THE

JOURNAL

Copyright

OF HISTOCHEMISTRY

© 1976 by The

Vol. 24, No. 9, pp.

CYTOCHEMISTRY

AND

Histochemical

Society,

Letters

EXTRAJUNCTIONAL

Di ffuse

ACETYLCHOLINE

extrajunct

ional

throughout

strable

in

the

RECEPTORS

denervated

(neurogenously

skeletal

muscle

choline

sensitivity

binding

of a-bungarotoxin

the

to

fibers

strict junction

With

an

in aBT

level

presence

diffuse muscle

lished

on

fibers

them

surviving

during

portions

crush

lesion

plate

zone

these

findings

muscle

separating of

or

myogenous

has

a broad

indicate

inadequately

same ing

should

between

vated To

clarify

zation

of

dysinnervated the

this

sized

male

rats

just

innervation,

Craggs

(4).

This

struct.irally

intact,

segment

ically.

technique modified

from

neural

muscle

were

light-microscopy AChR

extrajunctional

(2, 9).

an

Serial

trichrome,

sections

lesion

in

of

anesthe-

endplate

method

B

zone of

viable,

distal influence the

muscle myopath-

evaluated

1-17

days

presence

of

aBT-immunoperoxidase were

NADH-tetrazolium

also

stained

_________

FIG. 1. Rat sternohyoid muscle fibers, fresh-frozen, longitudinal sections, 7 days after a 1-mm crush was performed 2 mm distal to the zone of innervation (endplate region). Immunoperoxidase staining of aBT localizing AChR, no counterstain. (A) region distal to the crush, showing the muscle fibers to be morphologically intact and of normal diameter, and all having diffuse extrajunctional sarcolemmal staining. x 490. (B) regiQn proximal to the crush, showing staining at two neuromuscular junctions (endplates) but no extrajunctional sarcolemmal staining. x470.

Hall-

of a proximal apparently

for

by the

visuali-

crush

motor the

of

the

myogenously

nondegenerating

parted by

a

examination and

Fresh-frozen

thereafter

to the

by

distinguishdysinner-

performed to

allowed

segment

fiber

distal

If cor-

provide

in narrow

was

according

innervated

and

a central

muscle

by

states.

AChR

model,

sternohyoid

all bear

but,

myogenous

principle

The

AChR

as

noninnervated

extrajunctional

1A).

demonstrating

considered and

or primary further

that

extrajunctional fibers

neurogenous

states

(Fig.

together,

can

muscle

be

4 days

of this, through-

.4-

de-innervated

for

not

as

spite

end-

principle

of usefulness

innervated

token,

as early

In diffusely

to occa-

the

motor

AChRs.

that

spectrum

in

to a narrow

their

sarcolemmal

would

and

distal

mechanisms

extrajunctional

this

in devel-

Considered

noninnervated or

rect,

the

(6).

a general

sarcolemma

nucleus. AChR

an

proc-

is estab-

(3)

toward

internal

distal

although

indicated

influence

from

innervation point

neurogenous

of

them

an

extrajunctional

segments

the

is present

fibers

fiber normal,

electronmi-

have

embryogenesis

of muscle

fibers

diffuse

AChR

the

had

had

original essentially

to the

disease

neural

fiber all

and

as a method

studies

before

were

esterase

extra-

of using

neurogenic

extrajunctional

oping

out

“recovering”

crush

nonspecific

reactions.

fibers. the

and hope AChR

pharmacologic

neuromus-

ATPase,

phosphatase

neurogenously

light

to the

the

they

(2) in

at

led

identifying

However,

sional

localized

extrajunctional

specifically

that

which

by in contrast

the

precisely

fibers

(9),

the

muscle

complex

of diffuse

esses.

to

technique was

muscle

croscopic

7, 9),

innervated

membrane

de-innervated

(5,

myofibrillar

The

acetyl-

morphologically

of AChR

normally

binding

sarcolemma

by

MYOGENOUSLY

FIBERS

alkaline

demon-

de-innervated)

(aBT)

immunoperoxidase

junctional

for

8) and

confinement

cular

been

pharmacologically

(1,

tase,

receptor has

ON

MUSCLE

acetylcholine

sarcolemma

1976 in U.S.A.

to the Editor

DE-INNERVATED

(AChR)

1033-1041,

Printed

Inc.

with reduc-

1033

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1034

LETTERS

muscle

fiber segments

crush,

with

their

the

same

time

influence

AChR

at

When

in the zone proximal

neural

extrajunctional staining

the

and

the

intact,

had

the

neuromuscular

sternohyoid

junctional

was

lesion

appeared

normal

was

made,

rich

aBT1B). at

diffuse

in both

3.

no

(Fig.

denervated

equally

extra-

the

fiber

is necessarily

central

5.

This

work

Research

provided

was

in

Fellowship

Association

of

a-bungarotoxin and generously Mathew

done

technical

part

from America,

the

the

tenure

Muscular

Inc.

to

line

and antibody solutions provided by Drs.

Fambrough

diaphragm

after

sensitivity

9.

a

Ringel Vogel tion

Purified

SP, Z, and

complete or 151:1, 1960.

Bender

AN,

Daniels

MP:

analytical

2.

Daniels MP, of a-bungarotoxin

THE

partial

Z: Immunoperoxidase binding sites in

of acetylchoof

Festoff

BW,

Ultrastructural

application

skeletal

J

Engel

WK,

demonstraof

extrajunctional

rat skeletal

P. RINGEL

KING

ENGEL

National

staining muscle end-

of acetylcholine

and

Corn

and

Stroke

AGAINST

Disorders

Institutes

Bethesda,

THE

of Neurological

municative

of Health

Maryland

MICROSCOPICAL-IMMUNOHISTOCHEMICAL IN

Branch

Institute

National

recep-

LOCALIZATION ANTISERUM

60:248,

denervation.

N. BENDER Medical Neurology

Physiol (Lond) 147:178, 1959

ELECTRON

with

Physiol

ADAM

CITED

Axelsson J, Thesleff 5: A study of supersensitivity in denervated mammalian skeletal muscle. J

distribution

correlated Gen

receptors of denervated human and muscle fibres. Nature 255:730, 1975

were prepared Zvi Vogel and

1.

shows

J

STEVEN

Vogel

re-

density

7. Lee CY, Tseng LF, Chin TH: Influence of denervation on localization of neurotoxins from clapid venoms in rat diaphragm. Nature 215:1177, 1967 8. Miledi R: The acetylcholine sensitivity of frog mus-

W.

plates

denervation

in

P. Daniels.

LITERATURE

Acetylcholine

extrajunctional

sensitivity.

cle fibres after Physiol (Lond) of

DM:

and

nerve-free segments muscle. J Physiol (Lond) 170:389, 1964

Dystrophy

S.P.R.

HC,

Distribution

6. Katz B, Miledi R: The development

assistance.

during

Hartzell

ceptors. acetylcholine 1972

neurogenous.

Hubbard

tors. Nature 254:339, 1975 Diamond J, Miledi R: A study of foetal and newborn rat muscle fibres. J Physiol (Lond) 162:393, 1962

in rat

ACKNOWLEDGMENTS Gregory

EDITOR

4. Hall-Craggs ECB: Observations on the fate of muscle fibres temporarily isolated by transection of a muscle belly. Z Zellforsch 119:68, 1971

the

and distal segments. These findings provide visualization of the diffuse distribution of extrajunctional receptors on myogenously de-innervated fibers as well as on neurogenously de-innervated fibers and demonstrate that they appear identical in the two pathogenic mechanisms. Accordingly, the presence of extrajunctional AChR is not, by itself,evidence that the dys-innervation of a muscle

THE

to the

showed

junctions

muscle

crush

AChR

TO

20014

STUDY

ANTERIOR

A LOW

PITUITARY

MOLECULAR

ADENOHYPOPHYSIAL

OF

OF

AN

WEIGHT

CONSTITUENT

A peptide with a molecular weight of about 5,000 and with effects on the release of sperms in frogs has

been prepared from bovine anterior pituitaries by Kihlstr#{246}m and Danninge (2), Kihlstr#{246}m et al. (3) and

FIG. 1. An immunohistochemically stained cellwith antibody anti-SRS from a normal bovine pituitary. The cell contains numerous round stained secretory granules (RG), polymorphic stained granules (PG) as well as lysosomes (L). The Golgi complex (G) is prominent and contains a number of stained granules. Dilution antiserum: 1:50; picric acid formaldehyde solution fixation, Epon embedding. \5,800. FIG. 2. Higher magnification of Figure 1 with strong immunohistochemical staining over the round secretory granules (RG). The density of the granule is increased as the PAP molecules become more concentrated on the

granule. deposited.

So, the In the

stain appears diffusely stained

in

clumps of areas (around

mits the identification of isolated PAP

varying sizes the secretory

complex

molecules

(white arrows) granules) the

due slight

to the amount “background

of the staining”

as black flattened rings or pentagons

stain per-

(black

arrows). stained with antibody is specific for LH gonadotropin (ER) and unstained mitochondria (M) can also be seen. Fic. 4. Higher magnification of Figure 3 with stained The PAP complex molecules appear as clumps or spots: stain (white arrows). x 45,000. FIG.

sections.

3.

Immunohistochemically Antibody anti-LHfl

anti-LH$ on the same cell as in Figure 1 in serial producing cells. Unstained endoplasmic reticulum Dilution of antiseium: 1:50. \5,800. round granules (RG) and polymorphic granules (PG). the rings are, in this part of the figure filled up with

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