J. Cell Sci. 2, 573-586 (1967)Printed in Great Britain

AN ELECTRON-MICROSCOPE

STUDY OF SUBCELLULAR FRACTIONS

OF OCTOPUS BRAIN

D. G.JONES*Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge, andDepartment of Anatomy, University College, London

SUMMARY

Using subcellular fractionation techniques the primary fractions (P1; P2 and P3) have beenprepared from homogenates of the supraoesophageal lobes (brain) of Octopus vulgaris. A mor-phological study of the fractions was made, particular emphasis being placed upon the synapto-somes (nerve-ending particles), and the agranular and granular vesicles they contain. Theacetylcholine levels in the fractions were determined by bioassay.

Synaptosomes and isolated mitochondria are present in all the fractions examined. In addi-tion, the remains of blood vessels are found in P1( nuclei, vesicular structures and lamellatedbodies in Px and P2, and microsomes in P3 and the final supernatant. The synaptosomes aresimilar to those isolated from mammalian nervous tissue. The remains of the post-synapticendings are seen as recognizable processes around their periphery. They are more extensivethan the post-synaptic membranes of mammals, and thickenings on them are seen in tissuestained with phosphotungstic acid. The number of mitochondria outside synaptosomes varieswith the fixative used, being far greater with formalin than with permanganate. The agranularvesicles vary in diameter from 150 to 1200 A; over 70 % of them are between 250 and 500 A.In formalin-fixed preparations, the synaptosomes can be separated into two types: those con-taining mainly rounded vesicles, and those with many ellipsoidal vesicles. About 4 % of all thevesicles are granular; they have an average diameter of 720 A, with few of them measuringover 1000 A.

Acetylcholine is distributed throughout the fractions, the highest level being in P2, withappreciable levels in the others. The total recovered for the combined fractions of the supra-oesophageal lobes was 104-6 m/«nole/g tissue. For the combined fractions of the optic lobes,which are also rich in synaptosomes, the total was 32-3 m/tmole/g. tissue.

INTRODUCTION

Since the development of techniques for the isolation of nerve endings from braintissue (Whittaker, 1959; Gray & Whittaker, i960, 1962; De Robertis, Pellegrino deIraldi, Rodriguez de Lores Arnaiz & Salganicoff, 1962), many studies of differentregions of the brain in mammals have been undertaken. The aim of such work hasbeen, on the one hand to obtain as pure fractions as possible of the constituents of thenerve endings, and on the other to investigate the subcellular distribution of biogenicamines and of the enzymes involved in amine metabolism (see Whittaker, 1965).From this work it has become clear that there is a preferential localization of boundacetylcholine within the preparations derived from presynaptic nerve terminals (Gray

* Present address: Department of Anatomy, University College London, Gower Street,London, W.C.i.

37 Cell Sci. 2

574 D. G. Jones

& Whittaker, 1962; De Robertis et al. 1962) and synaptic vesicles (Whittaker, Michael-son & Kirkland, 1964; Whittaker & Sheridan, 1965; De Robertis, Rodriguez de LoresArnaiz, Salganicoff, Pellegrino de Iraldi & Zieher, 1963). However, as these prepara-tions have been derived from mixed populations of cholinergic and non-cholinergicneurons, their acetylcholine content is relatively low, being of the order of 12 m/tmole/gin the homogenates of guinea-pig (Gray & Whittaker, 1962) and rat (De Robertiset al. 1962) brains.

The cephalopod central nervous system has been known to be rich in acetyl-choline (ACh) since the work of Bacq (1935), who found 77 /ig (550 m/tmoles) ofACh/g of fresh cerebral ganglia in Octopus vulgaris. Subsequent work on Octopus tissueby Corteggiani (1938), Florey (1963), and Loe & Florey (1966) has confirmed theusefulness of cephalopods as a source of acetylcholine-rich material. It was thought,therefore, that it might be of value to apply to the brain of Octopus the technique ofsubcellular fractionation which has proved so successful in the case of the vertebratecentral nervous system. Such a study has been facilitated by the electron-microscopicalinvestigations of the nervous system of cephalopods by a number of workers (Barber,1966; Barber & Graziadei, 1965, 1967; Dilly, Gray & Young, 1963; Gray & Young,1964; Graziadei, 1965; Tonosaki, 1965; Yamamoto, Tasaki, Sugawara & Tonosaki,1965; Zonana, 1961).

The present report describes the separation of homogenates of the supraoesophagealganglia of the brain of Octopus vulgaris into their primary subcellular fractions. Men-tion is also made of the results obtained when the optic ganglia alone are used. Themain emphasis is placed upon the morphological appearance of the fractions, and anattempt has been made to identify as closely as possible their constituents. As a resultof the opportunity afforded to observe large numbers of synaptic vesicles, particularattention has been paid to them. In addition, the levels of acetylcholine in the prepara-tions have been estimated.

MATERIALS AND METHODS

All the experiments were carried out on preparations made from the brain ofOctopus vulgaris. Except in one experiment when the optic lobes alone were used, thewhole of the supraoesophageal region of the brain was employed.

The fractionation procedures were based upon those described by Gray & Whittaker(1962), with modifications to meet the different tonicity requirements of Octopusmaterial (see Results). After being dissected out, the tissue was homogenized in eithero-8 M sucrose, or 07M sucrose containing 0-33 M urea, to give a 10 % (w/v) suspension.The homogenizer was of the type described by Aldridge, Emery & Street (i960), hav-ing an all-round clearance of 0-25 mm, with a pestle rotation of 840 rev/min. Tostandardize the conditions, the pestle was moved up and down 12 times for eachhomogenization, with an interval of 30 sec after the first 6 times. The suspension wascentrifuged for 11 min at 1000 g to give the Px fraction, which was washed by re-suspension in the original sucrose or sucrose-urea medium and centrifuged as before.The combined supernatants were centrifuged at 17000 g for 1 h, giving the P2 fraction.

Fractionation of Octopus brain 575

The supernatant was centrifuged for a further hour at 100 000 g. The resulting pelletand supernatant were the P3 and S3 fractions, respectively. All the above procedureswere carried out at 0-4 °C.

For electron microscopy each pellet was resuspended in about 2 ml of the sucroseor sucrose-urea medium. To the resulting suspension were added 5 volumes of ice-cold fixative. The following fixatives were used: (1) o-6% potassium permanganate,buffered with veronal-acetate (Luft, 1956); (2) o-6% potassium permanganate,buffered with veronal-acetate, to which was added ic-2 g/100 ml of sodium chloride,to increase its tonicity to I M (modified veronal-acetate buffer); (3) 1 % osmiumtetroxide, buffered with veronal-acetate; and (4) cacodylate-buffered 4 % paraform-aldehyde (Sabatini, Bensch & Barrnett, 1963), followed by post-fixation in phosphate-buffered 1 % osmium tetroxide. With potassium permanganate, fixation lasted for30 min. The most satisfactory results with paraformaldehyde and osmium tetroxidewere obtained using periods of 30-60 min in each solution. The fixed suspensionswere centrifuged, usually at 5000 g for 10 min, and the pellets dehydrated in ethanoland embedded in Araldite. Sections were examined with a Siemens Elmiskop 1electron microscope after staining on the grids with lead hydroxide (Millonig, 1961),lead citrate (Reynolds, 1963) or uranyl acetate followed by lead citrate. Some blockswere stained with phosphotungstic acid before embedding.

Acetylcholine was assayed on small (8 x 0-25 mm) slips of the dorsal muscle of theleech, mounted in a small organ bath of 0-05 ml capacity (Szerb, 1962), according tothe procedure of Whittaker et al. (1964). Acetylcholine was released from its 'bound'state in particulate fractions before assay by acidifying to pH 4 with 0-33 N HC1 andheating at 100 °C for 10 min.

RESULTS

Fractionation conditions

Work was initially directed towards finding a homogenization medium suitable forOctopus tissue. A sucrose concentration of 0-32 M, being considerably hypo-osmotic tosea water, resulted in material which was severely damaged. This was judged by thedegree of distortion of the mitochondrial cristae, and by the presence of mainlyempty profiles, which were probably derived from nerve endings. A small number ofvesicular structures, 350-500 A in diameter, were present within and between a few ofthese profiles.

The use of a homogenization medium with a final molarity of about 1 M sucrose(roughly iso-osmotic with sea water) proved more satisfactory, but had a rather highdensity and viscosity which retarded sedimentation of particulate material. Conse-quently, a medium consisting of o-y M sucrose plus 0-33 M urea was adopted. It waslater found that o#8 M sucrose alone gave equally good results from a morphologicalstandpoint. A disadvantage of employing media containing such a high molarity ofsucrose is that there is a high degree of sucrose contamination in negative staining.Although Sheridan, Whittaker & Israel (1966) were successful in negatively stainingfractions of the electric organ of Torpedo, which had been homogenized in 0-5 M

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576 D. G. Jones

sucrose containing 0-33 M urea, the negative staining of Octopus fractions homogenizedin media of o-8 M or higher has so far been unsuccessful.

Cottrell (1966) used I-I M glucose in preference to sucrose for the homogenizationof clam ganglia. This was not found to have any obvious advantages over the sucroseand sucrose-urea solutions with Octopus.

Attempts to separate the P2 fraction into subfractions on a sucrose density gradienthave so far proved inadequate. The procedures used (Gray & Whittaker, 1962;Whittaker et al. 1964) resulted in considerable loss of acetylcholine, while the pelletswere too small for electron-microscope examination.

Morphology of the primary fractions

Fraction Pv Free nuclei are present in large numbers in this fraction. Generallythey are complete, and have a nuclear envelope.

In common with the other primary fractions, the most prominent feature of thisfraction is the presence of structures (Fig. 5) having a single outer membrane, enclosingone or more mitochondria, synaptic vesicles and a few larger vesicles. Their appearanceis similar to that of the synaptosomes (nerve-ending particles) of fractionated mammal-ian brain tissue (Gray & Whittaker, 1962; Whittaker et al. 1964; Whittaker &Sheridan, 1965; De Robertis et al. 1962, 1963; Barondes, 1966; Israel & Whittaker,1965). These structures are therefore regarded as synaptosomes. Unlike some mam-malian synaptosomes, isolated post-synaptic membranes are only rarely encountered.Instead, what are presumed to be pinched-off and much more complete post-synapticprocesses (Figs. 5, 10) are sometimes seen adhering at a number of points around thevesicle-filled bags. In some synaptosomes the intervening gap is occupied by granularmaterial, suggesting that the close proximity of the two components is a reflection oftheir synaptic relationship to each other rather than of a chance association (see Gray,1959; Gray & Young, 1964; Barber & Graziadei, 1966&).

In this fraction and in the P2 fraction lamellated bodies (Fig. 6) are found bothwithin membrane-bound profiles and free, in permanganate-fixed material. Theappearance of individual bodies varies.

Fig. 7. illustrates part of a group of spherical membrane-bound bodies. Similarstructures are in the P2 fraction. Their mean diameter is approximately 1500 A.Many of the larger ones are up to 2000 A in diameter and internally contain one, twoor three spherical vesicles. Although such structures are found scattered throughout theprimary fractions, they are commonly grouped together in large numbers. They maybe derived from the pericytes of blood vessels, the processes of neurites, either axonsor other neuronal processes, or glia. Further vesicular structures found in the Pjfraction are shown in Fig. 8. Intermingled with large, clear vesicles are synaptosomesand dense, fibrous debris.

Portions of small blood vessels are preserved with varying degrees of integrity.Barber & Graziadei (1965, 1966a, 1967) have described the structure of blood vesselsin the retina, arms and perioesophageal nerve ganglia of Octopus. In general they arecomposed of an incomplete endothelium, a complete basement membrane and a

Fractionation of Octopus brain 577

complete investment of pericytes. In the fractionated material the endothelium andbasement membrane are seen, but the layer of pericytes has been lost.

The cell in Fig. 9 has a prominent nucleus with, in the cytoplasm, groups of freeribosomes, endoplasmic reticulum, mitochondria and dense bodies which may belysosomes. These cells are approximately 7 /< in diameter and have a nucleus 5 /t indiameter. They are similar to the amoebocytes of Octopus.

Isolated mitochondria are present in this fraction.Fraction P2. This fraction presents a number of similarities to the F± fraction.

Nuclei, vesicular structures, lamellated bodies, isolated mitochondria and synaptosomesare all found, the synaptosomes being the dominant feature of the fraction. Nuclei areless frequent than in the Px fraction. Collagen has not been seen in this, or in any of theother, fractions. The spherical bodies have much the same appearance as their counter-parts in the Px fraction.

Large numbers of synaptosomes are contained in this fraction. Fig. 10 shows one ofthese, enclosing synaptic vesicles, mitochondria and a tubule. Post-synaptic processesare distributed around its periphery, fitting into depressions in the surface membraneof the synaptosome and separated from it by a gap of 140-280 A which is filled with adarkly-staining granular material. Membrane thickenings are seen in the region ofsome synaptic junctions in material stained with phosphotungstic acid. Tubules areseen in a number of synaptosomes and resemble those described by Israel & Whit-taker (1965) and Whittaker (1966) in the large synaptosomes in the nuclear fractionfrom the cerebellar cortex of the guinea-pig, rat, cat and pigeon. They may be formedby the intracytoplasmic fusion of vesicles.

The synaptosomes show a wide variation in their appearance, depending uponthe number, size and nature of the vesicles within them (Figs. 11, 12 and 14from P2). Figs. 11 and 12 give some indication of this variation. Fig. 12 showsa profile (a) which contains no or few vesicles. It resembles the profiles tentativelyidentified as synaptosome ghosts by Whittaker & Sheridan (1965). Empty profilesare also present in fraction Sj of the electric organ of Torpedo (Sheridan et al. 1966).The interiors of some profiles are granular, with few if any vesicles (Fig. 11),others appear clear and enclose a number of vesicles (c, Figs. 11, 12), while yetothers have a more granular background and a larger number of vesicles some ofwhich are large and granular (d, Figs. 11, 12). Granular or dense-core vesicles (gv,Figs. 13, 14) occur infrequently, most of them being confined to a small number ofsynaptosomes. The black body in Fig. 14 has a post-synaptic process associated withit, suggesting that it is a synaptosome or is derived from one (see Gray & Whittaker,1962). Large, dark vesicles with an average diameter of 670 A occupy the ending inFig. 15. Synaptic bags containing granules have been isolated from the heart ofAchatinidae (Nisbet & Plummer, 1966).

Of these variations in the synaptosomes, the predominant localization of granularvesicles in certain endings and of more electron-opaque vesicles in others is aconstant feature. The synaptosomes containing granular vesicles may correspond tothe type 2 endings in the retinal plexus of Octopus vulgaris described by Tonosaki

(I965)-

578 D. G. Jones

Fraction Pz. Synaptosomes and isolated mitochondria are readily recognizable(Fig. 16). Isolated membranes are present, while one synaptosome contains granularvesicles. Small vesicles lying free are probably microsomes (Fig. 17).

Fraction S3. Like fraction P3 this supernatant consists essentially of synaptosomes,mitochondria and small vesicles presumed to be microsomes (Fig. 18).

Morphology of optic lobe fractions. Preliminary studies on the morphology of theprimary fractions of the optic lobes reveal a general similarity of appearance to thefractions of the combined supraoesophageal lobes. The P3 fraction however is distin-guished by a relative paucity of synaptosomes.

Distribution of mitochondria

In permanganate-fixed material the proportion of mitochondrial profiles lying out-side synaptosomes in the combined fractions is approximately 20%. This compareswith rather more than 50 % lying free in fractions fixed with formalin, and with 40 %for the mitochondria of the P2 fraction of guinea-pig cerebral cortex (unpublishedobservations on formalin-fixed material). These figures suggest an increased fragilityof synaptic membranes when fixed by means of an aldehyde.

Morphology of the agranular and granular vesicles

The two synaptosomes in Fig. 19 are from a P2 fraction fixed in formalin followedby oxmium tetroxide. In one, many of the vesicles are rounded, while in the other themajority have an ellipsoidal shape and measure approximately 230 by 600 A. Thesignificance of these findings is discussed below.

The synaptosomes (Figs. 5 and 10-16) contain both clear and dark agranular(synaptic) vesicles, and granular or dense-core vesicles. By far the most numerousvesicles are the clear, agranular ones, the diameters of which range from 150-1200 A.This is in accordance with other studies on various parts of Octopus nervous systemby Barber (1966), Barber & Graziadei (19666) and Graziadei (1965), who describedsize ranges of 300-1200 A, 300-800 A and 200-800 A, respectively. Further analysis ofthese results (Fig. 1) shows that approximately 70% of the vesicles are between250 and 500 A in diameter. This agrees quite well with the figures given by Gray &Young (1964) on the vertical lobe of Octopus, and by Dilly et al. (1963) on the opticlobes of Octopus. The agranular vesicles of the optic lobes have a similar distribution,so that these vesicles cannot be distinguished morphologically from those in the com-bined supraoesophageal lobes. The distribution of these vesicles in each of theindividual primary fractions of the supraoesophageal lobes is again similar.

About 4 % of the vesicles in the fractions are granular, having dense cores (Figs. 13,14 and 16). There is little variation in the percentage between the primary fractions.Examination of the optic lobe fractions reveals that less than 1 % of these vesicles havedense cores. This agrees with the observations of Dilly et al. (1963), who describedonly occasional dense-core vesicles in this lobe. The granular vesicles in the combinedprimary fractions of the supraoesophageal lobes have an average diameter of 720 A.Their size range is shown in Fig. 2. Previous reports on the sizes of granular vesiclesin Octopus tissue have given values ranging from 500 to 1500 A (Gray & Young,

Fractionation of Octopus brain 5 79

1964; Tonosaki, 1965). Granular vesicles of 570 to 1250 A diameters are seen in theoptic lobe fractions, with an average value of 810 A. However, there could be a sub-population of agranular vesicles of the same diameter as the granular ones (Figs. 1, 2).

Synaptosome sizes

Synaptosome profiles vary in diameter from o-i to 3-2 [i. Although the meandiameter is greatest in the P2 fraction and lowest in S3, there is considerable overlapin the sizes of individual profiles within each fraction. The size ranges of the profilesin the combined primary fractions of the supraoesophageal lobes taken together andof the isolated optic lobes are shown in Fig. 3. The mean diameter of the synaptosomeprofiles is 0-93 fi (516 measured) in the supraoesophageal lobes, against 0-62 /* (578measured) in the optic lobes. These compare with a value of approximately 0-5 /i for

40 -

400 1000 1200600 800Diameter, A

Fig. 1. Distribution of profile diameters of 2377 agranular vesicles in the combinedPi, P2 and P3 fractions of the supraoesophageal lobes.

40 -

30

§ 20

10

200 800 1000400 600Diameter, A

Fig. 2. Distribution of profile diameters of 102 granular vesicles in the combinedP2 and P3 fractions of the supraoesophageal lobes.

580 D. G. Jones

the synaptosomes of the B fraction of the guinea-pig neocortex (Clementi, Whittaker &Sheridan, 1966), and with the large (2-5 /* diameter) synaptosomes which sediment inthe Px fraction of the cerebellar cortex of a number of mammalian species (Israel &Whittaker, 1965). The mean diameters calculated in this study may be on the lowside due to the difficulty encountered in deciding whether or not some of the smallervesicular structures are synaptosomes. Generally such smaller profiles have beenincluded in the results.

24

s§ 12

(a)

24

S 12

PL,

1-2Diameter, ft

2-4 1-2Diameter, fi

2-4

Fig. 3. Distribution of the synaptosome profile diameters in the combined Pu P2 andP3 fractions of (a) the supraoesophageal lobes, and (6) the optic lobes. The numbers ofprofiles counted were—516 and 578, respectively.

100 r

J3

u

50

100 r

U<

ooa

50

(b)

P, P2 P3 S , P3

Fig. 4. The subcellular distribution of acetylcholine in fractions of (a) the combinedsupraoesophageal lobes, and (b) the optic lobes.

Subcellular distribution of acetylcholine

The results of the assays are shown in Fig. 4. From this it can be seen that, inspite of the absence of an anticholinesterase, considerable amounts of acetylcholine(presumably in the bound form) were recovered from the various fractions. Theamount of acetylcholine in the optic lobes is considerably less than that in the rest ofthe brain. In each case the highest level of acetylcholine is in the P2 fraction, althoughsignificant amounts are present also in the other fractions.

The total acetylcholine recovered from the fractions of the supraoesophageal region

Fractionation of Octopus brain 581

of the brain is 104-6 m/imole/g tissue, and is lower than the amounts recovered byother workers from whole brain of Octopus (Bacq, 1935, in Octopus vulgaris; Florey,1963, and Loe&Florey, 1966, in Octopus dofleini). The corresponding amount of acetyl-choline recovered from the combined fractions of the optic lobes is 32-3 m/imole/gtissue, which is again lower than Bacq's figures for these lobes (Bacq, 1935). Theselower values might be due to the different extraction procedures used and failure toinactivate completely by the acid/heat extraction method the large amounts of cholin-esterase present in the tissue.

DISCUSSION

The fractionation procedures designed for use with mammalian brain tissue (Gray& Whittaker, 1962) have proved useful in dealing with Octopus nervous tissue. Afterappropriate modifications for the differing tonicity requirements of Octopus tissue,synaptosomes have been obtained in a good state of preservation. They are distributedthroughout all the primary fractions, unlike much mammalian brain tissue wherethey are largely restricted to the P2 fraction. This may be due to the greater size rangeof synaptosomes in the fractions of Octopus brain.

An adherent fragment of post-synaptic membrane is not found. Instead the post-synaptic process seems to remain much more nearly intact compared with mammalianbrain tissue, excepting the large synaptosomes of the cerebellar cortex (Whittaker,1965). Many such post-synaptic processes are small and closely related to synapto-somes. On other occasions there appears to be a close relationship between two or morelarge endings. These may be examples of chance close associations between pre-synaptic endings, or they may represent both pre- and post-synaptic endings. If thelatter accounts for any of these observations, the term ' synaptosome' (Whittaker et al.1964; Whittaker, 1965) should be extended to cover structures containing morecomplete post-synaptic components when these are present.

Cylindrical or ellipsoidal-shaped vesicles have been described in a number ofsituations, namely, cat cerebellum (Uchizono, 1965), cat spinal cord (Uchizono, 1966a),cat inferior olive (Walberg, 1965), rat olfactory cortex and superior colliculus (Lund &Westrum, 1966), goldfish brain (Robertson, Bodenheimer & Stage, 1963) and cray-fish stretch receptor muscle (Uchizono, 19666). The appearance of flattened vesiclesin a proportion of endings is seen only when formalin is used as the primary fixative.Uchizono (1965) concluded that the synapses containing rounded vesicles have anexcitatory function, in contrast to those with ellipsoidal vesicles which are inhibitoryin nature. The finding of ellipsoidal vesicles in a proportion of synaptosomes informalin-fixed fractions of Octopus brain is interesting, as it suggests two sorts ofnerve endings which might be excitatory and inhibitory, and so similar to those invertebrate tissue. Furthermore, in regard to their content of synaptic vesicles, it pro-vides further evidence for the correspondence of synaptosomes to intact nerve endings.It follows that if excitatory and inhibitory nerve endings can be distinguished morpho-logically, so can excitatory and inhibitory synaptosomes.

As yet little attention has been paid to the appearance of vesicles in formalin-fixed

582 D. G. Jones

whole tissue of Octopus. Although ellipsoidal vesicles have been noted in this tissue(E. G. Gray, personal communication), there are very few, and it is unlikely that twodistinct populations of vesicles are present in the lobes examined (optic and vertical).Possibly, in Octopus at least, the tissue must first be damaged—in this instance byhomogenization—before the factors required to produce ellipsoidal vesicles can operate.

If the granular vesicles are similar to the granular vesicles at vertebrate autonomicendings the numbers seen should be a fairly accurate estimate of the number present,as permanganate, which was used as a fixative, is thought to give good preservationof these vesicles (Richardson, 1966). The distribution frequency of the diameters ofthe granular vesicles (Fig. 2) indicates that only one population of these vesicles ispresent in the fractions of the supraoesophageal lobes. Gray & Young (1964), in thevertical and superior frontal lobes of Octopus, distinguished between dense-corevesicles of about 500 A diameter and those with a diameter of 1000-1500 A. In thepresent study, 2 % of the granular vesicles are more than 1000 A in diameter, and it isunlikely that these constitute a population distinct from that of the smaller granularvesicles. It is also unlikely that the granular vesicles are neurosecretory, as they aremainly smaller than the granular vesicles of neurosecretory tissues (Palay, 1957; Holmes,1964; Barber, 1967), while the regions examined do not correspond to those regions sofar identified as neurosecretory in Octopus (Alexandrowicz, 1964; Young, 1965).

In attempting to assess the significance of the levels of acetylcholine two factors areimportant. Large quantities of acetylcholine are present in each of the fractionsanalysed, these in turn being characterized at the electron-microscopic level by con-siderable numbers of synaptosomes. In the light of these findings it would seemreasonable to assume that the acetylcholine is located in at least some of these synapto-somes. Furthermore, there appears to be a correspondence between the numbers ofsynaptosomes and the level of acetylcholine. If this is the case most, or even all, of thesynaptosomes may contain acetylcholine. A similar conclusion was reached by Loe &Florey (1966), when they postulated that the distinction between cholinergic and non-cholinergic neurons in Octopus lies in the relative concentrations of acetylcholinecontained in each of them. The preparation of pure fractions of synaptosomes isrequired in order to test this hypothesis. Although the data presented do not excludethe possibility that acetylcholine is associated with tissue components other thansynaptosomes, they indicate that acetylcholine is largely confined to the nerve endings.This confirms the results of the biochemical work on Octopus dofleini by Loe & Florey(1966).

In spite of the high levels of acetylcholine compared with mammalian tissues, thereis no reason to suppose it is localized outside cholinergic neurons, since the number ofnerve cells which may be cholinergic is relatively small in mammals compared withOctopus.

The Octopus brain is exceedingly rich in synaptic contacts, and its fractions are veryrich in synaptosomes. It is therefore good tissue for the isolation of nerve endings, andlends itself to further fractionation studies.

The isolation of nerve endings from Octopus dofleini has been very recently reportedby Florey & Winesdorfer (1967). The homogenates contained 350 fig of ACh/g wet wt.

Fractionation of Octopus brain 583

I am grateful to Dr V. P. Whittaker for accommodation in his laboratory and for discussion.The work was supported in part by a U.S. Public Health Service Grant. I wish to thankProfessors J. Z. Young, F.R.S. and E. G. Gray for discussion and Miss L. Swales and Mr S.Waterman for technical assistance.

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{Received 9 May 1967)

For plates see overleaf.

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Fig. 5. Px fraction. Synaptosomes showing synaptic vesicles (sv), mitochondria (mit) andlarger vesicles (Iv). Post-synaptic processes (p) are separated from the outer membrane(m) of the synaptosome by a cleft (210-280 A wide) containing granular material. Thesynaptosomes are closely associated, the gap between them varying from 70 to 420 Ain width. Permanganate fixation.Fig. 6. P2 fraction. A lamellated body (Ib) within a membrane-bound structure.Permanganate fixation.Fig. 7. Pt fraction. A group of vesicular structures, amongst which are some havingan internal structure (vis). Permanganate fixation.Fig. 8. Pj fraction, showing vesicular structures, synaptosomes (s) and fibrous debris(fd). Permanganate fixation, using modified veronal-acetate buffer.

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Fig. 9. PL fraction. An amoebocyte, with its nucleus (n), endoplasmic reticulum (er),free ribosomes (r), mitochondria (wit) and lysosomes (/). Formalin and osmiumtetroxide fixation.Fig. 10. P2 fraction. The features of this synaptosome are similar to those of thesynaptosome from Pt shown in Fig. 5. Note also the tubule (t). (Iv, larger vesicles,m, membrane, p, post-synaptic process, sv, synaptic vesicle.) Permanganate fixation.Fig. 11. P2 fraction. Synaptosomes of various morphological appearances. For discus-sion, see text. Permanganate fixation.

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Fig. 12. P2 fraction. Further examples of synaptosomes; for discussion, see text.Permanganate fixation.Fig. 13. Px fraction. A synaptosome containing granular vesicles (gv). Permanganatefixation.Fig. 14. P2 fraction. Synaptosomes (s), an isolated mitochondrion (writ) and a blackbody (bb) are seen. Granular vesicles (gv) are present in one of the synaptosomes. Apost-synaptic process (p) is associated with the black body. Permanganate fixation.Fig. 15. Pi fraction. A synaptosome filled with large dark vesicles. Formalin and osmiumtetroxide fixation.

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Fig. 16. P3 fraction. Synaptosomes (s), isolated mitochondria (mit) and isolatedmembranes (mem) can be seen. Granular vesicles (gv) are present in one synaptosome.Permanganate fixation.Fig. 17. P3 fraction. Small vesicles which may be microsomes (mic) are shown. Per-manganate fixation, using modified veronal-acetate buffer.Fig. 18. S3 fraction. Synaptosomes (s), microsomes (mic) and an isolated mitochondrion(mit) are present. Permanganate fixation.Fig. 19. P2 fraction. Two synaptosomes, of which one (x) contains mainly roundedvesicles, while the other (y) has many ellipsoidal vesicles. Formalin and osmiumtetroxide fixation.

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