Autonomic control of heart rate during orthostasis and the importance of orthostatic-tachycardia in the snake Python molurus

Abstract

Orthostasis dramatically influences the hemodynamics of terrestrial vertebrates, especially large and elongated animals such as snakes. When these animals assume a vertical orientation, gravity tends to reduce venous return, cardiac filling, cardiac output and blood pressure to the anterior regions of the body. The hypotension triggers physiological responses, which generally include vasomotor adjustments and tachycardia to normalize blood pressure. While some studies have focused on understanding the regulation of these vasomotor adjustments in ectothermic vertebrates, little is known about regulation and the importance of heart rate in these animals during orthostasis. We acquired heart rate and carotid pulse pressure (P ^sub PC^) in pythons in their horizontal position, and during 30 and 60° inclinations while the animals were either untreated (control) or upon muscarinic cholinoceptor blockade and a double autonomic blockade. Double autonomic blockade completely eradicated the orthostatic-tachycardia, and without this adjustment, the P ^sub PC^ reduction caused by the tilts became higher than that which was observed in untreated animals. On the other hand, post-inclinatory vasomotor adjustments appeared to be of negligible importance in counterbalancing the hemodynamic effects of gravity. Finally, calculations of cardiac autonomic tones at each position revealed that the orthostatic-tachycardia is almost completely elicited by a withdrawal of vagal drive.[PUBLICATION ABSTRACT]

J Comp Physiol B (2014) 184:903912 DOI 10.1007/s00360-014-0841-0

ORIGINAL PAPER

Autonomic control of heart rate during orthostasis and the importance of orthostatictachycardia in the snake Python molurus

Vinicius Arajo Armelin Victor Hugo da Silva Braga Augusto Shinya Abe Francisco Tadeu Rantin

Luiz Henrique Florindo

Abstract Orthostasis dramatically inuences the hemo-dynamics of terrestrial vertebrates, especially large and elongated animals such as snakes. When these animals assume a vertical orientation, gravity tends to reduce venous return, cardiac lling, cardiac output and blood pressure to the anterior regions of the body. The hypotension triggers physiological responses, which generally include vasomotor adjustments and tachycardia to normalize blood pressure. While some studies have focused on understanding the regulation of these vasomotor adjustments in ectothermic vertebrates, little is known about regulation and the importance of heart rate in these animals during orthostasis. We acquired heart rate and carotid pulse pressure (PPC) in pythons in their horizontal position, and during 30 and 60 inclinations while the animals were either untreated (control) or upon muscarinic cholinoceptor

Received: 21 January 2014 / Revised: 10 June 2014 / Accepted: 20 June 2014 / Published online: 14 July 2014 Springer-Verlag Berlin Heidelberg 2014

blockade and a double autonomic blockade. Double autonomic blockade completely eradicated the orthostatictachycardia, and without this adjustment, the PPC reduction caused by the tilts became higher than that which was observed in untreated animals. On the other hand, post-inclinatory vasomotor adjustments appeared to be of negligible importance in counterbalancing the hemodynamic effects of gravity. Finally, calculations of cardiac autonomic tones at each position revealed that the orthostatictachycardia is almost completely elicited by a withdrawal of vagal drive.

Keywords Autonomic control Gravity Heart rate

Orthostasis Python molurus Snake

Introduction

The gravitational attraction exerted by planet Earth has strong inuence on all terrestrial organisms, placing special demands on the musculoskeletal system and dramatic alterations in the distribution of body uids in humans and animals alike (Lillywhite 1996; Morey-Holton 2003). When it comes to the cardiovascular system, gravity is responsible for establishing vertical pressure variation upon vascular beds, which depends on the body orientation that the animal is assuming. Because hydrostatic pressure is an important factor in the determination of uid dynamics, orthostasis in non-aquatic environments can jeopardize blood circulation (Lillywhite 1988). It is currently known that such effects are more pronounced in large and elongated animals, such as giraffes and snakes, because these characteristics favor the establishment of blood columns in their bodies. Inso-much, as evidenced by several studies, gravity-sensitive taxa that were widely exposed to gravitational stress during

Communicated by H.V. Carey.

V. A. Armelin F. T. Rantin

Department of Physiological Sciences, Federal University of So Carlos (UFSCar), Via Washington Luiz, km 235, So Carlos,

SP 13565-905, Brazil

V. A. Armelin V. H. da Silva Braga A. S. Abe F. T. Rantin L. H. Florindo

National Institute of Science and Technology in Comparative Physiology (INCT-FAPESP/CNPq), So Paulo, Brazil

V. H. da Silva Braga L. H. Florindo (*)

Department of Zoology and Botany, So Paulo State University (UNESP), Cristvo Colombo Street, 2265,

So Jos do Rio Preto, SP 15054-000, Brazile-mail: [email protected]

A. S. Abe

Department of Zoology, So Paulo State University (UNESP), 24A Avenue, 1515, Rio Claro, SP 13506-900, Brazil

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their evolutionary history demonstrate a number of adaptations to circumvent these issues (Seymour and Lillywhite 1976; Hargens et al. 1987; Lillywhite et al. 1997; Young et al. 1997; Brondum et al. 2009; Petersen et al. 2013).

The adaptations that maintain cardiovascular homeostasis during disturbances from postural changes in snakes can be separated into three subdivisions: morphological, behavioral and physiological. Comparisons between the hearthead distance of several species of snake revealed that this distance is highest in aquatic species and progressively decreases in terrestrial and arboreal serpents. It is possible that the heart position of snakes represents an anatomical adaptation to enhance cerebral perfusion during orthostasis (Seymour and Lillywhite 1976; Seymour 1987; Lillywhite et al. 2012), though this theory is controversial (Gartner et al. 2010, 2011; Lillywhite and Seymour 2011). Other morphological adaptations include reduced vascular compliance and tighter skin, which have been observed in scansorial species and may prevent caudal blood pooling while the animals are inclined (Lillywhite 1985a; Jayne 1988; Lillywhite and Henderson 1993). Behavioral control of arterial pressure has been reported in terrestrial and arboreal snakes as well: the behavior consists of undulations of the lateral muscles in a posterior-to-anterior direction, and it increases venous return, stroke volume and cardiac output during climbing (Lillywhite 1985b). Finally, physiological adjustments operate in conjunction with these features, and represent the most important adaptation for maintaining adequate cardiovascular parameters during orthostasis (Lillywhite 1996).

The physiological adjustments that counteract the effects of gravity on circulation essentially work through vaso-motion and changes in heart rate. When a snake assumes an upright posture, both systemic vasoconstriction and tachycardia occur in order to reduce caudal blood pooling, improving cardiac output and systemic arterial pressure (Lillywhite and Gallagher 1985; Lillywhite 1996). The vast majority of the studies on the regulation of these adjustments focus on vasomotor mediation. Donald and Lilly-white (1988) demonstrated that the semi-arboreal snake Elaphe obsoleta has denser adrenergic innervation of blood vessels in the posterior regions of the body than in anterior regions. Other authors attested the reactivity of systemic blood vessels to a variety of factors (Conklin et al. 1996), the importance of adrenergic and peptidergic regulation of vasomotion (Donald and Lillywhite 1988; Donald et al. 1990; Lillywhite and Donald 1994) and also the presence of extensive adrenergic, cholinergic and peptidergic inner-vation in the heart of snakes [as reviewed by Lillywhite and Donald (1994)].

Despite the studies on the innervation of snake hearts, little is known about how the changes in heart rate are

mediated in these animals during orthostasis. Young et al. (1997) disrupted the baroreexes of two species of snake with a vagotomy and abolished the orthostatic-tachycardia. Their achievement is evidence that this adjustment may be exclusively autonomic and predominantly cholinergic in Ahaetulla nasuta and Crotalus adamanteus; however, the experiments were performed with anesthetized snakes, a condition that may have suppressed other mechanisms involved. Lillywhite and Seymour (1978) tilted unanesthetized tiger snakes (Notechis scutatus) with and without -adrenergic blockade with propranolol, and demonstrated that the tachycardia associated with orthostasis is mediated only by the adrenergic subdivision of the autonomic nervous system in this speciesthese are, as far as we know, the only insights into the sympathovagal balance in snake hearts during orthostasis.

Given the lack of information about the relevance and control of orthostatic-tachycardia, the present work sought to verify the importance of this adjustment by means of its pharmacological abolition, and to describe its autonomic control in a long-bodied snake. The Python molurus is a snake that is well adapted to several habitats (eurytopic). As a remarkable representative of the Python genus, this species possesses an intracardiac separation of systemic and pulmonary circulations that cause higher systemic pressure and ow compared to the snakes of other genera (Jensen et al. 2010a). Thus, this serpent was chosen as our experimental model because of its cardiovascular peculiarities, size, shape and habitsthis decision was consistent with the August Krogh Principle: For many problems, there will be one or more animals on which it can be most conveniently studied [for detailed explanation of Kroghs Principle, please see the work of Krebs (1975)].

Materials and methods

Experimental animals

Eleven specimens of Python molurus (Linnaeus 1758) were legally obtained from a scientic breeding center in mid-2009 (Jacarezrio-UNESP, Rio Claro, So Paulo, Brazil) and transported to a serpentarium located at So Paulo State University (UNESP), So Jos do Rio Preto, So Paulo, Brazil. They were individually housed in 540-l plastic boxes and kept at 27 C (3) under natural photoperiod

conditions. The snakes weighed 7.23 0.63 kg and were

2.42 0.36 m in length (mean SEM). They were fed

laboratory rodents weekly and had continuous access to waterall animals appeared healthy from their acquisition on. The specimens sexes were not taken into consideration, and the serpents had been fasted for 3 weeks prior to experiments.

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Pre-experimental procedures

The snakes were anesthetized through the use of an inhalation mask over each specimens head. The mask was per-fused with a mixture of isourane (3 %) and oxygen until the animals became unresponsive (Mosley 2005). The glottis was then intubated with soft rubber tubing so the lungs could be directly ventilated (1 % isourane mixed with oxygen was used accordingly to Mosley (2005)) close to the patterns of an undisturbed python1.8 breaths per minute and a tidal volume of 17.5 ml kg1 using a manual ventilator (Colibri Inhalatory Anesthesia Apparatus, Brasmed Veterinary Products, Paulnia, SP, Brazil) (Secor et al. 2000). After that, the heart position was determined using palpation, and three electrocardiogram adhesive electrodes were placed around the heart (two of which were positioned laterally and 2 cm above the heart, and the third of which was disposed centrally and 5 cm below it). The electrodes were plugged into a BIOPAC SS2L cable and attached with adhesive tape.

A pulse plethysmogram (SS4LA, BIOPAC Systems Incorporated, Goleta, CA, USA) was positioned ventrally on the necks of the snakes. The xation point was standardized at 5 % of the animals total lengths. In addition, the device was always 20 % to the right of the center of the snakes necks; this distance was calculated using the overall neck width. The device was attached with cyanoacrylate glue and adhesive tape so that it would stay above the carotid artery and close to the entrance to the skull (one python carcass was previously dissected to estimate this location). To restrict the snakes movements and thus to prevent displacement of the plethysmogram, a 20-cm-long PVC splint was applied to the neck and attached with adhesive tape.

The electrocardiogram electrodes and pulse plethysmo-gram were connected to a BIOPAC MP36 (BIOPAC Systems Incorporated, Goleta, CA, USA) data acquisition system to continuously acquire and record the animals heart rates (fHderived from the electrocardiogram signal) and distal carotid pulse pressures (PPCderived from the pulse plethysmogram signal) [the difference between systolic and diastolic pressures, in mV, which is directly proportional to stroke volume (Panzer et al. 2008)], respectively. All aforementioned proceedings were accomplished in ~20 min.

Experimental protocol

After the pre-experimental procedures, all snakes were transferred to a tilting apparatus and allowed to recover from anesthesia (~30 min). This apparatus consisted of a PVC tube attached to an articulated bar, which enabled inclinations of up to 90. The tube dimensions were slightly larger than the animals and kept the serpents restrained. The

tube also had a tilt angle indicator and slits that allowed for ventilation, passage of the equipment cables, and intraperitoneal drug administration. After recovering from the anesthesia, the animals were permitted to rest for a 120-min period so that the fH and PPC could stabilize (these variables were stable in the nal 30 min of this period).

The fH and PPC were then acquired from seven pythons positioned at 0, 30 and 60, and the results constitute the data of untreated animals for each angle. Based on the methodology of Seymour and Arndt (2004), the inclination protocol consisted of tilting the serpents to the desired angle within ~5 s, maintaining the tilt for 1 min, and bringing them back to the horizontal position for at least 5 min. It is important to emphasize that it was not possible to continue the inclination for over 1 min or even to perform it in an angle above 60, because under these conditions, the animals tried to move excessively and prevented data collection.

Immediately the parameters were acquired from untreated animals, a blockade of muscarinic cholinergic receptors was performed on them with an intraperito-neal injection of atropine (2.5 mg kg1). The solution was allowed to take effect for 30 min before the inclination protocol was repeated. Upon completion of the experiments with atropine, the -adrenergic antagonist propranolol was intraperitoneally administered (3.5 mg kg1) and resulted in a complete autonomic blockade in the animals hearts (given the fact that atropine was still exerting its effects). The inclination protocol was conducted again after a 30-min wait. Both atropine and propranolol were diluted in 10 mg ml1 of an isosmotic saline solution.

Finally, the inclination protocol was performed on four other pythons after an intraperitoneal injection of isosmotic saline (0.35 ml kg1) and a 30-min wait in order to conrm that the previously obtained results were not induced by the drug vehicle or by the stress of their administration.

Calculation of cardiac autonomic tone

The adrenergic tone and cholinergic tone in the heart were quantied for each angle studied using the equations proposed by Altimiras et al. (1997) utilizing the RR intervals (60/fH; in s) that were obtained from the animals under control conditions, after the muscarinic receptor blockade and after the complete autonomic blockade. To calculate cardiac autonomic tone during inclinations, the moment of greatest change in fH observed at 30 and 60 was considered.

Data analysis and statistics

After plotting the data in descriptive graphics, inferential statistics were employed and considered the parameters

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acquired at the following moments: the stabilization period preceding the 30 inclinations (which constitutes the data of the animals in the horizontal position), and the moment of greatest change in cardiovascular parameters observed during the inclinations. The variation in PPC that occurred when animals were tilted was also calculated (0 vs 30/60 = PPC0 PPC30/60). All values are presented as

mean standard error. The statistical analyses mentioned

below were carried out using the GraphPad InStat 3.0 commercial software (GraphPad Software Inc.).

Within and between each of the three treatments, changes in PPC and fH were identied using a one-way

ANOVA for repeated measures followed by a Student NewmanKeuls multiple comparison test. Differences among PPC variations when the animals were positioned at 30 and 60 were assessed using Friedmans test succeeded by a Dunns post hoc. Changes in adrenergic tone and cholinergic tone induced by inclinations and differences existing between tone values observed at 30 and 60 were both evaluated via one-way ANOVA for repeated measures followed by a StudentNewmanKeuls post hoc. A one-way ANOVA followed by a Bonferroni post hoc was conducted to verify divergences between the parameters acquired in untreated and saline-treated animals. In all cases, a signicance level of P 0.05 was adopted.

Results

It was observed that PPC decreased immediately upon inclination in all treatments, and that this reduction was accompanied by a rise in fH in untreated animals but not in those that were atropinized or double blocked (Fig. 1af). Immediately after the animals were returned to the horizontal position, PPC reached values higher than those observed before tilting (Fig. 1bd), except untreated animals inclined to 30 (Fig. 1a)this phenomena was also observed in double-blocked pythons, but not in the two subsequent minutes shown in the descriptive graphics (Fig. 1e, f).

While the untreated animals were horizontal, their PPC values averaged 0.247 0.026 mV, and this value signi

cantly decreased to 0.145 0.010 and 0.126 0.006 mV

while the animals were inclined to 30 and 60 (Fig. 2a). In all body orientations, atropinized animals presented a PPC that was slightly higher than those of untreated animals; however, this difference was not statistically signicant (Fig. 2a, b). The values of PPC obtained after the muscarinic blockade were 0.301 0.037 mV in the hori

zontal position, which decreased to 0.195 0.028 and

0.170 0.017 mV while the animals were positioned at 30

and 60 (Fig. 2b). Upon double autonomic blockade, PPC rose to 0.565 0.083 mV at 0 and drastically decreased

to 0.245 0.034 and 0.203 0.028 mV when inclined to

30 and 60, respectively (Fig. 2c). The PPC values of double-blocked animals were signicantly higher than those of untreated pythons only in the case of the horizontal position (Fig. 2a, c). Figure 2 also depicts the PPC variation that occurred when the animals were tilted, a nding which shows that the drop in PPC induced by orthostasis is greater in double-blocked animals than in untreated specimens.

Untreated pythons were found to have a fH of 23.7 1.1 bpm in the horizontal position, and fH increased

to 33.2 1.2 and 35.2 1.1 bpm when tilted to 30

and 60, respectively (Fig. 3a). As depicted in Fig. 3b, atropine caused signicant tachycardia in the animals (31.9 1.6 bpm), but during inclinations, fH did not increase

signicantly and remained similar to those of untreated pythons (36.3 1.2 bpm at 30 and 36.5 1.2 bpm

at 60). Following the double autonomic blockade, a non-statistically signicant decrease in 0 fH occurred (18.4 0.8 bpm), and this parameter then became virtually

constant (18.7 0.9 bpm at 30, and 18.8 1.0 bpm at

60) (Fig. 3c). No differences were found between fH and PPC values at inclinations of 30 and 60 (Figs. 2a, 3a).

Figure 4 illustrates the calculated autonomic tone in the hearts of the snakes positioned at 0, 30, and 60. When in the horizontal position, the pythons exhibited adrenergic tone of 41.7 2.8 % and cholinergic tone of 20.5 3.7 %

(Fig. 4a). The cholinergic tone almost disappeared when the animals were tilted (4.9 1.5 % at 30, 1.7 1.7 %

at 60), whereas the adrenergic tone presented an inconsistent variation (increased to 48.2 2.4 % at 30 and

to 48.0 2.9 % at 60) (Fig. 4b, c). No differences were

found between 30 and 60 autonomic tones.

The comparison between the parameters of untreated and saline-treated animals did not show any statistical difference. The fH values of the pythons treated with isosmotic saline were 24.6 1.0 bpm at 0, 32.7 1.2 bpm

at 30, and 33.2 2.1 bpm at 60, and the PPC values were

0.270 0.051 mV at 0, 0.136 0.030 mV at 30, and

0.131 0.020 mV at 60 of inclination (Table 1).

Discussion

Critique of the methods

Previous studies (Rosenbluth and Simeone 1934; Altimiras et al. 1997; Wang et al. 2001) demonstrated that the order of the pharmacological manipulations can affect the calculated autonomic tone. This factor suggests an interaction between the autonomic subsystems in which the effects of cholinergic blockade are compensated by a change in adrenergic tone, and vice versa. In two species of sh, Altimiras et al. (1997) demonstrated that propranolol should be injected after atropine because it enhances cholinergic tone

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a b

c d

e f

Fig. 1 Descriptive graphs showing the heart rate (fH) and carotid pulse pressure (PPC) in the 2 min preceding the inclinations, the whole period that the animals were kept tilted, and the 2 min following the inclinations. The gure depicts the untreated control condition (a, b), the values obtained after muscarinic cholinoceptor blockade

with atropine (2.5 mg kg1) (c, d), and following the establishment of double autonomic blockade with propranolol (3.5 mg kg1; while atropine was still exerting its effects) (e, f). The solid line corresponds to fH, while the dotted line corresponds to PPC

when injected beforehand. However, although the order of administration was not reversed in our study, the inuence of this inversion on autonomic tone is irrelevant in Python molurus (V. A. Armelin and L. H. Florindo, unpublished observations).

Another possible critique is in the use of pulse plethysmography. Despite the inefciency of this technique in providing the raw values of arterial pressure, the acquisition of PPC proved to be sufcient for achieving the goals of this study. Moreover, this is a non-invasive technique, and therefore reduces the animals discomfort during the experiments and also prevents death.

Physiological consequences of tilting and the importance of orthostatic-tachycardia

The decrease in PPC during orthostasis indicates the occurrence of a gravity-induced caudal blood pooling, which

is primarily determined by the difculty imposed upon venous blood in its return to the heart. As previously observed in Python molurus and other reptiles, decreased venous return reduces cardiac lling and stroke volume by the ways of Frank-Starling law of the heart (Franklin 1994; Franklin and Axelsson 1994; Wang et al. 2002; Skals et al. 2005) and leads to a matched decrease in cardiac output and arterial blood pressure. This prediction of caudal blood pooling is corroborated by the sudden increase in PPC after the transitions from the upright position to the horizontal position (Fig. 1bd), in which the accumulated blood rapidly returns to the heart and causes a considerable elevation in cardiac lling and stroke volume.

It is well known that baroreexes are a prominent controller of arterial pressure in all groups of lower vertebrates (Burggren et al. 1997). In snakes, the baroreceptors that trigger these reexes seem to be mainly located in their central vasculature (Lillywhite and Donald 1994); thus,

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a b c

Fig. 2 Carotid pulse pressure (PPC) of untreated (a), atropinized (b) and double-blocked (c) Python molurus, when horizontal and when at full body inclinations of 30 and 60 (N = 7; paired data). The g

ure shows the untreated control condition, the values obtained after muscarinic cholinoceptor blockade with atropine (2.5 mg kg1), and following the establishment of double autonomic blockade with propranolol (3.5 mg kg1; while atropine was still exerting its effects).

The graph also depicts the PPC variation that occurred when the ani-

mals were tilted (0 vs 30 and 0 vs 60; for each treatment). Asterisks indicates a signicant difference from horizontal pythons of the same treatment; double daggers indicates a signicant difference from the same inclination of untreated animals; and ampersand indicates a signicant difference from the same repositioning of untreated animals. No statistical difference exists between the values of 30 and 60 inclinations in any treatment

a b c

Fig. 3 Heart rate (fH) of untreated (a), atropinized (b) and double-blocked (c) Python molurus, when horizontal and when at full body inclinations of 30 and 60 (N = 7; paired data). The gure shows

the untreated control condition, the values obtained posteriorly muscarinic cholinoceptor blockade with atropine (2.5 mg kg1), and following the establishment of double autonomic blockade with pro-

pranolol (3.5 mg kg1; while atropine was still exerting its effects). Asterisks indicates a signicant difference from horizontal pythons of the same treatment; double daggers indicates a signicant difference from the same inclination of untreated animals. No statistical difference exists between the values of 30 and 60 inclinations in any treatment

changes in central arterial pressure become a precondition for this reex to occur. Baroreexes are perhaps the most important artice for regulating blood pressure during disturbances related to postural changes in serpents (Lillywhite 1996); as expected, our results showed that the stroke volume impairment caused by the orthostatic-induced blood pooling (which consequently reduces blood pressure) rapidly generated the baroregulatory reex characterized by an increase in fH (Fig. 1a, b) that raises cardiac output, and also by vasomotor adjustments that reduce caudal blood accumulation. Together, these changes

improve venous return and stroke volume (note the slight PPC increase at the nal moments of the inclination periods in Fig. 1be).

Seymour and Arndt (2004) bent serpents in the region of the heart and tilted the two ends of their body independently (head-up and tail-down tilts). They noticed that arterial pressure of the head decreases more during a head-up tilt than during a tail-down tilt. Based on this nding, they argued that the vertical blood column above the heart is the most important factor in determining blood pressure in the heads of snakes. However, they overlooked the fact that

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Fig. 4 Calculated autonomic tones in the heart of Python molurus horizontally positioned (a), at a 30 inclination (b) and at a 60 inclination (c) (N = 7;

paired data). Asterisks indicates a signicant difference from the same type of tonus observedin horizontal animals. No signicant differences were found between 30 and 60 autonomic tones

a b c

Table 1 Heart rate and carotid pulse pressure of animals without treatment and under isosmotic saline treatment

Values presented as mean SEM. Unpaired data. No signicant differences were detected between untreated and saline-treated animals

fH heart rate, PPC carotid pulse pressure

0 fH 0 PPC 30 fH 30 PPC 60 fH 60 PPC

Untreated animals (N = 7) 23.7 1.1 0.247 0.026 33.2 1.2 0.145 0.010 35.2 1.1 0.126 0.006

Saline group (N = 4) 24.6 1.0 0.270 0.051 32.7 1.2 0.136 0.030 33.2 2.1 0.131 0.020

their results also showed that a tail-down position reduces central arterial pressure and promotes tachycardia, whereas a head-up position does not change these parameters. This factor can explain the lower decrease in arterial pressure of the head when the snakes were kept in a tail-down position. This response was probably triggered by the stimulation of baroreceptors, and suggests that caudal blood pooling may be more important than the blood column above the heart, regardless of the controversial existence of a siphon that counterbalances the effects of gravity in the anterior regions of these animals [for an overview on this controversy, please see the works of Seymour and Johansen (1987), Hicks and Badeer (1989), (1992); Seymour et al. (1993), Gisolf et al. (2005) and Hicks and Munis (2005)].

The muscarinic receptor blockade caused an increase in fH and practically prevented its variation during orthostasis (Fig. 3a, b). In addition, atropine failed to induce any signicant change in PPC (Fig. 2a, b). Because the same magnitude of fH alteration was observed during orthostasis, we can also conclude that the decrease in PPC observed after the inclinations was not a consequence of the increased fH.

On the other hand, complete autonomic blockade decreased fH, abolished orthostatic-tachycardia (Fig. 3c), and almost doubled PPC (Fig. 2a, c). This rise in PPC values was likely caused by an increase in cardiac lling derived from the lower fH, and/or by the occurrence of a systemic vasoconstriction elicited by the 2-adrenoreceptor blockade by propranolol, which may have recruited blood reservoirs [which is consistent with the ndings of Skals et al. (2005), who

reported that injections of the -adrenergic agonist isoproterenol reduced mean systemic blood pressure and raised fH

in Crotalus durissus].

Although the PPC values of double-blocked animals were higher than those found in untreated specimens in the horizontal position, a nding which hindered direct comparisons and deductions, the experiment revealed that the tilt-elicited PPC drop seen in double-blocked pythons exceeds that of untreated pythons (Fig. 2a, c), even with the vasoconstriction induced by propranolol (the reduction was ~45.14 and ~60.35 % in untreated and double-blocked animals, respectively). Systemic vasoconstriction limits capacitance of blood vessels, which, in turn, diminishes blood pooling (Lillywhite 1996). Thus, because the double autonomic blockade slightly reduced fH and also prevented its rise during orthostasis, it is conceivable that the extra blood that would be pumped to the body by this adjustment have been accumulated in the animals posterior regions. This accumulation may have generated a level of hydro-static pressure that could distend even constricted vessels and lead to blood pooling (i.e., if vasoconstriction had not been provoked by propranolol, the PPC reduction would have certainly been proportionally higher).

Stroke volume modications derived from vasomotor adjustments seemed to have particularly negligible effects when compared to changes in fH, as can be noted by the minimum PPC increase near the end of the inclination period (Fig. 1be). Therefore, the basal adrenergic vascular tone of these animals may be preventing a more drastic

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decrease in stroke volume during orthostasis. Similar relationships between fH and stroke volume have been reported for Python molurus during exercise (Secor et al. 2000) and for Trachemys scripta during several situations such as activity, temperature variation, and apnea (Wang and Hicks 1996; Krosniunas and Hicks 2003).

Lillywhite (1993) found a noteworthy extent of ortho-static intolerance in terrestrial viperid snakes, since both carotid blood ow and pulse pressure were practically nonexistent in Bitis arietans and Bitis nasicornis positioned at 45. The author also compared this intolerance to that of aquatic snakes and observed a very similar response in the aquatic colubrid Farancia erytrogamma tilted at 30 (it is important to emphasize that all animals possessed functioning barostatic reexes). Furthermore, Lillywhite and Seymour (1978) blocked the orthostatic-tachycardia of the scansorial snake Notechis scutatus and showed that changes in fH are not very important for the maintenance of satisfactory cardiovascular parameters during orthostasis in this species. Hence, it is reasonable to infer that the importance of orthostatic-tachycardia in sustaining blood circulation during vertical positions in snakes is directly related to cardiac morphology and cardiac physiology of the species, given that the hearts of pythons are more efcient in maintaining higher systemic blood ow and pressure compared to those of other snakes (Jensen et al. 2010a, b; Enok et al. 2014). Finally, our results show that, despite being a eurytopic snake with a very long body [a characteristic that increases the demands that gravity imposes upon the cardiovascular system (Lillywhite and Smits 1992)], the Python molurus does not suffer circulatory impairments as great as viperids or aquatic snakes suffer. This factor qualies Python molurus as a species that is tolerant to orthostasis.

Autonomic control of heart rate during orthostasis

The present study shows that orthostatic-tachycardia is exclusively mediated by the autonomic nervous system in Python molurus. Skovgaard et al. (2009) and Enok et al. (2012) demonstrated that non-adrenergic and non-cholinergic factors are strongly involved in fH regulation of digesting pythons, and although such factors were not considered in our experiments, they do not seem to play a role in orthostatic-tachycardia regulation because the fH of double blocked animals remained constant during inclinations (Fig. 3c). Such fH constancy also guarantees the trustworthiness of the autonomic tone values calculated and ensures that -adrenoreceptors play no chronotropic role in Python molurus, at least when they are acclimated to the conditions of this studysituation identical to that observed in Boa constrictor (Wang et al. 2001) acclimated to ~28 C and different from that observed in Rana tigrina acclimated to ~6 C (Chiu and Chu 1989).

At 0, the pythons exhibited an adrenergic tone value that was approximately twice as large as the cholinergic tone value (Fig. 4a). The tachycardia induced by orthostasis was essentially elicited by a retirement of vagal drive combined with a non-signicant increase in adrenergic tone (Fig. 4b, c), and we cannot distinguish whether it originated from the sympathetic nervous system or from circulating catecholamines of the adrenal gland (since our methodology involved the pharmacological blockade of -adrenoreceptors). Some studies have revealed that circulating catecholamines play an important role in cardiovascular regulation during exercise in reptiles (Stinner and Ely 1993) and amphibians (Wahlqvist and Campbell 1988; Axelsson et al. 1989). Wang et al. (2001) also speculated that a large proportion of the increased adrenergic tone in the heart of Boa constrictor under forced activity may be explained by circulating catecholamines. However, in the case of orthostasis, neither the sympathetic nervous system nor circulating catecholamines appeared to be of great importance to fH regulation relative to the parasympathetic nervous system.

Altimiras et al. (1997) and Wang et al. (2001) pointed out that the parasympathetic nervous system plays the dominant role in determining fH in animals at rest, and considering that the pythons remained at rest throughout our experiments (independently of body orientation), the acquired results are also in line with this prediction. Altimiras (1999) commented that the parasympathetic subdivision of the autonomic nervous system promotes more accurate and faster changes in fH compared to the sympathetic subdivision, an argument that gives sense to the hypothesis of cholinergic predominance in fH regulation in most vertebratesalthough there are exceptions, such as Notechis scutatus (Lillywhite and Seymour 1978). Nevertheless, our study unraveled the autonomic control of fH only during orthostasis, and in nature this situation is intrinsically associated with exercise, that is a condition in which the adrenergic systems are of considerable importance in cardiovascular regulation.

Acknowledgments This study was supported by the Brazilian National Council for Scientic and Technological Development (CNPq) and the So Paulo Research Foundation (FAPESP), through the Brazilian National Institute of Science and Technology in Comparative Physiology (INCT-FisC). We are grateful to Professors FP de Souza, MA Fossey, LPR Venancio and T Wang for the helpful discussions on several aspects of this work. We would also like to thank the INCT-FisC professors and two anonymous reviewers for their comments and suggestions, as well as the members of Florindos Laboratory for the assistance with the animal care.

Ethics in animal experimentation The experiments conducted in the present study were approved by UNESP/IBILCE Ethical Committee for Animal Research (Proc. 041/2011 CEUA), and were in accordance with all the regulations and ethical guidelines in Brazil.

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Conflict of interest The authors declare that they have no conict of interest.

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