Ralph Nelson - US grants

Neurons act in concert to perform remarkable computational feats. One of the more amazing of these is extracting information from light. In the eye, retinal neural circuits process images. One of the fundamental challenges for eyes is to maintain constancy of neural product over ambient illuminations ranging from starlight to the brightest noontime sun. To cover this range separate classes of photoreceptors have evolved, rods for nocturnal vision, and cones for diurnal vision. Each is served by dedicated sets of interneurons forming intraretinal rod and cone pathways. Mammals such as cats, rabbits,rats and mice provide excellent models for rod system circuitry. Cone system circuitry is relatively less well studied. Color vision is a characteristic of cones and their associated interneurons. The common mammalian models have a poorly developed color vision and a low density of cones. Zebrafish provides a lower vertebrate model with a cone dominated retina, rich color vision, and with the opportunity to study cone circuitry. The zebrafish is also well known for ease of genetic manipulations and an extensive library of mutants and transgenics. It is for these reasons that, over the past decade my lab and others have worked to develop zebrafish as a model for electrophysiological study of the visual system. Channels on neural membranes lead to circuitry properties. The earlier phases of this research program focused on isolated membrane properties of zebrafish retinal neurons, either dissociated or in retinal slice. Glutamatergic actions on cone bipolar cells revealed a variety of mechanisms, including metabotropic receptors, AMPA-kainate receptors, and transporter-associated chloride channels. In parallel with these studies, a morphological library of horizontal, bipolar, and amacrine cell types was developed, both through patch microelectrode staining, and through 'diolistic'staining, each technique in retinal slice. Recently the GABAergic properties of horizontal and bipolar cells have been added to the inventory of membrane properties on identified zebrafish retinal interneurons. There appear to be at least three GABA receptor types on bipolar cells, a GABA-A receptor and both picrotoxin sensitive and insensitive GABA-C receptors. Some bipolar cells express a GABA transporter, a signature for GABAergic neurons. Horizontal cells lack GABA receptors, but a subset transport GABA and are also GABAergic. As seen in voltage-probe techniques, GABA transporter signals are very large, and may serve as a GABA sensor mechanism. During the past review period, the physiological focus shifted to neural light responses. A flattened, perfused eyecup preparation was developed that provided access to horizontal cell light responses through sharp microelectrodes. Cell bodies, dendrites and axons of these horizontal cells were revealed in wet epifluorescence microscopy following injection of alexafluor 594. Spectral studies of horizontal cell light responses revealed 6 chromatic types, including trichromatic UV color opponent cells, with dominant UV cone signals being opposed by blue and green cone signals, but reinforced by red cone signals. The morphology of these UV trichromatic cells is characterized by long axons and comparatively wide dendritic fields. This color type has not previously been reported in horizontal cells of any other species. Several horizontal cell types receive inputs from all four cone types in zebrafish. These are 570nm-peaking red cones, 480nm-peaking green cones, 410nm-peaking blue cones, and 362nm-peaking UV cones. A linear model composed of the sum of four Hill functions, one for each cone signal, has been devised to measure all these color inputs. We call this the unified response-spectral-irradiance model. The model quantifies the stimulus color calculations that horizontal cells perform. The current research program has revisited the sharp electrode technique, but in modernized version. Amplifiers can now handle gigohm dye-filled electrodes, and micropositioners are vastly improved. In particular, the Alexa dyes and modern long focal length wet objectives allow visualization of live, stained neurons deep within the tissue. UV compliant objectives allow stimulation of zebrafish UV cone circuitry on the microscope stage. Computer driven optical benches provide time-efficient stimulus delivery. It is exciting to find that this combination of new and old techniques is well suited to the tiny neurons of zebrafish retina, and that it promises to open up knowledge of retinal circuitry in this animal model. The proposal is to continue to elaborate this new research tool to gain further insight into retinal cone system function.

Polar bears undergo a period of fasting in the summer and autumn, when sea-ice melting makes seals functionally unavailable as a food source. Pregnant polar bears also fast overwinter, which reduces the food- intake period to only 4 months/year for this group. While polar bears do not undergo true hibernation, preliminary data for both polar and black bears collected by Nelson suggests that polar bears have a faculative ability to withstand absence of food for long periods and at any time of the year by conserving body protein. Nelson now proposes to document clearly that protein recycling metabolic pathways are in operation in free-ranging polar bears, determine under what conditions of food availability this adaptation to fasting is invoked, compare the metabolic pathways involved in conserving protein with those determined for hibernating black bears, and consider the ecological framework in which facilitated fasting is adopted by polar bears, especially with regard to how it affects their life-history parameters. Results may offer significant insight into both medical and ecological questions and will contribute important data for undestanding the mechanisms used by mammals for conservation of body protein under a variety of ecological conditions. An understanding of polar bear fasting metabolism may also result in the development of useful heuristic models of starvation, associated weight changes, body weight setpoints and the long-term effects of dieting.

The long term objective of this research is to provide valuable information on heat loss and energy expenditure in infants under a variety of environmental and clinical conditions, with the goal of improving neonatal management and decreasing morbidity and mortality. The specific aims of this research are (1) to modify infrared thermography as a non-invasive, instant and accurate new method of measuring heat loss, and thus energy expenditure in infants, (2) to validate the new method, infrared thermography, by comparing it with an established method of determining energy expenditure in infants, indirect calorimetry, and (3) to determine if differences in energy expenditure are found in infants exposed to drug abuse in utero vs. unexposed infants both during the neonatal period and the first three months of life. Three experiments have been designed to achieve these aims. First, normal newborns (n=25), low birth weight (n=15), and very low birth weight (n=10) infants will be studied using infrared thermography to determine energy expenditure, and radiant, convective, evaporative and conductive heat losses. Heat loss equations will be modified to calculate energy expenditure in infants of varying birth weight and gestational age. Second, energy expenditure will be determined in normal birth weight (n=10), low birth weight incubated (n=10) and non-incubated (n=10), and incubated very low birth weight (n=10) infants by concurrent infrared thermography and indirect calorimetry. This study will determine the validity of infrared thermography to quantify energy expenditure in such infants, which are typical of the hospitalized infant population. Third, energy expenditure will be determined in infants born to drug abusing mothers (n--20) at 0, 4, 8 and 12 weeks of life. Energy expenditure will be determined in matched control infants born to drug-free mothers (n--20) paired with drug-exposed infants on the basis of birth weight and gestational age group. Infrared thermography may be a useful clinical and research tool for studying infant heat loss. Its use may result in better infant care, as well as more accurate predictions of infant energy expenditure and thermoregulatory ability. The use of this new technique in an informative study will determine if drug-exposed infants exhibit changes in energy expenditure, an unidentified and important clinical problem.

Seasonal food shortages constitute an important environmental challenge for many mammals. Some of the longest fasts undertaken by mammals occur when bears enter dens in autumn and remain there over winter without access to food or water. During their winter fast, bears maintain near normal body temperature, recycle nitrogenous wastes produced by metabolic processes associated with protein turnover, produce no net urine, and thus, protect body proteins. Unlike other species of bears, polar bears, can adopt these characteristic protein sparing mechanisms at any time of the year, not just during winter. In consequence, polar bears are one of the most proficient mammals at undertaking extended fasts. In order to meet the challenge of seasonal fasts, many mammals accrete energy as body fat during periods of food abundance, and draw on these stores when food is scarce. The energetic costs associated with the sophisticated protein sparing abilities of bears when fasting are presumably met from breakdown of such lipid stores. Polar bears eat heavily from late April through June, when recently weaned seal pups are available in large numbers. When prey is abundant, polar bears may consume only the blubber of the seals and leave the muscle mass untouched. During such feasting periods polar bears may have the highest dietary intake of lipids of any mammal and their fatty tissue depots expand rapidly. At times of maximal storage fat tissue depots may constitute more than 50% of the total body mass, but after extended fasting these may be reduced to less than 10% of body mass. The period of feasting may be only 6-8 weeks long; while fasting may exceed 8 months. Using stable-isotope labeled metabolites as tracers, we propose to carry out detailed, long-term monitoring of metabolic pathways in individual feeding and fasting polar bears throughout their annual cycle. This study will be the first to use state-of-the-art isotopic methodologies to investigate the means whereby free-ranging bears are able to switch so rapidly into a physiological state whereby long-term fasting is possible. This research will add significantly to our knowledge of the metabolic capabilities of one of the most important predators in the Arctic environment as well as advance our fundamental biochemical knowledge of feast and fast cycles.

In vertebrate animals, retinal neural circuits process images, extracting information about color, shape size and movement from visual surroundings. While laboratory animals such as cat, rat, mouse and rabbit provide plausible models of human nocturnal vision, zebrafish, like humans and other old-world primates, are remarkable for diurnal color vision. Zebrafish is a tetrachromat with 4 cone photoreceptor types selectively sensitive to red, green, blue and ultraviolet wavelengths. Studies of the neural circuitry through which this rich color information is processed can be aided through studies of fish lines with additional special-purpose genes. The zebrafish lines generated and maintained under this protocol were imported from other laboratories, where extra genes were added to, or deleted from, the DNA. In some cases new phenotypes are created in house by cross breeding. Adult and larval fish produced in this research program are studied according to goals and objectives described in other research programs.