Postnatal Maturation of Neurons in the Rabbit Superior Colliculus LAWHENCE H. MATHERS, JR. Department of'Structurul Biology, School of Medicine, Stunfwd University, Stanford, California 94305
The superior colliculi of Dutch-belted rabbits aged 1 to 30 days ABSTRACT were prepared according to Nissl and Golgi techniques and examined in comparison to the neuronal populations in the adult. It was found that the laminar dimensions of the adult are attained at 15 to 20 days, presumably due to glial and dendritic enlargement. The largest neuron in the superficial gray, the vertical cell, was found to mature earliest and to display the least evidence of dendritic development postnatally. Its dendrites do elongate from 0 to 15 days, but there are no growth cones or other unusual dendritic configurations. The intrinsic neurons of the superficial gray, the piriform and stellate cells show more evidence of postnatal dendritic development. Particularly in the case of stellate cells, there are dendritic growth cones present at birth, and early in development spines are found on the soma and large dendrites. Only at 15 to 20 days do the dendritic arbors of these cells assume normal adult shapes and sizes. These and other observations suggest that there are two phases of postnatal cellular maturation in the upper layers of the superior colliculus, one involving the smaller "interneurons." This phased maturation is compared with the known sequence of physiological maturation as studied by the method of receptive-field mapping.
Golgi studies of neuronal development in many areas of the mammalian nervous system, particularly the cerebral cortex (Marin-Padilla, '70; Scheibel and Scheibel, '71; Purpura, '71) have established the high degree of maturation which follows birth. It has been shown that artificial manipulations of the environment (Coleman and Riesen, '68; Valverde, '67; Pettigrew and Garey, '74) or, more drastically, removal of the receptor (Valverde, '68, '71) can affect the normal maturational sequence for neurons in the visual system. Much of this work has centered on neurons in the visual cortex of rodents, where visual deprivation or enucleation can be shown to have very specific effects on given portions of a neuron's morphology. Presented here is a description of the sequence of normal neuronal maturation in the superior colliculus of the rabbit. The development of neurons in the superior colliculus has not been studied to the extent which has the J. COMP. NEuR.. 173: 439.456.
striate cortex, although Tsang ('37), Lund and Lund ('71), and Mathers ('76a) have addressed the question of the effect of enucleation upon the growth of neurons in the superior colliculus. It is commonly understood that maturation of neurons in the superior colliculus is complete earlier than is maturation of neurons in the striate cortex. (Jacobsen, '30; Tsang, '37). However, as this paper shows, there is a considerable period of postnatal maturation in the superior colliculus as well, and its potential susceptibility to light deprivation cannot be ruled out. It is already known that infant enucleation does lead to disruption of normal growth in the superior colliculus (Tsang, '37; Mathers, '76a). Also previous physiological studies have revealed postnatal maturation of visual receptive fields in superior collicular cells of the rabbit (Spear et al., '72) and cat (Norton, '72; Stein et al., '73). While these changes are not as extensive as those in the
439
LAWRENCE H. MATHERS. JK.
440
visual cortex, they are nonetheless significant. The findings presented here are meant to form a basis for correlation of morphologic changes with those already described physiologically, and to complement ultrastructural studies of superior colliculus maturation (Mathers and Mercer, ’77). MATEHIALS AND METHODS
Tissues used in this study were taken from 28 rabbit pups aged 1, 5, 10, 15, 20, and 30 days. There was also available a series of Golgi-stained brains from adult rabbits. The young rabbits were raised in the laboratory. Nests were examined each day at 8:OO A.M., and since rabbit pups are usually born during the night, the first morning on which pups were observed was designated day 0. Animals of appropriate ages were sacrificed with an overdose of sodium pentobarbital. The brains were perfused with 10% buffered formalin and quickly removed. They were sliced to a suitable size, usually in the coronal plane, and placed in a 3.5% K2Cr2O7-0.2YoOso+ After several days in this solution they were transferred to 0.75% AgNo, for three to six days. They were then dehydrated and embedded in celloidin. Sections at 75-100 p were then cut and mounted in Permount. Drawings of selected neurons were made using a Zeiss drawing tube, and others were photographed. RESULTS
A. Normal ad& morphology Before proceeding to a description of the development undergone by the various cell types in the rabbit superior colliculus, it is necessary to report on the adult condition. The neuronal populations found in the rabbit superior colliculus are quite similar to those described by Langer and Lund (’74) and Valverde (’73),and much of their terminology has been adopted here. A more complete description of the rabbit superior colliculus is given in a separate publication (,Mathers, ’77). The largest cells found in the upper
layers of the superior colliculus are the vertical cells (fig. 2a), whose axons invariably leave the stratum griseum (SGS) and penetrate the stratum opticum (SO). The cell bodies of these neurons range from 122 0 p in diameter. As a general rule, the vertical cells lying deepest in the SGS have the largest cell bodies. The axon very commonly arises from a proximal dendrite and courses to the deep collicular layers with few or no branches. The dendrites of these neurons are also somewhat variable in their shape, but there is always a group of two to three stout branches coursing upward toward the surface of the superior colliculus. These branches, most commonly two in number, form an angle which is largest for those vertical cells lying deepest in the SGS, and smallest for those nearest the surface. In the case of the deeper-lying neurons, the “cylinder” of tissue in the SGS demarcated by these dendrites and their branches may exceed 4 0 0 p in diameter. The more superficial branches of these dendrites display a large number of knob-like protrusions, whereas the deeper-lying shaft portions of these dendrites have many fewer protrusions. There is occasionally a smaller dendritic ramification near the cell body, but this is rare. The majority of the dendritic arborization is seen within the upper 200 p of tissue in the superior colliculus. All the remaining neurons found in the superficial layers of the adult rabbit superior colliculus have axons which ramify locally, rather than traveling over relatively long distances. Near the surface of the colliculus is found a group of horizontal cells (fig. 3a) whose dendrites course for several hundred microns laterally less than 150 p below the surface of the colliculus. The dendrites are for the most part free of spines except for their near terminal portions. The axon, which has only rarely been stained, is found to arborize in the vicinity of the cell body. These neurons are of moderate size (10-14p diameter) and the shape of the soma is usually ovoid. A third major category of neurons is the stellute cells (fig. 4a), which are found
POSTNATAL COLLICULAH MAlURATION
throughout the depths of the superficial collicular layers and which show more variability in their shape than do the vertical cells or horizontal cells. These cells are small to medium in their soma1 dimensions (7-12p). Their dendritic arbors are all local and rarely extend beyond 100-150p from the soma. Those stellate cells which lie near the surface of the colliculus have dendritic trees which tend to be dispersed below the level of the soma, while those stellate cells whose cell bodies are deeper in the SGS have a radial dendritic arbor. The dendrites are heavily invested with spines in all but their near proximal segments. As with horizontal cells, the axons of stellate cells are local, and none has been seen to travel more than 200p from the soma. As numerous as the stellate cells are the piriform cells (fig. 8e), which form the fourth major category of neurons in the upper collicular layers. Piriform cells are found throughout the SGS, and cell bodies are usually 1 2 - 1 8 ~in diameter. The cell body is ovoid in the dorsoventral axis, and a large dendrite originates from both the dorsal and ventral side of the cell body. These dendrites usually give off two to four long branches which run in either direction away from the cell body. There are numerous smaller terminal branches, particularly in the upper 3 0 p of the SGS. Axons are infrequently stained well, but generally arise from the ventral dendrite and ramify locally. Piriform cells may be distinguished from vertical cells on the basis of the large ventral dendrite, and the fact that axon5 remain within the upper collicular layers.
13. Overall features of development At the time of birth, there are several distinctive features seen in the superior colliculus. First, the laminae of the superficial half of the colliculus are poorly delineated with cell stains (figs. la-d). With the Golgi technique, a stratum can often be distinguished as a lightly stained band of axons running principally in the mediolatera1 axis. This is taken to be the stratum opticum (SO), and in newborn rabbits it is
441
about 400p from the surface. In adulthood, this fiber stratum begins about 750800 p below the surface. In the newborn rabbits, some preparations have revealed the presence of very long, thin argyrophilic processes extending all the way from the lumen of the cerebral aqueduct to the pial surface of the superior colliculus (fig. 6a). These have not been stained in any animals five days postnatal or older. It would seem that these represent non-neuronal processes similar to those identified in the cerebral isocortex by Rakic ('711, and that migrating neurons may have used these to move to their appropriate positions. Work is currently being pursued to clarify this point. In favorable preparations it is often possible to see the cell bodies of these nonneuronal processes, and most of them which have been visualized lie near the surface of the cerebral aqueduct, or within 400-700 p of its lumen. In addition to the processes described in the preceding paragraph, there is from birth onward an increasingly dense population of axons running in a radial direction from the SO upward into the stratum griseum superficiale (SGS). These are thought to be the terminal branches of optic axons penetrating the SGS. They show an increasingly complex pattern of termination throughout the age sequence studied here, and by 15-20 days the number of stained end-terminal branches is more than double that seen at birth.
C . The vertical cell The vertical cell is quite mature at birth in comparison to other collicular neuron types. In the newborn, the cell bodies of these neurons are located most commonly 100-500p below the surface. The cell body is 7-12p in diameter, which is 60-70% of the average size in the adult. As in the adult, the dorsal surface of the neuron usually has two to three large dendrites coursing upward. However, unlike the adult, where dendrites run upward for 100-300p and branch extensively only in the upper 200 /.L, the terminal branches of these den-
442
LAWRENCE H. MATHERS. JR
drites in the newborn begin to appear 75100 p from the soma, and only rarely can a branch be traced to the surface (figs. 2b, 5a). The dendrites are relatively smooth proximally but heavily invested with spines in their terminal branches. The axon penetrates the stratum opticum and could be traced no further. It is difficult to state that the terminal branches of the dorsal dendrites of these vertical cells absolutely do not reach the surface of the colliculus; it can only be reported that they do not stain in these preparations. Another notable feature of this early developmental stage is the presence of spines on both the proximal dendrites, and occasionally on the soma itself. As development proceeds, the vertical cell soma becomes larger until it reaches its adult dimensions by the fifteenth postnatal day (figs. 5b,c). Between the day of birth and the fifteenth day, the number of spines on proximal dendrites and soma is markedly reduced, and the proximal dendritic shafts become wider and smoother. The axon is more easily impregnated during the 1- to 15-day age period than afterward, doubtless due to the myelination which begins on about the fifteenth day. From the outset, in well-stained neurons, it can be seen to penetrate the stratum opticum and enter the stratum griseum intermediale. The spines found on distal and terminal dendritic branches do not seem to undergo great alteration over the developmental period surveyed here. Because there is a greater number of distal dendritic branches in more mature animals, the total dendritic arbor of a given vertical cell does become more elaborate. But the density and morphology of spines on these branches do not seem to change significantly through the period studied. In the preparations studied here, it was much more common to find cell bodies of small vertical cells within 100-500p of the surface than is true in the adult (fig. 2b). These neurons usually had no more than two dendritic shafts coursing upward for 50-70p, where they branched into a few short extensions which rapidly lost their
stain. This was in contrast to the case of the vertical cells found at 300-500p , where dendritic development was more extensive, though still far short of that in the adult. It would appear that the more deeply-placed vertical cells are more highly developed than their cousins found nearer the surface at this stage. Presumably the more superficially placed vertical cells come to lie more deeply in the adult colliculus as a result of growth in the superficial neuropil.
D. The piriform cell Piriform cells are found in newborn rabbits, but their morphology is significantly different than that of the piriform cells of the adult. In the 1-day rabbits, piriform cells typically have soma 3-8 p in diameter and two groups of dendritic elaborations at the dorsal and ventral aspect (with respect to the surface). There is a bushy collection of dendrites at these two surfaces, with 3-8 larger branches extending dorsally and ventrally (fig. 4b). These dendritic growths extend no more than 25-3Op from the soma and then stop. At this point some very small dendritic twigs (
The superior colliculi of Dutch-belted rabbits aged 1 to 30 days ABSTRACT were prepared according to Nissl and Golgi techniques and examined in comparison to the neuronal populations in the adult. It was found that the laminar dimensions of the adult are attained at 15 to 20 days, presumably due to glial and dendritic enlargement. The largest neuron in the superficial gray, the vertical cell, was found to mature earliest and to display the least evidence of dendritic development postnatally. Its dendrites do elongate from 0 to 15 days, but there are no growth cones or other unusual dendritic configurations. The intrinsic neurons of the superficial gray, the piriform and stellate cells show more evidence of postnatal dendritic development. Particularly in the case of stellate cells, there are dendritic growth cones present at birth, and early in development spines are found on the soma and large dendrites. Only at 15 to 20 days do the dendritic arbors of these cells assume normal adult shapes and sizes. These and other observations suggest that there are two phases of postnatal cellular maturation in the upper layers of the superior colliculus, one involving the smaller "interneurons." This phased maturation is compared with the known sequence of physiological maturation as studied by the method of receptive-field mapping.
Golgi studies of neuronal development in many areas of the mammalian nervous system, particularly the cerebral cortex (Marin-Padilla, '70; Scheibel and Scheibel, '71; Purpura, '71) have established the high degree of maturation which follows birth. It has been shown that artificial manipulations of the environment (Coleman and Riesen, '68; Valverde, '67; Pettigrew and Garey, '74) or, more drastically, removal of the receptor (Valverde, '68, '71) can affect the normal maturational sequence for neurons in the visual system. Much of this work has centered on neurons in the visual cortex of rodents, where visual deprivation or enucleation can be shown to have very specific effects on given portions of a neuron's morphology. Presented here is a description of the sequence of normal neuronal maturation in the superior colliculus of the rabbit. The development of neurons in the superior colliculus has not been studied to the extent which has the J. COMP. NEuR.. 173: 439.456.
striate cortex, although Tsang ('37), Lund and Lund ('71), and Mathers ('76a) have addressed the question of the effect of enucleation upon the growth of neurons in the superior colliculus. It is commonly understood that maturation of neurons in the superior colliculus is complete earlier than is maturation of neurons in the striate cortex. (Jacobsen, '30; Tsang, '37). However, as this paper shows, there is a considerable period of postnatal maturation in the superior colliculus as well, and its potential susceptibility to light deprivation cannot be ruled out. It is already known that infant enucleation does lead to disruption of normal growth in the superior colliculus (Tsang, '37; Mathers, '76a). Also previous physiological studies have revealed postnatal maturation of visual receptive fields in superior collicular cells of the rabbit (Spear et al., '72) and cat (Norton, '72; Stein et al., '73). While these changes are not as extensive as those in the
439
LAWRENCE H. MATHERS. JK.
440
visual cortex, they are nonetheless significant. The findings presented here are meant to form a basis for correlation of morphologic changes with those already described physiologically, and to complement ultrastructural studies of superior colliculus maturation (Mathers and Mercer, ’77). MATEHIALS AND METHODS
Tissues used in this study were taken from 28 rabbit pups aged 1, 5, 10, 15, 20, and 30 days. There was also available a series of Golgi-stained brains from adult rabbits. The young rabbits were raised in the laboratory. Nests were examined each day at 8:OO A.M., and since rabbit pups are usually born during the night, the first morning on which pups were observed was designated day 0. Animals of appropriate ages were sacrificed with an overdose of sodium pentobarbital. The brains were perfused with 10% buffered formalin and quickly removed. They were sliced to a suitable size, usually in the coronal plane, and placed in a 3.5% K2Cr2O7-0.2YoOso+ After several days in this solution they were transferred to 0.75% AgNo, for three to six days. They were then dehydrated and embedded in celloidin. Sections at 75-100 p were then cut and mounted in Permount. Drawings of selected neurons were made using a Zeiss drawing tube, and others were photographed. RESULTS
A. Normal ad& morphology Before proceeding to a description of the development undergone by the various cell types in the rabbit superior colliculus, it is necessary to report on the adult condition. The neuronal populations found in the rabbit superior colliculus are quite similar to those described by Langer and Lund (’74) and Valverde (’73),and much of their terminology has been adopted here. A more complete description of the rabbit superior colliculus is given in a separate publication (,Mathers, ’77). The largest cells found in the upper
layers of the superior colliculus are the vertical cells (fig. 2a), whose axons invariably leave the stratum griseum (SGS) and penetrate the stratum opticum (SO). The cell bodies of these neurons range from 122 0 p in diameter. As a general rule, the vertical cells lying deepest in the SGS have the largest cell bodies. The axon very commonly arises from a proximal dendrite and courses to the deep collicular layers with few or no branches. The dendrites of these neurons are also somewhat variable in their shape, but there is always a group of two to three stout branches coursing upward toward the surface of the superior colliculus. These branches, most commonly two in number, form an angle which is largest for those vertical cells lying deepest in the SGS, and smallest for those nearest the surface. In the case of the deeper-lying neurons, the “cylinder” of tissue in the SGS demarcated by these dendrites and their branches may exceed 4 0 0 p in diameter. The more superficial branches of these dendrites display a large number of knob-like protrusions, whereas the deeper-lying shaft portions of these dendrites have many fewer protrusions. There is occasionally a smaller dendritic ramification near the cell body, but this is rare. The majority of the dendritic arborization is seen within the upper 200 p of tissue in the superior colliculus. All the remaining neurons found in the superficial layers of the adult rabbit superior colliculus have axons which ramify locally, rather than traveling over relatively long distances. Near the surface of the colliculus is found a group of horizontal cells (fig. 3a) whose dendrites course for several hundred microns laterally less than 150 p below the surface of the colliculus. The dendrites are for the most part free of spines except for their near terminal portions. The axon, which has only rarely been stained, is found to arborize in the vicinity of the cell body. These neurons are of moderate size (10-14p diameter) and the shape of the soma is usually ovoid. A third major category of neurons is the stellute cells (fig. 4a), which are found
POSTNATAL COLLICULAH MAlURATION
throughout the depths of the superficial collicular layers and which show more variability in their shape than do the vertical cells or horizontal cells. These cells are small to medium in their soma1 dimensions (7-12p). Their dendritic arbors are all local and rarely extend beyond 100-150p from the soma. Those stellate cells which lie near the surface of the colliculus have dendritic trees which tend to be dispersed below the level of the soma, while those stellate cells whose cell bodies are deeper in the SGS have a radial dendritic arbor. The dendrites are heavily invested with spines in all but their near proximal segments. As with horizontal cells, the axons of stellate cells are local, and none has been seen to travel more than 200p from the soma. As numerous as the stellate cells are the piriform cells (fig. 8e), which form the fourth major category of neurons in the upper collicular layers. Piriform cells are found throughout the SGS, and cell bodies are usually 1 2 - 1 8 ~in diameter. The cell body is ovoid in the dorsoventral axis, and a large dendrite originates from both the dorsal and ventral side of the cell body. These dendrites usually give off two to four long branches which run in either direction away from the cell body. There are numerous smaller terminal branches, particularly in the upper 3 0 p of the SGS. Axons are infrequently stained well, but generally arise from the ventral dendrite and ramify locally. Piriform cells may be distinguished from vertical cells on the basis of the large ventral dendrite, and the fact that axon5 remain within the upper collicular layers.
13. Overall features of development At the time of birth, there are several distinctive features seen in the superior colliculus. First, the laminae of the superficial half of the colliculus are poorly delineated with cell stains (figs. la-d). With the Golgi technique, a stratum can often be distinguished as a lightly stained band of axons running principally in the mediolatera1 axis. This is taken to be the stratum opticum (SO), and in newborn rabbits it is
441
about 400p from the surface. In adulthood, this fiber stratum begins about 750800 p below the surface. In the newborn rabbits, some preparations have revealed the presence of very long, thin argyrophilic processes extending all the way from the lumen of the cerebral aqueduct to the pial surface of the superior colliculus (fig. 6a). These have not been stained in any animals five days postnatal or older. It would seem that these represent non-neuronal processes similar to those identified in the cerebral isocortex by Rakic ('711, and that migrating neurons may have used these to move to their appropriate positions. Work is currently being pursued to clarify this point. In favorable preparations it is often possible to see the cell bodies of these nonneuronal processes, and most of them which have been visualized lie near the surface of the cerebral aqueduct, or within 400-700 p of its lumen. In addition to the processes described in the preceding paragraph, there is from birth onward an increasingly dense population of axons running in a radial direction from the SO upward into the stratum griseum superficiale (SGS). These are thought to be the terminal branches of optic axons penetrating the SGS. They show an increasingly complex pattern of termination throughout the age sequence studied here, and by 15-20 days the number of stained end-terminal branches is more than double that seen at birth.
C . The vertical cell The vertical cell is quite mature at birth in comparison to other collicular neuron types. In the newborn, the cell bodies of these neurons are located most commonly 100-500p below the surface. The cell body is 7-12p in diameter, which is 60-70% of the average size in the adult. As in the adult, the dorsal surface of the neuron usually has two to three large dendrites coursing upward. However, unlike the adult, where dendrites run upward for 100-300p and branch extensively only in the upper 200 /.L, the terminal branches of these den-
442
LAWRENCE H. MATHERS. JR
drites in the newborn begin to appear 75100 p from the soma, and only rarely can a branch be traced to the surface (figs. 2b, 5a). The dendrites are relatively smooth proximally but heavily invested with spines in their terminal branches. The axon penetrates the stratum opticum and could be traced no further. It is difficult to state that the terminal branches of the dorsal dendrites of these vertical cells absolutely do not reach the surface of the colliculus; it can only be reported that they do not stain in these preparations. Another notable feature of this early developmental stage is the presence of spines on both the proximal dendrites, and occasionally on the soma itself. As development proceeds, the vertical cell soma becomes larger until it reaches its adult dimensions by the fifteenth postnatal day (figs. 5b,c). Between the day of birth and the fifteenth day, the number of spines on proximal dendrites and soma is markedly reduced, and the proximal dendritic shafts become wider and smoother. The axon is more easily impregnated during the 1- to 15-day age period than afterward, doubtless due to the myelination which begins on about the fifteenth day. From the outset, in well-stained neurons, it can be seen to penetrate the stratum opticum and enter the stratum griseum intermediale. The spines found on distal and terminal dendritic branches do not seem to undergo great alteration over the developmental period surveyed here. Because there is a greater number of distal dendritic branches in more mature animals, the total dendritic arbor of a given vertical cell does become more elaborate. But the density and morphology of spines on these branches do not seem to change significantly through the period studied. In the preparations studied here, it was much more common to find cell bodies of small vertical cells within 100-500p of the surface than is true in the adult (fig. 2b). These neurons usually had no more than two dendritic shafts coursing upward for 50-70p, where they branched into a few short extensions which rapidly lost their
stain. This was in contrast to the case of the vertical cells found at 300-500p , where dendritic development was more extensive, though still far short of that in the adult. It would appear that the more deeply-placed vertical cells are more highly developed than their cousins found nearer the surface at this stage. Presumably the more superficially placed vertical cells come to lie more deeply in the adult colliculus as a result of growth in the superficial neuropil.
D. The piriform cell Piriform cells are found in newborn rabbits, but their morphology is significantly different than that of the piriform cells of the adult. In the 1-day rabbits, piriform cells typically have soma 3-8 p in diameter and two groups of dendritic elaborations at the dorsal and ventral aspect (with respect to the surface). There is a bushy collection of dendrites at these two surfaces, with 3-8 larger branches extending dorsally and ventrally (fig. 4b). These dendritic growths extend no more than 25-3Op from the soma and then stop. At this point some very small dendritic twigs (
