Steven C. McLoon

Ganglion cell death during normal retinal development in the chick: comparisons with cell death induced by early target field destruction.

Abstract Ganglion cell death during normal retinal development in the chick was observed in retinal whole mounts and in specimens sectioned for light and electron microscopy. Pyknotic cells were observed between the 12th and 16th days of incubation. These accounted for the loss of at least 20% of the cells from the ganglion cell layer. Early destruction of the primordial optic tectum produced an increased degeneration of ganglion cells (72%) during this same critical period when they evidently became dependent on central associations. Death of ganglion cells at this time in normal development suggests that significant numbers may ordinarily be unable to form sustaining connections. Normal and experimentally induced cell death immediately preceded a phase of cytological maturation which was generally evident in the ganglion cell layer by the 15th day of incubation. Developments with respect to the target field may not only maintain the cell but actually influence its late maturation. Counts from selected regions of retinal whole mounts revealed an overall temporal to nasal progression of cell death. This pattern was also seen in retinae when the tectum had been destroyed early in development. The pattern, therefore, was not regulated by interactions with target field, suggesting that ganglion cells in early phases of development proceed according to an intrinsic retinal program. The temporal to nasal retinal sequence, although independent of the tectum, appears to correspond to the progress of maturation in retinotopically related regions of the tectum. Coincident developments in the two fields could contribute to the retinotopic organization of the visual projection on region to region basis.

Specific projections of retina transplanted to rat brain.

SummaryRetinae were taken from fetal rats and transplanted adjacent to the superior colliculus of neonatal rats. After 1 month survival, the transplants were surgically removed from the hosts, locally damaged or injected with horseradish peroxidase (HRP) to determine the distribution of the transplant efferents in the host brains. Histological examination of the transplants revealed cell and plexiform layers characteristic of normal retinae. Since the retinae were undifferentiated at the time of transplantation, this layering developed within the host. The only obvious differences from normal retina were that the layers were organized in rosettes or folded sheets and lacked well developed photoreceptor outer segments. In animals which had lesions or HRP injections confined to the retinal transplant, proper staining of sections of the host brain revealed transplant projections. These projections were confined to the optic tract and nuclei which are normally retinorecipient such as the superior colliculus and dorsal lateral geniculate nucleus. Projections were found along the border of non-retinorecipient nuclei such as the lateral posterior nucleus, but did not appear to enter these nuclei. It was observed that within the superior colliculus the host retinal input had an effect on the distribution of the transplant projection. In one-eyed hosts the transplant projection was distributed throughout the stratum (s.) zonale, s. griseum superficiale, and s. opticum; whereas in the two-eyed hosts, the transplant projection was confined to the s. zonale and the border between s. griseum superficiale and s. opticum.We suggest that a special affinity exists between the axons of the retinal transplants and host visual structures. Furthermore, factors, such as competition and timing may be important in determining the distribution of the transplant axons within the specific target nuclei. Transplantation appears to be a useful technique for further studies on the mechanisms underlying the development of specific neuronal connections.

IMMEDIATE DIFFERENTIATION OF GANGLION CELLS FOLLOWING MITOSIS IN THE DEVELOPING RETINA

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.

Transient retinofugal pathways in the developing chick.

SummaryThe central retinal projections have been examined in normal chick embryos early in development to determine if a transient ipsilateral projection is present. Anterograde transport of HRP identified using tetramethyl benzidine as a chromogen and degeneration techniques were used on embryos of successive ages between 6 and 16 days of incubation to show the central distribution of the retinal axons from one eye. Besides the anticipated contralateral projection, a small projection was identified to primary visual nuclei on the side of the brain ipsilateral to the injected or lesioned eye in embryos between days 6 and 12 of incubation. A projection from the injected eye into the contralateral optic nerve was also identified in a number of embyos. By embryonic day 15 the retinal projection to the ipsilateral side of the brain and into the contralateral optic nerve had disappeared. This loss of the anomalous projections coincides with a period of substantial cell death in the retinal ganglion cell layer. It appears, therefore, that in the chick, like the rat, the ipsilateral retinofugal projection resulting from an embryonic enucleation may in part be due to retention of a normal ipsilateral projection.

Nitric oxide and the developmental remodeling of retinal connections in the brain

Publisher Summary The chapter focuses on the nitric oxide (NO) and the development remoting of retinal connections in the brain. The involvement of the postsynaptic cells with changes in the connections of retinal axons suggests that the postsynaptic neurons communicate retrogradely with the axons. This retrograde signal in some way instructs the axons as to their proper connections. The nature of this retrograde signal is not known, but recent studies suggest that NO is involved. NO has characteristics that could allow it to function as a retrograde messenger in the developing visual system. In some brain cells, when N-methyl-D-aspartate (NMDA) receptors are activated by the neurotransmitter glutamate, NO is synthesized and released. NO is a soluble gas and can diffuse across cell membranes without vesicle mediated release. In certain cells NO causes an increase in cyclic guanosine 3’, 5’-monophosphate levels. Retinal ganglion cells express a cyclic guanosine monophosphate (cGMP)-gated calcium channel responsive to NO suggesting that ganglion cells may respond to NO with an increase in intracellular calcium levels. An increase in intracellular calcium concentration has been shown to influence the extension and retraction of axons, activities that are essential to the refinement process.

Implantation of AtT-20 or genetically modified AtT-20/hENK cells in mouse spinal cord induced antinociception and opioid tolerance

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.

Cultured embryonic retinae transplanted to rat brain: Differentiation and formation of projections to host superior colliculus

Retinae of rats on embryonic day 14 were placed in explant culture for 2-14 days prior to transplantation adjacent to the superior colliculus of newborn rats. In explant culture cell division and neuronal differentiation continued unabated. One month after transplantation host brains were examined for transplant survival, differentiation and formation of projections to the host brain. The cultured retinal transplants survived and developed a morphology typical of mature retina, with normal cell and fiber laminae present. HRP injections into the host superior colliculus labeled neurons in the ganglion cell layer of the transplant which closely resembled ganglion cells in vivo. A small number of transplants received lesions. Degeneration material was traced into the superior colliculus and pretectal nuclei confirming that the cultured transplants had projections appropriate for retina entering the host brain. These results correlate closely with those seen after transplantation of embryonic rat retinae that had not been cultured. Thus, a period of explant culture of up to two weeks does not affect the ability of embryonic retinal transplants to differentiate and form projections into the host brain.

Loss of ganglion cells in fetal retina transplanted to rat cortex

Fetal retina survived and differentiated when placed into the cortex of newborn host rats. Two months after transplantation, anterograde tract tracing techniques failed to reveal any projections from these transplants into the host brain. Soma-size analysis of the cells in the ganglion cell layer of the transplants suggested that only displaced amacrine cells remained and that the ganglion cells had degenerated. The basis for these results is considered relative to trophic maintenance of ganglion cells, retinal axon guidance, and specificity of connections.

Plane of Cell Cleavage and Numb Distribution during Cell Division Relative to Cell Differentiation in the Developing Retina

Progenitor cells in the early developing nervous system can divide symmetrically, giving rise to two daughter cells that divide again, or asymmetrically, giving rise to one cell that differentiates and one that divides again. It has been suggested that the orientation of the cell cleavage plane during mitosis determines the type of division. A marker of early cell differentiation, the RA4 antigen, was used to identify regions of the developing chick retina with and without differentiating cells, and the orientation of the cleavage plane was characterized for mitotic figures in each region. No difference was found in the frequency of any orientation between the regions with or without differentiating cells. Furthermore, in the region of the retina with differentiating cells, the RA4 antigen was present in mitotic figures with every possible orientation. Thus, the orientation of the cleavage plane appears to be unrelated to whether or not a division produces a cell that differentiates. It has also been suggested that the intracellular protein Numb mediates neurogenesis via asymmetric localization during cell division. Numb localization was compared with expression of markers of early cell differentiation, the RA4 antigen and Delta. Differentiating and nondifferentiating cells were found both with and without Numb expression. Cells with a cleavage plane parallel to the retinal surface were polarized, such that Numb and/or the RA4 antigen, when present, were only in the daughter cell farthest from the ventricle. These findings indicate a need to reconsider current hypotheses regarding the key features underlying symmetric and asymmetric divisions in the developing nervous system.

Mechanisms involved in development of retinotectal connections: Roles of Eph receptor tyrosine kinases, NMDA receptors and nitric oxide

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.