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A technique for recording sympathetic nerve activity in unanaesthetized dogs
A TECHNIQUE FOR RECORDING SYMPATHETIC NERVE ACTIVITY IN UNANAESTHETIZED DOGS H. SCHMITT,HE!L& SCHMITTand SIMONEF~NARD Departement de Pharmacologic, Faculte de Medecine, Paris-Broussais-Hotel-Dieu, tine, Paris 6e, France (Accepted
17 October
15 rue de 1’Ecole de Mede-
1973)
Summary-A technique for recording splanchnic discharges chronically, in conscious dogs is described. Two small stainless steel electrodes were inserted into the splanchnic nerves and secured with dental cement. The wires were passed under the skin and attached to plugs. With this technique intravenous clonidine was shown to decrease splanchnic discharges, to induce bradycardia and biphasic changes in blood pressure: an increase followed by a decrease. By peroral route the effects were the same, except that the initial hypertension did not occur. No tolerance to this effect of clonidine was seen during the 10 days treatment. The method appears to be valuable in disclosing centrally or reflexly mediated decreases in the sympathetic tone.
Recording of activity in sympathetic nerves has been widely used to investigate central or reflex physiological or pharmacological influences on the sympathetic tone. However, the experiments have, as yet, been performed only on anaesthetized or paralyzed animals, precluding any investigation on longterm induced changes on this tone. WEIDINGER and KIRCHNER (1967) and BAUST, WEIDINGER and KIRCHNER (1968) recorded in conscious cats the activity of the renal postganglionic sympathetic nerve simultaneously with the EEG and EMG to investigate the influence of wakefulness, synchronized and desynchronized phases of sleep on the sympathetic tone. However, the technique did not allow distinction between ganglionic or central sites of action. It was therefore of interest to develop a technique to record chronically the discharges in a preganglionic sympathetic nerve together with changes in blood pressure. This paper describes such a technique and the results obtained with clonidine. and agent known to reduce sympathetic discharges in anaesthetized animals (SCHMITT, SCHMITT, BOISSIER and GIUDICELLI, 1967; SCHMITT, SCHMITT, BOISSIER,GIUDICELLIand FICHELLE,1968; KLUPP, KNAPPEN,OTSUKA, STRELLER and TEICHMANN,1970). MATERIALS
AND METHODS
Mongrel dogs of either sex weighing 5-l 1 kg were used. They were anaesthetized with pentobarbital (30 mg/kg i.v.). Under sterile conditions, a carotid artery was isolated and a polyethylene catheter 1.5 mm in external diameter (K-32 Feeding tube: Size 5 French) and 25 cm in length was introduced into the artery as far as the aorta. A second catheter was introduced into the jugular vein in order to perform the intravenous injections. Both catheters were closed with a rubber cap and passed under the skin to the back of the neck. In some experiments the right vertebral artery was isolated at the base of the neck and ligatured distally; a small polyethylene catheter was introduced into the cephalic end of the artery and sutured. The catheter was passed under the skin to the neck. In this way. 347
348
H.
SCHMITT,
H~LZNE SCHMITTand S. FENARD
administration of small doses of drugs could be performed in order to restrict the action to the rhombencephalon. The left splanchnic nerve was approached retroperitoneally by a paravertebral incision. The nerve was isolated at its exit from the diaphragm. A pair of special electrodes described below was introduced into the nerve; the electrodes were isolated and secured on the nerve with dental cement (Palaferm). The wires were passed under the skin to the back of the neck where plugs were attached for recording. To construct the electrodes, the end of a stainless steel dental broach (10 pm at the top) was cut at 34 mm from the tip and was soldered to a stainless steel wire coated with teflon (wire diameter: 0908; Medwire Corporation Vernon). Both electrodes, except for the tip (2-3 mm), as well as the soldering and l-2 mm of the wires were covered with dental cement (Palaferm). The tips of the electrodes were left bare for 2-3 mm and were spaced parallel 3 mm apart. The electrodes were handled with a nipper in order to introduce them into the isolated nerve. Antibiotic treatment (penicillin 100,000 I.U.) was started after surgery and continued over the following three days. The catheters were filled with heparinized saline and flushed each day with 2-3 ml of saline. For recording, the aortic catheter was connected to a Statham P23 dB pressure trans_ ducer fixed in a harness on the back of the animal, and blood pressure was displayed on a beam of an oscilloscope (Tektronix 502 A) using the d.c. channel, and simultaneously on a channel of a U.V.recorder (San’Ei). The wires of the electrodes were connected to a differentialpreamplifier, suitablyamplifiedand displayed simultaneously on the oscilloscope and on a channel of the U.V. recorder by using a special type of galvanometer which can record frequencies of up to 3000 Hz. The band between 100 and 1000 Hz was selected. Splanchnic action potentials were picked up at the output of the preamplifier, then fully rectified and smoothed with an R. C. operational amplifier. The smoothed signal was displayed on a third channel of the U.V.recorder and was approximately proportional to the mean electrical activity. In this way, it was possible to record splanchnic discharges for l-3 months. Forty-five dogs were used 3-5 days after surgery. Troubles were encountered with the catheters: in fact they were usually rejected lG30 days after the preparation, but without haemorrhage. RESULTS
Types of discharges
In unanaesthetized dogs, the pattern of preganglionic sympathetic discharges was identical with that observed under conditions of anaesthesia. Discharges synchronous with respiratory movements were frequently observed, but continuous or irregular patterns could also be recorded in the same animal. Arousal, emotions and excitement increased splanchnic discharges. The sympathetic discharges were usually higher when the dog was standing quiet than when the dog was lying on the table. For pharmacological experiments a long steady state was needed. For this purpose, the dogs were trained to rest quietly standing or lying. Under these conditions the heart rate usually showed a marked respiratory arrhythmia. Figure 1 shows the record of blood pressure and irregular splanchnic discharges which were not synchronized with heart rate or respiratory movements in a dog. At the end of the record, the splanchnic activity disappeared and blood pressure decreased slightly during a period of sedation.
Sympathetic
Conscious
200 0
nerve activity
in conscious
dog
dogs
349
Splanchnic
discharges
~~*~~~~~~~~~~~~~~~\ W\N ,*\\r* ‘*ur’-w@--* Blood
52
pressure
Fig. I. Records of blood pressure (in mm Hg) and splanchnic discharges in a conscious dog. Blood pressure shows marked respiratory arrhythmias. The splanchnic discharges were irregular without any synchronization with respiratory or heart rate. At the end of the record splanchnic discharges disappeared and blood pressure fell during a period of sedation.
This type of preparation could be checked and the results compared with those obtained in anaesthetized animals. After 15-30 min of recording steady blood pressure and splanchnic discharges, clonidine was injected intravenously*at the dose of 5-lOpg/kg. The effects were similar to those described in anaesthetized animals. Clonidine induced a marked reduction in splanchnic discharges; a brief hypertension occurred followed by a decrease of 10-20 mm Hg in blood pressure; heart rate was slowed. Vomiting usually occurred 3-5 min after the injection. The dogs were mildly sedated. Figure 2 shows the effects of clonidine (10 pg/kg) administered orally. No increase in blood pressure appeared. Ten minutes after the administration, blood pressure, heart rate and splanchnic discharges were reduced; thirty minutes later the effects were more pronounced. This experiment suggests a rapid absorption of clonidine by the gastrointestinal tract. It was of interest to look for an eventual tolerance to the effects of clonidine. Five dogs were treated twice a day with lOpg/kg of clonidine intravenously for 10 days. The effects were the same after each administration. It is clear that no tolerance to the activity of clonidine on central sympathetic structures occurred. 10 min
30 min
Clonidine 10ah? Fig. 2. Effects of clonidine administered orally on blood pressure (m mm Hg) and splanchnic discharges m a conscious dog. Clonidine (10 pgjkg orally) reduced blood pressure and heart rate, and abolished splanchnic discharges.
DISCUSSION
A technique for recording the activity in the preganglionic sympathetic fibres of the splanchnic nerve in conscious dogs and its application to some pharmacological problems are described. Recording sympathetic nerve activity in animals is a valuable technique in looking for the action of drugs on the sympathetic centres in acute experiments, especially when
350
H. SCHMITT. HELENE SCHMITT and S.
FENARD
the main reflex pathways have been ruled out. However the technique could not be used in chronic experiments. Some authors (BAUM,SHKOPSHIRE and VAKNEK,1972; TAUBERGER and KUHN, 1971) have tried to overcome the difficulty by comparing sjimpathetic nerve activity in groups of control and treated animals. Although possibly valuable when the changes are striking, this method needs large groups of animals for several reasons: (a) An absolute quantitative evaluation of the sympathetic nerve activity is difficult. The integrated sympathetic activity at the beginning of the experiments is taken as 100 per cent and the noise after death as 0 per cent. (b) The sympathetic nerve activity and the pattern of discharges vary widely in animals and therefore statistical analysis requires large groups of animals. Discharges of single sympathetic fibres could not have been used for this purpose, for in the same animal some fibres discharge and others are silent. (c) Recording of sympathetic discharges was performed after stripping the nerve from its sheath and using either the whole or a bundle of fibres for recording. Thus the skill with which the preparation is performed plays an essential role in the intensity of the discharges recorded. The technique described in this paper allows the recording of sympathetic nerve activity in the same animal with the electrodes remaining always in the same place. Recording could be performed in unanaesthetized animals. Due to the insertion of the electrodes into the nerve, the interelectrode resistance is low (4.0 Q), allowing easy recording. The records could be compared from day to day. This technique is therefore valuable for physiological and pharmacological purposes. The pattern of splanchnic discharges varied widely from animal to animal and sometimes in the same dog. Three main patterns of splanchnic discharges were recognized. The most frequent one was a continuous type of discharges. Respiratory groupings and irregular patterns of discharges were also encountered. These patterns were well recognized in anaesthetized animals (ADRIAN,BRONKand PHILLIPS,1932; BRONK,FERGUSON,MARGARIA and SOLANDT,1936; OKADAand Fox, 1967) and were described previously in unanaesthetized cats (BAUSTet al.. 1968). The method appears to be suitable for investigating the long term effects of drugs on the central control of the sympathetic tone. It has been applied to the study of the effects of clonidine, a drug known to centrally inhibit the sympathetic tone. Our results show that the effects of clonidine were similar in unanaesthetized and in anaesthetized animals. Clonidine has been shown to reduce pre- and postganglionic sympathetic discharges in animals (SCHMITTet al., 1967, 1968; HUKUHAKA,OTSUKA,TAKEDA and SAKAI,1968; KLUPP et al., 1970), but the importance of this effect for the antihypertensive action of the drug has been questioned (ZA’IMISand HANINGTON,1969). Our results show that clonidine induced a decrease in the sympathetic nerve activity in small doses. On the other hand, no tolerance to the effect of the drug after a lo-day treatment could be detected. It is therefore likely that the centrally-mediated decrease in the sympathetic tone plays a role in antihypertensive action of the drug, although peripheral sites of action are not entirely ruled out. The method described in this work allows an easy study of the effects of drugs on splanchnic discharges and blood pressure in conscious dogs. It is therefore valuable ih disclosing centrally or reflexly mediated changes in the sympathetic tone. Acknowledyrrilrnr-TRis work was carried de la Recherche MCdicale).
out with a grant
from I.N.S.E.R.M.
(Institut
National
de la Santt- et
Sympathetic
nerve activity
in conscious
dogs
3.51
REFERENCES ADRIAN, E. D., BRONK, D. W. and PHILLIPS, G. (1932). Discharges in mammalian sympathetic nerves. J. Physiol., Lond. 74: 11>133. BAUM, T.. SHROPSHIRE, A. T. and VARNER, L. L. (1972). Contribution of the central nervous system to the action of several antihypertensive agents (methyldopa, hydralazine and guanethidine), J. Phurmuc. exp. Thrr. 182: 135-144. BAUST, W., WEIDINGER, H. and KIRCHNER, F. (1968). Sympathetic activity during natural sleep and arousal. Arch. ital. Bio/. 106: 379-390. BRONK, D. W., FERGUSON, L. K.. MARGARIA, R. and SOLANIX, D. Y. (1936). The activity of the cardiac sympathetic centers. Am. J. Physiol. 117: 237-249. HUKUHARA, T., JR., OTSUKA, Y.. TAKEDA, R. und SAKAI, F. (1968). Die zentralen Wirkungen des 2-(2,6-Dichlorophenylamino)-2-imidazolin hydrochlorids. Arxeirnitteljbrsch. 18: 1147-l 153. KLUPP, H., KNAPPEN, F., OTSUKA, Y.. STRELLEK, I. and TEICHMANN, H. (1970). Effects of clonidine on central sympathetic tone. Eur. J. Pharmac. 10: 22>229. OKADA, H. and FOX, I. J. (1967). Respiratory grouping of abdominal sympathetic activity in the dog. Am. J. Physiol. 213: 48-56. SCHMITT, H., SCHMITT, H., BOISSIER, J. R. and GIUDICELLI, J. F. (1967). Centrally mediated decrease in sympathetic tone induced by 2-(2,6-dichlorophenylamino)-2-imidazoline (St 155, Catapresan). Eur. J. Pharmac. 2: 147-148. SCHMITT, H., SCHMITT. H., BOISSIER, J. R., GIUDICELLI, J. F. and FITHELLE. J. (1968). Cardiovascular effects of 2-(2,6-dichlorophenylamino)-2-imidazoline St 155): Central sympathetic structures. Eur. J. Pharmac. 2: 34(t 346. TAUBERGER, G. und KUHN, P. (1971). Untersuchungen der zentralnerviisen sympathicusdgmpfenden Wirkungen von a-Methyldopa. Arch. Pharmakol. 268: 33-43. WEIDINGER, H. und KIRCHNER, F. (1967). Sympathische Aktionspotentiale bei nicht narkotisierten, freibewegliechen Katzen. Arch. ges. Physiol. 294: R. 23. ZA~‘MIS,E. and HANNINGTON, E. (1969). A possible pharmacological approach to migraine. Lancrt 2: 298-300.
17 October
15 rue de 1’Ecole de Mede-
1973)
Summary-A technique for recording splanchnic discharges chronically, in conscious dogs is described. Two small stainless steel electrodes were inserted into the splanchnic nerves and secured with dental cement. The wires were passed under the skin and attached to plugs. With this technique intravenous clonidine was shown to decrease splanchnic discharges, to induce bradycardia and biphasic changes in blood pressure: an increase followed by a decrease. By peroral route the effects were the same, except that the initial hypertension did not occur. No tolerance to this effect of clonidine was seen during the 10 days treatment. The method appears to be valuable in disclosing centrally or reflexly mediated decreases in the sympathetic tone.
Recording of activity in sympathetic nerves has been widely used to investigate central or reflex physiological or pharmacological influences on the sympathetic tone. However, the experiments have, as yet, been performed only on anaesthetized or paralyzed animals, precluding any investigation on longterm induced changes on this tone. WEIDINGER and KIRCHNER (1967) and BAUST, WEIDINGER and KIRCHNER (1968) recorded in conscious cats the activity of the renal postganglionic sympathetic nerve simultaneously with the EEG and EMG to investigate the influence of wakefulness, synchronized and desynchronized phases of sleep on the sympathetic tone. However, the technique did not allow distinction between ganglionic or central sites of action. It was therefore of interest to develop a technique to record chronically the discharges in a preganglionic sympathetic nerve together with changes in blood pressure. This paper describes such a technique and the results obtained with clonidine. and agent known to reduce sympathetic discharges in anaesthetized animals (SCHMITT, SCHMITT, BOISSIER and GIUDICELLI, 1967; SCHMITT, SCHMITT, BOISSIER,GIUDICELLIand FICHELLE,1968; KLUPP, KNAPPEN,OTSUKA, STRELLER and TEICHMANN,1970). MATERIALS
AND METHODS
Mongrel dogs of either sex weighing 5-l 1 kg were used. They were anaesthetized with pentobarbital (30 mg/kg i.v.). Under sterile conditions, a carotid artery was isolated and a polyethylene catheter 1.5 mm in external diameter (K-32 Feeding tube: Size 5 French) and 25 cm in length was introduced into the artery as far as the aorta. A second catheter was introduced into the jugular vein in order to perform the intravenous injections. Both catheters were closed with a rubber cap and passed under the skin to the back of the neck. In some experiments the right vertebral artery was isolated at the base of the neck and ligatured distally; a small polyethylene catheter was introduced into the cephalic end of the artery and sutured. The catheter was passed under the skin to the neck. In this way. 347
348
H.
SCHMITT,
H~LZNE SCHMITTand S. FENARD
administration of small doses of drugs could be performed in order to restrict the action to the rhombencephalon. The left splanchnic nerve was approached retroperitoneally by a paravertebral incision. The nerve was isolated at its exit from the diaphragm. A pair of special electrodes described below was introduced into the nerve; the electrodes were isolated and secured on the nerve with dental cement (Palaferm). The wires were passed under the skin to the back of the neck where plugs were attached for recording. To construct the electrodes, the end of a stainless steel dental broach (10 pm at the top) was cut at 34 mm from the tip and was soldered to a stainless steel wire coated with teflon (wire diameter: 0908; Medwire Corporation Vernon). Both electrodes, except for the tip (2-3 mm), as well as the soldering and l-2 mm of the wires were covered with dental cement (Palaferm). The tips of the electrodes were left bare for 2-3 mm and were spaced parallel 3 mm apart. The electrodes were handled with a nipper in order to introduce them into the isolated nerve. Antibiotic treatment (penicillin 100,000 I.U.) was started after surgery and continued over the following three days. The catheters were filled with heparinized saline and flushed each day with 2-3 ml of saline. For recording, the aortic catheter was connected to a Statham P23 dB pressure trans_ ducer fixed in a harness on the back of the animal, and blood pressure was displayed on a beam of an oscilloscope (Tektronix 502 A) using the d.c. channel, and simultaneously on a channel of a U.V.recorder (San’Ei). The wires of the electrodes were connected to a differentialpreamplifier, suitablyamplifiedand displayed simultaneously on the oscilloscope and on a channel of the U.V. recorder by using a special type of galvanometer which can record frequencies of up to 3000 Hz. The band between 100 and 1000 Hz was selected. Splanchnic action potentials were picked up at the output of the preamplifier, then fully rectified and smoothed with an R. C. operational amplifier. The smoothed signal was displayed on a third channel of the U.V.recorder and was approximately proportional to the mean electrical activity. In this way, it was possible to record splanchnic discharges for l-3 months. Forty-five dogs were used 3-5 days after surgery. Troubles were encountered with the catheters: in fact they were usually rejected lG30 days after the preparation, but without haemorrhage. RESULTS
Types of discharges
In unanaesthetized dogs, the pattern of preganglionic sympathetic discharges was identical with that observed under conditions of anaesthesia. Discharges synchronous with respiratory movements were frequently observed, but continuous or irregular patterns could also be recorded in the same animal. Arousal, emotions and excitement increased splanchnic discharges. The sympathetic discharges were usually higher when the dog was standing quiet than when the dog was lying on the table. For pharmacological experiments a long steady state was needed. For this purpose, the dogs were trained to rest quietly standing or lying. Under these conditions the heart rate usually showed a marked respiratory arrhythmia. Figure 1 shows the record of blood pressure and irregular splanchnic discharges which were not synchronized with heart rate or respiratory movements in a dog. At the end of the record, the splanchnic activity disappeared and blood pressure decreased slightly during a period of sedation.
Sympathetic
Conscious
200 0
nerve activity
in conscious
dog
dogs
349
Splanchnic
discharges
~~*~~~~~~~~~~~~~~~\ W\N ,*\\r* ‘*ur’-w@--* Blood
52
pressure
Fig. I. Records of blood pressure (in mm Hg) and splanchnic discharges in a conscious dog. Blood pressure shows marked respiratory arrhythmias. The splanchnic discharges were irregular without any synchronization with respiratory or heart rate. At the end of the record splanchnic discharges disappeared and blood pressure fell during a period of sedation.
This type of preparation could be checked and the results compared with those obtained in anaesthetized animals. After 15-30 min of recording steady blood pressure and splanchnic discharges, clonidine was injected intravenously*at the dose of 5-lOpg/kg. The effects were similar to those described in anaesthetized animals. Clonidine induced a marked reduction in splanchnic discharges; a brief hypertension occurred followed by a decrease of 10-20 mm Hg in blood pressure; heart rate was slowed. Vomiting usually occurred 3-5 min after the injection. The dogs were mildly sedated. Figure 2 shows the effects of clonidine (10 pg/kg) administered orally. No increase in blood pressure appeared. Ten minutes after the administration, blood pressure, heart rate and splanchnic discharges were reduced; thirty minutes later the effects were more pronounced. This experiment suggests a rapid absorption of clonidine by the gastrointestinal tract. It was of interest to look for an eventual tolerance to the effects of clonidine. Five dogs were treated twice a day with lOpg/kg of clonidine intravenously for 10 days. The effects were the same after each administration. It is clear that no tolerance to the activity of clonidine on central sympathetic structures occurred. 10 min
30 min
Clonidine 10ah? Fig. 2. Effects of clonidine administered orally on blood pressure (m mm Hg) and splanchnic discharges m a conscious dog. Clonidine (10 pgjkg orally) reduced blood pressure and heart rate, and abolished splanchnic discharges.
DISCUSSION
A technique for recording the activity in the preganglionic sympathetic fibres of the splanchnic nerve in conscious dogs and its application to some pharmacological problems are described. Recording sympathetic nerve activity in animals is a valuable technique in looking for the action of drugs on the sympathetic centres in acute experiments, especially when
350
H. SCHMITT. HELENE SCHMITT and S.
FENARD
the main reflex pathways have been ruled out. However the technique could not be used in chronic experiments. Some authors (BAUM,SHKOPSHIRE and VAKNEK,1972; TAUBERGER and KUHN, 1971) have tried to overcome the difficulty by comparing sjimpathetic nerve activity in groups of control and treated animals. Although possibly valuable when the changes are striking, this method needs large groups of animals for several reasons: (a) An absolute quantitative evaluation of the sympathetic nerve activity is difficult. The integrated sympathetic activity at the beginning of the experiments is taken as 100 per cent and the noise after death as 0 per cent. (b) The sympathetic nerve activity and the pattern of discharges vary widely in animals and therefore statistical analysis requires large groups of animals. Discharges of single sympathetic fibres could not have been used for this purpose, for in the same animal some fibres discharge and others are silent. (c) Recording of sympathetic discharges was performed after stripping the nerve from its sheath and using either the whole or a bundle of fibres for recording. Thus the skill with which the preparation is performed plays an essential role in the intensity of the discharges recorded. The technique described in this paper allows the recording of sympathetic nerve activity in the same animal with the electrodes remaining always in the same place. Recording could be performed in unanaesthetized animals. Due to the insertion of the electrodes into the nerve, the interelectrode resistance is low (4.0 Q), allowing easy recording. The records could be compared from day to day. This technique is therefore valuable for physiological and pharmacological purposes. The pattern of splanchnic discharges varied widely from animal to animal and sometimes in the same dog. Three main patterns of splanchnic discharges were recognized. The most frequent one was a continuous type of discharges. Respiratory groupings and irregular patterns of discharges were also encountered. These patterns were well recognized in anaesthetized animals (ADRIAN,BRONKand PHILLIPS,1932; BRONK,FERGUSON,MARGARIA and SOLANDT,1936; OKADAand Fox, 1967) and were described previously in unanaesthetized cats (BAUSTet al.. 1968). The method appears to be suitable for investigating the long term effects of drugs on the central control of the sympathetic tone. It has been applied to the study of the effects of clonidine, a drug known to centrally inhibit the sympathetic tone. Our results show that the effects of clonidine were similar in unanaesthetized and in anaesthetized animals. Clonidine has been shown to reduce pre- and postganglionic sympathetic discharges in animals (SCHMITTet al., 1967, 1968; HUKUHAKA,OTSUKA,TAKEDA and SAKAI,1968; KLUPP et al., 1970), but the importance of this effect for the antihypertensive action of the drug has been questioned (ZA’IMISand HANINGTON,1969). Our results show that clonidine induced a decrease in the sympathetic nerve activity in small doses. On the other hand, no tolerance to the effect of the drug after a lo-day treatment could be detected. It is therefore likely that the centrally-mediated decrease in the sympathetic tone plays a role in antihypertensive action of the drug, although peripheral sites of action are not entirely ruled out. The method described in this work allows an easy study of the effects of drugs on splanchnic discharges and blood pressure in conscious dogs. It is therefore valuable ih disclosing centrally or reflexly mediated changes in the sympathetic tone. Acknowledyrrilrnr-TRis work was carried de la Recherche MCdicale).
out with a grant
from I.N.S.E.R.M.
(Institut
National
de la Santt- et
Sympathetic
nerve activity
in conscious
dogs
3.51
REFERENCES ADRIAN, E. D., BRONK, D. W. and PHILLIPS, G. (1932). Discharges in mammalian sympathetic nerves. J. Physiol., Lond. 74: 11>133. BAUM, T.. SHROPSHIRE, A. T. and VARNER, L. L. (1972). Contribution of the central nervous system to the action of several antihypertensive agents (methyldopa, hydralazine and guanethidine), J. Phurmuc. exp. Thrr. 182: 135-144. BAUST, W., WEIDINGER, H. and KIRCHNER, F. (1968). Sympathetic activity during natural sleep and arousal. Arch. ital. Bio/. 106: 379-390. BRONK, D. W., FERGUSON, L. K.. MARGARIA, R. and SOLANIX, D. Y. (1936). The activity of the cardiac sympathetic centers. Am. J. Physiol. 117: 237-249. HUKUHARA, T., JR., OTSUKA, Y.. TAKEDA, R. und SAKAI, F. (1968). Die zentralen Wirkungen des 2-(2,6-Dichlorophenylamino)-2-imidazolin hydrochlorids. Arxeirnitteljbrsch. 18: 1147-l 153. KLUPP, H., KNAPPEN, F., OTSUKA, Y.. STRELLEK, I. and TEICHMANN, H. (1970). Effects of clonidine on central sympathetic tone. Eur. J. Pharmac. 10: 22>229. OKADA, H. and FOX, I. J. (1967). Respiratory grouping of abdominal sympathetic activity in the dog. Am. J. Physiol. 213: 48-56. SCHMITT, H., SCHMITT, H., BOISSIER, J. R. and GIUDICELLI, J. F. (1967). Centrally mediated decrease in sympathetic tone induced by 2-(2,6-dichlorophenylamino)-2-imidazoline (St 155, Catapresan). Eur. J. Pharmac. 2: 147-148. SCHMITT, H., SCHMITT. H., BOISSIER, J. R., GIUDICELLI, J. F. and FITHELLE. J. (1968). Cardiovascular effects of 2-(2,6-dichlorophenylamino)-2-imidazoline St 155): Central sympathetic structures. Eur. J. Pharmac. 2: 34(t 346. TAUBERGER, G. und KUHN, P. (1971). Untersuchungen der zentralnerviisen sympathicusdgmpfenden Wirkungen von a-Methyldopa. Arch. Pharmakol. 268: 33-43. WEIDINGER, H. und KIRCHNER, F. (1967). Sympathische Aktionspotentiale bei nicht narkotisierten, freibewegliechen Katzen. Arch. ges. Physiol. 294: R. 23. ZA~‘MIS,E. and HANNINGTON, E. (1969). A possible pharmacological approach to migraine. Lancrt 2: 298-300.