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Aav Injection, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( a ) Three types of sighs observed in adult mice in vivo. A sigh is a spontaneous inspiratory effort that results in significantly increased inspiratory tidal volume, typically two to five times compared to normal breaths. Under anesthesia in mice, sighs in vivo can take multiple shapes. The most common shape is partial doublet, a biphasic double-sized breath with an initial phase that is identical to a normal breath (eupnea) and a later high-amplitude inspiration, coincident with a biphasic genioglossus EMG (GG EMG ) event (left). An in vivo sigh in mice can also present as one large breath, a monophasic augmented inspiration that has two to five times the volume of a normal breath, coincident with a monophasic GG EMG event (middle); or a double-peaked breath (doublets) with the first breath immediately followed by a similar amplitude second breath (right). ( b ) Raw and expanded traces show three types of shape observed in one mouse. ( c ) Sigh shapes are related to basal breathing frequency (45 spontaneous sighs from 3 mice). Different sigh shapes represent a continuum from ‘augmented breath’ on one side of the spectrum to ‘doublet’ on the other. Exact shape appears to be related to basal breathing frequency, with augmented breaths and occasional partial doublets exhibited during conditions with high basal respiratory rate/low amplitude; while doublets and partial doublets occur mostly with low basal respiratory rate/high amplitude. The latter is similar to conditions of vagotomy and in vitro, where breathing f is substantially reduced, and during which sighs often appeared as doublets. ( d ) Measurement of respiratory parameters of eupneic breath and three types of sighs. ( e ) Sighs exhibiting a doublet shape occur periodically at a frequency significantly lower than normal eupneic breaths after NMB and GRP microinjection into preBötC in ketamine/xylazine anesthetized mice. ( f ) Doublets are not artifacts resulting from any damage to preBötC, since doublet-shaped sighs induced by microinjection of NMB and GRP were blocked by subsequent preBötC microinjection of NMBR and GRPR antagonists BIM23042 and RC3095. ( g ) Chemogenetic excitation of SST preBötC neurons evokes frequent doublet-shaped sighs in ketamine/xylazine mice, while in awake mice it induces frequent sighs of augmented breath shape, suggesting that doublets are indeed sighs.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a ) Three types of sighs observed in adult mice in vivo. A sigh is a spontaneous inspiratory effort that results in significantly increased inspiratory tidal volume, typically two to five times compared to normal breaths. Under anesthesia in mice, sighs in vivo can take multiple shapes. The most common shape is partial doublet, a biphasic double-sized breath with an initial phase that is identical to a normal breath (eupnea) and a later high-amplitude inspiration, coincident with a biphasic genioglossus EMG (GG EMG ) event (left). An in vivo sigh in mice can also present as one large breath, a monophasic augmented inspiration that has two to five times the volume of a normal breath, coincident with a monophasic GG EMG event (middle); or a double-peaked breath (doublets) with the first breath immediately followed by a similar amplitude second breath (right). ( b ) Raw and expanded traces show three types of shape observed in one mouse. ( c ) Sigh shapes are related to basal breathing frequency (45 spontaneous sighs from 3 mice). Different sigh shapes represent a continuum from ‘augmented breath’ on one side of the spectrum to ‘doublet’ on the other. Exact shape appears to be related to basal breathing frequency, with augmented breaths and occasional partial doublets exhibited during conditions with high basal respiratory rate/low amplitude; while doublets and partial doublets occur mostly with low basal respiratory rate/high amplitude. The latter is similar to conditions of vagotomy and in vitro, where breathing f is substantially reduced, and during which sighs often appeared as doublets. ( d ) Measurement of respiratory parameters of eupneic breath and three types of sighs. ( e ) Sighs exhibiting a doublet shape occur periodically at a frequency significantly lower than normal eupneic breaths after NMB and GRP microinjection into preBötC in ketamine/xylazine anesthetized mice. ( f ) Doublets are not artifacts resulting from any damage to preBötC, since doublet-shaped sighs induced by microinjection of NMB and GRP were blocked by subsequent preBötC microinjection of NMBR and GRPR antagonists BIM23042 and RC3095. ( g ) Chemogenetic excitation of SST preBötC neurons evokes frequent doublet-shaped sighs in ketamine/xylazine mice, while in awake mice it induces frequent sighs of augmented breath shape, suggesting that doublets are indeed sighs.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: In Vivo, In Vitro, Microinjection

( a ) Sagittal section of medulla with Nmbr (white), Grpr (green), and Sst (red) RNAscope signals as well as DAPI (blue) at the level of preBötC; inset top left: location of image (line box) in mouse medulla. ( b ) Examples of colocalization between relevant markers in high magnification confocal images (100×100 μm) of tissues processed with RNAscope. ( c ), Venn diagrams representing relative number of preBötC neurons expressing relevant markers and their overlap, scaled according to total Vglut2 count (n=3 sections for each colocalization pair). VII: facial nucleus. NA: nucleus ambiguous. There were 22.6±2.3 Grpr + neurons and 14.6±1.4 Nmbr + neurons in each transverse section; 25/45 Nmbr + neurons coexpressed Grpr ; 25/77 Grpr + neurons coexpressed Nmbr . Majority of both Nmbr + and Grpr + co-expressed VgluT2 (47/55 and 63/76, respectively), but not Sst (6/46 and 5/54, respectively).

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a ) Sagittal section of medulla with Nmbr (white), Grpr (green), and Sst (red) RNAscope signals as well as DAPI (blue) at the level of preBötC; inset top left: location of image (line box) in mouse medulla. ( b ) Examples of colocalization between relevant markers in high magnification confocal images (100×100 μm) of tissues processed with RNAscope. ( c ), Venn diagrams representing relative number of preBötC neurons expressing relevant markers and their overlap, scaled according to total Vglut2 count (n=3 sections for each colocalization pair). VII: facial nucleus. NA: nucleus ambiguous. There were 22.6±2.3 Grpr + neurons and 14.6±1.4 Nmbr + neurons in each transverse section; 25/45 Nmbr + neurons coexpressed Grpr ; 25/77 Grpr + neurons coexpressed Nmbr . Majority of both Nmbr + and Grpr + co-expressed VgluT2 (47/55 and 63/76, respectively), but not Sst (6/46 and 5/54, respectively).

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: RNAscope, Expressing

( a ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Grpr Flp mice at the level of preBötzinger Complex (preBötC) shows Flp-dependent ChR2 expression (EYFP, green) targeted to preBötC GRPR neurons. ChR2-EYFP expression was detectable in preBötC neurons 4 wk after viral injection, with some expression in neuron axons outside the preBötC including in the BötC rostral to the preBötC; in the intermediate reticular formation (IRt) ventral to the Amb. We found very few ChR2-EYFP-expressed axons in neighboring ventral respiratory column (VRC) caudal to the preBötC, or at more distant brainstem sites, e.g., 7 N. No evidence of transfected neuron soma was found outside the preBötC. Section also labeled for ChAT (red) and NeuN (blue) immunoreactivity. Scale bar, 100 µm. ( b ) Representative confocal micrograph of coronal medullary section (rectangle segment in inset panel) of transgenic Nmbr -ChR2 mice at the level of preBötC shows ChR2 expression (tdTomato, red) in preBötC NMBR neurons. ChR2- tdTomato expression was detectable in the preBötC neurons, plus scattered neurons outside the preBötC including in the IRt ventral to the Amb and the gigantocellular reticular nucleus (Gi) dorsomedial to the preBötC. Section also labeled for SST (green) and NeuN (blue) immunoreactivity. Scale bar, 50 µm. ( c ), Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Nmbr Cre mice at the level of preBötC shows Cre-dependent HM3Dq expression (mCherry, red) targeted to preBötC NMBR neurons. HM3Dq-mCherry expression was detectable in preBötC neurons 4 wk after viral injection, with the highest density of transfected somas in the preBötC. No evidence of transfected neurons was found outside preBötC. Section also labeled for ChAT (green) immunoreactivity. Scale bar, 100 µm.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Grpr Flp mice at the level of preBötzinger Complex (preBötC) shows Flp-dependent ChR2 expression (EYFP, green) targeted to preBötC GRPR neurons. ChR2-EYFP expression was detectable in preBötC neurons 4 wk after viral injection, with some expression in neuron axons outside the preBötC including in the BötC rostral to the preBötC; in the intermediate reticular formation (IRt) ventral to the Amb. We found very few ChR2-EYFP-expressed axons in neighboring ventral respiratory column (VRC) caudal to the preBötC, or at more distant brainstem sites, e.g., 7 N. No evidence of transfected neuron soma was found outside the preBötC. Section also labeled for ChAT (red) and NeuN (blue) immunoreactivity. Scale bar, 100 µm. ( b ) Representative confocal micrograph of coronal medullary section (rectangle segment in inset panel) of transgenic Nmbr -ChR2 mice at the level of preBötC shows ChR2 expression (tdTomato, red) in preBötC NMBR neurons. ChR2- tdTomato expression was detectable in the preBötC neurons, plus scattered neurons outside the preBötC including in the IRt ventral to the Amb and the gigantocellular reticular nucleus (Gi) dorsomedial to the preBötC. Section also labeled for SST (green) and NeuN (blue) immunoreactivity. Scale bar, 50 µm. ( c ), Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Nmbr Cre mice at the level of preBötC shows Cre-dependent HM3Dq expression (mCherry, red) targeted to preBötC NMBR neurons. HM3Dq-mCherry expression was detectable in preBötC neurons 4 wk after viral injection, with the highest density of transfected somas in the preBötC. No evidence of transfected neurons was found outside preBötC. Section also labeled for ChAT (green) immunoreactivity. Scale bar, 100 µm.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Expressing, Injection, Transfection, Labeling, Transgenic Assay

( a–e ) Photostimulation of preBötC GRPR (brown) or NMBR (blue) neurons evoked sighs. ( a ) Top: Schematic of genetic strategy to target preBötC GRPR neurons. Schematic (right) depicting bilateral placement of optical cannula targeting preBötC. Middle and Bottom: Raw (middle) and expanded (bottom) traces show bilateral long pulse photostimulation (LPP) (gray box) of preBötC GRPR neurons that could elicit an ectopic sigh (red arrowhead). Gray arrowheads indicate endogenous sighs. ( b ) Photostimulation of preBötC GRPR induced ectopic sighs with similar V T and T E as endogenous sighs, but slightly lower T I (V T : t 3 =0.233, p=0.824; T E : t 3 =0.249, p=0.812; T I : t 3 =3.267, p<0.05). V T , T E , and T I of ectopic sighs were significantly greater than those of eupneic breaths (V T : t 3 =6.889, p=5 × 10 –4 ; T I : t 3 =3.183, p=0.019; T E : t 3 =8.675, p=3 × 10 –4 ). Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 2,9 = 32.744, p<0.001; T I : F 2,9 = 16.530, p<0.001; T E : F 2,9 = 48.771, p<0.001. ( c ) Top: Targeting scheme to generate Nmbr -ChR2 mice. Schematic (right) depicting bilateral placement of optical cannula targeting preBötC. Middle and Bottom: Raw (middle) and expanded (bottom) show bilateral LPP (gray box) of preBötC NMBR neurons could elicit an ectopic sigh (red arrowhead). Gray arrowheads indicate endogenous sighs. ( d ) Photostimulation of preBötC NMBR neurons induced ectopic sighs with similar V T and T E as endogenous sighs, but slightly lower T I (V T : t 3 =0.820, p=0.444; T E : t 3 =0.976, p=0.367; T I : t 3 =3.175, p=0.0192). V T , T, E and T I of ectopic sighs were significantly greater than those of eupneic breaths (V T : t 3 =9.756, p=7 × 10 –5 ; T I : t 3 =7.685, p=3 × 10 –4 ; T E : t 3 =7.326, p=3 × 10 –4 ). Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 2,9 = 58.564, p<0.001; T I : F 2,9 = 62.362, p<0.001; T E : F 2,9 = 31.646, p<0.001. ( e ) Latency from laser onset was negatively correlated with phase in Grpr Flp ( r =–0.980, p=4 × 10 –6 ) and Nmbr -ChR2 ( r =–0.994, p=6 × 10 –6 ) mice (Pearson Product Moment Correlation). Representative traces showing latency between laser onset (gray box indicates LPP) and ectopic sighs (red arrowheads) when LPP was applied in medial (0.4) and late (0.8) phase. Gray dots represent latency to ectopic sigh from stimulation onset in each phase in Grpr Flp (n=5 mice) and Nmbr -ChR2 (n=4 mice) mice, solid lines join the average latency in each phase (black circle). No sighs were generated by LPP in phase 0.0–0.1 (23±4 s from previous sigh, measured from 55 stimulus in 5 mice: basal sigh rate 16±2 hr, range 12–19; intersigh interval 230±41 s) in Grpr Flp and in phase 0.0–0.3 (61±21 s from previous sigh, measured from 50 stimulus in 4 mice: basal sigh rate 19±6 hr, range 12–25; intersigh interval 203±70 s) in Nmbr -ChR2 mice. ( f ) Chemogenetic activation of preBötC NMBR neurons induced sighs. Left top: schematic diagram of genetic strategy to selectively express DREADD receptor hM3Dq on preBötC NMBR neurons. Left bottom: representative traces of airflow and V T before and after application of clozapine-n-oxide (CNO) to brainstem surface. Middle: Activation of hM3Dq receptors expressed on NMBR neurons with CNO significantly increases sigh rate (paired two-tailed t-test, n=3 mice: t 2 =5.94, p=0.03). Right: Trace from a representative mouse illustrating the incidence of sighs before and after CNO application. Data are shown as mean ± SE. Asterisks indicate post-hoc multiple comparison test results or paired t-test results: *, significance with p<0.05.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a–e ) Photostimulation of preBötC GRPR (brown) or NMBR (blue) neurons evoked sighs. ( a ) Top: Schematic of genetic strategy to target preBötC GRPR neurons. Schematic (right) depicting bilateral placement of optical cannula targeting preBötC. Middle and Bottom: Raw (middle) and expanded (bottom) traces show bilateral long pulse photostimulation (LPP) (gray box) of preBötC GRPR neurons that could elicit an ectopic sigh (red arrowhead). Gray arrowheads indicate endogenous sighs. ( b ) Photostimulation of preBötC GRPR induced ectopic sighs with similar V T and T E as endogenous sighs, but slightly lower T I (V T : t 3 =0.233, p=0.824; T E : t 3 =0.249, p=0.812; T I : t 3 =3.267, p<0.05). V T , T E , and T I of ectopic sighs were significantly greater than those of eupneic breaths (V T : t 3 =6.889, p=5 × 10 –4 ; T I : t 3 =3.183, p=0.019; T E : t 3 =8.675, p=3 × 10 –4 ). Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 2,9 = 32.744, p<0.001; T I : F 2,9 = 16.530, p<0.001; T E : F 2,9 = 48.771, p<0.001. ( c ) Top: Targeting scheme to generate Nmbr -ChR2 mice. Schematic (right) depicting bilateral placement of optical cannula targeting preBötC. Middle and Bottom: Raw (middle) and expanded (bottom) show bilateral LPP (gray box) of preBötC NMBR neurons could elicit an ectopic sigh (red arrowhead). Gray arrowheads indicate endogenous sighs. ( d ) Photostimulation of preBötC NMBR neurons induced ectopic sighs with similar V T and T E as endogenous sighs, but slightly lower T I (V T : t 3 =0.820, p=0.444; T E : t 3 =0.976, p=0.367; T I : t 3 =3.175, p=0.0192). V T , T, E and T I of ectopic sighs were significantly greater than those of eupneic breaths (V T : t 3 =9.756, p=7 × 10 –5 ; T I : t 3 =7.685, p=3 × 10 –4 ; T E : t 3 =7.326, p=3 × 10 –4 ). Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 2,9 = 58.564, p<0.001; T I : F 2,9 = 62.362, p<0.001; T E : F 2,9 = 31.646, p<0.001. ( e ) Latency from laser onset was negatively correlated with phase in Grpr Flp ( r =–0.980, p=4 × 10 –6 ) and Nmbr -ChR2 ( r =–0.994, p=6 × 10 –6 ) mice (Pearson Product Moment Correlation). Representative traces showing latency between laser onset (gray box indicates LPP) and ectopic sighs (red arrowheads) when LPP was applied in medial (0.4) and late (0.8) phase. Gray dots represent latency to ectopic sigh from stimulation onset in each phase in Grpr Flp (n=5 mice) and Nmbr -ChR2 (n=4 mice) mice, solid lines join the average latency in each phase (black circle). No sighs were generated by LPP in phase 0.0–0.1 (23±4 s from previous sigh, measured from 55 stimulus in 5 mice: basal sigh rate 16±2 hr, range 12–19; intersigh interval 230±41 s) in Grpr Flp and in phase 0.0–0.3 (61±21 s from previous sigh, measured from 50 stimulus in 4 mice: basal sigh rate 19±6 hr, range 12–25; intersigh interval 203±70 s) in Nmbr -ChR2 mice. ( f ) Chemogenetic activation of preBötC NMBR neurons induced sighs. Left top: schematic diagram of genetic strategy to selectively express DREADD receptor hM3Dq on preBötC NMBR neurons. Left bottom: representative traces of airflow and V T before and after application of clozapine-n-oxide (CNO) to brainstem surface. Middle: Activation of hM3Dq receptors expressed on NMBR neurons with CNO significantly increases sigh rate (paired two-tailed t-test, n=3 mice: t 2 =5.94, p=0.03). Right: Trace from a representative mouse illustrating the incidence of sighs before and after CNO application. Data are shown as mean ± SE. Asterisks indicate post-hoc multiple comparison test results or paired t-test results: *, significance with p<0.05.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Comparison, Generated, Activation Assay, Two Tailed Test

( a ) Left: schematic of slice preparation with photograph of patched Nmbr -tdTomato preBötC neuron. Scale bar: 50 µm. Right: Whole cell current clamp recording from inspiratory-modulated preBötC NMBR neuron (top trace) that generated action potentials (APs) during each XII burst (bottom trace). Not every cluster of APs (*) is associated with a burst, but each burst was associated with one or more Aps. Far right: Two bursts shown at expanded timescale. ( b ) Left: schematic of slice preparation in 2 P experiment with image of GCaMP6f-expressing preBötC neurons. Scale bar 50 µm. Right: Representative Ca 2+ signal of neighboring preBötC NMBR neurons was correlated with XIIn bursts (9 mM [K + ] ext ). Large amplitude XIIn bursts represent sighs (green asterisks), XIIn bursts not associated with Ca 2+ signal indicated with black asterisks. ( c ) Percentage of rhythmically active (I-modulated), non-rhythmic and out-of-phase preBötC NMBR neurons in preBötC. ( d ) Ca 2+ transients in individual neurons were significantly larger during sighs than during eupneic bursts (paired two-tailed t-test: t 8 =7.14, p<0.001). ( e ) Intervals of sighs were significantly greater during sighs compared to eupneic bursts (same color-coded neurons as in d). ( f ) Left: schematic of slice preparation and configuration. Right: Simultaneous Ca2 +signal from NMBR neurons (n=7), preBötC field recording, and XIIn activity in control conditions ([K + ] ext = 6 mM) and after addition of NMB (30 nM). Burstlets (red asterisks) are seen in control but not with NMB. ( g ) Quantification of duration of eupneic and sigh bursts in 9 mM [K + ] ext and 6 mM [K + ] ext with or without NMB. Differences in duration between eupneic bursts in different conditions, and between sighs in different conditions were not significant (repeated measures ANOVA, F 5,19 = 20.1, p<0.001; asterisks indicate significance from Tukey post-hoc tests). ( h ) Percentage of bursts that were sighs during 5 min was significantly higher in 6 mM [K + ] ext +NMB than other conditions (repeated measures ANOVA, F 2,9 = 7.18, p=0.018). ( i ) Comparisons between XIIn burst frequency and preBötC burst frequency in 9 mM [K + ] ext and 6 mM [K + ] ext with or without NMB. XIIn and preBötC burst frequency were similar in 9 mM [K + ] ext and 6 mM [K + ] ext +NMB, and both conditions differ from 6 mM [K + ] ext alone. There is an increase in preBötC burst frequency in 6 mM [K+]ext due to the burstlets (shown in a), (repeated measures ANOVA, F 5,23 = 11.57, p<0.001). Data are shown as mean ± SE. Asterisks indicate significance from Tukey post-hoc tests or paired t-tests: *, significance with p<0.05; **, significance with p<0.01; ***, significance with p<0.001.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a ) Left: schematic of slice preparation with photograph of patched Nmbr -tdTomato preBötC neuron. Scale bar: 50 µm. Right: Whole cell current clamp recording from inspiratory-modulated preBötC NMBR neuron (top trace) that generated action potentials (APs) during each XII burst (bottom trace). Not every cluster of APs (*) is associated with a burst, but each burst was associated with one or more Aps. Far right: Two bursts shown at expanded timescale. ( b ) Left: schematic of slice preparation in 2 P experiment with image of GCaMP6f-expressing preBötC neurons. Scale bar 50 µm. Right: Representative Ca 2+ signal of neighboring preBötC NMBR neurons was correlated with XIIn bursts (9 mM [K + ] ext ). Large amplitude XIIn bursts represent sighs (green asterisks), XIIn bursts not associated with Ca 2+ signal indicated with black asterisks. ( c ) Percentage of rhythmically active (I-modulated), non-rhythmic and out-of-phase preBötC NMBR neurons in preBötC. ( d ) Ca 2+ transients in individual neurons were significantly larger during sighs than during eupneic bursts (paired two-tailed t-test: t 8 =7.14, p<0.001). ( e ) Intervals of sighs were significantly greater during sighs compared to eupneic bursts (same color-coded neurons as in d). ( f ) Left: schematic of slice preparation and configuration. Right: Simultaneous Ca2 +signal from NMBR neurons (n=7), preBötC field recording, and XIIn activity in control conditions ([K + ] ext = 6 mM) and after addition of NMB (30 nM). Burstlets (red asterisks) are seen in control but not with NMB. ( g ) Quantification of duration of eupneic and sigh bursts in 9 mM [K + ] ext and 6 mM [K + ] ext with or without NMB. Differences in duration between eupneic bursts in different conditions, and between sighs in different conditions were not significant (repeated measures ANOVA, F 5,19 = 20.1, p<0.001; asterisks indicate significance from Tukey post-hoc tests). ( h ) Percentage of bursts that were sighs during 5 min was significantly higher in 6 mM [K + ] ext +NMB than other conditions (repeated measures ANOVA, F 2,9 = 7.18, p=0.018). ( i ) Comparisons between XIIn burst frequency and preBötC burst frequency in 9 mM [K + ] ext and 6 mM [K + ] ext with or without NMB. XIIn and preBötC burst frequency were similar in 9 mM [K + ] ext and 6 mM [K + ] ext +NMB, and both conditions differ from 6 mM [K + ] ext alone. There is an increase in preBötC burst frequency in 6 mM [K+]ext due to the burstlets (shown in a), (repeated measures ANOVA, F 5,23 = 11.57, p<0.001). Data are shown as mean ± SE. Asterisks indicate significance from Tukey post-hoc tests or paired t-tests: *, significance with p<0.05; **, significance with p<0.01; ***, significance with p<0.001.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Slice Preparation, Generated, Expressing, Two Tailed Test, Activity Assay, Control

( a ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Nmbr Cre ; Grpr Flp double mutant mice at the level of preBötzinger Complex (preBötC) shows ChR2 expression (EYFP, green) targeted to preBötC NMBR-only neurons. ChR2-EYFP expression was detectable in preBötC neurons 4 wk after viral injection, with some expression in neuron axons in the BötC. No evidence of transfected neuron soma was found outside the preBötC. Section also labeled for ChAT (red) and NeuN (blue) immunoreactivity. Scale bar, 100 µm. ( b ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Nmbr Cre ; Grpr Flp double mutant mice at the level of preBötC shows ChR2 expression (EYFP, green) targeted to preBötC GRPR-only neurons. ChR2-EYFP expression was detectable in preBötC neurons 4 wk after viral injection, with very little expression in neuron axons in neighboring area outside the preBötC. No evidence of transfected neuron was found outside the preBötC. Section also labeled for ChAT (red) and NeuN (blue) immunoreactivity. Scale bar, 100 µm.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Nmbr Cre ; Grpr Flp double mutant mice at the level of preBötzinger Complex (preBötC) shows ChR2 expression (EYFP, green) targeted to preBötC NMBR-only neurons. ChR2-EYFP expression was detectable in preBötC neurons 4 wk after viral injection, with some expression in neuron axons in the BötC. No evidence of transfected neuron soma was found outside the preBötC. Section also labeled for ChAT (red) and NeuN (blue) immunoreactivity. Scale bar, 100 µm. ( b ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Nmbr Cre ; Grpr Flp double mutant mice at the level of preBötC shows ChR2 expression (EYFP, green) targeted to preBötC GRPR-only neurons. ChR2-EYFP expression was detectable in preBötC neurons 4 wk after viral injection, with very little expression in neuron axons in neighboring area outside the preBötC. No evidence of transfected neuron was found outside the preBötC. Section also labeled for ChAT (red) and NeuN (blue) immunoreactivity. Scale bar, 100 µm.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Mutagenesis, Expressing, Injection, Transfection, Labeling

$( a–c ) Effects of activating preBötC NMBR-only neurons on sigh generation. ( a ) Left: Schematic of the intersectional genetic strategy to target preBötC NMBR-only neurons. Right: Schematic depicting bilateral placement of optical cannula targeting preBötC. ( b ) Top: Raw (left) and expanded (right) traces show that preBötC SPP (gray box) of NMBR-only neurons induced an ectopic burst (red arrowhead) which did not reset the sigh rhythm. Bottom: preBötC SPP of NMBR-only neurons induced an ectopic burst (red arrowhead) in a sigh refractory period. Gray arrowheads indicate endogenous sighs. ( c ) V T of ectopic bursts evoked by NMBR-only photostimulation was smaller than that of endogenous sighs (n=4 mice, t 3 =4.278, p=0.005), but larger than of eupneic breaths (t 3 =6.588, p=6 × 10 –4 ); T E of evoked ectopic bursts was no different from endogenous sighs (t 3 =1.882, p=0.109), but higher than of eupneic breaths (t 3 =5.203, p=0.002); T I of evoked ectopic bursts was shorter than that of endogenous sighs (t 3 =3.917, p=0.008). Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 2,9 = 59.922, p<0.001; T I : F 2,9 = 25.206, p<0.001; T E : F 2,9 = 26.939, p<0.001. ( d-f ) Effects of activating preBötC GRPR-only neurons on sigh generation. ( d ) Left: Schematic of the intersectional genetic strategy to target preBötC GRPR-only neurons. Right: Schematic depicting bilateral placement of optical cannula targeting preBötC. ( e ) preBötC LPP of GRPR-only neurons in late phase elicited ectopic sighs and reset the sigh rhythm. Raw (left) and expanded (middle and right) show bilateral long pulse photostimulation (LPP) (gray box) of preBötC GRPR-only neurons elicited an ectopic sigh (red arrowhead). Gray arrowheads indicate endogenous sighs. ( f ) V T (t 3 =0.152, p=0.884), T I (t 3 =1.843, p=0.115), and T E (t 3 =1.619, p=0.157), of ectopic sighs were no different from endogenous sighs (n=4 mice), but increased compared to eupneic breaths (V T :, t 3 =12.855, p=10 –5 ; T I : t 3 =30.235, p=9 ×10 –8 ; T E : t 3 =9.007, p=4 × 10 –5 ), indicative of augmented breaths with postsigh apneas. Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 2,9 = 111.488, p<0.001; T I : F 2,9 = 648.855, p<0.001; T E : F 2,9 = 65.544, p<0.001. Data are shown as mean ± SE. Asterisks indicate post-hoc multiple comparison test or paired t-test results: *, significance with p<0.05.$

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a–c ) Effects of activating preBötC NMBR-only neurons on sigh generation. ( a ) Left: Schematic of the intersectional genetic strategy to target preBötC NMBR-only neurons. Right: Schematic depicting bilateral placement of optical cannula targeting preBötC. ( b ) Top: Raw (left) and expanded (right) traces show that preBötC SPP (gray box) of NMBR-only neurons induced an ectopic burst (red arrowhead) which did not reset the sigh rhythm. Bottom: preBötC SPP of NMBR-only neurons induced an ectopic burst (red arrowhead) in a sigh refractory period. Gray arrowheads indicate endogenous sighs. ( c ) V T of ectopic bursts evoked by NMBR-only photostimulation was smaller than that of endogenous sighs (n=4 mice, t 3 =4.278, p=0.005), but larger than of eupneic breaths (t 3 =6.588, p=6 × 10 –4 ); T E of evoked ectopic bursts was no different from endogenous sighs (t 3 =1.882, p=0.109), but higher than of eupneic breaths (t 3 =5.203, p=0.002); T I of evoked ectopic bursts was shorter than that of endogenous sighs (t 3 =3.917, p=0.008). Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 2,9 = 59.922, p<0.001; T I : F 2,9 = 25.206, p<0.001; T E : F 2,9 = 26.939, p<0.001. ( d-f ) Effects of activating preBötC GRPR-only neurons on sigh generation. ( d ) Left: Schematic of the intersectional genetic strategy to target preBötC GRPR-only neurons. Right: Schematic depicting bilateral placement of optical cannula targeting preBötC. ( e ) preBötC LPP of GRPR-only neurons in late phase elicited ectopic sighs and reset the sigh rhythm. Raw (left) and expanded (middle and right) show bilateral long pulse photostimulation (LPP) (gray box) of preBötC GRPR-only neurons elicited an ectopic sigh (red arrowhead). Gray arrowheads indicate endogenous sighs. ( f ) V T (t 3 =0.152, p=0.884), T I (t 3 =1.843, p=0.115), and T E (t 3 =1.619, p=0.157), of ectopic sighs were no different from endogenous sighs (n=4 mice), but increased compared to eupneic breaths (V T :, t 3 =12.855, p=10 –5 ; T I : t 3 =30.235, p=9 ×10 –8 ; T E : t 3 =9.007, p=4 × 10 –5 ), indicative of augmented breaths with postsigh apneas. Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 2,9 = 111.488, p<0.001; T I : F 2,9 = 648.855, p<0.001; T E : F 2,9 = 65.544, p<0.001. Data are shown as mean ± SE. Asterisks indicate post-hoc multiple comparison test or paired t-test results: *, significance with p<0.05.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Comparison

$( a ) Photostimulation of preBötC GRPR (brown) and NMBR (blue) neurons perturbs breathing frequency and inspiratory burst amplitude. Top: representative traces recorded during sigh refractory period show effects of bilateral preBötC long pulse photostimulation (LPP) of preBötC GRPR (brown) or NMBR (blue) neurons on eupneic breathing f and V T . Bottom: V T and f of eupneic breaths and during photostimulation. (paired t-tests, GrprF lp +AAV-ChR2, n=4 mice , f : t 3 =–8.13801, p=4 × 10 –3 ; V T : t 3 =–5.035, p=0.015; Nmbr -ChR2, n=5 mice, f : t 4 =7.33379, p=2 × 10 –3 , V T : t 4 =–5.875, p=4 × 10 –3 ). Normalized (norm) V T and f traces relative to stimulus onset measured prestimulation, during stimulation and poststimulation. ( b ) Distinct effects of activating preBötC GRPR-only (brown) and NMBR-only (blue) neurons on inspiratory burst amplitude. Top and bottom (left): representative traces show the effects of bilateral preBötC SPP and LPP of preBötC GRPR-only (brown) or NMBR-only (blue) neurons on breathing V T . Bottom (right): V T of eupneic breaths and during SPP stimulation. Increase in V T in response to bilateral preBötC SPP of preBötC GRPR-only (brown) or NMBR-only (blue) neurons (paired t-tests, GRPR-only, n=4 mice: t 3 =0.746, p=0.510; NMBR-only, n=4 mice: t 3 =–6.886, p=6 × 10 –3 ). ( c ) Activation of hM3Dq receptors expressed on NMBR neurons with clozapine-n-oxide (CNO) significantly increased breathing amplitude and decreased f (paired two-tailed t-tests, n=3 mice, f : t 2 =5.71, p=0.03; V T : t 2 =5.47, p=0.03). ( d ) Sighs can be induced after GRPR and NMBR inhibition. Left: schematic depicting bilateral placement of optical cannula targeting parafacial (pF) or preBötC. Middle: representative traces showing LPP of pF GRP neurons, or preBötC GRPR and NMBR neurons elicited ectopic sighs (doublet, red arrowhead), but not of pF NMB neurons, in the presence of GRPR antagonist RC3095 and NMBR antagonist BIM23042. Right: V T of doublets induced by pF LPP of GRP neurons was not significantly different from that of endogenous sighs (n=4 mice; t 3 =0.724, p=0.522); T I increased compared to endogenous sighs (t 3 =–7.493, p=5 × 10 –3 ), T E of the following respiratory cycle was unaffected (t 3 =–0.472, p=0.669). V T and T I of the doublets elicited by preBötC LPP of GRPR (n=5 mice, V T : t 4 =0.199, p=0.852; T I : t 4 =0.912, p=0.413) or NMBR (n=4 mice, V T : t 3 =–0.845, p=0.460; T I : t 3 =–0.376, p=0.732) neurons after blockade of both receptors were not different from spontaneous sighs. T E immediately following doublet increased in Grpr Flp (n=5 mice, t 4 =–3.144, p=0.035), but not lengthened in Nmbr -ChR2 mice (n=4 mice, t 3 =–0.801, p=0.482). Data are shown as mean ± SE. Asterisks indicate post-hoc multiple comparison test or paired t-test results: *, significance with p<0.05.$

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a ) Photostimulation of preBötC GRPR (brown) and NMBR (blue) neurons perturbs breathing frequency and inspiratory burst amplitude. Top: representative traces recorded during sigh refractory period show effects of bilateral preBötC long pulse photostimulation (LPP) of preBötC GRPR (brown) or NMBR (blue) neurons on eupneic breathing f and V T . Bottom: V T and f of eupneic breaths and during photostimulation. (paired t-tests, GrprF lp +AAV-ChR2, n=4 mice , f : t 3 =–8.13801, p=4 × 10 –3 ; V T : t 3 =–5.035, p=0.015; Nmbr -ChR2, n=5 mice, f : t 4 =7.33379, p=2 × 10 –3 , V T : t 4 =–5.875, p=4 × 10 –3 ). Normalized (norm) V T and f traces relative to stimulus onset measured prestimulation, during stimulation and poststimulation. ( b ) Distinct effects of activating preBötC GRPR-only (brown) and NMBR-only (blue) neurons on inspiratory burst amplitude. Top and bottom (left): representative traces show the effects of bilateral preBötC SPP and LPP of preBötC GRPR-only (brown) or NMBR-only (blue) neurons on breathing V T . Bottom (right): V T of eupneic breaths and during SPP stimulation. Increase in V T in response to bilateral preBötC SPP of preBötC GRPR-only (brown) or NMBR-only (blue) neurons (paired t-tests, GRPR-only, n=4 mice: t 3 =0.746, p=0.510; NMBR-only, n=4 mice: t 3 =–6.886, p=6 × 10 –3 ). ( c ) Activation of hM3Dq receptors expressed on NMBR neurons with clozapine-n-oxide (CNO) significantly increased breathing amplitude and decreased f (paired two-tailed t-tests, n=3 mice, f : t 2 =5.71, p=0.03; V T : t 2 =5.47, p=0.03). ( d ) Sighs can be induced after GRPR and NMBR inhibition. Left: schematic depicting bilateral placement of optical cannula targeting parafacial (pF) or preBötC. Middle: representative traces showing LPP of pF GRP neurons, or preBötC GRPR and NMBR neurons elicited ectopic sighs (doublet, red arrowhead), but not of pF NMB neurons, in the presence of GRPR antagonist RC3095 and NMBR antagonist BIM23042. Right: V T of doublets induced by pF LPP of GRP neurons was not significantly different from that of endogenous sighs (n=4 mice; t 3 =0.724, p=0.522); T I increased compared to endogenous sighs (t 3 =–7.493, p=5 × 10 –3 ), T E of the following respiratory cycle was unaffected (t 3 =–0.472, p=0.669). V T and T I of the doublets elicited by preBötC LPP of GRPR (n=5 mice, V T : t 4 =0.199, p=0.852; T I : t 4 =0.912, p=0.413) or NMBR (n=4 mice, V T : t 3 =–0.845, p=0.460; T I : t 3 =–0.376, p=0.732) neurons after blockade of both receptors were not different from spontaneous sighs. T E immediately following doublet increased in Grpr Flp (n=5 mice, t 4 =–3.144, p=0.035), but not lengthened in Nmbr -ChR2 mice (n=4 mice, t 3 =–0.801, p=0.482). Data are shown as mean ± SE. Asterisks indicate post-hoc multiple comparison test or paired t-test results: *, significance with p<0.05.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Activation Assay, Two Tailed Test, Inhibition, Comparison

RC3095 and BIM23042 effectively blocked GRPRs and NMBRs, as respiratory responses induced by GRP and NMB microinjection into the preBötC, sufficient to increase sigh rate by 585 ± 209% of control, were fully antagonized by the same concentration of RC3095 and BIM23042 (n=3). Air flow and V T traces show the changes in sigh rate under different conditions (from left to right) in anesthetized mice (note sighs are evident as single events significantly larger than normal breaths): control; GRP +NMB: after bilateral injection of GRP +NMB into preBötC; RC3095 +BIM23042: after bilateral injection of RC3095 +BIM23042 (300 µM each, 50–60 nl/side) into preBötC; GRP +NMB after antagonists: the second bilateral injection of GRP +NMB, i.e., same dose of GRP +NMB that resulted in the higher sigh rate before RC3095 +BIM23042 did not induce sighing after the antagonist injection.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: RC3095 and BIM23042 effectively blocked GRPRs and NMBRs, as respiratory responses induced by GRP and NMB microinjection into the preBötC, sufficient to increase sigh rate by 585 ± 209% of control, were fully antagonized by the same concentration of RC3095 and BIM23042 (n=3). Air flow and V T traces show the changes in sigh rate under different conditions (from left to right) in anesthetized mice (note sighs are evident as single events significantly larger than normal breaths): control; GRP +NMB: after bilateral injection of GRP +NMB into preBötC; RC3095 +BIM23042: after bilateral injection of RC3095 +BIM23042 (300 µM each, 50–60 nl/side) into preBötC; GRP +NMB after antagonists: the second bilateral injection of GRP +NMB, i.e., same dose of GRP +NMB that resulted in the higher sigh rate before RC3095 +BIM23042 did not induce sighing after the antagonist injection.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Microinjection, Control, Concentration Assay, Injection

( a ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Sst Cre mice at the level of preBötzinger Complex (preBötC) shows Cre-dependent ChR2 expression (EYFP, green) targeted to preBötC SST neurons. ChR2-EYFP expression was detectable in preBötC neurons 4 wk after viral injection, with some expression in neighboring neurons outside the preBötC, including in the BötC rostral to the preBötC. The peak density of transfected neurons was caudal to the BötC and ventral to the Amb. We found no evidence of transfected neurons at more distant brainstem sites. Section also labeled for NeuN (blue) immunoreactivity. Scale bar, 100 µm. ( b ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Sst Cre mice at the level of preBötC shows Cre-dependent HM3Dq expression (mCherry, red) targeted to preBötC SST neurons. HM3Dq-mCherry expression was detectable in preBötC neurons 4 wk after viral injection, with the highest density of transfected somas in the preBötC and minimal (<10% of transfected somas) found outside of preBötC. Section also labeled for ChAT (green) immunoreactivity. Scale bar, 100 µm. ( c ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Sst Cre mice at the level of preBötC shows Cre-dependent PSAM4 expression (EGFP, green) targeted to preBötC SST neurons. PSAM4-EGFP expression was detectable in preBötC neurons 4 wk after viral injection, with the highest density of transfected somas in the preBötC and minimal (<10% of transfected somas) found outside of preBötC. Section also labeled for ChAT (red) immunoreactivity. Scale bar, 100 µm.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Sst Cre mice at the level of preBötzinger Complex (preBötC) shows Cre-dependent ChR2 expression (EYFP, green) targeted to preBötC SST neurons. ChR2-EYFP expression was detectable in preBötC neurons 4 wk after viral injection, with some expression in neighboring neurons outside the preBötC, including in the BötC rostral to the preBötC. The peak density of transfected neurons was caudal to the BötC and ventral to the Amb. We found no evidence of transfected neurons at more distant brainstem sites. Section also labeled for NeuN (blue) immunoreactivity. Scale bar, 100 µm. ( b ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Sst Cre mice at the level of preBötC shows Cre-dependent HM3Dq expression (mCherry, red) targeted to preBötC SST neurons. HM3Dq-mCherry expression was detectable in preBötC neurons 4 wk after viral injection, with the highest density of transfected somas in the preBötC and minimal (<10% of transfected somas) found outside of preBötC. Section also labeled for ChAT (green) immunoreactivity. Scale bar, 100 µm. ( c ) Representative confocal micrograph of sagittal medullary section (rectangle segment in inset panel) of Sst Cre mice at the level of preBötC shows Cre-dependent PSAM4 expression (EGFP, green) targeted to preBötC SST neurons. PSAM4-EGFP expression was detectable in preBötC neurons 4 wk after viral injection, with the highest density of transfected somas in the preBötC and minimal (<10% of transfected somas) found outside of preBötC. Section also labeled for ChAT (red) immunoreactivity. Scale bar, 100 µm.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Expressing, Injection, Transfection, Labeling

( a–d ) Optogenetic activation of SST neurons generates sighs after blockade of NMBRs and GRPRs. ( a ) Top: Schematic of the genetic strategy to target preBötC SST neurons. Bottom: Schematic depicting bilateral placement of optical cannula targeting preBötC. ( b ) Ectopic sigh (red arrowhead) elicited by bilateral SPP (gray box) of preBötC SST neurons. ( c ) V T (t 4 =0.176, p=0.863), T I (t 4 =1.355, p=0.200), T E (t 4 =0.734, p=0.477) of ectopic sighs before blockade or V T (t 4 =0.856, p=0.409), T I (t 4 =0.894, p=0.389) of ectopic sighs after blockade of NMBR and GRPR were no different from endogenous sighs (n=5 mice). T E of ectopic sighs elicited after blockade was longer than that of endogenous sighs (t 4 =4.960, p=3 × 10 –4 ). V T , T I , and T E of ectopic sighs before (V T : t 4 =7.925, p=4 × 10 –6 ; T I : t 4 =11.194, p=10 –7 ; T E : t 4 =5.405, p=10 –4 ) or after (V T : t 4 =7.245, p=10 –5 ; T I : t 4 =13.442, p=10 –8 ; T E : t 4 =9.631, p=5 × 10 –7 ) blockade were increased compared with eupneic breaths (n=5 mice), indicative of augmented breaths with postsigh apneas. Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 3,16 = 30.357, p<0.001; T I : F 3,16 = 78.526, p<0.001; T E : F 3,16 = 31.134, p<0.001. ( d ) SPP elicits sighs in the presence of GRPR antagonist RC3095 and NMBR antagonist BIM23042. ( e, f ) Chemogenetic activation of SST neurons generated sighs after blockade of NMBRs and GRPRs. ( e ) Top: schematic diagram of genetic strategy to selectively express DREADD receptor hM3Dq on preBötC SST neurons. Bottom: representative trace of airflow and V T during baseline, after application of clozapine-n-oxide (CNO), and after microinjection of RC3095 and BIM23042. ( f ) Top: Activation of hM3Dq receptors expressed on preBötC SST + neurons significantly decreased breathing f , increased V T , and elevated sigh frequency; subsequent BIM23042 and RC3095 (B+R) microinjection into preBötC did not significantly affect breathing f , V T , nor sigh rate induced by CNO application (repeated measures ANOVA, n=3 mice; f : F 2,4 = 28.3, p=0.004; V T : F 2,4 = 25.7, p=0.005; sigh rate: F 2,4 = 26.1, p=0.005). Bottom: representative trace depicting sighs during baseline, after application of CNO, and after microinjection of NMBR and GRPR antagonists RC3095 and BIM23042. ( g ) Top: schematic diagram of genetic strategy to selectively express ultrapotent inhibitory DREADD receptor PSAM4-GlyR on preBötC SST + neurons. Bottom: representative trace of airflow and V T during baseline, after preBötC microinjection of peptides NMB and GRP (250 µM each, 50 nl/side), and application of uPSEM817 (10 mM, 30 µl applied to brainstem surface). ( h ) Top: Microinjection of peptides NMB and GRP into preBötC significantly decreased breathing f, increased V T , and elevated sigh frequency; subsequent inhibition of SST + preBötC neurons selectively eliminated any sighs, but preserves decreased f and V T (repeated measures ANOVA, n=3 mice; f: F 2,4 = 55.8, p=0.001; V T : F 2,4 = 19.0, p=0.009; sigh rate: F 2,4 = 83.8, p<0.001). Bottom: representative trace depicting sighs during baseline, after bilateral preBötC microinjection of NMB and GRP, and after application of PSAM4-GlyR ligand uPSEM817 to the brainstem surface. Data are shown as mean ± SE. Asterisks indicate post-hoc multiple comparison test or paired t-test results: *, significance with p<0.05; **, significance with p<0.01.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a–d ) Optogenetic activation of SST neurons generates sighs after blockade of NMBRs and GRPRs. ( a ) Top: Schematic of the genetic strategy to target preBötC SST neurons. Bottom: Schematic depicting bilateral placement of optical cannula targeting preBötC. ( b ) Ectopic sigh (red arrowhead) elicited by bilateral SPP (gray box) of preBötC SST neurons. ( c ) V T (t 4 =0.176, p=0.863), T I (t 4 =1.355, p=0.200), T E (t 4 =0.734, p=0.477) of ectopic sighs before blockade or V T (t 4 =0.856, p=0.409), T I (t 4 =0.894, p=0.389) of ectopic sighs after blockade of NMBR and GRPR were no different from endogenous sighs (n=5 mice). T E of ectopic sighs elicited after blockade was longer than that of endogenous sighs (t 4 =4.960, p=3 × 10 –4 ). V T , T I , and T E of ectopic sighs before (V T : t 4 =7.925, p=4 × 10 –6 ; T I : t 4 =11.194, p=10 –7 ; T E : t 4 =5.405, p=10 –4 ) or after (V T : t 4 =7.245, p=10 –5 ; T I : t 4 =13.442, p=10 –8 ; T E : t 4 =9.631, p=5 × 10 –7 ) blockade were increased compared with eupneic breaths (n=5 mice), indicative of augmented breaths with postsigh apneas. Statistical significance was determined with a One-Way RM ANOVA followed by All Pairwise Multiple Comparison Procedures (Holm-Sidak method), V T : F 3,16 = 30.357, p<0.001; T I : F 3,16 = 78.526, p<0.001; T E : F 3,16 = 31.134, p<0.001. ( d ) SPP elicits sighs in the presence of GRPR antagonist RC3095 and NMBR antagonist BIM23042. ( e, f ) Chemogenetic activation of SST neurons generated sighs after blockade of NMBRs and GRPRs. ( e ) Top: schematic diagram of genetic strategy to selectively express DREADD receptor hM3Dq on preBötC SST neurons. Bottom: representative trace of airflow and V T during baseline, after application of clozapine-n-oxide (CNO), and after microinjection of RC3095 and BIM23042. ( f ) Top: Activation of hM3Dq receptors expressed on preBötC SST + neurons significantly decreased breathing f , increased V T , and elevated sigh frequency; subsequent BIM23042 and RC3095 (B+R) microinjection into preBötC did not significantly affect breathing f , V T , nor sigh rate induced by CNO application (repeated measures ANOVA, n=3 mice; f : F 2,4 = 28.3, p=0.004; V T : F 2,4 = 25.7, p=0.005; sigh rate: F 2,4 = 26.1, p=0.005). Bottom: representative trace depicting sighs during baseline, after application of CNO, and after microinjection of NMBR and GRPR antagonists RC3095 and BIM23042. ( g ) Top: schematic diagram of genetic strategy to selectively express ultrapotent inhibitory DREADD receptor PSAM4-GlyR on preBötC SST + neurons. Bottom: representative trace of airflow and V T during baseline, after preBötC microinjection of peptides NMB and GRP (250 µM each, 50 nl/side), and application of uPSEM817 (10 mM, 30 µl applied to brainstem surface). ( h ) Top: Microinjection of peptides NMB and GRP into preBötC significantly decreased breathing f, increased V T , and elevated sigh frequency; subsequent inhibition of SST + preBötC neurons selectively eliminated any sighs, but preserves decreased f and V T (repeated measures ANOVA, n=3 mice; f: F 2,4 = 55.8, p=0.001; V T : F 2,4 = 19.0, p=0.009; sigh rate: F 2,4 = 83.8, p<0.001). Bottom: representative trace depicting sighs during baseline, after bilateral preBötC microinjection of NMB and GRP, and after application of PSAM4-GlyR ligand uPSEM817 to the brainstem surface. Data are shown as mean ± SE. Asterisks indicate post-hoc multiple comparison test or paired t-test results: *, significance with p<0.05; **, significance with p<0.01.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Activation Assay, Comparison, Generated, Microinjection, Inhibition

We confirmed that the application of uPSEM817 to brainstem surface (left) or saline infusion into the preBötzinger Complex (preBötC) (right) did not evoke changes in breathing parameters and did not evoke sighing in naïve mice.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: We confirmed that the application of uPSEM817 to brainstem surface (left) or saline infusion into the preBötzinger Complex (preBötC) (right) did not evoke changes in breathing parameters and did not evoke sighing in naïve mice.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Saline

NMB and GRP neurons in pF project to preBötC neurons expressing cognate receptors NMBR and GRPR. NMBR and GRPR preBötC neurons mediate sigh output via connections to downstream SST preBötC neurons, but also directly contribute to the pattern of eupneic breathing.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: NMB and GRP neurons in pF project to preBötC neurons expressing cognate receptors NMBR and GRPR. NMBR and GRPR preBötC neurons mediate sigh output via connections to downstream SST preBötC neurons, but also directly contribute to the pattern of eupneic breathing.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

Techniques: Expressing

( a ) No output effects and no sigh were produced by preBötzinger Complex (preBötC) photostimulation in Grpr -EGFP (top; scale bar, 100 µm) and Nmbr -RFP (bottom; scale bar, 100 µm) reporter mice. ( b ) Stimulating 500 µm rostral to preBötC in Grpr -Flp (top) and Nmbr -ChR2 (bottom) mice did not produce significant output effects.

Journal: eLife

Article Title: Sigh generation in preBötzinger complex

doi: 10.7554/eLife.100192

Figure Lengend Snippet: ( a ) No output effects and no sigh were produced by preBötzinger Complex (preBötC) photostimulation in Grpr -EGFP (top; scale bar, 100 µm) and Nmbr -RFP (bottom; scale bar, 100 µm) reporter mice. ( b ) Stimulating 500 µm rostral to preBötC in Grpr -Flp (top) and Nmbr -ChR2 (bottom) mice did not produce significant output effects.

Article Snippet: To express ChR2 selectively in preBötC neurons, AAV injections were performed on adult Sst Cre mice (Jackson Labs Strain #013044) or Grpr flp mice.

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