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Excitatory adrenergic innervation in the heart of the marine teleost fish Girella tricuspidata (Quoy and Gaimard)
J. Exp. Mar. Biol. EcoL, 1986, Vol. 95, pp. 87-93 Elsevier
87
JEM $614
Short Communication EXCITATORY ADRENERGIC INNERVATION IN THE HEART OF THE M A R I N E TELEOST FISH GIRELLA TRICUSPIDATA (Quoy and Gaimard)
J . C . MONTGOMERY,R . M . G . WELLS and M . J . PARRISH Department of Zoology. University of Auckland, Auckland, New Zealand (Received 30 September 1985; accepted 18 October 1985)
Abstract: Adrenergic innervation has been investigated in the heart of the marine teleost GireUatricuspidata (Quoy and Gaimard). Adrenergic fibres were demonstrated by fluorescence histochemistry in the atrium and sinus venosus. The cardiac branch of the vagus was shown to have inhibitory influences which gave way to positive inotropy and tachycardia following atropinization. These excitatory effects were blocked by the beta-adrenoceptor antagonist, propranolol. These observations are consistent with the activity of adrenergic fibres and were clearly demonstrated in all preparations (n = 8). Preliminary experiments using the anaesthetics benzocaine and MS 222 were discontinued because of inconsistent results possibly resulting from autonomic suppression. The relative importance of the neural mechanisms in the double antagonistic control of the fish heart is still unclear. Key words: Girella tricuspidata; teleost; heart; cardiac control; adrenergic innervation C a r d i a c regulatory m e c h a n i s m s function in fish to adjust cardiac output in response to changes in a range o f physical and organismic factors. Three regulatory m e c h a n i s m s have been recognised in fish: these involve the effects o f venous return pressure on cardiac output, the role o f circulating catecholamines, and vagal inhibitory innervation. M o r e recently, the occurrence o f sympathetic adrenergic fibres has been d e m o n s t r a t e d within the cardiac tissue o f some teleost species (Nilsson, 1984; Santer, 1985). As yet the n u m b e r of fish species studied in this respect is t o o small to k n o w h o w general the distribution o f excitatory innervation is a m o n g fish, and to be able to relate the distribution pattern to ecological or phylogenetic factors. The functional contributions o f the different regulatory m e c h a n i s m s in fish are also largely unknown. This study set out to establish the presence or absence o f excitatory neural control in the heart o f the active marine teleost fish Girella tricuspidata ( Q u o y and G a i m a r d ) using a perfused heart preparation, as an i m p o r t a n t c o m p o n e n t in u n d e r s t a n d i n g circulatory control within these fish during environmental and physiological stress. All experiments were performed on the perciform fish G. tricuspidata which were caught in set nets and then transferred to the sea water circulation system at the Leigh M a r i n e Laboratory, and kept at temperatures o f 17-19 ° C. All fish were mature adults o f between 0.5-1.5 kg, and were used within 2 w k o f capture. 0022-0981/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
88
J. C, MONTGOMERY E T AL.
Histochemical methods. Cardiac tissue removed from G. tricuspidata was processed for the fluorescent identification of monoamines. Best results were achieved using animals treated with two monoamine oxidase (MAO) inhibiting drugs 6 h before killing. These were Harmaline (Sigma), which in mammals specifically blocks the "A" form of MAO, and Pargyline (Sigma), which in mammals is specific for the "B" form (Youdim & Finberg, 1983). Alpha-methylated tyrosine was applied in conjunction with the MAO inhibitors. Neuronal uptake of the methylated amino acid results in its synaptic conversion to methylated adrenaline which is MAO resistant (Donald & Campbell, 1982). The combined effect of both MAO inhibitor and alpha-methylated tyrosine increased synaptic adrenaline concentration and therefore enhanced fluorescence. These drugs were administered via the caudal vein in 1 ml of isotonic saline at 1.0 x 10 4 g/kg. The fish were killed by a blow to the head, and the heart rapidly removed and placed in a liquid N 2 slush. Tissue samples were transferred in liquid N 2 onto the pre-cooled stage of an Edwards-Pearce D3 freeze dryer. Complete dehydration under P205 was achieved after 72 h at - 60 °C and 10- 2 mm Hg. Dry tissue was warmed to + 40 °C while still under vacuum. Dry heart tissue was placed in vacuum flasks which had previously been loaded with paraformaldehyde at a ratio of 5 g/l of flask volume (Falck & Owman, 1965). P205 (10 g/l) was also added to maintain a low humidity within the flask. Tissues were placed in the vacuum flask in small open vials, thereby avoiding direct chemical contact. Prepared flasks were subjected to a mild vacuum before incubation at 89 °C for 3 h. Incubated tissue samples were blocked in wax, 6-8/~m sections cut and mounted in paraffin oil. Sections were viewed on a Zeiss III RS fluorescence microscope at 450-490 nm wavelengths. Physiological methods. Fish were killed with a blow to the head and placed ventral surface upward in an operating sling. The gills were irrigated with aerated, filtered sea water (17-18 °C) at a rate of 1.5 1/min. The cardiac branch of the vagus was exposed by removal of the opercular plate, followed by incision of the membrane behind the 5th gill arch. The nerve was placed in a cuff electrode to permit electrical stimulation. The heart was then exposed and isolated from the circulation, the ventral aorta was occluded by an outflow cannula which provided a constant afterload of 7-12 cm H20, the sinus venosus was cannulated and the heart perfused with oxygenated Ringer (see Holmgren, 1977) at a constant rate of between 5 and 15 ml/min. The osmolarity of the perfusate was measured and found to be ~ 373 mOsm which compared favourably with values from unstressed fish, and the pH was adjusted to 7.85 by addition of NaHCO3 to approximate assumed normal physiological values. Drugs were administered to the heart by bolus injection into the perfusion line. Cardiac responses were recorded by attaching a heart clip to the apex of the ventricle and relaying the contractions through a 3 g/cm isotonic spring to a Grass (0.03FT) force-displacement transducer. Cardiac activity was monitored on a Grass polygraph
ADRENERGIC INNERVATION IN HEART OF TELEOST
89
from which inotropic (force) and chronotropic (rate) responses to stimulation were measured. In some instances a rate meter record was simultaneously displayed to monitor chronotropic responses. All observations were made on a minimum number of 8 fish. Short-wave (blue) fluorescence indicated the presence of adrenaline in cardiac tissue sections contained in varicose nerve fibres (Fig. 1). Fluorescence was observed only in
Fig. 1. Histochemical evidence for adrenergic innervation: fluorescence micrograph of atrial tissue after paraformaldehyde condensation of monoamines; B shows short-wave (blue) emission derived from adrenergic nerve fibres. cardiac tissue sections treated with monoamine oxidase inhibitors and alpha-methylated tyrosine. The density of innervation, indicated by the level of fluorescence, was higher in atrial and sinus venosus tissues. Stimulation of the cardiac branch of the vagus produced an immediate decrease in heart rate which lasted only for the duration of the stimulus train (Fig. 2A). Bolus injection of acetylcholine caused a dose-dependent chronotropic decrease, and a small decrease in the force of contraction (Table 1, Fig. 2B). The heart rate returned to the resting level after 2 - 3 min. Bolus injection of atropine, to block muscarinic cholinoceptors, produced a sustained 15~o increase in heart rate (Table 1) suggesting a degree of inhibitory tonus. After atropine treatment, neural stimulation of the cardiac branch of the vagus produced a further increase in heart rate and force of contraction (Fig. 3A). The response was slow to develop, with a latency of up to 4 s, and with a 10-s stimulus pulse train, the peak chronotropic response was not reached until 24 s after the beginning of stimulation. The response lasted for a period of 3-5 min (Fig. 3A). Isolation of the heart from the circulation precluded this response from being due to the humoral release of
90
J.C. M O N T G O M E R Y E T AL.
catecholamines. In preliminary experiments where the fish were anaesthetized with either benzocaine (0.3 g/l), or MS222 (0.15 g/l) before killing, no consistent excitation was observed in response to neural stimulation of the cardiac branch of the vagus. A
i
I
5
stim
sec
B
3 0 sec Ach
Fig. 2. Inhibitory cardiac responses: A, neural stimulation of the cardiac branch of the vagus nerve: a 10-s, 25-Hz pulse train was applied for the period shown; in this and all the other records of cardiac activity. systole is represented by the upwards deflection of the trace; B, bolus injection of acetylcholine (1 x 10- s g/l) into the heart, injection occurred at the time indicated by the arrow.
TABLE I Drug responses 30 s after bolus injection into the perfused, isolated heart. Cardiac responses (~,) Drug
g/l
Chronotropic
Inotropic
Acetylcholine
10 7 10 6 10 - s 10 4 10-6 10- 6
- 32 -51 -70 +15 +5 + 11
- 6 -6 -6 +5 +7 + 23
Atropine Adrenaline Isoprenaline
Isoprenaline produced a persistent positive chronotropic and inotropic response (Fig. 3B) which was more pronounced than that for adrenaline (Table 1). All excitatory responses were abolished by treatment with the beta-adrenoceptor antagonist, propranolol. The results presented here provide anatomical and physiological evidence for excitatory adrenergic innervation of the heart in the active marine teleost G. tricuspidata.
A D R E N E R G I C I N N E R V A T I O N IN H E A R T OF T E L E O S T
91
Of the species studied to date, adrenaline has been shown to be the predominant adrenergic neurotransmitter in teleost cardiac tissue (Von Euler & Fange, 1961" Abrahamsson & Nilsson, 1976; Holmgren, 1977). Adrenergic innervation has been
A
,oo[
f
75L
slim
60
sec
60
sec
B
I°° r 75~
f IsoDr
Fig. 3. Excitatory cardiac responses: A, neural stimulation of the cardiac branch of the vagus nerve, after atropine block of the inhibitory innervation; top trace shows the increased force of contraction (inotropic response), and the lower trace the output of a rate meter showing an unequivocal increase in heart rate (chronotropic response); neural stimulation was for 10 s at 20 Hz at the time indicated by the bar below the lower trace, vertical calibration of the rate meter output is in beats/min; B, bolus injection ofisoprenaline (1 × 10 5 g/l) into the isolated perfused heart; injection occurred at the time indicated by the arrow; note the positive inotropic and chronotropic responses; calibrations as in Fig. A.
demonstrated histochemically in trout (Gannon & Burnstock, 1969), cod (Holmgren, 1977), goldfish (Cameron, 1979), and a few other teleosts including eels (Donald & Campbell, 1982). In contrast, attempts to demonstrate adrenergic innervation in flatfish proved negative (Santer, 1972; Donald & Campbell, 1982). Neural stimulation of the cardiac branch of the vagus produced a strong inhibition of the heart. The control exerted through changes in vagal inhibitory tonus, mediated through muscarinic receptors, is considered the primary neural influence acting upon the teleost heart, and has been described in many other fish species (Laurent et al., 1983; Nilsson, 1984; Santer, 1985). After blockade of the inhibitory system by atropine, the heart rate rose 15~o indicating a degree of inhibitory tonus. Subsequent neural stimulation produced a substantial cardiac excitation which could be blocked by propranolol indicating that in G. tricuspidata the cardiac branch of the vagus carries
92
J.C. M O N T G O M E R Y E T AL.
excitatory fibres whose action is mediated by beta-adrenoceptors. Teleost fish are unusual in that they have an extension of sympathetic chain ganglia into the head. Ganglionic connections occur between these fibres and the cranial autonomic outflow thus providing a vago-sympathetic trunk (Nilsson, 1984). Physiological demonstration of excitatory cardiac control has been shown for some species for which anatomical evidence exists for adrenergic innervation (see Nilsson 1984; Santer, 1985). Studies of cardiac nerve stimulation in flatfish have failed to reveal adrenergic cardiac control (Santer, 1972; Cobb & Santer, 1973; Donald & Campbell, 1982). These results indicate that adrenergic innervation of the heart is probably common in teleost fish, but not universal. With the limited number of fish species examined in this respect it remains difficult to relate the pattern of distribution to either phylogeny, or the ecology of the fish. In addition to the dual antagonistic innervation of the heart demonstrated in this study, G. tricuspidata like other fish, will have intrinsic regulation resulting from venous return pressure ("Starling's Law") and humoral regulation resulting from changes in catecholamine concentration in the blood plasma (Laurent et aL, 1983). The functional interplay of these control mechanisms in fish remains poorly understood. From the control point of view, the advantage of direct neural control versus humoral control lies in its specificity to the target organ. Neural control provides the possibility for direct control of heart rate and force of contraction, and may be capable of modifying humoral influences intended for other target sites such as the gill vasculature. Antagonistic control may provide a greater range of cardiac outputs than relying on the modulation of inhibitory tonus alone, and in addition may allow differential inotropic and chronotropic control. An indication of this is the transient, predominantly chronotropic inhibitory responses to neural stimulation as opposed to the persistent combined inotropic and chronotropic excitatory responses. It seems that in this preparation neurally released catecholamines are not rapidly deactivated or removed from their site of action. In elasmobranch fish vagal branches innervate the heart, but adrenergic contributions to the vagi, or direct cardiac nerves are apparently absent (Nilsson, 1983). Teleost fish therefore occupy an interesting phylogenetic position between the elasmobranchs and the higher vertebrates, all of which have dual antagonistic innervation with vagal inhibitory fibres and sympathetic excitation. The relative functional importance of neural mechanisms in fish remains uncertain, but further study of species with and without dual innervation should provide interesting information on how circulatory control is achieved in response to environmental and organismic factors. ABRAHAMSSON, T. & S. NILSSON, 1976. Phenylethanolamine-N-methyl transferase (PNMT) activity and catecholamine content in chromaffin tissue and sympathetic neurons in the cod, Gadus morhua. Acta Physiol. Scand., Vol. 96, pp. 94-99. CAMERON, J.S., 1979. Autonomic nervous tone and regulation of heart rate in the goldfish, Carassius attratus. Comp. Biochem. Physiol., Vol. 63C. pp. 341-349. COBB. J.L.S. & R.M. SANTEr. 1973. Electrophysiology of cardiac function in teleosts: cholinergically mediated inhibition and rebound excitation. J. PhrsioL (London), Vol. 2311, pp. 561-574.
ADRENERGIC INNERVATION IN HEART OF TELEOST
93
DONALD, J. L. S. & G. CAMPBELL, 1982. A comparative study of the adrenergic innervation of the teleost heart. J. Comp. Physiol., Vol. 147, pp. 85-91. FALCK, B. & CH. OWMAN, 1965. A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines. Acta Univ. Lund Sect., 2, Vol. 7, pp. 1-23. GANNON, B.J. & BURNSTOCK,G., 1969. Excitatory adrenergic innervation of the fish heart. Comp. Biochem. Physiol., Vol. 29, pp. 765-773. HOLMGREN, S., 1977. Regulation of the heart of a teleost, Gadus morhua, by autonomic nerves and circulating catecholamines. Acta Physiol. Scand., Vol. 99, pp. 62-74. LAURENT,P., S. HOLMGREN & S. NILSSON, 1983. Nervous and humoral control of the fish heart: structure and function. Comp. Biochem. Physiol., Vol. 76A, pp. 525-542. NILSSON, S., 1983. Autonomic nerve function in the vertebrates. Zoophysiology series, Vol. 13. SpringerVerlag, Berlin-Heidelberg-New York, 253 pp. N1LSSON, S., 1984. Adrenergic control systems in fish. (Review). Mar. Biol. Lett., Vol. 5, pp. 127-146. S ANTER, R.M., 1972. Ultrastructural and histochemical studies on the innervation of the heart of a teleost (Pleuronectes platessa L.). Z. Zellforsch., Vol. 131, pp. 519-528. SANTER, R.M., 1985. Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell Biol., Vol. 89, pp. l-110. VON EULER, U. S. & R. F~NGE, 1961. Catecholamines in nerves and organs ofMyxine glutinosa, Squalus acanthias and Gadus morhua. Gen. Comp. Endocrinol., Vol. l, pp. 191-194. YOUDIM, M. B.H. & J.P.M. FINBERG, 1983. Monoamine oxidase inhibitor antidepressants. In, Psychopharmacology, Part 1, edited by G. Smith, Excerpta Medica, Amsterdam, pp. 38-70.
87
JEM $614
Short Communication EXCITATORY ADRENERGIC INNERVATION IN THE HEART OF THE M A R I N E TELEOST FISH GIRELLA TRICUSPIDATA (Quoy and Gaimard)
J . C . MONTGOMERY,R . M . G . WELLS and M . J . PARRISH Department of Zoology. University of Auckland, Auckland, New Zealand (Received 30 September 1985; accepted 18 October 1985)
Abstract: Adrenergic innervation has been investigated in the heart of the marine teleost GireUatricuspidata (Quoy and Gaimard). Adrenergic fibres were demonstrated by fluorescence histochemistry in the atrium and sinus venosus. The cardiac branch of the vagus was shown to have inhibitory influences which gave way to positive inotropy and tachycardia following atropinization. These excitatory effects were blocked by the beta-adrenoceptor antagonist, propranolol. These observations are consistent with the activity of adrenergic fibres and were clearly demonstrated in all preparations (n = 8). Preliminary experiments using the anaesthetics benzocaine and MS 222 were discontinued because of inconsistent results possibly resulting from autonomic suppression. The relative importance of the neural mechanisms in the double antagonistic control of the fish heart is still unclear. Key words: Girella tricuspidata; teleost; heart; cardiac control; adrenergic innervation C a r d i a c regulatory m e c h a n i s m s function in fish to adjust cardiac output in response to changes in a range o f physical and organismic factors. Three regulatory m e c h a n i s m s have been recognised in fish: these involve the effects o f venous return pressure on cardiac output, the role o f circulating catecholamines, and vagal inhibitory innervation. M o r e recently, the occurrence o f sympathetic adrenergic fibres has been d e m o n s t r a t e d within the cardiac tissue o f some teleost species (Nilsson, 1984; Santer, 1985). As yet the n u m b e r of fish species studied in this respect is t o o small to k n o w h o w general the distribution o f excitatory innervation is a m o n g fish, and to be able to relate the distribution pattern to ecological or phylogenetic factors. The functional contributions o f the different regulatory m e c h a n i s m s in fish are also largely unknown. This study set out to establish the presence or absence o f excitatory neural control in the heart o f the active marine teleost fish Girella tricuspidata ( Q u o y and G a i m a r d ) using a perfused heart preparation, as an i m p o r t a n t c o m p o n e n t in u n d e r s t a n d i n g circulatory control within these fish during environmental and physiological stress. All experiments were performed on the perciform fish G. tricuspidata which were caught in set nets and then transferred to the sea water circulation system at the Leigh M a r i n e Laboratory, and kept at temperatures o f 17-19 ° C. All fish were mature adults o f between 0.5-1.5 kg, and were used within 2 w k o f capture. 0022-0981/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
88
J. C, MONTGOMERY E T AL.
Histochemical methods. Cardiac tissue removed from G. tricuspidata was processed for the fluorescent identification of monoamines. Best results were achieved using animals treated with two monoamine oxidase (MAO) inhibiting drugs 6 h before killing. These were Harmaline (Sigma), which in mammals specifically blocks the "A" form of MAO, and Pargyline (Sigma), which in mammals is specific for the "B" form (Youdim & Finberg, 1983). Alpha-methylated tyrosine was applied in conjunction with the MAO inhibitors. Neuronal uptake of the methylated amino acid results in its synaptic conversion to methylated adrenaline which is MAO resistant (Donald & Campbell, 1982). The combined effect of both MAO inhibitor and alpha-methylated tyrosine increased synaptic adrenaline concentration and therefore enhanced fluorescence. These drugs were administered via the caudal vein in 1 ml of isotonic saline at 1.0 x 10 4 g/kg. The fish were killed by a blow to the head, and the heart rapidly removed and placed in a liquid N 2 slush. Tissue samples were transferred in liquid N 2 onto the pre-cooled stage of an Edwards-Pearce D3 freeze dryer. Complete dehydration under P205 was achieved after 72 h at - 60 °C and 10- 2 mm Hg. Dry tissue was warmed to + 40 °C while still under vacuum. Dry heart tissue was placed in vacuum flasks which had previously been loaded with paraformaldehyde at a ratio of 5 g/l of flask volume (Falck & Owman, 1965). P205 (10 g/l) was also added to maintain a low humidity within the flask. Tissues were placed in the vacuum flask in small open vials, thereby avoiding direct chemical contact. Prepared flasks were subjected to a mild vacuum before incubation at 89 °C for 3 h. Incubated tissue samples were blocked in wax, 6-8/~m sections cut and mounted in paraffin oil. Sections were viewed on a Zeiss III RS fluorescence microscope at 450-490 nm wavelengths. Physiological methods. Fish were killed with a blow to the head and placed ventral surface upward in an operating sling. The gills were irrigated with aerated, filtered sea water (17-18 °C) at a rate of 1.5 1/min. The cardiac branch of the vagus was exposed by removal of the opercular plate, followed by incision of the membrane behind the 5th gill arch. The nerve was placed in a cuff electrode to permit electrical stimulation. The heart was then exposed and isolated from the circulation, the ventral aorta was occluded by an outflow cannula which provided a constant afterload of 7-12 cm H20, the sinus venosus was cannulated and the heart perfused with oxygenated Ringer (see Holmgren, 1977) at a constant rate of between 5 and 15 ml/min. The osmolarity of the perfusate was measured and found to be ~ 373 mOsm which compared favourably with values from unstressed fish, and the pH was adjusted to 7.85 by addition of NaHCO3 to approximate assumed normal physiological values. Drugs were administered to the heart by bolus injection into the perfusion line. Cardiac responses were recorded by attaching a heart clip to the apex of the ventricle and relaying the contractions through a 3 g/cm isotonic spring to a Grass (0.03FT) force-displacement transducer. Cardiac activity was monitored on a Grass polygraph
ADRENERGIC INNERVATION IN HEART OF TELEOST
89
from which inotropic (force) and chronotropic (rate) responses to stimulation were measured. In some instances a rate meter record was simultaneously displayed to monitor chronotropic responses. All observations were made on a minimum number of 8 fish. Short-wave (blue) fluorescence indicated the presence of adrenaline in cardiac tissue sections contained in varicose nerve fibres (Fig. 1). Fluorescence was observed only in
Fig. 1. Histochemical evidence for adrenergic innervation: fluorescence micrograph of atrial tissue after paraformaldehyde condensation of monoamines; B shows short-wave (blue) emission derived from adrenergic nerve fibres. cardiac tissue sections treated with monoamine oxidase inhibitors and alpha-methylated tyrosine. The density of innervation, indicated by the level of fluorescence, was higher in atrial and sinus venosus tissues. Stimulation of the cardiac branch of the vagus produced an immediate decrease in heart rate which lasted only for the duration of the stimulus train (Fig. 2A). Bolus injection of acetylcholine caused a dose-dependent chronotropic decrease, and a small decrease in the force of contraction (Table 1, Fig. 2B). The heart rate returned to the resting level after 2 - 3 min. Bolus injection of atropine, to block muscarinic cholinoceptors, produced a sustained 15~o increase in heart rate (Table 1) suggesting a degree of inhibitory tonus. After atropine treatment, neural stimulation of the cardiac branch of the vagus produced a further increase in heart rate and force of contraction (Fig. 3A). The response was slow to develop, with a latency of up to 4 s, and with a 10-s stimulus pulse train, the peak chronotropic response was not reached until 24 s after the beginning of stimulation. The response lasted for a period of 3-5 min (Fig. 3A). Isolation of the heart from the circulation precluded this response from being due to the humoral release of
90
J.C. M O N T G O M E R Y E T AL.
catecholamines. In preliminary experiments where the fish were anaesthetized with either benzocaine (0.3 g/l), or MS222 (0.15 g/l) before killing, no consistent excitation was observed in response to neural stimulation of the cardiac branch of the vagus. A
i
I
5
stim
sec
B
3 0 sec Ach
Fig. 2. Inhibitory cardiac responses: A, neural stimulation of the cardiac branch of the vagus nerve: a 10-s, 25-Hz pulse train was applied for the period shown; in this and all the other records of cardiac activity. systole is represented by the upwards deflection of the trace; B, bolus injection of acetylcholine (1 x 10- s g/l) into the heart, injection occurred at the time indicated by the arrow.
TABLE I Drug responses 30 s after bolus injection into the perfused, isolated heart. Cardiac responses (~,) Drug
g/l
Chronotropic
Inotropic
Acetylcholine
10 7 10 6 10 - s 10 4 10-6 10- 6
- 32 -51 -70 +15 +5 + 11
- 6 -6 -6 +5 +7 + 23
Atropine Adrenaline Isoprenaline
Isoprenaline produced a persistent positive chronotropic and inotropic response (Fig. 3B) which was more pronounced than that for adrenaline (Table 1). All excitatory responses were abolished by treatment with the beta-adrenoceptor antagonist, propranolol. The results presented here provide anatomical and physiological evidence for excitatory adrenergic innervation of the heart in the active marine teleost G. tricuspidata.
A D R E N E R G I C I N N E R V A T I O N IN H E A R T OF T E L E O S T
91
Of the species studied to date, adrenaline has been shown to be the predominant adrenergic neurotransmitter in teleost cardiac tissue (Von Euler & Fange, 1961" Abrahamsson & Nilsson, 1976; Holmgren, 1977). Adrenergic innervation has been
A
,oo[
f
75L
slim
60
sec
60
sec
B
I°° r 75~
f IsoDr
Fig. 3. Excitatory cardiac responses: A, neural stimulation of the cardiac branch of the vagus nerve, after atropine block of the inhibitory innervation; top trace shows the increased force of contraction (inotropic response), and the lower trace the output of a rate meter showing an unequivocal increase in heart rate (chronotropic response); neural stimulation was for 10 s at 20 Hz at the time indicated by the bar below the lower trace, vertical calibration of the rate meter output is in beats/min; B, bolus injection ofisoprenaline (1 × 10 5 g/l) into the isolated perfused heart; injection occurred at the time indicated by the arrow; note the positive inotropic and chronotropic responses; calibrations as in Fig. A.
demonstrated histochemically in trout (Gannon & Burnstock, 1969), cod (Holmgren, 1977), goldfish (Cameron, 1979), and a few other teleosts including eels (Donald & Campbell, 1982). In contrast, attempts to demonstrate adrenergic innervation in flatfish proved negative (Santer, 1972; Donald & Campbell, 1982). Neural stimulation of the cardiac branch of the vagus produced a strong inhibition of the heart. The control exerted through changes in vagal inhibitory tonus, mediated through muscarinic receptors, is considered the primary neural influence acting upon the teleost heart, and has been described in many other fish species (Laurent et al., 1983; Nilsson, 1984; Santer, 1985). After blockade of the inhibitory system by atropine, the heart rate rose 15~o indicating a degree of inhibitory tonus. Subsequent neural stimulation produced a substantial cardiac excitation which could be blocked by propranolol indicating that in G. tricuspidata the cardiac branch of the vagus carries
92
J.C. M O N T G O M E R Y E T AL.
excitatory fibres whose action is mediated by beta-adrenoceptors. Teleost fish are unusual in that they have an extension of sympathetic chain ganglia into the head. Ganglionic connections occur between these fibres and the cranial autonomic outflow thus providing a vago-sympathetic trunk (Nilsson, 1984). Physiological demonstration of excitatory cardiac control has been shown for some species for which anatomical evidence exists for adrenergic innervation (see Nilsson 1984; Santer, 1985). Studies of cardiac nerve stimulation in flatfish have failed to reveal adrenergic cardiac control (Santer, 1972; Cobb & Santer, 1973; Donald & Campbell, 1982). These results indicate that adrenergic innervation of the heart is probably common in teleost fish, but not universal. With the limited number of fish species examined in this respect it remains difficult to relate the pattern of distribution to either phylogeny, or the ecology of the fish. In addition to the dual antagonistic innervation of the heart demonstrated in this study, G. tricuspidata like other fish, will have intrinsic regulation resulting from venous return pressure ("Starling's Law") and humoral regulation resulting from changes in catecholamine concentration in the blood plasma (Laurent et aL, 1983). The functional interplay of these control mechanisms in fish remains poorly understood. From the control point of view, the advantage of direct neural control versus humoral control lies in its specificity to the target organ. Neural control provides the possibility for direct control of heart rate and force of contraction, and may be capable of modifying humoral influences intended for other target sites such as the gill vasculature. Antagonistic control may provide a greater range of cardiac outputs than relying on the modulation of inhibitory tonus alone, and in addition may allow differential inotropic and chronotropic control. An indication of this is the transient, predominantly chronotropic inhibitory responses to neural stimulation as opposed to the persistent combined inotropic and chronotropic excitatory responses. It seems that in this preparation neurally released catecholamines are not rapidly deactivated or removed from their site of action. In elasmobranch fish vagal branches innervate the heart, but adrenergic contributions to the vagi, or direct cardiac nerves are apparently absent (Nilsson, 1983). Teleost fish therefore occupy an interesting phylogenetic position between the elasmobranchs and the higher vertebrates, all of which have dual antagonistic innervation with vagal inhibitory fibres and sympathetic excitation. The relative functional importance of neural mechanisms in fish remains uncertain, but further study of species with and without dual innervation should provide interesting information on how circulatory control is achieved in response to environmental and organismic factors. ABRAHAMSSON, T. & S. NILSSON, 1976. Phenylethanolamine-N-methyl transferase (PNMT) activity and catecholamine content in chromaffin tissue and sympathetic neurons in the cod, Gadus morhua. Acta Physiol. Scand., Vol. 96, pp. 94-99. CAMERON, J.S., 1979. Autonomic nervous tone and regulation of heart rate in the goldfish, Carassius attratus. Comp. Biochem. Physiol., Vol. 63C. pp. 341-349. COBB. J.L.S. & R.M. SANTEr. 1973. Electrophysiology of cardiac function in teleosts: cholinergically mediated inhibition and rebound excitation. J. PhrsioL (London), Vol. 2311, pp. 561-574.
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