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Sympathetic innervation of the spleen of the cane toad, bufo marinus
Comp. Biochem. Physiol. Vol. 61C, pp. 133-139
0306-4492/78/0901-0133502.00/0
© Pergamon Press Ltd 1978. Printed in Great Britain
SYMPATHETIC INNERVATION OF THE SPLEEN OF THE CANE TOAD, BUFO M A R I N U S STEFAN NILSSON* Department of Zoology, University of Melbourne, Parkville 3052, Victoria, Australia
(Received 7 December 1977) Abstract--1. The sympathetic nervous supply to the toad spleen was studied using perfused spleen preparations, isolated spleen strips and Faick-Hillarp fluorescent histochemistry. 2. The order of potency for adrenergic agonists indicates an alpha adrenoceptor mediated constriction of the spleen. 3. Acetylcholine gives variable responses on the perfused spleen, but contracts the isolated spleen strips. The effect of acetylcholine appears to be via muscarinic receptors, although no quantitative analysis could be made. 4. The constrictory innervation of the spleen appears to be solely adrenergic, and fluorescent histochemistry reveals fibres mainly around intrasplenic blood vessels and in the capsule. No evidence for a cholinergic innervation was obtained. 5. The sympathetic innervation of the spleen leaves the spinal chord in the ventral roots of the spinal nerves, and pre-synaptic fibres run to the coeliac ganglion. The vast majority of fibres to the spleen appear to have their cell bodies in the coeliac ganglion, since d-tubocurarine inhibits the effects of nerve stimulation proximal, but not distal, to this ganglion.
INTRODUCTION
MATERIALS AND METHODS
In mammals, the spleen receives post-ganglionic sympathetic constrictory fibres from the coeliac ganglion. These fibres are adrenergic, exerting their effect via alpha adrenoceptors of the arterial, trabecular and capsular smooth muscles of the spleen. Burn & Rand (1960) also concluded the presence of cholinergic spleno-dilatatory sympathetic fibres to the cat spleen. For references on the work on mammalian spleens, see Davies & Withrington (1973). Few accounts of the splenic innervation in lower vertebrates exist. The spleen of the spiny dogfish (Squalus acanthias) is constricted by adrenaline (Opdyke & Opdyke, 1971), and spleen strips from the tench (Tinca tinca) and a frog have been reported to contract by both adrenaline and noradrenaline (Vairel, 1933). Recent work on Squalus shows that the spleen of this species is controlled by solely adrenergic sympathetic fibres. Circulating catecholamines may also be important in constricting the spleen of Squalus, and even more so the spleen of the spotted dogfish, Scyliorhinus canicula (Nilsson, Holmgren & Grove, 1975). In the teleost, Gadus morhua, both cholinergic and adrenergic constrictory effects were recorded from sympathetic nerve stimulation (Nilsson & Grove, 1974), and the possibility that both types of transmitter are released from the same type of neurone has been suggested (Holmgren & Nilsson, 1976). This work forms part of a comparative study of the autonomic nervous control of the spleen in lower vertebrates, and is an attempt to obtain basic information on the mode of innervation of the toad spleen.
Cane toads, Bufo rrmrinus, of either sex with a body weight of 150-250 g were used in the experiments.
Experiments with the perfused toad spleen The animals were pithed and opened ventrally and 0.1~).2 ml heparin solution (5000 IU/ml) was injected into the left aortic arch (Fig. 1) This vessel-was then catheterized for inflow of perfusion fluid from a constant pressure head of 25-27 cm H20, using the perfusion apparatus of Davies & Rankin (1973) as described by Nilsson & Grove (1974). The perfusion fluid was a Mackenzie's saline of the following composition (in raM): NaCI 115, KCI 3.2,
am~~
"
~ dag
Fig. 1. Simplified diagram showing the vascular anatomy around the toad spleen: area--anterior mesenteric artery; cg---coeliac ganglion; da--dorsal aorta; hga--hepato-gas* Present address: Department of Zoophysiology, Uni- tric artery; iv--intestinal vein; ha--left aortic arch; raa-versity of G6teborg, Sweden. right aortic arch; sa--splenic artery; sv--splenic vein. 133
134
STEFAN NILSSON
NaHCO 3 20, NaH2PO4 3.1, MgSO4 1.4, CaCIz 1.3, glucose 16.7. The fluid was constantly bubbled with 95~ O2:5% C02. The venous effluent was collected by a catheter inserted in the intestinal vein, and the outflow rate determined by drop counting displayed as drops per minute by a GRASS 7P4 tachograph. All other vessels around the spleen were ligated to direct the flow of perfusion fluid through the spleen only (Fig. 1). The spleen with surrounding vascular and nervous supply was then carefully removed from the toad and pinned to a cork disc submersed in Mackenzie's solution at 25°C. The preparation was then left for at least 1 hr before the experiment was started. Addition of drugs was performed either by a syringe and catheter into the lower funnel of the perfusion apparatus (agonists), or by dissolving the drug in the entire bulk of perfusion fluid (antagonists) (Nilsson & Grove, 1974). Dose-response curves for single doses of agonists were obtained by plotting the flow decrease in per cent of initial flow rate against log dose. EDso values were estimated from these graphs as the dose producing 50~o flow reduction. In the case of acetylcholine, which rarely produced more than a 209/0 flow reduction, the dose producing 50~o of maximal effect was instead used as the EDso value. The affinities for the various agonists were then determined in pD2-units (-- - l o g ED~o) (van Rossum, 1963). pAx-values for the antagonists were estimated as described by Holmgren & Nilsson (1975). In experiments where stimulation of nerves to the spleen was performed, the right and left preganglionic branches of the splanchnic nerve leaving the chains at the level of~ the 4th spinal nerves were left attached to the coeliac ganglion (Fig. 2). Both nerve branches were placed over a pair of platinum hook electrodes, and the electrical stimulation was performed from a GRASS SD9 stimulator. In some experiments the nervous supply to the spleen was stimu-
5rain T
---I
lated distally to the coeliac ganglion by placing the entire splenic artery over the electrodes (Fig. 1). In an attempt to study the levels of outflow from the spinal chord of the spleno-constrictor fibres, dorsal and ventral roots of the lst-9th spinal nerves on the left side were electrically stimulated. In these experiments the animals were anaesthetized by immersion in MS 222 (tricaine methane sulphonate, Sandoz), ca. 0.1 mg/ml tap water until the corneal reflex had disappeared (ca. 15 rain). The spleen was prepared for perfusion as described above, and the spinal column with the sympathetic chains and the splanchnic outflow intact on the left side was dissected free and submersed together with the spleen in the Mackenzie's solution. The spinal roots were located, and placed over the electrodes for stimulation.
Experiments with isolated toad spleen strips The spleen was dissected out of pithed animals, and strips (ca. 10 mm long and 1-2 mm thick) were cut off the capsule and underlying tissue. The very minute size of the spleen allowed a maximum of two, rarely three, strips to be cut from each spleen. The strips were mounted in 50 ml of Mackenzie's solution in organ baths at 25°C and the tonus adjusted to ca. 50 mp (1 mp - vertical force of 1 rag). The preparations were then left for at least 1 hr before the start of the experiment. Cumulative concentration-response curves for the agonists were made and the affinities and intrinsic activities calculated and expressed as pD 2 - ( = - l o g ECso) and ~t-values respectively (van Rossum, 1963). A maximum of three consecutive concentration-response curves was made from each strip, and the strips were washed repeatedly and allowed to rest 1 hr between curves.
Fluorescent histochemistry Spleens were dissected out of pithed toads, quick-frozen in liquid propane cooled in liquid nitrogen, freeze-dried and prepared for fluorescent histochemistry as described by Falck & Owman (1965), using 3-hr treatment time in formaldehyde vapour at 80°C. Sections of 10#m were mounted in liquid paraffin and inspected in a Leitz Ortholux microscope equipped with a mercury burner, BG 12 excitation filter and a bai'rier filter at 510 nm. Photographs were taken with a Leitz Orthomat automatic camera on Kodak Tri-X film. In a few preparations noraflrenaline (5 mg/kg) was injected i.p. 1-2 hr before killing the toad.
Drugs The following drugs were used in this study: acetylcholine chloride, adenosine triphosphate di-sodium salt, L-adrenaline bitartrate, bretylium tosylate, 6-hydroxydopamine hydrobromide, l-hyoscine chloride, DL-isoprenaline hydrochloride, L-noradrenaline bitartrate, phentolamine methane sulphonate, L-phenylephrine hydrochloride, d-tubocurarine chloride, yohimbine hydrochloride. Amounts and concentrations of drugs are expressed in moles and moles/litre (M) respectively.
Statistics Fig. 2. Simplified diagram showing the sympathetic nervous supply to the coeliac ganglion in the toad: cg---coeliac ganglion; pgsn--pre-ganglionic splanchnic nerves. The spinal nerves are indicated by their Arabic numbers. The extension of the left sympathetic chain anterior to the 1st or posterior to the 7th spinal nerve is not shown. The splanchnic branches from the 3rd, 5th and or 6th ganglia are often missing. The tracings in the right part of the figure represent changes in flow rate through the spleen during stimulations of the ventral roots in the various segments with 20 Hz, 1 msec and 6 V for 10 sec. Horizontal marker = 5 rain; vertical marker = t h e range 20-60 drops/rain.
Values are given as means + S.E, Significance was tested by Student's t-test.
RESULTS
Experiments with the perfused toad spleen At the start of a perfusion, strong spontaneous rhythmical changes in perfusion rate occurred. This activity invariably ceased within the first 30 rain, leaving a quite steady flow of 0.4-1.0ml/min depending on spleen size.
I~5
Sympathetic innervation of the toad spleen
Table 2. pA:values for phentolamine and yohimbine estimated with noradrenaline as the agonist on the perfused toad spleen
,50mpI
Antagonist Phentolamine Yohimbine
pA2
pAto
n
7.9 + 0.3 6.6 + 0.1
6.8 + 0.3 5.5 + 0.1
4 4
Number of preparations = n. 30.
DF'M
~
•
~
t
io. 5 rain Fig. 3. Responses of the toad spleen to adrenaline. Upper tracing: isolated strip preparation, addition of adrenaline to give concentrations of 10 -7 , 3 x 10 -7, 10 -6 , 3 x 10 -6 M etc. Lower tracing: perfused spleen preparation, addition of a single dose of adrenaline (10-lo moles). The change in flow rate indicated in drops per minute (DPM). Time marker in both tracings 5 min. Addition of appropriate agonists to the inflowing perfusion fluid, or electrical stimulation of the nervous supply constricted the spleen visibly, and produced a decrease in the outflow rate. N o release of erythrocytes, as demonstrated in elasmobranchs and teleosts under similar experimental conditions (Nilsson & Grove, 1974; Nilsson et al., 1975), could be detected. Adrenergic agonists constricted the spleen (Fig. 3) with the order of potency adrenaline > noradrenaline/> phenylephrine > isoprenaline, where the sign > denotes significant (P < 0.05) difference between pD2-values (Table 1). The constrictory effect of noradrenaline could be competitively antagonized by phentolamine and yohimbine (Table 2), indicated by a parallel shift to the right of the nor2tdrenaline doseresponse curve in the presence of increasing concentrations of antagonist in the perfusion fluid. In a few cases a slight spleno-dilatation was evoked by isoprenaline (10-9-10 -8 mole).
Acetylcholine produced very inconsistent effects on the perfused spleen. The most frequent effect was a constriction (pD2 = 9.2 + 0.3, h = 8) which rarely gave a maximal flow reduction exceeding 209/0 of the initial flow rate. In a number of cases an increased flow resulted from the administration of acetylcholine, but this response could change to a spleno-constriction within the same preparation later during the experiment. Subsequent dose-response curves for acetylcholine showed marked desensitization to this agonist, and quantification of the effects of cholinergic antagonists was therefore not possible. Addition of hyoscine (10 -7 M) to the perfusion fluid, however, abolished all further responses to acetylcholine (up to 10- 5 moles), but did not detectably affect the response to noradrenaline. Addition of adenosine tri-phosphate (ATP) produced rapid constriction of the spleen (pD2 = 8.36 + 0.05, n = 4), and these responses were unaffected by yohimbine (10 -5 M). Spleno-constriction evoked by electrical stimulation of the sympathetic nervous supply to the spleen could be strongly diminished or abolished by yohimbine 3 x 10 -6 M, and was invariably abolished by yohimbine 10- 5 M. This concentration does not affect the response to ATP, and its effect on nerve stimulation is therefore not due to any spasmolytic side effects of the alpha adrenoceptor antagonist. The response to nerve stimulation was also abolished by bretylium (10 -6 M), in a concentration which did not affect the responses to noradrenaline (Fig. 4). 6-Hydroxy dopamine also abolished the nerve response in about 1 hr. Hyoscine, up to 10 -s M, did not detectably affect the nerve response. Pre-ganglionic stimulation was abolished by
Table 1. pD 2- and s-values for adrenergic agonists estimated on the perfused toad spleen (upper part of table) or isolated toad spleen strips (lower part of table) Agonist Adrenaline Noradrenaline Phenylephrine Isoprenaline
pD2 (= - l o g EDso) 9.65 8.80 8.60 5.72
___0.30 + 0.22 + 0.32 + 0.10
ct
m
n
Effect
5 5 4 4
Constriction Constriction Constriction Constriction
10 28 8 10
Contraction Contraction Contraction Contraction
pD2 (= - l o g ECso) Adrenaline Noradrenaline Phenylephrine Isoprenaline
6.26 5.61 5.38 4.34
+ + + +
0.08 0.04 0.04 0.08
1.05 _ 0.08 1.00 (ref.) 0.94 + 0.04 0.88 + 0.07
Note that the pD2-values refer to amounts of agonists (moles) in the perfused spleen, and to concentration of agonist (moles/litre) in the spleen strip experiments. Number of preparations = n.
136
STEFANNILSSON
spleen, and fluorescent fibres were also seen in the capsule (Fig. 5a, b). No evidence for fluorescent fibres along any trabeculae was obtained, but presence of autofluorescence could perhaps obscure finer fibres. No specifically fluorescent or autofluorescent ganglion cells could be seen in the spleen. The intensity of the fluorescent fibres was increased after pretreatment with noradrenaline, but no new structures were found.
100
o 0~ -ID
50
o m
o~
o
t'
2
,
DISCUSSION
Contrary to the situation in some other poikilothermic vertebrates (elasmobranchs and teleosts; Nilsson & Grove, 1974; Nilsson et al., 1975), the toad Fig. 4. Effect of d-tubocurarine (dTC) 3 x 10-s M and spleen does not appear to serve as an important reserbretylium (Bre) 10 -6 M on the constrictory response of the toad spleen evoked by electrical stimulation with voir for red blood cells. It is possible, however, that 20 Hz, 1 msec and 10~" for 10 sec every 8 min alternating already the strong rhythmic activity at the start of between stimulation proximal (O) and distal (0) to the coe- each perfusion expels all visible traces of erythrocytes liac ganglion. The diagram represents one experiment out from the spleen. of four similar. The results obtained with the adrenergic agonists, both on the perfused spleen preparation and the isolated strips, favour the view that these drugs act via an alpha adrenoceptor mechanism in the toad as in d-tubocurarine (3 x 10-5 M), while alternating post- other vertebrates (Bickerton, 1963; Takano, 1969; ganglionic stimulation of the same preparation was Nilsson & Grove, 1974; Holmgren & Nilsson, 1975; almost unaffected (Fig. 4). Nilsson et al., 1975). This is also supported by the Stimulation of the lst-9th ventral and dorsal spinal competitive nature of the blockade obtained with the roots revealed an outflow of sympathetic pathways alpha adrenoceptor antagonists phentolamine and to the spleen in the 2nd, 3rd, 4th, 5th and 6th ventral yohimbine. Their pA-values also compare well with roots (Fig. 2). No effect on the spleen was recorded values obtained with spleens from other animals while stimulating the dorsal roots of any segment. (Sheys & Green, 1972; Nilsson & Grove, 1974; Stimulations and cuttings of the left sympathetic Holmgren & Nilsson, 1975; Nilsson et al., 1975). The chain indicate that the fibres to the spleen may run possibility of a beta adrenoceptor mechanism producforwards or backwards in the chain from their seg- ing spleno-dilatation is suggested by the increase in ment of exit from the CNS, and join the splanchnic flow rate occasionally seen after low doses of nerves in other segments. The majority of fibres leave isoprenaline, but this response was not consistent the chain via the splanchnic nerve from the 4th sym- enough to allow further analysis. Acetylcholine gives very conflicting results, and pathetic ganglion. When the pre-ganglionic splanchnic nerves from the 3rd, 5th and 6th spinal nerves/ such variability in the splenic responses to this drug sympathetic ganglia are present, spleno-constrictor has also been encountered during experiments with mammals (see Davies & Withrington, 1973) and elasfibres also run in these. mobranchs (Nilsson et al., 1975) but not in a teleost (Gadus morhua) where acetylcholine acts consistently Experiments with isolated toad spleen strips via muscarinic receptors, producing constriction of The isolated strips showed no spontaneous activity. the spleen (Nilsson & Grove, 1974). The results presCumulative concentration-response curves (Fig. 3) for ented here may be taken to indicate an action mainly the adrenergic agonists revealed the following order via muscarinic receptors of the toad spleen, but the of potency in causing contraction: adrenaline > nor- interpretation is made unreliable by the lack of quantitative data, and the rapidly developing desensitizaadrenaline > phenylephrine > isoprenaline, where > indicates significant (P < 0.05)difference between tion to acetylcholine. The spleno-constrictor effect of pD2-values (Table 1). or-Values (Table 1) were esti- acetylcholine may, at least in part, be due to action mated with the test agonist before noradrenaline in on the splenic capsule, since this contracts in response half the number of preparations, and after noradrena- to acetylcholine in vitro. Mall (1902) suggested that line in the other half. The maximal tension developed acetylcholine could widen venous passages in the by one strip was, however, very constant from one mammalian spleen by contracting radial muscles, thus curve to the next when the same agonist was used producing an increased blood flow through the spleen. A similar explanation could perhaps be consecutively. Acetylcholine invariably contracted the strips applied to explain the spleno-dilatation sometimes (pD2 = 5.21 + 0.09, ~t = 0.24 __+0.03 with noradrena- observed with acetylcholine in the toad spleen. A line as reference, n = 10). Consecutive acetylcholine detailed study of the patterns of blood flow through this minute organ is obviously necessary. curves showed a marked desensitization. The responses to nerve stimulation of the perfused spleen can be explained by a solely adrenergic supply Fluorescent histochemistry from the coeliac ganglion. The pattern of innervation A dense innervation of intra-splenic blood vessels of the toad spleen thus resembles, in its main features, by fluorescent fibres could be seen in all parts of the that found in mammals. The possibility of cholinergic dTC
Bre
hr
Fig. 5. Fluorescent histochemistry (Falck-Hillarp) of the toad spleen, a--Varicose fluorescent fibres in the splenic capsule. Calibration = 100/~m. b---Dense innervation by fluorescent fibres of intrasplenic blood vessels. Calibration = 50#m.
Q
Sympathetic innervation of the toad spleen dilatatory fibres to mammalian spleens has been suggested (Burn & Rand, 1960; Daly & Scott, 1961), but the present experiment provided no evidence for the presence of such fibres in the toad spleen. After abolishment of the adrenergic nervous influence no measurable response to nerve stimulation remained. In the cod, Gadus morhua, both cholinergic and adrenergic effects of nerve stimulation on the spleen can be demonstrated by pharmacological methods (Nilsson & Grove, 1974), and it has been suggested tfiat both types of transmitters are released from the same neurone acting independently on appropriate receptors of the splenic smooth muscles (Holmgren & Nilsson, 1976). The sympathetic nervous supply to the toad spleen has the vast majority of its post-ganglionic cell bodies in the coeliac ganglion, since addition of the ganglionic blocker d-tubocurarine inhibits responses to pre-ganglionic stimulation in a concentration which does not have any great effect on the response to post-ganglionic stimulation. The outflow from the spinal chord of pre-ganglionic fibres is restricted to the ventral roots of the spinal nerves. Fibres may run forwards and backwards in the sympathetic chain before leaving in one of the pre-ganglionic splanchnic branches. The adrenergic nervous supply to the spleen is likely to exert its action mainly on two sites: intrasplenic blood vessels and the splenic capsule, both of which have a rich supply of fluorescent fibres. Acknowledgement--I wish to thank Professor G. D. Campbell for inviting me to his department in Melbourne where this work was carried out. REFERENCES
BICKERTON R. K. (1963) The response of isolated strips of cat spleen to sympathomimetic drugs and their antagonists. J. Pharmac. exp. Ther. 142, 99-110. BURN J. H. & RAND M. J. (1960) Sympathetic postganglionic cholinergic fibres. Br. J. Pharmac. 15, 56-66.
139
DALY M. DE BURGH & SCOTT M. J. (1961) The effects of acetyclcholine on the volume and vascular resistance of the dog's spleen. J. Physiol., Lond. 156, 246-259. DAVIES D. T. & R.~NKINJ. C. (1973) Adrenergic receptors and vascular responses to catecholamines of perfused dogfish gills. Comp. gen. Pharmac. 4, 139-147. DAVmS B. N. & WITHPdNGTON P. G. (1973) The actions of drugs on the smooth muscle of the capsule and blood vessels of the spleen. Pharmac. Rev. 25, 373-413. FALCK B. & OWMANCH. (1965) A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic amines. Acta Univ. Lurid. Sect. II, No. 7. HOLMGRENS. & NILSSONS. (1975) Effects of some adrenergic and cholinergic drugs on isolated spleen strips from the cod, Gadus morhua. Eur. J. Pharmac. 32, 163-169. HOLMGREN S. & NILSSONS. (1976) Effects of denervation, 6-hydroxydopamine and reserpine on the cholinergic and adrenergic responses of the spleen of the cod, Gadus morhua. Eur. J. Pharmac. 39, 53-59. MALL F. P. (1902) On the circulation through the pulp of the dog's spleen. Am. J. Anat. 2, 315-332. NILSSONS. & GROVE D. J. (1974) Adrenergic and cholinergic innervation of the spleen of the cod, Gadus morhua. Eur. J. Pharmac. 28, 135-143. NILSSON S., HOLMGREN S. ~ GROVE D. J. (1975) Effects of drugs and nerve stimulation on the spleen and arteries of two species of dogfish, Scyliorhinus canicula and Squalus acanthias. Acta physiol, scand. 95, 219-230. OPOYKE D. F. & OPDYKI~ N. E. (1971) Splenic responses to stimulation in Squalus acanthias. Am. J. Physiol. 221, 623-625. ROSStJMJ. M. VAN (1963) Cumulative dose-response curves I1. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch. int. Pharmacodyn. 143, 299-330. StaYS E. M. & GREEN R. D. (1972) A quantitative study of alpha adrenergic receptors in the spleen and aorta of the rabbit, d. Pharmac. exp. Ther. 180, 317-325. TAKANO S. (1969) A study of the contraction of splenic strips in kids and dogs with special reference to the cholinergic and adrenergic receptors. Jap. J. Pharmac. 19, 563-568. VAmELJ. (1933) Action de l'adr6naline et de rac6tylcholine sur la rate. J. Physiol. Path. gdn. 31, 42-52.
0306-4492/78/0901-0133502.00/0
© Pergamon Press Ltd 1978. Printed in Great Britain
SYMPATHETIC INNERVATION OF THE SPLEEN OF THE CANE TOAD, BUFO M A R I N U S STEFAN NILSSON* Department of Zoology, University of Melbourne, Parkville 3052, Victoria, Australia
(Received 7 December 1977) Abstract--1. The sympathetic nervous supply to the toad spleen was studied using perfused spleen preparations, isolated spleen strips and Faick-Hillarp fluorescent histochemistry. 2. The order of potency for adrenergic agonists indicates an alpha adrenoceptor mediated constriction of the spleen. 3. Acetylcholine gives variable responses on the perfused spleen, but contracts the isolated spleen strips. The effect of acetylcholine appears to be via muscarinic receptors, although no quantitative analysis could be made. 4. The constrictory innervation of the spleen appears to be solely adrenergic, and fluorescent histochemistry reveals fibres mainly around intrasplenic blood vessels and in the capsule. No evidence for a cholinergic innervation was obtained. 5. The sympathetic innervation of the spleen leaves the spinal chord in the ventral roots of the spinal nerves, and pre-synaptic fibres run to the coeliac ganglion. The vast majority of fibres to the spleen appear to have their cell bodies in the coeliac ganglion, since d-tubocurarine inhibits the effects of nerve stimulation proximal, but not distal, to this ganglion.
INTRODUCTION
MATERIALS AND METHODS
In mammals, the spleen receives post-ganglionic sympathetic constrictory fibres from the coeliac ganglion. These fibres are adrenergic, exerting their effect via alpha adrenoceptors of the arterial, trabecular and capsular smooth muscles of the spleen. Burn & Rand (1960) also concluded the presence of cholinergic spleno-dilatatory sympathetic fibres to the cat spleen. For references on the work on mammalian spleens, see Davies & Withrington (1973). Few accounts of the splenic innervation in lower vertebrates exist. The spleen of the spiny dogfish (Squalus acanthias) is constricted by adrenaline (Opdyke & Opdyke, 1971), and spleen strips from the tench (Tinca tinca) and a frog have been reported to contract by both adrenaline and noradrenaline (Vairel, 1933). Recent work on Squalus shows that the spleen of this species is controlled by solely adrenergic sympathetic fibres. Circulating catecholamines may also be important in constricting the spleen of Squalus, and even more so the spleen of the spotted dogfish, Scyliorhinus canicula (Nilsson, Holmgren & Grove, 1975). In the teleost, Gadus morhua, both cholinergic and adrenergic constrictory effects were recorded from sympathetic nerve stimulation (Nilsson & Grove, 1974), and the possibility that both types of transmitter are released from the same type of neurone has been suggested (Holmgren & Nilsson, 1976). This work forms part of a comparative study of the autonomic nervous control of the spleen in lower vertebrates, and is an attempt to obtain basic information on the mode of innervation of the toad spleen.
Cane toads, Bufo rrmrinus, of either sex with a body weight of 150-250 g were used in the experiments.
Experiments with the perfused toad spleen The animals were pithed and opened ventrally and 0.1~).2 ml heparin solution (5000 IU/ml) was injected into the left aortic arch (Fig. 1) This vessel-was then catheterized for inflow of perfusion fluid from a constant pressure head of 25-27 cm H20, using the perfusion apparatus of Davies & Rankin (1973) as described by Nilsson & Grove (1974). The perfusion fluid was a Mackenzie's saline of the following composition (in raM): NaCI 115, KCI 3.2,
am~~
"
~ dag
Fig. 1. Simplified diagram showing the vascular anatomy around the toad spleen: area--anterior mesenteric artery; cg---coeliac ganglion; da--dorsal aorta; hga--hepato-gas* Present address: Department of Zoophysiology, Uni- tric artery; iv--intestinal vein; ha--left aortic arch; raa-versity of G6teborg, Sweden. right aortic arch; sa--splenic artery; sv--splenic vein. 133
134
STEFAN NILSSON
NaHCO 3 20, NaH2PO4 3.1, MgSO4 1.4, CaCIz 1.3, glucose 16.7. The fluid was constantly bubbled with 95~ O2:5% C02. The venous effluent was collected by a catheter inserted in the intestinal vein, and the outflow rate determined by drop counting displayed as drops per minute by a GRASS 7P4 tachograph. All other vessels around the spleen were ligated to direct the flow of perfusion fluid through the spleen only (Fig. 1). The spleen with surrounding vascular and nervous supply was then carefully removed from the toad and pinned to a cork disc submersed in Mackenzie's solution at 25°C. The preparation was then left for at least 1 hr before the experiment was started. Addition of drugs was performed either by a syringe and catheter into the lower funnel of the perfusion apparatus (agonists), or by dissolving the drug in the entire bulk of perfusion fluid (antagonists) (Nilsson & Grove, 1974). Dose-response curves for single doses of agonists were obtained by plotting the flow decrease in per cent of initial flow rate against log dose. EDso values were estimated from these graphs as the dose producing 50~o flow reduction. In the case of acetylcholine, which rarely produced more than a 209/0 flow reduction, the dose producing 50~o of maximal effect was instead used as the EDso value. The affinities for the various agonists were then determined in pD2-units (-- - l o g ED~o) (van Rossum, 1963). pAx-values for the antagonists were estimated as described by Holmgren & Nilsson (1975). In experiments where stimulation of nerves to the spleen was performed, the right and left preganglionic branches of the splanchnic nerve leaving the chains at the level of~ the 4th spinal nerves were left attached to the coeliac ganglion (Fig. 2). Both nerve branches were placed over a pair of platinum hook electrodes, and the electrical stimulation was performed from a GRASS SD9 stimulator. In some experiments the nervous supply to the spleen was stimu-
5rain T
---I
lated distally to the coeliac ganglion by placing the entire splenic artery over the electrodes (Fig. 1). In an attempt to study the levels of outflow from the spinal chord of the spleno-constrictor fibres, dorsal and ventral roots of the lst-9th spinal nerves on the left side were electrically stimulated. In these experiments the animals were anaesthetized by immersion in MS 222 (tricaine methane sulphonate, Sandoz), ca. 0.1 mg/ml tap water until the corneal reflex had disappeared (ca. 15 rain). The spleen was prepared for perfusion as described above, and the spinal column with the sympathetic chains and the splanchnic outflow intact on the left side was dissected free and submersed together with the spleen in the Mackenzie's solution. The spinal roots were located, and placed over the electrodes for stimulation.
Experiments with isolated toad spleen strips The spleen was dissected out of pithed animals, and strips (ca. 10 mm long and 1-2 mm thick) were cut off the capsule and underlying tissue. The very minute size of the spleen allowed a maximum of two, rarely three, strips to be cut from each spleen. The strips were mounted in 50 ml of Mackenzie's solution in organ baths at 25°C and the tonus adjusted to ca. 50 mp (1 mp - vertical force of 1 rag). The preparations were then left for at least 1 hr before the start of the experiment. Cumulative concentration-response curves for the agonists were made and the affinities and intrinsic activities calculated and expressed as pD 2 - ( = - l o g ECso) and ~t-values respectively (van Rossum, 1963). A maximum of three consecutive concentration-response curves was made from each strip, and the strips were washed repeatedly and allowed to rest 1 hr between curves.
Fluorescent histochemistry Spleens were dissected out of pithed toads, quick-frozen in liquid propane cooled in liquid nitrogen, freeze-dried and prepared for fluorescent histochemistry as described by Falck & Owman (1965), using 3-hr treatment time in formaldehyde vapour at 80°C. Sections of 10#m were mounted in liquid paraffin and inspected in a Leitz Ortholux microscope equipped with a mercury burner, BG 12 excitation filter and a bai'rier filter at 510 nm. Photographs were taken with a Leitz Orthomat automatic camera on Kodak Tri-X film. In a few preparations noraflrenaline (5 mg/kg) was injected i.p. 1-2 hr before killing the toad.
Drugs The following drugs were used in this study: acetylcholine chloride, adenosine triphosphate di-sodium salt, L-adrenaline bitartrate, bretylium tosylate, 6-hydroxydopamine hydrobromide, l-hyoscine chloride, DL-isoprenaline hydrochloride, L-noradrenaline bitartrate, phentolamine methane sulphonate, L-phenylephrine hydrochloride, d-tubocurarine chloride, yohimbine hydrochloride. Amounts and concentrations of drugs are expressed in moles and moles/litre (M) respectively.
Statistics Fig. 2. Simplified diagram showing the sympathetic nervous supply to the coeliac ganglion in the toad: cg---coeliac ganglion; pgsn--pre-ganglionic splanchnic nerves. The spinal nerves are indicated by their Arabic numbers. The extension of the left sympathetic chain anterior to the 1st or posterior to the 7th spinal nerve is not shown. The splanchnic branches from the 3rd, 5th and or 6th ganglia are often missing. The tracings in the right part of the figure represent changes in flow rate through the spleen during stimulations of the ventral roots in the various segments with 20 Hz, 1 msec and 6 V for 10 sec. Horizontal marker = 5 rain; vertical marker = t h e range 20-60 drops/rain.
Values are given as means + S.E, Significance was tested by Student's t-test.
RESULTS
Experiments with the perfused toad spleen At the start of a perfusion, strong spontaneous rhythmical changes in perfusion rate occurred. This activity invariably ceased within the first 30 rain, leaving a quite steady flow of 0.4-1.0ml/min depending on spleen size.
I~5
Sympathetic innervation of the toad spleen
Table 2. pA:values for phentolamine and yohimbine estimated with noradrenaline as the agonist on the perfused toad spleen
,50mpI
Antagonist Phentolamine Yohimbine
pA2
pAto
n
7.9 + 0.3 6.6 + 0.1
6.8 + 0.3 5.5 + 0.1
4 4
Number of preparations = n. 30.
DF'M
~
•
~
t
io. 5 rain Fig. 3. Responses of the toad spleen to adrenaline. Upper tracing: isolated strip preparation, addition of adrenaline to give concentrations of 10 -7 , 3 x 10 -7, 10 -6 , 3 x 10 -6 M etc. Lower tracing: perfused spleen preparation, addition of a single dose of adrenaline (10-lo moles). The change in flow rate indicated in drops per minute (DPM). Time marker in both tracings 5 min. Addition of appropriate agonists to the inflowing perfusion fluid, or electrical stimulation of the nervous supply constricted the spleen visibly, and produced a decrease in the outflow rate. N o release of erythrocytes, as demonstrated in elasmobranchs and teleosts under similar experimental conditions (Nilsson & Grove, 1974; Nilsson et al., 1975), could be detected. Adrenergic agonists constricted the spleen (Fig. 3) with the order of potency adrenaline > noradrenaline/> phenylephrine > isoprenaline, where the sign > denotes significant (P < 0.05) difference between pD2-values (Table 1). The constrictory effect of noradrenaline could be competitively antagonized by phentolamine and yohimbine (Table 2), indicated by a parallel shift to the right of the nor2tdrenaline doseresponse curve in the presence of increasing concentrations of antagonist in the perfusion fluid. In a few cases a slight spleno-dilatation was evoked by isoprenaline (10-9-10 -8 mole).
Acetylcholine produced very inconsistent effects on the perfused spleen. The most frequent effect was a constriction (pD2 = 9.2 + 0.3, h = 8) which rarely gave a maximal flow reduction exceeding 209/0 of the initial flow rate. In a number of cases an increased flow resulted from the administration of acetylcholine, but this response could change to a spleno-constriction within the same preparation later during the experiment. Subsequent dose-response curves for acetylcholine showed marked desensitization to this agonist, and quantification of the effects of cholinergic antagonists was therefore not possible. Addition of hyoscine (10 -7 M) to the perfusion fluid, however, abolished all further responses to acetylcholine (up to 10- 5 moles), but did not detectably affect the response to noradrenaline. Addition of adenosine tri-phosphate (ATP) produced rapid constriction of the spleen (pD2 = 8.36 + 0.05, n = 4), and these responses were unaffected by yohimbine (10 -5 M). Spleno-constriction evoked by electrical stimulation of the sympathetic nervous supply to the spleen could be strongly diminished or abolished by yohimbine 3 x 10 -6 M, and was invariably abolished by yohimbine 10- 5 M. This concentration does not affect the response to ATP, and its effect on nerve stimulation is therefore not due to any spasmolytic side effects of the alpha adrenoceptor antagonist. The response to nerve stimulation was also abolished by bretylium (10 -6 M), in a concentration which did not affect the responses to noradrenaline (Fig. 4). 6-Hydroxy dopamine also abolished the nerve response in about 1 hr. Hyoscine, up to 10 -s M, did not detectably affect the nerve response. Pre-ganglionic stimulation was abolished by
Table 1. pD 2- and s-values for adrenergic agonists estimated on the perfused toad spleen (upper part of table) or isolated toad spleen strips (lower part of table) Agonist Adrenaline Noradrenaline Phenylephrine Isoprenaline
pD2 (= - l o g EDso) 9.65 8.80 8.60 5.72
___0.30 + 0.22 + 0.32 + 0.10
ct
m
n
Effect
5 5 4 4
Constriction Constriction Constriction Constriction
10 28 8 10
Contraction Contraction Contraction Contraction
pD2 (= - l o g ECso) Adrenaline Noradrenaline Phenylephrine Isoprenaline
6.26 5.61 5.38 4.34
+ + + +
0.08 0.04 0.04 0.08
1.05 _ 0.08 1.00 (ref.) 0.94 + 0.04 0.88 + 0.07
Note that the pD2-values refer to amounts of agonists (moles) in the perfused spleen, and to concentration of agonist (moles/litre) in the spleen strip experiments. Number of preparations = n.
136
STEFANNILSSON
spleen, and fluorescent fibres were also seen in the capsule (Fig. 5a, b). No evidence for fluorescent fibres along any trabeculae was obtained, but presence of autofluorescence could perhaps obscure finer fibres. No specifically fluorescent or autofluorescent ganglion cells could be seen in the spleen. The intensity of the fluorescent fibres was increased after pretreatment with noradrenaline, but no new structures were found.
100
o 0~ -ID
50
o m
o~
o
t'
2
,
DISCUSSION
Contrary to the situation in some other poikilothermic vertebrates (elasmobranchs and teleosts; Nilsson & Grove, 1974; Nilsson et al., 1975), the toad Fig. 4. Effect of d-tubocurarine (dTC) 3 x 10-s M and spleen does not appear to serve as an important reserbretylium (Bre) 10 -6 M on the constrictory response of the toad spleen evoked by electrical stimulation with voir for red blood cells. It is possible, however, that 20 Hz, 1 msec and 10~" for 10 sec every 8 min alternating already the strong rhythmic activity at the start of between stimulation proximal (O) and distal (0) to the coe- each perfusion expels all visible traces of erythrocytes liac ganglion. The diagram represents one experiment out from the spleen. of four similar. The results obtained with the adrenergic agonists, both on the perfused spleen preparation and the isolated strips, favour the view that these drugs act via an alpha adrenoceptor mechanism in the toad as in d-tubocurarine (3 x 10-5 M), while alternating post- other vertebrates (Bickerton, 1963; Takano, 1969; ganglionic stimulation of the same preparation was Nilsson & Grove, 1974; Holmgren & Nilsson, 1975; almost unaffected (Fig. 4). Nilsson et al., 1975). This is also supported by the Stimulation of the lst-9th ventral and dorsal spinal competitive nature of the blockade obtained with the roots revealed an outflow of sympathetic pathways alpha adrenoceptor antagonists phentolamine and to the spleen in the 2nd, 3rd, 4th, 5th and 6th ventral yohimbine. Their pA-values also compare well with roots (Fig. 2). No effect on the spleen was recorded values obtained with spleens from other animals while stimulating the dorsal roots of any segment. (Sheys & Green, 1972; Nilsson & Grove, 1974; Stimulations and cuttings of the left sympathetic Holmgren & Nilsson, 1975; Nilsson et al., 1975). The chain indicate that the fibres to the spleen may run possibility of a beta adrenoceptor mechanism producforwards or backwards in the chain from their seg- ing spleno-dilatation is suggested by the increase in ment of exit from the CNS, and join the splanchnic flow rate occasionally seen after low doses of nerves in other segments. The majority of fibres leave isoprenaline, but this response was not consistent the chain via the splanchnic nerve from the 4th sym- enough to allow further analysis. Acetylcholine gives very conflicting results, and pathetic ganglion. When the pre-ganglionic splanchnic nerves from the 3rd, 5th and 6th spinal nerves/ such variability in the splenic responses to this drug sympathetic ganglia are present, spleno-constrictor has also been encountered during experiments with mammals (see Davies & Withrington, 1973) and elasfibres also run in these. mobranchs (Nilsson et al., 1975) but not in a teleost (Gadus morhua) where acetylcholine acts consistently Experiments with isolated toad spleen strips via muscarinic receptors, producing constriction of The isolated strips showed no spontaneous activity. the spleen (Nilsson & Grove, 1974). The results presCumulative concentration-response curves (Fig. 3) for ented here may be taken to indicate an action mainly the adrenergic agonists revealed the following order via muscarinic receptors of the toad spleen, but the of potency in causing contraction: adrenaline > nor- interpretation is made unreliable by the lack of quantitative data, and the rapidly developing desensitizaadrenaline > phenylephrine > isoprenaline, where > indicates significant (P < 0.05)difference between tion to acetylcholine. The spleno-constrictor effect of pD2-values (Table 1). or-Values (Table 1) were esti- acetylcholine may, at least in part, be due to action mated with the test agonist before noradrenaline in on the splenic capsule, since this contracts in response half the number of preparations, and after noradrena- to acetylcholine in vitro. Mall (1902) suggested that line in the other half. The maximal tension developed acetylcholine could widen venous passages in the by one strip was, however, very constant from one mammalian spleen by contracting radial muscles, thus curve to the next when the same agonist was used producing an increased blood flow through the spleen. A similar explanation could perhaps be consecutively. Acetylcholine invariably contracted the strips applied to explain the spleno-dilatation sometimes (pD2 = 5.21 + 0.09, ~t = 0.24 __+0.03 with noradrena- observed with acetylcholine in the toad spleen. A line as reference, n = 10). Consecutive acetylcholine detailed study of the patterns of blood flow through this minute organ is obviously necessary. curves showed a marked desensitization. The responses to nerve stimulation of the perfused spleen can be explained by a solely adrenergic supply Fluorescent histochemistry from the coeliac ganglion. The pattern of innervation A dense innervation of intra-splenic blood vessels of the toad spleen thus resembles, in its main features, by fluorescent fibres could be seen in all parts of the that found in mammals. The possibility of cholinergic dTC
Bre
hr
Fig. 5. Fluorescent histochemistry (Falck-Hillarp) of the toad spleen, a--Varicose fluorescent fibres in the splenic capsule. Calibration = 100/~m. b---Dense innervation by fluorescent fibres of intrasplenic blood vessels. Calibration = 50#m.
Q
Sympathetic innervation of the toad spleen dilatatory fibres to mammalian spleens has been suggested (Burn & Rand, 1960; Daly & Scott, 1961), but the present experiment provided no evidence for the presence of such fibres in the toad spleen. After abolishment of the adrenergic nervous influence no measurable response to nerve stimulation remained. In the cod, Gadus morhua, both cholinergic and adrenergic effects of nerve stimulation on the spleen can be demonstrated by pharmacological methods (Nilsson & Grove, 1974), and it has been suggested tfiat both types of transmitters are released from the same neurone acting independently on appropriate receptors of the splenic smooth muscles (Holmgren & Nilsson, 1976). The sympathetic nervous supply to the toad spleen has the vast majority of its post-ganglionic cell bodies in the coeliac ganglion, since addition of the ganglionic blocker d-tubocurarine inhibits responses to pre-ganglionic stimulation in a concentration which does not have any great effect on the response to post-ganglionic stimulation. The outflow from the spinal chord of pre-ganglionic fibres is restricted to the ventral roots of the spinal nerves. Fibres may run forwards and backwards in the sympathetic chain before leaving in one of the pre-ganglionic splanchnic branches. The adrenergic nervous supply to the spleen is likely to exert its action mainly on two sites: intrasplenic blood vessels and the splenic capsule, both of which have a rich supply of fluorescent fibres. Acknowledgement--I wish to thank Professor G. D. Campbell for inviting me to his department in Melbourne where this work was carried out. REFERENCES
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