Breathing control center neurons that promote arousal in mice

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Science  31 Mar 2017:
Vol. 355, Issue 6332, pp. 1411-1415
DOI: 10.1126/science.aai7984
  • Fig. 1 Identification and genetic ablation of Cdh9/Dbx1 double-positive neurons in preBötC.

    (A) Cdh9 mRNA expression (blue) in section of E14.5 mouse embryo (11). Insets, ventrolateral medulla (VLM) and ventral cerebellum (Cer). (B) (Top) Cdh9 locus on Chromosome 15 (numbers indicate distance from centromere). (Middle) BAC RP23-318N6. Vertical lines indicate Cdh9 exons. (Bottom) Cdh9-LOSL-DTR BAC transgene: insertion at Cdh9 start codon of mOrange sequence and polyadenylation (pA) signals, flanked by loxP sites (triangles), followed by DTR sequence. (C and D) VLM sections of P0 Cdh9-LOSL-DTR mouse immunostained for mOrange to show Cdh9 expression (red) and P0 wild-type mouse immunostained for somatostatin (SST, green), shown aligned [registered by compact nucleus ambiguus (NAC), cranial nerve 7 (nVII), and ventral brainstem surface] in sagittal plane (upper panels) and transverse projection (lower panels). d, dorsal; c, caudal; m, medial. Scale bar, 200 μm. (E to I) preBötC of a P0 Cdh9-LOSL-DTR [(E) and (F)] or Cdh9-LOSL-DTR;Dbx1-lacZ [(G) to (I)] mouse immunostained for Cdh9-mOrange [(E) to (G) and (I), red], SST [(E), green], NEUN [(F), green], or β-galactosidase [Dbx1-LacZ, (H) and (I), blue]. Among Cdh9-expressing neurons, none coexpressed SST (n = 43 cells), all coexpressed NEUN (n = 57), and 56% coexpressed Dbx1 reporter (n = 292, arrowheads). Scale bar [(E) to (I)], 50 μm. (J and K) Whole-cell voltage-clamp recordings of Cdh9/Dbx1 preBötC neurons in slice Cdh9 preparations (top) and simultaneous integrated cranial nerve 12 (cnXII) activity (bottom). Neuron in (J) (neuron 1, table S1) shows bursts in all inspiratory events (inspiratory pattern). Neuron in (K) (neuron 3) shows more widespread activity but bursts only during some events (inspiratory-associated). Scale bars, 500 ms. (L) Schematic of VLM. Cdh9/Dbx1 neurons (blue border) intermingle with SST (green) and other Cdh9 neurons (red) in preBötC. (M and N) Intersectional genetic labeling of Cdh9/Dbx1 preBötC neurons with DTR (immunostain, green) in ~P35 Cdh9-LOSL-DTR;Dbx1-cre mice before (M) and after (N) intraperitoneal DT injection to ablate them. Scale bar, 50 μm.

  • Fig. 2 Respiratory and behavior changes after Cdh9/Dbx1 neuron ablation.

    (A) Plethysmography airflow traces of Cdh9-LOSL-DTR;Dbx1-cre mice before (black) and 2 days after (red) Cdh9/Dbx1 ablation. Note increased grooming and eupneic breaths and decreased active breaths and sniffing after ablation (see key). Scale bar, 2 s. (B) Distribution of respiratory rates (bin size 1 Hz) in 40-min assay of control (wild-type, Cdh9-LOSL-DTR, or Dbx1-cre; black, n = 5) and experimental (Cdh9-LOSL-DTR;Dbx1-cre; red, n = 5) animals before (dashed lines) and 2 days after (solid lines) Cdh9/Dbx1 ablation. (C) Percent of time in plethysmography chamber spent still-sitting (black), grooming (gray), or active (white) by control (n = 6) or experimental (n = 6) mice before (pre) or 2 days after (post) ablation or mock ablation. P values comparing pre- and postablation behavior: active (0.02), grooming (0.02), and still-sitting (0.07). (D) Grooming events in new chamber of Cdh9-LOSL-DTR;Dbx1-cre mice before (black) or after (red) ablation. Solid lines, individual mice (n = 6); dashed lines, average. (E) Duration of behaviors in (C) (mean ± SD, n = 6). After ablation, active episodes shortened (P = 0.005), grooming and still-sitting showed nonsignificant trend to lengthening (P = 0.24 and 0.21, respectively). (F and H) ECoG power spectral analysis [average (solid lines) ± SEM] of 20-min recording (trial 1) of Cdh9-LOSL-DTR;Dbx1-cre [(F), n = 5] or control Cdh9-LOSL-DTR [(H), n = 4] mice before (black) or 4 to 10 days after (red) ablation. δ, delta wave; V, voltage. Active behavior correlates with faster breathing (fig. S15, C to E). (G and I) Time spent in active (solid black line, mean ± SEM) and calm (dashed black line) behavioral states defined by electromyography (EMG) and ECoG (fig. S15) of individual animals in (F) and (H) (gray lines) during two 20-min assays pre- and post-Cdh9/Dbx1 ablation. Note decreased active and increased calm periods following ablation in experimental animals (P = 0.001 and 0.02, respectively, paired t test) and no change in controls (P = 0.86 and 0.81, respectively).

  • Fig. 3 Effect on breathing and behavior of ablation of Cdh9 neurons that project to and synapse on LC neurons.

    (A to D) Rabies virus monosynaptic retrograde trace from dopamine β-hydroxylase (Dbh)–expressing locus coeruleus (LC) neurons. Section through contralateral preBötC (A) to (C) of adult Cdh9-LOSL-DTR;Dbh-cre mouse 5 days after unilateral LC injection of rabies-GFP and helper virus, immunostained to show Cdh9-expressing neurons (mOrange, red). Arrowheads indicate colocalization of GFP and mOrange. Insets highlight boxed region. Scale bar, 50 μm. (D) Schematic of monosynaptic projection (red line) from Cdh9-expressing preBötC neurons (red circle) to contralateral LC, which projects to higher brain structures (arrow). (E) Scheme for ablating only Cdh9-expressing preBötC neurons that project to LC. CAV-Cre virus injected bilaterally into LC of adult Cdh9-LOSL-DTR mice (right) is taken up by Cdh9-expressing preBötC neurons that project there (red). Cre induces DTR expression, and DT injection induces ablation. (F and G) preBötC Cdh9-mOrange expression (white) in control uninjected [(F), mock ablation] and CAV-Cre injected [(G), ablation] Cdh9-LOSL-DTR mice 2 days after DT injection. Scale bar, 50 μm. Quantification showed 32% (mean) and 50% (maximal) reduction in mOrange neurons (n = 15 sections), close to the value expected if all Cdh9/Dbx1 preBötC neurons (50% of Cdh9-expressing neurons) project to LC. (H) Distribution of respiratory rates in 40-min assay (as in Fig. 2B) of CAV-Cre injected Cdh9-LOSL-DTR adult mice (red, n = 7) or wild-type littermates (black, n = 4) before (dashed) and 2 days after (solid) DT injection. (I) Behavioral analysis (as in Fig. 2C) of mice in (H). Pre- versus postablation P values: active (0.015), grooming (0.37), and still-sitting (0.015). The increased calm events in preablation experimental versus control mice was reproducible; it may be due to toxicity of DTR induced in adult neurons, which is not observed in Cdh9-LOSL-DTR;Dbx1-cre mice when DTR is expressed in early development, perhaps due to developmental compensation.

  • Fig. 4 Effect of Cdh9/Dbx1 neuron ablation on LC neuronal activity.

    (A to C, E, and F) c-FOS immunostaining (arrowheads) in LC of adult wild-type mouse in normal environment [home cage, (A)] and of control [wild-type, Cdh9-LOSL-DTR, or Dbx1-cre mice, (B) and (E)] and Cdh9/Dbx1-ablated mice [Cdh9-LOSL-DTR;Dbx1-cre mice 2 days after DT injection, (C) and (F)] after 1 hour in new chamber [(B) and (C)] or in a conical tube under physical restraint [(E) and (F)]. Scale bar, 50 μm. (D) Quantification of c-FOS+ neurons in (A) to (C) and (E) and (F) (mean ± SD) per 25-μm section of LC: (A) 0.4 ± 0.8 neurons (n = 39 sections, 6 animals); (B) 13.8 ± 6.5 neurons (14 sections, 4 animals); (C) 0.1 ± 0.3 neurons (17 sections, 6 animals); (E) 61.4 ± 31.4 neurons (5 sections, 3 animals); and (F) 63.8 ± 19.9 neurons (6 sections, 3 animals). c-FOS+/TH+ neurons, gray; c-FOS+/TH neurons embedded within TH+, black; c-FOS+/TH neurons directly surrounding TH+ LC region, white. (G) Ascending neural circuit from preBötC. Cdh9/Dbx1 preBötC neurons (red) provide monosynaptic excitatory input to noradrenergic LC neurons (red), which project throughout brain to promote arousal and active behaviors. Also shown is the classical circuit from preBötC rhythm-generating neurons (black) to premotorneurons in ventral respiratory group (VRG, black). (H) Model of preBötC with Cdh9/Dbx1 neurons distinct from, but regulated by, rhythm-generating neurons. This provides an ascending respiratory corollary signal to the LC and on to the rest of the brain, separate from classical descending motor circuit. Hence, when breathing speeds up or is otherwise altered, Cdh9/Dbx1 neurons activate LC to induce or maintain an aroused state. [Less direct circuits or downstream events from Cdh9/Dbx1 neurons could also contribute to LC activation, and because LC also regulates sensory modalities (27, 28), sensory alterations could also contribute to LC-induced behaviors. In addition, a direct contribution of Cdh9/Dbx1 neurons to preBötC breathing-rhythm generation cannot be excluded, because compensatory mechanisms may obscure them.]

Supplementary Materials

  • Breathing control center neurons that promote arousal in mice

    Kevin Yackle, Lindsay A. Schwarz, Kaiwen Kam, Jordan M. Sorokin, John R. Huguenard, Jack L. Feldman, Liqun Luo, Mark A. Krasnow

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S15
    • Table S1
    • Caption for Movie S1
    • References

    Images, Video, and Other Media

    Movie S1
    Representative video of Cdh9-LOSL-DTR;Dbx1-Cre mouse behavior before (left) and after (right) Cdh9/Dbx1 neural ablation. The videos are of the same mouse and are from the first 2 minutes of a plethysmography assay.

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