rabbit anti mafa pab  (Novus Biologicals)

Journal: Nature Communications

Article Title: Mafa-dependent GABAergic activity promotes mouse neonatal apneas

doi: 10.1038/s41467-022-30825-3

Figure Lengend Snippet: a Top, genetic scheme to introduce the Mafa4A mutation in neurons. Bottom, typical plethysmographic recordings (inspiration: upward) of control (black traces) and Mafa n4A/n4A (red traces) littermates at P0 + 12 h showing repetitive and apneic breathing cycles (the superposed traces are adjacent segments of a continuous record). Apneas (gray triangle in controls) are more frequent in the mutant (red diamonds). b Poincaré plots of the duration of consecutive respiratory cycles n + 1 (ordinates) as a function of n (abscissae) in a control (left) and a Mafa 4A/4A (right, 5 min recording samples). Repetitive short cycles (filled symbols) are clustered on the diagonal below the CORC (vertical and horizontal dotted lines), apneic cycles (empty symbols) give points away from the diagonal beyond the CORC. Note that the Mafa n4A/n4A mutant shows an increased incidence of apneas. c Evolution of the apneic time fraction (left ordinates axis, bar plot) and mortality (right inverted ordinates axis, vertical arrows) in control (gray, n = 29) and Mafa n4A/n4A mutants (red, n = 21) pups. Each symbol represents measurement of a single pup (One-way ANOVA between the two genotypes P < 0.0001, degree of freedom (df) = 1, F = 34.38.). d Percent relative change in the number of apneas (ordinates) compared to their controls for Mafa n4A/n4A (Statistical test between mutant and control littermates: n4A vs Cre +/+ littermates n = 17 for both genotypes, two-sided unpaired t -test P = 0.0004, df = 32, t = 3.965) and Mafa LacZ/LacZ ( null n = 13 vs wild type n = 10 littermates, two-sided Mann–Whitney test P = 0.0041, U = 20). e Example traces of the two types of apneas in Mafa n4A/n4A mutant pups. At left, breath holding apneas characterized by post-inspiratory onset (black arrowheads) of limited expiratory flow prolonging lung inflation (plateau above dotted baseline) followed by the resuming of inspiratory efforts of progressively increasing amplitudes (empty arrowheads). At right, central-like apneas characterized by normal inspiratory and expiratory phases separated by an abnormally long pause during which the lung is deflated. f Joint plethysmographic (top trace) and click audio recordings (bottom trace) during an epoch of breath holding apneas. Clicks that follow a post-inspiratory (arrowheads) breath holding apnea are associated to small upward pressure shifts (downward arrows) that immediately precede onset of an expiration (e below the click trace, 4 of 5 clicks) deflating the lung or occasionally an inspiration (i below the click trace, 1 of 5 clicks,) further inflating the lung. g Quantification of the apneic time fraction for apnea types in control pups ( Mafa flox4A/4A , n = 23) at P0 + 12 h (two-sided Mann–Whitney test P = 0.4100, U = 226.5). h Quantification of the breath holding apneic time fraction in Mafa n4A/n4A (nCre/+, n = 10 pups, red) , Mafa null ( n = 13 pups, blue) and that of respective control ( n +/+, n = 10 pups, gray, two-sided unpaired t -test P = 0.0145, df = 18, t = 2.704) and wild type (light gray, n = 9 pups, two-sided Mann–Whitney test P = 0.0052, U = 14) littermates. Plots represent mean ± sem.

Article Snippet: Endogenous expression of Mafa protein was detected, after demasking in citrate bufffer 10 mM pH6, using rabbit anti-Mafa polyclonal antibody (Bethyl IHC-00352; dilution 1/200 in PBS 0,05% Tween 20) followed either with a secondary goat anti-rabbit Cy3 antibody (Invitrogen, A10520).

Techniques: Introduce, Mutagenesis, Control, MANN-WHITNEY

Journal: Nature Communications

Article Title: Mafa-dependent GABAergic activity promotes mouse neonatal apneas

doi: 10.1038/s41467-022-30825-3

Figure Lengend Snippet: a Whole mount (ventral view) of a M afa LacZ/+ brain at E15 showing Mafa expression (Xgal staining, dark blue) restricted to the olfactory bulb (OB), to the pre-optic area of the hypothalamus (POA) to the brainstem (pons and medulla) and to the spinal cord. b Hemi-transverse section (medial left, dorsal top) of a M afa LacZ/+ pup at P0 showing that the preBötC area, below ChAT + neurons (white) of the nucleus ambiguous (nAmb), is devoid of gal+ cells (red). c Same as b showing the absence of bgal+ cells (red) in the RTN, below ChAT+ neurons (white) of the facial motor nucleus (Mo7). d Transverse section of a M afa LacZ/+ pup at P0 (at the d axial position indicated in a) showing Mafa + cells immunostained for bgal (black) in the caudal medulla in the spinal part of the trigeminal nucleus (sp5), in the peri12 reticular formation laterally to the hypoglossal motor nucleus (Mo12), in motoneurons of the accessory hypoglossal motor nucleus (acc-Mo12). e Close-up view of Mafa + peri12 neurons (red) counterstained for ChAT + (black) motoneurons at the same axial level as d . Transverse section (at the f axial position indicated in a ) showing Mafa + cells in the Sp5 and medial to the nucleus ambiguus in the peri-nAmb area. g Close up view of a hemi-transverse section (medial left, dorsal top) showing Mafa + cells (red labeling) combined to Gad1 in situ hybridization (black) at the same axial level as ( f ) showing Gad1 + /Mafa + cells in the peri12 and peri-nAmb. Close-up view from the ( h ) inset in ( g ). Close-up view from the i inset in ( g ). j Quantification of neurochemical types of Mafa + neurons of the caudal medulla reticular formation by in situ hybridization for VGlut2 (13 section counts), Glyt2 (11 section counts), Gad1 (10 section counts) and Gad2 (12 section counts). Note that Mafa + neurons of the caudal reticular formation (peri12 and peri-nAmb regions) are predominantly inhibitory. Plots represent mean ± sem. Scale bars (μm): 100 ( b – g ), 20 ( h , i ).

Article Snippet: Endogenous expression of Mafa protein was detected, after demasking in citrate bufffer 10 mM pH6, using rabbit anti-Mafa polyclonal antibody (Bethyl IHC-00352; dilution 1/200 in PBS 0,05% Tween 20) followed either with a secondary goat anti-rabbit Cy3 antibody (Invitrogen, A10520).

Techniques: Expressing, Staining, Labeling, In Situ Hybridization

Journal: Nature Communications

Article Title: Mafa-dependent GABAergic activity promotes mouse neonatal apneas

doi: 10.1038/s41467-022-30825-3

Figure Lengend Snippet: a The GABA A -receptor antagonist PTZ increases regular breathing rates in control (gray symbols, n = 10, two-sided paired Student’s t test, P < 0.0001, df = 9, t = 6.851) and Mafa n4A/n4A (red symbols, n = 10, two-sided paired Student’s t test P = 0.040, df = 9, t = 2.393) at P0 + 12 h (Student’s t test). b PTZ has no effect on the apneic time fraction of control pups ( n = 10, gray symbols, two-sided Wilcoxon paired test, P = 0.921) but PTZ and not NaCl reduces the apneic time fraction of Mafa n4A/n4A mutant pups ( n = 10, red symbols, two-way ANOVA F(1,18) = 8.176, P = 0.0104 followed by Bonferroni’s multiple comparison test, P = 0.0202 for PTZ; P = 0.5147 for NaCl). c PTZ reduces breath holding apneas in control ( n = 10 pups, two-sided Wilcoxon test P = 0.0039) and Mafa n4A/n4A mutant pups ( n = 10, two-sided paired Student’s t test P = 0,0098, df = 9, t = 3.264) but not central-like apneas of control (two-sided Wilcoxon test P = 0.0645) nor of Mafa n4A/n4A mutant pups (paired Student’s t -test P = 0.2791, df = 9, t = 1.152). d Genetic recombination to target the 4A mutation in inhibitory neurons. e Plethysmographic recordings of control (upper trace) and of VGAT Mafa4A mutant pups (lower trace) at P0 + 12 h showing apneic breathing and upwards pressure shifts (arrows) terminating breath holding apneas. f Poincaré plots of a control (left, black symbols) and of a Mafa VGAT4A mutant presenting with increased number of apneas (red symbols beyond the CORC). g The apneic time fraction of Mafa VGAT4A pups ( n = 10) is about double that of control littermates ( n = 13, two-sided unpaired t -test P = 0.0263, df = 22, t = 2.382). h The apneic time fraction corresponding to breath holding apnea is three-fold higher in Mafa VGAT4A ( VGAT Cre/+ , n = 10, red symbols) than controls ( VGAT +/+ , n = 13, gray symbols, two-sided Mann–Whitney test P = 0.002, U = 10) while that corresponding to central-like apneas is not changed by the mutation (unpaired t -test P = 0.5774, df = 20, t = 0.5663). Plots represent mean ± sem.

Article Snippet: Endogenous expression of Mafa protein was detected, after demasking in citrate bufffer 10 mM pH6, using rabbit anti-Mafa polyclonal antibody (Bethyl IHC-00352; dilution 1/200 in PBS 0,05% Tween 20) followed either with a secondary goat anti-rabbit Cy3 antibody (Invitrogen, A10520).

Techniques: Control, Mutagenesis, Comparison, MANN-WHITNEY

Journal: Nature Communications

Article Title: Mafa-dependent GABAergic activity promotes mouse neonatal apneas

doi: 10.1038/s41467-022-30825-3

Figure Lengend Snippet: a Left, transverse sections of P0 wild type pups showing the vagal jugular and nodose ganglia stained for DAPI (white) and by RNAscope using a probe against Mafa (red). Right, top, close-up views from the jugular ganglion probing Mafa (red), VGlut2 (green), Phox2b (blue) and the merge. Right, bottom, close-up views from the nodose ganglion. b Transverse sections of the trigeminal ganglion stained for DAPI (white) and Mafa (red). Right, close up views of the inset in ( b ) probing Mafa (red) , VGlut2 (green) and Gad1 & Gad2 (blue). Note the absence of Mafa expression in the nodose ganglion, expression of Mafa in the jugular (sparse) and trigeminal ganglia restricted to VGlut2 -expressing excitatory neurons. c Summary of RNAscope probing for co-expression of Mafa/VGlut2 - and Mafa/Gad1&2 -expression in vagal (5 sections, n = 3 ganglia) and trigeminal (7 sections, n = 3 ganglia) ganglia, nucleus tractus solitarius (nTS, 8 sections, n = 2 pups) and peri12 (8 sections, n = 2 pups). Plots represent mean ± sem. d Left, transverse hemi-section of the brainstem at pontine level of a VGAT Cre/+ ; Mafa FloxLacZ/+ P8 pup, showing absent Mafa + inhibitory neurons (black) in the PB/KF, Pr5 areas but their presence in the SPO and DLL. Right, representative transverse hemi-section of the brainstem at medullary level showing the presence of Mafa + inhibitory neurons in the peri12, peri-nAmb but their virtual absence in the Sp5, Pa5 and nTS ( n = 2 pups). e Monosynaptic tracing scheme showing unilateral injection of G-deleted Rabies viruses encoding mCherry and of a helper G- and YFP-encoding HSV virus into the geniohyoid muscle to transynaptically trace the position of premotor neurons. f Representative transverse section (from n = 4 VGAT cre/+ ; Mafa floxLacZ/+ pups) immunostained for βgal (white) in the caudal medulla showing mCherry + virally labeled premotor neurons (red) and rare (at this axial level) seeding mCherry + /YFP + geniohyoid motoneurons (yellow) in the accessory hypoglossal motor nucleus (arrowhead). g Close-up view of the inset in f showing that a fraction of mCherry + premotor neurons indicated by arrows are Mafa + (mCherry + /βgal + , white nuclear labeling). h 2D-reconstruction of the position of Mafa + geniohyoid premotor neurons (red dots) and Mafa + cells (gray dots) showing their location in the peri12 area. DLL dorsal nucleus of the lateral lemniscus, KF Kölliker-Fuse nucleus, PB parabrachial nuclei, Pa5 paratrigeminal nucleus, Pr5 principal sensory trigeminal nucleus, scp superior cerebellar peduncle, SPO superior paraolivary nucleus. Scale bars (µm): 100 ( a , b left, c ), 50 ( a , b right), 1000 ( d ), 500 ( f , h ), 100 (inset).

Article Snippet: Endogenous expression of Mafa protein was detected, after demasking in citrate bufffer 10 mM pH6, using rabbit anti-Mafa polyclonal antibody (Bethyl IHC-00352; dilution 1/200 in PBS 0,05% Tween 20) followed either with a secondary goat anti-rabbit Cy3 antibody (Invitrogen, A10520).

Techniques: Staining, RNAscope, Expressing, Injection, Virus, Labeling

Journal: Nature Communications

Article Title: Mafa-dependent GABAergic activity promotes mouse neonatal apneas

doi: 10.1038/s41467-022-30825-3

Figure Lengend Snippet: a Gad2 in situ hybridization (black) combined with anti-bgal staining (red) on P0 transverse sections of the caudal medulla of section of Mafa LacZ/+ (left) and Mafa LacZ/LacZ (right) pups. Note the reduced Gad2 signal surrounding βgal + nuclei in the absence of Mafa. Data from three mice for each genotype. In DAOY medulloblastoma cell line ( b ) or primary culture of granule cell progenitors ( c ) that do not express MAFA (Ctl, left plots in b and c ), transfection with either MAFA WT or MAFA 4A yields MAFA expression (left plots) that induces GAD2 transcription (middle plots). The right panel shows the fold induction of GAD2 , compared to MAFA expression ( n = 2 biological replicates each with n = 3 technical replicates). d The Gad2 promoter (878nt upstream of Gad2 transcription start, in between oligos used to clone Gad2 promoter in pGL3) contains MAF Responsive Elements (MARE, red boxes). e Luciferase assays in DAOY cells reporting Gad2 promoter (left) and control luciferase activity (right) when co-transfected with increasing doses of plasmids encoding MAFA WT or MAFA 4A. All assays were performed in triplicates. Two-way ANOVA was used to test that MAFA enhances Gad2 promoter activity in a dose-dependent manner ( P < 0.0001, df = 3; F = 218.4) and that MAFA 4A induces endogenous GAD2 transcription more efficiently than MAFA WT ( P < 0.0001, df = 1; F = 93.8). Plots represent mean ± sem. Scale bar (μm): 50 ( a ).

Article Snippet: Endogenous expression of Mafa protein was detected, after demasking in citrate bufffer 10 mM pH6, using rabbit anti-Mafa polyclonal antibody (Bethyl IHC-00352; dilution 1/200 in PBS 0,05% Tween 20) followed either with a secondary goat anti-rabbit Cy3 antibody (Invitrogen, A10520).

Techniques: In Situ Hybridization, Staining, Transfection, Expressing, Luciferase, Control, Activity Assay

Journal: Nature Communications

Article Title: Mafa-dependent GABAergic activity promotes mouse neonatal apneas

doi: 10.1038/s41467-022-30825-3

Figure Lengend Snippet: a Genetic scheme to introduce the hM3Dq DREADD in Mafa + inhibitory neurons. b Plethysmographic traces of the ventilation of a P1 Mafa Flpo/+ ; VGAT Cre/+ ; RC::hM3Dq/+ pups before (top) and 30 min after a subcutaneous injection of CNO (5 mg/kg). Note the presence of a click-like upward pressure shift terminating the breath holding apnea. c Injection of CNO in P1-2 mutants caused a five-fold increase of the apneic time fraction ( n = 12 pups, Two-way ANOVA comparing the CNO effect on mutants and on Cre +/+ siblings P = 0.001, df = 1, F = 14.7). This effect was usually reversible ( n = 10/12 pups) but caused irreversible ( n = 2/12 pups) apneic breathing (black traces filled and empty symbols) followed by death briefly after. d , e Experimental controls showing no effects of CNO on Cre +/+ pups ( d , n = 10 and no effect of saline (PBS) on mutant pups ( e , n = 4). f Quantification of the apneic time fraction of apnea types before and 60 min after injection of CNO showing significant increases for both types ( n = 12 pups; breath holding: two-sided Wilcoxon test P = 0.0005 and central-like: two-sided Wilcoxon test P = 0.0005). Plots represent mean ± sem.

Article Snippet: Endogenous expression of Mafa protein was detected, after demasking in citrate bufffer 10 mM pH6, using rabbit anti-Mafa polyclonal antibody (Bethyl IHC-00352; dilution 1/200 in PBS 0,05% Tween 20) followed either with a secondary goat anti-rabbit Cy3 antibody (Invitrogen, A10520).

Techniques: Introduce, Injection, Saline, Mutagenesis