Effect of Pharmacological Blockade on Cardiovascular Responses to Voluntary and Forced Diving in Muskrats

Cardiovascular changes evoked by forced submergence of birds and mammals have been extensively studied for more than a century (Bert, 1870; Richet, 1894). These changes include a vagally mediated bradycardia and an increase in peripheral vascular resistance brought about by the adrenergic system. These adjustments are presumed to be largely reflexogenic and result from a set of complex interactions (Daly, 1984; Jones et al. 1988). Input from nasal receptors initiates the response to forced submergence in mammals and diving ducks (Drummond and Jones, 1979; Dykes, 1974; Furilla and Jones, 1986a), whereas chemoreceptor drive initiates the response in dabbling ducks (Jones et al. 1982). The baroreflex also seems to be reset during forced dives (Daly, 1984; Smith and Jones, 1992) and apnoea is necessary for the response to occur (Daly, 1984; Drummond and Jones, 1979). More recent studies have shown that the diving response is also present in voluntarily diving animals but that it is much more labile (Jones et al. 1988). Observations such as cardiac deceleration and acceleration in anticipation of submersion and surfacing, respectively (Jones et al. 1973), habituation of diving bradycardia (Gabbot and Jones, 1987) and conditioning of bradycardia (Ridgway et al. 1975) suggest that the free-diving response may be controlled by higher centres within the central nervous system. Vagal outflow to the heart plays a major role in free-diving bradycardia (MacArthur and Karpan, 1989; Murdaugh et al. 1961), while [3-blockade has little effect on diving bradycardia in ducks (Furilla and Jones, 1987). However, a systematic study of the autonomic control of heart rate during voluntary diving and its effects on underwater endurance has not been carried out. In the present study, we investigated the autonomic nervous pathways involved in the free-diving response using pharmacological blockers. We acutely treated muskrats (Ondatra zibethicus) with the muscarinic blocker atropine, the α-adrenergic blocker phentolamine and the β-adrenergic blockers nadolol and propranolol, and studied, using telemetry, the effects of drugs on heart rate during voluntary dives. We also studied unrestrained forced dives for comparison. Finally, we investigated the effects of these drugs on voluntary diving performance as well as on maximum underwater endurance in forced dives.

Five adult muskrats (three males and two females), ranging in mass from 0.8 to 1.0 kg, were used in experiments. Muskrats Ondatra zibethicus (L.) were trapped in Surrey, British Columbia, and housed in pairs in 76 cmX51 cmX41 cm cages at the Animal Care Centre of the University of British Columbia. They were fed with laboratory rodent diet (LabDiet 5001, PMI Feeds, St Louis, Missouri) supplemented with carrots; each pair had access to a 28 cmX18 cmX13 cm tank filled with running water.

Muskrats were anaesthetized with a mixture of 2 mg kg⁻¹ acepromazine (AC Promazine, Austin Laboratories, Joliette, Quebec) and 40 mg kg⁻¹ ketamine (Ketalean, M.T.C. Pharmaceuticals, Cambridge, Ontario) injected subcutaneously. The eyes were protected with ophthalmic ointment (Neosporin, Burroughs Wellcome, Kirkland, Quebec). Fur was clipped from the area where incisions were to be made and the exposed skin was cleaned with Betadine (Purdue Frederick, Pickering, Ontario). Electrocardiogram (ECG) transmitters (Konigsberg Instruments, Pasadena, California) were implanted in the peritoneal cavity. Transmitter leads were threaded through the peritoneal wall and then subcutaneously over the thoracic cavity, where they were sutured to the ribs. Following surgery, antibiotic powder (Cicatrin, Burroughs Wellcome, Kirkland, Quebec) was applied to skin incisions and 50 mg kg⁻¹ oxytretracycline (Liquamycin, Rogar/STB, Montreal, Quebec) was injected subcutaneously. Experiments were not started until at least 1 week after surgery. All procedures were approved by the Animal Care Committee of the University of British Columbia.

All muskrats received the following treatments in randomized order: untreated (i.e. no injection), injection of saline (control) or injection of the muscarinic antagonist atropine sulphate (1 mg kg⁻¹, Sigma, St Louis, Missouri), the α-adrenergic antagonist phentolamine mesylate (1 mg kg⁻¹, Rogitine, Ciba-Geigy, Mississauga, Ontario) or the β-adrenergic antagonists DL-propranolol hydrochloride (4 mg kg⁻¹, Sigma) and nadolol (4 mg kg⁻¹, Sigma). In addition, a mixture of atropine (1 mg kg⁻¹ ) and phentolamine (1 mg kg⁻¹ ) was administered as the final treatment in all cases. Drugs were mixed in 0.9 % saline and injected subcutaneously at the beginning of diving sessions. Diving sessions for untreated, saline, propranolol, nadolol and atropine groups lasted 2 h, and sessions for phentolamine and atropine– phentolamine groups were for 1 h because of the short half-life of phentolamine. Different treatments were administered at least 2 days apart. To check for efficacy of blockade, heart rate (fH) was monitored after subcutaneous injection of the cholinergic agonist pilocarpine hydrochloride (1 mg kg⁻¹, Sigma), the α-adrenergic agonist l-phenylephrine hydrochloride (2 mg kg-1, Sigma) or the β-adrenergic agonist isoproterenol hydrochloride (0.05 mg kg⁻¹, Isuprel, Sterling-Winthrop, Markham, Ontario) at the end of diving sessions after atropine, phentolamine, and propranolol and nadolol injections, respectively. Effects of agonists injected alone, before blockade, were assessed in two animals. Pilocarpine decreased fH by 40 %, phenylephrine decreased fH by 60 % and isoproterenol increased fH by 40 %. In all cases, the agonists had no effect on fH in muskrats treated with the appropriate antagonist.

Voluntary dives were performed in an indoor 270 cmX122 cmX89 cm tank filled with water to a depth of 60 cm. Water temperature ranged from 8 to 12 ˚C. Muskrats could rest on a 61 cmX43 cm platform located above the water level in one corner of the tank. The tank was divided into four lanes which made an underwater maze when covered with plastic mesh. The animals entered the maze at one end, from the platform, and had to swim to the other end to reach food placed under water. The round trip was 21 m. Muskrats were first left overnight with a low level of water to learn their way around. In the second session, the water level was raised above the maze cover. Data were recorded from the third session. The animal was not given access to the underwater maze for 15 min at the beginning of each session. After drug or saline injection, access to the maze was allowed. There were no observers in the laboratory during free-diving sessions.

Forced dives were performed in a 58 cmX36 cmX23 cm plastic mesh cage submerged in a 91 cmX46 cmX43 cm aquarium. Water temperature ranged from 8 to 12 ˚C. Muskrats were left for 15 min in air in the cage at the beginning of the session. Drugs were injected and the first forced dive was carried out 5 min later. Dive length was varied randomly from 15 s to 2 min. At least 10 dives were performed within one session. Animals were left alone in the laboratory between dives. The experimenter entered the room about 30 s before each dive and left within the first 15 s after the dive.

The effects of the injected drugs on maximum dive time were estimated in three muskrats. To allow the animals to warm up and to get used to the procedure, they were first left in air for 10 min, then submerged for 2 min, left in air for 10 min, submerged for 4 min and again left in air for 10 min. Maximum dive time was then estimated on the third dive, which was stopped when the animals lost their balance. None of these dives was terminal. Saline, DL-propranolol hydrochloride (4 mg kg⁻¹, Sigma), atropine sulphate (1 mg kg⁻¹, Sigma), phentolamine mesylate (1 mg kg⁻ 1, Rogitine, Ciba-Geigy) and a mixture of atropine sulphate (1 mg kg⁻¹ ) and phentolamine mesylate (1 mg kg⁻¹ ) were administered in randomized order.

The ECG was monitored during diving sessions using a Konigsberg Instrument telemetry system (Pasadena, California). The ECG signal was recorded on the audio channel of a video recorder after modulation using a Vetter FM recording adaptor (A.R. Vetter Co., Rebersburg, Pennsylvania). The animal’ s behaviour was recorded on the video channel using a Panasonic camera (Secausus, New Jersey). When tapes were replayed, the ECG signal was demodulated and logged using Labtech Notebook software (Laboratory Technologies Corporation, Wilmington, Massachusetts) running on an IBM-compatible personal computer that calculated and stored inter-beat intervals. An event marker was connected to the computer to record the times of submersion and surfacing, judged from watching the animal on a television monitor. Subsequently, each inter-beat interval was converted to beats min⁻¹ . Mean heart rate over 1 min was calculated during periods of rest (resting fH) and routine activity such as grooming and eating (routine fH) in untreated animals. Routine fH was calculated before and after drug injection in treated muskrats. Mean values from each muskrat (N=5) were averaged to give a grand mean for resting fH and routine fH in the untreated group and for routine fH before and after injection in treated groups. To reduce variability caused by dive duration, only dives lasting more than 40 s and less than 75 s were analysed. The first four dives meeting these criteria were analysed for each animal in each group. Mean fH during diving (diving fH) as well as mean fH during the 15 s preceding a dive (pre-dive fH) and following a dive (post-dive fH) were calculated for each dive. 5 s period averages, starting 20 s before dives and ending 20 s after dives, were also computed, to study heart rate profiles during diving. Values from the four dives were averaged in each animal to give a mean for pre-dive fH, dive fH, post-dive fH and every 5 s period. Mean values from each animal (N=5) were then averaged in each group to give a grand mean for each variable. Maximum underwater times from three muskrats were averaged to give a mean for each treatment (N=3).

Values given in the text are the grand mean ± S.E.M. for heart rate and mean ± S.E.M. for maximum dive time. One-way and two-way analyses of variance (ANOVA) for repeated measures were computed, and multiple comparisons were performed using Student–Newman–Keuls tests. Overall effects and differences were considered significant when P<0.05. All statistics were calculated with SigmaStat software (Jandel Scientific, San Rafael, California).

Fig. 1 shows the distribution of voluntary dive durations in each group. In all groups, muskrats were able to perform dives for periods as long as or longer than the calculated aerobic dive limit for muskrats (approximately 50 s; MacArthur, 1990).

Fig. 2 shows the effects of diving on mean fH during voluntary and forced dives in untreated animals. Resting fH was significantly lower than routine fH (195±14 beats min⁻¹versus 252±13 beats min⁻¹ in voluntary dives and 217±11 beats min⁻¹versus 262±5 beats min⁻¹ in forced dives). Pre-dive fH (291±9 beats min⁻¹ ) was significantly higher than routine fH in voluntary dives and significantly lower (220±12 beats min⁻¹ ) in forced dives. Diving induced a marked decrease in fH in both voluntary and forced dives. Mean diving fH was 112±10 beats min⁻¹ in voluntary dives and 62±8 beats min⁻¹ in forced dives. The difference in fH between the two types of dives was significant. In voluntary dives, fH dropped in 15 s to 104±13 beats min⁻¹ and stayed around this value until the last 5 s of the dive, when it started to increase (Fig. 3). In forced dives, fH at 10 s into the dive was 61±14 beats min⁻¹ ; it remained at this rate until surfacing (Fig. 3). After surfacing, fH returned to pre-dive values within the first 5 s in voluntary dives (286±11 beats min⁻¹ ). Post-dive Fig. 2. Mean heart rate (±S.E.M., N=5) in untreated muskrats before, during and after voluntary and forced dives. Resting fH, routine fH, pre-dive fH, diving fH and post-dive fH are shown. * indicates a value significantly different from routine fH. fH after forced dives (283±6 beats min⁻¹ ) was not significantly different from post-dive fH after voluntary dives (Fig. 2).

The effects of injected drugs and diving on mean fH in the free-diving tank are presented in Fig. 4 and free-diving heart rate profiles are shown in Fig. 5. There was a marked effect of drug treatment on routine fH. Atropine and phentolamine significantly increased fH from 244±15 beats min⁻¹ to 288±2 beats min⁻¹ and from 258±7 beats min⁻¹ to 300±12 beats min⁻¹, respectively. Propranolol and nadolol significantly decreased routine fH from 264±8 beats min⁻¹ to 190±12 beats min⁻¹ and from 236±10 beats min⁻¹ to 183±7 beats min⁻¹, respectively. Saline injection had no effect on routine fH. Pre-dive fH did not differ significantly from routine fH in animals treated with any of the four drugs. Mean diving fH (304±7 beats min⁻¹ ) was not significantly different from pre-dive fH (303±3 beats min⁻¹ ) in atropine-injected muskrats (Fig. 4) and fH in these animals remained constant during the dive (Fig. 5). Diving fH after nadolol (93±12 beats min⁻¹ ) and phentolamine (117±17 beats min⁻¹ ) treatment was not significantly different from diving fH in saline-treated animals (92±10 beats min⁻¹ ) (Fig. 4), and fH profiles were similar during diving (Fig. 5). Diving fH in propranolol-treated animals (138±10 beats min⁻¹ ) was significantly higher than in saline-treated animals (Fig. 4). fH at 15 s into the dive was 130±12 beats min⁻¹ in this group and it remained around this value until the end of the dive, when it showed the same significant increase as in all the other groups except the atropine-treated group (Fig. 5). fH returned to predive values within the first 5 s after surfacing in all groups: 296±11 beats min⁻¹ in the saline group, 298±6 beats min⁻¹ in the atropine group, 307±11 beats min⁻¹ in the phentolamine group, 220±6 beats min⁻¹ in the nadolol group and 214±12 beats min⁻¹ in the propranolol group (Fig. 5).

The effects of injected drugs and submergence on mean fH in the forced diving cage are presented in Fig. 6 and forced diving heart rate profiles in Fig. 7. Atropine and phentolamine significantly increased routine fH from 260±12 beats min⁻¹ to 287±7 beats min⁻¹ and from 247±13 beats min⁻¹ to 304±7 beats min⁻¹, respectively. Propranolol and nadolol significantly decreased routine fH from 249±11 beats min⁻¹ to 196±7 beats min⁻¹ and from 238±5 beats min⁻¹ to 190±5 beats min⁻¹, respectively. Saline injection did not have any effect on routine fH. Routine fH in treated animals in the forced diving cage was not significantly different from routine fH in animals treated with the same drugs in the free-diving tank. Pre-dive fH dropped significantly compared with routine fH after drug treatment in every group except for those treated with atropine and propranolol. Nadolol-treated animals had the lowest pre-dive fH (153±7 beats min⁻¹ ). Saline-and phentolamine-treated groups had an intermediate pre-dive fH (228±12 beats min⁻¹ and 243±27 beats min⁻¹, respectively). Pre-dive fH was high in atropine-treated animals (292±6 beats min⁻¹ ) and low in propranolol-treated animals (181±11 beats min⁻¹ ). fH decreased significantly during diving in each group except for that treated with atropine (306±7 beats min⁻¹ ). Diving fH in propranolol-treated animals (88±9 beats min⁻¹ ) tended to be higher than in saline-(63±9 beats min⁻¹ ) and nadolol-(47±7 beats min⁻¹ ) treated groups but, unlike voluntary dives, differences between these three groups were not significant. Diving fH in phentolamine-treated animals (79±10 beats min⁻¹ ) did not differ from that in the saline-treated group. In every group except that treated with atropine, post-dive fH was significantly higher than pre-dive fH, but did not differ from routine fH.

To study the extent to which the diving response enables muskrats to survive under water, we investigated the effects of injected drugs on maximum dive time in forcibly submerged animals (N=3). Maximum underwater endurance was 12.0±1.1 min in untreated muskrats. Maximum dive time was not significantly different from untreated values in the saline (10.6±1.3 min) and propranolol (11.0±0.1 min) groups. Atropine significantly decreased maximum underwater time to 7.7±0.1 min (a 27 % decrease from the saline-treated group) and phentolamine further reduced maximum dive time to 5.2±0.4 min (a 51 % decrease from the saline-treated group). A mixture of atropine and phentolamine had the same effect on maximum dive time (5.2±0.5 min) as phentolamine alone.

Diving induced a marked and consistent decrease in fH in untreated muskrats. fH dropped to 36 % of pre-dive fH values or 53 % of resting fH values in 15 s in free-diving animals, values that compare well with earlier studies in muskrats (Drummond and Jones, 1979; MacArthur and Karpan, 1989; McCulloch and Jones, 1990). Diving bradycardia was consistently present in every dive, no matter how short. In fact, as soon as the muskrat’ s nose entered the water, fH slowed down, even if no dive was ultimately performed. This contrasts with responses in seals, which sometimes do not display any bradycardia during short dives (Jones et al. 1973), and dabbling ducks, which may not show any reduction in fH when the head is submerged (Furilla and Jones, 1986b). Cardiac deceleration was even more pronounced in forced dives (to 28 % of pre-dive fH and resting fH in 10 s). A difference in intensity of bradycardia between voluntary and forced dives has been reported for a number of species (Butler and Woakes, 1979; Furilla and Jones, 1986a; Kanwisher et al. 1981; Jones et al. 1988; MacArthur and Karpan, 1989; McCulloch and Jones, 1990). Intensified bradycardia during forced dives is sometimes explained by the attendant reduction in muscular activity. This was not the case in our experiments, because forcibly submerged muskrats usually swam in a circle or shook the side of the cage. The extreme forced dive bradycardia we observed may result from the presence of an experimenter during this type of dive and/or the absence of control of dive duration by the animals.

The effects of injected drugs on routine fH were as expected; that is, in agreement with their pharmacological action on the autonomic nervous system (Katzung, 1992). Atropine abolished bradycardia in voluntary and forced dives, confirming the essential role of the parasympathetic system (MacArthur and Karpan, 1989; Murdaugh et al. 1961). A surprising result was the reduced free-diving bradycardia in propranolol-treated animals. This effect was not seen with the other β-adrenergic antagonist nadolol. Propranolol is more liposoluble than nadolol and thus crosses the blood–brain barrier more readily (Katzung, 1992). A central effect of propranolol in causing sedation or perhaps blockade of central catecholaminergic pathways may explain the differential effect on diving fH of the two drugs. Indeed, muskrats treated with propranolol showed a reduced level of activity. They spent most of their time between dives resting on the platform, whereas muskrats in other treatment groups displayed more grooming, eating and exploratory activity. The anaesthetic properties of propranolol, which are not shared by nadolol (Katzung, 1992), could also cause a higher diving fH by reducing both afferent and efferent peripheral neural activity. A puzzling result is the absence of any effect of nadolol on diving bradycardia. If diving bradycardia is only due to increased vagal activity and not to sympathetic withdrawal, then blocking cardiac β-adrenergic receptors should further decrease fH during diving. The explanation that sympathetic tone collapses during diving does not hold because, in that case, atropine-treated animals would have shown a decrease in fH during submergence. We propose that an accentuated antagonism occurs between the two branches of the autonomic nervous system during diving (Levy, 1971). Intense vagal activity during diving may block sympathetic inputs to the heart, despite the persistence of sympathetic tone, so complete pharmacological sympathetic blockade will not result in a significant decrease of fH. This means that the parasympathetic system takes over cardiac control during diving and that sympathetic input to the heart is ineffective while the animal is under water. In fact, this would be a very efficient way of rapidly suppressing sympathetic influences because the effective response to changes in sympathetic activation occurs much more slowly than changes due to parasympathetic activity (Akselrod et al. 1985; Furilla and Jones, 1987; Japundzic et al. 1990). A cholinergically mediated reduction of the response of cardiac cells to adrenergic stimulation during diving may also explain why harbour seals develop a diving bradycardia despite a large increase in circulating epinephrine and norepinephrine levels (Hance et al. 1982). Also, mallard ducks forced to dive display the most remarkable increase in circulating catecholamine levels known, yet diving fH remains low and is unaffected by β-blockade (Butler and Jones, 1971; Lacombe and Jones, 1990). In free-diving ducks, diving fH increases with sympathetically driven increase in pre-dive fH. However, diving fH increases less than pre-dive fH, suggesting that accentuated antagonism between the two branches of the autonomic nervous system also occurs in voluntarily diving ducks.

Diving bradycardia occurs in association with peripheral vasoconstriction, which is effected by α-adrenergic control (Bron et al. 1966; Butler and Jones, 1971; Kobinger and Oda, 1969; Lacombe and Jones, 1991a). Some authors have argued that bradycardia in ducks forced to dive is a secondary cardiovascular adjustment to the change in peripheral resistance to maintain blood pressure (Blix et al. 1974; Andersen and Blix, 1974). Others have concluded that the baroreflex operates during forced dives but is not responsible for diving bradycardia (Jones et al. 1983; Kobinger and Oda, 1969; Smith and Jones, 1992). Our results show that diving bradycardia is unaffected by α-blockade with phentolamine, which extends to free-diving animals the conclusion that diving bradycardia occurs independently from vasoconstriction (Murdaugh et al. 1968). Although we could not monitor blood flow during voluntary dives, the increase in fH after injection of phentolamine together with the lack of effect of the α-adrenergic agonist phenylephrine in phentolamine-treated animals suggest that α-blockade was indeed effective in our animals. Furthermore, phentolamine markedly reduced maximum underwater endurance in forced dives.

The acceleration of fH before surfacing seen in our muskrats towards the end of voluntary dives has been previously reported in seals (Jones et al. 1973; Murdaugh et al. 1961; Thompson and Fedak, 1993). It is sometimes explained by a decompression reflex, but this cannot be the case in muskrats because they are shallow divers and swam in 60 cm of water in our experimental set-up. Cardiac acceleration before surfacing was not affected by α-and β-adrenergic blockade and was absent in atropine-injected animals, which suggests that it is caused by withdrawal of vagal inputs. Thus, an early reversal of the diving response seems to take place while the animal is still under water.

Untreated animals showed an increase in fH from resting to routine conditions which is easily explained by an increase in activity. There was a further increase from routine fH to pre-dive fH before voluntary dives. In this case, the level of activity does not appear to be the explanation because muskrats stayed motionless on the edge of the platform for a variable period ranging from a few seconds to 30 s before diving. Pre-dive tachycardia has been reported to occur simultaneously with hyperventilation in ducks (Butler and Woakes, 1979; Furilla and Jones, 1987), and there is ample evidence for an interaction between respiratory and cardiovascular control systems which would elevate fH (Daly, 1984). However, in our experiments, the elevation in pre-dive fH was absent in the propranolol and nadolol groups (no increase was seen in the atropine and phentolamine groups either, but routine fH was already high in these groups), indicating a strong influence of cardiac sympathetic innervation. Changes in fH due to lung inflation or increased activity of the inspiratory centres are unaffected by propranolol blockade and predominantly brought about by a reduction in cardiac vagal tone (Akselrod et al. 1985; Daly, 1984; Japundzic et al. 1990). Therefore, pre-dive tachycardia is of different origin. Pre-dive tachycardia may serve to increase oxygen delivery to the tissues before diving and thus to increase the aerobic capacity of the animal. It is interesting to note that an anticipatory bradycardia has been shown in harbour seals, suggesting a different strategy in preparation for diving in this species (Jones et al. 1973). Control muskrats showed a decrease in fH before forced dives, which was suppressed by atropine but not by nadolol, indicating the role of increased vagal activity. Propranolol-treated muskrats in the forced diving cage did not display as much grooming and exploratory activity as muskrats in other treatment groups. Also, they did not seem to react as strongly when the experimenter entered the room, which may explain why they did not exhibit any pre-dive bradycardia.

During the first seconds after surfacing, free-diving muskrats displayed the same high fH as just before diving. Post-dive tachycardia correlates with high oxygen consumption during this period (MacArthur and Krause, 1989). It probably reflects repayment of an oxygen debt incurred during diving, intense grooming and perhaps rewarming since body temperature may drop significantly during a dive (MacArthur and Karpan, 1989). Post-dive tachycardia was not seen in nadolol-and propranolol-treated animals, suggesting an increase in sympathetic activity during the recovery period.

Animals treated with atropine and/or phentolamine were still able to make the round trip in the free-diving tank, taking about 50 s, which is as long as their aerobic dive limit (ADL). The ADL was computed from estimated oxygen stores and measurement of post-dive oxygen consumption (MacArthur, 1990). Calculated this way, it represents a theoretical value, defined as the maximum amount of time that an animal can spend under water relying only on aerobic biochemical pathways. The ADL can also be determined empirically by measuring levels of blood lactate, the main metabolite of anaerobiosis. It is then defined as the maximum amount of time spent under water without any significant increase in blood lactate levels. The two values agree well in voluntarily diving Weddell seals (Kooyman et al. 1980). Since Weddell seals rarely dive for longer than their ADL in the wild, it has been concluded that this species performs most of its dives aerobically (Kooyman et al. 1980). Unfortunately, the ADL value derived from blood lactate measurements is not known for muskrats. However, if muskrats performed their dives primarily aerobically, suppressing the diving response should have significantly decreased the duration of their voluntary dives below their ADL. Since this was not the case in the present experiments, we hypothesize that muskrats may use anaerobic in addition to aerobic biochemical pathways throughout voluntary dives.

Blockade of the diving response with atropine significantly decreased maximum underwater time, and blockade with phentolamine decreased it further. Atropine and phentolamine given at the same time had the same effect as phentolamine alone. This suggests that peripheral vasoconstriction and bradycardia greatly improve underwater endurance in a non-additive manner and that vasoconstriction is more efficient, confirming findings in other species (Andersen and Blix, 1974; Lacombe and Jones, 1991b; Murdaugh et al. 1968). Nevertheless, muskrats without a cardiac and vasomotor response (atropine–phentolamine group) could stay under water for more than 5 min. This is unusual because harbour seals, for instance, cannot sustain as long a dive in the absence of a diving response (Murdaugh et al. 1968). The exceptional underwater endurance of muskrats in the absence of a diving response may be due to the essential role of anaerobic biochemical pathways during diving in this species.

In conclusion, these experiments emphasize the importance of vagal outflow to the heart during voluntary dives and of sympathetic outflow during the pre-dive and post-dive periods. They suggest an accentuated antagonism between the two branches of the autonomic nervous system during diving, so that parasympathetic influences on the heart predominate. Furthermore, they show that free-diving bradycardia may occur independently of peripheral vasoconstriction and that bradycardia and peripheral vasoconstriction greatly improve underwater endurance in muskrats. Finally, this study raises the fundamental question of understanding how muskrats can perform free dives as long as their ADL and survive 5 min forced dives without a diving response.

This research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada and the Heart and Stroke Foundation of British Columbia and Yukon. The authors wish to thank Dr A. T. Mosberg from R. J. Reynolds Tobacco Co. (Winston-Salem, NC) for lending us the ECG telemetry system.