Optic Fiber Layer Research Articles

EXPRESSION OF NEUROFILAMENTS IN THE RETINA IN EXPERIMENTAL DIABETIC RETINOPATHY AND THE INFLUENCE OF THE BENZODIAZEPINE RECEPTOR AGONIST CARBACETAM

Background. One of the typical neurochemical pathogenetic mechanisms causing the dysfunction of retinal neurons in diabetic retinopathy (DR) is a deficiency of GABA-ergic mediation. To some extent, this justifies the prospect of using GABA receptor modulators, including benzodiazepines.
 Aim: To determine the expression of neurofilaments in the initial stages of the experimental DR development and the influence of the benzodiazepine receptor agonist Carbacetam.
 Materials and methods. DR was modeled in 35 three-month-old male Wistar rats by a single injection of streptozotocin (50 mg/kg; Sigma-Aldrich, Co, China). The rats were divided into 3 groups: control, with the introduction of Insulin (30 Units; Novo Nordisk A/S, Bagsvaerd, Denmark) and with the introduction of Insulin and Carbacetam (5 mg/kg). NF-H neurofilaments (ThermoFisher Scietific, USA) were detected in the retina by immunohistochemistry.
 Results. Before DR modeling, NF-H-positive staining was detected in the inner and outer layers of the retina in mainly longitudinal fibers of different lengths, which corresponded to the axons of horizontal and amacrine cells; as well as in fibers and axonal hills of ganglion neurons. In the dynamics of the DR development in the plexiform layers of the retina, a progressive decrease in the intensity of staining was observed. In the optic fiber layer, staining was concentrated around dilated microvessels that tended to form microaneurysms. On the 28th day, NF-H-positive staining disappeared in the inner plexiform layer. Administration of Insulin with Carbacetam prevented the loss of NF-H-positive staining in the layers of the retina.
 Conclusion. The use of the benzodiazepine receptor agonist Carbacetam may be a promising direction for the correction of retinal neurodegeneration in DR.

Retinal development and the expression profiles of opsin genes during larval development in Takifugu rubripes.

The light-sensitive capacity of fish larvae is determined by the structure of the retina and the opsins expressed in the retinal and nonretinal photoreceptors. In this study, the retinal structure and expression of opsin genes during the early developmental stage of Takifugu rubripes larvae were investigated. Histological examination showed that at 1 days after hatching (dah), seven layers were observed in the retina of T. rubripes larva, including the pigment epithelial layer [retinal pigment epithelium layer (RPE)], photoreceptor layer (PRos/is), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL). At 2dah, optic fibre layer (OFL) can be observed, and all eight layers were visible in the retina. By measuring the thickness of each layer, opposing developmental trends were found in the thickness of ONL, OPL, INL, IPL, GCL and OFL. The nuclear density of ONL, INL and GCL and the ratios of ONL/INL, ONL/GCL and INL/GCL were also measured and the ratio of ONL/GCL ranged from 1.9 at 2dah to 3.4 at 8dah and no significant difference was observed between the different developmental stages (P > 0.05). No significant difference was observed for the INL/GCL ratio between the different developmental stages, which ranged from 1.2 at 2dah to 2.0 at 18 dah (P > 0.05). The results of quantitative real-time polymerase chain reaction (PCR) showed that the expression of RH1, LWS, RH2-1, RH2-2, SWS2, rod opsin, opsin3 and opsin5 could be detected from 1dah. These results suggest that the well-developed retina and early expression of the opsins of T. rubripes during the period of transition from endogenous to mixed feeding might be critical for vision-based survival skills during the early life stages after hatching.

Characterization of proliferative, glial and angiogenic responses after a CoCl2 -induced injury of photoreceptor cells in the adult zebrafish retina.

The adult zebrafish is considered a useful model for studying mechanisms involved in tissue growth and regeneration. We have characterized cytotoxic damage to the retina of adult zebrafish caused by the injection of cobalt chloride (CoCl2 ) into the vitreous cavity. The CoCl2 concentration we used primarily caused injury to photoreceptors. We observed the complete disappearance of cones, followed by rods, across the retina surface from 28 to 96hr after CoCl2 injury. The loss of 30% of bipolar cells was also observed by 50hr after lesion (hpl). CoCl2 injury provoked a strong induction of the proliferative activity of multipotent Müller glia and derived progenitors. The effect of CoCl2 on retina cells was significantly reduced by treatment with glutamate ionotropic receptor antagonists. Cone photoreceptor regeneration occurred 25days after injury. Moreover, a single dose of CoCl2 induced vascular damage and regeneration, whereas three injections of CoCl2 administered weekly provoked neovascular-like changes 20days after injury. CoCl2 injury also caused microglial reactivity in the optic disc, retina periphery and fibre layer. CoCl2 -induced damage enhanced pluripotency and proneural transcription factor gene expression in the mature retina 72 hpl. Tumour necrosis factor alpha, vascular endothelial growth factor (VEGF) and VEGF receptor mRNA levels were also significantly enhanced by 72 hpl. The injury paradigm we have described in this work may be useful for the discovery of signalling molecules and pathways that participate in the regenerative response and it may serve as a model to screen for compounds that could potentially treat aberrant angiogenesis.

Chronotopographical distribution patterns of cell death and of lectin‐positive macrophages/microglial cells during the visual system ontogeny of the small‐spotted catshark Scyliorhinus canicula

The patterns of distribution of TUNEL-positive bodies and of lectin-positive phagocytes were investigated in the developing visual system of the small-spotted catshark Scyliorhinus canicula, from the optic vesicle stage to adulthood. During early stages of development, TUNEL-staining was mainly found in the protruding dorsal part of the optic cup and in the presumptive optic chiasm. Furthermore, TUNEL-positive bodies were also detected during detachment of the embryonic lens. Coinciding with the developmental period during which ganglion cells began to differentiate, an area of programmed cell death occurred in the distal optic stalk and in the retinal pigment epithelium that surrounds the optic nerve head. The topographical distribution of TUNEL-positive bodies in the differentiating retina recapitulated the sequence of maturation of the various layers and cell types following a vitreal-to-scleral gradient. Lectin-positive cells apparently entered the retina by the optic nerve head when the retinal layering was almost complete. As development proceeded, these labelled cells migrated parallel to the axon fascicles of the optic fiber layer and then reached more external layers by radial migration. In the mature retina, lectin-positive cells were confined to the optic fiber layer, ganglion cell layer and inner plexiform layer. No evident correlation was found between the chronotopographical pattern of distribution of TUNEL-positive bodies and the pattern of distribution of lectin-labelled macrophages/microglial cells during the shark's visual system ontogeny.

Correlation of optic retinal nerve fiber layer thickness and visual function in patients with nonproliferative diabetic retinopathy

To investigate the change of optic retinal nerve fiber layer (RNFL) thickness in nonproliferative diabetic retinopathy (NPDR) and to evaluate the correlation between the optic RNFL structural change and visual function. A cross-sectional study. All cases came from ophthalmology department of Zhejiang Province Traditional Chinese Medical Hospital, and the First People's Hospital of Ningbo, and Zhongshan Ophthalmic Center, Sun Yat-Sen University from December 2009 to October 2012. All the disease and control cases were coming from the same hospitals at the same period. Subjects were divided into 3 different groups: patients with NPDR (n = 89, 89 eyes), patients with diabetic mellitus but without diabetic retinopathy (NRD) (n = 96, 96 eyes) and disease-free controls (n = 115, 115 eyes). One eye of each subject was randomly selected for study. Participants aged from 40 to 70 years at baseline and all diabetic patients had a diabetic history of at least 5 years. Optic RNFL thickness of each subject was measured by spectral domain optical coherence tomography(SD-OCT). Visual function examinations including contrast sensitivity test in spatial frequencies of 1.5, 3.0,6.0, 12.0, 18.0 cycles per degree (c/d), pattern electroretinograms (PERG) and best corrected visual acuity (BCVA) assay. The analysis of RNFL thickness in each group was performed at four preset locations of the optic disc (i.e., temporally, superiorly, nasally, and inferiorly). To assess the structure-function relationship, the BCVA,contrast sensitivity, PERG-P50 amplitude and latency to optic RNFL thickness at each quadrant were analyzed in both NPDR and NRD groups. The RNFL thickness at four preset locations of the optic disc (meanly, nasally, temporally, superiorly, and inferiorly) were (97.7 ± 13.0), (71.7 ± 10.3), (70.9 ± 13.3), (118.3 ± 19.7), and (123.1 ± 20.8) µm in the NPDR group; (98.6 ± 15.3), (74.8 ± 13.1), (71.8 ± 14.6), (119.5 ± 17.2), and (125.6 ± 19.9) µm in the NRD group;and (99.1 ± 11.8), (77.4 ± 12.6), (72.6 ± 13.2), (119.1 ± 18.1), and (127.1 ± 19.3) µm in the normal group. The nasally optic RNFL thickness was significantly different among the three groups (F = 8.56, P = 0.000). The thickness in the NPDR and in the NRD group were significantly thinner than that in the normal group (SNK-q test:q = 3.16, 3.11, respectively; both P < 0.05). In the NPDR group, the nasally optic RNFL thickness was significantly thinner than that in the NRD group (SNK-q test:q = 3.07, P < 0.05). The inferiorly optic RNFL thickness was also significantly different among the three groups (F = 3.841, P = 0.035). The thickness in the NPDR group was significantly thinner than that in the normal group (SNK-q test:q = 3.090, P < 0.05). Yet, the difference of inferiorly optic RNFL thickness between the NRD group and the normal or the NPDR group did not reach the level of statistical significance (SNK-q test:q = 2.101, 1.955, P > 0.05). The temporally or superiorly optic RNFL thickness,or the average thickness of optic RNFL did not differ significantly among the three groups (F = 0.985, P = 0.375; F = 0.333, P = 0.71; F = 0.975, P = 0.379, respectively). Contrast sensitivities (1.5, 3.0,6.0, 12.0, 18.0 c/d), PERG-P50 amplitude and latency were all significantly correlated with the RNFL thickness of the nasal quadrant (nasal = 0.28, 0.26, 0.3, 0.25, 0.45, 0.65, 0.48, respectively; P < 0.05) and the inferior quadrant (inferior = 0.25, 0.28, 0.27, 0.26, 0.28, 0.37, 0.71; P < 0.05) in the NPDR group. Contrast sensitivities in high spatial frequencies (6.0, 12.0, 18.0 c/d), PERG-P50 amplitude were also significantly correlated with the RNFL thickness of the nasal quadrant (nasal = 0.59, 0.45, 0.66, 0.33, respectively; P < 0.05) and the inferior quadrant (inferior = 0.46, 0.71, 0.52, 0.41, respectively; P < 0.05) in the NRD group. The optic RNFL thickness of the nasal and inferior quadrant have been already reduced in patients with NPRD and are significantly correlated with the change of vision function.

Change of retinal nerve fiber layer thickness in patients with nonarteritic inflammatory anterior ischemic optic neuropathy.

In this study, 16 patients (19 eyes) with nonarteritic anterior ischemic optic neuropathy in the acute stage (within 4 weeks) and resolving stage (after 12 weeks) were diagnosed by a series of complete ophthalmic examinations, including fundus examination, optical coherence tomography and fluorescein fundus angiography, and visual field defects were measured with standard automated perimetry. The contralateral uninvolved eyes were used as controls. The retinal nerve fiber layer thickness was determined by optical coherence tomography which showed that the mean retinal nerve fiber layer thickness and the retinal nerve fiber layer thickness from temporal, superior, nasal and inferior quadrants were significantly higher for all measurements in the acute stage than the corresponding normal values. In comparison, the retinal nerve fiber layer thickness from each optic disc quadrant was found to be significantly lower when measured at the resolving stages, than in the control group. Statistical analysis on the correlation between optic disc nerve fiber layer thickness and visual defects demonstrated a positive correlation in the acute stage and a negative correlation in the resolving stage. Our experimental findings indicate that optical coherence tomography is a useful diagnostic method for nonarteritic anterior ischemic optic neuropathy and can be used to evaluate the effect of treatment.

Expression of myelin genes in the developing chick retina

In submammalian animals including chicks, the retina contains oligodendrocytes (OLs), and axons in the optic fiber layer are wrapped with compact myelin within the retina; however, the expression of myelin genes in the chick retina has not been demonstrated yet. In the present study, we examined the expression of three myelin genes (proteolipid protein, PLP; myelin basic protein, MBP; cyclic nucleotide phosphodiesterase, CNP) and PLP in the developing chick retina, in comparison to the localization of Mueller cells. In situ hybridization demonstrated that all three myelin genes began to be expressed at E14 in the chick embryo retina. They are mostly restricted to the ganglion cell layer and the optic fiber layer, with a few exceptions in the inner nuclear layer where Mueller cells reside; however, PLP mRNA+ cells do not express glutamine synthetase, or vice versa. The present results elucidate that myelin genes are expressed only by OLs that are mostly localized in the innermost layer of the developing chick retina.

Performance of Imaging Devices versus Optic Disc and Fiber Layer Photography in a Clinical Practice Guideline for Glaucoma Diagnosis

To compare the performance of Moorfields Regression Analysis (MRA) and optical coherence tomography (OCT) with that of photographic evaluation of the optic nerve head and retinal nerve fiber layer (RNFL) in the application of the Finnish Evidence-Based Guideline for Open-Angle Glaucoma (FEBG-OAG). Patients referred for glaucoma evaluation (n=312) and subjects selected from the general population (n=41) were included in the study. All subjects underwent ophthalmic evaluation, optic nerve head stereophotography, monochromatic RNFL photography, Heidelberg retina tomography, OCT, and laser polarimetry evaluation. The subjects were classified based on stereophotographic or MRA and OCT results by applying the FEBG-OAG. The specificity of the FEBG-OAG for detecting normal patients (stereophotography and imaging devices) was 78% (strict criteria) and 100% (liberal criteria). Agreement between the stereophotographic evaluation and evaluation based on MRA and OCT was 70.2%. Classification of subjects with similar management advice based on these evaluations had 70.5% agreement. Central corneal thickness was a confounding factor in glaucoma diagnosis. Large optic disc sizes played a major role in misleading the diagnosis compared to small discs. Central corneal thickness and large optic disc size are confounding factors in glaucoma diagnosis. Moorfields Regression Analysis and OCT allow for objective implementation of the FEBG-OAG compared to conventional stereophotographic evaluation of the neuroretinal structures.

Myelin oligodendrocyte‐specific protein is expressed in Müller cells of myelinated vertebrate retinas

The retina of nonmammalian vertebrates has a loose myelin that enwraps the large axons of the ganglion cells in all areas, whereas that of mammals lacks myelin, with some exceptions, such as the rabbit retina, which shows compact myelin restricted to the myelinated streak. Electron microscopy studies in chicken retina showed processes of Müller cells (MCs) and oligodendrocytes enwrapping ganglion cell axons. How each of these cells contributes to chicken retina myelination and whether the MC of other myelinated retinas is involved in myelination remain unknown. By immunohistochemistry, with a monoclonal antibody against myelin oligodendrocyte-specific protein (MOSP), we show that MOSP is intensely expressed in the MC and the optic-fiber layer (OFL) in myelinated but not in unmyelinated retinas. By immunocytochemistry with isolated MCs from the chick and rabbit retinas, we show that MOSP is concentrated in the innermost domain of the vitread processes. By immunoblotting, we show that protein extracts from myelinated retinas, but not those from unmyelinated retinas, presented a single band labelled with anti-MOSP of molecular weight similar to that of brain MOSP. In addition, we show that the MC of the embryonic chicken retina starts to express MOSP just before myelination starts. Our results agree with those of electron microscopy studies showing myelin in chick retina formed by MC processes and with those of immunohistochemistry studies in rabbit and human retinas showing expression of other myelin molecules in the MC. Altogether, our results suggest that the MC in myelinated retinas might contribute MOSP to myelin.

Robo2 is required for Slit-mediated intraretinal axon guidance.

The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.

Early development of GABAergic cells of the retina in sharks: An immunohistochemical study with GABA and GAD antibodies

We studied the ontogeny and organization of GABAergic cells in the retina of two elasmobranches, the lesser-spotted dogfish ( Scyliorhinus canicula) and the brown shyshark ( Haploblepharus fuscus) by using immunohistochemistry for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). Both antibodies revealed the same pattern of immunoreactivity and both species showed similar organization of GABAergic cells. GABAergic cells were first detected in neural retina of embryos at stage 26, which showed a neuroepithelial appearance without any layering. In stages 27–29 the retina showed similar organization but the number of neuroblastic GABAergic cells increased. When layering became apparent in the central retina ( stage-30 embryos), GABAergic cells mainly appeared organized in the outer and inner retina, and GABAergic processes and fibres were seen in the primordial inner plexiform layer (IPL), optic fibre layer and optic nerve stalk. In stage-32 embryos, layering was completed in the central retina, where immunoreactivity appeared in perikarya of the horizontal cell layer, inner nuclear layer and ganglion cell layer, and in numerous processes coursing in the IPL, optic fibre layer and optic nerve. From stage 32 to hatching ( stage 34), the layered retina extends from centre-to-periphery, recapitulating that observed in the central retina at earlier stages. In adults, GABA/GAD immunoreactivity disappears from the horizontal cell layer except in the marginal retina. Our results indicate that the source of GABA in the shark retina can be explained by its synthesis by GAD. Such synthesis precedes layering and synaptogenesis, thus supporting a developmental role for GABA in addition to act as neurotransmitter and neuromodulator.

Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.

Growth hormone action in the developing neural retina: A proteomic analysis

The possible presence and action of growth hormone (GH) in the neural retina was investigated in newborn mice. The neural retina was found to be a site of GH gene expression, as GH mRNA was abundant in cells of the retinal ganglion cell layer, in which GH was also detected. It was also a site of GH action, since GH receptor (GHR) immunoreactivity mirrored that of GH. Actions of GH within the eye were indicated by a reduction in its axial length and retinal width (its neuroblastic, inner plexiform, and optic fiber layers) in GHR gene disrupted mice (GHR-/-), in comparison with wild type (GHR+/+) littermates. In the absence of GH signaling, four proteins in the retinal proteome of the GHR-/- mice (identified by 2-D gels and MS) differed in abundance with those in the wild type mice. Brain abundant membrane attached signal protein-1 (BASP-1) was down-regulated, whereas protein kinase C inhibitor 1, cyclophilin A, KH domain-containing, RNA-binding, signal transduction-associated protein 3 were up-regulated in GHR-/- mice. These proteins are involved in retinal vascularization, neural proliferation and neurite outgrowth. GH might thus have hitherto unsuspected roles in these processes during retinal development.

HCN4-like immunoreactivity in rat retinal ganglion cells

Antisera directed against hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels bind to somata in the ganglion cell layer of rat and rabbit retinas, and mRNA for different HCN channel isoforms has been detected in the ganglion cell layer of mouse retina. However, previous studies neither provided evidence that any of the somata are ganglion cells (as opposed to displaced amacrine cells) nor quantified these cells. We therefore tested whether isoform-specific anti-HCN channel antisera bind to ganglion cells labeled by retrograde transport of fluorophore-coupled dextran. In flat-mounted adult rat retinas, the number of dextran-backfilled ganglion cells agreed with cell densities reported in previous studies, and anti-HCN4 antisera bound to the somata of approximately 40% of these cells. The diameter of these somata ranged from 7 to 30 microm. Consistent with localization to cell membranes, the immunoreactivity formed a thin line that circumscribed individual somata. Optic fiber layer axon fascicles, and the proximal dendrites of some ganglion cells, also displayed binding of anti-HCN4 antisera. These results suggest that the response of some mammalian retinal ganglion cells to hyperpolarization may be modulated by changes in intracellular cAMP levels, and could thus be more complex than expected from previous voltage and current recordings.

Gene expression and protein localization of calmodulin‐dependent phosphodiesterase during ontogenesis of chick retina

Calmodulin-dependent phosphodiesterase (PDE1) is a key enzyme in cyclic nucleotides metabolism. We studied its gene expression and protein localization during retinal development in chick embryos. Western blot and densitometric analysis demonstrated that the expression of the three isoforms changed during development. PDE1A was highly expressed at the early stages and decreased as development proceeded. PDE1B expression remained relatively low and constant over time. PDE1C showed a prominent increase (13-fold) between embryonic day (E) 7 and E13, followed by a moderate increase between E13 and postnatal day (P) 1. The presence of the enzyme in the different retinal locations was strongly modulated by development. PDE1A immunostaining was first detected at the ganglion cell level (E7), then in the outer retina (E15-E21). At P5, the immunostaining was confined in the optic fiber layer. Isoform C immunolocalization followed the same inner-outer pattern as isoform A. At 5 days posthatching (P5), the immunoreactivity was restricted, as well as for the isoform A, in the optic fiber layer. The isoform B immunolabelling was low and evenly distributed across the retina at all stages. The different developmental profiles of PDE1A, PDE1B, and PDE1C induced a temporal modulation in cyclic nucleotides concentration, suggesting specific roles of this enzyme in the morphofunctional development of retinal circuitry.

Growth hormone and its receptor in projection neurons of the chick visual system: Retinofugal and tectobulbar tracts

Recent studies have shown the presence of growth hormone (GH) in the retinal ganglion cells (RGCs) of the neural retina in chick embryos at the end of the first trimester [embryonic day (E) 7] of the 21 day incubation period. In this study the presence of GH in fascicles of the optic fiber layer (OFL), formed by axons derived from the underlying RGCs, is shown. Immunoreactivity for GH is also traced through the optic nerve head, at the back of the eye, into the optic nerve, through the optic chiasm, into the optic tract and into the stratum opticum and the retinorecipient layer of the optic tectum, where the RGC axons synapse. The presence of GH immunoreactivity in the tectum occurs prior to synaptogenesis with RGC axons and thus reflects the local expression of the GH gene, especially as GH mRNA is also distributed within this tissue. The distribution of GH-immunoreactivity in the visual system of the E7 embryo is consistent with the distribution of the GH receptor (GHR), which is also expressed in the neural retina and tectum. The presence of a GH-responsive gene (GHRG-1) in these tissues also suggests that the visual system is not just a site of GH production but a site of GH action. These results support the possibility that GH acts as a local growth factor during early embryonic development of the visual system.

Contactin1a expression is associated with oligodendrocyte differentiation and axonal regeneration in the central nervous system of zebrafish

Contactin1a (Cntn1a) is a zebrafish homolog of contactin1 (F3/F11/contactin) in mammals, an immunoglobulin superfamily recognition molecule of neurons and oligodendrocytes. We describe conspicuous Cntn1a mRNA expression in oligodendrocytes in the developing optic pathway of zebrafish. In adults, this expression is only retained in glial cells in the intraretinal optic fiber layer, which contains ‘loose’ myelin. After optic nerve lesion, oligodendrocytes re-express Cntn1a mRNA independently of the presence of regenerating axons and retinal ganglion cells upregulate Cntn1a expression to levels that are significantly higher than those during development. After spinal cord lesion, expression of Cntn1a mRNA is similarly increased in axotomized brainstem neurons and white matter glial cells in the spinal cord. In addition, reduced mRNA expression in the trigeminal/anterior lateral line ganglion in erbb3-deficient mutant larvae implies Cntn1a in Schwann cell differentiation. These complex regulation patterns suggest roles for Cntn1a in myelinating cells and neurons particularly in successful CNS regeneration.

Intraretinal projection of retinal ganglion cell axons as a model system for studying axon navigation

The initial step of retinal ganglion cell (RGC) axon pathfinding involves directed growth of RGC axons toward the center of the retina, the optic disc, a process termed “intraretinal guidance”. Due to the accessibility of the system, and with various embryological, molecular and genetic approaches, significant progress has been made in recent years toward understanding the mechanisms involved in the precise guidance of the RGC axons. As axons are extending from RGCs located throughout the retina, a multitude of factors expressed along with the differentiation wave are important for the guidance of the RGC axons. To ensure that the RGC axons are oriented correctly, restricted to the optic fiber layer (OFL) of the retina, and exit the eye properly, different sets of positive and negative factors cooperate in the process. Fasciculation mediated by a number of cell adhesion molecules (CAMs) and modulation of axonal response to guidance factors provide additional mechanisms to ensure proper guidance of the RGC axons. The intraretinal axon guidance thus serves as an excellent model system for studying how different signals are regulated, modulated and integrated for guiding a large number of axons in three-dimensional space.

Single retinal changing contrast (third) detector elicits NMDA receptor response and higher activity level of frog tectum neuron network

The present study was designed to explore whether a discharge of a certain type of frog retinal ganglion cell [likely changing contrast (third) detector] can evoke NMDA response in frog tectum neurons and higher level of activity of tectal neuron network. Discharge of a single retinal ganglion cell was elicited by electrical stimulation of the retina. Evoked electrical activity of the tectum was recorded by the carbon-fiber microelectrode brought into the optic fiber layer F. We show that: (1) strong discharge of a frog individual retinal ganglion cell (third detector) has evoked NMDA response of tectal neurons and higher level of tectal neuron network activity characterized by prominent suprathreshold excitation of efferent neurons. Consequently, the firing of only one retinal ganglion cell (third detector) could lead to the activation of the tectobulbospinal tract and motor reaction. (2) The excitation of a retinotectal fiber of the first kind (axon of third detector) gave rise to the same effects as activation of a retinotectal fiber of the second kind (axon of fifth detector): the suprathreshold excitation of recurrent and efferent tectal neurons, the slow depolarizing potential (seen as the sNW), and the NMDA receptor activation were observed. However, stronger excitation (longer bursts of action potentials) was needed to evoke those effects in the considered case of the retinotectal input of the first kind. This difference could be attributed to the lower quantal size of neurotransmitter release in synapses of the retinotectal input of the first than second kind.

Expression, Translation, and Localization of a Novel, Small Growth Hormone Variant

A novel transcript of the GH gene has been identified in ocular tissues of chick embryos. It is, however, unknown whether this transcript (small chicken GH, scGH) is translated. This possibility was therefore assessed. The expression of scGH mRNA was confirmed by RT-PCR, using primers that amplified a 426-bp cDNA of its coding sequence. This cDNA was inserted into an expression plasmid to transfect HEK 293 cells, and its translation was shown by specific scGH immunoreactivity in extracts of these cells. This immunoreactivity was directed against the unique N terminus of scGH and was associated with a protein of 16 kDa, comparable with its predicted size. Most of the immunoreactivity detected was, however, associated with a 31-kDa moiety, suggesting scGH is normally dimerized. Neither protein was, however, present in media of the transfected HEK cells, consistent with scGH's lack of a signal sequence. Similar moieties of 16 and 31 kDa were also found in proteins extracted from ocular tissues (neural retina, pigmented epithelium, lens, cornea, choroid) of embryos, although they were not consistently present in vitreous humor. Specific scGH immunoreactivity was also detected in these tissues by immunocytochemistry but not in axons in the optic fiber layer or the optic nerve head, which were immunoreactive for full-length GH. In summary, we have established that scGH expression and translation occurs in ocular tissues of chick embryos, in which its localization in the neural retina and the optic nerve head is distinct from that of the full-length protein.