Aminergic transmission. Introduction and short review

Brain Research, 62 (1973) 441-460 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

441

AMINERGIC TRANSMISSION. INTRODUCTION AND SHORT REVIEW

ANNICA DAHLSTROM University of GSteborg, Institute of Neurobiology, GOteborg (Sweden)

(I) General introduction The term 'aminergic neurons' includes neuron systems in both the CNS and the peripheral nervous system (PNS). The central neurons synthesize, store and in all likelihood also release noradrenaline (NA), dopamine (DA) or 5-hydroxytryptamine (5-HT). The sympathetic adrenergic nerves in the PNS synthesize, store and release N A in mammals and adrenaline (A) in e.g. frogs. The localization and mapping of these central and peripheral aminergic neuron systems are rather well-knownZ0, ag,sl,99 and have been studied with the use of the histochemical fluorescence method of Hillarp and Falck (for ref. and description see e.g. refs. 15, 33). The general morphology of these neurons is similar in the CNS and in the PNS (Fig. 1). A medium-sized multipolar cell body gives rise to a thin, unmyelinated axon with or without collaterals. These preterminal or non-terminal axons give rise to a widespread nerve terminal network, with a summed length of the terminal branches reaching values of a few centimeters in the rat CNS 2 and up to 10 cm in the periph-

cellbody

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Fig. 1. Schematic illustration of a monoaminergic neuron. (From ref. 20.)

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ery 23. The terminal branches widen at intervals to regular swellings, the so-called varicosities which appear as bag-like structures from which the transmitters are presumed to be released upon the arrival of a nerve impulse. These varicosities contain large accumulations of synaptic vesicle structures, usually called amine storage granules, particles or vesicles. These amine storage particles store the transmitter which is thereby protected from de-amination by the mitochondrial enzyme monoamine oxidase (MAO). Also, only transmitter stored in the granules seems to be released upon nerve stimulation; evidence on this has been obtained in studies on peripheral adrenergic nerves by e.g. Hfiggendal and Malmfors 5~ who were unable to detect release of transmitter that was located freely in the cytoplasm following M A D inhibition and blockage of the amine storage mechanism of the granules by reserpine. It is likely that also in the central neurons the NA, D A or 5-HT must be stored in the amine storage vesicles in order to be released by nerve impulses. In addition to amines the storage particles also contain proteins. They are manufactured in the cell body and transported in a more or less mature state to the varicosities by an active, intra-axonal transport mechanism, operating at a rate of several mm/h. This transport has been studied by many authors with different methods and the exact figure has varied somewhat, depending upon the nerve studied, the animal and the method used 8,~4,43,72,73,76. However, the order of magnitude is OH

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443

AMINERGIC TRANSMISSION. A REVIEW

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Fig. 3. Schematic illustration of a noradrenergic nerve terminal varicosity. For explanation see text. similar in all studies and the rate of several mm/h generally observed categorizes the transport as fast, possibly depending upon the microtubule system in the neurons4, 5, 19,21 In all known types of MA neurons the granules, or precursors of granules, contain the transmitter already in the cell bodies. However, the amount of transmitter which the amine particles contain when they arrive in the nerve terminals by intraaxonal transport does not significantly contribute to the maintenance of the transmitter stores in the terminalsZ4, 43. The local transmitter stores in the terminals are normally maintained both by re-uptake of released transmitter through the varicosity membrane and by local synthesis of transmitter in the nerve terminals. The amino acids tyrosine and tryptophan are considered to be the natural precursors for catecholamine (CA) and 5-HT, respectively. The synthesis of CA is illustrated in Fig. 2. The last step, from NA to A, is thought to occur mainly in the suprarenal medulla in mammals, while in the frog, both peripheral adrenergic nerves and CNS neurons store A. As will be mentioned by H6kfelt in this session, some rat brain neurons appear to contain the enzyme phenylethanolamine-N-methyl-transferase (PNMT) and it is therefore possible that mammalian CNS neurons also form and store some A. The formation of 5-HT from tryptophan occurs via 5-hydroxylation of the amino acid by tryptophan hydroxylase to 5-hydroxytryptophan (5-HTP) which is then decarboxylated to 5-HT by an aromatic amino acid decarboxylase (AAD) which is immuno-cross-reactive with DOPA decarboxylase in CA neurons (see H6kfelt this session). Fig. 3 represents a schematic model of the transmitter turnover in NA nerve terminals. The small figures of Fig. 3 represent the following events: (1) uptake of tyrosine into the varicosity; (2) hydroxylation to dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase (TH) which has been presumed to be a soluble enzyme, and subsequent decarboxylation of L-DOPA to DA by DOPA-decarboxylase (DDC). Both these steps probably occur in the cytoplasm; (3) the DA is taken up into the amine storage granules; and (4) ~-hydroxylated to NA by DA-fl-hydroxylase (DBH) which is localized in the storage granules; (5) indicates a possible transfer of NA

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between storage granules which is probably operating as a spontaneous leakage of NA molecules which are then trapped by the uptake-storage mechanism of neighboring granules; (6) intragranular NA is released by nerve impulses; and (7) stimulates the effector cell receptor sites; (8) is metabolized by catechol-O-methyltransferase; (9) diffuses (overflow) to the blood stream (not in the CNS where the blood-brain barrier prevents the escape into the blood stream) ; or (10) is recaptured by the effective neuronal membrane pump; (l 1) the recaptured NA is then either taken up into a storage granule and can be re-used for release; or (t2) deaminated by the mitochondrial enzyme monoamine oxidase (MAO). The re-uptake of released transmitter is probably important for the economy of transmitter stores 6,7 and is also a mechanism for inactivation of released transmitter. Exogenous NA is taken up in adrenergically innervated tissues by one saturable, high affinity process (uptake 1 of Iversen) which corresponds to this re-uptake into the adrenergic nerve terminals and by one less saturable, low affinity process (uptake 2 of Iversen) which tSrobably corresponds to uptake of NA into non-neuronal cells like e.g. smooth muscle cells 66. In the DA- or 5-HT-containing varicosity, principally the same evenls are considered to occur with the exception of the final step of transmitter synthesis which does not occur in the storage granules by a granule-bound enzyme, but which is completed outside the granules by the decarboxylating enzymes. Therefore, as far as is known, the granules do not participate in the synthesis of transmitter but do take up, store and release the DA or 5-HT. (For review and references on transmitter turnover and pharmacology of aminergic neurons see e.g~:refs. 1, 39, 66.) t

(II) Localization and transport o f enzymes

In 1967, G i b b e t al. 44 demonstrated that DBH had antigenic properties. This provided a basis for immunological studies of mormamine-containing cells. Using an indirect immunofluorescent staining technique, Geffen et al. 41 in 1969 were able to localize both DBH and chromogranin A, a matrix protein of amine granules, in adrenal medulla and peripheral adrenergic neurons. These observations have now been extended by H6kfelt et aL ~aa, who have also purified D D C and P N M T (Fig. 2) and produced antibodies against all three enzymes. For practical reasons, the technical development of the method to study immunohistochemically the distribution of the enzymes has been performed using the adrenal medulla. Dr. HSkfelt is going to present this technique and also describe results obtained in the CNS. The development of immunohistochemical methods for enzymes involved in transmitter metabolism is important since it may open up new possibilities to localize and study specific neuron systems which cannot yet be visualized in the same elegant way as the monoamine neurons, with the Hillarp-Falck method. Also, DA and N A neurons, both green fluorescent with the Hillarp-Falck method, can be identified with immunofluorescence of DBH which occurs only in N A neurons. Other advantages will be discussed by Dr. H6kfelt (this session). As previously mentioned, T H has been presumed to be a soluble enzyme. Because of this one would expect T H to be transported from the soma to the terminals

AMINERGIC TRANSMISSION. A REVIEW

445

by the slow flow and not by the fast flow which is thought to carry mainly particulate substances 90. in accordance with this theory, Laduron and Belpaire 74 found that in ligated or crushed adrenergic nerves, T H accumulated very slowly, if at all, above the crush, while NA and DBH, both constituents of amine granules, accumulated rapidly. D D C was found to accumulate slowly, in a pattern quite different from that of DBH accumulation 72a. Studies on transport and accumulation of these enzymes - TH, D D C and DBH - - have recently been repeated by e.g. Jarrott and Geffen 69, Coyle and Wooten 16, Dairman et al. 27a and by Thoenen (see this session). The results seem to indicate that T H and D D C are transported faster than the slow flow but slower than the fast flow (around 0.7 mm/h) and may therefore to a certain degree be particulate at least in non-terminal axons. T H appears to be a difficult enzyme to characterize with respect to rate of transport. Some authors found it to be transported at approximately the same rate as DBH16, 69 while others found rates considerably slower than that of DBH (e.g. Thoenen et al., this session). One reason for the discrepancy between the results of Laduron 74 and those of other authors 16,69,96 may be the mode in which the accumulation of a substance above a crush is expressed. Since edema occurs around the site of crush and since a lot of protein-containing particles rapidly accumulate above the crush, any increase of a particular substance which is related to weight or protein content of the nerve may give falsely low figures. The most reliable way to express accumulation of a substance is therefore to relate it to a certain length of nerve which is relatively unchanged after crushing a nerve. As pointed out in the discussion following Dr. Thoenen's paper, another important point to study when estimating rates ofaxonal flow by crush experiments is the transportable fraction of an axonal substance. This may be done in double crush experiments or by determining the decrease in the nerve part distal to the crush soon (2-6 h) after operation. The segment (5 mm) just below the crush must in such experiments be omitted since retrograde accumulation of various substances may occur in this segment and interfere with the results. As demonstrated by Ochs and coworkers 83, the proximo-distal transport in axons continues in the distal part of a cut or crushed nerve. Since only continuous supply from the cell is interrupted by the axotomy while distally directed transport continues, a decrease, corresponding to the transportable fraction of the substance, is likely to occur. The transportable fraction o f axonal NA 52, acetylcholine (ACh) 22, cholineacetyltransferase (ChAc) 3s and acetylcholine esterase (ACHE)77, 86 has been estimated to be between 5 and 50% of the axonal content. This implies that the rate of transport of the respective substances (transportable fraction) is increased several-fold, since transport is often calculated from accumulated versus control content. This procedure, however, can only be used for substances that are transported comparatively rapidly; for substances transported more slowly, longer time intervals after crushing'would have to be used and the danger of interference from degeneration becomes more critical, as pointed out by Dr. Thoenen. (III) Regulation o f transmitter release

The release of MA at nerve stimulation has been investigated by various methods.

446

A. DAHLSTROM

Release of endogenous transmitter can be assayed either in blood or in the perfusion fluid from an isolated organ, e.g. the spleen. Thin tissues, e.g. irides or portal vein preparations, can be studied by assaying the NA released into organ bath fluids following field stimulation since diffusion routes are very short in these tissues. Brain tissue can be studied by cutting thin brain slices and incubating them in flasks of different media. The overflow of MA into the media upon field stimulation can then be assayed. It is often an advantage to preincubate tissues or perfuse organs with isotope labeled transmitters prior to stimulation. This exogenous, labeled transmitter is then taken up into nerve terminals by the membrane pump (uptake 1) and is subsequently released upon stimulation into perfusion fluid or incubation medium. It was observed early on that the amount of NA released per nerve impulse from an organ could vary considerably, depending upon the use of various methods to block the inactivation routes for released NA (of. Fig. 3). Inhibition of the membrane pump by e.g. imipramines resulted in increased NA overflow in perfusion fluid or incubation media. Efforts to block the a-receptors by e.g. phenoxybenzamine (PBZ) to diminish vasoconstriction and theoretically promote overflow into the perfusion fluid s7 caused an even greater release of NA 75,95. This enhanced release has also been suggested to be due to a membrane pump blocking effect of PBZ °5 or to inhibition of extraneuronal uptake 67. The postulated membrane pump blocking effect of PBZ is clearly less than that of desmethylimipramine (DMI) as judged from uptake studies with labeled NA (only slight blockage at 10-7 M compared to DMIa4), and as observed with fluorescence histochemistry by e.g. Malmfors 78, Hamber25

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447

AMINERGIC TRANSMISSION. A REVIEW

ger 55 and others. In experiments on cat's calf muscle, where a high blood flow was maintained throughout the experiment by muscular exercise (stimulation o f the motor nerves), the overflow of NA into the blood increased only slightly after Lu 3-010 (a potent membrane pump inhibitor) while addition of PBZ to the same preparation caused a large increase in the NA overflow (Fig. 4). Thus, neither vasodilator nor membrane pump blockade could explain the increased release following PBZ, nor can inhibition of extraneuronal uptake provide an explanation since normetanephrine which inhibits this uptake (uptake 2) does not influence NA overflowa4. In order to explain the variable NA release a theory for a local regulation of transmitter release depending upon receptor activation was presented by H/iggendal in 196947,48. He hypothesized that when enough transmitter was released to activate the receptors and cause effector cell response, some substance was released from the postsynaptic site to switch off further release. Interestingly, Polak s4 in the same year postulated a similar mechanism for central cholinergic transmission. The influence of receptor activation or blockade on transmitter release has been studied extensively by Farnebo and Hamberger who have shown in elegant experiments that the theory of a local feedback control of transmitter release may be valid for both PNS and CNS neurons, and that not only postsynaptic but possibly also presynaptic receptors may be involved in the regulatory mechanism (this session). During the last decade the prostaglandins (PG) (Fig. 5) have been demonstrated to possess powerful biological activities (e.g. ref. 59). Bergstrfm and von Euler in 19638 found that i.v. injections of PGEz caused a fall in blood pressure and an increased heart rate and cardiac output. In the same year it was reported that the pressor response to exogenously administered NA was diminished by P G in a dose-dependent manner 65,94. Later it was found that also the pressor response to endogenous NA released by electrical stimulation was depressed by PGSa,ss,e2, 71. These effects of PG on the adrenergic response were not due to the direct vasodilatory influence of PG on the vessels, since this effect did not last nearly as long as that on the adrenergic response TM. The idea that P G could mediate a physiological role in adrenergic transmission gained support when Ferreira and Vane 38 and Davies et aL 2s discovered that large amounts o f P G E were released from the spleen on stimulation of the splenic nerve. In 1969 Hedqvist56, 60 found that PGEs could modify the response

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of an adrenergically innervated organ both to exogenous NA and to NA released by nerve stimulation. Based on his own results and those of other investigators he formulated, independently of Hfiggendal in the same year, a theory of a feedback control mechanism for the release of the adrenergic transmitter 56. The substance which mediated this feedback control on release was suggested to be PG, liberated from the effector cells during nerve activity and NA release. Further studies have supported this view. Thus, for instance, inhibition of the local synthesis of PG by indomethacin, acetylsalicylic acid or the more specific inhibitor eicosatetraenoic acid (ETA) enhances NA release and effector cell response a~,61,89,1°°. The PBZ-induced increase of the fractional N A release can be blocked by the administration of PG 57 as well as the PBZ-induced rise in D B H release from nerve terminals 70. It is therefore possible that a-receptor sites are involved in the PG regulation of NA release 9a. Also Ca 2+ ions seem to be involved since the effect of P G on adrenergic transmission is inversely related to the external Ca 2+ concentration (cf. ref. 59). A proposed model of the role of P G in N A release, published by Smith 91, is shown in Fig. 6. In this figure, PG seems to be formed and released by non-neuronal cells, which indeed appears to be the case, atJeast in the denervated cat spleen from which large amounts of PG were released when N A was injected into the denervated spleen45,al, 10°. F r o m the discussion following Dr. Hedqvist's paper, it emerged that of the many series o f PG, the PGE2 and PGF2~ occur in most tissues. The A-series is interesting since it may :be related to the circulation of the kidneys. PG of different series have different actions on adrenergic transmission in isolated organs and their real role in physiology is therefore complex. However, since PG appear to occur in most tissues, including brain tissues, it is conceivable that they may play a general role in aminergic transmission. Heilbronn mentioned that she has also found effects of PGF2,~ on acetylcholine release, particularly after atropine administration.

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A M I N E R G I C TRANSMISSION. A R E V I E W

449

(IV) Different pools o f transmitter

For more than a decade, pharmacologists and physiologists have discussed the existence of at least two pools of transmitter in the nerve terminals; one small pool, easily available for release, and one large stable pool. Crout et al. 17 found a small pool of endogenous NA which was easily released by tyramine, and a larger pool which was not released by tyramine. [aH]NA taken up into adrenergic nerves was also distributed in two pools; if tyramine was given shortly after the [aH]NA injection much [3H]NA was released. If, on the other hand, tyramine was given about 24 h after [3H]NA, only very little of the stored [aH]NA was released es,sS. The [3H]NA was evidently initially stored in the tyramine-releasable pool and later transferred to the large, less releasable pool. These pools were demonstrated after release induced by a pharmacological agent, tyramine. What about the release induced by nerve stimulation? In 1963, Chidsey and Harrison la found that labelled NA, taken up by the adrenergic nerves of dog heart, was preferentially released if nerve stimulation was carried out shortly after the isotope injection. At later periods, less [SH]NA in relation to endogenous NA was released, and the authors suggested that the isotope had gradually mixed with a large stable pool of transmitter. Observations on reserpine-treated rabbits by H/iggendal and Lindqvist5~ further supported the idea of a functional, small, easily releasable pool of transmitter. After the injection of a large dose of reserpine, the storage capacity of the amine granules is irreversibly lost (cf. refs. 50, 82), and the amines which are no longer protected from metabolizing enzymes rapidly disappear from the tissue stores. Signs of MA depletion are diarrhea, enophthalmus, loss of central and peripheral activities. Full recovery of endogenous MA levels in nerve terminals requires several weeksZ5,50, but already, 24-48 h after reserpine, when tissue levels of MA have recovered by only a few per cent, the animal is almost back to alert, normal behavior with normal eyes and body posture 53. Much of the reserpine syndrome can be directly related to depleted MA stores and the rapid return to normal animal behavior indicates that only a few per cent of the normal transmitter stores are necessary for normal function, i.e. a small functional pool. Recently, the theory of a small, easily releasable transmitter pool has gained further support by electrophysiological studies by Bennett6, 7 in vas deferens preparations (Figs. 7, 8). If the preparation was stimulated at 10 Hz, the excitatory junctional potentials (EJP) recorded in smooth muscle cells tended to decline somewhat after the first few stimuli (Fig. 7B). The size of the EJP was considered to be related to the amount of NA release6, 7. Fig. 7B therefore may indicate that at high impulse frequencies, the initially high NA release is diminished after the first few impulses. Following DMI blockade of the 'membrane pump' re-uptake mechanism, the decline of the EJPs and possibly also of NA released per stimulus was clearly more pronounced (Fig. 7A), suggesting that a small releasable NA pool is maintained partly by re-uptake of previously released transmitter. Also de novo synthesis appears to maintain the releasable pool; following blockade of NA synthesis the biphasic decline in EJPs (Fig. 8) may conceivably be explained by mobilization of NA from at

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least two stores, one of which is more easily releasable (initial steep slope) than the other. These stores may be illustrated schematically in a simplified version by Fig. 9. Not only in in vitro preparations but also in vivo, newly recaptured transmitter is preferentially released during nerve activity. Also, newly synthesized MA in rat brains is preferentially released during shock stimulation, as demonstrated by Glowinski and co-workersgL

AMINERGIC TRANSMISSION. A REVIEW

451

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Fig. 9. Schematic illustration of the possible relationship between the two main stores of transmitter, as proposed by many authors. The small, easily releasable pool (store II) is supposed to be maintained by re-uptake of released transmitter and by de novo synthesis. The large stable pool (store I) is presumably loaded via store II, which may in turn be charged by store I by a transfer process.

(V) Heterogeneity o f amine storage particles

The population of amine storage granules in the nerve terminals was earlier thought to be homogenous in functional, morphological and biochemical aspects. Recent studies, however, indicate that there exist at least two types of NA storage particles in peripheral adrenergic neurons. Electron microscopical studies37,42,63,9s have demonstrated the presence of both large dense core vesicles (diameter 80-100 nm) and small dense core vesicles (diameter 40-50 nm) in adrenergic neurons. Both types have been shown to store NA (cf. refs. 12, 63). The large type can be demonstrated both in glutaraldehyde-fixed and in KMnO4-fixed tissues, but KMnO4 fixation is preferable for demonstrating the small type. The dense core in KMnO4-fixed material represents a precipitate with NA as shown by Hfkfelt and Johnson 64. The dense core in the large vesicles in glutaraldehyde-fixed material probably mainly represents a protein matrix of the particles. It should be noted that in glutaraldehyde-fixed tissues a dense core does not necessarily imply an amine storage particle; in many endocrine organs, and also in non-aminergic neurons, dense core vesicles are present which probably do not store a catecholamine. Sucrose gradient centrifugation studies14, ss have demonstrated two types of NA storage particles in adrenergically innervated organs; one dense type which contains both DBH and chromogranin A and one 'lighter' type, containing only very little DBH. Bisby and Fillenzl0,11 have found that the light type fraction contains mainly small dense core vesicles while the heavy fraction contains large dense core vesicles. In axons, and particularly crushed axons with accumulations of NA and DBH, mostly large dense core vesicles have been observed3, 42 and only particles of the heavy type, with DBH, are isolated from axonsH, ~9. In the terminals, on the other hand, most NA is stored in the light, DBH-poor fraction, the heavy type constituting a varying proportion of the total amine storage particles (Fig. 10). Bisby and Fillenz 11 have recently demonstrated that the spleen may contain up to 20 ~o of heavy particles, whereas in e.g. the vas deferens, the percentage is small. Several studies by e.g. Geffen et aL 4o, Smith et aL 92 and De Potter et aL a2 have

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shown that the release of NA on nerve stimulation is accompanied by a release of both chromogranin A and DBH. This supports the theory of exocytotic release of transmitter. However, exocytosis need not be total but may be partial. Thus, only a certain fraction of the protein content may be released each time (Fig. 11). In this way, proteins which probably cannot be formed in the nerve terminals but must be supplied by axonal transport from the perikarya are economized, and the DBH which is needed for transmitter synthesis is not totally lost by only a single nerve impulse. The anatomical compartmentalization of the two main transmitter stores is unknown. However, studies of reserpine-treated animals have provided some information. Following reserpine, one large dose of 10 mg/kg i.p. per rat, the endo-

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453

AMINERGIC TRANSMISSION. A REVIEW TIME FOR RECOVERY OF DIFFERENT FUNCTIONS IN THE ADRENERGIC NEURON AFTER A LARGE D O S E O F RESERPINE / "

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TRANSMISSION Release of 3H-NA

Fig. 12. Schematic illustration of the recovery of different functional parameters in a long adrenergic neuron after one large dose of reserpine (I0 mg/kg i.p. to rats). The recovery of the parameters was studied in in vivo preparations. Recovery of endogenous noradrenaline was studied with fluorescence histochemical and/or biochemical methods. (For references and further explanation see ref. 49).

genous NA is depleted, the capacity of the nerve terminals for uptake-storage of [aH]NA is blocked and transmission fails. After this initial period post-reserpine, the three above-mentioned functional parameters of nerve terminals (endogenous NA, uptake-retention of [3H]NA, and transmission) start to recover (Fig. 12). The onset of recovery (24-36 h after reserpine) is in all likelihood due to intra-axonal transport and arrival in the nerve terminals of functioning amine storage granules formed in the cell body, possibly after reserpine clearance from the circulation (for discussion on reserpine recovery, see refs. 18, 26, 49-51). Reserpine treatment induces TH and DBH in the nerve cell bodies via an increased preganglionic impulse activity (cf. Thoenen, this session, see also refs. 79, 80). Also the synthesis of amine storage particles seems to increase. The amount of NA that accumulates above a 6 h crush in rat sciatic nerve has been followed at different times after reserpine (Fig. 13, broken curve). Between the second and fifth days after reserpine an overshoot was noticed 26. The amount of accumulated NA is likely to be proportional to the relative number of amine storage granules which are arrested above the crush, and the results therefore indicate that during days 3-5 after reserpine, the number of young amine granules transported to the nerve terminals by intraaxonal transport is markedly increased. These granules are presumably of the large, DBH-rich type, like axonal granules in normal animals (cf. above). The recovery of the capacity of the nerve terminals to take up and store [3H]NA recovers in parallel with the increased transport of new granules (Fig. 13, solid curve) and overshoots when the number of new granules, arriving at the terminals, is supranorma151. It then declines in parallel with the normalization of granule transport. These findings indicate two things: (1) the [aH]NA storage capacity of nerve terminals occurs mainly in young amine granules; and (2) this capacity for uptake-storage of [3H]NA of the

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young granules is short-lasting. In fact, a t½ of about 12 h for this capacity has been estimated 51. De Potter et al. a° found that D B H in nerve terminals has a life-span of 26-40 h 31. This value is rather similar to the value for [8H]NA uptake-storage capacity. D B H is stored mainly in large, heavy, presumably young granules (cf. above) and if [aH]NA storage were to occur mainly in young granules, this capacity would decline in parallel with the decline of the protein content of the granules. The decline in protein content may be brought about by the release of proteins together with NA at nerve activity (Fig. 11) and would provide a basis for the conversion or aging of the heavy granules to small, DBH-poor, 'old' particles. Since [aH]NA is initially taken up into the 'small, easily releasable pool of transmitter', this pool may hypothetically be localized in the large, DBH-rich, young amine storage particles (Fig. 14). The large stable pool would accordingly be stored mainly in the large population of small vesicles which still contain A T P and Mg 2+. The molecular characteristics of such a storage mechanism are not understood. However, the in vitro experiments of Berneis et a l p indicate the possibility of such a storage complex. If the [aH]NA is initially taken up by the large, DBH-rich amine granules, autoradiography at the ultrastructural level might demonstrate silver grains over areas with large dense core vesicle profiles, provided that fixation was carried out shortly after the [3H]NA injection. This has not been found so far. In fact, as pointed out by Sotelo in the discussion, such a connection between large dense core vesicles and silver grains is very rarely seen. Dunant emphasized, however, the possibility that amines stored in the easily releasable pool of transmitter would, because of the

AMINERGIC TRANSMISSION. A REVIEW

455

@ @ Large stabl,e pool, : o l d a m i n e granules. Small. easibty releasable pool : young amine granules.

Fig. 14. A hypothetical model for the relation between amine-storage granules of different age in the nerve terminals, and the two pools of transmitter. The large circles may represent: (1) the large dense core vesicles observed electron microscopically, (2) the 'heavy noradrenaline (NA)-storage particles' isolated on sucrose gradients, and (3) the 'young amine granules'. The smaller circles may accordingly represent: (1) the small dense core vesicles, (2) the 'light NA-storage particles', and (3) the 'old amine granules'. The arrows indicate a hypothetical transfer of the transmitter between the pools, i.e. transfer between old and young granules according to the hypothesis presented in the text. A possible route for transfer of transmitter from the 'small easily releasable pool' to the 'large stable pool' by means of ageing of young granules is indicated to the right of the figure. (From ref. 27.)

characteristics of the pool, not stay in these storage positions during fixation or tissue fractionation. Since they are ready for release upon the entry of Ca 2+ into the terminals, the electrolyte movements during fixation or fractionation procedures might conceivably disrupt the bindings of the easily releasable pool. Since the light, small storage particles contain very little matrix DBH 29, they may be less active in synthesizing NA, and most of the NA synthesis would then occur in the large vesicles. If this is true, a transfer of transmitter between the different types of granules and the different pools must exist. For instance, after tetrabenazine, which depletes the NA stores by a mechanism similar to that of reserpine but blocks the granules for only a short period, the recovery to normal nerve terminal stores of e.g. NA is complete within a few hours 46. This rapid recovery is not due to a replacement of the blocked granules by intra-axonal transport of new granules but to the storage of NA in both pools; the NA presumably being synthesized by the large, heavy, DBH-rich particles. A transfer in the other direction, from the large stable pool to the small, easily releasable pool, probably also exists. This would be of physiological importance in situations where re-uptake and de novo synthesis cannot maintain the small, easily releasable pool. De Potter, the last speaker of this session, will discuss the fate of NA storage particles following electrical stimulation and the characteristics of the different populations of amine storage particles in dog splenic nerve and spleen.

456 S u p p o r t was o b t a i n e d f r o m the S w e d i s h M e d i c a l N o s . 14X-2207 and 04P-4173).

A. DAHLS]Rt)M Research

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1 ANDI~N, N.-E., CARLSSON, A., AND HAGGENDAL, J., Adrenergic mechanisms, Ann. Rev. Pharmacol., 9 (1969) 119-134. 2 AND,~N, N.-E., FUXE, K., HAMBERGER, B., AND HOKFELT, T., A quantitative study on the nigroneostriatal dopamine neuron system in rats, Acta physiol, scand., 67 (1966) 306-312. 3 BANKS, P., MANGNALL, D., AND MAYOR, D., The redistribution of cytochrome oxidase, noradrenaline and adenosine triphosphate in adrenergic nerves constricted at two points, J. Physiol. (Lond.), 200 (1969) 745-762. 4 BANKS, P., MAYOR, D., MITCHELL, M., AND TOMLINSON, D., Studies on the translocation of noradrenaline-containing vesicles in postganglionic sympathetic neurones in vitro. Inhibition of movement by colchicine and vinblastine and evidence for the involvement of axonal microtubules, J. Physiol. (Lond.), 216 (1971) 625-639. 5 BANKS, P., MAYOR, D., AND TOMLINSON,D. R., Further evidence for the involvement of microtubules in the intra-axonal movement of noradrenaline storage granules, J. Physiol (Lond.), 219 (1971) 755-761. 6 BENNETT,M. R., An electrophysiological analysis of the uptake of noradrenaline at sympathetic nerve terminals, J. Physiol. (Lond.), 229 (1973) 533-546. 7 BENNETT, M. R., An electrophysiological analysis of the storage and release of noradrenaline at sympathetic nerve terminals, J. Physiol. (Lond.), 229 (1973) 515-531. 8 BERGSTROM,S., AND EULER, U. S. VON, The biological activity of prostaglandin El, E2 and E3, Acta physiol, scand., 59 (1963) 493-494. 9 BERNEIS,K. H., PLETSCHER,A., AND DA PRADA, M., Metal-dependent aggregation of biogenic amines: A hypothesis for their storage and release, Nature (Lond)., 224 (1969) 281-283. l0 BIsav, M. A., AND FILLENZ, M., Isolation of two types of vesicles containing endogenous noradrenaline in sympathetic nerve terminals, J. Physiol. (Lond.), 210 (1970) 49-50P. l l BISBY, M. A., AND FILLENZ, M., The storage of noradrenaline in sympathetic nerve terminals, J. Physiol. (Lond.), 215 (1971) 163-179. 12 BLOOM, F.E., AND AGHAJANIAN, G. K., An electron microscopic analysis of large granular synaptic vesicles of the brain in relation to monoamine content, J. Pharmacol. exp. Ther., 159 (1968) 261-273. 13 CHIDSEY, C. A., AND HARRISON, D. C., Studies on the distribution of exogenous norepinephrine in the sympathetic neurotransmitter store, J. Pharmacol. exp. Ther., 140 (1963) 217-223. 14 CHUBB, I. W., DE POTTER W.P., AND DE SCHAEPDRYVER, A. F., TWO populations of noradrenaline-containing particles in the spleen, Nature (Lond.), 228 (1970) 1203-1204. 15 CORRODI, H., AND JOHNSSON,G., The formaldehyde fluorescence method for the histochemical demonstration of biogenic amines. A review on the methodology, J. Histochem. Cytochem., 15 (1967) 65-78. 16 COYLE, J., AND WOOTEN, F., Rapid axonal transport of tyrosine hydroxylase and dopamine-flhydroxylase, Brain Research, 44 (1972) 701-704. 17 CROUT,J. R., MUSKUS,A. J., AND TRENDELENBURG,U., Effect of tyramine in isolated guinea pig atria in relation to their noradrenaline stores, Brit. J. Pharmacol., 18 (1962) 600--611. 18 DAHLSTROM,A., The effect of reserpine and tetrabenazine on the accumulation of noradrenaline in the rat sciatic nerve after ligation, Acta physiol, scand., 69 (1967) 167-179. 19 DAHLSTROM, A., Effect of colchicine on transport of amine storage granules in sympathetic nerves of rat, Europ. J. Pharmacol., 5 (1968) 111-113. 20 DAHLSTROM,A., Fluorescence histochemistry of monoamines in the CNS. In H. H. JASPER, A. A. WARD AND A. POPE (Eds.), Basic Mechanisms of the Epilepsies, Little, Brown, Boston, Mass., 1969, pp. 212-227. 21 DAHLSTROM,A., Effects of vinblastine and colchicine on monoamine-containing neurons of the rat, with special regard to the axoplasmic transport of amine granules, Acta neuropath. (Berl.), Suppl. V (1971) 226-237. 22 DAHLSTROM,A., EVANS, C. A. N., H.~GGENDAL,C. J., HEIWALL, P. O., AND SAUNDERS,N. R., Rapid transport of acetylcholine in rat sciatic nerve proximal and distal to a lesion, J. Physiol. (Lond.), (1973) in press.

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23 DAHLSTR()M,A., AND H.~GGENDAL, J., Some quantitative studies on noradrenaline content in the cell bodies and terminals of a sympathetic adrenergic neuron system, Acta physiol, scand., 67 (1966) 271-277. 24 DAHLSTROM,A., AND H.~GGENDAL,J., Studies on the transport and life-span of amine storage granules in a peripheral adrenergic neuron system, Acta physiol, scand., 67 (1966) 278-288. 25 DAHLSTROM,A., AND H.~GGENDAL,J., Recovery of noradrenaline levels after reserpine compared with the life-span of amine storage granules in rat and rabbit, J. Pharm. Pharmacol., 18 (1966) 750-751. 26 DAHLSTROM, A., AND HAGGENDAL, J., Recovery of noradrenaline in adrenergic axons of rat sciatic nerves after reserpine treatment, J. Pharm. Pharmacol., 21 (1969) 633-638. 27 DAHLSTRI)M,A., AND H.~GGENDAL,J., On the possible relation between different pools of adrenergic transmitter and heterogeneity or amine storage granules in nerve terminals, Acta Physiol. poL, XXIII, Suppl. 4 (1972) 537-549. 27a DAIRMAN,W., GEFFEN,L., AND MARCHELLE,M., Axoplasmic transport of aromatic L-amino acid decarboxylase (EC 4.1. 1.26) and dopamine fl-hydroxylase (EC 1.14.2.1 ) in rat sciatic nerve, J. Neurochem., 20 (1973) 1617-1623. 28 DAVIES,B. N., HORTON,E. W., AND WITHRINGTON,P.-G., The occurrence of prostaglandin E2 in splenic venous blood of the dog following splenic nerve stimulation, J. Physiol. (Lond.), 188 (1967) 38P-39P. 29 DE POTTER, W. P., Noradrenaline storage particles in splenic nerve, Phil. Trans. B, 261 (1971) 313-317. 30 DE POTTER, W. P., AND CHUBB, I. W., The turn-over rate of noradrenergic vesicles, Biochem. J., 125 (1971) 375-376. 31 DE POTTER, W. P., CHUBB, I. W., AND DE SCHAEPDRYVER,A. F., Pharmacological aspects of peripheral noradrenergic transmission, Arch. int. Pharmacodyn., 196 (1972) 258-287. 32 DE POTTER, W. P., DE SCHAEPDRYVER,A. F., MOERMAN,E. J., AND SMITH, A. D., Evidence for the release of vesicle proteins together with noradrenaline upon stimulation of the splenic nerve, J. Physiol. (Lond.), 204 (1969) 102-104P. 33 FALCK,B., AND OWMAN, CH., Adetailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines, Acta Univ. Lund, Sect. I1, 7 (1965) 1-23. 34 FARNEBO,L.-O., AND HAMBERGER,B., Drug-induced changes in the release of aH-noradrenaline from field stimulated rat iris, Brit. J. Pharmacol., 43 (1971) 97-106. 35 FERREIRA,S. H., MONCADA,S., AND VANE, J. R., Indomethacin and aspirin abolish prostaglandin release from the spleen, Nature New Biol., 231 (1971) 237-239. 36 FERREIRA, S. H., AND VANE, J. R., Prostaglandins; their disappearance from and release into the circulation, Nature (Lond.), 216 (1967) 868-873. 37 FILLENZ, M., Fine structure of noradrenaline storage vecicles in nerve terminals of the rat vas deferens, Phil. Trans. B, 261 (1971) 319-323. 38 FONNUM, F., STORM-MATHISEN,J., AND WALBERG,F., Glutamate decarboxylase in inhibitory neurons. A study of the enzyme in Purkinje cell axons and boutons in the cat, Brain Research, 20 (1970) 259-275. 39 FUXE, K., HOKFELT,T., ANDUNGERSTEDT,U., Localization ofmonoamines in the central nervous system. In J. DE AJURIAGUERRAAND G. GAUTHIER(Eds.), Monoamines, Joyaux Gris Centraux et Syndrome de Parkinson, Thieme, Geneva, 1971, pp. 23-60. 40 GEFFEN,L. B., LIVETT,B. G., AND RUSH, R. A., Immunological localization of chromogranins in sheep sympathetic neurones, and their release by nerve impulses, J. Physiol. (Lond.), 204 (1969) 58-59P. 41 GEFEEN, L. B., LIVETT, B. G., AND RUSH, R. A., Immuno-histochemical localization of protein components on catecholamine storage vesicles, J. Physiol. (Lond.), 204 (1969) 593-606. 42 GEFFEN, L. B., AND OSTBERG, A., Distribution of granular vesicles in normal and constricted sympathetic neurons, J. Physiol. (Lond.), 204 (1969) 583-592. 43 GEFFEN, L.B., AND RUSH, R.A., Transport of noradrenaline in sympathetic nerves and the effect of nerve impulses on its contribution to transmitter stores, J. Neurochem., 15 (1968) 925-930. 44 GIBB, J. W., SPECTOR,S., AND UDENFRIEND,S., Production of antibodies to dopamine-fl-hydroxylase of bovine adrenal medulla, Molec. PharmacoL, 3 (1967) 473-478. 45 GILMORE,N., VANE, J. R., AND WYLLIE, J. H., Prostaglandins released by the spleen, Nature (Lond.), 218 (1968) 1135-1140. 46 H.~GGENDAL,J., The depletion and recovery of noradrenaline in the brain and some sympathetically innervated mammalian tissues after tetrabenazine, J. Pharm. Pharmacol., 20 (1968) 364-367.

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