rabbit anti chx10 antibody  (Novus Biologicals)

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Scheme illustrating our experimental protocol followed by single-nucleus RNA sequencing. Mice received upper-thoracic SCI. After 30 days, half of the mice underwent repetitive episodes of autonomic dysreflexia during 90 minutes. The lumbosacral spinal cord and the lower thoracic were dissected from the mice according to standard procedures. b , We obtained high-quality transcriptomes from 64,739 nuclei that were evenly represented across experimental conditions and spatial locations. c , Number of unique molecular identifiers (UMIs) per nucleus. Inset text shows the median number of UMIs. d , Number of genes detected per nucleus. Inset text shows the median number of genes detected. e , Proportion of mitochondrial counts per nucleus. Inset text shows the median proportion of mitochondrial counts. f , Number of UMIs quantified per nucleus in each major cell type of the mouse spinal cord. g , Number of genes detected per nucleus in each major cell type of the mouse spinal cord. h , Proportion of mitochondrial counts per nucleus in each major cell type of the mouse spinal cord. i , UMAP visualization of 64,739 nuclei colored by major cell type, segregated by the location of spinal cord tissues (L6, T12) and experimental conditions (SCI only, exposure to repeated episode of autonomic dysreflexia, AD). experimental condition. j , Proportions of nuclei from each major cell type depending on the location of spinal cord tissues and experimental conditions. k , UMAP visualization showing expression of key marker genes for the major cell types of the mouse spinal cord. l , UMAP visualization of 29,144 neuronal nuclei colored by neuronal subpopulations, split by experimental condition. m , UMAP visualization showing expression of key marker genes for the major neuronal subpopulation classifications of the mouse spinal cord. n , UMAP visualization and dendrograms showing cell type prioritizations assigned by Augur across the neuronal taxonomy of the lower thoracic ( Left ) and lumbosacral ( Right ) spinal cord. o , Photomicrographs of the lower thoracic and lumbosacral spinal cord after repetitive episodes of autonomic dysreflexia. Vsx2 ON neurons were labelled with immunohistochemistry. Long-distance projecting (Zfhx3, lumbosacral spinal cord) and locally-projecting (Nfib, lower thoracic spinal cord) were additionally colocalized with immunohistochemistry labelling of cFos.

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: RNA Sequencing Assay, Expressing, Marker, Immunohistochemistry

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of the single-nucleus sequencing experiment. Uniform manifold approximation and projection (UMAP) visualization of 64,739 neuronal nuclei, colored by neuronal subpopulation identity. Middle , UMAP visualizations of neuronal subpopulations in the lower thoracic (top) and lumbosacral (bottom) spinal cord. Right , Ranking neuronal subpopulations most responsive to autonomic dysreflexia with Augur. b , Schematic overview of the neuronal architecture of autonomic dysreflexia, including the nodes (numbers) that are dissected anatomically and functionally in the subsequent panels. c , Whole spinal cord visualization of projections from SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord that project to SC Hoxa7::Nfib::Vsx2 neurons located in the lower thoracic spinal cord. Insets illustrate the synaptic-like appositions of Calca ON projections labeled with immunohistochemistry onto SC Hoxa10::Zfhx3::Vsx2 neurons in the lumbosacral spinal cord. d , Calca ON projections labeled with immunohistochemistry onto SC Hoxa10::Zfhx3::Vsx2 neurons in the lumbosacral spinal cord, including insets showing synaptic-like appositions. e , Bar plot reporting the severity of autonomic dysreflexia, quantified as the mean change in systolic blood pressure in response to colorectal distension before and after the ablation of Calca ON neurons located in the dorsal root ganglia in Calca Cre ::Advil FlpO ::iDTR mice (n = 5; independent samples t-test; t = -6.0; p-value = 0.0006). f , Severity of autonomic dysreflexia before and after chemogenetic silencing of Vsx2 ON neurons located in the lumbosacral spinal cord in Vsx2 Cre (n = 5; paired samples t-test; t = -9.47; p-value = 0.00069). g , Projections from SC Hoxa10::Zfhx3::Vsx2 in the lower thoracic spinal cord co-labeled with SC Hoxa7::Nfib::Vsx2 neurons and their local projections as well as immunohistochemical labelling of Chat ON . Insets show synaptic-like appositions from SC Hoxa10::Zfhx3::Vsx2 neurons onto SC Hoxa7::Nfib::Vsx2 , and synaptic-like appositions of projections from SC Hoxa7::Nfib::Vsx2 to Chat ON sympathetic preganglionic neurons located in the intermediolateral column. h , Severity of autonomic dysreflexia before and after chemogenetic silencing of Vsx2 ON neurons located in the lower thoracic spinal cord in Vsx2 Cre (n = 5; paired samples t-test; t = -9.39; p-value = 0.00072). i , Severity of autonomic dysreflexia before and after chemogenetic silencing of Chat ON neurons located in the lower thoracic spinal cord in Chat Cre mice (n = 5; paired samples t-test; t = -8.03; p-value = 0.00048).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Sequencing, Labeling, Immunohistochemistry, Immunohistochemical staining

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of the neuronal architecture of autonomic dysreflexia. b , Zoom on the first node of the neuronal architecture of autonomic dysreflexia that involves the growth of projections from Calca ON neurons onto Vsx2 ON neurons with long-distance projections, named SC Hoxa10::Zfhx3::Vsx2 neurons. This growth was assessed on tissues collected 30 days after SCI in wild-type mice. c , Photomicrograph taken at L6 spinal segment from a mouse with an intact spinal cord and a mouse with a chronic SCI in which Calca ON axons were labelled with immunohistochemistry. d , Bar plots reporting the density of Calca ON axonal projections into the intermediate laminae of the spinal cord in uninjured mice and mice with chronic SCI (n = 4; independent samples t-test; t = 9.38; p-value = 0.000086). e , Overview of the experimental protocol to test the severity of autonomic dysreflexia after the ablation of Calca ON and PV ON neurons. To achieve the ablation of these neurons exclusively in the dorsal root ganglia, we used a Cre- and Flp-dependent strategy in Calca Cre ::Avil FlpO ::iDTR and PV Cre ::Avil FlpO ::iDTR mice that allowed the expression of diphtheria toxin receptors (DTR) in Calca ON and PV ON neurons located in the dorsal root ganglia, respectively. f , Pressor responses ( Left ; bold line represents mean trace ±standard error of mean (sem) for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia ( Right ) measured by the change in systolic blood pressure during colorectal distension in mice without diphtheria toxin-induced ablation of either Calca ON neurons or PV ON neurons, mice with diphtheria toxin-induced ablation of Calca ON neurons and mice with diphtheria toxin-induced ablation of PV ON neurons (n = 5; independent samples t-test; t= -5.9998; p-value = 0.00064, independent samples t-test; t= -9.3261; p-value = 0.00014). g , Zoom on the second node of the neuronal architecture of autonomic dysreflexia that involves SC Hoxa10::Zfhx3::Vsx2 neurons projecting to the low thoracic spinal cord. An intersectional viral labelling strategy was used to label the axons of SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord and that establish projections in the lower thoracic spinal cord. Vsx2 Cre mice received SCI and were injected with Retro-AAV-DIO-FlpO into the lower thoracic spinal cord and AAV8-Con/Fon-EYFP into the lumbosacral spinal cord. h , Photomicrograph of the L6 spinal segment from a Vsx2 Cre mouse with an intact spinal cord and a Vsx2 Cre mouse with a chronic SCI that received intersectional viral tracing to label SC Hoxa10::Zfhx3::Vsx2 neurons. Axons from Calca ON were also labelled with immunohistochemistry. Insets show synaptic-like appositions from Calca ON axons onto SC Hoxa10::Zfhx3::Vsx2 neurons. i , The necessary role of SC Hoxa10::Zfhx3::Vsx2 neurons in autonomic dysreflexia was evaluated using Cre-dependent expression of Gi DREADDs in SC Hoxa10::Zfhx3::Vsx2 neurons. j , Photomicrograph showing the expression of DREADD (G i ) receptors in SC Hoxa10::Zfhx3::Vsx2 neurons. k , Left , changes in systolic blood pressure in response to colorectal distension (shared area). Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia. Right , Severity of autonomic dysreflexia in Vsx2 Cre mice before and after chemogenetic silencing of Vsx2 ON neurons located in the lumbosacral spinal cord (n = 5; paired samples t-test; t = -9.47; p-value = 0.00069). l , The sufficient role of SC Hoxa10::Zfhx3::Vsx2 neurons in triggering autonomic dysreflexia was evaluated using optogenetic activation of SC Hoxa10::Zfhx3::Vsx2 neurons in Vsx2 Cre mice injected with AAV-Syn-flex-ChrimsonR-tdTomato into at the lumbosacral spinal cord. 30 days after SCI, blood pressure responses were monitored beat-by-beat using a blood pressure catheter inserted into the carotid artery. Red-shifted light was shined over the lumbosacral spinal cord for 60 seconds during each trial. m , Photomicrograph showing the expression of ChrimsonR in SC Hoxa10::Zfhx3::Vsx2 neurons. n , Left, Changes in systolic blood pressure in response to the photostimulation of SC Hoxa10::Zfhx3::Vsx2 neurons in mice with intact spinal cord and with chronic SCI. Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and blood pressure responses due to optogenetic activation of SC Hoxa10::Zfhx3::Vsx2 neurons. Right , Bar plots reporting mean changes in blood pressure in Vsx2 Cre mice with intact spinal cord and with SCI during optogenetic activation of SC Hoxa10::Zfhx3::Vsx2 neurons (n = 5; independent samples t-test; t = 5.14; p-value = 0.00496).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Immunohistochemistry, Expressing, Injection, Activation Assay

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Zoom on the third node of the neuronal architecture of autonomic dysreflexia that involves SC Hoxa7::Nfib::Vsx2 neurons located in the lower thoracic spinal cord. b , Overview of intersectional viral tracing strategy to label projections from SC Hoxa10::Zfhx3::Vsx2 into the lower thoracic spinal cord concomitantly to the labelling of SC Hoxa10::Zfhx3::Vsx2 Step 1 , AAV5-hSyn-flex-tdTomato was infused into the lower thoracic spinal cord of Vsx2-Cre mice to label SC Hoxa7::Nfib::Vsx2 . Step 2 , Retro-AAV-DIO-FlpO was infused into the lower thoracic spinal cord and AAV8-Con/Fon-EYP into the lumbosacral spinal cord to label the projections from SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord and that project in the lower thoracic spinal cord. c , Photomicrographs of the lower thoracic spinal cord with intersectional viral tracing labelling projections from SC Hoxa10::Zfhx3::Vsx2 , SC Hoxa7::Nfib::Vsx2 neurons and their projections from a representative mouse with an intact spinal cord and mouse with SCI. d , Bar plots reporting the mean density of projections from SC Hoxa10::Zfhx3::Vsx2 neurons in the grey matter of the lower thoracic spinal cord in mice with an intact spinal cord and with chronic SCI (n = 5; independent samples t-test; t = -3.09; p-value = 0.0162). e , Whole spinal cord visualization of projections from SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord (red) and visualization of SC Hoxa7::Nfib::Vsx2 neurons (blue) located in the lower thoracic spinal cord in mice with chronic SCI. f , The necessary role of SC Hoxa7::Nfib::Vsx2 neurons in autonomic dysreflexia was evaluated using Cre-dependent expression of G i DREADDs in SC Hoxa7::Nfib::Vsx2 neurons. g , Photomicrograph showing the expression of G i DREADD receptors in SC Hoxa7::Nfib::Vsx2 neurons. h , Left , changes in systolic blood pressure in response to colorectal distension. Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia. Right , Severity of autonomic dysreflexia in Vsx2 Cre mice before and after chemogenetic silencing of Vsx2 ON neurons located in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -9.39; p-value = 0.00072). i , The sufficient role of SC Hoxa7::Nfib::Vsx2 neurons in autonomic dysreflexia was evaluated using optogenetic activation of SC Hoxa7::Nfib::Vsx2 neurons in Vsx2 Cre mice injected with AAV-Syn-flex-ChrimsonR-tdTomato into the lower thoracic spinal cord. 30 days after SCI, blood pressure responses were monitored beat-by-beat using a blood pressure catheter inserted into the carotid artery. Red-shifted light was shine over the lumbosacral spinal cord for 60 seconds during each trial. j , Photomicrograph showing the expression of ChrimsonR in SC Hoxa7::Nfib::Vsx2 neurons. k , Left , changes in systolic blood pressure in response to colorectal distension. Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and blood pressure responses due to optogenetic activation of SC Hoxa7::Nfib::Vsx2 neurons. Right , Blood pressure responses in Vsx2 Cre mice with intact spinal cord and with chronic SCI during optogenetic activation of SC Hoxa7::Nfib::Vsx2 neurons (n = 5; independent samples t-test; t = 15.4; p-value = 0.0000148). l , Zoom on the fourth node of the neuronal architecture of autonomic dysreflexia that involves Chat ON sympathetic preganglionic neurons. m , Overview of experimental protocol to label projections from SC Hoxa7::Nfib::Vsx2 neurons located in the lower thoracic spinal cord in Vsx2 Cre mice with SCI. Thirty days after SCI and viral tracing, the spinal cord tissues were collected and processed. n , Photomicrograph of the lower thoracic spinal cord from a mouse with an intact spinal cord and a mouse with chronic SCI in which the projections of SC Hoxa7::Nfib::Vsx2 were labelled concomitantly to the immunohistochemical labelling of Chat ON neurons. o , The necessary role of Chat ON neurons in autonomic dysreflexia was evaluated using Cre-dependent expression of G i DREADDs in Chat ON neurons. p , Photomicrograph illustrating the expression of G i DREADD receptors in SC Hoxa7::Nfib::Vsx2 neurons. q , As in h , for Chat ON neurons located in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -8.03; p-value = 0.00048).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Expressing, Activation Assay, Injection, Immunohistochemical staining

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of experiments to trigger pressor responses with EES in mice with SCI. b , Pressor response induced by continuous (40 Hz) EES in a mouse with SCI. c , Uniform manifold approximation and projection (UMAP) visualization of 21,098 neuronal nuclei, colored by neuronal subpopulation identity. Right , Identification of perturbation-responsive neuronal subpopulations with Augur. d-f , Schematic overview of the successive nodes constituting the neuronal architecture though which EES applied over the low thoracic spinal cord induces pressor responses. d , EES-induced pressor responses before and after the ablation of PV ON neurons located in the dorsal root ganglia in PV Cre ::Advil FlpO ::iDTR mice (n = 5; independent samples t-test; t = -5.41; p-value = 0.0043). e , EES-induced pressor responses before and after chemogenetic silencing of Vsx2 ON located in the lower thoracic spinal cord in Vsx2 Cre mice (n = 5; paired samples t-test; t = -4.21; p-value = 0.014). f , EES-induced pressor responses before and after chemogenetic silencing of Chat ON neurons located in the lower thoracic spinal cord in Chat Cre mice (n = 5; paired samples t-test; t = -7.07; p-value = 0.0021). g , Photomicrograph of the lower thoracic spinal cord demonstrating vGlut1 ON synaptic puncta and synaptic-like appositions from large-diameter afferent neurons onto SC Hoxa7::Nfib::Vsx2 neurons labelled with in situ hybridization ( Left ) or viral tract tracing ( Right ) in the lower thoracic spinal cord of PV Cre ::Advil FlpO ::tdTomato mice.

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: In Situ Hybridization

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of the neuronal architecture through which EES induces pressor responses. b , Zoom on the first node of the neuronal architecture of EES-induced pressor responses that involves PV ON . c , Overview of the experimental protocol to test the involvement of afferent fibers from PV ON and Calca ON neurons in EES-induced pressor responses. To achieve the ablation of these neurons exclusively in the dorsal root ganglia, we used a Cre- and Flp-dependent strategy in Calca Cre ::Avil FlpO ::iDTR and PV Cre ::Avil FlpO ::iDTR mice that allowed the expression of diphtheria toxin receptors (DTR) in these specific neurons. d , EES-induced pressor responses ( Left ; bold line represents mean trace ±sem for each group and individual line traces are from each mouse) ( Right ) measured by the change in systolic blood pressure during EES in mice without any ablation, mice with diphtheria toxin-induced ablation of PV ON neurons and mice with diphtheria toxin-induced ablation of Calca ON neurons (n = 5; independent samples t-test; t= -5.4141; p-value = 0.0043, independent samples t-test; t= 6.3166; p-value = 0.0020). e , Overview of the experiment strategy to visualize large-diameter PV ON fibers in PV Cre ::Avil FlpO ::Ai9 (RCL-tdT) mice and confirmed that they established vGlut1 ON synaptic-appositions onto SC Hoxa7::Nfib::Vsx2 neurons. Thirty days after SCI, spinal cord tissues were collected and processed. f , Photomicrograph of the lower thoracic spinal cord showing vGlut1 synaptic puncta and synaptic-like appositions from large-diameter afferent neurons (PV Cre ::Advil FlpO ::Ai9 (RCL-tdT) mice) onto SC Hoxa7::Nfib::Vsx2 neurons labelled with in situ hybridization ( Left ) or viral tract tracing ( Right ). g , Photomicrograph of the lower thoracic spinal cord from a PV Cre ::Advil FlpO ::Ai9 (RCL-tdT) mouse combined with immunohistochemical labelling of ChatON neurons. h , Quantification of vGlut1 ON synaptic-appositions onto SC Hoxa7::Nfib::Vsx2 neurons and ChatON neurons in PV Cre ::Advil FlpO ::Ai9 (RCL-tdT) mice with an intact spinal cord and with a chronic SCI. i , Zoom on the second node of the neuronal architecture of EES-induced pressor responses that involves SC Hoxa7::Nfib::Vsx2 . The necessary role of SC Hoxa7::Nfib::Vsx2 neurons in EES-induced pressor response was evaluated using Cre-dependent expression of Gi DREADDs in SC Hoxa7::Nfib::Vsx2 neurons. j , EES-induced pressor responses ( Left ; bold line represents mean trace ±sem for each group and individual line traces are from each mouse) ( Right ) measured by the change in systolic blood pressure during EES in the same mice before and after chemogenetic silencing of Vsx2 ON neurons located in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -4.21; p-value = 0.014). k , Zoom on the third node of the neuronal architecture of EES-induced pressor responses that involves Chat ON sympathetic preganglionic neurons. As in h , for Chat ON neurons located in the lower thoracic spinal cord. l , As in j , for Chat ON neurons in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -7.07; p-value = 0.0021). m , Photomicrograph showing the expression of ChrimsonR in Vsx2 ON neurons and the tract resulting from the insertion of one electrode shank. n , Schematic overview of experiments to record the activity of SC Hoxa7::Nfib::Vsx2 during the application of EES and during episodes of autonomic dysre-flexia. o , Top , the waveforms display spikes and firing rate evoked by optogenetic stimulation of Vsx2 ON neurons by the application of continuous EES over the lower thoracic spinal cord, and by colorectal distention. Heatmap of neuronal clusters activated by EES, activated by EES and colorectal distension, activated by EES and tagged as Vsx2 ON neurons by optogenetic stimulation and EES, and activated by EES and colorectal distension and tagged as Vsx2 ON neurons activated by optogenetic stimulation.

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Expressing, In Situ Hybridization, Immunohistochemical staining, Activity Assay

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of autonomic neurorehabilitation and paradigm to quantify the severity of autonomic dysreflexia. b , Pressor responses ( Left ; individual mice and mean trace) and severity of autonomic dysreflexia ( Right ) in 5 with chronic SCI and 5 mice that underwent autonomic neurorehabilitation for 4 weeks, starting 1 week after SCI (independent samples t-test; t = -7.45; p-value = 0.00056). c , Schematic overview illustrating the competitive (overlapping) neuronal architectures of autonomic dysreflexia and EES-induced pressor responses, and their rearrangement after autonomic neurorehabilitation. d , vGlut1 ON synaptic puncta and synaptic-like appositions from SC Hoxa10::Zfhx3::Vsx2 neurons onto SC Hoxa7::Nfib::Vsx2 neurons in in mice with SCI and mice with SCI that underwent autonomic neurorehabilitation. ( top ) Bar plots reporting the mean density of axonal projections from SC Hoxa10::Zfhx3::Vsx2 neurons in the thoracic spinal cord in mice with SCI and mice with SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 2.51; p-value = 0.0369). ( bottom ) (bottom) Bar plots reporting the mean number of vGlut1 ON synaptic puncta apposing SC Hoxa7::Nfib::Vsx2 neurons (n = 5; independent samples t-test; t = 4.44; p-value = 0.0055). e , Schematic overview of experiments in which EES was applied daily over the lumbosacral spinal cord of mice with SCI, and paradigm to quantify the severity of autonomic dysreflexia. f , As in b , for mice with SCi that were subjected to the daily application of EES over the lumbosacral spinal cord (n = 5; independent samples t-test; t = 5.82; p-value = 0.00070).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques:

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Overview of the experimental protocol to deliver autonomic neurorehabilitation in mice with SCI. Step 1 . Mice received a complete transection of the spinal cord at the level of the T4 segment. Step 2 .Intersectional viral tracing by infusing Retro-AAV-DIO-FlpO into the lower thoracic spinal cord and AAV8-Con/Fon-EYP into the lumbosacral spinal cord to label SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord that project onto SC Hoxa7::Nfib::Vsx2 neurons located in the lower thoracic spinal cord. Step 3 . One week after SCI, electrodes were implanted over the T12 spinal segment to deliver EES. Step 4 . EES was applied for 30 minutes everyday for 4 weeks. Step 5 . F of autonomic dysreflexia was assessed during terminal experiments conducted in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation. Step 6 . Spinal cord tissues were collected and processed. b , Changes in systolic blood pressure ( Left ; bold line represents mean trace ± sem for each group and individual line traces are from each animal) and severity of autonomic dysreflexia ( Right ) measured by the change in systolic blood pressure during colorectal distension in mice with and without autonomic neurorehabilitation (n = 5; independent samples t-test; t = -7.45; p-value = 0.00056). c , Left , Photomicrographs of the lower thoracic spinal cord in mice with chronic SCI and mice with chronic that underwent autonomic neurorehabilitation in which SC Hoxa10::Zfhx3::Vsx2 neurons located in the lumbosacral spinal cord were labelled with an intersection virus strategy concomitantly to the labelling of SC Hoxa7::Nfib::Vsx2 neurons. (Right) Photomicrographs of the lower thoracic spinal cord with intersectional viral labelling combined with immunohistochemical labelling of vGlut1 ON synapses in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation. vGlut1ON synaptic puncta and synaptic-like appositions from SC Hoxa10::Zfhx3::Vsx2 neurons onto SC Hoxa7::Nfib::Vsx2 neurons in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation. d , Left , Bar plots reporting the mean number of vGlut1 ON synaptic puncta apposing SC Hoxa7::Nfib::Vsx2 neurons (n = 5; independent samples t-test; t = 4.44; p-value = 0.0055), right , and the mean density of axonal projections from SC Hoxa10::Zfhx3::Vsx2 neurons in the grey matter of the lower thoracic spinal cord in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 2.51; p-value = 0.0369). e , As in a , for mice subjected to daily application of EES over the lumbosacral spinal cord. f , As in b , for mice subjected to daily application of EES over the lumbosacral spinal cord (n = 5; independent samples t-test; t = 5.82; p-value = 0.00070). g , As in c , for mice subjected to daily application of EES over the lumbosacral spinal cord. h , As in d , for mice subjected to daily application of EES over the lumbosacral spinal cord.

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Virus, Immunohistochemical staining

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Schematic overview of autonomic neurorehabilitation in rats. A wireless telemetry system was implanted chronically to acquire longitudinal recordings of haemodynamic parameters. An electronic dura mater ( e-dura ) designed to target the dorsal roots projecting to the T11, T12, and T13 spinal segments was then implanted over the hemodynamic hotspot to regulate blood pressure. b , Augmentation of systolic blood pressure within a target range using a proportional integral (PI) controller that adjusts the amplitude of EES in closed-loop. c , Line graph reporting the severity of autonomic dysreflexia that was assessed weekly using colorectal distension in rats with SCI and rats with SCI that were undergoing autonomic neurorehabilitation. Raw data and statistics provided in Supplementary Table 1 . d , Whole spinal cord visualization of projections from neurons located in the lumbosacral spinal cord that establish neurons into the lower thoracic spinal cord. e , Axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord and projecting to the lower thoracic spinal cord, shown in rats with SCI and rats with SCI that underwent autonomic neurorehabilitation. f , Density of axonal projections from lumbosacral neurons in the thoracic spinal cord before and after autonomic neurorehabilitation (n = 5; independent samples t-test; t = -4.03; p-value = 0.0081). g , Synaptic-like appositions from neurons located in the lumbosacral spinal cord onto SC Hoxa7::Nfib::Vsx2 neurons combined with the labelling of vGlut1 ON synaptic puncta form large-diameter afferent fibers in a rat with chronic SCI and a rat with SCI that underwent autonomic neurorehabilitation. h , Bar plots reporting the mean density of vGlut1 ON synaptic puncta onto SC Hoxa7::Nfib::Vsx2 neurons in rats with SCI and rats with SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 12.71; p-value = 2.78e-06).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques:

Journal: bioRxiv

Article Title: The neuronal architecture of autonomic dysreflexia

doi: 10.1101/2024.05.06.592781

Figure Lengend Snippet: a , Overview of the experimental protocol to deliver autonomic neurorehabilitation in rats with SCI. Step 1 . Rats received a severe contusion (380 Kdyn) of the spinal cord at the level of T3 segment. Step 2 . AAV-DJ-hSyn-flex-mGFP-2A-Synaptophysin-mRuby and an AAV-Cre were co-infused into the the L6 segment of the spinal cord to label the projections from neurons located in the lumbosacral spinal cord. Step 3 . A wireless telemeter recording system, including a blood pressure cannula inserted into the abdominal aorta and microelectrodes sutured over the sympathetic renal nerve, was implanted chronically to monitor hemodynamics and sympathetic nerve activity, respectively. Step 4 . Seven days after SCI, an an electronic dura mater (e-dura) designed to target the dorsal roots projecting to the T11, T12, and T13 spinal segments was implanted over the hemodynamic hotspot to regulate blood pressure. Step 5 . EES was applied for 30 minutes everyday during 6 weeks using a proportional-integral (PI) controller that adjusted the amplitude of EES in closed-loop s to augment the systolic blood pressure to a target range. Step 6 . The severity of autonomic dysreflexia, induced by colorectal distension, was assessed every week for 6 weeks. Step 7 . After 6 weeks of autonomic neurorehabilitation, a final assessment was performed to test the severity of autonomic dysreflexia in all groups, which included rats with intact spinal cord, rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation. Step 8 . Spinal cords were collected and processed. b , Changes in systolic blood pressure in response to colorectal distension ( Left ; bold line represents mean trace ± sem for each group and individual line traces are from each rat) and bar plots reporting the severity of autonomic dysreflexia ( Right ) measured by the change in systolic blood pressure during colorectal distension over the course of 6 weeks in rats with intact spinal cord, rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation. Raw data and statistics provided in Supplementary Table 1 . c , Whole spinal cord visualization of projections from neurons located in the lumbosacral spinal cord. d , Plots reporting density of axonal projections ( top ) and synaptic punta ( bottom ) from neurons located in the lumbosacral spinal cord into the grey matter of the lower thoracic spinal cord in rats with intact spinal cord, rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation. e , Micrographs of the lower thoracic spinal cord in which the axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord are labelled for the three groups of rats. f , Bar plots reporting the mean density of axonal projections and synaptic puncta from neurons located in lumbosacral spinal cord into the grey matter of the lower thoracic spinal cord for the three groups of rats. Raw data and statistics are provided in Supplementary Table 1 . g , Micrographs of the lower thoracic spinal cord in which axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord are labelled concomitantly to vGlut1 ON synapses from large-diameter afferents and Vsx2 ON neurons. The density of vGlut1 ON synapses onto Vsx2 ON neurons is reconstructed for a rat with chronic SCI and a rat with chronic SCI that underwent autonomic neurorehabilitation. h , Bar plots reporting the density of synaptic-like appositions from neurons located in the lumbosacral spinal cord onto Vsx2 ON neurons in rats with intact spinal cord, rats with chronic SCI, and rats with chronic SCI that underwent autonomic neurorehabilitation. Raw data and statistics are provided in Supplementary Table 1 . i , As in i, for vGlut2 ON synaptic puncta onto SC Hoxa7::Nfib::Vsx2 neurons. Raw data and statistic provided are in Supplementary Table 1 . j , Quantification of vGlut1 ON synaptic puncta from large-diamter afferents Vsx2 ON in rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 12.71; p-value = 2.78e-06).

Article Snippet: The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh).

Techniques: Activity Assay

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles from organoid‐derived human retinal progenitor cells prevent lipid overload‐induced retinal pigment epithelium injury by regulating fatty acid metabolism

doi: 10.1002/jev2.12401

Figure Lengend Snippet: Isolation and characterisation of extracellular vesicles (EVs) derived from human embryonic stem cells (hESCs) and human retinal organoid‐derived retinal progenitor cells (hRPCs). (a) Induction of retinal organoids and schematic representation of the experimental design. (b) Typical morphology of hESCs and hRPCs under a light field. (c) Fluorescent staining of hESC markers (SSEA4, NANOG and OCT4) and hRPC markers (RAX, CHX10 and PAX6). (d, e) Size distribution and percentage of hESC‐EVs and hRPC‐EVs via nanoparticle tracking analysis. (f) Transmission electron micrographs showing the morphology of EVs derived from hESCs and hRPCs. (g) Identification of EV markers in hESCs, hRPCs, hESC‐EVs and hRPC‐EVs via western blotting. Data are presented as the mean ± SD, n = 6 (e). ** p < 0.01; *** p < 0.001; ns, not significant (Benjamini‐Hochberg corrected t ‐tests for e). Scale bar, 200 µm (b), 100 µm (c), 100 nm (f).

Article Snippet: The following primary antibodies and dilutions were used in this study: mouse anti‐SSEA4 (sc‐59368, 1:100; Santa Cruz Biotechnology, USA), mouse anti‐CHX10 (sc‐374151, 1:300; Santa Cruz Biotechnology), rabbit anti‐CHX10 (HPA003436 1:500; Sigma‐Aldrich), mouse anti‐RAX (sc‐271889, 1:500; Santa Cruz Biotechnology), mouse anti‐OCT4 (sc‐5279, 1:100; Santa Cruz Biotechnology), mouse anti‐NANOG (sc‐374103, 1:100; Santa Cruz Biotechnology), rabbit anti‐PAX6 (ab5790, 1:500; Abcam, UK) and mouse anti‐HuC/D (A21272, 1:200; Invitrogen, USA).

Techniques: Isolation, Derivative Assay, Staining, Transmission Assay, Western Blot