Anat. Embryo1.155, 1-14 (1978)
Anatomy and Embryology 9 by Springer-Verlag 1978
The Development of Non-Pyramidal Neurons in the Visual Cortex of the Rat J.G. Parnavelas*, R. Bradford, E.J. Mounty, and A.R. Lieberman Department of Anatomyand Embryology,UniversityCollegeLondon,GowerStreet, LondonWC1E 6BT, England
Summary. We have studied the maturation of non-pyramidal cells in layers IIVI of the visual cortex of albino rats from birth to maturity, using Golgi-Cox and rapid Golgi preparations. At birth, non-pyramidal cells are sparse, immature and concentrated in the deep part of the cortical plate: their number increases towards the end of the first week but they remain sparse and immature in the upper part of the cortical plate. During the second postnatal week, the number, size and extent of dendritic and axonal branching of these cells undergo considerable increases and the cells become conspicuous in layer IV and apparent in the supragranular layers: this 'growth spurt' occurs just after (and may be related to) the arrival and establishment in the cortex during the second half of the first postnatal week, of extrinsic afferents. During the third postnatal week, most of the cells complete their maturation. At the end of this week, the number of spinous cells is greater and the spine density of some cells is higher than in the adult, falling to adult values during the fourth postnatal week. It is noteworthy that the non-pyramidal cells appear to reach maturity at about the same time in all the layers studied, and at the same time as the pyramidal cells with which they are associated. These observations are not in accord with the prevalent view that non-pyramidal cells complete their differentiation much later than pyramidal cells. Key words: Visual cortex - Non-pyramidal neurons - Development - Rat. Studies of neuronal differentiation in the cerebral cortex have been concerned predominantly with pyramidal cells (Lorente de N6, 1933; Eayrs and Goodhead, 1959; Cajal, 1960; Noback and Purpura, 1961; Astr6m, 1967; Berry, 1974; Frotscher, 1975; Lund et al., 1977). Since these cells have a characteristic and consistent morphology, which can be recognized even when they are very immature, it is not surprising that they should have been the main focus of such * Present address and address for reprints." Dr. J.G. Parnavelas, Departmentof Cell Biology,The Universityof Texas, Health ScienceCenter at Dallas, Dallas, Texas 75235, U.S.A.
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studies. The differentiation of the vastly more heterogeneous population of nonpyramidal cells has received much less attention, although recently Lund et al., (1977) have provided a detailed description of the development of non-pyramidal cells in layer IV of the monkey visual cortex. In recent years, however, increasing attention has been directed towards the functional organization of non-pyramidal cells in adult cerebral cortex, especially in the visual cortex (e.g. Kelly and Van Essen, 1974), and there has been interesting speculation about their evolutionary and developmental significance (e.g. Altman, 1967; Jacobson, 1974, 1975). Such studies and speculations have emphasized the importance of obtaining accurate data on the morphology and synaptic relationships of non-pyramidal cells in the adult, as well as on their genesis, their differentiation, and the development of their synaptic connections. In previous studies of the visual cortex of adult rats, we described the morphology of Golgi-impregnated non-pyramidal cells (Parnavelas et al., 1977a) and their ultrastructure and synaptic relations (Parnavelas et al., 1977b). We have also examined the postnatal development of non-pyramidal cells, including RetziusCajal cells in layer I of the rat visual cortex (Bradford et al., 1977) and in the present study we describe the postnatal development of Golgi-impregnated non pyramidal cells by light microscopy, in layers II-VI of the visual cortex, and relate the findings to others concerning the development of pyramidal cells and of afferent fibres to the cortex.
Materials and Methods
Brains from a minimum of 10 female albino rats at each of the following ages were used: 12h, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 18, 20, 24, 28 and 35 days, and 3 months (youngadults). Nissl, GolgiCox and rapid Golgi procedureswere utilized as previouslydescribed(Parnavelasand Globus, 1976; Bradford et al., 1977; Parnavelas et al., 1977c).The Nissl-stained material was used to determinethe extentand positionin relationto the corticalsurface,of the corticallayersat each stage of development studied, and to assign to appropriatepositionsthosecellswhoselaminar positionwas not clearlydefined in the Golgi material. The analysis of the Golgi material was qualitative and was based on careful observations of several thousand developingand mature non-pyramidal neurons and camera lucida drawings of more than one thousand.
Results
First Postnatal Week All cortical neurons in the rat are generated before birth, but many do not attain their final positions until several days after birth (Berry and Rogers, 1965; Berry, 1974; Lund and Mustari, 1977), and the laminar organization characteristic of the adult cortex is not apparent in newborn animals. Only layers V and VI are clearly delineated by the 4th postnatal day in Nissl preparations, while the rest of the cortical plate appears as a homogeneous population of densely stained cells. The laminar differentiation of this portion of the cortical plate is achieved by the 6th day.
Development of Rat Visual Cortex
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d \
g \
/'h 50pm Fig. la-i. Camera lucida drawings, postnatal days 0-6. a Day 0, layer VI; b Day 0, from close to the middle of the cortical plate (presumptive layer IV); c Day 2, middle of cortical plate; d Day 2, layer VI; e Day 4, layer V; fDay 4, middle of cortical plate; g Day 4, layer V; h Day 6, layer IV; i Day 6, layer III
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I f
C
5oLm /
Fig. 2 all. Camera lucida drawings, postnatal days 8-15. a Day 8, layer IV; b Day 11, layer III; e Day 10, layer IV; d Day 8, layer V; e Day 15, layer IV; f Day 10, layer V; g Day 52, layer IV
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Non-pyramidal cells are sparse in the 12h animals and are present
predominantly in the deeper part of the cortex (Fig. I a, b). At this stage and for a few days thereafter, the perikarya of these cells are small and sometimes irregular in shape. Their dendrites are thin, short and beaded (Fig. 4a) and their axons course only for short distances; collaterals arise from the axons of a few cells as early as day 2 (Fig. 1 c, d). Signs of rapid growth (i.e. irregular enlargements, growth cones, filopodia; see M orest, 1969) are present on the perikarya, dendrites and axons of all neurons (Figs. 1,4a-c). The number of stained non-pyramidal cells increases considerably towards the end of the first week but they are still confined mainly to layers V and VI. At this stage, their dendrites are more branched, thicker and longer (Fig. 1 g-i, Fig. 4c). Growth cones and irregular enlargements are still recognizable on dendrites, although they are not as conspicuous as in younger rats. The axons of most cells are directed towards the pial surface and some give rise to fine terminal arborizations. It appears that most of the non-pyramidal neurons situated in the deeper layers are in a more advanced stage of differentiation than those situated more superficially. Second Postnatal Week
The number of clearly defined non-pyramidal neurons increases markedly and these cells can be identified as a distinctive population, particularly in layer IV, between days 8 and 10. The cell perikarya are larger than at the previous stages and continue to grow with time, as do the dendrites, which increase their branching and thickness and now resemble those of mature neurons (Fig. 2; Fig. 4a-f). Their axons display an elaborate arrangement of collaterals, many of which turn back and ramify amongst the dendrites and perikarya of the parent cells. The number of neurons possessing spinous dendrites and the density of spines increase towards the end of the second postnatal week (Fig. 2c, f,g; Fig. 4e; Fig. 5a). Some nonpyramidal cells are observed in which the dendrites directed towards the white matter bear spines while those directed towards the pial surface are beaded (Fig. 2 f). Finally, the gradient of maturation from deep to superficial layers seen during the first week of postnatal life is not apparent and there are virtually no growth cones present on the dendrites of any cells by the end of the second week. Third Postnatal Week
There are few differences in the appearance of non-pyramidal neurons between the end of the second and the third postnatal week, except that their dendrites are slightly longer and thicker and resemble those of mature non-pyramidal neurons (Fig. 3 and 5). The axons of many cells become more elaborate and appear to achieve full development at this stage (Fig. 2c; Fig. 5b). However, it is extremely difficult to comment with certainty on the extent of axonal ramifications based on Golgi preparations, especially Golgi Cox material, in which large portions, if not the entire axonal plexus of individual neurons, are often not impregnated. One final feature, particularly prominent in the latter half of this developmental stage, is the proportionally higher number of non-pyramidal neurons of the spinous variety present in all cortical layers. The spine density of some of these cells is
t~
lJJ
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Fig. 4a-f. Photomicrographs, postnatal days 0-10, x 450. a Day 0, layer VI; b Day 4, layer V; c Day 6, layer IV; d Day 8, layer II; e Day 8, layer II; f Day 10, layer VI
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Fig. 5a-c. Photomicrographs, postnatal days 14 and 20, x 450. a Day 14, layer IV; b Day 20, layer IV; c Day 20, layer II
Development of Rat Visual Cortex
Fig. 6a-d. Photomicrographs, postnatal day 18 - adult, x 450. a Day 18, layer VI; b Day 24, layer V; c 3 months, layer VI; d 3 months, layer IV
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markedly higher than that of the non-pyramidal neurons of the spinous variety present in adult animals (compare Fig. 3c, e and Fig. 6a to Fig. 3d).
Fourth Postnatal Week
All non-pyramidal neurons appear indistinguishable from their adult counterparts (Fig. 6c, d) by the middle of the fourth week of postnatal life (Fig. 3 a, b, d; Fig. 6b). The proportion of cells of the spinous variety begins to decrease at the end of the third week and falls to the proportion found in the adult during the fourth.
Discussion
It is widely held that cortical non-pyramidal cells complete their differentiation somewhat later than pyramidal cells, and indeed that throughout the nervous system the maturation of Golgi type II cells lags behind that of the Golgi type I cells with which they are associated (Jacobson, 1974, 1975; Glees, 1975; Rakic, 1975a). Observations not wholly consonant with this view are scattered through the literature (e.g. ~str6m, 1967), and a major conclusion of the present study, based on careful examination of a large number of neurons in a closely spaced time series, is that non-pyramidal cells in the visual cortex of the rat do not complete their differentiation significantly later than pyramidal cells. At birth very few nonpyramidal cells can be recognized, chiefly in the deeper part of the cortex: over the next 8-10 days progressively more become recognizable, the most mature (excepting the horizontal cells of layer I; see below) occupying the infragranular layers. Thereafter differences in the state of maturation of non-pyramidal cells at different cortical levels become less evident, and so far as can be deduced from Golgi preparations, the non-pyramidal cells acquire their mature perikaryal size, dendritic morphology and axonal ramification pattern more or less stimultaneously throughout the depth of the cortex and at the same time as our own observations (unpublished) and those of others (Eayrs and Goodhead, 1959; Frotscher, 1975) indicate that the pyramidal cells acquire theirs. Although these conclusions are based on qualitative analysis, quantitative analysis of 700 cells from the same material using a semi-automated dendrite tracking system strongly support them (Uylings et al., 1978a). It may be that detailed ultrastructural and functional studies will reveal aspects of non-pyramidal cell maturation that are not reflected in the Golgi preparations and that are in accord with the prevalent view. It should be said, however, that observations at this relatively gross level at least, do not support the suggestion that non-pyramidal cells are structurally less mature than pyramidal cells in the early postnatal period, and thus more modifiable by environmental, hormonal and metabolic influences (Jacobson, 1974, 1975). It may also be relevant in the context of Jacobson's suggestions concerning the modifiability of non-pyramidal cells, that experiments involving environmental manipulation have produced changes in both non-pyramidal cells and pyramidal cells (Globus and Scheibel, 1967; Volkmar and
Development of Rat Visual Cortex
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Greenough, 1972; Greenough and Volkmar, 1973; Globus et al., 1973; Borges and Berry, 1976; Parnavelas, 1978; Uylings et al., 1978b). With the conspicuous exception of the early-differentiating cells of layer I (Bradford et al., 1977; and see below), non-pyramidal cells of different types and in different cortical layers reach maturity at about the same time. The non-pyramidal cells generated late in the period of neurogenesis must therefore mature at a faster rate than pyramidal cells and other non-pyramidal cells generated earlier. In drawing this conclusion about the relatively rapid rate of differentiation of cells generated late in ontogeny, we do not imply acquiescence with the widely-held view that non-pyramidal cells are generated only after or towards the end of the period of pyramidal cell generation (Angevine, 1970; Berry, 1974; Rakic, 1975 a; Jacobson, 1975). Recent autoradiographic evidence indicates that non-pyramidal cells are produced throughout the period of neurogenesis and that they are added to all levels of the developing cortex throughout the period of its formation (Rickmann et al., 1977). It would appear, therefore, that non-pyramidal cells, or at least some non-pyramidal cells, are incorporated into the cortical anlage according to a developmental program different from that controlling the systematic disposition, layer by layer and from deep to superficial, of pyramidal cells (Angevine and Sidman, 1961; Berry and Rogers, 1965; Hicks and D'Amato, 1968; Berry, 1974; Brfickner et al., 1976; Lund and Mustari, 1977), a conclusion reached earlier by Astr6m (1967) on the basis of Golgi studies of the foetal sheep brain. It is also apparent from the autoradiographic data that the Retzius-Cajal cells, whose precocious development in layer I of the visual and other cortical areas has long been known and puzzled over (see Raedler and Sievers, 1976 and Bradford et al., 1977 for references), are generated very early (on embryonic days 12-14, K6nig et al., 1977; Rickmann et al., 1977), before most of the cells contributing to the cortical plate have been generated (embryonic day 13 until just before birth; Berry 1974; Brtickner et al., 1976; K6nig et al., 1977; Lund and Mustari, 1977). An extremely interesting question concerning the development of the cortex, is when, in relation to critical developmental stages, do extrinsic afferents reach their target neurons? There is now good evidence that the major extrinsic afferent systems to sensory areas of the neocortex in the rat reach their targets at relatively late stages. Thus an organized projection to the visual cortex is established only between days 4-8 postnatal, even though the projection fibres reach the subcortical white matter by embryonic day 18 (Lund and Mustari, 1977). The development of the commissural system follows a similar pattern: callosal axons, although present in the subcortical white matter at birth, only start to enter the cortex on postnatal day 3 and do not show and adult laminar distribution pattern until the end of the first week (Wise and Jones, 1976). Marin-Padilla (1970, 1978), on the other hand, maintains that as a general rule in mammals, fibres from the thalamus enter the cortical rudiment at a very early developmental stage, even before the cortical plate has formed, and that these afferents are responsible for inducing the differentiation of the cortical elements. Marin-Padilla's evidence is based solely upon the examination of Golgi preparations, whereas that of Wise and Jones (1976) and Lund and Mustari (1977) is based upon the more informative and reliable methods of autoradiography and experimental degeneration. Furthermore, the data of the latter authors are in accordance with findings in the monkey visual cortex (Rakic,
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1977) based on studies of the transneuronal transport of labelled tracers injected into the eye. It would seem reasonable, therefore, to accept the important implications of the latter studies, viz. that major aspects of cortical histogenesis particularly the development of the laminar pattern and the differentiation of the pyramidal cells - occur before the arrival of extrinsic inputs and that a major inductive influence of the afferents on these events is improbable. The situation with regard to the differentiation of the non-pyramidal cells, however, is less clear. In our material, the first part of the second postnatal week sees a marked increase in the distinctiveness and maturity of non-pyramidal cells, especially in layer IV and the supragranular layers. It may be, therefore, that the rapid growth and maturation of the non-pyramidal cells is triggered by the arrival in the cortex during the first postnatal week of subcortical and cortieo-cortical afferents. Such a process would be compatible with what is known of the inductive capabilities of afferents in certain sites in the C.N.S. (e.g. M orest, 1969; Berry, 1974; Rakic, 1975b), but would not provide a satisfactory explanation in the case of the Retzius-Cajal cells which mature at a much earlier stage. The acquisition by non-pyramidal cells of dendritic spines follows an interesting pattern, different from that shown by at least some of the pyramidal cells with which they are associated. Spine counts have shown that pyramidal cells of layers IV and V in rat visual cortex first acquire dendritic spines on postnatal day 4, and that spine density increases enormously between postnatal days 8 and 16, reaching adult values by the end of the fourth postnatal week (Parnavelas and Globus, 1976). Spine density in the non-pyramidal cell population, however, is maximal by the middle of the third postnatal week, and subsequently falls to adult values during the fourth week. Moreover, at the time of maximal spine density, the proportion of non-pyramidal cells with spinous dendrites is greater than in the adult. It would appear, therefore, that not only do most spinous non-pyramidal cells become less spinous during the period 3-4 weeks postnatal, but that some cells lose all or most of their spines to fall into the category of spine-free non-pyramidal cells. These results accord with previous studies by LeVay (1973) who found the spine density of non-pyramidal cells of the visual cortex to be higher in kitten than, cat, and by Lund et al., (1977) who found that the maximal spine density of layer IV non-pyramidal cells in the monkey occurred at 8 weeks postnatal and was followed by a period of spine loss. In contrast to the present findings and those of Parnavelas and Globus (1976), however, Lund et al. (1977) found that both non-pyramidal and pyramidal ceils showed a similar pattern with respect to the acquisition of dendritic spines, both showing a peak density at 8 weeks and a subsequent decline towards adult values. Irrespective of possible species differences in spine loss from maturing pyramidal cells, there is now abundant evidence that overproduction with subsequent resorption of supernumerary spines, together with loss or redistribution of the synaptic contacts associated with them, may be a phenomenon of general occurrence and considerable significance in the development of the C.N.S. (e.g. Cajal, 1960; Cragg, 1975; Lund et al., 1977; Ronnevi, 1977).
Acknowledgements. We thank the MedicalResearch Council and the WellcomeTrust for financial
support.
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References Altman, J.: Postnatal growth and differentiation of the mammalian brain, with implications for a morphological theory of memory. In: The neurosciences. (G.C. Quarton, T. Melnechuk, and F.O. Schmitt, eds.) pp. 723-743. New York: Rockefeller University Press 1967 Angevine, J.B. Jr., Sidman, R.L.: Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 192, 766-768 (1961) Astr6m, K-E.: On the early development of the isocortex in fetal sheep. Progr. in Brain Res. 26, 1-59 (1967) Berry, M.: Development of the cerebral neocortex of the rat. In: Studies on the development of behavior and the nervous system, Vol. 2, Aspects of neurogenesis. (G. Gottlieb, ed.) pp. %67. New York: Academic Press 1974 Berry, M., Rogers, A.W.: The migration of neuroblasts in the developing cerebral cortex. J. Anat. (Lond.) 99, 691-709 (1965) Borges, S., Berry, M.: Preferential orientation of stellate cell dendrites in the visual cortex of the darkreared rat. Brain Res. 112, 141-147 (1976) Bradford, R., Parnavelas, J.G., Lieberman, A.R.: Neurons in layer I of the developing occipital cortex of the rat. J. comp. Neurol. 176, 121-132 (1977) Briickner, G., Mareg, V., Biesold, D.: Neurogenesis in the visual system of the rat. An autoradiographic investigation. J. comp. Neurol. 166, 245-256 (1976) Cajal, S. Ramon y: Studies on vertebrate neurogenesis. (translated by L. Guth) pp. 325-335. Springfield, Illinois: Charles C. Thomas 1960 Cragg, B.G.: The development of synapses in the visual system of the cat. J. comp. Neurol. 160,147-166 (1975) Eayrs, J.T., Goodhead, B. :Postnatal development of the cerebral cortex of the rat. J. Anat. (Lond.) 93, 385-402 (1959) Frotscher, M.: Die postnatale Entwicklung corticaler Neurone und ihre Beeinflnssung durch ein Trauma bei Rattus norvegicus B. J. Hirnforsch. 16, 203221 (1975) Glees, P.: Maturation and aging of vertebrate neurons. In: Metabolic compartmentation and neurotransmission. (S. Berl, D.D. Clarke and D. Schneider, eds.) pp. 651-682. New York: Plenum 1975 Globus, A., Scheibel, A.B.: The effect of visual deprivation on cortical neurons: A Golgi studY. Exp. Neurol. 19, 331 345 (1967) Globus, A., Rosenzweig, M.R., Bennett, E.L., Diamond, M.C.: Effects of differential experience on dendritic spine counts in rat cerebral cortex. J. comp. physiol. Psychol. 82, 175-181 (1973) Greenough, W.T., Volkmar, F.R.: Pattern of dendrite branching in occipital cortex of rat reared in complex environments. Exp. Neurol. 40, 491-504 (1973) Hicks, S.P., D'Amato, C.J.: Cell migrations to the isocortex in the rat. Anat. Rec. 160, 619-634 (1968) Jacobson, M.: A plentitude of neurons. In: Studies on the development and behavior of the nervous system. Vol. 2, Aspects of neurogenesis. (G. Gottlieb, ed.)pp. 151-166. New York: Academic Press 1974 Jacobson, M.: Development and evolution of type II neurons: Conjectures a century after Golgi. In: Golgi centennial symposium: Perspectives in neurobiology. (M. Santini, ed.) pp. 14%151. New York: Raven Press 1975 Kelly, J.P., Van Essen, D.C.: Cell structure and function in the visual cortex of the cat. J. Physiol. (Lond.) 238, 515-547 (1974) K6nig, N., Valat, J., Fulcrand, J., Marty, R.: The time of origin of Cajal-Retzius cells in the rat temporal cortex. An autoradiographic study. Neurosci. Lett. 4, 21-26 (1977) LeVay, S.: Synaptic patterns in the visual cortex of the cat and monkey. Electron microscopy of Golgi preparations. J. comp. Neurol. 150, 53-86 (1973) Lorente de No, R.: Studies on the structure of the cerebral cortex. J. Psychol. Neurol. (Lpz.) 45, 381-438 (1933) Lund, R.D., Mustari, M.J.: Development of the geniculocortical pathway in rats. J. comp. Neurol. 173, 289-306 (1977) Lund, J.S., Boothe, R.G., Lund, R.D.: Development of neurons in the visual cortex (area 17) of the monkey (Macaca nemestrina): A Golgi study from fetal day 127 to postnatal maturity. J. comp. Neurol. 176, 149-188 (1977)
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Marin-Padilla, M.: Prenatal and early postnatal ontogenesis of the human motor cortex: A Golgi study. 1. The sequential development of the cortical layers. Brain Res. 23, 167-183 (1970) Marin-Padilla, M.: Dual origin of the mammalian neocortex and evolution of the cortical plate. Anat. Embryol. 152, 109-126 (1978) Morest, D.K.: The growth of dendrites in the mammalian brain. Z. Anat. Entwickl.-Gesch. 128, 2 9 ~ 317 (1969) Noback, C.R., Purpura, D.P.: Postnatal ontogenesis of neurons in cat neocortex. J. comp. Neurol. 117, 291-307 (1961) Parnavelas, J.G.: Influence of stimulation on cortical development. Progr. in Brain Res. 48, 247-260 (1978) Parnavelas, J.G., Globus, A.: The effect of continuous illumination on the development of cortical neurons in the rat: A Golgi study. Exp. Neurol. 51, 637-647 (1976) Parnavelas, J.G., Lieberman, A.R., Webster, K.E.: Organization of neurons in the visual cortex, area 17, of the rat. J. Anat. (Lond.) 124, 305-322 (1977a) Parnavelas, J.G., Sullivan, K., Lieberman, A.R., Webster, K.E.: Neurons and their synaptic organization in the visual cortex of the rat. Electron microscopy of Golgi preparations. Cell Tiss. Res. 183, 499-517 (1977b) Parnavelas, J.G., Mounty, E.J., Bradford, R., Lieberman, A.R.: The postnatal development of neurons in the dorsal lateral geniculate nucleus of the rat: A Golgi study. J. comp. Neurol. 171, 481-500 (1977c) Raedler, A., Sievers, J.: Light and electron microscopical studies on specific cells of the marginal zone in the developing rat cerebral cortex. Anat. Embryol. 149, 173-181 (1976) Rakic, P.: Effects of local cellular environments on the differentiation of LCN's. In: Local Circuit Neurons. Neurosci. Res. Progr. Bull. 13(3), 400-407 (1975a) Rakic, P.: Role of cell interaction in development of dendritic patterns. In: Physiology and pathology of dendrites. Advances in neurology. Vol. 12. (G.W. Kreutzberg, ed.) pp. 117-134. New York: Raven Press 1975b Rakic, P.: Prenatal development of the visual system in rhesus monkey. Phil. Trans. R. Soc. Lond. B. 278, 245-260 (1977) Rickmann, M., Chronwall, B.M., Wolff, J.R.: On the development of non-pyramidal neurons and axons outside the cortical plate: the early marginal zone as a pallial anlage. Anat. Embryol. 151, 285-307 (1977) Ronnevi, L-O.: Spontaneous phagocytosis of boutons on spinal motoneurons during early postnatal development. An electron microscopic study in the eat. J. Neurocytol. 6, 487-504 (1977) Uylings, H.B.M., Parnavelas, J.G., Veltman, W.A.M.: The postnatal differentiation of non-pyramidal neurons in the visual cortex of rats. Proceedings of the 18th Dutch Federation Meeting. p. 403 (1978a) Uylings, H.B.M., Kuypers, K., Veltman, W.A.M.: Environmental influences on the neocortex in later life. Progr. in Brain Res. 48, 261-274 (1978b) Volkmar, F.R., Greenough, W.T.: Rearing complexity affects branching of dendrites in the visual cortex of the rat. Science 176, 1445-1447 (1972) Wise, S.P., Jones, E.G.: The organization and postnatal development of the commissural projection of the rat somatic sensory cortex. J. comp. Neurol. 168, 313-344 (1976)
Accepted August 29, 1978
Anatomy and Embryology 9 by Springer-Verlag 1978
The Development of Non-Pyramidal Neurons in the Visual Cortex of the Rat J.G. Parnavelas*, R. Bradford, E.J. Mounty, and A.R. Lieberman Department of Anatomyand Embryology,UniversityCollegeLondon,GowerStreet, LondonWC1E 6BT, England
Summary. We have studied the maturation of non-pyramidal cells in layers IIVI of the visual cortex of albino rats from birth to maturity, using Golgi-Cox and rapid Golgi preparations. At birth, non-pyramidal cells are sparse, immature and concentrated in the deep part of the cortical plate: their number increases towards the end of the first week but they remain sparse and immature in the upper part of the cortical plate. During the second postnatal week, the number, size and extent of dendritic and axonal branching of these cells undergo considerable increases and the cells become conspicuous in layer IV and apparent in the supragranular layers: this 'growth spurt' occurs just after (and may be related to) the arrival and establishment in the cortex during the second half of the first postnatal week, of extrinsic afferents. During the third postnatal week, most of the cells complete their maturation. At the end of this week, the number of spinous cells is greater and the spine density of some cells is higher than in the adult, falling to adult values during the fourth postnatal week. It is noteworthy that the non-pyramidal cells appear to reach maturity at about the same time in all the layers studied, and at the same time as the pyramidal cells with which they are associated. These observations are not in accord with the prevalent view that non-pyramidal cells complete their differentiation much later than pyramidal cells. Key words: Visual cortex - Non-pyramidal neurons - Development - Rat. Studies of neuronal differentiation in the cerebral cortex have been concerned predominantly with pyramidal cells (Lorente de N6, 1933; Eayrs and Goodhead, 1959; Cajal, 1960; Noback and Purpura, 1961; Astr6m, 1967; Berry, 1974; Frotscher, 1975; Lund et al., 1977). Since these cells have a characteristic and consistent morphology, which can be recognized even when they are very immature, it is not surprising that they should have been the main focus of such * Present address and address for reprints." Dr. J.G. Parnavelas, Departmentof Cell Biology,The Universityof Texas, Health ScienceCenter at Dallas, Dallas, Texas 75235, U.S.A.
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studies. The differentiation of the vastly more heterogeneous population of nonpyramidal cells has received much less attention, although recently Lund et al., (1977) have provided a detailed description of the development of non-pyramidal cells in layer IV of the monkey visual cortex. In recent years, however, increasing attention has been directed towards the functional organization of non-pyramidal cells in adult cerebral cortex, especially in the visual cortex (e.g. Kelly and Van Essen, 1974), and there has been interesting speculation about their evolutionary and developmental significance (e.g. Altman, 1967; Jacobson, 1974, 1975). Such studies and speculations have emphasized the importance of obtaining accurate data on the morphology and synaptic relationships of non-pyramidal cells in the adult, as well as on their genesis, their differentiation, and the development of their synaptic connections. In previous studies of the visual cortex of adult rats, we described the morphology of Golgi-impregnated non-pyramidal cells (Parnavelas et al., 1977a) and their ultrastructure and synaptic relations (Parnavelas et al., 1977b). We have also examined the postnatal development of non-pyramidal cells, including RetziusCajal cells in layer I of the rat visual cortex (Bradford et al., 1977) and in the present study we describe the postnatal development of Golgi-impregnated non pyramidal cells by light microscopy, in layers II-VI of the visual cortex, and relate the findings to others concerning the development of pyramidal cells and of afferent fibres to the cortex.
Materials and Methods
Brains from a minimum of 10 female albino rats at each of the following ages were used: 12h, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 18, 20, 24, 28 and 35 days, and 3 months (youngadults). Nissl, GolgiCox and rapid Golgi procedureswere utilized as previouslydescribed(Parnavelasand Globus, 1976; Bradford et al., 1977; Parnavelas et al., 1977c).The Nissl-stained material was used to determinethe extentand positionin relationto the corticalsurface,of the corticallayersat each stage of development studied, and to assign to appropriatepositionsthosecellswhoselaminar positionwas not clearlydefined in the Golgi material. The analysis of the Golgi material was qualitative and was based on careful observations of several thousand developingand mature non-pyramidal neurons and camera lucida drawings of more than one thousand.
Results
First Postnatal Week All cortical neurons in the rat are generated before birth, but many do not attain their final positions until several days after birth (Berry and Rogers, 1965; Berry, 1974; Lund and Mustari, 1977), and the laminar organization characteristic of the adult cortex is not apparent in newborn animals. Only layers V and VI are clearly delineated by the 4th postnatal day in Nissl preparations, while the rest of the cortical plate appears as a homogeneous population of densely stained cells. The laminar differentiation of this portion of the cortical plate is achieved by the 6th day.
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d \
g \
/'h 50pm Fig. la-i. Camera lucida drawings, postnatal days 0-6. a Day 0, layer VI; b Day 0, from close to the middle of the cortical plate (presumptive layer IV); c Day 2, middle of cortical plate; d Day 2, layer VI; e Day 4, layer V; fDay 4, middle of cortical plate; g Day 4, layer V; h Day 6, layer IV; i Day 6, layer III
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I f
C
5oLm /
Fig. 2 all. Camera lucida drawings, postnatal days 8-15. a Day 8, layer IV; b Day 11, layer III; e Day 10, layer IV; d Day 8, layer V; e Day 15, layer IV; f Day 10, layer V; g Day 52, layer IV
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Non-pyramidal cells are sparse in the 12h animals and are present
predominantly in the deeper part of the cortex (Fig. I a, b). At this stage and for a few days thereafter, the perikarya of these cells are small and sometimes irregular in shape. Their dendrites are thin, short and beaded (Fig. 4a) and their axons course only for short distances; collaterals arise from the axons of a few cells as early as day 2 (Fig. 1 c, d). Signs of rapid growth (i.e. irregular enlargements, growth cones, filopodia; see M orest, 1969) are present on the perikarya, dendrites and axons of all neurons (Figs. 1,4a-c). The number of stained non-pyramidal cells increases considerably towards the end of the first week but they are still confined mainly to layers V and VI. At this stage, their dendrites are more branched, thicker and longer (Fig. 1 g-i, Fig. 4c). Growth cones and irregular enlargements are still recognizable on dendrites, although they are not as conspicuous as in younger rats. The axons of most cells are directed towards the pial surface and some give rise to fine terminal arborizations. It appears that most of the non-pyramidal neurons situated in the deeper layers are in a more advanced stage of differentiation than those situated more superficially. Second Postnatal Week
The number of clearly defined non-pyramidal neurons increases markedly and these cells can be identified as a distinctive population, particularly in layer IV, between days 8 and 10. The cell perikarya are larger than at the previous stages and continue to grow with time, as do the dendrites, which increase their branching and thickness and now resemble those of mature neurons (Fig. 2; Fig. 4a-f). Their axons display an elaborate arrangement of collaterals, many of which turn back and ramify amongst the dendrites and perikarya of the parent cells. The number of neurons possessing spinous dendrites and the density of spines increase towards the end of the second postnatal week (Fig. 2c, f,g; Fig. 4e; Fig. 5a). Some nonpyramidal cells are observed in which the dendrites directed towards the white matter bear spines while those directed towards the pial surface are beaded (Fig. 2 f). Finally, the gradient of maturation from deep to superficial layers seen during the first week of postnatal life is not apparent and there are virtually no growth cones present on the dendrites of any cells by the end of the second week. Third Postnatal Week
There are few differences in the appearance of non-pyramidal neurons between the end of the second and the third postnatal week, except that their dendrites are slightly longer and thicker and resemble those of mature non-pyramidal neurons (Fig. 3 and 5). The axons of many cells become more elaborate and appear to achieve full development at this stage (Fig. 2c; Fig. 5b). However, it is extremely difficult to comment with certainty on the extent of axonal ramifications based on Golgi preparations, especially Golgi Cox material, in which large portions, if not the entire axonal plexus of individual neurons, are often not impregnated. One final feature, particularly prominent in the latter half of this developmental stage, is the proportionally higher number of non-pyramidal neurons of the spinous variety present in all cortical layers. The spine density of some of these cells is
t~
lJJ
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Fig. 4a-f. Photomicrographs, postnatal days 0-10, x 450. a Day 0, layer VI; b Day 4, layer V; c Day 6, layer IV; d Day 8, layer II; e Day 8, layer II; f Day 10, layer VI
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Fig. 5a-c. Photomicrographs, postnatal days 14 and 20, x 450. a Day 14, layer IV; b Day 20, layer IV; c Day 20, layer II
Development of Rat Visual Cortex
Fig. 6a-d. Photomicrographs, postnatal day 18 - adult, x 450. a Day 18, layer VI; b Day 24, layer V; c 3 months, layer VI; d 3 months, layer IV
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markedly higher than that of the non-pyramidal neurons of the spinous variety present in adult animals (compare Fig. 3c, e and Fig. 6a to Fig. 3d).
Fourth Postnatal Week
All non-pyramidal neurons appear indistinguishable from their adult counterparts (Fig. 6c, d) by the middle of the fourth week of postnatal life (Fig. 3 a, b, d; Fig. 6b). The proportion of cells of the spinous variety begins to decrease at the end of the third week and falls to the proportion found in the adult during the fourth.
Discussion
It is widely held that cortical non-pyramidal cells complete their differentiation somewhat later than pyramidal cells, and indeed that throughout the nervous system the maturation of Golgi type II cells lags behind that of the Golgi type I cells with which they are associated (Jacobson, 1974, 1975; Glees, 1975; Rakic, 1975a). Observations not wholly consonant with this view are scattered through the literature (e.g. ~str6m, 1967), and a major conclusion of the present study, based on careful examination of a large number of neurons in a closely spaced time series, is that non-pyramidal cells in the visual cortex of the rat do not complete their differentiation significantly later than pyramidal cells. At birth very few nonpyramidal cells can be recognized, chiefly in the deeper part of the cortex: over the next 8-10 days progressively more become recognizable, the most mature (excepting the horizontal cells of layer I; see below) occupying the infragranular layers. Thereafter differences in the state of maturation of non-pyramidal cells at different cortical levels become less evident, and so far as can be deduced from Golgi preparations, the non-pyramidal cells acquire their mature perikaryal size, dendritic morphology and axonal ramification pattern more or less stimultaneously throughout the depth of the cortex and at the same time as our own observations (unpublished) and those of others (Eayrs and Goodhead, 1959; Frotscher, 1975) indicate that the pyramidal cells acquire theirs. Although these conclusions are based on qualitative analysis, quantitative analysis of 700 cells from the same material using a semi-automated dendrite tracking system strongly support them (Uylings et al., 1978a). It may be that detailed ultrastructural and functional studies will reveal aspects of non-pyramidal cell maturation that are not reflected in the Golgi preparations and that are in accord with the prevalent view. It should be said, however, that observations at this relatively gross level at least, do not support the suggestion that non-pyramidal cells are structurally less mature than pyramidal cells in the early postnatal period, and thus more modifiable by environmental, hormonal and metabolic influences (Jacobson, 1974, 1975). It may also be relevant in the context of Jacobson's suggestions concerning the modifiability of non-pyramidal cells, that experiments involving environmental manipulation have produced changes in both non-pyramidal cells and pyramidal cells (Globus and Scheibel, 1967; Volkmar and
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Greenough, 1972; Greenough and Volkmar, 1973; Globus et al., 1973; Borges and Berry, 1976; Parnavelas, 1978; Uylings et al., 1978b). With the conspicuous exception of the early-differentiating cells of layer I (Bradford et al., 1977; and see below), non-pyramidal cells of different types and in different cortical layers reach maturity at about the same time. The non-pyramidal cells generated late in the period of neurogenesis must therefore mature at a faster rate than pyramidal cells and other non-pyramidal cells generated earlier. In drawing this conclusion about the relatively rapid rate of differentiation of cells generated late in ontogeny, we do not imply acquiescence with the widely-held view that non-pyramidal cells are generated only after or towards the end of the period of pyramidal cell generation (Angevine, 1970; Berry, 1974; Rakic, 1975 a; Jacobson, 1975). Recent autoradiographic evidence indicates that non-pyramidal cells are produced throughout the period of neurogenesis and that they are added to all levels of the developing cortex throughout the period of its formation (Rickmann et al., 1977). It would appear, therefore, that non-pyramidal cells, or at least some non-pyramidal cells, are incorporated into the cortical anlage according to a developmental program different from that controlling the systematic disposition, layer by layer and from deep to superficial, of pyramidal cells (Angevine and Sidman, 1961; Berry and Rogers, 1965; Hicks and D'Amato, 1968; Berry, 1974; Brfickner et al., 1976; Lund and Mustari, 1977), a conclusion reached earlier by Astr6m (1967) on the basis of Golgi studies of the foetal sheep brain. It is also apparent from the autoradiographic data that the Retzius-Cajal cells, whose precocious development in layer I of the visual and other cortical areas has long been known and puzzled over (see Raedler and Sievers, 1976 and Bradford et al., 1977 for references), are generated very early (on embryonic days 12-14, K6nig et al., 1977; Rickmann et al., 1977), before most of the cells contributing to the cortical plate have been generated (embryonic day 13 until just before birth; Berry 1974; Brtickner et al., 1976; K6nig et al., 1977; Lund and Mustari, 1977). An extremely interesting question concerning the development of the cortex, is when, in relation to critical developmental stages, do extrinsic afferents reach their target neurons? There is now good evidence that the major extrinsic afferent systems to sensory areas of the neocortex in the rat reach their targets at relatively late stages. Thus an organized projection to the visual cortex is established only between days 4-8 postnatal, even though the projection fibres reach the subcortical white matter by embryonic day 18 (Lund and Mustari, 1977). The development of the commissural system follows a similar pattern: callosal axons, although present in the subcortical white matter at birth, only start to enter the cortex on postnatal day 3 and do not show and adult laminar distribution pattern until the end of the first week (Wise and Jones, 1976). Marin-Padilla (1970, 1978), on the other hand, maintains that as a general rule in mammals, fibres from the thalamus enter the cortical rudiment at a very early developmental stage, even before the cortical plate has formed, and that these afferents are responsible for inducing the differentiation of the cortical elements. Marin-Padilla's evidence is based solely upon the examination of Golgi preparations, whereas that of Wise and Jones (1976) and Lund and Mustari (1977) is based upon the more informative and reliable methods of autoradiography and experimental degeneration. Furthermore, the data of the latter authors are in accordance with findings in the monkey visual cortex (Rakic,
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1977) based on studies of the transneuronal transport of labelled tracers injected into the eye. It would seem reasonable, therefore, to accept the important implications of the latter studies, viz. that major aspects of cortical histogenesis particularly the development of the laminar pattern and the differentiation of the pyramidal cells - occur before the arrival of extrinsic inputs and that a major inductive influence of the afferents on these events is improbable. The situation with regard to the differentiation of the non-pyramidal cells, however, is less clear. In our material, the first part of the second postnatal week sees a marked increase in the distinctiveness and maturity of non-pyramidal cells, especially in layer IV and the supragranular layers. It may be, therefore, that the rapid growth and maturation of the non-pyramidal cells is triggered by the arrival in the cortex during the first postnatal week of subcortical and cortieo-cortical afferents. Such a process would be compatible with what is known of the inductive capabilities of afferents in certain sites in the C.N.S. (e.g. M orest, 1969; Berry, 1974; Rakic, 1975b), but would not provide a satisfactory explanation in the case of the Retzius-Cajal cells which mature at a much earlier stage. The acquisition by non-pyramidal cells of dendritic spines follows an interesting pattern, different from that shown by at least some of the pyramidal cells with which they are associated. Spine counts have shown that pyramidal cells of layers IV and V in rat visual cortex first acquire dendritic spines on postnatal day 4, and that spine density increases enormously between postnatal days 8 and 16, reaching adult values by the end of the fourth postnatal week (Parnavelas and Globus, 1976). Spine density in the non-pyramidal cell population, however, is maximal by the middle of the third postnatal week, and subsequently falls to adult values during the fourth week. Moreover, at the time of maximal spine density, the proportion of non-pyramidal cells with spinous dendrites is greater than in the adult. It would appear, therefore, that not only do most spinous non-pyramidal cells become less spinous during the period 3-4 weeks postnatal, but that some cells lose all or most of their spines to fall into the category of spine-free non-pyramidal cells. These results accord with previous studies by LeVay (1973) who found the spine density of non-pyramidal cells of the visual cortex to be higher in kitten than, cat, and by Lund et al., (1977) who found that the maximal spine density of layer IV non-pyramidal cells in the monkey occurred at 8 weeks postnatal and was followed by a period of spine loss. In contrast to the present findings and those of Parnavelas and Globus (1976), however, Lund et al. (1977) found that both non-pyramidal and pyramidal ceils showed a similar pattern with respect to the acquisition of dendritic spines, both showing a peak density at 8 weeks and a subsequent decline towards adult values. Irrespective of possible species differences in spine loss from maturing pyramidal cells, there is now abundant evidence that overproduction with subsequent resorption of supernumerary spines, together with loss or redistribution of the synaptic contacts associated with them, may be a phenomenon of general occurrence and considerable significance in the development of the C.N.S. (e.g. Cajal, 1960; Cragg, 1975; Lund et al., 1977; Ronnevi, 1977).
Acknowledgements. We thank the MedicalResearch Council and the WellcomeTrust for financial
support.
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Accepted August 29, 1978
