THE JOURNAL OF COMPARATIVE NEUROLOGY 319:519-535 (1992)

Ultrastructure of the Periaqueductal Grey Matter of the Rat: An Electron Microscopical and Horseradish Peroxidase Study PIETER BUMA, JAN VEENING, T H E 0 HAFMANS, HENK JOOSTEN, AND RUDOLF NIEUWENHUYS Department of Orthopaedics, University Hospital Nijmegen (P.B.) and Department of Anatomy and Embryology, Faculty of Medicine and Dentistry, University of Nijmegen (J.V., T.H., H.J., R.N.) 6500 HB Nijmegen, The Netherlands

ABSTRACT The neurons of the mesencephalic periaqueductal grey substance (PAG) in the rat are small and medium sized. The cells are frequently located in small clusters, without interdigitating glial elements and may be connected by direct membrane appositions or by gap junctions. The inner zone of the PAG is cell poor. In many cases, the cytoplasm of the cells is filled with extensive rough endoplasmic reticulum, free ribosomes, Golgi apparatus, and large lysosomelike granules. The nuclei show large indentations. The cells have a high nucleus-cytoplasm ratio. The neuropil is very extensive and particularly rich in large numbers of small unmyelinated axons, dendrites, axonal varicosities, and synaptic connections. Myelinated fibres are relatively scarce. The orientation of the fibres was studied in transverse and horizontal sections, in combination with HRP track tracing experiments. It appeared that throughout the PAG most of the fibres were orientated longitudinally. Quantitation showed that most fibres were present in the inner zones of the PAG. Moreover, the diameter of the fibres adjacent to the aqueduct was smaller than that of the fibres in the peripheral parts of the PAG. The thin unmyelinated fibres made extensive synaptic connections within the PAG. Many synaptic varicosities were found in the neuropil of the PAG. There were four types of synaptic varicosities, characterized by different populations of clear and dense-core secretory granules and by the different morphology of the synaptic specializations. In general, the different types of varicosity were homogeneously distributed in the different parts of the PAG. Electron dense secretory granules, when present, were located at some distance from the synaptic junction. Serial sections revealed varicosities which contained only dense-core secretory granules, without synaptic specializations. The dendrites of PAG neurons generally lacked synaptic spines. Many dendrites, particularly those of neurons located in the peripheral parts of the PAG, were directed toward the aqueduct. The present study shows that the PAG is a very complex brain area. The crisscrossing of s o n s and dendrites with synaptic connections at considerable distances from the cell bodies render it very difficult to unravel the relationships between the possible sources and destinations of ongoing information. This structure complicates the search for relationships between the functional organization and the cytoarchitectural borders in the PAG area. Key words: central grey, synapse, nonsynaptic varicosity, brain architecture, unmyelinated axon, quantitation

The midbrain mesencephaliccentral grey or periaqueductal grey (PAG) constitutes a compact zone of grey matter with relatively small neurons which surround the aqueduct. The PAG continues rostrally into the periventricular grey matter of the walls of the third ventricle and caudally o 1992 WILEY-LISS, INC.

into the periventricular grey matter of the rhombencephalon (Crosby and Woodbourne, '51). The PAG may be considered as the bed nucleus of the periventricular fibre ~ c ~ e p t December ed 16,1991.

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system, also called the fasciculus longitudinalis of Schutz brain regions are poorly understood. Gioia et al. ('83) studied the ultrastructural organization of the neuropil of (1891). Functionally, the PAG is a highly heterogeneous and the PAG in the cat. With respect to immunocytochemical complex brain region. In many behavioural, pharmacologi- investigations, many light microscopic studies have been cal, and physiological studies the numerous functions ex- published (for review see Nieuwenhuys, '85; e.g., glutamate erted by this midbrain region have been investigated. Its and aspartate, Clements et al., '87; neurotensin, Shipley et role in the central analgesic mechanisms, as originally al., '87). Electron microscopic observations have been made discovered by Reynolds in 1969, is well documented (for on immunostained fibres containing serotonin (Clements et review see Basbaum and Fields, '84). However, the recent al., '85: rat), enkephalin (Moss and Basbaum, '83: cat; literature indicates a wide spectrum of other behavioural Buma, '89b; Williams and Beitz, '90: rat), dynorphin (Buma, and autonomic effects that can be elicited in the PAG by '89b: rat), ACTH (Buma et al., '89; Lookeren Campagne et lesion experiments and by electrical or chemical stimula- al., '91: rat) and LHRH (Buma, '89a: rat), GABA (Reichling tion (for references see discussion). Tract tracing experi- and Basbaum, '90a,b: rat), and Substance P (Gioia and ments in the rat (Hardy and Leichnetz, '81; Morrell et al., Bianchi, '90: rat). '81; Mantyh, '82b; Marchand and Hagino, '83; Pechura and Thus, considerable advances have been made with reLiu, '86; Beitz et al., '86; Bandler and Tork, '87; Behbehani spect to the functional, light microscopic, physiological,and et al., '88; Akaishi et al., '88; Bianchi et al., '90; Chung et al., immunocytochemical characteristics of this brain region. '90; Ennis et al., '91; Herrero et al., '91; Veening et al., in In the light of the overwhelming amount of literature on press), monkey (Mantyh, '82b; Mantyh, '83a,b; Wiberg et the functions and relationships of the PAG, it is somewhat al., '87; Harmann et al., '88), cat (Abols and Basbaum, '81; surprising that the general ultrastructure of the PAG has Mantyh, '82b; Bragin et al., '84) and rabbit (Meller and not yet been studied in more detail. The present study aims Dennis, '86) have shown that the PAG is extensively to fill this gap. In an extensive electron microscopic investiconnected with many telencephalic, diencephalicand brain- gation, we studied four equidistant transverse sections of the PAG in the rat. stem areas. In an additional HRP electron microscopic study, some Classical studies on the architecture of the PAG with conventional staining methods mainly focused on the loca- features of the intrinsic fibres within the PAG were investition, orientation, diameter and density of the cell bodies gated in some detail. For this purpose, we performed a (Gillian, '43;Hamilton, '73; Liu and Hamilton, 'SO; Man- series of HRP injections into the rostral, middle, and caudal tyh, '82a; Gioia et al., '84; Beitz, '85; Beitz and Shepard, PAG and analysed the distribution of fibres containing '85; Gioia et al., '85; Meller and Dennis, '90a). More HRP in the other parts of the PAG on a light microscopic as recently, cytochrome oxidase histochemistry has been used well as on an ultrastructural level. as a tool for the visualization of axon terminal mitochondrial enzymes in the PAG (Conti et al., '88). Most of these studies focused on the characterization of subdivisions MATERIALS AND METHODS within the PAG, with variable results (see Gioia et al., '85 Microscopy for references). For routine electron microscopy, 5 adult male albino rats Very little is known about the ultrastructural organization of the PAG. Moreover, the ultrastructural features of (Wistar strain, body weight 250-270 g) were anaesthetized the afferent and efferent connections of the PAG with other with sodium pentobarbitone (60 mg/kg body weight) and

Abbreuiations 3 4 a ACTH BV CG cic CnF

CNS CP

cs

csc Ctp D

DAB DK DLG dlf DPWh DR dtg DTgP EW fr G G1 HRP IC

oculomotor nucleus trochlear nucleus cerebral aqueduct adrenocorticotropic hormone blood vessel central grey commissure of the inferior colliculus cuneiform nucleus central nervous system cerebral peduncle colliculus superior commissure of the superior colliculus central tegmental tract dendrite diamino-benzidine Darkschewitsch nucleus dorsal lateral geniculate nucleus dorsal longitudinal fasciculus deep white layer of superior colliculus dorsal raphe nucleus dorsal tegmental bundle dorsal tegmental nucleus, pericentral part Edinger-Westphal nucleus fasciculus retroflexus Golgi zone glial cell horseradish peroxidase inferior colliculus

InCo IMLF IPR LDTg 1FP LHRH M mcp Me5 Mf MGV ml mlf MN mP mt n PAG PC Pn PY RER s5 SER SG SNR su3 xcsp

intercollicular nucleus interstitial nucleus mlf interpeduncular nucleus, rostral subnucleus laterodorsal tegmental nucleus longitudinal fasciculus of the pons luteinizing hormone-releasinghormone mitochondria middle cerebellar peduncle mesencephalic trigeminal nucleus microfilament medial geniculate nucleus, ventral part medial lemniseus medial longitudinal fasciculus mammillary nucleus mammillary peduncle mammillothalamictract nucleolus periaqueductal grey substance posterior commissure pons pyramidal tract rough endoplasmic reticulum sensory root trigeminal nerve smooth endoplasmic reticulum secretory granules substantia nigra, reticular part supraoculomotor central grey decussation of superior cerebellar peduncle

ULTRASTRUCTURE OF THE PAG

Fig. 1. A-D. On the left is a series of line drawings to illustrate the levels of the mesencephalon that were studied in detail. Drawings were made of sections at a fixed interval of 1.3 mm, from Bregma -4.8 (A) to Bregma -8.72 (D) according to the atlas of Paxinos and Watson ('86). On the right side, camera lucida drawings of the PAG are shown. On the right side of the PAG, the distribution of myelinated fibres is depicted

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semidiagrammatically. Outlined areas were serially sectioned for electron microscopy, serially photographed and the number of axonal varicosities and thin fibres were quantified. Dotted circles on the left side of the PAG indicate the mirror image of the locations of HRP injections.

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perfused via the left ventricle of the heart with 0.9%NaCl (saturated with 95%0, and 5%CO, and adjusted to pH 7.4 with concentrated NaOH) until the liver was bleached (30 s-2 min). After perfusion with 500 ml phosphate-buffered (pH 7.4, 0.1M, 4°C) 1% paraformaldehyde and 1.25% glutaraldehyde,the rats were decapitated,the skulls opened, and the brains further fixed in situ for 24 h. Tissue blocks from the rostral part of the posterior commissure (anterior coordinates Bregma -4.0 according to the atlas of Paxinos and Watson, '861, to the caudal part of the locus coeruleus (bregma -9.O), were cut stereotactically with a sharp knife, carefully removed from the skull, and serially sectioned in phosphate-buffered saline (pH 7.4,50 km) in the coronal plane on a Lancer vibratome (Series 1000) at 4°C. The sections were postfixed for 2 h in 1%OsO, in 0.1M phosphate buffer (pH 7.41, washed in the buffer, dehydrated, and flat embedded in Epon 812 between glass slides and coverslips coated with dimethylchlorosilane (BDH Chemicals, Ltd). Following polymerization, the PAG was excised and mounted on preformed Epon blocks. Sections of the PAG were cut on a Reichert Ultracut E (for selection of areas see Fig. 1)and mounted on oval large one-holegrids in such a way that the ventricular surface could be used for orientation. To allow precise orientation of the fibres and cells within the PAG on an ultrastructural level, the following procedure was followed: camera lucida drawings were made of toluidine blue or paraphenylene diamine stained semithin sections (2 km) of 4 equidistant levels (from the commissura posterior, Bregma -4.8, to the caudal PAG posteriorly, Bregma -8.7 coordinates according to the atlas of Paxinos and Watson, '86); each overall section was photographed with a final magnification of X 290. From the photocompositions, the outlines of cells with a clearly visible nucleus were copied onto a transparent sheet. These sheets were photographically reduced to a magnification of ~ 6 5The . lateral borders of the PAG were defined as the transition zone between the area with a small number of myelinated fibres and areas containing numerous thick bundles of myelinated fibres. In each frontal section, three strips of tissue were excised at angles of 45", go", 135" to the median plane and 2 pm semithin section were made. The semithin sections of the strips were plotted into the drawings with the aid of low power light microscopic examination of the 2 km sections. Subsequently, adjacent serial ultrathin sections were prepared according to the method described by Buma et al. ('89). In order to be able to analyse the overall ultrastructure of the PAG as well as the regional differences and possible variations in the dorsal, lateral, or ventral aspects of the PAG, the following procedure was followed: serial photographs (between 40 and 70 of each strip), with a final magnification of ~20,000 were taken of the middle section of the serial electron microscopic sections of the strips, from the ventricular surface into the areas surrounding the PAG. The middle section of each series was photographed in order to allow

analysis of particular profiles in aqjacent sections. These series of photographs allowed structures within the boundaries of the PAG to be precisely located. On the basis of the total number of micrographs, and the precise light microscopic localization of the strips in the PAG, the lateral boundary of the PAG could be determined on the electron microscopic photographs. Next, series of micrographs of the strips within the borders of the PAG were divided into equal periaquedudal, intermediate, and peripheral parts. The photographs were used to encircle the profiles of the thin fibres and the different types of synaptic profile. With the aid of a contron videoplan system, profile areas, profile diameters and the length of the synaptic specializations (if present), were calculated from the tracings. Each photograph had a surface area of 30 p,m2.Before measuring, each photograph was covered by a test lattice and all the profiles of fibres or synaptic terminals lying within the test lattice and not touching the forbidden lines, were counted (Gundersen, '77).For thin fibres, this area was 25 km2;for the larger synaptic profiles, the central 15 km2was measured.

Fig. 2. A,C,E,F: Light microscopic aspects of PAG neurons, indi-

illustrating the paucity of cells and myelinated fibres in the direct vicinity of the aqueduct (B); the very characteristiethickenings of the ependymal lining in the middle PAG, with tanycyte processes to nearby blood vessels (D, arrow);and (G)the direct apposition of PAG neurons (arrows) without interdigitating neuropil elements. At low magnification, the profiles of dendrites are recognizable as relatively unstained structures (arrowheads).The outlined area in F indicates the location of G. ~ 4 6 0Aq, . aqueduct.Bars in B,D,G = 50 Fm.

cated by drawings made from photomontages (A,C,E,F: magnification, x65), same levels as shown in A. Note paucity of cells in the inner zone

of the PAG and the accumulation of larger cells in the ventro-lateral aspect of the drawing, indicating the location of the nucleus of Darkschewitsch. C,E:Note dorsal accumulationof largw neurons and the more dorso-lateral ammulation of relatively small neurons. F Distribution of cells in the caudal PAG. Note the absence of cells in the direct vicinity of blood vessels (asterisks). B,D,G Photomicrographs

HRP histochemistry The rats were anaesthetized for surgery with Nembutal (60 mg/kg) and placed in a stereotactic apparatus. Horseradin saline) ish peroxidase (HRP; Sigma grade VI;0.2 ~ 1 2 0 % was injected unilaterally, into the right or left side of the rostral, middle or caudal PAG (two successful injections on each level, same coordinates as the four levels described above), by a pressure technique through a Hamilton microsyringe. The HRP was injected slowly over a period of 15 min and the needle was kept in place for an additional 10 min to avoid spread of the label along the track. After 72 h, the animals were reanaesthetized and perfused with 500 ml ice cold 1%paraformaldehyde and 1.25%glutaraldehyde in 0.1 M phosphate-buffer (pH 7.4). Subsequently, the animals were perfused with 500 ml of a solution of 10%sucrose in the buffer. After 2 hs, the brains were sectioned on the vibratome. All sections were thoroughly rinsed and incubated with DAB (Graham and Karnovsky, '66). Alternate sections were mounted on chrome-alum coated slides, air dried, and coverslipped with or without counterstaining with cresyl violet. For electron microscopy, the sections were postfixed and further treated as described above.

RESULTS Light microscopy Four equidistant levels of the PAG were studied in detail (Bregma -4.8, -6.1, -7.4, -8.7,respectively). The first section studied was at the transition between the diencephd o n and the midbrain, a t the level of the posterior commissure (see Fig. 1, left row, for schematic representation of levels studied). Directly caudal to this section, the aqueduct and the PAG widened (Fig. 1B). The last section studied

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524 was just in front of the most caudal part of the colliculus inferior (Bregma -8.72; Fig. 1D). The outer border of the PAG was drawn at the transition between the myelin-poor PAG neuropil and the myelin-rich surrounding areas (the posterior commissure, Bregma -4.8; the commissure of the colliculus superior, the dorsal tegmental bundle, Bregma -6.1; the deep white layer of the superior colliculus and the dorsal tegmental nucleus, Bregma -7.4). Only the borders in the caudal PAG were somewhat arbitrary because the neuropil of the PAG was continuous with that of the cuneiform nucleus (Fig. 1D). All the levels of the PAG studied showed some common characteristics. The inner zone was always very cell poor (Fig. 2A-F). The cells in this part were small, spindleshaped, and orientated parallel to the ependymal lining (Fig. SA-C,E,F). In the outer parts of the PAG, the cells did not show any clear orientation with respect to the aqueduct. The cells appeared to be located in small clusters, in many cases in apparent, direct contact with each other (Fig. 2G). In general, great heterogeneity was found with respect to the size of the cells. The nucleus of Darkschewitsch was characterized by an accumulation of larger neurons (Fig. 2A). At the second and third levels studied, the inner, cell-poor zone extended into the lateral PAG, although it was not very pronounced. An accumulation of relatively small neurons was present, particularly at the dorsolateral aspect of the second and third levels (Fig. 2C,E). At the fourth level, a cell-poor area was found around the larger blood vessels (Fig. 2F). The adjacent laterodorsal tegmental nucleus was characterized by larger neurons. Myelinated fibres could be clearly visualized in the paraphenylene diamine-stained semithin sections (Fig. 2B,D,G). The myelinated fibres were generally very thin compared to fibres in compact bundles, such as the posterior commissure (Fig. 2B), or the commissure of the superior colliculus. The distribution of the myelinated fibres is semidiagrammatically depicted in Figure 1 (right side of PAG in the right row). The inner, cell-poor part of the PAG was almost devoid of myelinated fibres, except for its rostral part, immediately ventral to the posterior commissure (Fig. 2B). The myelinated fibres were fairly homogeneously dispersed over the PAG. The majority of the fibres showed round profiles in sections, thus indicating that they run in a rostrocaudal direction (Figs. 2B,D, 4D, 8A,D).In the lateral PAG, profiles of laterally running fibres were found, indicating that myelinated fibres connect the PAG with the pretectal region. At the second level, most of the profiles of cross-sectionedmyelinated fibres were round again, indicating a rostro-caudal orientation. Horizontally running fibres and crossing fibres were also present in the lateral part of the PAG, and directly ventral to the aqueduct. At the more caudal levels (Bregma -7.4) of the PAG, the architecture of the myelinated fibres was more complex. Again the inner zone of the PAG was almost free from myelinated fibres. An accumulation of profiles was only found in the dorsal midline region, extending from the ventricular surface to the outer border of the PAG. In the mid dorso-lateral region of the PAG, an accumulation of myelinated profiles occurred, while the outer portion of this region was almost completely devoid of fibres. Irrespective of the location within the PAG, again most of the fibre profiles were round, indicating a rostro-caudal orientation. In the last section studied, the dorsal fibres seemed to run preferentially in a ventro-lateral direction, towards or from the cuneiform nucleus. Ventrally, an accumulation of cross-sectioned fi-

bres was present. With respect to the other components of the neuropil, dendrites were light-microscopicallyrecognizable as relatively unstained structures (Fig. 2; arrowheads). In the direct vicinity of the aqueduct, these dendrites were mainly directed parallel to the ventricular lining. In the middle part of the PAG, the ependymal lining of the aqueduct formed two or three ventro-medial or ventrolateral, rostro-caudally orientated thickenings (Fig. 2D; previously described by Welsh and Beitz, '81; Buma, '89a,b). Tanycyte processes entered the neuropil of the PAG to terminate around nearby blood vessels, particularly from these thickenings (Fig. 2D).

Electron microscopy Neurons. The cells of the PAG appeared to be very heterogeneous with respect to their ultrastructure. In general, the cell bodies located in the inner zone were elongated in the plane of the section, with the axis through the largest diameter parallel to the surface of the ventricle. The nuclear envelope of most of the PAG cells had a very irregular outline (Fig. SA,B,E), due to deep cytoplasmic invaginations into the karyoplasm. These invaginations were filled with (po1y)ribosomes(Fig. 3E). The surface area occupied by the cytoplasm was generally not very large. Most of the cells contained some Golgi zones, moderate endoplasmic reticulum, mitochondria and lysosomes. The number of dense-core secretory granules, with a diameter of 100 nm, varied greatly between individual cells, but was generally very small. Many cells were located in small clusters, with their membranes in very close contact, without any interdigitating elements of the neuropil (Fig. 3A,C). In some cases, the membranes at these locations had a heptalaminar appearance, strongly suggesting the presence of gap junctions (Fig. 3C). The proximal dendritic processes were filled with rough endoplasmic reticulum, free ribosomes, mitochondria and neurofilaments. The cells were not intensely innervated by axo-somatic synapses. Only a few (1-3) synapses were present on a single somatic profile and were mainly of the symmetrical type, with many round electronlucent synaptic vesicles. Neuropil Dendrites. The dendrites of the PAG had a round to oval appearence in the sections (Fig. 4B,C). In crosssectioned dendrites, numerous microtubules were present (Fig. 4B,C). Profiles of cross-sectioned dendrites had a round or oval form, occasionally interconnected with small shafts (Fig. 5C). The shafts were always filled with microtubules (Fig. 5C). In the direct vicinity of the aqueduct, most of the profiles of dendritic varicosities were located parallel to the surface of the aqueduct. The mitochondria in the dendrites appeared to be round in transversely cut dendrites (Fig. 4A), but in longitudinally cut dendrites they were very long (up to 2 wm) and orientated along the long axis of the dendrites. Dendritic spines were extremely scarce (1-2 per section throughout the PAG; Fig. 5 C ) . They were always located on relatively thin dendritic processes. Most of the dendrites were at least partly enclosed by a thin glial membrane (Fig. 4A-C). The dendrites did not contain a clear vesicular population. Fibres. In transverse sections of the PAG, numerous thin unmyelinated fibres (from 80 to 570 nm) were found (Fig. 4B-D). The mean diameter of the thin fibres was 210 nm. Directly around the aqueduct, the mean diameter was 190 nm, but in the medial and outer parts of the PAG a

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Fig. 3. Ultrastructural aspects of PAG cells. A: Cluster of cells in the lateral part of the rostra1 PAG. Note close association of cell membranes without interdigitating neuropil, and invaginations of nuclear envelope. ~ 5 , 0 0 0B: . Cell with small rim of cytoplasm, filled with mitochondria (M), rough endoplasmic reticulum (RER) and ribosomes. x 11,000. C: Enlargement of the outlined area shown in A.

Arrows indicate the site of very intimate contact between the two cells. x 115,000.D Extensive rough endoplasmic reticulum in cell in lateral aspect of caudal PAG. Note free ribosomes. ~30,000.E: Cell in ventro-medial aspect of PAG with intense invaginations of the nuclear envelope, Golgi zones ( G )and lysosomes (arrows). x 12,000. N, nucleoli. Bars in A,B,D,E = 2 pm; bar in C = 0.2 p m .

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Fig. 4. Ultrastructure of neuropil elements of PAG. A Cytoplasm of astroglid 1x11. Note glial filaments (MF), rough endoplasmic reticulum (RER). D, dendrites, G1, glid processes. ~21,000. Inset ~ 3 0 , 0 0 0B .: Accumulation of thin unmyelinated fibres (stars) in the vicinity of a blood vessel (BV)in the dorso-lateral PAG. Note mitochondria (M)in

many of the thin fibres. X40,OOO. C: Part of neuropil in periaqueductal medio-caudal PAG. Note smooth endoplasmic cisternae (SER) in dendrites (D). x 15,000. D: Neuropil in the lateral PAG, halfway to the outer border. Note many thin fibres, intermingled with myelinated fibres. x 15,000.Bars = 1 km.

mean value of 230 nm was found. In general, the profiles of the fibres in the sections were round or polygonal (Figs. 4B-D, 8C) and frequently occurred in small or larger bundles (Fig. 4A-D). In many fibres, profiles of mitochondria were present (Fig. 4B,D). The number of microtubules

depended on the diameter of the fibres, with only one or two cross-sectioned microtubules in the very small fibres (Fig. 4B). The density of the fibres varied with respect to their location within the PAG. The larger blood vessels were

ULTRASTRUCTURE OF THE PAG always surrounded by large numbers of thin fibres, almost without other elements of the neuropil (Fig. 4B). A similar accumulation of thin fibres could be found in the dorsal part of the rostral PAG. Detailed analysis of the micrographs showed that from the ventricular surface to the outer border of the PAG, differences existed with respect to the number and diameter of the thin fibres, the myelinated fibres as well as the other neuropil elements. The cell-poor zone, directly surrounding the aqueduct, was characterized by many dendrites and axonal varicosities (Fig. 4C). Large numbers of thin fibres ran in a rostro-caudal direction. Regional differences with respect to the number of thin fibres were also found and a high density of fibres was found especially in the rostral PAG (Bregma -4.8). In the inner zone 121.33 ? 35.17 thin fibres were found per 25 km3,but the highest density was observed in the dorsal part, ventral to the posterior commissure (142.80 & 27.80/25 krn'). In the ventral inner zone of the PAG, lower numbers of fibres were present (86.60 5 22.03 fibre425 km'). In the intermediate and outer parts of the rostral PAG, 79.69 ? 30.13 and 65.47 ? 33.45 fibres125 km2were found, respectively. Also at the second level studied, the highest density of fibres (88.07 ? 15.89/25 km') occurred in the inner zone of the PAG. In the intermediate and especially in the outer part, lower numbers of fibres were present (58.40 f 26.84 and 31.87 2 18.87 fibred25 km2,respectively). Again the lowest numbers of fibres were found ventrally. At the third level studied, the same distribution was found. In the most caudal section, the situation was different again as the highest density of fibres was now located in the ventral inner zone of the PAG. Irrespective of the location of the parts of the sectors investigated, the density of thin fibres was much lower outside the PAG (mean 18.93 f 10.45 fibres/25 pm'), ranging from 15.23 to 23.34 fibres per 25 pm2. Axonal varicosities. It appeared that 6% of the surface area of the transverse sections of the PAG was occupied by varicose structures. Of all the varicosities measured (Fig. 5A), 43% were found in the inner sector directly around the aqueduct, 34% were found in the intermediate sector and 23% were located in the peripheral part of the PAG. These values indicate that the density of varicosities gradually decreased from the periventricular to peripheral parts of the PAG. No differences could be found in the rostro-caudal distribution of the varicosities. Axonal varicosities could be classified into five types: type I with asymmetrical densities (Fig. 5C) and round synaptic vesicles (23% of all the varicosities measured); type I1 contained symmetrical densities and round synaptic vesicles (14%; Fig. 5D);types I11 and IV were as types I and 11, respectively, but the varicosities contained dense-cored secretory granules, in addition to the electronlucent synaptic vesicles (42 and 17%, respectively; Fig. 5B). However, these dense-core secretory granules were nearly always located at some distance from the active synaptic site (Fig. 5B) and only a few dense-core secretory granules could occasionally be observed in the immediate vicinity of the synaptic specialization. Type V varicosity (Fig. 5E) was characterized by many large dense-core secretory granules (diameters of the secretory granules between 110 and 160 nm, surface area of the varicosities 0.34 ? 0.18 km2).Synaptic specializationswere not found on this type of varicosity, not even if the serial sectioning approach was used for detection (Fig. 5E). Of the total number of varicosities measured, 4% were of this type. In 49% of the profiles of types I-IV varicosities, the synaptic

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contact site was not present in the plane of the section. However, synaptic specializations of such varicosities could always be found after serial section analysis. The relative distributions of the different types of varicosity did not differ from the overall distribution, with the exception of type V varicosity (non-synaptic varicosities), which was exclusively located in the inner sector of the PAG. No differences could be found with respect to the length of the contact zone between the different types of varicosity (0.36 f 0.16and0.38 f 0.19,0.41 t 0.12,and0.41 2 0.16 km, respectively). Eight per cent of the varicosities showed more then one synaptic specialization (Fig. 5B). Also no differences could be found between the surface areas of the four types of varicosity (0.51 ? 0.24 and 0.53 ? 0.23, 0.47 2 21, and 0.56 f 18 pm2,respectively).Occasionally a varicosity with different types of vesicle were found 1%of all the varicosities contained clear vesicles that were not round but oval (Fig. 5F; mean surface area 0.53 5 0.37 pm', length of synaptic contact zone 0.38 ? 0.19 km). Of all the synaptic contacts, 0.9% were axo-somatic. These contacts did not differ from the axo-dendritic synapses. Synaptic contacts with spines were extremely scarce (2.3%of all the synaptic contacts). However, when present, such spines were located on the thin shafts of the dendrites (Fig. 5C). Glial. Regional differencesin the composition and characteristics of the glial of the PAG were not observed in any of the sections studied (Fig. 4A).

HRP experiments Labeled dendrites o f PAG neurons. The injection sites are schematically depicted in Figure 1and 6. Irrespective of the location of the injections, many cells and dendrites were completely filled with HRP (Fig. 7B,C,E). The dendrites contained numerous spindle-shaped swellings, interconnected by very thin shafts (Fig. 7B,C,E). These swellings represent propably an artefact induced by the HRP in the cells. From the central part of the injection site, numerous dendrites radiated away in the transversal plane through the neuropil of the PAG (Fig. 7A,B,E). Particularly from the lateral injections, it was evident that many PAG cells send their dendrites centrally towards the ventricular surface (Fig. 7A,B,E). In the vicinity of the aqueduct, the dendrites branched and many dendrites continued their course parallel t o the surface of the aqueduct, in some cases into the inner zone of the contralateral PAG. The HRPfilled dendrites did not show spines on a light or electron microscopiclevel. Fibre projections. Light microscopically, fibre projections from the injection sites could be observed to run through the PAG in rostral and caudal directions (Figs. 6, 7F,G). Electron microscopically, it appeared that both myelinated and thin unmyelinated fibres were labeled (Fig. 8A-D). Light microscopy showed that the central as well as the peripheral parts of the neuropil of the PAG seemded to be almost devoid of labeled fibres (Figs. 6A-D, 7F). However, on an electron microscopic level, particularly in the neuropil of the central zone, numerous labeled thin fibres could be seen (Fig. 8B,C).No labeled myelinated fibres were found in this central zone. Horizontal and sagittal sections indeed confirmed that almost all the fibres were orientated in a rostro-caudal direction (Fig. 8E-G). These sections also showed that the thin fibres were not completely filled with HRP (Fig. 8F). Careful analysis of the sections revealed

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Fig. 6. Injection sites in the right half of the PAG. The lucent central part of the injection site indicates the location of the tip of the needle. Black areas indicate the location of HRP filled cells. The right half (small dots) shows the semischematic distribution of light micro-

scopically visible labeled fibres; the left side (large dots) shows the mirror image of the retrogradely labeled cell bodies. No labeled cells were found in the contralateral neuropil of the PAG.

Fig. 5. Electron micrographs showing different types of varicosity taken from different parts of the PAG. A: Varicosity containing few dense-core secretory granules (SG) and only a few synaptic vesicles. The synaptic contact site and a cluster of synaptic vesicles are out of the plane of this section. Rostral, close to the ependymal lining of the aqueduct. PAC, ~ 4 0 , 0 0 0B: . Asymmetrical synaptic contact (Type 111) with a dendrite (D). Note the secretory granules located at a distance from the active site. x40,OOO. C : Synapse (Type 111) filled with many

synaptic vesicles and few dense-core secretory granules, making synaptic contact with a small dendritic spine. ~ 6 0 , 0 0 0 .D: Symmetrical synaptic contacts (Type, IV,arrowheads). ~35,000.E: Axonal profiles with dense-core secretory granules (Type V varicosity). ~50,000.F: Large varicosity with many pleomorphic synaptic vesicles. Note numerous mitochondria in this varicosity, compared to the absence of mitochondria in the varicosities shown in A-E. ~ 5 0 , 0 0 0 Bars . = 0.5

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Figure 7

Fig. 8. Electron micrographs of HRP injections. All micrographs are from ipsilateral PAG. A-D. Transverse sections. A: Below posterior commissure. Note mixture of unmyelinated and myelinated fibres. ~ 5 , 0 0 0B,C: . Labeled thin fibres in the inner part of middle and caudal PAG after injection in rostral PAG. x40,OOO and X27,000, respectively. D Fibres in middle PAG after rostral injection. X 10,000.E,F:Horizon-

tal sections. Note the absence of HRP in part of thin fibre shown in F (asterisk). x 15,000. G-J: Synaptic contacts in rostral, middle, and caudal PAG, after HRP injections in caudal (G), middle (J),and rostral PAG (H), respectively. ~ 1 4 , 0 0 0Bars . in A , D J = 2 prn. Bar in B,C = 0.5 pm,

Fig. 7. HRP tract tracing experiments. A-C,E details of the injection sites. A: Injection in rostral PAG (Bregma -6.3). ~ 4 5Dotted . line indicates the border of tho PAG. B,E: Details of injection shown in A. Note numerous dendritic processes (arrows) of HRP-filled cells, extending in the direction of the ventricle. B: Ventral PAG. E: Rostral PAG. ~280. C: Detail of dendrite labeled with HRP. Note varicosities. ~ 8 5 0 .

D Retrogradely labeled cells in the rostral PAG after an HRP injection in the caudal PAG. ~ 4 0 0F: . HRP-labeled fibres in rostral dorsolateral PAG. ~ 2 8 0G: . HRP-labeled fibres in the rostral PAG, at the transition with the diencephalon. Dotted line indicates the transition between the PAG and the posterior commissure. X280. Bar in A and C = 500 pm. Bar in B,D-G = 100 pm.

532

P. BUMA ET AL.

that intense fibre labeling occurred only in the immediate PAG. In PAG neurons, the following neuroactive peptides vicinity of the centre of the injection site (Figs. 6, 7A), have been found metenkephalin, dynorphin, somatostatin, suggesting that many of the fibres labeled were damaged corticotropin releasing hormone, neurotensin, vasoactive fibres en passant. Also many cell bodies were labeled in the intestinal polypeptide, substance P (for Refs. see Nieuwendirect vicinity of the injection site. The number of labeled huys, '85). In general, the distribution of the transmitterfibres gradually decreased with increasing distance from specified somata does not match the cytoarchitectural the injection sites. Horizontal sections revealed that occa- subdivisions of the PAG (Nieuwenhuys, '85; Buma, '89b). sionally, a thin fibre widened into a synaptic varicosity (Fig. Thus, the heterogeneity within a given subnucleus may 8G). Most of the fibres that traversed the PAG remained on even be far greater than expected on the basis of the the ipsilateral side, but a few fibres always crossed t o the classical cytoarchitecture alone. The fact that various transcontralateral neuropil of the PAG. mitter-specifiedcell types appear to be intermingled, makes More specifically, from the injection in the rostral PAG it even harder to indicate the relationship between the (Fig. 6, first row), at the transition between the PAG and cytoarchitectural boundaries and the functional organizathe diencephalon, fibres were observed descending into the tion of the PAG. PAG, laterally from the ventricle. At the more caudal levels In a previous study on the ultrastructure of the PAG of of the PAG (Bregma -7.8) the number of fibres diminished the cat, Gioia et al. (1983) reported that neurons are and they were located more ventrally. In the caudal PAG frequently located in the direct vicinity of one another. We (Bregma -8.3), the fibres were either running diffusely and found evidence for the existence of gap junctions between ventro-laterally towards the region of the cuneiform nu- individual PAG neurons. It is now generally accepted that cleus, or more ventrally into the transition area of the gap junctions are the sites of electrotonic coupling between central grey of the metencephalon. Injections in the middle neurons (Bennett and Goodenough, '78; Roubos et al., '85). of the PAG (Fig. 6B,C) showed projections in both the Therefore, the close apposition between individual PAG rostral and caudal direction. neurons and the occurrence of gap junctions strongly After all the HRP injections, labeled neurons were found suggests that electrotonic coupling is a common phenomethroughout the PAG, although the number and location non in the PAG of the rat. Small clusters of PAG neurons varied between individual injection sites. These cells were may act cooperatively. One other difference from the PAG characterized by many membrane-bound HRP granules of the rat is the nuclear inclusions that have been observed (Fig. 7D, electron microscopicobservations,data not shown). in PAG neurons of the cat (Gioia et al., '83). In the present The cells were found in a dorso-lateral (Bregma -5.8 to study, atypical nuclear inclusions were not found. -7.3) or ventro-lateral position (Bregma -8.3 and -8.7). Fibre composition However, the number and location (ventral or dorsal aspects of PAG) of the labeled cells was dependent on the To our knowledge, the present report is the first ultralocation of the injection. Many labeled neurons were found structural study on the fibre architecture of the PAG. The in the latero-dorsal aspect of the middle part of the PAG, variation in density of the thin fibres has important implicaparticularly after the HRP injection into the caudal ventro- tions for studies on the functional organisation of the PAG lateral neuropil of the PAG (Fig. 6D). with electrical stimulation. Both in the routine and the HRP material, it is evident that the majority of the fibres are very thin (-100-300 nm). In our study it was not DISCUSSION possible to discern the thin HRP-filled fibres on a light Cytoarchitecture microscopic level. On the basis of light microscopy it was Most studies on the structure of the PAG focused on the previously demonstrated that a layer of thin fibres is light microscopic aspects of this brain region. Controversy present immediately around the aqueduct (Mantyh, '82a). exists regarding the heterogeneity of the PAG and the In the present study it was demonstrated that high densinumber and demarcation of subnuclei within the PAG. On ties of fibres pass in a rostro-caudal or caudal-rostral the basis of quantitative cytoarchitectural studies on the direction through the PAG, parallel to the aqueduct in a orientation, diameter and density of cell bodies, the PAG very diffuse way and make up at least two thirds of the has been classically divided into three major nuclei: dorsal, PAG. This fibre condensation as a whole probably forms the medial, and lateral (Olszewski and Baxter, '54; Taber, '61; bundle of Schutz (1891), which was almost invisible at the Hamilton, '73; Liu and Hamilton, '80). Some studies indi- light microscopic level in the present study on the rat. In cated that these subnuclei are composed of the same cell the peripheral parts of the PAG, fewer fibres were present. types (Liu and Hamilton, '80; Gioia et al., '84; Beitz, '85; Functional aspects Meller and Dennis, '90b). Other authors could not find discrete subnuclei or subdivions and suggested that the The PAG can also be subdivided into its different parts on cells of the PAG are arranged in discrete groups of cells with the basis of the localization of different functions and variable cytoarchitectural characteristics (Hardy and Leich- behaviours. Electrical stimulation of the PAG may induce netz, '81; Mantyh, '82b; Mantyh, '83a,b; Gioia et al., '84). diverse reactions, such as defense reactions (McDoughallet According to these latter authors, the PAG could be de- al., '851, rage reactions (Hunsberger, '561, bladder distenscribed as a mosaic of clusters of possibly functionally sion (Skultety, '59), gastric motility (Skultety, '631, vocalizarelated neurons (Mantyh, '82a). tion (Kanai and Wang, '62; Jurgens and Pratt, '79; Larson, We also observed regional differences in the orientation, in press), emotional reactions (Hunsberger, ,561, aggressive density, and diameter of the cells; the differentiation has behaviour (Mos et al., '82), reproductive behaviour (Sacurrently been worked out in detail. However, the different kuma and Pfaff, '79; Sirinathsinghji et al., '83; Sirinathsingboundaries observed when cytoarchitectonic and immuno- hji, '85, '87; Ogawa, in press), (excessive)eating (Adametz cytochemical criteria are used constitute one of the compli- and O'Leary, '591, inhibition of the jaw opening reflex cating factors in understanding the organization of the (Oliveras et al., '74), inhibition of oxytocin release (Aulse-

ULTRASTRUCTUREOFTHEPAG

533

brook and Holland, '69),analgesia (Reynolds, '691, memory Spines may have a function in the regulatory mechanism storage (Kesner and Kalder, '801, slow wave sleep (Tissot, that controls the excitatory input to the cell (Palay and '801, and eye movements (Edwards and Henkel, '78). A Chan-Palay, '741, or they may function to extend the general problem with electrical stimulation is that the receptive surface of the cells. However the need for this precise action point is not known. Both neurons and latter function does not seem to be very large, because the passing myelinated and unmyelinated fibres can be acti- density of synaptic contacts on PAG dendrites in the rat is vated. The present study demonstrated that lateral PAG very low. Dendritic spines have also been considered to be a neurons send their dendrites transversely in the direction means of extending the surface area of dendrites for the of the ventricular region and that throughout most parts of reception of information from s o n s located at some disthe PAG, large numbers of unmyelinated fibres course in a tance, intermingled with other elements (Peters and Kaiserlongitudinal direction. These fibres are collectively known man-Abramof, '70). In the present study large areas of the as the bundle of Schutz (1891) or the periventricular fibre dendrites were not occupied by synaptic terminals. Thus, system. Thus, irrespective of the location of the stimulation apparently the receptive membrane surface of the dendrites electrode within the PAG, both neurons and passing fibres without spines is large enough for all the synaptic contacts. will always be stimulated, which makes it very difficult to In the present study, it was found that a small percentage localize the origin of a response after electrical stimulation. of the varicosities lacked synaptic specializations, It has More recently chemical stimulation has been applied locally previously been demonstrated with immunocytochemistry to the PAG, because of the drawbacks and complicating that ACTH and LHRH varicosities in the PAG are also of factors of electrical stimulation in an area such as the PAG. this nonsynaptic type (Buma, '89a,b; Buma et al., '89; Microinjections of excitatory amino acids had clear effects Lookeren Campagne et al., '91). Furthermore, the present on defensive behaviour. Detailed analysis showed that study and previous studies show that in many peptidergic injections into different parts of the PAG elicited clearly synaptic varicosities, the secretory granules in which the different behaviour patterns in the rat and cat (Bander et peptides are located are not in the direct vicinity of the al., '91). In addition, in a series of careful studies, the synaptic specialization (Barber et al., '79; Light et al., '83; functional roles of specific lateral parts of the PAG for Maxwell et al., '83; Clements et al., '85; Zhu et al., '86; cardio-vascular mechanisms has been elucidated by a com- Ulfhake et al., '87; Buma, '89a,b; Buma et al., '89; Lookeren bination of physiological and tract-tracing experiments Campagne et al., '91). Immunocytochemistry and electro(Carrive et al., '88, '89; Carrive and Bandler, '91; Bandler et physiological studies, particularly on invertebrate animals, al., '91). Thus, problems related to the highly complex have demonstrated that peptides released from unspecialanatomical organization of the PAG, the intermingled ized parts of the axolemma may be involved in the commufibres en passant, and dendrites of PAG neurons may in the nication between neurons (see Buma and Roubos, '86; future be resolved by combinations of physiological, behav- Buma and Nieuwenhuys, '88; Buma, '89b for references).A number of circumstantial arguments, mostly derived from ioural, and microinjection experiments. immunocytochemical, pharmacological and electrical stimulation studies on the function of transmitter-specified Dendritic organisation fibre contingents in the PAG, suggested that such nonsynAnother complication in the relationship between cytoar- aptic transfer of information also plays a role in the PAG of chitectural and functional subdivisions of the PAG, arises the rat (see Buma, '89a,b for references). However, the from the morphological characteristics of the dendrites of demonstration of nonsynaptic communication in a complex the PAG neurons. These dendrites radiate strongly, partic- brain region such as the PAG, will be very difficult. ularly in the direction of the aqueduct. HRP-filleddendrites Moreover, in the present study only 4% of the varicosities of lateral PAG neurons radiate not only to the ventricular were of the non-synaptic type. Therefore, it does not seem surface, but also, although to a lesser extent, in the opposite very plausible that nonsynaptic communication plays any direction outside the PAG. Ram6n-Moliner and Nauta ('66) significant role in the PAG. Additional studies on the classified the PAG as part of the "isodendritic core of the ultrastrucutural localization of release sites and receptor brain." According to their description, the area is populated molecules for the transmitters involved are needed to by neurons with long dendrites that radiate in all direc- further substantiate the concept that non-synaptic transmistions. Isodendritic neurons are believed to have undergone sion plays a role in the vertebrate brain. very little evolutionary differentiation and to receive the The further biochemical characterization of the PAG and afferent input of multiple origins. The present study con- the anatomical localization of receptors (serotonin: Pazos et firms the relatively undifferentiated appearance of PAG al., '85; Pazos and Palacios, '85; somatostatin: Reubi and neurons. The paucity of spines on the dendrites of PAG Maurer, '85; Uhl et al., '85; calcitonine: Fabbri et al., '85; cells, as found in the present study, is in contradiction with exitatory amino acids, glycine: Probst et al., '86; NMDA the observations of Gioia et al. ('85, cat) in their Golgi Jacquet, '88; GABA,: Mccarthy et al., '91a,b) is therefore studies and with the observations of Beitz and Shepard very important in elucidating the roles of the different ('85, rat). Both groups of authors described different types transmitter substances that are abundantly present in the of cell in the PAG, some of them rich in spines. The reason PAG (Nieuwenhuys, '85). Especially the combination of for this discrepancy is not clear, but the previous studies on immunocytochemical, pharmacological, and receptor studthe PAG in the rat did not include electron microscopy. As ies on the role of separate transmitters will be needed to the few spines observed in the present study were all elucidate the functional morphology of the PAG. smaller than one micron, it may be questioned whether the rapid Golgi method is sensitive enough for the light microLITERATURE CITED scopic demonstration of such small elements. 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