Types of neurons and some dendritic patterns of basolateral amygdala in humans — a Golgi study

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Types of neurons and some dendritic patterns of basolateral amygdala in humans - a Golgi study Jovo Tosevski 1, Aleksandar Malikovic 2, Jelena Mojsilovic-Petrovic 3, Vesna Lackovic 4, Miodrag Peulic 1, Predrag Sazdanovic 1, and Chris Alexopulos s 1Institute of Anatomy, School of Medicine, Svetozara Markovica 69, YU-34000 Kragujevac, Serbia, Yugoslavia, 2Institute of Anatomy, School of Medicine, Belgrade, Serbia, Yugoslavia, 3Institute of Biological Sciences, National Research Council, Ottawa, Canada, 4Institute of Histology and Embryology, School of Medicine, Belgrade, Serbia, Yugoslavia, and 5 Clinic of Neurology, School of Medicine, Kragujevac, Serbia, Yugoslavia

Summary. Classification of the neurons in the human basolateral amygdala is performed on preparations impregnated by the Golgi technique. Three different neuronal types are found in the nuclei of the basolateral amygdala: Type I - Pyramidal cells, with numerous dendritic spines and two subtypes (slender and squat); Type II Modified pyramidal cells, sparsely spinous with rare dendritic spines and two subtypes (single apical and double apical) and; Type I I I - Non-pyramidal cells, with few dendritic spines and three subtypes (bipolar, multipolar and gliaform). The analysis of the primary dendritic branches pointed out the occasional presence of dendritic bundles (fascicular dendritic arrangement) with their predomination in the parvicellular division of the basal nucleus and paralaminar nucleus. Additionally, the presence of dendrodendritic contacts, indicated by light microscopy, was also found in the parvicellular division of the basal nucleus and especially in the paralaminar nucleus. Key words: Amygdala - Neurons - Dendrites - Dendrodendritic contacts - Golgi - Humans

Introduction The amygdaloid complex represents a heterogeneous nuclear group located in the dorsomedial portion of the temporal lobe. This collection of the nuclei lies under the Correspondence to: J. Tosevski E-mail: [email protected]

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Ann Anat (2002) 184:93-103 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/annanat

uncal region, above and in front of the temporal horn of the lateral ventricle. The amygdaloid complex is divided into two main groups, the corticomedial and basolateral nuclei. The basolateral amygdala is a larger and better differentiated group in the human brain, and the smallcelled (the parvicellular) part of it represents the phylogenetically younger and more progressive part of the human amygdala (Stephan and Andy 1977). The early nomenclature and the parcellation of the basolateral amygdala originate from the comparative neuroanatomical investigations (Johnston 1923; Humphrey 1936). In spite of numerous and detailed studies on the parcellation of amygdala, difficulties in the terminology have still remained. In the present study, nomenclature and parcellation given by Ammaral et al. (1992) are accepted. This nomenclature agrees well with the previous separation (Price et al. 1987) which defined the basolateral amygdala as a nuclear mass situated more laterally and ventrally and composed of four nuclei: the lateral nucleus (LN), basal nucleus (BN), accessory basal nucleus (ABN) and paralaminar nucleus (PLN). However, reference data on the cell typing of the human basolateral amygdala are insufficient for a modern concept of the amygdala functions. They are usually reduced to basic classification of amygdaloid neurons to pyramidal and non-pyramidal types, failing to include subtyping which should be expected for such a complex structure. Additionally, there is only a small number of studies addressing dendritic organization which is particularly important from the functional point of view. High dependence of the amygdaloid functional properties may also be seen on the example of the characteristic organization of the afferent intranuclear and efferent amygda0940-96021021184/1-93 $15.00•0

upon autopsies performed within 10 hours post-mortem. Only normal brains with no visible malformations and without any neuropathological changes or neuropsychiatric history were used. The brains were fixed in phosphate buffered solution of 10% formalin (3.7% formaldehyde) over a period of at least 3 months. The 16 blocks, originating from 8 brains (16 hemispheres), comprising the amygdala were stained according to the GolgiKopsch method. Each of the remaining 8 blocks from 4 brains (8 hemispheres) was cut along the coronal plane and divided into 4 thinner slices. The slices were stained alternately with the Golgi-Kopsch, Nissl, and Kltiver-Barrera methods in order to enable confirmation of the exact topographical relationships between different nuclei of the amygdaloid complex. The application of the Nissl and Kltiver-Barrera method was necessary for further delineation of the amygdaloid nuclei on 20 pm thick coronal sections. The blocks and slices treated by the Golgi-Kopsch method were carefully trimmed and 80-100 gm thick coronal, horizontal and sagittal sections were cut. The transparency of the silver impregnation was the most favorable in 80 pm thick coronal sections. Along the two-thirds of the whole rostro-caudal extent of the amygdaloid complex, all of the investigated nuclei were presented and these sections were extensively used. The morphological landmark of the special topographic interest was the amygdaloid fissure. When the amygdaloid fissure was presented it clearly divided the amygdaloid complex from the hippocampal formation along the ventral surface of the temporal lobe. All of the basolateral amygdaloid nuclei were well impregnated on the prepared sections. The corticomedial amygdala, entorhinal and perirhinal cortex were well impregnated too. The only exception was the central amygdaloid nucleus which indicated lower impregnation quality on the sections used. On the other hand, the silver impregnation of the adjacent hippocampal formation was very good in quality but it characteristically showed lighter shade than the regions of the amygdaloid complex. Presented details in the staining sensitivity or selectivity, together with the obvious cytoarchitectural differences and lower impregnation quality of the central amygdaloid nucleus, may be Golgi-dependent characteristics observed in these regions. It is well known that the Golgi method is highly selective and picks out only a small proportion of the presented neurons. On the other hand, this technique provides a lot of very useful information on the neuronal processes and their branching patterns. All deep amygdaloid nuclei: the lateral, basal, accessory basal and paralaminar - known as the ventral part of the basal nucleus (Amaral et al. 1992) were investigated. The neurons, as well as the fibers from many well-stained areas were extracted from the preparations and reproduced in the camera lucida (ReichertJung, Polyvar) drawings. The total number of the neurons chosen from the basolateral amygdaloid nuclei was 300. The photographs of the selected neuronal types were also taken under different magnifications. The drawings of the neuronal types were first recorded by scanning, to be subsequently digitalized, and finally exposed to the measurements. The classification of neurons was performed according to the following criteria: a) shape and size of the cell bodies; b) dendritic organization - the position, number, length and its branching patterns; c) density of the spines covering dendrites; and d) axonal branching patterns. The diameters of the cell impregnation of the adjacent hippocampal formation was very good in quality but it characteristically showed lighter shade than the regions of the amygdaloid complex. Presented details in the staining sensitivity or selectivity, together with the bodies were measured in

loid fibers. So, fear conditioning of the experimental animals evidenced that all the inputs coming from the thalamo-amygdala pathways as well as from cortico-amygdala pathways were converged into the lateral amygdaloid nucleus, to be subsequently distributed through the internal amygdala pathways, converged into the central nucleus, leaving amygdaloid complex therefrom (Davis 1992; LeDoux 1993; Quirk et al. 1997). The results of the studies performed on the experimental animals have shown that the amygdaloid complex has heavy and reciprocal connections with the orbital and medial frontal cortices in the monkeys (Turner et al. 1980; Porino et al. 1981; Amaral and Price 1984). The reciprocal connections with cingulate, temporal and insular regions of the neocortex are also validated in the monkeys (Aggelton et al. 1980; Mufson et al. 1981). The recent functional imaging studies in humans have suggested a novel concept of amygdaloid complex as an integrative part of the cortical circuitry which has to do with the modulation of visual (Breiter et al. 1996), olfactory (Zald and Pardo 1997) and gustatory stimuli (Zald et al. 1998), arousal and facial expression of fear and anger (Davis 1992; L e D o u x 1996; Morris et al. 1996, 1998a; Phillips et al. 1997). A morphological basis of the functional properties of the amygdala is required for the assessment of the already established new functional concept proposing that the h u m a n amygdaloid complex is involved in decisionmaking and m e m o r y (Packard et al. 1994; Cahill et al. 1995; Adolphs et al. 1997), emotional and associative learning (Morris et al. 1998 b; Cahill and M c G a u g h 1998), emotional m e m o r y ( L e D o u x 1996), during conditioned fear acquisition and extinction (LaBar et al. 1998), planning and social judgment (Adolphs et al. 1998; Adolphs 1999), that could be collectively described as the cognitive-emotive processes (Gray 1999). The present study was aimed at a) analysis of morphological features of the neurons in the nuclei of basolateral amygdala; b) performing of morphological separation of these neurons according to the shapes and sizes of their somata, the position, number, length and branching patterns of their dendrites, presence or absence of dendritic spines and form of axons; c) classification of the neurons in the nuclei of the basolateral amygdala. Our research on the Golgi morphology of the h u m a n basolateral amygdala is the result of the need for better understanding of the morphological characteristics of neuronal subtypes and complexity of the dendritic organization as neural substrates of the complex functional properties presented in the recent imaging studies.

Material and methods The present study included 12 postmortem brains (24 hemispheres) belonging to adults of both genders (male to female ratio 6:6), aged 30-65 years. All the brains used were obtained 94

the sections throughout the nuclei of the basolateral amygdala. The measurements of the maximum length (Dmax) and width (Drain) of the somata, as well as the total dendritic length (TDL), were performed on all of the neurons belonging to each neuronal type using AutoLISP for AutoCAD 2000 on the Pentium PC.

the basis of their shape, size and dendritic patterns two subtypes of pyramidal cells were differentiated: slender and squat. The slender pyramidal cells were more than twice as high as they were broad. Their apical dendrite was long and robust. The sizes of their soma (21.5x16.0 gm in diameter) and their total dendritic length (1119.2 gm) were larger and longer than in the squat pyramidal cells. The squat pyramidal cells were approximately as high as they were broad. Their primary dendrites were roughly equal in caliber with no evident apical dendrite. The size of their soma (15.8 x 12.7 gm in diameter) and their total dendritic length (620.6 ~m) were smaller and shorter than in the slender pyramidal cells. The modified pyramidal cells had a rounder soma in comparison with the true pyramidal cells. They were less spinous than pyramidal cells. The spines covered much more distal parts of the primary dendrites and in particular the secondary dendrites. It seemed that the arborization of the initial segments of the modified pyramidal cells was higher in comparison with true pyramidal cells. O n the basis of their apical dendrites the modified pyramidal cells were further subdivided into two subtypes: the single apical and double apical. The single apical cells had one dominant apical dendrite which tapered gradually from the cell body. Their soma was 21.1 x 10.0 gm in size while their total dendritic length had 1341.9 gm in diameter. The double apical cells had two thick apical dendrites radiating away in the same direction from the cell body. Their soma was small (15.2x10.0 Bin in diameter) and their total dendritic length was 318.6 gin.

Results Classification of the neurons in the human basolateral amygdala

The neurons of the h u m a n basolateral amygdala were classified into three main types: type I - pyramidal cells, type II - modified pyramidal cells and; type III - nonpyramidal cells. The pyramidal cells were the predominant cell type in the nuclei of the basolateral amygdala. The outline of their soma was conical with one thick apical and many thinner basal dendrites. All pyramidal neurons were characterized by the presence of numerous dendritic spines. Most of the dendritic spines had thin and very short stalks with small terminal swellings. The dendritic spines were higher in density as distance from the soma increased. They covered more distal portions of the primary dendrites, as well as the secondary and tertiary dendrites. The axon of pyramidal cells arose from the base of the cell body, or from one of the primary dendrites near the cell body. It gave off several collaterals that ramified near the cell body. O n

Table 1. Neurons of the basolateral amygdala in human. Classification

The neurons of the basolateral amygdala in human Type Subtype

I: Pyramidal Slender Squat

II: Modified pyramidal Single apical Double apical

III: Nonpyramidal Bipolar Multipolar

Gliaform

Table 2. Neurons (N = 300) of the basolateral amygdala in human. Results are in micrometers (gin). The diameters of the cell bodies (+ SD) were measured in the sections throughout the nuclei of the basolateral amygdala. Two diameters were taken from each cell body: a) the maximum length (Dmax), and b) the maximum width (Dmin). In addition to these two diameters, the measurements of the total dendritic length (TDL) were performed too

Type

Subtype

Dmax

Dmin

TDL

I. Pyramidal

Slender Squat

21.5 + 1 15.8 + 0.8

216.0 + 0.7 12.7 + 0.7

1119.2 + 39.6 620.6 + 48.8

II. Modified pyramidal

Single apical Double apical

21.1 + 1.6 15.2 + 0.8

10.0 + 0.8 10.0 + 0.9

1341.9 + 56.0 318.6 + 48.5

III. Nonpyramidal

Bipolar large small Multipolar large small Gliaform

27.0 + 0.6 14.0 + 0.6

10.0 + 0.9 9.2 + 0.6

519.3 + 38.7 266.1 + 29.4

39.8 + 1.5 15.8 + 0.6 9.1 + 0.5

17.9 + 0.5 11.0 + 0.6 6.9 + 0.9

1502.6 + 47.6 688.1 + 52.4 266.1 + 39.3

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D

B V

Fig. 1. Drawings of Golgi impregnated pyramidal (Type I) and modified pyramidal (Type II) cells of the human basolateral amygdala. A: Pyramidal neuron - subtype slender in the lateral amygdaloid nucleus. B: Pyramidal neuron - subtype squat in the accessory basal amygdaloid nucleus. C: Modified pyramidal neuron - subtype single apical in the basal amygdaloid nucleus. D: Modified pyramidal neuron - subtype double apical in the paralaminar amygdaloid nucleus. Note the presence of the numerous dendritic spines in pyramidal cells and sparsely spinous modified pyramidal cells. Cross indicates orientation. Scale = 50 gm.

C

D

m

The nonpyramidal cells represented a morphologically heterogeneous type, situated among the pyramidal neurons in all nuclei of the basolateral amygdala. Their cell bodies varied in their shape and size. The dendrites radiated out in all directions from their cell bodies and branched modestly in the vicinity of their soma. The nonpyramidal cells had no dendritic spines or, at least, many fewer dendritic spines than pyramidal and modified pyramidal cells. The axon, difficult to impregnate satisfactorily, interrupted in the vicinity of the cell body into a larger number of collaterals. Among the nonpyramidal cells three subtypes occurred: bipolar, multipolar and gliaform cells. The bipolar cells had a fusiform or oval soma and single dendrites extending from the opposite poles of the cell body. Their dendritic trees were elongated and markedly narrow. The axon was given off from the soma or from the root of one of the dendrites. According to the size of their bodies there were large (27.0x10.0 gm in diameter) and small (14.0x9.2 gm in diameter) bipolar cells. The shape of the cell body in the large bipolar cells was fusiform, while in the small bipolar cells it was oval,

in most cases. The total dendritic length of the large bipolar cells (519.3 ~tm) was twice that of the small bipolar cells (266.1 gm). In some of the bipolar 10 cells, dendrites emerged from both poles and ramified considerably, forming two rather narrow plexuses. The multipolar cells had polygonal somata. Their dendrites (3-7 primary dendrites) arose from any part of the cell body and radiated out in all directions. The axon ramified considerably. On the basis of the size of their bodies there were large (39.8x17.9 gm in diameter) and small (15.8x11.0 ~tm in diameter) multipolar cells. The total dendritic length of the large multipolar cells (1502.6 ~tm) was more than twice that of the small multipolar cells (688.1 gin). The gliaform cells had a small and spherical outline of their somata. The dendrites of the gliaform cells (5-9 primary dendrites) were rather short and profusely branched. Their axons formed an extensive local axonal plexus interwoven with thin and delicate dendrites. The gliaform cells were among the smallest in the human basolateral amygdala. Their soma was sized only 9.1 x 6.9 gm while their total dendritic length was 266.1 gm. 96

Fig. 2. Microphotographs of Golgi impregnated pyramidal (Type I) and modified pyramidal (Type II) cells of the human basolateral amygdala. A: Pyramidal neuron - the subtype slender in the lateral amygdaloid nucleus. B: Pyramidal neuron - the subtype squat in the basal amygdaloid nucleus. C: Modified pyramidal neuron - subtype single apical in the basal amygdaloid nucleus. D: Modified pyramidal neuron - subtype double apical in the paralaminar amygdaloid nucleus. Magnification x 600.

Dendritic bundles (fascicular dendritic arrangement) in the nuclei of the basolateral amygdala

laterally, dorsally and ventrally. This kind of dendritic arborization was organized in such a manner that one apical dendrite ran directly into the bundle oriented laterally, while the second apical dendrite divided into two secondary branches that joined the bundles oriented dorsally and ventrally. The dendritic bundles were surrounded by numerous axonal terminals originating from the distant neurons. These axonal terminals were wrapped around dendrites in a "climber-like" manner (Fig. 6). The axons of the neurons in the basolateral amygdala showed profuse arborization in their proximal segments. The spines covered proximal axonal segments. Axonal terminals clustered around the dendrites were seen in some places. The origin of these axonal terminals is unknown.

In contrast to the usual axonal organization, arranged either in the form of intranuclear bundles (Fig. 6) or in the form of centrifugal amygdaloid pathways (the stria terminalis and ventral amygdalofugal pathway), an interesting dendritic arrangement in the nuclei of the basolateral amygdala, was observed. The dendritic organization of the basolateral amygdala showed remarkable features presented in the parvicellular division of the basal nucleus and paralaminar nucleus. In fact, well formed and robust primary dendritic branches were assembled in differently oriented bundles. In the parvicellular division of the basal nucleus, the majority of the dendrites assembled in the bundles run ventromedially while the axons run dorsolaterally. In contrast to the basal nucleus, dendritic bundles in the paralaminar nucleus mostly run mediolaterally. The dendritic bundles were mostly formed by the modified pyramidal (single apical and double apical) and the multipolar cells. The dendrites that originated from the double apical cell frequently run outward under the rectangles in order to form three different bundles oriented

Dendrodendritic contacts (DDC) in the nuclei of the basolateral amygdala The most striking aspect of the examined structure in the current study was the presence of the dendrodendritic contacts (DDC) between certain neuronal types of the basolateral amygdala in humans, indicated by the light

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Fig. 3. Drawings of Golgi impregnated nonpyramidal cells (Type III) of the human basolateral amygdala. A: Large bipolar neuron in the lateral amygdaloid nucleus. B: Small bipolar neuron in the accessory basal nucleus. C: Large multipolar neuron in the magnocellular division of the basal amygdaloid nucleus. D: Small multipolar neuron in the paralaminar amygdaloid nucleus. E: Gliaform neuron in the paralaminar amygdaloid nucleus. Nonpyramidal cells had no dendritic spines or, at least, deeidely fewer dendritic spines than pyramidal and modified pyramidal cells. Cross indicates orientation. Scale = 50 gm.

microscopy. It is well known that the Golgi method may be less reliable for detection of this kind of contacts. The reason is the wealth of the cytomorphological details on the sections that may occur inside the well impregnated and less transparent patches. In order to avoid any kind of confusion, our attention was focused on the D D C inside the sections and patches where the impregnation was transparent. By this way the D D C were clearly visible on the sections we used. The D D C were established between different types of the cells in the nuclei of the basolateral amygdala. However, it seemed that two forms of the D D C dominated. The first and more frequent form was established between the large bipolar cells, as the representative of the nonpyramidal cells and the double apical cells as the representative of the modified pyramidal cells. More frequently, this form of the D D C was formed by the secondary and tertiary dendritic branches and less frequently by the primary dendritic branches. The primary dendritic caliber of the bipolar cells tapered gradually running away from the soma in order to form the contact with the primary apical dendrite of the pyramidal cell.

The second and less frequent form of the D D C was established between the large bipolar cells and the pyramidal cells, either of the slender or squat subtype. The D D C are found to exist between the small bipolar and pyramidal cells as well. The D D C were observed in all nuclei of the human basolateral amygdala. Nevertheless, it seemed that their presence was more frequent in the parvicellular division of the basal nucleus and especially in the paralaminar nucleus.

Discussion Although a rapid increase in knowledge on the amygdala in subhumans has been achieved in the last few decades, it is a fact that data on the neuronal morphology of the amygdala are exceptionally rare in primates, particularly in humans. The basolateral amygdala in the subhuman mammals exhibited two main cell types: pyramidal and nonpyramidal cells. The pyramidal neurons of the basolateral amyg98

dala are characterized by the presence of pyramidal, "pyramidal like", "semipyramidal" or piriform somata that vary in size depending on the species and nucleus. This morphologically heterogeneous cell type is distinguished by the presence of numerous dendritic spines on the sec-

ondary and more distal dendritic branches. Pyramidal neurons have been described in all species and referred to as the P cells (Hall 1972), class I (McDonald and Culberson 1981; McDonald 1982) and pyramidal neurons (Millhouse and DeOlmos 1983). The nonpyramidal neu-

Fig. 4. Microphotographs of Golgi impregnated nonpyramidal cells (Type III) of the human basolateral amygdala. A: Large multipolar neuron in the magnocellular division of the basal amygdaloid nucleus. B: Small multipolar neuron in the paralaminar amygdaloid nucleus. C: Large bipolar neuron in the lateral amygdaloid nucleus. D: Small bipolar neuron in the accessory basal nucleus. E: Gliaform neuron in the paralaminar amygdaloid nucleus. Orig. magnif, x 600. 99

Fig. 5. Dendritic bundles (fascicular dendritic arrangement) in the human paralaminar amygdaloid nucleus. Well formed, robust and thick primary dendritic branches were assembled in the bundles that run mediolaterally, in most of cases. Dendritic bundles are indicated by the arrows in different magnifications: x 440 (A) and x 700 (B).

Fig. 6. Axonal arrangement in the nuclei of the human basolateral amygdala. A: Pyramidal neuron - subtype squat in the paralaminar amygdaloid nucleus. Axonal initial segment is clearly visible. Note the presence of axonal spines indicated by the arrow. Orig. magnif, x 1400. B: Axonal bundles that run parallel to the amygdaloid fissure. It seems that these axonal bundles (indicated by the arrows) belonging to the initial segments of the stria terminalis and ventral amygdalofugal pathway. Magnification x 100. C: Axonal terminals around dendritic bundles in the paralaminat amygdaloid nucleus. These terminals (indicated by the arrow) originate from a distant neuron. Orig. magnif, x 1400.

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Fig. 7. The dendrodendritic contacts (DDC) established between different neuronal types in the paralaminar amygdaloid nucleus. In this case DDC was established between a large bipolar cell (LB) and a pyramidal cell - subtype slender (P). DDC are indicated by the arrows in different magnifications: x700 (A) and x 1575 (B). rons of both main cell types of the basolateral amygdala are comparable to the cortical pyramidal and nonpyramidal neurons. However, our preparations clearly indicate that classification of neurons only to pyramidal and non-pyramidal is impossible, since there are modified forms of the pyramidal neurons as well as subtypes of the non-pyramidal neurons. The modified form of the pyramidal neurons Type II in our classification, represents an important proportion of the neuronal population of the basolateral amygdala and two subtypes - single apical and double apical are clearly distinguished, not only by one; i.e. two apical dendrites, but also by the larger soma in the single apical subtype when compared to double apical one. On the basis of the valuable pigmentoarchitectonic analysis of the basolateral amygdala in humans (Braak and Braak 1983) three neuronal classes are revealed. One of them (the class I cells) represents the cells with a stout main dendrite and presence of densely distributed dendritic spines. This neuronal class corresponds to our pyramidal and modified pyramidal neurons (the cell types I and II). On the other hand, the class II cells were characterized by variations in size and shape of their somata, and smoothly contoured dendrites with a few spines. This neuronal class is comparable to our nonpyramidal neurons (cell type III). The term "modified pyramidal cells" has been used to determine either semipyramidal spiny or spiny stellate neurons in the basolateral amygdala of the rat by the observation that in some cases pyramidal cells have two apical dendrites or exhibit apical dendrites with bifurcations of roughly equal caliber with no obvious apical dendrite (McDonald 1992). We believe that in humans, modified

pyramidal cells clearly indicate presence of the two subtypes. The cell bodies of both modified pyramidal cell subtypes (single apical and double apical) show a small number of dendritic spines and vary in shape from the pyramidal to ovoid outline of their somata. Anyway, the modified pyramidal cells have a rounder outline of their somata in comparison with true pyramidal cells. Although pyramidal cells (the type I cells) have many more dendritic spines than modified pyramidal cells (the type II cells), our impression is that in pyramidal cells dendritic spines are lower in density in comparison with the observations of some other authors. The fact is that pyramidal neurons in the human basolateral amygdala are moderate spinous rather than dense spinous cells. Also, pyramidal cells are characterized by the clear variability in the position, number, caliber and length of their apical and basal dendrites. The indicated variability was previously reported in some experimental mammals (Millhouse and DeOlmos 1983; McDonald, 1984). The immunohistochemical studies in the human amygdaloid complex using the calcium-binding protein parvalbumin (Sovari et al. 1995) suggest the existence of three different types of immunoreactive aspiny neurons ranging from small spherical cells (cell type I) to large multipolar cells (cell type II) and fusiform cells (cell type III). According to our results, large and aspiny multipolar neurons were found in the magnocellular division of the basal and accessory basal nucleus, while large and small bipolar aspiny neurons were observed in all of the investigated basolateral amygdaloid nuclei. On the basis of different expression and distribution of the calcium-binding proteins - ealbindin and calretinin (Setzer and Ulfig 1999), both small and large bipolar and small and large multipo-

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lar neurons are found in the human fetal amygdala. The results of both studies are in agreement with our descriptions of the nonpyramidal cells and their subtypes. Gliaform neurons are the smallest (only 9 x 7 gm in diameter) among the neurons of the basolateral amygdala. They have appeared in places, clearly impregnated, but the outline of their somata was slightly lighter stained in comparison with other neuronal types. Their dendrites (5-9 primary dendrites) are gracile and short with few spines. Axons branch profusely to form rich axonal plexus. The gliaform neurons (the neurogliaform cells) are observed in the basolateral amygdala of the cat (Tombol and Szafranska-Kosmal 1972; Kamal and Tombol 1975) and opossum (McDonald and Culberson 1981). The axons of the neurons in the basolateral amygdala show profuse arborization in all of the investigated nuclei. The pyramidal cells usually have more distinct and wider axonal hillock in comparison with the nonpyramidal neurons. In some of the pyramidal neurons, the axonal hillock and initial segment showed clearly visible spines presented in places. The axons of the pyramidal neurons give off several collaterals in their proximal segment. Most collaterals run fairly horizontally and contact perpendicularly directed axons of the neighboring or distant cells in order to form a "ladder-like" branching pattern. Similar findings have been described in the rat (McDonald 1982; Millhouse and DeOlmos 1983). The appearance of axonal terminals which tend to be clustered around the initial segments of the axons, verified previously in pyramidal cells of the rat (McDonald and Culberson 1981; McDonald 1982), was noticed in our sections too. There is evidence that these axonal clusters together with shafts of the initial segment represent postsynaptic contacts to the axon originating from the particular type of the nonpyramidal neurons (McDonald 1992). It seems that the basal and paralaminar nuclei represent cytomorphologically more complex nuclei in comparison to the other nuclei of the basolateral amygdala. The basal nucleus represents the largest amygdaloid nucleus in primates. Its main portion is composed of the parvicellular (ventral) division that extends along the whole length of the rostro-caudal axis. On the basis of the cytoarchitectonic criteria the paralaminar nucleus (the basal portion of the amygdaloid parvicellular division) may be estimated as a separate nucleus (Amaral et al. 1992). We find the present classification that estimates the paralaminar nucleus as a nucleus separated from the parvicellular (ventral) division of the basal nucleus acceptable. The remarkable feature of the parvicellular division of the basal nucleus and paralaminar nucleus is the presence of intranuclear dendritic bundles. Presented fascicular dendritic arrangement was not seen in other nuclei of the basolateral amygdala. The greater vitality of the paralaminar nucleus in comparison to the other nuclei was pointed out in the Alzheimer's disease in which amygdaloid nuclei show significant atrophy with the exception of the paralaminar portion of the basal nucleus (Scott et al. 1991).

Unfortunately, only little is known about the dendrodendritic contacts (DDC) in mammals. The presence of presynaptic dendrioles of unipolar brush cells that form dendrodendritic contacts to the granule cells was observed in the mammalian cerebellum (Muganaini et al. 1997). The DDC are also described in the rat hippocampus (Gulyas et al. 1996). The most interesting finding of DDC represents the reciprocal dendrodendritic synapses between the mitral cells and granule cells in the accessory olfactory bulb of the mouse (Kaba and Nakanishi 1995). In this case the D D C participate in the olfactory recognition memory formed to male pheromones by a female mouse at mating. The evidence about the presence of DDC in the human central nervous system is also rare. In one combined developmental study using Golgi impregnation, electronmicroscopy and immunocytochemical method the presence of DDC in the human lateral geniculate nucleus was observed (Wadhwa and Biljani 1988). The development of dendritic spines and the D D C starts in the 15 th week of gestation and spines appear first on the proximal segments of the dendrites and subsequently cover their distal segments. The DDC, indicated by the light microscopy on our preparations, occur between different neuronal types in the nuclei of the basolateral amygdala but in most cases they were established between the bipolar cells and modified pyramidal or pyramidal cells. Their presence should be expected in all the nuclei of the basolateral amygdala with particular attention on the parvicellular division of the basal nucleus and paralaminar nucleus. Further investigations of the D D C in the nuclei of the basolatereal amygdala should be performed following the multiple approach using different techniques that will point out the morphological and functional complexity of the human amygdaloid complex as one of the leading candidates involved in cognitive-emotive processes. Acknowledgments. The present research was supported by Serbian Ministry of Science and Technology Grants 13M15 and 8856.

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Accepted June 19, 2001

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