Journal: bioRxiv

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

doi: 10.1101/2023.05.22.541477

Figure Lengend Snippet: a. 3D model of the recording chamber implanted at the T12-L1 vertebrae. Skeleton created using microCT data from a naive mouse . Insets: a’ , optical window (cover glass) provides access to the dorsal spinal cord post-T13 laminectomy and a” , coronal view slightly off-axis shows a slice through side bars and highlights side bar placement adjacent to the articular processes and location of Teflon AF (green film). b. Diagram of chamber components that permit optical access to the lumbar (L4-L5) or lumbosacral (L5-S1) spinal cord. Super glue fixes the stabilizing plate to the two side bars. c. Two Teflon materials inhibit post-laminectomy fibrosis: opaque PRECLUDE is applied immediately after laminectomy (as in d ), then removed and replaced subsequently by transparent Teflon AF fluoropolymer (as in e ). d. Workflow for long-term spinal cord imaging includes three surgical steps. e. Overhead view of the spinal chamber one week after window placement. f. Using only silicone to cover the spinal cord leads to fibrosis and disappearance of the dorsal vein (black arrow) within a month (Mouse #1). Sequencing PRECLUDE and Teflon AF inhibits fibrosis, allowing visualization of the dorsal vein and ascending venules (black arrows) for months. Fluorescent imaging of mouse #2 and #3 in and , respectively. g. Whole body microCT sagittal max projection after chamber implantation. Chamber (green dashed line) fashioned using a 3D printed radiotransparent material (BioMed Clear). Scale bar, 5.0 mm. h. Coronal slice (yellow arrow and dashed line in g ) from microCT, post laminectomy, shows intact surrounding bone in relation to the glass window. A surface layer of radiotransparent dental cement secures the glass window to the side bars. Scale bar, 1.0 mm. i. Multi-vertebral 3D reconstruction of microCT data in g-h confirms T13 window placement and bone integrity. j. Two mice exhibiting normal behaviors after chamber implant (see ). k. Body weight of chamber-implanted mice (n = 16) pre- and post-surgery compared to age-matched controls (n = 2). l. Tracking of open field locomotion (30 min) after chamber implant. Inset: DeepLabCut markers of individual body parts used for openfield tracking. Scale bar, 10 cm. m. Locomotor speed of the same mouse during 30-min sessions, pre- and post-surgery (Stage 1, side bar). n. Mean open field locomotor speed comparing naïve (n = 7) and post-surgery mice at different stages (n = 7, 2, 18, and 19, respectively) with “Window late” indicating beyond 30 days post window procedure. Most mice in n-p are used for imaging. Bar plot and error bars in n - p are mean ± SD, gray lines indicate animals measured across multiple stages. o. Mean latency to fall in final (3rd) trial on an accelerating rotarod comparing naïve (n = 14) and post-surgery mice at different stages (n = 12, 2, 10, 5, 5, 5, 5, respectively). p. Von Frey mechanical thresholds comparing naïve (n = 9) and post-surgery mice at different stages (n = 7, 6, 13, and 19, respectively) with “Window late” indicating beyond 30 days post window procedure. q. Microglia (CX3CR1-EYFP) and astrocyte (α-GFAP) immunofluorescence of 100-μm thick L4 sections before and after chamber implant. Scale bars, 300 μm and 50 μm (zoom).

Article Snippet: To protect the glass coverslip window from scratching and damage, we 3D printed covers (Stratasys, uPrint ABS and MakerBot, Nylon 12 Carbon Fiber [375-0061A] or ABS-R [375-0071A]) to which we attached two neodymium magnets (McMaster, 5862K141) using cyanoacrylate glue (Henkel, Loctite 4311) or optical adhesive (Norland Products, NOA81) ( – ).

Techniques: Imaging, Sequencing, Immunofluorescence

Journal: bioRxiv

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

doi: 10.1101/2023.05.22.541477

Figure Lengend Snippet: a. Spinal cord imaging workflow, including steps for surgery preparation, spinal chamber implantation, in vivo imaging, and data analysis. Several steps, such as microCT validation, are optional. b. Spinal implant chamber components. A set of both 3D printed and laser cut stainless steel side bars and stabilizing plates are shown. Each 33G needle is clipped during chamber implant surgery after its placement through the spinous process. Scale bar, 1 cm. c. Spinal cord surgery setup made from commercially available components and 3D printed parts, see for a parts list. d. Side bars technical diagram with dimensions before and after (example taper angle) grinding during fabrication; units in mm. Scale bar, 1 cm. e. Stabilizing plate technical diagram with dimensions; units in mm. f. Several designs (top row, CAD; bottom row, real image) of the stabilizing plate that allows for different levels of cord exposure as well as locations where clamp can be applied or where the animal can be handled. Side bars included for size comparison. Scale bar, 1 cm. g. A 3D model showing a horizontal view of the spinal cord implant chamber and optional screws. h. Example of alternative design of spinal implant chamber (see f ) with miniature screws. i. A 3D render of spinal cord chamber window cover for protection of implant. j. Technical diagram of side bar cover; units in mm. k. Coronal view of the present implant with dorsal oriented attachment to the T12-L1 vertebrae, compared to prior strategies. Note that our design allows standard clamps to manipulate the chamber during surgery and imaging. Colors for items: same as in .

Article Snippet: To protect the glass coverslip window from scratching and damage, we 3D printed covers (Stratasys, uPrint ABS and MakerBot, Nylon 12 Carbon Fiber [375-0061A] or ABS-R [375-0071A]) to which we attached two neodymium magnets (McMaster, 5862K141) using cyanoacrylate glue (Henkel, Loctite 4311) or optical adhesive (Norland Products, NOA81) ( – ).

Techniques: Imaging, In Vivo Imaging, Biomarker Discovery, Comparison

Journal: bioRxiv

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

doi: 10.1101/2023.05.22.541477

Figure Lengend Snippet: a. Highlighted vertebral anatomy critical to chamber implantation, including lamina (blue shading), dorsal spinous process (DSP, red circle), and facet joint (green circle). b. 3D rendering of the T12-L1 vertebrae of the spinal column visualized from above. Different orientations of the laminae, DSPs, and facet joints are highlighted. Note, when side bars are placed in the correct position, only the two circled facet joints are surgically exposed. They rest above the side bars. c. After laser-cutting of the side bar, and prior to implantation, the side bar edges are manually tapered by a grinding wheel. Scale bar, 1 mm. d. The taper allows room for the spinal process needles to be inserted and the side bar to be fixated above the T13 transverse process (the image shown is perpendicular to the spinal column). e. After the spinal process needles are superglued above the tapered side bars (red dots), the laminae, and DSPs of T12 and L1 serve as binding substrates for the dental cement. To allow for future laminectomy, the T13 lamina is kept free of cement. f. A view from the side shows where the spinal process needles bore through the DSP of T12 and L1 (solid blue circles) and bind to dental cement above the side bars. g. Spinal column dissection of a chamber-implanted animal. From a dorsal view, the stabilizing plate, the spinal process needles, and the T13 lamina are visible. When viewed from below, the bottom surface of the side bars is visible. After removing the vertebral bodies, the location of the spinal cord in relation to the placement of the side bars is confirmed to be centered on T13. h. Laminectomy removes the T13 lamina. As a >300 micron distance from the midline is necessary for the dorsal horn to be visible for imaging, lateral offset of the lamina cut is important. i. A microCT-generated image shows a cross-section of the T13 vertebra. Red lines indicate the lateral extent to which the T13 lamina is transected during laminectomy to access the spinal cord residing under the lamina in the spinal canal. j. Confocal microscope images of PRECLUDE and Teflon AF collected using transmitted light or lasers (405,488,561,640 nm) demonstrate transparency and minimal autofluorescence of Teflon AF. Brightness and contrast matched across the rightmost four images. Scale bar, 2 mm. k. Mean projection image from one-photon imaging of 1-μm yellow-green microspheres before and after placing Teflon AF on top of the microspheres; brightness and contrast matched. Scale bar, 20 μm. l. As for k, except two-photon imaging of the same microsphere slide. Arrows indicate beads used for measurements in m . Scale bar, 20 μm. m. Profile through 10 beads matched in two-photon imaging (as in l ) with and without Teflon AF.

Article Snippet: To protect the glass coverslip window from scratching and damage, we 3D printed covers (Stratasys, uPrint ABS and MakerBot, Nylon 12 Carbon Fiber [375-0061A] or ABS-R [375-0071A]) to which we attached two neodymium magnets (McMaster, 5862K141) using cyanoacrylate glue (Henkel, Loctite 4311) or optical adhesive (Norland Products, NOA81) ( – ).

Techniques: Binding Assay, Dissection, Imaging, Generated, Microscopy

Journal: bioRxiv

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

doi: 10.1101/2023.05.22.541477

Figure Lengend Snippet: a. 3D printed phantom, used for microCT validation studies, representing skull and spinal column. A 3D printed spinal chamber (Surgical Guide) is implanted with radio-transparent and -opaque dental cement along with miniature brass and steel screws (to evaluate impact on microCT scans). b. Reconstructed horizontal view from microCT scan of phantom in a of 3D printed side bars showing radiotransparent properties of the materials. Yellow bars indicate acquisition planes with reconstruction artifacts; red arrows highlight reduced reconstruction of spinal chamber and column. Scale bars, 2 mm. c. Coronal view of scan as in b shows details of metal screws along with artifact scan lines aligned with areas of higher and lower density of the metal screws. Scale bars, 2 mm. d. Coronal view through sections of the phantom without (left) and with (right) metal screws in the acquisition plane shows the artifacts introduced by miniature steel screws. Scale bars, 2 mm. e. Coronal section from microCT scan (20 μm resolution) of a dissected mouse spinal column, with tissue and muscle attached, placed inside a 3D printed test piece, using the same material (BioMED Clear) as for the 3D printed spinal chamber. Note the ability to reconstruct details and internal geometry of the spinal column, along with surrounding soft tissue. Scale bars, 2 mm. f. Off-axis and sagittal views of 3D reconstructed microCT scan as in e . Note the detailed reconstruction of the spinal column, soft tissue, and geometry of the test piece, confirming that BioMed Clear is microCT compatible. g. Pipeline for 3D reconstruction of microCT scans; see for details. h. Coronal view of mouse with 3D printed spinal chamber (see – ) showing an acquisition plane at the T13 laminectomy location. Scale bars, 2 mm. i. 3D reconstruction of the mouse in – and h with bone (gray), spinal chamber (blue), and circular glass coverslip window (red). Inset: zoomed in view highlights the T13 laminectomy and placement of the circular coverglass. j. Model error (sum of score map cross-entropy and body part location L1-distance losses) as a function of DeepLabCut iterations for model trained using data from 3 mice. Model is trained for 600,000 iterations until convergence. k. Weights of individual animals after bar implant. l. Mean (per animal) latency to fall in all three trials on an accelerating rotarod, comparing naïve (n = 14) and post-surgery mice, at different stages (n = 12, 2, 10, 5, 5, 5, 5, respectively).

Article Snippet: To protect the glass coverslip window from scratching and damage, we 3D printed covers (Stratasys, uPrint ABS and MakerBot, Nylon 12 Carbon Fiber [375-0061A] or ABS-R [375-0071A]) to which we attached two neodymium magnets (McMaster, 5862K141) using cyanoacrylate glue (Henkel, Loctite 4311) or optical adhesive (Norland Products, NOA81) ( – ).

Techniques: Biomarker Discovery

Journal: bioRxiv

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

doi: 10.1101/2023.05.22.541477

Figure Lengend Snippet: a. Bulk expression of GCaMP6s throughout the spinal cord dorsal horn is achieved after intraspinal injection of AAV2retro-hSyn-Cre virus into Ai9 and Ai162 (Cre-dependent GCaMP6s) mice. b. GCaMP6s and tdTomato expression in neurons throughout the superficial and deep laminae of the sacral spinal cord after AAV intraspinal injection (as in a ). Scale bar, 100 μm. c. 3D model of vertebral- fixed setup for imaging and stimulus delivery. Behavior measurement uses a rotary encoder and high-speed cameras. Similar setup is used for awake imaging ( – ). d. Bilateral spinal cord imaging using widefield microscopy with low-magnification objectives and large-sensor sCMOS cameras. Miniscope #1 = Inscopix nVista, Miniscope #2 = Miniscope v4.4. e. Tail pinch (red bar) induces large increase in dorsal horn bulk GCaMP6s fluorescence, coincident with increased locomotion. Arrow points to the session time point in g . Scale bar, 300 μm. f. Widespread increase in stimulus-evoked dorsal horn GCaMP6s fluorescence can occur despite minimal locomotion. Arrow as in e . Scale bar, 300 μm. g. Neural activity (whole frame GCaMP6s fluorescence) aligned to locomotion, body part movement, and force of pinch applied to the tail (T) or back (B) or by innocuous tactile stimulation to the forepaw (F). Bottom heatmap is DLC-based detection of movement of individual body parts. Numbers below heat map highlight when: #1, neural activity correlated with increased behavior; #2, stimulus did not induce locomotion, but there was head movement. Gray portions of the heat map with stars are those where DLC had low confidence in predicting body part location, likely due to occlusion by the mouse moving out of the camera field of view. Pinch stimulus magnitude measured using force-sensitive resistor (values given in arbitrary units). h. Schematic illustrates location of body parts stimulated (recordings in i - k ). h’ , illustrates dorsal horn primary afferent terminal map based on anatomical tracing studies in mice and rats ( , ; ). i. One-photon fluorescence imaging field of view from GCaMP6s mouse (as in a ) recorded in j - k . Mean projection image after bandpass filtering highlights vasculature. Scale bar, 200 μm. j. Neural activity (mean projection image of Δ F / F ) in response to pinch of different parts of the body. Scale bar, 300 μm. k. Neural activity maps from j superimposed to show dorsal horn somatotopy. Scale bar, 300 μm.

Article Snippet: To protect the glass coverslip window from scratching and damage, we 3D printed covers (Stratasys, uPrint ABS and MakerBot, Nylon 12 Carbon Fiber [375-0061A] or ABS-R [375-0071A]) to which we attached two neodymium magnets (McMaster, 5862K141) using cyanoacrylate glue (Henkel, Loctite 4311) or optical adhesive (Norland Products, NOA81) ( – ).

Techniques: Expressing, Injection, Virus, Imaging, Microscopy, Fluorescence, Activity Assay

Journal: bioRxiv

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

doi: 10.1101/2023.05.22.541477

Figure Lengend Snippet: a. Visibly opaque (black) infrared acrylic allows imaging of animal behavior using near-IR light sources and cameras, while blocking animal observation of experimenters (e.g. during stimulus delivery). b. Model error (sum of score map cross-entropy and body part location L1-distance losses) as a function of DeepLabCut iterations for model trained using data from one mouse. 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 across 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 . 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. g. View of dorsal vein after procedure in f . h. Ambulating mouse after mounting procedure. i. General locomotion of a mouse in an open field during freely moving spinal cord imaging. Scale bar, 10 cm. j. Locomotor trace during the open field session in i (3.68 min, 10 Hz).

Article Snippet: To protect the glass coverslip window from scratching and damage, we 3D printed covers (Stratasys, uPrint ABS and MakerBot, Nylon 12 Carbon Fiber [375-0061A] or ABS-R [375-0071A]) to which we attached two neodymium magnets (McMaster, 5862K141) using cyanoacrylate glue (Henkel, Loctite 4311) or optical adhesive (Norland Products, NOA81) ( – ).

Techniques: Imaging, Blocking Assay, Microscopy