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Cholinergic nature of the primary afferent vagus synapsed in cross anastomosed superior cervical ganglia
Life Sciences Vol . 19, pp . Printed in the U .S .A .
1175-1180, 1976 .
Pergamon Press
CHOLINERGIC NATURE OF THE PRIMARY AFFERENT VAGUS SYNAPSED IN CROSS ANASTOMOSED SUPERIOR CERVICAL GANGLIA Motohatsu Fujiwara and Kazuyoshi Kurahashi Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan . (Received in final form August 26, 1976)
Summary The superior cervical ganglion was reinnervated by vagal afferent fibers following heterologous cross anastomoses between the superior cervical preganglionic trunk and the vagal trunk at the level of the supranodose ganglion in cats . The contractions of the nictitating membrane and the postganglionic action potentials from the external carotid sinus nerve in response to electrical stimulation of the vagal artificial preganglionic trunk in these operated cats were inhibited by treatment with tetraethylammonium and atropine . The choline acetyltransferase activities were measured by the radiometric method . The activities in cross anastomosed superior cervical ganglion were lower than those of normal superior cervical ganglion, but higher than those of chronically decentralized superior cervical ganglion . The activities in cross anastomosed nodose ganglion were lower than those of normal nodose ganglion, but higher than those of chronically decentralized superior cervical ganglion . These results further support the view that the primary afferent vagus artificially synapsed in the superior cervical ganglion is cholinergic . It has been suggested by De Castro (1,2) that the decentralized superior cervical ganglion reinnervated by the afferent vagus in cat is non-cholinergic in nature, because the contractions of the nictitating membrane induced by electrical stimulation of the artificial preganglionic trunk could not be potentiated by treatment with eserine . On the other hand, it was postulated by Fujiwara (formerly Matsumura) and Koelle (3), using the same type of preparation, that the artificial synapse in the reinnervated ganglion was cholinergic in nature, because contractions of the nictitating membrane induced by electrical stimulation of the vagal afferent fibers were blocked by treatment with tetraethylammonium (TEA) and potentiated by eserine, and because acetylcholinesterase was detected histochemically in the nodose and superior cervical ganglia reinnervated by the afferent vagus and activities were significantly higher than those in chronically decentralized superior cervical ganglion . Subsequent studies using electron microscopic observations and recording of action potentials from the postganglionic fibers confirmed the 1175
1176
Choliaergic Nature of Afferent Vagus
Vol . 19, No . 8
latter view (4) . The present experiments were an attempt to procure additional evidence to support the postulation of nature of the decentralized superior cervical ganglion reinnervated by the afferent vague fibers in cats . Methods For cross anastomosis, cats of either sex weighing 2 .0 to 4 .5 kq were anesthetized with sodium pentobarbital (35 mg/kg, i .p .) . Under aseptic conditions, tissues adjacent to both the nodose ganglion and the superior cervical ganglion were carefully dissected by forceps in such a way so as to minimize hervorrage and damage to the ganglia . The anastomosis between the peripheral vague, sectioned central to the nodose ganglion, and the peripheral cervical sympathetic trunk was performed according to the descriptions of De Castro (1,2), Fujiwara (formerly Matsumura) and Koelle (3), and Fujiwara and Kurahashi (4) . The central root of the nodose ganglion was dissected as far as possible, and then an epineural suture of ELP-eyeless needled, black braided silk FIG . 1
S.C .G.
STOMACH
STOMACH
Diagrammatic representation of the synaptic relationships in the normal (left) vagal nodose ganglion (NOD .G .) and superior cervical ganglion (S .C .G .), and in the cross anastomosed (right) cats . Thick line : vagal afferent fibers . Broken line : cholinergic fibers . Thin line adrenergic fibers . Just was placed approximately 2 to 5 mm from the ganglion . rostrad to the suture, the vagal trunk was cut with iridectomy scissors . The cervical sympathetic preganglionic trunk was then dissected free from adjacent tissues for a length of about
Vol. 19, No . 8
Cholinergic Nature of Afferent Vagua
117 7
2 .0 cm . The same suture was placed through the epineurium 1 .5 cm caudad to the superior cervical ganglion . After the sympathetic trunk had been cut just caudad to the suture, both ends of the suture were gently pulled to approximate the cut ends of the two trunks and knotted . Since the diameter of the vague is considerably greater than that of the cervical sympathetic trunk, special attension was paid to obtain good union of the two ends (Fig . 1) . After this procedure, all cats showed unilateral marked relaxation of the nictitating membrane and miosis . In most surviving 23 animals out of operated 103, the nictitating membrane became retracted and the pupil showed a tendency to return the original size within 2 to 3 months after anastomosis . At successive intervals following anastomosis, functional return was tested physiologically by noting the responses (unilateral mydriasis and contraction of the nictitating membrane) to inflation of the stomach with a balloon under pentobarbital anesthesia (35 mg/kg, i .p .) (Fig . 2) . For definitive experiments on anastomosed cats after over 6 months of operation, the cats were anesthetized with urethane (1 .5 g/kg, i .p .) and supplemental intraperitoneal injections were given as required . The trachea was cannulated for natural and artificial respirations . A segment of the corresponding vagal trunk, extending about 3 cm caudad from the nodose ganglion was dissected free from adjacent tissue and placed on platinum electrodes . Square-wave impulses were applied to the vagal artificial preganglionic trunk for periods of 10 seconds . FIG . 2
Functional test of the anastomosed cat : Unilateral right) mydriasis and contraction of the nictitating membrane in response to inflation of the stomach with a balloon or electrical stimulation of the vagal artificial preganglionic trunk in an anastomosed cat (123 days after surgery) . For decentralized superior cervical ganglion, cats were anesthetized with sodium pentobarbital (35 mg/kg, i .p .) . Under aseptic conditions, the cervical sympathetic trunk was excised about 2 cm caudad to the ganglion and the cut end was ligated to
1178
Choliaergic Nature of Afferent Vague
Vol . 19, No . 8
prevent regeneration of the preganglionic fibers . days were then allowed for degeneration .
Five to
28
The responses of the nictitating membrane induced by electrical stimulation of the vagal artificial preganglionic trunk were recorded on a pen-recorder via force-displacement transducer . The action potentials from the postganglionic external carotid sinus nerve in response to electrical stimulation of the afferent vague were monitored on the oscilloscope and recorded on film . All agents were injected through the lingual artery with clamping the external carotid artery . Choline acetyltransferase activities were measured by the radiometric assay procedures of Fonnum (5) . Results To evoke definite contractions of the nictitating membrane in response to electrical stimulation of the vagal artificial preganglionic trunk in anastomosed cats, stimuli of higher voltage FIG . 3
TEA (IOO~g1 ~~~~~ "
~~ 4 2mln
Atroplne (2 ;2oy~~)
J Effect of tetraethylammonium (TEA) (100 lzg, " , o) and atropine (2, 20 ug, " ,o ) on contractions of the nictitating membrane (upper trace) and action potentials in the postganglionic fibers (lower trace) induced by stimulation of the vagal artificial preganglionic trunk in anastomosed cats . A 10-sec tetanic stimulus of 10/sec was applied once every 3 min to produce contractions of the nictitating membrane and a 1-msec single stimulus once every 2 sec to evoke action potentials . than required with normal preganglionic sympathetic trunk were necessary . The mean contractile response of the nictitating membrane in 10 anastomosed cats (189 to 652 days after surgery) was 3 .6 ± 0 .5 g and that in 91 normal cats was 7 .3 ± 0 .2 g . The amplitude of postganglionic action potentials in anastomosed
Vol . 19, No . 8
Cholinergic Nature of Afferent Vagus
1179
The cats was about 20 uv and that in normal was over 200 Jav . contractions of the nictitating membrane and the postganglionic action potentials in response to electrical stimulation of the vagal trunk in anastomosed cats were inhibited dose-dependently by treatment with either TEA (5 to 100 }ig, i .a .) or atropine (2 to 20 Jag, i .a .) (Fig . 3) . Choline acetyltransferase activities were compared among the superior cervical ganglion reinnervated by the vagal afferent fibers, the normal superior cervical ganglion, and the chronically decentralized superior cervical ganglion . The anastomosed superior cervical ganglia were used for radiometric assay 175 to 475 days after operation and the chronically decentralized ganglia 5 to 15 days after operation . TABLE 1 Choline Acetyltranaferase Activities in Superior Cervical Ganglion (S .C .G .) and Nodose Ganglion (NOD .G .) in Anastomosed, Normal and Chronically Decentralized Cats . Nanom les/Ganglion/Hour Anastomosed S .C .G . NOD .G .
33 .5 t 10 .0 3 .2 t
(4)
0 .4 (4)
Normal
Decentralized
143 .2 t 14 .2*(16)
0 .7 t p .4*(4)
6 .2 t
1 .1 (13)
-------------
Number of observations in parentheses . Significantly different from the anastomosed cats (* P ~ 0 .001) . In anastomosed superior cervical ganglia, choline acetyltransferase activities were about one-fourth those in normal animals, but significantly higher than those in chronically decentralized ganglia which showed no mechanical or electrical responses following electrical stimulation of the preganglionic trunk . The activities in anastomosed nodose ganglia were lower than those in normal nodose ganglia, but significantly higher than in decentralized superior cervical ganglia (Table 1) . Discussion Signs indicating functional reinnervation of the afferent vagus, sectioned cranial to the nodose ganglion, in the superior cervical ganglion were as follows : (a) production of signs of unilateral sympathetic hyperactivity, in the area of distribution of the postganglionic fibers of the cross anastomosed superior cervical ganglion, in response to stimulation of the vagal afferent fibers by gastric dilatation, and contraction of the corresponding nictitating membrane following electrical stimulation of the vagal artificial preganglionic trunk (1,2,3,4), (b) recording of the action potentials which has been successfully performed from the carotid sinus branch of the postganglionic nerve in response to electrical stimulation of the vagal artificial preganglionic
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Cholinergic Nature of Afferent Vague
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trunk, (c) electron microscopic findings demonstrating establishment of synaptic connection of the regenerating fibers to the superior cervical ganglion (unpublished data) . The afferent vague, as well as other primary afferent fibers is generally considered to be non-cholinergic . This assumption is based chiefly on the relatively low activities of acetylcholinesterase (6), the extremely low amounts of acetylcholine (7) and choline acetyltransferase activities ($) found in dorsal roots and other nerves which consist almost exclusively of primary afferent fibers . In the present work, the postganglionic action potentials as well as contractions of the nictitating membrane in response to electrical stimulation of the vagal artificial preganglionic trunk were blocked by treatment with TEA and atropine . Such findings confirm the view that the nature of the transmission in the superior cervical ganglion artificially synapsed by the vagal afferent fibers is cholinergic and that the muscarinic as well as nicotinic receptors are involved in the transmission . It has been reported that choline acetyltransferase appears to be a specific marker for cholinergic nerves in the central nervous system (9,10,11,12) . Using sensitive radiometric assay procedures which have major advantages of enabling quantification of enzyme activity in low activity regions, and of being specific for choline acetyltransferase, the enzyme activities in the superior cervical ganglion reinnervated by the afferent vague and the nodose ganglion of anastomosed cats were measured . These activities were lower than those of normal superior cervical ganglion, but higher than those of chronically decentralized ganglion . These results further support the hypothesis (3) that the primary afferent vague synapsed in the superior cervical ganglion is at least in part cholinergic . Ackn owledg ement s This work was supported by a grant No . 010604 from the Ministry of Education of Japan . Thanks are due to M . Ohara, Kyoto University, for assistance with preparation of the manuscript . References 1. 2. 3. 4. 5. 6. 7.
8.
9. 10 . 11 . 12 .
F . DE CASTRO, Trab . Lab . Invest . biol . Univ . Madr . _34, 217 - 301 (194 F . DE CASTRO, Ar ch . int . Physiol . 59, 479 - 519 (1951) . M. MATSUMURA an~lc G . B . KOELLE, J . Pfiärmacol . exp. Ther . 134, 28 - 46 (1961) . Ff-FUJIWARA and K . KURAHASHI, Proc . West . Pharmacol . Soc . 18, 225 - 228 (1975) . F . FONNUM, Biochem. J . 115, 465 - 472 (1969) . 114, 296 A. S . V . BURGEN and L . M . CHIPMAN, J . Physiol . 305 (1951) . F . C . MACINTOSH, J . Physiol . 9~ 436 - 442 (1941) . 176, 505 - 507 (1954) . F . J . WOLFGRAM, Amer . J . Physiol . W. FELDBERG and M. VOGT, J . Physiol . 107, 372 - 381 (1948) . G . B . KOELLE, J . com . Ph siol . 100 211 - 235 (1954) . C . HEBB, Ph sio . Rev . , 196 -~ (1957) . C . HEBB, Nature Lon on)~52, 527 - 529 (1961) .
1175-1180, 1976 .
Pergamon Press
CHOLINERGIC NATURE OF THE PRIMARY AFFERENT VAGUS SYNAPSED IN CROSS ANASTOMOSED SUPERIOR CERVICAL GANGLIA Motohatsu Fujiwara and Kazuyoshi Kurahashi Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan . (Received in final form August 26, 1976)
Summary The superior cervical ganglion was reinnervated by vagal afferent fibers following heterologous cross anastomoses between the superior cervical preganglionic trunk and the vagal trunk at the level of the supranodose ganglion in cats . The contractions of the nictitating membrane and the postganglionic action potentials from the external carotid sinus nerve in response to electrical stimulation of the vagal artificial preganglionic trunk in these operated cats were inhibited by treatment with tetraethylammonium and atropine . The choline acetyltransferase activities were measured by the radiometric method . The activities in cross anastomosed superior cervical ganglion were lower than those of normal superior cervical ganglion, but higher than those of chronically decentralized superior cervical ganglion . The activities in cross anastomosed nodose ganglion were lower than those of normal nodose ganglion, but higher than those of chronically decentralized superior cervical ganglion . These results further support the view that the primary afferent vagus artificially synapsed in the superior cervical ganglion is cholinergic . It has been suggested by De Castro (1,2) that the decentralized superior cervical ganglion reinnervated by the afferent vagus in cat is non-cholinergic in nature, because the contractions of the nictitating membrane induced by electrical stimulation of the artificial preganglionic trunk could not be potentiated by treatment with eserine . On the other hand, it was postulated by Fujiwara (formerly Matsumura) and Koelle (3), using the same type of preparation, that the artificial synapse in the reinnervated ganglion was cholinergic in nature, because contractions of the nictitating membrane induced by electrical stimulation of the vagal afferent fibers were blocked by treatment with tetraethylammonium (TEA) and potentiated by eserine, and because acetylcholinesterase was detected histochemically in the nodose and superior cervical ganglia reinnervated by the afferent vagus and activities were significantly higher than those in chronically decentralized superior cervical ganglion . Subsequent studies using electron microscopic observations and recording of action potentials from the postganglionic fibers confirmed the 1175
1176
Choliaergic Nature of Afferent Vagus
Vol . 19, No . 8
latter view (4) . The present experiments were an attempt to procure additional evidence to support the postulation of nature of the decentralized superior cervical ganglion reinnervated by the afferent vague fibers in cats . Methods For cross anastomosis, cats of either sex weighing 2 .0 to 4 .5 kq were anesthetized with sodium pentobarbital (35 mg/kg, i .p .) . Under aseptic conditions, tissues adjacent to both the nodose ganglion and the superior cervical ganglion were carefully dissected by forceps in such a way so as to minimize hervorrage and damage to the ganglia . The anastomosis between the peripheral vague, sectioned central to the nodose ganglion, and the peripheral cervical sympathetic trunk was performed according to the descriptions of De Castro (1,2), Fujiwara (formerly Matsumura) and Koelle (3), and Fujiwara and Kurahashi (4) . The central root of the nodose ganglion was dissected as far as possible, and then an epineural suture of ELP-eyeless needled, black braided silk FIG . 1
S.C .G.
STOMACH
STOMACH
Diagrammatic representation of the synaptic relationships in the normal (left) vagal nodose ganglion (NOD .G .) and superior cervical ganglion (S .C .G .), and in the cross anastomosed (right) cats . Thick line : vagal afferent fibers . Broken line : cholinergic fibers . Thin line adrenergic fibers . Just was placed approximately 2 to 5 mm from the ganglion . rostrad to the suture, the vagal trunk was cut with iridectomy scissors . The cervical sympathetic preganglionic trunk was then dissected free from adjacent tissues for a length of about
Vol. 19, No . 8
Cholinergic Nature of Afferent Vagua
117 7
2 .0 cm . The same suture was placed through the epineurium 1 .5 cm caudad to the superior cervical ganglion . After the sympathetic trunk had been cut just caudad to the suture, both ends of the suture were gently pulled to approximate the cut ends of the two trunks and knotted . Since the diameter of the vague is considerably greater than that of the cervical sympathetic trunk, special attension was paid to obtain good union of the two ends (Fig . 1) . After this procedure, all cats showed unilateral marked relaxation of the nictitating membrane and miosis . In most surviving 23 animals out of operated 103, the nictitating membrane became retracted and the pupil showed a tendency to return the original size within 2 to 3 months after anastomosis . At successive intervals following anastomosis, functional return was tested physiologically by noting the responses (unilateral mydriasis and contraction of the nictitating membrane) to inflation of the stomach with a balloon under pentobarbital anesthesia (35 mg/kg, i .p .) (Fig . 2) . For definitive experiments on anastomosed cats after over 6 months of operation, the cats were anesthetized with urethane (1 .5 g/kg, i .p .) and supplemental intraperitoneal injections were given as required . The trachea was cannulated for natural and artificial respirations . A segment of the corresponding vagal trunk, extending about 3 cm caudad from the nodose ganglion was dissected free from adjacent tissue and placed on platinum electrodes . Square-wave impulses were applied to the vagal artificial preganglionic trunk for periods of 10 seconds . FIG . 2
Functional test of the anastomosed cat : Unilateral right) mydriasis and contraction of the nictitating membrane in response to inflation of the stomach with a balloon or electrical stimulation of the vagal artificial preganglionic trunk in an anastomosed cat (123 days after surgery) . For decentralized superior cervical ganglion, cats were anesthetized with sodium pentobarbital (35 mg/kg, i .p .) . Under aseptic conditions, the cervical sympathetic trunk was excised about 2 cm caudad to the ganglion and the cut end was ligated to
1178
Choliaergic Nature of Afferent Vague
Vol . 19, No . 8
prevent regeneration of the preganglionic fibers . days were then allowed for degeneration .
Five to
28
The responses of the nictitating membrane induced by electrical stimulation of the vagal artificial preganglionic trunk were recorded on a pen-recorder via force-displacement transducer . The action potentials from the postganglionic external carotid sinus nerve in response to electrical stimulation of the afferent vague were monitored on the oscilloscope and recorded on film . All agents were injected through the lingual artery with clamping the external carotid artery . Choline acetyltransferase activities were measured by the radiometric assay procedures of Fonnum (5) . Results To evoke definite contractions of the nictitating membrane in response to electrical stimulation of the vagal artificial preganglionic trunk in anastomosed cats, stimuli of higher voltage FIG . 3
TEA (IOO~g1 ~~~~~ "
~~ 4 2mln
Atroplne (2 ;2oy~~)
J Effect of tetraethylammonium (TEA) (100 lzg, " , o) and atropine (2, 20 ug, " ,o ) on contractions of the nictitating membrane (upper trace) and action potentials in the postganglionic fibers (lower trace) induced by stimulation of the vagal artificial preganglionic trunk in anastomosed cats . A 10-sec tetanic stimulus of 10/sec was applied once every 3 min to produce contractions of the nictitating membrane and a 1-msec single stimulus once every 2 sec to evoke action potentials . than required with normal preganglionic sympathetic trunk were necessary . The mean contractile response of the nictitating membrane in 10 anastomosed cats (189 to 652 days after surgery) was 3 .6 ± 0 .5 g and that in 91 normal cats was 7 .3 ± 0 .2 g . The amplitude of postganglionic action potentials in anastomosed
Vol . 19, No . 8
Cholinergic Nature of Afferent Vagus
1179
The cats was about 20 uv and that in normal was over 200 Jav . contractions of the nictitating membrane and the postganglionic action potentials in response to electrical stimulation of the vagal trunk in anastomosed cats were inhibited dose-dependently by treatment with either TEA (5 to 100 }ig, i .a .) or atropine (2 to 20 Jag, i .a .) (Fig . 3) . Choline acetyltransferase activities were compared among the superior cervical ganglion reinnervated by the vagal afferent fibers, the normal superior cervical ganglion, and the chronically decentralized superior cervical ganglion . The anastomosed superior cervical ganglia were used for radiometric assay 175 to 475 days after operation and the chronically decentralized ganglia 5 to 15 days after operation . TABLE 1 Choline Acetyltranaferase Activities in Superior Cervical Ganglion (S .C .G .) and Nodose Ganglion (NOD .G .) in Anastomosed, Normal and Chronically Decentralized Cats . Nanom les/Ganglion/Hour Anastomosed S .C .G . NOD .G .
33 .5 t 10 .0 3 .2 t
(4)
0 .4 (4)
Normal
Decentralized
143 .2 t 14 .2*(16)
0 .7 t p .4*(4)
6 .2 t
1 .1 (13)
-------------
Number of observations in parentheses . Significantly different from the anastomosed cats (* P ~ 0 .001) . In anastomosed superior cervical ganglia, choline acetyltransferase activities were about one-fourth those in normal animals, but significantly higher than those in chronically decentralized ganglia which showed no mechanical or electrical responses following electrical stimulation of the preganglionic trunk . The activities in anastomosed nodose ganglia were lower than those in normal nodose ganglia, but significantly higher than in decentralized superior cervical ganglia (Table 1) . Discussion Signs indicating functional reinnervation of the afferent vagus, sectioned cranial to the nodose ganglion, in the superior cervical ganglion were as follows : (a) production of signs of unilateral sympathetic hyperactivity, in the area of distribution of the postganglionic fibers of the cross anastomosed superior cervical ganglion, in response to stimulation of the vagal afferent fibers by gastric dilatation, and contraction of the corresponding nictitating membrane following electrical stimulation of the vagal artificial preganglionic trunk (1,2,3,4), (b) recording of the action potentials which has been successfully performed from the carotid sinus branch of the postganglionic nerve in response to electrical stimulation of the vagal artificial preganglionic
1180
Cholinergic Nature of Afferent Vague
Vol . 19, No . 8
trunk, (c) electron microscopic findings demonstrating establishment of synaptic connection of the regenerating fibers to the superior cervical ganglion (unpublished data) . The afferent vague, as well as other primary afferent fibers is generally considered to be non-cholinergic . This assumption is based chiefly on the relatively low activities of acetylcholinesterase (6), the extremely low amounts of acetylcholine (7) and choline acetyltransferase activities ($) found in dorsal roots and other nerves which consist almost exclusively of primary afferent fibers . In the present work, the postganglionic action potentials as well as contractions of the nictitating membrane in response to electrical stimulation of the vagal artificial preganglionic trunk were blocked by treatment with TEA and atropine . Such findings confirm the view that the nature of the transmission in the superior cervical ganglion artificially synapsed by the vagal afferent fibers is cholinergic and that the muscarinic as well as nicotinic receptors are involved in the transmission . It has been reported that choline acetyltransferase appears to be a specific marker for cholinergic nerves in the central nervous system (9,10,11,12) . Using sensitive radiometric assay procedures which have major advantages of enabling quantification of enzyme activity in low activity regions, and of being specific for choline acetyltransferase, the enzyme activities in the superior cervical ganglion reinnervated by the afferent vague and the nodose ganglion of anastomosed cats were measured . These activities were lower than those of normal superior cervical ganglion, but higher than those of chronically decentralized ganglion . These results further support the hypothesis (3) that the primary afferent vague synapsed in the superior cervical ganglion is at least in part cholinergic . Ackn owledg ement s This work was supported by a grant No . 010604 from the Ministry of Education of Japan . Thanks are due to M . Ohara, Kyoto University, for assistance with preparation of the manuscript . References 1. 2. 3. 4. 5. 6. 7.
8.
9. 10 . 11 . 12 .
F . DE CASTRO, Trab . Lab . Invest . biol . Univ . Madr . _34, 217 - 301 (194 F . DE CASTRO, Ar ch . int . Physiol . 59, 479 - 519 (1951) . M. MATSUMURA an~lc G . B . KOELLE, J . Pfiärmacol . exp. Ther . 134, 28 - 46 (1961) . Ff-FUJIWARA and K . KURAHASHI, Proc . West . Pharmacol . Soc . 18, 225 - 228 (1975) . F . FONNUM, Biochem. J . 115, 465 - 472 (1969) . 114, 296 A. S . V . BURGEN and L . M . CHIPMAN, J . Physiol . 305 (1951) . F . C . MACINTOSH, J . Physiol . 9~ 436 - 442 (1941) . 176, 505 - 507 (1954) . F . J . WOLFGRAM, Amer . J . Physiol . W. FELDBERG and M. VOGT, J . Physiol . 107, 372 - 381 (1948) . G . B . KOELLE, J . com . Ph siol . 100 211 - 235 (1954) . C . HEBB, Ph sio . Rev . , 196 -~ (1957) . C . HEBB, Nature Lon on)~52, 527 - 529 (1961) .