diameter between general anesthesia and awake states in a CX3CR1-EGFP mouse. Middle row illustrates the same frames after application of a Hessian-based Frangi vesselness filter that highlights the dorsal vein and a subset of dorsal ascending venules. These filtered images are used to calculate changes in vessel diameter. Scale bar, 300 µm. c, Procedure for determining diameter of dorsal vein and ascending venules: a Frangi filter was applied to highlight vessels and their local thickness was then calculated to determine vessel diameter. Example frames are illustrated across three major behavioral states of a Thy1-GFP mouse during a 25-min imaging session. Scale bar, 300 µm. d, Temporal change of vessel diameter and whole-frame fluorescence (normalized to 4-min awake baseline) within a single imaging session in a Thy1-GFP mouse before and after induction of general anesthesia (2% isoflurane). Same as Fig. 3p, but here additional right and left dorsal ascending venules are shown. e, Correlation of dorsal vein diameter and fluorescence during a 25-min imaging session across several behavioral states: awake (red), induction and maintenance of general anesthesia (green, isoflurane 2%), and waking up (emergence) from general anesthesia (blue). First order polynomial best-fit lines and R2 indicated by darker colored lines and associated text, respectively.

Extended Data Fig. 9 Behavior tracking of spinally fixed mice and freely moving spinal cord imaging with miniature microscopes.

a, Visibly opaque (black) infrared transmitting acrylic allows imaging of animal behavior using near-IR light sources and cameras, while blocking animal observation of experimenters (for example during stimulus delivery). b, Model error as a function of DeepLabCut iterations for a model trained using data from one mouse for each camera. Model training is terminated after 500,000 iterations, when the loss asymptotes. c, Part affinity fields for DeepLabCut networks across multiple cameras. d, Speed of individual body parts shows correlation of body part movement across cameras (#1–4). The mean speed across all cameras for each body part is used for display in Fig. 5g. Camera locations correspond to 1, left side of the body; 2, right side of the body; 3, right face; and 4, below the animal. Letters below each black arrow indicate the stimulus presented (C: cold; P: pinch; H; heat; A: air puff; S: sound); black bar denotes duration of the sound stimuli. e, 3D CAD of miniature microscope positioning above spinal implant chamber. f, Image of miniature microscope mounting (Inscopix, nVista). g, Image of miniature microscope mounting (Open Ephys, Miniscope V4.4). h, View of dorsal vein after procedure in g. Scale bar, 200 µm. i, Image of miniature microscope mounting in an awake animal (Inscopix, LScape module for nVue 2.0). j, Image of a miniature microscope mounted on the mouse using a clamp. k, Example of normal grooming behavior. l, Field of view from mouse in k. Scale bar, 200 µm. m, Ambulating mouse after mounting procedure in g. n, Locomotion of a mouse moving freely in an open field during spinal cord imaging (30 min, 10 Hz). Scale bar, 10 cm. o, Locomotor trace during the open field session in n. p, Multi-color miniature microscope imaging of both sides of the spinal cord 70 days after window placement in a Phox2a-Cre; Ai162 (GCaMP6s); Ai9 (tdTomato). Scale bar, 300 µm. q, Responses of SCPNsPhox2a (Phox2a-Cre; Ai162 [GCaMP6s]) to cold, hot, and air puff stimuli delivered to the left hindpaw during a ~1.8-hr continuous imaging session. Max projection of 5 s post-stimulus. Scale bar, 300 µm.

Extended Data Fig. 10 Imaging of spinal cord neuronal activity in awake and anesthetized animals.

a, Noxious stimulus-evoked SCPNPhox2a GCaMP6s activity in Phox2a-Cre; Ai162 (GCaMP6s) after 1st and 11th stimuli presentations in the same imaging session. Scale bar, 300 µm. b, Cell extraction outputs show cell (white, after manual sorting) compared to non-cell (red) outputs; the latter are excluded from further analysis. Scale bar, 300 µm. c, Activity of individual SCPNsPhox2a (GCaMP6s ΔF/F), as in a-b, on the left or right spinal cord during a single imaging session (5.61 min, 13.9 Hz). Black arrows point to noxious heat applied to the right hindpaw. d, Extended recording session (25.47 min, 20 Hz) for mouse as in Fig. 5d–g shows SCPNPhox2a stimulus-evoked activity (GCaMP6s) in response to 5 blocks of stimulus applications. e, ΔF/F processed GCaMP6s and raw tdTomato frames from Phox2a-Cre; Ai162 (GCaMP6s); Ai9 (tdTomato) mouse under general anesthesia (2% isoflurane) shows overlap in expression. Yellow arrows in e and g indicate the side that is stimulated. Scale bar, 300 µm. f, Activity of individual SCPNsPhox2a (GCaMP6s ΔF/F), as in e, on the left and right spinal cord during a single imaging session (7.74 min, 20 Hz) during application of various noxious and non-noxious stimuli. There is a ~2 min baseline period at the start of the session, prior to stimulus presentation. g, Same as e, except from a Phox2a-Cre; Ai162 (GCaMP6s) as in Fig. 5d-g. Scale bar, 300 µm. h, Same as f, but for the animal in g, during a single imaging session (6.72 min, 13.9 Hz). i, SCPNPhox2a activity (mean projection of ΔF/Fmin post-stimulus) after noxious heat applied to the left hindpaw across imaging sessions. Yellow dotted lines: dorsal vein. Yellow arrows: consistent SCPNPhox2a activity contralateral to the stimulated hindpaw. Insets: white arrows indicate enlarged areas showing consistent response of the same neurons across multiple imaging sessions. Scale bar, 300 µm. j, SCPNsPhox2a extracted (CELLMax) from individual awake animal imaging sessions (except day 8, which is under anesthesia) and aligned across days. Color indicates the same cell aligned across days; filled and open arrows indicate when that particular cell is or is not identified after cell extraction across imaging sessions, respectively. Scale bars, 100 µm.

Supplementary information

Supplementary Information

Supplementary Notes 1–14, Fig. 1–7, Tables 1–4 and references.

Reporting Summary

Supplementary Video 1

Behavior of mice with implanted spinal cord chambers. Top right: mice locomoting on a running disk. Bottom: mice in a home cage after chamber implant are very active.

Supplementary Video 2

Step-by-step guide for attachment of the spinal chamber to the vertebral column with playback speed for each surgical step indicated on top left.

Supplementary Video 3

Step-by-step guide for laminectomy and placement of the PRECLUDE membrane to inhibit fibrosis with playback speed for each surgical step indicated on top left.

Supplementary Video 4

Step-by-step guide for PRECLUDE removal, Teflon AF application and placement of circular glass coverslip window with playback speed for each surgical step indicated on top left.

Supplementary Video 5

MicroCT sagittal slices through a mouse with BioMed Clear spinal chamber implanted at T12–L1 and following laminectomy.

Supplementary Video 6

Three-dimensional rendering of microCT, postlaminectomy (T13), showing sequential removal of soft tissue (brown), spinal implant chamber (white) and glass window (red), leaving just the bone (gray).

Supplementary Video 7

Open field tracking of behavior postlaminectomy for DeepLabCut network trained (top row) and network naive (bottom row) movies.

Supplementary Video 8

Rostrocaudal shift motion correction using LD-MCM compared with TurboReg and NoRMCorre during SCPN recording in a Phox2a-Cre; Ai162 (GCaMP6s); Ai9 (tdTomato) mouse. The dots indicate features tracked using DeepLabCut in each movie and black bars indicate sections of no usable data.

Supplementary Video 9

Nonrigid deformation motion correction using displacement fields compared with NoRMCorre and TurboReg, during SCPN recording in a Phox2a-Cre; Ai162 (GCaMP6s); Ai9 (tdTomato) mouse.

Supplementary Video 10

Long-term imaging of a Thy1-GFP mouse after CS-MCM motion correction and with DeepLabCut tracking of vasculature features (colored dots).

Supplementary Video 11

Time-lapse imaging of increasing GFP and tdTomato expression before and after retro-orbital injection of AAV-PHP.eB-GFP and AAV-PHP.S-tdTomato.

Supplementary Video 12

Change in vasculature diameter and Thy1-GFP fluorescence during a single session (time bottom left, hours:minutes:seconds) as the mouse enters and exits general anesthesia (2% isoflurane).

Supplementary Video 13

Spinal cord somatotopic map identified using bulk GCaMP6s activity after stimulation of indicated caudal body parts in an Ai162 mouse injected with AAV2retro-hSyn-Cre into the spinal cord.

Supplementary Video 14

Awake spinal cord imaging session in response to noxious thermal and mechanical stimuli. Spinal cord projection neuron activity (Phox2a-Cre; Ai162 (GCaMP6s)) aligned to behavior from multiple camera angles (colored squares, body part tracking), locomotion (white, rotary encoder), head movement (cyan) and stimulus application (red).

Supplementary Video 15

Projection neuron activity across 20 days, before and after applying noxious heat to the right hindpaw (red bar on left side of movie). Days 7–8 are under isoflurane anesthesia.

Supplementary Video 16

Two-photon imaging of monocytes (CX3CR1-EGFP mouse under 2% isoflurane) showing multiple planes from the meninges to the spinal cord parenchyma (microglia) during a ~15 min session.

Supplementary Video 17

Time-lapse imaging for months of microglia dynamics before and after nerve injury (left hindpaw, SNI model) in a CX3CR1-EGFP mouse. The red regions indicate areas of greater fluorescence.

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Ahanonu, B., Crowther, A., Kania, A. et al. Long-term optical imaging of the spinal cord in awake behaving mice. Nat Methods 21, 2363–2375 (2024). https://doi.org/10.1038/s41592-024-02476-3

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