Journal: bioRxiv

Article Title: A cholinergic spinal pathway for the adaptive control of breathing

doi: 10.1101/2025.01.20.633641

Figure Lengend Snippet: (A) Transverse section of the cervical spinal cord showing retrograde CTB labeling (green) in PMNs (squared region). Scale bar = 100 µm. (B) Enlargement of CTB-labeled PMNs (green) shown in (A). Square region indicates a PMN shown in (C). Scale bar = 20 µm. (C) Enlargement of a single CTB-labeled PMN (CTB, green) shown in (B) from a Pitx2 tdTom adult mouse with synapses derived from Pitx2 Tdtom + interneurons (red) and cholinergic synapses (VAChT, blue). Numbers indicate labeled synapses on the PMN soma. Scale bar = 5 µm (D) Magnification of numbered synapses from (C) showing colocalization of tdTomato and VAChT. Scale bar = 1 µm. (E-F) Representative images of VAChT+ puncta on PMNs in P4 Pitx2 tdTom mice. Square region in (E) is enlarged in (F). Scale bar = 100 μm (E) and 20 μm (F). (G) Enlargement of the square region in (F). Numbers indicate examples of synapses on the PMN soma. Scale bar = 5 µm. (H) Magnification of numbered synapses from (G). Scale bar = 1 µm. (I) Percentage of VAChT+ puncta that are Pitx2 tdTom + on PMN somas at P4. (J-K) Representative images of VAChT+ puncta on PMNs in adult (P60) Pitx2 tdTom mice. Square region in (J) is enlarged in (K). Scale bar = 100 μm (J) and 20 μm (K). (L) Enlargement of the square region in (K). Numbers indicate examples of synapses on the PMN soma. Scale bar = 5 µm. (M) Magnification of numbered synapses from (L). Scale bar = 1 µm. (N) Percentage of VAChT+ puncta that are Pitx2 tdTom + on PMN somas at P60.

Article Snippet: The following primary antibodies were used in this study: goat anti-ChAT (Sigma, RRID: AB_2079751, 1:300), goat anti-VAChT (Millipore, RRID: AB_2630394, 1:1000), rabbit anti-VAChT (Synaptic Systems, RRID: AB_10893979, 1:500), chicken anti-RFP (Rockland, 600901379, 1:500), rabbit anti-DsRed (Takara Bio, RRID: AB_10013483, 1:1000), rabbit anti-c-Fos (Synaptic Systems, RRID: AB_2905595, 1:1000), goat anti-Scip (Santa Cruz Biotechnology, RRID:AB_2268536, 1:5000), rabbit anti-Pitx2 (1:16000) and rabbit anti-CTB (Novus Biologicals, NB100-63067, 1:500).

Techniques: Labeling, Derivative Assay

Journal: bioRxiv

Article Title: A cholinergic spinal pathway for the adaptive control of breathing

doi: 10.1101/2025.01.20.633641

Figure Lengend Snippet: (A) Schematic of the experimental setup showing extracellular ventral root recordings and intracellular whole-cell patch clamp recordings from individual Pitx2 tdTom + interneurons (red) in hemisected brainstem-spinal cord preparations from Pitx2 tdTom neonatal mice. (B) Pie charts showing the relative proportion of respiratory-related (red) and non-respiratory-related (gray) Pitx2+ interneurons within and below the C3-4 spinal segments. (C) Example trace of voltage-clamp recording from a respiratory-related Pitx2+ interneuron (top) during ongoing respiratory burst (bottom). Red dotted box showing zoomed-in traces during a respiratory burst. (D) as in (C) but regarding current-clamp recording.

Article Snippet: The following primary antibodies were used in this study: goat anti-ChAT (Sigma, RRID: AB_2079751, 1:300), goat anti-VAChT (Millipore, RRID: AB_2630394, 1:1000), rabbit anti-VAChT (Synaptic Systems, RRID: AB_10893979, 1:500), chicken anti-RFP (Rockland, 600901379, 1:500), rabbit anti-DsRed (Takara Bio, RRID: AB_10013483, 1:1000), rabbit anti-c-Fos (Synaptic Systems, RRID: AB_2905595, 1:1000), goat anti-Scip (Santa Cruz Biotechnology, RRID:AB_2268536, 1:5000), rabbit anti-Pitx2 (1:16000) and rabbit anti-CTB (Novus Biologicals, NB100-63067, 1:500).

Techniques: Patch Clamp

Journal: bioRxiv

Article Title: A cholinergic spinal pathway for the adaptive control of breathing

doi: 10.1101/2025.01.20.633641

Figure Lengend Snippet: (A) Experimental design for whole body plethysmography and schematic of a representative breath. A Pitx2 ΔChAT or Dbx1 ΔChAT mouse was paired with a sex-matched control littermate and the mice were exposed to normal air conditions (21% O 2 , 79% N 2 ) for 45 minutes, followed by 15 minutes of hypercapnia (5% CO 2 , 21% O 2 , 74% N 2 or 10% CO 2 , 21% O 2 , 69% N 2 ). (B-C) Examples of breath traces under normal air and 10% CO 2 in Pitx2 ΔChAT mice and their control littermates. A single breath was enlarged in (C). (D, H, L, and P) Breath frequency (D), minute ventilation (H), tidal volume (L), and PIF (P) distribution under normal air in Pitx2 ΔChAT and control mice (n = 14-17 per group). (E, I, M, and Q) Mean and normalized frequency (E), minute ventilation (I), tidal volume (M), and PIF (Q) under normal air in Pitx2 ΔChAT and control mice. (F, J, N, and R) Breath frequency (F), minute ventilation (J), tidal volume (N), and PIF (R) distribution under 10% CO 2 in Pitx2 ΔChAT and control mice (n = 14-17 per group). (G, K, O, and S) Mean and normalized frequency (G), minute ventilation (K), tidal volume (O), and PIF (S) under 10% CO 2 in Pitx2 ΔChAT and control mice.

Article Snippet: The following primary antibodies were used in this study: goat anti-ChAT (Sigma, RRID: AB_2079751, 1:300), goat anti-VAChT (Millipore, RRID: AB_2630394, 1:1000), rabbit anti-VAChT (Synaptic Systems, RRID: AB_10893979, 1:500), chicken anti-RFP (Rockland, 600901379, 1:500), rabbit anti-DsRed (Takara Bio, RRID: AB_10013483, 1:1000), rabbit anti-c-Fos (Synaptic Systems, RRID: AB_2905595, 1:1000), goat anti-Scip (Santa Cruz Biotechnology, RRID:AB_2268536, 1:5000), rabbit anti-Pitx2 (1:16000) and rabbit anti-CTB (Novus Biologicals, NB100-63067, 1:500).

Techniques: Control

Journal:

Article Title: Differential Localization of Vesicular Glutamate Transporters and Peptides in Corneal Afferents to Trigeminal Nucleus Caudalis

doi: 10.1002/cne.22414

Figure Lengend Snippet: Primary antibodies

Article Snippet: There was no immunolabeling in any of the mismatch combinations tested. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Source Immunogen Goat anti-CTb List Biological Laboratories, Inc. Purified cholera toxin beta subunit (manufacturer’s technical specifications) Rabbit anti-CTb Novus Biologicals Purified choleragenoid Rat anti-SP Accurate Chemical and Scientific Corporation Substance P peptide conjugated to bovine serum albumin Rabbit anti-CGRP Immunostar Full length synthetic rat alpha-CGRP Guinea pig anti-VGluT1 Chemicon Synthetic peptide corresponding to amino acid residues 542 – 560 of rat VGluT1 (GATHSTVQPPRPPPPVRDY) Guinea pig anti-VGluT2 Chemicon Synthetic peptide corresponding to amino acid residues 565 – 582 of rat VGluT2 (VQEAQDAYSYKDRDDYS) Open in a separate window Primary antibodies

Techniques: Purification