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Quantitative ultrastructural analysis of the periaqueductal gray in the rabbit.

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THE ANATOMICAL RECORD 236573-585 (1993)
Quantitative Ultrastructural Analysis of the
Periaqueductal Gray in the Rabbit
S.T. MELLER AND B.J. DENNIS
Department of Physiology, The University of Adelaide, South Australia, Australia
ABSTRACT
The fine structure of the periaqueductal gray (PAG) of the
rabbit was examined using the transmission electron microscope. On the
basis of synaptic polarity, vesicle size, and the nature of the pre- and postsynaptic elements, 10 essentially different synaptic types could be discerned (6 axo-dendritic, 2 axo-somatic, 1 axo-axonic, and 1 dendro-dendritic). Synaptic contacts on the soma of PAG neurons were small and
covered, on average, only 1.6%of the soma surface. The most striking feature of the synaptic structure of the PAG was that more than 94.1%of all
synapses were axo-dendritic. Of these, 83.5% were of the symmetrical type.
Most of these contacts occurred on buttons of small to medium size, and
contained either round vesicles of medium size or pleomorphic vesicles of
medium size. Boutons containing only flattened vesicles were quite rare.
Boutons contacting larger dendrites were generally small-to-medium in
size, made asymmetric-type synaptic contacts, and contained pleomorphic
vesicles of medium-to-large size. Medium-sized dendrites were contacted
principally by small boutons exhibiting either symmetrical or asymmetrical junctions containing medium-sized pleomorphic vesicles, and in addition a few of these boutons contained both large, and small, round vesicles.
Dendritic spines were generally provided with only one synaptic contact,
stretching the entire width of the spinous process. Boutons and the spines
on dendrites were approximately the same size. Synapses between two vesicle-containing structures (axo-axonicor dendro-dendritic synapses) were
rare (1.4%).They were generally asymmetric and contained round vesicles
of medium size. Complex synapses, where a glial sheet enclosed the synapse, were occasionally observed. Also seen were multiple synapses, with
up to 11 contacts on a single dendritic profile. Large dense-core vesicle
were seen in approximately 40% of all synapses, whereas small dense-core
vesicles were only found in about 3%. Data is provided on how different
synaptic features relate to ventral, lateral, dorsal, and medial PAG. Principally this is in relation to neuron size, glia cell content, axonal characterization, and vesicular type. o 1993 Wiley-Liss, Inc.
Key words: Neuropil, Synapse, Quantitative, Neuron, Vesicle
The periaqueductal gray (PAG) is a region of recent
interest due to the great variety of functional roles
which have been attributed to it (see Beitz, 1985;
Meller and Dennis, 1986; Meller and Dennis, 1991).
There have been a number of reports on the ultrastructure of the PAG in the cat and rat (Hamilton et al.,
1973; Liu and Hamilton, 1976; Liu, 1978; Gioia et al.,
1983; Bianchi and Gioia, 1984; Moss and Basbaum,
1983; Williams and Beitz, 1989, 1990a,b). Although
most of these reports have focused on individual features of structures in the PAG, to date, there has been
no systematic analysis of the PAG in any species. In
addition, while there are detailed descriptions of the
neuronal types, subdivisions, and afferent and efferent
connections of the rabbit PAG (Meller and Dennis,
1986, 1990a,b, 1991), and there is good reasons to use
the rabbit as an experimental animal in neuroscience
0 1993 WILEY-LISS, INC.
research (Dennis and Meller, in press), there is no ultrastructural analysis of the rabbit PAG.
Considering the large range of neurotransmitters,
neuromodulators, and peptides found in neurons of the
CNS, the manner in which synapses should be classified to account for functional differences is an obvious
challenge still to be met. It has long been suggested
that synapses with a symmetrical arrangement of preand post-synaptic membrane thickenings may serve as
Received July 2, 1992; accepted January 13, 1993.
Address reprint requests to Dr. B.J. Dennis, Department of Physiology, The University of Adelaide, South Australia, Australia.
Dr. S.T. Meller’s present address is Department of Pharmacology,
College of Medicine, University of Iowa, Iowa City, IA 52242.
574
S.T. MELLER AND B.J. DENNIS
inhibitory synapses, and the asymmetric type synapse
may be excitatory (Gray, 1959; Eccles, 1964). Also, it
has been proposed that round vesicles are associated
with excitatory synapses, and that flattened and pleomorphic vesicles are associated with inhibitory synapses (Uchizono, 1965). Further, dense-core vesicles, of
different sizes, have been implicated as containing a
variety of neuroactive peptides (Pickel et al., 1977a;
Beaudett and Descarries, 1981; Gabella, 1976; Pickel
et al., 197713; Johansson et al., 1980). Most recently,
immunocytochemicalanalysis of ultrathin sections has
provided further analysis of the excitatory and inhibitory transmitters localized within different vesicle and
synapse types.
Whereas there is an increasing number of reports
concerning the cellular and functional organization of
the PAG (see Beitz, 1985; Meller and Dennis, 1986,
1990a,b), as yet there is no general agreement on ultrastructural details of the PAG. Therefore, the present
study was initiated to provide a detailed ultrastructural analysis of the entire PAG on which to base subsequent functional studies. Portions of these data have
been previously reported (Meller and Dennis, 1987).
MATERIALS AND METHODS
Three adult male lop-eared rabbits (OrctoEagus cuniculus) weighing between 2.1 and 2.7 kg were used in
order to examine the ultrastructural characteristics of
previously described PAG subdivisions (Meller and
Dennis, 1986, 1990b):dorsal, lateral, ventral, and medial. Rabbits were housed on a 12/12hr light/dark cycle
and provided with food and water ad libitum for 1-2
weeks prior to use.
Rabbits were deeply anaesthetized by an intravenous injection of sodium pentobarbital (Ciba-Geigy,
Pendle Hill, New South Wales, Australia). The descending aorta was clamped, and 2,500 IU heparin was
injected intracardiacally before transcardiac perfusion
via a cannula tied into the ascending aorta. A prefixation rinse of 500 ml phosphate buffered heparinized
saline was followed by fixation, using 800 ml, 0.05M
3% glutaraldehyde, 2% paraformaldehyde, pH 7.2. In
each case, the brain was removed, trimmed of cortex,
and the regions rostra1 to the third ventricle and caudal to the fourth ventricle were also removed. The remainder of each brain was sectioned into 3-mm-thick
coronal blocks. The resultant blocks encompassed the
entire length of the PAG surrounding the aqueduct.
Block 1 contained the PAG from the level of the posterior commissure to the level of the red nucleus, block 2
extended from the level of the red nucleus to the level
of the caudal oculomotor nucleus, and block 3 extended
from the level of the caudal oculomotor nucleus to the
level of the caudal inferior colliculus. In each block, at
least 3 core samples were taken from each of the 4 PAG
subdivisions previously described (Meller and Dennis,
1986, 199Ob). This ensured that the samples did not
overlap boundaries between divisions.
The dissected blocks were washed in 0.1M phosphate
buffer, pH 7.2 (2 x 10 min), postfixed in 1%osmium
tetroxide (1hr) and embedded in a TAAB plastic resin
(TAAB Laboratories Equipment Ltd., Reading, UK).
Thin sections (1-2 pm) from each block were cut using
an ultramicrotome, mounted on glass slides, and
stained with toluidine blue to permit light microscopic
structural examination of each region. At least 10 (and
up to 30) ultrathin sections from each block were subsequently cut, placed on a metal grid, and stained with
a saturated alcoholic solution of uranyl nitrate solution
in the dark for 15-20 min, followed by a lead citrate
solution for 12 min. The grids were washed, dried, and
subsequently viewed with a JEOL transmission electron microscope (TEM) a t 60 Kv.
Soma, dendrites, axons, synapses, and glia from
more than 360 cut sections within the PAG were examined quantitatively with a light microscope and
TEM. Soma, dendrite, and axonal shape and size, the
arrangement and size of all cytoplasmic organelles,
and nuclear details were recorded. All synaptic contacts were counted and classified according to: location
of the contact (soma, axon, or dendrite), vesicle type
(round, flattened, or pleomorphic), vesicle size (small,
medium, or large), vesicle density (low, medium, high,
or crystalline), bouton size (small, medium, or large),
type of synaptic contact (symmetrical or asymmetric),
whether they made single or multiple contacts, the
presence of large and small dense-core vesicles, and
whether subsynaptic dots were found. Vesicle size
(small = 20-28 nm, medium = 34-42 nm, large =
50-60 nm) and density (low = 1-10 vesicles, medium
= 10-20 vesicles, high = >20 vesicles) and bouton size
(small = <1 pm, medium = 1-2 pm, large = > 2 pm)
were arbitrarily classified according to the sizes indicated. All size measurements were made directly on
the TEM. All measured and descriptive parameters for
all synapses analyzed were subjected to cluster analysis using BioMedical Data Package (BMDP, Statistical
Software Inc., Los Angeles, California) whereby,
through progressive levels of clustering, significant relationships can be extracted. Basically, this permitted
the identification of synapses having similar features
to be grouped together.
RESULTS
General Cytologica/ Features
Soma
Some shape varied from round to spindle to polygonal. The only regional difference in the distribution of
cells was a predominance of round and ovoid cells in
the medial PAG and more polygonal cells in lateral and
ventral PAG. Neuronal size varied from the smallest
cells (11pm in diameter), generally found within medial PAG, through to the largest cells (44 pm in diameter), found within the outer limits of both lateral and
ventral regions of the PAG. Some cells (located predominantly in the medial division, but present also in
smaller numbers in other PAG regions) appeared to
have a much denser cytoplasm due t o the predominantly greater number of ribosomes and rough endoplasmic reticulum (RER) within them.
The nucleus, which was generally found to occupy a
central position in the cell, contained large clumps of
chromatin accumulated on the inner surface of its
membrane, with small interchromatic granules and
perichromatic particles scattered throughout (Fig. 1).
Nuclear inclusions were found in about half of the cells
examined. The majority of these inclusions, up to 1.6
pm in diameter, had a crystalline-like appearance (Fig.
lb). Fibrillar-like inclusions were also detected in some
ULTRASTRUCTURE OF THE RABBIT PAG
Fig. 1. Nuclear inclusions in PAG cells. (A), Low-power magnification of the general cytological features of the soma of a PAG neuron
and its surrounding neuropil. The nucleus within the soma shows
clearly examples of both crystalline (C) and fibrillar (F) nuclear inclusions. x 4,000. Bar = 1 pm. (B), The most common type of inch-
575
sion is one with a crystalline appearance, seen here (arrowed) immediately below the nucleolus (N) and its nuclear satellites (NS).
X 18,000. Bar = 1 pm. (C), Occasionally, the nuclear inclusion had a
distinctly fibrillar appearance (F). x 24,000. Bar = 1 pm. These examples are enlargements of the inclusions in a.
576
S.T. MELLER AND B.J. DENNIS
Fig. 2. This electron micrograph illustrates the general ultrastructural features of the organelles to be
found within the soma and neuropil of PAG neurons. Note within the soma randomly-dispersedchromatin (C) internal to the nuclear envelope (E). The cytoplasm contains several Golgi complexes (G),
lysozomes (L),rough endoplasmic reticulum (RER), and RER aggregated into well-defined Nissl bodies
(N), plus numerous mitochondria (M).x 26,000. Bar = 1 Km.
nuclei (Fig. lc). The nucleolus was roughly spherical,
contained numerous dense granules, and had a latticelike appearance. Occasionally, one nuclear satellite
(two on rare occasions) was attached to the nucleolus.
Ribosomes, observed around the cytoplasmic surface of
the nuclear envelope, tended to accumulate in the cytoplasm within nuclear invaginations. The intermembranous distance of the nucleus was 22-33 nm. Nuclear pores were seldom observed.
In the cytoplasm, Nissl substance was observed to be
either dust-like, with numerous ribosomes appearing
either singly or in rosettes, or clumped with a dense
accumulation of RER giving rise to a Nissl body (Fig.
2). At least one, and sometimes up to 4 Nissl bodies
were observed in the cytoplasm of larger cells. The
Golgi apparatus was often located in close proximity to
the nucleus. It generally consisted of 4-5 flattened cisternae with no ribosomes, and numerous associated
small vesicles (about 30-100 nm diameter). Occasionally up to 3 Golgi apparatuses were observed within a
cell. Spherical or ovoid shaped lysozomes (100-370 nm)
were observed both near the Golgi apparatus and at
distant sites within the cytoplasm (Fig. 2). Large multivesicular bodies (500-600 nm) which contained small
round and elongated vesicles were observed in all cells,
often in close proximity to the Golgi apparatus. Numerous round (120-400 nm) and elongated (120-400 X
270-520 nm) mitochondria, which displayed a dense
matrix, were found distributed at random throughout
the cytoplasm. Microtubules (20-25 nm) and neurofilaments (10-12 nm) were also found scattered throughout the cytoplasm. At the junction of soma and primary
dendrites, neurofilaments and microtubules were very
prominent where they funnelled into the dendrite.
Neuropil
Dendrites varied in diameter from small (< 2 Fm),
medium (2-4 Fm) to large (>4 Fm) (Table 1).The dendrites (especially the larger ones) were ultrastructurally similar to the soma, except that microtubules were
far more prominent in dendritic profiles. Mitochondria
were observed in most dendritic profiles, as were neu-
ULTRASTRUCTURE OF THE RABBIT PAG
TABLE 1. Representation of the total numbers of
synapses (expressed as a %) contacting small (<2 am),
medium (2-4pm), and large (>4 pm) diameter
dendrites in each subdivision of the PAG
577
PAG (Fig. 3):round, elongated, and flattened (Table 2).
Variations in vesicle shape within boutons located in
dorsal, lateral, ventral, and medial PAG are indicated
in Table 2. Although larger dendrites tended to have
larger boutons contacting them, there was no signifiDendrite size
PAG region
Small
Medium
Large cant difference in the size or types of boutons contacting a proximal dendrite, a distal dendrite, or a dendritic
Dorsal
58.1
36.1
5.8
spine. In addition to the vesicle types described above,
Lateral
57.7
28.2
14.1 large and small dense-core vesicles (which were generVentral
50.6
29.1
20.3
ally round with an electron-dense core) were distribMedial
61.5
29.4
9.1
uted throughout boutons of the PAG (Fig. 4;Table 3).
Average
57.4
31.0
11.6 Large dense-core vesicles were found to be present in
approximately 40% of all synapses, always in combination with electron-lucent vesicles. There were more
rofilaments. Other features, not always apparent boutons containing large dense-core vesicles in ventral
within a dendrite, were the endoplasmic reticulum, and medial PAG than in dorsal and lateral PAG (Table
Golgi apparatus, multivesicular bodies, and lysozomes. 3). Small dense-core vesicles were only observed in
Generally, the smaller the dendrite, the fewer the about 3% of PAG synapses and never in boutons within
number of organelles observed within them. The num- ventral PAG (Table 3).
ber of spines on the soma of neurons in the PAG were
Vesicle size: The size of electron-lucent vesicles
very low, especially when compared with the numbers ranged from 30-120 nm in diameter (Fig. 3).The large
observed on dendrites. Spines were about 1-2 pm in majority of these vesicles fell into two main groups
length, had variable diameters and shapes, and typi- ranging between 34-38 nm or 40-42 nm in diameter
cally contained a spine apparatus. No microtubules or (Table 4).A third group of round vesicles were much
neurofilaments were observed in dendritic spines.
smaller, averaging between 20 and 28 nm in diameter.
Unmyelinated axons contained mitochondria, many These vesicles were not prevalent and tended to be loneurofilaments, a few microtubules, smooth endoplas- cated (along with other vesicles) in boutons which
mic reticulum, small vesicles, and multivesicular bod- showed a relatively greater density of vesicles, and also
ies. No ribosomes or RER were found in axons. Mito- in those containing flattened vesicles. A fourth group
chondria were thinner and more elongated than in the of round vesicles were quite large, with diameters in
cytoplasm, and were generally orientated parallel to the range of 50-60 nm. Very occasionally, an excepthe length of the axon. At the site of synaptic boutons, tionally large round electron-lucent vesicle was found,
mitochondria were smaller and associated with many approximately 90-120 nm in diameter. Large elonsmall, round, or flattened vesicles (0.4-2.5pm). My- gated vesicles were also observed which were in the
elinated axons had the same characteristic structure as range of 73-77 x 40-42 nm, whereas medium sized
unmyelinated axons except for their larger size and the elongated vesicles were about 50-60 x 25-35 nm. It
presence of myelin sheaths surrounding them.
was a consistent finding that the neuropil contained
Glia outnumbered neurons by 4.2:l.Glia generally more than twice as many boutons exhibiting large
contained a well-developed Golgi apparatus and large round vesicles and twice as many small vesicles of both
numbers of granules, ribosomes, and microtubules round and elongated types, as did axo-somatic synwhich gave them a dense appearance. Glia were repre- apses.
sented as astrocytes, oligodendrocytes, and microglia.
Approximately 40% of all somatic synapses and
Astrocytes did not have a very extensive RER and con- 30.2% of all neuropil synapses contained a t least 1
tained relatively few ribosomes. Consequently, they large dense-core vesicle and, quite frequently, several
were generally lighter in appearance than other glia were present. Some large dense-core vesicles were apand neuronal cells. Also, their cell bodies were quite proximately 70-85 nm in diameter while another
usually irregular in outline. They were found t o send group of even larger vesicles measured about 100-120
out sheets of cytoplasmic processes which on occasions nm in diameter, with the dense core occupying a t least
enclosed almost an entire neuronal soma or many syn- 75% of the vesicle. Smaller dense core vesicles were
aptic contacts. Oligodendrocytes were usually darker also observed in which the average diameter was
than other cells. In contrast to astrocytes, they pos- 40-50 nm, with a dense core occupying about 50% of
sessed a well-developed RER, and each had a large the vesicle.
number of free ribosomes near their outer perikaryal
Vesicle density: Vesicle density varied markedly
membrane surface. The nucleus of these cells was usu- within different boutons (Table 5). Density was considally found in an eccentric position within the cyto- ered medium where there was an accumulation of vesplasm. Oligodendrocytes were usually associated in a icles at the site of the synaptic contact with a moderate
satellite position with larger neurons in the PAG, with number of vesicles distributed throughout the rest of
the membranes of the two cells in direct apposition. the bouton. This was the case in 50.9% of synapses on
There were no specializations observed on the mem- the soma and 47.8% of synapses in the neuropil. Lowbranes between glia and neuronal cells.
density boutons were those containing only a few vesicles at the synaptic contact and few or no vesicles in
Synaptic characteristics
the rest of the synaptic bouton (15.9% of synapses on
Presynaptic profiles. Vesicle shape: Three different the soma and 7.9% of synapses in the neuropil). A highshapes of electron-lucent vesicles were distinguished in density bouton was evaluated where the vesicles over
presynaptic boutons of axons in the neuropil of the the synaptic contact were moderate to dense in number
578
S.T. MELLER AND B.J. DENNIS
Fig. 3.The most common synaptic types observed in the rabbit PAG.
(A), Two adjacent axo-somatic synapses of the symmetric type, containing predominantly round vesicles. x 44,000. (B), Two axo-dendritic synapses of the asymmetrical type both containing round vesicles. The one in the center of the micrograph has subsynaptic dots
below the post-synaptic membrane. X 22,000. (C), Two axo-axonal
profiles (both labelled A) on either side of a dendritic spine (D) show-
ing a n axodendritic synaptic contact. X 29,000. (D), An example of a
complex synapse in the PAG, with four well-defined synapses contacting a single dendritic profile. Two of the synapses (small arrows) are
symmetrical. The other two (large arrows) are asymmetrical. All synapses contain round vesicles with the one at the lower right containing, additionally, several dense-core vesicles. X 22,500.
579
ULTRASTRUCTURE OF THE RABBIT PAG
TABLE 2. Summary of the distribution of vesicles by shape (expressed as a %) in boutons contacting either the
soma or elements in the neuropil within the four subdivisions of the PAG
Vesicle shape
Round
Pleomorphic
Flattened
Medial
Soma
Neuropil
9.7
37.3
86.7
61.0
3.6
1.7
Dorsal
Soma
Neuropil
12.0
46.6
84.0
52.3
4.0
1.1
and the rest of the bouton was filled with vesicles, as
was the case for 32.5%of synapses on the soma and for
42.9% of synapses in the neuropil. Occasionally, boutons were observed where the vesicles were packed
very closely together so that their structure took on a
crystalline-like appearance (0.7% of synapses on the
soma and 1.4%of synapses in the neuropil).
Bouton size: A number of different-sized presynaptic boutons could be recognized within the PAG (Table 6): small boutons (<1 pm on their longest axis),
medium boutons (1-2 pm), and large boutons (>2 pm).
Of the boutons contacting the soma, 75.5%fell into the
small- and medium-sized range, and over 85%of these
were between 0.8 and 1.3 pm in length. In the neuropil,
92.4% of boutons were of the small and medium size,
again with over 85% between 0.8 and 1.3 pm in size.
Thus, the large majority of boutons fell into a relatively
narrow range of sizes. Those contacting somas tended
to be slightly larger than those contacting dendrites.
Postsynaptic specializations. Both symmetrical and
asymmetrical thickening of the synaptic membrane up
to 60 nm in thickness were observed, but they generally averaged about 35 nm (Fig. 3). Although length of
synapses generally varied between 230 nm and 450
nm, some were occasionally observed to be up to 700
nm in length. Of synapses contacting the soma, 83.5%
were symmetrical and 16.5% were asymmetrical,
whereas in the neuropil 46.4%were symmetrical and
53.6%were asymmetrical junctions. Occasionally, rows
of subsynaptic dots were observed beneath the postsynaptic thickening of asymmetric synapses. These
dots were 19 nm in diameter, evenly spaced a t 18 nm,
and they were continuous across the entire length of
the synapse. Synaptic clefts varied from about 13 to 28
nm in width; the majority being between 20 and 24 nm.
Dense areas of granular or filamentous material were
also associated with the cytoplasm at the synapse.
PAG region
Lateral
Soma
Neuropil
16.7
57.5
76.6
39.8
6.7
2.7
Ventral
Soma
Neuropil
32.4
41.9
64.7
56.9
2.9
1.2
Soma
17.0
78.7
4.3
Total
Neuropil
42.2
56.2
1.6
soma surface was covered by synapses; never more
than 4.9% was covered. There were usually only 1-4
synaptic contacts on the soma. Most axo-somatic contacts (83.5%)were of the symmetrical type and the boutons associated with these were one of several different
types. The most common type of synapse involved boutons of small and intermediate size containing round
vesicles of medium size. Boutons of small and medium
size containing pleomorphic vesicles of medium size
were also quite prevalent. Boutons with pleomorphic
vesicles contained only a relatively small percentage of
elongated vesicles where ellipticity (length divided by
width) of these pleomorphic vesicles varied from 1.23.4. Boutons containing only elongated vesicles were
very rare (4.3%).Of all somatic synapses, 37.1% contained large dense-core vesicles and only 0.7% of somatic synapses contained small dense-core vesicles.
Soma-soma synaptic contacts were never observed.
Axo-dendritic synapses. Dendrites were classified as:
proximal dendrites (when they were seen to leave the
soma), large dendrites which were probably proximal
dendrites but could not be positively identified as such,
medium-sized dendrites, and small dendrites (which
were actually classified as spines because of their small
size or the fact that they were quite clearly seen to be
spine appendages on dendrites). Boutons contacting
larger dendrites were generally small to medium in
size, of the asymmetric type, and contained pleomorphic vesicles of medium-to-large size. The medium
sized dendrites were contacted principally by small
boutons exhibiting both symmetrical and asymmetrical junctions containing pleomorphic vesicles of medium size. In addition a few of these boutons showed
both large, and small, round vesicles. Dendritic spines
generally showed only one synaptic contact stretching
the entire width of the spinous process. Spines on dendrites, and the bouton, were approximately the same
size, although sometimes the bouton had a larger diSynaptic types
ameter than the dendritic spine. Basically the same
All PAG synapses were assumed to be chemical, al- types of synaptic contacts were observed on small and
though nonsynaptic junctions were also found. These large dendrites. Of synapses on dendrites, 57.4% occontacts, the puncta adherens, showed membrane curred on small dendrites, 30.7% were on mediumthickenings in a symmetrical arrangement, yet not as- sized dendrites, and only 11.9% were on large dendrites. The characteristic features of the 6 major
sociated with vesicles.
Using the criteria of type of contact, bouton size, and axo-dendritic synapses are listed in Table 7 and the
vesicle size, density and shape, it proved possible to distribution of synapses on different regions of the dencategorize synapses into a number of groups (Table 7). drite are listed in Table 1.
Axo-axonic and dendro-dendritic synapses. Synapses
Ten distinctly different synaptic types were distinguished: 2 axo-somatic synaptic types, 6 axo-dendritic between two vesicle-containing structures were rarely
synaptic types, 1 axo-axonic synaptic type, and 1 den- observed (1.4%).Those that were found appeared to be
of the asymmetric type with the presynaptic profile
dro-dendritic synaptic type (Table 7).
Axo-somatic synapses. Axo-somatic synapses covered containing round vesicles of medium size. The postsynonly a small area of the soma. On average, 1.6%of the aptic profile generally contained fewer vesicles, and
580
S.T.MELLER AND B.J. DENNIS
Fig. 4. Illustration of some of the various types of vesicles observable
in synapses within the PAG. (A), An axodendritic asymmetric synapse showing a wide variety of round vesicles ranging from small to
the largest. x 60,000. (B), An axon synapsing on a dendritic spine
containing a spine apparatus (large arrow). Nearby, to the left (small
arrow) is an axonal profile containing many dense-core vesicles,
though not at a synaptic junction. x 27,000. (C), Two asymmetric,
axodendritic synapses (arrowed) on a single dendrite. The axonal bouton at the large arrow contains mainly round, clear vesicles. The other
contains fewer, flattened vesicles. x 27,000. (D), An example of two
neighbouring axo-dendritic, asymmetric synapses containing small,
round vesicles, in contact with the same dendritic spine 6).
x 68,000.
these were almost always of the flattened or pleomorphic type. Occasionally, the presynaptic bouton contained pleomorphic vesicles and the postsynaptic bouton contained round vesicles. These synaptic junctions
were either obvious axo-axonic connections, specified
by their morphology, or, more infrequently, presumed
to be reciprocal dendro-dendritic synapses.
Complex synapses. Synapses were very occasionally
581
ULTRASTRUCTURE OF THE RABBIT PAG
TABLE 3. Summary of the total numbers of boutons of
different sizes, within each subdivision of the PAG, that
contain large or small dense-core vesicles (1arge:small)'
~~
PAG region
Dorsal
Lateral
Ventral
Medial
Average
Small
43.1:2.6
28.6:4.8
48.7:O.O
44.3:7.1
42.1:3.9
Bouton
Medium
34.4:8.3
40.9:O.O
37.5:O.O
43.8:O.O
39.1:1.8
size
Large
20.0:6.3
77.7:O.O
50.0:O.O
50.0:O.O
50.0:2.5
Average
37.0:5.0
38.4:7.1
45.6:O.O
45.5:4.5
42.2:3.1
'Within each division and within each bouton size the remainder of the
percentage indicates vesicles that were electron-lucent and did not contain dense-core vesicles.
TABLE 4. Summary of the distribution of vesicle size (expressed as a %) in boutons contacting either the soma
or elements in the neurovil within the four subdivisions of the PAG'
~
Vesicle size
Medium
Small
Large
Small & med
Med & lge
Small, med, & lge
Medial
Soma Neuropil
48.1
43.0
11.5
17.8
0.0
2.8
19.2
23.4
15.4
6.5
5.8
6.5
'Small = 20-28 nm diameter, medium (med)
Soma
72.0
16.0
0.0
8.0
4.0
0.0
=
Dorsal
Neuropil
38.0
16.2
4.5
24.2
12.6
4.5
PAG region
Lateral
Soma Neuropil
47.5
54.0
7.5
16.1
2.5
4.1
27.5
14.9
10.0
9.5
5.0
1.4
34-42 nm, and large (lge)
=
Ventral
Soma Neuropil
55.9
41.9
5.9
23.5
5.9
7.4
11.7
16.0
14.7
8.7
5.9
2.5
Soma
53.7
9.9
1.9
18.0
11.9
4.6
Total
Neuropil
43.6
18.2
4.2
20.8
8.6
4.6
50-60 nm.
TABLE 5. Summary of the distribution of vesicle density (expressed as a %) in boutons contacting either the
soma or elements in the neuropil within the four subdivisions of the PAG'
Vesicle density
Medium
High
Low
Crystalline
Medial
Soma
Neuropil
48.5
44.5
40.7
47.0
10.8
6.8
0.0
1.7
Dorsal
Soma
Neuropil
40.0
53.5
40.0
42.0
20.0
4.5
0.0
0.0
PAG region
Lateral
Soma
Neuropil
55.0
48.6
20.0
36.5
22.5
14.9
2.5
0.0
Ventral
Soma
Neuropil
58.8
50.6
29.4
38.3
11.8
8.6
2.5
0.0
Soma
50.9
32.5
15.9
0.7
Total
Neuropil
47.8
42.9
7.9
1.4
lSee text for explanation of density criteria.
observed where astrocytic processes within the neuropil, either completely or nearly completely, enveloped
synaptic profiles. Such an arrangement suggests that
glia may serve, as one of their functions, to separate
regions of the neuropil. What functional significance is
reflected in this remains to be seen.
Variations With Respect to PAG Subdivisions
Obvious differences between neurons in different
subdivisions of the PAG were: neurons in medial PAG
were the smallest; lateral and ventral PAG contained
the greatest number and diversity of cells; dorsal PAG
contained the largest number of glia; dendrites in the
medial region were of the smallest diameter. Within
medial PAG, axons were generally unmyelinated,
though additionally, isolated myelinated fibres (0.51.2 pm) were observed. In the other regions of the PAG,
myelinated fibers more often were grouped in bundles
and were larger, varying from 0.4-2.6 pm in diameter.
Synapses also showed some slight variation in differ-
ent regions of the PAG. Synapses on the soma of neurons in ventral PAG contained more round vesicles and
proportionally fewer pleomorphic-shaped vesicles.
Also, a greater number of synaptic boutons containing
large vesicles contacted the soma of cells in ventral
PAG than in other regions. As a general rule, there
was a lower density of vesicles at soma synaptic contacts than a t contacts in the neuropil. This was particularly the case for cells in dorsal and lateral PAG. Medial and dorsal PAG showed far more boutons
containing densely-packed vesicles on the soma. Medial PAG contained more axo-somatic synapses than
other regions of the PAG. In the neuropil, the medial
PAG showed most boutons with more pleomorphic vesicles, and lateral PAG the least. Vesicle size was relatively constant in synapses on the neuropil, except in
ventral PAG where a slightly larger number of both
smaller and larger vesicles were found within synaptic
boutons. Similar proportions of small, medium, and
large boutons were found throughout the neuropil of all
582
S.T.MELLER AND B.J. DENNIS
TABLE 6. Summary of the distribution of bouton size (expressed as a %) in boutons contacting either the soma
or elements in the neuropil within the four subdivisions of the PAG'
Bouton size
Small
.
medium
Large
~~
ISmall
=
Medial
Soma
Neuropil
23.1
42.7
51.9
50.9
25.0
6.4
i l km diameter, medium
=
Dorsal
Soma
Neuropil
36.0
60.4
52.0
35.1
12.0
4.5
1-2 pm, and large
=
PAG region
Lateral
Soma
Neuropil
27.5
44.6
40.0
47.3
32.5
8.1
Ventral
Soma
Neuropil
20.6
39.5
55.9
45.7
23.5
14.8
Soma
25.8
49.7
24.5
Total
Neuropil
46.9
45.5
7.6
>2 pm.
TABLE 7.A summary of the features of the 10 synaptic types which were discerned
within the PAG'
Synaptic type
Axo-somatic
Axo-somatic
Axo-dendritic
Axo-dendritic
Axo-dendritic
Axo-dendritic
Axo-dendritic
Axo-dendritic
Axo-axonic
Dendro-dendritic
Membrane
Symmetrical
Asymmetrical
Symmetrical
Symmetrical
Symmetrical
Asymmetrical
Asymmetrical
Asymmetrical
Asymmetrical
Svmmetrical
Vesicle
sham
Round
Pleomorphic
Round
Pleomorphic
Pleomorphic
Round
Round
Pleomorphic
Round
Round
Vesicle
size
Medium
Medium
Medium
Medium
Small
Medium
Small
Medium
Medium
Medium
Vesicle
density
Medium
Medium
Medium
Medium
High
Medium
High
Medium
Low
Low
Bouton size
Medium & large
Medium & large
Small
Small & medium
Small & medium
Small & medium
Small & medium
Small
Small
Small
'See text for an explanation of the vesicle size and density and bouton size.
regions, except dorsal PAG which showed far greater difference between the descriptions for the rabbit and
numbers of small boutons and fewer medium-sized cat was the presence, in the rabbit, of soma profiles
which had an electron-dense appearance characterized
ones.
by a large accumulation of a granular-type of material
in the cytoplasm. A similar appearance was also noted
DISCUSSION
in isolated dendritic profiles which would
This study has provided a concise description of the occasionally
presumably have belonged to cells whose soma would
ultrastructure of PAG neurons, dendrites, axons, glia, have
a similar structure. Another group of small neuand synaptic contacts in the rabbit. While there have
were observed, predominantly in the medial divibeen a number of ultrastructural investigations of the rons
which was characterized by a large amount of
PAG in other species, most have focused on one feature sion,
RER in their cytoplasm. Neither of these cell types
of the PAG and none have incorporated the extent of were
as having been observed in the cat PAG
the analysis provided here. In addition, there has not (Gioiareported
et
al.,
1983;
and Gioia, 1984; Moss and
been any ultrastructural analysis of the rabbit PAG. Basbaum, 1983). InBianchi
other
experiments
demThe most striking feature of the synaptic ultrastruc- onstrated that some axons and dendritesweofhave
PAG
neuture of the rabbit PAG was that over 95% of the con- rons enter the aqueduct (Meller and Dennis, 1990a),
tacts between neuronal elements occurred within the
the increased amount of RER in some cells of the
neuropil with axo-dendritic contacts providing 94.1%of thus
medial
could conceivably be associated with an
the total synapses indicating that integration takes increasedivision
in
synthetic
which might therefore be
place at the level of the neuropil. The variations in the involved somehow in activity
secretion
into or absorption from
size of synaptic boutons, and the density and size of the CSF.
vesicles observed, presumably reflect functional differThe appearance of cytoplasmic organelles, the arences.
rangement of the nucleus and nucleolus, and the high
nucleus/cytoplasmic ratio were similar to that deFine Structure of the Soma of PAG Neurons and
scribed for the cat (Gioia et al., 1983). Earlier studies in
the Neuropil
the cat indicated the presence of a large number of
The soma of PAG neurons were generally round, cytoplasmic inclusions in the nucleus of PAG neurons
spindle, or polygonal in shape. Soma sizes of rabbit (Hamilton et al., 1973; Liu and Hamilton, 1976; Liu,
PAG neurons were found to be larger than those ob- 1978; Gioia et al., 1983; Bianchi and Gioia, 1985). In
served for the cat (Gioia et al., 1983), falling within the contrast, the present study has shown that cells within
range of 12-45 p,m diameter. There did not appear to the PAG generally have only one crystalline-like inbe a clear cut-off point which permitted groups of cells clusion; on rare occasions, a fibrillar inclusion was also
to be differentiated as either small or large neurons, as observed. Whether these structures play a role in celGioia et al. (1983) reported for the cat. One significant lular control as suggested by Liu and Hamilton (1976)
ULTRASTRUCTURE OF THE RABBIT PAG
and Bianchi and Gioia (1985) remains to be determined.
The number of synaptic contacts on the soma of PAG
neurons was found to be quite low when compared with
those made on dendrites. On the average there were
only 3-4 contacts per soma per section which accounted for 8-10% of the total number of synaptic contacts. There was very little variation in the numbers of
synaptic contacts on the soma with respect to cells in
dorsal, lateral, ventral, or medial PAG. Gioia et al.
(1983) and Bianchi and Gioia (1984) came to a similar
conclusion for the cat PAG. Moss and Basbaum (19831,
also in the cat, described the number of synaptic contacts on the soma of caudal PAG neurons to be even
lower (about 2-3% of total synapses). The number of
soma synaptic contacts is probably limited in relation
to the area available for synaptic contact on the receptive surfaces. A similar suggestion has been made for
the pyramidal cells of the somatosensory cortex where
the soma accounts for 4% of the surface area of the cell
and 43%of the total area is taken up solely by dendritic
spines (Mungai, 1967). In contrast, the soma surface
area of large cells in the reticular formation accounts
for 20% of the total surface area of neurons (Mannen,
1966).
Within the neuropil, the large number of dendrites,
cut in any of a number of planes, were observed to
range from very large to very small in size and, as the
findings of our Golgi study also suggested, dendritic
spines were not very prevalent (Meller and Dennis,
1990b). In contrast, studies on the ultrastructure of the
PAG in the cat have suggested that dendritic spines
are quite numerous (Gioia et al., 1983; Bianchi and
Gioia, 1984). Myelinated and unmyelinated axons,
which made up a large percentage of the neuropil, are
more numerous and larger in both number and size
with distance from the aqueduct. The number and size
of the myelinated axons was far greater in dorsal, lateral, and ventral PAG regions than in medial PAG,
whereas the proportion of unmyelinated axons to myelinated axons was greater in medial PAG, suggesting
that neurons of the small-sized medial PAG may be
predominantly locally-projecting cells or interneurons.
The types and ultrastructural analysis of glia within
the PAG has not previously been reported. In the
present study, numerous glia were observed in the neuropil, with oligodendrocytes frequently being found in
direct apposition to the soma of PAG neurons. Whether
they perform a purely nutritive or supportive role is
not yet clear, although astrocytes and their processes
were observed penetrating the neuropil, where they
were occasionally found to either encapsulate a synaptic contact or to contact and surround blood vessels. It
has been suggested that this arrangement may limit
the free diffusion of neurotransmitters, or alter the distribution of ions around synapses (Schubert, 19841, or
control the distribution and density of synapses on the
soma and dendrites of cells (Meshul et al., 1987).
Synaptic Features of the PAG
The major characteristic ultrastructural feature of
any synapse is the presence of synaptic vesicles within
the presynaptic bouton associated with varied thickenings of the pre- and post-synaptic membranes. On the
583
soma, the most prominent synaptic contacts were symmetric or multiple symmetric synapses containing
pleomorphic vesicles (80%) which agrees quite well
with the number (88%)assessed by Bianchi and Gioia
(1984) for the cat PAG. The size of the synaptic boutons
varied from small to large with the most prominent
types being the intermediate and large boutons. In the
neuropil in the cat, Bianchi and Gioia (1984) reported
that 88% of the synapses contained pleomorphic vesicles whereas our estimate for the rabbit was 53.2%,
more closely agreeing with the 59.3%proposed for caudal PAG in the cat by Moss and Basbaum (1983). Both
of these reports in the cat and our results for the rabbit
agree that, in the neuropil, approximately half of the
synapses have symmetrical and half asymmetrical
junctions. Small and medium-sized boutons were the
most prominent contacts, and these were mostly on the
middle and distal dendrites. The synaptic density on
proximal dendrites is less than that found on more
distal dendrites, and the type of synapses found on
proximal dendrites are similar in size and morphology
to those found at soma contacts. The presence of a large
number of axo-dendritic synapses, together with
smaller numbers of presumed axo-axonic, dendro-dendritic, and complex synapses suggest that the neuropil
is indeed the main target of afferent input to the PAG.
Four general categories of vesicles have been found
in the presynaptic profiles of the PAG: round, flattened, pleomorphic, and dense-core vesicles. In the
PAG of the cat, Gioia et al. (1983) and Bianchi and
Gioia (1984) found these same four basic vesicle types,
while Moss and Basbaum (1983) recognized only round,
pleomorphic, and dense-core vesicles. Not only were
four categories recognized in this study, but a range of
different sizes within each population has been described.
Different vesicle shapes and sizes have been proposed to reflect different functional properties of synapses. Symmetrical contacts have been suggested to be
associated with inhibitory synapses and asymmetrical
contacts associated with excitatory synapses (Gray,
1959; Eccles, 1964). Further, a correlation between
round vesicles and excitation, as against pleomorphic
or flattened vesicles and inhibition, has also been proposed (Uchizono, 1965; Bodian, 1970). Of the axo-dendritic synapses in the neuropil (which account for
94.1% of all synapses), a clear majority of them displayed symmetrical contacts containing pleomorphic
vesicles or asymmetrical contacts containing round
vesicles. This is in line with there being basically inhibition a t the level of the soma, whereas in the neuropil half the synapses result in inhibition and half in
excitation. A report by Penny et al. (1984) demonstrated that glutamic acid decarboxylase activity (an
indicator of the presence of the inhibitory transmitter,
gamma amino butyric acid) is spread relatively evenly
throughout the PAG of the rabbit. This present account
indicates that presumed inhibitory synapses are also
spread evenly throughout the PAG. Although this situation may provide a good correlation between morphological features of a synapse and its supposed functional role, it remains to be seen whether vesicle shape
and the polarity of the synapse support the concept of
the nature of inhibitory and excitatory synapses. Such
features should be closely examined in subsequent
584
S.T. MELLER AND B.J. DENNIS
studies where immunocytochemical evidence for
known transmitters is available at the TEM level.
Boutons containing flattened vesicles and displaying
symmetrical contacts were few, though found consistently in the rabbit. This was also the case for the PAG
of the cat (Gioia et al., 1983; Bianchi and Gioia, 1984;
Moss and Basbaum, 1983). Another consistent finding
was the presence of a few large dense-core vesicles of
varying sizes spread amongst the usual population of
vesicles in a presynaptic profile. These were found in
approximately 40% of all synaptic profiles. A smaller
number of synapses (3%) contained small dense-core
vesicles which appeared similar to those identified a s
catecholamine-containing profiles in the cerebellum
(Chan-Palay, 19771. Although dense-core vesicles were
also reported in the cat (Moss and Basbaum, 1983) and
rat (Clements et al., 1985), they were not nearly as
prevalent as we have found here in the rabbit. A number of additional endings containing up to a dozen large
dense-core vesicles were also observed in the neuropil
of the PAG of the rabbit, but these were never observed
to form synaptic contacts in the PAG. These profiles
resembled the serotonergic axons found by Beaudett
and Descarries (1981) and could conceivably be derived
from the raphe nuclei. A similar feature was also reported by Clements et al. (1985) who suggested that
such axons in the rodent PAG were either travelling
through the PAG without releasing transmitters or
that they were involved in some type of non-synaptic
transmitter release. That, together with the finding by
Clements et al. (1985) that very few synapses in the
PAG showed serotonin immunoreactivity, may explain
why investigators such as Aghajanian et al. (1972)
were unable to observe much of a n effect with iontophoretically-applied serotonin.
Synapses in the neuropil were found to consist
largely of axodendritic synapses (sometimes up to 11
synapses on one dendrite); however, a small number of
presumed axo-axonic and dendro-dendritic synapses
were also found. The presence of these, together with
other complex synapses also observed, would indicate
that the PAG is intricately involved in complex processing of afferent information within the neuropil.
The existence of axo-axonic connections, though small
in number, is indicative of presynaptic inhibition,
which has already been proposed to be active in the
PAG in pharmacological and physiological studies
(Hara et al., 1961; Sakuma and Pfaff, 1980; Soper and
Melzack, 1982; Handwerker and Sack, 1982; Reichling
et al., 1984).
In summary, the present study has provided a comprehensive ultrastructural analysis of the rabbit PAG.
The PAG of the rabbit shows qualitatively and quantitatively similar synaptic types to those shown for the
cat. However, there also are differences shown between
these two species. For example, the PAG of the rabbit
shows larger neurons that only contain one cytoplasmic inclusion; other neurons in the PAG are more electron-dense and contain large amounts of RER (typically in medial PAG). In addition, neurons and
dendrites have far fewer dendritic spines that neurons
and dendrites in the cat. Further, this study has provided descriptions of the ultrastructure of glia in the
PAG and also of variations in different subdivisions of
the PAG. In general, though, many of the cytological
features are similar between the cat and rabbit, further
validating and justifying the use of the rabbit in neuroscience research (Dennis and Meller, 1993). These
results have provided a clear picture of normal synaptology at all levels, and in each subdivision, of the rabbit PAG. This information, together with burgeoning
knowledge of neurotransmitters present within the
PAG (see Moss and Basbaum, 1983; Beitz, 1985; Clements et al., 1985) and of the ultrastructural localization
of excitatory and inhibitory neurotransmitters to different synapse types (Williams and Beitz 1989,
1990a,b), should be of assistance in determining the
specifics of neuronal circuitry whereby the PAG performs its numerous functional roles.
ACKNOWLEDGMENTS
This study was made possible with support from The
Australian Brain Foundation.
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