EXPERIMENTAL
52, 189-205 (1976)
NEUROLOGY
Dendritic
Patterns
TOKUNAGA
AKIRA The
Third
of Neurons
Dcpartnwnt
Received
January
of Anatomy, Chiba, 23,1976;
in the Rat Superior AND KATSUMI School of 280, Japan revisioti
OTANI
Medicine,
received
April
Colliculus l
Chiba
University,
19,1976
The cell type of the rat superior colliculus was investigated using the modified Golgi-Cox method. The superior colliculus was found to be composed of two laminar sets, which develop different neuronal organizations. The superficial layers, containing the zonal-to-optic strata, consisted of many neurons of variable size and shape. However, on the basis of the profile and the orientation of the dendritic fields, cells of the superficial layers were classified into four types: the horizontal, the cylindrical, the reversed conical, and the multipolar cells. Some of the cells were further subdivided according to the orientation and the width of dendritic fields. The deep layers which included the middle gray to deep white laminae were characterized by medium-sized and large multipolar neurons. In addition to these cells with global or ellipsoidal dendritic fields, another two types of cells, vertical and horizontal neurons, were also included.
INTRODUCTION Many behavioral experiments have disclosed that the superior colliculus plays an important role in visuomotor control of the visual spatial orientation or visual attention (3, 6, 8, 9, 29). Casagrande et al. (6) claimed that the behavioral effects due to a small superficial lesion were very different from these resulting from a deep lesion, The small superficial ablation produced deficits of visual pattern discrimination, while the deep lesion caused inability to track, follow, and orient objects or visual inattentiveness. Electrophysiological studies (7, 10, 1.5, 19, 27) have revealed that the units in the superficial layers respond to a small spot stimulation, and that some units in the deep layers are responsive not only to such visual 1 The authors wish to thank Mr. Kazuo Miyama and Mrs. Fumiyo Saito for photographic assistance and for histological preparations, respectively. Critical reading of the manuscript by Dr. Hiroharu Noda; Brain Research Institute, University of California, is also gratefully acknowledged. 189 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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stimulation but also to auditory and/or the somatic stimulations as well. In previous anatomical studies on its efferent projections, the superior colliculus has been regarded as one nuclear mass. Recently, however, considerable attention has been devoted to different destinations of the fiber projections between superficial and deep layers (1, 4, 11-13, 24). Before attempting to correlate these physiological data with the histological findings, however, a more detailed study on the neuronal organization of the colliculus is necessary.
,’
.’
2
,,*’
FIG. 1. A drawing of all cell processes obtained from tions which were cut coronally through the middle level l-The first (superficial) layer; 2-Th’c second layer; central gray matter. The cell numbers 1 to 12 indicate
three successive 200 pm-secof the rat superior colliculus. 3-The third layer; SGCthe cell types (see Table 1).
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FIG. 2. The lateral part of the rat superior colliculus. A drawing of all cell branches obtained from three successive 200 firn-coronal sections. l-The first (superficial) layer, 2-the second layer, 3-the third layer; SGC-central gray matter. Th’e cell numbers 1 to 12 indicate the cell (types (see Table 1).
A number of Golgi studies on the superior colliculus have been published (5, 17, 33-35). In this paper, we attempted to classify the cell types of the rat superior colliculus with the modified Golgi-Cox method. We used three characteristics of dendritic arbolizations as criteria to classify the collicular cells: the profile and orientation of the dendritic zone, and the width of the dendritic field.
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MATERIALS
AND
AND
OTANI
METHODS
Young white rats, weighing 40 to 70 g (6 to 8 weeks old), were used. Small blocks of 7 to S-mm thickness containing the superior colliculus were taken for impregnation with a tungstate modification of the Golgi-Cox method (26). They were kept in the fixative for 20 to 30 days, then alkalinized within 24 hr. Then they were soaked for 24 hr in two changes of the 0.05% acetic acid and washed for 1 day in many changes of distilled water. All blocks were dehydrated through a series of alcohols, embedded in celloidin, cut at 120 or 200 q, then mounted with balsam and. a coverslip. A total of 32 series of rat superior colliculi was examined in this study. Ten were sectioned sagittally, five horizontally, and 17 coronally. All cell processes were drawn with a camera lucida (Hamano Co.). In Figs. 1 through 3, the drawings were made by overlapping three successive sections with magnifications of x270. The form and distribution of the dendritic spines were observed with Xl00 oil immersion lens.
FIG. 3. A sagittal section of the rat superior colliculus. A drawing of all cell processes obtained from three successive 200 pm-sections. In this section, the boundary between the 2nd and 3rd layers is not distinct. l-The first (superficial) layer; 2, 3-the second and third (d’eep) layers. The cell numbers 1 to 12 indicate the cell ty.pes (see Table 1).
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TABLE Cell Superficial
Types
in the Superior
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COLLICULUS
1 Colliculus
of the Rat Deep
layers
Horizontal type (1)” Cylindrical type With dorso-ventrally oriented dendrites Wide-field neurons (2) Narrow-field neurons (3) With dorsally oriented dendrites Narrow-field neurons (5) Reversed conical type (4) Multipolar type Wide-field neurons (6) Narrow-field neurons (7) [Ventrally oriented type5 (S)]
layers
Multipolar type Medium-size field Wide-field neurons Vertical type (11) Horizontal type (12)
a (1) through (12) correspond with cell type numbers b This type corresponds to the marginal cell of Cajal
neurons (10)
in Figs. 1 to 3. (5) and Langer et al.
(9)
(17).
RESULTS The superior colliculus impregnated with the Golgi-Cox method contained many neurons which were variable in size and shape of the cell bodies and dendritic trees (Figs. l-3). However, based on the orientation, profile, and width of the dendritic field, these neurons could be classified into several types (Table 1). The cells of the upper layers were found to be of four types: horizontal cells, cylindrical cells, reversed conical cells, and multipolar cells, The neurons of the deep layers were classified into three types: multipolar cells, vertical cells, and horizontal cells. Some of these were further subdivided according to the width of the dendritic field. The
Superficial
Layers
The Horizontal Type (Figs. 1-3, No. 1). Horizontal cells were found in the upper half of the superficial layers (within 260 to 270 pm of the surface). In typical cells, one thick primary dendrite arose from both ends of the fusiform cell body of about 10 x 20 pm and ran parallel or tangential to the collicular surface, taking a fairly straight course for a long distance (400 to 1000 pm in length). Usually the primary dendrite was separated in the proximity of the cell body into several daughter branches which extended obliquely in the dorsal or ventral direction. The primary dendrites were covered with a few spines. While the distal portions possessed a considerable number of spines in horizontal cells, the density of the spine was lowest among neurons of the superficial layers. The long axis of the
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ovoidal or fusiform cell body was not always oriented parallel to the collicular surface. Some cell bodies showed vertical orientation (Fig. 2). The main dendrites of these cells were emitted along the extension of the long axis of the cell body for a short distance and then bent to run parallel or tangential to the collicular lamina, branching off their secondary dendrites. There existed some atypical horizontal cells whose primary branches arose from one end of the oval cell soma (Figs. 1 and 4a). In horizontal sections, the main dendrites changed their course at a large angle and the daughter branches distributed only occasionally in the same plane. We examined whether these dendrites exhibited any preferential orientation. The dendrites of 20 horizontal cells from the horizontal sections were superimposed, aligning the anteroposterior axis and the cell soma (Fig. 4b). It was demonstrated that the branches of horizontal cell dendrites as a whole distributed evenly without any specific orientation. Therefore, the upper half of the superficial layers was covered evenly with the horizontal cell dendrites woven into a dense network, though it was a local or spotty area to which the dendrites of each horizontal cell could distribute. The axon arose from the soma or thick dendritic trunk and pursued an irregular course in the superficial layers, branching profusely. The Cylindrical Type (Figs. l-3, Nos. 2, 3, 5 ; Figs. 5a, 5b). These neurons were common in the stratum griseum superficiale. They were classified into two subtypes on the basis of the orientation of the dendritic field, one with dorsoventrally and the other with dorsally oriented dendrites. The former subgroup was further subdivided according to the width of the field. (A) The cylindrical neurons with dorsoventrally oriented dendrites (Figs. 13, No. 2; Fig. 5a). In these neurons, the dendrites issued in a vertical axis from both ends of the fusiform cell body (15 X 30 @n). As a whole, the cell bodies of the wide-field neurons were located in the ventral region of the upper layers, i.e., from the middle portion of the stratum griseum superficiale to the stratum opticum. One to four primary dendrites arose from each pole of the fusiform cell soma and soon ramified into many secondary branches. The ascending or apical dendrites extended to the collicular surface, branching more frequently to form a complex intermingling bouquet. The ascending dendrites were longer than the descending ones. The proportion of the length of the apical to the basal branches was estimated of about 3 or 4: 1. Both dendritic components of the cells with cell bodies lying in the middle of the upper layers had almost the same length. Some reflexive dendrites, such as a descending process from the apical dendrite or an ascending branch from the basal one, could be frequently observed in this group (Figs. 5a, 5b). A few branches derived from the middle portion of the cell body and ran parallel to the lamina for a short distance. As a rule, the spines of the cylindrical neurons were
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very densely packed and of many types, showing variation ranging from the simple “spherical head with a thin neck” form (with 1 pm length) to the multipolar form (7 to 12 pm ; Fig. SC). In coronal and sagittal sections, the dendritic arborization of these cells exhibited a similar pattern. Therefore, it can clearly be assumed that the three-dimensional reconstruction of the dendritic field showed a cylindrical form, with an estimated diameter of 170 pm and a length of 300 to 350 pm.
FIG. 4a. A drawing of the horizontal cell processes from a horizontal section through the top of the colliculus. The arrow indicates the anteroposterior axis of the superior colliculus. A-Anterior, P-pasterior, M-medial, L-lateral. (b) A tracing of all dendrites from 20 horizontal cells drawn from horizontal sections, aligning the anteroposterior axis of each cell and each cell soma. The horizontal cell dendrites were distributed as a whole evenly without preferred orientation.
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The axon arose from either of the basal dendritic branches or cell soma, and descended to the lower layers of the colliculus. The narrow-field cylindrical cells (Figs. l-3, No. 3 ; Fig. 5b) were similar to the wide-field ones in many respects. They have a small elongated cell
FIG. 5 (a). Wide-field cylindrical neurons. Cells B, D, E, and cells A, C ,are drawn from coronal and sagittal sections, ,respectively. The calibration bar is 100 pm. (b) Narrow-field cylindrical neurons. Cells A, B, E, and cells C, D are drawn from coronal and sagittal sections, respectively. The calibration bar i,s 100 pm. (c) Mosaic reconstruction of a wide-field cylindrical cell, with a combination of reflecting and ordinary substage illuminations. X ZOO. FIG. 6. Dorsally oriented cylindrical neurons. Cells A, B, D and the cell C are drawn from sagittal and coronal sections, respectively. Calibration mark is 100 pm.
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FIG. 7. Mosaic reconstruction
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of a reversed conical neuron, with combinedillumi-
nation.X 200. FIG.
8. Mosaic reconstruction
of a vertical
neuron in the second layer, with corn-
binedillumination.X 200. body of about 10 x 20 pm, located in the relatively dorsal part of the upper layers. Their dendritic patterns exhibited a wide variety, from complex arborization to simple bipolar appearance (Fig. 5b). Their dendritic fields showed a small cylindrical form with a maximum diameter of 60 pm and a vertical length less than 200 pm. The ratio of the length of the ascending branches to that of descending branches was from Z-5 :l. (B) The cylindrical nezlrons zvith dorsally oriented dendrites (Figs. l3, No. 5, Fig. 6). These neurons were scattered throughout the upper half of the superficial layers, Two to three main dendrites arose from the dorsal surface of a round cell body of about 30 pm in diameter, extending toward the surface of the colliculus as they branch out. Because the dendritic tree extends less laterally, the dendritic field showed a slender cylindrical form of less than 100 pm in diameter. They resembled somewhat the narrowfield cylindrical neurons which lacked basal dendrites. The axon projected to the lower layers from the ventral surface of the cell body. There was a great deal of variability among these neurons wit11 respect to dendritic arborization, from the complex bouquet to several ascending branches with few daughter dendrites. The dendrites were covered with numerous spinesof variable form.
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The Reversed Conical Type (Figs. 1-3, No. 4, Fig, 7). The mediumsized stellate cell bodies lay in the deep part of the superficial layers, i.e., from the ventral region of the superficial gray and the optic layers. Three to six primary dendrites coursed dorsally and dorsolaterally. The branches, arsing from the side of the cell body, extended obliquely and radially toward the collicular surface. The dendrites from the dorsal surface of the soma ran upward in a relatively straight course. They bifurcated several times coursing as long as 400 w up to the central border of the horizontal cell zone. Regardless of the direction of cutting planes, the profile of the dendritic field of these cells exhibited a fan-like form. Therefore, the field did not extend in the two-dimensional plane, as is seen in the Purkinje cell, but they occupied a three-dimensional region. The profile of the dendritic tree appeared as an inverted cone. The base of the circular cone faced toward the collicular surface and it extended more than 700 pm in the diameter. No dendritite projected from the ventral surface of the cell body. The proximal portion of the primary dendrites appeared fairly smooth contouring a small number of ball-on-a-neck type spines. Spines of various types were more frequently seen at increasing distances from the cell body. The axon descended to the deep layers, ramifying many collaterals at its origin. The Multipolar Type (Figs. l-3, No. 6 and 7). (A) Wide-field w&ipolar cells (Figs. 1-3, No. 6). The 25-to-30 pm polygonal cell bodies lying within the optic layer sent, multipolarly, five to seven primary dendrites. They resembled the reversed conical neurons in many respects. But they were different from these neurons in that some dendrites projected below the cell soma to penetrate the optic layer. The axon arose from either the ventral somal surface or from a thick dendritic trunk and coursed to the lower layers. (B) Narrow-field multipolar neurons (Figs. l-3, No. 7). The small neurons which were found all over the upper layers sent spiny dendrites from any part of the round or elliptical cell body (about 15 pm in diameter). The number of dendrites and the branching patterns were variable from cell to cell. Besides these cells, some neurons with ventrally oriented branches were impregnated. The small, round cell body, less than 15 pm in diameter, was located just beneath the collicular surface and/or the zonal layer. Many spiny and gnarled thin processes issued from the ventral surface of the soma to form the complex intermingling tufts (Figs. 1 and 3, No. 8). The distribution area of the processes extended less than 70 pm in diameter and showed a small ellipsoidal shape. These cells may correspond to the marginal cells of Cajal (5) and Langer et al. (17). As a rule, neurons which are located beneath the surface tend to escape impregnation with the
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Golgi-Cox technique. Moreover, the axons could not be clearly identified from their processes. Therefore, it is hardly decided whether these cells are real neurons or glial elements. Each cell type in the superficial layers, except for the aforementioned “marginal cells,” were impregnated with almost equal frequency in every section. The Deep Layers The deep layers mainly consisted of multipolar and vertical neurons. The Multipolar Type (Figs. 1-3, No. 9 and 10). Four to six primary thick dendrites issued from any part of the polygonal or elongated cell body and spreading more or less straightly on every side, and bifurcating to several higher ordered branches. This category was divided into two subtypes based on the width of the dendritic field. (A) The wzediwz-sized field neurons (Figs. l-3, No. 9). The cell bodies were oval or elongated in shape and about 20 pm in diameter. The dendritic field exhibited somewhat of an ellipsoid shape with a 150 to 200-pm long axis. As a whole, the dendritic branching pattern of the medium-sized cells was slightly simpler than that of the large multipolar cells. Moreover, the dendrites of large multipolar neurons distributed much more evenly and densely than did the medium-sized cell dendrites. The cells with medium-sized fields were scattered throughout the deep layers. (B) The wide-field cells (Figs. 1-3, No. 10). The profile of the field of these cells displayed a spherical shape more than 400 pm in diameter. A considerable number of dendrites extended from any part of the polygonal or stellate cell body 30 to 40 pm in diameter, lying in the lateral part of the deep layers. In some multipolar neurons in the deeper zone of the stratum album profundum, the dendrites projecting from the ventral part of the cell body invaded the central gray matter. The density of the spines on the primary dendritic trunks was apparently reduced in comparison with that on the higher ordered dendrites, which were covered with many spines about 1 pm in length of the ball-on-a-neck form. A few spines of the multipolar type were found on the multipolar cell dendrites. The axons arose from the soma or from a thick dendritic trunk close to the cell body. They ran in parallel to the third layer or descended for a short distance and then bent laterally or medially to join with the fiber bundle of the stratum album profundum. The axons from some mediumsized multipolar neurons projected dorsally toward the vicinity of the optic layer (Figs. 1 and 3; arrows). We could not confirm that they transverse the optic layer to distribute in the superficial layers. The Ye&cat Cells (Figs. 1-3, No. 11, Fig. 8). In longitudinal sections, one or two dendrites projected from the ends of the spindle-like cell body (10 X 15 pm) which had a dorsolateral orientation. Some secondary
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dendrites branched from the parent trunks at a considerably larger angle. The obliquely extending secondary branches were shorter than the main vertical dendrites. The dendritic arborizations of the deep vertical cells were simpler ‘than those of superficial cylindrical neurons. Most of the axons of the vertical cells intermingled with fibers of the third layer. However, some climbed toward the optic layer (Fig. 3 ; arrows). The horizontal neurons were also considered in these cell types, although they were only rarely impregnated. A thick primary dendrite arose from each pole of the fusiform cell body the long axis (20 pm in length) of which oriented in parallel to the layers. They ran tangential or parallel to the third layer. Several daughter dendrites branched at a wide angle from the primary dendrites. DISCUSSION In Golgi studies, any of the following neuronal properties may be useful in classifying cell-types; the form or location of the soma, the destination of the axon, and the pattern of dendritic ramification. In any nucleus in the central nervous system, no nerve cell appears identical to another in the size and shape of the cell body and in the number and detailed branching form of the processes. However, the orientation, profile, and width of the whole dendritic zone including the cell soma offers considerable similarities among neurons in a given region. These characteristics may also reflect functional analogies, including the area and the direction from which the neurons collect their afferent information. In this paper, we have utilized these three characteristics of the dendritic zone as criteria to classify the cells of the superior colliculus. Valverde (33) reported that the horizontal cells in the superficial layers of the mouse superior colliculus are localized only in the zonal layer. We found that the horizontal cells were located commonly in the upper half of the superficial layers in agreement with Cajal (5) and Langer et al. (17). Those authors plotted the length of horizontal cell dendrites versus the angle of the orientation. They concluded that the horizontal cell had a preferred orientation of their dendrites along the vertical and horizontal axes of the visual field projection upon the colliculus. We traced the course of the horizontal cell dendrites aligning the anteroposterior axis and the cell soma. It was found that the cell processes as a whole distributes evenly without showing preferred orientation. Each horizontal cell emits a small number of long dendrites in every direction. The sum of the dendritic distribution of each horizontal cell can form a dense network upon the superficial layers. The cylindrical neurons are divided into two subtypes ; cells with dorsoventrally oriented dendrites and others with dorsally oriented branches.
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The former group is further divided into wide- and narrow-dendritic field neurons. The wide-field neurons correspond to Cajal’s large fusiform cells in the vertical fusiform cell zone, to his voluminous fusiform cells in the zone of optic fibers (S), to some of the short axon cells (33) and to pyramidal cells of Langer et al. (17). Most investigators have described that axons of these cells descend to the lower layers (5, 17, 34, 35), while Valverde (33) observed ascending axons from these neurons. Cajal’s small fusiform cells (5)) and the superficial and intermediate vertical fusiform cells (17) could be classified into the same category as the small-field neurons of this type. Cajal’s short ovoidal tufted cells in the zone of horizontal cells (5), some cells with long axons found in Viktorov’s figures (34, 35) and piriform cells (17) correspond to the neurons with dorsally oriented dendrites. Their dendrites and axons commonly arise from the dorsal and the ventral surface of the cell body, respectively, although the detailed branching pattern varies from neuron to neuron, The reversed conical neurons correspond to the triangle and ovoidal cell of Cajal (5), to some of the long projecting neurons (33), and to the type II ganglionic cells in the wide vertical neurons (17). The axons of these cells travel down to to the deeper layers, projecting some collaterals to the superficial layers. These characteristics are in accord with the findings of Valverde (33) and Langer et al. (17). With regard to the density of spines in reversed conical neurons, Langer and his coworkers (17) separated them as spiny and smooth cell types. However, our Golgi-Cox preparations revealed that the primary dendrites were covered with a few spines of simple form, and that the density and complexity of the shape of spines were remarkably increased in the distal portions of the higher ordered dendrites. The wide-field multipolar neurons in the superficial layers were described as the triangle or stellate cells of Cajal (5) and as the type III ganglionic cells ( 17). The cell body of these neurons was located mainly in the stratum opticum. They resembled the reversed conical neurons, except that a small number of dendrites from the lower surface of the cell body projected toward the deep layers. The narrow-field multipolar neurons corresponded to the short-axon cells and the fusiform cells in the zone of the optic layer (5), and to the stellate cells (17). Neurons of this type were scattered throughout the superficial layers. The profile and the width of their dendritic field were quite different from cell to cell. With respect to the terminal region of retinotectal and corticotectal fibers in the superficial layers of the superior colliculus, it has been reported that the retinal axons terminated in a wide region ranging from the optic to zonal layers, while the afferent fibers from the visual cortex distributed
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in the deeper half of the upper layers (22). It has been also demonstrated that a retinotopical organization exists among retinal ganglionic cells, visual cortical neurons, and collicular cells (18, 20, 28). Langer et al. (17) showed the terminal plexus of a presumed retinal axon which formed a field of about 50 pm in diameter. On the basis of the width of dendritic field, it can be estimated that more than eight retinal axons terminate on a dendrite of a single horizontal cell, and that at least two to seven axons are projected to the apical dendritic field of the cylindrical neurons. On the other hand, the reversed conical and the large multipolar neurons can be connected with 24 to 37 optic fibers. It is also assumed that corticotectal fibers from area 17 project to the basal dendrites of wide-field cylindrical neurons and the processes of the reversed conical and large-field multipolar cells. Many electrophysiologic experiments (7, 15, 19, 27) have revealed that neurons in the superior colliculus can respond to a small light stimulus moving within their receptive fields. The units which are responsive to the movement stimulus may correspond to wide-field neurons which are connected with many retinal fibers, while the collicular neurons with narrow dendritic field, which receive a small number of retinal axons, can respond preferably to stimulation of a small visual field. Taking the retinotopic representation in the colliculus into consideration, these cells may play an important role in detecting the stimulated site in the visual field. The units in the superficial layers can be separated into two classes on the basis of the size of receptive field; small and large field units. The small fields, 2 to 15” in diameter, were circular and the large ones, 30 to 90” in diameter, were irregular and often patchy, as if they consisted of a conglomerate of smaller fields (15). The former units distributed to the superficial gray layer and the latter to the optic lamina. Two of our superficial neuronal types, i.e., cylindrical and multipolar types, could be further divided into subtypes of narrowand wide-field cells. The physiological data of Humphrey (15) are supported by our anatomical findings. Moreover, seven types of selected receptive fields were reported in the superficial layers of rabbit superior colliculus (19). For an understanding of the collicular functions it is necessary to identify each unit with the cell type classified by the Golgi method. The electrophoretical intracellular dye injection method (30) is a useful and effective means of correlating physiologic and morphologic findings. In a previous paper (32), the superior colliculus was divided into three major layers on the basis of the myeloarchitecture and its afferent .connections. The first layer (from the zonal to the optic layers) is primarily connected with the visual system. The second layer (from the intermedial gray to the deep gray laminae) and the third layer (stratum album pro-
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fundum) receive afferent fibers from multiple origins. Viktorov (34, 35) claimed that the deep layers are divided into two independent nuclear complexes on the basis of neuronal organization and fiber connections. However, it is clearly evident from the present observations that the superior colliculus consists of two sets of strata : superficial (first) and deep (second and third) layers. The former layers contain many neurons with variable profiles and orientations of dendritic trees, while the latter are formed mainly of large and medium-sized multipolar neurons and additionally contain vertical and horizontal cells. In the present paper, the deep layers still remain divided into two parts. This subdivision of the deep layers is not based on neuron type, but on the fiber connections. As mentioned in the previous paper (32)) the third layer formed the main fiber pathway in the superior colliculus. Regarding efferent projections from the superior colliculus, it has been reported that neurons in the superficial layers project to structures different from those which receive terminals of efferent fibers of the deep layers (1, 4, 11-13, 24). The origin of the superficial projection fibers may possibly be found in both reversed conical and wide-field multipolar neurons. From this observation, the axons of these cells do not travel through the optic layer to the collicular brachium, but most of them descend toward the deep layers. On the other hand, it was shown that axons from some deep cells, namely, the medium-sized multipolar and vertical cells, projected dorsally to the vicinity of the optic layer. We do not deny the possibility that such climbing axons may extend through the optic layer to pretectal and thalamic regions. It has already been shown that the deep layers of the colliculus receive many different fibers from the superficial colliculus layers and the spinal cord ( 16), the inferior colliculus (25), the fastigial nucleus (2, 31) and nonvisual neocortices (23). Electrophysiological studies (7, 9, 10) have confirmed that some collicular units in the deep layers were able to respond not only to visual but also to auditory and somatic stimuli. Moreover, it has been found that most of the deep neurons have visual receptive fields which are similar to those of the superficial cells. Furthermore, a correlation exists between the visual fields and the auditory or somatic receptive fields (9, 10). Drager et al. (7) pointed out in the mouse superior colliculus that there was a striking interrelation between the position of whiskers and the visual receptive fields. It should be emphasized that the deep layers are composed not only of multipolar cells but also of some vertical neurons with rather narrower dendritic fields. It is reasonable to speculate that axons from the several superficial neurons may terminate retinotopically on the dendrites of the deep vertical neurons. Assuming that these cells receive converging nonvisual fibers, they may be involved in a geometric
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correlation between visual and somatosensory receptive fields. Some of the vertical and some of the medium-sized multipolar neurons may have a role as interneurons. Impulses from the vertical neurons pass to the multipolar cells. After complex integrating processes in these relay cells, impulses may arrive at collicular outputs and transfer to other parts of the brain. Our cytoarchitectonic study on the rat superior colliculus (32) has shown that large polygonal cells measuring over 40 pm in diameter are distributed mainly to the lateral part of the deep layers. Moreover, investigations using the Cajal method revealed that axons from these cells coursed to the bottom of the stratum album profundum and then formed a predorsal bundle, after turning laterally at nearly a right angle. Therefore, it can be concluded that the large multipolar neurons, located in the lateral part of the deep layers of the colliculus, are the origin of the tectospinal tract. REFERENCES P. 1971. The neuroanatomical organization of the visual system in the tree ‘shrew. Folia Primatol. 16 : l-34. 2. ANGAUT, P. 1969. The fastigo-tectal projections. An anatomical experimental study. Brain, Res. 13: 186-189. 3. BARNES, P. J., L. M. SMITH, and R. M. LATTO. 1970. Orientation to visual stimuli and the superior colliculus in the rat. Quart. J. Exp. Psychol. 22: 239-247. 4. BENEVENTO, L. A., and J. H. FALLON. 1975. The ascending projections of the superior colliculus in the rhesus monkey (Macaca mulatta). J. Comp. Neurol. 1860: 339-362. 5. CAJAL, S. RAM~N Y. 1909-1911. “Histologic du systeme nerveux de l’homme et des vert6br&,” pp. 174-195. L. Azoulay [trans.]. Maloine, Paris. 6. CASAGRANDE, V. A., and I. T. DIAMOND. 1974. Ablation study of the superior colliculus in the tree shrew (Tupaia glis). J. Comp. Neural. 156 : 207-238. 7. DRAGER, U. C., and D. H. HUBEL. 1975. Physiology of visual cells in mouse superior colliculus and correlation with somatosensory and auditory input. Nature (London) 253 : 203-204. 8. GOODALE, M. A., and R. C. C. MURISON. 1975. The effects of lesions of the superior colliculus on locomotor orientation and the orienting reflex in the rat. Brain Res. 88: 243-261. 9. GORDON, B. 1972. The superior colliculus of the brain. Sci. Amer. 227: 72-82. 10. GORDON, B. 1973. Receptive fields in deep layers of cat superior colliculus. J. Neurophysiol. 36 : 157-178. 11. GRAYBIEL, A. M. 1972. Some extrageniculate visual pathways in the cat. Invest. Ophthamol. 11: 322-332. 12. HARTING, J. K., K. K. GLENDENNING, I. T. DIAMOND, and W. C. HALL. 1973. Evolution of the primate visual system: Anterograde degeneration studies of the tecto-pulvinar system. Amer. J. Phys. Alzthropol. 38: 383-392. 13. HARTING, J. K., W. C. HALL, I. T. DIAMOND, and G. F. MARTIN. 1973. Anterograde degeneration study of the superior colliculus in tupaia glis: Evidence for a subdivision between superficial and deep layers. J. Comp. Neural. 148: 361386. 1. ABPLANALP,
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21. 22. 23. 24. 25. 26. 27. 28.
29.
30. 31.
32. 33.
34.
35.
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W. R., A. SEFTON, and C. WEBB. 1962. Primary optic centers of the rat in relation to the terminal distribution of the crossed and uncrossed optic nerve fibers. J. Camp. Nrzrrol. 118: 295-321. HUMPHREY, N. K. 1968. Responses to visual stimuli of units in the superior cofliculus of rats and monkeys. Exp. Nczcrol. 20: 312-340. KOBAYASHI, M. 1970. Long asmcending fiber tracts from the spinal cord in the cat. 1. Chiba Med. Sot. 46: 279-287 (in Japanese). LANGER, T. P., and R. D. LUND. 1974. The upper layers of the superior colliculus of the rat: A Golgi study. J. Camp. Ncurol. 158: 405-436. LUND, R. D. 1966. The occipitotectal pathway of the rat. J. Anat. 100: 51-62. MASLAND, R. H., K. L. CHOW, and D. L. STEWART. 1971. Receptive-field characteristics of superior colliculus neurons in the rabbit. I. Neurophysiol. 34: 148-156. MEIKLE, T. H., JR., and J. M. SPRAGUE. 1964. The neural organization of the visual pathways in the cat. Znf. Rev. Newobiol. 16: 149-189. OTANI, K. 1964. Projection fibers from the visual cortex of the cat. J. Chiba Med. Sot. 40 : 125-138. (in Japanese). OTANI, K., T. TAKAHASHI, and T. SHIRAI. 1970. Terminal degeneration in the stratum griseum superficiale and the stratum opticum of the superior colliculus of the rat. Adv. Neural. Sci. 13 : 753-762 (in Japanese). OTANI, K. 1971. Visual system, pp. 438-4.55. In “The Anatomy of the Brain.” M. Okamoto, and T. Kusama [Fds.]. Asakura shoten, Tokyo (in Japanese). OTANI, K., M. KIMURA, and J. YAMADA. 1975. The tectofugal projections in the rat and cat. pp. 183. “Proceedings, 10th International Congress Anatomy,” Tokyo. POWELL, E. W., and J. B. HATTON. 1%9. Projections of the inferior colliculus in cat. J. Camp. NcuroZ. 136: 183-192. RAMON-MOLINER, E. 1958. A tungstate modification of the Golgi-Cox method. Staiw Tcchnol. 33 : 19-29. SCHAEFER, K. P. 1970. Unit analysis and electrical stimulation in the optic tectum of rabbits and cats. Brain Behav. Evol. 3 : 222-240. SIMINOFF, R., H. 0. SCHWASSMANN, and L. KRUGER. 1966. An eleotrophysiological study of th’e visual projection to the superior colliculus of the rat. J. Cow@. Newel. 127 : 435-444. SPRAGUE, J. M. 1972. The superior colliculus and pretectum in visual behavior. Invest. Ophthalmol. 11: 473-482. STRETTON, A. 0. W., and E. A. KRAVITZ. 1968. Neuronal geometry: Determination with a technique of intracellular dye injection, Science 162: 132-134. THOMAS, D. M., R. P. KAUFMAN, J. M. SPRAGUE, and W. W. CHAMBERS. 1956. Experimental
52, 189-205 (1976)
NEUROLOGY
Dendritic
Patterns
TOKUNAGA
AKIRA The
Third
of Neurons
Dcpartnwnt
Received
January
of Anatomy, Chiba, 23,1976;
in the Rat Superior AND KATSUMI School of 280, Japan revisioti
OTANI
Medicine,
received
April
Colliculus l
Chiba
University,
19,1976
The cell type of the rat superior colliculus was investigated using the modified Golgi-Cox method. The superior colliculus was found to be composed of two laminar sets, which develop different neuronal organizations. The superficial layers, containing the zonal-to-optic strata, consisted of many neurons of variable size and shape. However, on the basis of the profile and the orientation of the dendritic fields, cells of the superficial layers were classified into four types: the horizontal, the cylindrical, the reversed conical, and the multipolar cells. Some of the cells were further subdivided according to the orientation and the width of dendritic fields. The deep layers which included the middle gray to deep white laminae were characterized by medium-sized and large multipolar neurons. In addition to these cells with global or ellipsoidal dendritic fields, another two types of cells, vertical and horizontal neurons, were also included.
INTRODUCTION Many behavioral experiments have disclosed that the superior colliculus plays an important role in visuomotor control of the visual spatial orientation or visual attention (3, 6, 8, 9, 29). Casagrande et al. (6) claimed that the behavioral effects due to a small superficial lesion were very different from these resulting from a deep lesion, The small superficial ablation produced deficits of visual pattern discrimination, while the deep lesion caused inability to track, follow, and orient objects or visual inattentiveness. Electrophysiological studies (7, 10, 1.5, 19, 27) have revealed that the units in the superficial layers respond to a small spot stimulation, and that some units in the deep layers are responsive not only to such visual 1 The authors wish to thank Mr. Kazuo Miyama and Mrs. Fumiyo Saito for photographic assistance and for histological preparations, respectively. Critical reading of the manuscript by Dr. Hiroharu Noda; Brain Research Institute, University of California, is also gratefully acknowledged. 189 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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stimulation but also to auditory and/or the somatic stimulations as well. In previous anatomical studies on its efferent projections, the superior colliculus has been regarded as one nuclear mass. Recently, however, considerable attention has been devoted to different destinations of the fiber projections between superficial and deep layers (1, 4, 11-13, 24). Before attempting to correlate these physiological data with the histological findings, however, a more detailed study on the neuronal organization of the colliculus is necessary.
,’
.’
2
,,*’
FIG. 1. A drawing of all cell processes obtained from tions which were cut coronally through the middle level l-The first (superficial) layer; 2-Th’c second layer; central gray matter. The cell numbers 1 to 12 indicate
three successive 200 pm-secof the rat superior colliculus. 3-The third layer; SGCthe cell types (see Table 1).
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FIG. 2. The lateral part of the rat superior colliculus. A drawing of all cell branches obtained from three successive 200 firn-coronal sections. l-The first (superficial) layer, 2-the second layer, 3-the third layer; SGC-central gray matter. Th’e cell numbers 1 to 12 indicate the cell (types (see Table 1).
A number of Golgi studies on the superior colliculus have been published (5, 17, 33-35). In this paper, we attempted to classify the cell types of the rat superior colliculus with the modified Golgi-Cox method. We used three characteristics of dendritic arbolizations as criteria to classify the collicular cells: the profile and orientation of the dendritic zone, and the width of the dendritic field.
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TOKUNAGA
MATERIALS
AND
AND
OTANI
METHODS
Young white rats, weighing 40 to 70 g (6 to 8 weeks old), were used. Small blocks of 7 to S-mm thickness containing the superior colliculus were taken for impregnation with a tungstate modification of the Golgi-Cox method (26). They were kept in the fixative for 20 to 30 days, then alkalinized within 24 hr. Then they were soaked for 24 hr in two changes of the 0.05% acetic acid and washed for 1 day in many changes of distilled water. All blocks were dehydrated through a series of alcohols, embedded in celloidin, cut at 120 or 200 q, then mounted with balsam and. a coverslip. A total of 32 series of rat superior colliculi was examined in this study. Ten were sectioned sagittally, five horizontally, and 17 coronally. All cell processes were drawn with a camera lucida (Hamano Co.). In Figs. 1 through 3, the drawings were made by overlapping three successive sections with magnifications of x270. The form and distribution of the dendritic spines were observed with Xl00 oil immersion lens.
FIG. 3. A sagittal section of the rat superior colliculus. A drawing of all cell processes obtained from three successive 200 pm-sections. In this section, the boundary between the 2nd and 3rd layers is not distinct. l-The first (superficial) layer; 2, 3-the second and third (d’eep) layers. The cell numbers 1 to 12 indicate the cell ty.pes (see Table 1).
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TABLE Cell Superficial
Types
in the Superior
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COLLICULUS
1 Colliculus
of the Rat Deep
layers
Horizontal type (1)” Cylindrical type With dorso-ventrally oriented dendrites Wide-field neurons (2) Narrow-field neurons (3) With dorsally oriented dendrites Narrow-field neurons (5) Reversed conical type (4) Multipolar type Wide-field neurons (6) Narrow-field neurons (7) [Ventrally oriented type5 (S)]
layers
Multipolar type Medium-size field Wide-field neurons Vertical type (11) Horizontal type (12)
a (1) through (12) correspond with cell type numbers b This type corresponds to the marginal cell of Cajal
neurons (10)
in Figs. 1 to 3. (5) and Langer et al.
(9)
(17).
RESULTS The superior colliculus impregnated with the Golgi-Cox method contained many neurons which were variable in size and shape of the cell bodies and dendritic trees (Figs. l-3). However, based on the orientation, profile, and width of the dendritic field, these neurons could be classified into several types (Table 1). The cells of the upper layers were found to be of four types: horizontal cells, cylindrical cells, reversed conical cells, and multipolar cells, The neurons of the deep layers were classified into three types: multipolar cells, vertical cells, and horizontal cells. Some of these were further subdivided according to the width of the dendritic field. The
Superficial
Layers
The Horizontal Type (Figs. 1-3, No. 1). Horizontal cells were found in the upper half of the superficial layers (within 260 to 270 pm of the surface). In typical cells, one thick primary dendrite arose from both ends of the fusiform cell body of about 10 x 20 pm and ran parallel or tangential to the collicular surface, taking a fairly straight course for a long distance (400 to 1000 pm in length). Usually the primary dendrite was separated in the proximity of the cell body into several daughter branches which extended obliquely in the dorsal or ventral direction. The primary dendrites were covered with a few spines. While the distal portions possessed a considerable number of spines in horizontal cells, the density of the spine was lowest among neurons of the superficial layers. The long axis of the
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ovoidal or fusiform cell body was not always oriented parallel to the collicular surface. Some cell bodies showed vertical orientation (Fig. 2). The main dendrites of these cells were emitted along the extension of the long axis of the cell body for a short distance and then bent to run parallel or tangential to the collicular lamina, branching off their secondary dendrites. There existed some atypical horizontal cells whose primary branches arose from one end of the oval cell soma (Figs. 1 and 4a). In horizontal sections, the main dendrites changed their course at a large angle and the daughter branches distributed only occasionally in the same plane. We examined whether these dendrites exhibited any preferential orientation. The dendrites of 20 horizontal cells from the horizontal sections were superimposed, aligning the anteroposterior axis and the cell soma (Fig. 4b). It was demonstrated that the branches of horizontal cell dendrites as a whole distributed evenly without any specific orientation. Therefore, the upper half of the superficial layers was covered evenly with the horizontal cell dendrites woven into a dense network, though it was a local or spotty area to which the dendrites of each horizontal cell could distribute. The axon arose from the soma or thick dendritic trunk and pursued an irregular course in the superficial layers, branching profusely. The Cylindrical Type (Figs. l-3, Nos. 2, 3, 5 ; Figs. 5a, 5b). These neurons were common in the stratum griseum superficiale. They were classified into two subtypes on the basis of the orientation of the dendritic field, one with dorsoventrally and the other with dorsally oriented dendrites. The former subgroup was further subdivided according to the width of the field. (A) The cylindrical neurons with dorsoventrally oriented dendrites (Figs. 13, No. 2; Fig. 5a). In these neurons, the dendrites issued in a vertical axis from both ends of the fusiform cell body (15 X 30 @n). As a whole, the cell bodies of the wide-field neurons were located in the ventral region of the upper layers, i.e., from the middle portion of the stratum griseum superficiale to the stratum opticum. One to four primary dendrites arose from each pole of the fusiform cell soma and soon ramified into many secondary branches. The ascending or apical dendrites extended to the collicular surface, branching more frequently to form a complex intermingling bouquet. The ascending dendrites were longer than the descending ones. The proportion of the length of the apical to the basal branches was estimated of about 3 or 4: 1. Both dendritic components of the cells with cell bodies lying in the middle of the upper layers had almost the same length. Some reflexive dendrites, such as a descending process from the apical dendrite or an ascending branch from the basal one, could be frequently observed in this group (Figs. 5a, 5b). A few branches derived from the middle portion of the cell body and ran parallel to the lamina for a short distance. As a rule, the spines of the cylindrical neurons were
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very densely packed and of many types, showing variation ranging from the simple “spherical head with a thin neck” form (with 1 pm length) to the multipolar form (7 to 12 pm ; Fig. SC). In coronal and sagittal sections, the dendritic arborization of these cells exhibited a similar pattern. Therefore, it can clearly be assumed that the three-dimensional reconstruction of the dendritic field showed a cylindrical form, with an estimated diameter of 170 pm and a length of 300 to 350 pm.
FIG. 4a. A drawing of the horizontal cell processes from a horizontal section through the top of the colliculus. The arrow indicates the anteroposterior axis of the superior colliculus. A-Anterior, P-pasterior, M-medial, L-lateral. (b) A tracing of all dendrites from 20 horizontal cells drawn from horizontal sections, aligning the anteroposterior axis of each cell and each cell soma. The horizontal cell dendrites were distributed as a whole evenly without preferred orientation.
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The axon arose from either of the basal dendritic branches or cell soma, and descended to the lower layers of the colliculus. The narrow-field cylindrical cells (Figs. l-3, No. 3 ; Fig. 5b) were similar to the wide-field ones in many respects. They have a small elongated cell
FIG. 5 (a). Wide-field cylindrical neurons. Cells B, D, E, and cells A, C ,are drawn from coronal and sagittal sections, ,respectively. The calibration bar is 100 pm. (b) Narrow-field cylindrical neurons. Cells A, B, E, and cells C, D are drawn from coronal and sagittal sections, respectively. The calibration bar i,s 100 pm. (c) Mosaic reconstruction of a wide-field cylindrical cell, with a combination of reflecting and ordinary substage illuminations. X ZOO. FIG. 6. Dorsally oriented cylindrical neurons. Cells A, B, D and the cell C are drawn from sagittal and coronal sections, respectively. Calibration mark is 100 pm.
DENDRITES
FIG. 7. Mosaic reconstruction
IN
SUPERIOR
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of a reversed conical neuron, with combinedillumi-
nation.X 200. FIG.
8. Mosaic reconstruction
of a vertical
neuron in the second layer, with corn-
binedillumination.X 200. body of about 10 x 20 pm, located in the relatively dorsal part of the upper layers. Their dendritic patterns exhibited a wide variety, from complex arborization to simple bipolar appearance (Fig. 5b). Their dendritic fields showed a small cylindrical form with a maximum diameter of 60 pm and a vertical length less than 200 pm. The ratio of the length of the ascending branches to that of descending branches was from Z-5 :l. (B) The cylindrical nezlrons zvith dorsally oriented dendrites (Figs. l3, No. 5, Fig. 6). These neurons were scattered throughout the upper half of the superficial layers, Two to three main dendrites arose from the dorsal surface of a round cell body of about 30 pm in diameter, extending toward the surface of the colliculus as they branch out. Because the dendritic tree extends less laterally, the dendritic field showed a slender cylindrical form of less than 100 pm in diameter. They resembled somewhat the narrowfield cylindrical neurons which lacked basal dendrites. The axon projected to the lower layers from the ventral surface of the cell body. There was a great deal of variability among these neurons wit11 respect to dendritic arborization, from the complex bouquet to several ascending branches with few daughter dendrites. The dendrites were covered with numerous spinesof variable form.
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The Reversed Conical Type (Figs. 1-3, No. 4, Fig, 7). The mediumsized stellate cell bodies lay in the deep part of the superficial layers, i.e., from the ventral region of the superficial gray and the optic layers. Three to six primary dendrites coursed dorsally and dorsolaterally. The branches, arsing from the side of the cell body, extended obliquely and radially toward the collicular surface. The dendrites from the dorsal surface of the soma ran upward in a relatively straight course. They bifurcated several times coursing as long as 400 w up to the central border of the horizontal cell zone. Regardless of the direction of cutting planes, the profile of the dendritic field of these cells exhibited a fan-like form. Therefore, the field did not extend in the two-dimensional plane, as is seen in the Purkinje cell, but they occupied a three-dimensional region. The profile of the dendritic tree appeared as an inverted cone. The base of the circular cone faced toward the collicular surface and it extended more than 700 pm in the diameter. No dendritite projected from the ventral surface of the cell body. The proximal portion of the primary dendrites appeared fairly smooth contouring a small number of ball-on-a-neck type spines. Spines of various types were more frequently seen at increasing distances from the cell body. The axon descended to the deep layers, ramifying many collaterals at its origin. The Multipolar Type (Figs. l-3, No. 6 and 7). (A) Wide-field w&ipolar cells (Figs. 1-3, No. 6). The 25-to-30 pm polygonal cell bodies lying within the optic layer sent, multipolarly, five to seven primary dendrites. They resembled the reversed conical neurons in many respects. But they were different from these neurons in that some dendrites projected below the cell soma to penetrate the optic layer. The axon arose from either the ventral somal surface or from a thick dendritic trunk and coursed to the lower layers. (B) Narrow-field multipolar neurons (Figs. l-3, No. 7). The small neurons which were found all over the upper layers sent spiny dendrites from any part of the round or elliptical cell body (about 15 pm in diameter). The number of dendrites and the branching patterns were variable from cell to cell. Besides these cells, some neurons with ventrally oriented branches were impregnated. The small, round cell body, less than 15 pm in diameter, was located just beneath the collicular surface and/or the zonal layer. Many spiny and gnarled thin processes issued from the ventral surface of the soma to form the complex intermingling tufts (Figs. 1 and 3, No. 8). The distribution area of the processes extended less than 70 pm in diameter and showed a small ellipsoidal shape. These cells may correspond to the marginal cells of Cajal (5) and Langer et al. (17). As a rule, neurons which are located beneath the surface tend to escape impregnation with the
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Golgi-Cox technique. Moreover, the axons could not be clearly identified from their processes. Therefore, it is hardly decided whether these cells are real neurons or glial elements. Each cell type in the superficial layers, except for the aforementioned “marginal cells,” were impregnated with almost equal frequency in every section. The Deep Layers The deep layers mainly consisted of multipolar and vertical neurons. The Multipolar Type (Figs. 1-3, No. 9 and 10). Four to six primary thick dendrites issued from any part of the polygonal or elongated cell body and spreading more or less straightly on every side, and bifurcating to several higher ordered branches. This category was divided into two subtypes based on the width of the dendritic field. (A) The wzediwz-sized field neurons (Figs. l-3, No. 9). The cell bodies were oval or elongated in shape and about 20 pm in diameter. The dendritic field exhibited somewhat of an ellipsoid shape with a 150 to 200-pm long axis. As a whole, the dendritic branching pattern of the medium-sized cells was slightly simpler than that of the large multipolar cells. Moreover, the dendrites of large multipolar neurons distributed much more evenly and densely than did the medium-sized cell dendrites. The cells with medium-sized fields were scattered throughout the deep layers. (B) The wide-field cells (Figs. 1-3, No. 10). The profile of the field of these cells displayed a spherical shape more than 400 pm in diameter. A considerable number of dendrites extended from any part of the polygonal or stellate cell body 30 to 40 pm in diameter, lying in the lateral part of the deep layers. In some multipolar neurons in the deeper zone of the stratum album profundum, the dendrites projecting from the ventral part of the cell body invaded the central gray matter. The density of the spines on the primary dendritic trunks was apparently reduced in comparison with that on the higher ordered dendrites, which were covered with many spines about 1 pm in length of the ball-on-a-neck form. A few spines of the multipolar type were found on the multipolar cell dendrites. The axons arose from the soma or from a thick dendritic trunk close to the cell body. They ran in parallel to the third layer or descended for a short distance and then bent laterally or medially to join with the fiber bundle of the stratum album profundum. The axons from some mediumsized multipolar neurons projected dorsally toward the vicinity of the optic layer (Figs. 1 and 3; arrows). We could not confirm that they transverse the optic layer to distribute in the superficial layers. The Ye&cat Cells (Figs. 1-3, No. 11, Fig. 8). In longitudinal sections, one or two dendrites projected from the ends of the spindle-like cell body (10 X 15 pm) which had a dorsolateral orientation. Some secondary
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dendrites branched from the parent trunks at a considerably larger angle. The obliquely extending secondary branches were shorter than the main vertical dendrites. The dendritic arborizations of the deep vertical cells were simpler ‘than those of superficial cylindrical neurons. Most of the axons of the vertical cells intermingled with fibers of the third layer. However, some climbed toward the optic layer (Fig. 3 ; arrows). The horizontal neurons were also considered in these cell types, although they were only rarely impregnated. A thick primary dendrite arose from each pole of the fusiform cell body the long axis (20 pm in length) of which oriented in parallel to the layers. They ran tangential or parallel to the third layer. Several daughter dendrites branched at a wide angle from the primary dendrites. DISCUSSION In Golgi studies, any of the following neuronal properties may be useful in classifying cell-types; the form or location of the soma, the destination of the axon, and the pattern of dendritic ramification. In any nucleus in the central nervous system, no nerve cell appears identical to another in the size and shape of the cell body and in the number and detailed branching form of the processes. However, the orientation, profile, and width of the whole dendritic zone including the cell soma offers considerable similarities among neurons in a given region. These characteristics may also reflect functional analogies, including the area and the direction from which the neurons collect their afferent information. In this paper, we have utilized these three characteristics of the dendritic zone as criteria to classify the cells of the superior colliculus. Valverde (33) reported that the horizontal cells in the superficial layers of the mouse superior colliculus are localized only in the zonal layer. We found that the horizontal cells were located commonly in the upper half of the superficial layers in agreement with Cajal (5) and Langer et al. (17). Those authors plotted the length of horizontal cell dendrites versus the angle of the orientation. They concluded that the horizontal cell had a preferred orientation of their dendrites along the vertical and horizontal axes of the visual field projection upon the colliculus. We traced the course of the horizontal cell dendrites aligning the anteroposterior axis and the cell soma. It was found that the cell processes as a whole distributes evenly without showing preferred orientation. Each horizontal cell emits a small number of long dendrites in every direction. The sum of the dendritic distribution of each horizontal cell can form a dense network upon the superficial layers. The cylindrical neurons are divided into two subtypes ; cells with dorsoventrally oriented dendrites and others with dorsally oriented branches.
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The former group is further divided into wide- and narrow-dendritic field neurons. The wide-field neurons correspond to Cajal’s large fusiform cells in the vertical fusiform cell zone, to his voluminous fusiform cells in the zone of optic fibers (S), to some of the short axon cells (33) and to pyramidal cells of Langer et al. (17). Most investigators have described that axons of these cells descend to the lower layers (5, 17, 34, 35), while Valverde (33) observed ascending axons from these neurons. Cajal’s small fusiform cells (5)) and the superficial and intermediate vertical fusiform cells (17) could be classified into the same category as the small-field neurons of this type. Cajal’s short ovoidal tufted cells in the zone of horizontal cells (5), some cells with long axons found in Viktorov’s figures (34, 35) and piriform cells (17) correspond to the neurons with dorsally oriented dendrites. Their dendrites and axons commonly arise from the dorsal and the ventral surface of the cell body, respectively, although the detailed branching pattern varies from neuron to neuron, The reversed conical neurons correspond to the triangle and ovoidal cell of Cajal (5), to some of the long projecting neurons (33), and to the type II ganglionic cells in the wide vertical neurons (17). The axons of these cells travel down to to the deeper layers, projecting some collaterals to the superficial layers. These characteristics are in accord with the findings of Valverde (33) and Langer et al. (17). With regard to the density of spines in reversed conical neurons, Langer and his coworkers (17) separated them as spiny and smooth cell types. However, our Golgi-Cox preparations revealed that the primary dendrites were covered with a few spines of simple form, and that the density and complexity of the shape of spines were remarkably increased in the distal portions of the higher ordered dendrites. The wide-field multipolar neurons in the superficial layers were described as the triangle or stellate cells of Cajal (5) and as the type III ganglionic cells ( 17). The cell body of these neurons was located mainly in the stratum opticum. They resembled the reversed conical neurons, except that a small number of dendrites from the lower surface of the cell body projected toward the deep layers. The narrow-field multipolar neurons corresponded to the short-axon cells and the fusiform cells in the zone of the optic layer (5), and to the stellate cells (17). Neurons of this type were scattered throughout the superficial layers. The profile and the width of their dendritic field were quite different from cell to cell. With respect to the terminal region of retinotectal and corticotectal fibers in the superficial layers of the superior colliculus, it has been reported that the retinal axons terminated in a wide region ranging from the optic to zonal layers, while the afferent fibers from the visual cortex distributed
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in the deeper half of the upper layers (22). It has been also demonstrated that a retinotopical organization exists among retinal ganglionic cells, visual cortical neurons, and collicular cells (18, 20, 28). Langer et al. (17) showed the terminal plexus of a presumed retinal axon which formed a field of about 50 pm in diameter. On the basis of the width of dendritic field, it can be estimated that more than eight retinal axons terminate on a dendrite of a single horizontal cell, and that at least two to seven axons are projected to the apical dendritic field of the cylindrical neurons. On the other hand, the reversed conical and the large multipolar neurons can be connected with 24 to 37 optic fibers. It is also assumed that corticotectal fibers from area 17 project to the basal dendrites of wide-field cylindrical neurons and the processes of the reversed conical and large-field multipolar cells. Many electrophysiologic experiments (7, 15, 19, 27) have revealed that neurons in the superior colliculus can respond to a small light stimulus moving within their receptive fields. The units which are responsive to the movement stimulus may correspond to wide-field neurons which are connected with many retinal fibers, while the collicular neurons with narrow dendritic field, which receive a small number of retinal axons, can respond preferably to stimulation of a small visual field. Taking the retinotopic representation in the colliculus into consideration, these cells may play an important role in detecting the stimulated site in the visual field. The units in the superficial layers can be separated into two classes on the basis of the size of receptive field; small and large field units. The small fields, 2 to 15” in diameter, were circular and the large ones, 30 to 90” in diameter, were irregular and often patchy, as if they consisted of a conglomerate of smaller fields (15). The former units distributed to the superficial gray layer and the latter to the optic lamina. Two of our superficial neuronal types, i.e., cylindrical and multipolar types, could be further divided into subtypes of narrowand wide-field cells. The physiological data of Humphrey (15) are supported by our anatomical findings. Moreover, seven types of selected receptive fields were reported in the superficial layers of rabbit superior colliculus (19). For an understanding of the collicular functions it is necessary to identify each unit with the cell type classified by the Golgi method. The electrophoretical intracellular dye injection method (30) is a useful and effective means of correlating physiologic and morphologic findings. In a previous paper (32), the superior colliculus was divided into three major layers on the basis of the myeloarchitecture and its afferent .connections. The first layer (from the zonal to the optic layers) is primarily connected with the visual system. The second layer (from the intermedial gray to the deep gray laminae) and the third layer (stratum album pro-
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fundum) receive afferent fibers from multiple origins. Viktorov (34, 35) claimed that the deep layers are divided into two independent nuclear complexes on the basis of neuronal organization and fiber connections. However, it is clearly evident from the present observations that the superior colliculus consists of two sets of strata : superficial (first) and deep (second and third) layers. The former layers contain many neurons with variable profiles and orientations of dendritic trees, while the latter are formed mainly of large and medium-sized multipolar neurons and additionally contain vertical and horizontal cells. In the present paper, the deep layers still remain divided into two parts. This subdivision of the deep layers is not based on neuron type, but on the fiber connections. As mentioned in the previous paper (32)) the third layer formed the main fiber pathway in the superior colliculus. Regarding efferent projections from the superior colliculus, it has been reported that neurons in the superficial layers project to structures different from those which receive terminals of efferent fibers of the deep layers (1, 4, 11-13, 24). The origin of the superficial projection fibers may possibly be found in both reversed conical and wide-field multipolar neurons. From this observation, the axons of these cells do not travel through the optic layer to the collicular brachium, but most of them descend toward the deep layers. On the other hand, it was shown that axons from some deep cells, namely, the medium-sized multipolar and vertical cells, projected dorsally to the vicinity of the optic layer. We do not deny the possibility that such climbing axons may extend through the optic layer to pretectal and thalamic regions. It has already been shown that the deep layers of the colliculus receive many different fibers from the superficial colliculus layers and the spinal cord ( 16), the inferior colliculus (25), the fastigial nucleus (2, 31) and nonvisual neocortices (23). Electrophysiological studies (7, 9, 10) have confirmed that some collicular units in the deep layers were able to respond not only to visual but also to auditory and somatic stimuli. Moreover, it has been found that most of the deep neurons have visual receptive fields which are similar to those of the superficial cells. Furthermore, a correlation exists between the visual fields and the auditory or somatic receptive fields (9, 10). Drager et al. (7) pointed out in the mouse superior colliculus that there was a striking interrelation between the position of whiskers and the visual receptive fields. It should be emphasized that the deep layers are composed not only of multipolar cells but also of some vertical neurons with rather narrower dendritic fields. It is reasonable to speculate that axons from the several superficial neurons may terminate retinotopically on the dendrites of the deep vertical neurons. Assuming that these cells receive converging nonvisual fibers, they may be involved in a geometric
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