Mammalian central nervous system axolemma: histochemical evidence for axonal plasma membrane origin of bovine and rat axolemma-enriched membrane fractions

MAMMALIAN AXOLEMMA:

CENTRAL

NERVOUS

HISTOCHEMICAL

PLASMA AND

EVIDENCE

MEMBRANE RAT

ORIGIN

OF

SYSTEM FOR

AXONAL

BOVINE

AXOLEMMA-ENRICHED

MEMBRANE

FRACTIONS

J. STANLEY, R. G. SAUL, M. G. HADFIELD and G. H. DEVRIES Department of Biochemistry and Neuropathology. Medical College of Virginia. Virginia Commonwealth University. Richmond. VA 23298. U.S.A. Abstract-Purified

myelinated axons were isolated from fresh bovme corpus callosum and rat braInstem and osmotically shocked to strip myelin and axolemma from axon filaments. Two axolemma-enriched fractions, myelin, and a pellet of myelin-free axons were harvested by discontinuous sucrose centrifugathe axolemma-enriched fraction wa\ tion of the shocked myelinated axons. By electron microscopy composed of trilaminar membrane vesicles (diameter 50.-1000 nm) and linear membrane fragment<. myelin fragments were seldom seen but some mitochondrial membrane contamination nas evldeni The classical myelin stain Luxol Fast Blue never counter-stained the myelin-free axons. but drcl stain myelin, myelinated axons, and to a lesser extent, both axolemmal fractions. The staining of the axolemma-enriched fraction was amorphous and seemed incommensurate with the level of mjelin contamination. Acetylcholinesterase staining of the axolemma-enriched fraction counterstained with Luxol Fast Blue revealed a number of acetylcholinesterase-positive membrane vesicles by light microscopy. Occasional enzyme-positive vesicles were also evident in the myelin while myelin-free axons showed vIrtualI> no Luxol Fast Blue stain but were heavily stained for the enzyme. Higher resolution histochemicai staining by electron microscopy confirmed the light microscopy, showing acetylcholinesterase rcuction product lining the axolemma of bovine and rat CNS neurons. Myelin. mitochondria. and other diverse membranes showed no reaction product. About 30”,, of bovme corpus callosal axons show4 some acetylcholinesterase staining but only 39,, of the total lengths of all the bovine CNS axolcmma showed reaction product. Positive acetylchoiinesterase staining could also be demonstrated in a small proportion of the bovine axolemma-enriched membranes, with reaction product lining the membrane fragments and captured within their vesicles. The percentage of vesicles that were stained closely agreed with the percentage of axolemmal length stained in the whole white matter Since acetylcholinesterase staining was highly localized to the axonal plasma membrane. the acetylcholinesterase-positive membrane fragments in the axolemma-enriched membranes could only originate from the axolemma. We conclude on the basis of the operational behavior of these axolemmal vesicles that many of the membranes found in these fractions are derived from the axonal plasma membram~

THE MOLECULAR properties of electrically excitable membrane in the mammalian CNS are virtually unknown. This is largely due to the lack of a suitable procedure to isolate the axolemma from the myriad of other membranes which comprise the CNS. Large, readily dissectible, principally unmyelinated nerves of invertebrates such as the walking leg nerves of lobster (DENBURG, 1972) and crab (BALERNA,FOSSET,CHICHEPORTICHE,ROMEY & LAZDUNSKI, 1975), squid retinal Abbreriations: AChE, acetylcholinesterase: AcThCh, acetylthiocholine, iodide: BP, myelin basic protein: BuThCh, butyrylthiocholine iodide: BuChE, butyrylcholinesterase of pseudocholinesterase; BW 284~51, 1,5 bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide; CNS, central nervous system: EGTA, ethyleneglycol-bis (fi-aminoethyl ether) N,N’-tetraacetic acid; isoOMPA, tetraisopropylpyrophosphoramide; LFB, Luxol Fast Blue: TES. N-tris (Hydroxymethyl) methyl-2aminoethane sulfonic acid. 155

axons (FISCHER, CELLINO, ZAMBRANO,ZAMPKJHI. TLI LES-NAGEL.MARCUS & CANESSA-FISCHER.1970). squid stellar nerves (CAMEJO, VILLEGAS. BARNOLA & VILLEGAS, 1969). and garfish olfactory nerves (CHA(.KO. GOLDMAN, MALHO~RA & DEWEY, 1974) have been used as the source of axolemmal preparations. We have recently described a method to separate axonal plasma membrane contained within the myelinatcd axon from other white matter constituents (DEVRIIX, 1976; DEVRIES. MATTHIEU, BENY. CHKHEPORTICH~:, LAZDUNSKI

& DOLIVO,

ZETUSKY, ANDERSON &

1978:

ZETUSKY. CALAHRI;SI:,

DEVRIES,

1978:

MATYHIEI’,

DOLIVO, 1977). The axolemmaenriched fractions are enriched in surface membrane marker enzymes such as sodium, potassium activated adenosine triphosphatase, acetylcholinesterase (AChE) and 5’-nucleotidase (DEVRIES rt LI/.. 1978), contain high specific activities of several enzymes involved in glycolipid metabolism (H 4RFORl). WEBSTER, BENY &

I56

.I SI

W.AE:(.HTI.K, SAC I. & D~VKIE:S. (‘ARINI.

~I.S.I.FL1.I

glycoprotein

&

(DEVKI~S

metabolism

VRI~S. MATTHIEII. tally

bind

1978).

BE:~\‘Y &

tetrodotoxin

DOI.I\O.

arc

;Iclt\e

Ill

vf trl..

1978:

Db-

1976)

, 1

<‘I

0:

c-

1978: C’osr,\~TtNo-Ct

h:VRlES.

\\I

and speciti-

(DI VHILS c’t trl.. 1978).

In this

describe histochemical evidence which supports the axonal plasma membrane origin of the axolemma-enriched fractions isolated from bo\ ine and rat CNS using AChE. an enqme Nhich had been demonstrated to be present in the axolemma of the peripheral ner\,ous system ( Ko~I_I.E. 19501. In addition. we describe the results of Luxol fast blue staining of these axolemma-enriched membrane fractions. A preliminary report of these results has been presented (ST~NI I:Y. SAM.. HAIXKI I) & DI.VKIES. 1976). report

we

EXPERIMENTAL

PROCEDURCS

A n irnul.5 The

corpus

combined

collosum

for wch

Sprague- Dawlq from Flow

rats

brains

wet-c

Isolation.

Malt

were obtained

Rats were decapitated

and

were qulcklq dlssccted and chilled h) im-

in 0.X5 %?-sucrose. 0. from

bovine

300 400g

VA.

butTcr (pH 7.0). Brainstemh. medulla,

three

axolemma

weighing

Labs. Dublin.

their brainstems mersion

from

bovine

I5 wNa(‘l.

0.05 \t-KH,PO,

di\\ectcd

to the lcccl of the

three rats wcrc combined

wt.). Each rat axolemma

preparation

(about

required

2g

wet

36 ratb,

Several

control

\tudie\

were conducted

speciticity of .AChF histochrmvzal

to cnhurc the

~~alning. TISSUV was ill-

cuhatcd III media from which the substrates. ac~‘t)Ithlocholine iodldc. cupric Bo\lne

CNS

axolemmal

minor

modifications

bovine

axolemma,

minced

with

conducted

fractions

of our method 12~ 24g

were

(DtVRti?s.

of fresh

a Stadie-Riggs

blade.

al 4 C. Myelinated

prepared

white All

1976). For

matter

were

operations

were

axons were Isolated as pre-

viously described (DEVRI~S ~‘1t/l.. 1972). The purified linated axons were osmotically pH 7.5 (instead

hhocked

of the previously

used distilled

pH7.5

containing to

!leltl

axons were centrifuged

I rnbt-TES.

four

fractions:

(II

homogenized

and

axons.

re-centrifuged

tinuous gradients to free them of cro\\ rat

axolemma-enriched

viously

described

reported

fractions

(Dr VRII.S

t’i

here were selected from

12-g bovine preparations

Hosting

interfaces

the

\amc

were

prepared

197X).

more

than

and re-

discon-

contamination.

were u/..

on

membranes

All fraction\ on

on three

I rnM-EGTA.

m>el~n

1.0 v I.2 ZI gradient

(iL) a pellet of m!elin-free

to

sucrose density

pH 7.5 and

0.X M-sucrose: (ii) and (iii) axolemma-enrlched at the 0.X M I.0 II and

water)

been omitted.

The

as pre-

Experlmentk 10 scparatc

and four 2-t-g rat preparations.

~ulfatc. or poras\tum

But!rylthlochol~ne

i\ not hqdrol)/ed concemratlon

bq AC’hE,

H;I\

huh,titutcd

terases \\a\

‘The

linal

docholtnehtcrase AcThCh

(I\~-OMPAI.

actiblt)

and ua\

acetylcholine thiocholine

were obtained bromide.

*hlch

Specllic acetylcholinehterass may bc blocked

by selectlcc

determined

with

acrivit)

of neuronal

origin

inhibitors.

284~51

Blue, MBS deNemours):

OCT@

tories. Naperville,

Chemical

embedding

of non-specific

fractions were Incubated laining

IO ’\I-escrine,

ln acetylthiocholmc

a rc\ersihle

vzctiom

wcrc

incubated

11,

media

one dlhromide InhibItor.

(BW

B

284~51).

ILL).

selective. re\cr\ible

tdentically

C‘o.): Luxol

Fast

omitted

and

docholinesterasc)

and other (‘NS

(BW

284~51.

hlockcrs

and

neostigmine.

iodide had been

AChE

active sites before an> reaction

inhihltorh product

media were then changed

AcThCh.

plus or minus AChE

wah expressly

frac‘I‘lssue

for 30 min at 25 c‘ In media. plu$

inhibitors

This ensured the prior access of the BuChE

formed. Incubation AcThCh

ACht.

and stained together

and eserine). from which acetylthiocholine

taining

5 *

pen!an-i-

which will not hloch Bu<‘hE or non-spccltic cho-

IIons were treated

enqmc

that

activil!.

containing

IO- ’ r+1.5 his 4-;~llyldimcth~l;Immoniumphcn~l)

Co.. E. 1. duPont

Lahor~l-

inhlhitor.

To dcmonstratc

staining reyuhed from \pecilic acet~lcholinc~ternse

or minus the AChE

(Miles

csterases.

medium co+

cholinehtcra\e

which w11l not block other cctcraae\.

slices were pre-incubated

medium

demon\~rate

callo~um and msmhrnnc

?ectmns of M hole corpu\

butyryl-

(Burroughs-Wellcome

(J. T. Baker

To

that staining wa5 not the product

tetraiso-

(Sigma Chemical

~SL’LI-

~a\

control

iodide.

(‘iso-OMPA’)

Iso-OMPA

as the substrate.

bromide.

acetylthiocholine

iodide. eserine. neostigmine

propylpqrophosphoramide Co.); BW

from the following companies’

omlttcd c~nI>

of

to block the non-apecllic

linesteravz acti\il>. Whole corpus callosal. axolemmal Chemicals

a ~clcc-

pacudochollnc\-

ctrnccntration

’\I) wansufficient

(8 x IO

which

ln I 7 HIM final

of non-spccilic

added to all tncuhatmn\

\peclficd

id

anldc

iodide

Tetraisoprop)Ipyroph~l~phor;lmtde inhihnor

fcrrq

iodldc (BuThChl.

for acetylthiocholinc

tlve, Irre\,erslhle as

from each other.

step (0.8 M. 1.0 M and I.2 vi discontinuous gradients

myc-

in lOmM-EGTA.

separate the axons. myelin. and axolemma The shocked myelinated

by

omitted.

Normal

al)

could bc

to solutions inhibitors. and

(pseuto

conunles\

rnhihitor-

Mammalian treated

sections

were incubated

simultaneously

CNS axolemma m separate

157

hlstochemlatry

chopper.

Cut sections

were washed

briskly

In the same

20Oml dishes for 4 18 h at 37 C with gentle agitation on a DubnotT shaker. At 2- 3 h. the reddish, golden-brown

cacodylate buffer on ice for l--3 h. Final incubation medium contained

AChE reaction product was clearly discernible in uninhibited sections hut was quite faint. Longer incubation periods of 8 IO h were used here due to known low levels of endogenous AChE activity in bovine corpu\ callosum. Beyond 13 h t&sue sections began to fragment and float off plahs slides. When the incubation was complete, all sections were remobcd and washed carefully in two changes of cold distilled water for 5 min. Sections were then either (i) counterstained to reveal nuclei in freshly filtered Harris hematoxylin (I min): (ii) counter-stained with Luxol Fast Blue (ride in/r(i): or (iii) dehydrated in graded alcohols and xylene. mounted In Permount, and examined microscopically. Lirr,,/ t‘lr\r Rllrc Following AChE hlstochemical staining about half the tlssuc
I.8 mu-CuSO,. 75 mM-succinate buffer (pH 6.4). 40 mMNa2S0,. 10~ ’ r+iso-OMPA (a hutyrylcholinesterase inhibitor), and 12 mM-acetqlthiocholine iodide. To demonstrate Bu< hE acttvitl. 13 mat-BuThCh was substituted for AcThC‘h in incubation medium from which the BuChE inhibitor. iso-OMPA. had been omitted. The medium appeared clear and brilliant blue. About IO ml of medium was used per 100 300 mg wet wt. of tissue. To emure the enzyme specificity of AChE stainmg, control sectlons were Incubated in media lackmg AcThCh or copper aulfatc. AChE inhlhitors (5 x 10 ‘u-BW 2x4~51. 10~ ’ M-e\erine. and IO ” bt-neostigmine) were added to pretncuhation and incubation media of half the sections to control for non-specific reaction products. Thin tissue slices were pre-incubated on ice for I h in media lacking AcThCh (or BuThCh), plus or minus AChE Inhibitor\ Incubation media were then poured ol? and replaced ulth identical media containing AcThCh (01 BuThChl. Tissue was Incubated at 0 and 37 C from 30min to 19 h with gentle shaking. AChE staining appears to the unaided eye as haz! streaks of brown black discoloration in the tissue section. Staining was much more Intense. debeloped faster. and was more diecult to block with the reversible inhibitor BW 284~51 at 37 C than at 0 C. The results reported here represent lncuhatmns conducted at 37 C for 6 IOh. Further processing followed the outline of Lruts & StrrlTt- (19691. Two changes of cold freshly prepared I”,, Na,S in 50musuccinic acid buffer (pH 5.3) oker 1.5 2.0 h were used in the reduction step. After \cveral hashes in cold succinic acid buffer. tissue was post-fixed in ?“,, 0~0, in 50 mM-sodium cacodylate buffer (pH 7.4) for I h. washed. and serially dehydrated over 2 h m graded alcohols and propylene oxide. Tissue was embedded in Epon mixture. cut at silver and grey interference colors, and was not post-stained with uranyl acetate or lead citrate to avoid possible heaq metal precipitate: resembling histochemical reaction product. Sections were examined with a Hitachi HS-8 electron microscope.

l~~~c!~/~/zo/inr.s/~r~~.s~.The copper thiocholine method of Lturs & SHI-TI (1969) was applied to bovine and rat whole white matter and CNS fractions with minor modifications. Fresh bobme corpus callosum or centrum semiovale white matter or fresh rat brainstem, from which most of the cortex had been carefully dissected, were sliced by razor blade into ,ections roughly 250pm thick as described by LEWIS & SHI,TE (19661. Whole white matter was fixed shortly after dissection from whole brain. Fractions from the axolemma isolation procedure were resuspended in 10 mM-TES buffer. pH 7.5. centrifuged at 82,500g for 30min (SW 27 Rotor), carefully removed with a curved spatula, and sliced with a razor blade Into about 3OOpm sections. All tissue was fixed on Lee for 1.5-~3 h in 2”;, glutaraldehyde-I’?,> paraformaldehyde in 0.1 M-cacodylate buffer (pH 7.0). Sections were then washed in 2-4 changes of the same cacodylate buffer for 4-24 h. Tissue nas then placed in agar at less than 45 C and cut into 25 /lrn sections with a Sorval tissue

33 mhl-glycme.

RESULTS

morphology of the bovine CNS fracThe general tions at the level of electron microscopy is shown in Fig. 1. The myelin has the expected multilamellar appearance (Fig. 1C). The myelin-free axons obtained by this procedure have an electron microscopic appearance similar to that previously described for myelin-free axons (DEVRIES et ul., 1972). The 0.8/1.0 fraction (Fig. 1A) represents a heterogeneous popuiation of linear and vesicular membranes with some identifiable mitochondrial and multilamellar myelin profiles present. The 1.0~7.2 fraction (Fig. 1B) is also heterogeneous but contains less identifiable multilamellar myelin than the 0.8/1.0 fraction whereas mitochondrial and synaptosomal profiles are more evident. Light

microscopic

histochemistr~

Figure 2 demonstrates

the light microscopic

histo-

FIG. 1. Bovine C‘NS fractions. (A). Axolemma-enriched 0.8 bf;l.O M membranes. Many 2O&fOOOnm diameter trilaminar membrane vesicles and fragments are evident. An occasional mitochondrial shadow (M) and multi-lamellar myelin-appearing membrane (My) are seen. (B). Axolemma-enriched 1.0 ~/l.? M membranes. Numerous 100~ 1000 nm membrane vesicles. A few mitochondrial fragments (M) and dense synaptosome-like vesicles (DV) can be seen. Multi-lamellar membranes characteristic of myelin are absent. (C). Myelin. Multi-lamellar myelin membranes unraveling FK,. 2. Bovine CNS fractions-- light microscopic histochemistry. (A). Whole corpus callosum section (AChE. Hematoxylin). Several axons stained AChE-positive along their lengths. Counter-stained with hematoxylin to reveal primary glia and supporting tissue nuclei. (B). Whole corpus callosum section (AChE. LFB). Numerous long. tubular AChE-positive axons. Tissue in background is counterstained brilliant blue with LFB. (C). Whole corpus callosum section (AChE). Reddish-brown copper ferricyanide AC‘hE reaction product lines several axons. No counterstain. (D). Purified myelinated axons isolated by three flotations (AChF, LFB). Isolated curvilinear myelinated axons stained AChE-positive. Occa\ion,tl cherry red hues are phase contrast photographic artifact. (E). Myelin (AChE. LFB) with numerous LFB-positive vesicles. (F). Axolemma-enriched 0.8 M:‘I.OM membranes (AChE and LFB). Scale: Bar in G applies. (G). Axolemma-enriched 1.O Ml I .2 M membranes (AChE and LFB). Two sections showing several larger 1 5 /urn AChE-positive fragments and numerous. smaller AChE-containing vesicles tn the background. (H). Axolemma-enriched 0.8~il.OM membranes (AChE w/BW 284~51 and LFB). Scale: Bar in G applies. (I). Myelin-free axons (AChE and LFB). Vigorous AChE staining in tubular configuration. Counterstaining with LFB produced virtually no LFB staining. A rare LFB-positive shadow

was the only one found

in more than

20 such microscopic

fields.

Ftc;. 3. Bovine corpus callosum sectton AChE EM histochemistry (A). AChE-staining neatly lures the ~‘rt p~~.s~ur axolemma of tw’o bovine axons (7). Linear, continuous patches of AChE staining are not associated with myelin (MY), mitochondria (M) or other membranes. About 3”,, of visible axolemma \tarn AChE-positive under these conditions. (B). Axolemmal AChE staining. AChE reaction product courses along the axonal plasma membrane. No other membranes are similarly stained, though in the corpus callosal sections many other diverse membranes are exposed to reaction substrates. The blach spechling (*) of the open loops of myelin represent an artifactual chemical precipitate (probably (‘~1). ((‘I. Most AChE staining is blocked by the specific, reversible AChE inhibitor. 5 x 10 s M-BW 284c5 1.

I-I<; 3. Bovine axolemma-enrtched

1.0 M 1.2 M membranes-AChE EM histochemistry. Bovine fractions stained for AChE (I9 h, 37 C) without and with inhibitors. (A). Numerous AChE-positive membrane vesicles and fragments show black. copper sulfide deposits lining their surfaces and captured within them. (B). AChE staining was inhtbited by about 80”,, by 5 x 10. s M-BW 284~51 based on count of stained membranes per utut area. (C F). Axolemmal AChE staining. Higher power views of experiment \hown in (AI. Profuse reaction product is deposited linearly along putative axolemmal membranes. Scale: Bar in (F) applies to (C F). FIG. 5. Rat brarnstem~ AChE EM histochemistry. (A). Black, AChE reaction product neatly lines only the en ptr,\.\~ur axolemma of the myelinated rat axon. The AChE-containing axolemma can also be seen clearly at bottom left (“I. Adjacent myelinated axons (AX) show almost no staining AChE activity. Myelin (MY). mitochondria (M), and other diverse membranes do not stain. TWO small AChEposittve membranes

at lower right appear

(RI AChE-reaction

product appears attached to radiating neurofilament bundles (t), Small length of axolemma is also stained (AXL).

Ftc;. 6. activity. vesicles (D).

on subsequent

micrographs

to be axonal

plasma (NF)

membranes. at 5 points

Rat axolemma-enrtched 0.8 M/ 1.O M membranes. (A,B). Many membrane fragments show AChE Mitochondria-like membranes (M) never stained. (C). Two trilaminar AChE-positive membrane from same experiment. The multi-lamellar myelin-like membrane (*) shows no AChE staining. Axolemmal AChE staining has been blocked by more than 95”,, by 5 x lo- ’M-BW 284~51.

159

FIG. I

161

FIG. 3

163

lb4

165

Mammalian CNS axolemma histochemisrry chemistry of the bovine CNS fractions. Membranes have been isolated from bovine corpus callosum and stained for acetylcholinesterase (AChE 12 h, 37 C). AChE activity produces a golden-rust colored reaction precipitate which appears somewhat more yellow under certain phase microscopic conditions (D, F-H). As indicated in the figure legend, some sections have been post-stained with Luxol Fast Blue (LFB, 1 h. 56 C). It is remarkable that only a few of the myelinated fibres present in whole white matter stain positively for AChE (Fig. 2A-C). It should be noted that the LFB staining shown in Fig. 2(B) is amorphous. The LFB staining of the myelin present in the myelinated axon fraction tends to obscure the AChE stain which coincides with the tubular myelinated axon profiles (Fig. 2B). Although the specific activity of AChE in the myelin fraction is quite low, numerous AChE positive membrane vesicles are evident by light level histochemistry (Fig. 2E). Similar AChE-positive vesicles have been observed by McILWAIN (1974) in isolated myelin. As shown in Fig. 6(F) the 0.811.0 fraction contains an abundance golden AChE-positive vesicles. The LFB counterstain of the 1.0:‘1.2 fraction is also positive but less intense than in any of the other isolated fractions. The specific AChE inhibitor BW 284~51 (5 x 10m5M) is shown to mhlblt more than 95”,, of all histochemically discernible AChE activity in this and all bovine CNS fractions. The myelin-free axons are heavily stained for AChE with a virtual absence of LFB stain corroborating previous biochemical data for the absence of myelin in this fraction (DEVRIES et ~1.. 1972). Electron

microscopic

histochemistr!

The AChE histochemistry of the bovine corpus callosum and axolemma-enriched membranes at the electron microscopic level are shown in Figs 3 and 4. In Fig. 3 whole bovine white matter has been stained for AChE (19 h. 37°C) with and without AChE inhibitors. The paucity of fibers and axolemma which show positive AChE staining confirms the light level observations. Since not all the axolemma in the starting material has stained positively for AChE it is expected that only a certain proportion of the membranes in the axolemma-enriched fractions would also stain positively as is indeed the case (Fig. 4). The activit! of AChE is more readily demonstrated in tissue from rat CNS which has a higher level of AChE activity (Figs 5 and 6). Whole rat brain stem has been stained for AChE for 6 h at 37-C to obtain the results shown in Fig. 6. AChE reaction products are almost completely absent from the myelin and mitochondria evident in Fig. 5. In Fig. 6 the rat axolemma-enriched fractions have been stained for AChE for 4 h at 4 C with and without AChE inhibitors, Once again a fair number of membrane vesicles demonstrate positive staining which reflects the relative abundance of stain in the tissue in situ. The specificity of the reaction in the rat CNS is demonstrated by the blocking with BW 284~51.

DISCUSSION The goal of this investigation was to provide direct morphological evidence for the in situ origin of the axolemma-enriched fractions. Our inability to demonstrate positive staining in all the axolemma present in whole white matter precludes unequivocal evidence for the awonal plasma membrane origin of all the membrane vesicles. However. since we could demonstrate that in mammalian CNS white matter only the axolemma exhibits a positive reaction for AChE, any AChE-positive membrane vesicle found in the axolemma-enriched fractions can only be derived from the axonal plasma membrane. Although the total amount of such vesicles found in either the rat or bovine axolemma-enriched fractions is small it relates rather well to the percentage of AChE-positive axolemma in total white matter. This implies that we are not isolating a select population of the axolemma present in the starting homogenate but a random population representative of the total axolemma m the whole white matter. We view the AChE-positive membrane vesicles as valid markers of the axonai plasma membrane and indicative of the localization of such membrane after fractionation. By this criteria the isolation scheme optimizes the separation of axolemma from other white matter membrane constituents. When considered together with the low levels of marker enzymes for non-axolemmal membranes in these fractions (DEVRIES. 1976: DEVRIES et ul.. 19781, we believe this evidence supports our contention that the bulk of these membrane fractions are indeed of an axonal plasma membrane origin. As shown in Table 1 the axolemmal preparations which have been isolated and characterized to date show remarkable variations in the level of AChE both in the starting homogenate and in the axolemmal preparations. The invertebrates generally have the highest AChE specific activities while there is a 10-500 fold drop in specific activity in the vertebrates from garfish to human. This variation can be attributed in part to the freshness of the tissue since the activit! of AChE is known to decrease with time (FAHN & Co-rt.. 1976). However. there is a trend for the higher vertebrates to have lower AChE specific activities. The present study marks the first time that AChE positive membranes have been histochemically demonstrated at the electron microscopic level in both the starting material from which the axolemmaenriched fractions were prepared and the isolated fractions themselves. Thus, as shown in Table 1, although high specific activities for AChE have been demonstrated biochemically in a number of isolated axolemma preparations, there has been no attempt to demonstrate such activity histochemically at the electron microscopic level in the isolated preparations. Therefore, it is not known whether the biochemically observed AChE is a property of all membrane vesicles in the preparation or of only a select subpopulation. Although CHACKO et ul. ( 1976) could demonstrate AChE at the electron microscopic level

TARL~ 1. SPE(.II;I(. h(-r~viry

OI 4~ I‘I-\I (~HOI.INESIERASII\ AXOLEMMAI PKI 1~4~41KJ~\S ___~

Source

Reference

Whole* homogenate

~..__ _ __~~

.Axolcmma+

Bovcne CNS Rat CNS Human CNS Rabbit CNS Lobster walking leg nerve Crab walking leg nerve Garfish olfactory nerve Squid optic nerve Squid stellar nerve *All values are reported as pmol acetylcholine hydrolyzed per mg protein per h at 37 C. Dash (- -I indicates data are not available. tAll CNS axolemma values are the 1.0:1.2 fraction. A: DEVRIES. 1976. B: DEVRIES, MATTHIEU. BENY. CHICHEPORTICHE, LAZDUNSKI & DOLIVO, 1977. C: ZETCSKY. CALABRESE. ZFTUSKY. ANUERSON. CLUEN. DFVRIES, 1978. D: MATTHII-.U, WEBSTER. BENY & DOLIVO, 1977. E: BARNOLA. VILLECIAS& CAMEJO, 1973. F: BALERNA. FOSSET,CHKHEPORTKHE. R~MEY & LAZDUNSKI, 1975. G: CHA(XO. VII.I.F
in lobster axons, they failed to show such activity in garfish olfactory nerve from which an AChE-containing axolemmal fraction was isolated. LFB is considered a classical myelin stain yet the intensity of staining in the axolemma-enriched fractions is much greater than can be expected on the basis of myelin contamination. The molecular component of myelin which is responsible for positive LFB stain is not known although evidence has been presented to suggest that it is a lipid such as cerebroside (CLASEN. SIMON’. FKOTT. PANI)ALFI. LAIKG & axolemma-enriched fractions LESPF;, 1973). These have been shown to contain appreciable levels of this glycolipid (DEVRIES & SAL‘L. 1976). it is possible that cerebroside is responsible for the positive LFB stain in the axolemma-enriched fractions. It is clear that the component responsible for the positive LFB stain is uniformly present throughout the fraction rather than being restricted to a few specialized membrane vesicles. This would indicate that if the axolemmaenriched fractions are representative of axolemma as a w’hole, the component responsible for the positive LFB stain is also an intrinsic component of the entire axolemma. We judge that the results of this investigation support our contention that we indeed are isolating membrane fractions which are derived from the axonal membrane in si~u. More recent freeze-fracture

analysis of these axolemma-enriched fractions has also demonstrated that the morphology of the isolated fractions is consistent with the axonal plasma membrane in situ (CULLEN & DEVRIES, 1978). When taken as a whole, the biochemical and present morphological data strongly indicate that these membrane fractions contain a significant amount of membrane derived from the axonal plasma membrane. Future approaches toward defining the exact proportion of the fraction which is derived from the axonal plasma membrane must not depend on a capricious enzymatic activity but rather reside in an inherent molecular property of the membrane such as immunological reactivity or interaction with specific lectins. Such experiments are currently in progress in this laboratory. We are hopeful that these membrane fractions will aid our understanding of the molecular architecture of this vital CNS membrane. 4ckn01v/rdyumenr,s This research was supported by N.I.H. Grant NS 10821-03 and A. D. Williams Fund 3558-549. We thank Drs. JOHN L. P(IVI.ISHO(-K and D. L. MCII.WAIN for EM histochemical advice. Dr. F. MONG for loan of cryostat. and Dr. CARSON J. CORNRR~KS and Dr. MAKY Ltt Pr~ri~s for helpful discussions and support. For expert technical assistance we thank Ms. CAI ARMSTKONC;. Ms. DORIS Fr:~rz. Ms. DEHHX L~wrs and Ms. KA~‘HI GI.IIIN~;FR. We thank Ms. Jr DY WATTS for typing the manuscript.

REFERENCES BAL~RNA M.. FOSS~I- M.. CHITHEPORTI(‘HE R., ROMEY G. & LAZDUNSKI M. (1975) Constitution and properties of axonal membranes of crustacean nerves. Bioc,hemistr!, 14. 5500 551 I. CAMEJO G.. VILLFZAS G.. BARNOLA F. V. & VIIL.I%AS R. (1969) Characterization of two different membrane fractions isolated from the first stellar nerves of the squid Do.\ir/icu.s gigus. Biochim. Bioph~~.\. Acfa (Ahsr.) 193, 247 ~259. CHACKO G.. GOLDMAN D. E., MALHOTRA H. C. & DEWEY M. M. (1974) Isolation and characterization of plasma membrane fractions from garfish Lepbosfrus osseus olfactory nerve. /. Cell Biol. 62. 831-843. CHACKO G. K.. VILLE(;AS G. M.. BARNOLA F. V.. VILLEC~ASR. & GOLDMAN D. E. (1976) The polypeptide and the phospholipid components of axon plasma membranes. Biochim. Biophrs. Actor 443, 19. 32.

Mammalian

CNS axolemma

167

histochemlatrq

CLASEN R. A.. SIMON R. G., SCOTT R., PANDOLFI S., LAtNc

I. & LESNK A. (1973) The staining of the myelin sheath by Luxol dye techniques. J. Neuropath. cup. Neural. 32, 271-283. COSTANTINO-CECCARINI E.. CESTELLIA. & DEVRIES G. H. (1978) Characterization and developmental changes of UDPgalactose : ceramide galactosyl transferase in a rat CNS axolemma-enriched fraction. Differences and similarities of the enzyme associated with the microsomal and myelin fractions. J Yeurochem. In press. CULLEN M. & DEVRIES G. H. (1978) Freeze fracture characterization of CNS myelin and axolemma-enriched fractions isolated from rabbit optic nerve. Anat. Rec. 190, 372. DENBURG J. L. (1972) An axon plasma membrane preparation from the walkmg legs of the lobster. Homcw~c.\umer~( <>nu\ Biochim. Biophrs. Actct (Ahst.) 282, 453-458 CNS. Sci@ncr 175. 1370-I 371 DEVRIES G. H.. NORTON W. T. & RAINE C. S. (1972) Axons: isolation from mammallan DEVRIES G. H. (1976) Isolation of axolemma-enriched fractions from bovine central nervous system. .Yrrrnuc? ktt 3, 117~122. DEVRIES G. H. & SAUL R. G. (1976) Lipid composition of bovine CNS axolemma. Neurosci. Ahst. 2, 409. DEVRIES G. H.. MATTHIFU J.-M., BENY M. & DOLIVO M. (1976) Glycoprotein m metabolism in rat CNS axolemma. Truns. Am. Sot. Neurochrm. 7, 199. DEVRIES G. H.. MATTHIEU J.-M., BENY M.. CHICHEPORTI~HF R.. LA~IIUNSKI M. & Dot IVO M. (1978) Bruin Ret. 147, 339. El.-BADAWI A. & SCHENK E. A. (1967) Histochemical methods for separate, consecutive, and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections. J. Histochem. Cytwhem. 15, 58&58X. FAHN S. & COTE L. J. (1976) Stability of enzymes in post-mortem rat brain. J. Stwrochem. 26. 1034~1042. FISCHER S., C~LLINO M., ZAMBRANO F., ZAMPIGHI G., TELLEZ-NAC;EI. M., MAKC~IS D. & CAIVESSA-FISCHERM. (1970) Isolation and characterization of plasma membranes from the retinal axons of the squid: an axolemma-rich preparation. Archs Biochettl. 138, l-15. HARFORD J. B.. WAECHTER C. J., SAUI. R. G. & DEVRIES G. H. (197X) Evidence for the synthesis of mannosylphosphoryldolichol and N-acetylgltrcosaminylpyrophosphoryldolichol by an awolemma-enriched membrane preparation from bovine white matter. .J. .Veurochem. 32: 91 98. KARNOVSKY M. & ROOTS L.. (1964) A “drrect-coloring” thiocholine method for cholinesterases. J. Hi.~toc~hrm. Crro&nl 12, 219-226. KL~XER H. & BARRERA E. (1953) A method for the combined staining of cells and fibers in the nervous system. .I. Neuropath. exp. Neural. 12, 40&403. KOELI.E G. B. (1950) The histochemical differentiation of types of cholinesterases and their localizations in tissues of the cat. J Phurmuc. rup. Ther. 100, 158-179. LEWIS P. R. & SHI:TE C. C. D. (1966) The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. J. Cell .%i. 1, 381 390. LFWIS P. R. & SHUTE C. C. D. (1969) An electron-microscopic study of cholinesterase distribution in the rat adrenal medulla. J. ,Microscopy 89, 181.-193. MATTHIEI; J. M.. WEBSTER H., BENV M. & DOLIVO M. (1977) Characterization of two subcellular fractions isolated from myelinated axons. Bruin Res. Bull. 2, 289-298. M&WAIN D. L. (1974) Localization of the acetylcholinesterase-containing membranes in purified myelin Bruin Res. 69, 182- 187. STANLEY J., SAUL R.. HADFIELD M. G. & DEVRIES G. H. (1976) Mammalian CNS axolemma: histochemtcal for neuronal origin of “axolemma-enriched” membrane fractions. Neuroscience Abst. 2, 616. Z~TUSKY W., CAI.ABRESE V.. ZETUSKY A., ANDERSON G., CULLEN M. & DEVRIES G. H. (1978) Isolation characterization of human CNS axolemma-enriched fractions, .I. Xc’wochem. In press.

(4ccepreri

21 Max 197X)

fractions evidence and

partial