EXPERIMENTAL
NEUROLOGY
Cellular
247-268 (1977)
Morphology in the Visual Layer Developing Rat Superior Colliculus
ANNA Dcpartrrlmt
55,
R. LABRIOLA
of L-lrlatomy,
AND LOIS Ii.
Collcgc Newark,
of Mrdici~~c New Jcrsry
Rcccivcd
Novonbcr
of the
LAE_RILE 1
and Dc?ltistry 07103
of New
Jersey,
2,19?6
Golgi studies were undertaken in the superficial (visual) superior colliculus of 53 rat pups (106 colliculi) from birth to 31 days postnatal. Golgi-Cox techniques revealed discrete periods of development based upon changes in cell morphology. Major orientation of cells occurred between birth and 3 days postnatal. From 4 to 5 days postnatal, major cell populations could be identified, as (1) marginal cells; (2) horizontal cells; (3) piriform cells; (4) narrow-field vertical cells; (5) wide-field vertical cells ; and (6) stellate cells. The period from 6 to 8 days was marked by a significant increase in dendritic growth cones, spicules, and varicosities, accompanied by some dendritic elongation and branching. From 9 to 10 days postnatal, further increases in dendritic elongation and branching occurred, with disappearance of dendritic growth cones, spicules, and varicosities. Further dendritic growth and branching was observed between 15 and 23 days. The period between 23 and 31 days was characterized by the appearance of spiny types of all cell populations. Correlations between these periods of cellular differentiation and stages of synaptogensis are discussed.
INTRODUCTION Roth physiological and anatomical studies indicate that the three most superficial collicular layers form a region where visual information from the retina is reorganized and integrated (1, G, 9, 11, 13, 16, 32, 23, 25, 32). Not only has such work implicated the superficial superior colliculus in mediating visual attention and fovea1 vision, but it has also placed the tectal system on the same level of importance as the geniculostriate system. Not until recent years has the tectal system received the recognition that is due. As such, the superior colliculus is of considerable importance in 1 Dr. Labriola was supported by a grant from the Graduate School of Biomedical Sciences. Dr. Laemle was supported by National Institutes of Health Grant No. EY11074.
Copyright All rights
@ 1977 by Academic Press. of rep,roduction in any form
Inc. reserved.
ISSN
0014-4886
248
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normal visual function. Examination of the anatomy of the neonatal superior colliculus is essential to the understanding of collicular activity in vision. One goal of the present study has been to provide the morphologic basis for elucidating the role of the colliculus in the visual process. MATERIALS
AND
METHODS
Fifty-three Wistar albino rat pups at ages from birth to 31 days postnatal were anesthetized with ether or by intraperitoneal injection of 3.5% chloral hydrate (35 mg/g body weight). The animals were killed by intracardiac perfusion with dilute 170 sodium nitrite in physiological saline followed by 20% buffered formalin. The brains were left in sit26 from 2 to 3 hr and then both colliculi were carefully removed for further fixation and subsequent impregnation according to the Golgi-Cox technique and celloidin embedding. Transverse and tangential sections were cut at 120 pm. Camera lucida drawings of individual neurons in the visual superior colliculus were prepared on calibrated graph paper using a Wild microscope at a magnification of X 500. RESULTS General Trends Study of the developing superficial superior colliculus in rat pups revealed the following general trends. An increase in thickness of the mid rostrocaudal one-third of the superficial superior colliculus was observed from birth, day 0 (thickness, 350 to 390 pm), to 4 to 5 days postnatal (thickness, 590 to 640 pm). Birth to Postnatal Day 3. Neurons of the superficial superior colliculus were present at birth. They had relatively few processes. These were not fully branched as compared to their eventual size and extent in the adult. The collicular zones (Fig. l), as defined by Cajal, could not be observed at this time. There was a preponderance of disoriented, bipolar cells distributed throughout the superior colliculus (Fig. 8A) . Their somata were relatively smooth, some occasionally exhibiting elevations ; only a few processes were observed at their respective perikaryal poles. The cell body size was approximately 7 X 9 pm to 8 X 14 pm. The length of cell processes varied from 8 to 40 pm or longer in some cases. As most cells were disoriented (i.e., the bipolar projections from the somata were not arranged in any preferential direction with respect to the collicular surface), it was difficult to assess a preferential growth pattern of the processes, for example, apical versus basal pole growth rates, as would be possible if the cell were oriented perpendicular to the collicular surface. At this point in development it was difficult to identify cellular processes as either axon or dendrite using mor-
I)E\:ELOPING
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SUPERIOR
FIG. 1. Schemata of the six cell types in the respective zones and strata of the superficial superior colliculus of 15 to 16-day-old rats. Abbreviations: a-axon: hhorizontal cell ; m-marginal cell ; p-piriform cell ; s-stellate cell ; v-narrow-field, vertical cell; w-wide-field vertical cell; ZH-zone of horizontal cells; ZV-zone of vertical cells ; ZOF-zone of optic fibers ; SZ-stratum zonale ; SGS-stratum griseum superficiale ; and SO-stratum opticum.
phologic criteria. Processes did not differ significantly with respect to (i) diameter, (ii) angle of origin from the soma, or (iii) elaboration of branching, if in fact branching did occur at that time. Secondary branching was observed infrequently and tertiary branching only rarely. A cell process
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occasionally appeared to exhibit long, attenuated neurofilamentous, hairlike projections from the shaft. The average length of these neurofilamentous projections was 7 to 20 pm. The shaft of a cell process sometimes exhibited dilatations or varicosites. The diameter of the shafts decreased distally from the cell body, with branching, which was usually minimal, occurring relatively near the soma, 4 to 13 pm from the cell body. Bundles or “nests” of from three to six bipolar perikarya and processes were commonly observed during this period of development. Although the majority of cells were relatively primitive, two definite cell types such as found in the adult could be distinguished: the narrow-field vertical cells and the stellate cells. Narrow-field vertical cells were situated in the deeper portion of the superior colliculus, corresponding to the more mature zone of vertical cells of older animals. Stellate cells were situated in the deeper portion of the superior colliculus, corresponding to both the zone of vertical cells and the zone of optic fibers of older animals. Postnatal Days 4 to 5. Between 4 and 5 days postnatal there was a dispersion of the neuronal somata and a decrease in the packing density of the cells. Growth of the somata and extension and proliferation of the cell processes were observed. A variety of cells could be distinguished according to (i) position of the cell, (ii) shape of the soma, (iii) orientation of soma and processes with respect to the collicular surface, (iv) direction, extension, and branching pattern of dendrites, and (v) direction and extension of the axon. Consequently, the collicular zones as defined according to specific cell types became evident. From superficial to deep, these included the zone of horizontal cells, the zone of vertical cells, and the zone of optic fibers. Early forms of all cell types were distinguishable at this time: the (i) marginal cells, (ii) horizontal cells, (iii) piriform cells, (iv) narrow-field vertical cells. (v) wide-field vertical cells, and (vi) stellate cells, could be identified. Within the none of horizontal cells, the following major cell populations were present : (i) marginal cells, most superficially located, (ii) horizontal cells, throughout the zone, and (iii) piriform cells, in the deepest portion of this zone. Narrow-field vertical cells and stellate cells were occasionally seen as well. Cell populations in the ,-one of vertical cells included (i) narrow-field vertical cells, clispersed throughout the zone, (ii) wide-field vertical cells, in the deepest portion of the zone, and (iii) stellate cells, situated throughout the zone of vertical cells. The predominant cell of the zone uf optic fibers was the stellate cell. Most cells appeared to be in the same stage of maturation as the stellate cells of
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the more superficial zone of vertical cells, Occasionally, completely mature stellate cells were present. Post~tal L>ays 6 to 8. In the neuropil of 6- to S-day-old rats, the extracellular space was decreased and the density of cell processes increased. Most characteristic of this period of development was a significant increase in dendritic growth cones, spicules, and varicosities (Fig. SB, C) in all layers of the superficial superior colliculus. Posfnnfal Days 9 to 10. At 9 to 10 days, the neuropil had become dense and had expanded to occupy extracellular spaces observed at earlier ages. Only in the zone of optic fibers were the extracellular spaces a noteworthy feature. Further elongation of and increase in the number of dendrites occurred with an increase in dendritic branching. Growth cones and most varicosities and spicules along the dendritic shafts were no longer present. Collateral branching of axons was observed. Postnatal Days 1.5 to 16. The preceding observations (from birth to 10 days postnatal) were representative of the superior colliculus prior to eye opening. After eye opening, in the period from 15 to 16 days postnatal, the extracellular space was markedly reduced by the progressive development of the neuropil so that not only were the zones of horizontal cells, vertical cells, and optic fibers observed to be most definitive, but also the described alternating fiber-cell-fiber (stratum zonale-stratum griseum superficialestratum opticum) lamination was clearly evident (Fig. 1). The general histology of the superior colliculus was observed to correspond to that of the adult central nervous system. Further elongation of dendrites was observed in this period of development. There was a significant increase in the number of stellate cells in the zone of optic fibers. Rats 23 to 31 Days Old. In the postnatal period from 23 to 31 days, there was a notable increase in the number and overall development of spines along the dendritic shafts of collicular neurons. Both spiny and smooth varieties of all six kinds of neurons were observed at this time. Matwation
of Cell Pojulations
Marginal Cells Postnatal Days 3 to 5 (Fig. 2A j . Marginal cells appearedmost definitively at postnatal Day 5; they were the most superficial cells of the superior colliculus, the smooth, round-to-ovoid cell bodies usually within 15 to 25 pm of the colIicular surface. This range of depth spanned the stratum zonale and the upper limit of the stratum griseum superficiale. The perikarya measured 5 to 8 pm in width and 7 to 10 pm in length. One or two primary dendrites projected from the deeper portions of the perikaryal surface; some secondary branching was evident. The dendrites extended 10 to 20
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FIG. 2. Camera lucida drawings of developing marginal cells in transverse sections of superior colliculus. Golgi-Cox, X375. Abbreviation : a-axon. A-Early marginal cell observed in an animal 5 days postnatal; primary dendrites arise from the deep surface of the perikaryon. B-Cell observed at 6 days postnatal. Note varicosities (arrow) along dendritic shaft. C-Marginal cell of lo-day-old rat. Note absence of dendritic varicosities. D-Marginal cell of 15day-old rat. Note extensive dendrite and small uniform dilatations along the axon. E-Marginal cell of Sday-old rat. Note dense dendritic arbor and development of spines.
pm in length from the cell body. Dendritic shafts sometimes exhibited varicosities and neurofilamentous extensions. The axon, with a finer, more delicate diameter than the dendrites, originated from either the cell body or a primary dendrite and projected deeply or laterally with respect to the perikaryon. The axonal ramification was restricted to the same local distribution as the dendrites. Postnatal Days 6 to 8 (Figs. ZB, SD). Marginal cell bodies averaged 5 to 8 pm in width and 12 to 15 pm in length. The cell orientation and location were identical to those observed in the previous period. Dendrites increased in length, averaging 50 to 60 ,pm. There was a significant increase in dendritic growth cones, as well as varicosities, spicules, and neurofilamentous projections. The axon originated from either cell body or primary dendrite. Postnatal Days 9 to 10 (Fig. 2C). Cell location, orientation, and somal dimensions were identical to those of the previous age group and were identical to those of the fully mature adult marginal cells. Dendritic shafts no longer exhibited any varicosities, spicules, or growth cones, and the diameter increased and was more uniform. There was a significant increase in secondary branching, and tertiary branching appeared. Very few stubby and ball-on-stalk dendritic spines were observed at this time. Postnatal Days 15 to 16 (Fig. 20). Dendrites of marginal cells could be followed from the perikaryon for at least 50 to 70 pm. Some dendrites extending laterally along the surface of the colliculus were traced from 70 to 160 pm. Two types of branching of primary dendrites were observed: marginal cell dendrites either branched extensively, creating a dense arbor in close proximity to the cell body, or branched less densely with thinner
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patterns of projection. Primary dendrites measured 1.5 to 3.0 pm in diameter, secondary dendrites 0.5 to 1.0 pin. I Iiglier-order dendrites tapered gradually. Along the axons, followed 80 to 90 /*iii from the cell body, uniform dilatations were observed. At no time was the marginal cell found to project beyond the zone of horizontal cells. Postnatal Days 23 to 31 (Fig. ZE). Marginal cells of this period exhibited an abmidance of dendritic spines. Types of spines included stubby, mushroom, and bail-on-stalk varieties. Few spines were observed on the cell body and primary dendrites in comparison to the density of spines on higher-order dendrites. It was not unusual to observe a single cell with relatively smooth dendrites interspersed among spiny dendrites, comprising a gnarled, extremely spiny dendritic arbor. The spines were evenly and thickly distributed along the shafts distal to the cell body. No change was noted in the axon which originated from either the cell body or a primary dendrite, and its projection was restricted to the same local area as the dendrites. The axon arborization and growth were identical to those observed from 15 to 16 clays postnatal and reached adult parameters at this time. Horizolztal
Cells
Postnatal Days J to 5 (Figs. 3A, 8E). Horizontal cells had a fusiform body 8 to 15 /*“i in width and 12 to 18 ,.~m in length. There was a bipolar projection of the primary dendrites, with cell body and dendrites aligning themselves parallel with the surface of colliculus. Dendritic length could be followed for an average of 15 to 25 pm from the cell body. The cells were within 200 to 250 pm of the surface, within the zone of horizontal cells (equivalent to the stratum zonale and upper stratum griseum superficiale). Primary dendrites were found to be in the same plane with the cell body. Perikaryal elevations were observed, and secondary branching was evident at this time. Axons originated from either the perikaryon or a primary dendrite and were traced superficially, laterally, or deeply to the cell body. Postnatal Days 6 to 8 (Fiy. 3B). The location and orientation of horizontal cells was identical to those at 4 to 5 days postnatal. Perikaryal dimensions remained within the same range. Dendrites could be followed from 40 to 50 pm. There was a notable increase in growth cones, varicosities, and spicules on the dendritic shafts during this period. Postmtnl Days 9 fo 10 (Figs. 3C, SF). Horizontal cell location, orientation, and somal dimensions were identical to those previously described in the earlier age group from 4 to 5 days postnatal and were identical to adult parameters. Further elongation of dendrites occurred ; lengths were traced from 70 to 80 pm or more. Secondary branching increased, and tertiary branching was evident. Primary dendrites were followed S to 20 pm (and
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2ou
15-16
1
FIG. 3. Camera lucida drawings of horizontal cells observed in transverse sections of superior colliculus. Golgi-Cox, X375. Abbreviation : a-axon. A-Early horizontal cell observed at 5 days postnatal. Note bipolar projection of primary dendrites (arrows). Secondary branching is evident (small arrow). B-Cell observed at 6 days demonstrating abundant dendritic varicosities (arrow). C-Horizontal cell of lo-dayold rat. Note absence of growth cones and varicosities, and increased branching of dendrites. D-Horizontal cell of 16-day-old rat. Note elongation of dendrites and proximity of axonal projection to the dendritic projection field. E-Cell observed in a 31-day-old rat. Note cluster of spines alongthe dendriticshaft and at branchpoints.
sometimes50 pm) before secondary branching was observed. Higher-order branching occurred at more frequent intervals ; e.g., secondary branches were sometimestraced only 5 to 10 pm before tertiary branching. Dendritic shafts displayed no varicosities, growth cones, or spicules; the diameter was increased and more uniform. Collateral axonal branching was observed. Postnatal Days 1.5 fo 16 (Fig. 30). Horizontal cell dendrites were oriented parallel to the collicular surface and could be followed for 120 to 1000 pm laterally. The range of the dorsoventral spread of the dendritic arbor was 20 to 50 pm. Secondary or higher-order dendrites were observed projecting superficially or deeply from branch points. These dendrites were no longer in the same plane as the cell body and primary dendrites. Infrequently, secondary and, more often, tertiary and higher-order dendrites would deviate from the general pattern of projection and project to the deeper
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portion of the zone of horizontal cells. The diameter of the primary dendrites was 2 to 3 pm with gradual tapering of higher-order dendrites. Primary dendrites extended 5 to 30 pm from the cell body before secondary branching. Very few dendritic spines of the stubby, mushroom, or ball-on-stalk variety were observed. The axonal projection was similar to dendritic patterns, and both were also similar to adult parameters. Dilatations were sometimes observed along the extent of the axon. Horizontal cell processes usually did not extend into the zone of vertical cells. Pustnatul Days 23 to 31 (Fig 3E). A SI‘g m‘fi cant number of spiny horizontal cells appeared in this period. Dendritic spines included stubby, mushroom, and ball-on-stalk varieties, as was observed during the same period in marginal cell development. However, spines were not evenly distributed but were observed to occur quite frequently in clusters not only at branch points but also along certain points of unbranched dendritic shafts. Overall, horizontal cells were relatively less spiny than other spiny cell tyl)es observed. No change in axonal morphology or projection was evident. Piviforrlc
Cells
I’ostmltal flags 3: to 5 (Fig. Id ) . Piriforin cells had a smooth, triangular to cup-shaped body S to 1.2 pm in width and 12 to 18 pm in length. The location of the piriform cell bodies was restricted to the boundary between the zone of horizontal cells and the next deeper layer, the zone of vertical
FIG. 4. Catnera lucida drawings of piriform cells observed in transverse sections of superior colliculus. Golgi-Cox. X375. Abbreviation : a-axon. A-Early piriform cell observed in animal 5 days postnatal. Note cup-shaped body, varicosities, and spicules along superficially projecting dendrites (arrows). Axon projects deeply. B-Piriform cell at 6 days postnatal. Note varicosities, spicules, and growth cone (arrows) along dendritic shafts. C-Piriform cell of lo-day-old rat. Note increase in dendritic branching and absence of growth cones. D-Piriform cell of 16-day-old rat. Note increase in dendritic length and arborization. E-Piriform cell of 31-day-old rat. Yote the extremely spiny dendritic shafts.
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cells. During this period of development. two primary dendrites projected superficially from the cell body and could be followed 130 to 30 Pl~l. The dendrites extended vertically or obliquely with respect to the collicular surface and did not project below the cell body. Denclritic shafts exhibited varicosities. spicules. and neurofilanlentous fibers. The thin, smooth axon originated from the base of the sonla and projectecl deeply ; no collaterals were evident. P~&tal Days 6 fo 8 (Fig. IB j. The location. orientation, and somal dimensions of the piriform cell were identical to those previously described ill the period from 4 to 5 days postnatal. Elevations were observed on the cell body. Dendrites could be followed for 40 ~111.Dendritic growth cones appeared. There was also a significant increase in clendritic varicosities and spicules. Axons were followed 25 to 30 ,~.m from the base of the cell body, and collateral branching was evident. I’ostnatal Days Y to IO (Figs. #C, 8G). The location. orientation, and somal dimensions described in the previous age group were identical to that of piriforni cells in this period of development and were identical to adult parameters. Denclritic length increased to 100 pm or longer, and there was an increase in the number of dendritic branches. Secondary branches originated from 4 to 20 ,ml from the cell body. Tertiary branching increased in frequency. No growth cones and few if any varicosities and spicules were observed on the dendritic shafts. Very few stubby or ball-onstalk types of clendritic spines were observed. Axons originated strictly from the somal base and could be followed deeply to the cell body for 50 to 60 pm. I’osfnatal Days 15 fo 16 (Fig. JD). Piriform cell dendrites extended superficially to the cell body for 120 to 200 pm The entire tlendritic arbor extended at the level of and superficial to the cell body ; very rare11 did it extend below the perikaryon. The width of this superficially oriented tlendritic arbor measured 100 to 250 q. The diameter of primary dendrites measured 3 to 3 pm with gradual tapering of higher-order dendrites. The dendrites were observed to divide by equipartition ; i.e., dendrites branched at equal intervals. Most axons of piriform cells were thin and smooth. infrequently exhibiting dilatations along their lengths. Axons projected into the zone of vertical cells, sometimes giving off a collateral into the deep portion of the zone of horizontal cells. Postnatal Da,ys 23 to 31 (Figs. JE, 8H). There was an even and extremely dense distribution of spines throughout the dendritic arbor. Cell 1)oclies of spiny piriform cells displayed numerous elevations. All types of spines were observed ; however, the ball-on-stalk variety occurred much more frequently than other types of secondary and higher-order dendrites. Axons of this period fit the description of those in animals from 15 to 16
days old frequently.
with
one
exception
: Collateral
branching
was
ohser\-ed
mm-e
I’osfnaful IJaJ5 f fo 5 (Figs. LTL-l, 81). Xarrowfield vertical cells tlisplayed an elongated fusiform body 10 to 20 pi11 in u%lth and 20 to 30 pm in length. The long axis of the cell \vas oriented vertically, i.e., perpentlicular to the collicular surface. Cell hotlies were not smooth : elevations along the contours of the somata \vere frequently ohservetl. Narrowfield vertical cells \vere predominantly situated throughout the zone of vertical cells icorresponding to the lower stratum griseum superficiale). The cells tlisplayed two primary dendrites. one ascending toward the collicular surface ant1 one descending toward deeper layers. The long axis of the hipolar dendritic projections was aligned l~erpendicular to the surface. IIentlritic shafts could he followetl 80 pm in both directions, ant1 they displayed TraYicosities, spicules, and neurofilanientous projections. Secondary branching occurred. -4 relatively smooth axon originated from the cell body or a priniary dendrite and projected laterally and deeply. AZany axons codtl he traced 100 pin in length at this time.
6-k
4-5 days
9 -10
A
f
FIG.
?OU 5. Camera
eiltlrites were followed for 20 to 40 pm in length. Secontlary lx-anching was ohserved. I’aricosities, spicules. and neurofilaiiieiitous projections were present on dendritic shafts. (;rowth cones were occasionall\ohervetl. The ason originated froiii the cell body or a 1”iiiiary tleiitlrite and projected into the tleel)er portions of the superior colliculus. Xsons coultl he traced as far as 60 pni. I’ostm7fal Days 6 fo S (Pip. /‘I?. VII ). Stellate cell location and orieiitation were identical with those in the I)rex%ns description. Sonial tliniensions remained the mile. Elongation of dendrites was ohserved ; tlic length increased to ,40 to 60 pin. Deiitlritic ~aricosities. sl)icules, ant1 growth cones occurred in much greater ahntlatice than previously 01~ served ill animals 4 to 5 (lays of age. X0 sigiiificatit change in asod characteristics was ohserved. ~c~strltltal Ijays 9 to 10 ( ITi{gs. ic‘, ()E ) . The location. orientation. and s0lnal dimensions of the stellate cell were itlelltical to those described iii aninlals 6 to S days of age and in tlie adult cell. Xii increase in length and niiml)er of tlendritic hraiiclies was observetl. Growth cones, varicosities. and spicuies no longer apl)earetl 011 the tlendritic shafts. Dendrites, although more uniform in diameter, inaintainetl a delicate appearance. Axons could he followed for 60 to 100 pm. I’ost,7atal Jhzys 15 to 16 (Figs. 7l2, 9F). There was a iiotalde numeric increase in the stellate cell population. The radial spread of the deiidritic arlmr measured 100 to I50 pm in diameter. ‘C:erJ~ few tlenclritic spines, like those described for the other cells, were ohserved at this tinie. The ason sometimes esliibited dilatations but was illore often notably smooth. Axonal length could he traced up to 110 pm. The stellate cell processes were restricted to the zone iii which its cell hotl~~ was situated, as it1 the adult. I’ostlfntal Da)5 23 fo 37 stulhy spines were observed evenI\- distributed throughout
iE. 9G ). Ball-on-stalk, iiiusliroom. ant1 on stellate cell dendrites. Spine density \vas the tlentlritic arhr. Higher-order dendrites
(I;i{ys.
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displayed a greater number of ball-on-stalk spines. Stellate cells were less spiny than all other cell types with the exception of the horizontal cells. Xo significant change was observed in axonal characteristics. DISCUSSION To date, few histological and cptological studies have concentrated on the development of the visual superior colliculus in neonatal nianinials. Studies have reported cytogenesis and migration of neurons in the superficial superior colliculus of rats (2, .?O) priniarilv during the prenatal period. The present investigation is the first to describe the earl\- development of specific cell populations in the superficial superior colliculus and makes the following contributions to the literature. At birth, the superior colliculus is populated with disoriented. bipolar cells, many of which are arranged in bundles or “nests.” However. some definite cell types have been observed. Major cell orientation seemsto occur between birth and 5 days postnatal. By 5 days postnatal, collicular cells can be identified as (i) marginal cells, ( ii ) horizontal cells, (iii ) piriforin cells, [iv) narrow-field vertical cells, (v) wide-field vertical cells, and (vi ) stellate cells. Major dendritic development is observed between 6 and 15 to 16 days postnatal and is characterized by an elongation of and an increase in the number of dendrites. The period between 23 and 31 davs is characterized by the appearance of spiny types of all cell populations. The preceding observations are closely correlated with developmental stagesdefined on the basis of synaptogenesis ( 10). Stage I (10) extends from birth to 12 to l-i days postnatal and is characterized by a major wave of optic axon synapse formation and a minor wave of new intrinsic synapses. Present data based on changes in FIG. 8. Photomicrographs taken from transverse sections of the superior colliculus. Golgi-Cox. Abbreviation : a-axon. A-Disoriented bipolar cell observed in rat pup at birth. Note varicosities (V) and neurofilamentous projection (KF). X500 BPiriform cell of a 6-day-old rat illustrating growth cone on the proximal portion of the dendrite (arrow ). Inset shows same growth cone focused to demonstrate filopodia (f). X500. C-Dendrite of same cell. Note numerous varicosities (\‘) and spicules (S) along dendritic shaft. X1250. D-Marginal cell of 6-day-old rat. Kate the abundant varicosities (V) along the dendritic shaft. X500. E-Early horizontal cell of a S-day-old rat. X500. F-Horizontal cell of lo-day-old rat. Note increased dendritic branching. ~625. G-Piriform cell of a lo-day-old rat. x500. H-Piriform cell of 31day-old rat. Note the numerous elevations on the cup-shaped perikaryon (arrows). The initial lengths of the superficially directed primary dendrites are relatively smooth. X625. I-Early narrow-field vertical cell of a S-day-old rat. X500. J--Sarrow-field vertical cell of a lo-day-old rat. Note varicosities ( V 1 and spicules (S) along elongated dendrites. X500.
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cell morphology indicate that stage I actually consists of four discrete periods of development as follows: (i) birth to 3 days postnutal, major orientation of cells; (ii) 4 to 5 days postnatal, emergence of major cell populations; (iii) 6 to 8 days postnatal, significant increase in dendritic growth cones, spicules, and varicosities accompanied by dendritic shaft elongation and branching; and (iv) 9 to 10 days poshtatal, further increase in dendritic elongation and branching and disappearance of dendritic growth cones, varicosities, and spicules, with a small number of dendritic spines, collateral asonal branching, and the presence of uniform axonal dilatations. Stage II (10) extends from 15 or 16 to 23 days postnatal and is characterized by a second wave of optic axon synapses. This stage coincides with present observations of further dendritic growth and branching between eye opening and 23 days and an increased occurrence of axonal dilatations. X mature morphological organization, identical to that of the adult, appears to be established at this time. Stage III (IOj continues from 25 to between 30 and 10 days postnatal and is characterized by a major wave of intrinsic axon synapse formation. Within this stage, present data indicate a widespread appearance of mature clendritic spines in all cell populations of the superficial superior colliculus. The present study found bundles or “nests” of immature cells throughout the superficial superior colliculus at the time of birth. Similar observations of developing cells in the central nervous system have been reported (7, lS, 31). During development of the superior colliculus in rat pups from 5 to 31 days old, the presence of (i) marginal cells, (ii) horizontal cells, (iii ) piriform cells, (iv) narrow-field vertical cells, (v) wide-field vertica1 cells, and (vi j stellate cells concurs with similar findings of all these cell populations in the young adult rat (8). Using the Golgi method, Tokunaga and Otani (27) classified cell types in the superficial superior colliculus. They distinguished five different neurons which correspond closely with cell populations identified in the present investigation. They are “the horizontal type,” which corresponds to the horizontal cells of the present study; “the cylindrical type with dorsoventrally oriented dendrites,” which corresponds to the narrow-field vertical cells ; “the cylindrical neurons with dorsally oriented dendrites,” which correspond to piriform cells ; “the reversed conical types,” which correspond to the wide-field vertical cells ; and “the multipolar type” which corresponds to the stellate cell. A number of studies, all in adult animals, do not report the presence of the following cells: marginal cells (Z-l, -37, 30, 31) ; horizontal cells (24) ; and narrow-field vertical cells (30:)
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In a light and electron microscopic study, three classes of neurons in the more superficial superior colliculus (i.e., the stratum zonale and stratum griseum superficiale) in the adult chimpanzee have been reported (26). Two types correspond most closely with neurons identified in the present study. They are “marginal cells” in the stratum zonale, which correspond to marginal cells of the present investigation, and “presynaptic dendrite cells” in the stratum griseum superficiale, which correspond to the horizontal cells. The third class of neurons, the “collicular relay cells” are situated in the stratum griseum superficiale. These cells project primary dendrites toward the collicular surface and/or the stratum opticum, or they extend dendritic branches parallel with or oblique to the collicular surface. This group of cells may correspond to piriform cells and narrowand wide-field vertical cells. More specific correlation is limited as a further ultrastructural subclassification of “collicular relay cells” is not available. Ultrastructural observations (26) have not only verified the light microscopic description of marginal and horizontal cells [present investigation and (8) ] but, in addition, have presented ultrastructural criteria for the identification of presynaptic denclritic cells (i.e., the horizontal cells). Such correlative data between light and electron microscopic studies are invaluable in assessing the functional role of cell populations. To date, collicular recordings on neonatal rat pups from birth to 31 days postnatal have not been reported. The present study has attempted to establish an anatomic substrate for unit studies of the developing superior colliculus in order to broaden our knowledge of collicular physiology. The variety of spines (stubby, mushroom, and ball-on-stalk) are well defined in all cell populations of the superficial superior colliculus from 33 to 31 days postnatal. This observed spine development may be correlated with the marked wave of intrinsic axon synapse observed from day 25 to between days 30 and 40. Growth cones, spicules, and varicosities along dendritic shafts are well established as major morphologic characteristics of immature dendritic growth processes (13-15, 19). The overwhelming abundance of these features present in all cell populations of the superficial superior colliculus from 6 to 8 days postnatal may represent a step in dendritic branching and elongation. What appears to be a disappearance of these early denand spicules) may in dritic characteristics (growth cones, varicosities, fact have been the result of two concomitant processes : (i) the progression of some of these structures into the more mature dendritic branches observed by days 9 to 10 and (ii) the regression of other structures into a totally retracted state. On the basis of light and electron microscopic findings, the same case in point has been reported (4) : “varicose den-
drites and beaded processes” were observed lo emanate from early inner marginal neurons of the developing trigeminal nucleus caudalis in newborn kittens, followed by lengthening of dendrites and a decrease in the number of ‘thin, short dendrites’ and ‘reduction in the number of beaded processes’. From ultrastructural evidence, it was concluded that the retraction of those esisting neural structures is linked with the postnatal differentiation of dendrites. In light of the findings from deprivation experiments involving cell populations of the visual cortex (3, 5, 25, 29), it is not unreasonable to expect “sensitive” or “critical” periods of cell development in the superior colliculus also. The present study has revealed that ontogenetic events in emerging cell populations of the superior colliculus can be identified with discrete time periods between birth and 31 days postnatal, suggesting possible stages of vulnerability. To date, no morphological studies, at least in the albino rat, have attempted to relate the effects produced by light deprivation or deafferentation with abnormalities in cell development in the superior colliculus. The present investigation provides a background for exploring the relative sensitivity of different stages of collicular development and for evaluating the theory of a “critical period” in the cytological development of the superior colliculus. REFERENCES 1.
J. 1962. Some fiber projections to the superior colliculus in the cat. J. Camp. Ncwrol. 119 : 77-95. 2. BRUCKNER, G., MARES, V., AND BIESOLD, D. 1976. Neurogenesis in the visual system of the rat. An autoradiographic investigation. J. Camp. Ncurol. 166: 245-256. 3. COLEMAN, P. D., AND RIESAN, A. H. 1968. Environmental effects on cortical ALTMAN,
dendritic fields. 1. Rearing in the dark. J. .4nat. 102 : 363-375. 4. FALLS, W., AND GOBEL, S. 1976. A Golgi and EM study of the marginal and substantia gelatinosa layers of trigeminal nucleus caudalis in newborn kittens. Amt. Rec. 184 : 399. 5. GLOBUS, A., AND SCHEIBEL, A. B. 1967. The effect of visual deprivation on cortical neurons: A Golgi study. Exp. Ncurol. 19: 331-345. 6. INGLE, D., AND SCHNEIDER, G. E., Eds. 1970. Sz~bcortical ~‘isual Sq’stcws. Brain Behav. Evol. 3: 1-352. 7. LAEMLE, L. K. 1974. The development of the human visual system. I. The visual cortex. Awt. Kcc. 178: 449. 8. LANCER, T. P., AND LUND, R. D. 1974. The upper layers of the superior colliculus of the rat : A Golgi study. J. Cowtp. New-ol. 158 : 405-436. 9. LUND, R. D. 1969. Synaptic patterns of the superficial layers of the superior colliculus of the rat. J. Cowp. Neural. 135: 179-208. 10. LUND, R. D., AND LUND, J. S. 1972. Development of synaptic patterns in the superior colliculus of the rat. Brain RES. 42: l-20. 11. MCILWAIN, J. T., AND BUSER, P., 1968. Receptive fields of single cells in the cat’s superior colliculus. Exp. Brain Rcs. 5 : 314-325. 12. MICHAEL, C. R. 1967. Integration of visual information in the superior colliculus. J. GEL Physiol. 50 : 2584-2586.
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13. Mokes,r, 1). K. 1969. The differentiation of cerebral dendrites : A study of the postmigratory nemoblast in the medial nucleus of the trapezoid body. Z. Amzf. E~~t”Le~icklz~lrgs,~csch. 128 : 271-289. 14. MOHEST, D. K. 1969a. The growth of dendrites in the mammalian brain. Z.Annt. E,1tzc,icklzlrlgsgescR. 128 : 290-317. 15. MOREST, D. K. 1970. A study of neurogenesis in the forebrain of opossum pouch young. 2. -4 wt. Elrtzoicklzrlrgsgcsrk. 130 : 265-305. 16. NAUTA, W. J. H., AND VAN STRAATEN, J. J. 1947. The primary optic centers of the rat, an experimental study by the “bouton” method. /. Anat. 81 : 127-134. 17. PETERS, A., AND KAISERI\ZAN-ABRAMOF, I. R. 1975. The small pyramidal neurons of the rat cerebral cortex. The perikaryon, dendrites, and spines. J. Anat. 127: 321-356. 18. PETERS, A., AND WALSH, T. M. 1972. A study of the organization of apical dendrites in the somatic sensory cortex of the rat. J. Camp. NEZWOZ. 144: 253-268. 19. PURPURA, D. P. 1975. Normal and aberrant neuronal development in the cerebral cortex of human fetus and young infant. Pages 141-169 in N. BUCHWALD AND M. A. B. BRAZIER, Eds., Mechaxisms in Mental Retardation. Academic Press, New York. 20. RAEDLER, A., AND SIEVERS, J. 1975. The development of the visual system of the albino rat. AdzI. Alzat. Embryol. Crll Biol. 50, Fast. 3. 21. SCHEIBEL, M. E., AND SCHEIBEL, A. B. 1970. bz K. L. CHOW AND A. L. LEIMAN, Eds., The Structural and Fzrrlctional Orgaxization of the Neocortcx. Ncz~rosci. Rcs. Prog. Bull. 8, Vol. 2. MIT Press, Cambridge, Massachusettes. 22. SIMINOFF, R., SCHWASSMANN, H. O., AND KRUGER, L. 1966. An electrophysiological study of tile visual projection to the superior colliculus of the rat. J. Comp. Ncwol. 127: 435-444. 23. SPRAGUE, J. M., BERLUCCHI, G., AND RIZZOLATTI, G. 1973. The role of the superior colliculus and pretectum in vision and visually guided behavior. Pages 27-101 ijt R. JUNG, Ed., Handbook of S~?lsory Physiology, VII, 3, Central Visual Information, B. Springer-V
NEUROLOGY
Cellular
247-268 (1977)
Morphology in the Visual Layer Developing Rat Superior Colliculus
ANNA Dcpartrrlmt
55,
R. LABRIOLA
of L-lrlatomy,
AND LOIS Ii.
Collcgc Newark,
of Mrdici~~c New Jcrsry
Rcccivcd
Novonbcr
of the
LAE_RILE 1
and Dc?ltistry 07103
of New
Jersey,
2,19?6
Golgi studies were undertaken in the superficial (visual) superior colliculus of 53 rat pups (106 colliculi) from birth to 31 days postnatal. Golgi-Cox techniques revealed discrete periods of development based upon changes in cell morphology. Major orientation of cells occurred between birth and 3 days postnatal. From 4 to 5 days postnatal, major cell populations could be identified, as (1) marginal cells; (2) horizontal cells; (3) piriform cells; (4) narrow-field vertical cells; (5) wide-field vertical cells ; and (6) stellate cells. The period from 6 to 8 days was marked by a significant increase in dendritic growth cones, spicules, and varicosities, accompanied by some dendritic elongation and branching. From 9 to 10 days postnatal, further increases in dendritic elongation and branching occurred, with disappearance of dendritic growth cones, spicules, and varicosities. Further dendritic growth and branching was observed between 15 and 23 days. The period between 23 and 31 days was characterized by the appearance of spiny types of all cell populations. Correlations between these periods of cellular differentiation and stages of synaptogensis are discussed.
INTRODUCTION Roth physiological and anatomical studies indicate that the three most superficial collicular layers form a region where visual information from the retina is reorganized and integrated (1, G, 9, 11, 13, 16, 32, 23, 25, 32). Not only has such work implicated the superficial superior colliculus in mediating visual attention and fovea1 vision, but it has also placed the tectal system on the same level of importance as the geniculostriate system. Not until recent years has the tectal system received the recognition that is due. As such, the superior colliculus is of considerable importance in 1 Dr. Labriola was supported by a grant from the Graduate School of Biomedical Sciences. Dr. Laemle was supported by National Institutes of Health Grant No. EY11074.
Copyright All rights
@ 1977 by Academic Press. of rep,roduction in any form
Inc. reserved.
ISSN
0014-4886
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normal visual function. Examination of the anatomy of the neonatal superior colliculus is essential to the understanding of collicular activity in vision. One goal of the present study has been to provide the morphologic basis for elucidating the role of the colliculus in the visual process. MATERIALS
AND
METHODS
Fifty-three Wistar albino rat pups at ages from birth to 31 days postnatal were anesthetized with ether or by intraperitoneal injection of 3.5% chloral hydrate (35 mg/g body weight). The animals were killed by intracardiac perfusion with dilute 170 sodium nitrite in physiological saline followed by 20% buffered formalin. The brains were left in sit26 from 2 to 3 hr and then both colliculi were carefully removed for further fixation and subsequent impregnation according to the Golgi-Cox technique and celloidin embedding. Transverse and tangential sections were cut at 120 pm. Camera lucida drawings of individual neurons in the visual superior colliculus were prepared on calibrated graph paper using a Wild microscope at a magnification of X 500. RESULTS General Trends Study of the developing superficial superior colliculus in rat pups revealed the following general trends. An increase in thickness of the mid rostrocaudal one-third of the superficial superior colliculus was observed from birth, day 0 (thickness, 350 to 390 pm), to 4 to 5 days postnatal (thickness, 590 to 640 pm). Birth to Postnatal Day 3. Neurons of the superficial superior colliculus were present at birth. They had relatively few processes. These were not fully branched as compared to their eventual size and extent in the adult. The collicular zones (Fig. l), as defined by Cajal, could not be observed at this time. There was a preponderance of disoriented, bipolar cells distributed throughout the superior colliculus (Fig. 8A) . Their somata were relatively smooth, some occasionally exhibiting elevations ; only a few processes were observed at their respective perikaryal poles. The cell body size was approximately 7 X 9 pm to 8 X 14 pm. The length of cell processes varied from 8 to 40 pm or longer in some cases. As most cells were disoriented (i.e., the bipolar projections from the somata were not arranged in any preferential direction with respect to the collicular surface), it was difficult to assess a preferential growth pattern of the processes, for example, apical versus basal pole growth rates, as would be possible if the cell were oriented perpendicular to the collicular surface. At this point in development it was difficult to identify cellular processes as either axon or dendrite using mor-
I)E\:ELOPING
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SUPERIOR
FIG. 1. Schemata of the six cell types in the respective zones and strata of the superficial superior colliculus of 15 to 16-day-old rats. Abbreviations: a-axon: hhorizontal cell ; m-marginal cell ; p-piriform cell ; s-stellate cell ; v-narrow-field, vertical cell; w-wide-field vertical cell; ZH-zone of horizontal cells; ZV-zone of vertical cells ; ZOF-zone of optic fibers ; SZ-stratum zonale ; SGS-stratum griseum superficiale ; and SO-stratum opticum.
phologic criteria. Processes did not differ significantly with respect to (i) diameter, (ii) angle of origin from the soma, or (iii) elaboration of branching, if in fact branching did occur at that time. Secondary branching was observed infrequently and tertiary branching only rarely. A cell process
250
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occasionally appeared to exhibit long, attenuated neurofilamentous, hairlike projections from the shaft. The average length of these neurofilamentous projections was 7 to 20 pm. The shaft of a cell process sometimes exhibited dilatations or varicosites. The diameter of the shafts decreased distally from the cell body, with branching, which was usually minimal, occurring relatively near the soma, 4 to 13 pm from the cell body. Bundles or “nests” of from three to six bipolar perikarya and processes were commonly observed during this period of development. Although the majority of cells were relatively primitive, two definite cell types such as found in the adult could be distinguished: the narrow-field vertical cells and the stellate cells. Narrow-field vertical cells were situated in the deeper portion of the superior colliculus, corresponding to the more mature zone of vertical cells of older animals. Stellate cells were situated in the deeper portion of the superior colliculus, corresponding to both the zone of vertical cells and the zone of optic fibers of older animals. Postnatal Days 4 to 5. Between 4 and 5 days postnatal there was a dispersion of the neuronal somata and a decrease in the packing density of the cells. Growth of the somata and extension and proliferation of the cell processes were observed. A variety of cells could be distinguished according to (i) position of the cell, (ii) shape of the soma, (iii) orientation of soma and processes with respect to the collicular surface, (iv) direction, extension, and branching pattern of dendrites, and (v) direction and extension of the axon. Consequently, the collicular zones as defined according to specific cell types became evident. From superficial to deep, these included the zone of horizontal cells, the zone of vertical cells, and the zone of optic fibers. Early forms of all cell types were distinguishable at this time: the (i) marginal cells, (ii) horizontal cells, (iii) piriform cells, (iv) narrow-field vertical cells. (v) wide-field vertical cells, and (vi) stellate cells, could be identified. Within the none of horizontal cells, the following major cell populations were present : (i) marginal cells, most superficially located, (ii) horizontal cells, throughout the zone, and (iii) piriform cells, in the deepest portion of this zone. Narrow-field vertical cells and stellate cells were occasionally seen as well. Cell populations in the ,-one of vertical cells included (i) narrow-field vertical cells, clispersed throughout the zone, (ii) wide-field vertical cells, in the deepest portion of the zone, and (iii) stellate cells, situated throughout the zone of vertical cells. The predominant cell of the zone uf optic fibers was the stellate cell. Most cells appeared to be in the same stage of maturation as the stellate cells of
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the more superficial zone of vertical cells, Occasionally, completely mature stellate cells were present. Post~tal L>ays 6 to 8. In the neuropil of 6- to S-day-old rats, the extracellular space was decreased and the density of cell processes increased. Most characteristic of this period of development was a significant increase in dendritic growth cones, spicules, and varicosities (Fig. SB, C) in all layers of the superficial superior colliculus. Posfnnfal Days 9 to 10. At 9 to 10 days, the neuropil had become dense and had expanded to occupy extracellular spaces observed at earlier ages. Only in the zone of optic fibers were the extracellular spaces a noteworthy feature. Further elongation of and increase in the number of dendrites occurred with an increase in dendritic branching. Growth cones and most varicosities and spicules along the dendritic shafts were no longer present. Collateral branching of axons was observed. Postnatal Days 1.5 to 16. The preceding observations (from birth to 10 days postnatal) were representative of the superior colliculus prior to eye opening. After eye opening, in the period from 15 to 16 days postnatal, the extracellular space was markedly reduced by the progressive development of the neuropil so that not only were the zones of horizontal cells, vertical cells, and optic fibers observed to be most definitive, but also the described alternating fiber-cell-fiber (stratum zonale-stratum griseum superficialestratum opticum) lamination was clearly evident (Fig. 1). The general histology of the superior colliculus was observed to correspond to that of the adult central nervous system. Further elongation of dendrites was observed in this period of development. There was a significant increase in the number of stellate cells in the zone of optic fibers. Rats 23 to 31 Days Old. In the postnatal period from 23 to 31 days, there was a notable increase in the number and overall development of spines along the dendritic shafts of collicular neurons. Both spiny and smooth varieties of all six kinds of neurons were observed at this time. Matwation
of Cell Pojulations
Marginal Cells Postnatal Days 3 to 5 (Fig. 2A j . Marginal cells appearedmost definitively at postnatal Day 5; they were the most superficial cells of the superior colliculus, the smooth, round-to-ovoid cell bodies usually within 15 to 25 pm of the colIicular surface. This range of depth spanned the stratum zonale and the upper limit of the stratum griseum superficiale. The perikarya measured 5 to 8 pm in width and 7 to 10 pm in length. One or two primary dendrites projected from the deeper portions of the perikaryal surface; some secondary branching was evident. The dendrites extended 10 to 20
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FIG. 2. Camera lucida drawings of developing marginal cells in transverse sections of superior colliculus. Golgi-Cox, X375. Abbreviation : a-axon. A-Early marginal cell observed in an animal 5 days postnatal; primary dendrites arise from the deep surface of the perikaryon. B-Cell observed at 6 days postnatal. Note varicosities (arrow) along dendritic shaft. C-Marginal cell of lo-day-old rat. Note absence of dendritic varicosities. D-Marginal cell of 15day-old rat. Note extensive dendrite and small uniform dilatations along the axon. E-Marginal cell of Sday-old rat. Note dense dendritic arbor and development of spines.
pm in length from the cell body. Dendritic shafts sometimes exhibited varicosities and neurofilamentous extensions. The axon, with a finer, more delicate diameter than the dendrites, originated from either the cell body or a primary dendrite and projected deeply or laterally with respect to the perikaryon. The axonal ramification was restricted to the same local distribution as the dendrites. Postnatal Days 6 to 8 (Figs. ZB, SD). Marginal cell bodies averaged 5 to 8 pm in width and 12 to 15 pm in length. The cell orientation and location were identical to those observed in the previous period. Dendrites increased in length, averaging 50 to 60 ,pm. There was a significant increase in dendritic growth cones, as well as varicosities, spicules, and neurofilamentous projections. The axon originated from either cell body or primary dendrite. Postnatal Days 9 to 10 (Fig. 2C). Cell location, orientation, and somal dimensions were identical to those of the previous age group and were identical to those of the fully mature adult marginal cells. Dendritic shafts no longer exhibited any varicosities, spicules, or growth cones, and the diameter increased and was more uniform. There was a significant increase in secondary branching, and tertiary branching appeared. Very few stubby and ball-on-stalk dendritic spines were observed at this time. Postnatal Days 15 to 16 (Fig. 20). Dendrites of marginal cells could be followed from the perikaryon for at least 50 to 70 pm. Some dendrites extending laterally along the surface of the colliculus were traced from 70 to 160 pm. Two types of branching of primary dendrites were observed: marginal cell dendrites either branched extensively, creating a dense arbor in close proximity to the cell body, or branched less densely with thinner
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patterns of projection. Primary dendrites measured 1.5 to 3.0 pm in diameter, secondary dendrites 0.5 to 1.0 pin. I Iiglier-order dendrites tapered gradually. Along the axons, followed 80 to 90 /*iii from the cell body, uniform dilatations were observed. At no time was the marginal cell found to project beyond the zone of horizontal cells. Postnatal Days 23 to 31 (Fig. ZE). Marginal cells of this period exhibited an abmidance of dendritic spines. Types of spines included stubby, mushroom, and bail-on-stalk varieties. Few spines were observed on the cell body and primary dendrites in comparison to the density of spines on higher-order dendrites. It was not unusual to observe a single cell with relatively smooth dendrites interspersed among spiny dendrites, comprising a gnarled, extremely spiny dendritic arbor. The spines were evenly and thickly distributed along the shafts distal to the cell body. No change was noted in the axon which originated from either the cell body or a primary dendrite, and its projection was restricted to the same local area as the dendrites. The axon arborization and growth were identical to those observed from 15 to 16 clays postnatal and reached adult parameters at this time. Horizolztal
Cells
Postnatal Days J to 5 (Figs. 3A, 8E). Horizontal cells had a fusiform body 8 to 15 /*“i in width and 12 to 18 ,.~m in length. There was a bipolar projection of the primary dendrites, with cell body and dendrites aligning themselves parallel with the surface of colliculus. Dendritic length could be followed for an average of 15 to 25 pm from the cell body. The cells were within 200 to 250 pm of the surface, within the zone of horizontal cells (equivalent to the stratum zonale and upper stratum griseum superficiale). Primary dendrites were found to be in the same plane with the cell body. Perikaryal elevations were observed, and secondary branching was evident at this time. Axons originated from either the perikaryon or a primary dendrite and were traced superficially, laterally, or deeply to the cell body. Postnatal Days 6 to 8 (Fiy. 3B). The location and orientation of horizontal cells was identical to those at 4 to 5 days postnatal. Perikaryal dimensions remained within the same range. Dendrites could be followed from 40 to 50 pm. There was a notable increase in growth cones, varicosities, and spicules on the dendritic shafts during this period. Postmtnl Days 9 fo 10 (Figs. 3C, SF). Horizontal cell location, orientation, and somal dimensions were identical to those previously described in the earlier age group from 4 to 5 days postnatal and were identical to adult parameters. Further elongation of dendrites occurred ; lengths were traced from 70 to 80 pm or more. Secondary branching increased, and tertiary branching was evident. Primary dendrites were followed S to 20 pm (and
254
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2ou
15-16
1
FIG. 3. Camera lucida drawings of horizontal cells observed in transverse sections of superior colliculus. Golgi-Cox, X375. Abbreviation : a-axon. A-Early horizontal cell observed at 5 days postnatal. Note bipolar projection of primary dendrites (arrows). Secondary branching is evident (small arrow). B-Cell observed at 6 days demonstrating abundant dendritic varicosities (arrow). C-Horizontal cell of lo-dayold rat. Note absence of growth cones and varicosities, and increased branching of dendrites. D-Horizontal cell of 16-day-old rat. Note elongation of dendrites and proximity of axonal projection to the dendritic projection field. E-Cell observed in a 31-day-old rat. Note cluster of spines alongthe dendriticshaft and at branchpoints.
sometimes50 pm) before secondary branching was observed. Higher-order branching occurred at more frequent intervals ; e.g., secondary branches were sometimestraced only 5 to 10 pm before tertiary branching. Dendritic shafts displayed no varicosities, growth cones, or spicules; the diameter was increased and more uniform. Collateral axonal branching was observed. Postnatal Days 1.5 fo 16 (Fig. 30). Horizontal cell dendrites were oriented parallel to the collicular surface and could be followed for 120 to 1000 pm laterally. The range of the dorsoventral spread of the dendritic arbor was 20 to 50 pm. Secondary or higher-order dendrites were observed projecting superficially or deeply from branch points. These dendrites were no longer in the same plane as the cell body and primary dendrites. Infrequently, secondary and, more often, tertiary and higher-order dendrites would deviate from the general pattern of projection and project to the deeper
DEVELOPIXG
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portion of the zone of horizontal cells. The diameter of the primary dendrites was 2 to 3 pm with gradual tapering of higher-order dendrites. Primary dendrites extended 5 to 30 pm from the cell body before secondary branching. Very few dendritic spines of the stubby, mushroom, or ball-on-stalk variety were observed. The axonal projection was similar to dendritic patterns, and both were also similar to adult parameters. Dilatations were sometimes observed along the extent of the axon. Horizontal cell processes usually did not extend into the zone of vertical cells. Pustnatul Days 23 to 31 (Fig 3E). A SI‘g m‘fi cant number of spiny horizontal cells appeared in this period. Dendritic spines included stubby, mushroom, and ball-on-stalk varieties, as was observed during the same period in marginal cell development. However, spines were not evenly distributed but were observed to occur quite frequently in clusters not only at branch points but also along certain points of unbranched dendritic shafts. Overall, horizontal cells were relatively less spiny than other spiny cell tyl)es observed. No change in axonal morphology or projection was evident. Piviforrlc
Cells
I’ostmltal flags 3: to 5 (Fig. Id ) . Piriforin cells had a smooth, triangular to cup-shaped body S to 1.2 pm in width and 12 to 18 pm in length. The location of the piriform cell bodies was restricted to the boundary between the zone of horizontal cells and the next deeper layer, the zone of vertical
FIG. 4. Catnera lucida drawings of piriform cells observed in transverse sections of superior colliculus. Golgi-Cox. X375. Abbreviation : a-axon. A-Early piriform cell observed in animal 5 days postnatal. Note cup-shaped body, varicosities, and spicules along superficially projecting dendrites (arrows). Axon projects deeply. B-Piriform cell at 6 days postnatal. Note varicosities, spicules, and growth cone (arrows) along dendritic shafts. C-Piriform cell of lo-day-old rat. Note increase in dendritic branching and absence of growth cones. D-Piriform cell of 16-day-old rat. Note increase in dendritic length and arborization. E-Piriform cell of 31-day-old rat. Yote the extremely spiny dendritic shafts.
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cells. During this period of development. two primary dendrites projected superficially from the cell body and could be followed 130 to 30 Pl~l. The dendrites extended vertically or obliquely with respect to the collicular surface and did not project below the cell body. Denclritic shafts exhibited varicosities. spicules. and neurofilanlentous fibers. The thin, smooth axon originated from the base of the sonla and projectecl deeply ; no collaterals were evident. P~&tal Days 6 fo 8 (Fig. IB j. The location. orientation, and somal dimensions of the piriform cell were identical to those previously described ill the period from 4 to 5 days postnatal. Elevations were observed on the cell body. Dendrites could be followed for 40 ~111.Dendritic growth cones appeared. There was also a significant increase in clendritic varicosities and spicules. Axons were followed 25 to 30 ,~.m from the base of the cell body, and collateral branching was evident. I’ostnatal Days Y to IO (Figs. #C, 8G). The location. orientation, and somal dimensions described in the previous age group were identical to that of piriforni cells in this period of development and were identical to adult parameters. Denclritic length increased to 100 pm or longer, and there was an increase in the number of dendritic branches. Secondary branches originated from 4 to 20 ,ml from the cell body. Tertiary branching increased in frequency. No growth cones and few if any varicosities and spicules were observed on the dendritic shafts. Very few stubby or ball-onstalk types of clendritic spines were observed. Axons originated strictly from the somal base and could be followed deeply to the cell body for 50 to 60 pm. I’osfnatal Days 15 fo 16 (Fig. JD). Piriform cell dendrites extended superficially to the cell body for 120 to 200 pm The entire tlendritic arbor extended at the level of and superficial to the cell body ; very rare11 did it extend below the perikaryon. The width of this superficially oriented tlendritic arbor measured 100 to 250 q. The diameter of primary dendrites measured 3 to 3 pm with gradual tapering of higher-order dendrites. The dendrites were observed to divide by equipartition ; i.e., dendrites branched at equal intervals. Most axons of piriform cells were thin and smooth. infrequently exhibiting dilatations along their lengths. Axons projected into the zone of vertical cells, sometimes giving off a collateral into the deep portion of the zone of horizontal cells. Postnatal Da,ys 23 to 31 (Figs. JE, 8H). There was an even and extremely dense distribution of spines throughout the dendritic arbor. Cell 1)oclies of spiny piriform cells displayed numerous elevations. All types of spines were observed ; however, the ball-on-stalk variety occurred much more frequently than other types of secondary and higher-order dendrites. Axons of this period fit the description of those in animals from 15 to 16
days old frequently.
with
one
exception
: Collateral
branching
was
ohser\-ed
mm-e
I’osfnaful IJaJ5 f fo 5 (Figs. LTL-l, 81). Xarrowfield vertical cells tlisplayed an elongated fusiform body 10 to 20 pi11 in u%lth and 20 to 30 pm in length. The long axis of the cell \vas oriented vertically, i.e., perpentlicular to the collicular surface. Cell hotlies were not smooth : elevations along the contours of the somata \vere frequently ohservetl. Narrowfield vertical cells \vere predominantly situated throughout the zone of vertical cells icorresponding to the lower stratum griseum superficiale). The cells tlisplayed two primary dendrites. one ascending toward the collicular surface ant1 one descending toward deeper layers. The long axis of the hipolar dendritic projections was aligned l~erpendicular to the surface. IIentlritic shafts could he followetl 80 pm in both directions, ant1 they displayed TraYicosities, spicules, and neurofilanientous projections. Secondary branching occurred. -4 relatively smooth axon originated from the cell body or a priniary dendrite and projected laterally and deeply. AZany axons codtl he traced 100 pin in length at this time.
6-k
4-5 days
9 -10
A
f
FIG.
?OU 5. Camera
eiltlrites were followed for 20 to 40 pm in length. Secontlary lx-anching was ohserved. I’aricosities, spicules. and neurofilaiiieiitous projections were present on dendritic shafts. (;rowth cones were occasionall\ohervetl. The ason originated froiii the cell body or a 1”iiiiary tleiitlrite and projected into the tleel)er portions of the superior colliculus. Xsons coultl he traced as far as 60 pni. I’ostm7fal Days 6 fo S (Pip. /‘I?. VII ). Stellate cell location and orieiitation were identical with those in the I)rex%ns description. Sonial tliniensions remained the mile. Elongation of dendrites was ohserved ; tlic length increased to ,40 to 60 pin. Deiitlritic ~aricosities. sl)icules, ant1 growth cones occurred in much greater ahntlatice than previously 01~ served ill animals 4 to 5 (lays of age. X0 sigiiificatit change in asod characteristics was ohserved. ~c~strltltal Ijays 9 to 10 ( ITi{gs. ic‘, ()E ) . The location. orientation. and s0lnal dimensions of the stellate cell were itlelltical to those described iii aninlals 6 to S days of age and in tlie adult cell. Xii increase in length and niiml)er of tlendritic hraiiclies was observetl. Growth cones, varicosities. and spicuies no longer apl)earetl 011 the tlendritic shafts. Dendrites, although more uniform in diameter, inaintainetl a delicate appearance. Axons could he followed for 60 to 100 pm. I’ost,7atal Jhzys 15 to 16 (Figs. 7l2, 9F). There was a iiotalde numeric increase in the stellate cell population. The radial spread of the deiidritic arlmr measured 100 to I50 pm in diameter. ‘C:erJ~ few tlenclritic spines, like those described for the other cells, were ohserved at this tinie. The ason sometimes esliibited dilatations but was illore often notably smooth. Axonal length could he traced up to 110 pm. The stellate cell processes were restricted to the zone iii which its cell hotl~~ was situated, as it1 the adult. I’ostlfntal Da)5 23 fo 37 stulhy spines were observed evenI\- distributed throughout
iE. 9G ). Ball-on-stalk, iiiusliroom. ant1 on stellate cell dendrites. Spine density \vas the tlentlritic arhr. Higher-order dendrites
(I;i{ys.
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displayed a greater number of ball-on-stalk spines. Stellate cells were less spiny than all other cell types with the exception of the horizontal cells. Xo significant change was observed in axonal characteristics. DISCUSSION To date, few histological and cptological studies have concentrated on the development of the visual superior colliculus in neonatal nianinials. Studies have reported cytogenesis and migration of neurons in the superficial superior colliculus of rats (2, .?O) priniarilv during the prenatal period. The present investigation is the first to describe the earl\- development of specific cell populations in the superficial superior colliculus and makes the following contributions to the literature. At birth, the superior colliculus is populated with disoriented. bipolar cells, many of which are arranged in bundles or “nests.” However. some definite cell types have been observed. Major cell orientation seemsto occur between birth and 5 days postnatal. By 5 days postnatal, collicular cells can be identified as (i) marginal cells, ( ii ) horizontal cells, (iii ) piriforin cells, [iv) narrow-field vertical cells, (v) wide-field vertical cells, and (vi ) stellate cells. Major dendritic development is observed between 6 and 15 to 16 days postnatal and is characterized by an elongation of and an increase in the number of dendrites. The period between 23 and 31 davs is characterized by the appearance of spiny types of all cell populations. The preceding observations are closely correlated with developmental stagesdefined on the basis of synaptogenesis ( 10). Stage I (10) extends from birth to 12 to l-i days postnatal and is characterized by a major wave of optic axon synapse formation and a minor wave of new intrinsic synapses. Present data based on changes in FIG. 8. Photomicrographs taken from transverse sections of the superior colliculus. Golgi-Cox. Abbreviation : a-axon. A-Disoriented bipolar cell observed in rat pup at birth. Note varicosities (V) and neurofilamentous projection (KF). X500 BPiriform cell of a 6-day-old rat illustrating growth cone on the proximal portion of the dendrite (arrow ). Inset shows same growth cone focused to demonstrate filopodia (f). X500. C-Dendrite of same cell. Note numerous varicosities (\‘) and spicules (S) along dendritic shaft. X1250. D-Marginal cell of 6-day-old rat. Kate the abundant varicosities (V) along the dendritic shaft. X500. E-Early horizontal cell of a S-day-old rat. X500. F-Horizontal cell of lo-day-old rat. Note increased dendritic branching. ~625. G-Piriform cell of a lo-day-old rat. x500. H-Piriform cell of 31day-old rat. Note the numerous elevations on the cup-shaped perikaryon (arrows). The initial lengths of the superficially directed primary dendrites are relatively smooth. X625. I-Early narrow-field vertical cell of a S-day-old rat. X500. J--Sarrow-field vertical cell of a lo-day-old rat. Note varicosities ( V 1 and spicules (S) along elongated dendrites. X500.
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cell morphology indicate that stage I actually consists of four discrete periods of development as follows: (i) birth to 3 days postnutal, major orientation of cells; (ii) 4 to 5 days postnatal, emergence of major cell populations; (iii) 6 to 8 days postnatal, significant increase in dendritic growth cones, spicules, and varicosities accompanied by dendritic shaft elongation and branching; and (iv) 9 to 10 days poshtatal, further increase in dendritic elongation and branching and disappearance of dendritic growth cones, varicosities, and spicules, with a small number of dendritic spines, collateral asonal branching, and the presence of uniform axonal dilatations. Stage II (10) extends from 15 or 16 to 23 days postnatal and is characterized by a second wave of optic axon synapses. This stage coincides with present observations of further dendritic growth and branching between eye opening and 23 days and an increased occurrence of axonal dilatations. X mature morphological organization, identical to that of the adult, appears to be established at this time. Stage III (IOj continues from 25 to between 30 and 10 days postnatal and is characterized by a major wave of intrinsic axon synapse formation. Within this stage, present data indicate a widespread appearance of mature clendritic spines in all cell populations of the superficial superior colliculus. The present study found bundles or “nests” of immature cells throughout the superficial superior colliculus at the time of birth. Similar observations of developing cells in the central nervous system have been reported (7, lS, 31). During development of the superior colliculus in rat pups from 5 to 31 days old, the presence of (i) marginal cells, (ii) horizontal cells, (iii ) piriform cells, (iv) narrow-field vertical cells, (v) wide-field vertica1 cells, and (vi j stellate cells concurs with similar findings of all these cell populations in the young adult rat (8). Using the Golgi method, Tokunaga and Otani (27) classified cell types in the superficial superior colliculus. They distinguished five different neurons which correspond closely with cell populations identified in the present investigation. They are “the horizontal type,” which corresponds to the horizontal cells of the present study; “the cylindrical type with dorsoventrally oriented dendrites,” which corresponds to the narrow-field vertical cells ; “the cylindrical neurons with dorsally oriented dendrites,” which correspond to piriform cells ; “the reversed conical types,” which correspond to the wide-field vertical cells ; and “the multipolar type” which corresponds to the stellate cell. A number of studies, all in adult animals, do not report the presence of the following cells: marginal cells (Z-l, -37, 30, 31) ; horizontal cells (24) ; and narrow-field vertical cells (30:)
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In a light and electron microscopic study, three classes of neurons in the more superficial superior colliculus (i.e., the stratum zonale and stratum griseum superficiale) in the adult chimpanzee have been reported (26). Two types correspond most closely with neurons identified in the present study. They are “marginal cells” in the stratum zonale, which correspond to marginal cells of the present investigation, and “presynaptic dendrite cells” in the stratum griseum superficiale, which correspond to the horizontal cells. The third class of neurons, the “collicular relay cells” are situated in the stratum griseum superficiale. These cells project primary dendrites toward the collicular surface and/or the stratum opticum, or they extend dendritic branches parallel with or oblique to the collicular surface. This group of cells may correspond to piriform cells and narrowand wide-field vertical cells. More specific correlation is limited as a further ultrastructural subclassification of “collicular relay cells” is not available. Ultrastructural observations (26) have not only verified the light microscopic description of marginal and horizontal cells [present investigation and (8) ] but, in addition, have presented ultrastructural criteria for the identification of presynaptic denclritic cells (i.e., the horizontal cells). Such correlative data between light and electron microscopic studies are invaluable in assessing the functional role of cell populations. To date, collicular recordings on neonatal rat pups from birth to 31 days postnatal have not been reported. The present study has attempted to establish an anatomic substrate for unit studies of the developing superior colliculus in order to broaden our knowledge of collicular physiology. The variety of spines (stubby, mushroom, and ball-on-stalk) are well defined in all cell populations of the superficial superior colliculus from 33 to 31 days postnatal. This observed spine development may be correlated with the marked wave of intrinsic axon synapse observed from day 25 to between days 30 and 40. Growth cones, spicules, and varicosities along dendritic shafts are well established as major morphologic characteristics of immature dendritic growth processes (13-15, 19). The overwhelming abundance of these features present in all cell populations of the superficial superior colliculus from 6 to 8 days postnatal may represent a step in dendritic branching and elongation. What appears to be a disappearance of these early denand spicules) may in dritic characteristics (growth cones, varicosities, fact have been the result of two concomitant processes : (i) the progression of some of these structures into the more mature dendritic branches observed by days 9 to 10 and (ii) the regression of other structures into a totally retracted state. On the basis of light and electron microscopic findings, the same case in point has been reported (4) : “varicose den-
drites and beaded processes” were observed lo emanate from early inner marginal neurons of the developing trigeminal nucleus caudalis in newborn kittens, followed by lengthening of dendrites and a decrease in the number of ‘thin, short dendrites’ and ‘reduction in the number of beaded processes’. From ultrastructural evidence, it was concluded that the retraction of those esisting neural structures is linked with the postnatal differentiation of dendrites. In light of the findings from deprivation experiments involving cell populations of the visual cortex (3, 5, 25, 29), it is not unreasonable to expect “sensitive” or “critical” periods of cell development in the superior colliculus also. The present study has revealed that ontogenetic events in emerging cell populations of the superior colliculus can be identified with discrete time periods between birth and 31 days postnatal, suggesting possible stages of vulnerability. To date, no morphological studies, at least in the albino rat, have attempted to relate the effects produced by light deprivation or deafferentation with abnormalities in cell development in the superior colliculus. The present investigation provides a background for exploring the relative sensitivity of different stages of collicular development and for evaluating the theory of a “critical period” in the cytological development of the superior colliculus. REFERENCES 1.
J. 1962. Some fiber projections to the superior colliculus in the cat. J. Camp. Ncwrol. 119 : 77-95. 2. BRUCKNER, G., MARES, V., AND BIESOLD, D. 1976. Neurogenesis in the visual system of the rat. An autoradiographic investigation. J. Camp. Ncurol. 166: 245-256. 3. COLEMAN, P. D., AND RIESAN, A. H. 1968. Environmental effects on cortical ALTMAN,
dendritic fields. 1. Rearing in the dark. J. .4nat. 102 : 363-375. 4. FALLS, W., AND GOBEL, S. 1976. A Golgi and EM study of the marginal and substantia gelatinosa layers of trigeminal nucleus caudalis in newborn kittens. Amt. Rec. 184 : 399. 5. GLOBUS, A., AND SCHEIBEL, A. B. 1967. The effect of visual deprivation on cortical neurons: A Golgi study. Exp. Ncurol. 19: 331-345. 6. INGLE, D., AND SCHNEIDER, G. E., Eds. 1970. Sz~bcortical ~‘isual Sq’stcws. Brain Behav. Evol. 3: 1-352. 7. LAEMLE, L. K. 1974. The development of the human visual system. I. The visual cortex. Awt. Kcc. 178: 449. 8. LANCER, T. P., AND LUND, R. D. 1974. The upper layers of the superior colliculus of the rat : A Golgi study. J. Cowtp. New-ol. 158 : 405-436. 9. LUND, R. D. 1969. Synaptic patterns of the superficial layers of the superior colliculus of the rat. J. Cowp. Neural. 135: 179-208. 10. LUND, R. D., AND LUND, J. S. 1972. Development of synaptic patterns in the superior colliculus of the rat. Brain RES. 42: l-20. 11. MCILWAIN, J. T., AND BUSER, P., 1968. Receptive fields of single cells in the cat’s superior colliculus. Exp. Brain Rcs. 5 : 314-325. 12. MICHAEL, C. R. 1967. Integration of visual information in the superior colliculus. J. GEL Physiol. 50 : 2584-2586.
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13. Mokes,r, 1). K. 1969. The differentiation of cerebral dendrites : A study of the postmigratory nemoblast in the medial nucleus of the trapezoid body. Z. Amzf. E~~t”Le~icklz~lrgs,~csch. 128 : 271-289. 14. MOHEST, D. K. 1969a. The growth of dendrites in the mammalian brain. Z.Annt. E,1tzc,icklzlrlgsgescR. 128 : 290-317. 15. MOREST, D. K. 1970. A study of neurogenesis in the forebrain of opossum pouch young. 2. -4 wt. Elrtzoicklzrlrgsgcsrk. 130 : 265-305. 16. NAUTA, W. J. H., AND VAN STRAATEN, J. J. 1947. The primary optic centers of the rat, an experimental study by the “bouton” method. /. Anat. 81 : 127-134. 17. PETERS, A., AND KAISERI\ZAN-ABRAMOF, I. R. 1975. The small pyramidal neurons of the rat cerebral cortex. The perikaryon, dendrites, and spines. J. Anat. 127: 321-356. 18. PETERS, A., AND WALSH, T. M. 1972. A study of the organization of apical dendrites in the somatic sensory cortex of the rat. J. Camp. NEZWOZ. 144: 253-268. 19. PURPURA, D. P. 1975. Normal and aberrant neuronal development in the cerebral cortex of human fetus and young infant. Pages 141-169 in N. BUCHWALD AND M. A. B. BRAZIER, Eds., Mechaxisms in Mental Retardation. Academic Press, New York. 20. RAEDLER, A., AND SIEVERS, J. 1975. The development of the visual system of the albino rat. AdzI. Alzat. Embryol. Crll Biol. 50, Fast. 3. 21. SCHEIBEL, M. E., AND SCHEIBEL, A. B. 1970. bz K. L. CHOW AND A. L. LEIMAN, Eds., The Structural and Fzrrlctional Orgaxization of the Neocortcx. Ncz~rosci. Rcs. Prog. Bull. 8, Vol. 2. MIT Press, Cambridge, Massachusettes. 22. SIMINOFF, R., SCHWASSMANN, H. O., AND KRUGER, L. 1966. An electrophysiological study of tile visual projection to the superior colliculus of the rat. J. Comp. Ncwol. 127: 435-444. 23. SPRAGUE, J. M., BERLUCCHI, G., AND RIZZOLATTI, G. 1973. The role of the superior colliculus and pretectum in vision and visually guided behavior. Pages 27-101 ijt R. JUNG, Ed., Handbook of S~?lsory Physiology, VII, 3, Central Visual Information, B. Springer-V
