Unequal alternating monocular deprivation causes asymmetric visual fields in cats.

Kittens were reared so that each eye received normal patterned vision on alternate days. If the eyes received equal periods of stimulation, the visual fields were normal. If one eye received much more experience than the other, the field of the less experienced eye was restricted to the temporal hemifield. This change, which differs from that observed when one or both eyes are deprived continuously of patterned input, suggests that an imbalance in the duration of stimulation can influence the outcome of the normal competitive interaction between pathways from the two eyes and can cause a selective suppression of a portion of the input from the less experienced eye. This suppression may involve the ipsilateral retino-geniculo-cortical pathways or it may involve the entire cortical pathway from the less experienced eye, leaving the colliculus to control responses to visual targets.

Effects of monocular deprivation on geniculocortical synapses in the cat.

In monocularly deprived (MD) cats, many cells in the lateral geniculate nucleus (LGN) but few cells in the visual cortex respond to input from the deprived eye, suggesting that the connections to visual cortex from the deprived geniculate laminae may have been disrupted. I have examined these connections in MD cats by using electron microscopic autoradiography of visual cortex after injections of tritiated lysine into single laminae of LGN. After injections into either deprived or experienced laminae, there was label over terminals that contained mitochondria and round synaptic vesicles and that made asymmetric contacts with dendritic profiles. However, the terminals of deprived afferents differed from those of experienced afferents. They were 25% smaller, contained 33% fewer mitochondria, were more likely to make synapses that were presynaptically convex (and thus, perhaps, immature), and synapsed onto smaller spines. These morphological changes were greater for afferents to upper layer IV than for afferents to lower layer IV. The geniculocortical synapses from deprived laminae were also reduced in number. To correct for variations in injection size and for a probable reduction in protein synthesis by cells in the deprived laminae, I computed the ratio of labeled synaptic terminals to labeled myelinated axons. Injections into the deprived laminae labeled 43% fewer synaptic terminals per labeled myelinated axon than did injections into the experienced lamina. The finding that the synaptic terminals of deprived afferents are both abnormal morphologically and fewer in number can help to explain the reduced effectiveness of the deprived eye in driving cortical cells.

Relay cells, not interneurons, of cat's lateral geniculate nucleus contain N-acetylaspartylglutamate.

N-acetylaspartylglutamate (NAAG) is an endogenous brain dipeptide that satisfies many of the criteria for a neurotransmitter. We have previously identified NAAG immunoreactivity in neurons of the lateral geniculate nucleus (LGN) of the cat and monkey. To determine whether all LGN neurons contain NAAG, we treated sections of cat LGN with affinity-purified antibodies to NAAG and counterstained them with thionin. The larger neurons contained NAAG, but the smaller neurons did not. We treated other sections with antiserum to glutamic acid decarboxylase (GAD), the rate-limiting enzyme in the synthesis of gamma-aminobutyric acid (GABA), in order to label interneurons of the LGN. In these sections, the smaller cells were labeled; the larger neurons were not. We hypothesized that NAAG was present in relay cells, but not interneurons. We used two double-labeling paradigms to test this hypothesis. We combined immunocytochemistry for NAAG using a fluorescent secondary antibody with either (1) fluorescent retrograde tracers (true blue, granular blue, rhodamine beads, or propidium iodide) injected into areas 17 and/or 18 or (2) immunocytochemistry for GAD using a second fluorescent secondary antibody. In the LGN, over 99% of retrogradely labeled cells contained NAAG, but few GAD-positive neurons did. In contrast, neurons of the perigeniculate nucleus contained both NAAG and GAD, demonstrating that staining by one set of antisera did not inhibit staining by the other and that perigeniculate neurons are chemically distinct from the interneurons of the LGN. We conclude that in LGN, the relay cells, which project to visual cortex, contain NAAG, whereas most of the interneurons, which contain GABA, do not.

Development of the dendritic fields of layer 3 pyramidal cells in the kitten's visual cortex.

The cat's visual cortex is immature at birth and undergoes extensive postnatal development. For example, cells of layers 2 and 3 do not complete migration until about 3 weeks after birth. Despite the importance of dendritic growth for synaptic and functional development, there have been few studies of dendritic development in the cat's visual cortex to correlate with numerous studies of functional and synaptic development. Accordingly, we used the Golgi method to study the development of the dendrites of layer 3 pyramidal cells in the visual cortex of a series of cats ranging in age from 2 days to 3 years. Blocks of visual cortex were impregnated by the Golgi-Kopsch method and sectioned in the tangential plane. Layer 3 pyramidal cells were drawn with a camera lucida and analyzed by Sholl diagrams and vector addition. In kittens < 1 week old, these cells were very immature, with only an apical dendrite and no basal dendrites. Basal dendrites appeared during the second week. By 2 weeks, all of the basal dendrites had emerged from the soma, but they had few branches and were tipped with growth cones. By 4 weeks, they had finished branching but continued to grow in length until, by 5 weeks, they reached their adult size. Examination of the basal dendritic fields in the tangential plane revealed that their dendritic fields were more elongated at 2 weeks than at later ages, perhaps because of their smaller size. The distribution of dendritic field orientations was uniform at all ages except 3 and 4 weeks, when there was a preponderance of fields oriented in the rostrocaudal direction. Because dendritic growth and branching occurred very rapidly over a period that precedes and overlaps with the peak periods of synaptogenesis and of sensitivity to the effects of early visual experience, they may depend on afferent visual activity. The early emergence of primary dendrites, however, suggests that this process is independent of afferent activity. The coincident timing of dendritic branching with the presence of dendritic growth cones suggests that branching may occur at growth cones.

Exposure to lines of only one orientation modifies dendritic morphology of cells in the visual cortex of the cat.

To determine whether selective exposure to lines of one orientation modifies the shape of the dendritic fields of cells in visual cortex, we examined the dendritic morphology of neurons in area 17 of five normally reared cats, five cats reared viewing only vertical lines, and three cats reared viewing only horizontal lines. Kittens were placed with their mothers into a totally dark room before their eyes had opened. Beginning at 4 weeks of age, the kittens were brought out for daily periods of exposure wearing masks that limited the vision of each eye to a field of three vertical lines or three horizontal lines. After a minimum of 170 hours of exposure, the animals were killed and blocks of visual cortex were impregnated by the Golgi-Kopsch procedure and cut tangential to the pial surface. Complete neurons from layers III and IV were drawn with the aid of a camera lucida, and the orientations of the dendritic fields wer analyzed using Sholl diagrams. In normal cats, the distributions of the orientations of dendritic fields were uniform, whereas in strip-reared cats, the distributions for the layer III pyramidal cells were shifted. The direction of this shift varied with the experience of the cat: In cats reared viewing only vertical lines, the dendritic fields were oriented orthogonal to the representation of the vertical meridian, and in cats reared viewing only horizontal lines, the fields were oriented parallel to the representation of the vertical meridian. In contrast, the distribution of dentritic orientations for the stellate cells was not affected by stripe-rearing. These results demonstrate a morphological effect of early visual experience that is specific to the particular stimulus presented during rearing and suggest that (1) cortical cells differ in the degree to which they can be modified by such experience, and (2) the dendritic morphology of cortical neurons is related to their preferred orientations.

N-acetylaspartylglutamate immunoreactivity in neurons of the monkey's visual pathway.

The acidic dipeptide N-acetylaspartylglutamate (NAAG) was identified immunohistochemically within neurons of the visual pathways of two adult macaque monkeys which had undergone midsagittal sectioning of the optic chiasm 6 or 9 years earlier. In both temporal and nasal retinae, amacrine cells, including some displaced amacrine cells, expressed NAAG immunoreactivity. In temporal but not nasal retina, retinal ganglion cells were stained, as were their dendrites in the inner plexiform layer, and their axons in the optic nerve fiber layer. In nasal retina, the ganglion cells had degenerated because they were axotomized by the optic chiasm section. In the target regions of the retinal ganglion cells, the superior colliculus and the lateral geniculate nucleus (LGN), both neuropil and cell bodies were stained. In LGN, staining was confined to layers 2, 3, and 5, that is, to the layers innervated by the intact ipsilateral pathway. Immunoreactivity was also seen in the cells of layers 2, 3A, 4B, 5, and 6 of area 17 and layers 3 and 5 of area 18. The neuropil was stained in all layers of area 17, but more heavily in layers 1, 2, 4B, the bottom of 4C beta, 5B, and 6B. Within 4C the staining was patchy; in tangential sections there were alternating bands of light and dark label which matched the ocular dominance bands demonstrated by cytochrome oxidase histochemistry in adjacent sections. This banding pattern is consistent with the presence of NAAG in geniculocortical terminals of the intact ipsilateral pathway and the absence of such terminals for the contralateral pathway, which had undergone transneuronal degeneration due to the optic chiasm sectioning. Overall, our results for monkey are very similar to those in cat and suggest that NAAG or a structurally related molecule may have a prominent role in the communication of visual signals at retinal, thalamic, and cortical levels.

Effects of unequal alternating monocular exposure on the sizes of cells in the cat's lateral geniculate nucleus.

In unequal alternating monocular exposure, each eye receives normal patterned input, but on alternate days and for unequal periods. This imbalance in stimulation produces a behavioral deficit for the less-experienced eye and alters the ability of that eye to activate cortical cells. To determine whether unequal alternating exposure also affects the sizes of cells in the lateral geniculate nucleus (LGN), we measured the cross-sectional areas of geniculate neurons in seven normally reared cats, 14 cats reared with equal alternating exposure, and 17 cats reared with unequal alternating exposure. We found that, in the LGNs of cats reared with unequal alternating monocular exposure, cells in layers that received their input from the less-experienced eye were smaller than those in layers that received their input from the more-experienced eye. This effect was restricted to the binocular segments of the nucleus, and the difference in cell size was a function of the imbalanced exposure, rather than the length of exposure per se. In control groups given balanced alternating exposure, cell size was not correlated with the length of daily exposure. In cats reared with unequal exposure, the change in cell size was greater in the nucleus ipsilateral to the less-experienced eye. Further, the size of the effect was correlated with the size of the imbalance imposed during rearing: Cats reared with a moderate imbalance (8 hours/day vs. 4 hours/day) showed less change in cell size than cats reared with a large imbalance (8 hours/day vs. 1 hour/day). These results are consistent with those of behavorial and physiological studies and strongly suggest (1) that unequal alternating monocular exposure affects the sizes of cells in the LGN by altering the normal competitive balance between the retinogeniculocortical pathways from the left and right eyes, and (2) that the contralateral pathway has some inherent advantage in this competition. We also found a slight shrinkage of cells in the LGNs of cats reared with equal alternating monocular exposure. Since this effect was restricted to the binocular segments of the nucleus, and was not related to the length of exposure given, it was probably caused by the imbalanced binocular competition that occurred during each day's monocular exposure.

Binocular exposure causes suppression of the less experienced eye in cats previously reared with unequal alternating monocular exposure.

In unequal alternating monocular exposure (unequal AME), each eye receives normal patterned visual input but on alternate days and for unequal periods. It has been shown previously that this imbalance in stimulation produces a deficit in the nasal visual field of the less experienced eye (LEE). The effect of subsequent binocular exposure on these visual deficits has now been examined. No evidence of recovery was found. Instead, visual fields remained the same or became smaller. In cats reared with little or no imbalance (8 hr/day vs 7 hr/day or 1 hr/day vs 1 hr), subsequent binocular exposure had no effect on visual fields. In cats reared with a moderate or large imbalance (8 hr/day vs 4 hr/day or 8 hr/day vs 1 hr/day), subsequent binocular exposure led to a suppression of the LEE: when tested binocularly, these cats rarely responded to targets presented in the monocular field of the LEE. The deficits became progressively more severe throughout the period of binocular exposure, until eventually they could be observed even when the LEE was tested monocularly. Most of these cats were clearly esotropic but not all esotropic cats showed suppression. The degree of suppression was correlated with the degree of the imbalance imposed during unequal AME. Our results suggest that when the eyes are misaligned, binocular exposure does not permit recovery of visual function in a disadvantaged eye, but may exacerbate the existing imbalance.

Morphological changes in the geniculocortical pathway associated with monocular deprivation.

To summarize (Fig. 10), the structural consequences of monocular deprivation include the following changes: the relay cells in the binocular segments of the deprived geniculate layers shrink and contain less of the possible neurotransmitter NAAG. These changes appear to be secondary to a loss of terminal synaptic arbor. Certainly, deprived geniculocortical cells project to smaller ocular dominance patches in layer IV of visual cortex, where they make fewer and abnormal synapses. As a result, they activate ocular activation columns that, in addition to being small, are faint and usually fail to extend into extragranular layers. This failure to extend to other layers probably results from a failure of the poorly activated deprived-eye cells in layer IV to compete successfully with neighboring experienced-eye cells in layer IV, resulting in a loss of connections from layer IV to other layers (Fig. 11). Thus, the primary effect of monocular deprivation is probably the disruption of the geniculocortical synapse, with the other changes, such as cell size, and possibly the change in neurotransmitter content, being secondary. The disrupted synapse would result in poorly driven cortical cells and faint ocular activation columns, which in turn would bias a secondary competition for access to cells in extragranular layers. There are certain general principles that unite the findings presented in this chapter with the others in this session. First, there are similarities in the types of morphological changes observed, for example, changes in the number and size of synaptic terminals, as well as mitochondrial changes. This implies that there are similar changes during development and adult plasticity and also similar changes in vertebrates and invertebrates. Second, it is not so much the amount of activity that determines these changes, but the pattern of activity. In my results, the relative imbalance in activity is important, but not the absolute amount (for example, the columns activated by the 8-hr eye of an AME 8/1 are different from those activated by the 8-hr eye of an AME 8/8). Similarly, the binocular segment, where there was an imbalance and competition could occur, was affected, whereas the monocular segment, where there was no imbalance and competition could not occur, was not. Finally, the recent results of Reiter and Stryker suggest that monocular deprivation produces changes only when the activity of the presynaptic cell and the postsynaptic cell are correlated.(ABSTRACT TRUNCATED AT 400 WORDS)

A computer-assisted video technique for preparing high resolution pictures and stereograms from thick specimens.

A computer-assisted video technique is presented for rapidly and accurately gathering, storing and depicting three-dimensional anatomical structures in thick specimens. Several optical sections through the specimen are combined to produce high-resolution photographs with essentially infinite depth-of-field. Further, the depth information implicit in the series of optical sections makes the creation of stereoscopic pairs relatively simple. The technique employs a real-time digitizing frame store and a computer. A video camera is attached to a microscope and successive optical sections are stored digitally as the plane of focus is systematically changed. After storage, the image of each optical section is enhanced to emphasize elements that are sharply focussed. The final two-dimensional image is generated by selecting for each point in the final picture the darkest grey value occurring at the corresponding point in any of the pictures in the through-focus series. A picture with essentially infinite depth-of-field is produced when points of correspondence in the series are determined by a ray passing normal to the plane of optical section. Right and left pictures for a stereoscopic pair are produced when points of correspondence are determined by a ray slanting either left or right of normal. This technique is illustrated with cobalt chloride-filled neurons from whole-mounted cricket ganglia, with HRP-filled axons from whole-mounted goldfish tectum, with Golgi-Kopsch-impregnated neurons from cat visual cortex, and with sections of cobalt chloride-filled antennal afferents in cricket.

[14C]2-deoxyglucose demonstration of the organization of ocular dominance in areas 17 and 18 of the normal cat.

The purpose of this study was to demonstrate the spatial organization of ocular dominance in the visual cortex of the cat. We administered [14C]2-deoxyglucose ([14C]2-DG) to 4 alert, monocularly stimulated cats; one eye had previously been removed from 3 of these cats, and the other cat had received a uniocular injection of tetrodotoxin (TTX). In areas 17 and 18, but not in area 19, we observed alternating regions of heavy and light label, which were clearest in layer IV. Near the representation of the area centralis, especially in the hemisphere ipsilateral to the stimulated eye, the labeled regions formed columns that extended from the pial surface to the white matter. In the representation of peripheral retina, especially in the hemisphere contralateral to the stimulated eye, the pattern was often (but not always) restricted to the middle layers. We conclude that this pattern of label reflects the organization of ocular dominance because: (1) we never observed this pattern in control cats in which both eyes were stimulated or neither eye was stimulated; (2) many characteristics of the pattern are consistent with physiological studies of ocular dominance, and (3) the width and spacing of the alternating label was consistent with the size of the patches of geniculocortical afferents representing the left and right eyes in layer IV of areas 17 and 18.

Removal of the more-experienced eye decreases visual field deficits in cats reared with unequal alternating monocular exposure.

Unequal alternating monocular exposure produces a nasal field deficit for the less-experienced eye, which persists despite prolonged unrestricted binocular exposure. We now report that this deficit decreases after the more experienced eye is removed. Prior to enucleation, the visual field of the less-experienced eye was restricted to the temporal hemifield; 5 months after enucleation of the more-experienced eye, this field extended into the nasal field. Our results are consistent with those in monocularly-deprived cats, and with the occasional recovery of human amblyopes after loss of the fixating eye.

Behavioral and physiological effects of monocular deprivation: a comparison of rearing with diffusion and occlusion.

To compare the effects of monocular deprivation produced by occlusion and diffusion, 9 cats were reared in the dark from birth to 4 weeks of age, when they were brought out for periods of exposure with one eye covered. For 3 cats, the left eye was covered with a white diffuser while the right eye received 8 h of normal patterned exposure (MD/D-8). For 2 cats, the left eye was covered with a black occluder while the right eye was exposed for 8 h (MD/O-8), and for 4 cats, the right eye was covered with a black occluder while the left eye was exposed for 1 h (MD/O-1). Monocular exposure continued until the cats were 3 months old, when they began receiving binocular exposure. For all cats, the visual field of the exposed eye was normal. For the MD/D cats, the field of the pattern-deprived eye was restricted to the monocular crescent, and resembled the fields of monocularly lid-sutured cats. In contrast, for the MD/O cats, the field of the pattern-deprived eye was much larger, extending nearly to the midline. Thus, monocular diffusion produced more restricted visual fields than did monocular occlusion. Preliminary physiological data from the MD/D-8 and MD/O-8 cats showed that more cortical cells responded to stimulation of the pattern-deprived eye in the MD/O-8 cats than in the MD/D-8 cats. Taken together with our earlier results on cats reared with unequal patterned input to the two eyes, these results further suggest that there is a temporal-to-nasal gradient in sensitivity to the effects of an imbalance in stimulation to the two eyes.

N-acetylaspartylglutamate immunoreactivity in neurons of the cat's visual system.

The acidic dipeptide, N-acetylaspartylglutamate (NAAG) was identified immunohistochemically within neurons of the cat's visual system. In the retina, NAAG-like immunoreactivity was observed in some horizontal and amacrine cells at the inner and outer margins of the bipolar cell layer. NAAG-like immunoreactivity was also observed in many retinal ganglion cell bodies, their neurites, and the neuropil of their target areas, the lateral geniculate nucleus (LGN) and the superior colliculus. Additionally, peptide immunoreactivity was also seen in the projection neurons of the LGN, in cells of the pulvinar nucleus, and in the pyramidal cells of layers III and V in areas 17, 18 and 19 of the cerebral cortex. These data suggest that NAAG or a structurally related molecule may have a prominent role in the communication of visual signals at retinal, thalamic and cortical levels.

Effect of eye removal on N-acetylaspartylglutamate immunoreactivity in retinal targets of the cat.

The endogenous brain dipeptide N-acetylaspartylglutamate (NAAG) has previously been demonstrated in the somata of retinal ganglion cells and the neuropil of retinal targets. In this paper we report that the NAAG immunoreactivity of the neuropil in the retinal targets is dependent on an intact optic pathway. Removal of one eye produced a marked decrease in the staining of the neuropil in layer A of the contralateral geniculate nucleus (LGN) and layer A1 of the ipsilateral LGN. There was also decreased staining in the superficial layers of the superior colliculus contralateral to the removal. These results suggest that NAAG is present in the terminals of retinal ganglion cells and is consistent with a role for NAAG in visual synaptic transmission.

Dark-rearing fails to affect the basal dendritic fields of layer 3 pyramidal cells in the kitten's visual cortex.

The development of the cat's visual cortex is incomplete at birth and is influenced by the cat's early visual experience. We have previously demonstrated that the basal dendritic fields of layer 3 pyramidal cells grow substantially during the first 5 weeks after birth and that stripe-rearing affects their orientation. In this paper we determined the effects on these dendritic fields of visual deprivation (dark-rearing) during the first 3 months of life. The visual cortices of both normally reared and dark-reared cats were impregnated by the Golgi method, sectioned in the tangential plane and counterstained. The basal dendritic fields of completely impregnated pyramidal cells from layer 3 were drawn with the aid of a camera lucida, and compared in terms of number and length of primary dendrites, branching, size, elongation, and distribution of dendritic field orientations. Surprisingly, we observed no significant differences in any parameter measured. Thus, although stripe-rearing can specifically alter the orientation of the dendritic fields of the layer 3 pyramidal cells, and dark-rearing has been shown by others to alter the size of layer 4 stellate cells, dark-rearing failed to affect the dendritic fields of layer 3 pyramidal cells.

N-acetylaspartylglutamate immunoreactivity in human retina.

The acidic dipeptide N-acetylaspartylglutamate (NAAG), which satisfies many of the criteria for a neurotransmitter, was identified immunohistochemically within two human retinae. We observed NAAG immunoreactivity in retinal ganglion cells, their dendrites in the inner plexiform layer, and their axons in the optic nerve fiber layer. The vast majority of ganglion cells were stained, including displaced ganglion cells, ganglion cells of different sizes, and those whose dendrites arborized in the inner and outer sublaminae of the inner plexiform layer, that is, presumed On- and Off- cells. The sizes of labeled and unlabeled cells in the ganglion cell layer, as measured in counterstained material, suggest that the unlabeled cells consist primarily or only of displaced amacrine cells. We also saw immunoreactivity in small cells along the inner margin of the inner nuclear layer, presumably amacrine cells, and in small cells with little cytoplasm in the inner plexiform and ganglion cell layers, presumably displaced amacrine cells. These results are consistent with a role for NAAG in the transmission of visual information from the retina to the rest of the brain. Further, they are similar to those reported previously in rat, cat and monkey, thus demonstrating the relevance of previous studies to humans.