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.

Exuberant development of connections, and its possible permissive role in cortical evolution.

The callosal visual connections of the cat provide a model for studying the phenotypes of cortical axons and their differentiation. The terminal arbor of a callosal axon develops in several successive stages. At each stage, the arbor approximates the adult phenotype more closely. This is achieved through two mechanisms: (1) exuberant, but increasingly constrained, growth and (2) partial deletion of previously generated parts of the arbor. This differentiation is controlled by interactions of the axon with its cellular environment, and by visual experience. It might have played a permissive role in the evolution of the cerebral cortex by enabling adjustments of cortical connectivity to changes in the number, size, internal organization and cellular composition of cortical areas.

Organization of immature intrahemispheric connections.

In the adult cat injections of retrograde fluorescent tracers near the border between areas 17 and 18 and extending to the underlying white matter label neurons in restricted parts of nine other ipsilateral visual areas. A very similar, restricted distribution of retrograde labeling is found in newborn kittens when injections near the 17/18 border are confined to the cortical gray matter. When, however, the neonatal 17/18 border injection reaches the underlying white matter, more visual areas and numerous nonvisual areas become labeled, each of them over nearly its whole tangential extent. Labeled nonvisual areas include the primary and secondary auditory areas, the auditory areas of the posterior ectosylvian gyrus, areas 7 and 5, the cingulate gyrus, and the primary and secondary somatosensory areas. The widespread labeling in kittens was not due to larger or differently placed injections, since the distribution and extent of retrograde labeling in the ipsilateral lateral geniculate nucleus were similar at all ages. The transitory projections from the auditory and somatosensory areas are not reciprocated by a projection from areas 17 or 18. In kittens injected around the end of the first postnatal month the distribution of labeled association neurons is similar to that found in the adult; i.e., many of the juvenile projections have been eliminated. Only a few of the transitory axons to areas 17 and 18 enter the gray matter; the others remain confined to the white matter. Some of these axons were anterogradely labeled with rhodamine-B-iso-thiocyanate from the auditory cortex; they show bulbous endings, some of which are probably growth cones. Retrograde double-labeling experiments showed that, in the newborn, some neurons on the lateral sulcus have at least two long collaterals, one running rostrally, the other caudally; such branching is not observed in adults. In conclusion, areas 17/18 receive at birth from a large, continuous territory including areas, or parts of areas, which will later eliminate these projections. Most of the transitory projections do not appear to enter the cortex to any great extent. The major reshaping of association projections occurs before the end of the first postnatal month. The development of association projections resembles that of callosal projections.

The development of the corpus callosum in cats: a light- and electron-microscopic study.

Changes in the size and shape of the corpus callosum (CC)--and in number, size, and structure of callosal axons--between embryonic day 38 (E38) and postnatal day 150 (P150) were studied by light and electron microscope in 25 kittens. The development of the CC was divided into three phases: 1. Embryonic development (E38, 53, 58): At E38, only part of the body of the CC was formed. At E53 and E58, the CC was still very short, but its different parts (genu, body, and splenium) had formed. The cross-sectional callosal area (CCA) was 5.4 mm2 at E53 and 5.6 mm2 at E58. The CC contained 46.3 and 56.4 million axons at E53 and E58 respectively. Mean axon diameters were 0.26 micron at E53 and 0.27 micron at E58. 2. Early postnatal development (P4, 9, 15, 18, 21, 26): The CC at P4 was much longer than at E58 and still slightly elongated during this phase; CCA reached 8.55 mm2 at P4 and 8.88 mm2 at P26. There was a substantial axonal loss (66.8 million at P4 and 52.6 million at P26). From P15 onward, premyelinated and myelinated axons were seen. Mean axon diameter increased from 0.30 micron at P4 to 0.33 micron at P26. 3. Late postnatal development (P39, 57, 92, 107, 150). The CC grew dramatically in both length and thickness, the latter especially in the genu. CCA was 10.1 mm2 at P39 and 15.3 mm2 at P150. The number of axons still decreased (46.5 million at P39 and 31.9 million at P150). The growth of the CCA paralleled the increase of myelinated axons (0.5% at P26 and 29.6% at P150 and in the mean axon diameters (0.34 micron at P39 and 0.42 micron at P150). A number of axonal ultrastructural peculiarities (electron-dense bodies, large vacuoles, lamellated bodies, etc., including those mentioned below) were noticed; their frequency at different ages was estimated as the percent of total axons. Interestingly, accumulations of vesicles inside axons increased from 4.1% at E53 to 8.9% at P26, dropped to 0.2% at P39, and remained below 1% thereafter. Swollen mitochondria increased from 0.2% at E53 to 0.9% at P26 and dropped to 0.06% (on the average) from P39 onward. Accumulations of vesicles and swollen mitochondria increased during the phase of rapid axonal elimination; thus, they may indicate axonal retraction and/or degeneration. Microglia-gitter cells and astrocytes showing signs of phagocytosis were found during the embryonic and early postnatal development and may be involved in axon elimination.

Typology, early differentiation, and exuberant growth of a set of cortical axons.

The corpus callosum interconnects both corresponding (homotopic) and noncorresponding (heterotopic) cortical sites of the two hemispheres. We have studied the axons that establish heterotopic connections from visual areas 17 and 18 (E axons) by using anterogradely transported biocytin and three-dimensional serial reconstructions in adult cats and in kittens. Their site of termination distinguished four types of axons. Type EI ends near the border between areas 19/21a or 7, and type EII near the PMLS/PLLS border (posteromedial and posterolateral lateral suprasylvian areas). Type EIII and EIV terminate the first near the PMLS/PLLS and PMLS/21a borders, and the second near the PMLS/PLLS and 19/21a or 7 borders. Taking into account the previously studied homotopic axons (O axons; Houzel et al. [1994] Eur. J. Neurosci. 6:898-917), it can be concluded that areas 17 and 18 are interhemispherically connected by at least five types of axons, three of which (O, EI, and EII) terminate near one areal border, the other two (types EIII and EIV), near two areal borders. All types terminate near representations of the vertical meridian of the visual field. The different types of axons can be identified already during the first postnatal week; at this age, unlike in the adult, they originate not only near the 17/18 border, but also, transiently, in area 17. This suggests that the developing cortex contains sets of neurons destined to send their axon to different targets; however, the axons grow beyond their sites of adult termination. Indeed, exuberant growth takes place at the stage of axonal elongation, and at subsequent stages of axonal differentiation, i.e., during subcortical branching, intracortical branching and synaptogenesis. The growth is progressively more constrained in its topographic distribution and the axons are subsequently reshaped by regressive events.

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.

Development of projections from auditory to visual areas in the cat.

In newborn kittens, cortical auditory areas (including AI and AII) send transitory projections to ipsi- and contralateral visual areas 17 and 18. These projections originate mainly from neurons in supragranular layers but also from a few in infragranular layers (Innocenti and Clarke: Dev. Brain Res. 14:143-148, '84; Clarke and Innocenti: J. Comp. Neurol. 251:1-22, '86). The postnatal development of these projections was studied with injections of anterograde tracers (wheat germ agglutinin-horseradish peroxidase [WGA-HRP]) in AI and AII and of retrograde tracers (WGA-HRP, fast blue, diamidino yellow, rhodamine-labeled latex beads) in areas 17 and 18. It was found that the projections are nearly completely eliminated in development, this, by the end of the first postnatal month. Until then, most of the transitory axons seem to remain confined to the white matter and the depth of layer VI; a few enter it further but do not appear to form terminal arbors. As for other transitory cortical projections the disappearance of the transitory axons seems not to involve death of their neurons of origin. In kittens older than 1 month and in normal adult cats, retrograde tracer injections restricted to, or including, areas 17 and 18 label only a few neurons in areas AI and AII. Unlike the situation in the kitten, nearly all of these are restricted to layers V and VI. A similar distribution of neurons projecting from auditory to visual areas is found in adult cats bilaterally enucleated at birth, which suggests that the postnatal elimination of the auditory-to-visual projection is independent of visual experience and more generally of information coming from the retina.

Forms and measures of adult and developing human corpus callosum: is there sexual dimorphism?

The sexual dimorphism of the human corpus callosum (CC) is currently controversial, possibly because of difficulties in morphometric analysis. We have reinvestigated the issue by using morphometric techniques specially designed to yield objective measurements of CC size and shape. The development of the CC was studied with similar techniques in order to investigate whether its final shape and size might be influenced by axonal elimination, as could be expected from previous animal studies. We have measured the CCs of 32 men and 26 women; 27 male and 19 female CCs were from brain tissue, the others were from magnetic resonance imaging graphs. Women tended to have 1) a smaller cross-sectional callosal area (CCA); 2) a larger fraction of CCA in the posterior fifth of the CC; 3) more slender CCs; and 4) more bulbous splenia. These differences could not be detected by simple inspection but were demonstrated by measurement and statistical analysis. However, CCA was correlated with the other sexually dimorphic parameters, and the sex-related differences in the latter became nonsignificant when variations in CCA were factored out or when male and female populations with similar CCA were compared. In addition, we analyzed CCs of 16 male and 16 female fetuses and of 13 male and 15 female infants and children. This sample ranged in age between 20 weeks of gestation and 14 years but covered in detail the period up to 14 months after birth. CCA increased throughout the latter period but decreased slightly between about 33 weeks of gestation and the beginning of the second postnatal mouth. This decrease coincided with thinning of the CC and a marked increase in bulbosity of the splenium. No sexual dimorphism could be demonstrated until the beginning of the postnatal period. In the age group between birth (at term) and the 14th month, CCA was, as in the adult, larger in males. Unlike in the adults, the CC was longer in males and the bulbosity index was the same in the two sexes. Axonal elimination may play a role in the perinatal pause in CCA growth and in the concomitant changes in callosal shape.

Cellular aspects of callosal connections and their development.

Detailed visualization, three-dimensional reconstruction, and quantification of individual callosal axons interconnecting the visual areas 17 and 18 of the cat was undertaken in order to clarify the structural basis for interhemispheric interaction. These studies have generated the notion of macro- vs micro-organization of callosal connections. The first refers to the global distribution of callosal connections in the hemisphere as well as to the pattern of area-to-area connections. The latter refers to the fine radial and tangential distributions of individual callosal axons. A discrete disjunctive, 'columnar' pattern of termination of callosal axons, previously unknown for the visual areas, was found. The consequence of caliber and distribution of callosal axons and their branches on the dynamic properties of interhemispheric interactions were analyzed by computer simulations. These studies suggested that callosal axons could synchronize activity within and between the hemispheres in ways relevant for the 'binding' of perceptual features. These new concepts prompted a reexamination of the normal development of callosal connections. The central issue is whether intrinsic developmental programs, or else cellular interactions open to environmental information specify the morphological substrate of interhemispheric interactions. The answer to this question is still incomplete. In development, transient, widespread arbors of callosal axons, which could provide the basis for plastic changes of callosal connections were found in the white matter and the deep cortical layers. On the other hand, growth into the cortex and synaptogenesis of callosal axons appear to be highly, topographically specific albeit not necessarily independent of visual experience.

On nature and limits of cortical developmental plasticity after an early lesion, in a child.

MS is a little girl who suffered severe, bilateral destruction of her primary visual areas at six weeks, after premature birth at 30 weeks. Between the ages of 4.5 and 5.5 years she partially recovered different aspects of visual function, and, in particular, the ability to segregate figures from background, based on texture cues. The recovery might have been due to the compensatory role of the remaining visual areas that could have acquired response properties similar to those of the primary visual areas. This is not supported by the available FMRI (functional magnetic resonance imaging) responses to visual stimuli. Instead, abnormalities in the pattern of stimulus-induced changes of interhemi-spheric EEG-coherence in this patient suggest that her visual callosal connections, and possibly other cortico-cortical connections have re-organized abnormally. Since cortico-cortical connections, including the callosal ones appear to be involved in perceptual binding and figure-background segregation, their reorganization could be an important element in the functional recovery after early lesion, and/or in the residual perceptual impairment.

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.

Maxsim, software for the analysis of multiple axonal arbors and their simulated activation.

In order to analyze the structural organization of complex axonal arbors reconstructed from histological serial sections, and to investigate the functional implications of their geometrical properties, we developed software providing the following facilities: (1) direct importation of data files generated by a commercially available 3-D light-microscopic reconstruction system, including routine procedures for identification and correction of data acquisition errors; (2) real-time 3-D rotations of the arbors in the stack of serial sections; (3) multiple interactive display modes; (4) possibility of modifying diameter and/or connectivity of different branches; (5) simulation of the invasion of the arbor by a single action potential initiated at any chosen point, and visualization of spatio-temporal profiles of activation; (6) extraction of quantitative data converted to standard file formats compatible with available mathematical software. All these tools can be applied to single or multiple axons, individually or simultaneously. The software, called Maxsim, is a highly flexible C-written program running on graphical workstations using the UNIX operating system and X-Window environment.

Some new trends in the study of the corpus callosum.

In recent years the corpus callosum has provided a model for the study of cortical connections in the adult and developing brain. In particular, aspects of development originally described in the corpus callosum could be generalized to other cortical connections. New frontiers include the analysis of the human corpus callosum, studies of callosal connections at the cellular level and the analysis of dynamic interactions between the hemispheres. Gross morphological parameters of the human corpus callosum have been measured and related to gender, handedness etc. The detailed dendritic and axonal morphology of individual callosal neurons and their development is being defined. Electrophysiological investigations and computer stimulations are stressing temporal aspects of the interactions between the hemispheres.

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.

Differential expression of neurofilament subunits in the developing corpus callosum.

In the course of development, corticocortical axons seem to first appear in a labile state from which they either mature into a stable state or are eliminated. These state transitions may be related to cytoskeletal modifications. By immunohistochemistry and immunobiochemistry we found that, in the corpus callosum of the cat, the heavy (200 kDa) subunit of neurofilaments (NF) becomes progressively more visible during the first postnatal month. This aspect of cytoskeletal maturation parallels the developmental loss of callosal axons, i.e. probably the stabilization of the axons which are not eliminated. A similar maturation of the heavy subunit was observed in the visual cortical areas 17 and 18. The medium (150 kDa) and to a lesser extent the light (70 kDa) NF subunits are already present a few days after birth.

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.

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.

Difference in distribution of microtubule-associated proteins 5a and 5b during the development of cerebral cortex and corpus callosum in cats: dependence on phosphorylation.

MAP5, a microtubule-associated protein characteristic of differentiating neurons, was studied in the developing visual cortex and corpus callosum of the cat. In juvenile cortical tissue, during the first month after birth, MAP5 is present as a protein doublet of molecular weights of 320 and 300 kDa, defined as MAP5a and MAP5b, respectively. MAP5a is the phosphorylated form. MAP5a decreases two weeks after birth and is no longer detectable at the beginning of the second postnatal month; MAP5b also decreases after the second postnatal week but more slowly and it is still present in the adult. In the corpus callosum only MAP5a is present between birth and the end of the first postnatal month. Afterwards only MAP5b is present but decreases in concentration more than 3-fold towards adulthood. Our immunocytochemical studies show MAP5 in somata, dendrites and axonal processes of cortical neurons. In adult tissue it is very prominent in pyramidal cells of layer V. In the corpus callosum MAP5 is present in axons at all ages. There is strong evidence that MAP5a is located in axons while MAP5b seems restricted to somata and dendrites until P28, but is found in callosal axons from P39 onwards. Biochemical experiments indicate that the state of phosphorylation of MAP5 influences its association with structural components. After high speed centrifugation of early postnatal brain tissue, MAP5a remains with pellet fractions while most MAP5b is soluble. In conclusion, phosphorylation of MAP5 may regulate (1) its intracellular distribution within axons and dendrites, and (2) its ability to interact with other subcellular components.

Developmental changes in the heavy subunit of neurofilaments in the corpus callosum of the cat.

In the corpus callosum of the cat, the heavy subunit of neurofilaments (NFH) can be demonstrated with the monoclonal antibody NE14, as early as P11, not at P3, and only in a few axons. At P18-19 and more markedly at P29, many more callosal axons have become positive to NE14 and this is similar to what is found in the adult. In contrast, callosal axons become positive to the neurofilament antibody SMI-32 only between P29 and P39 and remain positive in the adult. Treatment with alkaline phosphatase prevents axonal staining with NE14, but results in SMI-32 staining of a few callosal axons as early as P11, but not at P3. Between P11 and P19 the number of axons stained with SMI-32 after alkaline phosphatase treatment increases, in parallel with that of axons stained with NE14. Thus NE14 appears to recognize a phosphorylated form of NFH, while SMI-32 appears to recognize an epitope of NFH which is either masked by phosphate or inaccessible until between P29 and P39, unless the tissue is treated with alkaline phosphatase. These two forms of NFH appear towards the end of the period of massive developmental elimination of callosal axons. They are also synchronous with changes in the spacing of neurofilaments quantified in a separate ultrastructural study. These cytoskeletal changes may terminate the juvenile-labile state of callosal axons and allow further axial growth of the axon.

The development of the anterior commissure in normal and hypothyroid rats.

The development of axon number in the anterior commissure (AC) was analyzed in 39 normal and 37 hypothyroid rats using conventional electron microscopy. Hypothyroid rats underwent antithyroid treatment with methimazole from embryonic day (E) 14 onwards, followed in a fraction of the animals by thyroidectomy at postnatal day (P) 6. In normal rats, the midsagittal cross-sectional anterior commissure area (ACA) increased throughout their life; in hypothyroid rats, ACA was stationary from P4 onwards and at P174-180 it was reduced by 39% relative to normal rats. In normal rats, the number of AC axons increased rapidly from 168,500 at E18 to, on average, 1,049,000 from P4 onwards. Similarly, in hypothyroid rats, the number of axons increased from 135,000 at E18 to, on average, 1,052,000 from P4 onwards. At all ages, the number of axons was similar in normal and hypothyroid rats. During development of the AC, the evolution of axon number observed in normal and hypothyroid rats is different from what was reported for other telencephalic commissures, including the AC of the monkey, where an important fraction of the axons are eliminated postnatally. Antithyroid treatment dissociated ACA from total number of AC axons.