Exp. Brain Res. 30, 493-510 (1977)
Experimental Brain Research 9
Springer-Verlag1977
Axonal Transport in the Electromotor Nerves of Torpedo Marmorata L.P. Davies 1, V.P. Whittaker and H. Z i m m e r m a n n Abteilung ffir Neurochemie, Max-Planck-Institut ffir biophysikalische Chemie, Am Fagberg, D-3400 G6ttingen, Federal Republic of Germany
Summary. Studies on the axonal transport of cholinergic cell components were made on the electromotor nerves of Torpedo marmorata. Choline acetyltransferase was rapidly accumulated at ligatures on Torpedo nerves, both in vivo and in segments incubated in vitro. In vivo accumulation was maximal approximately one month after nerve interruption. Orthograde transport (both in vitro and in vivo) is calculated to have a velocity of 5 0 - 1 4 0 mm/day, if, as double-ligature experiments suggest, only about 15 % of the axoplasmic enzyme is mobile. A small retrograde accumulation of the transferase was demonstrated. Lactate dehydrogenase did not accumulate but a slight reduction of its activity at ligatures was observed. In contrast to mammalian cholinergic nerves, no accumulation of esterase was observed. A C h accumulation proximal to a cut was apparent and may result in part from local synthesis in the presence of elevated levels of its synthesizing enzyme. Measurements have been made on the activity of choline acetyltransferase in the brain and all parts of the "electric system". In view of these results it is difficult to see how the measured rate of axonal translocation is sufficient to supply the levels of the enzyme found within the electric organ. Within the electromotor cells, choline acetyltransferase is highly concentrated in the axon terminals. Key words: T o r p e d o m a r m o r a t a - Axonal transport - Cholinergic enzymes - E n z y m e distribution
Elasmobranch fish of the family Torpedinidae possess bilateral electric organs which receive extensive cholinergic innervation from paired nuclei (electric lobes) in the brain stem. This specialised electric apparatus is used as a model system for the study of cholinergic cell function since it provides cell bodies, 1 Present address: Roche Research Institute of Marine Pharmacology, P.O. Box 255 Dee Why, N.S.W. 2099, Australia
494
L.P. Davies et al.
axons, n e r v e - t e r m i n a l sacs a n d n e r v e - t e r m i n a l c o m p o n e n t s in sufficient q u a n t i t i e s for b i o c h e m i c a l i s o l a t i o n a n d analysis. Studies with T o r p e d o e l e c t r o m o t o r n e r v e s h a v e s h o w n that c h o l i n e a c e t y l t r a n s f e r a s e ( C h A c ) ( H e i l b r o n n a n d P e t t e r s s o n , 1973), a c e t y l c h o l i n e ( A C h ) ( H e i l b r o n n a n d P e t t e r s s o n , 1973; Z i m m e r m a n n a n d W h i t t a k e r , 1973; W h i t t a k e r et al., 1975) a n d vesicle m e m b r a n e p r o t e i n ( U l m a r a n d W h i t t a k e r , 1974) a c c u m u l a t e a b o v e a single l i g a t u r e in vivo. T o d a t e , no studies h a v e e x a m i n e d e s t e r a s e d i s t r i b u t i o n in l i g a t u r e d T o r p e d o nerves. W e h a v e i n v e s t i g a t e d t r a n s p o r t o f a c e t y l c h o l i n e s t e r a s e ( A C H E ) a n d C h A c in d o u b l e l i g a t u r e e x p e r i m e n t s b o t h in vivo a n d in vitro i n c u b a t i o n s with i s o l a t e d n e r v e segments. T h e m e a s u r e d r a t e o f t r a n s p o r t of an e n z y m e is d e p e n d e n t on t h e o b s e r v e d a c c u m u l a t i o n o f t h e e n z y m e activity p r o x i m a l to a l i g a t u r e a n d t h e p r o p o r t i o n o f e n z y m e which is m o b i l e in t h e axon. I n d o u b l e - l i g a t u r e e x p e r i m e n t s ( F o n n u m et al., 1973) it is p o s s i b l e to e s t i m a t e b o t h t h e r a t e o f t r a n s p o r t a n d t h e p r o p o r t i o n o f e n z y m e w h i c h is m o v i n g , since t h e u p p e r ligature prevents newly-synthesised enzyme from entering the section of axon b e t w e e n t h e ligatures i.e. in t h e a b s e n c e of e n z y m e d e g r a d a t i o n t h e r e is a k n o w n a m o u n t o f e n z y m e in t h e l i g a t u r e d s e g m e n t . F u r t h e r m o r e , such e x p e r i m e n t s m a k e it p o s s i b l e to d i f f e r e n t i a t e b e t w e e n c h a n g e s c a u s e d b y a c c u m u l a t i o n at a l i g a t u r e a n d t h o s e c h a n g e s i n d u c e d b y a x o n a l d a m a g e at t h e l i g a t u r e site. I n o t h e r e x p e r i m e n t s s e v e r a l fish w e r e l i g a t u r e d a n d left for p e r i o d s of o n e to two m o n t h s to check e n z y m e a c c u m u l a t i o n in n e r v e s with c h r o n i c single ligatures.
Materials and Methods Experiments were performed on Torpedo marmorata supplied by the Institut de Biologie Marine, Arcachon, France, and kept in tanks of circulating sea-water at 17~ C. For most of these studies, female fish were used. Ligation of Nerves. In these studies all four electromotor nerves were used, since there is no evidence to suggest that they differ in any way, apart from the number of axons within each bundle (Whittaker and Zimmermann, 1976). Nerves were constricted by tightly-applied ligatures of Terylene thread. Surgical procedures on fish anaesthetised with Tricalne methane sulphonate (MS-222) were carried out as described (Zimmermann and Whittaker, 1974). Ligatures were applied at a point between the skull and gill arches. For double-ligature studies, a second constriction was placed on the nerves at a point between the gill arches and the electric organ. The wounds were treated with bacitracin, closed with Michel clips and tissue glue (Cyanolit; Bostik, Niirnberg, F.R.G.) and fish returned to the aquarium. For the lobe-nerve preparation the nerves were dissected free of the gill arches and removed with the brain, which was contained in a "cup" of surrounding cartilage. Incubation of Nerves. For in vitro incubations, double-ligatured nerve segments (2-5 cm long) were placed in sealed conical flasks containing 10 ml oxygenated Torpedo ringer (Babel-Gu6rin, 1974; with NaHCO3 increased to 20 mM and added penicillin (250 U/ml) and streptomycin (250 mcg/ml)) and shaken in a water bath kept at 14-17 ~ C. Extraction of Nerves. After the incubation period, nerves were frozen on a metal plate that was kept cold on a block of dry-ice and uniform segments (2.3 or 2.9 mm) cut using a multi-blade scalpel. Individual segments were homogenized in 10 mM EDTA containing 0.5 % Triton X-100 (250-500 p.l); a Teflon-Perspex microhomogenizer (Ernst Schiitt, G6ttingen) was initially used, but in subsequent experiments it was found more efficient and convenient to crush the pieces, frozen in liquid N2, with a stainless steel mortar and pestle (Ladinsky et al., 1972). The powder was transferred to the homogenization solution contained in 1.5 ml Eppendorf reaction vials and
Axonal Transport in Torpedo Nerves
495
vigorously vortexed. Extracts were centrifuged (3000xgav. for 5 min) to remove unhomogenized tissue and aliquots taken for ACHE, ChAc and protein. Assay of Enzymes and Substrates. ChAc was assayed by a slight modification of the method of Fonnum (1975). The cpm in the blanks were only 0 . 6 - 0 . 7 % of the original amount of [1-14C]acetyl-CoA in the incubation medium, while 99.0% of added [1-14C]acetylcholine was extracted. Activity was measured for acetyl-CoA concentrations in the range 0.1-0.4 raM. At 0.4 mM there was a slight increase (8%) in activity over 0.2 mM; 0.2 mM was used routinely in the nerve extract assays. The partially purified enzyme from the electric organ has a Km for acetyl-CoA of 50 ~M (Baker and Dowdall, 1976). The activity in the nerve extracts was linear with incubation time for 1 h and proportional to the amount of extract. Increasing the temperature from 21-37 ~ C increased the rate of enzymic acetylation. Measurement of activity against a range of choline concentrations from 2-16 mM gave an apparent Km at 30 ~ C of 2.4 mM by Lineweaver-Burk or Hofstee plot. Like Bull et al. (1969) we found that temperature had a large effect on the Km of electric organ ChAc (crude extract) for choline, with an increase from 1.14 + 0.02 mM at 21 ~ C to 2.60 + 0.04 mM at 29.6 ~ C and 6.25 + 0.14 mM at 37 ~ C (linear regression analysis of Hofstee plots). With such large changes in K m and Vm it is thus important to ensure that incubation conditions, particularly temperature, are carefully controlled in any comparative experiments of ChAc activity. Routine measurements were made with 8 mM choline and a temperature of 30 ~ C. AChE was initially measured by using [1-I4C]acetylcholine essentially according to the procedure of Tu6ek (1974), but in later experiments the spectrophotometric method of Ellman et al. (1961) was found more convenient. The concentrations of acetylthiocholine iodide and 5.5'-dithiobis-(2-nitrobenzoic acid) used resulted in maximal activity of the Torpedo enzyme. Lactate dehydrogenase (LDH) was measured by following the oxidation of NADH2 in the presence of pyruvate (Johnson, 1960) and protein by the method of Lowry (Lowry et al., 1951) 0,1% using bovine serum albumin as standard (Ez78 nm= 0.666). ACh was estimated using the leech bioassay as described by Whittaker and Barker (1972). Efficiency of Extraction of Enzymes. Results for enzyme studies are expressed on a basis of extracted protein rather than total activity per segment as is common for transport studies. Because of the large diameter of the nerves, the effect of the ligature is to compress the axon bundles and reduce the diameter, thus altering the amount of material in a segment of fixed length. Furthermore, some branching of the nerves within the gill arches of the Torpedo results in a small proximo-distal decrease in nerve diameter. In tying double ligatures in vivo it was often not possible to apply the lower ligature above the point of initial branching of the nerve in the organ; thus those fascicles which were not included in the ligature had to be dissected away before sectioning and assay. Expression of results on the basis of solubilized protein also takes into account variations in the efficiency of extraction of each nerve piece and losses during transfer of powdered frozen material. Laduron (1973) has discussed the inherent problems in expressing accumulation of a product per unit length of nerve. The efficiencies of extraction of the nerve enzymes into the low-speed supernatants of the crude homogenates were determined by resuspending and assaying the activities remaining in the pellet. For LDH an average of 93.4 + S.E.M. 0.76%(12) was in the supernatant, for AChE 93.1 _+ 1.51, for ChAc 54 + 2.87 and protein (extracted from the pellet by 0.2 N NaOH with overnight warming), 37.8 _+ 2.21. For short term ligature experiments in vitro there were no significant differences in the extraction efficiency of the enzymes from pieces near the ligatures compared with pieces in the middle of the nerve. However, for longer term experiments (40 h and 1 week in vivo) it was found that there were some differences in the proportions of total ChAc extracted, depending on the position of the nerve piece relative to the ligature. Thus the measurement of ChAc in the low-speed supernatants of homogenates was not a completely accurate reflection of ChAc activity in the nerve segments and experiments were repeated to compare "total" and "supernatant" ChAc specific activity. These changes in extraction efficiency are discussed in "Results". Materials. Chemicals were purchased as follows: [1-14C] acetylcoenzyme A ( 3 - 5 mCi/mmol) and [1-14C]acetylchollne chloride (12.2 mCi/mmol) (Amersham-Buchler, Brannschweig, F.R.G.); penicillin/streptomycin solution (Flow, Irvine, Scotland); tetraphenylboron (Aldrich, Beerse, Belgium); acetylthiocholine iodide and 2,2 dinitro-5,5-dithiodibenzoic acid (Merck, Darmstadt, F.R.G.), bacitracin (Sigma, St. Louis, U.S.A) and disodium 4-nitro-phenylphosphate (B.D.H. Ltd., Poole, U.K.). Other chemicals were purchased from J. T. Baker (Deventer, Holland).
496
L. P. Davies et aL
INCUBATED
CONTROL (a} 60
~.0
20[ 0
(b)
E
"J0-5I 0 t-
o0.6~ f
(c)
Experimental Brain Research 9
Springer-Verlag1977
Axonal Transport in the Electromotor Nerves of Torpedo Marmorata L.P. Davies 1, V.P. Whittaker and H. Z i m m e r m a n n Abteilung ffir Neurochemie, Max-Planck-Institut ffir biophysikalische Chemie, Am Fagberg, D-3400 G6ttingen, Federal Republic of Germany
Summary. Studies on the axonal transport of cholinergic cell components were made on the electromotor nerves of Torpedo marmorata. Choline acetyltransferase was rapidly accumulated at ligatures on Torpedo nerves, both in vivo and in segments incubated in vitro. In vivo accumulation was maximal approximately one month after nerve interruption. Orthograde transport (both in vitro and in vivo) is calculated to have a velocity of 5 0 - 1 4 0 mm/day, if, as double-ligature experiments suggest, only about 15 % of the axoplasmic enzyme is mobile. A small retrograde accumulation of the transferase was demonstrated. Lactate dehydrogenase did not accumulate but a slight reduction of its activity at ligatures was observed. In contrast to mammalian cholinergic nerves, no accumulation of esterase was observed. A C h accumulation proximal to a cut was apparent and may result in part from local synthesis in the presence of elevated levels of its synthesizing enzyme. Measurements have been made on the activity of choline acetyltransferase in the brain and all parts of the "electric system". In view of these results it is difficult to see how the measured rate of axonal translocation is sufficient to supply the levels of the enzyme found within the electric organ. Within the electromotor cells, choline acetyltransferase is highly concentrated in the axon terminals. Key words: T o r p e d o m a r m o r a t a - Axonal transport - Cholinergic enzymes - E n z y m e distribution
Elasmobranch fish of the family Torpedinidae possess bilateral electric organs which receive extensive cholinergic innervation from paired nuclei (electric lobes) in the brain stem. This specialised electric apparatus is used as a model system for the study of cholinergic cell function since it provides cell bodies, 1 Present address: Roche Research Institute of Marine Pharmacology, P.O. Box 255 Dee Why, N.S.W. 2099, Australia
494
L.P. Davies et al.
axons, n e r v e - t e r m i n a l sacs a n d n e r v e - t e r m i n a l c o m p o n e n t s in sufficient q u a n t i t i e s for b i o c h e m i c a l i s o l a t i o n a n d analysis. Studies with T o r p e d o e l e c t r o m o t o r n e r v e s h a v e s h o w n that c h o l i n e a c e t y l t r a n s f e r a s e ( C h A c ) ( H e i l b r o n n a n d P e t t e r s s o n , 1973), a c e t y l c h o l i n e ( A C h ) ( H e i l b r o n n a n d P e t t e r s s o n , 1973; Z i m m e r m a n n a n d W h i t t a k e r , 1973; W h i t t a k e r et al., 1975) a n d vesicle m e m b r a n e p r o t e i n ( U l m a r a n d W h i t t a k e r , 1974) a c c u m u l a t e a b o v e a single l i g a t u r e in vivo. T o d a t e , no studies h a v e e x a m i n e d e s t e r a s e d i s t r i b u t i o n in l i g a t u r e d T o r p e d o nerves. W e h a v e i n v e s t i g a t e d t r a n s p o r t o f a c e t y l c h o l i n e s t e r a s e ( A C H E ) a n d C h A c in d o u b l e l i g a t u r e e x p e r i m e n t s b o t h in vivo a n d in vitro i n c u b a t i o n s with i s o l a t e d n e r v e segments. T h e m e a s u r e d r a t e o f t r a n s p o r t of an e n z y m e is d e p e n d e n t on t h e o b s e r v e d a c c u m u l a t i o n o f t h e e n z y m e activity p r o x i m a l to a l i g a t u r e a n d t h e p r o p o r t i o n o f e n z y m e which is m o b i l e in t h e axon. I n d o u b l e - l i g a t u r e e x p e r i m e n t s ( F o n n u m et al., 1973) it is p o s s i b l e to e s t i m a t e b o t h t h e r a t e o f t r a n s p o r t a n d t h e p r o p o r t i o n o f e n z y m e w h i c h is m o v i n g , since t h e u p p e r ligature prevents newly-synthesised enzyme from entering the section of axon b e t w e e n t h e ligatures i.e. in t h e a b s e n c e of e n z y m e d e g r a d a t i o n t h e r e is a k n o w n a m o u n t o f e n z y m e in t h e l i g a t u r e d s e g m e n t . F u r t h e r m o r e , such e x p e r i m e n t s m a k e it p o s s i b l e to d i f f e r e n t i a t e b e t w e e n c h a n g e s c a u s e d b y a c c u m u l a t i o n at a l i g a t u r e a n d t h o s e c h a n g e s i n d u c e d b y a x o n a l d a m a g e at t h e l i g a t u r e site. I n o t h e r e x p e r i m e n t s s e v e r a l fish w e r e l i g a t u r e d a n d left for p e r i o d s of o n e to two m o n t h s to check e n z y m e a c c u m u l a t i o n in n e r v e s with c h r o n i c single ligatures.
Materials and Methods Experiments were performed on Torpedo marmorata supplied by the Institut de Biologie Marine, Arcachon, France, and kept in tanks of circulating sea-water at 17~ C. For most of these studies, female fish were used. Ligation of Nerves. In these studies all four electromotor nerves were used, since there is no evidence to suggest that they differ in any way, apart from the number of axons within each bundle (Whittaker and Zimmermann, 1976). Nerves were constricted by tightly-applied ligatures of Terylene thread. Surgical procedures on fish anaesthetised with Tricalne methane sulphonate (MS-222) were carried out as described (Zimmermann and Whittaker, 1974). Ligatures were applied at a point between the skull and gill arches. For double-ligature studies, a second constriction was placed on the nerves at a point between the gill arches and the electric organ. The wounds were treated with bacitracin, closed with Michel clips and tissue glue (Cyanolit; Bostik, Niirnberg, F.R.G.) and fish returned to the aquarium. For the lobe-nerve preparation the nerves were dissected free of the gill arches and removed with the brain, which was contained in a "cup" of surrounding cartilage. Incubation of Nerves. For in vitro incubations, double-ligatured nerve segments (2-5 cm long) were placed in sealed conical flasks containing 10 ml oxygenated Torpedo ringer (Babel-Gu6rin, 1974; with NaHCO3 increased to 20 mM and added penicillin (250 U/ml) and streptomycin (250 mcg/ml)) and shaken in a water bath kept at 14-17 ~ C. Extraction of Nerves. After the incubation period, nerves were frozen on a metal plate that was kept cold on a block of dry-ice and uniform segments (2.3 or 2.9 mm) cut using a multi-blade scalpel. Individual segments were homogenized in 10 mM EDTA containing 0.5 % Triton X-100 (250-500 p.l); a Teflon-Perspex microhomogenizer (Ernst Schiitt, G6ttingen) was initially used, but in subsequent experiments it was found more efficient and convenient to crush the pieces, frozen in liquid N2, with a stainless steel mortar and pestle (Ladinsky et al., 1972). The powder was transferred to the homogenization solution contained in 1.5 ml Eppendorf reaction vials and
Axonal Transport in Torpedo Nerves
495
vigorously vortexed. Extracts were centrifuged (3000xgav. for 5 min) to remove unhomogenized tissue and aliquots taken for ACHE, ChAc and protein. Assay of Enzymes and Substrates. ChAc was assayed by a slight modification of the method of Fonnum (1975). The cpm in the blanks were only 0 . 6 - 0 . 7 % of the original amount of [1-14C]acetyl-CoA in the incubation medium, while 99.0% of added [1-14C]acetylcholine was extracted. Activity was measured for acetyl-CoA concentrations in the range 0.1-0.4 raM. At 0.4 mM there was a slight increase (8%) in activity over 0.2 mM; 0.2 mM was used routinely in the nerve extract assays. The partially purified enzyme from the electric organ has a Km for acetyl-CoA of 50 ~M (Baker and Dowdall, 1976). The activity in the nerve extracts was linear with incubation time for 1 h and proportional to the amount of extract. Increasing the temperature from 21-37 ~ C increased the rate of enzymic acetylation. Measurement of activity against a range of choline concentrations from 2-16 mM gave an apparent Km at 30 ~ C of 2.4 mM by Lineweaver-Burk or Hofstee plot. Like Bull et al. (1969) we found that temperature had a large effect on the Km of electric organ ChAc (crude extract) for choline, with an increase from 1.14 + 0.02 mM at 21 ~ C to 2.60 + 0.04 mM at 29.6 ~ C and 6.25 + 0.14 mM at 37 ~ C (linear regression analysis of Hofstee plots). With such large changes in K m and Vm it is thus important to ensure that incubation conditions, particularly temperature, are carefully controlled in any comparative experiments of ChAc activity. Routine measurements were made with 8 mM choline and a temperature of 30 ~ C. AChE was initially measured by using [1-I4C]acetylcholine essentially according to the procedure of Tu6ek (1974), but in later experiments the spectrophotometric method of Ellman et al. (1961) was found more convenient. The concentrations of acetylthiocholine iodide and 5.5'-dithiobis-(2-nitrobenzoic acid) used resulted in maximal activity of the Torpedo enzyme. Lactate dehydrogenase (LDH) was measured by following the oxidation of NADH2 in the presence of pyruvate (Johnson, 1960) and protein by the method of Lowry (Lowry et al., 1951) 0,1% using bovine serum albumin as standard (Ez78 nm= 0.666). ACh was estimated using the leech bioassay as described by Whittaker and Barker (1972). Efficiency of Extraction of Enzymes. Results for enzyme studies are expressed on a basis of extracted protein rather than total activity per segment as is common for transport studies. Because of the large diameter of the nerves, the effect of the ligature is to compress the axon bundles and reduce the diameter, thus altering the amount of material in a segment of fixed length. Furthermore, some branching of the nerves within the gill arches of the Torpedo results in a small proximo-distal decrease in nerve diameter. In tying double ligatures in vivo it was often not possible to apply the lower ligature above the point of initial branching of the nerve in the organ; thus those fascicles which were not included in the ligature had to be dissected away before sectioning and assay. Expression of results on the basis of solubilized protein also takes into account variations in the efficiency of extraction of each nerve piece and losses during transfer of powdered frozen material. Laduron (1973) has discussed the inherent problems in expressing accumulation of a product per unit length of nerve. The efficiencies of extraction of the nerve enzymes into the low-speed supernatants of the crude homogenates were determined by resuspending and assaying the activities remaining in the pellet. For LDH an average of 93.4 + S.E.M. 0.76%(12) was in the supernatant, for AChE 93.1 _+ 1.51, for ChAc 54 + 2.87 and protein (extracted from the pellet by 0.2 N NaOH with overnight warming), 37.8 _+ 2.21. For short term ligature experiments in vitro there were no significant differences in the extraction efficiency of the enzymes from pieces near the ligatures compared with pieces in the middle of the nerve. However, for longer term experiments (40 h and 1 week in vivo) it was found that there were some differences in the proportions of total ChAc extracted, depending on the position of the nerve piece relative to the ligature. Thus the measurement of ChAc in the low-speed supernatants of homogenates was not a completely accurate reflection of ChAc activity in the nerve segments and experiments were repeated to compare "total" and "supernatant" ChAc specific activity. These changes in extraction efficiency are discussed in "Results". Materials. Chemicals were purchased as follows: [1-14C] acetylcoenzyme A ( 3 - 5 mCi/mmol) and [1-14C]acetylchollne chloride (12.2 mCi/mmol) (Amersham-Buchler, Brannschweig, F.R.G.); penicillin/streptomycin solution (Flow, Irvine, Scotland); tetraphenylboron (Aldrich, Beerse, Belgium); acetylthiocholine iodide and 2,2 dinitro-5,5-dithiodibenzoic acid (Merck, Darmstadt, F.R.G.), bacitracin (Sigma, St. Louis, U.S.A) and disodium 4-nitro-phenylphosphate (B.D.H. Ltd., Poole, U.K.). Other chemicals were purchased from J. T. Baker (Deventer, Holland).
496
L. P. Davies et aL
INCUBATED
CONTROL (a} 60
~.0
20[ 0
(b)
E
"J0-5I 0 t-
o0.6~ f
(c)
