David W. Marshak - US grants

The long-term goal of the proposed experiments is to understand how neurons containing peptides, an important class of neurotransmitter candidates, contribute to information processing in the retina of the macaque monkey. The experiments in the first 3-year period will focus on the 4 groups of peptides in the macaque retina: somatostatin, vasoactive intestinal peptide, neuropeptide Y, substance P, and the peptides structurally related to these. Extracts of macaque retina will be analyzed by high pressure liquid chromatography and radioimmunoassay. Light microscopic immunohistochemical studies will determine how many morphologically distinct types of neurons contain each of the peptides and their regional distribution. Subsequent experiments will combine immunohistochemical localization with another immunohistochemical or autoradiographic technique; the double-label experiments would indicate whether related peptides, unrelated peptides, or conventional neuro transmitters are found in the same cells as these peptides. Similar techniques modified for electron microscopy will identify the synaptic inputs and outputs of the peptidergic cells in the central retina. Computer-assisted 3-dimensional reconstruction from serial electron micrographs will be used to describe the labeled cells more completely and identify the unlabeled cells which are also contacted. These experiments combining biochemical and anatomical techniques would provide the basis for physiological studies of peptide release and actions in the primate retina. Of all the models used for vision research, the macaque monkey is the species in which both the structures of the peptides and synaptic connections of the neurons which contain them are most likely to be similar to the human. Recent studies indicate that peptidergic neurons are selectively affected by degenerative brain diseases. If this were also the case in some types of retinal diseases, this basic research on the function of primate retinal peptides would be valuable for understanding and, perhaps, treating these diseases.

The long-term goal of the proposed experiments is to understand how neurons containing peptides, an important class of neurotransmitter candidates, contribute to information processing in the retina of the macaque monkey. The experiments in the first 3-year period will focus on the 4 groups of peptides in the macaque retina: somatostatin, vasoactive intestinal peptide, neuropeptide Y, substance P, and the peptides structurally related to these. Extracts of macaque retina will be analyzed by high pressure liquid chromatography and radioimmunoassay. Light microscopic immunohistochemical studies will determine how many morphologically distinct types of neurons contain each of the peptides and their regional distribution. Subsequent experiments will combine immunohistochemical localization with another immunohistochemical or autoradiographic technique; the double-label experiments would indicate whether related peptides, unrelated peptides, or conventional neuro transmitters are found in the same cells as these peptides. Similar techniques modified for electron microscopy will identify the synaptic inputs and outputs of the peptidergic cells in the central retina. Computer-assisted 3-dimensional reconstruction from serial electron micrographs will be used to describe the labeled cells more completely and identify the unlabeled cells which are also contacted. These experiments combining biochemical and anatomical techniques would provide the basis for physiological studies of peptide release and actions in the primate retina. Of all the models used for vision research, the macaque monkey is the species in which both the structures of the peptides and synaptic connections of the neurons which contain them are most likely to be similar to the human. Recent studies indicate that peptidergic neurons are selectively affected by degenerative brain diseases. If this were also the case in some types of retinal diseases, this basic research on the function of primate retinal peptides would be valuable for understanding and, perhaps, treating these diseases.

This is a proposal for an Exploratory Research Grant (R21), submitted in response to RFA DK 98 009 on the Pathogenesis and Therapy of Complications of Diabetes. The long-term goal of the proposed research is to understand the role of histamine released from centrifugal axons in the etiology of small vessel damage to the retina of diabetic patients. In preliminary experiments, axons from the optic nerve, but not cell bodies, in the macaque retina were found to contain histamine, and their morphology and contacts with retinal blood vessels were described more completely than was possible with other, less-selective staining methods. These results provide additional evidence that centrifugal axons actually exist in primates. The only neurons in the brain that contain histamine are found in the posterior hypothalamus, where retrograde labeling studies indicate that the centrifugal axons originate. The results also suggest that histamine and its antagonists could be used to study the functions of the centrifugal axons in the normal primate retina, and they suggest explanations for the histamine effects that have been observed already. Finally, these findings may be clinically-significant since histamine antagonists apparently prevent the breakdown in the blood-retina barrier in diabetic patients. The first specific aim of the proposed experiments is to develop a method for labeling the centrifugal axons for electron microscopy and to study their ultrastructure. The exact number and diameters of the centrifugal axons in macaque retinas will be determined, and if they make specialized contacts with retinal blood vessels or retinal neurons, these will be described. Three possible marker for electron microscopic immunohistochemistry are: the neuropeptide galanin, which has been localized to histamine-containing neurons in primates and to axons, but not cell bodies, in the macaque retina; histidine decarboxylase, the enzyme that synthesizes histamine, and histamine, itself. All three have been localized previously by electron microscopy, but not in the retina. The second specific aim is to determine whether human centrifugal axons also contain histamine, as expected from the preliminary data, and to describe any changes in these axons in eyes of diabetic donors. The working hypothesis is that these axons are more active than normal in diabetics and that this is why antihistamines are effective in preventing vascular leakage in these patients. If this is correct, there may be a morphological correlate of this increased activity. The human material will also be studied using the electron microscopic technique developed in the first set of experiments.

The visual system remains responsive over an enormous range of ambient illumination, and this adaptation is accomplished entirely within the retina. While some of these changes in sensitivity are mediated by the rods and cones, themselves, there are also important contributions to adaptation from the other neurons in the retina. Dopamine will be the focus in the first grant period because it is thought to play a critical role in light adaptation, changing the strength of chemical and electrical synapses so that the retina remains sensitive to contrast as the intensity of the background light increases. The major targets of the dopaminergic neurons in mammals, All amacrine cells are known to be uncoupled both by dopamine and b photopic light stimulation. However, it is still uncertain which other effects of light are attributable to dopamine in mammalian retinas and what role, if any, dopamine plays in dark adaptation. Last year, the membrane properties of dopaminergic neurons were described for the first time, and they were found to fire action potentials spontaneously in vitro. Two other surprising, new findings were that, in total darkness, All amacrine cells are uncoupled and dopamine is released from the retina. These data led to the hypothesis that dopaminergic neurons are spontaneously active in total darkness, tonically inhibited in scotopic backgrounds and receive both excitatory and inhibitory input in brighter background. The goal of the proposed experiments is to test elements of this hypothesis more rigorously using computer models. It is clear that dopaminergic retinal neurons pla an important role in human vision since the electroretinogram is abnormal in patients with Parkinson's disease, in which dopaminergic neurons degenerate, and in patients taking neuroleptic drugs that block dopamine receptors. The results of these experiments may also be applicable to dopaminergic neurons elsewhere in the Central nervous system. For example, the effect of dopamine on electrical coupling was discovered in the retina and later found to occur in the brain, as well.

DESCRIPTION (provided by applicant): The goal of this training program is to encourage medical students of the University of Texas, Houston to pursue research in Neuroscience. The PI will oversee recruitment and selection of trainees and then monitor their progress. During the summer following their first year, students will participate in a Summer Research Program, which has been supported by NIH since 1985 and is directed by the Co-Pi. The key personnel are the Pi's of NINDS grants and investigators supported by other agencies who do research that supports the NINDS mission. In the spring of their first year, students select mentors, who suggest research topics and help prepare a proposal describing the project, which may be clinical, translational or basic research that supports the mission of the NINDS. The proposals are then evaluated by a Faculty Advisory Committee, and stipends are awarded based on the quality of the proposals. The program begins with mandatory workshops in laboratory safety, bioethics and the responsible conduct of research, regulations related to the use of human subjects in research, and the proper use and care of experimental animals. Each week, trainees are also required to attend research seminars and enrichment sessions describing career options. The major activity is full-time research for 10 weeks with guidance from the mentors. In addition to the space and equipment available in the laboratories of the mentors, there are many core facilities available to support student research. Trainees are invited to attend grand rounds in neurology, pediatric neurology and neurosurgery and encouraged to attend research seminars in basic neuroscience. Trainees prepare an abstract and a poster summarizing their results and present them during the fall of their second year. Some also present their results at national meetings and co-author publications. Students who wish to continue their research training are encouraged to select research as an elective, to take a year off from medical school or to apply for admission to the M.D./Ph.D. program.

DESCRIPTION (provided by applicant): The goal of these experiments is to understand the processing of visual information in primate retinas. The focus in this grant period will be on midget ganglion cells, which mediate both high acuity vision and red-green color vision. Midget ganglion cells are the most common type in primate retinas, but despite many years of research, a number of important questions about the neural circuit providing their input remain unanswered. Two of these questions will be addressed in the proposed anatomical experiments. The first question deals with the source of the input from rods to midget ganglion cells. It is uncertain whether midget ganglion cells receive highly-sensitive input from rods via synapses from local circuit neurons, AII amacrine cells, onto midget bipolar cells and if so, where in the retina this first appears. Midget bipolar cells and AII amacrine cells will be labeled using whole mount preparations of macaque retina, and their contacts will be labeled using a third marker for either chemical or electrical synapses. The working hypothesis is that these synapses appear just outside the rod-free, central fovea. An alternative hypothesis is that central midget ganglion cells receive rod input only via relatively insensitive rod-cone gap junctions, but peripheral midget ganglion cells receive more sensitive rod input via AII cells. The second question deals with neural circuit that generates opposing responses of midget ganglion cells to stimulation of red and green cones. In the central retina, the excitation is selective because midget ganglion cells receive input from a single red or green cone via a single midget bipolar cell. But it is uncertain how selective excitation would be generated in the periphery, where midget ganglion cells receive input from more than one midget bipolar cell. It is unclear how selective inhibition arises anywhere in the retina because the inhibitory local circuit neurons, horizontal cells and amacrine cells, are unselective in their connections. The working hypothesis to account for the selectivity of midget ganglion cell responses is based on results from physiological experiments in other mammalian retinas and a linear model of the neural circuit developed during the last grant period. According to the model, amacrine cells with relatively narrow dendritic fields and branches throughout the inner plexiform layer make the responses of midget ganglion cells more specific than would be predicted by the distribution of the red and green cones. Although individual amacrine cells use the inhibitory neurotransmitter glycine and are unselective in their connections, their net effect is to enhance excitation of the midget ganglion cell in response to stimulation of one cone type. The working hypothesis is that the underlying mechanism is inhibition of a tonic, inhibitory input by a second type of amacrine cell. This hypothesis will be tested by identifying the glycinergic amacrine cells presynaptic to midget bipolar cells and midget ganglion cells and studying their interactions with other amacrine cells in the circuit. Because the retinas of humans and macaques are so similar, the results of the proposed experiments would be relevant to human vision. PUBLIC HEALTH RELEVANCE: This research deals with the neural circuit that generates the light responses of midget ganglion cells. These are, by far, the most common type of ganglion cells in humans and other primates, and they mediate both high acuity vision and red-green color vision. The experiments on the origin of rod inputs to midget ganglion cells would help to understand vision in dim light, when both rods and cones are active. In the United States, this is particularly important for driving at night, and problems with vision in dim light are an early sign for many eye diseases. These experiments would also help to explain the mechanism underlying the electroretinogram, a widely-used method to diagnose eye diseases and monitor the effects of treatments.