352
Brain Research, 124 (1977) 352 356 () Elsevier/North-Holland Biomedical Press, Amsterdam Printed in The Netherlands
AChE positive electromotoneurons in the electric lobe of Torpedo
marmoroto SHIGERU TSUJI Laboratoire de Biologie Animale, Universitd Paris VI, 12 rue Cuvier, Paris 5 (France)
(Accepted December 17th, 1976)
There is now little doubt that except in Sternarchus all electric organs are embryologically muscles. The electric organs are innervated, according to the origins of embryological muscles, either by cranial or by spinal neurons, called electromotoneurons2, 4. If the homology between muscle and electric organ leaves little doubt, the homology between motoneurons and electromotoneurons gives rise to some histological problems. In fact, Tsuji et al. 18 and Tsuji 16 demonstrated histochemically AChE positive and AChE negative electromotoneurons in the spinal cord of Electrophorus. Tsuji 17 further showed with electron microscopy that the AChE positive cells are surrounded by numerous chemical synapses while AChE negative cells are characterized by electrical synapses. It is supposed that AChE positive cells with chemical synapses might be the primary motoneurons for the muscles and the electric organs, while AChE negative cells with electric synapses might be the interneurons for the synchronization of organ discharge. Contrary to most of the electric fishes, the electric organ of Sternarchus is of neurogenic origin2,3,1L The organ is formed by spinal neurons without postsynaptic cells and the organ lacks AChE 2. Sandri et al. 1°, working on the spinal cord of Sternarchus, found AChE positive and negative motoneurons such as in the spinal cord of Electrophorus. In contrast to Tsuji's hypothesis they put forward the hypothesis that these AChE negative neurons might be electromotoneurons, because this neurogenic organ does not need any synthesis of ACHE. Furthermore they interpreted the AChE negative neurons of Electrophorus to be electromotoneurons in spite of the high level AChE activity in the electric organ. Although definitive marking experiments have not been carried out either with Electrophorus or with Sternarchus electromotoneurons, we have tried to obtain more examples of AChE positive and negative electromotoneurons. The electric lobe of Torpedo marmorata was chosen for its exclusive innervation to electric organ by means of 4 large symmetrical electric nervesl,a,H,1L The electric lobe contains no motoneurons for the muscles but only electromotoneurons for the electric organ. Cholinergic transmission from electric nerves to the electric organ is well known z,4. Gautron has localized the synaptic AChE of this electric organ s.
353 Electric lobes
Cerebellum
ii:i::iii::it::!::iii:i::iiiiiiii~i~i: ........i"
: ...........
I
fig. 3
;:"I'-"~ i ll~lteeell
I
Control A
-
:
'
nucleus P
Fig. 1. Schematic drawing of the electric lobe of Torpedo marmorata. Paramedian and sagittal section passing through the command nucleus. The total length of the lobe is 15 ram. The two squares indicate approximately the parts of the electric lobe shown in micrography. AP, anterior-posterior direction. The present paper shows by a histochemical method that all electromotoneurons of the electric lobe are AChE positive. It constitutes thus an example of AChE positive electromotoneurons. Cryostat sections (16/zm thick) of the electric lobe of Torpedo marmorata were obtained. After fixation (30 min) in 2.5 ~ glutaraldehyde in phosphate buffer (0.1 M, pH 7.2), the AChE activity of each section was revealed with the.modified thiocholine method of Koelle 15. Incubation time was 90 min at p H 5.5. The sections were counterstained with toluidine blue. The controls with butyrylthiocholine iodide were negative and those with iso-OMPA (10 -5 M) were positive. Transversal and longitudinal sections of any part of the electric lobe showed a homogeneous group of AChE positive electromotoneurons ~Fig. 1). The AChE negative neurons were never observed with counterstaining. Part of the longitudinal section shows such intensely stained electromotoneurons (Fig. 2). It is also interesting to note that the neurons of the control nucleus (Szabo's 1~ 'noyau oval') which innervate the electric lobe are AChE negative (Fig. 3). The present work shows that all the electromotoneurons of the electric lobe of Torpedo are AChE positive. Since all the neurons of the lobe are pure electromotoneurons, this is a clear demonstration for the existence of AChE positive electromotoneurons. It is well known that the vertebrate motoneurons are endowed with AChE of perikaryon 6. It is also known that, in spite of numerous chemical synapses, electrical synapses are difficult to find on vertebrate motoneurons 14. This rarity of the electrical synapses of vertebrate motoneurons differs from the ease of location of electrical synapses on electric fish electromotoneurons as has been reportede,7, 8.
354
Fig. 2. AChE positive electromotoneurons of electric lobe of Torpedomarmorata. Modified thiocholinc method of Koelle followed by a secondary staining with toluidine blue. All electromotoneurons arc positive. • 40. The question then arises as to how the homology between electromotoneurons and motoneurons could be described on a enzymatical and ultrastructural basis. According to some authors the electromotoneurons are AChE negative ~0 and bear electrical synapses 7,8. This implies that the histogenesis of the electromotoneurons is accompanied by the loss of AChE and the gain of electrical synapses in comparison with histodifferentiation of the motoneurons. Working on Electrophorus spinal cord 16-1s we have reported that there are two kinds of electromotoneurons: one type is AChE positive and provided with chemical synapses, the other is AChE negative with electrical synapses. We supposed that the AChE positive neurons with chemical synapses might be the actual electromotoneutons16,17. According to our hypothesis the histogenesis of the electromotoneurons might be fundamentally the same as that of the motoneurons. This idea was not retained by Sandri et al. 1° working on Sternarchus, whose electric organ is a peculiar case of neurogenic origin. The present paper demonstrates that all the electromotoneurons of the Torpedo electric lobe are AChE positive. Electron microscopic study of the electric lobe by Roberts and Ryan 9 has shown chemical but no electrical synapses on electromotoneurons. Our preliminary observations on the same material confirm their observations. Since the electromotoneurons are the only neurons in the electric lobe, Torpedo's case is a clear example of AChE positive electromotoneurons having only chemical synapses.
355
Fig. 3. AChE positive electromotoneurons of electric lobe of Torpedo marmorata and AChE negative control nucleus ('noyau oval') which innervate the electromotoneurons, x 50.
356 T h e e n z y m a t i c a n d u l t r a s t r u c t u r a l p r o p e r t i e s o f Torpedo e l e c t r o m o t o n e u r o n s are n o t c o n t r a d i c t o r y to t h e i r h o m o l o g y w i t h m o t o n e u r o n s . F u r t h e r m o r e these h i s t o c h e m i c a l a n d s y n a p t i c c h a r a c t e r i s t i c s o f Torpedo e l e c t r o m o t o n e u r o n s are t h e s a m e as t h o s e f o u n d in A C h E p o s i t i v e e l e c t r o m o t o n e u r o n s o f Electrophorus a n d p r o m p t us to s u p p o s e t h a t t h e y are the real e l e c t r o m o t o n e u r o n s . T h e Torpedos were k i n d l y g i v e n by D r . M. Isra61 a n d Dr. J. Massouli6.
1 Albe-Fessard, D. et Buser, P., Analyse microphysiologique de la transmission r6flexe au niveau
du lobe 61ectrique de la Torpille (Torpedo marmorata), J. Physiol. (Paris), 46 (1954) 923-946. 2 Bennett, M. V. L., Electric organs. In W. S. Hoar and D. J. Randall (Eds.), Fish Physiology, Vol. 5, Academic press, New York, 1971, pp. 347-491. 3 De Oliveira Castro, G., Differentiated nervous fibers that constitute the electric organ of Stertrarchus albifrons Linn., An. Acad. bras. Cien., 27 (1955) 557-562. 4 Fessard, A., Les organes 61ectriques. In P.-P. Grass6 (Ed.), Traitd de Zoologie, Vot. 13, Masson, Paris, 1958, pp. 1143-1238. 5 Gautron, J., Localisation des cholin6sterases au niveau de la jonction nerf-61ectroplaque de la Torpille marbr6e, C.R. Acad. Sci. (Paris), 271 (1970) 714-717. 6 Koelle, G. B., Cytological distributions and physiological functions of cbolinesterases. In G. B. Koelle (Ed.), Handbuch der experimentellen Pharmakologie, XV, Springer, Berlin, 1963, pp. 187-298. 7 Meszler, R. M., Pappas, G. D. and Bennett, M. V. L., Morphology of the electromotor system in the spinal cord of the electric eel. Electrophorus electricus, J. Neurocytol., 3 (1974) 251-261. 8 Pappas, G. D., Waxman, S. G. and Bennett, M. V. L., Morphology of spinal electromotor neurons and presynaptic coupling in the gymnotid Sternarchus albifrons. J. Neuroeytol., 4 (1975) 469-478. 9 Roberts, B. L. and Ryan, K. P., Cytological features of the giant neurons controlling electric discharge in the ray, Torpedo, J. mar. biol. Ass. (U.K.), 55 (1975) 123-131. 10 Sandri, C., Akert, K., Kristol, C., Van Buren, J. and Bennett, M. V. L., AChE-positive and -negative motoneurons in the spinal cord of Sternarchus albifrons, Brain Research, 111 (1976) 157-161. 11 Szabo, T., Anatomo-physiologie Compar~e de Divers Organes t~lectriques et de Leurs Centres Nerveux de Commande, Thesis, Paris, 1957. 12 Szabo, T., Anatomo-physiologie des centres nerveux sp6cifiques de quelques organes 61ectriques. In C. Chagas and A. Paes de Carvalho (Eds.), Bioelectrogenesis, Elsevier, Amsterdam, 1961, pp. 185-201. 13 Szabo, T., Un relais darts le syst6me des connexions du lobe 61ectrique de la Torpille, Arch. Anat. micr. Morph. exp., 43 (1954) 187-201. 14 Sz6kely, G. and Kosaras, B., Dendro-dendritic contacts between frog motoneurons shown with the cobalt labeling technique, Brain Research, 108 (1976) 194-198. 15 Tsuji, S., On the chemical basis of thiocholine methods for demonstration of acetylcholinesterase activities, Histochemistry, 42 (1974) 99-110. 16 Tsuji, S., Histochemical demonstration of acetylcholinesterase-rich cells in spinal electromotor nucleus of Electrophorus electricus, Brain Research, 88 (1975) 499-501. 17 Tsuji, S., Les ultrastructures des 61ectromotoneurons spinaux de rElectrophorus electricus, avec ou sans activit6 acetylcholinesterasiques, C.R. Acad. Sci. (Paris), 280 (1975) 737-739. 18 Tsuji, S., Rieger, F., Peltre, G., Massouli6, J. et Benda, P., Acetylcholinest6rase du muscle de la moelle 6pini6re et du cerveau de gymnote, J. Neurochem., 19 (1972) 989-997. 19 Waxman, S. G., Pappas, G. D. and Bennett, M. V. L., Morphological correlates of functional differentiation of nodes of Ranvier along single fibers in the neurogenic electric organ of the knife fish Sternarehus, J. Cell Biol., 53 (1972) 210-224.
Brain Research, 124 (1977) 352 356 () Elsevier/North-Holland Biomedical Press, Amsterdam Printed in The Netherlands
AChE positive electromotoneurons in the electric lobe of Torpedo
marmoroto SHIGERU TSUJI Laboratoire de Biologie Animale, Universitd Paris VI, 12 rue Cuvier, Paris 5 (France)
(Accepted December 17th, 1976)
There is now little doubt that except in Sternarchus all electric organs are embryologically muscles. The electric organs are innervated, according to the origins of embryological muscles, either by cranial or by spinal neurons, called electromotoneurons2, 4. If the homology between muscle and electric organ leaves little doubt, the homology between motoneurons and electromotoneurons gives rise to some histological problems. In fact, Tsuji et al. 18 and Tsuji 16 demonstrated histochemically AChE positive and AChE negative electromotoneurons in the spinal cord of Electrophorus. Tsuji 17 further showed with electron microscopy that the AChE positive cells are surrounded by numerous chemical synapses while AChE negative cells are characterized by electrical synapses. It is supposed that AChE positive cells with chemical synapses might be the primary motoneurons for the muscles and the electric organs, while AChE negative cells with electric synapses might be the interneurons for the synchronization of organ discharge. Contrary to most of the electric fishes, the electric organ of Sternarchus is of neurogenic origin2,3,1L The organ is formed by spinal neurons without postsynaptic cells and the organ lacks AChE 2. Sandri et al. 1°, working on the spinal cord of Sternarchus, found AChE positive and negative motoneurons such as in the spinal cord of Electrophorus. In contrast to Tsuji's hypothesis they put forward the hypothesis that these AChE negative neurons might be electromotoneurons, because this neurogenic organ does not need any synthesis of ACHE. Furthermore they interpreted the AChE negative neurons of Electrophorus to be electromotoneurons in spite of the high level AChE activity in the electric organ. Although definitive marking experiments have not been carried out either with Electrophorus or with Sternarchus electromotoneurons, we have tried to obtain more examples of AChE positive and negative electromotoneurons. The electric lobe of Torpedo marmorata was chosen for its exclusive innervation to electric organ by means of 4 large symmetrical electric nervesl,a,H,1L The electric lobe contains no motoneurons for the muscles but only electromotoneurons for the electric organ. Cholinergic transmission from electric nerves to the electric organ is well known z,4. Gautron has localized the synaptic AChE of this electric organ s.
353 Electric lobes
Cerebellum
ii:i::iii::it::!::iii:i::iiiiiiii~i~i: ........i"
: ...........
I
fig. 3
;:"I'-"~ i ll~lteeell
I
Control A
-
:
'
nucleus P
Fig. 1. Schematic drawing of the electric lobe of Torpedo marmorata. Paramedian and sagittal section passing through the command nucleus. The total length of the lobe is 15 ram. The two squares indicate approximately the parts of the electric lobe shown in micrography. AP, anterior-posterior direction. The present paper shows by a histochemical method that all electromotoneurons of the electric lobe are AChE positive. It constitutes thus an example of AChE positive electromotoneurons. Cryostat sections (16/zm thick) of the electric lobe of Torpedo marmorata were obtained. After fixation (30 min) in 2.5 ~ glutaraldehyde in phosphate buffer (0.1 M, pH 7.2), the AChE activity of each section was revealed with the.modified thiocholine method of Koelle 15. Incubation time was 90 min at p H 5.5. The sections were counterstained with toluidine blue. The controls with butyrylthiocholine iodide were negative and those with iso-OMPA (10 -5 M) were positive. Transversal and longitudinal sections of any part of the electric lobe showed a homogeneous group of AChE positive electromotoneurons ~Fig. 1). The AChE negative neurons were never observed with counterstaining. Part of the longitudinal section shows such intensely stained electromotoneurons (Fig. 2). It is also interesting to note that the neurons of the control nucleus (Szabo's 1~ 'noyau oval') which innervate the electric lobe are AChE negative (Fig. 3). The present work shows that all the electromotoneurons of the electric lobe of Torpedo are AChE positive. Since all the neurons of the lobe are pure electromotoneurons, this is a clear demonstration for the existence of AChE positive electromotoneurons. It is well known that the vertebrate motoneurons are endowed with AChE of perikaryon 6. It is also known that, in spite of numerous chemical synapses, electrical synapses are difficult to find on vertebrate motoneurons 14. This rarity of the electrical synapses of vertebrate motoneurons differs from the ease of location of electrical synapses on electric fish electromotoneurons as has been reportede,7, 8.
354
Fig. 2. AChE positive electromotoneurons of electric lobe of Torpedomarmorata. Modified thiocholinc method of Koelle followed by a secondary staining with toluidine blue. All electromotoneurons arc positive. • 40. The question then arises as to how the homology between electromotoneurons and motoneurons could be described on a enzymatical and ultrastructural basis. According to some authors the electromotoneurons are AChE negative ~0 and bear electrical synapses 7,8. This implies that the histogenesis of the electromotoneurons is accompanied by the loss of AChE and the gain of electrical synapses in comparison with histodifferentiation of the motoneurons. Working on Electrophorus spinal cord 16-1s we have reported that there are two kinds of electromotoneurons: one type is AChE positive and provided with chemical synapses, the other is AChE negative with electrical synapses. We supposed that the AChE positive neurons with chemical synapses might be the actual electromotoneutons16,17. According to our hypothesis the histogenesis of the electromotoneurons might be fundamentally the same as that of the motoneurons. This idea was not retained by Sandri et al. 1° working on Sternarchus, whose electric organ is a peculiar case of neurogenic origin. The present paper demonstrates that all the electromotoneurons of the Torpedo electric lobe are AChE positive. Electron microscopic study of the electric lobe by Roberts and Ryan 9 has shown chemical but no electrical synapses on electromotoneurons. Our preliminary observations on the same material confirm their observations. Since the electromotoneurons are the only neurons in the electric lobe, Torpedo's case is a clear example of AChE positive electromotoneurons having only chemical synapses.
355
Fig. 3. AChE positive electromotoneurons of electric lobe of Torpedo marmorata and AChE negative control nucleus ('noyau oval') which innervate the electromotoneurons, x 50.
356 T h e e n z y m a t i c a n d u l t r a s t r u c t u r a l p r o p e r t i e s o f Torpedo e l e c t r o m o t o n e u r o n s are n o t c o n t r a d i c t o r y to t h e i r h o m o l o g y w i t h m o t o n e u r o n s . F u r t h e r m o r e these h i s t o c h e m i c a l a n d s y n a p t i c c h a r a c t e r i s t i c s o f Torpedo e l e c t r o m o t o n e u r o n s are t h e s a m e as t h o s e f o u n d in A C h E p o s i t i v e e l e c t r o m o t o n e u r o n s o f Electrophorus a n d p r o m p t us to s u p p o s e t h a t t h e y are the real e l e c t r o m o t o n e u r o n s . T h e Torpedos were k i n d l y g i v e n by D r . M. Isra61 a n d Dr. J. Massouli6.
1 Albe-Fessard, D. et Buser, P., Analyse microphysiologique de la transmission r6flexe au niveau
du lobe 61ectrique de la Torpille (Torpedo marmorata), J. Physiol. (Paris), 46 (1954) 923-946. 2 Bennett, M. V. L., Electric organs. In W. S. Hoar and D. J. Randall (Eds.), Fish Physiology, Vol. 5, Academic press, New York, 1971, pp. 347-491. 3 De Oliveira Castro, G., Differentiated nervous fibers that constitute the electric organ of Stertrarchus albifrons Linn., An. Acad. bras. Cien., 27 (1955) 557-562. 4 Fessard, A., Les organes 61ectriques. In P.-P. Grass6 (Ed.), Traitd de Zoologie, Vot. 13, Masson, Paris, 1958, pp. 1143-1238. 5 Gautron, J., Localisation des cholin6sterases au niveau de la jonction nerf-61ectroplaque de la Torpille marbr6e, C.R. Acad. Sci. (Paris), 271 (1970) 714-717. 6 Koelle, G. B., Cytological distributions and physiological functions of cbolinesterases. In G. B. Koelle (Ed.), Handbuch der experimentellen Pharmakologie, XV, Springer, Berlin, 1963, pp. 187-298. 7 Meszler, R. M., Pappas, G. D. and Bennett, M. V. L., Morphology of the electromotor system in the spinal cord of the electric eel. Electrophorus electricus, J. Neurocytol., 3 (1974) 251-261. 8 Pappas, G. D., Waxman, S. G. and Bennett, M. V. L., Morphology of spinal electromotor neurons and presynaptic coupling in the gymnotid Sternarchus albifrons. J. Neuroeytol., 4 (1975) 469-478. 9 Roberts, B. L. and Ryan, K. P., Cytological features of the giant neurons controlling electric discharge in the ray, Torpedo, J. mar. biol. Ass. (U.K.), 55 (1975) 123-131. 10 Sandri, C., Akert, K., Kristol, C., Van Buren, J. and Bennett, M. V. L., AChE-positive and -negative motoneurons in the spinal cord of Sternarchus albifrons, Brain Research, 111 (1976) 157-161. 11 Szabo, T., Anatomo-physiologie Compar~e de Divers Organes t~lectriques et de Leurs Centres Nerveux de Commande, Thesis, Paris, 1957. 12 Szabo, T., Anatomo-physiologie des centres nerveux sp6cifiques de quelques organes 61ectriques. In C. Chagas and A. Paes de Carvalho (Eds.), Bioelectrogenesis, Elsevier, Amsterdam, 1961, pp. 185-201. 13 Szabo, T., Un relais darts le syst6me des connexions du lobe 61ectrique de la Torpille, Arch. Anat. micr. Morph. exp., 43 (1954) 187-201. 14 Sz6kely, G. and Kosaras, B., Dendro-dendritic contacts between frog motoneurons shown with the cobalt labeling technique, Brain Research, 108 (1976) 194-198. 15 Tsuji, S., On the chemical basis of thiocholine methods for demonstration of acetylcholinesterase activities, Histochemistry, 42 (1974) 99-110. 16 Tsuji, S., Histochemical demonstration of acetylcholinesterase-rich cells in spinal electromotor nucleus of Electrophorus electricus, Brain Research, 88 (1975) 499-501. 17 Tsuji, S., Les ultrastructures des 61ectromotoneurons spinaux de rElectrophorus electricus, avec ou sans activit6 acetylcholinesterasiques, C.R. Acad. Sci. (Paris), 280 (1975) 737-739. 18 Tsuji, S., Rieger, F., Peltre, G., Massouli6, J. et Benda, P., Acetylcholinest6rase du muscle de la moelle 6pini6re et du cerveau de gymnote, J. Neurochem., 19 (1972) 989-997. 19 Waxman, S. G., Pappas, G. D. and Bennett, M. V. L., Morphological correlates of functional differentiation of nodes of Ranvier along single fibers in the neurogenic electric organ of the knife fish Sternarehus, J. Cell Biol., 53 (1972) 210-224.
