Anita E. Hendrickson - US grants

The primate retina is unusual among mammals in having a highly specialized central region, the fovea, which is formed by tightly-packed small cones, a displacement of the inner retina away from these cones, and a high concentration of ganglion cells which receive their input from the foveal cones. This organization is directly related to a high visual acuity characteristic of normal humans and monkeys. The peripheral retina has a complex topography formed by cone and rod photoreceptors (PR), with both cell types decreasing in spatial density with eccentricity. Rod and con circuits to ganglion cells in peripheral retina are still not well understood. How does this complex PR topography develop? When do inner retinal circuits become morphologically mature. The details of primate foveal development are gradually being revealed, in large prt due to the ready availability of well-preserved human and monkey retinal tissue available to our group at the University of Washington. In past years we have shown that both monkey and human fovea a long postnatal developmental maturation, and that cone spatial density increases with age, mainly due cell migration into the fovea. In this proposal we will continue to study primate fetal, infant and adult retinal morphology, using modern neuroanatomical techniques, to delineate the sequence of neuronal generation, neurotransmitter expression, and synaptic maturation in central vs peripheral retina. Our goal is to understand the cell types participating in the development of neuronal circuits, and to identify when cone rod circuits become mature. We also will examine whether neonatal visual deprivation affects any of these developmental parameters. Projects will include studies to show: 1) how PR cell size relates to changes in PR spatial density and retinal area; 2) how changes in foveal PR cell size, shape and density relate to visual performance; 3) what the distance is that PR migrate to cause the postnatal increase in foveal cone density; 4) when PR contain outer segment-related intracellular and extracellular molecules which are involved in phototransduction or vitamin A transport between PR and pigment epithelium; 5) when PR synapses are mature; 6) how the number, packing density, and size of pigment epithelial cells changes over development; 7) what effect visual deprivation has on PR development; 8) in what order neurotransmitter-specific inner retinal neurons are generated, and 9) how and when neurotransmitter-specific synaptic circuits develop in the retina.

The primary goal of this research is to better understand the role that individual neurotransmitters plan in the processing of visual information and whether individual neurotransmitters may be major factors in visual deprivation. One major aim is to identify what neurotransmitters are preent in monkey dorsal lateral geniculate (LGN) and striate cortex, and then to delineate what position they play within the neuronal circuitry of these regions. Neuroanatomical techniques will be used for all studies. We have already documented the presence of the known neurotransmitters gamma amino butyric acid (GABA), serotonin and catecholeamines in both LGN and cortex, as well as the peptides substance P in LGN and neuropeptide Y in cortex. We will continue our work 1. to localize the source of these pathways by double labeling with retrogradelytransported 3H-GABA or horseradish peroxidase followed by light microscopic (LM) immunocytochemistry (Imc), 2. to identify the synaptic type and postsynaptic position of each transmitter using eletron microscopic (EM) Imc, and 3. to follow their prenatal and postnatal development in striate cortex using EM, LM and EM Imc, and LM receptor binding autoradiography. Using the same techniques, we also will study the possible deletion, increase or rearrangement of thse neurotransmitters in the LGN and cortex in animals that have been visually deprived by perinatal monocular lid suture or atropine and adult monocular enucleation. We have shown that perinatal monocular atropinization induces a deprivation specific for high spatial frequencies. Striate cortex in these monkeys shows an alteration in cytochrome oxidase (CO) staining patterns. We will determine what neurophile changes underlie this alteration by means of CO staining for LM and EM. LMImc of GABA localization and LMImc based on antisera to specific synaptic proteins. We also will continue to study this deprivation model using behavioral and eletrophysiological techniques to determine the functional changes which may lead to this processing deficiency. These studies will gain better understanding of the mechanisms that lead to normal and abnormal development in the primate visual system, with the goal of providing more insight into the treatment of children who suffer from visual deprivation.

The primate retina is unusual among mammals in having a highly specialized fovea with a tightly-packed, all cone foveola; a high concentration of ganglion cells; and displacement of the inner retinal layers away from the fovea. High acuity and color vision are characteristic of and directly related to this organization. The details of primate retinal organization, especially of its development, are incomplete. In part, this is due to the lack of well preserved pre- and post-natal tissue. In contrast, a great deal is known about the behavioral development of primate visual function which creates a discrepancy in trying to relate behavioral to structural development. We have access to a large volume of human and monkey retinal tissue at the University of Washington. This proposal will utilize this valuable material to study primate fetal, infant, and adult retinal morphology. Modern neuroanatomical techniques will be used including light and EM, immunocytochemistry, in vitro uptake of 3H-labeled neurotransmitter candidates, Golgi impregnation, retrograde labeling of ganglion cells, computer morphometry, and double-label combinations of these methods. Projects will include a) an analysis of the neuronal, synaptic and neurotransmitter organization of the sublaminae within the inner plexiform layer, including their development; b) identification of neuronal pathways containing the putative neurotransmitters GABA, glycine, dopamine, glutamate and various peptides; c) determining the developmental sequence of the neuronal pathways in b; d) testing for the coexistence of two or more neurotransmitter candidates in single neurons; e) charting the development of the monkey fovea including a quantitative analysis of photoreceptor packing and the number and distribution of synapses, and a qualitative analysis of the change in cell morphology and the composition of the transient layer of Chievitz during maturation; and f) completing an anatomical study of human foveal development to ascertain when it becomes fully adult.

A combined morphological and electrophysiological study of adult and developing Macaca nemestrina visual cortex is planned. The developmental anatomy of thalamic input layer 4 will be studied: a) by EM quantitative analysis of synaptic distribution in layers 4A, 4Ca and 4Cb during the first 12 postnatal weeks compared to the adult. This study will utilize labeled thalamic and cortical synapses to determine possible differences in developmental rates of extrinsic vs intrinsic pathways: b) determining the developmental sequence within layer 4 in which the calcium-binding protein, parvalbumin (PV), voltage-gated calcium channels, and NMDA glutamate receptors appear. Ligand binding, light and EM immunocytochemistry will be used to detect these markers of functional maturity in layer 4 neurons. The electrophysiology of adult and infant cortex will be studied using in vitro cortex slices from rat and monkey. Extracellular recording will be used to obtain the transmembrane voltage response to intracellularly and synaptically evoked currents in order to describe the electrophysiological properties of identified neurons through development. The same neurons will then be filled intracellularly with biocytin so that their morphology can be determined. Double label immunocytochemistry will then be applied to correlate electrophysiology with the presence or absence of PV. The functional role of intracellular calcium buffers in neurons of adult cortex will be tested by injecting PV into neurons which do not normally contain it, and by injecting calcium chelators into both PV+ and PV-neurons. These correlated approaches will provide important new anatomical and functional information on intrinsic and extrinsic pathway circuits in monkey layer 4 and relevant biophysical information, now totally lacking, on how these circuits develop during the most critical period for layer 4 development. This new information may provide much needed insight into what goes wrong when visual input is abnormal or deprived during this period.

microscopy; growth factor; eye; Primates; Mammalia;

We have obtained well-labeled monkey fetuses using intravenous injections of 3Hthymidine (3HT) into the pregnant female on a known day of gestation. Retinal sections have been successfully double labeled for cell birth date (3HT autoradiography) and cell markers (immunocytochemistry). Currently three series are being collected at Fd45, Fd55 and Fd72 with 4-5 fetuses in each series. This material will provide an invaluable resource for the analysis of retinal growth and for determining the sequence of cell generation and differentiation within subpopulations of retinal neurons. An analysis of double-labeled dopamine amacrine cells has shown that these large cells are born somewhere in the middle of the amacrine types. They are never heavily labeled for 3HT, which would show that they are the first to be generated; the first amacrine cells to be generated are large amacrines, which contain the neurotransmitter GABA. However, dopamine amacrines are significantly m ore labeled than other amacrines, putting them in the middle of the cascade of amacrine generation. FUNDING NIH grants RR00166, EY04536, EY07031 and EY01730, and by a Kayser Award. Hendrickson, A., Sears, S., and Bumsted, K. Expression of photoreceptor-specific proteins in primate retina. Med. Sci. Monitor 4 11-16, 1998. Bumsted, K. and Hendrickson, A. Distribution and development of short-wavelength cones differ between Macaca monkey and human fovea. J. Comp. Neurol. 403 502-516, 1999.

microscopy; growth factor; eye; Primates; Mammalia;

We have studied the spatial and temporal development of photoreceptor-specific proteins in the macaque retina from early fetal life to adulthood. These include Red/Green (R/G) and Blue (B) opsin for cones; rhodopsin for rods; proteins involved in the visual phototransduction cascade such as transducin, rhodopsin kinase and phosphodiesterase; and the structural protein peripherin. Sequential waves of opsin expression move across the retina, beginning in the fovea at Fd66-70, with rhodopsin expressed first, biopsin slightly later (Fd70), and R/G opsin expressed last with a significant delay. Several tests of expression patterns suggest that each opsin is expressed independently of the others and does not influence neighboring photoreceptor opsin phenotypic choice. Peripherin is expressed at the same time as opsin in each cell type, consistent with its structural role in stabilizing the fluid outer segment (OS) membrane. Transduction cascade proteins appear shortly aft er R /G opsin, but 1-2 weeks after S opsin. All cones at a given point express transduction proteins at the same time, suggesting that some local signal initiates their expression and coordinates the onset of the dark current within cone neural circuits. FUNDING NIH grants RR00166, EY04536, EY07031 and EY01730, and a Kayser Award. Hendrickson, A., Sears, S., and Bumsted, K. Expression of photoreceptor-specific proteins in primate retina. Med. Sci. Monitor 4 11-16, 1998. Bumsted, K. and Hendrickson, A. Distribution and development of short-wavelength cones differ between Macaca monkey and human fovea. J. Comp. Neurol. 403 502-516, 1999.

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