Fine-structural Identification of Autonomic Nerves and their Relation to Smooth Muscle

389

Fine-structural Identification of Autonomic Nerves and their Relation to Smooth Muscle GEOFFREY BURNSTOCK

AND

TAKASHI IWAYAMA

Department of Zoology, University of Melbourne, Parkville, Victoria (Australia)

G E N E R A L MODEL O F T H E A U T O N O M I C N E U R O M U S C U L A R JUNCTION

The application of modern cellular techniques has given a clearer picture of the nature of the autonomic neuro-effector junction. In particular, electron microscope and fluorescent histochemical studies of the precise relationship of individual nerves to single smooth muscle cells (see Burnstock, 1970) have supported and extended the earlier concept of the 'autonomic ground plexus' put forward by Hillarp (1946); these morphological results, together with studies of the electrophysiology of transmission (see Bennett and Burnstock, 1968) allow a model of the autonomic neuromuscular junction to be proposed, which is illustrated in Fig. 1. The essential features are: (1) smooth muscle cells, connected by low resistance pathways represented by specialised areas of close apposition or 'nexus' (Dewey and Barr, 1962) are arranged in efector bundles. (2) Transmitfqr is released 'en passage' from large numbers of

''I!IRXC T L Y - I N N F R V A T L D " CELI XITI1 CLOSE ( Z O O

n") NEUROMU5CLI.AR

JONCTIONS

I "C
T -1

a-

"INDIRECTLY-COUPLED" C E L L EXHIBITS ACTION POTENfLALS ONLY OW RCSISTANCE PATHWAY

\ARICOSI: NERVE FIBRE

Fig. 1. Schematic representation of autonomic innervation of smooth muscle. For explanation see text. References p p . 402-404

390

G . B U R N S T O C K , T. I W A Y A M A

preterminal varicosities as well as from the terminal varicosity. (3) In most organs, some, but not all, the smooth muscle cells are directly innervated, i.e. have close (less than 500 A) apposition with axon varicosities naked of Schwann cell investment (Fig. 2); these have been termed ‘directly-innervated cells’. (4) The cells adjoining ‘directly innervated cells’ are coupled electrotonically to them by low resistance pathways, so that passive potential changes, in particular excitatory junction potentials, are observed in these cells, which have been termed ‘coupled cells’. (5) When the muscle cells in an area of the effector bundle become depolarised simultaneously, an all-or-none action potential is initiated which propagates through the tissue. Thus, in some tissues, many cells, called here ‘indirectly-coupled’ cells, are neither directly innervated nor directly coupled to innervated cells, yet contract on stimulation of the nerves which supply the organ. It should be pointed out that it is unlikely that the three cell types described in this model are different in structure and properties; on the contrary it is probable that many cells might play the role of a ‘directly-innervated’, ‘coupled’ or ‘indirectly-coupled’ cell at different times during the normal physiological pattern of nervous control of the organ in the intact animal. This model appears to be applicable to all systems, although there is considerable variation in the density of innervation of smooth muscle in different organs. For example, all the muscle cells of the mouse and rat vas deferens and probably some other organs, appear to be directly-innervated with at least one and probably up to six close (200 A) neuromuscular junctions (Richardson, 1962; Taxi, 1965; Yamauchi and Burnstock, 1969b). In other organs, such as the guinea-pig vas deferens, urinary bladder, nictitating membrane and dog retractor penis, between one quarter and threequarters of the muscle cell population appear to be directly-innervated (see Merrillees, 1968; Burnstock, 1970), so that only a small proportion of ‘indirectly-coupled‘ cells are likely to be present. Finally, in systems such as the ureter, uterus, arteries and longitudinal muscle coat ofthe gut, only a small proportion of muscle cells are directlyinnervated, so that a large number of cells are ‘indirectly coupled’ and are activated via a well developed nexus system for intermuscle fibre spread of activity. SMOOTH M U S C L E A N D S Y M P A T H E T I C N E R V E S G R O W N I N TISSUE C U L T U R E

Nexus between smooth muscle cells Nexus are likely to form the morphological basis of the low resistance pathways which allow electrotonic coupling of activity in muscle effector bundles. They are a prominent feature in sparsely innervated organs, such as ureter and gut, where spread of activity between muscle cells is of considerable importance (see Burnstock, Holman and Prosser, 1963a). It was surprising that nexus were also found in the mouse and rat vas deferens, where every cell appears to be directly-innervated (Richardson, 1962; Lane and Rhodin, 1964; Yamauchi and Burnstock, 1969b), so that activation of cells by interfibre spread of current would not appear to be necessary. However, evidence for interfibre spread of activity has been demonstrated in the mouse vas deferens (Furness and Burnstock, 1969).

FINE-STRUCTURAL IDENTIFICATION OF AUTONOMIC NERVES

391

It was of interest therefore to see if the formation of the nexus was dependent on the presence of nerves. It has been shown recently in our laboratory and illustrated in the time-lapse movie film shown at this conference, that smooth muscle cells separated enzymatically prior to culture, aggregate to form muscle bundles, which exhibit coordinated contractions, implying interfibre spread of activity. Electron-microscopic examination of the bundles formed in culture have shown nexus between neighbouring muscle cells (Campbell et al., 1971). Thus it appears that nexus are an inherent feature of normal development of smooth muscle and do not depend on the density of innervation by nerves. That is to say, the presence of nexus in heavily innervated smooth muscle may not play an important physiological role, but rather represent a general feature of smooth muscle development which, being of no selective disadvantage, has not been eliminated by the process of evolution. Varicose nerve fibres One might also ask the question : are the varicosities of the extensive terminal regions of autonomic nerves induced by the presence of the muscles they supply or are they a genetically determined feature of these axons? Electron micrographs of longitudinal sections through considerable lengths of sympathetic axon grown in tissue culture in the absence of muscle eflectors, have shown the typical in situ varicose appearance (Campbell et al., 1971). This suggests that varicosities are part of the inherent make-up of the nerve, and also negates the view that varicosities are formed by the peristaltic movements of the Schwann cells, since none were present in these sections. Comparable varicose profiles and inclusions have been demonstrated in early outgrowths from cultures of chick spinal cord (Grainger et al., 1968), and in developing unmyelinated nerve (Robertson, 1962). Neuromuscular junctions Joint cultures of smooth muscle and sympathetic nerves have recently been grown in our laboratory and some electron micrographs showing close apposition of nerve terminals and muscle membranes have been demonstrated (Campbell et al., 1971). Both the nerve and muscle membranes in the regions of closest apposition are free of basement membrane, so that separation of opposing membranes of as little as 50 8,is visible in limited areas. The axon in these regions contains a few large (900-1600 A) granular vesicles and mitochondria, but is mainly occupied by an extensive irregular smooth membranous network A few microtubules are also present. It is not known yet whether these junctions are functional, but it seems unlikely in view of the results of studies of the normal development of autonomic innervation of the mouse vas deferens (Yamauchi and Burnstock, 1969a, b; Furness et al., 1970b). Although the density of innervation of the vas deferens continued to increase up to at least 6 months, the intra-axonal contents of the axon profiles was comparable to that of adult nerves even at 15 days. However, functional transmission was not established until several days later. This phasing of structure and function also occurs in the normal developReferences p p . 402-404

Fig. 2. (a) Close (- 200 A) neuromuscular junction in the vas deferens of a mouse injected for 30 min with 5-OHDA (50 mg/kg). Note subsynaptic cysternae (arrows). Osmium fixation. x 82000. (b) Close (- 200 A) neuromuscular junctions in large intestine of toad (Bufomurinus). Note aggregations of micropinocytotic vesicles (arrows) in muscle beneath the nerve profiles. Glutaraldehyde and osmium fixation. x 64000. (c) lntracellular noradrenergic nerve profiles (N) deep inside a smooth muscle cell of mouse vas deferens treated for 30 min with 5-OHDA (50 mg/kg). Note subsynaptic cysternae (arrows). Osmium fixation. x 26000.

FINE-STRUCTURAL IDENTIFICATION OF AUTONOMIC NERVES

393

ment of synapses in the human gut (Read and Burnstock, 1970), and of autonomic neuromuscular junctions in anterior eye chamber transplants of vas deferens and taenia coli (Burnstock et al., 1970b). S Y N A P T I C CLEFT

There is considerable variation in the precise relationship of nerve varicosities and muscle cells in different organs, and the whole question of what constitutes the autonomic neuromuscular cleft needs resolution, particularly in view of the physiological and pharmacological evidence of ‘en passage’ release of transmitter from varicosities in extensive preterminal axons (Malmfors, 1965; Bennett and Burnstock, 1968; Furness, 1970). Nerve-muscle separation in the regions of closest apposition in the vas deferens is about 200 A (Richardson, 1962; Merrillees et al., 1963; Lane and Rhodin, 1964; Yamauchi and Burnstock, 1969b; and Fig. 2a, c). From an analysis of a serial electron microscope study of the nervous environment of single smooth muscle cells in the guinea pig vas deferens (Merrillees, 1968), it was concluded that it was unlikely that transmitter released from varicosities further than 1000 A away would have a significant effect on muscle cells (Bennett and Merrillees, 1966). This conclusion was supported by the results of an entirely different approach, namely, acetylcholinesterase (AChE) staining (Robinson, 1969). In this study it was shown that about 15-20% of the axon profiles in the guinea pig vas deferens showed heavy positive staining for AChE, but that only muscle membranes within 1200 of these profiles showed matching AChE staining. Furthermore, Schwann cell processes intervened between muscle and nerve membranes in 80% of all the cases where nerves were separated from muscle by greater than 1100 A. The closest apposition between nerves and smooth muscle in most blood vessels studied is consistently greater (500-800 A) than in the vas deferens (see Burnstock, 1970). A study of AChE staining in the guinea pig uterine artery, coniparable to that described above for the vas deferens, showed that in this case there was matching muscle staining for AChE within 10000 A of stained nerve profiles (Bell, 1969). This could be taken as an indication that in blood vessels the effective neuromuscular cleft is about 10 000 A (i.e. about 10 times wider than the neurornuscular cleft in the vas deferens). In the longitudinal muscle coat of the alimentary tract most varicose nerves are confined in bundles and only occasionally run singly, free of Schwann cell investment (Taxi, 1965; Lane and Rhodin, 1964; Rogers and Burnstock, 1966b). The closest approach of intramural nerves to muscle cells in the taenia coli of the guinea pig is about 1000 A and in a combined analysis of electrophysiological and fine structural studies, it was suggested that the effective synaptic cleft in the gut might be at least 3000 A (Bennett and Rogers, 1967). However, in the circular muscle coat of the gut, there are many examples of closer (200 A) apposition of nerve and muscle membranes (Rogers and Burnstock, 1966b; Thaemert, 1963, 1966; Nagasawa and Mito, 1967; and Fig. 2b). Rrfercnccs pp. 402-404

394

G. B U R N S T O C K , T. I W A Y A M A

POST-SYNAPTIC STRUCTURE

A number of authors have examined the question of post-synaptic specialisation of smooth muscle membranes in the regions of closest apposition with terminal varicosities, but usually no increase in membrane thickening or density has been seen (Richardson, 1962; Merrillees et aE., 1963; Lane and Rhodin, 1964; Nagasawa and Mito, 1967; Taxi, 1969). An elongated sac of endoplasmic reticulum (sub-synaptic cysternae) has been noted in the vas deferens (Fig. 2a and Richardson, 1962; Merrillees, et al., 1963; Lane and Rhodin, 1964), but since this is not a consistent feature of all areas of close apposition, it is still not certain that it can be regarded as a feature of autonomic post-synaptic specialisation. Similarly, aggregations of micropinocytotic vesicles are often seen in post-synaptic regions in the gut (Fig. 2b), but again this is not a consistent feature. ‘IN T R A C E L L U LAR’ N E R V E TER M I N ALS

In some organs, notably the rat and mouse vas deferens, nerve profiles were occasionally observed within deep grooves in smooth muscle cells (Richardson, 1962; Lane and Rhodin, 1964; Taxi, 1965). More recently, with improved methods of preservation of granular vesicles, including the use of short-term injection of 5- and 6hydroxydopamine (Tranzer and Thoenen, 1968b; Furness et al., 1970a; Bennett et al., 1970), it has been shown that considerable numbers of both adrenergic and cholinergic axons penetrate and perhaps terminate deep inside smooth muscle cells (Fig. 2c, and Watanabe, 1969; Furness and Iwayama, 1971). Varicosities containing abundant vesicles are often characteristically in close apposition to the nucleus and are enveloped by the perinuclear organelles. The functional significance of this extraordinary finding is not known, but their inaccessibility may partly explain the difficulty in altering neuromuscular responses with various autonomic blocking and potentiating drugs (Holman, 1970). MULTIAXONAL JUNCTIONS

Groups of 4 to 7 axons have been shown to be in close apposition (150-200 A) with single muscle cells in the small intestine of mammals where they were termed ‘multiterminal synapses’ (Brettschneider, 1962) and of toad where they were called ‘multiaxonal junctions’ (Rogers and Burnstock, 1966b). This is illustrated in Fig. 2b. It is not known whether each axon profile that comes into contact with a single muscle cell represents a different axon from separate neurones or whether they are branches of one axon. However, junction potentials resulting from stimulation of different nerves have been recorded recently from single cells in the guinea pig and bird gut, although coupling of activity between neighbouring muscle cells complicates the interpretation (Furness, 1969; Bennett, 1970). The terminal regions of adrenergic and cholinergic nerves are often closely associated within the same Schwann process (Thoenen et al., 1966; Thaemert, 1966; Iwayama,

F I N E - S T R U C T U R A L I D E N T I F I C A T I O N OF A U T O N O M I C N E R V E S

395

et al., 1970). The concept that there is a cholinergic link in the adrenergic transmission process (Burn and Rand, 1959) has been discussed at some length (Ferry, 1966 ;Burn and Rand, 1965 ; Campbell, 1970). The close association of adrenergic and cholinergic terminals has been proposed recently (Burn, 1968) as a morphological basis for this theory. INTRA-AXONAL FEATURES O F DIFFERENT AUTONOMIC NERVES

A diagrammatic representation of the essential intra-axonal features of the main nerve types is presented in Fig. 3. b. NORADRENERCIC.

a. CHOLINERGIC

c.(?) NON-ADRENERGIC , NON -CHOLIMERGIC

d.(?) SENSORY

n

0AGRANULAR

(SYNAPTIC) VESICLES

i)

(300-600

(NEUROSECRETORY) Type

@ SMALL GRANULAR

VESICLES (SGV)

VESICLES (LGV)

(1000 - 2 o o y

(300-600

Fig. 3. Diagrammatic representation of sections through the terminal varicosities of autonomic nerves. For explanation see text. References pp. 402-404

Fig. 4. Intra-axonal structure of different autonomic nerves: (a) Cholinergic (C) and noradrenergic (N) nerve profiles in the circular muscle coat of the vas deferens of a rat treated for 30 min with 5-OHDA (50 nig/kg). Note that both small and large-granular vesicles in the adrenergic profile have taken up the drug, but not the agranular or large-granular vesicles in the cholinergic profile. Osmium fixation. x 38000. (b) Adrenergic axon profile in Auerbach’s plexus of chicken gizzard. In this case large granular vesicles (which take up 5- and 6-OHDA) are predominant. Note halo between intravesicular, granular and limiting vesicle membrane. Osmium fixation. x 42000. (c) Non-adrenergic, non-cholinergic axon profile (asterisk) in large intestine of toad, treated for 45 rnin with 6-OHDA (100 mgikg). The large-granular vesicles (characterised by granulation throughout the vesicle) contained in these nerves do not take up 6-OHDA. Note also the cholinergic nerve profile (C). Glutaraldehyde-osmium fixation. x 48000. (d) Nerve profile (S) probably representing the terminal portion of

FINE-STRUCTURAL IDENTIFICATION OF AUTONOMIC NERVES

397

Cholinergic nerves It is generally accepted that profiles containing predominantly small agranular vesicles (300-600 A) represent cholinergic nerves (De Robertis and Bennett, 1955; Whittaker et al., 1964; Grillo, 1966; Burnstock and Robinson, 1967; and Fig. 4a, d). A few large-granular vesicles (900-1200 A) are usually also present; they do not take up catecholamines, 5- or 6- hydroxydopamine (OHDA) (Tranzer et al., 1969; Bennett et al., 1970) and their function is not known.

Adrenergic nerves Considerable evidence has accumulated that nerves containing noradrenaline are characterised by the predominance of small-granular vesicles (300-600 A) with a dense core (see Grillo, 1966; Burnstock and Robinson, 1967; Farrell, 1968; Hokfelt, 1968; Taxi, 1969; Geffen and Livett, 1971 ; and Figs. 2a, c; 4a). Some large-granular vesicles (900-1200 A) are also present. However in this case, there is evidence that they are also capable of taking up and storing catecholamines (Tranzer and Thoenen, 1968a, b; Hokfelt, 1968; Bennett et al., 1970; Furness et al., 1970a), although they are relatively resistant to depletion by reserpine (Bloom and Barrnett, 1966; Bondareff and Gordon, 1966; Clementi et al., 1966; Hokfelt, 1966). It seems more likely that the low percentage of small agranular vesicles present in varicosities of adrenergic nerves represent ‘empty’ small-granular vesicles rather than providing evidence for the cholinergic link hypothesis (Burn and Rand, 1959) since drug treatment leading to increase in levels of noradrenaline in the nerves is associated with a decrease in the percentage of agranular vesicles (Thoenen et al., 1966; Van Orden et al., 1966; Tranzer et al., 1969). While fixation of the tissue with potassium permanganate readily preserves the granules within vesicles in adrenergic nerves, other fixatives do not always do so (Richardson, 1966; Hokfelt and Nilsson, 1965; Hokfelt and Jonsson, 1968; Tranzer et al., 1969). An important observation has been made recently in our laboratory (Iwayama and Furness, 1971) that incubation of the tissue in Krebs’ solution prior to fixation in osmium or glutaraldehyde gives consistent preservation of granules. This method may be of particular value in studies of the action of drugs on intra-axonal stores of catecholamines (see below). In birds, some adrenergic profiles contain predominantly large granular vesicles and take up 6-OHDA (Bennett et al., 1971 and Fig. 4 b) and are therefore presumably adrenergic. Similar adrenergic profiles have been found in reptiles (Baumgarten and Braak, 1968).

Non-adrenergic, non-cholinergic (purinergic) nerves The existence of non-adrenergic inhibitory nerves in the gut has been established with both pharmacological (Burnstock et al., 1966; Campbell, 1966; Martinson, 1965; Day and Warren, 1968) and electrophysiological (Burnstock et al., 1963b, 1964; Bennett et al., 1966; Biilbring and Tomita, 1967; Kuriyama et al., 1967) methods, and Rcfermcrs p p . 402-404

398

G. B U R N S T O C K , T . I W A Y A M A

evidence has been presented that ATP or some related purine nucleotide is the neurotransmitter substance released from these nerves (Burnstock et al., 1970a), which have been tentatively termed ‘purinergic’ (Burnstock, 1971). In a recent study of the fine structural features of the non-adrenergic inhibitory nerves which supply the toad lung, it has been shown that axon profiles containing predominantly largegranular vesicles (1000-2000 A) (characterised by the absence of an electron-opaque halo between the granular core and the boundary membrane) remain in the lung as do non-adrenergic inhibitory responses following degeneration of adrenergic inhibitory nerves with 6-OHDA (Robinson et al., 1971). These profiles are identical with those seen in the toad intestine (Fig. 4c; Rogers and Burnstock, 1966b) which also contains non-adrenergic, non-cholinergic autonomic nerves (see Burnstock, 1969). A study of the fine structure of nerve profiles in the highly localised and concentrated Auerbach’s plexus of the avian gizzard has been made (Bennett and Cobb, 1969; Bennett et al., 1971). Cholinergic, adrenergic and non-adrenergic inhibitory nerves have been identified in this plexus by physiological means (Bennett, 1969) and two types of profile containing predominantly large-granular vesicles demonstrated (Bennett et al., 1970b). The large-granular vesicles in one type of profile are characterised by a size range of 900-1200 A with a dense granular core and a light halo between the granule and the membrane (Fig. 4b); these vesicles are associated with catecholamines and take up 6-OHDA (Bennett et al., 1970). The other type of profile contains granular vesicles which are usually larger (1000-2000 A), do not have a clear halo between the granule and boundary membrane and are closely comparable to those described for non-adrenergic inhibitory nerves in the toad lung and intestine. 5-Hydroxytryptamine-containingnerves

A specific cytochemical method for the localisation of 5-HT at the ultrastructural level has been developed (Tranzer and Thoenen, 1967a; Etcheverry and Zieher, 1968a, b) and, together with the use of p-chlorophenylalanine as a depletor of 5-HT, smallgranular vesicles (400-600 A) have been shown to be associated with 5-HT in autonomic nerve terminals in the pineal gland (Bloom and Giarman, 1967). These results suggest that both 5-HT and catecholamines are normally contained within smallgranular vesicles in pineal nerves, but whether the two amines are stored in the same or different vesicles is not known. In either case, the 5-HT contained in the intrapineal portions of the sympathetic nerves cannot be regarded as true autonomic transmitter substance, since it is synthesised outside the nerves in the pineal parenchymal cells and merely taken up and stored by the nerves (Zweig and Axelrod, 1969). Sensory nerves

In view of the many sensory nerves which are known to supply most autonomically innervated tissues, it is surprising that these profiles have not previously been recognised. Axon profiles which are quite different from those characteristic of cholinergic and

F I N E - S T R U C T U R A L I D E N T I F I C A T I O N OF A U T O N O M I C N E R V E S

399

adrenergic nerves, have been observed in a variety of tissues, including vas deferens (Merrillees, 1968), pial artery (Hagen and Wittkowski, 1969), rat anterior cerebral artery (Burnstock et al., 1970c), bird ureter (Uehara and Burnstock, 1971) and cat bladder (Campbell and Uehara, personal communication). They contain few, if any, vesicles and are packed with small, oval mitochondria with characteristically only two cristae seen in transverse sections; such a profile in the ureter of the finch is shown in Fig. 4d. It should be pointed out that mitochondria of this kind are often also seen in adrenergic and cholinergic nerve profiles. In the rat cerebral artery, these profiles have been traced back by serial sampling to myelinated nerve fibres, suggesting that they may represent sensory nerves (Burnstock et al., 1970c), a feature which has also been demonstrated in a longitudinal section by Hagen and Wittkowski, 1969.

Non-nervous proJiles in smooth muscle Apart from the nerve types described above, a number of other profiles are sometimes found in close relation to smooth muscle. These include sections through: ( I ) Chromafin or ‘chromafin-type’ cells. These cells are characterised by many large membrane-bound granules, comparable to those seen in the adrenal medulla (see Elfvin, 1965). The granules in dopamine-containing cells described in many tissues in ruminants (Hebb et al., 1968), often show a substructure made up of 150-w granules (Burnstock et al., 1970~). ( 2 ) ‘Interstitial’ cells. These are found particularly in the gut and their status was in doubt for many years, until ultrastructural studies showed clearly that they were probably fibroblast-like cells and/or macrophages (Richardson, 1960; Rogers and Burnstock, 1966a). They usually partially envelop bundles of axons, Schwann cell processes and sometimes form close relationships to muscle cells ; but their function is still not known. ( 3 ) Muscle cell intrusions and Schwann cells. Occasionally sections through Schwann cell processes unaccompanied by nerves are seen in close relation to muscle cells. Intrusions of processes of one smooth muscle into its neighbour are characteristic of many tissues (Merrillees et al., 1963; Thaemert, 1963). These profiles are often relatively free of organelles, incuding myofilaments, and therefore difficult to identify. The use of 5- or 6-OHDA as a marker for sympathetic nerves is often a useful way of distinguishing these nerves from muscle or Schwann cell profiles (see Figs. 2a, c; 4a). EFFECT OF D R U G S O N A D R E N E R G I C N ER V ES

The acute and chronic actions of a large number of drugs on the fine structure of sympathetic nerves has been reported (see Van Orden et al., 1966; Burnstock and Robinson, 1967; Tranzer et al., 1969; Hokfelt, 1968; Geffen and Livett, 1971). In these studies the main observations were of alterations in the number and proportion of granular vesicles. This approach, because of the problem of obtaining reliable control values with the fixation procedures available, has been largely disappointing and inconclusive. In this report, discussion will be confined to changes in fine structure References p p . 402-404

400

G. B U R N S T O C K , T. I W A Y A M A

produced by only two drugs, 6-OHDA and guanacline, which do not involve vesicle counts. 6-OHDA 6-OHDA depletes the catecholamines from sympathetically innervated tissues (Porter et al., 1963; Laverty et al., 1965; Porter et al., 1965). More recently, it has been shown that, within one hour of injection of the drug, there is a marked increase in the density of cores of both large and small intra-axonal vesicles in sympathetic nerves (Bennett et al., 1970a; Furness et al., 1970a); by 24 hours, if the concentration of injected drug is sufficient, there is specific degeneration of adrenergic nerve terminals (Tranzer and Thoenen, 1967b, 1968a; Thoenen and Tranzer, 1968; Malmfors and Sachs, 1968). There has been a recent analysis of the mechanism of action of 6-OHDA in producing these changes (Bennett et al., 1970a; Furness et al., 1970a). The first sign of nerve damage is seen a t 1-2 hours, consisting of electron transparency of the axoplasm and a decrease in number of organelles. From 2-10 h, the axoplasm becomes generally electron dense, reminiscent of the appearance of nerves following surgical denervation (Birks et al., 1960; Taxi, 1965; Pluchino et al., 1970; Roth and Richardson, 1969; Iwayama, 1970) and is accompanied by failure of neuromuscular transmission (Furness et al., 1970a). While entry of 6-OHDA into vesicles occurs, the important factor in degeneration appears to be the extragranular (axoplasmic) concentration of the drug in the nerve. Damage to the membrane could account for the potentiation of packaged release of noradrenaline (measured as spontaneous junction potentials) which is known to occur under these conditions (Furness et al., 1970a), and lead to degeneration of the nerve. Guanacline

Guanacline (N-2-guanedinoethyl-4-methyl-1,2,3,6-tetrahydropyridine sulphate) has catecholamine-depleting and adrenergic-blocking actions similar to guanethidine (Kroneberg et al., 1967; Schiimann and Philippu, 1968). It has been used in the treatment of hypertension (Gross et al., 1965), but it was reported that postural hypotension persists in humans even several months after withdrawal of therapy (Dawborn et al., 1969). In a recent ultrastructural and histochemical study of sympathetic neurones both during and after cessation of chronic treatment of rats with guanacline (5 mg/ kg/day), it has been shown that a massive deposition of lipoprotein granules (comparable to ‘ageing pigment’) is produced (Burnstock et al., 1971). This persists for up to at least 12 weeks following cessation of treatment. No comparable changes were observed in animals treated with the same dose of guanethidine. SUMMARY

(1) A general model of the autonomic neuromuscular junction has been proposed,

F I N E - S T R U C T U R A L I D E NT I P I c A T I O N O F A U T O N O M I c N E R V E S

401

based on both structural and physiological data. It takes into account the spread of activity between neighbouring smooth muscle cells via nexus and ‘en passage’ release of transmitter from varicosities in extensive terminal regions of the nerves. (2) Nexus between smooth muscle cells grown in tissue cultures suggest that they are an inherent feature of smooth muscle and do not depend on innervation by nerves. Similarly, varicosities appear in tissue cultures of nerves, which suggests that they are a genetically determined feature of axons and are not induced by the organs they innervate. Nerve profiles in close apposition to muscle are described in joint cultures of nerves and smooth muscle. (3) The synaptic cleft is difficult to define precisely at autonomic neuromuscular junctions: close approaches of nerve and muscle membranes in the vas deferens and circular muscle coat of the intestine are of the order of 200 A, but transmitter released from up to 1200 8,appears to be effective; in most arteries the minimum neuromuscular separation is of the order of 800 8, and the functional cleft appears to be about l p . Other preparations show intermediate separations. (4) No consistent post-synaptic specialisation of muscle membranes has been seen at autonomic neuromuscular junctions. However, subsynaptic cysternae are often seen in muscle in the vas deferens, and aggregations of micropinocytotic vesicles in the muscle beneath terminal axons in gut and some blood vessels are also common. (5) Terminal varicosities penetrate deep inside some smooth muscle cells of the vas deferens, sometimes in close relation to perinuclear organelles; the significance of this finding is unknown. (6) Multiaxonaljunctions with single smooth muscle cells, particularly in the circular muscle coat of the intestine, have been described and the close relationship of terminal regions of some adrenergic and cholinergic nerves discussed. (7) The intra-axonal features of different nerves have been described. Cholinergic axons contain a predominance of small (300-600 8,) agranular vesicles, and a small proportion of large (900-1200 A) granular vesicles of unknown function. Adrenergic axons contain a predominance of small (300-600 A) granular vesicles, but some large (900-1200 A) granular vesicles (which also take up monoamines) and small agranular vesicles (which probably represent ‘empty’ small granular vesicles) are also present. Some adrenergic profiles in birds and reptiles contain predominantly large (900-7200 8,) granular vesicles. Non-adrenergic, non-cholinergic autonomic axons in gut and lung contain a predominance of large (1000-2000 A) granular vesicles, which, unlike those described in adrenergic and cholinergic nerves, are completely filled with granular material. Some small agranular vesicles are usually also present. Some nerve profiles, which probably represent sensory nerves, are characterised by large numbers of small, oval mitochondria and few, if any, vesicles. (8) Non-nervous profiles in close relation to smooth muscles have also been described, including chromaffin and ‘chromaffin-type’ cells, ‘interstitial cells’, and Schwann cells. (9) Ultrastructural changes produced in sympathetic nerves by various drugs have been discussed. 5- and 6-OHDA displace noradrenaline from small- and large-granular vesicles within 1 hour of injection. When the concentration of 6-OHDA reaches a References p p . 402-404

402

G. B U R N S T O C K , T. I W A Y A M A

critical concentration in the axoplasm, membrane damage seems to occur that may lead to the structural changes associated with degeneration of the terminal regions of the nerves, which are comparable to those seen following surgical denervation. Chronic treatment with the anti-hypertensive drug, guanacline, causes massive deposition of lipoprotein granules in sympathetic neurones, which is long-lasting and perhaps irreversible.

REFERENCES BAUMGARTEN, H. G. AND BRAAK,H. (1968) Z . Zellforsch., 86, 574602. BELL,C. (1969) Circulation Res., 24, 61-70. BENNETT, M. R. AND BURNSTOCK, G. (1968) Handbook OfPhysiology, Secfion6 . Alimentary Canal, IV. Motility, Publ. American Physiological Society, Washington, pp. 1709-1732. BENNETT, M. R. A N D MERRILLEES, N. C .R. (1966) J. Physiol., 185, 520-535. BENNETT, M. R. AND ROGERS, D. C. (1967) J. Cell Biol., 33, 573-596. BENNETT, M., BURNSTOCK, G. A N D HOLMAN, M. E. (1966) J. Physiol., 182, 541-558. BENNETT, T. (1969) J. Physiol., 204, 669-686. BENNETT, T. (1970) Comp. Biochem. Physiol., 32, 669-680. BENNETT, T., BURNSTOCK, G., COBB,J. L. S. AND MALMFORS, T. (1970) Brit. J. Pharmacol., 38, 802-809. BENNETT, T. AND COBB,J. L. S. (1969) Z . Zellforsch., 99, 109-120. BENNETT, T., COBB,J. L. S. AND MALMFORS, T. (1971) Experimental studies on Auerbach's plexus, with particular reference to the adrenergic component, To be published. BIRKS,R., HUXLEY, H. E. AND KATZ,B. (1960) J. Physiol., 150, 134-144. BLOOM, F. E. AND BARRNETT, R. J. (1966) Nature, 210, 599-601. BLOOM, F. E. A N D GIARMAN, N. J. (1967) Anat. Rec., 157, 351. BONDAREFF, W. AND GORDON, B. (1966) J. Pharmacol. Exp. Therup., 153,42-47. BRETTSCHNEIDER, H. (1 962) 2. Mikroskop.-Anat. Forsch., 683, 333-360. BULBRING, E. AND TOMITA, T. (1967) J. Physiol., 189, 299-315. BURN,J. H., (1968) In WOLSTENHOLME AND O'CONNOR (Eds.), Adrenergic Neurotrunsmission Ciba Foundation Study Group N o . 33, Churchill. London, p. 39 in: Discussion of the mechanism of the release of noradrenaline. BURN,J. H. AND RAND,M. J. (1959) Nature, 184, 163-165. RURN,J. H. AND RAND,M. J. (1965) Ann. Rev. Pharmacol., 5 , 163-182. BURNSTOCK, G. (1968) Proc. 24th Intern. Congr. Physiol. Sci., Washington, 6, 7-8. BURNSTOCK, G. (1969) Pharmacol. Rev., 21, 247-324. BURNSTOCK, G. (1970) In BRADING, JONESAND TOMITE(Eds.), Smooth Muscle, Edward Arnold Publ. Ltd. London, pp. 1-69. BURNSTOCK, G. (1971) Nature, 229, 282-283. BURNSTOCK, G., DOYLE, A. E., GANNON, B. J., GERKINS, J. F., IWAYAMA, T. AND MASHFORD, M. L. (1971) Europ. J . Pharrnacol., 13, 175-187. BURNSTOCK, G., CAMPBELL, G., BENNETT, M. AND HOLMAN, M. E. (1963b) Nature, 200,581-582. BURNSTOCK, G., CAMPBELL, G., BENNETT, M. AND HOLMAN, M. E. (1964) Intern. J . Neuropharmacol., 3, 163-166. BURNSTOCK, G., CAMPBELL, G. AND RAND,M. J. (1966) J. Physiol., 182, 504526. BURNSTOCK, G., CAMPBELL, G., SATCHELL, D. G. AND SMYTHE, A. (1970a) Brit. J . Pharmacol., 40, 668-688. BURNSTOCK, G., CAMPBELL, G. R., UEHARA, Y . , FURNESS, J. B. AND MALMFORS, T. (1970b) Proc. Austr. Physiol. Pharrnacol. Soc., May 1970, 1,70-71P. BURNSTOCK, G., GANNON, B. AND IWAYAMA, T., (1970~)Circulation Res., 27 (Suppl. II), S-24. BURNSTOCK, G., HOLMAN, M. E. A N D PROSSER, C. L. (1963a) Physiol. Rev., 43, 482-527. BURNSTOCK, G. AND ROBINSON, P. M. (1967) Circulation Res., 21, Suppl. 3, 43-55. CAMPBELL, G. (1966) J. Physiol., 185, 600-612.

F I N E - s T R U CT U R A L I D E N T I F I c A T I O N OF A U T O NO M I c N E R V E s

403

CAMPBELL, G. (1970) In BRADING, JONESAND TOMITE(Eds.), Smooth Muscle Edward Arnold Publ. Ltd., London, pp. 451495. CAMPBELL, G. R., UEHARA,Y . , MARK,G. AND BURNSTOCK, G. (1971) J. Cell Biol., 49, 21-34. CLEMENTI, F., MANTEGAZZA, P. AND BOTTURI,M. (1966) Intern. J. Neuropharmacol., 5, 281-285. J. K., DOYLE,A. E., EBRINGER, A., HOWQUA, J., JERUMS, G., JOHNSTON, C. I., MASHFORD, DAWBORN, M. L. AND PARKIN,J. D. (1969) Pharn7acol. Clin., 2, 1-5. DAY,M. D. AND WARREN, P. R. (1968) Brit. J. Pharmacol., 32, 227-240. DE ROBERTIS, E. AND BENNETT, H. S. (1955) J. Biophys. Biochem. Cytol., 1, 47-65. DEWEY,M. M. AND BARR,L. (1962) Science, 137, 670-672, ELFVIN,L. G. (1965) J. Ultrastruct. Res., 12, 263-286. ETCHEVERRY, G. J. AND ZIEHER,L. M. (1968a) J. Histochem. Cytochem., 16, 162-171. ETCHEVERRY, G. J. AND ZIEHER,L. M. (1968b) Z. ZeNforsch., 86, 393-400. FARRELL, K. E. (1968) Nature, 217, 279-281. FERRY,C. B. (1966) Physiol. Rev., 46, 420-456. FURNESS, J. B. (1969) J. Physiol., 205, 549-562. FURNESS, J. B. (1970) Pfhigers Arch. Ges. Physiol., 314, 1-13. FURNESS, J. AND BURNSTOCK, G. (1969) Comp. Biochem. Physiol., 31, 337-345. FURNESS, J. B., CAMPBELL, G . R., GILLARD,S. M., MALMFORS, T. AND BURNSTOCK, G. (1970a) J. Pharmacol. Exp. Therap., 174, 111-122. FURNESS, J. B. A N D IWAYAMA, T. (1970) Z. Zellforsch., 113, 259-270. FURNESS, J. B., MCLEAN,J. R. AND BURNSTOCK, G. (1970b) Develop. Biol., 21,491-505. GEFFEN,L. B. AND LIVETT,B. G . (1971) Physiol. Rev., 51, 98-157. GRAINGER, F., JAMES,D. W. AND TRESMAN, R. L.(1968) Z. Zellforsch., 90, 53-67. GRILLO,M. A. (1966) Pharmacol. Rev., 18, 387-399. GROSS,W., BRACHARZ, H., AND LAAS,H. (1965) In Hochdruckforschung Fortschritte auf dem Gebiete der Inneren Medizin, 11, Symposium held in Freiburg i. Br., July 18 and 19, 1964, Georg Thieme Verlag, Stuttgart . HAGEN,E. AND WITTKOWSKI, W. (1 969) Z. Zellforsch., 95, 429-444. HEBB,C., KLSA,P. AND MANN,S. (1968) Histochemical Journal, 1, 166175. HILLARP,N. A. (1946) M.D. Thesis, Acta Anat., 2, Suppl. 4, 1-153. HOKFELT,T. (1966) Experientia, 22, 56. HOKFELT, T. (1968) Z. Zellforsch., 91, 1-74. HOKFELT, T. AND JONSSON, G. (1968) Histochemie, 16, 45-67. HOKFELT,T. AND NILSSON,0.(1965) 2. Zelvorsch., 66, 848-853. HOLMAN, M. E. (1970) In E. BULBRING (Ed.), Smooth Muscle, Edward Arnold Publ. Ltd., Londm. IWAYAMA, T. (1970) 2. Zelgorsch., 109, 465480. J. B. (1971) J. Cell Biol., 48, 699-703. IWAYAMA, T. AND FURNESS, IWAYAMA, T., FURNESS, J. B. AND BURNSTOCK, G. (1970) Circulation Res., 26, 635-646. KRONEBERG, G., SCHLOSSMANN, K. AND STOEPEL, K. (1967) Arzneimittel Forsch., 17, 199-207, KURIYAMA, H., OSA,T. AND TOIDA,N. (1967) J. Physiol., 191, 257-270. LANE,B. P. AND RHODIN,J. A. G. (1964) J. Ultrastruct. Res., 10, 470-488. LAVERTY, R., SHARMAN, D. F. AND VOGT,M. (1965) Brit. J. Pharmacol., 24, 549-560. MALMFORS, T. (1965) Acta Physiol. Scand., 61, (Suppl. 2481, 1-93. MALMFORS, T. AND SACHS,CH. (1968) Europ. J. Pharmacol., 3, 89-92. MARTINSON, J. (1965) Acta Physiol. Scand., 64, 453-462. MERRILLEES, N. C. R. (1968) J. Cell Biol., 37, 794-817. MERRILLEES, N., BURNSTOCK, G. AND HOLMAN, M. b. (1963) J. Cell Biol., 19, 529-550. NAGASAWA, J. AND MITO, S. (1967) Tohoku J. Exp. Med., 91, 277-293. NORBERG, K.-A. AND HAMBERCER, B. (1964) Acta Physiol. Scand., 63, Suppl. 238, 1-42. PLUCHINO, S., VANORDEN111, L. S., DRASKOCZY, P. R., LANGER,S. Z. AND TRENDELENBURG, V. (1970) J. Pharmacol. Exp. Therap., 172, 77-90. PORTER,C. C., TOTARO,J. A. AND BURCIN,A. (1965) J. Pharmacol. Exp. Therap., 150, 17-22. PORTER,C, C., TOTARO,J. A. AND STONE,C. A. (1963) J. Pharmacol. Exp. Therap., 140, 308-316. READ,J . B. AND BURNSTOCK, G. (1970) Develop. Biol., 22, 513-534. RICHARDSON, K. C. (1960) J. Anat., 94, 457472. RICHARDSON, K. C. (1962) J. Anat., 96, 421-442. RICHARDSON, K. C. (1966) Nature, 210, 756. ROBERTSON, J. D. (1962) In Ultrastructure and Meiabolim of the Nervous System, 40, 94-158. ROBINSON, P. M. (1969) J. Cell Biol., 41, 462-476.

404

G. B U R N S T O C K , T. I W A Y A M A

ROBINSON, P. M., MCLEAN,J. R. AND BURNSTOCK, G. (1970) J. Pharmacol. Exp. Therap., in press. ROGERS, D. AND BURNSTOCK, G. (1966 a); J. Comp. Neurol., 126, 255-284. ROGERS, D. AND BURNSTOCK, G. (1966 b) J. Comp. Neurol., 126, 625-652. ROTH,C. D. AND RICHARDSON, K. C. (1969) Amer. J. Anat., 124, 341-360. SCHUMANN, H. J. AND PHILIPPU,A. (1968) Arzneimittel-Forsch., 18, 1571-1574. TAXI,J. (1965) Naturelles Zoologie, 12e serie, VII, 413-674. TAXI,J. (1969) Progr. Brain R e x , 31, 5-20. THAEMERT, J. C. (1963) J. Cell Biol., 16, 361-377. THAEMERT, J. C. (1966) J. Cell Biol., 28, 3749. THOENEN, H. AND TRANZER, J. P. (1968) Arch. Exp. Pathol. Pharmakof.,261, 271-288. THOENEN, H., TRANZER, J. P., HURLIMANN, A. AND HAEFELY, W. (1966) Helv. Physiol. Pharmacol. Acta, 24, 229-246. TRANZER, J. P. AND THOENEN, H. (1967a) Experientia, 23, 743-745. TRANZER, J. P. AND THOENEN, H. (1967 b) Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol., 257, 343. TRANZER, J. P. AND THOENEN, H. (1968 a) Experientia, 24, 155-156. TRANZER, J. P. AND THOENEN, H. (1968 b) Experientia, 24,484486. TRANZER, J. P., THOENEN, H., SNIPES,R. L. AND RICHARDS, J. G. (1969) Progr. Brain Res., 31,33-46. UEHARA, Y . AND BURNSTOCK, G. (1971) 2.Zellforsch., submitted. VANORDEN,L. S. 111, BLOOM, F. E., BARRNETT, R. J. AND GIARMAN, N. J. (1966) J. Pharmacol. Exp. Therap., 154, 185-199. WATANABE, H. (1969) Acta Anat. Nipgon, 44, 189-202. WHITTAKER, V. P., MICHAELSON, I. A. AND KIRKLAND, R. J. A. (1964) Biochem. J., 90, 293-303. YAMAUCHI, A. AND BURNSTOCK, G. (1969 a) J. Anat., 104, 1-15. YAMAUCHI, A. AND BURNSTOCK, G. (1969 b) J. Anat., 101,17-32. ZWEIG,M. AND AXELROD, J. (1969) J. Neurobiol., 1, 87-97.