The motor innervation of the gill blood vessels of the dogfish Scyliorhinus canicula L. has been investigated by electrical stimulation of (1) the branchial branches of the IXth and Xth cranial nerves in isolated perfused 1st holobranch preparations and (2) both Xth cranial nerves in whole anaesthetized fish. The observed vascular responses to nerve stimulation appear to be entirely due to contraction of the striated muscles of the gill arch and not to any direct motor innervation of the major gill blood vessels since the responses were blocked only by the drug pancuronium, which blocks striated muscle motor end-plates. The specificity of pancuronium for the motor end-plate of striated muscle in the dogfish was established by showing that it did not block nervous transmission across the cardiac ganglia.

The results from the nerve stimulation studies have been investigated further by pharmacological studies on isolated perfused gill preparations. Acetylcholine produces an atropine-sensitive increase in resistance to perfusion, while both adrenalin and noradrenalin decrease the resistance to perfusion.

The gills of elasmobranchs receive parasympathetic innervation via the branchial branches of the IX th and Xth cranial nerves (Young, 1933), and fibres from these nerves are reported to innervate the branchial blood vessels (Boyd, 1936). However, sympathetic innervation of the elasmobranch gill has yet to be demonstrated and it is considered unlikely that any exists (Nilsson, Holmgren & Fange, 1983). The branchial nerves contain both sensory fibres (Lutz & Wyman, 1932; Irving, Solandt & Solandt, 1935; Satchell & Way, 1962) and motor fibres which innervate the striated muscles of the gill arch (Gaskell, 1886; Norris & Hughes, 1920). These nerves may also contain motor fibres which innervate the branchial blood vessels and regulate blood flow within the branchial vascular bed. However, little has so far been reported concerning the nervous control of gill blood flow in either elasmobranch or teleost fish.

In his review of the autonomic innervation of the visceral and cardiovascular systems of vertebrates, Burnstock (1969) notes that no account was available on the effect of nerve stimulation on the resistance to blood flow in any vascular bed in fish, yet that it seemed likely that vagal, cholinergic, vasoconstrictor fibres innervate the gills, at least in teleosts.

Vasoconstriction in response to acetylcholine in the branchial vascular bed has been repeatedly demonstrated in both teleosts (Östlund & Fange, 1962; Reite, 1969; Wood, 1974; Smith, 1977; Pettersson & Nilsson, 1979) and elasmobranchs (Davies & Rankin, 1973). Acetylcholine is only ever active locally (Koelle, 1963); it does not circulate in the blood. Consequently it has often been assumed that the vasoactive effects of acetylcholine in fish gills provide evidence for a cholinergic vasomotor innervation (Belaud, Peyraud-Waitzenegger & Peyraud, 1971; Smith, 1977). However, acetylcholine may produce a vasomotor response in vascular smooth muscle even when no cholinergic innervation appears to be present (Koelle, 1975). It is, therefore, necessary to demonstrate the existence of a vasomotor innervation of the gills directly by examining the effects of branchial nerve stimulation on vascular function.

Recently, a few reports on the effects of branchial nerve stimulation on the branchial vascular resistance to blood flow in teleosts (Nilsson, 1973; Pettersson & Nilsson, 1979) have demonstrated the existence of both sympathetic and parasympathetic vasomotor innervation. A vasoconstrictor response to branchial nerve stimulation has also been reported for the dogfish S. canicula (Davies, quoted by Bolis & Rankin, 1975), although this report is only very brief. The present study was undertaken to examine qualitatively the nervous regulation of gill blood flow in the dogfish by electrical stimulation of the branchial nerves in both whole animals and in isolated, saline-perfused, gill preparations.

Isolated, saline-perfused gill arches

The effects of branchial nerve stimulation on branchial vascular function were examined in isolated gill arches, perfused with physiological saline, from a total of seven dogfish, S. canicula. Both sexes were used and their masses ranged from 0·638 kg to 1·125 kg. The results from these experiments have been further qualified by pharmacological studies on similar isolated gill preparations from a further 11 dogfish. The majority of these experiments were conducted at the Plymouth laboratories of the Marine Biological Association of the U.K. during January and February, 1980. The fish were caught by trawl and held in large sea water aquaria for between 3 to 4 days at approximately 15 °C prior to any experiment.

A dogfish was pithed and the 2nd right afferent branchial artery, which supplies blood to the 1st right holobranch, was cannulated with a 30 cm length of Polythene tube (o.d. 1·2 mm, Portex) for the inflow of aerated elasmobranch physiological saline (Capra & Satchell, 1977a). This had previously been filtered (0·2μm pore filter, Gelman) and contained 20 units i.u. sodium heparin ml-1 (Weddel). The 1st holobranch was used because its afferent artery was the most accessible and afforded rapid cannulation. Perfusion was commenced immediately from a constant flow, pulsatile pump (Metcalfe & Butler, 1982) at a rate of 0·1 ml stroke-1 kg-1 of whole fish × 30 strokes min-1. This is approximately the flow rate to a single holobranch that might be expected in an intact dogfish in which cardiac output is about 30ml min-1 kg-1 (Short, Taylor & Butler, 1979), if it is assumed that all holobranchs receive an equal proportion of cardiac output. The orobranchial cavity was opened on the left side, and the 1st and 2nd right efferent branchial arteries, which drain the 1st holobranch of efferent arterial blood, were cannulated separately with short lengths of Polythene tube (o.d. 0·9 mm). The distal ends of these two cannulae were joined via a ‘Y’ junction to a single outflow cannula. The communicating vessels between the 1st and 2nd hemibranchs and between the 3rd and 4th hemibranchs, which are located on the dorsal edge of the 1st and 2nd gill clefts respectively, were ligated close to the 1st holobranch on the right side so that all efferent arterial flow left the preparation via the single outflow cannula. The distal end of this cannula was inserted tightly into the barrel of a glass Pasteur pipette which allowed it to be rigidly mounted. In those preparations in which the branchial nerves were to be electrically stimulated, the IXth and Xth cranial nerves were isolated in the anterior cardinal sinus and bilaterally sectioned close to the chondrocranium. The isolated holobranch, together with its nerves, was dissected free from the animal and placed in a water jacketed organ bath containing aerated elasmobranch saline maintained at 15 °C (Fig. 1).

Fig. 1.

The experimental arrangement used for perfusing isolated 1st holobranch preparations of the dogfish during electrical stimulation of the branchial nerves. O, organ bath; R, saline reservoir; P, pulsatile saline pump; PT, pressure transducer; S, physiological stimulator; E, stimulating electrodes; D, infra red drop counter; PR, pen recorder; B, syringe barrel fordrug infusion; T, three-way tap; arrows show direction of saline flow; ▸, air flow.

Fig. 1.

The experimental arrangement used for perfusing isolated 1st holobranch preparations of the dogfish during electrical stimulation of the branchial nerves. O, organ bath; R, saline reservoir; P, pulsatile saline pump; PT, pressure transducer; S, physiological stimulator; E, stimulating electrodes; D, infra red drop counter; PR, pen recorder; B, syringe barrel fordrug infusion; T, three-way tap; arrows show direction of saline flow; ▸, air flow.

Afferent perfusion pressure was measured via a pressure transducer (S.E. Labs, S.E.M. 4·86) connected to the inflow cannula proximal to the preparation (Fig. 1) and its output displayed on a rectilinear pen recorder (Ormed Limited). Afferent perfusion pressure measurements were adjusted to make allowance for the measured resistance of the inflow cannula between the junction with the pressure transducer and the gill preparation. Efferent arterial pressure was maintained at about 0·7 kPa by adjusting the level of the outflow cannula tip relative to the preparation, having accounted for the measured resistance of the outflow cannula. Zero pressure was taken as the pressure at the surface of the saline in the organ bath. Efferent arterial flow was recorded by an infra-red drop counting device. This was connected to a linear display (Neurolog) which operated as an instantaneous rate meter, the output from which was displayed on the pen recorder. Efferent arterial flow rate was calculated as drop rate×drop volume. The latter was measured frequently during experiments, but did not appear to alter measurably over the range of dropping rates observed.

Electrical stimulation of the branchial nerves

In seven isolated gill preparations, the peripheral cut ends of the IXth and Xth cranial nerves were picked up on silver hook electrodes and lifted clear of the saline in the organ bath. The nerves were stimulated at intensities ranging from 0·2 to 10 V, at frequencies ranging between 2 and 500 Hz and with a pulse duration of 1 ms for periods of between 1 and 5 s from a physiological stimulator (Neurolog). The functioning of at least somatic motor fibres in the nerves was substantiated by virtue of the fact that their electrical stimulation caused contraction of the striated muscles of the gill arch.

During nerve stimulation the effects of the following pharmacological blocking agents were investigated : the cholinergic receptor antagonists pancuronium bromide (Pavulon; Organon Labs) and atropine sulphate (Sigma), the adrenergic receptor antagonists phentolamine mesylate (Rogitine, Ciba) and propranolol (Inderal, I.C.I.). All drugs were added, singly or together, to 10 or 20 ml of physiological saline contained in a 20 ml syringe barrel, connected to the perfusion system by a three-way tap situated between the saline reservoir and the pump (Fig. 1). In this way the perfusion could be changed from drug-free saline to saline containing drugs without interrupting the perfusion flow. This method of drug administration also obviated the dilution of the drug by drug-free perfusate which would have occurred if drugs had been administered as bolus injections into drug-free perfusate. Drug doses are given either as g ml-1 of perfusate, or as molar concentrations.

Pharmacological studies

In seven similar isolated perfused gill preparations in which the branchial nerves were not stimulated, the effects of the cholinergic receptor antagonists (as above) upon the vascular response of the preparation to acetylcholine chloride (Sigma) were investigated. In these studies, perfusion of the preparations with the antagonist preceded perfusion with the agonist. Finally, in a further four such preparations, the vascular responses to the adrenergic receptor agonists adrenalin bitartrate and noradrenalin bitartrate (Sigma) were investigated.

Branchial nerve stimulation in whole dogfish

In view of the physiological limitations of the isolated, perfused gill preparation (see Results), the study was extended by examining the effects of electrical stimulation of the Xth cranial nerve on pre-and post-branchial blood pressure in intact, anaesthetized fish. This preparation was most convenient since it also permitted a study of possible ganglion-blocking properties of the neuromuscular blocking drug pancuronium bromide used in the isolated, perfused gill studies. This was achieved by examining the effects of pancuronium on the cardiac slowing caused by electrical stimulation of the branchial branch of the cardiac vagus.

These experiments were performed in Birmingham on eight dogfish of either sex the mass of which ranged from 0·540 kg to 0·920 kg. The fish were obtained from the Plymouth laboratories of the Marine Biological Association of the U.K. and transported to Birmingham and maintained as described by Metcalfe & Butler (1982).

Each fish was anaesthetized in unbuffered sea water containing about 0–04 g I-1 tricane methyl sulphonate (Sigma) and placed on an operating table in a constant temperature room maintained at 15 °C. The gills were irrigated with recirculating, aerated sea water containing anaesthetic. The caudal artery was cannulated for the measurement of post-branchial blood pressures, with a 30 cm length of Polythene tube (o.d. 1·22mm, Portex) filled with heparinized elasmobranch saline as described by Metcalfe & Butler (1982). The ventral aorta was cannulated, for the measurement of pre-branchial blood pressures, by a method similar to that described for the Lemon shark N. brevirostris by Bushnell et al. (1982), although in the present study the cannula was led out through the spiracle rather than through the lower jaw.

The anterior cardinal sinus on one side was exposed and opened, care being taken that no air entered the circulation during this part of the procedure by temporarily placing a tissue paper plug in the opening of the Cuvierian duct. The visceral branch of the vagus posterior to the 4th branchial division (Fig. 2) was sectioned to prevent subsequent stimulation of the vagus from affecting systemic blood flow, or from affecting heart rate via the visceral cardiac vagus (Short, Butler & Taylor, 1977). The branchial cardiac vagus was sectioned at a point close to where it leaves the 4th branchial division (Fig. 2) and cleared of connective tissue for a length of 5·7 mm to allow subsequent stimulation. The anterior cardinal sinus was then closed by a suture after removal of the tissue paper plug and any air trapped in the sinus. The procedure was then repeated on the other side of the fish.

Fig. 2.

A diagrammatic illustration of the right and left Xth cranial nerves of the dogfish Scyliorhinus canicula (dorsal view) showing the positions of nerve section (=) and the positions of nerve stimulation (▾). bcv, branchial cardiac vagus.

Fig. 2.

A diagrammatic illustration of the right and left Xth cranial nerves of the dogfish Scyliorhinus canicula (dorsal view) showing the positions of nerve section (=) and the positions of nerve stimulation (▾). bcv, branchial cardiac vagus.

The roots of the right and left vagi were exposed by removing a portion of the chondrocranium above the medulla from which these nerves arise. The spinal cord was sectioned at a point posterior to the medulla to prevent it subsequently being stimulated. Silver stimulating electrodes were pushed into the foramina through which the vagi leave the cartilaginous chondrocranium on both sides of the fish (Fig. 2). The anterior cardinal sinus on either the right or left side was partly reopened to allow access to the branchial cardiac vagus which was picked up on small silver hook electrodes for subsequent stimulation (Fig. 2). Both right and left vagal roots were stimulated simultaneously at intensities ranging between 0·1 and 10 V, frequencies ranging between 2 and 100 Hz and with a pulse width of 1 ms from the physiological stimulator. The branchial cardiac vagus was stimulated at intensities ranging between 0·1 and 3·0 V at a frequency of 50 Hz with a pulse width of 1 ms. 50 Hz is reported to be the optimal frequency for stimulation of the branchial cardiac vagus for causing maximum reductions in heart rate (Short et al. 1977). The effects of pancuronium bromide (2 mg kg-1) and atropine sulphate (0·15 mg kg-1) on the responses to both paired vagal root and branchial cardiac vagal nerve stimulation were investigated.

The branchial vascular response to electrical stimulation of the branchial nerves in isolated, saline-perfused gills

In the isolated, saline-perfused gill preparation prior to nerve stimulation, the mean afferent arterial perfusion pressure was about 2·5 kPa, and efferent arterial flow rate generally only accounted for 10-20% of the afferent arterial flow. In vivo afferent arterial blood pressure is usually about 5·0kPa in this species at 15 °C (Short et al. 1979), and efferent arterial flow rate might be expected to be 60-90% of afferent arterial flow (Metcalfe & Butler, 1982). The features of the present preparation were presumably due to leakage of perfusate via the cut ends of the holobranch, even though efferent arterial back pressure was low in comparison with that recorded in vivo (about 3·9 kPa, Short et al. 1979). Since some venous drainage from the gill arch must normally occur in vivo, which may be important for any ability to alter the regional distribution of branchial blood flow, it would be unphysiological to ligate completely these cut ends of the holobranch. However, there appeared to be no repeatable method of partially occluding the leakage route so none was attempted with the final preparations. These factors, combined with physiological afferent arterial flow rates, resulted in the low afferent arterial perfusion pressure.

The magnitude of the vascular responses elicited by nerve stimulation varied between preparations, although the general nature of the response was similar in all cases. For this reason, and in consideration of the limitations of the preparation (above), a quantitative analysis of the results would not be applicable and only a qualitative analysis has been attempted.

Electrical stimulation of the 1st branchial branch of the Xth cranial nerve which innervates the 1st holobranch (the pre-trematic branch) at all intensities and at all frequencies in all preparations failed to elicit any changes in either afferent arterial perfusion pressure or efferent arterial flow rate (Fig. 3). Consequently all the results presented refer to the electrical stimulation of the branchial branch of the IXth cranial nerve (the post-trematic branch) which innervates the 1st holobranch.

Fig. 3.

Traces of efferent arterial flow (F : ml min-1) and afferent arterial pressure (P: kPa) obtained from isolated, saline-perfused 1st holobranch preparations. (A) During electrical stimulation of the IXth and Xth cranial nerves before pancuronium; and (B) during electrical stimulation (at the same intensity) of the IXth cranial nerve after pancuronium (20μgml-1). S, periods of electrical stimulation at frequencies indicated in Hz, at voltages of between 0·1 and 10 V. Bars marked X show stimulation of the Xth cranial nerve. Time marker at top indicates minute intervals.

Fig. 3.

Traces of efferent arterial flow (F : ml min-1) and afferent arterial pressure (P: kPa) obtained from isolated, saline-perfused 1st holobranch preparations. (A) During electrical stimulation of the IXth and Xth cranial nerves before pancuronium; and (B) during electrical stimulation (at the same intensity) of the IXth cranial nerve after pancuronium (20μgml-1). S, periods of electrical stimulation at frequencies indicated in Hz, at voltages of between 0·1 and 10 V. Bars marked X show stimulation of the Xth cranial nerve. Time marker at top indicates minute intervals.

In all preparations, electrical stimulation of the branchial branch of the IXth cranial nerve at all intensities above 0·1-0·3 V resulted in a vigorous contraction of the gill arch musculature. This response was associated with an increase in efferent arterial flow rate in six of the seven preparations, and with an increase in afferent arterial perfusion pressure in all seven preparations (Fig. 3). This response was generally maximal at intensities between 0·3 and 3·0 V and at frequencies of 30-50 Hz and above; the maximum increase in efferent arterial flow rate varied between 20-100 % of the pre-stimulation value. The stimulation frequency at which the maximal response was observed appeared to approximate to the fusion frequency of the striated muscle contractions of the gill arch.

Paralysis of the striated muscles of the gill arch with the neuromuscular blocking drug pancuronium bromide (20μgml-1) reduced or abolished all changes in both afferent arterial perfusion pressure and efferent arterial flow rate in response to nerve stimulation. This occurred at all frequencies and intensities of stimulation in all four preparations in which the drug was used (Fig. 3). In those instances where some small residual changes in afferent arterial perfusion pressure and/or efferent arterial flow rate persisted after the application of pancuronium, these were associated with some slight, observable, contraction of the gill arch, presumably due to incomplete paralysis of the skeletal muscles.

In all five preparations investigated without paralysis with pancuronium, the vascular response to nerve stimulation was not modified by the muscarinic receptor antagonist atropine (22μgml-1) (Fig. 4) or by the alpha-adrenergic receptor antagonist phentolamine (50μgml-1) or by the beta-adrenergic receptor antagonist propranolol (10μgml-1) (Fig. 4).

Fig. 4.

Traces of efferent arterial flow (F : ml min-1) and afferent arterial pressure (P: kPa) obtained from isolated saline-perfused 1st holobranch preparations. Upper traces: (A) during electrical stimulation of the IXth cranial nerve before atropine, and (B) during electrical stimulation (at the same intensity) of the IXth cranial nerve after atropine (22μgml-1). Lower traces: (A) during electrical stimulation of the IXth cranial nerve before alpha-and beta-adrenoceptor blockade, and (B) during electrical stimulation (at the same intensity) of the IXth cranial nerve after alpha-and betaadrenoceptor blockade (phentolamine 50μg gml-1, propranolol 10μg gml-1). S, periods of electrical stimulation at frequency indicated in Hz, at voltages of between 0·1 and 10 V. Time marker at top indicates minute intervals.

Fig. 4.

Traces of efferent arterial flow (F : ml min-1) and afferent arterial pressure (P: kPa) obtained from isolated saline-perfused 1st holobranch preparations. Upper traces: (A) during electrical stimulation of the IXth cranial nerve before atropine, and (B) during electrical stimulation (at the same intensity) of the IXth cranial nerve after atropine (22μgml-1). Lower traces: (A) during electrical stimulation of the IXth cranial nerve before alpha-and beta-adrenoceptor blockade, and (B) during electrical stimulation (at the same intensity) of the IXth cranial nerve after alpha-and betaadrenoceptor blockade (phentolamine 50μg gml-1, propranolol 10μg gml-1). S, periods of electrical stimulation at frequency indicated in Hz, at voltages of between 0·1 and 10 V. Time marker at top indicates minute intervals.

Pharmacological studies

In seven isolated, saline-perfused 1st holobranch preparations in which the branchial nerves were not stimulated, acetylcholine (0·2μgml-1) caused a small reversible increase in afferent arterial perfusion pressure and a marked reduction, or even complete abolition of efferent arterial flow (Fig. 5). This response to acetylcholine could not be prevented by pancuronium (20μgml-1) (Fig. 5) but was either much reduced or completely prevented by atropine (22μgml-1) (Fig. 5); taken together with pancuronium’s effects on nerve stimulation these findings confirm the specificity of the drug to nicotinic receptors in the dogfish gill. In a further four preparations adrenalin (two) and noradrenalin (two) produced marked increases in efferent arterial flow rate and this was associated with a reduction in afferent arterial perfusion pressure at concentrations of 10−6moll-1 and above (Fig. 6).

Fig. 5.

Traces of efferent arterial flow (F : ml min-1) and afferent arterial pressure (P: kPa) obtained from isolated saline-perfused 1st holobranch preparations. (A) In response to acetylcholine (ACh) (0·2μgml-1) before and after atropine (At) (22μg ml-1). (B) In response to acetylcholine (ACh) (0·2μg ml-1) before and after pancuronium (P) (20μg g ml-1). Time marker at bottom indicates minute intervals. ◂ ▸ indicates administration of drug.

Fig. 5.

Traces of efferent arterial flow (F : ml min-1) and afferent arterial pressure (P: kPa) obtained from isolated saline-perfused 1st holobranch preparations. (A) In response to acetylcholine (ACh) (0·2μgml-1) before and after atropine (At) (22μg ml-1). (B) In response to acetylcholine (ACh) (0·2μg ml-1) before and after pancuronium (P) (20μg g ml-1). Time marker at bottom indicates minute intervals. ◂ ▸ indicates administration of drug.

Fig. 6.

Traces of efferent arterial flow (F : ml min-1) and afferent arterial pressure (P: kPa) obtained from isolated saline-perfused 1st holobranch preparations. (A) In response to noradrenalin (NA) (10−6 mol 1-1). (B) In response to adrenalin (Ad) (10−6mol 1-1). Time marker at top indicates minute intervals. ◂ ▸ indicates administration of drug.

Fig. 6.

Traces of efferent arterial flow (F : ml min-1) and afferent arterial pressure (P: kPa) obtained from isolated saline-perfused 1st holobranch preparations. (A) In response to noradrenalin (NA) (10−6 mol 1-1). (B) In response to adrenalin (Ad) (10−6mol 1-1). Time marker at top indicates minute intervals. ◂ ▸ indicates administration of drug.

The branchial vascular response to electrical stimulation of the Xth cranial nerves in whole, anaesthetized dogfish

In whole fish prior to nerve stimulation, ventral, aortic and dorsal aortic blood pressures were 3·l±0·2kPa and 1·9 ±0·1 kPa respectively. These values are somewhat lower than the values reported for unanaesthetized dogfish (Short et al. 1979) at 15 °C. This is probably due to a loss of systemic vascular tonus as a result of anaesthesia. These values are however higher than those in the isolated, saline-perfused gill preparations reported in the first part of this study. Mean heart rate was 43 ± 2·0 beats min-1 ; this value was higher than that reported for this species at 15 °C (Short et al. 1979) and this is probably due to the removal of a vagal inhibition of the heart after section of the cardiac branches of the vagus (Taylor, Short & Butler, 1977).

Simultaneous stimulation of the right and left roots of the Xth cranial nerves which innervate the gills posterior to the 1st holobranch at intensities above threshold, and at frequencies between 10 and 100 Hz, caused an immediate and marked increase in ventral aortic blood pressure which either decreased gradually during the period of stimulation (Fig. 7) or was maintained throughout (Fig. 8). This was associated with an immediate but much smaller rise in dorsal aortic blood pressure which rapidly returned to normal during the period of stimulation. This response was observed in all eight fish, although the magnitude of the increases varied between animals. In five fish in which the branchial branch of the cardiac vagus was stimulated, this resulted in an immediate and dramatic cessation of the heart beat (Figs 7, 8). In all five fish in which the drug was used, pancuronium (2 mg kg-1) abolished all vascular responses to paired vagal root stimulation, but had no effect on the cardiac response to branchial cardiac vagal stimulation (Fig. 7). This observation indicates that at the dose level used, pancuronium does not block the nicotinic receptors present in the cardiac ganglia and is specific to the nicotinic receptors on striated muscle.

Fig. 7.

Traces of ventral aortic blood pressure (VAbp: kPa) and dorsal aortic blood pressure (DAbp: kPa) obtained from whole, anaesthetized dogfish during electrical stimulation of either both vagal roots (bv) (30-50 Hz) or one branchial branch of the cardiac vagus (cv) (50 Hz) before and after the administration of pancuronium (Pan) (2 mg kg-1). Marker at top indicates periods of electrical stimulation.

Fig. 7.

Traces of ventral aortic blood pressure (VAbp: kPa) and dorsal aortic blood pressure (DAbp: kPa) obtained from whole, anaesthetized dogfish during electrical stimulation of either both vagal roots (bv) (30-50 Hz) or one branchial branch of the cardiac vagus (cv) (50 Hz) before and after the administration of pancuronium (Pan) (2 mg kg-1). Marker at top indicates periods of electrical stimulation.

Fig. 8.

Traces of ventral aortic blood pressure (VAbp: kPa) and dorsal aortic blood pressure (DAbp: kPa) obtained from whole, anaesthetized dogfish during electrical stimulation of either both vagal roots (bv) (30-50 Hz) or one branchial branch of the cardiac vagus (cv) (50 Hz) before atropine (At) (0·15mgkg-1), after atropine, and after pancuronium (Pan) (2mgkg-1). Marker at top indicates periods of electrical stimulation.

Fig. 8.

Traces of ventral aortic blood pressure (VAbp: kPa) and dorsal aortic blood pressure (DAbp: kPa) obtained from whole, anaesthetized dogfish during electrical stimulation of either both vagal roots (bv) (30-50 Hz) or one branchial branch of the cardiac vagus (cv) (50 Hz) before atropine (At) (0·15mgkg-1), after atropine, and after pancuronium (Pan) (2mgkg-1). Marker at top indicates periods of electrical stimulation.

In three fish, atropine (0-15 mg kg-1) had no effect on the vascular response to paired vagal root stimulation (Fig. 8), but in two of these fish in which the branchial cardiac vagus was stimulated, no change in heart rate was observed after atropine, indicating that the dose was effectively blocking muscarinic acetylcholine receptors (Fig. 8). The subsequent administration of pancuronium at the above dose abolished all branchial vascular responses to paired vagal root stimulation (Fig. 8).

The control of branchial blood flow

From the present study it appears that all vascular responses to electrical stimulation of the branchial nerves in both isolated, perfused 1st holobranch preparations and in whole anaesthetized dogfish are entirely the result of the contraction of the striated muscles of the gill arch, rather than the result of contraction of the smooth muscle in the major blood vessels themselves. These vascular responses could not be prevented either by atropine (in both perfused holobranchs and whole fish) or by adrenergic receptor blockade (in perfused holobranchs), but were consistently prevented in both preparations by paralysis of the striated muscles of the gill arch with pancuronium bromide. The specificity of pancuronium to striated muscle motor end-plates has been demonstrated in the present study since it did not affect the vascular responses to acetylcholine in perfused holobranchs (a muscarinic response blocked by atropine) nor did it block transmission at the cardiac ganglia (Young, 1933; Burnstock, 1969) at doses effective in abolishing the branchial vascular responses to nerve stimulation.

Despite the rather unphysiological perfusion conditions in the isolated, 1st holobranch preparations, the vascular responses to branchial nerve stimulation in both whole fish and perfused holobranchs were qualitatively similar. In both studies, branchial nerve stimulation resulted in a marked and rapid increase in afferent perfusion pressure. In perfused holobranchs this response was associated with increases in efferent arterial flow rate, although in whole fish little change in dorsal aortic blood pressure was observed in response to branchial nerve stimulation. This may be caused either by capacitance effects within the systemic circulation as a result of reduced systemic vascular tonus during anaesthesia, or by a redistribution of afferent blood flow from the gills innervated by the Xth cranial nerve (hemibranchs 4–9) to the gills innervated by the IXth cranial nerve (hemibranchs 1-3 ; note: the IXth cranial nerve was not stimulated in these experiments). In the isolated 1st holobranch preparation, efferent arterial flow rate was low in comparison with afferent arterial flow rate. This appeared to be due to leakage of perfusate via the extensive venous sinuses of the gill arch and interbranchial septum (Cooke, 1980; J. D. Metcalfe & P. J. Butler, in preparation). Electrical stimulation of the branchial nerves causes contraction of the striated muscle of the gill arch which presumably compresses the venous sinuses within the interbranchial septum, increasing the resistance to flow of perfusate via this route. This would favour the flow of perfusate through the efferent arterial route, resulting in an increase in efferent arterial flow, particularly in perfused holobranchs in which afferent arterial flow remained constant. This is entirely consistent with the present observations of the responses to branchial nerve stimulation prior to paralysis of the striated muscles of the gill arch with pancuronium.

In the isolated 1st holobranch preparation, only the post-trematic branchial branch of the IXth cranial nerve appears to contain fibres which, on being stimulated electrically, are capable of affecting perfusion. Presumably, the pre-trematic branchial branch of the Xth nerve, which also innervates the 1st holobranch, contains only sensory nerve fibres.

In the pharmacological studies on isolated holobranchs, acetylcholine caused and increase in afferent arterial perfusion pressure and a decrease in efferent arterial flow, presumably due to constriction of the main arterial vessels in response to stimulation of muscarinic receptors since the response was blocked by atropine but not by pancuronium. This vasoconstriction observed in response to acetylcholine confirms similar reports of studies on perfused gills of both 5. canicula (Davies & Rankin, 1973) and trout (Smith, 1977). However, the actions of acetylcholine cannot alone be regarded as evidence for cholinergic vasomotor innervation (see Introduction).

In perfused holobranchs, both adrenalin and noradrenalin caused overall vasodilatation, and this confirms previous reports of similar studies conducted on elasmobranch gills (Davies & Rankin, 1973; Capra & Satchell, 1977b). A similar vasodilatation might have been expected in response to branchial nerve stimulation following paralysis of the skeletal muscles of the gill arch if any adrenergic vasomotor fibres were present in these nerves. The threshold concentrations of these drugs required to elicit a response in the present study (about 10−6 mol I-1 for both adrenalin and noradrenalin) are much higher than that reported by previous authors (about 10−11moll-1, Davies & Rankin, 1973). However, 10−6moll-1 is close to (within an order of magnitude) the levels of circulating adrenalin and noradrenalin found in 5. canicula during hypoxic stress (about 2·8×10−7 mol I-1 for adrenalin, 4·5 × 10−7 mol I-1 for noradrenalin, Butler, Taylor, Capra & Davison, 1978). It is assumed that the vascular responses of the isolated perfused holobranchs observed in the pharmacological studies are representative of the responses that might have been observed in similar studies on whole fish. However, such studies were not performed since the branchial vascular responses would have been obscured by systemic vascular responses.

Pettersson & Nilsson (1979) report that both cholinergic and adrenergic vasomotor nerve fibres innervate the branchial vascular bed in the cod Gadus morhua, and that electrical stimulation of the entire vago-sympathetic nerve trunk in which these fibres pass causes vasoconstriction which could be reversed by atropine to a vasodilatation mediated by beta-adrenergic receptors. However, in all but one of their experiments these authors found a persistent vasoconstrictor response to nerve stimulation which in some cases obscured the beta-adrenergic receptor mediated dilatory response, and which in others was revealed after beta-adrenergic receptor blockade. This vasoconstrictor response could not be abolished by any of the pharmacological agents employed by these authors, apart from tetrodotoxin. However, these authors did not investigate the effects of striated muscle blockade during nerve stimulation. It is possible that the persistent vasoconstriction observed may have been the result of contraction of striated muscles in the gill arch, similar to the response observed in the present study.

In S. canicula, sphincters have been observed in the afferent lamellar arterioles (Wright, 1973) but no innervation was apparent. Both these observations have been confirmed in a more recent study by Dunel & Laurent (1980). Sphincters have also been reported to exist in the efferent filament artery just prior to its junction with the efferent arch artery in this species (Wright, 1973), and these sphincters are reported to receive motor innervation which is probably cholinergic (Dunel & Laurent, 1980). This appears to contradict the results obtained in the present study. However, the observations of these authors are based upon histological, rather than physiological, studies.

The results presented here strongly indicate that, unlike those of the cod, the major blood vessels of the branchial vascular bed of the dogfish are devoid of any direct motor innervation which may control the regional distribution of branchial blood flow. Presumably, any direct control must be either via humoral agents such as the circulating catecholamines adrenalin and noradrenalin, or via intrinsic mechanisms, possibly similar to that reported by Satchell (1962) for Squalus acanthias in which brief periods of anoxia caused branchial vasoconstriction. These responses could not be abolished by section of the branchial nerves and Satchell (1962) concluded that this was an intrinsic response of the branchial vascular bed to anoxia. A similar response to hypoxia has been reported for the cod (Pettersson & Johansen, 1982).

From the present study it has not been possible to determine whether or not the nervously mediated contraction of the striated muscles of the gill arch in vivo may act as a mechanism to direct blood flow from the venous sinuses of the interbranchial septum to the respiratory vascular network of the gill filament. However, Hughes & Ballintijn (1965) report that these muscles (the septal constrictor branchials) contract towards the end of each ventilatory cycle, just before the gill slits close. Though the activity of these muscles is presumably essential to ventilation, it may also serve to enhance blood flow across the respiratory surface during those periods of highest oxygen availability in each ventilatory cycle.

Financial support was provided by the Science and Engineering Research Council.

Belaud
,
A.
,
Peyraud-Waitzenegger
,
M.
&
Peyraud
,
C.
(
1971
).
Étude comparée réactions vasomotrise des branchies perfusées de deux Teleosteen: La Carpe et Le Congre
.
C. r. Séanc. Soc. Biol
.
165
,
1114
1118
.
Boils
,
L.
&
Rankin
,
J. C.
(
1975
).
Adrenergic control of blood flow through fish gills: environmental implications
.
In Comparative Physiology - Functional Aspects of Materials
, (eds
L.
Bolis
,
S. H. P.
Maddrell
&
K.
Schmidt-Nielsen
), pp.
223
233
.
Amsterdam
:
North Holland
.
Boyd
,
J. D.
(
1936
).
Nerve supply to the branchial arch arteries of vertebrates
.
J. Anal
.
71
,
157
158
.
Burnstock
,
G.
(
1969
).
Evolution of the autonomic innervation of visceral and cardiovascular systems in vertebrates
.
Pharmac. Rev
.
21
,
247
324
.
Bushnell
,
P. G.
,
Lutz
,
P. L.
,
Steffensen
,
J. F.
,
Oikari
,
A.
&
Gruber
,
S. H.
(
1982
).
Increases in arterial blood oxygen during exercise in the Lemon Shark (Negaprion brevirostris)
.
J. comp. Physiol
.
147
,
41
47
.
Butler
,
P. J.
,
Taylor
,
E. W.
,
Capra
,
M. F.
&
Davison
,
W.
(
1978
).
The effect of hypoxia on the levels of circulating catecholamines in the dogfish Scyliorhinus canicula L
.
J. comp. Physiol
.
127
,
325
330
.
Capra
,
M. F.
&
Satchell
,
G. H.
(
1977a
).
Adrenergic and cholinergic responses of the isolated saline-perfused heart of the elasmobranch fish Squalus acanthias
.
Gen. Pharmac
.
8
,
56
65
.
Capra
,
M. F.
&
Satchell
,
G. H.
(
1977b
).
The adrenergic response of the isolated saline perfused pre-branchial arteries and gills of the elasmobranch Squalus acanthias
.
Gen. Pharmac
.
8
,
67
71
.
Cooke
,
I. C. R.
(
1980
).
Functionalaspects of the morphology and vascular anatomy of the gills of the Endeavour dogfish, Centrophorus scalpratus (McCulloch) (Elasmobranchii: Squalidae)
.
Zoomorphologie
94
,
167
183
.
Davies
,
D. T.
&
Rankin
,
J. C.
(
1973
).
Adrenergic receptors and vascular responses to catecholamines in perfused dogfish gills
.
Comp. gen. Pharmac
.
8
,
67
71
.
Dunel
,
S.
&
Laurent
,
P.
(
1980
).
Functional organisation of the gill vasculature in different classes of fish
.
In Epithelial Transport in the Lower Vertebrates
, (ed.
B.
Lahlou
), pp.
37
58
.
Cambridge
:
Cambridge University Press
.
Gaskell
,
W. H.
(
1886
).
Structure, distribution and function of the nerves which innervate the visceral and vascular systems
.
J. Physiol., Lond
.
7
,
1
80
.
Hughes
,
G. M.
&
Ballintijn
,
C. M.
(
1965
).
The muscular basis of the respiratory pumps in the dogfish (Scyliorhinus canicula)
.
J. exp. Biol
.
43
,
363
383
.
Irving
,
L. D.
,
Solandt
,
O. Y.
&
Solandt
,
D. M.
(
1935
).
Nerve impulses from branchial pressure receptors in the dogfish
.
J. Physiol., Lond
.
84
,
187
190
.
Koelle
,
G. B.
(
1963
).
Cytological distribution and pharmacological functions of cholinesterases
.
InHandbuch der experimentellen Pharmakologie. XV. Cholinesterase and Anticholinesterase Agents
, (ed.
G. B.
Koelle
), pp.
187
298
.
Berlin
:
Springer-Verlag
.
Koelle
,
G. B.
(
1975
).
Parasympathomimetic agents
.
In The Pharmacological Basis of Therapeutics
, (eds
L. S.
Goodman
&
A.
Gilman
), 5th Edn, pp.
467
476
.
New York, Toronto, London
:
Macmillan & Co
.
Lutz
,
B. R.
&
Wyman
,
L. C.
(
1932
).
Reflex cardiac inhibition of branchiovascular origin in the elasmobranch Squalus acanthias
.
Biol. Bull. mar. biol. Lab., Woods Hole
62
,
10
16
.
Metcalfe
,
J. D.
&
Butler
,
P. J.
(
1982
).
Differences between directly measured and calculated values for cardiac output in the dogfish: a criticism of the Fick method
.
J. exp. Biol
.
99
,
255
268
.
Nilsson
,
S.
(
1973
).
On the autonomic nervous control of organs in teleostean fishes
.
In Comparative Physiology
, (eds
L.
Bolis
,
K.
Schmidt-Nielsen
&
S. H. P.
Maddrell
), pp.
323
331
.
Amsterdam
:
North Holland
.
Nilsson
,
N.
,
Holmgren
,
S.
&
Fänge
,
R.
(
1983
).
Autonomic nerve function in fish
.
In Control Processes in Fish Physiology
, (eds
J. C.
Rankin
,
T. J.
Pitcher
&
R.
Duggan
), pp.
1
22
.
Beckenham
:
Croom Helm
.
Norris
,
H. W.
&
Hughes
,
S. P.
(
1920
).
The cranial, occipital and anterior spinal nerves of the dogfish Squalus acanthias
.
J. comp. Neurol
.
31
,
293
400
.
Ostlund
,
E.
&
Fange
,
R.
(
1962
).
Vasodilatation by adrenaline and noradrenaline and the effects of some other substances on the perfused fish gill
.
Comp. Biochem. Physiol
.
5
,
307
309
.
Pettersson
,
K.
&
Johansen
,
K.
(
1982
).
Hypoxic vasoconstriction and the effects of adrenaline on gas exchange efficiency in fish gills
.
J. exp. Biol
.
97
,
263
272
.
Pettersson
,
K.
&
Nilsson
,
S.
(
1979
).
Nervous control of the branchial vascular resistance of the Atlantic cod Gadus morhua
.
J. comp. Physiol
.
129
,
179
183
.
Reite
,
O.
(
1969
).
The evolution of vascular smooth muscle responses to histamine and 5HT. 1. Occurrence of stimulatory actions in fish
.
Acta physiol, scand
.
75
,
221
239
.
Satchell
,
G. H.
(
1962
).
Intrinsic vasomotion in the dogfish gill
.
J. exp. Biol
.
39
,
503
512
.
Satchell
,
G. H.
&
Way
,
H. K.
(
1962
).
Pharyngeal proprioceptors in the dogfish Squalus acanthias
.
J. exp. Biol
.
39
,
243
250
.
Short
,
S.
,
Butler
,
P. J.
&
Taylor
,
E. W.
(
1977
).
The relative importance of nervous humoral and intrinsic mechanisms in the regulation of heart rate and stroke volume in the dogfish Scyliorhinus canicula
.
J. exp. Biol
.
70
,
77
92
.
Short
,
S.
,
Taylor
,
E. W.
&
Butler
,
P. J.
(
1979
).
The effectiveness of oxygen transfer during normoxia and hypoxia in the dogfish (Scyliorhinus canicula) before and after cardiac vagotomy
.
J. comp. Physiol
.
132
,
289
295
.
Smith
,
D. J.
(
1977
).
Sites of cholinergic vasoconstriction in trout gills
.
Am. J. Physiol
.
233
,
222
229
.
Taylor
,
E. W.
,
Short
,
S.
&
Butler
,
P. J.
(
1977
).
The role of the cardiac vagus in the response of the dogfish Scyliorhinus canicula to hypoxia
.
J. exp. Biol
.
70
,
57
75
.
Wood
,
C. M.
(
1974
).
A critical examination of the physical and adrenergic factors affecting blood flow through the gills of the rainbow trout
.
J. exp. Biol
.
60
,
241
265
.
Wright
,
D. E.
(
1973
).
The structure of the gills of the elasmobranch Scyliorhinus canicula
.
Z. Zellforsch. mikrosk. Anat
.
144
,
489
509
.
Young
,
J. Z.
(
1933
).
The autonomic nervous system of selachians
.
Q.Jl microsc. Sci
.
75
,
571
624
.