Research ArticleEVOLUTIONARY BIOLOGY

Cardiorespiratory interactions previously identified as mammalian are present in the primitive lungfish

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Science Advances  21 Feb 2018:
Vol. 4, no. 2, eaaq0800
DOI: 10.1126/sciadv.aaq0800
  • Fig. 1 Cardiorespiratory and metabolic recordings from a lungfish, L. paradoxa, at 25°C.

    Recordings of ventilation [recorded as surfacing events (mV)], oxygen uptake (measured as changes in %O2), and heart rate (min−1) during consecutive air-breathing cycles in a single lungfish (220 g). Recordings from 1 to 4 hours after surgery (A) and in the recovered, undisturbed animal 24 hours after instrumentation (B).

  • Fig. 2 Cardiac and metabolic indexes of recovery after instrumentation in lungfish, L. paradoxa, at 25°C.

    (A) Mean heart rate calculated from instantaneous electrocardiogram (ECG) recordings from eight lungfish 6 hours after implantation of electrodes and then every 24 hours for 72 hours. (B) Oxygen uptake from air for eight lungfish after 6, 24, 48, and 72 hours after surgical procedures. (C) A tachogram plot of 8192 consecutive RRi from a single L. paradoxa (196 g) 6 and 24 hours after surgery. (D) PSD derived from RRi collected from eight fish 6 hours after implantation of electrodes and then every 24 hours for 72 hours. Data in (A), (B), and (D) are plotted as means ± SEM. Different letters denote significant differences between mean values (ANOVA with Tukey’s post hoc test, P < 0.05).

  • Fig. 3 Tachogram of RRi and power spectral density L. paradoxa at 25°C.

    (A) A tachogram plot of 8192 consecutive RRi from a single L. paradoxa (260 g) in untreated condition before drug injection (black trace) and following autonomic blockade with atropine (dark gray trace) and atropine + propranolol (gray trace). (B) Average spectral amplitude density from lungfish in an untreated condition (Control: before drug injection, 48 hours after surgery; n = 8, black area), after cholinergic receptor blockade (Atropine; n = 6, small dark gray area), and after total blockade of β-adrenergic and cholinergic receptors (Atropine + Propranolol; n = 6, small gray area). The mean breathing frequency (fR) of the Control (fR, ~1.7 breaths hour−1), Atropine (fR, ~7.3 breaths hour−1), and Atropine + Propranolol (fR, ~5.9 breaths hour−1) groups are represented by black, dark gray, and gray arrows, respectively.

  • Fig. 4 Effects of hypoxia and hypercarbia on respiratory frequency and HRV in L. paradoxa at 25°C.

    Histograms of breathing frequency in 0.0006-Hz bins from a lungfish and HRV within 8192 consecutive heartbeat intervals under normoxia (263 g) (A and B) and hypoxia plus hypercarbia (gas mixture of 95% N2, 5% CO2, and 3% O2) (318 g) (C and D). The spectra represent the relative distribution of frequency components within the HRV and breathing signals. The single, clear, respiration-related peak seen in the HRV signal from normoxic lungfish disappears from the lungfish under hypoxia plus hypercarbia due to the irregular nature of its air-breathing cycles. Spectral amplitude axes are up to 4 × 107 in (B) and 2 × 107 in (D).

  • Fig. 5 Conduction velocity and transmission electron micrographs of cardiac vagus in L. paradoxa (505 g).

    (A) A typical example of a compound action potential recorded following electrical stimulation of the cardiac vagus nerve with a single pulse (0.5 ms, 70 V). Three components were identified (black arrowheads), with conduction velocities of 10.5, 0.23, and 0.16 m s−1. Top left: Action potential recorded from an additional stimulation (0.5 ms, 50 V), recorded with a faster time base to identify the stimulus artifact (black arrow). (B) Photomicrographs obtained from the cross section of the cardiac vagus, which illustrates a representative range of fiber diameters. Arrows point to unmyelinated fibers, and asterisks indicate myelinated fibers. (C) The detail of the layering of the myelin sheath is readily observed.

  • Fig. 6 Transverse sections and topographical illustration of the brainstem of the lungfish.

    (A to C) Micrographs of transverse 40-μm sections of the brainstem rostral to obex, showing preganglionic motoneurons (VPN) labeled with the retrograde fluorescent tracer FG [(A and B) taken 4.9 and 2 mm caudal to 1 mm rostral to obex, respectively; scale bar, 50 μm] and CVPN located by the retrograde transport of the fluorescent tracer DiI [(C) taken 2.1 mm rostral to obex; scale bar, 20 μm]. There are three distinct cell groups (DVNv, DVNd, and DVNl) within the DVN and an SLVN. (D) Schematic diagram of VPN groups in the DVN in relation to the fourth ventricle (v). The cell bodies of VPN and CVPN were located in four groups. The main group comprised the ventrally located DVNv, which divided rostrally to form a separate dorsal subgroup (DVNd). This division merged again more rostrally. A third group of neuron cell bodies appeared at the rostral extent of the DVN (DVNl), and a scattered group of smaller cells was located ventrolaterally, outside DVN (SLVN). SLVN is homologous to the mammalian NA.

  • Table 1 Cardiorespiratory and HRV parameters in L. paradoxa.

    Experimental conditions: after instrumentation (Post-surgery; n = 9), recovered resting fish (Control; n = 8), with cholinergic receptor blockade (Atropine; n = 6), or total blockade of β-adrenergic and cholinergic receptors (Atropine + Propranolol; n = 6). Following instrumentation, lungfish was allowed to recover for at least 24 hours before measurements. Data are means ± SEM. Different superscript letters indicate significant differences [analysis of variance (ANOVA) with Tukey’s post hoc test, P < 0.05]. SDNN, SD of all normal-to-normal intervals; RMSSD, square root of the mean squared differences between each successive RRi and the mean interval; PSD, power spectral density.

    ParametersExperimental groups
    Post-surgeryControlAtropineAtropine + Propranolol
    O2 uptake per breath (ml breath−1 kg−1)0.11 ± 0.02A0.83 ± 0.07B0.29 ± 0.05A,C0.43 ± 0.08C
    Embedded ImageO2 (ml kg−1 hour−1)23.6 ± 1.8A10.6 ± 0.8B24.2 ± 3.1A20.4 ± 2.5A
    fR (breaths hour−1)11.5 ± 1.7A1.7 ± 0.1B7.3 ± 1.5C5.9 ± 1.6C
    Tidal volume (ml kg−1)27.9 ± 1.8A25.3 ± 1.6A26.7 ± 1.7A34.1 ± 1.9B
    Air-breathing cycle (s)8.8 ± 0.4A9.5 ± 0.4A8.6 ± 0.3A9.7 ± 0.4A
    fH (beats min−1)38.8 ± 1.4A29.5 ± 1.5B39.2 ± 1.9A36.3 ± 2.2A
    RR mean (ms)1547.4 ± 58.8A1,947.7 ± 74.7B1548.8 ± 69.8A1682.8 ± 90.4A
    RR variance (ms2)4953.2 ± 1187.8A28,244.1 ± 3,344.7B1834.9 ± 491.5A3690.9 ± 1311.4A
    SDNN (ms)66.6 ± 8.1A192.4 ± 16.5B40.4 ± 6.3A54.9 ± 11.7A
    RMSSD (ms)40.7 ± 4.2A102.8 ± 9.8B22.5 ± 5.5A19.9 ± 4.9A
    PSD (ms2)3664.0 ± 437.3A26,957.5 ± 2,791.1B998.9 ± 364.7A2903.7 ± 970.1A

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/2/eaaq0800/DC1

    fig. S1. Spectral amplitude density from two lungfish (L. paradoxa) with different breathing frequencies.

    fig. S2. Changes in the heart rate in L. paradoxa during an air-breathing event at 25°C.

    fig. S3. The effect of autonomic modulation of HRV on mean oxygen uptake per breath in L. paradoxa at 25°C.

    fig. S4. The rostrocaudal distribution of cell bodies of VPN in the brainstem of L. paradoxa.

    table S1. Morphometric variables in the ultrastructure of transverse sections of the cardiac branches of the vagus nerve in L. paradoxa.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Spectral amplitude density from two lungfish (L. paradoxa) with different breathing frequencies.
    • fig. S2. Changes in the heart rate in L. paradoxa during an air-breathing event at 25°C.
    • fig. S3. The effect of autonomic modulation of HRV on mean oxygen uptake per breath in L. paradoxa at 25°C.
    • fig. S4. The rostrocaudal distribution of cell bodies of VPN in the brainstem of L. paradoxa.
    • table S1. Morphometric variables in the ultrastructure of transverse sections of the cardiac branches of the vagus nerve in L. paradoxa.

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