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1.
J Neurosci ; 21(12): 4318-25, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11404417

ABSTRACT

The axonal projection from the retina to the tectum exhibits a precise topographic order in the mature chick such that neighboring ganglion cells send axons to neighboring termination zones in the contralateral tectum. The initial pattern formed during development is much less organized and is refined to the adult pattern during a discrete period of development. Refinement includes elimination of radically aberrant projections, such as those from the temporal side of the retina to posterior regions of the tectum, as well as a more subtle improvement in the topographic precision of the projection. The enzyme that synthesizes nitric oxide is expressed at high levels in the tectum during the developmental period in which the topography improves. Pharmacological blockade of nitric oxide synthesis during this period prevented elimination of topographically inappropriate retinotectal projections in a dose-dependent manner. This effect could not be duplicated by treatment of embryos with a vasoconstrictor, indicating that vascular changes were not a factor. These results show that nitric oxide is involved in refinement of the topography of the retinotectal projection as well as in other aspects of refinement of this projection in developing chick.


Subject(s)
Nitric Oxide/metabolism , Retina/metabolism , Superior Colliculi/metabolism , Visual Pathways/metabolism , Animals , Chick Embryo , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Microspheres , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitroarginine/pharmacology , Phenylephrine/pharmacology , Retina/drug effects , Retina/embryology , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/metabolism , Superior Colliculi/drug effects , Superior Colliculi/embryology , Vasoconstrictor Agents/pharmacology , Visual Pathways/drug effects , Visual Pathways/embryology
2.
J Neurosci ; 20(4): 1458-69, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10662836

ABSTRACT

The pattern of axonal projections early in the development of the nervous system lacks the precision present in the adult. During a developmental process of refinement, mistargeted projections are eliminated while correct projections are retained. Previous studies suggest that during development nitric oxide (NO) is involved in the elimination of mistargeted retinal axons, whereas brain-derived neurotrophic factor (BDNF) may stabilize retinal axon arbors. It is unclear whether these neuromodulators interact. This study showed that NO induced growth cone collapse and retraction of developing retinal axons. This effect was not attributable to NO-induced neurotoxicity. BDNF protected growth cones and axons from the effects of NO. This effect was specific to BDNF, because neither nerve growth factor (NGF) nor neurotrophin-3 (NT-3) prevented NO-induced growth cone collapse and axon retraction. Exposure to both BDNF and NO, but not either factor alone, stabilized growth cones and axons. Stabilized axons exhibited minimal retraction or extension. This response appears to be a new axon "state" and not simply a partial amelioration of the effect of NO, because lower doses of BDNF or NO allowed axon extension. Furthermore, BDNF/NO-induced growth cone stabilization correlated with the appearance of a cytochalasin D-resistant population of actin filaments. BDNF protection from NO likely was mediated locally at the level of the growth cone, because growth cones or individual filopodia in contact with BDNF-coated beads were protected from NO-induced collapse. These findings suggest a cellular mechanism by which some axonal connections are stabilized and some are eliminated during development.


Subject(s)
Axons/physiology , Brain-Derived Neurotrophic Factor/physiology , Ganglia, Spinal/physiology , Neurons/physiology , Nitric Oxide Donors/pharmacology , Nitric Oxide/physiology , Retina/physiology , Animals , Axons/drug effects , Axons/ultrastructure , Brain-Derived Neurotrophic Factor/pharmacology , Cells, Cultured , Chick Embryo , Ganglia, Spinal/cytology , Ganglia, Spinal/embryology , Heart/embryology , Hydrazines/pharmacology , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Neurons/cytology , Neurons/drug effects , Neurotrophin 3/pharmacology , Organ Culture Techniques , Retina/cytology , Retina/embryology , Signal Transduction , Visual Pathways/physiology
3.
J Neurosci ; 19(1): 229-35, 1999 Jan 01.
Article in English | MEDLINE | ID: mdl-9870953

ABSTRACT

A transient ipsilateral retinotectal projection is normally eliminated during embryonic development of the chick visual system. Administration of the NMDA receptor antagonist 5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) during the developmental period in which this projection normally disappears prevented its complete elimination. Previous studies showed that tectal cells express nitric oxide synthase during development, and blocking synthesis of nitric oxide also prevented elimination of the ipsilateral retinotectal projection. The effect of NMDA receptor blockade on nitric oxide synthase activity in tectal cells was assessed biochemically in chick embryos. Increasing concentrations of MK-801 resulted in a dose-dependent decrease in nitric oxide synthase activity. This result suggests that NMDA receptor activation can regulate nitric oxide synthase activity in the tectum. The degree of rescue of the ipsilateral retinotectal projection was compared in embryos treated either with MK-801 or with an inhibitor of nitric oxide synthesis, Nomega-nitro-L-arginine (L-NoArg). At comparable levels of inhibition of nitric oxide synthesis, no significant difference was observed in the degree of rescue mediated by NMDA receptor blockade or nitric oxide synthesis blockade. These results suggest that NMDA receptor-mediated elimination of the ipsilateral retinotectal projection is completely mediated via nitric oxide.


Subject(s)
Chick Embryo/physiology , Nitric Oxide/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Retina/physiology , Superior Colliculi/physiology , Visual Pathways/physiology , Analysis of Variance , Animals , Cell Death , Chick Embryo/growth & development , Dizocilpine Maleate/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/antagonists & inhibitors , Nitroarginine/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Retina/drug effects , Retina/embryology , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/physiology , Superior Colliculi/drug effects , Superior Colliculi/embryology , Time Factors , Visual Pathways/drug effects , Visual Pathways/embryology
4.
Development ; 125(6): 1059-66, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9463352

ABSTRACT

The different retinal cell types arise during vertebrate development from a common pool of progenitor cells. The mechanisms responsible for determining the fate of individual retinal cells are, as yet, poorly understood. Ganglion cells are one of the first cell types to be produced in the developing vertebrate retina and few ganglion cells are produced late in development. It is possible that, as the retina matures, the cellular environment changes such that it is not conducive to ganglion cell determination. The present study showed that older retinal cells secrete a factor that inhibits the production of ganglion cells. This was shown by culturing younger retinal cells, the test population, adjacent to various ages of older retinal cells. Increasingly older retinal cells, up to embryonic day 9, were more effective at inhibiting production of ganglion cells in the test cell population. Ganglion cell production was restored when ganglion cells were depleted from the older cell population. This suggests that ganglion cells secrete a factor that actively prevents cells from choosing the ganglion cell fate. This factor appeared to be active in medium conditioned by older retinal cells. Analysis of the conditioned medium established that the factor was heat stable and was present in the <3 kDa and >10 kDa fractions. Previous work showed that the neurogenic protein, Notch, might also be active in blocking production of ganglion cells. The present study showed that decreasing Notch expression with an antisense oligonucleotide increased the number of ganglion cells produced in a population of young retinal cells. Ganglion cell production, however, was still inhibited in cultures using antisense oligonucleotide to Notch in medium conditioned by older retinal cells. This suggests that the factor secreted by older retinal cells inhibits ganglion cell production through a different pathway than that mediated by Notch.


Subject(s)
Retina/cytology , Retina/embryology , Retinal Ganglion Cells/cytology , Animals , Base Sequence , Cell Differentiation , Cell Division , Cells, Cultured , Chick Embryo , Culture Media, Conditioned , Gene Expression Regulation, Developmental , Membrane Proteins/genetics , Membrane Proteins/metabolism , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , Receptors, Notch , Retina/metabolism , Retinal Ganglion Cells/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Time Factors
5.
Prog Brain Res ; 118: 115-31, 1998.
Article in English | MEDLINE | ID: mdl-9932438

ABSTRACT

Axons of retinal ganglion cells exhibit a specific pattern of connections with the brain. Within each visual nucleus in the brain, retinal connections are topographic such that axons from neighboring ganglion cells have neighboring synapses. Research is beginning to shed light on the mechanisms responsible for development of topographic connections in the visual system. Much of this research is focused on the axonal connections of the retina with the tectum. In vivo and in vitro experiments indicate that the pattern of retinotectal connections develops in part due to positional labels carried by the growing retinal axons and by the tectal cells. Evidence suggests that gradients of Eph receptor tyrosine kinases serve as positional labels on the growing retinal axons, and gradients of ligands for these receptors serve as positional labels in the tectum. Blocking expression of EphA3, a receptor tyrosine kinase, in the developing retina resulted in disruption of the topography of the retinotectal connections, further supporting the role of these, molecules. Although positional labels appear to be important, other mechanisms must also be involved. The initial pattern of retinotectal connections lacks the precision seen in the adult. The adult pattern of connections arises during development by activity dependent refinement of a roughly ordered prepattern. The refinement process results in elimination of projections to the wrong side of the brain, to non-visual nuclei and to inappropriate regions within a nucleus. Blocking NMDA receptors during the period of refinement preserved anomalous retinotectal projections, which suggests that elimination of these projections is mediated by NMDA receptors. Furthermore, tectal cells normally express high levels of nitric oxide synthase (NOS) during the period of refinement, and blocking nitric oxide (NO) synthesis also preserved inappropriate projections. Thus, both NMDA receptors and NO appear to be involved in refinement. Blocking NMDA receptor activation reduced NOS activity in tectal cells, which suggests the possibility that NO is the downstream mediator of NMDA function related to refinement. A quantitative comparison of blocking NMDA receptors, NO synthesis or both showed that all three treatments have comparable effects on refinement. This indicates that the role of NMDA receptor activation relative to refinement may be completely mediated through nitric oxide. Quantitative analysis also suggests that other mechanisms not involving NMDA receptors or NO must be involved in refinement. Other mechanisms appear to include cell death.


Subject(s)
Nitric Oxide/physiology , Receptor Protein-Tyrosine Kinases/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Retina/growth & development , Superior Colliculi/growth & development , Animals , Axons/physiology , Humans , Nitric Oxide/biosynthesis , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, EphA3 , Receptors, N-Methyl-D-Aspartate/metabolism , Retina/metabolism , Substrate Specificity , Superior Colliculi/metabolism
7.
J Comp Neurol ; 352(4): 594-606, 1995 Feb 20.
Article in English | MEDLINE | ID: mdl-7722002

ABSTRACT

Previous investigations have identified proteoglycans in the central nervous system during development and have implicated some proteoglycans as axon guidance molecules that act by inhibiting axon extension. The present study investigated the pattern of immunoreactivity for several glycosaminoglycans common to certain proteoglycans relative to growing retinal axons in the developing chick visual system and in retinal explant cultures. Immunostaining for chondroitin-6-sulfate, chondroitin-4-sulfate, and keratan sulfate was observed to colocalize with retinal axons throughout the retinofugal pathway during the entire period of retinal axon growth. The proteoglycan form of collagen IX, however, was only observed in the retina, primarily peripheral to the areas with actively growing axons. The pattern of immunostaining for chondroitin sulfate in tissue sections suggested that the retinal axons might be a source for some of the chondroitin sulfate immunostaining in the developing visual pathway. This was confirmed in that chondroitin sulfate immunostaining was also observed on neurites emanating from cultured retinal explants. These findings indicate that retinal axons grow in the presence of chondroitin sulfate and keratan sulfate proteoglycans and that these proteoglycans in the developing chick visual pathway have functions other than to inhibit axon growth.


Subject(s)
Chondroitin Sulfates/genetics , Keratan Sulfate/genetics , Proteoglycans/genetics , Retina/cytology , Visual Pathways/cytology , Animals , Cell Division , Chick Embryo , Chondroitin Sulfates/metabolism , Gene Expression , Immunohistochemistry , Keratan Sulfate/metabolism , Proteoglycans/immunology , Proteoglycans/metabolism , Retina/physiology , Visual Pathways/physiology
8.
Neuron ; 14(1): 117-24, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7826629

ABSTRACT

The aim of this study is to gain insight into the time during the life history of a retinal neuron that it becomes committed to a particular phenotype. At this point, it is not possible to identify the time of commitment, but the time that differentiation begins can be identified. Bromodeoxyuridine labeling coupled with immunohistochemistry with a ganglion cell-specific antibody was used to fix the time of the beginning of ganglion cell differentiation relative to the time of mitosis in the developing chick retina. It was found that ganglion cells can begin to differentiate in less than 15 min after the end of mitosis. This suggests that the retinal ganglion cell fate may be determined before or during mitosis.


Subject(s)
Cell Differentiation , Mitosis , Retina/embryology , Retinal Ganglion Cells/cytology , Animals , Bromodeoxyuridine/metabolism , Chick Embryo , Immunohistochemistry , Kinetics , Time Factors
9.
Science ; 265(5178): 1593-6, 1994 Sep 09.
Article in English | MEDLINE | ID: mdl-7521541

ABSTRACT

The adult pattern of axonal connections from the eye to the brain arises during development through the refinement of a roughly ordered set of connections. In the chick visual system, refinement normally results in the loss of the ipsilateral retinotectal connections. Inhibition of nitric oxide synthesis reduced the loss of these transient connections. Because nitric oxide is expressed by tectal cells with which retinal axons connect and because reduction of nitric oxide synthesis by tectal cells resulted in a change in the connections of retinal axons, nitric oxide probably serves as a messenger from tectal cells back to retinal axons during development.


Subject(s)
Axons/metabolism , Neurons/metabolism , Nitric Oxide/metabolism , Retina/embryology , Retinal Ganglion Cells/metabolism , Superior Colliculi/embryology , Amino Acid Oxidoreductases/metabolism , Animals , Arginine/analogs & derivatives , Arginine/pharmacology , Chick Embryo , NG-Nitroarginine Methyl Ester , Nitric Oxide/biosynthesis , Nitric Oxide Synthase , Nitroarginine , Retinal Ganglion Cells/cytology , Superior Colliculi/metabolism , Visual Pathways
10.
J Neurosci ; 14(8): 4806-14, 1994 Aug.
Article in English | MEDLINE | ID: mdl-8046451

ABSTRACT

AtT-20 cells, which make and release beta-endorphin, or AtT-20/hENK cells, an AtT-20 cell line transfected with the human proenkephalin gene and secreting enkephalin as well as presumably beta-endorphin, were implanted in mouse spinal subarachnoid space. Cell implants did not affect the basal response to thermal nociceptive stimuli. Administration of isoproterenol, believed to stimulate secretion from these cells, produced antinociception in groups receiving AtT-20 or AtT-20/hENK cell implants but not in control groups receiving no cells. The antinociceptive effect of isoproterenol was dose related and could be blocked by the opioid antagonist naloxone. Implantation of these cells offers a novel approach for the study of tolerance. Mice receiving AtT-20 cell implants developed tolerance to beta-endorphin and the mu-opioid agonist DAMGO, whereas mice receiving genetically modified AtT-20/hENK cell implants developed tolerance to the delta-opioid agonist DPDPE. Genetically modified AtT-20/hENK cell implants, but not AtT-20 cell implants, reduced the development of acute morphine tolerance in the host mice. This finding is consistent with the suggestion that enkephalin alters development of opioid tolerance. These results suggest that opioid-releasing cells implanted around mouse spinal cord can produce antinociception and may provide an alternative therapy for chronic intractable pain.


Subject(s)
Enkephalins/metabolism , Narcotics/pharmacology , Nociceptors/drug effects , Protein Precursors/metabolism , beta-Endorphin/metabolism , Animals , Cell Line , Cell Transplantation , Dose-Response Relationship, Drug , Drug Tolerance , Enkephalin, Ala(2)-MePhe(4)-Gly(5)- , Enkephalin, D-Penicillamine (2,5)- , Enkephalins/genetics , Enkephalins/pharmacology , Isoproterenol/pharmacology , Male , Mice , Mice, Inbred ICR , Pituitary Neoplasms/metabolism , Protein Precursors/genetics , Spinal Cord/metabolism , Spinal Cord/surgery , Transfection , Tumor Cells, Cultured
11.
J Neurosci ; 14(3 Pt 2): 1746-55, 1994 Mar.
Article in English | MEDLINE | ID: mdl-7510335

ABSTRACT

A precise pattern of connections between the retina and central visual nuclei in the brain is established during development. Activity-dependent presynaptic mechanisms and NMDA receptor-mediated postsynaptic mechanisms are thought to play important roles in this developmental process. A model proposed for production of the newly described neurotransmitter, nitric oxide, involves presynaptic activity and activation of postsynaptic NMDA receptors. If present in the developing visual system, nitric oxide could represent a form of retrograde communication from postsynaptic to presynaptic cells that mediates the formation of the proper pattern of connections. This study used the diaphorase histochemical technique to detect the presence of nitric oxide synthase (NOS), the enzyme responsible for the production of nitric oxide, in the developing chick optic tectum. Results from this study showed that NOS is present in the developing tectum and that its expression coincides temporally with innervation by retinal axons. NOS expression reaches a peak at the time that refinement of the initial pattern of connections is occurring. WGA/HRP labeling of retinal axons confirmed that processes of NOS-positive cells in the tectum extend well into the area of the ingrowing retinal axons. Histochemical results from eyeless chick embryos indicate that NOS expression is dependent on the presence of retinal axons, which suggests that retinal axons synapse on cells that express nitric oxide. Northern blot analysis using a cDNA probe to NOS from rat brain verified the histochemical results. These results are consistent with nitric oxide having a role in development of the proper pattern of connections in the chick retinotectal system.


Subject(s)
Amino Acid Oxidoreductases/metabolism , Axons/physiology , Synaptic Transmission , Visual Pathways/embryology , Animals , Blotting, Northern , Chick Embryo , Embryonic and Fetal Development , Eye Enucleation , Histocytochemistry , NADPH Dehydrogenase/metabolism , Nitric Oxide Synthase , Reference Values
12.
J Neural Transplant Plast ; 4(1): 15-26, 1993.
Article in English | MEDLINE | ID: mdl-8110861

ABSTRACT

AtT-20 cells, which produce beta-endorphin, and AtT-20/hENK cells, which are AtT-20 cells transfected with a proenkephalin gene, were implanted in the rat spinal subarachnoid space in an effort to produce an antinociceptive effect. Host rats were tested for antinociceptive activity by standard nociceptive tests, tail flick and hot plate. Although cell implants had minimal effect on the basal response to thermal nociceptive stimuli, administration of the beta 2-adrenergic agonist isoproterenol produced antinociception in the cell-implanted group but not in the control group. The antinociceptive effect of isoproterenol was dose-related and could be blocked by the opioid antagonist naloxone. Immunohistochemical analysis of spinal cords revealed the presence of enkephalin-negative cells surrounding the spinal cord of rats receiving AtT-20 cell implants, and enkephalin-positive cells surrounding the spinal cord of rats receiving AtT-20/hENK cell implants. These results suggest that opioid-releasing cells implanted around rat spinal cord can produce antinociception and may provide an alternative therapy for chronic pain.


Subject(s)
Cell Transplantation , Enkephalins/metabolism , Pain Management , Pituitary Gland, Anterior/pathology , Pituitary Neoplasms/pathology , Protein Precursors/metabolism , Spinal Cord/physiopathology , beta-Endorphin/metabolism , Animals , Enkephalins/genetics , Isoproterenol/antagonists & inhibitors , Isoproterenol/pharmacology , Male , Mice , Naloxone/pharmacology , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Pain/drug therapy , Pain/physiopathology , Pain Measurement , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/metabolism , Pituitary Neoplasms/metabolism , Protein Precursors/genetics , Pyrrolidinones/pharmacology , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism , Rolipram , Secretory Rate/drug effects , Subarachnoid Space , Transfection , Tumor Cells, Cultured/transplantation
13.
Biotechniques ; 12(6): 842-7, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1642892

ABSTRACT

Traditional techniques for the production of monoclonal antibodies usually result in generation of monoclonal antibodies to immunodominant molecules. To enhance the production of monoclonal antibodies to rare or less immunodominant antigens, subtractive immunization techniques have been employed. This study compared the ability of several subtractive immunization techniques to suppress the immune system to a given antigen. Neonatal tolerization, chemical immunosuppression with cyclophosphamide, a combination of the two and various permutations of these techniques were compared. The results from this study indicated that chemical immunosuppression with cyclophosphamide was the most effective subtractive immunization technique and that the cyclophosphamide regime employed was a critical determinant in the success of chemical immunosuppression.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antigens/immunology , Immunization/methods , Animals , Animals, Newborn , Antibodies, Monoclonal/immunology , Chick Embryo , Cyclophosphamide/pharmacology , Immune Tolerance/drug effects , Immunosuppression Therapy , Mice
14.
J Neurosci ; 11(5): 1470-7, 1991 May.
Article in English | MEDLINE | ID: mdl-2027056

ABSTRACT

A monoclonal antibody was developed that recognizes an antigen with an asymmetric distribution in the chick retina. Immunohistochemistry showed that this antigen, temporal retinal axon protein (TRAP), was present on most if not all axons that arose from the temporal side of the retina. Very few of the axons from the nasal side of the retina were positive for TRAP. The nasal-temporal difference appeared to be in the number of axons that stained with this antibody rather than in the intensity of staining. The transition between nasal and temporal retina based on TRAP distribution appeared to be a vertical line centered on the optic fissure. A competition-based ELISA was developed to quantify the average amount of TRAP on axons in different regions of the retina. This assay also suggested that the pattern of TRAP distribution across the retina was a step function, though the results did not completely rule out the possibility of a continuous concentration gradient oriented circumferentially around the retina. Explants of embryonic nasal and temporal retina had a similar dichotomy in TRAP expression during the first 1 or 2 d in culture. The antibody to TRAP bound to retinal neurites in culture without the cell membrane being made permeable, which suggests that TRAP is a cell-surface molecule. In culture, TRAP was also expressed on the growth cones. Immunoblots showed that TRAP is trypsin sensitive and has an approximate molecular mass of 135 kDa. This is the first molecule identified with an asymmetric distribution in the nasal-temporal axis of the retina.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Antibodies, Monoclonal , Avian Proteins , Retina/cytology , Retinal Ganglion Cells/cytology , Animals , Antigens, Surface/analysis , Cells, Cultured , Chick Embryo , Enzyme-Linked Immunosorbent Assay , Eye Proteins/analysis , Immunoblotting , Mice , Mice, Inbred BALB C , Organ Culture Techniques
15.
J Neurosci ; 11(2): 445-53, 1991 Feb.
Article in English | MEDLINE | ID: mdl-1846908

ABSTRACT

During development of the projection from the retina to the brain in the chick, a transient ipsilateral retinotectal projection forms and disappears. This disappearance is coincident with a wave of ganglion cell death in the retina. The contribution of cell death to the disappearance of this projection, as opposed to another mechanism such as axon retraction, was examined. Retinal ganglion cells with a projection to the ipsilateral tectum were retrogradely labeled by injection of long-lasting fluorescent dyes into the tectum prior to the onset of ganglion cell death. Large injections of fast blue labeled approximately 1800 ganglion cells in the ipsilateral retina before the period of cell death began. If the injected embryos were allowed to survive past the peak period of ganglion cell death, the average number of labeled ganglion cells in the ipsilateral retina was reduced by somewhat more than half. It is possible that the remaining labeled ganglion cells projected only to nontectal visual nuclei and were labeled by fast blue that had diffused out of the tectum. This was tested by repeating the study using very localized injections of 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate or fluorescent microspheres into the tectum. These small injections confirmed that cells with transient projections to the ipsilateral tectum survived past the elimination of this projection. Thus, ipsilaterally projecting ganglion cells have, at most, a slightly greater propensity for death than the average ganglion cell, and elimination of the transient ipsilateral retinotectal projection in chick can be explained only, in part, by the mechanism of cell death.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Retina/physiology , Retinal Ganglion Cells/physiology , Superior Colliculi/physiology , Synaptic Transmission , Animals , Cell Survival , Chick Embryo , Fluorescent Dyes , Microspheres , Retina/cytology , Retina/embryology , Superior Colliculi/embryology , Time Factors
16.
Anat Embryol (Berl) ; 184(5): 433-40, 1991.
Article in English | MEDLINE | ID: mdl-1741476

ABSTRACT

Changes in the morphology of the early optic cup were observed in embryos of two distantly-related vertebrate species, a teleost fish, northern pike (Esox lucius), and chicken (Gallus gallus). A similar morphological pattern was noted to appear in both species shortly after the involution of the optic vesicle and the formation of the inner retinal layer. At a gross level, three notches were observed in the retinal margin at approximately nasal, dorsal, and temporal positions, while in histological sections a sharp constriction was found in the thickness of the dorsal retinal layer. In both species, this dorsal constriction appeared to be continuous with the central or dorsal notch. The time of appearance and configuration of this morphological pattern is intriguingly similar to the specification and polarity of retinal positional markers, and suggest a segmentation hypothesis for the origin of retinal polarity.


Subject(s)
Fishes/embryology , Retina/embryology , Animals , Chick Embryo , Eye/anatomy & histology , Eye/embryology
17.
J Neurosci ; 9(4): 1424-32, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2703885

ABSTRACT

A monoclonal antibody, RA4, was developed that recognizes retinal ganglion cell axons in the mature retina. Between embryonic days 3 and 9, the RA4 antigen was associated with cell bodies in certain regions of the retina in addition to the ganglion cell axons. The RA4-positive cells were of 3 types: an apolar cell adjacent to the ventricular surface, a bipolar cell that spanned the thickness of the retina, and a monopolar cell in the ganglion cell layer. Evidence suggests that these cells are premigratory and migrating retinal ganglion cells. The expression of the RA4 antigen is the earliest indicator of ganglion cell differentiation yet reported. The existence of RA4-positive apolar cells along the outer surface of the retina suggests that the ganglion cell phenotype is expressed as soon as the cell becomes postmitotic. Approximately 20% of the migrating ganglion cells were in pairs. The paired cells most likely arose from the terminal division of a germinal cell. One possibility suggested by these data is that a ganglion cell-specific germinal cell arises from a pluripotent germinal cell. Immunoblots and other analyses revealed the RA4 antigen to be a 140 kDa cytoplasmic protein in the retina. RA4 also recognized many long tract axons in the brain. In the brain, the RA4 epitope was observed on proteins with at least 7 different molecular weights. Evidence suggests that different cell types may express the RA4 antigen with slightly different molecular weights.


Subject(s)
Axons/metabolism , Nerve Tissue Proteins/metabolism , Retina/cytology , Retinal Ganglion Cells/cytology , Animals , Antibodies, Monoclonal , Brain/metabolism , Cell Differentiation , Cell Movement , Chick Embryo , Immunoblotting , Immunohistochemistry , Retina/metabolism , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/physiology
18.
J Neurosci ; 8(6): 1981-90, 1988 Jun.
Article in English | MEDLINE | ID: mdl-3385486

ABSTRACT

The optic nerve of the developing rat was examined for the presence of laminin, an adhesive glycoprotein, to assess whether it might serve as a substrate for retinal axon growth in vivo. The optic stalk and nerve of developing rats were screened immunohistochemically for the presence of laminin before, during, and after the period of retinal axon growth. On embryonic day 14 (E14), laminin immunoreactivity was present in the ventral portion of the optic stalk, the same region in which the first retinal axons grow. Between E16 and postnatal day 10 (P10), cells positive for laminin were distributed throughout the cross-sectional area of the nerve. There was a progressive appearance of glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for astrocytes, from the optic nerve head towards the chiasm beginning on E20. At the advancing front of GFAP immunoreactivity, cells were positive for both laminin and GFAP. Behind the front, laminin immunoreactivity disappeared from the cells. By P12, the only laminin immunoreactivity that remained within the optic nerve surrounded the vasculature. This is a time after the last retinal axons grow through the optic nerve. Monolayer cell cultures were prepared from perinatal rat optic nerves and processed for immunohistochemistry to determine which astrocyte type was laminin-positive. Type 1 astrocytes, which primarily compose the immature nerve, are GFAP-positive, A2B5-negative, and laminin-positive. Type 2 astrocytes, a major component of the mature optic nerve, were GFAP-positive, A2B5-positive and laminin-negative. An extract of developing optic nerve was analyzed by immunoblot along with laminin purified from Engelbreth-Holm-Swarm (EHS) sarcoma. Purified laminin ran with SDS-PAGE under reducing conditions as 2 bands with Mrs of 200,000 and 4000,000. Both bands reacted with antibodies to laminin. A low-salt extraction of whole optic nerve from E18 rats resulted in 2 bands with the same Mr as seen with laminin from EHS sarcoma. When only the inside of the optic nerve (which lacked the basal lamina and meninges that surround the outside) was processed, there was a dark 200,000 D band, but the 400,000 D band was virtually absent. These results are consistent with the hypothesis that laminin, or a variant form of laminin, serves as a substrate for retinal axon growth in the developing rat optic nerve.


Subject(s)
Animals, Newborn/metabolism , Fetus/metabolism , Laminin/metabolism , Optic Nerve/metabolism , Animals , Animals, Newborn/growth & development , Cells, Cultured , Immunohistochemistry , Neuroglia/metabolism , Optic Nerve/embryology , Optic Nerve/growth & development , Rats , Rats, Inbred Strains
19.
Brain Res ; 467(1): 61-8, 1988 Mar 01.
Article in English | MEDLINE | ID: mdl-3359331

ABSTRACT

We have examined the ability of Schwann cell-conditioned medium (SCCM) to promote neurite growth from embryonic retina and tectum in explant culture. Both retinal and tectal explants adhered to polylysine substrates, but neurite outgrowth from the explants was minimal when grown in the presence of non-conditioned serum-free defined medium. The addition of SCCM resulted in a significant extension of neurites from both types of explants. The neurite outgrowth from the retinal explants was mainly radial and appeared to be in fascicles of small diameter. Neurite outgrowth from the tectal explants stimulated by the SCCM tended to be in larger fascicles and in a more convoluted pattern than that seen from the retinal explants. After serial passaging of the SCCM to remove any substrate-adsorbable factors all neurite-promoting activity was lost. Neurite growth from 100% of the explants was seen on the tissue culture plates that had been preincubated with the SCCM. SCCM was analyzed by immunoblot to determine whether it contained laminin, a potent promoter of neurite outgrowth. Samples of media were electrophoresed on a sodium dodecyl sulphate (SDS)-polyacrylamide gel alongside purified laminin, transferred to nitrocellulose and stained with an antibody to laminin. Purified laminin ran in two bands at 400 kDa and 200 kDa. The conditioned media had a laminin positive band at 200 kDa. Antibody to laminin, when added to the conditioned media, resulted in a loss of the neurite promoting activity of the SCCM. These results suggest that the conditioned media contained laminin or a laminin-like molecule.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Culture Media , Dendrites/physiology , Nerve Growth Factors/pharmacology , Retina/cytology , Schwann Cells/metabolism , Superior Colliculi/cytology , Animals , Cells, Cultured , Dendrites/drug effects , Laminin/analysis , Nerve Growth Factors/metabolism , Rats , Rats, Inbred Strains , Retina/drug effects , Retina/physiology , Schwann Cells/physiology , Superior Colliculi/drug effects , Superior Colliculi/physiology
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