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The FESN-sponsored follow-up meeting on neural development highlighted progress toward understanding several central issues in developmental neurobiology with particular emphasis on investigation into the mechanisms of cell fate determination. In systems as diverse as the HSN neurons of C. elegans, the photoreceptor cells of the Drosophila eye, the wide range of cell types within the vertebrate retina and the neurons of the cerebral cortex, hindbrain and spinal cord, the importance of environment in the determination and maintenance of cell fate was clearly established. Advances in cell marking techniques, including fluorescent dye and retroviral tagging, have enabled the fates of cells in normal and heterotypic environments to be followed and have demonstrated the initial plasticity of the progenitor cell population in many systems. The recent establishment of in vitro systems for studying neural development should further define the precise nature and identity of the environmental signals that act to establish and maintain cell fate. Of course, establishment of cell identity is only the initial phase in the formation of the mature nervous system. Once the fate of individual cells is determined, migration of cells to appropriate locations, extension of axons to appropriate targets and refinement of neuronal circuitry must occur. Both the definition of genes that influence these processes in nematodes and recent advances in imaging techniques that provide a means of observing these later, dynamic processes in 'living' brain slices promise to significantly advance understanding of the complexities of development of functional nervous systems.

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The adult brain has a remarkable ability to adjust to changes in sensory input. Removal of afferent input to the somatosensory, auditory, motor or visual cortex results in a marked change of cortical topography. Changes in sensory activity can, over a period of months, alter receptive field size and cortical topography. Here we remove visual input by focal binocular retinal lesions and record from the same cortical sites before and within minutes after making the lesion and find immediate striking increases in receptive field size for cortical cells with receptive fields near the edge of the retinal scotoma. After a few months even the cortical areas that were initially silenced by the lesion recover visual activity, representing retinotopic loci surrounding the lesion. At the level of the lateral geniculate nucleus, which provides the visual input to the striate cortex, a large silent region remains. Furthermore, anatomical studies show that the spread of geniculocortical afferents is insufficient to account for the cortical recovery. The results indicate that the topographic reorganization within the cortex was largely due to synaptic changes intrinsic to the cortex, perhaps through the plexus of long-range horizontal connections.

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Pyramidal neurons within the cerebral cortex are known to make long-range horizontal connections via an extensive axonal collateral system. The synaptic characteristics and specificities of these connections were studied at the ultrastructural level. Two superficial layer pyramidal cells in the primate striate cortex were labeled by intracellular injections with horseradish peroxidase (HRP) and their axon terminals were subsequently examined with the technique of electron microscopic (EM) serial reconstruction. At the light microscopic level both cells showed the characteristic pattern of widespread, clustered axon collaterals. We examined collateral clusters located near the dendritic field (proximal) and approximately 0.5 mm away (distal). The synapses were of the asymmetric/round vesicle variety (type I), and were therefore presumably excitatory. Three-quarters of the postsynaptic targets were the dendritic spines of other pyramidal cells. A few of the axodendritic synapses were with the shafts of pyramidal cells, bringing the proportion of pyramidal cell targets to 80%. The remaining labeled endings were made with the dendritic shafts of smooth stellate cells, which are presumed to be (GABA)ergic inhibitory cells. On the basis of serial reconstruction of a few of these cells and their dendrites, a likely candidate for one target inhibitory cell is the small-medium basket cell. Taken together, this pattern of outputs suggests a mixture of postsynaptic effects mediated by consequence the horizontal connections may well be the substrate for the variety of influences observed between the receptive field center and its surround.

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The binding of RPE-1, a mouse monoclonal antibody selective for newt retinal pigment epithelium, was followed in eyes undergoing embryonic development and retinal regeneration. Using the indirect immunofluorescence technique on frozen sections, we observed bright and continuous labelling exclusively in the retinal pigment epithelium (RPE) of normal adult newts, but labelling became diminished near the ora serrata region and stopped abruptly at the ciliary margin. During development, labelling was not detected in the retinal pigment epithelium (RPE) until the formation of photoreceptor outer segments and was not observed in any other ocular tissue. There was no correlation between the appearance of pigment in retinal pigment epithelial cells and their labelling with the RPE-1 antibody. Furthermore, albino salamander embryos showed the same pattern of labelling with RPE-1 as that seen in age-matched pigmented animals. During retinal regeneration, RPE cells were labelled less intensely, but heavy labelling was observed in the newly formed retinal cells. With time, labelling in regenerated retina receded, so that by the end of regeneration, labelling by RPE-1 was once more restricted to the RPE cells. The identification of RPE-1 as a marker for postmitotic retinal neurons about to undergo differentiation provides a promising approach for further studies of regeneration with the help of molecular tools.

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Perception of a visual attribute, such as orientation, is strongly dependent on the context within which a feature is presented, such as that seen in the tilt illusion. The possibility that the neurophysiological basis for this phenomenon may be manifest at the level of cells in striate cortex is suggested by anatomical and physiological observations of orientation dependent long range horizontal connections which relate disparate points in the visual field. This study explores the dependency of the functional properties of single cells on visual context. We observed several influences of the visual field area surrounding cells' receptive field on the properties of the receptive field center: inhibition or facilitation dependent on the orientation of the surround, shifts in orientation preference and changes in the bandwidth of orientation tuning. To relate these changes to perceptual changes in orientation we modeled a neuronal ensemble encoding orientation. Our results show that the filter characteristics of striate cortical cells are not necessarily fixed, but can be dynamic, changing according to context.

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Macaque monkeys become myopic when raised with fused lids to expose the retina to formless shadows during the period of postnatal eye development. The effect of the abnormal visual input is an excessive expansion of the posterior segment of the eye, a process that seems to be controlled by the nervous system. The mechanism by which the nervous system influences eye growth appears to be different in the stumptailed macaque (Macaca arctoides) and the rhesus macaque (M. mulatta). Lid-fused arctoides monkeys do not develop myopia when the ciliary muscle is paralysed or the optic nerve is cut, suggesting that the abnormal growth is caused by excessive accommodation. In contrast, paralysis of the ciliary muscle or optic nerve section does not prevent the development of myopia in the rhesus macaque, suggesting that in this species the axial growth is controlled by the retina. In both species neonatal lid fusion causes a marked increase in retinal vasoactive intestinal polypeptide (VIP). VIP is contained in a single type of amacrine cell whose dendrites spread in the middle of the inner plexiform layer. It remains to be determined whether the increase in the level of VIP is related to the abnormal axial elongation caused by lid fusion. At present we are also exploring the effects of accommodation on the growth of the eye by training juvenile arctoides monkeys to work on complex visual discrimination paradigms. Preliminary results show that performing a visual task at close range may influence the axial length and refraction in this macaque species.

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The findings presented in these studies have brought out different ideas concerning the mechanisms of processing in primary visual cortex than were held at the outset. Rather than thinking of receptive fields as being restricted in their extent, with the process of integration of the components of an image occurring at a much later stage along the visual pathway, we have shown that the integrative process is a progressive one, beginning in the primary visual cortex (or perhaps even earlier) and building up in a cascading series of converging and diverging connections. Rather than thinking of the filter characteristics of a cell as being fixed, it is apparent that they are dynamic and can be modified by the context in which features are presented. Finally, rather than a cortex with a functional architecture that is fixed after a critical period ending in infancy, we find that perturbing the system can lead to long-term topographical reorganization. Other examples of contextual interactions have been demonstrated in the submodalities of motion, where a cell's directional selectivity is modulated by the presence of movement in the surround (Allman et al. 1985; Tanaka et al. 1986; Gulyas et al. 1987; Orban et al. 1987). In the domain of color, the phenomenon of color constancy, reported for cells in visual area V4 (Zeki 1983), also requires lateral interactions in visual space, comparing the wavelength distribution of light coming from surfaces in different parts of the visual field. The influences presented in these studies, as in our own work in the domain of orientation, are modulatory. The long-term changes in cortical topography following removal of somatosensory input (Merzenich et al. 1984, 1988) or by retinal lesions suggest that with the appropriate manipulations the lateral interactions can be enhanced to the point of activating the postsynaptic cells. Although retinal lesions clearly represent an abnormal disruption of sensory input, they may nevertheless be representative of long-term reorganizations of neural networks occurring under normal circumstances, such as those required for memory.

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The cortical circuitry of the visual cortex has been worked out in great detail. Anatomical investigations reveal stereotyped connections within cortical columns and specific long-range connections between distant columns. Pharmacological techniques for blocking the activity in individual cortical layers or columns allow the microdissection of the cortical circuit. These studies could relate specific functional roles to particular cortical connections.

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A prominent and stereotypical feature of cortical circuitry in the striate cortex is a plexus of long-range horizontal connections, running for 6-8 mm parallel to the cortical surface, which has a clustered distribution. This is seen for both intrinsic cortical connections within a particular cortical area and the convergent and divergent connections running between area 17 and other cortical areas. To determine if these connections are related to the columnar functional architecture of cortex, we combined labeling of the horizontal connections by retrograde transport of rhodamine-filled latex microspheres (beads) and labeling of the orientation columns by 2-deoxyglucose autoradiography. We first mapped the distribution of orientation columns in a small region of area 17 or 18, then made a small injection of beads into the center of an orientation column of defined specificity, and after allowing for retrograde transport, labeled vertical orientation columns with the 2-deoxyglucose technique. The retrogradely labeled cells were confined to regions of orientation specificity similar to that of the injection site, indicating that the horizontal connections run between columns of similar orientation specificity. This relationship was demonstrated for both the intrinsic horizontal and corticocortical connections. The extent of the horizontal connections, which allows single cells to integrate information over larger parts of the visual field than that covered by their receptive fields, and the functional specificity of the connections, suggests possible roles for these connections in visual processing.

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The relationships between ocular dominance columns and intrinsic cortical circuitry were examined in brain slices prepared from the striate cortex of macaques. Ocular dominance columns in layer 4C beta were visualized in vitro following anterograde transport of rhodamine injected into the lateral geniculate nucleus in vivo. The axonal and dendritic arborizations of individual layer 4C beta cells were revealed by intracellular fluorescent dye injections. Both qualitative observations and quantitative analysis showed that the dendrites of cells close to borders remained preferentially, although not absolutely, in the "home" column (the column containing the cell body). Thus, the segregated pattern of afferent input appears to have considerable influence on the pattern of dendritic arbors. Similarly, while axon collaterals within layer 4C beta could cross into the adjacent column, their limited lateral spread produced arbors that remained primarily within the home column. The terminal arbors of collaterals that travelled from layer 4C beta to layer 3 had a larger lateral spread, and the termination pattern appeared to be independent of column borders. Thus, our observations indicate that, while the course of many layer 4C beta dendrites appears to be guided by columnar boundaries as defined by geniculate afferents, there exist morphological substrates for intercolumnar interactions even between 4C beta cells. Intercolumnar interactions are seen more commonly in layer 3, however, where larger, denser axon arbors originating from 4C beta cells can freely cross ocular dominance column boundaries.

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Lids were fused in six neonatal and one adult macaque monkey (Macaca mulatta and Macaca arctoides) and were kept fused for 1 to 18.5 months. The juvenile macaques, but not the adult one, developed myopia due to excessive elongation of the eye. In all animals, the immunohistochemical reactivity of the retina for vasoactive intestinal polypeptide (VIP) was much higher in the closed than in the open eyes. The neuropeptide was localized to the perikaryon and dendrites of amacrine cells. No difference was observed in substance P immunoreactivity between open and closed eyes, suggesting that the observed effect is selective. The change in VIP immunoreactivity could be the result of an increase in peptide synthesis, a decrease in peptide release, or a combination of the two. These results indicate that VIP may play a part in the regulation of postnatal ocular growth.

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Optical imaging of cortical activity offers several advantages over conventional electrophysiological and anatomical techniques. One can map a relatively large region, obtain successive maps to different stimuli in the same cortical area and follow variations in response over time. In the intact mammalian brain this imaging has been accomplished with the aid of voltage sensitive dyes. However, it has been known for many years that some intrinsic changes in the optical properties of the tissue are dependent on electrical or metabolic activity. Here we show that these changes can be used to study the functional architecture of cortex. Optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex, obtained by reflection measurements of the intrinsic signal, were confirmed with voltage sensitive dyes or by electrophysiological recordings. In addition, we describe an intrinsic signal originating from small arteries which can be used to investigate the communication between local neuronal activity and the microvasculature. One advantage of the method is that it is non-invasive and does not require dyes, a clear benefit for clinical applications.

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Anatomical studies in the visual cortex have shown the presence of long-range horizontal connections with clustered axonal collaterals, suggesting interactions over distances of several millimeters. We used cross-correlation analysis in cat striate cortex to detect interactions between cells over comparable distances. Using one cell as a reference, we recorded from other cells with a second electrode at varying distances and looked for correlated firing between the two recording sites. This technique allowed us to combine a physiological measure of the strength and type of connection between cells with a characterization of their receptive field properties. The observed interactions were excitatory, and extended over horizontal distances of several millimeters. Furthermore, the interactions were between orientation columns of like specificity, resulting in a waxing and waning in the strength of interaction as the electrodes passed through different orientation columns. We studied relationships between strength of correlation and other receptive field properties and found a tendency for facilitatory interactions between cells sharing the same eye preference. A large proportion of our correlations was due to common input. This feature, and the similarity of interactions between cells in the same column with the reference cell, suggest a high degree of interconnectivity between and within the columns. As the distance between the two electrodes increased, the overlap of the receptive fields of the cells participating in the interactions gradually diminished. At the furthest distances recorded, the cell pairs had nonoverlapping receptive fields separated by several degrees. The distribution and range of these interactions corresponded to the clustering and extent of the horizontal connections observed anatomically.

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This study examines the extent to which the restriction of visual experience to lines of a single orientation influences the organization of the striate cortex in infant monkeys (Macaca mulatta). Previous studies of kittens raised with monocular exposure to a single line orientation have consistently shown the response preference of cells driven by that eye to be biased towards the experienced orientation. Studies of binocular exposure to restricted orientations have been equivocal. In the infant monkey cortex responses to oriented lines have virtually all the specificity of responses seen in the adult animal. In an effort to clarify the phenomenon and the mechanism by which orientation bias might be obtained, we examined the effects of monocular exposure to a restricted orientation in infant macaques. Three monkeys were used. Each monkey was raised with one open eye exposed to lines of a single orientation and one eye occluded by lid suture. As in other cases of monocular deprivation in either cat or monkey, few binocularly driven cells were recorded and the majority of cells were dominated by the open eye. Cells driven by the open eye had normal representation of all orientation preferences and there was no overall increase in the number of cells preferring the orientation to which the eye had been exposed. The cells dominated by the occluded eye, however, showed a lack of cells responding to orientations to which the open eye had been exposed. These findings suggest that a competitive mechanism operates between the two eyes to provide an orientation selective advantage to the open eye.

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Myopia develops in macaque monkeys when their lids are surgically fused at birth and kept closed for one year. This experimental refractive error has many features in common with human myopia: It is caused by progressive axial elongation of the eye, is often accompanied by fundus changes, and can only be induced before eye growth has been completed. Myopia does not develop in animals raised in the dark; thus, it is triggered by an alteration of the visual input and is presumably mediated by the nervous system. In Macaca arctoides, atropine administration prevents abnormal eye elongation, and this suggests that lid-fusion myopia is caused by excessive accommodation. In M. mulatta, atropine is ineffective; furthermore, myopia develops when lids are sutured after interruption of the optic pathways. Thus, in this species accommodation can be ruled out as a determinant of eye elongation, and other neural mechanisms may be responsible for the refractive error. Our experiments suggest that the refractive state is largely programmed on a genetic basis, but that an abnormal visual experience can disrupt the process of postnatal eye growth and induce axial myopia.

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