Acta physiol. scand. 1976. 96. 478-485 From the Institute of Neurobiology and the Dept. of Pharmacology, University of Goteborg, Sweden, and the Dept. of Physiology, University College London, England
The Effect of Local Application of Vinblastine or Colchicine on Acetylcholine Accumulation in Rat Sciatic Nerve' BY P.-0. HEIWALL, N. R. SAUNDERS, A. DAHLSTROM and J. HAGGENDAL Received 24 July 1975
Abstract HEIWALL, P.-0.. N. R. SAUNDERS, A. DAHLSTROM and J. HAGGENDAL. The effect of local application of vinblastine or colchicine on acetylcholine accumulation in rat sciatic nerve. Acta physiol. scand. 1976. 96. 478-485. The accumulation of acetylcholine (ACh) following nerve crush was inhibited by high concentrations of M) i n a dose-dependent manner. The two mitotic colchicine (IO-l, lo-' M) and vinblastine (IO-'-lO-* inhibitors were effective both when applied to axons (subepineural injections) and to perikarya (injections into the lumbar intumescence). The results indicate that mitotic inhibitors, when applied locally to cholinergic motor neurons, may block the proximo-distal transport of ACh. The possibility is discussed that this blockade is caused by an interference with a microtubule mechanism in the axons. Evidence for an increased local synthesis of ACh near the site of injection into nerves was also obtained with the highest concentrations of either substance used.
The significance of the presence of acetylcholine (ACh) in axons of cholinergic neurons is not clear, in contrast to its well-known involvement in impulse transmission at axon terminals (Katz 1966). One possible explanation which has been suggested for its presence in nonterminal axons may be intra-axonal transport of ACh from proximal parts of the neuron into more distal segments (cf. Haggendal et al. 1971, Dahlstrom et al. 1974). By using nerve crushes and estimating the changes in ACh in segments proximal and distal to the crush, results were obtained which indicated that (1) a fraction of the axonal ACh content was transported proximo-distally in the nerves at a rate of 4-5 mm/h (about 120 mm/day); (2) the transportable fraction of ACh was about 20% of the total axonal ACh (Dahlstrom et al. 1974). Since acetylcholinesterase (AChE) is also transported distally and accumulates in large amounts proximal to a crush (Lubinska and Niemierko 1971), the ACh which accumulates in the same segment is presumably localized in a separate compartment, protecting it from hydrolysis by AChE. It is possible that this compartment may be similar to that in periphReported in part at the Wennergren Symposium "Dynamics of Degeneration and Growth in Neurons", Stockholm, May 1973.
478
VINBLASTINE ON NERVE ACH
479
era1 adrenergic nerves where storage organelles containing the transmitter noradrenaline (NA) are transported distally in the axons towards the nerve terminals (cf. Dahlstrom and Haggendal 1970). The proximo-distal transport of amine storage granules in rat adrenergic nerves is rapid (8-9 mm/h, 200 mm/day; Haggendal et al. 1973, and is probably dependent on the presence of intact microtubules. If mitotic inhibitors like vinblastine (VIN) and colchicine (COL) are administered to nerves or soaked on peripheral sympathetic ganglia, the transport of NA storage organelles is inhibited in a dose-dependent manner (cf. Dahlstrom 1970, Banks et a/. 1971, Hokfelt and Dahlstrom 1971). This effect is likely to be due to the effect of the two drugs on the microtubules, since inhibition of transport at increasing concentrations of e.g. COL was parallelled by a decreasing number of visible microtubules (Banks e t a / . 1971). In order to test the possibility that microtubules may be involved in axonal transport of ACh in rat motor neurons, the two mitotic inhibitors COL or VIN were injected either into nerves or into the spinal cord close to the nerve cell bodies, and their effect on ACh accumulation was studied.
Methods Operative procedures
Male Sprague-Dawley rats (180-200 g) were anesthetized with ether. Their sciatic nerves were exposed 5-10 mm distal to f o r . infrapiriformis. 5 {cl subepineural injections were made at the site of a nerve side
branch (see Fig. I ) using a Hamilton microsyringe and 30 gauge ncedle. Into one nerve COL (Sandoz) M) dissolved in 0.9”” saline, was injected. In the opposite nerve 0.9% saline was injected as control. 2 h later the rats were reanesthetized with ether, and the sciatic nerves were crushed at a level 15 mm distal to the site of injection by compressing the nerve for 5 s between a fine silk thread and a glass-rod (Lubiiiska 1959). After a further 6 h the rats were killed by a blow on the head and both sciatic nerves were removed rapidly, blotted on filter paper on an ice-cooled plate, and divided into segments as shown in Fig. I , i.e. 5 mm proximal to the site of crushing (C) and 10 mm proximal (A) and around (B) the site of injection. In some experiments the nerves were neither injected nor crushed (“normal”) and in others they were crushed but not injected (“crushed”). In these last experiments the nerves were either crushed and the ACh levels estimated after 6 h of accumulation (“6 h crush”), o r the animals were sham-operated, i.e. the nerves were exposed but neither injected nor crushed; 2 h later the rats were reoperated and their sciatic nerves crushed as already described (“sham-op.”). 2-4 pieces of nerve were pooled for each estimation. In another series of expts., injections were made into the lumbar spinal cord following laminectomy. 4 injections (Fig. 3) of 5 ,tcl of COL (10-1-10-3M),V I N M), 0.9% saline or 0.15 M citrate buffer (pH 4.5) were given. 6-7 h later the sciatic nerves were crushed as described above. After a further 12 h the rats were killed, and 5 m m of nerve immediately proximal to the crush was dissected out and extracted for ACh. (IO-l, lo-’ M) or V I N (Eli-Lilly) (10-2-10-4
Extraction and assay of ACIt The methods used have hem described p:cviously ( D a h l ~ r o m e t a / . 1974). ACh was extracted by homogenizing the nerv3 segments in ice-cold lo”,, trichloroccetic acid, usirg the method of Maclntosh and Perry (1950). ACh was esti,nated according to Blaber and Cuthbert (1961). !using the terminal ileum of the guineapig and Schild’s (1942) 4-point assay. ACh was identified by inactivation with purified acetylcholinesterase (Worthington Biochemical Corporation, New Jersey) and by atropine inhibition. Student’s t-test was used for statistical analysis of the results.
Results The effect of different concentrations of COL and VIN upon the level of ACh accumulating proximal to a crush is shown in Fig. 1 and Fig. 2. Normal uncrushed, uninjected sciatic
480
P.-0.HEIWALL, N. R . SAUNDERS, A. DAHLSTROM AND J. HAGGENDAL
crush
05cm C crush
100
6 hr before
I
C normal
A B C
A B C
6hr crush
shrmop
A B C N a C I in1
A B C collO.*M
i
A B C colto-’M
Fig. I . The effect of colchicine (c0l.j on acetylcholine (AChj accumulation and transport in rat sciatic nerve. The ordinate indicates the amount of ACh in pmol per 5 or 10 mm piece of nerve following treatments which are indicated along the abscissa. Mean hS.E. are given. Number of observations is 3 except the normal segment C where n = 5. The injections were done 2 h prior to the crush as indicated in the upper right corner of the figure. Asterisks indicate levels of significance in differences against controls, injected with saline prior to crushing or only crushed for 6 h (p
The Effect of Local Application of Vinblastine or Colchicine on Acetylcholine Accumulation in Rat Sciatic Nerve' BY P.-0. HEIWALL, N. R. SAUNDERS, A. DAHLSTROM and J. HAGGENDAL Received 24 July 1975
Abstract HEIWALL, P.-0.. N. R. SAUNDERS, A. DAHLSTROM and J. HAGGENDAL. The effect of local application of vinblastine or colchicine on acetylcholine accumulation in rat sciatic nerve. Acta physiol. scand. 1976. 96. 478-485. The accumulation of acetylcholine (ACh) following nerve crush was inhibited by high concentrations of M) i n a dose-dependent manner. The two mitotic colchicine (IO-l, lo-' M) and vinblastine (IO-'-lO-* inhibitors were effective both when applied to axons (subepineural injections) and to perikarya (injections into the lumbar intumescence). The results indicate that mitotic inhibitors, when applied locally to cholinergic motor neurons, may block the proximo-distal transport of ACh. The possibility is discussed that this blockade is caused by an interference with a microtubule mechanism in the axons. Evidence for an increased local synthesis of ACh near the site of injection into nerves was also obtained with the highest concentrations of either substance used.
The significance of the presence of acetylcholine (ACh) in axons of cholinergic neurons is not clear, in contrast to its well-known involvement in impulse transmission at axon terminals (Katz 1966). One possible explanation which has been suggested for its presence in nonterminal axons may be intra-axonal transport of ACh from proximal parts of the neuron into more distal segments (cf. Haggendal et al. 1971, Dahlstrom et al. 1974). By using nerve crushes and estimating the changes in ACh in segments proximal and distal to the crush, results were obtained which indicated that (1) a fraction of the axonal ACh content was transported proximo-distally in the nerves at a rate of 4-5 mm/h (about 120 mm/day); (2) the transportable fraction of ACh was about 20% of the total axonal ACh (Dahlstrom et al. 1974). Since acetylcholinesterase (AChE) is also transported distally and accumulates in large amounts proximal to a crush (Lubinska and Niemierko 1971), the ACh which accumulates in the same segment is presumably localized in a separate compartment, protecting it from hydrolysis by AChE. It is possible that this compartment may be similar to that in periphReported in part at the Wennergren Symposium "Dynamics of Degeneration and Growth in Neurons", Stockholm, May 1973.
478
VINBLASTINE ON NERVE ACH
479
era1 adrenergic nerves where storage organelles containing the transmitter noradrenaline (NA) are transported distally in the axons towards the nerve terminals (cf. Dahlstrom and Haggendal 1970). The proximo-distal transport of amine storage granules in rat adrenergic nerves is rapid (8-9 mm/h, 200 mm/day; Haggendal et al. 1973, and is probably dependent on the presence of intact microtubules. If mitotic inhibitors like vinblastine (VIN) and colchicine (COL) are administered to nerves or soaked on peripheral sympathetic ganglia, the transport of NA storage organelles is inhibited in a dose-dependent manner (cf. Dahlstrom 1970, Banks et a/. 1971, Hokfelt and Dahlstrom 1971). This effect is likely to be due to the effect of the two drugs on the microtubules, since inhibition of transport at increasing concentrations of e.g. COL was parallelled by a decreasing number of visible microtubules (Banks e t a / . 1971). In order to test the possibility that microtubules may be involved in axonal transport of ACh in rat motor neurons, the two mitotic inhibitors COL or VIN were injected either into nerves or into the spinal cord close to the nerve cell bodies, and their effect on ACh accumulation was studied.
Methods Operative procedures
Male Sprague-Dawley rats (180-200 g) were anesthetized with ether. Their sciatic nerves were exposed 5-10 mm distal to f o r . infrapiriformis. 5 {cl subepineural injections were made at the site of a nerve side
branch (see Fig. I ) using a Hamilton microsyringe and 30 gauge ncedle. Into one nerve COL (Sandoz) M) dissolved in 0.9”” saline, was injected. In the opposite nerve 0.9% saline was injected as control. 2 h later the rats were reanesthetized with ether, and the sciatic nerves were crushed at a level 15 mm distal to the site of injection by compressing the nerve for 5 s between a fine silk thread and a glass-rod (Lubiiiska 1959). After a further 6 h the rats were killed by a blow on the head and both sciatic nerves were removed rapidly, blotted on filter paper on an ice-cooled plate, and divided into segments as shown in Fig. I , i.e. 5 mm proximal to the site of crushing (C) and 10 mm proximal (A) and around (B) the site of injection. In some experiments the nerves were neither injected nor crushed (“normal”) and in others they were crushed but not injected (“crushed”). In these last experiments the nerves were either crushed and the ACh levels estimated after 6 h of accumulation (“6 h crush”), o r the animals were sham-operated, i.e. the nerves were exposed but neither injected nor crushed; 2 h later the rats were reoperated and their sciatic nerves crushed as already described (“sham-op.”). 2-4 pieces of nerve were pooled for each estimation. In another series of expts., injections were made into the lumbar spinal cord following laminectomy. 4 injections (Fig. 3) of 5 ,tcl of COL (10-1-10-3M),V I N M), 0.9% saline or 0.15 M citrate buffer (pH 4.5) were given. 6-7 h later the sciatic nerves were crushed as described above. After a further 12 h the rats were killed, and 5 m m of nerve immediately proximal to the crush was dissected out and extracted for ACh. (IO-l, lo-’ M) or V I N (Eli-Lilly) (10-2-10-4
Extraction and assay of ACIt The methods used have hem described p:cviously ( D a h l ~ r o m e t a / . 1974). ACh was extracted by homogenizing the nerv3 segments in ice-cold lo”,, trichloroccetic acid, usirg the method of Maclntosh and Perry (1950). ACh was esti,nated according to Blaber and Cuthbert (1961). !using the terminal ileum of the guineapig and Schild’s (1942) 4-point assay. ACh was identified by inactivation with purified acetylcholinesterase (Worthington Biochemical Corporation, New Jersey) and by atropine inhibition. Student’s t-test was used for statistical analysis of the results.
Results The effect of different concentrations of COL and VIN upon the level of ACh accumulating proximal to a crush is shown in Fig. 1 and Fig. 2. Normal uncrushed, uninjected sciatic
480
P.-0.HEIWALL, N. R . SAUNDERS, A. DAHLSTROM AND J. HAGGENDAL
crush
05cm C crush
100
6 hr before
I
C normal
A B C
A B C
6hr crush
shrmop
A B C N a C I in1
A B C collO.*M
i
A B C colto-’M
Fig. I . The effect of colchicine (c0l.j on acetylcholine (AChj accumulation and transport in rat sciatic nerve. The ordinate indicates the amount of ACh in pmol per 5 or 10 mm piece of nerve following treatments which are indicated along the abscissa. Mean hS.E. are given. Number of observations is 3 except the normal segment C where n = 5. The injections were done 2 h prior to the crush as indicated in the upper right corner of the figure. Asterisks indicate levels of significance in differences against controls, injected with saline prior to crushing or only crushed for 6 h (p
