on the nervous regulation of gill blood flow in …branchial nerve stimulation in whole dogfish in...

J. exp. Biol. 113, 253-267 (1984) 2 5 3Printed in Great Britain © The Company of Biologists Limited 1984

ON THE NERVOUS REGULATION OF GILL BLOODFLOW IN THE DOGFISH (SCYLIORHINUS CANICULA)

BY J. D. METCALFE AND P. J. BUTLER

Department of Zoology and Comparative Physiology, University ofBirmingham, P.O. Box 363, Birmingham

Accepted 28 March 1984

SUMMARY

The motor innervation of the gill blood vessels of the dogfish Scyliorhinuscanicula L. has been investigated by electrical stimulation of (1) the bran-chial branches of the IXth and Xth cranial nerves in isolated perfused 1stholobranch preparations and (2) both Xth cranial nerves in wholeanaesthetized fish. The observed vascular responses to nerve stimulationappear to be entirely due to contraction of the striated muscles of the gill archand not to any direct motor innervation of the major gill blood vessels sincethe responses were blocked only by the drug pancuronium, which blocksstriated muscle motor end-plates. The specificity of pancuronium for themotor end-plate of striated muscle in the dogfish was established by showingthat it did not block nervous transmission across the cardiac ganglia.

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

INTRODUCTION

The gills of elasmobranchs receive parasympathetic innervation via the branchialbranches of the IXth and Xth cranial nerves (Young, 1933), and fibres from thesenerves are reported to innervate the branchial blood vessels (Boyd, 1936). However,sympathetic innervation of the elasmobranch gill has yet to be demonstrated and it isconsidered unlikely that any exists (Nilsson, Holmgren & Fange, 1983). The bran-chial nerves contain both sensory fibres (Lutz & Wyman, 1932; Irving, Solandt &Solandt, 1935; Satchell & Way, 1962) and motor fibres which innervate the striatedmuscles of the gill arch (Gaskell, 1886; Norris & Hughes, 1920). These nerves mayalso contain motor fibres which innervate the branchial blood vessels and regulateblood flow within the branchial vascular bed. However, little has so far been reportedconcerning the nervous control of gill blood flow in either elasmobranch or teleostfish.

In his review of the autonomic innervation of the visceral and cardiovascular sys-tems of vertebrates, Burnstock (1969) notes that no account was available on the effect

|Key words: Fish, gills, blood flow.

254 J. D. METCALFE AND P. J. BUTLER

of nerve stimulation on the resistance to blood flow in any vascular bed in fish, yet thaiit seemed likely that vagal, cholinergic, vasoconstrictor fibres innervate the gills, arleast in teleosts.

Vasoconstriction in response to acetylcholine in the branchial vascular bed has beenrepeatedly demonstrated in both teleosts (Ostlund & 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 notcirculate in the blood. Consequently it has often been assumed that the vasoactiveeffects of acetylcholine in fish gills provide evidence for a cholinergic vasomotorinnervation (Belaud, Peyraud-Waitzenegger & Peyraud, 1971; Smith, 1977). How-ever, acetylcholine may produce a vasomotor response in vascular smooth muscleeven when no cholinergic innervation appears to be present (Koelle, 1975). It is,therefore, necessary to demonstrate the existence of a vasomotor innervation of thegills directly by examining the effects of branchial nerve stimulation on vascularfunction.

Recently, a few reports on the effects of branchial nerve stimulation on the branchialvascular resistance to blood flow in teleosts (Nilsson, 1973; Pettersson & Nilsson,1979) have demonstrated the existence of both sympathetic and parasympatheticvasomotor innervation. A vasoconstrictor response to branchial nerve stimulation hasalso been reported for the dogfish 5. canicula (Davies, quoted by Bolis & Rankin,1975), although this report is only very brief. The present study was undertaken toexamine qualitatively the nervous regulation of gill blood flow in the dogfish byelectrical stimulation of the branchial nerves in both whole animals and in isolated,saline-perfused, gill preparations.

MATERIALS AND METHODS

Isolated, saline-perfused gill arches

The effects of branchial nerve stimulation on branchial vascular function wereexamined in isolated gill arches, perfused with physiological saline, from a total ofseven dogfish, 5. canicula. Both sexes were used and their masses ranged from0-638 kg to 1-125 kg. The results from these experiments have been further qualifiedby pharmacological studies on similar isolated gill preparations from a further 11dogfish. The majority of these experiments were conducted at the Plymouthlaboratories of the Marine Biological Association of the U.K. during January andFebruary, 1980. The fish were caught by trawl and held in large sea water aquaria forbetween 3 to 4 days at approximately 15 °C prior to any experiment.

A dogfish was pithed and the 2nd right afferent branchial artery, which suppliesblood to the 1st right holobranch, was cannulated with a 30 cm length of Polythenetube (o.d. 1 -2 mm, Portex) for the inflow of aerated elasmobranch physiological saline(Capra & Satchell, 1977a). This had previously been filtered (0-2 jUm pore filter,Gelman) and contained 20 units i.u. sodium heparin ml"1 (Weddel). The 1stholobranch was used because its afferent artery was the most accessible and affordedrapid 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 wholefish X 30 strokes min"1. This is approximately the flow rate to a single holobranc^

Gill blood flow in the dogfish 255

at might be expected in an intact dogfish in which cardiac output is aboutmlmin-'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 onthe left side, and the 1st and 2nd right efferent branchial arteries, which drain the 1stholobranch of efferent arterial blood, were cannulated separately with short lengthsof Polythene tube (o.d. 0-9mm). The distal ends of these two cannulae were joinedvia a 'Y' junction to a single outflow cannula. The communicating vessels between the1st and 2nd hemibranchs and between the 3rd and 4th hemibranchs, which are locatedon the dorsal edge of the 1st and 2nd gill clefts respectively, were ligated close to the1st holobranch on the right side so that all efferent arterial flow left the preparationvia the single outflow cannula. The distal end of this cannula was inserted tightly intothe barrel of a glass Pasteur pipette which allowed it to be rigidly mounted. In thosepreparations in which the branchial nerves were to be electrically stimulated, the IXthand Xth cranial nerves were isolated in the anterior cardinal sinus and bilaterallysectioned close to the chondrocranium. The isolated holobranch, together with itsnerves, was dissected free from the animal and placed in a water jacketed organ bathcontaining aerated elasmobranch saline maintained at 15 °C (Fig. 1).

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) andits output displayed on a rectilinear pen recorder (Ormed Limited). Afferent per-fusion pressure measurements were adjusted to make allowance for the measuredresistance of the inflow cannula between the junction with the pressure transducer andthe gill preparation. Efferent arterial pressure was maintained at about 0-7 kPa byadjusting the level of the outflow cannula tip relative to the preparation, havingaccounted for the measured resistance of the outflow cannula. Zero pressure was takenas the pressure at the surface of the saline in the organ bath. Efferent arterial flow wasrecorded by an infra-red drop counting device. This was connected to a linear display

s

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


(Neurolog) which operated as an instantaneous rate meter, the output from which vi^idisplayed on the pen recorder. Efferent arterial flow rate was calculated as droprateXdrop volume. The latter was measured frequently during experiments, but didnot appear to alter measurably over the range of dropping rates observed.

Electrical stimulation of the branchial nervesIn 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 salinein 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 forperiods of between 1 and Ss from a physiological stimulator (Neurolog). Thefunctioning of at least somatic motor fibres in the nerves was substantiated by virtueof the fact that their electrical stimulation caused contraction of the striated musclesof the gill arch.

During nerve stimulation the effects of the following pharmacological blockingagents were investigated: the cholinergic receptor antagonists pancuronium bromide(Pavulon; Organon Labs) and atropine sulphate (Sigma), the adrenergic receptorantagonists phentolamine mesylate (Rogitine, Ciba) and propranolol (Inderal,I.C.I.). All drugs were added, singly or together, to 10 or 20 ml of physiological salinecontained in a 20 ml syringe barrel, connected to the perfusion system by a three-waytap situated between the saline reservoir and the pump (Fig. 1). In this way theperfusion could be changed from drug-free saline to saline containing drugs withoutinterrupting the perfusion flow. This method of drug administration also obviated thedilution of the drug by drug-free perfusate which would have occurred if drugs hadbeen administered as bolus injections into drug-free perfusate. Drug doses are giveneither as gml"1 of perfusate, or as molar concentrations.

Pharmacological studiesIn 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) wereinvestigated. In these studies, perfusion of the preparations with the antagonistpreceded perfusion with the agonist. Finally, in a further four such preparations, thevascular responses to the adrenergic receptor agonists adrenalin bitartrate andnoradrenalin bitartrate (Sigma) were investigated.

Branchial nerve stimulation in whole dogfishIn view of the physiological limitations of the isolated, perfused gill preparation (see

Results), the study was extended by examining the effects of electrical stimulation ofthe Xth cranial nerve on pre- and post-branchial blood pressure in intact,anaesthetized fish. This preparation was most convenient since it also permitted astudy of possible ganglion-blocking properties of the neuromuscular blocking drugpancuronium bromide used in the isolated, perfused gill studies. This was achievedby examining the effects of pancuronium on the cardiac slowing caused by electricalstimulation of the branchial branch of the cardiac vagus.

These experiments were performed in Birmingham on eight dogfish of either


phe mass of which ranged from 0-540 kg to 0-920 kg. The fish were obtained from thePlymouth laboratories of the Marine Biological Association of the U.K. and transpor-ted 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 constanttemperature room maintained at 15 °C. The gills were irrigated with recirculating,aerated sea water containing anaesthetic. The caudal artery was cannulated for themeasurement of post-branchial blood pressures, with a 30 cm length of Polythene tube(o.d. 1-22 mm, Portex) filled with heparinized elasmobranch saline as described byMetcalfe & Butler (1982). The ventral aorta was cannulated, for the measurement ofpre-branchial blood pressures, by a method similar to that described for the Lemonshark N. brevirostris by Bushnell et al. (1982), although in the present study thecannula 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 takenthat no air entered the circulation during this part of the procedure by temporarilyplacing a tissue paper plug in the opening of the Cuvierian duct. The visceral branch

bcvbcv

Fig. 2. A diagrammatic illustration of the right and left Xth cranial nerves of the dogfish Scyliorhinuscanicula (dorsal view) showing the positions of nerve section (=) and the positions of nerve stimula-tion ( T ) . bcv, branchial cardiac vagus.


of the vagus posterior to the 4th branchial division (Fig. 2) was sectioned to preventsubsequent stimulation of the vagus from affecting systemic blood flow, or fromaffecting heart rate via the visceral cardiac vagus (Short, Butler & Taylor, 1977). Thebranchial cardiac vagus was sectioned at a point close to where it leaves the 4thbranchial division (Fig. 2) and cleared of connective tissue for a length of 5-7 mm toallow subsequent stimulation. The anterior cardinal sinus was then closed by a sutureafter removal of the tissue paper plug and any air trapped in the sinus. The procedurewas then repeated on the other side of the fish.

The roots of the right and left vagi were exposed by removing a portion of thechondrocranium above the medulla from which these nerves arise. The spinal cordwas sectioned at a point posterior to the medulla to prevent it subsequently beingstimulated. Silver stimulating electrodes were pushed into the foramina throughwhich 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 toallow access to the branchial cardiac vagus which was picked up on small silver hookelectrodes for subsequent stimulation (Fig. 2). Both right and left vagal roots werestimulated simultaneously at intensities ranging between 0-1 and 10 V, frequenciesranging between 2 and 100 Hz and with a pulse width of 1 ms from the physiologicalstimulator. The branchial cardiac vagus was stimulated at intensities ranging between0-1 and 3-0 V at a frequency of 50 Hz with a pulse width of 1 ms. 50 Hz is reported tobe the optimal frequency for stimulation of the branchial cardiac vagus for causingmaximum reductions in heart rate (Short et al. 1977). The effects of pancuroniumbromide (2mgkg~') and atropine sulphate (0-15 mgkg"1) on the responses to bothpaired vagal root and branchial cardiac vagal nerve stimulation were investigated.

RESULTS

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

In the isolated, saline-perfused gill preparation prior to nerve stimulation, the meanafferent arterial perfusion pressure was about 2-5 kPa, and efferent arterial flow rategenerally only accounted for 10-20% of the afferent arterial flow. In vivo afferentarterial blood pressure is usually about 50 kPa in this species at 15 °C (Short et al.1979), and efferent arterial flow rate might be expected to be 60-90% of afferentarterial flow (Metcalfe & Butler, 1982). The features of the present preparation werepresumably due to leakage of perfusate via the cut ends of the holobranch, eventhough efferent arterial back pressure was low in comparison with that recorded invivo (about 3-9 kPa, Shorten al. 1979). Since some venous drainage from the gill archmust normally occur in vivo, which may be important for any ability to alter theregional distribution of branchial blood flow, it would be unphysiological to ligatecompletely these cut ends of the holobranch. However, there appeared to be norepeatable method of partially occluding the leakage route so none was attempted withthe final preparations. These factors, combined with physiological afferent arterialflow rates, resulted in the low afferent arterial perfusion pressure.

The magnitude of the vascular responses elicited by nerve stimulation varied be-tween preparations, although the general nature of the response was similar in alfl


»pases. 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 aqualitative analysis has been attempted.

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

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

Paralysis of the striated muscles of the gill arch with the neuromuscular blockingdrug pancuronium bromide (20jUgml~') reduced or abolished all changes in bothafferent arterial perfusion pressure and efferent arterial flow rate in response to nervestimulation. This occurred at all frequencies and intensities of stimulation in all fourpreparations in which the drug was used (Fig. 3). In those instances where some smallresidual changes in afferent arterial perfusion pressure and/or efferent arterial flowrate persisted after the application of pancuronium, these were associated with someslight, observable, contraction of the gill arch, presumably due to incompleteparalysis of the skeletal muscles.

In all five preparations investigated without paralysis with pancuronium, thevascular response to nerve stimulation was not modified by the muscarinic receptorantagonist atropine (22/igml"1) (Fig. 4) or by the alpha-adrenergic receptor

S 50 50 100 100 200 10 S 5 S 5 10 20 100 5 2050 50 200 50

H I 1X X

Fig. 3. Traces of efferent arterial flow (F: mlmin"1) and afferent arterial pressure (P: kPa) obtainedfrom isolated, saline-perfused 1st holobranch preparations. (A) During electrical stimulation of theIXth and Xth cranial nerves before pancuronium; and (B) during electrical stimulation (at the sameintensity) of the IXth cranial nerve after pancuronium (20/igmr1). S, periods of electrical stimula-tion at frequencies indicated in Hz, at voltages of between 0-l and 10 V. Bars marked X showstimulation of the Xth cranial nerve. Time marker at top indicates minute intervals.


B -

0-6

0

5

S 50 SO SO 20

A-1-

1I

S 10 SO 200 20 SO 200

Fig. 4. Traces of efferent arterial flow (F: mlmin"1) and afferent arterial pressure (P: kPa) obtainedfrom isolated saline-perfused 1st holobranch preparations. Upper traces: (A) during electricalstimulation of the IXth cranial nerve before atropine, and (B) during electrical stimulation (at thesame intensity) of the IXth cranial nerve after atropine (22 ng m l ) . Lower traces: (A) duringelectrical 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 beta-adrenoceptor blockade (phentolamine 50,Ugmr', propranolol 10/lgml"'). S, periods of electricalstimulation at frequency indicated in Hz, at voltages of between O'l and 10 V. Time marker at topindicates minute intervals.

antagonist phentolamine (50/igmr1) or by the beta-adrenergic receptor antagonistpropranolol (10/igmr1) (Fig. 4).

Pharmacological studies

In seven isolated, saline-perfused 1st holobranch preparations in which the bran-chial nerves were not stimulated, acetylcholine (0-2 ̂ g ml"1) caused a small reversibleincrease in afferent arterial perfusion pressure and a marked reduction, or even com-plete abolition of efferent arterial flow (Fig. 5). This response to acetylcholine couldnot be prevented by pancuronium (20 figm\~l) (Fig. 5) but was either much reducedor completely prevented by atropine (22/tig ml"1) (Fig. 5); taken together with pan-curonium's effects on nerve stimulation these findings confirm the specificity of thedrug to nicotinic receptors in the dogfish gill. In a further four preparations adrenalin(two) and noradrenalin (two) produced marked increases in efferent arteriajj


At ACh

0-6

ACh P ACh• •

Fig. 5. Traces of efferent arterial flow (F: mlmin ') and afferent arterial pressure (P: kPa) obtainedfrom isolated saline-perfused 1st holobranch preparations. (A) In response to acetylcholine (ACh)(OZ^gmr 1 ) before and after atropine (At) (22/igmr1). (B) In response to acetylcholine (ACh)(02 ^g ml"1) before and after pancuronium (P) (20 )J% ml"1). Time marker at bottom indicates minuteintervals. ^ • indicates administration of drug.

flow rate and this was associated with a reduction in afferent arterial perfusionpressure at concentrations of 10~6moll~1 and above (Fig. 6).

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

In whole fish prior to nerve stimulation, ventral, aortic and dorsal aortic bloodpressures were 3*l±0-2kPa and l -9±0-lkPa respectively. These values aresomewhat 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 ofanaesthesia. 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 was43 ± 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 theheart 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 whichinnervate the gills posterior to the 1st holobranch at intensities above threshold, andat frequencies between 10 and 100 Hz, caused an immediate and marked increase inventral aortic blood pressure which either decreased gradually during the period ofstimulation (Fig. 7) or was maintained throughout (Fig. 8). This was associated withan immediate but much smaller rise in dorsal aortic blood pressure which rapidlyreturned to normal during the period of stimulation. This response was observed inall eight fish, although the magnitude of the increases varied between animals. In five

Ksh in which the branchial branch of the cardiac vagus was stimulated, this resulted


t I I I 1 I I 1

0-6 r-

/

{4

1 1 ] 1 1 1 1 1 T t 1

0

Fig. 6. Traces of efferent arterial flow (F: ml min"') and afferent arterial pressure (P: kPa) obtainedfrom isolated saline-perfused 1st holobranch preparations. (A) In response to noradrenalin (NA)(10"6mol I"1). (B) In response to adrenalin (Ad) (10"6mol 1"'). Time marker at top indicates minuteintervals. ^ • indicates administration of drug.

in an immediate and dramatic cessation of the heart beat (Figs 7, 8). In all five fishin which the drug was used, pancuronium (2 mg kg"1) abolished all vascular responsesto paired vagal root stimulation, but had no effect on the cardiac response to branchialcardiac vagal stimulation (Fig. 7). This observation indicates that at the dose levelused, pancuronium does not block the nicotinic receptors present in the cardiacganglia and is specific to the nicotinic receptors on striated muscle.

In three fish, atropine (0-15 mg kg"1) had no effect on the vascular response topaired vagal root stimulation (Fig. 8), but in two of these fish in which the branchialcardiac 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 abolishedall branchial vascular responses to paired vagal root stimulation (Fig. 8).


VAbp(kPa)

DAbp(kPa) 2 n

0-1

v—

Time (min)

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 vagalroots (bv) (30-50 Hz) or one branchial branch of the cardiac vagus (cv) (50 Hz) before and after theadministration of pancuronium (Pan) (2mgkg~'). Marker at top indicates periods of electrical sti-mulation.

bv bv bv bv f,v•* -v -w

6-1

VAbp 4 '(kPa) 2 .

0 -

DAbp(kPa)

Time (min)

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 vagalroots (bv) (30-50 Hz) or one branchial branch of the cardiac vagus (cv) (50 Hz) before atropine (At)(0-15 mgkg"'), after atropine, and after pancuronium (Pan) (2mgkg~'). Marker at top indicatesperiods of electrical stimulation.


DISCUSSION

The control of branchial blood flow

From the present study it appears that all vascular responses to electrical stimula-tion of the branchial nerves in both isolated, perfused 1st holobranch preparations andin whole anaesthetized dogfish are entirely the result of the contraction of the striatedmuscles of the gill arch, rather than the result of contraction of the smooth muscle inthe major blood vessels themselves. These vascular responses could not be preventedeither by atropine (in both perfused holobranchs and whole fish) or by adrenergicreceptor blockade (in perfused holobranchs), but were consistently prevented in bothpreparations by paralysis of the striated muscles of the gill arch with pancuroniumbromide. The specificity of pancuronium to striated muscle motor end-plates hasbeen demonstrated in the present study since it did not affect the vascular responsesto 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, 1stholobranch preparations, the vascular responses to branchial nerve stimulation inboth whole fish and perfused holobranchs were qualitatively similar. In both studies,branchial nerve stimulation resulted in a marked and rapid increase in afferent per-fusion pressure. In perfused holobranchs this response was associated with increasesin efferent arterial flow rate, although in whole fish little change in dorsal aortic bloodpressure was observed in response to branchial nerve stimulation. This may be causedeither by capacitance effects within the systemic circulation as a result of reducedsystemic vascular tonus during anaesthesia, or by a redistribution of afferent bloodflow from the gills innervated by the Xth cranial nerve (hemibranchs 4-9) to the gillsinnervated by the IXth cranial nerve (hemibranchs 1-3; note: the IXth cranial nervewas not stimulated in these experiments). In the isolated 1st holobranch preparation,efferent arterial flow rate was low in comparison with afferent arterial flow rate. Thisappeared to be due to leakage of perfusate via the extensive venous sinuses of the gillarch and interbranchial septum (Cooke, 1980; J. D. Metcalfe & P. J. Butler, inpreparation). Electrical stimulation of the branchial nerves causes contraction of thestriated muscle of the gill arch which presumably compresses the venous sinuseswithin the interbranchial septum, increasing the resistance to flow of perfusate via thisroute. This would favour the flow of perfusate through the efferent arterial route,resulting in an increase in efferent arterial flow, particularly in perfused holobranchsin which afferent arterial flow remained constant. This is entirely consistent with thepresent observations of the responses to branchial nerve stimulation prior to paralysisof the striated muscles of the gill arch with pancuronium.

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

In the pharmacological studies on isolated holobranchs, acetylcholine caused


^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 stimulationof muscarinic receptors since the response was blocked by atropine but not by pan-curonium. This vasoconstriction observed in response to acetylcholine confirmssimilar reports of studies on perfused gills of both 5. canicula (Davies & Rankin,1973) and trout (Smith, 1977). However, the actions of acetylcholine cannot alone beregarded as evidence for cholinergic vasomotor innervation (see Introduction).

In perfused holobranchs, both adrenalin and noradrenalin caused overallvasodilatation, and this confirms previous reports of similar studies conducted onelasmobranch gills (Davies & Rankin, 1973; Capra & Satchell, 19776). A similarvasodilatation might have been expected in response to branchial nerve stimulationfollowing paralysis of the skeletal muscles of the gill arch if any adrenergic vasomotorfibres were present in these nerves. The threshold concentrations of these drugsrequired to elicit a response in the present study (about 10~6 mol I"1 for both adrenalinand noradrenalin) are much higher than that reported by previous authors (aboutlO^ 'moi r 1 , Davies & Rankin, 1973). However, KT'moir 1 is close to (withinan order of magnitude) the levels of circulating adrenalin and noradrenalin foundin iS. canicula during hypoxic stress (about 2-8x 10~7mol I"1 for adrenalin, 4-5 X10~7 mol I"1 for noradrenalin, Butler, Taylor, Capra & Davison, 1978). It is assumedthat the vascular responses of the isolated perfused holobranchs observed in thepharmacological studies are representative of the responses that might have beenobserved in similar studies on whole fish. However, such studies were not performedsince the branchial vascular responses would have been obscured by systemic vascularresponses.

Pettersson & Nilsson (1979) report that both cholinergic and adrenergic vasomotornerve fibres innervate the branchial vascular bed in the cod Gadus morhua, and thatelectrical stimulation of the entire vago-sympathetic nerve trunk in which these fibrespass causes vasoconstriction which could be reversed by atropine to a vasodilatationmediated by beta-adrenergic receptors. However, in all but one of their experimentsthese authors found a persistent vasoconstrictor response to nerve stimulation whichin some cases obscured the beta-adrenergic receptor mediated dilatory response, andwhich in others was revealed after beta-adrenergic receptor blockade. This vasocon-strictor response could not be abolished by any of the pharmacological agents em-ployed by these authors, apart from tetrodotoxin. However, these authors did notinvestigate the effects of striated muscle blockade during nerve stimulation. It ispossible that the persistent vasoconstriction observed may have been the result ofcontraction of striated muscles in the gill arch, similar to the response observed in thepresent study.

In 5. canicula, sphincters have been observed in the afferent lamellar arterioles(Wright, 1973) but no innervation was apparent. Both these observations have beenconfirmed in a more recent study by Dunel & Laurent (1980). Sphincters have alsobeen reported to exist in the efferent filament artery just prior to its junction with theefferent arch artery in this species (Wright, 1973), and these sphincters are reported toreceive motor innervation which is probably cholinergic (Dunel & Laurent, 1980). Thisappears to contradict the results obtained in the present study. However, the observa-

tions of these authors are based upon histological, rather than physiological, studies.


The results presented here strongly indicate that, unlike those of the cod, the majoi|blood vessels of the branchial vascular bed of the dogfish are devoid of any directmotor innervation which may control the regional distribution of branchial bloodflow. Presumably, any direct control must be either via humoral agents such as thecirculating catecholamines adrenalin and noradrenalin, or via intrinsic mechanisms,possibly similar to that reported by Satchell (1962) for Squalus acanthias in whichbrief periods of anoxia caused branchial vasoconstriction. These responses could notbe abolished by section of the branchial nerves and Satchell (1962) concluded that thiswas an intrinsic response of the branchial vascular bed to anoxia. A similar responseto hypoxia has been reported for the cod (Pettersson & Johansen, 1982).

From the present study it has not been possible to determine whether or not thenervously mediated contraction of the striated muscles of the gill arch in vivo may actas a mechanism to direct blood flow from the venous sinuses of the interbranchialseptum to the respiratory vascular network of the gill filament. However, Hughes &Ballintijn (1965) report that these muscles (the septal constrictor branchials) contracttowards the end of each ventilatory cycle, just before the gill slits close. Though theactivity of these muscles is presumably essential to ventilation, it may also serve toenhance blood flow across the respiratory surface during those periods of highestoxygen availability in each ventilatory cycle.

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

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on the nervous regulation of gill blood flow in …branchial nerve stimulation in whole dogfish in...

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