A spatially dynamic network underlies the generation of inspiratory behaviors

Fig. 1.. The intact ventral medullary column produces a more robust inspiratory rhythm than the isolated preBötC. (A) Comparison of population spiking activity recorded from the preBӧtC in transverse (magenta) and horizontal (cyan) slice preparations. Example raw population spiking (below) and integrated (above) activity is shown for each slice preparation. (B) Group data from n = 40 transverse and n = 41 horizontal slices comparing inspiratory period and (C) burst-to-burst irregularity scores. (D) Representative optogenetic stimulations in transverse and horizontal slice preparations from Dbx1CreERT2;Rosa26ChR2 mice showing differences in the refractory period. (E) Quantified probability of photoevoking an inspiratory burst relative to elapsed time following a spontaneous burst in n = 4 transverse and n = 5 horizontal slices (*P > 0.05, ***P > 0.001, ****P > 0.0001; means ± SEM).

Fig. 2.. Spatial and functional characterization of respiratory neurons in the horizontal slice. (A) Locations of 47 inspiratory neurons recorded from 12 horizontal slices (200-µm grid), and three example excitatory (red; 1–3) and inhibitory (blue; 4–6) neurons. Raster plots show spiking activity over 30 consecutive inspiratory bursts; solid lines show integrated spiking activity (population burst onset at t = 0). Note the decrementing spiking pattern of inhibitory neurons. (B) Locations of 12 expiratory and 6 postinspiratory neurons recorded from 6 horizontal slices. Raster plots (Right) show a representative expiratory neuron with modest postinhibitory rebound activity (7), an excitatory postinspiratory neuron that is also active during inspiration (8), and an inhibitory postinspiratory PiCo neuron. Note that strong postinspiratory bursts only occur during some cycles in the absence of norepinephrine. (C) Quantified rostrocaudal locations of excitatory (red) and inhibitory (blue) inspiratory, expiratory, and postinspiratory neurons relative to the VII nerve (*P < 0.05; means ± SEM).

Fig. 3.. Inhibitory mechanisms regulate spatial reconfiguration of the inspiratory network. (A) Representative integrated population recordings from the preBӧtC center and 534 ± 40 µm rostral before (purple) and following suppression of GABAergic and glycinergic inhibition (red). (B) Group data showing changes in burst amplitude and frequency (n = 12). (C) Heatmaps (200-µm grid) of inspiratory burst amplitude, across the horizontal slice under control conditions (n = 8) and following suppression of synaptic inhibition with strychnine and gabazine (n = 4). Differences rostral to the preBӧtC are shown as a map of Bonferroni-corrected P values. (D) Locations of silent or tonic spiking excitatory (red; n = 9) or inhibitory (blue; n = 4) neurons recorded rostral of the preBӧtC. The gray dots indicate the location of neurons with phasic inspiratory activity shown in Fig. 2A. (E) Spike raster plots, for example, excitatory and inhibitory neurons during 15 consecutive inspiratory bursts at baseline and following coapplication of strychnine and gabazine; the solid lines show integrated spiking activity. (F) Average spiking activity of excitatory (n = 9) and inhibitory (n = 4) neurons at baseline…

Fig. 4.. Rhythmogenic properties along the inspiratory column. (A) Bright-field (Left) and EYFP fluorescence (Right) images of a Dbx1CreERT2;Rosa26ChR2EYFP horizontal slice showing optogenetic stimulation sites extending from 1,500 µm rostral to 1,000 µm caudal of the preBӧtC and representative integrated population activity (light purple) of the contralateral preBӧtC during 50 consecutive 200-ms light stimulations (the solid colored lines are averaged traces). (B) Quantified probability of evoking a contralateral preBӧtC burst at each stimulus location, which was (C) dependent on the elapsed time following a spontaneous preBӧtC burst (0.5-s bins) (n = 6 slices). (D) Representative bright-field and fluorescent images of consecutive transverse slices from a Dbx1CreERT2;Rosa26ChR2EYFP mouse; two rostral (R1 and R2) to the preBӧtC (pBC) and one caudal (C). (E) Representative integrated population activity and raster plots of bursting during ipsilateral (Left) and contralateral (Right) optogenetic stimulations yoked to preBӧtC bursting activity, and (F) quantified probability of evoking bursts in each region (n = 7 of each slice). (G) Representative population activity and raster plots of…

Fig. 5.. Vagal sensory feedback restricts inspiratory activity rostral of the preBӧtC. (A) Schematic of the anesthetized in vivo preparation used to perform paired recordings from the ventral medulla and motor output from the XII nerve. (B) Representative experiment showing integrated population recordings of inspiratory activity from 600 µm rostral to the preBӧtC (cyan), near the preBӧtC center (magenta), and from the hypoglossal (XII) nerve (black) under baseline conditions (purple) and following bilateral vagotomy (red). Breathing frequency is shown below (10-s bins). (C) Group data showing changes in burst amplitude and frequency (n = 6). (D) Heatmaps of inspiratory activity, as a percentage of preBӧtC burst amplitude, across the ventral medulla under control conditions (n = 5) and following vagotomy (n = 7), and the difference shown as Bonferroni-corrected P values (*P > 0.05, **P < 0.01, ****P > 0.0001; means ± SEM).

Fig. 6.. The spatial distribution of inspiratory activity expands during gasping and sighing in vivo. (A) Representative experiment showing integrated population recordings of inspiratory activity from 600 µm rostral of the preBӧtC, near the preBӧtC center, and from the hypoglossal (XII) nerve during a brief bout of anoxia to evoke gasping. Breathing frequency is shown below (10-s bins). (B) Group data showing changes in burst amplitude and frequency (n = 7). (C) Representative eupnea and sigh recorded from the preBӧtC center, 600 µm rostral, and the hypoglossal (XII) nerve, and (D) overlaid eupneic bursts relative to the sigh at each location. (E) Group data of sigh burst amplitude, as a percentage of eupnea amplitude, recorded at the preBӧtC center and 200, 400, and 600 µm rostral (*P > 0.05, **P > 0.01, ***P > 0.001; means ± SEM).

Fig. 7.. Hypothesized model for dynamic spatial reconfiguration of inspiratory activity based on a distributed network of excitatory and inhibitory neurons. Gradients of functional connectivity and inhibition determine the threshold for rhythmogenesis, which is lowest in the preBӧtC. During eupneic breathing (purple), a balance of excitation and inhibition restricts inspiratory activity and permits fast, dynamic rhythmic activity. Suppression of inhibition or removal of vagal sensory feedback (gray) leaves recurrent excitatory mechanisms unrestrained, leading to a rostral expansion of inspiratory activity, hypersynchronization of the network, and a slow inflexible rhythm. During sighs (yellow), a transient increase in excitation during the sigh burst expands the network rostrally resulting in a postsigh apnea. During severe hypoxia or asphyxia, the network is reconfigured to produce gasping (green). Excitatory drive to the network increased and inhibition is suppressed, leading to a large rostral expansion of inspiratory activity and a very robust, but slow, rhythm.