Exp Brain Res (1985) 61:204-209 Exp, erimental Brain Research �9 Springer-Verlag 1985

Cytochrome oxidase activity in the striate cortex and lateral geniculate nucleus of the newborn and adult macaque monkey

H. Kennedy, J. Bullier, and C. Dehay

Laboratoire de Neuropsychologie Expdrimentale, INSERM-Unit6 94, 16, Avenue du Doyen L6pine, F-69500 Bron, France

Summary. The laminar location of cytochrome oxi- dase staining has been compared in the lateral geniculate nucleus and area 17 in newborn and adult macaque monkeys. In area 17 of the adult, the distribution of cytochrome oxidase activity confirmed published findings. In the newborn animals, the tissue reacted as strongly for cytochrome oxidase as in the adult but the pattern of labelling was different in two respects. Firstly in layer 1 activity was stronger and occupied a wider portion of this layer. Secondly, cytochrome oxidase staining in layer 4C occupied two separate bands, a small narrow band at the bottom of 4C~ and a wider one occupying the full width of 4C~ and spilling over into 4t3. The pattern of cytochrome oxidase activity did not appear to be influenced by eccentricity in the newborn whereas, in the adult, label in 4C was more intense in cortex subserving central vision. In the lateral geniculate nucleus of the adult, the magnocellular layers and the most dorsal parvocellular layer reacted most strongly for cytochrome oxidase. In the newborn, parvocellu- lar layers were more uniformly labelled and the difference between parvo- and magnocellular layers more pronounced. These results are discussed in relationship to the development of thalamo-cortical projections in the monkey.

Key words: Monkey - Striate cortex - L G N - Cytochrome oxidase - Development

Introduction

Cytochrome oxidase (Cyt. Ox.) is a mitochondrial enzyme used in the cell for the production of energy. The spatial distribution of Cyt. Ox. activity in

Offprint requests to: H. Kennedy (address see above)

histological sections reflects local differences in metabolic activity and has proved especially useful for exploring afferent connections as well as regional functional specialization (Wong Riley 1979; Living- stone and Hubel 1984). Within area 17 of the macaque zones of high Cyt. Ox. activity have been found to correspond to regions of thalamic terminals (Horton and Hubel 1981; Livingstone and Hubel 1982). In the past few years, there has been consider- able interest in the termination of the lateral genicu- late nucleus (LGN) in area 17 of the developing monkey. During the first two months of life the extent of the cortical territory occupied by the afferents from each eye can be influenced by visual experience (Hubel et al. 1977) and it has been shown that the phenomena of plastic rearrangment of the inputs from the two eyes and the normal process of development are synchronised (LeVay et al. 1980). We have therefore examined the distribution of Cyt. Ox. activity to see if this would suggest differences in thalamo-cortical projections between newborn and adult monkeys.

Methods

Three newborn cynomolgus monkeys (Macaca Irus) aged 30 to 48 h were used in these experiments. Three adult monkeys served as controls. Two of the newborns were part of a series investigating callosal connectivity in the neonate using horseradish peroxidase. The third animal received an injection of wheat germ agglufinin conjugated to horseradish peroxidase (WGA-HRP) in the white matter underlying area 17, 20 h prior to sacrifice. The concentra- tion of WGA-HRP was 2% in saline and the injections were made using micropipettes with a broken tip of 50-100 ~xna diam.

Animals were perfused under deep anaesthesia with isotonic saline followed by one liter of 1% paraformaldehyde and 1.25% glutaraldehyde in phosphate buffer. This aldehyde mixture was passed for 45 min and followed with a 10% sucrose solution for a similar length of time. The brains were blocked and stored for at least 12 h in sucrose. The brain was then sectioned (60 ~tm) on a

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Fig. 1A-D. Laminar location of Cyt. Ox. activity in newborn and adult monkeys. Nissl stain of newborn (A) and adult (C) monkey. Cyt. Ox. activity in newborn (B) and adult (D) monkey. Photomicrographs of Nissl stain taken at same scale (scale bar = 308 ~tm). Enlargement of photomicrographs of Cyt. Ox. have been made so as to compensate for differential shrinkage

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Fig. 2A-D. Photomicrographs of Cyt. Ox. activity in newborn (A, B) and adult (C, D) monkey. A and C show Cyt. Ox. activity in layer 1 and B and D show Cyt. Ox. activity in layer 4. Scale bar" 154 ~tm

freezing microtome either in the horizontal or parasaggital plane. Adjacent sections were processed for HRP histochemistry (Mesulam et al. 1980) and for Cyt. Ox. activity (Wong-Riley 1979).

The comparison between Cyt. Ox. staining in adults and newborns was made in the cortex in sections which were cut approximate by perpendicular to the cortical surface within 1 cm of the representation of the fovea. Nissl stained sections were projected onto high power photomicrographs of adjacent Cyt. Ox. reacted sections. The two histochemical procedures cause different degrees of shrinkage, so that the laminar location of Cyt. Ox. activity was inferred in those adjacent sections where blood vessels could be accurately matched. The intensity of Cyt. Ox. activity in different regions of layer 4C was compared at different cortical eccentricities by measuring the optical density threshold using a PC Eye image digitaliser on an IBM PC computer.

Results

In area 17 of the newborn animal the cytological laminae can be distinguished (Lurid 1973; O 'Kusky and Colonnier 1982). Although the overall thickness of the cortex is approximately the same in the newborn and adult, there are differences in the width of individual layers. The most notable change is in

layers 5 and 6. Layer 5 in the newborn animal accounts for 15% of the total width of the cortex and for only 7.5% in the adult (Fig. 1). Conversely lamina 6 in the newborn was about 10% of the cortical width and over 20% in the adult. The other cortical layers were about the same width in new- borns and adults.

In the adult, high levels of Cyt. Ox. activity were found in all layers, except layer 5. In layers 1, 4 and 6, Cyt. Ox. stain was found as a more or less continuous band parallel to the cortical surface whereas, in layers 2/3 separate patches of high activity were observed. Compar ison of a section processed for Cyt. Ox. activity and the adjacent Nissl-stained section showed that the distribution of Cyt. Ox. activity within the cortical laminae is different in the adult and newborn (Fig. 1). The principal differences occur in the sublaminae 4C and 4B of layer 4. Whereas in the adult, Cyt. Ox. staining fills both 4Ca and 4C~ with a continuous band of high activity (Hor ton and Hubel 1981), this is not the case in the newborn where it produces a bilaminar distribution. The bo t tom band of high activity

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Fig. 3. Dark field photomicrograph of area 17 showing axonal termination following WGA-HRP injection into the white matter in a newborn monkey. Scale bar: 1 rJlrfl

Fig. 4A and B. Digitized image at equivalent threshold of optical density in layer 4C in cortex subserving the central visual field (A) and peripheral visual field (B) in the same animal. In both A and B the termination of layer 4C at the V1/V2 border is on the far left. Scale bar: 0.5 mm

occupies the base of layer 4C[3 and the upper band fills the whole of 4Ca and clearly spills over into 4B. Between these two zones of high Cyt. Ox. activity there is a narrow band of low activity which corre- sponds to the upper two thirds of layer 4C~ (Fig. 1).

In Fig. 1, the photomicrographs of Cyt. Ox. activity have been adjusted so as to correspond to the Nissl stain, so that it is not possible to compare directly either the density or the dimensions of the Cyt. Ox. rich regions in the adult and newborn. This comparison is provided by Fig. 2 which presents high power photomicrographs of those regions which showed differences between the two sets of animals. In the newborn, Cyt. Ox. activity was as vigourous as in the adult. In the newborn, the thicker band occupies the whole of layer 4Ca and extends for about 90 ~m in layer 4B. In the adult, the single band of activity occupies 4Ca and 4C[~ and grazes much less into 413 (Fig. 2B and 2D). The other major difference in Cyt. Ox. staining between the newborn and adult is in the top of layer 1. In the newborn animal, this corresponds to a thickness of about 50 ~tm while in the adult it is about 20 ~m (Fig. 2A and 2C).

We investigated whether the distribution of Cyt. Ox. label in layer 4C in the newborn has any

relationship to the pattern of thalamic terminals. WGA-HRP was injected into the white matter underlying area 17 of one animal and we observed the pattern of anterograde filling in layer 4. It is known that this layer receives afferents from the LGN and not from pulvinar or other cortical areas (Hubel and Wiesel 1972; Ogren and Hendrickson 1977; Maunsell and Van Essen 1983). The labelling in layer 4 therefore reflects the distribution of LGN afferents. Near the injection site, local reaction product of HRP obscured any differential labelling in cortical laminae. However, at a distance of 2-4 mm from the injection site a characteristic distribution of HRP reaction product was observed (Fig. 3). Very few retrogradely labelled cells were observed and the reaction product of the WGA-HRP was a fine dust- like deposit typical of axon terminals. At the bottom of layer 6 filled axons can be seen coursing up from the white matter but are not filled above this layer, presumably because of the fine calibre of immature axons. After photographying, the section was coun- terstained so as to allocate regions of dense axon terminals to particular cortical layers. Layers 2 and 3 were free of reaction product despite the presence of Cyt. Ox. patches. Two bands could be observed in layer 4C, one at the bottom of 4C[3 and the other in

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Fig. 5A and B. Parasaggital sections of the lateral geniculate nucleus showing cytochrome oxidase activity in parvo- and magnocellular layers. A newborn monkey. B adult monkey. Scale bar: 1 mm

4Ca and the lower part of 4B. These results suggest that the bands of Cyt. Ox. activity in layer 4C might correspond to regions of dense LGN input separated by a region of sparse LGN input.

It has been reported that there is a denser band of Cyt. Ox. activity along the base of layer 4C in the adult (Wong-Riley and Carroll 1984). This is not readily apparent upon visual inspection of our mate- rial (Fig. 2). By looking at optical density thresholds, we found that there is an increase in activity which, however, only occurs in cortex subserving central vision (Fig. 4). These results might reflect the richer input from the parvocellular layers of the LGN to cortex subserving the fovea (Connolly and Van Essen 1984) and are consistent with other differences in Cyt. Ox. staining in layers 2 and 3 between cortex subserving different regions of the visual field (Livingstone and Hubel 1984). Optical density mea- surements therefore show that the pattern of Cyt. Ox. staining observed in layers 4C of the newborn has a more pronounced similarity with that found in the adult cortex subserving central vision.

Cyt. Ox. activity in the LGN is not uniformly distributed in the different laminae (Fig. 5). In the adult, the two magnocellular layers and layer 6 showed marginally higher levels of Cyt. Ox. activity, confirming previous findings (Wong-Riley and Car- roll 1984). In the newborn, the magnoceUular layers were much more densely stained and the parvocellu- lar layers more weakly stained than in the adult, so that the overall difference between the two sets of layers was greater in the younger animals (Fig. 5).

On some sections, layer 6 did appear in the newborn to be slightly more deeply stained than the other parvocellular layers,

Discussion

The present results show that the pattern of Cyt. Ox. activity in the LGN and striate cortex changes during development. In the adult animal, the magnocellular laminae of the LGN project to layer 4Cc~ while the parvocellular layers project mainly to 4C[3 (Hubel and Wiesel 1972). Our results show that in the newborn, the magnocellular pathway exhibits a higher Cyt. Ox. staining than the parvocellular pathway. This suggests that, during early stage of development, there is a difference in the activity of neurons in these two parallel pathways.

In the cortex, the outstanding differences in Cyt. Ox. activity are in layers I and 4C. The stronger label in layer 1 could mean that inputs to this layer (e.g. from the pulvinar) are relatively more active in the newborn. Within layer4, major developmental changes concerning afferents from the LGN take place during the first six weeks of life. During this period, there is a sorting out of afferents from the LGN laminae innervated by each eye (Hubel et al. 1977; LeVay et al. 1980). Our results show that the pattern of Cyt. Ox. activity and thalamic input into layer 4 in the newborn (Fig. 3) is different from the adult pattern. It is possible to interpret this difference in terms of the developmental processes taking place in this layer.

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The difference between newborn and adult observed in layer 4C15 suggests that the input to this layer is more immature at birth than is the input to layer 4Cc~. There is evidence that the magnocellular layers of the LGN are established earlier (Rakic 1976) and might mature quicker than do the par- vocellular layers (Garey and Saini 1981; Gottlieb et al. 1985) so that input to 4Ca could preceed that to layer 4C[L Studies on axons terminating in layer 4C also support a late development of 4C[3 (Blasdel and Lund 1983). There is also evidence from experiments on the plasticity of ocular dominance columns to suggest major developmental differences between the two subdivisions of layer 4C. LeVay and co- authors (1980) concluded from reverse suture experi- ments that anatomical plasticity is terminated earlier in layer 4Ca than it is in 4C[L They interpreted these results as indicating either that the differential effect is the consequence of a relatively greater loss of terri- tory by the deprived eye in 4Ca (i.e. a greater susceptibility of magnocellular terminals to depriva- tion) or that plasticity is terminated earlier in the magnocellular afferents. Our results indicating a later development of the parvocellular input to 4C[3 recon- cile both these interpretations: the precocious mag- nocellular input to layer 4Ca would make it more susceptible to early deprivation and the late develop- ment of input to 4C~ would prolong plasticity in this layer.

Acknowledgements. We are grateful to Norlle Boyer and Pascale Giroud for technical assistance and to Fran~oise Girardet for typing the manuscript. Howard Cooper provided helpful assist- ance with the optical density measurements. Funding by M. R. T. grant no 83. C. 0907 is gratefully acknowledged.

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Received June 21, 1985 / Accepted August 19, 1985