Journal: eLife

Article Title: ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the lamprey central canal

doi: 10.7554/eLife.73114

Figure Lengend Snippet: ( A–C ) Stimulated emission depletion (STED) images of dopamine-containing dense-core vesicles (DCVs) in the soma of CSF-c neurons in normal (pH 7.4), acidic (pH 6.5), and alkaline (pH 8.5) extracellular solution. Scale bar, 1 µm. ( D ) Quantification of the number of dopamine DCVs number density in cell area (µm −2 ) in the different conditions ( n = 10). Student’s t -test: non-significant (n.s.) between pH 7.4 and 6.5 (p = 0.27, t 9 = 1.12), and 7.4 and 8.5 (p = 0.29, t 9 = 1.08). ( E ) Whole-cell patch recording of a CSF-c neuron, showing firing spontaneous action potentials in control (pH 7.4), acidic (p H 6.5), and alkaline (pH 8.5) conditions in the presence of gabazine (20 mM) and kynurenic acid (2 mM). ( F–H ) Photomicrographs of the CSF-c neurons recorded in ( E ) intracellularly filled with Neurobiotin (arrow) during recording. The labelled cell showed immunoreactivity to tyrosine hydroxylase (TH, arrow). Scale bar, 10 µm. ( I ) Action potential frequency during 1 min in CSF-c neurons at pH 7.4, 6.5, and 6.8, respectively ( n = 15). Student’s paired t -test: non-significant difference (n.s.) between pH 7.4 and 6.5 (p = 0.24, t 14 = −1.22), and 7.4 and 8.5 (p = 0.1, t 14 = −1.75). The bar graph data are represented as the means, with error bars representing standard deviation (SD). cc, central canal. Figure 4—source data 1. Effect of acidic or alkaline pH on dopamine dense-core vesicles (DCVs) number density in cell area of dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons. Quantification of dopamine DCVs number density in cell area (µm −2 ) in dopaminergic CSF-c neurons in the different pH. Figure 4—source data 2. Effect of acidic and alkaline pH on action potential frequency in dopaminergic cerebrospinal fluid-contacting (CSF-c) neurons. Quantification of action potential frequency in dopaminergic CSF-c neurons at different pH conditions during 1 min.

Article Snippet: Neurons were intracellularly labelled by injection of 0.5% Neurobiotin (Vector Laboratories) during whole-cell recordings.

Techniques: Standard Deviation

Journal: eLife

Article Title: ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the lamprey central canal

doi: 10.7554/eLife.73114

Figure Lengend Snippet:

Article Snippet: Neurons were intracellularly labelled by injection of 0.5% Neurobiotin (Vector Laboratories) during whole-cell recordings.

Techniques: Isolation, Recombinant, Plasmid Preparation, Injection, Software, Labeling, In Situ Hybridization

Journal: Journal of Neurophysiology

Article Title: Muscle proprioceptors in adult rat: mechanosensory signaling and synapse distribution in spinal cord

doi: 10.1152/jn.00497.2017

Figure Lengend Snippet: Mechanosensory signaling by muscle proprioceptors. A: key features of in vivo recording paradigm: attachment of triceps surae muscles through Achilles tendon to motor operating in length servo mode to produce controlled muscle stretch; electrode on triceps surae nerve for electrical stimulation of orthodromic action potentials and isometric muscle twitches; glass micropipette penetrating single afferent axon in dorsal root (rostral to dorsal root ganglion) for recording action potentials and labeling axon with Neurobiotin. B and C: action potentials (gray traces) with superimposed instantaneous firing rates (black dots) responding to different waveforms of 3-mm muscle length changes (black traces). B: ramp-hold-release stretch with relatively fast ramp velocity (bottom trace; 20 mm/s). C: 3 successive triangular stretches at slow ramp velocity (bottom trace; 4 mm/s, 3 mm). 1 and 3, 1st and 3rd slow triangular stretches, respectively. Measurements identified (B) instantaneous firing rates (fr) for initial burst IB(fr) and for peak dynamic Dyn(pfr) and mid-static firing rate Stat(mfr); static firing duration Stat(fd); length threshold (ThrL).

Article Snippet: Tissue sections were first incubated in blocking buffer (5% normal donkey serum diluted in 0.01 M PBS containing 0.1% Triton X-100) for 30–60 min. Neurobiotin was revealed by incubating the sections for 2–3 h at room temperature in blocking buffer containing a 1:100 dilution of streptavidin conjugated to Alexa Fluor 488 (Life Technologies).

Techniques: In Vivo, Muscles, Labeling

Journal: Journal of Neurophysiology

Article Title: Muscle proprioceptors in adult rat: mechanosensory signaling and synapse distribution in spinal cord

doi: 10.1152/jn.00497.2017

Figure Lengend Snippet: Group Ib axon collaterals and varicosities (afferent 7 in Fig. 5, Tables 2 and ​and3)3) A: group Ib classification: action potentials (gray traces) recorded during mechanical stimulation of triceps surae muscles (black traces). A1: firing accelerated during the rising phase of isometric twitch contraction. A2: absence of high-frequency initial bursting at the onset of fast muscle stretch. A3: failure to fire during high-frequency muscle vibration (≥100 Hz). B: axon collateral distribution: Neurobiotin-filled afferent collaterals in sequential tissue sections projected in 2 dimensions and superimposed in relation to the gray matter borders and the central canal (dashed yellow lines, L4/L5) to create the appearance of “looking through” several tissue sections. Collateral branches roughly spanned medial lamina V/VI and VII. C: each green dot represents a Neurobiotin-labeled varicosity (1,087/1,167 varicosities stained positive for VGLUT1 ir). Greatest density in medial LVI. D: postsynaptic contacts: examples of double-labeled varicosities aligned with soma and dendrites of postsynaptic neurons (gray, NeuN).

Article Snippet: Tissue sections were first incubated in blocking buffer (5% normal donkey serum diluted in 0.01 M PBS containing 0.1% Triton X-100) for 30–60 min. Neurobiotin was revealed by incubating the sections for 2–3 h at room temperature in blocking buffer containing a 1:100 dilution of streptavidin conjugated to Alexa Fluor 488 (Life Technologies).

Techniques: Muscles, Labeling, Staining

Journal: Journal of Neurophysiology

Article Title: Muscle proprioceptors in adult rat: mechanosensory signaling and synapse distribution in spinal cord

doi: 10.1152/jn.00497.2017

Figure Lengend Snippet: Group Ia axon collaterals and varicosities (afferent 1 in Fig. 5, Tables 2 and ​and3;3; same format as Fig. 2). A: group Ia classification. A1: firing interrupted during the rising phase of isometric twitch contraction. A2: high-frequency initial bursting at the onset of fast muscle stretch. A3: perfect firing entrainment to high-frequency muscle vibration. B: collateral branches: dense in LV/VI, sparse in LVII, and expansive in LIX. C: each green dot represents a Neurobiotin-labeled varicosity (all varicosities, n = 2,926, stained positive for VGLUT1 ir). D: postsynaptic contacts: examples of double-labeled varicosities aligned with soma and dendrites of postsynaptic neurons in 3 laminar territories.

Article Snippet: Tissue sections were first incubated in blocking buffer (5% normal donkey serum diluted in 0.01 M PBS containing 0.1% Triton X-100) for 30–60 min. Neurobiotin was revealed by incubating the sections for 2–3 h at room temperature in blocking buffer containing a 1:100 dilution of streptavidin conjugated to Alexa Fluor 488 (Life Technologies).

Techniques: Labeling, Staining

Journal: Journal of Neurophysiology

Article Title: Muscle proprioceptors in adult rat: mechanosensory signaling and synapse distribution in spinal cord

doi: 10.1152/jn.00497.2017

Figure Lengend Snippet: Group II axon collaterals and varicosities (afferent 4 in Fig. 5, Tables 2 and ​and3;3; same format as Fig. 2). A: group II classification. A1: firing interrupted during the rising phase of isometric twitch contraction. A2: absence of high-frequency initial bursting at the onset of fast muscle stretch. A3: incomplete entrainment of firing to high-frequency muscle vibration. B: collateral branches: relatively dense in medial LV/VI and dorsolateral LIX, sparse in LVII. C: each green dot represents a Neurobiotin-labeled varicosity (all varicosities, n = 1,836, stained positive for VGLUT1 ir). D: postsynaptic contacts: examples of double-labeled varicosities aligned with soma and dendrites of postsynaptic neurons in 3 laminar territories.

Article Snippet: Tissue sections were first incubated in blocking buffer (5% normal donkey serum diluted in 0.01 M PBS containing 0.1% Triton X-100) for 30–60 min. Neurobiotin was revealed by incubating the sections for 2–3 h at room temperature in blocking buffer containing a 1:100 dilution of streptavidin conjugated to Alexa Fluor 488 (Life Technologies).

Techniques: Labeling, Staining

Journal: Journal of Neurophysiology

Article Title: Muscle proprioceptors in adult rat: mechanosensory signaling and synapse distribution in spinal cord

doi: 10.1152/jn.00497.2017

Figure Lengend Snippet: Distribution of afferent varicosities. Columns illustrate different views of the spatial distributions for varicosities from group Ia, II, and Ib afferents segregated in rows. Left: dots representing Neurobiotin-filled varicosities superimposed and color coded for afferent number. Two-dimensional images were constructed from sequential tissue sections oriented with respect to the spinal gray matter border and central canal in L4/5, with no attempt to demarcate lamina or shifting boundaries of gray matter from L6 to L3. Center: territory contour and density (grayscale intensity coded) of varicosities for each afferent. Right: contour maps of varicosities combined across all 3 afferents for each afferent class. Contour maps were created by calculating the density of varicosities and outlining areas above a threshold.

Article Snippet: Tissue sections were first incubated in blocking buffer (5% normal donkey serum diluted in 0.01 M PBS containing 0.1% Triton X-100) for 30–60 min. Neurobiotin was revealed by incubating the sections for 2–3 h at room temperature in blocking buffer containing a 1:100 dilution of streptavidin conjugated to Alexa Fluor 488 (Life Technologies).

Techniques: Construct

Journal: Journal of Neurophysiology

Article Title: Muscle proprioceptors in adult rat: mechanosensory signaling and synapse distribution in spinal cord

doi: 10.1152/jn.00497.2017

Figure Lengend Snippet: Firing response properties of Neurobiotin-labeled afferents

Article Snippet: Tissue sections were first incubated in blocking buffer (5% normal donkey serum diluted in 0.01 M PBS containing 0.1% Triton X-100) for 30–60 min. Neurobiotin was revealed by incubating the sections for 2–3 h at room temperature in blocking buffer containing a 1:100 dilution of streptavidin conjugated to Alexa Fluor 488 (Life Technologies).

Techniques:

Journal: bioRxiv

Article Title: Functional development of eye movements and visuomotor circuits in lampreys

doi: 10.1101/2023.09.06.556551

Figure Lengend Snippet: The colored rectangles group results belonging to the developmental stage indicated under the schematic of the experimental preparation (blue: stage 5 metamorphic; green: stage 7 metamorphic/postmetamorphic). (A) Schematic drawing (left) indicating the location of the photomicrograph (right) showing retrogradely labeled neurons in the pretectum (PT) of a stage 5 metamorphic lamprey after a Neurobiotin injection in the middle rhombencephalic reticulospinal nucleus (MRRN). Projection neurons can be observed both in the periventricular region (dashed line oval), and in lateral aspects (arrows). (B) Extracellular responses in the MRRN after stimulation of the PT (red trace) and optic tectum (OT, green trace). The onset of the extracellular activity is indicated by a dashed red line for PT stimulation, and a dashed green line for OT stimulation. (C) Photomicrograph showing that no retrogradely labeled neurons can be seen in the OT (indicated by a dashed line) of a stage 5 metamorphic lamprey after a Neurobiotin injection in the MRRN. (D) Graph showing that the onsets of MRRN responses evoked by PT stimulation (red) were significantly shorter than those evoked by OT stimulation (green; unpaired t-test). (E-F) Graphs showing the mean responses evoked in the MRRN of a stage 5 metamorphic animal evoked by stimulation of the PT (E) and OT (F) in response to 4 pulses (10 Hz). Values are normalized to the first local field potential (LFP). (G) Schematic drawing (left) indicating the location of the photomicrograph (right) showing a few retrogradely labeled neurons from the MRRN (arrows) in the OT of a late metamorphic animal (stage 7/recent postmetamorphic). (H) Extracellular responses in the MRRN of a late metamorphic animal (stage 7/recent postmetamorphic) in response to OT stimulation (4 pulses, 10 Hz). A two-components response can be observed: a fast onset weak response (indicated by a dashed line rectangle) followed by a stronger signal. In the electrophysiological traces, stimulation artifacts were removed for clarity. In all graphs, data are shown as mean ± s.d. Abbreviations: nMLF Nucleus of the Medial Longitudinal Fasciculus, pc Posterior commissure, SNc Substantia Nigra pars compacta, TS Torus Semicircularis, nIII Oculomotor Nucleus, M5 Retinopetal Nucleus of Schöber. Scale bar = 100 µm in A, G and M; 200 µm in C and I.

Article Snippet: To detect the Neurobiotin tracer, sections were incubated in Cy2-conjugated streptavidin (Jackson ImmunoResearch) 1:1000 in blocking solution (1 % bovine serum albumin, 10 % sheep serum, 0.1 % sodium azide and 0.3 % Triton X-100 in PBS).

Techniques: Labeling, Injection, Activity Assay