Journal: Nature Communications

Article Title: A medullary centre for lapping in mice

doi: 10.1038/s41467-021-26275-y

Figure Lengend Snippet: a Rosa FTLG allele used for intersectional transgenic labeling of boutons from vGlut2/Phox2b interneurons (left) and schematic of the results (right). b Coronal sections through the hindbrain of a Phox2b::Flpo ; vGlut2::Cre ; Rosa FTLG mouse at P4, showing synaptic boutons (black) from vGlut2/Phox2b interneurons in relation to motor nuclei (ChAT + , blue) at low (left), and higher (middle) magnifications, and close-ups of boutons (green) on motoneurons (right), which are either Phox2b + (purple) or Phox2b − (blue). c (left) Strategy for mono-synaptically restricted transsynaptic labeling of premotor neurons from the posterior digastric muscle (PD) in a Phox2b::Cre ; Rosa nlsLacZ mouse, with G-deleted rabies virus (RV) encoding mCherry and complemented by a G-encoding helper HSV virus ( HSV-YFP - G ), and summary of the results. (right panel) The only seed neurons are Acc7 motoneurons, double-labeled by the HSV-G and RV-mCherry viruses. d , e Coronal sections through the hindbrain at P8 showing labeled premotor neurons (black on the left panels) in the IRt ( d ) and Peri5 ( e ), which for the most part (72.7% ± 3.5 SEM, n = 4 animals) express Phox2b (right panels). AD anterior digastric, IRt intermediate reticular formation, nTS nucleus of the solitary tract, PD posterior digastric, Peri5 peritrigeminal area, RF reticular formation, RTN retrotrapezoid nucleus. Scale bars, b 1 mm for the left column, c 250 μm, d , e 500 μm.

Article Snippet: Primary antibodies used were: goat anti- Phox2b (RD system AF4940, diluted 1:100), rabbit anti-peripherin (Abcam ab4666, 1:1000), guinea pig anti-Lmx1b (Müller et al., 2002, 1:1000), goat anti-ChAT (Millipore AB144p), 1:100), chicken anti-βGal (Abcam, ab9361, 1:1000), chicken anti- GFP (Aves Labs, GFP-1020,1:1000), goat anti-ChAT (Millipore, AB144p, 1:100), rabbit anti- GFP (Invitrogen, A11122, 1:1000), rabbit anti- Phox2b (Pattyn et al., 1997,1:500), rat anti-RFP (Chromotek, 5F8, 1:1000), and goat anti-CTB (List Labs, #703, 1:500).

Techniques: Transgenic Assay, Labeling, Virus

Journal: Nature Communications

Article Title: A medullary centre for lapping in mice

doi: 10.1038/s41467-021-26275-y

Figure Lengend Snippet: a Two schematic hemisections of the embryonic medulla (left) or pons (right), showing the origin of branchiomotor nuclei (Mo5, MoA, and Mo10), Peri5 Phox2b and IRt Phox2b in progenitor (p) domains of the ventricular layer (VL), their settling sites in the mantle layer (ML), and their transcriptional codes. b – d Coronal sections through the pons at E18.5, showing Peri5 Phox2b ( b ) or Peri5 Atoh1 ( c , d ) labeled with the indicated antibody or probe. Peri5 Atoh1 cells co-express Phox2b and Atoh1 (arrowheads in d ). e Coronal sections through Mo5 in a Phox2b::Flpo;Atoh1::Cre ; Fela mouse at P0, showing the doubly recombined (nlsLacZ + ) cells of Peri5 Atoh1 (red). f Coronal section through Mo5 in a Phox2b::Flpo;Atoh1::Cre ; Fela mouse, where Phox2b + motoneurons are GFP + (cyan) and Phox2b + /Atoh1 + neurons are nlsLacZ + (red), counterstained for Lbx1 (gray at low magnification, green in the close-ups). g Coronal section through the pons at E11.5 showing the migrating Phox2b + Mo5 and dB2 precursors (black and brown arrowheads, respectively) and, at their meeting point, Peri5 Atoh1 cells that have switched on Atoh1 . Asterisk: lateral recess of the IVth ventricle (IV). h Coronal sections through nTS (yellow arrowhead) and IRt Phox2b (blue arrowhead) at E18.5, at low magnification (upper) or at high magnification for the IRt (lower), stained with the indicated antibodies. A history of Olig3 expression is revealed by recombination of the histone-GFP ( hGFP ) reporter in the Olig3::Cre ERT2 background (left). Mosaicism is likely due to incomplete induction of Cre. Virtually all cells of IRt Phox2b (98% ± 0.2 SEM, n = 3 animals) co-expressed Lmx1b with Phox2b . i Coronal sections through nTS (brown arrowhead) and IRt Phox2b (blue arrowhead) at indicated stages at low magnification (upper) and high magnification for the IRt (lower), immunostained for Phox2b and in situ hybridized for Cited1 . j Coronal section at E15.5 showing that nTS and IRt Phox2b are separated by the medullary root of the vagus nerve (nX). Sp5 spinal trigeminal tract. k Coronal section through the nTS and IRt Phox2b of an adult, showing the central boutons of epibranchial ganglia (that express Foxg1 and are labeled by SypGFP in a Foxg1 iresCre ; Phox2b::Flpo;Rosa FTLG background) in the nTS, but not IRt Phox2b (left). Magnified details (right). Scale bars, b , c 125 μm, d 50 μm, e , f 100 μm, g , h , j , k 200 μm, and i , 250 μm.

Article Snippet: Primary antibodies used were: goat anti- Phox2b (RD system AF4940, diluted 1:100), rabbit anti-peripherin (Abcam ab4666, 1:1000), guinea pig anti-Lmx1b (Müller et al., 2002, 1:1000), goat anti-ChAT (Millipore AB144p), 1:100), chicken anti-βGal (Abcam, ab9361, 1:1000), chicken anti- GFP (Aves Labs, GFP-1020,1:1000), goat anti-ChAT (Millipore, AB144p, 1:100), rabbit anti- GFP (Invitrogen, A11122, 1:1000), rabbit anti- Phox2b (Pattyn et al., 1997,1:500), rat anti-RFP (Chromotek, 5F8, 1:1000), and goat anti-CTB (List Labs, #703, 1:500).

Techniques: Labeling, Staining, Expressing, In Situ

Journal: Nature Communications

Article Title: A medullary centre for lapping in mice

doi: 10.1038/s41467-021-26275-y

Figure Lengend Snippet: a Strategy for the transgenic labeling of projections from Peri5 Atoh1 (and RTN) and summary of the results; b – f Coronal ( b – d , f ) or parasagittal ( e ) sections through a P8 hindbrain showing GFP -labeled boutons (black) on motoneurons (blue) at medium (left) and high (right) magnification. g Strategy for the viral tracing of projections from IRt Phox2b and summary of the results (left), and mGFP-labeled infected cells of IRt Phox2b (right); h – l Coronal ( h – j , l ) or parasagittal ( k ) sections through a P56 hindbrain showing the GFP -labeled fibers (black) of IRt Phox2b neurons at low (left) and medium (middle) magnifications, and in extreme close-ups (right), together with Syp-mRuby labeled boutons (yellow) on motoneurons (blue). m schematic for retrograde tracing of premotor neurons for the right posterior digastric muscle, in a Phox2b::Cre ; Rosa SypGFP , and summary of the results. n – q (left) Coronal sections through the hindbrain at P8 showing the mCherry + projections (black) of premotor neurons on the motor nuclei (ChAT + , blue); (right) close-ups on motoneurons receiving double-labeled Syp-GFP / mCherry boutons (yellow). Scale bars, b – f 200 μm for the left column, h – l 500 μm for the left column, n – q 200 μm for the left column.

Article Snippet: Primary antibodies used were: goat anti- Phox2b (RD system AF4940, diluted 1:100), rabbit anti-peripherin (Abcam ab4666, 1:1000), guinea pig anti-Lmx1b (Müller et al., 2002, 1:1000), goat anti-ChAT (Millipore AB144p), 1:100), chicken anti-βGal (Abcam, ab9361, 1:1000), chicken anti- GFP (Aves Labs, GFP-1020,1:1000), goat anti-ChAT (Millipore, AB144p, 1:100), rabbit anti- GFP (Invitrogen, A11122, 1:1000), rabbit anti- Phox2b (Pattyn et al., 1997,1:500), rat anti-RFP (Chromotek, 5F8, 1:1000), and goat anti-CTB (List Labs, #703, 1:500).

Techniques: Transgenic Assay, Labeling, Infection, Retrograde Tracing

Journal: Nature Communications

Article Title: A medullary centre for lapping in mice

doi: 10.1038/s41467-021-26275-y

Figure Lengend Snippet: a (Upper left) Schematic of the viral injection and fiber-optic implantation for stimulation of IRt Phox2b , and transverse section through the hindbrain showing transduced IRt Phox2b neurons and position of optical fiber (OF, asterisk); scale bar 100 μm. (Upper right) Example frames of the mouse face before and during stimulation including DeepLabCut tracked position of the jaw (blue) and tongue (red). (Lower) Individual traces of tracked jaw and tongue position on the Y-axis upon 50 ms stimulation (five trials). b (Upper left) Schematic of the viral injection and fiber-optic implantation for stimulation of Peri5 Atoh1 and transverse section through the hindbrain showing transduced Peri5 Atoh1 neurons and position of optical fiber (asterisk); scale bar 200 μm. (Upper right) Example frames of the mouse face before and during stimulation including DeepLabCut tracked position of jaw (blue). (Lower) Individual traces (five trials) of tracked jaw position on the Y-axis upon 50 ms stimulation. c Individual traces (five trials) of the tracked jaw (left) and tongue (right) position on the Y-axis upon stimulation of IRt Phox2b of increasing length. A repetitive movement is triggered by stimulation beyond 300 ms (arrowhead). d Individual traces (five trials) of tracked jaw position on the Y-axis upon a 1000 ms stimulation of Peri5 Atoh1 . The jaw remains open and quivers non-rhythmically during the stimulus. e (Left) Schematic of viral injection and optical fiber implantation for observation of IRt Phox2b activity, and transverse section through the hindbrain showing transduced IRt Phox2b neurons and position of optical fiber (asterisk); scale bar 100 μm. (Right) Example frames of the mouse face before and during a bout of licking from a lick port (arrowhead), during a photometry recording, including DeepLabCut tracked position of the jaw (blue) and tongue (red). f Example trace of change in bulk fluorescence of IRt Phox2b during a recording session (∼2 min) of unitary licking events and licking bouts (red arrowheads), contact events with the lick port, and movements of the tongue and the jaw on the Y-axis. g (left) Superimposed correlation curves between licking activity and calcium activity (each curve corresponding to one of 15 recording sessions, each 1–5 min, in one mouse) which peaked at 1.2 s after lick port contact; (right) no peak was observed after shuffling the data.

Article Snippet: Primary antibodies used were: goat anti- Phox2b (RD system AF4940, diluted 1:100), rabbit anti-peripherin (Abcam ab4666, 1:1000), guinea pig anti-Lmx1b (Müller et al., 2002, 1:1000), goat anti-ChAT (Millipore AB144p), 1:100), chicken anti-βGal (Abcam, ab9361, 1:1000), chicken anti- GFP (Aves Labs, GFP-1020,1:1000), goat anti-ChAT (Millipore, AB144p, 1:100), rabbit anti- GFP (Invitrogen, A11122, 1:1000), rabbit anti- Phox2b (Pattyn et al., 1997,1:500), rat anti-RFP (Chromotek, 5F8, 1:1000), and goat anti-CTB (List Labs, #703, 1:500).

Techniques: Injection, Activity Assay, Fluorescence

Journal: Nature Communications

Article Title: A medullary centre for lapping in mice

doi: 10.1038/s41467-021-26275-y

Figure Lengend Snippet: a Strategy for the retrograde transsynaptic labeling of input neurons to IRt Phox2b with exemplar sites of input. b Bar graph of the relative percentage of monosynaptic input neurons labeled from IRt Phox2b starter neurons, displayed per brain region as defined in the Allen Brain Atlas ( n = 4063 input cells and n = 598 starter cells, from n = 3 animals; individual values as circles; seeding efficiency = 7.4 inputs/starters ± 1.8 SEM). Rabies labeled input neurons were largely (74.5 ± 1.1% SEM) restricted to the medulla (pink, MY) and exhibited a slight but consistent ipsilateral bias (55.6 ± 3.0% SEM). Major sources of these medullary inputs were the intermediate, gigantocellular, and parvocellular reticular nuclei. Inputs from the cortex, midbrain, and pons represented a minority of rabies-labeled neurons (1.6 ± 0.3% SEM, 6.4 ± 0.5% SEM, and 12.3 ± 1.1% SEM respectively). c – g Images at low magnification (left) and high magnification (right) of monosynaptic input neurons in the IRt, Peri5, mesencephalic nucleus of the trigeminal nerve, contralateral superior colliculus, and motor cortex. Green arrowheads: seed neurons; red arrowheads: ( n -1) IRt neurons. CB cerebellum, CNU caudoputamen, CTX cortex, DCN deep cerebellar nucleus, MB midbrain, Mes5 mesencephalic nucleus of the trigeminal nerve, MY myelencephalon, P pons, SC superior colliculus. Scale bar c – g 1 mm.

Article Snippet: Primary antibodies used were: goat anti- Phox2b (RD system AF4940, diluted 1:100), rabbit anti-peripherin (Abcam ab4666, 1:1000), guinea pig anti-Lmx1b (Müller et al., 2002, 1:1000), goat anti-ChAT (Millipore AB144p), 1:100), chicken anti-βGal (Abcam, ab9361, 1:1000), chicken anti- GFP (Aves Labs, GFP-1020,1:1000), goat anti-ChAT (Millipore, AB144p, 1:100), rabbit anti- GFP (Invitrogen, A11122, 1:1000), rabbit anti- Phox2b (Pattyn et al., 1997,1:500), rat anti-RFP (Chromotek, 5F8, 1:1000), and goat anti-CTB (List Labs, #703, 1:500).

Techniques: Labeling