The environment of axonal migration in the developing chick retina: a scanning electron microscopic (SEM) study.

We have made a SEM study of the basal intercellular spaces of the retina in chick embryos of different developmental stages. Since this is the environment where optic axons grow, the structural characteristics of this region might play some role in the orientation of axonal migration towards the choroid fissure. The basal region of undifferentiated retinas is formed by the vitreal expansions of neuroepithelial cells. In pre-axonal stages, the intercellular spaces between these expansions do not show any preferential orientation towards the fissure. The growth cones of ganglion cell axons appear in an apicobasal direction and turn towards the fissure immediately beneath the vitreal surface. Fasciculation is an early event during development and, in the more advanced stages, the vitreal expansions from retinal cells are placed in rows following the same orientation as the axon bundles. These observations are discussed in relationship to current hypotheses on axonal migration and orientation.

Expression of neurotrophins and trk receptors in the avian retina.

Using the RNase protection assay, we have found that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are expressed in the avian retina during development. The expression peaks around embryonic days 12-15, with decreasing levels at later stages of development. Abundant levels of NGF and BDNF but low levels of NT-3 mRNA were found in the adult retina. We also found that light/darkness regulated the levels of NGF and BDNF mRNAs but not the levels of NT-3 mRNA in the 5-day-old chicken retina. It was demonstrated that NGF and BDNF mRNA levels were up-regulated by light exposure. The cellular localization of mRNA expression for the neurotrophins and neurotrophin receptors TrkA, TrkB, and TrkC in the retina was studied using in situ hybridization. The patterns of NGF and trkA mRNA expression were very similar and were localized to the external part of the inner nuclear layer on the border with the outer plexiform layer and corresponded to the localization of horizontal cells. NT-3 labeling was also found over the external part of the inner nuclear layer, whereas trkC mRNA was found over all layers in the retina. BDNF labeling was found over all layers in the retina, whereas TrkB labeling was intense over cells in the ganglion cell layer, which is in agreement with the response of ganglion cells to BDNF stimulation. Functional neurotrophin receptors were suggested by the response of retinal explants to neurotrophin stimulation. These data indicate that the neurotrophins play local roles in the retina that involve interactions between specific neuronal populations, which were identified by the localization of the Trk receptor expression. The data also suggest that NGF and BDNF expression is regulated by normal neuron usage in the retina.

Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid.

PURPOSE: In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. METHODS: Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. RESULTS: Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [(3)H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. CONCLUSIONS: These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: impact on hormone secretion.

OBJECTIVE: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/beta-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli beta-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). DESIGN: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/beta-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. METHODS: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for beta-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. RESULTS: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/beta-gal showed widespread expression of the beta-galactosidase transgene around the injection areas. CONCLUSIONS: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.

BMPR-II expression and OP-1 effects in developing chicken retinal explants.

Low-stringency PCR was used to isolate the chicken homologue of the human bone morphogenetic protein receptor type II (BMPR-II). In situ hybridization localized mRNA expression for this serine/threonine kinase receptor. It was weakly expressed in the vitreal side of the neural retina at E6. In the E7 chicken, fairly strong labelling was found in cells of the internal part of the neural retina. At E9, strong labelling was found in the region of the retinal ganglion cells. Explants of E6 retina exposed to osteogenic protein-1 (OP-1/BMP-7), exhibited dense retinal fibre outgrowth. This suggests that BMPR-II may form a signalling receptor complex important for retinal development. OP-1 and related ligands may serve functions supplementary to those of neurotrophins.

Target-field specificity in the induction of retinal neurite outgrowth.

Nasal and temporal microexplants from the retina of the chicken embryo (E6; White Leghorn) were cultured on a collagen gel in the presence of extract from anterior or posterior E18 optic tectum. After 4 days of incubation maximum neurite lengths were measured as a function of protein concentration (1-500 micrograms/ml). Temporal retina was stimulated by both extracts but maximum neurite growth was obtained with anterior optic tectum at 500 micrograms/ml. Conversely, nasal retinal fiber outgrowth was best stimulated by posterior tectum, with maximum neuritic extension occurring at a concentration of 60 micrograms/ml. The stimulation was exerted by soluble protein fractions. The results show that topographically distinct axonal target fields of the optic tectum can selectively stimulate growth of neurites from the appropriate region of the retina.

Organotypic cultures of neural retina: neurite outgrowth stimulated by brain extracts.

A bioassay for the growth of retinal neurites was designed. Circular plugs 0.5-1.5 mm in diameter were excised from the chick neural retina at embryonic day 6 and cultured as organotypic explants on a collagen gel. After addition of medium or various tissue extracts cultures were incubated and the length of extending neurites measured. In control medium with 10% serum, neurites were sparse and reached less than 0.2 mm after 4 days. Extract of the optic lobe from 18-day-old chick embryos distinctly increased the density and length of neurites in a dose-dependent manner reaching 1.2 mm after 4 days. A unit of outgrowth activity corresponding to half the maximum length of fibers was defined. The activity of the optic lobe extracts was retained in fractions with nominal molecular weights over 100,000 daltons upon pressure dialysis. Extract from the forebrain hemispheres had nearly the same effect on retinal neurite growth. Mouse submandibular gland NGF failed to evoke retinal fiber growth. Our data imply that macromolecules of the developing brain can support the growth of optic axons.

Neuritogenesis of retinal ganglion cells is differentially promoted by target extract.

Labeled retinal ganglion cells from neonatal rats extended neurites in dissociated cell culture as a cell type-specific response to the influence of a superior collicular extract. The molecule responsible for this neuritogenic effect is soluble and non-dialysable (> 12 kDa). Nerve growth factor had a neuritogenic effect both on ganglion cells and other types of retinal cells.

Choroid coat extract and ciliary neurotrophic factor strongly promote neurite outgrowth in the embryonic chick retina.

Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retinal explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the E18 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose-dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways.

Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen.

Extracellular matrix molecules such as laminin, fibronectin and collagen promote neurite outgrowth in vitro. We have investigated the capacity of hydrated gels of collagen types I-III and monomeric collagen types I-VI on plastic surfaces to support neuritogenesis. The attachment and survival of explants from the day 6 chick embryo were studied and neurite outgrowth measured as mean elongation rate and maximal neurite length. Collagen types I and III, both as three-dimensional gels or as native monomers supported neuritogenesis equal to or better than laminin. Collagen type V also supported neurite out-growth although less effectively. Collagen types II, IV and VI, as well as denatured collagens of all types tested, did not support outgrowth. The monoclonal anti-beta 1 integrin antibody (CSAT), as well as rabbit polyclonal antibodies directed to the integrin beta 1-chain, effectively inhibited neurite outgrowth on permissive collagenous substrata, indicating that collagen-binding integrins were involved in the neuritogenesis. These beta 1-integrins were independent of Arg-Gly-Asp (RGD) since neurite formation proceeded in the presence of synthetic RGD-containing peptides. Fluorescence immunohistochemistry revealed the presence of the integrin beta 1-chain on the outgrowing neurites. The results suggest a possible function of collagen and collagen-binding integrins in the development of the visual system.

Effects of docosahexaenoic acid on retinal development: cellular and molecular aspects.

We have recently shown that docosahexaenoic acid (DHA) is necessary for survival and differentiation of rat retinal photoreceptors during development in vitro. In cultures lacking DHA, retinal neurons developed normally for 4 d; then photoreceptors selectively started an apoptotic pathway leading to extensive degeneration of these cells by day 11. DHA protected photoreceptors by delaying the onset of apoptosis; in addition, it advanced photoreceptor differentiation, promoting opsin expression and inducing apical differentiation in these neurons. DHA was the only fatty acid having these effects. Mitochondrial damage accompanied photoreceptor apoptosis and was markedly reduced upon DHA supplementation. This suggests that a possible mechanism of DHA-mediated photoreceptor protection might be the preservation of mitochondrial activity; a critical amount of DHA in mitochondrial phospholipids might be required for proper functioning of these organelles, which in turn might be essential to avoid cell death. Müller cells in culture appeared to be involved in DHA processing: they took up DHA, incorporated it into glial phospholipids, and channeled it to photoreceptors in coculture. Both Müller cells, when cocultured with neuronal cells, and the glial-derived neurotrophic factor (GDNF) protected photoreceptors from cell death. These results suggest that glial cells may play a central role in regulating photoreceptor survival during development through the provision of trophic factors. The multiple effects of DHA on photoreceptors suggest that, in addition to its structural role, DHA might be one of the trophic factors required by these cells.

Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats.

Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.

Transcription factor XBP-1 is expressed during osteoblast differentiation and is transcriptionally regulated by parathyroid hormone (PTH).

X-box-binding protein 1 (XBP-1) is a basic-region leucine zipper protein in the cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors involved in different cell-differentiation processes. We have investigated the expression of XBP-1 in differentiating MC3T3-E1 osteoblastic cells. Cultures were treated with ascorbic acid (AA) and beta-glycerophosphate (BGP) to induce differentiation. Under these conditions, the basal transcription of xbp-1 increases at day 2 following induction, peaks at day 5 and decreases thereafter. This result showed that xbp-1 gene is differentially expressed during MC3T3-E1 cell differentiation. Detection of XBP-1 by immunofluorescence at days 0 (control culture without AA and BGP), 8 and 21 showed that the protein has a major cytoplasmic perinuclear location. In addition, xbp-1 is transcriptionally upregulated by parathyroid hormone within 2.5 h of treatment and decreases thereafter.

Scanning electron microscopy of frozen sections: a method for studying selected planes in small specimens.

A procedure for studying small biological specimens with the SEM, using frozen sections of fixed material is described. Controlled fixation schedules and convenient handling of sections insure optimal preservation of cell shape and size. The greatest advantage of the method presented is to allow scanning through any desired plane of the specimen, even in those planes parallel to an epithelial face. SEM of frozen sections is thus helpful for the study of cell distribution and organization within a tissue.

A method for explantation of selected areas of the neural retina.

A method of obtaining explants of selected areas of the embryonic neural retina is described. Small plugs of the tissue are sucked into a glass capillary tube connected to a micrometer syringe via flexible tubing. The plugs of tissue are separated into their components (neural retina, pigmented layer, and mesenchyme). Retinal explants so obtained have similar shape and size. Polarity of the explants is easily determined. The procedure is simple, rapid, and precise. The method is suitable for studies of retina based on explanted or transplanted tissue.

Differential outgrowth of retinal neurites on purified extracellular matrix molecules.

Organotypic cultures of the embryonic retina were used to study the influence of extracellular matrix molecules on neurite elongation during development of the central nervous system. Microexplants from the chick retina (embryonic day 6) were grown in medium containing appropriate trophic support on purified matrix molecules adsorbed to plastic at various concentrations. The maximum neurite length obtained on each type of substratum was measured on day 4 of culture. No fiber outgrowth occurred on substrata of vitronectin or a hyaluronate-binding chondroitin sulfate proteoglycan. In contrast, neurite elongation was strongly promoted on laminin in a dose-dependent manner. Fibronectin elicited a neurite outgrowth corresponding to about one-third the length of the outgrowth on laminin. A 31,000-dalton fibronectin fragment representing the heparin-binding domain elicited neurite elongation comparable to that promoted by the intact fibronectin molecule. Other isolated domains of fibronectin, including the 105,000-dalton "cell-binding" domain, did not allow neurite outgrowth. Furthermore, preincubation of fibronectin substratum with antibodies to the heparin-binding fibronectin fragment entirely prevented outgrowth. Fiber outgrowth was also evoked on substrata of platelet factor 4, a protein binding heparan sulfate. Adding increasing concentrations of heparin progressively inhibited the neurite extension on laminin, whereas similar addition of soluble chondroitin sulfate proteoglycan had no effect. The results indicate that growing retinal neurites show strong preference for laminin versus fibronectin. Moreover, the outgrowth-promoting activity of both cell adhesion proteins seems to be localized to their heparin-binding regions. It is suggested that during development of the visual system, elongating retinal neurites can actively discriminate between different extracellular molecules by a mechanism that may involve participation of cell surface heparan sulfate proteoglycans.

Growth inhibition, morphological differentiation and stimulation of survival in neuronal cell type (Neuro-2a) treated with trophic molecules.

Trophic molecules are key regulators of survival, growth and differentiation of neural cells. Neuronal cell type Neuro-2a is a good model to study development and molecules modulating this process, and retinoic acid (RA) and neurotrophins (NGF, BDNF, NT-3 and NT-4) have been shown to be active in this modulation. The purpose of the present study was the functional analysis of these trophic molecules in our short-term bioassay of Neuro-2a cells, an immortalised murine neuroblastoma cell line. Through cell counting, image process and arithmetic combination of digital parameters of treated and untreated cultures, we show that RA inhibits growth and induces morphological neuronal phenotype of treated cells. Through DNA labelling with BrdU we also show that NGF, BDNF, and NT-3 increase survival and proliferation of cells, grown in serum-deprived media. From these results we conclude that neurotrophins have manifest trophic effects on cells improving survival, growth and proliferation and we also confirm the growth arrest and differentiation properties of RA on Neuro-2a cells.

Neurotrophin-3 enhances neurite outgrowth in cultured hippocampal pyramidal neurons.

Low density dissociated cultures of embryonic rat hippocampal cells were used to study the effects of neurotrophin-3 (NT-3) on neuronal morphogenesis. The results obtained indicate that NT-3 enhances neurite outgrowth and branching; this is a dose-dependent effect, detected in approximately 50% of the neurons, and prevented by K-252a, an inhibitor of the trk family of receptor protein kinases. NT-3 also accelerates the development of neuronal polarity, a phenomenon preceded by a dramatic accumulation of bundles of looped microtubules within axonal growth cones; these microtubule bundles contain tyrosinated, detyrosinated, and acetylated alpha-tubulin. Taken collectively, our data suggest that even though the basic shape of hippocampal neurons may be endogenously determined, critical aspects of their morphological development may be modulated by trophic factors such as NT-3. In addition, our observations suggest that at least some of the neuritogenic effects of NT-3 involve a stimulation of microtubule assembly and/or transport.

MAP-1B/TAU functional redundancy during laminin-enhanced axonal growth.

When cultured cerebellar macroneurons develop attached to a laminin-containing substrate or after the acute addition of laminin to the tissue culture medium, there is an acceleration in the rate and extent of axonal elongation. Furthermore, laminin is capable of inducing axonal formation and microtubule stabilization in neurons arrested at stage II of neuritic development by tau suppression (Caceres and Kosik, 1990; Caceres et al., 1991). Laminin-enhanced or induced axonal extension is paralleled by a selective and dramatic incorporation of phosphorylated MAP-1b into axonal microtubules. Axonal formation in neurons growing in the presence of laminin is prevented by treatment of the cultures with a mixture of MAP-1b and tau antisense oligonucleotides, but not by the single suppression of any one of these MAPs. However, suppression of MAP-1b, but not of tau, greatly reduces the increase in the rate and extent of axonal elongation induced by laminin. No such effects are elicited by MAP-1b antisense oligonucleotides in neurons growing in the absence of laminin, e.g. polylysine alone, where most of the MAP-1b present in the cells is dephosphorylated and not associated with the cytoskeleton. Taken collectively, these data suggest that, with regard to axonal elongation, MAP-1b and tau can be functionally substituted, and that extracellular matrix molecules, such as laminin, affect axonal extension by promoting the in vivo utilization of MAP-1b.