0022-1554/78/261 1-0973$02.00/0 THE JOURNAL OF HISTOCHEMISTRY Copyright © 1978 by The Histochemical

HISTOCHEMISTRY

AND

OF

JAN

Received

Inc.

RAT INTRAFUSAL MUSCLE MOTOR INNERVATION

KUCERA,

Medical

Vol. 26. No. 11, pp. 973-988, Printed in

CYTOCHEMISTRY

Society,

KATERINA

Neurology

DOROVINI-ZIS

Branch,

for publication

May

NINCDS

W. KING

AND

National

15, 1978, and in revised

FIBERS

Institutes

form July

AND

1978

S. A.

(

THEIR

ENGEL

of Health

13, 1978 (MS

78-157)

Muscle spindles were followed in serial transverse sections of freshly frozen rat soleus muscles. Adenosine triphosphatase (ATPase) histochemical staining reaction was used to identify nuclear bagi, nuclear bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along bagi and bag2 fibers but not along chain fibers. Bag1 fibers displayed ultrastructural heterogenity when their intraand extracapsular regions were compared. Simple “diffuse” and more elaborate “plate” motor nerve terminals were demonstrated histochemically along the poles of bagi and bagl fibers by staining for cholinesterase. One motor terminal of the “plate” appearance was present on a chain fiber pole. There was no consistent spatial correlation between the intensity of regional ATPase staining along the nuclear bag fibers and the location, number and type of motor endings. Other factors, such as intrafusal fiber sensory innervation and regional differences in active and passive functional recruitment of nuclear bag fibers during muscle activity, may contribute to the ATPase staining variability along the intrafusal fibers.

Intrafusal

muscle

fibers

in rat

muscle

spindles

Though three types of motor nerve terminals have been identified in light and electron microscopic studies of mammalian muscle spindles (3), information is lacking on the number, type

can be separated into nuclear bag and nuclear chain fibers according to the appearance of their nucleated equatorial regions (3). Two types of nuclear bag muscle fiber, called bag, and bag2, have

been

distinguished

by

morphology,

histochemical

ture, and intensity

development of “myofibrillar”

phatase tion can

(ATPase) be used

differences

and

in their

profile,

ultrastruc-

(2, 4). Differences adenosine

histochemical to identify nuclear

rat

in the triphos-

staining bag,,

and

along fibers

other

reacnuclear

fibers

the metabolic multiaxonal

remains

reactions intrafusal muscle

maintain histochemical entire length (13). The variability along the is not

gested that the ences may relate apparatus along

However, because

staining

the length of individual (2, 22, 34). Extrafusal

contrast, over their histochemical trafusal

enzyme

known.

Yellin

regional histochemical to variations in the the fibers, possibly

and muscle

functional spindle

as

yet

incompletely

present property

endings nuclear

and

each

skeletofusimotor

of the

three

investigation to study

along chain

the

nerve terminals can staining techniques

nuclear fibers

of

sites

of

be detected (19, 26). In

we took advantage of distribution of motor

the of

types

bag,, the

nuclear bag2 and rat soleus muscle

spindle. The location of motor terminals on the three intrafusal fiber types, revealed histochemically by staining for cholinesterase (ChE), was compared with the corresponding fiber histo-

muscle fibers, by

uniformity cause of the course of in-

chemical (ATPase) serial transverse

(32,

extracapsular bag fibers

34)

by

intrafusal muscle fiber. Like extrafusal motor endplates,

the this

vary

spindle.

sug-

differcontractile reflecting

consequences motor innervation.

this suggestion is difficult the motor innervation of the

of fusimotor

received

intrafusal motor by cholinesterase

bag2 and nuclear chain intrafusal fibers in serial transverse sections of a spindle with a high degree of consistency and reliability (2, 4, 22). Interestingly, histochemical profiles of ATPase

location

endings

Special

quite unlike tracapsular

and ultrastructural sections of the attention

was

regions where assume ATPase those course

they (22).

profile in muscle

same paid

to

spindle

freely lying nuclear staining properties

exhibit

during

their

in-

of METHODS

to assess rat spindle

Histochemistry:

borne-Mendel diately post

understood.

Soleus

rats (200-250 mortem and

973

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

muscles

of four

g) were quenched

female

Os-

removed immein isopentane

974

KUCERA,

DOROVINI-ZIS

cooled to -160#{176}C with liquid nitrogen. The frozen specimens were cut serially in a cryostat at 7 or 8 im. The transverse sections were incubated for 1 min at 24#{176}C with a-naphthyl acetate as substrate and hexazonium pararosaniin as coupler for the histochemical demonstration of esterases (24); the sections were counterstained with hematoxylin. Every eighth or ninth section was processed for “myofibrillar” adenosine triphosphatase (ATPase) at pH 9.4 (15) after alkali (pH 10.4) or acid (pH 4.3) preincubation in alternate order, employing the staining technique of Dubowitz and Brooke (9). Separately, 10 m serial transverse sections of two soleus muscles were stained for the two forms of ATPase only to study the staining of intrafusal muscle fibers in detail and to prepare Figure 1. Muscle spindles were located, intrafusal fiber types identified, and ATPase staining profiles established over the full extents ofintrafusal fibers. Staining intensities

were

expressed

as light,

moderate,

or dark.

The appearance of dense, red reaction product for esterases on intrafusal fibers was noted; the length of each stained area and its distance from the spindle equator was recorded. Motor nerve endings on intrafusal fibers contain both nonspecific and specific cholinesterase (19). aNaphthyl acetate is hydrolyzed by nonspecific esterases, nonspecific cholinesterase and acetylcholinesterase

(12,

contributed experiments.

24).

One

or

all

of

these

enzymes

may

have

to the patches of reaction product in our However, at short incubation times, such as those used in our studies, most of the reaction product with a-naphthyl acetate is considered to be due to cholinesterases ( 12). Therefore, the term cholinesterase (ChE) will be used in this paper when referring to the enzyme activity of the stained areas. Enzyme activity is localized mainly in the postsynaptic region of the rat neuromuscular junction (12). Accordingly, the stained areas on intrafusal fibers in our experiments were assumed to correspond to the sites of motor nerve endings. Electron microscopy: For ultrastructural studies, a technique similar to that used by Banks et al. (2) was adopted. In six muscle spindles, a region of interest was identified in 10 itm cryostat sections of the frozen specimen stained with hematoxylin and eosin. A single 5O-rn thick frozen section was then taken and this was followed by several 10 tm sections subsequently used for classification of the types of intrafusal muscle fibers and to determine fiber ATPase and ChE staining in the region selected. The 50 rn frozen section was transferred to cold 5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2 and fixed for 16-20 hrs supported by a plastic cover-slip. After fixation, the section was washed in buffer, post-fixed for 2 hr in 1% osmium tetroxide buffered at pH 7.2 in 0.1 M sodium cacodylate, dehydrated through a graded series of methanol solutions and embedded in Epon. Sections were cut on an LKB ultrotome, stained with uranyl acetate and lead citrate and examined with an AEI

AND

ENGEL

EM 801 electron of 60 kv.

microscope

at an accelerating

voltage

RESULTS

Spindle

morphology:

Twenty

dles were located in rat lengths were calculated verse

sections

stained

of two

consists of the

spindle

soleus from

for ATPase.

poles

extending

equator

(3).

a spindle pole measured equator to the termination fusal fiber was 1980 j.tm The and

nuclear bag2 fiber the longest spindle

being fiber.

was

930 m

from the of the spindle

end m

(range

sule

tm);

840 jm

the

(range

of intrafusal

mean

spent

fibers

(ac-ATPase), as described

one

infrequently with other

nuclear

three fiber

bag2. third

spindle

one

bag1, two ifiustrated

bag2 fibers and in Figure 4C.

during for

the

minor were most

ofibrils are absent ing was present. where

spindle

extracapsular of the two

example,

differences noted along

of their

nucleated

cap-

from

50-60%

and low majority

ac-ATPase of spindles

one

and

bag2

two

or

fibers. Spindles were rarely ena spindle

one

with

chain

one

fiber

of ATPase the intrafusal

except

where

or no ATPase in tile region and

is

staining fibers

course,

terminates,

polar regions types of nuclear

Fi-

ac-ATPase bag1; those ac-ATPase

nuclear chain type and they

regions

and little However,

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

identified

were classified ATPase after and acid

intracapsular equatorial

capsule

the

of their Three

were

The fiber

bag,,

countered;

Only intensity

mean to the was 250

tim)

and low as nuclear and high

nuclear chain combinations

such

bag1 fiber

in the encapsulated previously (22).

high alk-ATPase (Fig. 1A, B). The

possessed

The

histochemistry:

muscle

bers with low alk-ATPase staining were recognized with high alk-ATPase

displayed staining

thickest its pole

equator space

visible

of

of the intratm).

of the chain

zm).

660-1200

fibers

staining were nuclear fibers represented the

side

length

the fiber,

in every spindle examined. They according to their staining, with alkali preincubation (alk-ATPase) preincubation spindle regions

spindle

either

than that of a nuclear 540-1320

equator. Nuclear bag course extracapsularly. Intrafusal fiber types

The

center of the periaxal fluid

140-400

ended

Each from

was usually intrafusal

(range

distance

spin-

from the center of the longest (range 1360-2650

10-400 jun longer The mean length

pole

muscle

muscles and their 10 m serial trans-

mystainnear in

the

of the spindle, each bag fiber in a spindle

INNERVATION

FIG.

1. Periodic

sections

of a muscle

OF

spindle

RAT

INTRAFUSAL

from

rat

soleus

muscle

MUSCLE

through

975

FIBERS

capsular

(A, B) and

extracapsular

(C-F) spindle sections were

regions stained for ATPase after pH 10.4 (A, C, E) and pH 4.3 (B, D, F) preincubation. The taken 360gm (A, B), 970 zm (C, D), 1530 jIm (E) and 1410 im (F) from spindle equator. Extrafusal muscle separates into type I and type II fibers as marked in frames C and D. b,-nuclear bag, fiber, b2-nuclear bag2 fiber, c-nuclear chain fiber, sc-spindle capsule. All x900.

demonstrated characteristic staining properties. A bag, lightly with alk-ATPase thirds of its intracapsular erately dark or as dark proaching the termination remained so while This alk-ATPase encompassed

changes fiber that along the

in ATPase had stained inner two-

course became modas bag2 fiber when apof spindle capsule and

passing beyond the capsule. dark region of the bag, fiber

200-600

,tm.

Further

along

its

ex-

tracapsular course, light alk-ATPase

the bag, fiber regained staining and remained

its light

until its termination. The bag, fiber characteristically stained lightly with ac-ATPase in the encapsulated spindle regions but invariably became fiber

dark in its extracapsular lost its high staining with

it passed invariably

the

termination

became

light

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

course. The alk-ATPase

of spindle for

the

rest

capsule

bag2 once and

of its extra-

976

KUCERA,

DOROVINI-ZIS

capsular course. However, the bag2 fiber stained darkly with ac-ATPase both in its intracapsular and extracapsular portions (Fig. 1). Thus in the extreme polar regions nuclear fibers always stained alike with reactions

and

resembled

extrafusal

fibers which are also dark with ac-ATPase cal ATPase staining fested teristic tions

being situated

The

type

chain fibers, with the two

retained

up

to their

their ATPase endings

pattern

of

ATPase

staining

along

ent

soleus

capsule fusal

which

muscles.

The

alk-ATPase

bag1

staining

fiber

in the

termination was the fibers to demonstrate

the

alike or differ-

the

fiber

pole

(Fig.

region

of of

only area variability

of intrain its

3B).

A tandem spindle has two encapsulated regions spaced apart along the length of a common intrafusal fiber (3). Two such spindles were encountered; gions were

the two encapsulated equatorial respaced along the length of a nuclear

bag2 fiber of twice normal length. In one tandem spindle (Fig. 5A) the two contributing equatorial regions were widely separated (3500 m) and the common bag2 fiber had a regular histochemical profile in both intraand extracapsular regions. One of the regions of altered ATPase was, therefore, in the fiber,

between

other tandem two contributing arated (2350 m) not demonstrate

the

two

staining middle

equatorial

spindle (Fig. equatorial and the altered

6).

5B), units

common alk-ATPase

with of this

regions.

alklong In the

however, were less

the sep-

bag2

did in

fiber staining

fiber

Fiber by

sections

by

longitudinal

division the

progressively

was

of

the

the

ATPase

division in serial

a cleft

diameter

and dividing it ultimately of the spindles (Fig. 6B) tamed

fiber

recognized

presence

across

extending

of bag1

fiber

into two parts. In one the daughter fiber re-

staining

properties

of

the

parent bag1 fiber and had a fiber diameter appropriate for a bagi fiber (smaller than bag2) throughout its length. This daughter fiber passed through the spindle equatorial region acquiring by

of fiber

the

sensory

lack

nuclei

opposite 6A), the

vicinity

ENGEL bag,

(Fig.

without

length and degree of ATPase staining among different muscle spindles. Bag, fiber in one of the spindles encountered manifested two separate regions of increased alk-ATPase staining along

nuclear

cated

muscle fibers was remarkably muscle spindles from the same

rat

characreac-

extracapsularly.

intrafusal among increased

I muscle

light with alk-ATPase and (9). Regional histochemidifferences were not mani-

along nuclear appearance

were

bagi and bag2 the two ATPase

AND

(3) and

spindle daughter

equatorial

as

mdi-

aggregation

terminated

pole. fiber

nection with the ing characteristics

innervation

of the

midway

in the

In the other spindle (Fig. likewise retained its con-

parent and

bag, fiber diameter

and had staintypical of the

bagi fiber in the pole of its origin. However, this daughter fiber presumably acquired primary sensory nerve innervation while passing through the spindle equator, as indicated by the appearance of the central aggregation of fiber nuclei into “nuclear bags” (3). The fiber then continued through onstrated large bag2

the

fiber fiber.

opposite ATPase

spindle staining

diameter

characteristic

Intrafusal five complete spindle sections variable reaction

fiber and

pole where properties

motor several

it demand a

of the endings: incomplete

nuclear Twentymuscle

poles were examined in serial transverse stained for cholinesterase activity. A number of discrete deposits of the ChE product were visible along the length of

every were

intrafusal assumed

nerve

endings.

Nuclear richness

fiber followed. to indicate

bag, fibers were motor endings.

of

areas

of ChE

forms pole.

and lengths Some active

activity

The the

stained sites of

characterized From two

with

deposits

were visible areas were

areas motor

to

by a nine

in various

along every represented

bag, by

comparable locations between the two equators. Intrafusal muscle fibers characteristically do not branch during their course (3). Nevertheless, two spindles were encountered where a new,

thin, short, arched lines reaction product closely surface without deforming

or small spots of the applied to the fiber its round contours

(Fig.

no

daughter

underlying

fiber

appeared

to

arise

from

a parent

2A,

B). the

There

were

precipitate

muscle

though

fiber a fiber

nuclei nucleus

FIG. 2. Appearance of motor nerve endings on nuclear bag, (b,), nuclear bag2 (b2), nuclear chain (c), and extrafusal (ex) muscle fibers in 8 tm transverse sections through intracapsular (A-F, I) and extracapsular (H) regions of several muscle spindles. Spindle capsule is about to terminate in section G. Sections A, B and D, F are through the same spindles. Stained for cholinesterase and counterstained with hematoxylin. “Diffuse” endings (arrows) on nuclear bag, (A, B) and bag2 (C, D) fibers. “Plate” endings on nuclear bag, (E, G, H), bag2 (F) and nuclear chain fibers (I). Myoneural junction on an extrafusal fiber (J). Distances from spindle equator (pin): A-240, B-280, C-290, D-370, E-560, F-620, G-910, H-1520, 1-420. Length of the endings (tm): A-16, B-16, C24, D-24, E-32, F-64, G-24, H-24, 1-32, J-40. All x900.

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

INNERVATION

OF

RAT

INTRAFUSAL

MUSCLE

.1

.

977

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0

978

KUCERA, to the

adjacent dark

nucleus,

cell,

was

active

area

possibly

often

above

endings were were invariably

could

a part the

designated present

DOROVINI-ZIS

be

present.

of the

A

Schwann

stained

area.

Such

as “diffuse” and they on bagi fibers. Their

AND

ENGEL

represented a separate “plate” on a bagi fiber last

spindle

lowing nations.

motor

the

gions

inner

endings, similar were fewer foci

ofthe

an

occasional

Although

third more

of the robust,

provided nuclei. bag1

fiber

fiber-encapsulated it was still

there were Characteristically,

fiber

were

encapsulated region. ending in the middle region appeared classified as “diffuse”

no underlying “diffuse”

first

the

motor

muscle endings

endings

fiber on

encoun-

of bag,

ings. Nuclear

about

fuse” bers, vation ent.

one

The

tened, present neural

or underlying suggesting apparatus,

fuse”

nated lengths

“plate” and

found regions without

in the outer of bagi underlying

The

mean

sulated 24-56

endings. appearance

of bagi of

=

“plates” of variable could sometimes be

two-thirds ofthe encapsulated fibers, some with and others muscle fiber nuclei (Fig. 2E).

length

region jim; N

Such

the

8).

“plates” fiber

in the

was

However,

a

38

pin

“plate”

encap(range ending

that was consistently present, possibly representing a separate entity, was located on bagi fibers just before or more frequently just past the The

termination mean length

of the of the

muscle spindle capsule. bagi “plate” in this fiber

region was 24 m (range 16-40 rim; N a nucleus was often present underneath cipitate curred

=

25) and the pre-

(Fig. 2G). Usually one such “plate” on a bag1 fiber, mostly in an extracapsular

location distance

800-1300 related

oc-

,im from the equator, the exact mainly to overall spindle length.

Occasionally two such adjacent “plates” were present on bag1 fiber, located on the same area of fiber perimeter which suggested collateral branching

of the

parent

motor

axon

(Fig.

3C, E).

this bagi “plate” in the vicinity of capsule termination was absent (Fig. 4B). In several spindles, there was an additional “plate” of simRarely,

The

mean

“plates” (range

in this peripheral location 14-36 tm; N = 5). It could

tained

whether

this

most

peripheral

length

of bag1

As

with

“diffuse”

endings

multiple

bagi

motor there pole,

fibers,

mostly

farther away from often more robust

“dif-

occurred

on

the spindle than those

equator on bag,

of comparable lengths fiber poles were devoid

(Fig. 2C, of “dif-

endings, if present, of the encapsulated mean length was

were re42 jim

bag2 fibers were ings. There were would correspond consistently

capsule terminates, extracapsular regions

found

in their

extracapsular

regions.

Where it was difficult to classify ChE activity on a bag fiber as either “plate”, such areas were designated plate” may

endings indicate

rather

than

to indicate limitations the

ending. Nuclear

chain

consistent

in their

chain fiber the midpolar

presence fibers

of a separate had

motor

appearance

possessed region

activity

21-48 traversed

did not on

jim;

possess a fiber

incubation

kind

endings

and

and was

fairly

location.

thick, often

The

N close

50). to the

=

more

than

one

area

of ChE

with

prolonged

times.

We

have

encountered

pole

endings

34

A myelinated ending. Chain

even

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

in re-

and the flattened,

pole

of motor

of

fiber nuclei subjacent of the endings was

bag

devoid

This used

a “plate” type ending of each pole. The ChE

one or more muscle 21). The mean length

jim (range axon often

a patch of “diffuse” or as “diffuse-

this uncertainty. of the methods

action product was dense underlying fiber contour with (Fig.

and of

invariably without motor endno endings on bag2 fibers which to the “plates” of bag, fibers

several examples of a nuclear of endings on one of its two

“plate”

end-

However, on a fiber

the

was 21 sm be ascer-

not

fol-

termire-

of motor

possessed

the region where spindle never beyond it. The

mm)

4B).

six.

when

(range 24-64 jim; N = 20) and underlying muscle fiber nuclei were usually present (Fig. 2F). Rarely, there was a “plate” ending on bag2 near

2H,

and

devoid

to bag1 fibers. of ChE activity

endings. “Plate” in the outer two-thirds gion of the fiber. Their

fibers

3E

fibers

fibers, although D). Some bag2

ilar appearance located on bagi fibers more peripherally, 1300-1600 tm from the equator (Figs. 3D,

were

A the

and “plate” endings occurred on bag2 fibut some differences in the motor innerof the two types of bag fiber were appar-

bag2 fibers and were

fiber nuclei were developed subareas were desig-

to

encountered

fibers towards their polar extracapsular

fibers

bag2

tered on intrafusal fibers when proceeding from the spindle equator towards the poles. When the deposition of ChE reaction product was thicker, denser and the fiber contour flataggregated a variably the active

ending

intrafusal The extreme

mean length (extent along the length of the fiber) was 23 tm (range 8-72 tm; N = 100). One to six such areas occurred on a bagi pole in the two-thirds

kind of nerve ending. was characteristically

(5

chain fiber devoid poles (Fig. 4E). The had

a regular

his-

OF

INNERVATION

RAT

INTRAFUSAL

A

MUSCLE

979

FIBERS

A

B1 B2Z C( A

C(

Sc B

#{163}

B1

-

D2

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B2 UPPER: LOWER: #{163}

LIGHT

C(

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sc 0

0.5

10.4 4.3

1.0

1.5

FIG. 3. Distribution bag2 (B2) and nuclear

MODERATE DARK mm

2.0

of motor endings stained for cholinesterase along the poles of nuclear bag, (B,), nuclear chain (C) fibers in five muscle spindles. Intrafusal fiber types were identified in periodic spindle sections stained for ATPase after pH 10.4 (upper of the two bars representing each intrafusal fiber) and pH 4.3 (lower bar) preincubation. Staining intensities are expressed as light, moderate, and dark. Symbols: -“diffuse” endings; t-”diffuse-plate” endings; A-”plate” endings. The length of the symbols indicates the length of motor endings. SC-spindle capsule; the thick part of the line represents the periaxial fluid space. Scale indicates distances from spindle equators, which are at zero mark. Empty circles in the equatorial region of intrafusal fibers symbolize their nuclei.

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

980

KUCERA,

DOROVINI-ZIS

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FIG. in five

4. Distribution muscle spindles.

of motor Symbols

tochemical

appearance

sometimes its innervated

somewhat thinner counterpart.

fibers

of

unusual

although

lengths

the

fiber

and shorter Two nuclear were

1.5

1.0

endings in staining as in Figure 3.

observed.

for cholinesterase

was than chain Al-

4.3

EJLIGHT MODERATE DARK

sc 0

10.4

though chain profiles chain

along

as long fibers and

the

poles

as bag1

mm of intrafusal

fibers,

these

2.0 muscle

long

displayed histochemical motor innervation similar

fibers.

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

fibers

nuclear ATPase to other

INNERVATION Occasionally

areas

of ChE

OF

activity

at the same different intrafusal

sections of a particular ings involved could

spindle be of the

and

bag2

a “plate” chain given

fibers.

ending

More

and a nuclear bag transverse section.

tained endings

whether at the

this same

motor

axonal

branching

“Plate”

endings

on

there on

fiber (mostly It could not

the

or

was or

regions

and

nerve

devoid of ChE (19) and Mayr surrounded

axons

by periaxial

fluid

space

981

There

was

of intrafusal

extrafusal

of cholinesterases

fibers at the

no ChE

fibers,

The Figures

having

accumulations

myotendinous

junction

distribution fibers

of motor

of several

3-5,

and

endings

spindle

6. There

along

poles

was

intra-

is shown

considerable

iability in the motor supply among nuclear and bag2 fibers. Generally, only one pole spindle could be studied in its entirety. This

motor fusi-

not

nuclear

detract

from

the

validity

of our

in varbag, of a does

observations

because contractions of intrafusal fibers involve each pole independently (20). When both poles of a spindle were visualized, little symmetry of

at

equatorial

the

them

Processing serial muscle sections ternately with periodic sections for

were

with Hess fiber regions were

stain-

in distinction

(8).

was

occur

FIBERS

endings.

ends

to some

bag2) in a be ascer-

not

surrounding

activity, in agreement (26). The intrafusal

in

coincidental.

chain fibers in a spindle usually did the same transverse level. It should be noted that spindle

of motor

fusal

three

MUSCLE

ing at the

a nuclear

of several level reflected

two

free

The endtype on

frequently,

occurrence spindle

ob-

transverse fibers

region. “diffuse”

concomitantly

INTRAFUSAL

were

served concomitantly level of two or three

bag1

RAT

sites

of motor

lowed us distribution

mostly

to

innervation

compare of motor

was

in the endings

apparent. for ChE ATPase

alal-

same spindle the along the intra-

sc c

-

A =

-

=7

) 62

B2 #{163}

B1

sc

2.0

-

1.5

1.0

2.0

2.5

1

1.0

1.5

mm

sc B

-..

=

#{163} ‘7/1777//A

....

B2Z

B2 #{163}

Bi

( UPPER: 10.4 LOWER: 4.3 EJ LIGHT MODERATE

C( Sc

1.0 1.0

FIG.

tandem equatorial staining tandem

0.5 1.5

DARK

mm

5. ATPase

staining and motor innervation of intrafusal fibers in the central extracapsular region of two spindles (A and B). In each of the two tandem spindles, a common nuclear bag2 fiber links two regions, which are separated 3500 jim in spindle A and 2350 jim in spindle B. Note different ATPase of nuclear bag2 fibers in spindle A and B. Scale indicates distances from the two equators of each

spindle,

which

are

not

shown.

Other

symbols

as in Figure

3.

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

KUCERA,

982

DOROVINI-ZIS

A

AND

ENGEL

A

BiBj

--

Bi Z

a

-

.

:

.

#{163}

V/7,’/////l V7/7/.

:-:-:

A ‘7/7//A

‘II’

7

B2._

‘22’

‘

#{163}

C( sc 0.5

1.0

0.5

0

mm

1.5

B

r BiZ B2.

-RE’

.-

.

‘II. A

UPPER: 10.4 LOWER: 4.3 LIGHT MODERATE DARK

A

C...

EJ #{163}

A

c____________________________

-

sc 0.5

FIG.

fiber

6. ATPase

derived

bag, fiber “parent”

0

staining

and

by longitudinal

motor

division

0.5 mm

innervation

from

of two

a “parent”

muscle

nuclear

division is shown to the left of the spindle equators bag, fiber. Equatorial nucleation is present in the

“daughter”

fiber

of spindle

B. Note

different

ATPase

spindles

(A,

bag, fiber. and

the

B)

In both

with

spindles,

“daughter”

fibers

a “daughter” intrafusal the site of the nuclear are depicted above the

“daughter” fiber of spindle of the two “daughter” fibers.

staining

A and absent in the Symbols as in Figure

3.

fusal

fibers

with

profile gions

(Figs. of the

3-5 and 6). The nuclear bag2 fiber

their

regional

stant and similar histochemical two ATPase reactions in every

histochemical

encapsulated displayed profile spindle

rea conwith the studied

regardless of the number, motor endings. Likewise,

type and location in the extracapsular

spindle

fiber

regions

ATPase

every

staining

bag2

properties

and

reversed the

staining

profile

in all

spindles

“plate” region

of

its alk-

changeover

occurred in no apparent spatial relation to the sites of motor endings on bag2 fibers. Similarly, the encapsulated inner two-thirds of bag, fibers and the extreme extracapsular polar regions of the bag, fiber were of constant and similar ATPase

though devoid of motor endings. In other spindles, several “plates” were present on the bag, fiber extracapsularly and the most peripheral

regardless

of

the

ending staining

was darkly

Extrafusal

fusal were

situated with

fiber

muscle separable

fibers into

outside alk-ATPase.

histochemistry:

stained light (type

Extra-

II) fibers. The type I fibers comprised about 80% of total fiber population in the rat soleus muscle, as

described

by

Extrafusal romuscular located Dense

others

fiber junctions

(10).

motor endings: of extrafusal

the location and type of motor endings nuclear bag fibers. The location of

Intrafusal scribed above, nuclear bag

fiber ultrastructure: the ATPase staining fibers during their

course

different

often staining

“plate” coincident of bag,

ending

on

bag,

fibers

with the region of the fiber with the alk-ATPase

tion. However, in some region was moderately

was dark reac-

spindles this bag, fiber dark with alk-ATPase

occurred

in thick

lines

nucleated endplate. The esterase-defmed endplates 24-48

jim;

dle

N

=

were

exhibited

tural

The fibers

in the midlength region of soleus accumulation of the ChE reaction

uct

peripheral

bag,

with alk-ATPase I) and dark (type

exact condition of bag, motor endings. However, there may be an exception to the otherwise apparent lack of correlation between the regional intensities of ATPase staining and along the the most

the

when

appearance intra-

and

or

arcades

mean was

neuwere

muscle. prodwithin

length 37 jim

of

a the

(range

50).

from

the

encapsulated.

of bag, extracapsular

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

As properties extracapsular staining The

and

bag2

deof they

ultrastrucfibers

regions

in

was

spin-

also

INNERVATION compared

in the

same

spindle.

chemical evaluation sured proper fiber

of the identification

two

fiber regions, jim from the

jim

representative and 1800-2000

OF

RAT

Parallel

spindle and

histo-

studied selection

about equator,

INTRAFUSAL

asof

500-600 for the

MUSCLE

ultrastructural

examination.

were fibers

in this manner. two different

sampled displayed

pearances intracapsular

983

FIBERS

along their length, region myofibrils

Three

spindle

Individual ultrastructural namely, lacked

poles bag, ap-

that in the an M-line,

FIG. 7. Representative longitudinal sections of a nuclear bag, fiber in its intracapsular (A) and extracapsular (B) regions. An extracapsular “plate” ending on nuclear bag, fiber in another spindle (C). The M-line is present in section B and absent in A and C (arrowheads). Distances from spindle equators (jim): A-650, B-1960, C-1210. Symbols: A-motor axon, N-sole plate nucleus, JF-junctional folds. Frames A, B enlarged 1.5 times compared to C.

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

984

KUCERA,

but

possessed

capsular in both

a distinct

regions. sampled

appearance other fiber

M-line

DOROVINI-ZIS

in extreme

Bag2 fiber possessed regions. Unlike the

extra-

the

an M-line adequate

of myofibrils, the preservation organelles was not optimal

detailed comparative study of their appearance along the length of individual intrafusal fibers. In three spindles the “plate” ending on bag, fibers die for

situated

just

past

capsule was first ChE and then

regions

of the

turally.

In

equator, of the “plate”

termination

of spin-

localized in sections the immediately

fiber

this

the

were

region,

examined

stained adjoining ultrastruc-

900-1200

jim

from

the

the bag, fibers lacked an M-line. In one spindles, thin sections through the bag, at 1210 jim were obtained. It was charof a sole plate folds (Fig. 7).

acterized by the presence and irregular junctional

nucleus

been have

types

of motor

recognized in the been designated

nerve

have

spindle. They P2 plate and

trail motor endings on the basis of their microscopic appearance in teased spindle arations impregnated with silver, and by tron microscopic studies (3, 4). Investigations with the rat, though much less detailed those with the cat, have also suggested the ence plate ings and

of trail endings

motor in the

terminals spindle

light prepelec-

than pres-

and two kinds of (14, 28). These end-

are distinguished by shape, length, location there are differences in the complexity of

their subneural apparatus. tor axons of different origin 28, 30).

Our

study

of the

They and

rat

represent function

spindle

mo(3, 26,

was

chiefly

poles

was

devoid

or three of similar

chain ChE

of motor fibers in appearance,

a

suggesting

that their innervation may be of the type. It is likely that the chain “plate” endings observed by us with ChE staining correspond to “nuclear chain plates” described by

same

Ovalle

(28)

studies Similarly,

of

tochemical lumbrical “plates”, fibers.

in the Mayr

light

and

electron

rat lumbrical (26) in a light

study of teased muscles described in a midpolar location, Mayr

(26)

“plates” had neuromuscular

also

microscopic

muscle spindles. microscopic hisspindles from rat the presence of on nuclear chain

noticed

that

a certain resemblance junctions. Some

the

chain

to extrafusal nuclear chain

cat spindle may have plate endings as p, plates), with axon terminals

in the

(designated ramifying

terminals

cat muscle as p, plate,

fiber

chain

The two had endings

fibers

DISCUSSION

Three

ENGEL

two

endings. spindle of a

for

AND

upon

a nucleated

endplate

and

sup-

plied by branches of extrafusal motor axons (18). No such mixed skeletofusimotor innervation has been demonstrated for the rat soleus muscle spindle as yet (3). Alternatively, the “plate” endings on rat soleus chain fibers could be supplied by fusimotor An unexpected

axons outcome

with static function of our study was

(3). that

each nuclear chain fiber pole possessed not more than one motor ending area identifiable with the ChE staining. The chain fibers of cat and rabbit spindles are multiterminally innervated, and both

plate fiber

same and

and trail endings (3). In fact, Ovalle

Smith

endings

(30) occur

lumbrical differences different

thought on

can occur on the (28) and Porayko

that

chain

both

fibers

trail

and

in spindles

plate of rat

muscles. Conceivably, there may be in spindle motor innervation among rat muscles. Our study has dealt only

concerned with the distribution of motor endings along the three types of intrafusal muscle fibers and with the correlation of their location with the ATPase staining profiles of the fibers. This

with the soleus muscle. Unlike nuclear chain fibers, nuclear bag, and bag2 fibers were multiterminally innervated and endings of different histochemical appearance

necessitated

coexisted

a serial-section

histochemical

proach with its inherent limitations to motor ending identification.

will

comments respondence

motor The

between

endings motor

was

the

fiber

types.

ChE

received

a “plate” aggregation

least

as to the possible

be made

endings on intrafusal and the conventional

the

appearance

variable pole

a nerve appearance, of fiber

and trails. of nuclear

among of a chain terminal

the

the

cor-

of motor

fibers in staining classification

into plates innervation Each

ap-

with respect Nevertheless,

for ChE of spindle chain

rat

fiber

intrafusal stained

associated

and generally nuclei. Occasionally,

fibers for with with an one of

on

simple

a

given

intrafusal

It is likely

fiber

pole.

fiber

endings

that

classified

by

us as “diffuse” in ChE staining correspond pleomorphic fusimotor trail endings as described in teased

silver

preparations

of the

cat

spindle (3, 28). Trail endings are known in comparable locations in juxta-equatorial dle regions and static fusimotor naked without apparatus

axons the and

post-junctional suggests that

are innervated axons (3, 5).

and

to rat

to occur spin-

by branches They consist

of of

closely applied to intrafusal fibers presence of a discrete subneural having

no,

folds (3, both nuclear

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

or only 5,

14, bag,

rudimentary, 28). and

Our bag2

study fibers

INNERVATION receive the

trail

larger

vation

in the

terminals

share

going

rat

to bag,

is of interest

bag1 fibers in the cat spindle endings, an assertion disputed

5).

“Diffuse”

on

bag2

to

other

the

often

ap-

of bag, fibers. is reflected by of the endings

factors,

as greater

such

distance

from

fusimotor on both

axons can branch to form trail endings bagi and bag2 fibers in the cat spindle

It is not

(5).

applies The

spindle

to Boyd

fibers

equator.

whether

known

a similar

rat spindle. of our “plate”

to the identity

Single

bag1 and described

bag2 fibers is less certain. plate endings on bag

lumbrical

muscle

spindle.

plex

light

and

than poorly

his

“nuclear

light

endplate

com-

with

area.

only

Mayr

material nuclear capsulated

of cat

appearance

longer

than

plates

on

de-

fibers.

some of the “plates” that bags in the outer two-thirds regions of spindles were

than those on “plates” located

chain fibers. extracapsularly

istically

shorter.

This

separate

kinds

might

bers. Correlative and functional “plate”

that

present

and In our

of two

rat

bag

on

rat

bag

fibers

into

fi-

extracapsular

fiber

may

axons appeared and

in

represent

spindle

intracapsular to represent static

the

endings

Nuclear responses

on

distinct

the

because bag2 of comparable

bag2

fibers

to

“plate” endings the termination

bag,

(3)

in

and

the

on them are likely of motor axons with

actions.

In the cat spindle, an occasional trail fusimotor ending on bag fibers in the outer two thirds of

spindle-encapsulated

region

may

be

rather

motor

and

rather

endings

is

robust

of

histochemical

appearance

suggests

different

appearance

in ChE staining every bag1 pole

that

multiterminally

innervated

innervated.

but

Although

muscle

to

fiber

this

along is not

also

poly-

may

properties

apply

responsible

reactions are neuron trophic histochemistry

be similarly

to one ending clear bag fibers,

in this

dependent

on a fiber the dark

thought to influences was sug-

on

its

motor

pole. Considering alk-ATPase staining

nuof

fiber region close to the termination of capsule may have been in some way to the “plate” ending frequently present so, the

occasional

fiber

bagi

absence

region

might

by a postulated ongoing struction of spindle motor this

bag1

degree

fiber of

of an ending

be accounted

process of terminals

region, ATPase

for

the recon(3). Apart

however,

a consid-

staining

uniformity

among rat spindles was maintained despite variability in the number, type and location of motor endings along the bag, and bag2 fibers. It appears that factors other than the aspects studied of spindle motor innervation, may govern the regional nuclear bag staining

fibers length

participate

stretch

fiber

were

It

endings

innervation (32, 34). This may apply to nuclear chain fibers which have a homogenous histochemical profile and motor innervation limited

erable

of dynamic

termination

the rat spindle devoid of endings

location.

static

“plate”

gested

from

entities. Some aspects of muscle spindle response to applied stretch are controlled by dynamic motor axons which terminate exclusively on bag1 fibers in plate endings (4, 6). Our study suggests that the

6).

of our “diffuse-plate”

for the ATPase staining be mediated by motor (17). Intrafusal fiber

there.

on

a plate

(3,

are considered to represent morphologically and functionally different parent motor axons (3, 4, 5, 6). The invariable occurrence ofmotor endings

the bag, characterplates

from

microscopy

classification

morphological

a bag, spindle related

histochemical, morphological studies will be needed to separate

endings

of uncertain trail endings. Intrafusal

occurred on of the enalso longer

However, were

suggests be

plates

P2

chain

some

Extrafusal a

(26)

to differentiate light

to the majority of bag2 fibers also, some poles of bag2 fibers had only one ChE-active area. A nuclear chain fiber pole had never more than one ChE-active area.

structure

plates”,

that

neuronally

plates in the intraand extracapsular on nuclear bag fibers (not further in teased preparations of spindles of rat muscles. He noted that they had the

microscopic

were

ofless

possible

985

FIBERS

difficult

through

only

Ovalle (28) fibers of rat

were

and

ending

of different bag1 fibers

on nuclear

microscopic

chain

developed

scribed locations typed) lumbrical

They

electron

static

situation

endings

MUSCLE

robust

obser-

do not receive by others (4,

peared more robust than those We do not know if this difference some variation in the innervation is related

INTRAFUSAL

with

This

according

(6), trail

or

RAT

spindle,

fibers.

because,

endings

OF

ATPase fibers

differences

staining especially between

differences in regard their

along to the

intra-

and

tracapsular regions. We have studied intrafusal ATPase reaction because of the

fibers with consistency

reproducibility

But,

of the

staining.

chemical profile of intrafusal fibers along their length with phosphorylase

the also

exthe and

histo-

varies staining

and the reaction for glycogen (2, 4). In twitch muscles of the cat, the intensity of the histochemical ATPase reaction is correlated with the contraction time of individual motor

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

KUCERA,

986

units

with

darker

the

in the rat

and

faster

nuclear

Thus

the

in

comparative bers

with

fibers

bags, times

encapsulated intensity

chain

regions,

the fithe

parallels

the bag staining

fiber

poles

fibers intra-

can

in view of and extra-

contract

simi-

ENGEL

equator

There

were in the

ences bag,

fibers

Barker

devoid when

the

of

known

regions

whether

of nuclear

the

bag

motor endings participate more central, innervated fiber

shorten. It has been suggested that fiber polar regions away from motor may be activated passively by stretch

fibers

actively regions some

bag

terminals imposed

by actively contracting regions (1). The pattern of extrafusal muscle fiber activity alone has been proposed to account for neurotrophic influences, and fibers may undergo alterations in histochemical appearance as a result of exercise (25, 29). If similar considerations apply to intrafusal differences in the during contraction

fibers, regional participation flected

in ATPase

staining

clear bag fibers. The degree of stretch on a muscle is known staining

properties

under

certain

sion parts,

than and

their In

ATPase staining. each of the two two

gle

nuclear

dle

with

the

two the

fiber.

muscle (33).

tandem

ATPase

or

attain

The

bag fior ten-

spindles

Only

the

bag,

gions ering

fiber.

M-line

to be absent

equator, more

although distant rat

variability or to “plate” ending on

change

between

differences of interest

in the

the

bag2

to

M line fiber

other

re-

Considthe ATP-

along nuclear bag to know if regional subcellular

fibers, vari-

organelles

the

supply (23). Among bag1 fiber splits most

denervation divided study

transient represented fiber. In two

ferent differed

(23).

It is not

bag, fibers had originated

disturbance primary the spindles

the intrafusal readily after clear

whether

encountered in the as a result of a

of spindle motor supply or formation of a branched with divided bag, fibers,

daughter

fibers

ATPase in their

staining sensory

displayed

strikingly

properties. innervation,

dif-

They also only one of

them being innervated by a primary sensory axon. Since the daughter fiber with sensory innervation present was the histochemically unu-

more

widely

the

similar

noted

applies

spindle

regions

from

of my(2) and

that differed in ATPase staining. the morphological substrate for

dle motor fibers, the

have about

distance

a feature et al.

a feature of normal spindles (3) although splitting of intrafusal fibers can be induced experimentally in adult rats by interfemng with spin-

sual

do extrathat the of certain

the

in the

fiber myofibrillar ATPase, but also by mitochondrial or sarcotubular ATPase activities, an impression not shared by others (21). Longitudinal division of intrafusal fibers is not

spinits two

were Banks

the

lacking

also. Guth (16) has suggested that the staining intensity of the histochemical “myofibrillar” ATPase reaction may be influenced not only by

a sin-

fiber change between the

differalong

The basis of the M-line varibut it does not appear to

No

was

ability

tandem

bag2 staining

a certain

encounalong

but

were

relate to fiber ATPase staining the location of the peripheral

in the

occurred

equators in the way bag2 fibers usually capsularly. This observation suggests bag2 fiber may need to be, either length,

fibers

M-lines

bag, fiber regions. ability is unknown,

the two present

encapsulated countermight be reflected in

equatorial common

nu-

imposed ATPase

conditions

units

equatorial

apart did extracapsular

along

processes of nuclear to different stretch

central differences

bag2

of fiber be re-

and/or tension to influence the

experimental

extracapsular may be subject

tered,

differences

of extrafusal

long bers

their such

degree could

stain-

ultrastructural of myofibrils

et al. (4) reported

ase staining it would be

It is not

in ATPase

up to about 1500 jim from the they apparently did not sample

contractions

(6, 20).

regional

intracapsular regions ofibrils extracapsularly.

condition

extracapsular

change

appearance in that

larly to extrafusal fibers (20) in response to propagated action potentials. Nuclear bag fibers are thought to be capable only oflocal, graded tonic entire

for the

ing to occur.

rap-

of intrafusal

reaction

speed occur along different ATPase

capsularly. Nuclear

more

AND spindle

cat

fibers in the same manfibers. It would be of differences in contrac-

contraction speed of the ner as with extrafusal interest to know whether tion their

being Both

bag2 fibers have bag, fibers (6, 31).

staining alk-ATPase

(7).

contract

and than

spindle

the

fibers

reaction

chain

than nuclear contraction

idly faster

contracting

alk-ATPase

DOROVINI-ZIS

one, been the

Sensory velopment in new-born

it appears

the

sensory

axon

rats

terminals

are

immature,

not differentiate 12 days after are

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on June 18, 2015

found

may

way in bringing profile.

axons are thought to initiate of rat muscle spindles (3).

muscle fibers do fiber types until fusimotor

that

instrumental in some unusual histochemical

and into birth

the deSpindles intrafusal distinct (27). First

on developing

in-

INNERVATION trafusal

myotubes

Spindles

in rat

their

motor

acquire

within

12 hr

muscles

after

at

their

RAT

birth

irreversibly

innervation

during

OF

INTRAFUSAL

(27).

deprived

birth

of

subsequently

postnatal

development

a

degree of histochemical and ultrastructural differentiation into distinct intrafusal fiber types (35). That observation led Zelena and Soukup (35) to suggest that differential ATPase staining of intrafusal

fibers

as ultrastructural

as well

ferences among the fiber types the spindle sensory innervation, preserved in their experiments. time

of Zelena

and

Soukup’s

cal and lengths

ultrastructural of the intrafusal

appreciated,

and

study,

not

the not

along were

known

and morphological develop despite neo-

natal

deprivation

de-efferentation

and

ent nerve impulse is the possibility nerve influences distance account logical

and extracapsular nuclear bag2 to develop, a primary

fiber with interest

that

contacted

staining Our and

dark

the

by

of nuclear

unrelated

and

to

the

location

role fiber

of other activity,

interaction

and to

suggests a large type

be that

9.

11.

12.

13.

14. the that 15.

extent of

motor

factors such as intratension, or sensory fiber

influences to

histochemical along

vary

intrafusal

remain conditions

8.

bag2

if further

as

and

with

7.

occur.

that

are

in determining the

motor nerves experimental rate

not

fibers

variations

endings. The fusal muscle nerves

bag

of nuclear

axons, did

study demonstrates ultrastructural properties regional

contact It is of

regions with the two to staining displayed (11) at the time they

sensory

6.

intrafusal

in close (3, 35).

staining

differentiation

lengths

first

and it forms sensory axon the

as

10.

regions. fiber is the

fibers in the encapsulated ATPase reactions is similar by developing myotubes are

there sensory a limited

from spindle equator. If so, that could for different histochemical and morphoproperties of nuclear bag fibers in the

intraThe

1-190

of effer-

activity. Nevertheless, that morphogenetic may extend only for

16.

properties mediated

elucidated allow their

by

17.

under sepa-

study.

18.

LITERATURE

CITED

1. Arbuthnott ER, Boyd IA, Gladden MH, McWilliam PN: Real and apparent -y-axon contraction sites in intrafusal fibers. J Physiol 268:25P, 1977 2. Banks RW, Harker DW, Stacey MJ: A study of mammalian intrafusal fibers using a combined his-

987

4. Barker D, Banks RW, Harker DW, Milburn A, Stacey MJ: Studies of the histochemistry, ultrastructure, motor innervation, and regeneration of mammalian intrafusal muscle fibers. Prog Brain Res 44:67, 1976 5. Barker D, Emonet-Denand F, Harker DW, Jami F, Laporte Y: Distribution of fusimotor axons to intrafusal muscle fibers in cat tenuissimus spindles

whether

the complex histochemical profile of nuclear bags can

FIBERS

tochemical and ultrastructural technique. J Anat 123:783, 1977 3. Barker D: The morphology of muscle receptors, Handbook of Sensory Physiology, Vol. 111/2. Edited by CC Hunt, Springer-Verlag, Berlin, 1974, p

histochemi-

differences fiber types

it is still

dif-

is determined by which remained However, at the

MUSCLE

19.

20.

determined

by

the

glycogen-depletion

method.

J Physiol 261:49, 1976 Boyd IA: The mechanical properties of dynamic nuclear bag fibers, static nuclear bag fibers and nuclear chain fibers in isolated cat muscle spindles. Prog Brain Res 44:33, 1976 Burke RE, Levine DN, Zajac FE, Tsairis P, Engel WK: Mammalian motor units: Physiological-histochemical correlation in three types in cat gastrocnemius. Science 174:709, 1971 Couteaux R: Particularit#{233}s histochemiques des zones d’insertion du muscle stri#{233}. C r S#{233}anc Soc Biol 147:1974, 1953 Dubowitz V, Brooke MH: Histological and histochemical stains and reactions. Muscle Biopsy. W. B. Saunders Co., London, 1973, p 20-33 Eisen A, Karpati G, Carpenter 5: Reserpine induced alteration of physiological properties and histochemical fiber types in rat skeletal muscle. Exp Neurol 46:554, 1975 Engel , Karpati G: Impaired skeletal muscle maturation following neonatal neurectomy. Dev Biol 17:713, 1968 Er#{228}nkd 0, Ter#{228}v#{228}inen H: Distribution of esterases in the myoneural junction of the striated muscle of the rat. J Histochem Cytochem 15:399, 1967 Farrell PR, Fedde MR: Uniformity of structural characteristics throughout the length of skeletal muscle fibers. Anat Rec 164:219, 1969 Gladden MH: Muscle spindle innervation in the intertransverse caudal muscles of the rat. Experientia 25:604, 1969 Guth L, Samaha FJ: Procedure for the histochemical demonstration of actomyosin ATPase. Exp Neurol 28:365, 1970 Guth L: Fact and artifact in the histochemical procedure for myofibrillar ATPase. Exp Neurol 41:440, 1973 Guth L, Samaha FJ, Albers RW: The neural regulation of some phenotypic differences between the fiber types of mammalian skeletal muscle. Exp Neurol 26:126, 1970 Harker DW, Jami L, Laporte Y, Petit J: Fast conducting skeletofusimotor axons supplying intrafusal chain fibers in the cat peroneus tertius muscle. J Neurophysiol 40:791, 1977 Hess A: Two kinds of motor endings on mammalian intrafusal muscle fibers revealed by the cholinesterase technique. Anat Rec 139:173, 1961 Hunt CC: The Physiology of Muscle Receptors. Handbook of Sensory Physiology. Vol. 111/2. Ed-

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KUCERA, ited

by CC Hunt,

Springer-Verlag,

Berlin,

DOROVINI-ZIS

AND

1974, p

191-234 21.

22.

23.

24.

25.

26.

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28.

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29.

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34.

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ENGEL

fusal muscle fibers, a correlated light and electron microscopic study. J Anat 111:239, 1972 Pette D, Smith ME, Staudte HW, Vrbova G: Effects of long-term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles. Pfluegers Arch 338:257, 1973 Porayko 0, Smith RS: Morphology of muscle spindles in the rat. Experientia 24:588, 1968 Smith RS: Properties of intrafusal muscle fibers, Muscular Afferents and Motor Control. Edited by R Granit, Almqvist & Wiksell, Stockholm, 1966, p 69-80 Yellin H: Unique intrafusal and extraocular muscle fibers exhibiting dual actomyosin ATPase activity. Exp Neurol 25:153, 1969 Yellin H: Changes in fiber types of the hypertrophying denervated hemidiaphragm. Exp Neurol 42:412, 1974 Yellin H: Regional differences in the contractile apparatus of intrafusal muscle fibers. Am J Anat 139:147, 1974 Zelena J, Soukup T: The differentiation of intrafusal fiber types in rat muscle spindles after motor denervation. Cell Tissue Res 153:115, 1974

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