Recovery of the baroreflex and autonomic modulation after anesthesia with MS-222 in bullfrogs.

The time course for recovery after anesthesia is poorly described for tricaine methanesulfonate (MS-222). We suggest that the baroreflex and the heart rate variability (HRV) could be used to index the recovery of the autonomic modulation after anesthesia. We analyzed the recovery profile of behavioral and physiological parameters over time to analyze the progression of recovery after anesthesia of American bullfrogs with MS-222. Mean heart rate stabilized after 17 h, whereas the baroreflex efficiency index took 23 h and the baroreflex operating gain, 29 h. Mean arterial pressure recovered after 26 h. Power spectral density peaked at 23 h and again after 40 h. Baroreflex was a relevant component of the first phase of HRV, while autonomic modulation for resting may take longer than 40 h. We suggest that physiological recovery is a complex phenomenon with multiple progressive phases, and the baroreflex may be a useful tool to observe the first substantial recovery of post-instrumentation capacity for autonomic modulation.

Evaluation of sex-based differences in central control of breathing in American bullfrogs.

The neural control of breathing exhibits sex differences. There is now a large effort to account for biological sex in mammalian research, but the degree to which ectothermic vertebrates exhibit sex differences in the central control of breathing is not well-established. Therefore, we compared respiratory-related neural activity in brainstem-spinal cord preparations from female and male bullfrogs to determine if important aspects of the central control of breathing vary with sex. We found that the breathing pattern was similar across males and females, but baseline frequency of the respiratory network was faster in females. The magnitude of the central response to hypercapnia was similar across sexes, but the time to reach maximum burst rate occurred more slowly in females. These results suggest that sex differences may account for variation in traits associated with the control of breathing and that future work should carefully account for sex of the animal in analysis.

A comparison of four common mathematical models to assess baroreflex sensitivity.

Different methods have been used to assess baroreflex gain in experiments where changes in the carotid sinus pressure or the arterial blood pressure using different techniques provoke a baroreflex response, usually a rapid variation of heart rate. Four mathematical models are most used in the literature: the linear regression, the piecewise regression, and two different four-parameter logistic equations: equation 1, Y = (A1-D1)/[1 + eB1(X - C1) ] + D1; equation 2, Y = (A2-D2)/[1 + (X/C2)B2 ] + D2. We compared the four models regarding the best fit to previously published data in all vertebrate classes. The linear regression had the worst fit in all cases. The piecewise regression generally exhibited a better fit than the linear regression, though it returned a similar fit when no breakpoints were found. The logistic equations showed the best fit among the tested models and were similar to each other. We demonstrate that equation 2 is asymmetric and the level of asymmetry is accentuated according to B2. This means that the baroreflex gain calculated when X = C2 is different from the actual maximum gain. Alternatively, the symmetric equation 1 returns the maximum gain when X = C1. Furthermore, the calculation of baroreflex gain using equation 2 disregards that baroreceptors may reset when individuals experience different mean arterial pressures. Finally, the asymmetry from equation 2 is a mathematical artifact inherently skewed to the left of C2, thus bearing no biological meaning. Therefore, we suggest that equation 1 should be used instead of equation 2.

Ontogenetic scaling of the baroreflex function in the green iguana (Iguana iguana).

Large body mass (Mb) in vertebrates is associated with longer pulse intervals between heartbeats (PI) and thicker arterial walls. Longer PI increases the time for diastolic pressure decay, possibly resulting in loss of cardiac energy as "oscillatory power," whereas thicker arterial walls may affect the transmission of impulses and sensing of pressure fluctuations thus impairing baroreflex function. We aimed to investigate the effect of growth on the relative cardiac energy loss and baroreflex function. We predicted that 1) the relative use of cardiac energy should be preserved with increased time constant for pressure decay (τ = vascular resistance × compliance) and 2) if arterial circumferential distensibility does not change, baroreflex function should be unaltered with Mb. To test these hypotheses, we used green iguanas (Iguana iguana) weighing from 0.03 to 1.34 kg (43-fold increment in Mb). PI (P = 0.037) and τ (P = 0.035) increased with Mb, whereas the oscillatory power fraction (P = 0.245) was unrelated to it. Thus, the concomitant alterations of τ and PI allowed the conservation of cardiac energy in larger lizards. Larger animals had thicker arterial walls (P = 0.0007) and greater relative collagen content (P = 0.022). Area compliance scaled positively to Mb (P = 0.045), though circumferential distensibility (P = 0.155) and elastic modulus (P = 0.762) were unaltered. In addition, baroreflex sensitivity, measured by both the pharmacological (P = 0.152) and sequence methods (P = 0.088), and the baroreflex effectiveness index (P = 0.306) were also unrelated to Mb. Therefore, changes in arterial morphology did not affect circumferential distensibility and presumably sensing of pressure fluctuation, and the cardiovagal baroreflex is preserved across different Mb.

Baroreflex responses of decerebrate rattlesnakes (Crotalus durissus) are comparable to awake animals.

A decerebrate rattlesnake, Crotalus durissus, has previously been used as a model Squamate for cardiovascular studies. It enabled instrumentation for concomitant recordings of diverse variables that showed autonomic responses. However, to validate the preparation and its scope for use, it is necessary to assess how close its cardiovascular variables are to non-decerebrate snakes and the effectiveness of its autonomic responses. Similarly, it is important to analyze its recovery profile after instrumentation and observe if it maintains stability throughout the duration of experimental protocol. Here we have objectively assessed these points by comparing decerebrate preparations and non-decerebrate snakes, after the occlusive cannulation of the vertebral artery. We have assessed cardiovascular variables and the baroreflex to analyze the presence, magnitude and stability of complex autonomic-controlled parameters as indicators of autonomic nervous system (ANS) functionality. After instrumentation, mean heart rates were high but recovered to stable values within 24 h. Mean arterial pressure stabilized within 24 h in control snakes and 48 h in decerebrate preparations. After that, both parameters remained stable. The operational gain and effectiveness index of the baroreflex recovered within the first 6 h after instrumentation in both experimental groups. In addition, the baroreflex capacities and its limits were also equivalent between the groups. These experiments demonstrated that decerebrate preparations and inactive, non-decerebrate snakes showed comparable recovery profiles following anesthesia and cannulation, maintained similar values of cardiovascular variables during experimental manipulation and exhibited functional, ANS modulated reflexes. Accordingly, the present results attest the relevance of this decerebrate preparation for studies on cardiovascular modulation.

The mechanical and morphological properties of systemic and pulmonary arteries differ in the Madagascar ground boa, a snake without ventricular pressure separation.

The walls of the mammalian aorta and pulmonary artery are characterized by diverging morphologies and mechanical properties, which have been correlated with high systemic and low pulmonary blood pressure, as a result of intraventricular pressure separation. However, the relationship between intraventricular pressure separation and diverging aortic and pulmonary artery wall morphologies and mechanical characteristics is not understood. The snake cardiovascular system poses a unique model for the study of this relationship, as representatives both with and without intraventricular pressure separation exist. In this study, we performed uniaxial tensile testing on vessel samples taken from the aortas and pulmonary arteries of the Madagascar ground boa, Acrantophis madagascariensis, a species without intraventricular pressure separation. We then compared these morphological and mechanical characteristics with samples from the ball python, Python regius, and the yellow anaconda, Eunectes notaeus - species with and without intraventricular pressure separation, respectively. Our data suggest that although the aortas and pulmonary arteries of A. madagascariensis respond similarly to the same intramural blood pressure, they diverge in morphology, and that this attribute extends to E. notaeus. In contrast, P. regius aortas and pulmonary arteries diverge both morphologically and in terms of their mechanical properties. Our data indicate that intraventricular pressure separation cannot fully explain diverging aortic and pulmonary artery morphologies. Following the law of Laplace, we propose that pulmonary arteries of small luminal diameter represent a mechanism to protect the fragile pulmonary vasculature by reducing the blood volume that passes through, to which genetic factors may contribute more strongly than physiological parameters.

Role of Nitric Oxide in the Cardiovascular System of South American Rattlesnakes (Crotalus durissus).

AbstractUnderstanding the basis of vascular tonus regulation is fundamental to comprehending cardiovascular physiology. In the present study, we used the recently developed decerebrate rattlesnake preparation to investigate the role of nitric oxide (NO) in the control of vascular tonus in a squamate reptile. This preparation allowed multiple concomitant cardiovascular parameters to be monitored, while avoiding the deleterious effect of anesthetic drugs on autonomic modulation. We observed that both systemic and pulmonary circuits were clearly responsive to NO signaling. NO increased vascular conductance in the systemic and pulmonary systems. Vasodilation by NO of the systemic circulation was compensated by cardiovascular alterations involving venous return, cardiac output, and cardiac shunt adjustments. The cardiac shunt seemed to be actively used for hemodynamic adjustments via modulation of the pulmonary artery constriction. N(ω)-nitro-L-arginine methyl ester injection demonstrated that NO contributes to modulating resting vasodilation in the systemic circuit. In contrast, NO-mediated vasodilation did not have an important role in the pulmonary circulation in inactive decerebrated snakes at 25°C. These responses vary importantly from those described for anesthetized snakes.

Baroreflex responses to activity at different temperatures in the South American rattlesnake, Crotalus durissus.

In humans, physical exercise imposes narrower limits for the heart rate (fH) response of the baroreflex, and vascular modulation becomes largely responsible for arterial pressure regulation. In undisturbed reptiles, the baroreflex-related fH alterations at the operating point (Gop) decreases at elevated body temperatures (Tb) and the vascular regulation changes accordingly. We investigated how the baroreflex of rattlesnakes, Crotalus durissus, is regulated during an activity at different Tb, expecting that activity would reduce the capacity of the cardiac baroreflex neural pathway to buffer arterial pressure fluctuations while being compensated by the vascular neural pathway regulation. Snakes were catheterized for blood pressure assessment at three different Tb: 15, 20 and 30 °C. Data were collected before and after activity at each Tb. Baroreflex gain (Gop) was assessed with the sequence method; the vascular limb, with the time constant of pressure decay (τ), using the two-element Windkessel equation. Both Gop and τ reduced when Tb increased. Activity also reduced Gop and τ in all Tb. The relationship between τ and pulse interval (τ/PI) was unaffected by the temperature at resting snakes, albeit it reduced after activity at 20 °C and 30 °C. The unchanged τ/PI and normalized Gop at different Tb indicated those variables are actively adjusted to work at different fH and pressure conditions at rest. Our data suggest that during activity, the baroreflex-related fH response is attenuated and hypertension is buffered by a disproportional increase in the rate which pressure decays during diastole. This compensation seems especially important at higher Tb where Gop is already low.

Prenatal hypoxia affects scaling of blood pressure and arterial wall mechanics in the common snapping turtle, Chelydra serpentina.

In reptiles, exposure to hypoxia during embryonic development affects several cardiovascular parameters. These modifications may impose different mechanical stress to the arterial system, and we speculated that the arterial wall of major outflow vessels would be modified accordingly. Since non-crocodilian reptiles possess a partially divided ventricle, ensuing similar systemic and pulmonary systolic pressures, we investigated how morphological and mechanical properties of segments from the left aortic arch (LAo) and the proximal and distal segments of the left pulmonary artery (LPAp and LPAd, respectively) change as body mass (Mb) increases. Eggs from common snapping turtles, Chelydra serpentina, were incubated under normoxia (21% O2; N21) or hypoxia (10% O2; H10), hatched and maintained in normoxia thereafter. Turtles (0.11-6.85 kg) were cannulated to measure arterial pressures, and an injection of adrenaline was used to increase pressures. Portions of the LAo, LPAp and LPAd were fixed under physiological hydrostatic pressures for histology and mechanical assessment. Arterial pressures increased with Mb for N21 but not for H10. Although mechanical and functional characteristics from the LPAp and LPAd were similar between N21 and H10, wall thickness from LAo did not change with Mb in the H10 group, thus wall stress increased in larger turtles. This indicates that larger H10 turtles probably experience an elevated probability of arterial wall rupture without concomitant changes in the cardiovascular system to prevent it. Finally, collagen content of the LPAp and LAo was smaller than in LPAd, suggesting a more distensible arterial wall could attenuate higher pressures from larger turtles.

Arterial wall thickening normalizes arterial wall tension with growth in American alligators, Alligator mississippiensis.

Arterial wall tension increases with luminal radius and arterial pressure. Hence, as body mass (Mb) increases, associated increases in radius induces larger tension. Thus, it could be predicted that high tension would increase the potential for rupture of the arterial wall. Studies on mammals have focused on systemic arteries and have shown that arterial wall thickness increases with Mb and normalizes tension. Reptiles are good models to study scaling because some species exhibit large body size range associated with growth, thus, allowing for ontogenetic comparisons. We used post hatch American alligators, Alligator mississippiensis, ranging from 0.12 to 6.80 kg (~ 60-fold) to investigate how both the right aortic arch (RAo) and the left pulmonary artery (LPA) change with Mb. We tested two possibilities: (i) wall thickness increases with Mb and normalizes wall tension, such that stress (stress = tension/thickness) remains unchanged; (ii) collagen content scales with Mb and increases arterial strength. We measured heart rate and systolic and mean pressures from both systemic and pulmonary circulations in anesthetized animals. Once stabilized alligators were injected with adrenaline to induce a physiologically relevant increase in pressure. Heart rate decreased and systemic pressures increased with Mb; pulmonary pressures remained unchanged. Both the RAo and LPA were fixed under physiological hydrostatic pressures and displayed larger radius, wall tension and thickness as Mb increased, thus, stress was independent from Mb; relative collagen content was unchanged. We conclude that increased wall thickness normalizes tension and reduces the chances of arterial walls rupturing in large alligators.

Baroreflex gain and time of pressure decay at different body temperatures in the tegu lizard, Salvator merianae.

Ectotherms may experience large body temperature (Tb) variations. Higher Tb have been reported to increase baroreflex sensitivity in ectotherm tetrapods. At lower Tb, pulse interval (PI) increases and diastolic pressure decays for longer, possibly resulting in lower end-diastolic pressures and mean arterial pressures (Pm). Additionally, compensatory baroreflex-related heart rate modulation (i.e. the cardiac branch of the baroreflex response) is delayed due to increased PI. Thus, low Tb is potentially detrimental, leading to cardiovascular malfunctioning. This raises the question on how Pm is regulated in such an adverse condition. We investigated the baroreflex compensations that enables tegu lizards, Salvator merianae, to maintain blood pressure homeostasis in a wide Tb range. Lizards had their femoral artery cannulated and pressure signals recorded at 15°C, 25°C and 35°C. We used the sequence method to analyse the heart rate baroreflex-related corrections to spontaneous pressure fluctuations at each temperature. Vascular adjustments (i.e. the peripheral branch) were assessed by calculating the time constant for arterial pressure decay (τ)-resultant from the action of both vascular resistance and compliance-by fitting the diastolic pressure descent to the two-element Windkessel equation. We observed that at lower Tb, lizards increased baroreflex gain at the operating point (Gop) and τ, indicating that the diastolic pressure decays at a slower rate. Gop normalized to Pm and PI, as well as the ratio τ/PI, did not change, indicating that both baroreflex gain and rate of pressure decay are adjusted according to PI lengthening. Consequently, pressure parameters and the oscillatory power fraction (an index of wasted cardiac energy) were unaltered by Tb, indicating that both Gop and τ modulation are crucial for cardiovascular homeostasis.

Cholinergic regulation along the pulmonary arterial tree of the South American rattlesnake: vascular reactivity, muscarinic receptors, and vagal innervation.

Vascular tone in the reptilian pulmonary vasculature is primarily under cholinergic, muscarinic control exerted via the vagus nerve. This control has been ascribed to a sphincter located at the arterial outflow, but we speculated whether the vascular control in the pulmonary artery is more widespread, such that responses to acetylcholine and electrical stimulation, as well as the expression of muscarinic receptors, are prevalent along its length. Working on the South American rattlesnake (Crotalus durissus), we studied four different portions of the pulmonary artery (truncus, proximal, distal, and branches). Acetylcholine elicited robust vasoconstriction in the proximal, distal, and branch portions, but the truncus vasodilated. Electrical field stimulation (EFS) caused contractions in all segments, an effect partially blocked by atropine. We identified all five subtypes of muscarinic receptors (M1-M5). The expression of the M1 receptor was largest in the distal end and branches of the pulmonary artery, whereas expression of the muscarinic M3 receptor was markedly larger in the truncus of the pulmonary artery. Application of the neural tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) revealed widespread innervation along the whole pulmonary artery, and retrograde transport of the same tracer indicated two separate locations in the brainstem providing vagal innervation of the pulmonary artery, the medial dorsal motor nucleus of the vagus and a ventro-lateral location, possibly constituting a nucleus ambiguus. These results revealed parasympathetic innervation of a large portion of the pulmonary artery, which is responsible for regulation of vascular conductance in C. durissus, and implied its integration with cardiorespiratory control.

Cardiovascular shunting in vertebrates: a practical integration of competing hypotheses.

This review explores the long-standing question: 'Why do cardiovascular shunts occur?' An historical perspective is provided on previous research into cardiac shunts in vertebrates that continues to shape current views. Cardiac shunts and when they occur is then described for vertebrates. Nearly 20 different functional reasons have been proposed as specific causes of shunts, ranging from energy conservation to improved gas exchange, and including a plethora of functions related to thermoregulation, digestion and haemodynamics. It has even been suggested that shunts are merely an evolutionary or developmental relic. Having considered the various hypotheses involving cardiovascular shunting in vertebrates, this review then takes a non-traditional approach. Rather than attempting to identify the single 'correct' reason for the occurrence of shunts, we advance a more holistic, integrative approach that embraces multiple, non-exclusive suites of proposed causes for shunts, and indicates how these varied functions might at least co-exist, if not actually support each other as shunts serve multiple, concurrent physiological functions. It is argued that deposing the 'monolithic' view of shunting leads to a more nuanced view of vertebrate cardiovascular systems. This review concludes by suggesting new paradigms for testing the function(s) of shunts, including experimentally placing organ systems into conflict in terms of their perfusion needs, reducing sources of variation in physiological experiments, measuring possible compensatory responses to shunt ablation, moving experiments from the laboratory to the field, and using cladistics-related approaches in the choice of experimental animals.

Evaluation of the sequence method as a tool to assess spontaneous baroreflex in reptiles.

The sequence method is an alternative to the traditional pharmacological approach (i.e., the Oxford technique) used to calculate baroreflex gain (G) in mammals. Although the sequence method assesses baroreflex by measuring spontaneous events of blood pressure regulation, the pharmacological method relies on the injection of vasoactive drugs that impact the baroreflex mechanism itself. The sequence method might be relevant for dynamic measurement of baroreflex modulation but it was never validated for vertebrates with low heart rate. Hence, we tested the sequence method in three species of reptiles and compared the results with those provided by the classic pharmacological method. G was similar between both methods and values correlated when parameters for the sequence method were set at delay 0 or 1 (i.e., the baroreflex system responds immediately to blood pressure changes or after 1 heartbeat). Calculation of the baroreflex effectiveness index was adequate at a minimum of 300 cycles and a delay of 1 for the three species. Therefore, the sequence method has been validated to investigate baroreflex regulation in reptiles, enabling studies during dynamic alterations in homeostasis.

Long term effects of chronic prenatal exposure to hypercarbia on organ growth and cardiovascular responses to adrenaline and hypoxia in common snapping turtles.

Reptilian embryos often face challenging environmental gas compositions during incubation, which may inflict long-lasting effects in the individuals' physiological responses. These conditions can have a lasting effect on the animal into juvenile life as chronic prenatal exposure to hypercarbia results in enlarged hatchling organ size, higher growth rate and resting metabolic rate, although relatively smaller increment in metabolic scope during digestion. Therefore, we wanted to verify whether prenatal hypercarbia exposure would cause persistent effects on morphology and physiological responses in C. serpentina. We measured organ masses and cardiovascular parameters in five years old turtles incubated either under 3.5% hypercarbia (H3.5) or normoxia (N21). We expected that: i) organ masses of H3.5 would be bigger than N21; ii) acute exposure to hypoxia should decrease blood flows in H3.5, since metabolic scope is presumably reduced in this group. As hypoxia exposure elicits catecholamine release, we also tested cardiovascular responses to adrenaline injection. Lungs and stomach exhibited higher growth rates in H3.5. Divergent cardiovascular responses between groups to adrenaline injection were observed for heart rate, pulmonary blood flow, pulmonary mean arterial pressure, blood shunt, systemic stroke volume, and stomach perfusion. Hypoxia caused decreased systemic blood flow and cardiac output, systemic and total stroke volume, and systemic vascular conductance in H3.5. These variables were unaffected in N21, but pulmonary flow and stroke volume, and stomach blood perfusion were reduced. These data support the hypothesis that exposure to hypercarbia during embryonic development has long term effects on organ morphology and cardiovascular responses of C. serpentina.

The electrocardiogram of vertebrates: Evolutionary changes from ectothermy to endothermy.

The electrocardiogram (ECG) reveals that heart chamber activation and repolarization are much faster in mammals and birds compared to ectothermic vertebrates of similar size. Temperature, however, affects electrophysiology of the heart and most data from ectotherms are determined at body temperatures lower than those of mammals and birds. The present manuscript is a review of the effects of temperature on intervals in the ECG of ectothermic and endothermic vertebrates rather than a hypothesis-testing original research article. However, the conclusions are supported by the inclusion of original data (Iguana iguana, N = 4; Python regius, N = 5; Alligator mississippiensis, N = 4). Most comparisons were of animals of approximately 1 kg. Compared to mammals and birds, the reptiles at 35-37 °C had 4 fold lower heart rates, 2 fold slower atrial and ventricular conduction (longer P- and QRS-wave durations), and 4 fold longer PR intervals (atrioventricular delay) and QT intervals (total ventricular repolarization). We conclude that the faster chamber activation in endotherms cannot be explained by temperature alone. Based on histology, we show that endotherms have a more compact myocardial architecture. In mammals, disorganization of the compact wall by fibrosis associates with conduction slowing and we suggest the compact tissue architecture allows for faster chamber activation. The short cardiac cycle that characterizes mammals and birds, however, is predominantly accommodated by shortening of the atrioventricular delay and the QT interval, which is so long in a 1 kg iguana that it compares to that of an elephant.

Analysis of vascular mechanical properties of the yellow anaconda reveals increased elasticity and distensibility of the pulmonary artery during digestion.

In animals with functional division of blood systemic and pulmonary pressures, such as mammals, birds, crocodilians and a few non-crocodilian reptiles, the vessel walls of systemic and pulmonary arteries are exquisitely adapted to endure different pressures during the cardiac cycle, systemic arteries being stronger and stiffer than pulmonary arteries. However, the typical non-crocodilian reptile heart possesses an undivided ventricle that provides similar systolic blood pressure to both circuits. This raises the question whether in these species the systemic and pulmonary mechanical vascular properties are similar. Snakes also display large organ plasticity and increased cardiac output in response to digestion, and we speculate how the vascular circuit would respond to this further stress. We addressed these questions by testing the mechanical vascular properties of the dorsal aorta and the right pulmonary artery of fasted and fed yellow anacondas, Eunectes notaeus, a snake without functional ventricular separation that also exhibits large metabolic and cardiovascular responses to digestion. Similar to previous studies, the dorsal aorta was thicker, stronger, stiffer and more elastic than the pulmonary artery. However, unlike any other species studied so far, the vascular distensibility (i.e. the relative volume change given a pressure change) was similar for the two circuits. Most striking, the pulmonary artery elasticity (i.e. its capacity to resume its original form after being stretched) and distensibility increased during digestion, which suggests that this circuit is remodeled to accommodate the larger stroke volume and enhance the Windkessel effect, thus providing a more constant blood perfusion during digestion.

Ventilatory responses of the clown knifefish, Chitala ornata, to hypercarbia and hypercapnia.

The aim of the present study was to determine the roles of externally versus internally oriented CO2/H+-sensitive chemoreceptors in promoting cardiorespiratory responses to environmental hypercarbia in the facultative air-breathing fish, Chitala ornata (the clown knifefish). Fish were exposed to environmental acidosis (pH ~ 6.0) or hypercarbia (≈ 30 torr PCO2) that produced changes in water pH equal to the pH levels of the acidotic water to distinguish the relative roles of CO2 versus H+. We also injected acetazolamide to elevate arterial levels of PCO2 and [H+] in fish in normocarbic water to distinguish between internal and external stimuli. We measured changes in gill ventilation frequency, air breathing frequency, heart rate and arterial blood pressure in response to each treatment as well as the changes produced in arterial PCO2 and pH. Exposure to normocarbic water of pH 6.0 for 1 h did not produce significant changes in any measured variable. Exposure to hypercarbic water dramatically increased air breathing frequency, but had no effect on gill ventilation. Hypercarbia also produced a modest bradycardia and fall in arterial blood pressure. Injection of acetazolamide produced similar effects. Both hypercarbia and acetazolamide led to increases in arterial PCO2 and falls in arterial pH although the changes in arterial PCO2/pH were more modest following acetazolamide injection as were the increases in air breathing frequency. The acetazolamide results suggest that the stimulation of air breathing was due, at least in part, to stimulation of internally oriented CO2/H+ chemoreceptors monitoring blood gas changes.

Reply to the commentary by Hillman et al. on: "Vascular distensibilities have minor effects on intracardiac shunt patterns in reptiles" by Filogonio et al. (2017).

Our meta-analysis (Filogonio et al., 2017) on central vascular blood flows in a snake (Crotalus durissus) and a turtle (Trachemys scripta) was motivated by Hillman et al.'s (2014) analysis on amphibians to investigate whether cardiac shunt patterns depend on cardiac output and vascular distensibilities. In contrast to Hillman et al. (2014), we did not uncover a general trend that supports the notion that cardiac shunts in reptiles are dictated by vascular distensibilities. In addition to our response to the criticism raised by Hillman et al. (2017), we suggest that future experiments should consider (i) both compliance and distensibility of the major arteries; (ii) differences in volume of the systemic and pulmonary circuits to account for the accommodation of stroke volume; and (iii) an evaluation of the pulsatile pressures in both the ventricle and the major arteries to consider the timing of the ventricular ejection provided by opening of the ventricular valves. We hope these suggestions may help future clarification of the relative importance of passive arterial mechanical properties compared to autonomic regulation in determining intracardiac shunts in both amphibians and reptiles.

Vascular distensibilities have minor effects on intracardiac shunt patterns in reptiles.

The different vascular distensibilities of systemic and pulmonary circuits were recently proposed as an important mechanism defining the direction of cardiac shunt and the distribution of cardiac output in vertebrates with undivided cardiac ventricles. In short, the more distensible pulmonary vascular bed was proposed to accommodate a larger portion of the blood ejected from the heart when cardiac output increases. To evaluate this hypothesis, we performed a meta-analysis based on fourteen previously published studies in two species of reptiles (the freshwater turtle, Trachemys scripta, and the South American rattlesnake, Crotalus durissus) to describe how cardiac shunt patterns change when cardiac output increases. We found no general linear relation between total blood flow and systemic or pulmonary blood flows. Thus, cardiac output alterations cannot be used as a reliable reference for prediction of shunt patterns in both T. scripta and C. durissus. Hence, differential distensibilities appear to be of minor importance for the determination of cardiac shunt patterns in the species analyzed. We speculate that the lack of correlation in these phylogenetically distant species may indicate that this is a common trend within non-crocodilian reptiles.