Comparative studies in synaptosome formation: Preparation of synaptosomes from the ventral nerve cord of the lobster (Homarus americanus)

Brain Research, 101 (1976) 103-111 (© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

103

C O M P A R A T I V E STUDIES IN S Y N A P T O S O M E F O R M A T I O N : P R E P A R A TION OF S Y N A P T O S O M E S F R O M T H E V E N T R A L N E R V E C O R D OF THE LOBSTER ( H O M A R U S A M E R I C A N U S )

R, F. NEWKIRK*, E. W. BALLOU**, G. VICKERS*** ANDV. P. WHITTAKER*** Marine BiologiealLaboratory, WoodsHole, Mass. 02543 (U.S.A.) (Accepted June 27th, 1975)

SUMMARY

A flotation method for preparing synaptosomes, previously developed for work with squid nervous tissue, has now been successfully applied to the ventral nerve cord of lobster. Perhaps due to the greater content of connective tissue, homogenization of the lobster nerve cord was more difficult than with squid optic lobes and the yield of synaptosomes was lower. The synaptosomes fraction showed a 3.8-fold enrichment of bound acetylcholine relative to the homogenate and was almost 10 times richer in acetylcholine than a guinea pig cerebral cortical synaptosome fraction. The lobster synaptosomes accumulated choline rapidly when incubated at room temperature in sea water, and showed a high degree of occlusion of lactate dehydrogenase, thus confirming that they are sealed structures. The lobster can thus be added to the wide range of species from whose nervous systems synaptosomes can be isolated, and merits further study as a possibly rich source of cholinergic synaptosomes.

1N T R O D U C T I O N

Apart from work with cephalopods (octopusT, 9 and squid 6) very little work has been done on synaptosome formation in invertebrates. Yet the ventral nerve cord of lobster has been reported to have relatively high levels of acetylcholine 1°,12, and the sensory input into the ventral ganglia is now believed to be largely cholinergic 2,s. Thus many, perhaps most, of the nerve terminals in the ventral ganglia must be

* Life ScienceDepartment, Virginia State College, Petersburg, Va. 23803, U.S.A. ** Department of Physiology,Washington University, St. Louis, Mo. 63117, U.S.A. *** Abteilung f/_ir Neurochemie, Max-Planck-Institut for biophysikalische Chemie, D-3400 GSttin~ gen-Nikolausberg, Postfach 968, G.F.R..

104 cholinergic. Homogenates of the ventral cord are rich in bound acetylcholinc ~z >,tiggesting synaptosome formation, and such acetylcholine is reported to bc released b\ K + (ref. 12), a property of mammalian synaptosomes, at least under metabolizing conditions a One difficulty in working with marine invertebrates is the relatively high osmotic pressure of their body fluids. In the case of squid optic ganglia, homogenization in 0.7 M sucrose (though somewhat hypo-osmotic) was compatible with the preservation of much of the acetylcholine of the tissue in bound form and centrifugation of the homogenate (or of the low-speed supernatant prepared from it) at intermediate g-forces led to the formation of a floating pellicle highly enriched in cholinergic nerve terminals~L We have now attempted to apply this fractionation scheme to homogenates of lobster nerve cord in 0.8 M sucrose. The nerve cord, even when linely chopped, was much more difficult to homogenize than optic ganglia of squid, but the homogenate did yield a floating pellicle in small yield which was rich in synaptosomes and in bound acetylcholine. This preparation avidly took up choline; although not as rich in cholinergic terminals as squid preparations, it should be useful in investigating cholinergic function in the lobster. METHODS

Homogenization and j'ractionation The head ganglion and thoracic chain were dissected from lobsters anesthetized by chilling in ice, finely chopped and homogenized in 0.8 M sucrose to give an approximately 1 0 ~ w/v homogenate. Usually the ganglia from 4 lobsters were combined giving a mean weight (rag) of tissue in each experiment of 947 ~ S.E.M. 35 (3). Homogenization and fractionation conditions were approximately as previously described for squid 6. A pellet was collected at 1000 x g (all g values are averages) for 10 min and resuspended in 0.8 M sucrose for assay (fraction P1). The supernatant was centrifuged at 10,000 -," g for 30 min to give a pellet, a relatively clear zone (fraction S~) and a floating pellicle. Pellet and pelticle were separated as cleanly as possible and resuspended in 0.8 M sucrose to give fractions PzH and P2L respectively. In two experiments, the supernatant from the low-speed spin was centrifuged at 17,000 >,~ g for 60 rain. The pellicle and pellet so obtained are designated P2H' and P2L'. The volume of the particulate fractions equivalent to 1 g of tissue ranged from l to 8 ml/g of original tissue, but was usually around 3 ml/g.

Assays Lactate dehydrogenase was measured spectroscopically by observing the change in extinction at 340 nm when N A D H is oxidized by pyruvate, and fumarase by the change in extinction at 250 nm when the elements of water are removed from imalate. Total tissue acetylcholine was measured by microbioassay on a thin slip of leech muscle after extraction with trichloroacetic acid according to a standard method 11. Acetylcholine present in fractions was released by acidifying them to p H 4.0 with 0.5 M HCI and heating them at 100 °C for 10 rain before assay. Protein

5 3

2

3

Fumarase

Acetylcholine

Experiments

No. of

Protein Lactate d e h y d r o g e n a s e

Component

nmol

AE2~o/min

mg AE3a0/min

Units ( U)

44 ~ 12

0.28 ± 0.19

70 ± 13 1.58 ± 0.43

(U/g)

Homogenate

26 ± 11 9 ~ 4 (0.34) 21 ± 2 (0.80) 21 ± 2 (0.80)

P1 11 ± 4 4 ± 1 (0.36) 27 ~ 8 (2.45) 18 ~ 7 (1.54)

P2H

5 ± 1 3 ± 1 (0.60) 7 ~ 3 (1.40) 18 ~ 0 (3.80)

P2L

58 ~ 4 84 ± 9 (1.45) 45 ± 2 (0.77) 43 : 6 (0.74)

$2

Distributionof component (as °o of recovered)

14 2

53 :

11

142 ± 100

59 ~ 116 ±

Recovery (% of homogenate)

Values are m e a n s :~ r a n g e (2 experiments) or S.E.M. (3 or more). Values in p a r e n t h e s e s are relative specific c o n c e n t r a t i o n s (i.e. t h e a m o u n t of c o m p o nent, as ~ o f that recovered, in each fraction divided by the a m o u n t o f protein, as ~ o f that recovered, in that fraction) a n d relate to the line above. Fractions P2L a n d P~H were prepared u s i n g a n integrated centrifugal field o f 300,000 :J g" rain.

DISTRIBUTION OF PROTEIN~ LACTATE DEHYDROGENASE, FUMARASE AND ACETYLCHOLINE IN SUBCELLULAR FRACTIONS OF LOBSTER NERVE CORD

TABLE I

106 TABLI= I1 EFFECT OF IN('R|'.ASED ('[NTRIFLJ(jAI_. I:II:LD ()N D I S T R I B U T I O N

OF P R O T E I N A N [ ) A ( l : r ' l I.( ttf)I.IN[

IN ~4i|]-

('T-LLI.!LAR FRA('T1ONS OF IA)BSTI R N E R V E ('OR I)

Values arc means of two experiments tegrated centrifugal field of about 10~

range. Fractions P2L' and P,)H" were prepared using an i~e ' rain.

Component

Unit (U)

Homogenate Distribution of component (as % of recovered) ( U/Z of . . . . . . . . . . . . . . . . P~ P2H' P2L" $2' l i,~ ~'llC ;

Recover.v (as % ~([ ht;nlogenate)

Protein Acetylcholine

mg nmole

22 25

46 50

5 5

22 7 11 ~ 4 (0.50)

16 i 9 15 :i 6 (0.94t

17 7 38 ! 11 (2.24)

45 23 36 i I (0.80}

I 19

was d e t e r m i n e d by the m e t h o d o f L o w r y a n d is e x p r e s s e d in t e r m s o f a b o v i n e p l a s m a a l b u m i n s t a n d a r d . Full details o f the m e t h o d s h a v e b e e n given in a p r e v i o u s public a t i o n 13. Electron m i c r o s c o p y P e l l e t t e d f r a c t i o n s were fixed o v e r n i g h t in c o l d 2.5~o g l u t a r a l d e h y d e - l . 0

M

Fig. 1. Electron micrograph of a representative portion of P2L' showing synaptosomes (thick arrowsL with internal synaptic vesicles and mitochondrion, contaminated with membranous sacs (thin arrows). Magnification ~< 42,000.

107

Fig. 2. Electron micrograph of a portion of P2H' containing several synaptosome profiles. Note also the presence of free mitochondria (thick arrows) and cellular debris. Magnification ~'~ 13,500. Fig. 3. Electron micrograph of another portion of P2H' showing numelous vesicular membrane fragments, often with cytoplasmic inclusions (arrows). Magnification × 21,000.

108 CaC12-0.1 M collidine buffer, pH 7.4, rinsed several times in collidine buffet and post-fixed for 2 h in 27,',; aqueous osmium tetroxide. Dehydration was achieved in graded ethanols; preparations were then transferred via propylene oxide to gpon 812 for embedding.

Choline uptake Choline uptake was measured at 10/tM choline concentration as previously described for squid synaptosomes ~'.

RESULTS

Characterization of fractions Table l shows the distribution (as a percentage of recovered protein or activity) of the various markers in the fractions. Acetylcholine, a marker for synaptosomes derived from cholinergic nerve terminals, is mainly recovered in fraction $2; nevertheless, the fraction with the highest relative specific concentration (RSC) is fraction P2L; this shows an almost 4-fold enrichment of the transmitter relative to the original homogenate. Fraction P2H also has a relative specific concentration of acetylcholine greater than unity showing some enrichment of this fraction also. When the pellicle and pellet fractions were prepared in a more intense integrated field (17,1300 :~ g 60 rain) (Table ll) higher proportions of protein and acetylcholine were recovered in the peilicle and pellet fractions (P2L' and P2H'), but the relative specific concentration of acetylcholine in the pellicle, while still showing considerable enrichment, was lower than before. Total tissue acetylcholine, determined by the trichloracetic acid extraction method, was 61 :~ S.E.M. 30 (4) nmole/g, i.e. about 70~o of the total tissue acetylcholine remains bound under our conditions. Electron microscopy (Figs. 1-3) showed that both fraction P,~L' and P.~H' contained numerous synaptosomes. Neither fraction was homogeneous, but whereas fraction P2L' contained as its main contaminant large featureless membranous sacs, P2H' contained in addition free mitochondria and numerous membrane-bound bodies with cytoplasmic inclusions. The latter might have been derived from dendrites, axons or glial cell processes. The activity of fumarase (Table l), a reputed mitochondrial marker, was low in the homogenate and all fractions; however, the fraction with the highest RSC was fraction P2H. The presence of free mitochondria in this fraction, as in the corresponding fraction from squid 6, as well as synaptosomal mitochondria, was confirmed by electron microscopy (Fig. 2). However the RSC of fraction P2L was also above 1.0, which may be due to the intrasynaptosomal mitochondria in this fraction. Lactate dehydrogenase, as would be expected of a soluble cytoplasmic marker, is recovered mainly in fraction S,), which also has the highest RSC with respect to this component.

Choline uptake The uptake of choline by fractions PzL and P2L' was linear lbr 30 min and is

109 TABLE IlI CHOLINE U P T A K E BY SYNAPTOSOME PREPARATIONS FROM LOBSTER NERVE C O R D

Species

Lobster Squid (ref. 5) Guinea pig***

Fraction

P2L PzL" P2L B

Uptake of ,;r3H]choline* pmole/min/g of tissue

pmole/min/mg of protein

297 361"* 12,000 610

145 210"* 600 40

Choline uptake (pmole/ min) -- acetylcholine content (nmole) 71 76 40 138

* The uptake of choline was measured at 10/~M (** 1 i~M) choline concentration as the difference between the uptake at 24 26 °C and that at 0 ~C during the first I0 rain of incubation. *** M. J. Dowdall, personal communication;results for 37 cC.

given in Table i l l . It is calculated in two ways, per vol. of fraction equivalent to l g of tissue (column 3) or per mg of p r o t e i n ( c o l u m n 4). The uptake is lower t h a n in squid (compare line 1 with line 3) but not so m u c h lower when the latter basis of c o m p a r i s o n , which takes into a c c o u n t the lower yield of the fraction in the lobster, is adopted. F u r t h e r m o r e , the differences in rate of uptake of choline parallel the differences in acetylcholine c o n t e n t o f the two fractions ( c o l u m n 5). This suggests that the uptake o f choline is a property of the cholinergic synaptosomes i n the two fractions a n d that the synaptosomes in the fraction derived from lobster are as well, or better sealed t h a n those in the fraction from squid. The uptake of choline was inhibited 93 ~,, by i n c u b a t i o n at 0 °C a n d 96 ~ by h y p o - o s m o t i c shock.

TABLE IV COMPARISON OF ACETYLCHOLINE C O N T E N T OF V A R I O U S SYNAPTOSOME F R A C T I O N S

Species

Tissue

Synaptosome fraction

Acetylcholine concen- Source tration (nmole/mg of protein)

Squid Lobster

optic ganglia nerve cord

P2L P~L

13.52 2.03

P2L'

2.76

olfactory lobe P2L

0.25

Dogfish

Guinea pig cerebral cortex B Theoretical for cholinergic synaptosomes*

0.29 25-31

calcd, from Table 3, ref. 6 calcd, from Table I, this paper calcd, from Table II, this paper unpublished result of Simon and Whittaker calcd, from Table II, ref.l

* Calculated (calcd.) on assumption that guinea-pig cortex homogenates contain (per g) 90 mg of protein, 12-15 nmole of acetylcholine (ref. 1) and 3.88 × 1011synaptosomes of mean radius 0.56/tin (ref. 4) of which 15 ~ are cholinergic (ref. 14) and that protein content of synaptosomes is the same as that of whole tissue.

110 DISCUSSION

Lobster nerve cord is clearly a much less suitable source of cholinergic synaptosomes than squid optic ganglia. The yield of nervous tissue per animal is much lower and the ganglia contain so much connective tissue that homogenization is difficult. Recoveries of protein and acetylcholine were often low, suggesting that the fairly drastic chopping and homogenization conditions were not optimal. Furthermore, considerable amounts of acetylcholine and fumarase were recovered in fraction $2 suggesting that either separation of synaptosomes and mitochondria from the supernatant had not reached completion under the centrifugation conditions used or that these organelles had been in part further comminuted under the fairly rigorous homogenization conditions used. The reason for flotation of squid synaptosomes in 0.7 M sucrose is not fully understood; presumably well-sealed synaptosomes have a lower density than that of the surrounding medium, but if sealing is not as efficient in the lobster, some synaptosomes may equilibrate with the suspension medium and thus fail to float on centrifuging. A similar phenomenon has been observed in the dogfish (Simon and Whittaker, unpublished observation). Though the yield of the floating pellicle fraction was small (the fraction contained only 5-17 ~o of the total recovered protein of the fractions depending on how it was prepared) and its specific acetylcholine concentration was only about one-fifth to one-sixth of that of the corresponding fraction from squid (Table IV), nevertheless, the only organized structures present in it are synaptosomes and its specific acetylcholine corzcentration is considerably higher than that of synaptosomes from vertebrate brains (Table IV). It thus represents a reasonable enriched preparation of isolated cholinergic terminals, persumably derived from the sensory input of the ganglia. Improved fractionation methods, including softening the ganglia with trypsin or collagenase, could probably be devised. The lobster can thus be added to the wide range of species from whose nervous system it is possible to isolate synaptosomes and merits further study as a possibly rich source of cholinergic synaptosomes. ACKNOWLEDGEMENTS

This work was carried out as a project in the 1974 Neurobiology Course at the Marine Biological Laboratory, Woods Hole. Some results obtained by V. Clark, C. Cornwell, P. Huizar, S. Olson and G. Wheaton during the 1973 course have also been included. We are grateful to Miss F. Henderson and Frl. H. Ellermeier for the electron micrographs. REFERENCES 1 BARKER,L. A., DOWDALL, M. J., AND WHITTAKER,V. P., Choline metabolism in the cerebral cortex of guinea pigs: stable-bound acetylcholine,Biochem. J., 130 (1972) 1063-1080. 2 BARKER,D. L., HERBERT,E., HILDEBRAND,J. G., ANDKRAVITZ,E. A., Acetylcholineand lobster sensory neurones, J. Physiol. (Lond.), 226 (1972) 205-229. 3 BELLEROCHE,J. S. DE, AND BRADFORD,H. F., The stimulus-induced release of actylcholine from synaptosome beds and its calcium dependence,J. Neurochem., 19 (1972) 1817-1819.

111 4 CLEMENTI, F., WHITTAKER, V. P., AND SHERIDAN, M. N., Th~ yield of synaptosomes from the cerebral cortex of guinea pigs estimated by a polystyrene bead 'tagging' procedure, Z. Zellforsch., 72 (1966) 126-138. 5 DOWDALL, M. J., AND SIMON, E. J., Comparative studies on synaptosomes: uptake of [N-Me-:~H] choline by synaptosomes from squid optic lobes, J. Neurochem., 21 (1973) 969-982. 6 DOWDALL, M. J., AND WHITTAKER, V. P., Comparative studies on synaptosome formation: the preparation of synaptosomes from the head ganglion of squid, Loliffo pealii, J. Neurochem., 20 (1973) 921-935. 7 FLOREY, E., AND WINESDORFER, F., Cholinergic nerve endings in octopus brain, J. Neurochern., 15 (1968) 169-177. 8 HILDEBRAND, J. G., TOWNSEL, J. G., AND KRAVI3Z, E. A., DiStlibution of acetylcholine, choline, choline acetyltransferase and acetylcholinesterase in regions and single identified axons of the lobster nervous system, J. Neurochem., 23 (1974) 951--963. 9 JONES, D. G., An electron-microscope study of subcellular fractions of Octopus brain, J. Cell Sci., 2 (1967) 573-586. 10 KEYL, M. J., M1CHAELSON, I. A., AND WHITTAKER, V. P., Physiologically active choline esters in certain marine gastropods and other invertebrates, J. Physiol. (Lond.), 139 (1951) 434 454. 11 MACINTOSH, F. C., AND PERRY, W. M. L., Biological estimation of acetylcholine, Meth. reed. Res., 3 (1950) 78-92. 12 SCHALLEK, W., Action of potassium on bound acetylcholine in lobster nerve cord, J. cell. comp. Physiol., 26 (1945) 15-24. 13 WHITTAKER, V. P., AND BARKER, L. A., The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. In R. FRIED (Ed.), Methods in Neurochemistry, Vol. 2, Marcel Dekker, New York, 1972, pp. 1-52. 14 WHITTAKER, V. P., AND SHERIDAN, M. N., The morphology and acetylcholine content of cerebral cortical synaptic vesicles, J. Neurochem., 12 (1965) 363 372.