SC I ENCE ADVANCES | R E S EARCH ART I C L E
EVOLUT IONARY B IOLOGY
1Department of Physiological Sciences, Federal University of São Carlos (UFSCar), São
Carlos, 13565-905 São Paulo, Brazil. 2National Institute of Science and Technology in
Comparative Physiology (INCT FisComp), São Carlos, São Paulo, Brazil. 3School of Bio-
sciences, University of Birmingham, Edgbaston, BirminghamB15 2TT, UK. 4Department
of Zoology, São Paulo State University (UNESP), Rio Claro, São Paulo, Brazil. 5Institute of
Biology, Federal University of Bahia (UFBA), Salvador, Bahia, Brazil.
*These authors contributed equally to this work.
†Corresponding author. Email: cleo.leite@ufscar.br

Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
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Cardiorespiratory interactions previously identified
as mammalian are present in the primitive lungfish
Diana A. Monteiro,1,2* Edwin W. Taylor,1,3* Marina R. Sartori,4 André L. Cruz,2,5

Francisco T. Rantin,1,2 Cleo A. C. Leite1,2†

The present study has revealed that the lungfish has both structural and functional features of its system for phys-
iological control of heart rate, previously considered solely mammalian, that together generate variability (HRV).
Ultrastructural and electrophysiological investigation revealed that the nerves connecting the brain to the heart
are myelinated, conferring rapid conduction velocities, comparable to mammalian fibers that generate instanta-
neous changes in heart rate at the onset of each air breath. These respiration-related changes in beat-to-beat cardiac
intervals were detected by complex analysis of HRV and shown to maximize oxygen uptake per breath, a causal
relationship never conclusively demonstrated in mammals. Cardiac vagal preganglionic neurons, responsible for
controlling heart rate via the parasympathetic vagus nerve, were shown to have multiple locations, chiefly within
the dorsal vagalmotor nucleus thatmay enable interactive control of the circulatory and respiratory systems, similar
to that described for tetrapods. The present illustration of an apparently highly evolved control system for HRV in a
fishwith a proven ancient lineage, based onpaleontological, morphological, and recent genetic evidence, questions
much of the anthropocentric thinking implied by somemammalian physiologists and encouraged bymany psycho-
biologists. It is possible that some characteristics of mammalian respiratory sinus arrhythmia, for which functional
roles havebeen sought, are evolutionary relics that had their physiological role defined in ancient representatives of
the vertebrates with undivided circulatory systems.
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INTRODUCTION
In vertebrates, respiratory and cardiovascular systems are functionally
linked and often exhibit tight interactions that optimize the delivery
and removal of respiratory gases, producing coordinated responses
to variations in metabolic demand or altered gas composition in the
environment (1, 2). In mammals, one form of these cardiorespiratory
interactions (CRIs) is respiratory sinus arrhythmia (RSA). This phe-
nomenon is characterized by heart rate ( fH) increasing during inspira-
tion and decreasing during expiration (3). Power spectral analysis
(PSA) of fH variability (HRV) reveals a peak at a relatively high fre-
quency (HF), which is related to ventilation (4, 5). RSA is, in large part,
mediated by fluctuations of the inhibitory input from the brain to the
heart through the vagus nerve in response to both centrally generated
(feed-forward) influences from respiratory neurons and afferent input
from pulmonary stretch receptors (feedback) that gate baroreceptor
inputs (6, 7). Beat-by-beat control is dependent on rapid conduction
of bursts of efferent activity down the cardiac vagus, which contains
myelinated “B-fibers” (7, 8). These CRIs are generated centrally by va-
gal preganglionic neurons (VPNs) and, in particular, cardiac VPNs
(CVPNs), located primarily in the ventrolateral nucleus ambiguus
(NA),where they interactwith the ventral group of inspiratory neurons
(1, 9–12). The existence of the NA and the central interactions gener-
ating RSA have been described frommammalian studies, and as a con-
sequence, biomedical physiologists are apt to consider them uniquely
mammalian, whereas a group of psychobiologists have erected a “poly-
vagal theory” that considers themammals tohave a “smart vagus,”denied
to other vertebrates, that provides a highly orchestrated set of physiolog-
ical and behavioral functions (13).We have questioned the uniqueness of
the structural and functional features of the mammalian cardiac vagus
and its consequent role in the control of CRIs, as we consider these
features to have evolved in the early phylogeny of the vertebrates.

This study set out to trace the evolutionary origins of mammalian
RSA in the patterns of CRI typical of early air-breathing vertebrates by
investigating its control in the South American lungfish (“piramboia”),
Lepidosiren paradoxa. Lungfishes (Dipnoi) have previously been iden-
tified as a key group for the study of the evolution of the mechanisms
that led to effective air breathing in vertebrates (11, 14). They aremem-
bers of a group of lobe-finned fishes that constitute the Sarcopterygii,
a group identified from a continuous fossil record originating in the
Devonian period around 400 million years ago. The sarcopterygians
split into twomain lineages—the coelacanths that never left the oceans,
presently represented by the “living fossil” Latimeria, and the rhipidis-
tians thatmigrated into freshwater habitats. They, in turn, split into two
major groups: the lungfishes and the tetrapodomorphs (15, 16). The
lungfish evolved the first proto-lungs and proto-limbs, developing the
ability to survive the temporary loss of their water environment,
whereas the tetrapodomorphsmigrated onto land to become committed
air breathers, giving rise to present-day amphibians, reptiles, birds, and
mammals. Thus, the lungfishes represent the earliest stages in the evo-
lutionary record of air-breathing vertebrates. L. paradoxa hasmany ple-
siomorphic features characteristic of early tetrapods such as limb and jaw
structure and an independent circulation for its primitive lung (16).
L. paradoxa is the single representative of the Dipnoi in South America.
It inhabits freshwater pools and slow-flowing rivers, which often contain
very low levels of dissolved oxygen and are prone to dry out completely
during dry seasons. The lungfish is an obligatory air breather, rising to
the surface at regular intervals to ventilate its lung-like air-breathing or-
gan (16), and can survive for long periods in burrows during drought.
Thus, Lepidosiren depends almost exclusively on its lungs for O2 uptake
and shares a periodic breathing pattern with the tetrapod vertebrates.
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The aim of the present work was to study in detail the nature and
control of the CRIs that occur during air breathing in L. paradoxa, to
elucidate the role of the autonomic nervous system in generating these
interactions, and to identify their functional characteristics. We hy-
pothesized that furthering our understanding of these relationships
in this primitive vertebratemay uncover their origins and present func-
tional roles inmammals. The investigation encompassed physiological
studies on unrestrained fish that used PSA of HRV plus ultrastructural
and functional studies on the cardiac branches of the vagus nerve and
neuroanatomical studies on the projections of the vagus nerve into the
brainstem.
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RESULTS
Recovery from surgery
After instrumentation, each animal was left in the respirometer to re-
cover from the effects of anesthesia and surgery, as described in Mate-
rials and Methods. General recovery was attested as stabilization of
cardiorespiratory and metabolic variables: mean heart rate ( fH), fre-
quency of air breaths ( fR), O2 uptake ( _VO2), and HRV (Figs. 1 and
2 and Table 1).
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Cardiorespiratory parameters
Whenfirst inserted into the respirometer, the lungfish,L.paradoxa, showed
elevated fH (38 beatsmin−1), with fR both elevated (11 respirationsmin−1;
Table 1) and irregular (Fig. 1A shows these variables from 1 to 24 hours
after instrumentation). When allowed to settle for over 24 hours, both
fH and fR fell to lower stable levels (Table 1), with fH showing regular,
marked increases associatedwith each air breath (Fig. 1B).Undisturbed,
recovered lungfish displayed a regular episodic breathing pattern ( fR,
1.7 breaths hour−1). Mean tidal volume and duration of each exhala-
tion and inhalation cycle (mean ± SEM) were 25.3 ± 5.1 ml kg−1 and
10.7 ± 1.2 s, respectively (Table 1). Mean fH was stable, with a value of
29.7 ± 1.0 beats min−1, and O2 uptake fell to a stabilized level (Fig. 2, A
and B, and Table 1). O2 uptake was restricted to the period of visits to
the air-water interface, and heart rate increased markedly at each air
breath (Fig. 1B). A tachogram of R-R intervals (RRi) revealed that they
were relatively stable 6 hours after surgery but became variable and
showed a marked reduction at each air breath after a recovery period
of 24 hours (Fig. 2C). Recovery ofHRV after 24 hours was accompanied
by an increase in total power, as revealed by PSA to a stable high value
(~575 to 975%) in comparison with immediately post-operative
values (Fig. 2D and Table 1).

HRV and CRIs at rest in recovered fish
PSA of RRi in lungfish, following 24 hours of recovery, revealed pro-
nounced RRi variation at each air breath identified as a large peak in
the spectrum of PSA from each individual fish (Fig. 3, A and B, and
fig. S1, A and B). These lungfish showed a clearly defined peak in the
power spectrum at their individual breathing frequencies, demon-
strating clearly defined CRIs. Because of the low respiratory rate of
lungfish (in the range of 0.0003 to 0.0005 Hz, equivalent to 1.08 to
1.80 breaths hour−1), the peaks are located at relatively “very low fre-
quencies” (VLF), according to mammalian criteria (Task Force, 1996;
see Materials and Methods). These respiration-related peaks likely
incorporate other sources of variability, but the regular air breaths
dominate the recordings.

Role of the autonomic nervous system in control of HRV
A tachogramof 8192 consecutiveRRi froma single normalL. paradoxa
(before drug injection; 48 hours after surgery) revealed a high level of
HRV accompanied by a sharp reduction in cardiac interval at each air
breath. Autonomic blockade reducedHRV and eliminated the changes
associatedwith air breathing (Fig. 3, A and B). The effects of autonomic
blockade on the mean of power spectra derived from eight lungfish are
shown in Fig. 3B. Pharmacological blockade with atropine and pro-
pranolol reduced total PSD by about 2.5 times. There was no difference
between atropine and double autonomic blockade.

The administration of atropine alone or when combined with pro-
pranolol significantly decreased HRV to levels similar to those mea-
sured in fish before their recovery from instrumentation. Consequently,
the highest variance, SDNN, RMSSD, and total power were found in
recovered fish 24 hours after instrumentation (Table 1).

The time course of changes in fH at a series of air breaths was ana-
lyzed from recordings in eight lungfish following a 24-hour recovery.
Instantaneous heart rate was recorded over 100 heartbeats around each
air breath, and the beat-to-beat changes were related to the moment at
which each fish exhaledbefore inspiring air. Thesedata revealed amarked
and rapid increase in mean fH by 22%, from 29.5 ± 1.5 to 35.6 ± 1.8 at
each air breath in normal, inactive fish (fig. S2, A and B). This increase
seemed to be divided into two phases: an initial rapid increase in fH at
Fig. 1. Cardiorespiratory andmetabolic recordings froma 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 instrumen-
tation (B).
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surfacing (within one heartbeat) and a further progressive increase in fH
during the breathing episode. Injection of the cholinergic receptor an-
tagonist atropine into six lungfish eliminated beat-to-beat fluctuations
and raisedmean fH to 39.2 ± 1.9. Total autonomic blockade, achieved by
Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
injection of the b-adrenergic receptor antagonist propranolol, returned
fH to 36.3 ± 2.2 (fig. S2 and Table 1), the fH value following an air breath
before autonomic blockade (fig. S2), implying that a normal air breath is
accompanied by suppression of autonomic tonic control.
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).
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 b-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.
Parameters

Experimental groups
Post-surgery
 Control
 Atropine
 Atropine + Propranolol
O2 uptake per breath (ml breath−1 kg−1)
 0.11 ± 0.02A
 0.83 ± 0.07B
 0.29 ± 0.05A,C
 0.43 ± 0.08C
_VO2 (ml kg−1 hour−1)
 23.6 ± 1.8A
 10.6 ± 0.8B
 24.2 ± 3.1A
 20.4 ± 2.5A
fR (breaths hour−1)
 11.5 ± 1.7A
 1.7 ± 0.1B
 7.3 ± 1.5C
 5.9 ± 1.6C
Tidal volume (ml kg−1)
 27.9 ± 1.8A
 25.3 ± 1.6A
 26.7 ± 1.7A
 34.1 ± 1.9B
Air-breathing cycle (s)
 8.8 ± 0.4A
 9.5 ± 0.4A
 8.6 ± 0.3A
 9.7 ± 0.4A
fH (beats min−1)
 38.8 ± 1.4A
 29.5 ± 1.5B
 39.2 ± 1.9A
 36.3 ± 2.2A
RR mean (ms)
 1547.4 ± 58.8A
 1,947.7 ± 74.7B
 1548.8 ± 69.8A
 1682.8 ± 90.4A
RR variance (ms2)
 4953.2 ± 1187.8A
 28,244.1 ± 3,344.7B
 1834.9 ± 491.5A
 3690.9 ± 1311.4A
SDNN (ms)
 66.6 ± 8.1A
 192.4 ± 16.5B
 40.4 ± 6.3A
 54.9 ± 11.7A
RMSSD (ms)
 40.7 ± 4.2A
 102.8 ± 9.8B
 22.5 ± 5.5A
 19.9 ± 4.9A
PSD (ms2)
 3664.0 ± 437.3A
 26,957.5 ± 2,791.1B
 998.9 ± 364.7A
 2903.7 ± 970.1A
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Resting and undisturbed lungfish exhibited a large cholinergic to-
nus of 32% and a relatively small adrenergic tonus of 7.5%, indicating
that any increase in fH could be due towithdrawal of vagal tone. Hence,
both the maintenance of mean fH and the modulation of RRi around
each air breath show major dependence on vagal activity. This mech-
anism is evident in the effects of atropine injection on the elevation in
heart rate and consequent abolition of the increase in RRi at each air
breath in normal lungfish (Fig. 3 and fig. S2).

Following a 24-hour recovery from instrumentation, O2 uptake per
breath increased seven times compared to the value immediately after
instrumentation. Autonomic modulation of RRi proved essential for
normal levels of O2 uptake, as injection of autonomic antagonists
markedly decreased O2 uptake per breath, by 65% after cholinergic re-
ceptor blockade and by 48% after total blockade (Table 1 and fig. S3).

Effects of changes in respiratory gas composition
The power spectrum representation of HRV and histogram of the
breathing frequency of a L. paradoxa, following recovery from surgery,
are presented in Fig. 4. The regularity of surfacing events for air breaths
under normoxia resulted in clear coincident peaks in the histogram and
power spectrum of HRV (Fig. 4, A and B). Following exposure to com-
Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
bined hypoxia and hypercarbia, the frequency of air breaths markedly
increased and became irregular (mean value, 11.2 ± 0.9 breaths hour−1

or ~0.0031 Hz; Fig. 4C). Also, mean tidal volume and duration of each
exhalation and inhalation cycle were 80.1 ± 3.51 ml kg−1 and 19.8 ±
0.9 s, respectively, which represented significant increases by 3.3 and
2.1 times, respectively, when compared to the normoxic condition (re-
covery group, 24 hours after surgery; Table 1). So, exposure to a com-
bination of hypoxia and hypercarbia caused an increase in respiratory
drive and disruption of the regularity of air breathing. As a conse-
quence, the characteristic respiration-related peak on the power
spectrum for HRV disappeared. The spectrum presented a diffuse col-
lection of peaks, scattered over the spectral bandwidth (Fig. 4D).

Conduction velocity and structure of the cardiac vagus
Peripheral electrical stimulation of the left cardiac branch of the vagus
(at 24°C) evoked a compound action potential with a predominant fast
component (9.0 to 10.5 m s−1) plus discrete, considerably smaller and
slower components (0.15 to 0.25 m s−1; Fig. 5A). These can be sep-
arated into “B-fiber” and “C-fiber” responses on the basis of mamma-
lian criteria, with the slow components possibly recorded from the
unmyelinated fibers identified in Fig. 5A that may be sensory fibers
stimulated antidromically in this preparation.

Transmission electron microscopy (TEM) of serial transverse sec-
tions of the cardiac branch of the vagus nerve in L. paradoxa (Fig. 5, B
and C) revealed the occurrence of large and small myelinated fibers,
intermingled with bundles of unmyelinated ones. The mean values
for the number and density of myelinated and unmyelinated fibers,
their area and diameter, the percentage of occupancyof types, themean
myelin thickness, and the myelinated/unmyelinated fiber ratio are
provided in table S1. There is a predominance of myelinated fibers in
the cardiac vagus of L. paradoxa, as revealed by the ratio ofmyelinated/
unmyelinated fibers. These fibers displayed relatively large diameters
(up to 9.8 mm) and myelin thicknesses (up to 1.6 mm). Myelinated
nerve fibers accounted for at least 55% of the tissue volume, and their
distribution is not symmetric in a nerve section, constituting clusters of
fast fibers. Myelinated fiber density and its percentage of occupancy in
the nerve section were both significantly higher in the left cardiac
branch, denoting a possible bilateral asymmetry of cardiac innervation.
Unmyelinated fibers with fiber diameters ranging from 2.2 to 4.8 mm
were equally distributed in the right and left cardiac vagus (table S1).

Neuroanatomy of VPN in the brain stem
Injection of the neural tracer fluorogold (FG) revealed VPN cell bodies
distributed in four distinct locations in either side of the midline in the
brainstem of lungfish (Fig. 6 and fig. S4A). Three of these locations
were positioned close to the fourth ventricle, within the dorsal motor
nucleus of the vagus (DVN): a large ventral group (DVNv), containing
a total of 3000 labeled cells, extending from 12.4 mm caudal to 4.6 mm
rostral to obex (Fig. 6, C andD, and fig. S4B). A smaller group of dorsal
DVN (DVNd) formed by the separation of the large group of DVN
midway through its rostrocaudal extent and found from 8 mm caudal
to 4.8 mm rostral to obex, with a total of 300 cells (Fig. 6, B and D, and
fig. S4C). Also, there is a separate, more rostral group characterized as
the lateral DVN (DVNl) because of their location close to the much
expanded fourth ventricle composed of 100 cells from 1.9 to 4.6 mm
rostral to obex with a large number of cells in each section (4 to 20 cells;
Fig. 6, A and D, and fig. S4D). The fourth location was composed of a
group of smaller cells with a diffuse distribution ventrolaterally outside
of the DVN, identified as scattered lateral vagal motoneurons (SLVNs)
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 b-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.
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and found from 4.9 to 6.0 mm rostral to obex, with a total of about
300 cell bodies (Fig. 6, B and D, and fig. S4E).

CVPN, labeled by DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-
carbocyanine perchlorate) applied topically to the heart, constituted
about 23% of the VPN-labeled cells (Fig. 6C). CVPNs were distributed
from 3.4 mm caudal to 5.8 mm rostral to obex, partially overlapping
the distribution of VPN (fig. S4, B to E). They were located within all
cell groups described above, with CVPN constituting 20% of VPN in
the DVNv, 67% in the DVNd, 6% in the DVNl, and 11% in the SLVN
(fig. S4, B to E).
DISCUSSION
Morphological and paleontological evidence has long placed the
lungfishes phylogenetically close to the origin of the lung-breathing tet-
rapod vertebrates. A recent study of limb regeneration has extended
this evidence by revealing morphological steps similar to those seen
in salamanders (17). These similarities are mirrored in the processes
of differential gene expression that initiate and regulate limb regenera-
tion in both groups. This recent evidence strongly supports the hypoth-
esis that lungfish occupy the bona fide transition point from fish fin to
tetrapod limb during the move onto land, and we have explored the
complementary hypothesis that they have a primitive version of the
control systems enabling effective air breathing that is conserved in tet-
rapod vertebrates, including mammals. Our investigation has revealed
Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
that L. paradoxa has both structural and functional features controlling
CRIs that have previously been considered solely mammalian.

Monitoring respiratory and cardiovascular variables over 72 hours
revealed that recovery from the combined effects of anesthesia and in-
strumentation was complete after 24 hours. Recovery was assessed by
stabilization of several factors: cardiorespiratory parameters (mean fH
and fR), an index of metabolic rate _V

�
O2), andHRV, which is an index

of normal autonomic modulation of the heart. Analysis of HRV by
PSA of RRi revealed that the highest variance, assessed as SDNN,
RMSSD, or total power, was found in recovered fish 24 hours after sur-
gery. A tachogram of RRi from a L. paradoxa 48 hours after surgery
revealed a high level of HRV due largely to a sharp reduction in cardiac
interval at each air breath. There was a clear peak in the spectrum for
HRV at the air-breathing frequency for different fish. The marked rise
in fH at each air breath in settled, recovered L. paradoxa contrasts with
measurements on other air-breathing fishes that typically show a sharp
reduction in fH at the moment of gulping air that precedes an equally
sharp rise in fH as they inflate the air-breathing organ (18). Our data
reveal a two-stage increase in heart rate during an air breath that com-
menced at or immediately before surfacing. This implies that the initial
increase is independent of inflation of the lung and is likely to be cen-
trally generated.

Oxygen uptake per breath was low before recovery, whereas fH,
_VO2, and air-breathing frequency were high. The high, unvarying fH
and increased fR together constitute the immediate response of each
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 andD). 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).
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fish to the stresses imposed by handling, anesthesia, and operative pro-
cedures. The increased _VO2may reflect a general increase inmetabolic
rate but, in part, will fuel the energy required for the frequent visits to
the water surface. Following recovery, 24 hours after re-release into the
holding tank, breathing frequency became slow and regular, and each
breath was associated with a marked increase in both fH and oxygen
uptake per breath. Thus, the present study has provided evidence of
a clear link between the marked acceleration in fH at each air breath,
identified in the analysis of HRV, and the consequent _VO2 in un-
restrained, settled lungfish. This increase in _VO2 per breath in recov-
ered lungfish represents an essential increase in efficiency (high rates of
gas exchange in relation to work done) because the energy required to
breathe is relatively high in air-breathing fishes that must gulp air from
the water surface, using feedingmuscles inserted on the pectoral girdle
and innervated by the hypobranchial nerves (19, 20). Also, each visit
requires energy for movement and orientation.

The infusion of atropine in L. paradoxamarkedly elevated fH to the
rate achieved following an air breath, revealing a parasympathetic to-
nus of 32%, whereas the subsequent injection of propranolol revealed a
sympathetic tonus of 8%. Virtually identical levels of cardiac autonom-
ic tone were recorded from the African lungfish Protopterus annectens
(21). Given the lack of adrenergic innervation of the lungfish heart
(22, 23), the relatively low levels of adrenergic tone could be due to local
Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
release of catecholamine from endogenous chromaffin cells, which line
the atrial lumen (22, 23).

Autonomic blockadewith atropine andpropranolol elevated fH and
eliminated the changes associated with air breathing. The total power
spectra were decreased by about 2.5 times, with HRV similar to that
measured in fish before their recovery from surgery. So, the lack of
HRV recorded 6 hours after surgery can be attributed to the loss of a
predominant parasympathetic inhibitory tone, responsible for the loss
of beat-to-beat control of fH in the lungfish. The loss of autonomic con-
trol of the cardiovascular system commonly results from invasive
instrumentation or anesthesia (24). In addition, exposure to experi-
mentally induced hypoxia plus hypercarbia in L. paradoxa generated
markedly irregular breathing cycles. The loss of the regular bouts of air
breathing was accompanied by an increased fH and by a decrease in
the PSD ofHRV from the levels characteristic of settled normoxic and
normocapnic lungfish.

Our interpretation of the role of HRV in maximizing the oxygen
uptake per breath must consider previous knowledge of the nature
of peripheral control of perfusion of the dual respiratory gas exchang-
ers in lungfish. Whereas heart rate increased during the intermittent
ventilation of the lungs in both the South American and Australian
lungfishes, L. paradoxa and Neoceratodus forsteri, in the African
lungfish, Protopterus sp., there were very small changes in heart rate
(25). However, in each of these species, pulmonary ventilation is asso-
ciated with a marked rise in pulmonary blood flow, which is accom-
plished by vascular shunt control (25). Flow through the branchial
Fig. 5. Conduction velocity and transmission electron micrographs of cardiac
vagus in L. paradoxa (505 g). (A) A typical example of a compound action po-
tential 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 mye-
linated 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-mm 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 mm] and CVPN located by the retrograde
transport of the fluorescent tracer DiI [(C) taken 2.1 mm rostral to obex; scale bar,
20 mm]. 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 divisionmerged 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.
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circulation is controlled by the ductus arteriosus, whereas perfusion of
the lung is controlled by the pulmonary arterial vasomotor segment
(PAVS), a muscular segment of the pulmonary artery with vasomotor
activity (16, 23, 26, 27). Cholinergic stimulus dilates the ductus while
constricting PAVS, indicating that both structures are controlled by
cholinergic fibers, possibly of vagal origin (26, 28). During water
breathing, cholinergic activation causes decreased pulmonary blood
flow due to constriction of the pulmonary artery and dilation of both
the ductus and brachial shunts, thus directing blood to the dorsal aorta
and the branchial circulation (26, 29). During each air breath, a con-
siderable portion of blood flow shifts from the systemic to the pulmo-
nary circulation, with 20 to 70% of the total cardiac output directed to
the lungs (30, 31).

Thus, CRI adjustments in fH are an important part of a complex
mechanism for the control of gas exchange that involves several
cardiovascular adjustments, including venous return, myocardial con-
tractility, net shunt control, and the control of perfusion in the respi-
ratory tissue in the lungs. Notwithstanding the complex cardiovascular
interactions underlyingCRI, HRV is a fundamental part of it because it
links directly with cardiac output. If the importance of the changes
in fH, expressed as peaks in the HRV spectrum at each air breath, is
emphasized, then parallels between these change in CRI and similar
respiration-related changes in CRI in other vertebrates can be com-
pared. In water-breathing fish, perfusing a branchial circulation in se-
ries with the systemic circulation, several forms of CRI have been
described, including cardiorespiratory synchrony, hypoxic bradycar-
dia, or an increase in fH during ram ventilation (1, 11, 32). In amphib-
ians and reptiles, CRI is often associated with bouts of discontinuous
ventilation, accompanied by cardiac shunt alterations (2, 33–35),
whereas in mammals, CRI exists as RSA (1, 7, 11, 12, 20, 36).

Mammalian RSA shows an increase in fH during inhalation and a
reduction during expiration. It is more prevalent in healthy, fit individ-
uals, arises close to term in fetal development, providing an indicator of
normal development of the brainstem (11, 37), and is reduced in old
age (5). All of these features imply a functional role in cardiorespiratory
integration. Central control arises fromchanges in the activity of CVPN
located outside of the DVN, in the ventrolateral NA (1, 7, 11, 12). In
mammals, about 30% of VPNs, but up to 80% of CVPNs, are in the
NA. Here, they are influenced by activity in the neighboring group of
respiratory neurons that, in turn, are affected by afferent input from
pulmonary stretch receptors that gate baroreceptor inputs (1). Activity
in the respiratory neurons inhibits activity in CVPN, eliminating their
tonic effect on the heart and causing the respiration-related changes fH
that constituteRSA (5, 7, 38). The beat-to-beatmodulation of heart rate
in mammals that constitutes RSA has been shown to be reliant on the
rapid conduction velocity of efferent cardiac fibers arising from CVPN
in the NA, conferred upon them by myelination (8). In mammals,
myelinated/unmyelinated fiber ratios range from1:3 to 1:7, and a larger
diameter (up to 10 mm) is commonly reported for myelinated fibers
(39, 40). The existence of dual locations for VPN outside of the
DVN and, in particular, the location of CVPN in the ventrolateral
NA, having myelinated efferent axons, have been considered uniquely
mammalian, constituting their smart vagus and providing the physio-
logical basis for the polyvagal theory recently reviewed by Porges (13).

However, these central interactions are not unique to mammals. In
an elasmobranch fish, the dogfish, or catshark (Scyliorhinus canicula),
CVPNs in the DVN show respiration-related activity that is driven
rather than inhibited by activity inneighboring respiratorymotoneurons
(1, 11, 12, 19, 41, 42). The resultant respiration-related efferent activity
Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
in the cardiac vagi is able to influence instantaneous fH, generating car-
diorespiratory synchrony. In L. paradoxa, each air breath is accompa-
nied by an increase in heart rate, induced by a reduction in vagal tone,
implying that activity in CVPN is inhibited during “inspiration,” as de-
scribed in mammals. So, this primitive air-breathing fish has a control
system generatingCRI that ismammalian-like rather than piscine. The
present study describedVPN in four separate nuclei in the brainstemof
Lepidosiren, all of them containing CVPN. All major groups were lo-
cated in the DVN, but we describe a small group of VPN (identified as
SVLN) in scattered locations ventrolateral to these groups that may
constitute a primordial NA. Measurement of conduction velocities in
the cardiac nerves revealed that a dominant component of the com-
pound action potential had a high conduction velocity (~10 m s−1),
resembling the fast conducting B-fibers described from the mamma-
lian vagus. Ultrastructural examination of transverse sections of these
nerves revealed a large proportion of myelinated fibers. This represent-
ative of a primitive vertebrate group, with its origins located close to
those of the tetrapods, has the structural and functional equipment that
enables it to exert instantaneous, beat-to-beat control of the heart,
which could be considered reasonably “smart.”

There is plenty of evidence for fine and rapidly exerted control of
the heart in other nonmammalian vertebrates. Those with dis-
continuous breathing patterns, such as air-breathing fish or diving tet-
rapods, typically display bradycardia during apnea and a pronounced
cardiac acceleration immediately upon the first air breath, implying
overriding central nervous integration of their cardiorespiratory sys-
tems (10, 11). The increased heart rate immediately at the onset of pe-
riodic air breathing results mainly from withdrawal of vagal activity to
the heart due to central interactions between motor neurons driving
both systems (1, 11). The rapid response with a marked tachycardia
often developing between two heartbeats implies a B-fiber response
dependent on myelinated efferent fibers. Data from S. canicula (41, 42)
showed that the cardiac vagus exhibited conduction velocities between
4.75 and 16.3 m s−1 similar to those described as B-fibers in mammals
(10, 32). Myelination of the vagal branches has been described in fish,
mammals, and birds (5, 40–43). Two locations for CVPN were de-
scribed for S. canicula, with those in DVN showing respiration-related
activity that could recruit the heart into cardiorespiratory synchrony,
whereas a scattered ventrolateral group showed sporadic activity linked
to hypoxic bradycardia (32, 42). The proportion of VPN located outside
of the DVN varies markedly between groups of nonmammalian verte-
brates, from30 to 40% in some amphibians and reptiles, whereas in fish,
other reptiles, and birds, it can be as low as 2 to 5% (11, 20). This may
reflect the relative importance of central, feed-forward versus reflex,
feedback control exerted on the heart via the vagus nerve (11, 12).

Porges (44) has promoted the polyvagal theory, ascribing to itmany
behavioral and even social functions in humans such as “love.”We do
notwish to intervene in the debate around these extensions of the poly-
vagal theory; however, we do contest its physiological basis and evolu-
tionary assumptions. According to Porges (13), CVPN originating in
theNA and supplying the heart withmyelinated efferent axons, having
high conduction velocities, constitutes the smart vagus restricted to
mammals, whereas the “lower” vertebrates retain the ancient vagus,
restricted to the DVN and supplying unmyelinated, slow fibers that
generate slowly developing, defensive changes in heart rate. He identi-
fies a phylogenetic progression from the regulation of the heart by
endocrine communication, to unmyelinated nerves, and finally to my-
elinated nerves found exclusively in mammals (13) and persists in
stating that “only mammals have a myelinated vagus,” linking this to
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the evolution of the NA (13). The present study reveals that the mech-
anisms he identifies as solelymammalian are undeniably present in the
lungfish that sits at the evolutionary base of the air-breathing verte-
brates. These include (i) a predominant role for vagal control of heart
rate, as described for many other vertebrates, including mammals;
(ii) close coupling between respiration and heart rate recorded as in-
stantaneous changes in heart rate due to changes in vagal control at the
onset of each air breath, detected by using PSA to characterize HRV;
(iii) efferent vagal fibers with fast conduction rates (similar tomamma-
lian B-fibers) conferred upon them by myelination; and (iv) CVPN,
responsible for controlling heart rate via the parasympathetic vagus
nerve having multiple locations within the dorsal vagal motor nucleus,
plus scattered cells in a more ventrolateral location that could conceiv-
ably be a primordial NA. Together, these enable interactive control of
the circulatory and respiratory systems, similar to that described for
tetrapods, including mammals. A closer examination of the available
literature reveals that other groups of vertebrates also presage the
mechanisms required for fine control of heart rate (1, 11). Several
authors have shown that HRV related to respiration is present in spe-
cies of amphibians (2), reptiles [for example, rattlesnakes (45)], and
birds [ducks (10, 11) and shearwaters (43)]. Thus, the repeated conten-
tion, central to the polyvagal theory, that the structural and functional
bases of RSA are solely mammalian is clearly fallacious.

It has been suggested that RSA or HRV in synchrony with respira-
tion may increase the effectiveness of pulmonary gas exchange in
mammals bymatching perfusion to ventilation within each respiratory
cycle (5, 46). Although there is robust evidence that the incidence of a
clear RSA is correlated with health, age, and several particular cardio-
vascular problems in human subjects (47–49), we have lacked direct
experimental evidence of its physiological role. Hayano et al. (50) de-
veloped amodel of RSA in anesthetizeddogs to examine the hypothesis
that RSA improves the effectiveness of pulmonary gas exchange. The
authors demonstrated that artificial RSA could decrease the ratio of
physiological dead space to tidal volume and the fraction of intra-
pulmonary shunt by 10 and 51%, respectively, and increase O2 con-
sumption by 4% compared with control. This small improvement
obtained from such an extreme preparation does not support a clear
physiological role for RSA in the normally functioning mammal. By
contrast, the present data from lungfish show that HRV maximizes
oxygen uptake per breath in settled, normal animals, indicating a pos-
itive influence on gas exchange at the level of the lung via effective
ventilation/perfusion matching.

It is possible that investigation of a clear functional role for RSA in
mammals failed to experimentally record an important quantitative
improvement in gas exchange because of the completely divided circu-
lation, characteristic of the group. This possibility invites a provocative
evolutionary hypothesis: that RSA inmammals is a primitive character
with a minimal quantitative role in respiratory gas exchange because
their completely divided circulation obviates a cardiac or circulatory
shunt between systemic and pulmonary circulations. In air-breathing
vertebrates having undivided circulations, the elevated heart rate during
inspiration is accompanied by a reduction in the peripheral resistance
of the lung circuit (both under vagal control), causing increased perfu-
sion and exchange of respiratory gases over the lung. This does not re-
fute the alternative hypothesis that RSA in mammals can save cardiac
work.While disputing the evidence for RSA improving respiratory gas
transport, Ben-Tal et al. (51), using complex modeling procedures,
raised the idea that RSA could minimize cardiac work while maintain-
ing physiological levels of the partial pressure of CO2. Thus, it is possible
Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
to propose that the changes in heart rate observed during inspiration
and characterized as RSA can only play a substantial role in improving
the effectiveness of respiratory gas exchange over the lung in non-
mammalian, non-avian species, having an undivided circulation with
a functional cardiac or circulatory shunt. Hence, until further experi-
mental validation, we cannot refute the hypothesis that RSA may be
a relic character in mammals that has been retained over evolutionary
time, having a rather limited role in improving respiratory gas exchange
in fit, young individuals while providing some degree of protection by
reducing cardiac work. Thus, the present study has illustrated that, ap-
parently, highly evolved control systems can have primitive roots. This
finding should give pause for thought tomany biomedical physiologists
and, in particular, to a faction within psychobiology (13), questioning
their often anthropocentric assumptions.
MATERIALS AND METHODS
Animals
Specimens of piramboia, the South American lungfish, L. paradoxa, of
either sex and a mean body mass of 0.26 ± 0.2 kg (mean ± SD) were
obtained fromcommercial suppliers inMatoGrosso do Sul, Brazil. The
fish were transported to a holding facility in the Department of Physi-
ological Sciences, FederalUniversity of SãoCarlos (UFSCar), SãoCarlos,
São Paulo state, Brazil, where they weremaintained indoors, in 500-liter
holding tanks, supplied with a flow of well water at 24 ± 2°C, under
controlled photoperiod (12 hours:12 hours). The fish were fed to sati-
ation every 2 days with fish pellets (40% of protein) but were fasted for
48 hours before use in experiments. This study was performedwith the
approval of the Committee of Ethics in Animal Experimentation
(CEUA; approval no. 2032180216) of UFSCar and in accordance with
national guidelines for the care and use of laboratory animals.

Physiological experimentation: Surgery, recovery,
and recording conditions
The fish used for cardiorespiratory recording were anesthetized by im-
mersion in a solution of benzocaine (0.5 g liter−1) and then placed on a
surgical tray with access to air for breathing with the gills irrigated by a
flow of water containing the anesthetic (0.25 g liter−1). Using sterile
techniques, two ECG electrodes were inserted under the skin in the
ventral midline: one just anterior to the heart and the second electrode
approximately 2 to 3 cm caudal to that position and posterior to the
heart. Electrodes and wires were secured on the skin by cotton sutures.
Lidocaine (2%) was injected at the site of electrode insertion. The
procedure took about 5 min. Recovery from anesthesia was achieved
by gill/skin irrigation with anesthetic-free water. After recovery of
righting reflexes and normal movement, each fish was released into
anesthetic-free water with access to air to complete recovery (20 to
30min). Each fishwas thenplaced in a purpose-built respirometerwith
both aquatic and aerial phases. The chamber contained 7 liters of con-
tinuous flow normoxic water (140 mmHg) maintained at 25°C. The
fish had open access from the water surface to an air space (200 ml)
composed of a translucent plastic funnel attached to the top of the res-
pirometer. Recordings started 1 hour after each fish was placed into the
respirometer.

The experiments were conducted with the aim ofmonitoring settled
fish, showing routine levels of activity. Mean heart rate ( fH), oxygen
uptake ( _VO2), and HRV were recorded continuously for 8 to 10 hours
each day for up to 3 days to follow and evaluate the time course of re-
covery from instrumentation. The experimental protocol was followed
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on fish reckoned as fully recovered from anesthesia and the operative
procedures, based on the overall metabolic and autonomic indices, re-
flected by _VO2 and HRV.

Recordings of cardiorespiratory parameters
The ECG was recorded at 1 kHz, amplified and filtered using a bioam-
plifier and data acquisition unit (PowerLab system, ADInstruments),
and used as ameasure ofmean fH. Instantaneous fHwas calculated from
RRi on the ECGwaveform and used to obtain the sequence of intervals
required for derivation of HRV, as described in the following sections.

To record the incidence and volume of air breaths, a Fleisch tube
(pneumotachograph) connected to a differential pressure transducer
[adapted from Glass et al. (52)] was connected to the open end of the
funnel. The air chamber was continuously flushed at 400mlmin−1 via a
mass flow controller (Sable Systems International). The excurrent air
passed through columns of Drierite (Sigma-Aldrich) and an O2 ana-
lyzer (FC-10A; Sable Systems International). All signals were recorded
continuously by a data acquisition system (ADInstruments) and used
to calculate the frequency of air breaths ( fR) and O2 uptake per breath.

Heart rate variability
To characterize HRV using PSA, the raw ECG signal was converted
into a tachogramofRRi froma continuous trace consisting of 8192 con-
secutive heartbeats, containing no artifacts. This long time series was
necessary to assemble proper recordings of variations in RRi through
6 to 10 consecutive ventilatory cycles ( fR = 1 to 2 respirations hour−1,
0.0003 to 0.0005 Hz, in resting normoxic lungfish at 25°C). The se-
quence of RRiwas analyzed using purpose-built software (CardioSeries
v.2.6.2, www.danielpenteado.com) designed to perform time-frequency
analysis of cardiovascular variability, allowing precise adjustment of
parameters related to frequency domain analysis (for example, inter-
polation rate, segment length, and boundaries of frequency bands),
using aHanningwindow tominimize spectral leakage and interpolated
at 2Hz. The spectrum of variability was calculated for segments of data
using a direct fast Fourier transform algorithm for discrete time series.
HRV was represented on both time and frequency domains, and var-
iability was quantified by the average SDNN and RMSSD. The spectra
were also integrated for total PSD. Nonstationary data were not taken
into consideration for PSD calculation. Spectral integration for the sep-
aration of LF and HF bands has no relation to the three main spectral
components distinguished in the PSA spectrum from resting humans,
which are HF (0.15 to 0.4 Hz), LF (0.04 to 0.15 Hz), and VLF (0.003 to
0.04 Hz) (37). So, band separation was not considered. The resultant
output is plotted graphically, where cyclic oscillations in fH appear as
peaks at their relative oscillatory frequencies in the power spectrum.

CRIs in recovered fish
After recovery from instrumentation, mean routine levels of cardio-
respiratory variables were recorded from a continuous trace of 10 to
12 hours. Fish were then subjected to pharmacological blockade of
sympathetic and parasympathetic influences on fH. On the fourth day
of recording, atropine sulfate (1.2 mg kg−1) (Sigma-Aldrich) was in-
jected into the peritoneal cavity to blockmuscarinic receptors (21). Sub-
sequently, on the fifth day of recording, atropine plus propranolol
(2.7 mg kg−1) (Sigma-Aldrich) was injected to block cholinergic and
b-adrenergic receptors (21). The drugs were dissolved in 0.9% sterile
saline. The maximum bolus injection volume was 1 ml kg−1. The cho-
linergic and adrenergic tones on the heart were calculated from com-
bined mean RRi for individual fish (45).
Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
CRIs on exposure to hypoxia and hypercarbia
To further explore the cardiac responses to air breathing observed by
PSA of RRi, we generated alterations in ventilation by chemoreceptor
stimulation. Following recovery in HRV under normoxic conditions,
the air space in the experimental chamber was gassedwith a preformu-
lated mixture of 3% O2, 2% CO2, and 95% N2 (São Carlos Gases)
delivered through a gas flow meter at 400 liters min−1. The alterations
in fH were plotted as interval histograms. Although air-breathing ver-
tebrates are seldom exposed to aerial hypoxia and hypercarbia, exper-
imental exposure to varying gasmixtures is a commonmethod used to
induce changes in fH (53, 54). The procedure aimed to evoke changes in
ventilation, making it possible to validate that the LF peak revealed by
PSA on normoxic lungfish was a result of CRIs.

Measurement of conduction velocity
Reflex control of heart rate requires close coordination between affer-
ent and efferent arms of reflex arcs and its connections in the central
nervous system. Hence, of greatest importance in the beat-to-beat con-
trol of the heart is rapid conduction of activity in the efferent (motor)
arm of the reflex. The conduction velocities of the efferent axons in the
cardiac nerves of the lungfish were measured in vivo in decerebrated
lungfish. The cardiac vagus was exposed close to the pericardium by a
lateral incision, dissected free of connective tissue, and mounted on
bipolar silver electrodes for recording. The electrodes were connected
via a headstage to a biological amplifier (Neuro Amp EX, PowerLab,
ADInstruments). The vagus nerve was then sectioned cranially, close
to the brainstem, and raised onto a pair of platinum hook electrodes
connected to a physiological stimulator (Grass S48, Grass Medical
Instruments). Diverse bursts of pulses of electrical stimulation (dura-
tion, 0.2 to 1.5 ms; 5 to 70 V) were delivered downstream at 20-s inter-
vals. The velocity of the compound potentials generated in the nerve
was calculated based on the time lapse between the artifact caused by
stimulation and the nerve’s response, with reference to the length of the
nerve between the points of stimulation and recording (63mm). These
electrical recordingswere performed inside a screenedmetal “Faraday”
cage to reduce electrical interferencewith the recorded signals. Record-
ing sample rate was set at 40 kHz.

Transmission electron microscopy
Conduction velocities in nerve fibers relate to their diameter and, for
rapid conduction, to the presence of myelination (55). To count the
numbers of vagal fibers and to record the incidence ofmyelinated fibers
in the cardiac vagus, the right and left branches were dissected out of
a fish under terminal anesthesia and fixed in Karnovsky’s solution
[2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate
buffer (pH7.4)] and postfixed in 1%osmium tetroxide, 0.8%potassium
ferrocyanide, and 5 mM calcium chloride in 0.1 M sodium cacodylate
buffer (pH 7.4) for 1 hour. Portions of the fixed nerves were washed
three times for 10 min each in the same buffer and dehydrated in a
graded series of acetone, followed by three successive baths of 100%
acetone and subsequent replacement of the acetone with Polybed resin
(1:1) for 6 hours. Ultrafine sections of around 80 nm were obtained
using an automatic ultramicrotome (Leica EMUC7) and collectedwith
200-mesh copper grids. The sections were then treated with uranyl ac-
etate and lead citrate to increase contrast (56). Photomicrographs were
obtained using a transmission electron microscope (JEOL 1230 TEM).
These images were analyzed by dedicated software (ImageJ) tomeasure
the area, major fiber diameter, myelin thickness, and number of mye-
linated and nonmyelinated fibers. The density of the unmyelinated or
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myelinated fibers and the ratio ofmyelinated/unmyelinated fibers were
calculated, including the percentage of the nerve area occupied (per-
centage of occupancy) by the unmyelinated and myelinated fibers.

Neuroanatomy
Having shown that central control of variations in heart rate is exercised
predominantly by the parasympathetic arm of the autonomic nervous
system,with efferent supply provided by theXth cranial nerve, the vagus,
we traced its projections into the brainstem. Fluorescent tracers were
used to map the distribution of VPN in the brainstem. For retrograde
labeling of VPN, FG (hydroxystilbamidine bis-methanesulfonate;
Sigma-Aldrich) was dissolved in sterile saline at 3 mg ml−1 (57), and
a volume of 5 ml g−1 of the solution was injected intraperitoneally. The
animals were then kept in holding tanks under routine conditions (see
above) for 70 days at 24°C to enable transport of the tracer along the
axons and its accumulation in the vagal neuron cell bodies.

To locate the areas in the brainstem containing CVPN, DiI (Sigma-
Aldrich)was topically applied to the heart, as described byCorbett et al.
(58). Following anesthesia, as described above, the pericardial sac was
exposed, and a 3- to 4-mm piece of sterile hemostatic sponge impreg-
nated with DiI crystals (0.5 mg) was placed on the heart, close to the
sinoatrial node region, and secured in place by suturing the pericar-
dium. Each animal was then recovered from anesthesia and returned
to a holding tank for a period of 40 days, during which the tracer was
transported to cell bodies of CVPN in the brainstem. Lungfish brains
were fixed, as described below.

Fish from either protocol were then anesthetized with benzocaine
and perfused transcardially, first with heparinized saline solution and
then with 4% paraformaldehyde in 0.1 M sodium phosphate buffer at
pH 7.4. The brain and the initial portion of the spinal cord were care-
fully dissected out and stored in the fixative solution for up to 8 hours at
4°C. The sample was then washed with buffered saline and placed
overnight in 20% sucrose phosphate-buffered solution as a cryopro-
tectant at 4°C. Each brain was then frozen and sectioned (transverse
sections of 40 mm) on a cryostat (Microm HM 505 E), and serial
sectionsweremounted on gelatin-coated slides, coverslipped over glyc-
erine. Sections were examined under a photomicroscope (Olympus
BX50 illuminator UV U-ULH) with a video camera attached to an
image analysis system (Image-Pro Plus), enabling the images of fluo-
rescing neuron cell bodies to be captured. Cell bodies of labeled neu-
rons were counted and mapped according to their proximity to the
fourth ventricle and their rostrocaudal distance from obex, the point
on the brainstem from which the rostral extent of the fourth ventricle
is covered dorsally by the choroid plexus that contains no nervous
tissue.We report the results of the distribution ofVPNandCVPNneu-
rons from specimens, providing the highest cell counts following ap-
plication of FG or DiI.

Analysis of data
Cardiorespiratory and HRV parameters are presented as means ±
SEM. One-way ANOVA complemented by Tukey’s multiple compar-
ison test (GraphPad Instat version 3.00, GraphPad Software) was per-
formed to check the possible existence of significant variations between
the values obtained at different times in recovery protocol or to verify
the occurrence of possible differences among experimental groups
(Control, Atropine, andAtropine + Propranolol groups).Morphomet-
ric data from the vagus nerve were tested using the unpaired Student’s
t test, with the variables being between the right and left side. All as-
sumptions to perform the statistical analysis were taken into account
Monteiro et al., Sci. Adv. 2018;4 : eaaq0800 21 February 2018
(that is, independence, normality, and homoscedasticity). All differ-
ences between means at a 5% (P < 0.05) level were considered signif-
icant. For clarity, the number of animals used in each procedure is
indicated in the relevant figures and tables.
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.
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Acknowledgments: This project benefited from a valuable collaboration with G. S. F. Silva
[Universidade Estadual Paulista (UNESP)]. The authors are grateful to the Gonçalo Moniz
Research Center, Oswaldo Cruz Foundation (CPQGM/FIOCRUZ, Salvador, Bahia, Brazil) for
the use of its transmission electron microscope. We are also grateful to the Department of
Genetics and Evolution of UFSCar (São Carlos, São Paulo, Brazil) and the Department of
Biology of Biosciences Institute (UNESP, Rio Claro, São Paulo, Brazil) for the use its fluorescence
microscopes. Manipulators used to record from cardiac nerves were borrowed from
S. Publicover, School of Biosciences, University of Birmingham, UK. Funding: This research
was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(postdoctoral fellowship to D.A.M.) and the National Institute of Science and Technology in
Comparative Physiology (INCT-FisComp). E.W.T. was a senior visiting researcher under the
Science Without Borders Program (CNPq 401061/2014-0). Author contributions: C.A.C.L.
and E.W.T. conceived the study and designed the experiments; D.A.M. performed the
experiments; M.R.S. and E.W.T. performed the neuroanatomy study; A.L.C. provided the TEM;
D.A.M. analyzed the data; C.A.C.L. and F.T.R. provided support and supervised the project;
E.W.T., D.A.M., and C.A.C.L. wrote the manuscript; all authors edited and commented on
the manuscript. Competing interests: The authors declare that they have no competing
interests. Data and materials availability: All data needed to evaluate the conclusions in the
paper are present in the paper and/or the Supplementary Materials. Additional data related
to this paper may be requested from the authors.

Submitted 29 September 2017
Accepted 19 January 2018
Published 21 February 2018
10.1126/sciadv.aaq0800

Citation: D. A. Monteiro, E. W. Taylor, M. R. Sartori, A. L. Cruz, F. T. Rantin, C. A. C. Leite,
Cardiorespiratory interactions previously identified as mammalian are present in the
primitive lungfish. Sci. Adv. 4, eaaq0800 (2018).
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lungfish
Cardiorespiratory interactions previously identified as mammalian are present in the primitive

Diana A. Monteiro, Edwin W. Taylor, Marina R. Sartori, André L. Cruz, Francisco T. Rantin and Cleo A. C. Leite

DOI: 10.1126/sciadv.aaq0800
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