Overall patterns of eye-specific retino-geniculo-cortical projections to layers III, IV, and VI in primary visual cortex of the greater galago (Otolemur crassicudatus), and correlation with cytochrome oxidase blobs.

Studies in the greater galago have not provided a comprehensive description of the organization of eye-specific retino-geniculate-cortical projections to the recipient layers in V1. Here we demonstrate the overall patterns of ocular dominance domains in layers III, IV, and VI revealed following a monocular injection of the transneuronal tracer wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). We also correlate these patterns with the array of cytochrome oxidase (CO) blobs in tangential sections through the unfolded and flattened cortex. In layer IV, we observed for the first time that eye-specific domains form an interconnected pattern of bands 200-250 µm wide arranged such that they do not show orientation bias and do not meet the V1 border at right angles, as is the case in macaques. We also observed distinct WGA-HRP labeled patches in layers III and VI. The patches in layer III, likely corresponding to patches of K lateral geniculate nucleus (LGN) input, align with layer IV ocular dominance columns (ODCs) of the same eye dominance and overlap partially with virtually all CO blobs in both hemispheres, implying that CO blobs receive K LGN input from both eyes. We further found that CO blobs straddle the border between layer IV ODCs, such that the distribution of CO staining is approximately equal over ipsilateral and contralateral ODCs. These results, together with studies showing that a high percentage of cells in CO blobs are monocular, suggest that CO blobs consist of ipsilateral and contralateral subregions that are in register with underlying layer IV ODCs of the same eye dominance. In macaques and humans, CO blobs are centered on ODCs in layer IV. Our finding that CO blobs in galago straddle the border of neighboring layer IV ODCs suggests that this novel feature may represent an alternative way by which visual information is processed by eye-specific modular architecture in mammalian V1.

Ocular dominance columns in V1 are more susceptible than associated callosal patches to imbalance of eye input during precritical and critical periods.

In Long Evans rats, ocular dominance columns (ODCs) in V1 overlap with patches of callosal connections. Using anatomical tracers, we found that ODCs and callosal patches are present at postnatal day 10 (P10), several days before eye opening, and about 10 days before the activation of the critical period for ocular dominance plasticity (~P20). In rats monocularly enucleated at P10 and perfused ~P20, ODCs ipsilateral to the remaining eye desegregated, indicating that rat ODCs are highly susceptible to monocular enucleation during a precritical period. Monocular enucleation during the critical period exerted significant, although smaller, effects. Monocular eye lid suture during the critical period led to a significant expansion of the ipsilateral projection from the nondeprived eye, whereas the contralateral projection invaded into, and intermixed with, ipsilateral ODCs innervated by the deprived eye. We propose that this intermixing allows callosal connections to contribute to the effects of monocular deprivation assessed in the hemisphere ipsilateral to the nondeprived eye. The ipsilateral and contralateral projections from the deprived eye did not undergo significant shrinkage. In contrast, we found that callosal patches are less susceptible to imbalance of eye input. In rats monocularly enucleated during either the precritical or critical periods, callosal patches were maintained in the hemisphere ipsilateral to the remaining eye, but desegregated in the hemisphere ipsilateral to the enucleated orbit. Callosal patches were maintained in rats binocularly enucleated at P10 or later. Similarly, monocular deprivation during the critical period had no significant effect on callosal patches in either hemisphere.

Blockade of retinal or cortical activity does not prevent the development of callosal patches normally associated with ocular dominance columns in primary visual cortex.

Callosal patches in primary visual cortex of Long Evans rats, normally associated with ocular dominance columns, emerge by postnatal day 10 (P10), but they do not form in rats monocularly enucleated a few days before P10. We investigated whether we could replicate the results of monocular enucleation by using tetrodotoxin (TTX) to block neural activity in one eye, or in primary visual cortex. Animals received daily intravitreal (P6-P9) or intracortical (P7-P9) injections of TTX, and our physiological evaluation of the efficacy of these injections indicated that the blockade induced by a single injection lasted at least 24 h. Four weeks later, the patterns of callosal connections in one hemisphere were revealed after multiple injections of horseradish peroxidase in the other hemisphere. We found that in rats receiving either intravitreal or cortical injections of TTX, the patterns of callosal patches analyzed in tangential sections from the flattened cortex were not significantly different from the pattern in normal rats. Our findings, therefore, suggest that the effects of monocular enucleation on the distribution of callosal connections are not due to the resulting imbalance of afferent ganglion cell activity, and that factors other than neural activity are likely involved.

Influence of ocular dominance columns and patchy callosal connections on binocularity in lateral striate cortex: Long Evans versus albino rats.

In albino rats, it has been reported that lateral striate cortex (V1) is highly binocular, and that input from the ipsilateral eye to this region comes through the callosum. In contrast, in Long Evans rats, this region is nearly exclusively dominated by the contralateral eye even though it is richly innervated by the callosum (Laing, Turecek, Takahata, & Olavarria, 2015). We hypothesized that the inability of callosal connections to relay ipsilateral eye input to lateral V1 in Long Evans rats is a consequence of the existence of ocular dominance columns (ODCs), and of callosal patches in register with ipsilateral ODCs in the binocular region of V1 (Laing et al., 2015). We therefore predicted that in albino rats input from both eyes intermix in the binocular region, without segregating into ODCs, and that callosal connections are not patchy. Confirming our predictions, we found that inputs from both eyes, studied with the transneuronal tracer WGA-HRP, are intermixed in the binocular zone of albinos, without segregating into ODCs. Similarly, we found that callosal connections in albino rats are not patchy but instead are distributed homogeneously throughout the callosal region in V1. We propose that these changes allow the transcallosal passage of ipsilateral eye input to lateral striate cortex, increasing its binocularity. Thus, the binocular region in V1 of albino rats includes lateral striate cortex, being therefore about 25% larger in area than the binocular region in Long Evans rats. Our findings provide insight on the role of callosal connections in generating binocular cells.

The Effect of Onset Age of Visual Deprivation on Visual Cortex Surface Area Across-Species.

Blindness early in life induces permanent alterations in brain anatomy, including reduced surface area of primary visual cortex (V1). Bilateral enucleation early in development causes greater reductions in primary visual cortex surface area than at later times. However, the time at which cortical surface area expansion is no longer sensitive to enucleation is not clearly established, despite being an important milestone for cortical development. Using histological and MRI techniques, we investigated how reductions in the surface area of V1 depends on the timing of blindness onset in rats, ferrets and humans. To compare data across species, we translated ages of all species to a common neuro-developmental event-time (ET) scale. Consistently, blindness during early cortical expansion induced large (~40%) reductions in V1 surface area, in rats and ferrets, while blindness occurring later had diminishing effects. Longitudinal measurements on ferrets confirmed that early enucleation disrupted cortical expansion, rather than inducing enhanced pruning. We modeled the ET associated with the conclusion of the effect of blindness on surface area at maturity (ETc), relative to the normal conclusion of visual cortex surface area expansion, (ETdev). A final analysis combining our data with extant published data confirmed that ETc occurred well before ETdev.

Visual Interhemispheric and Striate-Extrastriate Cortical Connections in the Rabbit: A Multiple Tracer Study.

Previous studies in rabbits identified an array of extrastriate cortical areas anatomically connected with V1 but did not describe their internal topography. To address this issue, we injected multiple anatomical tracers into different regions in V1 of the same animal and analyzed the topography of resulting extrastriate labeled fields with reference to the patterns of callosal connections and myeloarchitecture revealed in tangential sections of the flattened cortex. Our results extend previous studies and provide further evidence that rabbit extrastriate areas resemble the visual areas in rats and mice not only in their general location with respect to V1 but also in their internal topography. Moreover, extrastriate areas in the rabbit maintain a constant relationship with myeloarchitectonic borders and features of the callosal pattern. These findings highlight the rabbit as an alternative model to rats and mice for advancing our understanding of cortical visual processing in mammals, especially for projects benefiting from a larger brain.

Topography of striate-extrastriate connections in neonatally enucleated rats.

It is known that retinal input is necessary for the normal development of striate cortex and its corticocortical connections, but there is little information on the role that retinal input plays in the development of retinotopically organized connections between V1 and surrounding visual areas. In nearly all lateral extrastriate areas, the anatomical and physiological representation of the nasotemporal axis of the visual field mirrors the representation of this axis in V1. To determine whether the mediolateral topography of striate-extrastriate projections is preserved in neonatally enucleated rats, we analyzed the patterns of projections resulting from tracer injections placed at different sites along the mediolateral axis of V1. We found that the correlation between the distance from injection sites to the lateral border of V1 and the distance of the labeling patterns in area 18a was strong in controls and much weaker in enucleates. Data from pairs of injections in the same animal revealed that the separation of area 18a projection fields for a given separation of injection sites was more variable in enucleated than in control rats. Our analysis of single and double tracer injections suggests that neonatal bilateral enucleation weakens, but not completely abolishes, the mediolateral topography in area 18a.

Deafferentation-induced plasticity of visual callosal connections: predicting critical periods and analyzing cortical abnormalities using diffusion tensor imaging.

Callosal connections form elaborate patterns that bear close association with striate and extrastriate visual areas. Although it is known that retinal input is required for normal callosal development, there is little information regarding the period during which the retina is critically needed and whether this period correlates with the same developmental stage across species. Here we review the timing of this critical period, identified in rodents and ferrets by the effects that timed enucleations have on mature callosal connections, and compare it to other developmental milestones in these species. Subsequently, we compare these events to diffusion tensor imaging (DTI) measurements of water diffusion anisotropy within developing cerebral cortex. We observed that the relationship between the timing of the critical period and the DTI-characterized developmental trajectory is strikingly similar in rodents and ferrets, which opens the possibility of using cortical DTI trajectories for predicting the critical period in species, such as humans, in which this period likely occurs prenatally. Last, we discuss the potential of utilizing DTI to distinguish normal from abnormal cerebral cortical development, both within the context of aberrant connectivity induced by early retinal deafferentation, and more generally as a potential tool for detecting abnormalities associated with neurodevelopmental disorders.

Diffusion tensor imaging detects early cerebral cortex abnormalities in neuronal architecture induced by bilateral neonatal enucleation: an experimental model in the ferret.

Diffusion tensor imaging (DTI) is a technique that non-invasively provides quantitative measures of water translational diffusion, including fractional anisotropy (FA), that are sensitive to the shape and orientation of cellular elements, such as axons, dendrites and cell somas. For several neurodevelopmental disorders, histopathological investigations have identified abnormalities in the architecture of pyramidal neurons at early stages of cerebral cortex development. To assess the potential capability of DTI to detect neuromorphological abnormalities within the developing cerebral cortex, we compare changes in cortical FA with changes in neuronal architecture and connectivity induced by bilateral enucleation at postnatal day 7 (BEP7) in ferrets. We show here that the visual callosal pattern in BEP7 ferrets is more irregular and occupies a significantly greater cortical area compared to controls at adulthood. To determine whether development of the cerebral cortex is altered in BEP7 ferrets in a manner detectable by DTI, cortical FA was compared in control and BEP7 animals on postnatal day 31. Visual cortex, but not rostrally adjacent non-visual cortex, exhibits higher FA than control animals, consistent with BEP7 animals possessing axonal and dendritic arbors of reduced complexity than age-matched controls. Subsequent to DTI, Golgi-staining and analysis methods were used to identify regions, restricted to visual areas, in which the orientation distribution of neuronal processes is significantly more concentrated than in control ferrets. Together, these findings suggest that DTI can be of utility for detecting abnormalities associated with neurodevelopmental disorders at early stages of cerebral cortical development, and that the neonatally enucleated ferret is a useful animal model system for systematically assessing the potential of this new diagnostic strategy.

Role of interstitial branching in the development of visual corticocortical connections: a time-lapse and fixed-tissue analysis.

We combined fixed-tissue and time-lapse analyses to investigate the axonal branching phenomena underlying the development of topographically organized ipsilateral projections from area 17 to area 18a in the rat. These complementary approaches allowed us to relate static, large-scale information provided by traditional fixed-tissue analysis to highly dynamic, local, small-scale branching phenomena observed with two-photon time-lapse microscopy in acute slices of visual cortex. Our fixed-tissue data revealed that labeled area 17 fibers invaded area 18a gray matter at topographically restricted sites, reaching superficial layers in significant numbers by postnatal day 6 (P6). Moreover, most parental axons gave rise to only one or occasionally a small number of closely spaced interstitial branches beneath 18a. Our time-lapse data showed that many filopodium-like branches emerged along parental axons in white matter or deep layers in area 18a. Most of these filopodial branches were transient, often disappearing after several minutes to hours of exploratory extension and retraction. These dynamic behaviors decreased significantly from P4, when the projection is first forming, through the second postnatal week, suggesting that the expression of, or sensitivity to, cortical cues promoting new branch addition in the white matter is developmentally down-regulated coincident with gray matter innervation. Together, these data demonstrate that the development of topographically organized corticocortical projections in rats involves extensive exploratory branching along parental axons and invasion of cortex by only a small number of interstitial branches, rather than the widespread innervation of superficial cortical layers by an initially exuberant population of branches.

Topography and axon arbor architecture in the visual callosal pathway: effects of deafferentation and blockade of TV-methyl-D-aspartate receptors

Visual callosal fibers link cortical loci in opposite hemispheres that represent the same visual field but whose locations are not mirror-symmetric with respect to the brain midline. Presence of the eyes from postnatal day 4 (P4) to P6 is required for this map to be specified. We tested the hypothesis that specification of the callosal map requires the activation of A'-methyl-D-aspartate receptors (NMDARs). Our results show that blockade of NMDARs with MK-801 during this critical period did not induce obvious abnormalities in callosal connectivity patterns, suggesting that retinal influences do not operate through NMDAR-mediated processes to specify normal callosal topography. In contrast, we found that interfering with NMDAR function either through MK801-induced blockade of NMDARs starting at P6 or neonatal enucleation significantly increases the length of axon branches and total length of arbors, without major effects on the number of branch tips. Our results further suggest that NMDARs act by altering the initial elaboration of arbors rather than by inhibiting a later-occurring remodeling process. Since the callosal map is present by P6, just as axonal branches of simple architecture grow into gray matter, we suggest that regulation of arbor development by NMDAR-mediated processes is important for maintaining the precision of this map.

Topography and axon arbor architecture in the visual callosal pathway: effects of deafferentation and blockade of N-methyl-D-aspartate receptors.

Visual callosal fibers link cortical loci in opposite hemispheres that represent the same visual field but whose locations are not mirror-symmetric with respect to the brain midline. Presence of the eyes from postnatal day 4 (P4) to P6 is required for this map to be specified. We tested the hypothesis that specification of the callosal map requires the activation of N-methyl-D-aspartate receptors (NMDARs). Our results show that blockade of NMDARs with MK-801 during this critical period did not induce obvious abnormalities in callosal connectivity patterns, suggesting that retinal influences do not operate through NMDAR-mediated processes to specify normal callosal topography. In contrast, we found that interfering with NMDAR function either through MK801-induced blockade of NMDARs starting at P6 or neonatal enucleation significantly increases the length of axon branches and total length of arbors, without major effects on the number of branch tips. Our results further suggest that NMDARs act by altering the initial elaboration of arbors rather than by inhibiting a later-occurring remodeling process. Since the callosal map is present by P6, just as axonal branches of simple architecture grow into gray matter, we suggest that regulation of arbor development by NMDAR-mediated processes is important for maintaining the precision of this map.

Development of callosal topography in visual cortex of normal and enucleated rats.

In normal rats callosal projections in striate cortex connect retinotopically corresponding, nonmirror-symmetric cortical loci, whereas in rats bilaterally enucleated at birth, callosal fibers connect topographically mismatched, mirror-symmetric loci. Moreover, retina input specifies the topography of callosal projections by postnatal day (P)6. To investigate whether retinal input guides development of callosal maps by promoting either the corrective pruning of exuberant axon branches or the specific ingrowth and elaboration of axon branches at topographically correct places, we studied the topography of emerging callosal connections at and immediately after P6. After restricted intracortical injections of anterogradely and retrogradely transported tracers we observed that the normal, nonmirror-symmetric callosal map, as well as the anomalous, mirror-symmetric map observed in neonatally enucleated animals, are present by P6-7, just as collateral branches of simple architecture emerge from their parental axons and grow into superficial cortical layers. Our results therefore do not support the idea that retinal input guides callosal map formation by primarily promoting the large-scale elimination of long, nontopographic branches and arbors. Instead, they suggest that retinal input specifies the sites on the parental axons from which interstitial branches will grow to invade middle and upper cortical layers, thereby ensuring that the location of invading interstitial branches is accurately related to the topographical location of the soma that gives rise to the parental axon. Moreover, our results from enucleated rats suggest that the cues that determine the mirror-symmetric callosal map exert only a weak control on the topography of fiber ingrowth.

Neonatal enucleation reduces the proportion of callosal boutons forming multiple synaptic contacts in rat striate cortex.

Although bilateral enucleation at birth produces marked abnormalities in the overall distribution and topography of interhemispheric callosal connections in rat visual cortex, it is not known whether it also alters the morphology of callosal synapses. Here we report on the effect of neonatal enucleation on the proportion of callosal boutons making multiple postsynaptic contacts. Synapses were analyzed in adult rats after injections of the anterograde tracer biotinylated dextran amine into the opposite striate cortex. Results show that neonatal enucleation produces a significant reduction in the proportion of callosal boutons making multiple postsynaptic contacts.

Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons.

Cortical projection neurons exhibit diverse morphological, physiological, and molecular phenotypes, but it is unknown how many distinct types exist. Many projection cell phenotypes are associated with laminar fate (radial position), but each layer may also contain multiple types of projection cells. We have investigated two hypotheses: (1) that different projection cell types exhibit characteristic molecular expression profiles and (2) that laminar fates are determined primarily by molecular phenotype. We found that several transcription factors were differentially expressed by projection neurons, even within the same layer: Otx1 and Er81, for example, were expressed by different neurons in layer 5. Retrograde tracing showed that Er81 was expressed in corticospinal and corticocortical neurons. In contrast, Otx1 has been detected only in corticobulbar neurons [Weimann et al., Neuron 1999;24:819-831]. Birthdating demonstrated that different molecularly defined types were produced sequentially, in overlapping waves. Cells adopted laminar fates characteristic of their molecular phenotypes, regardless of cell birthday. Molecular markers also revealed the locations of different projection cell types in the malformed cortex of reeler mice. These studies suggest that molecular profiles can be used advantageously for classifying cortical projection cells, for analyzing their neurogenesis and fate specification, and for evaluating cortical malformations.

Retinal influences specify cortico-cortical maps by postnatal day six in rats and mice.

Studies of callosal projections in striate cortex show that the retina is involved in the development of topographical connections. In normal animals callosal fibers connect retinotopically corresponding, nonmirror-symmetric cortical loci, whereas in animals bilaterally enucleated at birth, callosal fibers connect topographically mismatched, mirror-symmetric loci. Moreover, in rodents the overall pattern of visual callosal connections is adult-like by postnatal day 12 (P12). In this study we delayed the onset of retinal deafferentation in rats and mice in order to determine the period when retinal influences are critically needed for the development of retinotopically matched callosal linkages. Callosal maps were revealed by placing small injections of retrogradely and anterogradely transported tracers into different loci of lateral striate cortex. We found that the patterns of callosal linkages in rats enucleated at P12, P8, and P6 were nonmirror-symmetric, as in normally reared rats. In contrast, the patterns of linkages in rats enucleated at P4 closely resembled the mirror-symmetric pattern seen in rats enucleated at birth (P0). A similar reversal in topography (from symmetric to nonsymmetric) occurred in mice when enucleation was delayed from P4 to P6. These findings indicate that retinal input prior to P6, but not prior to P4, is sufficient for specifying normal callosal topography. Moreover, they suggest that development of retinotopically matched callosal linkages depends critically on retinal influences during a brief period between P4 and P6, when callosal connections are still very immature.