Leny A. Cavalcante

Postnatal development of retinogeniculate, retino-pretectal and retinotectal projections in the opossum

The postnatal development of retinogeniculate, retinopretectal and retinotectal projections has been studied by the anterograde transport of proline-labeled proteins in 20 pouch young opossums aged from 10 to 60 days. Radioautographical findings suggest delayed development of uncrossed as compared to crossed projections. There is a phase of overlapping projections from both eyes in thalamic and tectal target sites. Partial segregation of projections to the dorsal lateral geniculate nucleus (GLD) is preceded by differential distribution of crossed and uncrossed terminal fields along its dorsoventral axis (at age 23 days). The quasilaminar pattern of projections in the dorsocaudal region of GLD pars alpha is incipient by 42 days and is fully established at 60 days of age, as eye opening starts. The mature pattern of projections to the ventral lateral geniculate nucleus (GLV) is established much earlier, at 23 days of age. The development of retinopretectal projections is assessed mostly from the analysis of the olivary pretectal nucleus (PO). Distribution of silver grains into discrete areas coextensive with PO is relatively delayed (by 23 days of age) as compared to the nuclei of the lateral geniculate body. Soon after, however, the mature pattern of projections to PO is established (at 33 days of age). The early development of retinotectal projections (from 10 to 23 days) is compatible with an initial tangential course of crossed optic fibers in the superior colliculus (CS) but other alternatives remain open, such as a sequential outside-in arrangement of terminal fields of deeply coursing fibers. Arborization of uncrossed fibers is delayed at extreme rostromedial and caudolateral portion of the territory of the main uncrossed retinotectal projection. Segregation of uncrossed projections at different depths of CS is nearly complete by 42 days. Differences in the development of terminal fields in different target nuclei or in regions of a given target site are discussed in relation to retinal and local factors.

Alterations in metabolism and gap junction expression may determine the role of astrocytes as “good samaritans” or executioners

Our knowledge of astroglia and their physiological and pathophysiological role(s) in the central nervous system (CNS) has grown during the past decade, revealing a complex picture. It is becoming increasingly clear that glia play a significant role in the homeostasis and function of the CNS and that neurons should no longer be considered the only cell type that responds, both rapidly and slowly, to electrochemical activity. We discuss recent advances in the field with an emphasis on the impact of hypoxia and ischemia on astrocytic metabolism and the functional relationship between glucose metabolism and gap junctions in astrocytes. We also address the controversy over whether astrocytic gap junctions mediate protection or killing of neurons during or after hypoxic or ischemic insults.

Contribution of heparan sulfate to the non-permissive role of the midline glia to the growth of midbrain neurites

Radial glial cells and astrocytes are heterogeneous with respect to morphology, cytoskeletal‐ and membrane‐associated molecules and intercellular interactions. Astrocytes derived from lateral (L) and medial (M) midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in coculture (Garcia‐Abreu et al. J Neurosci Res 40:471, 1995). There is a correlation between these abilities and the differential patterns of laminin (LN) organization that is fibrillar in growth‐permissive L astrocytes and punctate in the non‐permissive M astroglia (Garcia‐Abreu et al. NeuroReport 6:761, 1995). There are also differences in the production of glycosaminoglycans (GAGs) by L and M midbrain astrocytes (Garcia‐Abreu et al. Glia 17:339, 1996). We show that the relative amounts of the glycoproteins laminin LN, fibronectin (FN) and tenascin (TN) are virtually identical in L and M glia, thus, confirming that an abundant content of LN is not sufficient to promote neurite growth. To further analyze the role of GAGs in the properties of M and L glia, we employed enzymatic degradation of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS). Treatment with chondroitinase has little effect on the non‐permissive properties of M glia but reduces the growth‐supporting ability of L glia. By contrast, heparitinase I produces no significant changes on L glia but leads to neurite growth promotion by M glia. Taken together, these results suggest that glial CS helps to promote neurite growth and, more importantly, they indicate that a HS proteoglycan is, at least, partially responsible for the non‐permissive role of the midline glia to the growth of midbrain neurites. GLIA 29:260–272, 2000.

Expression and function of ganglioside 9-O-acetyl GD3 in postmitotic granule cell development.

We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-O-acetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dose-dependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum.

Retinofugal projections in the opossum, An anterograde degeneration and radioautographic study.

A study of anterograde degeneration and anterograde transport was undertaken in the opossum primary optic system in order to clarify several points regarding fiber organization and patterns of terminal fields. Through the radioautographic technique of axon tracing, it was demonstrated that the accessory optic system follows the generalized scheme of Hayhow, consisting of two fascicles the three terminal nuclei.

Compartmental distribution of sulfated glycosaminoglycans in lateral and medial midbrain astroglial cultures

Sulfated glycosaminoglycans (S‐GAGs) were isolated from the pericellular (P), intracellular (I), and extracellular (E) compartments of astrocytes cultures from lateral (L) and medial (M) sectors of embryonic mouse midbrain; these sectors differ in their ability to support neurite growth (L, permissive, M, non‐permissive for growth) and laminin deposition patterns (L, fibrillar; M, punctate pattern). The total amount of S‐GAGs in M cultures was twice that in L cultures and was particularly high in the P compartment of M glia. Both glial cultures showed heparan sulfate (HS) in the three cellular compartments but chondroitin sulfate (CS) GAGs were vestigial in I and P compartments of L glia. Our results suggest that M and L astrocytes are heterogeneous concerning the ability to synthesize GAGs and distribute them among the different cellular compartments. Together with other data (Garcia‐Abreu et al: J Neurosci Res 40:471, 1995; Garcia‐Abreu et al: Neuroreport 6:761, 1995), the present results suggest that this heterogeneous features might be at least partially responsible for the differential effects of L and M glial cultures on the growth of midbrain neurons and may also be involved in complex ways in the guidance of axons at the brain midline.

Growth and restriction of the ipsilateral retinocollicular projection in the opossum

The distribution of optic nerve fibers and terminals in the superior colliculus (SC) was followed throughout its development in pouch young opossums in order to establish the normal sequence of events leading to the formation of mature patterns. Up to 7 days of life in the pouch, labeled fibers can be followed only as far as the rostral aspect of the optic tract. The earliest evidence for crossed retinal projections in the SC is found at 10 days of age. In parasagittal sections, the label extends along the rostrocaudal tectal axis from the rostral border to the presumptive caudal pole of the SC. Unequivocal evidence for ipsilateral retinocollicular projection is found at 15 days extending to all but the caudal 5th of the rostrocaudal extent of the SC. The projections from both eyes overlap extensively in the SC at 22 days and after this age significant changes occur, mostly at the ipsilateral side: a sub-pial tier of fine label develops excluding both rostral and caudal collicular poles; a deeper tier of coarse label extends from the rostral to the caudal pole and a third, patchy tier of label is found at the prospective strata griseum superficiale and griseum intermediate. By 47 and 60 days the tangential distribution of the projections is virtually indistinguishable from the adult pattern although laminar segregation does not seem as sharp as in the adult. Comparisons of the changeable patterns of ipsilateral retinocollicular projections from 22 to 34 days with the invariant, aberrant pattern in adult animals submitted to uniocular enucleation at either age suggests that the preservation of a juvenile pattern does not provide a comprehensive explanation for the formation of aberrant projections.

Olfactory ensheathing cells as putative host cells for Streptococcus pneumoniae: Evidence of bacterial invasion via mannose receptor-mediated endocytosis

Olfactory ensheathing cells (OECs) are a special glia that ensheath olfactory receptor axons that enter the brain via olfactory phila, thus, providing a potential route for access of pathogens. Streptococcus pneumoniae (Sp), that has a capsule rich in mannosyl residues, is the most common cause of rhinosinusitis that may evolve to meningitis. We have tested whether OECs in vitro express the mannose receptor (MR), and could internalize Sp via MR. Cultures were infected by a suspension of Sp (ATCC 49619), recognized by an anti-Sp antibody, in a 100:1 bacteria:cells ratio. Competition assays, by means of mannan, showed around a 15-fold reduction in the number of internalized bacteria. To verify whether MR could be involved in Sp uptake, OECs were reacted with an antibody against the MR C-terminal peptide (anti-cMR) and bacteria were visualized with Sytox Green. Selective cMR-immunoreaction was seen in perinuclear compartments containing bacteria whereas mannan-treated cultures showed an extremely low percentage of internalized bacteria and only occasional adhered bacteria. Our data suggest the involvement of MR in adhesion of bacteria to OEC surface, and in their internalization. Data are also coherent with a role of OECs as a host cell prior to (and during) bacterial invasion of the brain.

Synemin expression in developing normal and pathological human retina and lens.

Synemin (Syn) is an intermediate filament (IF) protein. To gain insight into a morphogenetic role of Syn, we have studied its expression patterns in the developing human retina and lens and compared it with those of other IF proteins. In addition, we have tested Syn expression in fetuses (23 and 28 weeks) affected by Walker-Warburg syndrome (WWS), Meckel syndrome, and trisomy 13. In the retina, Syn expression starts in the nerve fiber and ganglion cell layers (NFL and GCL) at 15 weeks, remains there in up to 20 weeks, and spreads to other layers and may be colocalized with vimentin, GFA, or neurofilaments in the subsequent 16 weeks. This expansion of Synemin expression from 20 to 28 weeks is not observed in WWS in which Syn immunoreactivity in NFL is reduced and Vim expression is increased. Changes are seen in Syn or vimentin expressions in the retinae of 23-week-old Meckel syndrome or 28-week-old trisomy 13 fetuses. Syn expression in the lens is, at first (16 weeks), uniformly distributed, becoming stronger in the epithelium of the anterior part at 25 weeks and later. As in the retina, Syn expression in lens is also selectively affected in WWS. The colocalization of Synemin with vimentin, GFA, or NF supports the idea that Syn is a key cross-linking protein that connects different cytoskeletal structures. Moreover, stagnant Syn expression in WWS retina and lens reinforces the notion of a significant role of this protein in morphogenesis.

Genesis of neurons of the retinal ganglion cell layer in the opossum.

SummaryIn this study, we have examined the genesis of neurons of the retinal ganglion cell layer of the opossum Didelphis marsupialis by [3H]-thymidine autoradiography. Our results suggest that most neurons surviving to adulthood are generated in postnatal life from day 1 to day 23. Cells are generated according to a coarse gradient from the retinal geometric center to the periphery. Regional analysis of soma size distributions in different cohorts suggest that this gradient is actually formed by two partially-overlapping, concentric waves of cell proliferation. Most medium and large ganglion cells are formed during the early wave, whereas most displaced amacrine cells and small ganglion cells are formed during the late wave. Our results confirm the appropriateness of the opossum as a model for studies of development of the mammalian visual system.