Functional development of intrinsic properties in ganglion cells of the mammalian retina

Senosory neurons manifest pronounced changes in excitability during maturation, but the factors contributing to this ubiquitous developmental phenomenon are not well understood. To assess the contribution of intrinsic membrane properties to such changes in excitability, in the present study whole cell patch-clamp recordings were made from developing ganglion cells in the intact retina of postnatal rats. During a relatively brief developmental period (postnatal days P7-P27) ganglion cells exhibited pronounced changes in the discharge patterns generated by depolarizing current injections. The youngest cells (P7-P17) typically responded to maintained depolarizations with only a single spike or a rapidly adapting discharge pattern. In contrast, the predominant response mode of more mature cells (P21-P27) was a series of repetitive discharges that lasted for the duration of the depolarization period, and by P25 all cells responded in this manner. These functional changes characterized all three morphologically defined cell classes identified by intracellular labeling with Lucifer yellow. To determine if expression of the potassium current (Ia) and the kinetics of the Na-channel related to the increased excitability of developing ganglion cells described above, current- and voltage-clamp recordings were made from individual neurons. The different firing patterns manifested by developing retinal ganglion cells did not reflect the presence or absence of the Ia conductance, although cells expressing Ia tended to generate spikes of shorter duration. With maturation the speed of recovery from inactivation of the Na current increased markedly and this related to the increased excitability of developing ganglion cells. Neurons yielding only a single spike to maintained depolarization were characterized by the slowest speed of recovery; cells with rapidly adapting discharges showed a faster recovery and those capable of repetitive firing recovered fastest from Na-channel inactivation. It is suggested that these changes in intrinsic membrane properties may relate to the different functional roles subserved by ganglion cells during development.     

Changes in the immunoreactivity for inhibin in mouse retina during development of photoreceptor cells

INTRODUCTION: A costovertebral paraganglioma with a long history of recurrences and metastases is reported. MATERIAL AND METHODS: The lesion is documented on histological ground and with immunocytochemistry and electron microscopy. RESULTS: The patient is a 42 year old man which presented with a 5 cm axillary mass and enlarged mediastinal lymph nodes. The former lesion has been interpreted as a metastasis of paraganglioma. The patient underwent surgery because of a costovertebral paraganglioma at the age of 19 and showed locoregional metastases and a recurrence over a period of 23 years. Twenty-seven years from removal of the primitive tumor, the patient is alive and well. All the neoplasms showed identical histological features as they were composed of neoplastic elements arranged in nests surrounded by dendritic cells. Neoplastic cells immunoreacted with anti-chromogranin antiserum whereas sustentacolar cells were positive with S-100 antiserum. DISCUSSION: The differential diagnosis between a multicentric paraganglioma a metastasis has been particularly taken into consideration. In addition, axillary tumor was distinguished from a metastatic renal cell carcinoma and melanoma. The literature regarding costovertebral paraganglioma has been also revised.     

Fibroblast growth factor receptor function is required for the orderly projection of ganglion cell axons in the developing mammalian retina

During the early stages of development various cell adhesion molecules (CAMs) and fibroblast growth factor receptors (FGFR) are expressed throughout the retinal neuroepithelium. The ability of retinal ganglion cells to project their axons to the optic fissure depends, in part, on cell-cell interactions mediated by cell adhesion molecules. In the present study we show that the ability of the firstborn rat retinal ganglion cells to extend axons in vitro can be stimulated by NCAM and L1, but not N-cadherin. Both CAM responses can be fully inhibited by antibodies that block neuronal fibroblast growth factor receptor function and by agents that block defined steps in the FGFR signal transduction cascade. When added to living E13.5 rat retinal whole-mount preparations the same agents induced errors in the orderly establishment of young axon patterns in the retinal periphery and caused axons in the retinal center to defasciculate. These results suggest that the activation of the fibroblast growth factor receptor signal cascade not only promotes survival and proliferation of various cell types but can also mediate intraretinal axon guidance.     

Expression of GATA-4 in migrating gonadotropin-releasing neurons of the developing mouse

Epidemiological studies indicating that nitrite inhalant abuse is a co-factor in HIV infection and in Kaposi's sarcoma are supported by recent experimental studies, described in this review. Inhalation exposure to the nitrites produce a nonspecific cytotoxicity, depleting many cells of the immune system. Apparently distinct from this cytotoxicity, inhalation of the nitrites impairs a variety of immune mechanisms, affecting both humoral and cell-mediated immunity. In addition, the inhalant-increased macrophage production of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), can directly stimulate HIV replication and can also stimulate the growth of Kaposi's sarcoma cells. Thus, nitrite inhalants may impair immune resistance to infection and actively promote viral replication and tumor growth.     

Neurotrophin-3 antibodies disrupt the normal development of the chick retina

When chick embryos are treated with a monoclonal antibody specifically blocking the activity of neurotrophin-3 (NT-3), the development of the retina is profoundly affected. Fewer axons are found in the optic nerve, and the retina shows abnormalities in all layers. Early during retinogenesis, the proportion of dividing cells is higher in NT-3-deprived embryos compared with age-matched controls and that of differentiated neurons is smaller. The NT-3 receptor trkC is expressed early by a majority of retinal cells, and NT-3 is present in the retina at the earliest stage studied. Initially, it is located mainly in the pigmented epithelium, with a shift toward the neural retina as development proceeds. Thus, NT-3 seems to be an essential intrinsic signal acting early in development to promote the differentiation and survival of many retinal neurons.     

The number of unidentified amacrine cells in the mammalian retina

The three largest known populations of amacrine cells in the rabbit retina were stained with fluorescent probes in whole mounts and counted at a series of retinal eccentricities. The retinas were counterstained using a fluorescent DNA-binding molecule and the total number of nuclei in the inner nuclear layer were counted in confocal sections. From the total number of inner nuclear layer cells and the known fraction of them occupied by amacrine cells, the fraction of amacrine cells made up by the stained populations could be calculated. Starburst cells made up 3%, indoleamine-accumulating cells made up 4%, and AII cells made up 11% of all amacrine cells. By referring four smaller populations of amacrine cells to the number of indoleamine-accumulating cells, they were estimated to make up 4% of all amacrine cells. Thus, 78% of all amacrine cells in the rabbit's retina are known only from isolated examples, if at all. This proportion is similar in the retinas of the mouse, cat, and monkey. It is likely that a substantial fraction of the local circuit neurons present in other regions of the central nervous system are also invisible as populations to current techniques.     

The localization of guanylyl cyclase-activating proteins in the mammalian retina

PURPOSE: To explore the distribution of guanylyl cylase-activating proteins 1 and 2 (GCAP1 and GCAP2) in the mammalian retina. METHODS: Cryostat and vibratome vertical sections and wholemount retinas from mouse, rat, cat, bovine, monkey, and human eyes were prepared for immunocytochemistry and viewing by light and confocal microscopy. RESULTS: In all mammalian retinas investigated, intense GCAP1 immunoreactivity (GCAP1-IR) was seen in cone photoreceptor inner and outer segments, cell bodies, and synaptic regions. Intensity of the GCAP1-IR was strong in inner segments of rods in all species but weaker in outer segments-particularly so in primates and cats. GCAP2 immunoreactivity (GCAP2-IR) was weak in bovine, mouse, and rat cones but was intense in human and monkey cones. In all species except primates, GCAP2 staining was intense in rod inner and outer segments. In primates GCAP2-IR was intense in the rod inner segment but faint in the rod outer segment. A striking difference from the GCAP1 pattern of immunoreactivity was seen with GCAP2 antibodies as far as the inner retina was concerned. GCAP2-IR was evident in certain populations of bipolar, amacrine, and ganglion cells in all species. CONCLUSIONS: GCAP1 and GCAP2, which are involved in Ca2+-dependent stimulation and inhibition of photoreceptor guanylyl cyclase, can be detected in mammalian photoreceptor inner and outer segments, consistent with their physiological function. The occurrence of both GCAPs in the synaptic region of the photoreceptors indicates participation of these proteins in pathways other than regulation of phototransduction. The occurrence of GCAP2 in inner retinal neurons is indicative of second-messenger chemical transduction, possibly in metabotropic glutamate, gamma-aminobutyric acid (GABA) receptor, and nitric oxide-activated neural circuits.     

Distribution of transforming growth factor-beta isoforms in the mammalian retina

The distribution of transforming growth factor-beta (TGF-beta) was examined in the posterior segment of the monkey, human, and feline eye using antisera to TGF-beta 1, TGF-beta 2, or TGF-beta 3. A number of different antibodies, tissue processing methods, immunolocalization techniques, and microscopic imaging systems were used in an attempt to gain a more comprehensive picture of TGF-beta isoform distribution in the retina and retinal pigmented epithelium (RPE). The results are generally consistent in identifying one or more of the three TGF-beta isoforms in the cytoplasm of a small, overlapping subset of cells. RPE cells, photoreceptors, Mueller cells, ganglion cells, hyalocytes, and cells associated with choroidal and retinal vessels are all represented in this immunoreactive population. No evidence of extracellular labeling was noted. The intracellular distribution of the three isoforms is distinctly different in photoreceptors. Anti-TGF-beta 1 precursor and anti-TGF-beta 2 immunoreactivity is confined primarily to rod outer segments, whereas anti-TGF-beta 3 immunoreactivities are restricted to mitochondria within inner segments. In the RPE, clusters of anti-TGF-beta 2 positive cytoplasmic granules are located near the cells' lateral borders, whereas anti-TGF-beta 3 labeling is concentrated apically. These results provide baseline information from which new hypotheses regarding the function(s) of TGF-beta isoforms in the retina can be formulated.     

Transient, high levels of SNAP-25 expression in cholinergic amacrine cells during postnatal development of the mammalian retina

In the present study, we have examined the development of cholinergic amacrine cells in the retina of the Brazilian opossum, Monodelphis domestica. An antibody directed against choline acetyltransferase (ChAT) revealed that ChAT-like immunoreactivity (ChAT-IR) was first observed at 15 days postnatal (15PN). By 25PN, ChAT-IR identified two matching populations of amacrine cells in the inner nuclear and ganglion cell layer. Bromodeoxyuridine birth-dating analysis coupled with immunolabeling with the anti-ChAT antibody revealed that the cholinergic amacrine cells are born postnatally, between 2PN and 15PN. In addition, we have examined the differentiation of the cholinergic amacrine cells by using an antibody directed against a presynaptic terminal-associated protein, synaptosomal-associated protein of 25 kDa (SNAP-25). Double-labeling analysis revealed that relatively high levels of SNAP-25-IR were selectively present in cholinergic amacrine cells prior to eye opening. However, in the mature retina, high levels of SNAP-25-IR were no longer observed in the ChAT-IR amacrine cells. These results reveal a distinct period in development, prior to eye opening, when high levels of SNAP-25-IR are selectively expressed in cholinergic amacrine cells. The specificity and time course of the high levels of SNAP-25 in cholinergic amacrine cells may be critical in mediating the transient properties of these cells during visual system development.     

Evidence of decreased adhesion between the neural retina and retinal pigmented epithelium of the Mitfvit (vitiligo) mutant mouse

Secretoneurin, derived from the chromogranin secretogranin II, triggers the selective migration of human monocytes, eosinophils, fibroblasts, endothelial and smooth muscle cells. More recently, we located specific binding sites on the human monocytic cell line MonoMac-6. Differentiated U937 transendothelial diapedesis was evaluated using an in vitro model of the vascular wall and specific monoclonal antibodies against CD11/CD18 and the alpha-chains of the very late activation antigen (VLA)-4 were used to evaluate involved adhesion molecules. Results showed a significant migratory response to secretoneurin between 10(-8) to 10(-10) M. Migration was comparable to a maximal effect induced by the monocyte chemotactic agent N-formyl-Met-Leu-Phe (fMLP, 10(-8) M). Rabbit anti-secretoneurin antibodies were able to block the neuropeptide effect but not of fMLP and a trypsinized secretoneurin preparation as well as the secretogranin II-fragment EL-17 were ineffective in eliciting migration. Transmigration of U937 across endothelial-layers toward secretoneurin is inhibited by antibodies to CD11/CD18 adhesion molecules. The novel neuropeptide secretoneurin may play a role in regulating migration of monocytes into the subendothelial space, supposing a role in inflammatory responses.     

Postnatal development and the differential expression of presynaptic terminal-associated proteins in the developing retina of the Brazilian opossum, Monodelphis domestica

In the present study we have characterized the postnatal (PN) development of the retina in the Brazilian opossum, Monodelphis domestica. Monodelphis, a small, pouchless marsupial, undergoes a protracted period of postnatal development. Using bromodeoxyuridine immunohistochemistry, we have investigated postnatal neurogenesis of the retina. In addition, we have examined the differentiation of the retina by using antibodies directed against the presynaptic terminal-associated proteins synaptotagmin, Rab3A, synaptophysin and synaptosomal-associated protein-25 (SNAP-25), and have characterized their spatial and temporal distribution during postnatal development. This study is the first systematic comparison of the developmental expression of multiple presynaptic terminal-associated proteins in relation to retinal neurogenesis and differentiation. At birth (1PN), the Monodelphis retina was relatively undifferentiated morphologically and birthdating analysis revealed mitotically active cells throughout the retina. The 8PN retina was organized into two cellular layers: an outer region of mitotically active neuroepithelial cells and an inner region of postmitotic cells. The inner plexiform layer formed between 5PN and 10PN, and exhibited unique patterns of immunoreactivity with the antibodies used in this analysis. By 25PN the retina was well laminated, and synaptotagmin-, Rab3A-, synaptophysin- and SNAP-25-like immunoreactivities exhibited distinct and specific patterns within the plexiform layers, although they had not yet achieved their mature, adult patterns. These results indicate that each of these proteins exhibits developmentally regulated changes in its cellular localization, and therefore may play important roles during morphogenesis and synaptogenesis of the vertebrate retina.     

Expression and function of the type 3 complement receptor in tissues of the developing mouse

Macrophage (Mphi) expression of the leukocyte integrins has been implicated in their adhesion and migration in the adult. Little is known, however, of the expression or function of these molecules during development. This study defines the spatial and temporal sequences of expression of the type 3 complement receptor (CR3) in the developing mouse; establishes the functional efficacy of this molecule in spreading, adhesion, and phagocytosis; and investigates its role in inflammatory and constitutive migration. Expression of CR3 on monocytes occurred early compared to Mphi-restricted glycoprotein F4/80, but expression on stellate tissue Mphi appeared later than F4/80 and was transient. Expression of CR3 on resident tissue Mphi is more widespread during development, being retained on only very specific Mphi populations in the adult. Neutrophil polymorphs expressed CR3 from day 17 of gestation onward. The anti-CR3 mAb 5C6 was used to investigate the role of CR3 in adhesion, spreading, and phagocytosis by neonatal Mphi. Neonatal macrophages were found to adhere, spread, and phagocytose by CR3-dependent mechanisms, and a CR3-independent system was implicated in the spreading of neonatal Mphi. The role of CR3 in migration during development was then investigated. 5C6 had potent effects on the early stages of the migration of myelomonocytic cells to an inflammatory stimulus in vivo. Despite efficient transplacental transfer of the Ab from pregnant mother to fetus, the process by which monocytes generate populations of resident tissue Mphi was undisrupted, indicating the existence of CR3-independent mechanisms of monocyte migration during development.     

Role of astrocytes in the control of developing retinal vessels

PURPOSE: To assess the role of astrocytes in controlling the growth of developing retinal vessels. METHODS: Growth of retinal vessels in the neonatal rat retina was examined in three conditions: normal development, cyclic hyperoxia, and normoxia (1 day 70% to 75% oxygen, 1 day room air for up to seven cycles from birth, and room air for up to 16 days), and direct hypoxia (10% oxygen from postnatal day 3 [P3]). Retinas were examined as wholemounts labeled for astrocytes, microglia, and blood vessels and in some experiments for the fragmentation of DNA characteristic of apoptosis. RESULTS: In normoxia, superficial retinal vessels formed to the processes of astrocytes. In cyclic hyperoxia, the depletion of superficial retinal vessels and subsequent neovascularization described by others were confirmed. The neovascularization was preceded by the depletion by apoptotic death of the astrocyte population, first between vessels but eventually breaching the glia limitans along vessels. The earliest forms of neovascularization resembled microaneurysms, each protruding through a defect in the glia limitans of a capillary. Neurons of the ganglion cell layer survived. Direct hypoxia from P3 caused hypertrophy of superficial vessels. Between P3 and P6, some vessels accelerated past the still-spreading astrocytes, often growing out of the retina into the vitreous humor. Direct hypoxia also caused astrocyte degeneration, but capillaries retained astrocyte investment and were not the site of vascular damage. By P8, breaches in the astrocytic glia limitans became prominent but were restricted to large veins. At such breaches, bleeding into the vitreous humor was common. CONCLUSIONS: Retinal vessels normally develop in close association with astrocytes. Where that association is broken, preretinal vessels may grow or bleed into the vitreous humor. Astrocytes play important roles in constraining retinal vessels to the retina and in maintaining their integrity.     

Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina

Synapse-associated proteins are the scaffold for the selective aggregation of ion channels at synapses; they provide the link to cytoskeletal elements and possibly are involved with the regulation of synaptic efficacy by electrical activity. The localization of the postsynaptic density protein PSD-95 was studied in different mammalian retinae (rat, monkey, and tree shrew) by using immunocytochemical methods. Immunofluorescence for PSD-95 was most prominent in the outer plexiform layer (OPL). The axon terminals of rods and cones, the rod spherules and cone pedicles, were strongly labeled. Electron microscopy, using preembedding immunocytochemistry, showed PSD-95 localized presynaptically within the photoreceptor terminals. Distinct PSD-95 labeling was also present in the inner plexiform layer (IPL). It had a punctate appearance suggesting the synaptic clustering of PSD-95 in the IPL. Electron microscopy showed that PSD-95 was concentrated in processes that were postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for PSD-95. Double-labeling experiments were performed for PSD-95 and for SAP 102 or PSD-93, respectively, two other members of the family of synapse-associated proteins. All three were found to be colocalized in the synaptic hot spots in the IPL. In the OPL, however, PSD-95 and PSD-93 were found presynaptically, whereas SAP 102 was located postsynaptically at photoreceptor synapses. Double-labeling experiments also were performed for PSD-95 and for the NR1 subunit of the NMDA receptor. They were found to be colocalized in synaptic hot spots in the IPL.     

Emerging roles for SH2/PTB-containing Shc adaptor proteins in the developing mammalian brain

In mammalian systems, SH2-containing cytoplasmic signalling molecules are known to play an important role in determining cell responsiveness to the environment. In particular, following activation of a receptor protein tyrosine kinase (RPTK), proteins like Shc and Grb2 bind to phosphotyrosine residues of stimulated receptors, thereby activating downstream components of specific signalling pathways. The ShcA gene was identified in 1992 and was found to encode three proteins with properties of adaptor molecules coupling RPTKs to Ras. Early data obtained in non-neuronal cells have revealed that Shc and Grb2 proteins are highly expressed and activated in all cells. However, recent analyses of ShcA mRNA and protein in the developing brain revealed progressive downregulation of their expression during differentiation from neuroblasts to neurons. Conversely, the two newly identified Shc homologues (ShcB/Sli and ShcC/Rai) are highly expressed in the mature brain.Thus, variations in the intracellular levels of adaptor proteins might represent one of the mechanisms by which a differentiating cell changes its ability to respond to a given factor, allowing a cell to choose between proliferation and differentiation.     

Differential distribution of agrin isoforms in the developing and adult avian retina

At the developing and regenerating neuromuscular junction, agrin is responsible for the formation of aggregates containing the acetylcholine receptor (AChR) and other molecules. Multiple isoforms of agrin are generated by alternative splicing, and the presence of an 8, 11, or 19 (8 + 11) amino acid insert at splice site B is required for agrin's AChR aggregation activity. An antiserum was generated against the 19 amino acid peptide which reacted specifically with the B11 and B19 agrin isoforms. The antiserum blocked the formation of agrin-induced AChR aggregates on myotubes, but the peptide itself had no aggregation activity, suggesting that agrin's active site is close to the splice site, but not the peptide itself. In the embryonic and adult retina anti-peptide immunoreactivity was concentrated in the synapse-containing layers. In contrast, the inner limiting membrane and radial cells, which express strong immunoreactivity with a pan-specific anti-agrin antiserum, were not stained by the anti-peptide antiserum, showing that agrin isoforms are differentially distributed in the retina. In addition, agrin B11 and B19 isoforms were associated with ganglion cell axons, particular at early developmental stages before synapse formation, indicating additional functions for these isoforms in the developing CNS.