Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord
- Department of System Pathology for Neurological Disorders, Brain Research Institute, Niigata University, Japan
- Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, Japan
- Section of Electron Microscopy, Supportive Center for Brain Research, National Institute for Physiological Sciences, Japan
- Department of Developmental and Regenerative Neurobiology, Institute of Brain Science, Nagoya City University Graduate School of Medical Sciences, Japan
- Center for Advanced Medicine and Clinical Research, Kashiwaba Neurosurgical Hospital, Japan
- Center for Integrated Human Brain Science, Niigata University, Japan
- Department of Anatomy, Division of Histology and Cell Biology, Jichi Medical University, School of Medicine, Japan
- Division of Ultrastructural Research, National Institute for Physiological Sciences, Japan
Figures
Figure 1 with 1 supplement
Intracerebroventricular injection of adeno-associated virus (AAV) with a neuron-specific promoter specifically labels cerebrospinal fluid-contacting neurons (CSF-cNs).
(A) Schema of AAV-Syn-mCherry injections into the sensorimotor cortex (left) or lateral ventricle (right). (B) mCherry-labeled neurons around the central canal (Cc) occasionally observed after …
Figure 1—figure supplement 1
Specific labeling of cerebrospinal fluid-contacting neurons (CSF-cNs) by intracerebroventricular injections of adeno-associated virus (AAV).
(A–D) Representative images of AAV-Syn-mCherry labeled CSF-cNs and their fibers (red) in the ventral funiculus at the cervical (A), thoracic (B), lumbar (C), and sacral levels (D). PKD2L1, green. …
Figure 2 with 1 supplement
Single-cell tracing in a cleared spinal cord reveals structures and projection patterns of cerebrospinal fluid-contacting neurons (CSF-cNs).
(A, B) Clearing of adeno-associated virus (AAV)-Syn-mCherry-injected cervical cord with CUBIC. Cleared cervical cord (A) and a fluorescent image (B) shown in a side view under a stereomicroscope. (C,…
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Figure 2—source data 1
Raw data for direction, length, and collaterals of CSF-cN projections.
- https://cdn.elifesciences.org/articles/83108/elife-83108-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Projections of single-cell traced cerebrospinal fluid-contacting neurons (CSF-cNs).
(A) Representative images of single-cell traced tdTomato+ CSF-cNs (white) at the cervical level of Rosa26lsl-tdTomato mice injected with a low dose of adeno-associated virus-Syn-Cre. …
Figure 3 with 1 supplement
Injection of adeno-associated virus (AAV)-Syn-DIO construct into Pkd2l1-Cre mice increases labeling specificity of cerebrospinal fluid-contacting neurons (CSF-cNs).
(A–D) mCherry labeled cells (red, arrowheads) in the lateral ventricle (A), SFO (B), third ventricle (C), and AP (D) following AAV-Syn-mCherry intracerebroventricular injection into wild-type (WT) …
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Figure 3—source data 1
Summary of adeno-associated virus-Syn-mCherry and -DIO-hM4Di-mCherry-labeled cells in the brain and spinal cord of wild-type and Pkd2l1-Cre mice (related to Figure 3M).
- https://cdn.elifesciences.org/articles/83108/elife-83108-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Adeno-associated virus (AAV)-Syn-DIO injections into Pkd2l1-Cre mice do not label ventricular cells in the brain.
(A–D) mCherry-labeled cells (red, arrowheads) in the third ventricle (A), OVLT (B), Aq (PAG) (C), and cerebellum (D) following intracerebroventricular AAV-Syn-mCherry injection into wild-type (WT) …
Figure 4 with 1 supplement
Serial block-face scanning electron microscopy (SBF-SEM) analyses with COX4-dAPEX2 and SYP-HRP labeling reveal structures and recurrent connections of cerebrospinal fluid-contacting neurons (CSF-cNs).
(A, B) Representative light microscopic images of 3, 3'-diaminobenzidine (DAB)-stained spinal cord sections of Pkd2l1-Cre mice with injection of adeno-associated virus (AAV)-Syn-DIO-COX4-dAPEX2 (A) …
Figure 4—figure supplement 1
Serial block-face scanning electron microscopy (SBF-SEM) and transmission electron microscope (TEM) analyses in the spinal cord of Pkd2l1-Cre mice injected with adeno-associated virus (AAV)-Syn-DIO-COX4-dAPEX2 and SYP-HRP.
(A, B) Representative images of cerebrospinal fluid-contacting neurons (CSF-cNs) labeled with COX4-dAPEX2 (A) and SYP-HRP (B) following AAV-Syn-DIO-COX4-dAPEX2 and -DIO-SYP-HRP …
Figure 5
Electrophysiological recording with optogenetic stimulation reveals recurrent cerebrospinal fluid-contacting neuron (CSF-cN) connections.
(A) Diagram of electrophysiological recording of Pkd2l1-GFP+ CSF-cNs and blue light stimulation of ChR2-mCherry+ axons of caudally located CSF-cNs in spinal slices of Pkd2l1-Cre;CAG-lcl-EGFP mice. …
Figure 6 with 1 supplement
Cerebrospinal fluid-contacting neuron (CSF-cN) connections with premotor and motor neurons in the spinal cord.
(A) Diagram of Chat-GFP+ cholinergic neurons and adeno-associated virus (AAV)-Syn-mCherry labeled CSF-cNs in ChatCre;CAG-lcl-EGFP mice. Cc, central canal; CCC, central canal cluster cells; MN, motor …
Figure 6—figure supplement 1
Connections of cerebrospinal fluid-contacting neurons (CSF-cNs) with premotor and motor neurons (MNs) in the spinal cord.
(A) Diagram of Chat-EGFP+ cells and adeno-associated virus (AAV)-Syn-mCherry-labeled CSF-cNs in ChatCre;CAG-lcl-EGFP mice. CCC, central canal cluster cells. Image positions of (B, C) are indicated. …
Figure 7 with 1 supplement
Chemogenetic inhibition of cerebrospinal fluid-contacting neurons (CSF-cNs) impairs treadmill locomotion.
(A) The experimental system to analyze treadmill locomotion. Trajectories of fore- and hind-paw were tracked by DeepLabCut and kinematic parameters including step and stride lengths were analyzed by …
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Figure 7—source data 1
Raw data for maximum treadmill speeds, circular phase plots, step frequency, stride lengths, step lengths, the ladder walk test, and the beam walk test.
- https://cdn.elifesciences.org/articles/83108/elife-83108-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Schematic model of cerebrospinal fluid-contacting neuron (CSF-cN) connections in the spinal circuits.
(A) Sagittal view of rostrally projecting CSF-cNs comprising recurrent connections with rostral CSF-cNs and with axial motor neurons (MNs), Chat+ interneurons (central canal cluster cells or V0c), …
Videos
Video 1
Magnified z-stack images of mCherry-labeled cerebrospinal fluid-contacting neurons in a cleared cervical cord.
Video 2
Z-stack images of a tdTomato+ cell body of single-cell traced cerebrospinal fluid-contacting neuron.
Video 3
Z-stack images of tdTomato+ projections of single-cell traced cerebrospinal fluid-contacting neuron in a cleared cervical cord.
Video 4
Z-stack images of tdTomato+ projections of single-cell traced cerebrospinal fluid-contacting neurons (CSF-cNs) innervating to GFP+ CSF-cNs in a cleared cervical cord.
Video 5
Serial section serial block-face scanning electron microscopy images of cerebrospinal fluid-contacting neurons and the subependymal axon bundle around the central canal.
Video 6
Treadmill locomotion during a programmed increase in speeds from 5.0 to 35 m/min in an hM4Di-introduced Pkd2l1-Cre mouse pre- and post-clozapine-N-oxide (CNO) treatment.
Video 7
