J. Phy8iol. (1969), 204, pp.

583-592 583
With 3 plate and 2 text-figurem
Printed in Great Britain

DISTRIBUTION OF
GRANULAR VESICLES IN NORMAL AND CONSTRICTED
SYMPATHETIC NEURONES
BY L. B. GEFFEN AND ANNA OSTBERG
From the Department of Physiology, Monash University,
Clayton, Victoria, Australia
(Received 19 February 1969)
SuMARY
1. A quantitative analysis has been made of the characteristics of
granular vesicles in different parts of normal and ligated post-ganglionic
sympathetic neurones supplying the cat spleen. The least diameter of the
vesicle membrane and its electron-dense core was measured from electron
micrographs with an automated particle size analyzer.
2. Vesicles of average diameter 443A, with and without electron-dense
cores (192 A), were concentrated in the axon terminals of the spleen, whereas
in the cell bodies in the coeliac ganglion and their axons there were only a
few large vesicles (787A) with large granules (562A).
3. Ligation of the splenic nerves for 24 hr resulted in an accumulation
proximal to the constriction of granular vesicles significantly larger
(688 A) and more completely granulated (478 A) than those in the terminals.
4. Reserpine reduced the number of vesicles with granular cores without
discrimination between the two populations.
5. Since the distribution of noradrenaline in sympathetic neurones can
be correlated with the presence of large as well as small granular vesicles,
it is suggested that the larger, more granular type found mainly proximally
in the neurone is in transit from its perikaryal site of synthesis to the axon
terminals, where it is transformed into the smaller synaptic vesicles.

INTRODUCTION
Although it is widely recognized that there are at least two types of
granular vesicles in post-ganglionic sympathetic nerve terminals, only the
smaller variety 300-600A in diameter, which form the vast majority, have
been thought to be important storage sites for noradrenaline (see Potter,
1967). Noradrenaline, however, is distributed not only in the axon ter-
minals but also in the cell bodies and preterminal axons of post-ganglionic
584 L. B. GEFFEN AND ANNA OSTBERG
sympathetic neurones, where granular vesicles occur much more sparsely
and have diameters of between 600 and 1500 A (Taxi, 1964; Grillo, 1966).
In ligated sympathetic nerves, granular vesicles accumulate just
proximal to the ligature (Kapeller & Mayor, 1967), as does noradrenaline
fluorescence (Dahlstrom, 1965). The present study compares the sizes of
granular vesicles in different parts of normal and constricted splenic
neurones to examine whether the distribution of noradrenaline also corre-
sponds to that of the larger type of granular vesicle.

METHODS
Tissues for electron microscopy were taken from six cats. The major branch of the
splenic nerves had been constricted under ether anaesthesia 18-24 hr previously, by
crushing the nerves with a fine silk ligature against a glass rod (see Geffen & Rush,
1968). The coeliac ganglion, the constricted and normal branches of the splenic
nerves, together with the corresponding portions of spleen, were removed and fixed
for 15 min in cold 2-5 % (v/v) osmic acid, buffered with ice-cold modified Dalton's
chromate buffer (Richardson, 1962). The tissue was then trimmed and returned to
the cold fixative for a total fixation time of 4-4k hr. After washing in distilled water
and dehydrating rapidly in graded solutions of acetone, it was embedded in Araldite
and polymerized at 370 C.
Initially, thick sections (2 #c) were cut to establish the orientation of the tissue
and they were stained with toluidine blue to check the quality of the fixation and
impregnation. Thin sections (600-800 A) were cut on a Cambridge ultra-microtome,
mounted on carbon-coated 75- or 200-mesh copper grids, stained with uranyl acetate
followed by lead citrate, and then viewed and photographed in a Siemens Elmiskop
I electron microscope at magnifications of 10,000, 20,000 and 30,000.
Every vesicular structure in the axons, irrespective of whether it contained a
definite granule or not, was measured provided the 'electron density' of the vesicle
contents exceeded that of the surrounding axoplasm. Measurements were made from
prints on document paper (Ilford, Document 60) with a Zeiss Particle Size Analyser
(TGZ 3) using the reduced scale and linear bin size. The enlargement of document
prints was adjusted to give a vesicle size on print of 1-9 mm. The vesicle was measured
by fitting the size of the circular light spot to the smallest outside diameter of the
vesicle. Each measured vesicle was perforated and its size automatically recorded in
one of 48 bins. The accuracy of the measurement by the method ranges from 30 %
for the smallest to 2 % for the largest bin. The interval limits were then divided by
the total magnification of the print, and the distribution histogram plotted in
Angstrom units.
Reserpine (Serpasil, Ciba) was given intraperitoneally in a single dose of 5 mg/kg
6 hr after ligating the splenic nerves and 18 hr before removal of the tissues.

RESULTS
Normal and constricted axons. The normal unmyelinated axons of cat
splenic nerves have been described in detail by Elfvin (1958, 1961). They
are characterized by mitochondria, neurofilaments and neurotubules and a
very sparse vesicle population. The vesicular elements seen in cross-section
 ORIGIN OF ADRENERGIC SYNAPTIC VESICLES 585
varied greatly in size and occasionally had an electron-dense core. These
granulated vesicles had a mean diameter of 700A, which is greater than
the average diameter of 450A noted by Elfvin (1958) and close to 750A
as reported by Kapeller & Mayor (1967).
When the splenic nerves were ligated for 24 hr their appearance was
very similar to that described by Kapeller & Mayor (1967). For 1 mm
proximal to the constriction there was an accumulation of mitochondria,
tubular and vesicular elements in the swollen axon profiles (PI. la). The
number of larger, granulated vesicles was greatly increased in many of the
axons. Their mean diameter was 688A and their core size averaged 478A.
Some of these had ill-defined cores but were easily distinguished from the
electron-lucid structures, whose diameters usually exceeded 1000A. The
latter may have been swollen neurotubules since they often had elliptical
or cylindrical profiles.
The axons for 1 mm distal to the site of constriction were empty by com-
parison (PI. 1 b). They contained only a few granulated vesicles and mito-
chondria and there were none of the blown-up tubular elements which were
so prominent proximal to the constriction.
Axon terminal of intact and constricted nerve. The axon varicosities of
the spleen contained, in addition to mitochondria and neurotubules, both
granular and agranular vesicles (PI. 2b). The granular variety included
two types: a few large vesicles of comparable size and morphology to those
seen in the axon, but with less dense cores, and a majority of smaller vesicles
of mean diameter 443 A, with irregular, intensely osmiophilic cores
averaging 192A in diameter. The few agranular vesicles were the same size
as the small granular ones.
The morphology of the vesicles in portions of the spleen whose nerves
were intact or ligated for 24 hr appeared identical. The innervation of the
smooth muscle of the spleen was sparse (Fillenz, 1966), and it was difficult
to find large numbers of vesicles in the varicose portions of the axons.
Since there was no decrease within 24 hr of the noradrenaline content of
the ligated nerves (Geffen & Rush, 1968) the micrographs of axon terminals
from both portions of spleen were pooled for vesicle size estimations.
Ganglion cell bodice. The cytoplasm of the sympathetic ganglion cell
bodies was packed with organelles. There were numerous membrane-
bound lysosomes (up to 0-4 , diameter), dense granules without mem-
branes (about 0-2 iu), mitochondria, and smooth and rough endoplasmic
reticulum with rosettes of small granules. Occasional vesicles with large
dense granules were found, particularly in association with the Golgi
apparatus (PI. 2a). They were similar to those seen in the constricted axons
but were slightly larger, with a mean membrane diameter of 787A and a
mean core diameter of 562A.
586 L. B. GEFFEN AND ANNA OSTBERG
There was also a large number of agranular 'vesicles' which could have
been cross-sections of convoluted tubules in continuity with the mem-
branes of the Golgi apparatus since they were often aligned in rows. Their
diameters varied greatly (mean 453 A) and they had agranular but generally
more electron-dense interiors than the surrounding cytoplasm.

TABLE 1. Size distribution of vesicles and their granules in

different parts of cat splenic neurones
Membrane
Soma Axon Terminal
Mean diameter (A) 787 688 443
+ S.D. 175 136 113
n 21 661 679
Relative size (%) 100 87 62
P 0.001 0'001
Proportion of vesicles - 0-61 0.46
with granules

Granule
Soma Axon Terminal
Mean diameter (A) 562 478 192
+ S.D. 120 102 87
n 15 360 267
Relative size (%) 100 85 34
P 0 001 0'001
Granule-to-membrane 0 70 0-43
ratio
P is the level of significance of the difference between the means on a Student t test.
The values given for the axon are from constricted nerves.

Reserpinized nerves. Reserpine decreased the number of large granulated

vesicles occurring in constricted axons and decreased the core density of
those surviving (PI. 3). There were also numerous multivesicular bodies
and lysosomes as well as degenerating structures difficult to identify. The
problem of locating nerve varicosities in the spleen made it hard to evaluate
the extent to which reserpine affected the smaller vesicles. However, in
those nerve profiles definitely identified, reserpine in the dose used did not
have a differential effect on the smaller vesicles alone.
Size distribution of vehicles and granules. The analysis of the vesicle
 ORIGIN OF ADRENERGIC SYNAPTIC VESICLES 587
diameters and their granular cores, based on the measurement with the
Zeiss particle analyser, is given in Table 1. The frequency histograms of the
vesicle and granule diameters are shown in Figs. 1 and 2 respectively, with
the terminal axons' distribution above and the constricted axons' below.
The mean diameters of both the membranes and granules of the vesicles
in the ganglia and in the constricted axons were significantly larger than
those in the axon varicosities of the spleen. In the axon terminals of the
spleen there were a few larger vesicles whose membrane and granule
diameters clustered near the mode of the corresponding histogram from the
constricted axons, but there were insufficient numbers to test whether they
constituted a separate population.
100 -
a

75 -

50 -

U 25
0Iv

01
LL.
I
-
I .. *.........
I 1
|l|XrI I
00 300 500 700 900 1100 1300
b

25

0 100 500300 700 900 1100 1300

Vesicle diameter (A)
Text-fig. 1. Frequency distribution histograms of the vesicle membrane
diameters from (a) axon terminals, (b) ligated axons and ganglion cell
bodies (shaded) of splenic neurones.

A higher proportion (54 %) of the vesicles measured in the terminal

varicosities lacked a definite granule compared to those in the constricted
axons (39 %), and in those with granules the core occupied 70 % of the
volume of the vesicles above the constriction compared to only 43 % of the
interior of those in the axon terminals.
588 L. B. GEFFEN AND ANNA OSTBERG

DISCUSSION
The results indicate that the granular vesicles of the cell bodies and con-
stricted axons are significantly larger than those commonly occurring in
the axon terminals, in terms of the diameter of both the vesicles and their
electron-dense cores. The average diameters of the membrane and granule
of the vesicles decreased significantly from the soma to the axon to the
terminal varicosities. This tendency was more marked peripherally, espe-
cially for the granules.

U
Ar Th-

25 -
-50-
b

25 -

12 Stir..
Granule diameter (A)
Il

Text-fig. 2. Frequency distribution histograms of granular core diameters

from vesicles in (a) axon terminals, (b) ligated axons and ganglion cell
bodies (shaded) of splenic neurones.

One interpretation of the results is that the two populations of vesicles

represent different states of maturity. The larger vesicle, more completely
filled with osmiophilic material, which occurs in the perikarya and preter-
minal axons, may be a precursor of the synaptic vesicle, in transit from its
site of synthesis in the ganglion along the nerves to the axon terminals.
Here it could either bud or gradually become smaller in the process of
repeatedly turning over its noradrenaline content during impulse trans-
mission, for, while the noradrenaline stores in peripheral nerves turn over
at least daily (Axelrod, 1963), a turnover time for the vesicles of the order
 ORIGIN OF ADRENERGIC SYNAPTIC VESICLES 589
of 50-100 days has been calculated (Dahlstrom & Haggendal, 1966;
Geffen & Rush, 1968). Similar explanations have been invoked to explain
the variation in the morphology of granular vesicles during activity in the
cat pineal gland (Bondareff, 1965) and the guinea-pig intestine (Tafuri,
1964).
Evidence in support of somatofugal transport of granular vesicles con-
taining noradrenaline comes from studies of doubly ligated nerves in
which endogenous noradrenaline (Dahlstrdm & Haggendal, 1966),
granular vesicles (Mayor & Kapeller, 1967), and radioactive noradrenaline
and protein (Livett, Geffen & Austin, 1968), all accumulated above the
proximal but not the distal ligature. Reserpine depleted both the accumu-
lated noradrenaline (Dahlstr6m, 1967) and the granular contents of the
vesicles (Kapeller & Mayor, 1967). Recovery after reserpine of nor-
adrenaline (Dahlstr6m, Fuxe & Hillarp, 1965) and of granular vesicles
(Hassler & Bak, 1966) occurred first in the cell body and later in the nerve
terminals in unconstricted nerves. In doubly ligated nerves, recovery
occurred only above the proximal ligature, and was faster the closer the
ligature was to the cell bodies (Dahlstr6m, 1967).
Additional support for the concept that the large granular vesicles are
involved in noradrenaline storage comes from subcellular fraction studies.
Noradrenaline-containing particles of various sizes and densities with
many properties in common have been isolated from adrenal medulla
(Blaschko & Welch, 1953), sympathetic ganglia (Fischer & Snyder, 1965;
Phillipu, Pfeiffer, Schumann & Lickfield, 1967) and sympathetic nerve
trunks (Euler & Hillarp, 1956) as well as from sympathetically innervated
tissues (see Potter, 1967). The particles isolated from bovine splenic nerve
trunks are denser than those from axon terminals in tissues like the rat
heart, although a corresponding 'heavier' noradrenaline storage particle
has recently been found in rat heart homogenates (Roth, Stjirne, Bloom
& Giarman, 1968).
Among the reasons why large granular vesicles have been thought to
differ qualitatively from the smaller type is that they have been found
resistant to reserpine depletion (Bondareff, 1965; Bloom & Barrnett,
1966; Hdkfelt, 1966; Clementi, Mantegazza & Botturi, 1966). However,
Van Orden, Bloom, Barrnett & Giarman (1966), in their experiments on
depletion of granular vesicles in the vas deferens with reserpine and with
nerve stimulation, could not detect a differential effect on the two types.
A similar result was obtained in the present study. The reported relative
resistance to reserpine of the larger granular vesicles may be due to greater
initial quantities of transmitter and its binding substances.
Alternative explanations for the presence of large granular vesicles in
590 L. B. GEFFEN AND ANNA OSTBERG
sympathetic neurones have been proposed. They occur throughout the
autonomic nervous system in both pre- and post-ganglionic nerves, of all
species studied, whereas the smaller type has been found only in post-
ganglionic mammalian nerves (see Grillo, 1966). The large variety may
contain other biogenic amines (Burnstock & Robinson, 1967) or they may
have a role to play in the regeneration of nerves since they also occur in
ligated myelinated axons (Pellegrino de Iraldi & de Robertis, 1968).
At present it is not possible to choose with confidence among these and
other explanations, and recourse may eventually have to be made to
electron-microscope radioautography using labelled antibodies prepared
against components of the small granular vesicles. The use of such anti-
bodies for fluorescence staining is described in the following paper (Geffen,
Livett & Rush, 1969).
We thank Dr Mollie E. Holman for her interest and help, and Messrs R. Rush,
R. Simmonds and Miss D. Harrison for technical assistance. The work was supported
by grants from the National Heart Foundation of Australia.

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592 L. B. GEFFEN AND ANNA OSTBERG

EXPLANATION OF PLATES
PLATE 1
Cat splenic nerves ligated for 24 hr and sectioned less than 1 mm (a) proximal and
(b) distal to the constriction. Proximally, the axons are very swollen and packed
with organelles including large granular vesicles (lgv), electron-lucid tubules (elt),
mitochondria (m), and a few myelin figures (mf ). Distally, the axons are not distended
and contain only a few mitochondria and collapsed tubular elements, but no granular
vesicles. x 28,000.
PLATE 2
A comparison of the granular vesicles in (a) the region of the Golgi apparatus of the
perikaryon and (b) axon terminals in the capsule of the spleen. There are both large
(lgv) and small (8gv) granular vesicles in the axon terminals, whereas in the peri-
karyon there are a few large granular vesicles and a population of smaller agranular
vesicles (av). x 80,000.
PLATE 3
The effect of reserpine on the granular vesicles proximal to an axonal constric-
tion: (a) untreated showing numerous large granular vesicles (Igv) almost filled with
electron-dense cores; (b) 18 hr after reserpine, the granules have gone and have often
been replaced by one or more membrane-bound droplets within the vesicle. The mito-
chondria (m) and other organelles show damage and there is an increase in the
number of multi-vesicular bodies (mvb) and myelin figures (mf). x 40,000.
 The Journal of Physiology, Vol. 204, N.7o. 3 Plate 1

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L. B. GEFFEN AND ANNA OSTBERG (Facin~g p. 592)

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L. B. GEFFEN AND ANNA OSTBERG

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L. B. GEFFEN' AND ANNA OSTBERG