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Cytological aspects of the axonal migration of catecholamines and of their storage material
Brain Research, 62 (1973) 431-437
431
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
C Y T O L O G I C A L ASPECTS OF T H E A X O N A L M I G R A T I O N OF C A T E C H O L A M I N E S A N D OF T H E I R S T O R A G E M A T E R I A L
J. TAXI AND C. SOTELO Laboratoire de Biologie Animale, Universit~ de Paris VI, 75005 Paris and Laboratoire de Neuromorphologie, U-106 LN.S.E.R.M., 75014 Paris (France)
Ligated sympathetic nerves have been largely used to study axonal transport of catecholamines and their related storage organelles (see references in Dahlstr6ma). In the present communication, different kinds of experiments were carried out on the ligated sciatic nerve of the rat in order to re-study with another approach - - high resolution radioautography - - the problem of axonal transport of catecholamines. Two main questions were analyzed: (A) What is the contribution of axonal transport to the large accumulation of noradrenaline (NA) observed with fluorescence histochemistry 1 in the proximal portion of ligated sympathetic fibers? (B) What are the cytological features in these proximal portions which can be correlated with NA storage? (A) To answer the first question the following experiments were performed (a detailed description of the methods used here are already published12,16): 5 mCi of [3H]NA or of a precursor ([3H]DOPA or [aH]DA) were injected intravenously into a 80-100 g rat. It is already established14,15,17 that noradrenergic neurons are, under these conditions, immediately loaded with the [SH]NA. After a delay of 2 h, necessary to obtain a decrease of the blood concentration in [aH]NA to a negligible level, the sciatic nerves were ligated. From this moment, substances migrating inside the axons must accumulate above the ligature, and among such substances the [3H]NA. The rats were killed and fixed 3 h or 20 h after ligation. No labeling was observed on the proximal portion of the fibers after a period of migration of 20 h, but a moderate labeling was encountered in the 3 h experiments. The disappearance of the labeling between 3 and 20 h in our experiments means that the bulk of fluorescence NA visible after 24 h of ligation t cannot be accounted for by the migration of NA manufactured in the neuronal perikarya, but must be related to local synthesis and storage. When similar experiments were made in animals pretreated with a monoamineoxidase inhibitor (IMAO), a weak to moderate labeling was observed on proximal fibers not only in rats killed 3 h after ligation, but also in those killed after 20 h. The presence of labeling in these instances is certainly due to the slowing down of NA catabolism. These results, similar to those obtained by Geffen et al. 4 in quite different
Z
E
AXONAL MIGRATIONOF CATECHOLAMINES
433
experiments in the splenic nerve of the cat, tend to prove that the axonal migration of catecholamines is only an epiphenomenon related to the distal migration of enzymatic and storage proteins from the perikaryon. (B) To answer the second question, that is to say, the study of the ultrastructural features of the proximal portion of intensely fluorescent fibers, the following experiments were carried out: the sciatic nerves of rats were ligated. The labeled substance ([aH]NA or its precursors) was intravenously injected, 3 h or 22 h after the ligation. Within 30 min after the labeled administration, the sciatic nerves were fixed (a detailed description of this type of experiment has been already published12). The radioautographic method was then used to identify the sympathetic axons of the sciatic nerve, on the basis of their specificity for uptake and storage of exogenous [aH]NA (Fig. 1). Fibers retaining the [aH]NA exhibit a large heterogeneity in their contents. The most frequently observed aspect corresponds to fibers containing numerous vesicular profiles, measuring 40-100 nm in diameter (Fig. 1). The large majority of such vesicles appear empty. However, some of them exhibit a dense core. Among these dense-cored vesicles, and according to their size, the two well-known types present in the peripheral autonomic nervous system can be recognized: (1)vesicles with an average diameter of 60-80 nm (large granulated vesicles: LGV); and (2) vesicles of about 45 nm in diameter (small granulated vesicles: SGV). The former are much more numerous than the latter. Radioautographic silver grains can also be superimposed on axonal profiles almost full of tubular structures (Fig. 2) without dense contents, or with clusters of lysosomes, or even, less often, with mitochondria. Thus, the morphological features of ligated sympathetic fibers of the rat sciatic nerve are quite different from those described for the splenic nerve of the cat 7,s. To ascertain that this discrepancy is not due to a matter of fixation, similar experiments were made using 3 ~o potassium permanganate (KMnO4) as a fixative, since its ability to preserve the dense core in vesicles is well established ~,1°. Despite the poor value of this fixative for radioautography, because it apparently disrupts noradrenaline molecules during its reduction to produce the dense core in vesicles, a moderate labeling, enough for the identification of sympathetic axons, was obtained (Fig. 3). Although with this fixation the number of granulated vesicles, especially of SGV, is obviously increased, the heterogeneity in the contents of the labeled axons is still well marked. Thus, axonal profiles containing only few granulated vesicles (Fig. 3), or only agranular vesicles, or even tubular structures were significantly labeled. According to these results, there is not a constant correlation between the labelFig. 1. Rat. Proximal segment of a sciatic nerve ligated 22 h before osmic acid fixation. The [aH]NA was administered 30 rain before the fixation. Only one of the unmyelinated fibers is labeled, attesting the high specificity of the reaction. The labeled sympathetic nerve fiber contains mainly empty vesicular profiles of various diameters. Few SGV are also present (arrows). x 14,000. Fig. 2. Rat. Similar material and experimental conditions as in Fig. 1. The largest labeled axon mainly contains tubular structures (T). The arrow points to the zone of continuity between a tubular and a vesicular profile exhibiting a dense core. These pictures indicate that at least part of the vesicles originate locally by a process of outgrowth and budding off from the smooth endoplasmic reticulum. x 31,000.
434
J. I'AXI A N I ) C. SOIEI_O
AXONAL MIGRATIONOF CATECHOLAMINES
435
ing and the presence of granulated vesicles, even SGV, in the ligated fibers. In order to prove that under these pathological conditions--constriction of a n e r v e - - t h e presence of granulated vesicles (LGV or even SGV) in KMnO4 fixed material is not a histochemical test to identify catecholaminergic fibers, some control tests have been carried out on the preganglionic trunk of the rat superior cervical ganglion. Twenty hours after ligation o f this almost purely cholinergic nerve, axonal profiles of its proximal stump contain not only LGV but also SGV (Fig. 4). These results prove that under the present experimental conditions, the vesicular content is not a valid criterion to identify noradrenergic fibers. The action of some drugs (IMAO, reserpine) on the ultrastructural features and uptake capability of the proximal portion of ligated sympathetic fibers has been studied: (I) Action of IMAO: this drug does not induce any change in the morphological heterogeneity of labeled fibers. From a quantitative point of view the labeling seems to be heavier than the one obtained in non-treated animals. Untortunately, precise quantitative comparisons between experiments performed in different animals cannot be achieved due to technical limitations 15. (2) Action of reserpine (5 mg/kg i.p.): this drug induces the depletion of the dense core of the granulated vesicles. However, it is to be noticed that a certain number of LGV keep their normal appearance. Concerning the labeling, after 3 or 6 h of drug action it is greatly reduced or even absent. These morphological observations, closely related to those obtained by Kapeller and Mayor s about the action of reserpine on LGV content in the ligated splenic nerve of the cat, sustain the idea that LGV constitute a heterogeneous population. In axon terminal varicosities of normal sympathetic peripheral fibers, SGV and perhaps LGV are considered as the specific storage organelles for catecholamines. A problem still in controversy concerns the origin of such vesicles. Some authors2, s have suggested that granulated vesicles are elaborated in the perikaryon, and that they reach the axon terminal varicosities by axonal transport. Several arguments can be raised against this concept. Despite the fact that in the neuronal perikarya of sympathetic ganglia LGV and SGV can be presentS, 13 and that after KMn04 fixation 6 SGV are more numerous than has been originally described, SGV and partly LGV are probably little involved in axonal transport for the following reasons. (a) Radioautographic studies in sympathetic ganglia (Taxi and Droz15; Taxi, unpublished observations) using exogenous [3H]NA have shown that only clusters of SGV exhibit a reliable labeling in rats not treated with an IMAO. However, in rats pretreated with an IMAO, in which there is an intense labeling o f ganglionic neurons,
Fig. 3. Rat. Similar experimental conditions as in Fig. 1, but the sciatic nerve was fixed with 3 KMnO4. The labeled fiber mainly contains empty vesicular profiles of different sizes. × 25,000. Fig. 4. Rat. Proximal end of the preganglionic trunk of a superior cervical ganglion 20 h after crush, fixed with 3 ~ KMnO4. In this cholinergic modified nerve fiber, LGV (large arrows) and even SGV (small arrows) are intermixed with tubular profiles of the smooth endoplasmic reticulum. × 22,500.
436
J. TAXI AND C. SOTELO
no special affinity was observed between labeling and the Golgi region with its associated LGV, indicating that such vesicles are probably not involved in catecholamine storage, but that they probably represent primary lysosomes 11. Clusters of SGV, retaining the [3H]NA, are more numerous in dendrites. In both instances, in perikarya and in dendrites, such SGV clusters are mainly localized near the plasma membrane, being associated with it by peculiar morphological differentiations resembling the cytoplasmic dense patches characterizing the presynaptic zones. For this reason, it may be suggested that such vesicles are involved in a somatic or dendritic release mechanism, rather than in a phenomenon of axonal migration. (b) The characteristics of the [3H]NA storage in ganglionic perikarya 15 is not only quantitatively different from that of the axon terminal varicosities, but also qualitatively. (As indicated above, it is possible to obtain a specific labeling of the perikarya only in rats pretreated with an IMAO.) For this reason the setting of storage organelles at the periphery must not be a simple translation of such organelles already manufactured in the perikaryon. Another possible explanation for the presence of granulated vesicles in peripheral axonal varicosities may be their formation in situ, as has been postulated for agranular vesicles in central axon terminals 9. If this is the case, the enzymes involved in the synthesis of catecholamines and their proteinaceous storage material are synthetized in the perikaryon, where a part of them is already assembled as SGV. Few SGV and most LGV can migrate, since they can be observed in non-synaptic segments of axons (1 SGV/10 LGV, on average). However, the majority of these proteins are transported by axonal flow, not as a distinct organelle, but as a non-particulate protein, probably inside the smooth endoplasmic reticulum. The assemblage of such proteins into distinct storage organelles occurs only at the axon terminal varicosities, and leads directly to the formation of SGV from the smooth endoplasmic reticulum. The artificial stoppage of axonal migration by nerve ligation induces dramatic local changes due to the high concentration of substances and the subsequent local remodeling. This may lead to the appearance of granulated vesicles of various sizes, some of them being related not to catecholamine storage organelles, but to lytic processes. F r o m a morphological point of view, the proximal stump of a ligated nerve cannot be compared with an axon terminal where the axonal transport is naturally stopped.
1 DAHLSTROM,A.,
Observations on the accumulation of noradrenaline in the proximal and distal parts of peripheral adrenergic nerve after compression, J. Anat. (Lond.), 99 (1965) 677-689.
2 DAHLSTR~M,A., The Intraneuronal Distribution of Noradrenaline and the Transport and Life-span of Amine Storage Granules in the Sympathetic Adrenergic Neuron, M.D. Thesis, Stockholm, 1966. 3 DAHLSTR()M, A., Axoplasmic transport (with particular respect to adrenergic neurons), Phil. Trans. B, 261 (1971) 325-358. 4 GEFFEN, L. B., DESCARRIES,L., AND DROZ, B., Intra-axonal migration of [aH]norepinephrine
injected into the coeliac ganglion of cats: radioautographic study of the proximal segment of constricted splenic nerves, Brain Research, 35 (1971) 315-318. 5 GRILLO, M. A., Electron microscopy of sympathetic tissues, Pharmacol. Rev., 18 (1966) 387-399. 6 HOKFELT,T., In vitro studies on central and peripheral monoamine neurons at the ultrastructural level, Z. Zellforsch., 91 (1968) 1-74.
AXONAL MIGRATION OF CATECHOLAMINES
437
7 KAPELLER,K., AND MAYOR,D., Ultrastructural changes proximal to a constriction in sympathetic axons during first 24 hours after operation, J. Anat. (Lond.), 100 (1966) 439-441. 8 KAPELLER, K., AND MAYOR, D., The accumulation of noradrenaline in constricted sympathetic nerve as studied by fluorescence and electron microscopy, Proc. roy. Soc. B, 167 (1967) 282-292. 9 PALAY,S. L., The morphology of synapses in the central nervous system, Exp. Cell Res., Suppl. 5 (1958) 275-293. 10 RICHARDSON, K. C., Electron microscopic identification of autonomic nerve endings, Nature (Lond.), 210 (1966) 756. 11 SOTELO,C., The fine structural localization of norepinephrine-aH in the substantia nigra and area postrema of the rat: An autoradiographic study, J. Ultrastruct. Res., 36 (1971) 824-841. 12 SOTELO,C., AND TAXI, J., On the axonal migration of catecholamines in constricted sciatic nerve of the rat: A radioautographic study, Z. Zellforsch., 138 (1973) 345-370. 13 TAxi, J., Contribution/t l'6tude des connexions des neurones moteurs du syst~me nerveux autonome, Ann. Sci. nat. Zool., 7 (1965) 413-674. 14 TAXI, J., ET DROZ, B., Etude de l'incorporation de noradr6naline-aH (NA-SH) et de 5-hydroxytryptophane-aH (5-HTP-3H) dans l'6piphyse et le ganglion cervical sup6rieur, C.R. Acad. Sci. (Paris), 263 (1966) 1326-1329. 15 TAXI,J., AND DROZ, B., Radioautographic study of the accumulation of some biogenic amines in the autonomic nervous system. In S. H. BARONDES(Ed.), Cellular Dynamics of the Neuron, Symposia Int. Soc. Cell Biol., Vol. 8, Academic Press, New York, 1969, pp. 175-190. 16 TAXI, J., ET SOTELO, C., Le probl~me de la migration des cat6cholamines dans les neurones sympathiques, Rev. neurol., 127 (1972) 23-36. 17 WOLFE, D. E., POTTER, L. T., RICHARDSON,K. C., AND AXELROD,J., Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography, Science, 138 (1962) 440-442.
431
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
C Y T O L O G I C A L ASPECTS OF T H E A X O N A L M I G R A T I O N OF C A T E C H O L A M I N E S A N D OF T H E I R S T O R A G E M A T E R I A L
J. TAXI AND C. SOTELO Laboratoire de Biologie Animale, Universit~ de Paris VI, 75005 Paris and Laboratoire de Neuromorphologie, U-106 LN.S.E.R.M., 75014 Paris (France)
Ligated sympathetic nerves have been largely used to study axonal transport of catecholamines and their related storage organelles (see references in Dahlstr6ma). In the present communication, different kinds of experiments were carried out on the ligated sciatic nerve of the rat in order to re-study with another approach - - high resolution radioautography - - the problem of axonal transport of catecholamines. Two main questions were analyzed: (A) What is the contribution of axonal transport to the large accumulation of noradrenaline (NA) observed with fluorescence histochemistry 1 in the proximal portion of ligated sympathetic fibers? (B) What are the cytological features in these proximal portions which can be correlated with NA storage? (A) To answer the first question the following experiments were performed (a detailed description of the methods used here are already published12,16): 5 mCi of [3H]NA or of a precursor ([3H]DOPA or [aH]DA) were injected intravenously into a 80-100 g rat. It is already established14,15,17 that noradrenergic neurons are, under these conditions, immediately loaded with the [SH]NA. After a delay of 2 h, necessary to obtain a decrease of the blood concentration in [aH]NA to a negligible level, the sciatic nerves were ligated. From this moment, substances migrating inside the axons must accumulate above the ligature, and among such substances the [3H]NA. The rats were killed and fixed 3 h or 20 h after ligation. No labeling was observed on the proximal portion of the fibers after a period of migration of 20 h, but a moderate labeling was encountered in the 3 h experiments. The disappearance of the labeling between 3 and 20 h in our experiments means that the bulk of fluorescence NA visible after 24 h of ligation t cannot be accounted for by the migration of NA manufactured in the neuronal perikarya, but must be related to local synthesis and storage. When similar experiments were made in animals pretreated with a monoamineoxidase inhibitor (IMAO), a weak to moderate labeling was observed on proximal fibers not only in rats killed 3 h after ligation, but also in those killed after 20 h. The presence of labeling in these instances is certainly due to the slowing down of NA catabolism. These results, similar to those obtained by Geffen et al. 4 in quite different
Z
E
AXONAL MIGRATIONOF CATECHOLAMINES
433
experiments in the splenic nerve of the cat, tend to prove that the axonal migration of catecholamines is only an epiphenomenon related to the distal migration of enzymatic and storage proteins from the perikaryon. (B) To answer the second question, that is to say, the study of the ultrastructural features of the proximal portion of intensely fluorescent fibers, the following experiments were carried out: the sciatic nerves of rats were ligated. The labeled substance ([aH]NA or its precursors) was intravenously injected, 3 h or 22 h after the ligation. Within 30 min after the labeled administration, the sciatic nerves were fixed (a detailed description of this type of experiment has been already published12). The radioautographic method was then used to identify the sympathetic axons of the sciatic nerve, on the basis of their specificity for uptake and storage of exogenous [aH]NA (Fig. 1). Fibers retaining the [aH]NA exhibit a large heterogeneity in their contents. The most frequently observed aspect corresponds to fibers containing numerous vesicular profiles, measuring 40-100 nm in diameter (Fig. 1). The large majority of such vesicles appear empty. However, some of them exhibit a dense core. Among these dense-cored vesicles, and according to their size, the two well-known types present in the peripheral autonomic nervous system can be recognized: (1)vesicles with an average diameter of 60-80 nm (large granulated vesicles: LGV); and (2) vesicles of about 45 nm in diameter (small granulated vesicles: SGV). The former are much more numerous than the latter. Radioautographic silver grains can also be superimposed on axonal profiles almost full of tubular structures (Fig. 2) without dense contents, or with clusters of lysosomes, or even, less often, with mitochondria. Thus, the morphological features of ligated sympathetic fibers of the rat sciatic nerve are quite different from those described for the splenic nerve of the cat 7,s. To ascertain that this discrepancy is not due to a matter of fixation, similar experiments were made using 3 ~o potassium permanganate (KMnO4) as a fixative, since its ability to preserve the dense core in vesicles is well established ~,1°. Despite the poor value of this fixative for radioautography, because it apparently disrupts noradrenaline molecules during its reduction to produce the dense core in vesicles, a moderate labeling, enough for the identification of sympathetic axons, was obtained (Fig. 3). Although with this fixation the number of granulated vesicles, especially of SGV, is obviously increased, the heterogeneity in the contents of the labeled axons is still well marked. Thus, axonal profiles containing only few granulated vesicles (Fig. 3), or only agranular vesicles, or even tubular structures were significantly labeled. According to these results, there is not a constant correlation between the labelFig. 1. Rat. Proximal segment of a sciatic nerve ligated 22 h before osmic acid fixation. The [aH]NA was administered 30 rain before the fixation. Only one of the unmyelinated fibers is labeled, attesting the high specificity of the reaction. The labeled sympathetic nerve fiber contains mainly empty vesicular profiles of various diameters. Few SGV are also present (arrows). x 14,000. Fig. 2. Rat. Similar material and experimental conditions as in Fig. 1. The largest labeled axon mainly contains tubular structures (T). The arrow points to the zone of continuity between a tubular and a vesicular profile exhibiting a dense core. These pictures indicate that at least part of the vesicles originate locally by a process of outgrowth and budding off from the smooth endoplasmic reticulum. x 31,000.
434
J. I'AXI A N I ) C. SOIEI_O
AXONAL MIGRATIONOF CATECHOLAMINES
435
ing and the presence of granulated vesicles, even SGV, in the ligated fibers. In order to prove that under these pathological conditions--constriction of a n e r v e - - t h e presence of granulated vesicles (LGV or even SGV) in KMnO4 fixed material is not a histochemical test to identify catecholaminergic fibers, some control tests have been carried out on the preganglionic trunk of the rat superior cervical ganglion. Twenty hours after ligation o f this almost purely cholinergic nerve, axonal profiles of its proximal stump contain not only LGV but also SGV (Fig. 4). These results prove that under the present experimental conditions, the vesicular content is not a valid criterion to identify noradrenergic fibers. The action of some drugs (IMAO, reserpine) on the ultrastructural features and uptake capability of the proximal portion of ligated sympathetic fibers has been studied: (I) Action of IMAO: this drug does not induce any change in the morphological heterogeneity of labeled fibers. From a quantitative point of view the labeling seems to be heavier than the one obtained in non-treated animals. Untortunately, precise quantitative comparisons between experiments performed in different animals cannot be achieved due to technical limitations 15. (2) Action of reserpine (5 mg/kg i.p.): this drug induces the depletion of the dense core of the granulated vesicles. However, it is to be noticed that a certain number of LGV keep their normal appearance. Concerning the labeling, after 3 or 6 h of drug action it is greatly reduced or even absent. These morphological observations, closely related to those obtained by Kapeller and Mayor s about the action of reserpine on LGV content in the ligated splenic nerve of the cat, sustain the idea that LGV constitute a heterogeneous population. In axon terminal varicosities of normal sympathetic peripheral fibers, SGV and perhaps LGV are considered as the specific storage organelles for catecholamines. A problem still in controversy concerns the origin of such vesicles. Some authors2, s have suggested that granulated vesicles are elaborated in the perikaryon, and that they reach the axon terminal varicosities by axonal transport. Several arguments can be raised against this concept. Despite the fact that in the neuronal perikarya of sympathetic ganglia LGV and SGV can be presentS, 13 and that after KMn04 fixation 6 SGV are more numerous than has been originally described, SGV and partly LGV are probably little involved in axonal transport for the following reasons. (a) Radioautographic studies in sympathetic ganglia (Taxi and Droz15; Taxi, unpublished observations) using exogenous [3H]NA have shown that only clusters of SGV exhibit a reliable labeling in rats not treated with an IMAO. However, in rats pretreated with an IMAO, in which there is an intense labeling o f ganglionic neurons,
Fig. 3. Rat. Similar experimental conditions as in Fig. 1, but the sciatic nerve was fixed with 3 KMnO4. The labeled fiber mainly contains empty vesicular profiles of different sizes. × 25,000. Fig. 4. Rat. Proximal end of the preganglionic trunk of a superior cervical ganglion 20 h after crush, fixed with 3 ~ KMnO4. In this cholinergic modified nerve fiber, LGV (large arrows) and even SGV (small arrows) are intermixed with tubular profiles of the smooth endoplasmic reticulum. × 22,500.
436
J. TAXI AND C. SOTELO
no special affinity was observed between labeling and the Golgi region with its associated LGV, indicating that such vesicles are probably not involved in catecholamine storage, but that they probably represent primary lysosomes 11. Clusters of SGV, retaining the [3H]NA, are more numerous in dendrites. In both instances, in perikarya and in dendrites, such SGV clusters are mainly localized near the plasma membrane, being associated with it by peculiar morphological differentiations resembling the cytoplasmic dense patches characterizing the presynaptic zones. For this reason, it may be suggested that such vesicles are involved in a somatic or dendritic release mechanism, rather than in a phenomenon of axonal migration. (b) The characteristics of the [3H]NA storage in ganglionic perikarya 15 is not only quantitatively different from that of the axon terminal varicosities, but also qualitatively. (As indicated above, it is possible to obtain a specific labeling of the perikarya only in rats pretreated with an IMAO.) For this reason the setting of storage organelles at the periphery must not be a simple translation of such organelles already manufactured in the perikaryon. Another possible explanation for the presence of granulated vesicles in peripheral axonal varicosities may be their formation in situ, as has been postulated for agranular vesicles in central axon terminals 9. If this is the case, the enzymes involved in the synthesis of catecholamines and their proteinaceous storage material are synthetized in the perikaryon, where a part of them is already assembled as SGV. Few SGV and most LGV can migrate, since they can be observed in non-synaptic segments of axons (1 SGV/10 LGV, on average). However, the majority of these proteins are transported by axonal flow, not as a distinct organelle, but as a non-particulate protein, probably inside the smooth endoplasmic reticulum. The assemblage of such proteins into distinct storage organelles occurs only at the axon terminal varicosities, and leads directly to the formation of SGV from the smooth endoplasmic reticulum. The artificial stoppage of axonal migration by nerve ligation induces dramatic local changes due to the high concentration of substances and the subsequent local remodeling. This may lead to the appearance of granulated vesicles of various sizes, some of them being related not to catecholamine storage organelles, but to lytic processes. F r o m a morphological point of view, the proximal stump of a ligated nerve cannot be compared with an axon terminal where the axonal transport is naturally stopped.
1 DAHLSTROM,A.,
Observations on the accumulation of noradrenaline in the proximal and distal parts of peripheral adrenergic nerve after compression, J. Anat. (Lond.), 99 (1965) 677-689.
2 DAHLSTR~M,A., The Intraneuronal Distribution of Noradrenaline and the Transport and Life-span of Amine Storage Granules in the Sympathetic Adrenergic Neuron, M.D. Thesis, Stockholm, 1966. 3 DAHLSTR()M, A., Axoplasmic transport (with particular respect to adrenergic neurons), Phil. Trans. B, 261 (1971) 325-358. 4 GEFFEN, L. B., DESCARRIES,L., AND DROZ, B., Intra-axonal migration of [aH]norepinephrine
injected into the coeliac ganglion of cats: radioautographic study of the proximal segment of constricted splenic nerves, Brain Research, 35 (1971) 315-318. 5 GRILLO, M. A., Electron microscopy of sympathetic tissues, Pharmacol. Rev., 18 (1966) 387-399. 6 HOKFELT,T., In vitro studies on central and peripheral monoamine neurons at the ultrastructural level, Z. Zellforsch., 91 (1968) 1-74.
AXONAL MIGRATION OF CATECHOLAMINES
437
7 KAPELLER,K., AND MAYOR,D., Ultrastructural changes proximal to a constriction in sympathetic axons during first 24 hours after operation, J. Anat. (Lond.), 100 (1966) 439-441. 8 KAPELLER, K., AND MAYOR, D., The accumulation of noradrenaline in constricted sympathetic nerve as studied by fluorescence and electron microscopy, Proc. roy. Soc. B, 167 (1967) 282-292. 9 PALAY,S. L., The morphology of synapses in the central nervous system, Exp. Cell Res., Suppl. 5 (1958) 275-293. 10 RICHARDSON, K. C., Electron microscopic identification of autonomic nerve endings, Nature (Lond.), 210 (1966) 756. 11 SOTELO,C., The fine structural localization of norepinephrine-aH in the substantia nigra and area postrema of the rat: An autoradiographic study, J. Ultrastruct. Res., 36 (1971) 824-841. 12 SOTELO,C., AND TAXI, J., On the axonal migration of catecholamines in constricted sciatic nerve of the rat: A radioautographic study, Z. Zellforsch., 138 (1973) 345-370. 13 TAxi, J., Contribution/t l'6tude des connexions des neurones moteurs du syst~me nerveux autonome, Ann. Sci. nat. Zool., 7 (1965) 413-674. 14 TAXI, J., ET DROZ, B., Etude de l'incorporation de noradr6naline-aH (NA-SH) et de 5-hydroxytryptophane-aH (5-HTP-3H) dans l'6piphyse et le ganglion cervical sup6rieur, C.R. Acad. Sci. (Paris), 263 (1966) 1326-1329. 15 TAXI,J., AND DROZ, B., Radioautographic study of the accumulation of some biogenic amines in the autonomic nervous system. In S. H. BARONDES(Ed.), Cellular Dynamics of the Neuron, Symposia Int. Soc. Cell Biol., Vol. 8, Academic Press, New York, 1969, pp. 175-190. 16 TAXI, J., ET SOTELO, C., Le probl~me de la migration des cat6cholamines dans les neurones sympathiques, Rev. neurol., 127 (1972) 23-36. 17 WOLFE, D. E., POTTER, L. T., RICHARDSON,K. C., AND AXELROD,J., Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography, Science, 138 (1962) 440-442.