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G M Innocenti

Publications and source records attributed to G M Innocenti.

At least 55 records · Page 3Linked to original sources

A combination of Golgi impregnation and fluorescent retrograde labeling.

Central nervous system structures containing neurons labeled by the fluorescent tracers Fast blue (FB), Diamidino yellow dihydrochloride (DY), Rhodamine B isothiocyanate (RITC) and Rhodamine-labeled latex microspheres (RLM) were processed with the Golgi method. The goal was to improve the visualization of the fluorescent labeled neurons and to allow their ultrastructural examination. While the fluorescence of FB and RITC is greatly attenuated by the Golgi method, RLM and DY are still visible in Golgi-impregnated neurons. However, it is usually necessary to remove the silver precipitate by gold-toning.

Amidines↗

Interchange of callosal and association projections in the developing visual cortex.

Neurons projecting transitorily into the corpus callosum from area 17 of the cat were retrogradely labeled by the fluorescent tracer Fast Blue (FB) injected into contralateral areas 17 and 18 on postnatal days 1-5. During the second postnatal month these neurons were still labeled by the early injection, although they had eliminated their callosal axon. At this time, 15-20% of these neurons could be retrogradely relabeled by injections of Diamidino Yellow (DY) into ipsilateral areas 17 and 18, but few or none by similar injections in the other areas that receive from area 17 (19, 21a, PMLS, 20a, 20b, DLS). Similarly, area 17 neurons projecting transitorily to contralateral area PMLS during the first postnatal week could be relabeled by DY injections in ipsilateral areas 17 and 18 but not in PMLS. Already around birth, many transitorily callosal neurons in area 17 send bifurcating axons both to contralateral areas 17 and 18 and ipsilateral area 18. It is probable that during postnatal development some of these neurons selectively eliminate their callosal axon collaterals and maintain the projection to ipsilateral area 18. In fact, some transitorily callosal neurons in area 17 can be double-labeled by simultaneous perinatal injections of FB in contralateral areas 17 and 18 and of a new long-lasting retrograde tracer, rhodamine-conjugated latex microspheres, in ipsilateral area 18. The same neurons can then be relabeled by reinjecting ipsilateral area 18 with DY during the second postnatal month. This finding, however, does not exclude the possibility that some transitorily callosal neurons send an axon to ipsilateral area 18 after eliminating their callosal axon. In conclusion, area 17 neurons that project transitorily through the corpus callosum later participate, probably permanently, in ipsilateral corticocortical projections but selectively to areas 17-18. The mechanism responsible for this selectivity is unknown, but it may be related to the differential radial distribution (i.e., to birth date) of area 17 neurons engaged in the various corticocortical projections. The problems raised by the use of long-lasting retrograde fluorescent tracers in neurodevelopmental studies and by the quantification of results of double- and triple-labeling paradigms are also discussed.

Action Potentials↗

Postnatal development of corticocortical connections.

In the course of normal development transitory corticocortical projections are formed within each hemisphere and between the two hemispheres. The organization of the transitory projections and the main events in their elimination have been investigated. Experiments on visual areas of the cat indicate that the elimination/stabilization of a fraction of the juvenile projections depends on activity.

Animals↗

Comparison of the distributions of ipsilaterally and contralaterally projecting corticocortical neurons in cat visual cortex using two fluorescent tracers.

Using the retrograde fluorescent tracers Fast Blue and Diamidino Yellow we have studied the callosal and ipsilateral corticocortical connections between the cat's area 17/18 border region and the posteromedial lateral suprasylvian visual area (PMLS), as well as the callosal connections of each of these regions with its contralateral homologue. The main goal was to determine whether single cortical neurons project with branching axons to more than one cortical target. In addition, the double-labeling technique enabled us to examine, within a single section of cortical tissue, the relative distributions of neurons with different cortical targets. Most corticocortical neurons labeled in the area 17/18 border region and in area PMLS projected to only one of the cortical injection sites tested. When two callosal neuron types were labeled in the same area, no double-labeled neurons were found. When ipsilateral corticocortical and callosal neurons were labeled in combination, a few double-labeled neurons were found in both cortical regions examined. The most common type of double-labeled neuron was located in area PMLS and projected bilaterally to the area 17/18 border region. Our findings regarding the laminar distributions of ipsi- and contralaterally projecting neurons are in agreement with previous studies. In addition, we have found that, for callosal neurons within the upper layers of areas 17 and 18, neurons projecting to the contralateral area 17/18 border are located in the lower half of layer II/III and in upper layer IV, whereas neurons projecting to contralateral area PMLS are restricted to the lower portion of layer II/III. In addition, for callosal neurons within the deep layers of area PMLS, neurons projecting to contralateral area PMLS are located throughout layers V and VI, whereas neurons projecting to the contralateral area 17/18 border are restricted to layer VI. There are numerous other possible targets for axon collaterals not examined in this paper. However, the scarcity of neurons with multiple projections demonstrated in this study reflects the high degree of specificity of cortical connectivity. This anatomical organization may be the basis for a precise channeling of differential information at the single neuron level.

Amidines↗

Maturation of visual callosal connections in visually deprived kittens: a challenging critical period.

The number of callosally projecting neurons (callosal neurons) which can be labeled in cortical areas 17 and 18 by horseradish peroxidase (HRP), injected in the contralateral visual cortex, is reduced to about 50% of normal in cats reared with their eyelids bilaterally sutured. In the same animals the density of HRP anterogradely transported to areas 17 and 18 is also decreased. The apparent loss of callosal neurons is limited to layers III and IV (subzone a), whereas layer VI (subzone c) is unaffected. The effect is obtained after 3 months or more but not after 1 month of deprivation. Two months of visual experience following deprivation do not restitute a normal number of callosal neurons. However, 10 days of normal visual experience preceding the deprivation are sufficient to prevent the effects of the latter. Animals deprived of vision after a short period of normal visual experience and animals allowed normal vision after 1 month of visual deprivation have a more widespread distribution of callosal neurons than do normal animals; in this way they are similar to previously described cats reared with convergent or divergent strabismus, monocular enucleation, or monocular eyelid suture. The results suggest that: vision is actively responsible for both the maintenance and the elimination of fractions of the juvenile callosal connections; the elimination which normally takes place during the second postnatal month requires normal binocular vision; and activity-dependent competition between callosal and other axons can explain the role of vision.

Animals↗

The organization of immature callosal connections.

In newborn kittens, the anterograde transport of horseradish peroxidase, alone or bound to wheat-germ agglutinin, indicates that callosal axons have entered selectively the restricted portions of the neocortical gray matter (e.g., the area 17/18 border) which receive callosal afferents in adults. The callosal axons do also reach regions where they lack in the adult, but there they seem not to penetrate far into the gray matter. Neonatal injections of retrograde fluorescent tracers restricted to the gray matter in areas 17, 18, and posteromedial lateral suprasylvian area (PMLS) label neurons in the contralateral hemisphere only when the tracers were directed into regions known to receive callosal axons. In particular, injections near the 17/18 border label neurons in the contralateral hemisphere at the homologous site and at restricted, retinotopically corresponding locations in other visual areas: a pattern similar to the adult one. In contrast, an injection reaching the white matter of areas 17 or 18 labels a wider, continuous territory extending mediolaterally over most visual areas from 17 to posterolateral lateral suprasylvian area (PLLS) and including regions which later become acallosal; in addition, labeled neurons are found in the limbic cortex medial to area 17 and in the auditory cortex lateral to PLLS, none of which is known to project to either 17 or 18 in the adult. In flattened reconstructions of the cortex, the shape of the territory labeled by each of these injections is characteristically, although somewhat irregularly, crescent shaped; its rostrocaudal position varies with that of the injection. An injection extending into the white matter of more lateral visual areas (19, 21a, PMLS) labels callosal neurons over a similar territory, which extensively overlaps that labeled by the 17/18 border injections and likewise includes regions which are acallosal in the adult. In spite of the overlapping distribution of labeling obtained from separate injection sites, as in adults, each cytoarchitectonically (or retinotopically) defined area seems to receive from a different set of neurons, although a few neurons send bifurcating axons to more than one area. In conclusion, injections restricted to the cortical gray matter reveal a topographic organization of juvenile callosal connections similar to that of the adult. In contrast, injections extending into the white matter and adequate to reach the transitory callosal axons which appear to be confined there reveal what appears to be an earlier organization. These two organizations probably reflect different morphogenetic factors.

Animals↗

Bilateral transitory projection to visual areas from auditory cortex in kittens.

A transitory projection from primary and secondary auditory areas to the contralateral and ipsilateral areas 17 and 18 exists in newborn kittens. Distinct neuronal populations project to ipsilateral areas 17-18, contralateral areas 17-18 and contralateral auditory cortex; they are at different depth in layers II, III, and IV. By postnatal day 38 the auditory to visual projections have been lost, apparently by elimination of axons rather than by neuronal death. While it was previously reported that the elimination of transitory axons is responsible for focusing the origin of callosal connections to restricted portions of sensory areas it now appears that similar events play a more general role in the organization of cortico-cortical networks. Indeed, the elimination of juvenile projections is largely responsible for determining which areas will be connected in the adult.

Animals↗

Multiple sets of visual cortical neurons projecting transitorily through the corpus callosum.

In areas 17 and 18 of adult cats only a few neurons send bifurcating axons to more than one contralateral area or to areas in both hemispheres; most neurons seem to project to only one area. We now found that such a selective connectivity is already present at birth, i.e. several weeks before transitory callosal axons are eliminated. This is true even for those portions of cortex which will lose access to the corpus callosum: in particular different neurons project transitorily from medial area 17 to different contralateral areas. Thus cortical neurons may be induced to project to specific targets by a mechanism operating long before (and possibly independent of) the axon elimination. The latter may, however, be responsible for the final topographic organization of the connections.

Animals↗

Is there a genuine exuberancy of callosal projections in development? A quantitative electron microscopic study in the cat.

The main tract of interhemispheric connections, the corpus callosum, is now suspected to contain more axons at birth than in adulthood. This notion is based on results obtained with retrograde pathway tracing techniques, but this indirect approach has several shortcomings. Since the elimination of projections during development now seems to be a general phenomenon, probably a crucial one in the establishment of connections, we have examined the development of the corpus callosum using quantitative electron microscopy. An average of 70% of the callosal axons present at birth are eliminated by adulthood in the cat. We have also calculated a new figure of 23 million axons in the adult cat corpus callosum, which is over 4 times greater than the currently accepted figure.

Animals↗

Transitory macrophages in the white matter of the developing visual cortex. I. Light and electron microscopic characteristics and distribution.

Injections of horseradish peroxidase (HRP) into the occipital cortex of the kitten and diffusing to the white matter label a widely distributed microglial population and in addition, cells with light and electron microscopic features of 'gitter cells'. The latter are concentrated in a complex and highly consistent system of interconnected clusters in the white matter of the lateral, postlateral, middle suprasylvian and posterior ectosylvian gyri, as well as on the roof of the lateral ventricle. The 'gitter cells' have the ultrastructural and (as described by others) chemical characteristics of macrophages, and may be involved in the elimination of transitory axons.

Aging↗

Transitory macrophages in the white matter of the developing visual cortex. II. Development and relations with axonal pathways.

Clusters of 'gitter cells' develop in the white matter of the occipital cortex of the cat at the end of the first postnatal week. These clusters, and others already present at birth, disappear by the end of the first postnatal month. The life span of the clusters in the occipital white matter corresponds to the period when transitory callosal axons are eliminated. The clusters have close contact with callosal axons and can be labeled by HRP injected in the contralateral hemisphere and transported through the corpus callosum. One of the clusters clearly forms in a part of the white matter crossed by transitory callosal axons. The 'gitter cells' might be involved in the elimination of these axons. Consistent with this hypothesis, ultrastructural observations show groups of axons completely surrounded by 'gitter cell' cytoplasm as if they were being phagocytosed.

Aging↗

Growth and reshaping of axons in the establishment of visual callosal connections.

The visual cortical areas in the two hemispheres are interconnected by axons running through the corpus callosum. In adult cats, these axons originate from, and terminate in, tangentially restricted portions of each area. In young kittens, however, callosal axons originate from the entire extent of each area, although they apparently enter the gray matter only in the restricted regions where they will also be found in adults. In kittens, but not in adults, callosal axons also reach other regions, but there they appear to be confined to the lowest part of layer VI. During the first two postnatal months, the callosal efferent zones become progressively restricted to their adult locations. During this process, many neurons eliminate the axons (or axon collaterals) that they had formerly sent through the corpus callosum and form permanent connection ipsilaterally.

Age Factors↗

The arborization of single callosal axons in the mouse cerebral cortex.

After several large cortical injections of horseradish peroxidase, individual callosal axons could be observed in most cortical areas contralateral to the injected hemisphere. They left the white matter and travelled for various distances (up to 2 mm) deep in layer VI, then turned to penetrate the cortex radially or obliquely, giving collaterals to several layers and forming narrow terminal arborisations in supragranular layers. In addition, callosal fibers were seen predominantly in deep cortical layers, which fibers could be interpreted either as collaterals of the thick fibers or as a distinct class of callosal afferents.

Afferent Pathways↗

The postnatal development of somatosensory callosal connections after partial lesions of somatosensory areas.

The distribution of S1 (first somatosensory area) and S2 (second somatosensory area) neurons projecting to the contralateral S2 was studied with horseradish peroxidase in normal adult cats and in cats aged between 129 and 248 days in which the injected S2 area had been deprived of some of its input by an earlier lesion (on postnatal days 3 to 30; day of birth = day 1) of ipsilateral S1, alone or combined with a lesion of contralateral S2. In animals with S1 lesions, as in the normal controls, labeled neurons were selectively distributed to the regions of the trunk representation and to parts of the forelimb and hindlimb representations; however, the normally acallosal region in the forepaw representation contained scattered labeled neurons in three of the four animals whose S1 had been lesioned during the first postnatal week. In these animals, the distribution of labeled neurons in the contralateral S2 was apparently normal. Furthermore, the additional lesion of this area during the first postnatal week (one animal) did not increase the degree of filling-in of the normally acallosal parts of S1. The partial filling-in of the acallosal parts of S1 is probably due to the preservation to adulthood of some of the callosal neurons which are present in these regions during the early postnatal life. Possibly, these neurons did not disappear (or lose their callosal axons) because the neonatal lesion (i) allowed their successful competition for terminal space in contralateral S2 or (ii) induced a reorganization of the peripheral input to this area.

Afferent Pathways↗

Postnatal shaping of callosal connections from sensory areas.

Horseradish peroxidase (HRP) was injected unilaterally into the first and second visual areas (V1 and V2; areas 17 and 18) of 20 kittens aged between 2 and 90 days and into the second somatosensory area (S2) of 16 kittens aged between 1 and 52 days. The radial and tangential (normal and parallel to the pial surface, respectively) distributions of neurones giving origin to callosal axons (callosal neurones) were studied. In adult cats, callosal efferent zones (CZs) are defined by the distribution of callosal neurones. CZs occupy in the visual cortices, tangentially and radially restricted parts of areas 17, 18, 19 of the lateral suprasylvian gyrus and in the somatosensory cortices, parts of S1 and S2. At birth, callosal neurones are distributed throughout the tangential extent of visual and somatosensory areas; they are also more widespread in depth than in the adult. During the first postnatal month, as a result of the gradual disappearance of callosal neurones from parts of the visual and somatosensory areas, the adult CZs emerge. The CZ in areas 17 and 18 undergoes a further tangential reduction during the second and third postnatal months.

Aging↗

The postnatal development of visual callosal connections in the absence of visual experience or of the eyes.

Counts of callosal neurons retrogradely labeled by horseradish peroxidase (visualized using multiple substrates) were obtained in areas 17 and 18 of five kittens reared with their eyelids bilaterally sutured and of three kittens which had undergone bilateral enucleation on postnatal days 1--4. These counts were compared with those obtained in normal adult cats. The normal adult distribution of the callosal neurons results from the gradual postnatal reduction of a more widespread juvenile population. Binocular visual deprivation by lid suturing dramatically decreases the final number of callosal neurons and narrows their region of distribution (callosal zone) in areas 17 and 18. A less severe reduction in the final number of callosal neurons is caused by bilateral enucleation, which also increases the width of the callosal zone compared to that of normal cats. Thus, visual experience is necessary for the normal stabilization of juvenile callosal connections. However, since some callosal neurons form connections in the absence of vision, other influences capable of stabilizing juvenile callosal neurons also exist. These influences are probably antagonized by destabilizing influences or inhibited, when the eyes are intact.

Aging↗