Sharon L. Juliano

Regulation of neural progenitor cell development in the nervous system.

The mammalian telencephalon, which comprises the cerebral cortex, olfactory bulb, hippocampus, basal ganglia, and amygdala, is the most complex and intricate region of the CNS. It is the seat of all higher brain functions including the storage and retrieval of memories, the integration and processing of sensory and motor information, and the regulation of emotion and drive states. In higher mammals such as humans, the telencephalon also governs our creative impulses, ability to make rational decisions, and plan for the future. Despite its massive complexity, exciting work from a number of groups has begun to unravel the developmental mechanisms for the generation of the diverse neural cell types that form the circuitry of the mature telencephalon. Here, we review our current understanding of four aspects of neural development. We first begin by providing a general overview of the broad developmental mechanisms underlying the generation of neuronal and glial cell diversity in the telencephalon during embryonic development. We then focus on development of the cerebral cortex, the most complex and evolved region of the brain. We review the current state of understanding of progenitor cell diversity within the cortical ventricular zone and then describe how lateral signaling via the Notch‐Delta pathway generates specific aspects of neural cell diversity in cortical progenitor pools. Finally, we review the signaling mechanisms required for development, and response to injury, of a specialized group of cortical stem cells, the radial glia, which act both as precursors and as migratory scaffolds for newly generated neurons.

Astroglial permissivity for neuritic outgrowth in neuron-astrocyte cocultures depends on regulation of laminin bioavailability.

The molecular determinants underlying the failure of axons to regenerate in the CNS after injury were studied in an in vitro model of astrogliosis and neuronal coculture. Mechanically lesioned neuron–astrocyte mouse cortical cocultures were treated with antisense glial fibrillary acidic protein (GFAP)‐mRNA in order to inhibit the formation of gliofilaments that occurs in response to injury. This inhibition relieves the blockage of neuron migration and neuritic outgrowth observed after lesion, and migrating neurons reappeared, supported by a laminin‐labeled extracellular network (permissive conditions). We then questioned the relationship between this permissivity and laminin production. Follow‐up studies on the concentration of laminin indicated that, after antisense treatment, the laminin level was increased in the cocultures and was under the control of astrocyte–neuron interactions. The addition of exogenous laminin favored neuronal migration and neurite outgrowth, whereas neutralizing laminin bioavailability with antibodies recognizing the astroglial laminin resulted in an inhibition of both neuronal access to the lesion site and neurite outgrowth, suggesting an active role for laminin in the permissive process. This permissive process could be associated with modulation of extracellular matrix (ECM) molecule degradation by proteinases. Among the latter, matrix metalloproteinases (MMPs) are involved in the breakdown of the ECM component. Our investigation showed a net decrease of the matrix metalloproteinase MMP‐2 expression and activity and an increase of its endogenous inhibitor TIMP‐2 expression. Both proteins associated with permissivity should be involved in the laminin stabilization and cell‐matrix interactions. High levels of laminin and laminin bioavailability, consequent to a reduction in astrogliosis, may be important permissive elements for neuronal migration and neurite outgrowth postlesion. GLIA 37:105–113, 2002.

Histogenesis of ferret somatosensory cortex

The ferret has emerged as an important animal model for the study of neocortical development. Although detailed studies of the birthdates of neurons populating the ferret visual cortex are available, the birthdates of neurons that reside in somatosensory cortex have not been determined. The current study used bromodeoxyuridine to establish when neurons inhabiting the somatosensory cortex are generated in the ferret; some animals also received injections of [3H]thymidine. In contrast to reports of neurogenesis in ferret visual cortex, most neurons populating the somatosensory cortex have been generated by birth. Although components of all somatosensory cortical layers have been produced at postnatal day 0, the layers are not distinctly formed but develop over a period of several weeks. A small number of neurons continue to be produced for a few days postnatally. The majority of cells belonging to a given layer are born over a period of approximately 3 days, although the subplate and last (layer 2) generated layer take somewhat longer. Although neurogenesis of the neocortex begins along a similar time line for visual and somatosensory cortex, the neurons populating the visual cortex lag substantially during the generation of layer 4, which takes more than 1 week for ferret visual cortex. Layer formation in ferret somatosensory cortex follows many established principles of cortical neurogenesis, such as the well‐known inside‐out development of cortical layers and the rostro‐to‐caudal progression of cell birth. In comparison with the development of ferret visual cortex, however, the generation of the somatosensory cortex occurs remarkably early and may reflect distinct differences in mechanisms of development between the two sensory areas. J. Comp. Neurol. 387:179–193, 1997.

Fine-tuning of Neurogenesis Is Essential for the Evolutionary Expansion of the Cerebral Cortex

We used several animal models to study global and regional cortical surface expansion: The lissencephalic mouse, gyrencephalic normal ferrets, in which the parietal cortex expands more than the temporal cortex, and moderately lissencephalic ferrets, showing a similar degree of temporal and parietal expansion. We found that overall cortical surface expansion is achieved when specific events occur prior to surpragranular layer formation. (1) The subventricular zone (SVZ) shows substantial growth, (2) the inner SVZ contains an increased number of outer radial glia and intermediate progenitor cells expressing Pax6, and (3) the outer SVZ contains a progenitor cell composition similar to the combined VZ and inner SVZ. A greater parietal expansion is also achieved by eliminating the latero-dorsal neurogenic gradient, so that neurogenesis displays a similar developmental degree between parietal and temporal regions. In contrast, mice or lissencephalic ferrets show more advanced neurogenesis in the temporal region. In conclusion, we propose that global and regional cortical surface expansion rely on similar strategies consisting in altering the timing of neurogenic events prior to the surpragranular layer formation, so that more progenitor cells, and ultimately more neurons, are produced. This hypothesis is supported by findings from a ferret model of lissencephaly obtained by transiently blocking neurogenesis during the formation of layer IV.

A normal radial glial scaffold is necessary for migration of interneurons during neocortical development

The relationship between radial glia and neurons migrating tangentially from the ganglionic eminence (GE) has been suggested but not firmly established. To study this relationship we used a ferret model of cortical dysplasia where radial glia are highly disorganized. To produce this, an antimitotic, methylazoxy methanol (MAM) is injected on the 24th day of gestation (E24 MAM). Neurons migrating away from the GE in MAM‐treated animals tend to remain in the intermediate zone (IZ) and do not reach the cortical plate (CP) as they do in normal ferret slices. We recently observed that the disrupted radial glia after MAM treatment could be restored toward their normal morphology by exogenous application of neuregulin1 (NRG1). We demonstrate here that when E24 MAM slices are treated with NRG1, the distribution of cells arising from the GE was similar to normal slices. In a second paradigm, we disrupted radial glia by adding ciliary neurotrophic factor (CNTF) to the culture media of normal ferret slices; CNTF induces acute differentiation of radial glia into astrocytes. After CNTF exposure, few tangentially migrating cells reach the CP compared to untreated slices. These results show that interneurons fail to reach the CP by disrupted normal radial glia and restoring the normal radial glial scaffold is sufficient to allow migrating cells to invade the CP. Our results suggest an important role for radial glia by controlling directly or indirectly the migration of interneurons to the CP, their main target.

Development of local connections in ferret somatosensory cortex

Ferrets have become recognized as a useful and interesting model for study of neocortical development. Because of their immaturity at birth, it is possible to study very early events in the ontogeny of the brain. We used living slices of ferret somatosensory cortex to study the formation and development of intrinsic elements within the neocortex. A small number of fixed, hemisected brains injected with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) were also used. The slices were obtained from ferret kits aged postnatal day (P)1 to P62 and maintained in a chamber; each slice received injections of fluorescent‐labeled dextrans. The injections were made at different ages in several distinct sites, which included the proliferative ventricular zone, the intervening white matter (or intermediate zone), and different sites of developing cortex, including the deeper cortical plate, which incorporated the subplate in young animals, and more superficial cortical sites, depending on the age of the animal. Several animals also received injections into the ventrobasal thalamus. Injections into young animals (P1–7) produced a dominant radial pattern that extended from the ventricular zone into the cortex. Injections into the ventricular zone labeled many cells that appeared morphologically like radial glia as well as presumptive neurons. Although the predominant pattern was radial, injections in the ventricular zone often produced tangentially oriented cells and horizontally arranged fibers at the outer edge of the proliferative zone. These cells and fibers may provide a substrate for tangential dispersion of neurons within the neocortex. More superficial injections within the slice labeled lines of cells that appeared to be stacked upon one another in a radial pile in the cortex; the cortical plate received very few lateral projections. Data obtained from more mature slices indicated that, although the overall pattern of staining remained radial, the precise character of the pattern changed to include more lateral spread into surrounding cortex, which eventually refined and developed into distinct patches by P28, when the overall cortical architecture appeared adult like. The data involving thalamocortical connections were more limited, but they indicated that the thalamus projects precisely to the somatosensory cortex in a point‐to‐point fashion from the earliest date studied (P0) and that the ventrobasal nucleus terminates upon the somatosensory cortex in a patchy manner during the early postnatal days of development. This study of the development of the somatosensory cortex confirms the ubiquitous nature of column‐like connections throughout the neocortex and provides a novel view of the radial nature of early neocortical maturation.

Organization of the forepaw representation in ferret somatosensory cortex

Little is known about the function and structure of ferret somatosensory cortex. We used anatomical methods and multi-unit recordings to characterize the cytoarchitecture, functional responses and topography of the forepaw representation in ferret somatosensory cortex. The representation of the cutaneous ferret forepaw encompasses approximately the caudal half of the posterior sigmoid gyrus. The posterior sigmoid gyrus and coronal sulcus contain unique cytoarchitectonic fields that conform in large part to earlier descriptions of somatosensory regions in the cat. The cytoarchitectonic regions form mediolateral bands that comprise areas 4, 3a, 3b, 1, and 2 (from rostral to caudal). We studied most extensively areas 3a and 3b for functional responses to somatic stimuli; our data indicate that ferret somatosensory cortex contains at least two representations of the forepaw in these two areas. Our data also suggest that within ferret somatosensory cortex, the morphological and submodality features gradually, rather than abruptly, distinguish themselves as unique cortical fields.

A radialization factor in normal cortical plate restores disorganized radial glia and disrupted migration in a model of cortical dysplasia

Treatment of pregnant ferrets on embryonic day 24 (E24) with the antimitotic methylazoxy methanol (MAM) leads to a specific constellation of effects in newborn kits, which include a very thin and poorly laminated neocortex, disruption of radial glial cell morphology with early differentiation into astrocytes, and abnormal positioning of Cajal–Retzius cells. We suggest that MAM treatment on E24 results in this model of cortical dysplasia by eliminating a population of cells that produce a factor capable of maintaining radial glia in their normal morphology. The abnormal radial glia, either alone or in combination with other abnormal features, are likely to prevent proper migration into the cortical plate. To test the possibility that normal cortex can provide the missing substance that influences radial glia, slices of E24 MAM‐treated cortex were removed at postnatal day 0 (P0) and cultured adjacent to explants of P0 normal cortical plate. By labelling a small number of cells with injections of fluorescent dextrans into the cultured slices, we found that abnormal radial glia in MAM treated slices cocultured adjacent to normal cortical plate were restored toward normal, in comparison to E24 MAM treated slices cultured alone and in other control conditions. We also found that abnormally positioned Cajal–Retzius cells move into the marginal zone and that neurons are able to migrate into the cortical plate more effectively in the coculture condition. These data indicate that normal cortical plate of ferrets contains a factor causing radial glia to maintain their elongated morphology; the improved position of radial glia encourages repositioning of Cajal–Retzius cells and improved neuronal migration into the cortical plate.

Basal forebrain lesions alter stimulus-evoked metabolic activity in rat somatosensory cortex

Acetylcholine (ACh) has been suggested to play a crucial role in normal cortical functioning. To assess the impact of cortical ACh depletion on metabolic activity in the barrel field of rat somatosensory cortex, unilateral lesions of the basal forebrain were made. One to 9 weeks later, a 2-deoxyglucose experiment was conducted. Stimulus-evoked metabolic activity in the barrel field ipsilateral to the lesion was significantly reduced compared with the contralateral side. These results suggest that ACh plays a significant role in processing stimulus-evoked sensory information.

Endogenous Neuregulin Restores Radial Glia in a (Ferret) Model of Cortical Dysplasia

Radial glia are integral components of the developing neocortex. During corticogenesis, they form an important scaffold for neurons migrating into the cortical plate. Recent attention has focused on neuregulin (NRG1), acting through erbB receptors, in maintaining their morphology. We developed a model of developmental radial glial disruption by delivering an antimitotic [methylazoxy methanol (MAM)] to pregnant ferrets on embryonic day 24 (E24). We previously found that normal ferret cortex contains a soluble factor capable of realigning the disorganized radial glia back toward their normal morphology. Characterization of the reorganizing activity in normal cortex demonstrated that the probable factor mediating these responses was a 30–50 kDa protein. To test whether this endogenous soluble factor was NRG1, we used organotypic cultures of E24 MAM-treated ferret neocortex supplemented with the endogenous factor obtained from normal cortical implants, exogenous NRG1β, antibodies that either blocked or stimulated erbB receptors, or a soluble erbB subtype that binds to available NRG1. We report that exogenous NRG1 or antibodies that stimulate erbB receptors dramatically improve the morphology of disrupted radial glia, whereas blockade of NRG1-erbB signaling prevents the radial glial repair. Our results suggest that NRG1 is an endogenous factor in ferret neocortex capable of repairing damaged radial glia and that it acts via one or more erbB receptors.