The sympathetic nervous system in man — aspects derived from microelectrode recordings

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The sympathetic nervous system in man - aspects derived from microelectrode recordings B. Gunnar Wallin and Jan Faglus In man the study o f autonomic nervous system function has been restricted to the succeeded by changes of sympathetic observation o f effector organ responses during certain manoeuvres. From such 'effector organ activities2-5. studies the image of a slowly reacting regulatory system has evolved, while the concept o f a diffuse 'sympathetic tone' has been widely used to describe the Sympathetic outflow in human presumed generalized ouqlow o f impulses in sympathetic nerves. The extremity nerves introduction o f microneurography, allowing direct microelectrode recordings o f In both muscle and skin fascicles, sympathe.~c discharges in human extremity nerves 1, has contributed to changing sympathetic outflow is seen as multithis view. unit volleys of impulses with interToday the sympathetic nervous system plethysmogram (photo-electric) are posed intervals of neural silence; there can be seen to be a highly differ- often included. is no evidence of continuous activity. entiated, rapidly activated system, with Evidence of the sympathetic origin The temporal pattern of activity, subdivisions specialized to regulate of the recorded impulses are: (1) the however, is entirely different in the different organ functions in response to activity is efferent, as shown by two systems. the changing demands o f the external application of local anaesthesia proxiand internal milieu. The two sub- mal and distal to the recording site; (2) Muscle nerve sympathetic activity (MSA ) divisions that can be studied with the impulses are conducted with a Bursts of MSA are time-locked to microneurography are muscle nerve velocity of about 1 m s-1; (3) the activity the cardiac rhythm2. There is a close sympathetic activity (MSA) and skin is reversibly abolished by the sympa- inverse correlation between blood nerve sympathetic activity (SSA) - thetic ganglion-blocker trimetaphan; pressure variations and MSA; the visceral sympathetic nerves and para- and (4) changes of nerve activity are bursts occur during transient reducsympathetic nerves are still inaccesARTERIAL ILMIOIEFLEX sible. In spite o f this limitation, and although recordings are made from peripheral postganglionic fibres, it has been possible to draw conclusions, not only about reflex mechanisms, but also more generally about central sympathetic control in man. The recording microelectrode is made from insulated tungsten, with an uninsulated tip a few microns in diameter. This is inserted manually through the intact and unanaesthetized skin into the underlying nerve in an alert and co-operating subject. The reference electrode is placed subcutanNORMOI"ENSIVE HYPERTENSIVE eously 1-2 cm away. Usually multiunit activity is obtained (and indeed is preferred), but occasionally single unit activity is encountered. Most recordings are made in large nerves such as median, tibial or peroneal nerves, but smaller cutaneous nerves have been used. Recordings cause only minimal discomfort and no permanent aftereffects have been reported, although ills * paraesthesiae may occur in 10-20% of the subjects for a few days after the Fig. 1. Muscle nerve sympathetic activity (MSA), relation to blood pressure. Peroneal nerve experiment. In most experiments recordings. Mean voltage neurograms (upper tracings) and intraarterial blood pressure recordings (A) ECG and respiratory movements are MSA appears predominantly during the falling phase o f spontaneous blood pressure fluctuations. recorded and sometimes intra-arterial Pulse synchrony is due to baroreflex modulation. Asterisk indicates sudden A V block, followed by strong burst o f MSA. Reflex latency between corresponding heart beats and subsequent bursts blood pressure is monitored. Changes indicated by skew line and shaded areas. (B) MSA is not correlated to long-term level of blood of electrical skin resistance (evoked by pressure: similar level of MSA in normotensive and hypertensive subject despite clearly different blood sweating) and a finger or toe pulse pressure levels. (A) T a k e n , with permission, from Ref. 42 and (B) modified from Ref. 16.

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~) 1986, Elsevier Science Publishers B.V., Amsterdam 0378 - 5912/86/$02.00

64 tions and disappear when blood pressure increases 1,6. As shown in Fig. 1A a blood pressure fall during a prolonged diastole is followed by a particularly strong MSA burst v. Electrical stimulation of the carotid sinus nerve inhibits MSA with accompanying reduction of muscle vascular resistance 8. The cardiac rhythmicity is due to arterial baroreceptor modulation of the neural outflow. With each systolic blood pressure peak, sympathetic activity is inhibited, but reappears when diastolic blood pressure falls below a certain level. Bilateral local anaesthesia of glossopharyngeal and vagus nerves in the neck evoked a pronounced increase of MSA (accompanied by high blood pressure and tachycardia), and the pulse synchrony was replaced by a 0.4-0.7 Hz irregular rhythm (similar to but not identical with sympathetic activity in skin nerves; see below) 9. Thus it seems that central sympathetic outflow to muscles consists of irregular discharges and that the characteristic temporal pattern is brought about by recurrent baroreceptor inhibition entraining the bursts in the cardiac rhythm. A detailed analysis of the relationship between MSA and blood pressure shows that the outflow of impulses correlates with fluctuations of diastolic blood pressure, and not to the longterm level of blood pressure (Fig. 1B) 6. This relationship between MSA and diastolic blood pressure variations clearly follows from the fact that the baroreflex is inhibitory and since systolic inhibition of sympathetic activity is complete, diastolic blood pressure variations are the basis of regulation. This reflex influence from arterial baroreceptors on MSA has dynamic properties. For example, at a given value of diastolic blood pressure, sympathetic discharges are stronger and more common during the falling phase of a blood pressure change than during the rising phase (Fig. 1A) 6. If afferent baroreceptor nerve traffic is altered by step changes of transmural carotid pressure there is only a transient change of MSA; adaptation is more or less complete after 1-2 s even if the stimulus is maintained ~°.H. Together with the lack of correlation between mean levels of MSA and diastolic blood pressure these findings suggest that arterial baroreflex effects on M S A are important predominantly for buffering changes of arterial blood presst~re but have much less importance for maintaining the long-term blood pressure level.

FINS- ~ebruary 1986 Intrathoracic volume receptors (which can be considered equivalent to cardiopulmonary baroreceptors or low pressure receptors) exert more tonic reflex effects on MSA. Application of weak subatmospheric pressure around the lower body displaces blood from the chest to the lower body, thereby unloading volume receptors without changing arterial blood pressure. This causes an increase of M S A which is maintained as long as the stimulus is applied lz. Many changes of MSA which occur in daily life no doubt result from complex interaction of arterial and cardiopuimonary baroreceptor stimuli. For example, the Valsalva manoeuvre* causes a fall of arterial and central venous pressure. This regularly leads to a strong increase of M S A followed by inhibition which coincides with the blood pressure overshoot after the manoeuvre 3. Transition from supine to upright posture also unloads both types of receptors and evokes an increase in MSA 13. Arterial baroreceptor unloading may be important for the initial compensatory burst sequence in conjunction with the actual change of posture but for the persisting higher level of MSA, influence from low pressure receptors probably dominate. A poorly understood feature of MSA is the profound difference in strength of activity between individuals 14. In a given subject the pattern outflow of sympathetic impulses shows a high degree of similarity in different extremity nerves and the level of activity at rest (expressed as number of bursts/ 100 heart beats or bursts/minute) is reproducible from day to day even with years between recordings ~4.15. Between individuals, however, the level of activity at supine rest ranges from below 10 to more than 90 bursts/ 100 heart beats. As mentioned above the differences are not related to differences in arterial blood pressure 6'16 and they are neither related to differences in degree of physical conditioning ~7. The level of MSA does increase with age but age cannot be the main factor since there are wide inter-individual differences at all ages between 18-55 years. There is also a weak inverse relationship between levels of MSA and heart rate suggesting interaction between neural control of cardiac and peripheral vascular

* Increase of intrathoracic pressure by forcible exhalation effort against the closed glottis.

function. Although there is at present no explanation for the inter-individual variation, it probably reflects basic inter-individual differences in cardiovascular homeostatic mechanisms. Possibly the inter-individual differences are related to differences in the degree of arterial and/or cardiopulmonary baroreflex inhibition rather than to differences in central sympathetic drive 9. As seen from Fig. 1A, there is a reflex delay from a cardiovascular event to a resulting effect on MSA. Largely this delay is due to conduction time in postganglionic C fibres and consequently the reflex delay provides an indirect measure of conduction velocity in these fibres TM. The heart rate has no influence on this delay. Burst duration, on the other hand, correlates closely to the length of the cardiac interval (cf. Fig. 1A), which again illustrates the importance of systolic-diastolic blood pressure fluctuations for the temporal appearance of MSA.

Skin nerve sympathetic activity (SSA) The temporal pattern of sympathetic activity seen in skin nerve fascicles differs markedly from that in muscle nerves. In the initial phase of a recording at room temperature, there is usually a fairly intense resting activity with bursts of varying duration and strength occurring in a slow, irregular rhythm (Fig. 2A) 4. With prolonged recordings at rest, the activity gradually subsides and the nerve may become more or less silent. The spontaneous activity is regularly followed by sweating and skin vasoconstriction, i.e. the discharges are made up of a mixture of sudomotor and vasoconstrictor impulses 4. The two types of impulses are propagated with different velocity TM and can be regularly recorded from the same intrafascicular site. Changes of alertness causes changes of the strength of SSA and during general anaesthesia spontaneous SSA is virtually absent ~'~. A sudden deep breath and a variety of arousal stimuli regularly elicit a strong burst of SSA (Fig. 2B) and the reflex delay correlates with conduction time in postganglionic fibres TM. Emotional excitement usually induces more long-lasting increase of outflow -~. The above-mentioned decay of spontaneous activity at rest during a recording probably reflects declining emotional stress from the experimental procedure. Thus SSA is involved in emotional reactions, and the stress-induced

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long-lasting simultaneous activation of pathetic activity in skin and muscle stable from one occasion to another24. sudomotor and vasoconstrictor fibres nerve fascicles, and even of sudomotor Similarly MSA both tends to be stable constitutes the neurophysiological and vasoconstrictor activity in skin in an individual and vary markedly basis of cold sweat. nerves, emphasize that there is no between individuals. Moreover a signiPhysiologically, the most important common overall sympathetic tone. ticant correlation between levels of function of SSA is thermoregulation. Instead the sympathetic nervous sys- MSA at rest and plasma noradrenaline During body cooling, at resting condi- tem should be regarded as a number of from antecubital (i.e. in front of tions, an increase of activity is functionally separate subdivisions, elbow) venous blood was also found evoked. At the same time plethysmo- each of which is modulated by its own in normotensive subjects2s and in grapby displays reduced digital pulse central connections and afferent in- patients with essential hypertension26. amplitudes, whereas there is no sign of fluences. This view is in agreement A possible explanation for this finding sweating, showing that the increase of with the finding of different contribu- could be that striated muscles, comSSA is due to selective activation of tion of individual organs to total prising 40% of total body weight, vasoconstrictor fibres. During moder- noradrenaline release23. For MSA, contain such a large number of symate warming outflow of impulses is evidently an individual, characteristic pathetic terminals that spiUover from reduced to a minimum, and when body resting tone can be defined, which these will be a major contribution to heating is associated with sweating seems to be similar for a large part of plasma noradrenalin¢, especially when there is again an increase of SSA, this body musculature. For SSA the term blood is sampled from an antecubital time consisting of sudomotor impul- 'tone' is of little value in view of the vein, where 45% of the noradrenaline ses2°. Thus selective activation of either profound variations according to en- is estimated to derive from local the sudomotor or vasoconstrictor fibres vironmental circumstances. production27. If otber sympathetic with simultaneous suppression of the outflows (e.g. to the kidneys) are other fibre population occurs during RelationbetweenMSAand plasma similar to that of muscle, the correlatemperature changes. At extreme noradrenaline tion would be strengthened. temperatures weak arousal stimuli may At rest, plasma levels of noradrenaSince resting SSA is low at comfortlead to selective activation of only one line, the transmitter released from able ambient temperature5,20, and cell type 2°, presumably because in that postganglionic sympathetic neurons, since sudomotor postganglionic fibres situation the stimulus is subthreshold vary markedly between individuals, are cholinergic, it is not surprising that for the other cell population. but in a given subject the level is fairly t h e noradrenaline spillover rate from Simultaneous recordings from two nerves supplying glabrous skin (palm of hand and/or sole of foot) have shown a strict correlation between the ECG I i outflow from the two nerves but regional differences have been observed between nerves supplying glabrous skin of the hand and hairy skin act of the forearm2L No overt cardiac ! ! rhythmicity is present in SSA (Fig. 5s 2A), but detailed rhythm analysis with ECG-triggered averaging showed a weak temporal relationship, the relevance of which in unclear22. Carotid sinus nerve stimulation had no reproducible effect of SSAs, suggesting that there is little or no baroreceptor influence on SSA. The lack of change of SSA following block of afferent baroreceptor pathways9 agrees with this interpretation. It is noteworthy that during transient baroreceptor deafferentation the temporal pattern of MSA became similar (but not identical) to that of SSA, and in addition arousal stimuli evoked reflex discharges in parallel in SSA and MSA9. This raises the ~ act possibility that SSA and MSA have related or common intrinsic rhythm • i generators with the normal differences 5s in pattern to a large extent being due Fig. 2. Skin nerve sympathetic activity (SSA) recorded in right median nerve. (A) Spontaneous SSA in to baroreflex modulation (and sup- the typical slow, irregular rhythm; no overt cardiac rhythmicity is seen. ECG and mean voltage pression of arousal reflexes) in MSA neurogram. (B) Strong bursts of SSA elicited by arousal stimulus (sudden shout indicated by arrow in

A

B

Resp

Skin resi st. _ ~

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Theconceptof sympathetictone The different properties of sym-

leJ~ panel) and deep inspiration (right panel). Tracings from top: respiration, finger paise plethysmogram, palmar skin resistance (reduction upwards) and mean voltage neurogram. The burst o f SSA is followed by digital vasoconstriction and palmar sweating (indicated by decrease of skin resistance).

~#~ skin is found to be low 23. Due to difficulties in quantifying SSA, however, its precise relationship to noradrenaline levels is unknown.

Other applications of sympathetic nerve recordings

Complex physiological manoeuvres The strong influence of mental stress on SSA is evident from the above findings, but the effect on MSA is less pronounced and less clear. Both weak increases and decreases of MSA may occur during mental arithmetic3; a systematic study of these effects is in progress. Isometric muscle work is accompanied by increased heart rate and blood pressure 2s. A recent study of the effect of 2 min sustained hand grip on MSA recorded in a peroneal nerve showed that sympathetic activity was unchanged during the first but increased during the second minute. Several pieces of evidence suggested that the increase was a reflex effect induced by ischaemia in the contracting muscles 29. Since blood pressure increased at the same time resetting of the baroreceptor regulatory level must have occurred during this manoeuvre. The cold pressure test, i.e. immersion of one hand into ice water for 1-2 minutes, caused an increase of both SSA 3° and MSA 31 although with a marked interindividual variation for the latter (Fagius, J., unpublished observations).

Pharmacological studies The recording method is well suited to study drug effects. Intravenous administration of the selective 13adrenoreceptor antagonist metoprolol caused an overall increase of MSA in most subjects 32, but the underlying mechanism is unclear. In contrast, long-term metoprolol treatment was accompanied by a slight reduction of MSA in comparison with control recordings before treatment 33. The vasodilator sodium nitroprusside induces an increase of MSA (Wallin, B. G., Sundl~f, G., M6rlin, C., unpublished observations), whereas clonidine in a low dose increased and in a high dose decreased MSA 34.

Pathophysiological studies Several pathophysiological studies have been published recently. Polyneuropathy is sometimes associated with reduced autonomic functions. In a study of polyneuropathies of different aetiologies, MSA and/or SSA of

f I N S - t~ehruary 1986 normal appearance could often be recorded despite a pronounced somatic polyneuropathy and no evidence of reduced postganglionic conduction velocity was found 35. In diabetic polyneuropathy failure to detect sympathetic impulses was much more common than in other types of polyneuropathy :~'. The findings suggest that in polyneuropathy, sympathetic failure is due to cessation of conduction in an increasing number of postganglionic fibres, resulting in weaker activity, which can finally no longer be detected in the nerve recordings 36. In acute inflammatory polyradiculoneuropathy (the Guillain-Barr6 syndrome) some patients develop tachycardia and temporary hypertension. Recently three such cases were studied and all had a marked increase of MSA during the acute illness ~5. The underlying mechanism is probably a lesion of afferent baroreflex pathways, an interpretation that is supported by experimental block of glossopharyngeal and vagus nerves, where the clinical picture was mimicked and a strong increase of MSA was evoked `). Ira traumatic quadraplegia, autonomic dysfunction is a well recognized feature. In such patients, sympathetic outflow to the extremities below the level of lesion has been studied 37"38, and both in skin and muscle nerves, spontaneous activity was much more sparse than in normal subjects. Deep breaths, abdominal pressure over the bladder and mechanical or electrical stimuli to the skin, caudal to the spinal transection, induced single sympathetic bursts and such reflex discharges occurred in parallel in skin and muscle nerve fascicles. There were no signs of thermoregulatory or baroreceptor influence on the activity. Digital vasoconstriction following a single sympathetic burst lasted longer than in normals. Hence, attacks of autonomic dysreflexia cannot be explained by sympathetic hyperactivity of spinal origin, but the prolonged vasoconstrictions and the parallel activation of skin and muscle sympathetic activity may contribute to episodes of high blood pressure in these patients. Two cases of vasovagal syncope were associated with a sudden disappearance of MSA:~L The result was similar in a patient who suffered from glossopharyngeal neuralgia with fainting 4t', The sympathetic nervous system has been suggested to be involved in migraine and Raynaud's phenomenon. In u study of MSA in migraineurs in

headache-free intervals, and during migraine attacks, no abnormality was seen (Fagius, J., unpublished observa+ tions), an involvement of other parts of the sympathetic nervous system is not ruled out, however. In Raynaud's phenomenon there were normal digital vasoconstrictor responses lo strong bursts of SSA evoked by arousal stimuli and no evidence of increased outflow of SSA to the hand when the contralateral hand was exposed to ice water 30.

intraneural stimulation Intraneural microstimulation of skin nerves has been used to elicit cutaneous sympathetic effector responses 41+ Direct stimulation of sudomotor fibres evoked reduction of skin electrical resistance (i.e. sweating) which became maximal at a stimulation frequency of 15 Hz. Vasomotor responses were complex due to simultaneous activation of vasconstrictor and vasodilator effects. Recently two noncholinergic and non-13-adrenergic vasodilator mechanisms were identified on the dorsal side of the foot, one being a reflex and the other a local vasodilatation probably due to direct stimulation of (afferent?) unmyelin+ ated axons (Blumberg, H. and Wallin, B.G., unpublished observations), Interestingly, in the sympathetic recordings, impulses from vasodilator fibres have never been identified. Future applications The microneurographic recording technique is well suited to studying the physiology and pathophysiology of the sympathetic nervous system in man. The method is safe and recordings cause only little discomfort. Probably, however, it is too complex for routine diagnostic purposes except in specially selected cases. Recently it has been introduced in several laboratories in Europe and the U S A and hopefully this will promote knowledge about sympathetic regulatory mechanisms in man. Acknowledgements The work of the authors is supported by the Swedish Medical Research Council, grant number B85-04X-03546-14A and the Swedish Society of Medical Sciences. Selected references I Hagbarth, K.-['. and Vallbo, A+ B. (1968) Acta Physiol. Scand. 74, 96-11)8 2 l)clius, W.. Hagbarth, K.-E.+ Hongell, A+ and Wallin, B . G . (1972) Acta PhysioL S~and. 84.65 81 3 l)clius, W., Hagbarth, K+E., Hongell, A, and Wallin. B G. (19721 Acta Physiol

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T I N S - February 1986 &rand. 84, 82-94 4 Hagbarth, K.-E., Hallin, R. G., Hongell, A., Torebj6rk, H.E. and Wallin, B.G. (1972) Acta Physiol. Scand. 84, 164-176 5 Defius, W., Hagbarth, K.-E., Hongell, A. and Wallin, B.G. (1972) Acta Physiol. Scand. 84, 177-186 6 Sundl6f, G. and Wallin, B.G. (1978) J. Physiol. (London) 274, 621--637 7 WaUin, B. G., Delius, W. and Sundl6f, G. (1974) Seand. J. Clin. Lab. Invest. 34, 293-

3OO 8 Wallin, B. G., Sundl6f, G. and Delius, W. (1975) Pflr'gers Arch. 358, 101-110 9 Fagius, J., Wallin, B.G., Sundl6f, G . , Nerhed, C. and Englesson, S. (1985) Brain 108, 42.3--438 l0 B/lth, E., Lindblad, L.-E. and Wallin, B. G. (1981) J. Physiol. (London) 311,551-564 11 Wallin, B. G. and Eckberg, D. L. (1982) Am. J. Physiol. 242, HI85-H190 12 Sundl6f, G. and Wallin, B.G. (1978) J. Physiol. (London) 278, 525-532 13 Burke, D., Sundl6f, G. and Wallin, B. G. (1977) I. Physiol. (London) 272, 399--414 14 Sundl6f, G. and Wallin, B.G. (1977) J. Physiol. (London) 272, 383-397 15 Fagius, J. and Wallin, B. G. (1983) Brain 106, 589--600 16 Wallin, B. G. and Sundl6f, G. (1979) Hypertension 1, 67-77 17 Svedenhag, J., Wallin, B. G., Sundl6f, G. and Hendksson, J. (1984) Acta Physiol. Scand. 120, 499-504 18 Fagius, J. and Wallin, B.G. (1980) J. Neurol. Sci. 47, 433--448

19 Wallin, B. G. and K6nig, U. (1976) Brain Res. 103, 157-160 20 Bini, G., Hagbarth, K.-E., Hynninen, P. and Wanin, B. G. (1980) J. Physiol. (London) 306, 537-552 21 Bini, G., Hagbarth, K.-E., Hynninen, P. and Wallin, B. G. (1980) J. Physiol. (London) 306, 553-565 22 Bini, G., Hagbarth, K.-E. and WaUin, B. G. (1981) J. Auton. Nerv. Syst. 4, 17-24 23 Esler, M., Jennings, G., Leonard, P., Sacharias, N., Burke, F., Johnns, J. and Blombery, P. (1984) Acta Physiol. Scand. Suppl 527, 11-16 24 Lake, C. R., Ziegler, M. G. and Kopin, I. J. (1976) Life Sci. 18, 1315-1326 25 Wallin, B.G., Sundl6f, G., Eriksson, B.-M., Dominiak, P., Grobecker, H. and Lindblad, L.E. (1984) Acta Physiol. Scand. 111, 69-73 26 M6rlin, C., Wallin, B.G. and Eriksson, B.-M (1983) Acta Physiol. Scand. 119, 117-121 27 Hjemdahl, P., Freysehuss, U., Juhlin-Dannfelt, A. and Linde, B. (1984) Acta Physiol. Scand. Suppl 527, 25-29 28 Lind, A. R., Taylor, S. H., Humphreys, P. W., Kennelly, B. M. and Donald, K. W. (1964) Clin. Sci. 27, 229-244 29 Mark, A. L., Victor, R. G., Nerhed, C. and Wallin, B. G. Circ. Res. (in press) 30 Fagius, J. and Blumberg, H. Cardiovasc. Res. (in press) 31 Victor, R. G., Leimbach, W. N., Wallin, B. G. and Mark, A. L. (1985) J. Am. Coll. Cardiol. 5, 415

32 Sundl6f, G., Wallin, B. G., Str6mgren, E. and Nerhed, C. (1983) Hypertension 5,749756 33 Wallin, B. G., Sundl6f, G., Str6mgren, E. and ,~berg, H. (1984) Hypertension 6, 557562 34 Wallin, B. G. and Frisk-Holmberg, M. (1981) Hypertension 3, 340-346 35 Fagius, J. and Wallin, B.G. (1980) J. Neurol. Sci. 47, 449--461 36 Fagius, J. (1982) Diabetologia 23,415-420 37 Stjernberg, L. and Wallin, B. G. (1983) J. Auton. Nerv. Syst. 7, 313-318 38 Wallin, B. G. and Stjernberg, L. (1984) Brain 107, 183-198 39 Wallin, B. G. and Sundl6f, G. (1982) J. Auton. Nerv. Syst. 6, 287-291 40 Wallin, B. G., Westerberg, C.-E. and Sundl6f, G. (1984) Neurology 34, 522-524 41 Wanin, B. G., Blumberg, H. and Hynninen, P. (1983) Neurosci. Lett. 36, 189-194 42 Wallin, B. G., Sundl6f, G. and Lindblad, L.-E. (1980) in Arterial Baroreceptors and Hypertension (Sleight, P., ed.), pp. 101-107, Oxford University Press

B. Gunnar Wallinis at the Department of Clinical Neurophysiology, University of G6teborg, Sahlgrenska Sjukhuset, S-413 45 G6teborg, Sweden. Jan Fagius is at the Department of Neurology, University of Uppsala, Akademiska Sjukhuset, S-75185 Uppsala, Sweden.

Quick-Freeze, deep-etch visualization of the axonal cytoskeleton Nobutaka Hirokawa The recently developed quick-freeze, freeze-fracture, deep-etch technique has revealed the existence, in unfixed axons, o f an extensive crosslinked system o f neurofilaments, microtubules, membrane organelles and axolemma. Using I D P N intoxicated axons, it has been shown that membrane organelles conveyed by fast axonal f l o w are closely associated with microtubules, and crossbridges have been visualized both between microtubules and between membrane organelles and microtubules. Combined with antibody decoration, the quick-freeze technique has demonstrated that a 200 kDa polypeptide is a component of neurofilamentassociated crossbridges and that a microtubule-associated protein, M A P I , is a major component o f crossbridges between microtubules in axons. N e r v e cells d e v e l o p a c o m p l i c a t e d s h a p e with an intrinsic polarity d e t e r m i n e d by the direction o f i m p u l s e c o n d u c t i o n . I n o r d e r to c o n d u c t impulses, t h e a x o n m u s t utilize a w e a l t h o f different p r o t e i n s , but b e c a u s e p r o t e i n s are n o t s y n t h e s i z e d in t h e axon, all the material necessary for t h e axon a n d its s y n a p s e s m u s t be carried t h e r e by cytoplasmic axonal flow 1-3. R e c e n t l y m u c h interest has f o c u s e d o n t h e c y t o s k e l e t o n in relation to this p r o c e s s , a n d especially in relation to t h e fast flow that c o n v e y s m e m b r a n e organelles 4. T w e n t y years ago e l e c t r o n

microscopists f o u n d n e u r o f i l a m e n t s a n d m i c r o t u b u l e s to b e t h e cytoskeletal e l e m e n t s in the axon 5. Fuzzy filamentous materials b e t w e e n t h e s e cytoskeletal e l e m e n t s and b e t w e e n cytoskeletal e l e m e n t s a n d m e m b r a n e organelles w e r e also described. B a s e d o n t h e s e structural studies, several h y p o t h e s e s have b e e n p r o p o s e d for t h e m e c h a n i s m o f fast flow, such as t h e m i c r o s t r e a m m e c h a n i s m 7, the microtubule-dynein-like A T P a s e system 6'8, the n e u r o f i l a m e n t as a m o t o r hypothesis 9, the a c t o m y o s i n - b a s e d transp o r t i n g filament t h e o r y ~°, and r e c e n t

m i c r o t r a b e e u l a r lattice t h e o r i e s n . A p r e r e q u i s i t e o f c o n v e n t i o n a l elect r o n m i c r o s c o p y is that s a m p l e s b e chemically fixed a n d d e h y d r a t e d before o b s e r v a t i o n . H o w e v e r , it has b e e n p o i n t e d out that osmification could affect actin filaments 12 a n d that deh y d r a t i o n could cause distortion o f cytoskeletal e l e m e n t s 13. T h e r e f o r e , t h e r e is a possibility that very fine structures such as crossbridges a m o n g cytoskeletal e l e m e n t s a n d m e m b r a n e organelles are artifacts c a u s e d by chemical fixation o r d e h y d r a t i o n .

Three-dimensional images of the unfixed axonai cytoskeleton O n e o f t h e m a i n objectives o f structural biologists is to o b s e r v e cellular structures in living cells as precisely as possible. T h e recently d e v e l o p e d quick-freeze, d e e p - e t c h t e c h n i q u e e n a b l e s us to o b s e r v e not only t h e t h r e e - d i m e n s i o n a l architecture o f cytoskeletal e l e m e n t s , but also the true i n n e r a n d o u t e r surface o f cell

~) 1986. Elsevier Science Publishers B.V., Amsterdam

0378 - 5912/86/$02.00