Cardiovascular responses and the role of the neurohumoral cardiac regulation during digestion in the herbivorous lizard Iguana iguana.

Carnivorous reptiles exhibit an intense metabolic increment during digestion, which is accompanied by several cardiovascular adjustments responsible for meeting the physiological demands of the gastrointestinal system. Postprandial tachycardia, a well-documented phenomenon in these animals, is mediated by the withdrawal of vagal tone associated with the chronotropic effects of non-adrenergic and non-cholinergic (NANC) factors. However, herbivorous reptiles exhibit a modest metabolic increment during digestion and there is no information about postprandial cardiovascular adjustments. Considering the significant impact of feeding characteristics on physiological responses, we investigated cardiovascular and metabolic responses, as well as the neurohumoral mechanisms of cardiac control, in the herbivorous lizard Iguana iguana during digestion. We measured oxygen consumption rate (O2), heart rate (fH), mean arterial blood pressure (MAP), myocardial activity, cardiac autonomic tone, fH/MAP variability and baroreflex efficiency in both fasting and digesting animals before and after parasympathetic blockade with atropine followed by double autonomic blockade with atropine and propranolol. Our results revealed that the peak of O2 in iguanas was reached 24 h after feeding, accompanied by an increase in myocardial activity and a subtle tachycardia mediated exclusively by a reduction in cardiac parasympathetic activity. This represents the first reported case of postprandial tachycardia in digesting reptiles without the involvement of NANC factors. Furthermore, this withdrawal of vagal stimulation during digestion may reduce the regulatory range for short-term fH adjustments, subsequently intensifying the blood pressure variability as a consequence of limiting baroreflex efficiency.

Cardiorespiratory reflexes in white sturgeon (Acipenser transmontanus): Lack of cardiac baroreflex response to blood pressure manipulation?

Arterial pressure (Pa) regulation is essential to adequately distribute nutrients to metabolizing tissues, remove wastes and avoid lesions associated with hypertension. In vertebrates, short-term Pa regulation is achieved through the baroreflex, which elicits inversely proportional changes in heart rate (fH) and vascular resistance to restore Pa. The cardiac limb of this reflex has been reported in all vertebrate groups studied to date: teleosts, amphibians, snakes, lizards, crocodiles, birds and mammals - which led to the suggestion that the baroreflex is an ancient trait present in all vertebrate species. However, it is not clear whether more basal groups of vertebrates, such as cyclostomes, elasmobranchs and chondrosteans, manifest baroreflex regulation of fH. Thus, the aim of this study was to determine whether the white sturgeon (Acipenser transmontanus; Chondrostei: Acipenseridae) exhibits a cardiac baroreflex. To do so, we induced Pa perturbations through injections of phenylephrine, sodium nitroprusside (SNP) and saline solution (hypervolemia), and examined possible fH baroreflex responses. We also investigated whether fH responses triggered by fright and chemoreflex were present in this species, in order to confirm the potential of sturgeon to perform reflexive cardiac adjustments. The findings indicate that A. transmontanus exhibits reflex bradycardia in response to fright and chemoreceptor stimulation, illustrating its capacity for short-term cardiac regulation. However, this species does not display baroreflex control of fH across its physiological range. This dissociation suggests that while the nervous and cardiovascular systems of A. transmontanus are primed for rapid reflex responses, a cardiac baroreflex mechanism remains absent.

Do spatial and recognition memories have a lateralized processing by the dorsal hippocampus CA3?

The present study evaluated the function of the right and left CA3 of the dorsal hippocampus (dHPC) in the processing of (i) recognition memory, (ii) recent and remote spatial memory, (iii) working memory and (iv) navigation strategy. Wistar rats were divided into four experimental groups: vehicle group (VG), animals received a bilateral injection of phosphate-saline buffer (PBS) in both right and left dorsal CA3; dHPC-R group, animals received an injection of ibotenic acid (IBO) in the right dorsal CA3; dHPC-L group, animals received an IBO injection in left dorsal CA3; and dHPC-Bi group, animals received bilateral injections of IBO in both dorsal CA3. Rats were submitted to a sequence of behavioral tests: Morris water maze (MWM), object recognition test (ORT), forced T-maze and MWM 30 days after the first exposure. The results showed no evidence of functional lateralization and the dorsal CA3 does not seem to be essential for learning and memory (recent and remote) processing and allocentric navigation analyzed in the MWM and T-maze, respectively. However, rats with right or bilateral lesions in the dorsal CA3 failed to recognize the familiar object in the ORT, suggesting a lateralized processing of recognition memory. That result is unprecedented and contributes to the knowledge about the compartmentalization of HPC functions.

The baroreflex in aquatic and amphibious teleosts: Does terrestriality represent a significant driving force for the evolution of a more effective baroreflex in vertebrates?

All vertebrates have baroreflexes that provide fast regulation of arterial blood pressure (PA) to maintain adequate tissue perfusion and avoid vascular lesions from excessive pressures. The baroreflex is a negative feedback loop, where altered PA results in reciprocal changes in heart rate (fH) and systemic vascular conductance to restore pressure. In terrestrial environments, gravity usually leads to blood pooling in the lower body reducing venous return, cardiac filling, cardiac output and PA. Conversely, in aquatic environments, the hydrostatic pressure of surrounding water mitigates blood pooling and prevents vascular distensions. In this context, we aimed to test the hypothesis that vertebrate species that were exposed to gravity-induced hemodynamic disturbances throughout their evolutionary histories have a more effective barostatic reflex than those that were not. We examined the cardiac baroreflex of fish that perform (Clarias gariepinus and Hoplerythrinus unitaeniatus) and do not perform (Hoplias malabaricus and Oreochromis niloticus) voluntary terrestrial sojourns, using pharmacological manipulations of PA to characterize reflex changes in fH using a four-variable sigmoidal logistic function (i.e. the "Oxford technique"). Our results revealed that amphibious fish exhibit higher baroreflex gain and responsiveness to hypotension than strictly aquatic fish, suggesting that terrestriality and the gravitational circulatory stresses constitute a relevant driving force for the evolution of a more effective baroreflex in vertebrates. We also demonstrate that strictly aquatic teleosts have considerable baroreflex gain, supporting the view that the baroreflex is an ancient cardiovascular trait that appeared before vertebrates colonized the gravity-dominated realm of land.

Postprandial cardiorespiratory responses and the regulation of digestion-associated tachycardia in Nile tilapia (Oreochromis niloticus).

Cardiorespiratory adjustments that occur after feeding are essential to supply the demands of digestion in vertebrates. The well-documented postprandial tachycardia is triggered by an increase in adrenergic activity and by non-adrenergic non-cholinergic (NANC) factors in mammals and crocodilians, while it is linked to a withdrawal of vagal drive and NANC factors in non-crocodilian ectotherms-except for fish, in which the sole investigation available indicated no participation of NANC factors. On the other hand, postprandial ventilatory adjustments vary widely among air-breathing vertebrates, with different species exhibiting hyperventilation, hypoventilation, or even no changes at all. Regarding fish, which live in an environment with low oxygen capacitance that requires great ventilatory effort for oxygen uptake, data on the ventilatory consequences of feeding are also scarce. Thus, the present study sought to investigate the postprandial cardiorespiratory adjustments and the mediation of digestion-associated tachycardia in the unimodal water-breathing teleost Oreochromis niloticus. Heart rate (fH), cardiac autonomic tones, ventilation rate (fV), ventilation amplitude, total ventilation and fH/fV variability were assessed both in fasting and digesting animals under untreated condition, as well as after muscarinic cholinergic blockade with atropine and double autonomic blockade with atropine and propranolol. The results revealed that digestion was associated with marked tachycardia in O. niloticus, determined by a reduction in cardiac parasympathetic activity and by circulating NANC factors-the first time such positive chronotropes were detected in digesting fish. Unexpectedly, postprandial ventilatory alterations were not observed, although digestion triggered mechanisms that were presumed to increase oxygen uptake, such as cardiorespiratory synchrony.

Cardiovascular and ventilatory interactions in the facultative air-breathing teleost Pangasianodon hypophthalmus.

All vertebrates possess baroreceptors monitoring arterial blood pressure and eliciting reflexive changes in vascular resistance and heart rate in response to blood pressure perturbations imposed by, e.g., exercise, hypoxia, or hemorrhage. There is considerable variation in the magnitude of the baroreflex amongst vertebrate groups, making phylogenetic trends and association with major evolutionary events such as air-breathing and endothermy, difficult to identify. In the present study, we quantified the baroreflex in the facultative air-breathing catfish Pangasianodon hypophthalmus. Using a pharmacological approach, we quantified the cardiac limb of the baroreflex and by subjecting fish to hypoxia and by stimulation with NaCN with and without pharmacological autonomic blockade; we also examined the cardiovascular regulation associated with air-breathing. As in most other air-breathing fish, air-breathing elicited a substantial tachycardia. This tachycardia was abolished by cholinergic muscarinic pharmacological blockade, which also abolished the cardiac limb of the baroreflex, and consequently such fish failed to maintain their arterial blood pressure when air-breathing. In higher vertebrate classes, baroreceptors elicit ventilatory changes; however, whether this is the case in fish has not previously been investigated. Pangasianodon hypophthalmus demonstrated a prominent increase in ventilation during imposed hypotension. Collectively, these results demonstrate, for the first time, an efficient baroreflex in an air-breathing fish, point towards involvement of baroreceptors in blood pressure regulation during air-breathing, and show a correlation between blood pressure and ventilation, providing additional information on the origin of this link.

Autonomic control of cardiovascular adjustments associated with orthostasis in the scansorial snake Boa constrictor.

Orthostatic hypotension is a phenomenon triggered by a change in the position or posture of an animal, from a horizontal to a vertical head-up orientation, characterised by a blood pooling in the lower body and a reduction in central and cranial arterial blood pressure (PA). This hypotension elicits systemic vasoconstriction and tachycardia, which generally reduce blood pooling and increase PA Little is known about the mediation and importance of such cardiovascular adjustments that counteract the haemodynamic effects of orthostasis in ectothermic vertebrates, and some discrepancies exist in the information available on this subject. Thus, we sought to expand our knowledge on this issue by investigating it in a more elaborate way, through an in vivo pharmacological approach considering temporal circulatory changes during head-up body inclinations in unanaesthetised Boa constrictor To do so, we analysed temporal changes in PA, heart rate (fH) and cardiac autonomic tone associated with 30 and 60 deg inclinations, before and after muscarinic blockade with atropine, double blockade with atropine and propranolol, and α1-adrenergic receptor blockade with prazosin. Additionally, the animals' fH variability was analysed. The results revealed that, in B. constrictor: (1) the orthostatic tachycardia is initially mediated by a decrease in cholinergic tone followed by an increase in adrenergic tone, a pattern that may be evolutionarily conserved in vertebrates; (2) the orthostatic tachycardia is important for avoiding an intense decrease in PA at the beginning of body inclinations; and (3) α1-adrenergic orthostatic vasomotor responses are important for the maintenance of PA at satisfactory values during long-term inclinations.

Control of air-breathing in fishes: Central and peripheral receptors.

This review considers the environmental and systemic factors that can stimulate air-breathing responses in fishes with bimodal respiration, and how these may be controlled by peripheral and central chemoreceptors. The systemic factors that stimulate air-breathing in fishes are usually related to conditions that increase the O2 demand of these animals (e.g. physical exercise, digestion and increased temperature), while the environmental factors are usually related to conditions that impair their capacity to meet this demand (e.g. aquatic/aerial hypoxia, aquatic/aerial hypercarbia, reduced aquatic hidrogenionic potential and environmental pollution). It is now well-established that peripheral chemoreceptors, innervated by cranial nerves, drive increased air-breathing in response to environmental hypoxia and/or hypercarbia. These receptors are, in general, sensitive to O2 and/or CO2/H+ levels in the blood and/or the environment. Increased air-breathing in response to elevated O2 demand may also be driven by the peripheral chemoreceptors that monitor O2 levels in the blood. Very little is known about central chemoreception in air-breathing fishes, the data suggest that central chemosensitivity to CO2/H+ is more prominent in sarcopterygians than in actinopterygians. A great deal remains to be understood about control of air-breathing in fishes, in particular to what extent control systems may show commonalities (or not) among species or groups that have evolved air-breathing independently, and how information from the multiple peripheral (and possibly central) chemoreceptors is integrated to control the balance of aerial and aquatic respiration in these animals.

The autonomic control of upright posture tachycardia in the arboreal lizard Iguana iguana.

In terrestrial environments, upright spatial orientation can dramatically influence animals' hemodynamics. Generally, large and elongated species are particularly sensitive to such influence due to the greater extent of their vascular beds being verticalized, favoring the establishment of blood columns in their bodies along with caudal blood pooling, and thus jeopardizing blood circulation through a cascade effect of reductions in venous return, cardiac filling, stroke volume, cardiac output, and arterial blood pressure. This hypotension triggers an orthostatic-(baroreflex)-tachycardia to normalize arterial pressure, and despite the extensive observation of this heart rate (fH ) adjustment in experiments on orthostasis, little is known about its mediation and importance in ectothermic vertebrates. In addition, most of the knowledge on this subject comes from studies on snakes. Thus, our objective was to expand the knowledge on this issue by investigating it in an arboreal lizard (Iguana iguana). To do so, we analyzed fH , cardiac autonomic tones, and fH variability in horizontalized and tilted iguanas (0°, 30°. and 60°) before and after muscarinic blockade with atropine and double autonomic blockade with atropine and propranolol. The results revealed that I. Iguana exhibits significant orthostatic-tachycardia only at 60o inclinations-a condition that is primarily elicited by a withdrawal of vagal drive. Also, as in humans, increases in low-frequency fH oscillations and decreases in high-frequency fH oscillations were observed along with orthostatic-tachycardia, suggesting that the mediation of this fH adjustment may be evolutionarily conserved in vertebrates.

The influence of midazolam on heart rate arises from cardiac autonomic tones alterations in Burmese pythons, Python molurus.

The GABAA receptor agonist midazolam is a compound widely used as a tranquilizer and sedative in mammals and reptiles. It is already known that this benzodiazepine produces small to intermediate heart rate (HR) alterations in mammals, however, its influence on reptiles' HR remains unexplored. Thus, the present study sought to verify the effects of midazolam on HR and cardiac modulation in the snake Python molurus. To do so, the snakes' HR, cardiac autonomic tones, and HR variability were evaluated during four different experimental stages. The first stage consisted on the data acquisition of animals under untreated conditions, in which were then administered atropine (2.5mgkg-1; intraperitoneal), followed later by propranolol (3.5mgkg-1; intraperitoneal) (cardiac double autonomic blockade). The second stage focused on the data acquisition of animals under midazolam effect (1.0mgkg-1; intramuscular), which passed through the same autonomic blockade protocol of the first stage. The third and fourth stages consisted of the same protocol of stages one and two, respectively, with the exception that atropine and propranolol injections were reversed. By comparing the HR of animals that received midazolam (second and fourth stages) with those that did not (first and third stages), it could be observed that this benzodiazepine reduced the snakes' HR by ~60%. The calculated autonomic tones showed that such cardiac depression was elicited by an ~80% decrease in cardiac adrenergic tone and an ~620% increase in cardiac cholinergic tone - a finding that was further supported by the results of HR variability analysis.

The Effects of Feeding on Cardiac Control of the Broad-Nosed Caiman (Caiman latirostris): The Role of the Autonomic Nervous System and NANC Factors.

The metabolic increment that occurs after feeding demands cardiovascular adjustments to be maintained, as increased heart rate (fH ) and cardiac output. In mammals, postprandial tachycardia seems to be triggered by an increase in adrenergic activity and by nonadrenergic noncholinergic (NANC) factors, while in ectothermic vertebrates, this adjustment seems to be linked to a withdrawal of vagal drive as well as to NANC factors. Because the factors behind postprandial tachycardia have not yet been investigated in crocodilians, the present study sought to evaluate the postprandial tachycardia mediators in the broad-nosed caiman. To this end, fasting and digesting animals were instrumented with intraperitoneal cannula and subcutaneous electrocardiogram electrodes (for the measurement of fH , cardiac autonomic tones, and total fH variability, as well as for a power spectral analysis of fH ). Data were then collected with the animals in an untreated state, as well as after muscarinic cholinergic blockade with atropine (2.5 mg kg-1 ) and after double autonomic blockade with atropine and propranolol (5.0 mg kg-1 ). Fasting animals' fH was ∼18 bpm, a value which increased to ∼30 bpm during digestion. After the double autonomic blockade, fasting animals exhibited an fH of ∼15 bpm, while digesting animals' fH was ∼23 bpm. This result is evidence of the presence of NANC factors with positive chronotropic effects acting during digestion. The calculated autonomic tones showed that, after feeding, the adrenergic tone increased while the cholinergic tone remained unchanged. Finally, fH variability analyses revealed that this adrenergic increase is primarily derived from circulating catecholamines.

Gill denervation eliminates the barostatic reflex in a neotropical teleost, the tambaqui (Colossoma macropomum).

The baroreflex is one of the most important regulators of cardiovascular homeostasis in vertebrates. It begins with the monitoring of arterial pressure by baroreceptors, which constantly provide the central nervous system with afferent information about the status of this variable. Any change in arterial pressure relative to its normal state triggers autonomic responses, which are characterized by an inversely proportional change in heart rate and systemic vascular resistance and which tend to restore pressure normality. Although the baroreceptors have been located in mammals and other terrestrial vertebrates, their location in fish is still not completely clear and remains quite controversial. Thus, the objective of this study was to locate the baroreceptors in a teleost, the Colossoma macropomum. To do so, the occurrence and efficiency of the baroreflex were both analyzed when this mechanism was induced by pressure imbalancements in intact fish (IN), first-gill-denervated fish (G1), and total-gill-denervated fish (G4). The pressure imbalances were initiated through the administration of the α1-adrenergic agonist phenylephrine (100 µg kg(-1)) and the α1-adrenergic antagonist prazosin (1 mg kg(-1)). The baroreflex responses were then analyzed using an electrocardiogram that allowed for the measurement of the heart rate, the relationship between pre- and post-pharmacological manipulation heart rates, the time required for maximum chronotropic baroreflex response, and total heart rate variability. The results revealed that the barostatic reflex was attenuated in the G1 group and nonexistent in G4 group, findings which indicate that baroreceptors are exclusively located in the gill arches of C. macropomum.

Autonomic control of post-air-breathing tachycardia in Clarias gariepinus (Teleostei: Clariidae).

The African catfish (Clarias gariepinus) is a teleost with bimodal respiration that utilizes a paired suprabranchial chamber located in the gill cavity as an air-breathing organ. Like all air-breathing fishes studied to date, the African catfish exhibits pronounced changes in heart rate (f H) that are associated with air-breathing events. We acquired f H, gill-breathing frequency (f G) and air-breathing frequency (f AB) in situations that require or do not require air breathing (during normoxia and hypoxia), and we assessed the autonomic control of post-air-breathing tachycardia using an infusion of the ß-adrenergic antagonist propranolol and the muscarinic cholinergic antagonist atropine. During normoxia, C. gariepinus presented low f AB (1.85 ± 0.73 AB h(-1)) and a constant f G (43.16 ± 1.74 breaths min(-1)). During non-critical hypoxia (PO2 = 60 mmHg), f AB in the African catfish increased to 5.42 ± 1.19 AB h(-1) and f G decreased to 39.12 ± 1.58 breaths min(-1). During critical hypoxia (PO2 = 20 mmHg), f AB increased to 7.4 ± 1.39 AB h(-1) and f G decreased to 34.97 ± 1.78 breaths min(-1). These results were expected for a facultative air breather. Each air breath (AB) was followed by a brief but significant tachycardia, which in the critical hypoxia trials, reached a maximum of 143 % of the pre-AB f H values of untreated animals. Pharmacological blockade allowed the calculation of cardiac autonomic tones, which showed that post-AB tachycardia is predominantly regulated by the parasympathetic subdivision of the autonomic nervous system.

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

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 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 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.

Branchial O(2) chemoreceptors in Nile tilapia Oreochromis niloticus: Control of cardiorespiratory function in response to hypoxia.

This study examined the distribution and orientation of gill O(2) chemoreceptors in Oreochromis niloticus and their role in cardiorespiratory responses to graded hypoxia. Intact fish, and a group with the first gill arch excised (operated), were submitted to graded hypoxia and their cardiorespiratory responses (oxygen uptake - V˙O(2) , breathing frequency - fR, ventilatory stroke volume - VT, gill ventilation - V˙G, O(2) extraction from the ventilatory current - EO(2) , and heart rate - fH) were compared. Their responses to bolus injections of NaCN into the bloodstream (internal) or ventilatory water stream (external) were also determined. The V˙O(2) of operated fish was significantly lower at the deepest levels of hypoxia. Neither reflex bradycardia nor ventilatory responses were completely abolished by bilateral excision of the first gill arch. EO(2) of the operated group was consistently lower than the intact group. The responses to internal and external NaCN included transient decreases in fH and increases in fR and Vamp (ventilation amplitude). These cardiorespiratory responses were attenuated but not abolished in the operated group, indicating that chemoreceptors are not restricted to the first gill arch, and are sensitive to oxygen levels in both blood and water.

Hypercarbic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): the role of branchial CO2 chemoreceptors.

The aim of the present study was to determine the roles that externally versus internally oriented CO(2)/H(+)-sensitive chemoreceptors might play in promoting cardiorespiratory responses to environmental hypercarbia in the air-breathing fish, Hoplerythrinus unitaeniatus (jeju). Fish were exposed to graded hypercarbia (1, 2.5, 5, 10 and 20% CO(2)) and also to graded levels of environmental acidosis (pH approximately 7.0, 6.0, 5.8, 5.6, 5.3 and 4.7) equal to the pH levels of the hypercarbic water to distinguish the relative roles of CO(2) versus H(+). We also injected boluses of CO(2)-equilibrated solutions (5, 10 and 20% CO(2)) and acid solutions equilibrated to the same pH as the CO(2) boluses into the caudal vein (internal) and buccal cavity (external) to distinguish between internal and external stimuli. The putative location of the chemoreceptors was determined by bilateral denervation of branches of cranial nerves IX (glossopharyngeal) and X (vagus) to the gills. The data indicate that the chemoreceptors eliciting bradycardia, hypertension and gill ventilatory responses (increased frequency and amplitude) to hypercarbia are exclusively branchial, externally oriented and respond specifically to changes in CO(2) and not H(+). Those involved in producing the cardiovascular responses appeared to be distributed across all gill arches while those involved in the gill ventilatory responses were located primarily on the first gill arch. Higher levels of aquatic CO(2) depressed gill ventilation and stimulated air breathing. The chemoreceptors involved in producing air breathing in response to hypercarbia also appeared to be branchial, distributed across all gill arches and responded specifically to changes in aquatic CO(2). This would suggest that chemoreceptor groups with different orientations (blood versus water) are involved in eliciting air-breathing responses to hypercarbia in jeju.

Hypoxic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): role of branchial O2 chemoreceptors.

In one series of experiments, heart frequency (f (H)), blood pressure (P (a)), gill ventilation frequency (f ( R )), ventilation amplitude (V (AMP)) and total gill ventilation (V (TOT)) were measured in intact jeju (Hoplerythrinus unitaeniatus) and jeju with progressive denervation of the branchial branches of cranial nerves IX (glossopharyngeal) and X (vagus) without access to air. When these fish were submitted to graded hypoxia (water PO(2) approximately 140, normoxia to 17 mmHg, severe hypoxia), they increased f ( R ), V (AMP), V (TOT) and P (a) and decreased f (H). In a second series of experiments, air-breathing frequency (f (RA)), measured in fish with access to the surface, increased with graded hypoxia. In both series, bilateral denervation of all gill arches eliminated the responses to graded hypoxia. Based on the effects of internal (caudal vein, 150 microg NaCN in 0.2 mL saline) and external (buccal) injections of NaCN (500 microg NaCN in 1.0 mL water) on f (R), V (AMP), V (TOT), P (a) and f (H) we conclude that the O(2) receptors involved in eliciting changes in gill ventilation and associated cardiovascular responses are present on all gill arches and monitor the O(2) levels of both inspired water and blood perfusing the gills. We also conclude that air breathing arises solely from stimulation of branchial chemoreceptors and support the hypothesis that internal hypoxaemia is the primary drive to air breathing.