Binocularity in the cat visual cortex is reduced by sectioning the corpus callosum.

In the normal cat, most cells in area 17 can be binocularly driven. Sectioning the corpus callosum results in a significant reduction in binocularly driven cells. Normal binocular vision is thus dependent on the corpus callosum.

Distribution of transitory corpus callosum axons projecting to developing cat visual cortex revealed by DiI.

Functional studies of the development of the corpus callosum in the cat have shown that an intact callosum during postnatal month 1 is necessary for normal visual development. In vivo tracing techniques have not provided enough information on corpus callosum connectivity to fully evaluate the evidence for a morphological mechanism for the functional effects of neonatal callosum section. However, lipophilic in vitro membrane tracers permit a more detailed search for such evidence because the entire limit of many cells can be labeled simultaneously. To investigate the morphological basis for the observed functional results in cats, the corpus callosum was labeled in vitro with the carbocyanine dye, DiI. Crystals of DiI were placed in the midsagittal callosum in tissue from 2 to 277-day-old cats. Tissue was coronally sectioned 3-22 months later. Sections were photographed and reconstructed to show the overall distribution of corpus callosum projections, as well as the locations of individual corpus callosum axons and their presumed terminals. The distribution of corpus callosum projections, examined in cortical areas 17-19, 7, and posterior medial lateral suprasylvian cortex, changes significantly during development. During postnatal week 1, callosal axons extend throughout these cortical areas to layer I. Numerous varicosities on callosal axons are located en passant and at axon terminals in layer I. During postnatal week 2, the density of callosal projections is reduced in all cortical areas, although many axons still extend to layer I. By postnatal month 2, the callosal axons extending to layer I are predominantly near the border with adjacent cortical areas; in the nonborder regions of these areas, many axons extend to layer VI while a much smaller number of axons extend to layers II-V. By postnatal month 3, the callosal projections to supragranular layers are almost exclusively restricted to cytoarchitectonic border regions; in the remaining regions, including medial area 17, there are occasional axons extending to the supragranular layers and only a moderate number of axons extending to infragranular layers. Thus, a substantial number of elaborately formed transitory corpus callosum axons, distributed throughout visual cortex, exist for several weeks during postnatal development; in area 17, these axons are found in central through peripheral visual field representations. The transitory callosal axons appear to have axon terminals in layer I as well as en passant terminals while extending through layers II-VI. If some of these terminals were to form synapses, there would be extensive opportunities for the corpus callosum to provide input to layers I-VI throughout visual cortex during the period of development in which cortical microcircuitry is being established.

Myelination of the corpus callosum in the cat: time course, topography, and functional implications.

The parameters of myelin development were ascertained in two specific regions of the corpus callosum in a series of cats aged 12 postnatal days through adult. The posteriormost portion of the splenium and the anterior-most portion of the genu were examined in cross section by using the electron microscope. Measurements were made to determine the age at which myelin first appeared, the number and distribution of myelinated fibers, the number and distribution of myelin lamellae, and cross-sectional area of ensheathed axons during development. The results indicate that myelination begins and ends earlier in the anterior region of the callosum. Measurements of myelin lamellae indicate similarities between anterior and posterior regions of the callosum, although development occurs earlier in the genu than in the splenium. No evidence was found for a sequence in the size of axons acquiring myelin sheaths, except that extremely small fibers are the last to begin myelinating. Myelination of the splenium of the corpus callosum begins at the very end of the behavioral and physiological critical period for the corpus callosum's role in visual functional development (Elberger: Behav. Brain Res. 11:223-231, '84; Elberger and Smith: Exp. Brain Res. 57:213-223, '85). Since myelination of a pathway is used as an index of functional reliability, this indicates that the basis for the callosal role in developing visual functions is probably not based on its physiological input to visual cortex.

Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry.

The fluorescent carbocyanine dye DiI can be used for retrograde and anterograde labeling of neuronal pathways. To investigate the possible neurochemical identity of DiI-labeled neuronal cell bodies and terminals, we used a procedure for double-labeling of the same tissue with antisera to specific neuroactive substances. This procedure involves visualizing the immunohistochemical label with an FITC-conjugated secondary antiserum. Both labels can be viewed in the same tissue by fluorescence microscopy, and individual cell bodies and processes double-labeled with DiI and antiserum can be identified by switching between filter sets appropriate for rhodamine (to see the DiI labeling) and for fluorescein (to see the immunhistochemical labeling). The method has been used with primary antisera to excitatory and inhibitory amino acid neurotransmitters, as well as to neuropeptides, and is likely to be useful with antibodies against a wide variety of substances. Several other immunocytochemical methods were found to be incompatible with DiI labeling.

A modification of biotinylated dextran amine histochemistry for labeling the developing mammalian brain.

Biotinylated dextran amine (BDA) has proven to be an excellent anterograde tracer in adult mammalian brains, having some advantages over other anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biocytin. However, results are inferior when BDA is used in neonatal mammals. To improve the sensitivity and quality of BDA labeling in neonatal mammalian brains, the tetramethylbenzidine-sodium tungstate (TMB-ST) method for horseradish peroxidase (HRP) histochemistry was modified and used in BDA histochemistry. After BDA application to the visual cortex of neonatal rat and cat, contralateral and ipsilateral cortical and subcortical regions were examined for BDA-labeled exons and terminals. The modified BDA histochemistry produced corpus callosum (CC) axons in neonatal rat and cat that were heavily and continuously labeled. The distribution, trajectories, branching and termination of individual CC axons, and even possible axon-axon contracts, were clearly identified in exquisite detail, even at low magnification. The quality of BDA labeling in the ipsilateral lateral geniculate nucleus and superior colliculus was similar to that of the CC axonal labeling. These results indicate that the modified BDA histochemistry provides a very sensitive and reliable approach to revealing the detailed distribution and morphology of projecting axons and terminals in the developing mammalian nervous system.

HRP reacted with the chromogen o-tolidine produces whole-cell reaction product at light and electron microscope levels: negative effects of sucrose and Golgi staining on benzidine reactions.

Studies of pathway microcircuitry often require electron microscope analysis. To facilitate these analyses, methods for labeling cells in their entirety are extremely useful. Furthermore, such a method would be most useful if the label would be completely confined by the cell membrane so that second labels for synapse identification could be used. No existing method reliably and repeatably produces this kind of a result. In seeking to develop such a method, a seldom-used chromogen for horseradish peroxidase (HRP) was found which produced superlative results for light and electron microscope analysis. o-Tolidine (3,3'-dimethylbenzidine) reacted with HRP produces a very electron-dense reaction product distributed uniformly throughout the cytoplasm and nucleoplasm; membranes are unobscured so that mitochondria, lysosomes, Golgi apparati and endoplasmic reticula are well defined. The reaction product extends into cellular processes of all sizes, including processes with cell bodies not within the plane of section, and is easily visualized at even the lowest electron microscope magnification. The HRP reaction product is completely confined by the cell membrane, thus terminals presynaptic to labeled cells remain distinct. However, the o-tolidine/HRP reaction product is negatively affected by exposure to oxidizers. In tissue exposed to sucrose before or after being reacted with o-tolidine, the HRP reaction product is less electron dense and is found only in lysosomes outside the nucleus or occasionally in proximal cellular processes. The o-tolidine/HRP reaction product is similarly affected when exposed to potassium dichromate for Golgi staining. For some other benzidine compounds, the chromogen/HRP reaction product is also negatively affected by exposure to these chemicals. Therefore, o-tolidine is a superior chromogen for HRP, labeling cells with details similar to that found for cells intracellularly injected with HRP.

Developmental interactions between the corpus callosum and the visual system in cats.

The neonatal corpus callosum is involved in the development of pathways or mechanisms that coordinate the inputs from the two eyes. Several related visual functions are permanently altered by the absence of the callosum during early development. As determined behaviorally and by visual evoked potentials, the normal amount of 90 degrees of the visual field in which both eyes respond to stimulation is almost completely eliminated. Also, there is a reduced number of binocular cells in striate cortical regions representing most of the visual field. In addition, behaviorally measured visual acuity is reduced. Changes in acuity and striate binocularity only result when a callosal section occurs during a critical period of the first postnatal month, and the earlier the surgery, the greater the changes. The lack of myelination during the first postnatal month indicates that the conduction properties of the callosum are poorly developed during its critical period. The pattern of callosal connectivity is probably significant for its role in development, but not all callosal fibers are necessary for normal visual development. Developmental plasticity of callosal connections has been demonstrated for striate cortex, but now it has also been demonstrated for the claustrum. Thus, the callosal role in regions representing both central and peripheral visual field, in neocortical and non-neocortical brain areas should be reassessed.

The existence of a separate, brief critical period for the corpus callosum to affect visual development.

The critical period for the role of the corpus callosum in visual development was explored in terms of the length of time during which the callosum has any influence on the development of visual acuity. Cats were given a surgical section of the posterior corpus callosum at 1, 2, 3, 4 and 29 weeks of age. These, as well as normal and operated control cats, were behaviorally tested for visual acuity thresholds from 5 through 29 weeks of age. Only the 1, 2 and 3 week callosum-sectioned cats showed deficits in visual acuity; the 4 and 29 week callosum-sectioned cats had acuity thresholds equivalent to those of the control cats. These results define a relatively brief critical period of time during which the corpus callosum effectively interacts with the developing visual system. This critical period ends during the fourth postnatal week; throughout the first postnatal month the corpus callosum's effectiveness in altering subsequent visual development gradually declines. Using the amount of acuity deficit as the measure of alteration of visual development, the input of the corpus callosum during the first postnatal month is as critical to visual development as is normal visual input during the first 4-6 postnatal months.

The corpus callosum provides a massive transitory input to the visual cortex of cat and rat during early postnatal development.

Studies of corpus callosum development in cat revealed that the callosum must be intact during postnatal month 1 if normal visual development is to occur [11-20,25]. The use of DiI, a lipophilic carbocyanine dye that is an in vitro membrane tracer, permits a detailed search for morphological evidence to account for these functional results because many cells can be simultaneously labeled in their entirety. To search for morphological evidence, the corpus callosum was labeled in vitro with DiI in tissue from cats aged 2-277 days old [21]. To determine whether there was consistent callosal development in mammals, similar studies were carried out in tissue from rats aged 0 days old through adult [22]. Hemispheres were coronally sectioned 1-24 months later. Sections were reconstructed in photomontages to show the overall distribution of corpus callosum projections, as well as provide details about the locations of individual corpus callosum axons and their presumed terminals. The distribution of corpus callosum projections, examined in visual cortex of cat and rat, changed significantly during development. During early postnatal development, callosal axons extended throughout visual cortex to layer I. Numerous varicosities on callosal axons were located en passant and at axon terminals in layer I. In the following weeks, the density of callosal projections was reduced in all cortical areas, although many axons still extended to layer I. By postnatal month 2 the callosal axons were predominantly near the borders between adjacent cortical areas. Thus, for several postnatal weeks, many elaborately formed transitory corpus callosum axons are distributed throughout visual cortex. The transitory callosal axons appear to have terminals in layers I-VI. If some of these terminals were to for synapses, the corpus callosum could provide an extensive input to layers I-VI throughout visual cortex while the majority of cortical microcircuitry is being established.

Divergent strabismus following neonatal callosal section is due to a failure of convergence.

Eye alignment was measured in neonatal callosum-sectioned cats that were 1-3 years old. Alignment was measured from photographs of the cat's corneal reflex when alert, anesthetized and paralyzed, and by plotting to optic disc separation during paralysis. The callosal alignment was equally divergent when alert and paralyzed and was identical to the control alignment under paralysis. Therefore, the alert callosal divergence results from a failure to converge the eyes.

Binocular properties of lateral suprasylvian cortex are not affected by neonatal corpus callosum section.

The lateral suprasylvian cortex (LS) was physiologically examined in cats with neonatal section of the posterior corpus callosum (CC) at 2 or 3 weeks after birth. All receptive field properties of the LS cells, including ocular dominance, were found to be normal, despite a reduction in binocular activation in area 17 in the 2 week CC cats. Therefore, binocular activation of LS cells is not dependent on callosal input at any age, nor is it dependent on normal levels of binocular activation of striate cortex.

Optically induced strabismus results in visual field losses in cats.

Kittens were reared using goggles containing a prism that simulated a convergent strabismus. A bilateral loss of 30 degrees of the contralateral monocular visual field resulted, indicating that the components of the visual pathways representing the nasal visual field are more sensitive to spatially conflicting visual inputs than the components representing the temporal field. Differences in the extent of binocular visual fields occurred, with alternating fixators demonstrating a full field and unilateral fixators demonstrating a binocular field reduction ipsilateral to the deviating eye.

Neuropeptide Y- and somatostatin-immunoreactive axons in the corpus callosum during postnatal development of the rat.

Corpus callosum (CC) projections in adult mammals were generally thought to be excitatory and to use excitatory amino acids as their transmitters. Little information has been available about the electrical properties and neurochemical status of developing CC connections. The present study investigated the chemical status of rat CC axons during postnatal development by using antibodies to neuropeptide Y (NPY) and to somatostatin (SOM). Both NPY-immunoreactive (ir) and SOM-ir axons were found in the CC of the rat from newborn through adult; however, the number of SOM-ir CC axons is less than that of NPY-ir CC axons at corresponding ages. The density of both NPY-ir and SOM-ir CC axons initially increased, then peaked, and finally decreased to the mature level. In the adult, only a few NPY-ir and SOM-ir CC axons were found in the CC. These results indicate that many NPY-ir and SOM-ir CC axons are transitory during early postnatal development. The results also suggest that the functions of CC connections in adult mammals may be different from that of developing ones. The present results as well as the previous results demonstrate that both developing and mature CC connections are chemically heterogeneous.

Plasticity of claustroneocortical connections via the corpus callosum in the cat.

The cat's distribution of claustral cells that project to the contralateral visual cortex via the corpus callosum was examined. Horseradish peroxidase (HRP) was applied to severed callosal axons to label a heterogeneous population of callosal connections. Cats reared with optically induced strabismus, and Siamese cats, had HRP-filled cells extending more ventrally in the claustrum than in controls. In these groups the compaction of labeled cells was higher than in controls and the amount of increased labeled area was not dependent on the resulting eye alignment. This indicates possible plasticity of visual claustrocallosal connectivity in cats.

Norepinephrine is a presynaptic input to visual corpus callosum cells.

The neurochemical identity of presynaptic inputs to cell bodies of corpus callosum fibers (callosal cells) located in visual cortex of the cat was investigated using a double labeling technique. Callosal cells were labeled by horseradish peroxidase applied to the severed ends of callosal fibers, and [3H]norepinephrine (NE) was injected into the crown of the lateral gyrus corresponding to the visual cortical area 17/18 border to label synapses. The tissue surrounding the injection sites was processed for electron microscope autoradiography. The results show that NE can be localized to cortical synapses contacting callosal cells in visual cortex of the cat.

Confirmation of the existence of transitory corpus callosum axons in area 17 of neonatal cat: an anterograde tracing study using biotinylated dextran amine.

Corpus callosum (CC) axons in visual cortex were labeled anterogradely by in vivo biotinylated dextran amine (BDA) in neonatal cat at postnatal day (PND) 6, 10 and 15. Labeled CC axons were distributed throughout the visual cortex including medial area 17. The number of CC axons in medial area 17 increased from PND 6 to PND 10, and then decreased from PND 10 to PND 15. At PND 15, few CC axons could be followed into the grey matter in medial area 17. Thus, BDA labels transitory CC axons that extend through all cortical layers in medial area 17, confirming the results revealed by in vitro DiI labeling.

Spatial dissociation of visual inputs alters the origin of the corpus callosum.

The distribution of the origin of corpus callosum neurons was investigated in cats reared with an optically induced strabismus by applying horseradish peroxidase (HRP) to severed callosal axons. These animals demonstrated an enlargement of the region of callosal connectivity compared to normal cats. Bilaterally there was an expanded efferent zone, with callosal cell bodies widely distributed in area 17, extending down the medial bank of the lateral gyrus halfway to the fundus of the splenial sulcus. This suggests that rearing a cat with visual spatial dissociation requires additional communication between the hemispheres in the form of increased callosal connections between cortical regions representing more peripheral portions of the visual field.

Comparative commissure function: interocular transfer of successive visual discriminations in cats.

Intact and chiasm-sectioned cats were tested for interocular transfer after monocular training of a successive two-choice discrimination task. Similar tasks were previously employed to demonstrate complete failure of interocular transfer in both commissure-intact pigeons and goldfish. In contrast, both groups of cats showed clear interocular transfer. The results provide evidence for differing functional capacity in analogous interhemispheric pathways among vertebrates, and suggest that interocular transfer of successive visual discriminations may be a suitable paradigm for study of phyletic differences in behavioral ability.

The role of pioneer neurons in the development of mouse visual cortex and corpus callosum.

The primordial plexiform neuropil is very critical to neocortical development. The pioneer neurons, mainly Cajal-Retzius cells in the marginal zone, and subplate neurons in the subplate, differentiate from the primordial plexiform neuropil. In this study, the development of corpus callosum, visual cortex, and subcortical pathways has been observed in C57BL/6 mice with various methods, such as DiI labeling in vitro and in vivo, Dil and DiA in vitro double labeling, immunocytochemistry, and in vivo BrdU and Fast Blue labeling. As early as E14, the primordial plexiform neuropil can be found in the telencephalic wall, and it contains many pioneer neurons. On E15 the primordial plexiform neuropil differentiates into the marginal zone and the subplate. Cajal-Retzius cells exist in the marginal zone, and subplate neurons are in the subplate. Either Cajal-Retzius cells or subplate neurons have long projections toward the ganglionic eminence, suggesting that they migrate tangentially from the ganglionic eminence. Cajal-Retzius cells are involved in radial migration, and subplate neurons participate to guide pathfinding of subcortical pathways. This study reveals how the pioneer neurons, through radial and tangential migration, play an important role in neocortical formation and in the pathfinding of the corpus callosum and subcortical pathways. Furthermore, DiI labeling in vivo has demonstrated the presence of pioneer neurons all along the corpus callosum pathway, especially in the midline. This suggests that pioneer neurons may also play a role in guiding the pathfinding of the corpus callosum.

Neuropeptide Y immunoreactive axons in the corpus callosum of the cat during postnatal development.

Many immunocytochemical studies have identified different types of neurotransmitters localized in the corpus callosum (CC) axons in the adult mammal. Few studies have looked at the development of different neurochemically identified CC systems. Previous studies on the development of cat CC axons have indicated that a large number of transitory CC axons project to the cortex during early postnatal development. The present study focuses on the development of one neurochemically identified group of CC axons in the cat, labeled with an antibody against neuropeptide Y (NPY), to determine if this group participates in transitory CC axonal growth. Cats at specified ages from birth to adulthood were studied with a routine method of immunocytochemistry for antiserum to NPY. NPY-immunoreactive (ir) CC axons were detected at all stages examined, from newborn to adult; the peak density occurred during postnatal weeks (PNW) 3-4. During PNW 1-2, the density of NPY-ir CC axons increased gradually; some NPY-ir axons at this age had growth cones located within the CC bundle between the cerebral hemispheres. The density of the NPY-ir CC axons decreased gradually during PNW 5-7, and from PNW 8 to maturity only a few NPY-ir CC axons were observed. These results indicate that at least two types of NPY-ir CC axons (i.e., transitory and permanent) exist during development, and that most of these axons are eliminated or only express NPY-ir for a short period during development. The results also indicate that neurochemical subsets of CC axons participate in the extensive transitory growth observed by means of the membrane tracer DiI but they may follow unique developmental timetables.