Jaime F. Olavarria

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.

Non-mirror-symmetric patterns of callosal linkages in areas 17 and 18 in cat visual cortex

In the cat, callosal connections in area 17 are largely confined to a 5–6‐mm‐wide strip at the 17/18 border. It is commonly thought that callosal fibers extending from between the 17/18 border regions interconnect loci that are mirror‐symmetric with respect to the midline of the brain, but this idea has not been tested experimentally. The present study examined the organization of callosal linkages in the 17/18 border region of normal adult cats by analyzing the patterns of connections revealed in one hemisphere after small injections of different fluorescent tracers into the opposite 17/18 callosal region. The location of the injection sites within areas 17 and 18 was assessed by examining architectonic data and by inspecting the labeling pattern in the ipsilateral visual thalamus. Area 17 and 18 were separated by a 1 –1.5‐mm‐wide zone of cytoarchitectonic transition rather than by a sharp border.

The retinal input to calbindin-D28k-defined subdivisions in macaque inferior pulvinar

Several studies have provided evidence for direct retinal input to the pulvinar of macaques monkeys, but there is no general agreement regarding the extent of this projection. Moreover, it is not known how retinal input correlates with chemoarchitectonic subdivisions recently recognized within the large, classical divisions of the pulvinar. The potential implications of this correlation have become more evident after reports that chemoarchitectonic subdivisions of the inferior pulvinar (PI) have specific patterns of connections with cortical visual areas. We have therefore re-examined the retino-PI projection using intraocular injections of horseradish peroxides, and correlated it with pulvinar subdivisions revealed using an antibody for calbindin-D28k. Retinal projections were found preferentially within the medial subdivision of the PI, with some involvement of the posterior and central calbindin-D28k defined subdivisions.

Callosal connections correlate preferentially with ipsilateral cortical domains in cat areas 17 and 18, and with contralateral domains in the 17/18 transition zone

Previous studies have shown that the distribution of callosal connections in the 17/18 callosal zone of the cat is patchy at a small scale, but the mechanisms that determine this periodic pattern remain unclear. The present study investigated this issue by correlating the distribution of retrogradely labeled callosal cells with the underlying patterns of ocular dominance columns (ODCs) revealed transneuronally after intraocular injections of wheat germ agglutinin‐horseradish peroxidase. The density of labeled callosal cells was found to vary significantly between adjacent territories dominated by different eyes, indicating that the distribution of callosal cells is significantly biased toward domains that are eye specific. Moreover, callosal connections relate to the pattern of ODCs in a rather unique way: callosal cells correlate preferentially with contralateral ODCs within the 17/18 transition zone (TZ), and with ipsilateral ODCs in regions of areas 17 and 18 located outside the TZ. Similar results were obtained in cats raised with strabismus, indicating that the overlap between right and left ODCs present in normal cats does not influence the correlation between callosal neurons and ODCs. The results are consistent with the hypothesis that callosal linkages are stabilized during development by interhemispheric correlated activity driven by bilateral projections from temporal retina. It is proposed that developmental constraints imposed by both this retinally driven mechanism and the pattern of ODCs are likely to determine not only the association of callosal clusters with specific sets of ODCs, but also important aspects of the functional characteristics of the callosal pathway in cat striate cortex. J. Comp. Neurol. 433:441–457, 2001.

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.

Organization of callosal linkages in visual area V2 of macaque monkey

In visual area V2 of the macaque monkey callosal cells accumulate in finger‐like bands that extend 7–8 mm from the V1/V2 border, or approximately half the width of area V2. The present study investigated whether or not callosal connections in area V2 link loci that are located at the same distance from the V1/V2 border in both hemispheres. We analyzed the patterns of retrograde labeling in V2 resulting from restricted injections of fluorescent tracers placed at different distances from the V1/V2 border in contralateral area V2. The results show that varying the distance of V2 tracer injections from the V1/V2 border led to a corresponding variation in the location of labeled callosal cells in contralateral V2. Injections into V2 placed on or close to the V1 border produced labeled cells that accumulated on or close to the V1 border in contralateral V2, whereas injections into V2 placed away from the V1 border produced labeled cells that accumulated mainly away from the V1 border. These results provide evidence that callosal fibers in V2 preferentially link loci that are located at similar distances from the V1/V2 border in both hemispheres. Relating this connectivity pattern to the topographic map of V2 suggests that callosal fibers link topographically mirror‐symmetrical regions of V2, i.e., callosal fibers near the V1/V2 border interconnect areas representing visual fields on, or close to, the vertical meridian, whereas callosal connections from regions away from the V1/V2 border interconnect visuotopically mismatched visual fields that extend onto opposite hemifields. J. Comp. Neurol. 428:278–293, 2000.

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. J. Comp. Neurol. 459:156–172, 2003.

Distribution of neurons projecting to the superior colliculus correlates with thick cytochrome oxidase stripes in macaque visual area V2.

In visual area V2 of macaque monkeys, cytochrome oxidase (CO) histochemistry reveals a pattern of alternating densely labeled thick and thin stripe compartments and lightly labeled interstripe compartments. This modular organization has been associated with functionally separate pathways in the visual system. We examined this idea further by comparing the pattern of CO stripes with the distribution of neurons in V2 that project to the superior colliculus. Visually evoked activity in the superior colliculus is known to be greatly reduced by blocking magnocellular but not parvocellular layers of the lateral geniculate nucleus (LGN). From previous evidence that V2 thick stripes are closely associated with the magnocellular LGN pathway, we predicted that a significant proportion of V2 neurons projecting to the superior colliculus would reside in the thick stripes.

Reduced retinal activity increases GFAP immunoreactivity in rat lateral geniculate nucleus.

Dynamic regulation of astrocytic processes by the electrical activity of local neurons has been previously described in chick cochlear nucleus. The present study extends this observation by showing that astrocytes in the rat lateral geniculate nucleus (LGN) also increase their immunoreactivity for glial fibrillary acidic protein (GFAP) soon after deprivation of afferent visual neuronal activity. Within 6 h of enucleation, which eliminates a major source of afferent input to the contralateral LGN, GFAP immunoreactivity increases relative to the ipsilateral LGN. A similar increase in GFAP immunoreactivity can be induced by intraocular injections of tetrodotoxin, demonstrating that a reversible manipulation of optic nerve electrical activity is sufficient to regulate LGN astrocytes. This rapid response to activity deprivation is less dramatic than the gliotic reaction observed 3 weeks following deafferentation, by which time afferent terminals have degenerated. These results support the notion that regulation of astrocytic processes by neural activity may play an important role in activity-dependent synaptic regulations in the various sensory systems of vertebrates.

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. J. Comp. Neurol. 496:495–512, 2006.