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Cardiovascular reflexes in conscious toads
Journal of the Autonomic Nervous System, 5 (1982) 345-355
345
Elsevier Biomedical Press
Cardiovascular reflexes in conscious toads Anette Hoffmann and Maria Bernardete Cordeiro de Souza Department of Physiology, School of Medicine of Ribeirgto Preto, USP, 14100 Ribeir~o Preto, SP, (Brazil)
(Received February 24th, 1981) (Accepted October 26th, 1981)
K e y words: toad--pulmocutaneous arterial trunk--cardiovascular reflexes--auto-
nomic effectors
Abstract Methods used for implanting sensors and catheters in temporarily ether-anesthetized toads ( B u f o paracnemis) are described. Following recovery it was found that distension of the pulmocutaneous arterial trunk and high frequency electrical stimulation of the laryngeal nerve of conscious toads induce an abrupt fall in arterial pressure accompanied or not by bradycardia or cardiac arrest. A brief suppression of throat movements may occur but this is not a constant finding. The response is blocked by atropine or methyl-homatropine and persists in animals with high spinal sectioning, thus indicating its cholinergic parasympathetic nature. However a certain amount of sympathetic inhibition is not ruled out. Perfusion of the artery with lobeline and electrical stimulation of the laryngeal nerve at low frequency (l/s) induces a rise in arterial pressure which is blocked by phentolamine. The hypertension is followed by enhancing of both throat oscillations and electromyographic discharges. The occurrence of chemoreceptors in the pulmocutaneous arterial wall in these animals is discussed. Blockage of the laryngeal nerve with lidocaine or perfusion of the pulmocutaneous arterial trunk with the same solution elicited a blood pressure rise, tachycardia and enhanced ventilatory movements. This was attributed to suppression of the baroreceptor tonus.
Introduction
lshii and Ishii [3] demonstrated cardiovascular reflexes in anesthetized toads caused by distension of the pulmucutaneous artery, and also recorded impulses in 0165-1838/82/0000-0000/$02.75 © 1982 ElsevierBiomedicalPress
346
the peripheral cut end of the laryngeal nerve (the vagus branch which innervates the mentioned artery) which were synchronous with the cardiac systole. Nikiforowsky [7] and Kuno [4] had previously reported the occurrence of a fall in arterial pressure after stimulation of the vagus, and Neil et al. [5] had observed activity in the laryngeal nerve which was synchronous with the increase in arterial pressure induced in the trunk of the pulmocutaneous artery. All these studies, however, were carried out on anesthetized animals, a condition which may have altered the patterns of the reflexes. In the present investigation, we studied cardiovascular reflexes in conscious unrestrained toads. These reflexes were elicited by mechanical distension of the isolated pulmocutaneous trunk, by perfusion of this artery with lobeline, and by electrical stimulation of the central cut end of the laryngeal nerve. What led us to explore this reflexogenous area was our inability to obtain cardiovascular reflexes by distension of the carotid labyrinth in conscious toads. It is well known that this organ is considered to be homologous with the carotid sinus in mammals. We were also interested in determining the autonomic mechanisms involved in bringing about the responses, and to this purpose we utilized pharmacological blocking agents and spinal preparations.
Materials and Methods Forty-five toads (Bufo paracnemis) weighing 200-400 g were used in this study. The experiments were not performed in winter. Surgery was performed under ether anesthesia and consisted of cannulation of the abdon-dnal aorta with a silastic segment (to record arterial pressure), and of implantation of a pair of electrodes into the skin covering the floor of the mouth (to record throat movements). This last type of recording permits evaluation of the respiratory cycle in the toads [1]. Arterial pressure and throat movements were recorded with a Nihon Kohden polygraph model RM-45. In addition, a long silastic cannula was introduced into the anterior abdominal vein, secured to the skin of the back. and ufdized for systemic drug injection. In addition to being submitted to this general suxgical procedure, each experimental group underwent specific inlerventions. In the animals of group t (n = 16), the pulmonary and cutaneous arteries were cannulated with long sitastic segments, later secured to the skin of the back. Access to the arteries was obtained by means of a skin incision in the axillary region. The pulmocutaneous trunk was occluded near its origin. In this way, the arterial segment in question was vasctl!~rly isolated, which permitted its perfusion with Ringer solution under pressure to distend the artery, or with Ringer solution containing drugs capable of stimulating the chemoreceptors. Distension was effected with the aid of a syringe attached to a T-tube by polyethylene tubing. The arms of the T-tube were connected with the silastic c a n m ~ in the cutaneous artery on one side, and with ~ pressure transducer (Statham P23-AA) attached to a polygraph chmmel (Physiogrttph Four, E & M Inmmment,, Houston) on the other. Thus perfimion pressure could be ccmlpared with the c~bration scale
347 of a mercury manometer, also attached to the transducer. The chemoreceptors were stimulated with lobeline (Sandoz) diluted in Ringer solution (0.4 or 0.6 mg/ml). In this case, perfusion was also carried out with the aid of a syringe connected with the cutaneous artery, at a sufficiently slow speed (0.15 m l / m i n ) so as not to elicit the baroreceptor reflex. Some animals were perfused with 2% lidocaine. Once the responses due to chemical stimulation began to occur, the artery was washed with normal Ringer solution. A pair of stainless-steel electrodes was implanted into the back muscles in this group of animals for electromyographic recording because we observed that chemoreceptor stimulation elicited also a motor response. In another group of animals (group 2, n = 18), the laryngeal nerve was isolated on one side by the same axillary route and distally sectioned. The proximal segment of the nerve was laid on a pair of stainless-steel electrodes located at the bottom of a 0.5 mm wide slit in one of the facets of a small (3 mm) plexiglass cube. Contact of the nerve with the electrode was assured by a silastic segment introduced into the slit over the nerve and having the same width as the slit thus preventing dislocation from its position. The electrode continued into a long flexible cable of the same material, but insulated, which was secured to the skin of the animal's back and attached to the insulating unit of a stimulator at the time of the experiment. The contralateral laryngeal nerve was left intact. Rectangular unidirectional waves were used for stimulation, varying in duration according to the frequency employed: at low frequencies ( l / s ) , duration was 5 ms, and at high frequencies (50 or 100/s), 0.5 ms. The voltages used varied according to the response threshold in each animal, but were all within the 1-4 V range. Stimulation time was 15 or 30 s. Electric stimulation of the laryngeal nerve was also performed in 3 animals whose brain stem had been completely sectioned rostrally on the rhombencephalon. Two toads were submitted to electric stimulation of the central stump of the laryngeal nerve, though the stimulation was applied to the more distal end of the nerve, before it reaches the pulmocutaneous artery. Finally, group 3 (n = 5) consisted of animals whose laryngeal nerves were bilaterally isolated and surrounded by long silastic segments which left the thoracic cavity through the axiUary incision. A little hole was opened where the silastic touched the nerve so that a leakage of solution occurred each time the cannula was perfused with lidocaine. Only arterial pressure was recorded for this group. In view of the difficulty in interpreting the results where the autonomic responses were pharmacologically blocked, a subgroup of animals (n = 6) was submitted to high sectioning of the spinal cord with the object of observing whether the baroreceptor reflex caused by electric stimulation of the laryngeal nerve persisted or not. In this subgroup also only arterial pressure reactions were studied. An incision was made through the skin and the bone drilled above the dorsal region located on the medial line immediately below the articulation of the skull with the first cervical vertebra, and the nervous system was sectioned between the rhombencephalon and the spinal cord under visual control, with the aid of a Zeiss stereomicroscope. After the effects of anesthesia had completely disappeared, the animals of the 3 groups were placed in a rectangular wooden box with openings on one of the side walls for passage of the cannulae and cables. The toads in groups 1 and 2 were
348
submitted to a single experimental session which lasted an average of 3 h. Each animal in group 1 was submitted to 3 distensions of the pulmocutaneous artery and the mean of the 3 resulting responses was used in the general computation of the reaction for the group. In general, lobeline and lidocaine were injected only once into each animal. The number of electrical stimulations varied for each toad because of the need to establish adequate parameters for obtaining maximum response. Once these parameters had been established the stimulation was repeated 3 consecutive times and the mean of the cardiovascular responses obtained was considered as representative for the animal and calculated in the computation for the group. Mean arterial pressure as well as heart rate were calculated from the recording of pulsatile pressure. The results were analyzed quantitatively by the Student paired t-test. When the animals were submitted to systemic application of an autonomic blocking drug, a sufficient amount of time (60 min) for dispersion of the effects was allowed to elapse before a new substance was injected. The drugs used were: atropine sulfate (Merck) (3 m g / 5 0 0 g body weight), homatropine methylbromide (Searle) (0.5 m g / 5 0 0 g body weight), phentolamine (Ciba) (0.3 m g / 5 0 0 g body weight) and propranolol (ICI) (2 m g / 5 0 0 g body weight).
Results
Group 1: Perfusion of the pulmocutaneous arterial trunk Distension of the arterial trunk by perfusion with Ringer solution under pressure induced an abrupt fall in arterial pressure (31% below control mean arterial pressure) with bradycardia or cardiac arrest (Fig. 1). The graph in Fig. 2 shows that the hypotension obtained by distension of the pulmocutaneous artery is statistically significant when compared to the control. The response lasted an average of 170 s. even when the stimulus was maintained for more than the usual time (5 s). The alterations in frequency and amplitude of throat movements did not follow a regular pattern, with no possibility of relating them to the stimulus. Intravenous injection of atropine abolished this reflex. Since this drug crosses the blood-brain barrier, we switched to the use of methyl-homatropine as a blocker, but obtained the same results.
1U--
Fig. 1. Fall in arterial pressure and cardiac arrest caused by perfusionof the pulmocutaneousartery under the arterial t ~ was distended.
pressure. The arrow indicates the moment when
349
Perfusion of the pulmocutaneous artery with Ringer's solution containing lobeline induced a gradual increase in arterial pressure (40% above control level) and heart rate (Fig. 3). The hypertension obtained is statistically significant when compared to
6(2
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E E
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C
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C
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C
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Fig. 2. Graphic presentation of the fall in arterial pressure obtained in the following situations: D, distension of the pulmocutaneous artery; ES, electrical stimulation of the laryngeal nerve; and SA, electrical stimulation of the laryngeal nerve in a spinal animal. Each group is compared with its control (C). The vertical bars represent the standard error of the mean and the asterisks significance for P <0.001. 30 S
GM
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Fig. 3. Rise in arterial pressure (BP), in throat movements (GM) and in the firings of the back muscles (EMG) caused by perfusion of the pulmocutaneous artery (between arrows) with lobeline (0.6 mg/ml). The blood pressure variation precedes GM and EMG responses. The asterisk indicates the moment when the artery was washed with Ringer's solution and the time intervals indicate minutes after washing. The high deflection in EMG tracing is due to animal's movement.
350 the control, as shown by the graph in Fig. 4. Once the response was under way, the artery was washed with normal Ringer's solution, after which the arterial pressure took an average of 240 s to recover control values. The rises in arterial pressure were
:¢
c
L
Fig. 4. Graphic presentation of the rises in mean arterial pressure obtained by perfusion of the pulmocutaneous artery with lobeline (L) and by electrical stimulation of the laryngealnerve (ES) at 10w frequency. The vertical bars represent the standard error of the mean and the asterisk significance in relation to the control (C) for P <0.01.
accompanied by increased frequency and amplitude of throat movements and of electromyographic firings (Fig. 3). While this response was present distension of the pulmocutaneous artery did not induce a baroreceptor reflex, Previous phentolamine injection by the systemic route p r c v c n ~ the onset of the chemoreceptor response. Perfusion with 2~ l i d ~ produced a c a ~ o v a s o ! d a r ~ respiratory response identical to, but considerably more intense than, that o b t a i ~ w i ~ lobeline,
Group 2." Electrical stimulation of the laryngeal nerve All stimulated animals suffered a fall in arterial pressure (Fig. 2), and 55% of them also exhibited increases (Fig. 4). All responses were statistically s i ~ i c a n t with respect to their control values (Figs. 2 and 6). The falls were generally ~ t a i n e d with high-frequency stimuli (50 or 100/s), although in 33% of the animals they occurred also at low frequency ( l / s ) . The rises in pressure occurred only with low-frequency stimuli ( l / s ) . In 42~ of the cases in w h i ~ arrest, while only bradycardia was observe hypotension occurred without any alc,'rations in ~ ~ q m m c y . However, by heightening the stimulus intensity a bradycardia or cardiac arrest was ~ s e r v e d .
351
, GM
. :~.~, L.!..J , ! ,l
I '1
I
b
BP ~70-
-I" ,dAdJt~i~i~ E E 30-
,liill,,,,,,,,ii,,,t,,.i,.~lllIliiliiBllillliiIOiil~llll~li~
Fig. 5. Fall in arterial pressure (BP) due to high frequency electrical stimulation (50 Hz) of the laryngeal nerve for 15 s (15 s). The brief suppression of throat movements (GM) during maximal hypotension is followed by a rise in amplitude and frequency during blood pressure return to control values.
Mean response duration was 143 s. Some toads presented a bradycardia without blood pressure alteration. As in the case of the baroreceptor reflexes obtained by distension, those elicited by electrical stimulation of the laryngeal nerve produced the highest pressure fall at the beginning of stimulation, with control levels soon reached again even in the presence of prolonged stimulation. In contrast, the onset of a pressure rise was gradual and maximum values were maintained during prolonged stimulation. The stimuli applied to most animals in this group, however, were of short duration and the response did not reach its highest peak. The pressure rise was accompanied by tachycardia and increased throat movements, with a mean duration of 140s (Fig. 6). Responses persisted also in anesthetized toads (chloralose/urethane), and in the animals with transection of the brain stem rostrally to the rhombencephalon. Stimulation of the central stump of the distal end of the laryngeal nerve, where it does not yet innervate the pulmocutaneous artery, caused very slight and short falls in pressure that had no statistical significant and that
m 70-"r BP r-
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,~ "" '"
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Fig. 6. Rise in arterial pressure (BP) and in throat movements (GM) due to low frequency electrical stimulation (1 Hz) of the laryngeal nerve for 40 s (40 s).
352 o~60"I"
E
20-
15 S
Fig. 7. Fall in arterial pressure due to electric stimulation of the laryngeal nerve of a toad with high spinal sectioning.
could be in no way compared to those obtained by stimulating the s a ~ portion where it incorporated fibers of the p u l m ~ t a n e o u s artery'
nerve in the
Pharmacological blockage of the responses Atropine and methyl-homatropine blocked the falls in arterial pressure and cardiac arrest. Phentolarnine reduced the basal pressure and the hypotensions obtained by electric stimulation. However, the values of the pressure falls continued to be statistically significant. In addition, an increase in the voltage of the applied stimulus caused the initial amplitude of the response to reappear. In animals which only exhibited pressure falls, blockage by methyl-homatropine elicited the opposite response when the nerve was stimulated. Hypertensions were blocked by phentolamine. Propranolol did not prevent the occurrence Of blood pressure fall or increase.
Animals with cut spinal cord Despite high sectioning of the spinal cord, electrical stimulation of the laryngeal nerve in the toads of this subgroup continued to induce falls in arterial pressure
1( "ro} E E~
Fig. 8. Rise in arterial blood pressure (BP) and in throat movements(GM) due to bilateral blockage~f the laryngeal nerves (2~ lidocaine) in awake unrestrained toadS, b: 4 rain aher beginningof S t ~ ;
353 (Figs. 2 and 7), accompanied by cardiac arrest or by bradycardia. These responses were obtained by the use of both high and low frequencies, even though the former were more effective. Group 3: Bilateral treatment of the laryngeal nerve with lidocaine The response was similar to that obtained when perfusing the pulmocutaneous arterial trunk with the same solution: a blood pressure rise, tachycardia and enhanced throat movements (Fig. 8).
Discussion The carotid labyrinth of amphibians, considered to be homologous with the carotid sinus of mammals, appears to be a chemoreceptor reflexogenous zone because its distension does not elicit systemic cardiovascular effects in anesthetized toads [2]. No baroreceptor reflex was observed in conscious toads either (A. Hoffmann, unpublished results). A baroreceptor reflex, however, can be easily obtained by distension of the pulmocutaneous artery, a fact already reported by Ishii and Ishii [3] for anesthetized toads and confirmed by us for conscious animals. The falls in mean arterial pressure obtained by distension of the pulmocutaneous artery (31% below control mean arterial pressure) were more marked than those observed after stimulation of the laryngeal nerve (24% below mean basal pressure). Similarly, bradycardia was more intense (24%below control level) in the group submitted to natural stimulation (distension) than in the group submitted to electrical stimulation of the nerve (10% below control level). Also, mean response duration was longer in the animals submitted to distension of the arterial trunk (170s) than in the animals submitted to electrical stimulation (143 s). It should be pointed out here that electrical stimulation of the nerve is scarcely a physiological procedure and must cause simultaneous activation of chemoreceptor and baroreceptor fibers. This was demonstrated for the animals in which the arterial pressure response was inverted from a fall to a rise after systemic injection of methyl-homatropine. Despite these limitations, electrical stimulation of nervous structures is a valid experimental procedure, and variation in the parameters of the stimulus frequently permits demonstration of a system with opposite functions. Thus, the use of high-frequency stimuli mainly elicited blood pressure falls, while blood pressure rises were obtained only with the use of low frequencies. What we observe here is exactly the opposite of what occurs at the central level, where stimulation of the visceral receptor zone of the bulb in anesthetized toads, i.e. of the region where the baroreceptor and chemoreceptor fibers establish their first synapsis, induces falls in arterial pressure only with low-frequency stimulation. Pressure rises are obtained both with high- and low-frequency stimulation [11 ]. These results were obtained with toads anesthetized with chloralose/urethane; however, we do not believe this fact to alter the nature of the response, since some of our animals in this group were anesthetized with the same drug combination without any alterations in the results
354
described. The situation observed in amphibians contrast with that described for mammals, where the maximum fall in arterial pressure obtained by stimulation of the sinus nerve occurred with the use of frequencies between 20 and 30 Hz. On the other hand, electric stimulation of the solitary tract nucleus determined maximum falls with frequencies from 30 to 120 Hz [8]. The response obtained by electric stimulation of the laryngeal nerve can be considered to be a rhombencephalic reflex, because it occurs in animals with transection of the brain stem at the caudal end of the midbrain. The unanesthetized toads hypotensive responses reach maximum values at the beginning of stimulation and gradually return to control values despite persistence of the stimulus. In contrast, the pressure rises observed both with perfusion of the pulmocutaneous artery with lobeline and with stimulation of the laryngeal nerve occur gradually and maintain maximum levels while the stimulus persists. The two responses seem to involve neurons that react in different ways in the presence of a persistent and unchanging stimulus. Perfusion with lobeline did not only alter blood pressure, but also induced increased throat movements and increasing firing of the tonic back muscles. The animal, which had been quiet before perfusion, would move around in the experimental box. This fact shows that the stimulation of chemoreceptors induces behavioral arousal also in amphibians. Blockage of the laryngeal nerves or of the receptors located in the pulmocutaneous arterial wall induced a blood pressure rise. A similar response was described by Ishii and Ishii [3] as a consequence of the laryngeal nerve section in anesthetized toads. We agree with these authors that the response is related to the suppression of the baroreceptor tonus. The nature of pharmacological blockage of the baroreceptor reflex obtained by systemic injection of atropine and methyl-homatropine could not be adequately clarified. In amphibians, the sympathetic system releases acetylcholine with vasoconstricting action on certain vascular beds (the lung, for example). If the baroreceptor reflex were due to sympathetic inhibition, atropine could partially block it by acting on these beds. As the response is still present in spinal animals and is blocked by atropine, we think it is mainly due to an active parasympathetic vasodilatation. The occurrence of choline@ vasodilating innervation was observed in the arterioles of the retrolingual membrane of frogs by Siggins and Bloom [9] and by Siggins and Weitsen [lo]. Neurogenic vasodilation persists in this region after chemical sympathectomy produced by 6-OHDA injection [ 12], thus confirming the nonadrenergic origin of dilating nerves. We observed that after systemic injection of phentolamine a reduction in the amplitude of falls obtained by electric stimulation of the laryngeal nerve occurs. This may be due to a reduction in mean basal arterial pressure produced by this drug, or to the lack of specificity of the drugs which block the sympathetic response, a fact already described by Nickerson (61. In any case, we cannot rule out the presence of a certain amount of sympathetic inhibition in the genesis of the baroreceptor reflex. The contribution of P-dilator receptor, if present, did not affect the systemic blood pressure, for propranolol injection did not prevent the hypotensions due to laryngeal nerve stimulation.
355
Acknowledgement T h i s s t u d y w a s s u p p o r t e d b y g r a n t s f r o m the F u n d a ~ o de A m p a r o ~ P e s q u i s a d e S~o P a u l o ( F A P E S P ) a n d F i n a n c i a d o r a d e E s t u d o s ae P r o j e t o s ( F I N E P ) .
References 1 Costa, F.B.R., Somatoautonomic Responses to Electrical and Chemical Stimulation of the Toad's Tegmentum, Dissertation, School of Medicine of Ribeirao Preto, University of S~to Paulo, 1977, (in Portuguese). 2 Ishii, K., Honda, K. and Ishii, K., The function of the carotid labyrinth in the toad, Tohoku J. exp. Med., 88 (1966) 103-116. 3 Ishii, K. and Ishii, K., A reflexogenic area for controlling the blood pressure in toad (Bufo vulgaris formosa), Jap. J. Physiol., 28 (1978) 423-431. 4 Kuno, Y., Einige Beobachtungen tiber den Blutdruck des Frosches, Pfliigers Arch., 158 (1914) 1-18. 5 Neil, E., Str/Sm, L. and Zotterman, Y., Action potential studies afferent fibres in the IX th and X th cranial nerves of the frog, Acta physiol, scand., 20 (1950) 338-350. 6 Nickerson, M., The pharmacology of adrenergic blockade, Pharmacol. Rev., I (1949) 27-41. 7 Nikiforowsky, P.M., On depressor nerve fibres in the vagus of the frog, J. Physiol. (Lond.), 45 (1912) 459-461. 8 Seller, H. and lllert, M., The localization of the first synapses in the carotid sinus baroreceptor reflex pathway and its alteration of the afferent input, Pfltigers Arch., 306 (1969) 1-19. 9 Siggins, G.R. and Bloom, F.E., Cytochemical and physiological effects of 6-hydroxydopamine on periarteriolar nerves of frogs, Circulat. Res., 27 (1970) 23-38. 10 Siggins, G.R. and Weitsen, H.A., Cytochemical and physiological evidence for cholinergic, neurogenic vasodilation of amphibian arterioles and precapillary sphincters. I. Light microscopy, Microvasc. Res., 3 (1971) 308-322. 11 Souza, M.B.C., Autonomic Responses to Stimulation of the Visceral Sensory Zone of the Rhomboencephalon in Anesthetized Toads, Dissertation. School of Medicine of Ribeir~o Preto, University of S~to Paulo, 1980, (in Portuguese). 12 Thoenen, H. and Tranzer, J.P., Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine, Naunyn-Schmiedeberg's Arch. Pharmak., 261 (1968) 271-278.
345
Elsevier Biomedical Press
Cardiovascular reflexes in conscious toads Anette Hoffmann and Maria Bernardete Cordeiro de Souza Department of Physiology, School of Medicine of Ribeirgto Preto, USP, 14100 Ribeir~o Preto, SP, (Brazil)
(Received February 24th, 1981) (Accepted October 26th, 1981)
K e y words: toad--pulmocutaneous arterial trunk--cardiovascular reflexes--auto-
nomic effectors
Abstract Methods used for implanting sensors and catheters in temporarily ether-anesthetized toads ( B u f o paracnemis) are described. Following recovery it was found that distension of the pulmocutaneous arterial trunk and high frequency electrical stimulation of the laryngeal nerve of conscious toads induce an abrupt fall in arterial pressure accompanied or not by bradycardia or cardiac arrest. A brief suppression of throat movements may occur but this is not a constant finding. The response is blocked by atropine or methyl-homatropine and persists in animals with high spinal sectioning, thus indicating its cholinergic parasympathetic nature. However a certain amount of sympathetic inhibition is not ruled out. Perfusion of the artery with lobeline and electrical stimulation of the laryngeal nerve at low frequency (l/s) induces a rise in arterial pressure which is blocked by phentolamine. The hypertension is followed by enhancing of both throat oscillations and electromyographic discharges. The occurrence of chemoreceptors in the pulmocutaneous arterial wall in these animals is discussed. Blockage of the laryngeal nerve with lidocaine or perfusion of the pulmocutaneous arterial trunk with the same solution elicited a blood pressure rise, tachycardia and enhanced ventilatory movements. This was attributed to suppression of the baroreceptor tonus.
Introduction
lshii and Ishii [3] demonstrated cardiovascular reflexes in anesthetized toads caused by distension of the pulmucutaneous artery, and also recorded impulses in 0165-1838/82/0000-0000/$02.75 © 1982 ElsevierBiomedicalPress
346
the peripheral cut end of the laryngeal nerve (the vagus branch which innervates the mentioned artery) which were synchronous with the cardiac systole. Nikiforowsky [7] and Kuno [4] had previously reported the occurrence of a fall in arterial pressure after stimulation of the vagus, and Neil et al. [5] had observed activity in the laryngeal nerve which was synchronous with the increase in arterial pressure induced in the trunk of the pulmocutaneous artery. All these studies, however, were carried out on anesthetized animals, a condition which may have altered the patterns of the reflexes. In the present investigation, we studied cardiovascular reflexes in conscious unrestrained toads. These reflexes were elicited by mechanical distension of the isolated pulmocutaneous trunk, by perfusion of this artery with lobeline, and by electrical stimulation of the central cut end of the laryngeal nerve. What led us to explore this reflexogenous area was our inability to obtain cardiovascular reflexes by distension of the carotid labyrinth in conscious toads. It is well known that this organ is considered to be homologous with the carotid sinus in mammals. We were also interested in determining the autonomic mechanisms involved in bringing about the responses, and to this purpose we utilized pharmacological blocking agents and spinal preparations.
Materials and Methods Forty-five toads (Bufo paracnemis) weighing 200-400 g were used in this study. The experiments were not performed in winter. Surgery was performed under ether anesthesia and consisted of cannulation of the abdon-dnal aorta with a silastic segment (to record arterial pressure), and of implantation of a pair of electrodes into the skin covering the floor of the mouth (to record throat movements). This last type of recording permits evaluation of the respiratory cycle in the toads [1]. Arterial pressure and throat movements were recorded with a Nihon Kohden polygraph model RM-45. In addition, a long silastic cannula was introduced into the anterior abdominal vein, secured to the skin of the back. and ufdized for systemic drug injection. In addition to being submitted to this general suxgical procedure, each experimental group underwent specific inlerventions. In the animals of group t (n = 16), the pulmonary and cutaneous arteries were cannulated with long sitastic segments, later secured to the skin of the back. Access to the arteries was obtained by means of a skin incision in the axillary region. The pulmocutaneous trunk was occluded near its origin. In this way, the arterial segment in question was vasctl!~rly isolated, which permitted its perfusion with Ringer solution under pressure to distend the artery, or with Ringer solution containing drugs capable of stimulating the chemoreceptors. Distension was effected with the aid of a syringe attached to a T-tube by polyethylene tubing. The arms of the T-tube were connected with the silastic c a n m ~ in the cutaneous artery on one side, and with ~ pressure transducer (Statham P23-AA) attached to a polygraph chmmel (Physiogrttph Four, E & M Inmmment,, Houston) on the other. Thus perfimion pressure could be ccmlpared with the c~bration scale
347 of a mercury manometer, also attached to the transducer. The chemoreceptors were stimulated with lobeline (Sandoz) diluted in Ringer solution (0.4 or 0.6 mg/ml). In this case, perfusion was also carried out with the aid of a syringe connected with the cutaneous artery, at a sufficiently slow speed (0.15 m l / m i n ) so as not to elicit the baroreceptor reflex. Some animals were perfused with 2% lidocaine. Once the responses due to chemical stimulation began to occur, the artery was washed with normal Ringer solution. A pair of stainless-steel electrodes was implanted into the back muscles in this group of animals for electromyographic recording because we observed that chemoreceptor stimulation elicited also a motor response. In another group of animals (group 2, n = 18), the laryngeal nerve was isolated on one side by the same axillary route and distally sectioned. The proximal segment of the nerve was laid on a pair of stainless-steel electrodes located at the bottom of a 0.5 mm wide slit in one of the facets of a small (3 mm) plexiglass cube. Contact of the nerve with the electrode was assured by a silastic segment introduced into the slit over the nerve and having the same width as the slit thus preventing dislocation from its position. The electrode continued into a long flexible cable of the same material, but insulated, which was secured to the skin of the animal's back and attached to the insulating unit of a stimulator at the time of the experiment. The contralateral laryngeal nerve was left intact. Rectangular unidirectional waves were used for stimulation, varying in duration according to the frequency employed: at low frequencies ( l / s ) , duration was 5 ms, and at high frequencies (50 or 100/s), 0.5 ms. The voltages used varied according to the response threshold in each animal, but were all within the 1-4 V range. Stimulation time was 15 or 30 s. Electric stimulation of the laryngeal nerve was also performed in 3 animals whose brain stem had been completely sectioned rostrally on the rhombencephalon. Two toads were submitted to electric stimulation of the central stump of the laryngeal nerve, though the stimulation was applied to the more distal end of the nerve, before it reaches the pulmocutaneous artery. Finally, group 3 (n = 5) consisted of animals whose laryngeal nerves were bilaterally isolated and surrounded by long silastic segments which left the thoracic cavity through the axiUary incision. A little hole was opened where the silastic touched the nerve so that a leakage of solution occurred each time the cannula was perfused with lidocaine. Only arterial pressure was recorded for this group. In view of the difficulty in interpreting the results where the autonomic responses were pharmacologically blocked, a subgroup of animals (n = 6) was submitted to high sectioning of the spinal cord with the object of observing whether the baroreceptor reflex caused by electric stimulation of the laryngeal nerve persisted or not. In this subgroup also only arterial pressure reactions were studied. An incision was made through the skin and the bone drilled above the dorsal region located on the medial line immediately below the articulation of the skull with the first cervical vertebra, and the nervous system was sectioned between the rhombencephalon and the spinal cord under visual control, with the aid of a Zeiss stereomicroscope. After the effects of anesthesia had completely disappeared, the animals of the 3 groups were placed in a rectangular wooden box with openings on one of the side walls for passage of the cannulae and cables. The toads in groups 1 and 2 were
348
submitted to a single experimental session which lasted an average of 3 h. Each animal in group 1 was submitted to 3 distensions of the pulmocutaneous artery and the mean of the 3 resulting responses was used in the general computation of the reaction for the group. In general, lobeline and lidocaine were injected only once into each animal. The number of electrical stimulations varied for each toad because of the need to establish adequate parameters for obtaining maximum response. Once these parameters had been established the stimulation was repeated 3 consecutive times and the mean of the cardiovascular responses obtained was considered as representative for the animal and calculated in the computation for the group. Mean arterial pressure as well as heart rate were calculated from the recording of pulsatile pressure. The results were analyzed quantitatively by the Student paired t-test. When the animals were submitted to systemic application of an autonomic blocking drug, a sufficient amount of time (60 min) for dispersion of the effects was allowed to elapse before a new substance was injected. The drugs used were: atropine sulfate (Merck) (3 m g / 5 0 0 g body weight), homatropine methylbromide (Searle) (0.5 m g / 5 0 0 g body weight), phentolamine (Ciba) (0.3 m g / 5 0 0 g body weight) and propranolol (ICI) (2 m g / 5 0 0 g body weight).
Results
Group 1: Perfusion of the pulmocutaneous arterial trunk Distension of the arterial trunk by perfusion with Ringer solution under pressure induced an abrupt fall in arterial pressure (31% below control mean arterial pressure) with bradycardia or cardiac arrest (Fig. 1). The graph in Fig. 2 shows that the hypotension obtained by distension of the pulmocutaneous artery is statistically significant when compared to the control. The response lasted an average of 170 s. even when the stimulus was maintained for more than the usual time (5 s). The alterations in frequency and amplitude of throat movements did not follow a regular pattern, with no possibility of relating them to the stimulus. Intravenous injection of atropine abolished this reflex. Since this drug crosses the blood-brain barrier, we switched to the use of methyl-homatropine as a blocker, but obtained the same results.
1U--
Fig. 1. Fall in arterial pressure and cardiac arrest caused by perfusionof the pulmocutaneousartery under the arterial t ~ was distended.
pressure. The arrow indicates the moment when
349
Perfusion of the pulmocutaneous artery with Ringer's solution containing lobeline induced a gradual increase in arterial pressure (40% above control level) and heart rate (Fig. 3). The hypertension obtained is statistically significant when compared to
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Fig. 2. Graphic presentation of the fall in arterial pressure obtained in the following situations: D, distension of the pulmocutaneous artery; ES, electrical stimulation of the laryngeal nerve; and SA, electrical stimulation of the laryngeal nerve in a spinal animal. Each group is compared with its control (C). The vertical bars represent the standard error of the mean and the asterisks significance for P <0.001. 30 S
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Fig. 3. Rise in arterial pressure (BP), in throat movements (GM) and in the firings of the back muscles (EMG) caused by perfusion of the pulmocutaneous artery (between arrows) with lobeline (0.6 mg/ml). The blood pressure variation precedes GM and EMG responses. The asterisk indicates the moment when the artery was washed with Ringer's solution and the time intervals indicate minutes after washing. The high deflection in EMG tracing is due to animal's movement.
350 the control, as shown by the graph in Fig. 4. Once the response was under way, the artery was washed with normal Ringer's solution, after which the arterial pressure took an average of 240 s to recover control values. The rises in arterial pressure were
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Fig. 4. Graphic presentation of the rises in mean arterial pressure obtained by perfusion of the pulmocutaneous artery with lobeline (L) and by electrical stimulation of the laryngealnerve (ES) at 10w frequency. The vertical bars represent the standard error of the mean and the asterisk significance in relation to the control (C) for P <0.01.
accompanied by increased frequency and amplitude of throat movements and of electromyographic firings (Fig. 3). While this response was present distension of the pulmocutaneous artery did not induce a baroreceptor reflex, Previous phentolamine injection by the systemic route p r c v c n ~ the onset of the chemoreceptor response. Perfusion with 2~ l i d ~ produced a c a ~ o v a s o ! d a r ~ respiratory response identical to, but considerably more intense than, that o b t a i ~ w i ~ lobeline,
Group 2." Electrical stimulation of the laryngeal nerve All stimulated animals suffered a fall in arterial pressure (Fig. 2), and 55% of them also exhibited increases (Fig. 4). All responses were statistically s i ~ i c a n t with respect to their control values (Figs. 2 and 6). The falls were generally ~ t a i n e d with high-frequency stimuli (50 or 100/s), although in 33% of the animals they occurred also at low frequency ( l / s ) . The rises in pressure occurred only with low-frequency stimuli ( l / s ) . In 42~ of the cases in w h i ~ arrest, while only bradycardia was observe hypotension occurred without any alc,'rations in ~ ~ q m m c y . However, by heightening the stimulus intensity a bradycardia or cardiac arrest was ~ s e r v e d .
351
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Fig. 5. Fall in arterial pressure (BP) due to high frequency electrical stimulation (50 Hz) of the laryngeal nerve for 15 s (15 s). The brief suppression of throat movements (GM) during maximal hypotension is followed by a rise in amplitude and frequency during blood pressure return to control values.
Mean response duration was 143 s. Some toads presented a bradycardia without blood pressure alteration. As in the case of the baroreceptor reflexes obtained by distension, those elicited by electrical stimulation of the laryngeal nerve produced the highest pressure fall at the beginning of stimulation, with control levels soon reached again even in the presence of prolonged stimulation. In contrast, the onset of a pressure rise was gradual and maximum values were maintained during prolonged stimulation. The stimuli applied to most animals in this group, however, were of short duration and the response did not reach its highest peak. The pressure rise was accompanied by tachycardia and increased throat movements, with a mean duration of 140s (Fig. 6). Responses persisted also in anesthetized toads (chloralose/urethane), and in the animals with transection of the brain stem rostrally to the rhombencephalon. Stimulation of the central stump of the distal end of the laryngeal nerve, where it does not yet innervate the pulmocutaneous artery, caused very slight and short falls in pressure that had no statistical significant and that
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Fig. 6. Rise in arterial pressure (BP) and in throat movements (GM) due to low frequency electrical stimulation (1 Hz) of the laryngeal nerve for 40 s (40 s).
352 o~60"I"
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Fig. 7. Fall in arterial pressure due to electric stimulation of the laryngeal nerve of a toad with high spinal sectioning.
could be in no way compared to those obtained by stimulating the s a ~ portion where it incorporated fibers of the p u l m ~ t a n e o u s artery'
nerve in the
Pharmacological blockage of the responses Atropine and methyl-homatropine blocked the falls in arterial pressure and cardiac arrest. Phentolarnine reduced the basal pressure and the hypotensions obtained by electric stimulation. However, the values of the pressure falls continued to be statistically significant. In addition, an increase in the voltage of the applied stimulus caused the initial amplitude of the response to reappear. In animals which only exhibited pressure falls, blockage by methyl-homatropine elicited the opposite response when the nerve was stimulated. Hypertensions were blocked by phentolamine. Propranolol did not prevent the occurrence Of blood pressure fall or increase.
Animals with cut spinal cord Despite high sectioning of the spinal cord, electrical stimulation of the laryngeal nerve in the toads of this subgroup continued to induce falls in arterial pressure
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Fig. 8. Rise in arterial blood pressure (BP) and in throat movements(GM) due to bilateral blockage~f the laryngeal nerves (2~ lidocaine) in awake unrestrained toadS, b: 4 rain aher beginningof S t ~ ;
353 (Figs. 2 and 7), accompanied by cardiac arrest or by bradycardia. These responses were obtained by the use of both high and low frequencies, even though the former were more effective. Group 3: Bilateral treatment of the laryngeal nerve with lidocaine The response was similar to that obtained when perfusing the pulmocutaneous arterial trunk with the same solution: a blood pressure rise, tachycardia and enhanced throat movements (Fig. 8).
Discussion The carotid labyrinth of amphibians, considered to be homologous with the carotid sinus of mammals, appears to be a chemoreceptor reflexogenous zone because its distension does not elicit systemic cardiovascular effects in anesthetized toads [2]. No baroreceptor reflex was observed in conscious toads either (A. Hoffmann, unpublished results). A baroreceptor reflex, however, can be easily obtained by distension of the pulmocutaneous artery, a fact already reported by Ishii and Ishii [3] for anesthetized toads and confirmed by us for conscious animals. The falls in mean arterial pressure obtained by distension of the pulmocutaneous artery (31% below control mean arterial pressure) were more marked than those observed after stimulation of the laryngeal nerve (24% below mean basal pressure). Similarly, bradycardia was more intense (24%below control level) in the group submitted to natural stimulation (distension) than in the group submitted to electrical stimulation of the nerve (10% below control level). Also, mean response duration was longer in the animals submitted to distension of the arterial trunk (170s) than in the animals submitted to electrical stimulation (143 s). It should be pointed out here that electrical stimulation of the nerve is scarcely a physiological procedure and must cause simultaneous activation of chemoreceptor and baroreceptor fibers. This was demonstrated for the animals in which the arterial pressure response was inverted from a fall to a rise after systemic injection of methyl-homatropine. Despite these limitations, electrical stimulation of nervous structures is a valid experimental procedure, and variation in the parameters of the stimulus frequently permits demonstration of a system with opposite functions. Thus, the use of high-frequency stimuli mainly elicited blood pressure falls, while blood pressure rises were obtained only with the use of low frequencies. What we observe here is exactly the opposite of what occurs at the central level, where stimulation of the visceral receptor zone of the bulb in anesthetized toads, i.e. of the region where the baroreceptor and chemoreceptor fibers establish their first synapsis, induces falls in arterial pressure only with low-frequency stimulation. Pressure rises are obtained both with high- and low-frequency stimulation [11 ]. These results were obtained with toads anesthetized with chloralose/urethane; however, we do not believe this fact to alter the nature of the response, since some of our animals in this group were anesthetized with the same drug combination without any alterations in the results
354
described. The situation observed in amphibians contrast with that described for mammals, where the maximum fall in arterial pressure obtained by stimulation of the sinus nerve occurred with the use of frequencies between 20 and 30 Hz. On the other hand, electric stimulation of the solitary tract nucleus determined maximum falls with frequencies from 30 to 120 Hz [8]. The response obtained by electric stimulation of the laryngeal nerve can be considered to be a rhombencephalic reflex, because it occurs in animals with transection of the brain stem at the caudal end of the midbrain. The unanesthetized toads hypotensive responses reach maximum values at the beginning of stimulation and gradually return to control values despite persistence of the stimulus. In contrast, the pressure rises observed both with perfusion of the pulmocutaneous artery with lobeline and with stimulation of the laryngeal nerve occur gradually and maintain maximum levels while the stimulus persists. The two responses seem to involve neurons that react in different ways in the presence of a persistent and unchanging stimulus. Perfusion with lobeline did not only alter blood pressure, but also induced increased throat movements and increasing firing of the tonic back muscles. The animal, which had been quiet before perfusion, would move around in the experimental box. This fact shows that the stimulation of chemoreceptors induces behavioral arousal also in amphibians. Blockage of the laryngeal nerves or of the receptors located in the pulmocutaneous arterial wall induced a blood pressure rise. A similar response was described by Ishii and Ishii [3] as a consequence of the laryngeal nerve section in anesthetized toads. We agree with these authors that the response is related to the suppression of the baroreceptor tonus. The nature of pharmacological blockage of the baroreceptor reflex obtained by systemic injection of atropine and methyl-homatropine could not be adequately clarified. In amphibians, the sympathetic system releases acetylcholine with vasoconstricting action on certain vascular beds (the lung, for example). If the baroreceptor reflex were due to sympathetic inhibition, atropine could partially block it by acting on these beds. As the response is still present in spinal animals and is blocked by atropine, we think it is mainly due to an active parasympathetic vasodilatation. The occurrence of choline@ vasodilating innervation was observed in the arterioles of the retrolingual membrane of frogs by Siggins and Bloom [9] and by Siggins and Weitsen [lo]. Neurogenic vasodilation persists in this region after chemical sympathectomy produced by 6-OHDA injection [ 12], thus confirming the nonadrenergic origin of dilating nerves. We observed that after systemic injection of phentolamine a reduction in the amplitude of falls obtained by electric stimulation of the laryngeal nerve occurs. This may be due to a reduction in mean basal arterial pressure produced by this drug, or to the lack of specificity of the drugs which block the sympathetic response, a fact already described by Nickerson (61. In any case, we cannot rule out the presence of a certain amount of sympathetic inhibition in the genesis of the baroreceptor reflex. The contribution of P-dilator receptor, if present, did not affect the systemic blood pressure, for propranolol injection did not prevent the hypotensions due to laryngeal nerve stimulation.
355
Acknowledgement T h i s s t u d y w a s s u p p o r t e d b y g r a n t s f r o m the F u n d a ~ o de A m p a r o ~ P e s q u i s a d e S~o P a u l o ( F A P E S P ) a n d F i n a n c i a d o r a d e E s t u d o s ae P r o j e t o s ( F I N E P ) .
References 1 Costa, F.B.R., Somatoautonomic Responses to Electrical and Chemical Stimulation of the Toad's Tegmentum, Dissertation, School of Medicine of Ribeirao Preto, University of S~to Paulo, 1977, (in Portuguese). 2 Ishii, K., Honda, K. and Ishii, K., The function of the carotid labyrinth in the toad, Tohoku J. exp. Med., 88 (1966) 103-116. 3 Ishii, K. and Ishii, K., A reflexogenic area for controlling the blood pressure in toad (Bufo vulgaris formosa), Jap. J. Physiol., 28 (1978) 423-431. 4 Kuno, Y., Einige Beobachtungen tiber den Blutdruck des Frosches, Pfliigers Arch., 158 (1914) 1-18. 5 Neil, E., Str/Sm, L. and Zotterman, Y., Action potential studies afferent fibres in the IX th and X th cranial nerves of the frog, Acta physiol, scand., 20 (1950) 338-350. 6 Nickerson, M., The pharmacology of adrenergic blockade, Pharmacol. Rev., I (1949) 27-41. 7 Nikiforowsky, P.M., On depressor nerve fibres in the vagus of the frog, J. Physiol. (Lond.), 45 (1912) 459-461. 8 Seller, H. and lllert, M., The localization of the first synapses in the carotid sinus baroreceptor reflex pathway and its alteration of the afferent input, Pfltigers Arch., 306 (1969) 1-19. 9 Siggins, G.R. and Bloom, F.E., Cytochemical and physiological effects of 6-hydroxydopamine on periarteriolar nerves of frogs, Circulat. Res., 27 (1970) 23-38. 10 Siggins, G.R. and Weitsen, H.A., Cytochemical and physiological evidence for cholinergic, neurogenic vasodilation of amphibian arterioles and precapillary sphincters. I. Light microscopy, Microvasc. Res., 3 (1971) 308-322. 11 Souza, M.B.C., Autonomic Responses to Stimulation of the Visceral Sensory Zone of the Rhomboencephalon in Anesthetized Toads, Dissertation. School of Medicine of Ribeir~o Preto, University of S~to Paulo, 1980, (in Portuguese). 12 Thoenen, H. and Tranzer, J.P., Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine, Naunyn-Schmiedeberg's Arch. Pharmak., 261 (1968) 271-278.