Ian Davison - US grants

This study concerns the mechanisms that enable certain types of seaweed common to high latitude North American waters (specifically kelp belonging to the Laminaria genus) to maintain rapid rates of photosynthesis at low temperatures (0-20 degrees centigrade). This phenomenon of low temperature photosynthetic adaptation is poorly understood. The results of the proposed work will have a major impact on our understanding of photosynthesis in marine algae that account for over a third of the plant growth on the planet. Also, a better understanding of the adaptations these kelp have made in their photosynthetic machinery at extreme conditions may have applications ranging from the aquaculture of these plants to the improved understanding of how crop plants on land can be made more resistent to cold.

Intertidal algae in sub-polar and temperate regions experience frequent and severe freezing during tidal emersion in the winter. The ability to survive repeated freezing is an important adaptation, and may greatly influence vertical zonation. In contrast to higher plants, relatively little is known about the ecological or physiological effects of freezing on marine macroalgae. This research will focus on the role of freezing as a sub-lethal stress, which reduces growth and perhaps competitive ability, by examining the effect of freezing on photosynthesis. The research will address the following questions: (1) what mechanisms are responsible for freezing resistance, (2) does the same mechanism confer resistance to both freezing and desiccation, (3) are interspecific differences in freezing resistance correlated with tidal height due to genetic adaptation or phenotypic acclimation, and (4) does the relative importance of freezing acclimation vary between upper-shore, stress tolerant, and lower-shore, competitive algae? This investigation will contribute to our knowledge of freezing as an ecological factor, and also address questions of general importance to all scientists studying the intertidal zone.

Marine macroalgae exhibit phenotypic responses of their photosynthetic metabolism when grown at different temperatures. This thermal acclimation involves changes in cellular contents of photosynthetic enzymes and pigments that enable seaweeds to maintain similar rates of light-limited and light-saturated photosynthesis over a range of growth temperatures. Although these responses are undoubtedly a major reason for the biological success of seaweeds in low temperature environments, little is known about either the effect of temperature on the organization of the photochemical apparatus (e.g., number and size of chlorophyll- pigment complexes), or the processes that regulate thermal acclimation. This project will permit Dr. Davison to spend a year's sabbatical in 1993 working in the laboratories of Dr. P. G. Falkowski (Brookhaven National Laboratory) and Dr. E. Gantt (university of Maryland) to learn modern molecular, biochemical and biophysical techniques. These techniques will then be used to address the following questions about thermal acclimation of photosynthesis. 1. How is thermal acclimation regulated at the molecular level (i.e., transcription or translation)? 2. How rapidly does the acclimation response occur. 3. How does thermal acclimation affect the organization and function of the photochemical apparatus? For example, how does temperature affect the stoichiometry of the various components of the photosystems and the size and efficiency of energy transfer within the light- harvesting chlorophyll protein complexes? 4. Does the apparent similarity between thermal acclimation and photoacclimation indicate that they are regulated by the same mechanism? In particular, this project will test the hypothesis that thermal acclimation and photoacclimation are both regulated by the cellular energy budget. The techniques used will include: (i) antibodies and cDNA probes for measuring the cellular content and rates of synthesis (transcription) of mRNA that codes for the key proteins of the photosynthetic apparatus and contents and synthesis (translation) of these proteins, (ii) a pump-probe fluorometer and other biophysical techniques to measure the functional size of the light-harvesting antennae associated with photosystems I and II and to study energy transfer within and between chlorophyll-protein complexes, and (iii) immunocytochemistry to study the localization of components of the photochemical apparatus on the thylakoid membranes. The information obtained through this research should substantially increase our understanding of the physiological processes that enable seaweeds to achieve high rates of productivity in low temperature environments.

9521341 Davison The study will examine stress-tolerance in two major groups of perennial intertidal macroalgae, the red and brown seaweeds. The research will test the hypothesis that active oxygen is involved in emersion stress of intertidal seaweeds. Damage due to active oxygen will be determined in stress-tolerant and stress- susceptible species exposed to emersion stress by measuring the peroxidation of membrane lipids. Plants will be grown in laboratory culture under conditions that increase their ability to withstand emersion stress. If the research hypothesis is correct, increases in stress tolerance should be associated with increased levels of antioxidants and/or protective enzymes. The proposed research will also determine if intertidal seaweeds express specific stress-proteins in response to emersion stress. The results of the physiological studies will be used to design field experiments to measure the occurrence and importance of sublethal emersion stress in natural communities and to compare the allocation of resources to stress-tolerance in low and high shore species. This research represents one of the first attempts to understand the processes that confer stress tolerance in intertidal seaweeds and will provide valuable insights into the ecology and physiology of these plants. Information on the mechanism of stress tolerance will allow ecologists to assess the costs of stress tolerance and relate these to reproductive output, growth and competitive ability. ***

Davison 9418033 The genetic adaptations that enable certain plant species to survive and grow in polar environments where temperatures are near or below 0oC year-round are poorly understood. Low- temperature adaptation is complicated in terrestrial plants by freezing, desiccation and stomatal conductance, and in marine phytoplankton by a variable and unpredictable physical environment. Polar macroalgae provide an experimental system that is not subject to these complications and that is well- suited to the study of cold-adaptation in plants. Cold- adaptation is particularly well developed in Antarctic macroalgae, in which rates of photosynthesis and growth at OoC are comparable to rates achieved at 10-15oC by temperate species. The proposed research uses endemic Arctic and Antarctic seaweeds to answer the question, "What adaptations do polar algae possess that enable them to assimilate carbon and grow rapidly at very low temperatures?" The research focusses on carbon-metabolism characteristics of three closely related polar-temperate pairs of brown algae: Arctic Laminaria solidungula and temperate Laminaria saccharina; Antarctic Desmarestia anceps and temperate Desmarestia aculeata, and Antarctic Himantothallus grandifolius that is related to Desmarestia aculeata and D. anceps, but is morphologically similar to Laminaria saccharina. Carbon-metabolism processes (photosynthesis, respiration and light-independent carbon fixation) that are important in cold-adaptation will be identified in sporophytes of each species pair acclimated to the same temperature. Specific mechanisms of adaptation will be determined by comparing components of the photosynthetic apparatus as well as contents, activities and thermal properties of key enzymes involved in photosynthesis, respiration and light- independent carbon fixation. Comparisons of multiple species pairs and a broad suite of carbon-assimilation parameters will provide a comprehensive analysis of the mechanisms o f low- temperature adaptation in algal species endemic to both the Arctic and Antarctic Oceans and increase an overall understanding of low temperature adaptation in all plants.

Davison - 9907305

Physical factors such as light, temperature and nutrient availability are known to limit marine productivity and play an important role in determining species distribution and community structure. Most understanding of the role of physical factors is based on studies with a single variable with other conditions being optimized for growth. Consequently, little information is available on physiological responses to the natural environment where several physical factors may be suboptimal. The ability to understand the constraints on marine productivity requires not only an understanding of potential synergistic or antagonistic interactions but also an analysis of their effects on algae with different ecological strategies. This investigation will examine the response of marine red macroalgae to simultaneous nitrogen limitation and ultraviolet radiation stress. Both factors are known to be important determinants of marine primary productivity and they frequently co-occur. This is true not only of the tropics, but also in cold- temperate oceans such as the Gulf of Maine. Red macroalgae were selected for this research because they provide the opportunity to study interactions between UV and nitrogen limitation in a group of algae with similar physiological and morphological characteristics but with well-defined differences in UV-tolerance and well-characterized contents of mycosporine-like amino acids. Furthermore, in contrast to phytoplankton, benthic macroalgae experience a more predictable light climate and are relatively long-lived, exposing individuals to a wider range of environmental conditions. The red algae studied from the Gulf of Maine will include Porphyra umbilicalis, Chondrus crispus, Membranoptera alata and Phycodry rubens. These species range from UV-tolerant intertidal and shallow sublittoral species to UV-susceptible species from the deeper sublittoral. The investigators anticipate that the costs and benefits of UV-tolerance and the impact of nitrogen limitation will vary between UV-tolerant and UV-susceptible species. The research will focus on the effect of nitrogen metabolism (limited or replete) on (a) short and long-term effects of UV-stress (e.g., short-tem inhibition of photosynthesis, lipid peroxidation, and growth), (b) UV-photoprotection including contents of sunscreens such as mycosporine-like amino acids (MAAs), antioxidants such as ascorbate, gluathione and tocopherols, and enzymes of reactive oxygen metabolism such as catalase, superoxide dismutase and ascorbate peroxidase, and (c) ability to recover from UV-stress (e.g., the role of protein synthesis in recovery). The research will also examine the effect of UV-stress on nitrogen metabolism (e.g., nitrogen content and rates of uptake and assimilation). The research will involve measurements on field-collected material, laboratory experiments under controlled conditions and outdoor experiments in flowing seawater and natural radiation manipulated by a variety of UV and photosynthetically active radiation (PAR)-filters. The research will substantially contribute to an understanding of the effect of UV and inorganic nitrogen availability on marine productivity and, in particular, elucidate the importance of interactions between these factors. The research involves collaboration between faculty at an undergraduate teaching institution (Westfield State College, MA) and a Land and Sea Grant research University (University of Maine). In addition to graduate education, the project has a substantial research experience for undergraduate component, providing undergraduates from Westfield State College with the opportunity to become involved in research.

The Central Michigan University is awarded a grant to renovate a former boat house and install a series of 12 mesocosm tanks that will allow CMU faculty, students, and visiting investigators at the CUM Biological Station (http://www.cst.cmich.edu/CMUBS/) to conduct controlled and replicated ecological experiments on the pelagic and benthic ecosystems of Lake Michigan as well as beaches and wetlands and to address key ecological questions dealing with the response of these habitats to multiple stressors (e.g., climate change, nutrient enrichment and introduction of non-indigenous species). The proposed renovations will directly impact the research of at least ten faculty members at Central Michigan University, as well as collaborators from other institutions and visitors to CMUBS. For more than 30 years Central Michigan University has operated a Biological Station (CMUBS) on Beaver Island in northern Lake Michigan. Until recently the primary focus of research at CMUBS has been on the Island's diverse array of undisturbed terrestrial ecosystems, and the station has also supported an active summer undergraduate and graduate education program.

The improvements will support the University's growing strength in freshwater biology and related disciplines (e.g., geochemistry, remote sensing and GIS) as well as the importance of the lakes to Michigan and the mid-western and north-eastern US. The proposed renovations will significantly enhance the ability of faculty, graduate students and visitors to CMUBS to conduct research on a variety of aquatic and wetland organisms and to address ecological questions related to key topics such as non-indigenous species, endangered species, lake level cycles and global climate change. The new facility at CMUBS will provide additional opportunities to engage interested students in STEM learning and encourage their progress toward STEM-related research and graduate study. It will further enhance the academic success of underrepresented and first-generation college students through many CMU programs including Upward Bound, GEAR UP, Ronald E. McNair Baccalaureate Achievement Program, NSF's URM: "BUMP Into Research at CMU!". The mesocosm facilities will provide enhanced opportunities for interdisciplinary approaches and collaborations with other institutions. The new facilities will significantly improve the overall scientific infrastructure dedicated to understanding the Great Lakes, and hence augment our understanding of, and ability to protect and manage, one of the most important freshwater systems in the world. Users of the facility will integrate research findings and perspectives into the diverse array of university courses and K-12 presentations that they routinely offer, and the research projects and underlying philosophy will be broadly disseminated through publications in the primary literature, reviews/overviews, seminars, symposia and meetings on an international scale.

DESCRIPTION (provided by applicant): Memories of social experiences with other individuals can be extremely powerful, especially those produced during heightened arousal states such as reproductive encounters. The way the brain stores information about social experience, however, remains enigmatic. The broad goal of our proposal is to define the neural changes that occur during adult pheromonal imprinting, a potent form of individual recognition memory triggered by mating in rodents. We aim to establish the precise synaptic mechanisms and circuit-level effects of the chemosensory memory trace instilled by sexual experience. We first use genetic tools for fluorescently tagging the cells activated by stud pheromones during mating, allowing targeted analysis of these neurons after sensory experience with ex vivo cellular electrophysiology (Aim 1). We also develop novel approaches for visualizing how learning affects pheromone processing circuits during behavior, which should ultimately help illuminate the neural underpinnings of a much wider range of social behaviors (Aim 2). Together, this work bridges complementary cellular and network scales to provide new views of a long- standing model of chemosensory learning.

ABSTRACT Multiphoton microscopy is one of the preferred techniques for high-resolution functional brain imaging because of its remarkable depth penetration in thick tissue. In standard configurations, such imaging involves scanning a femtosecond laser focus in 3D throughout a sample. The laser power is fixed during the scan and image information is contained in the time dependence of the detected fluorescence signal. Several problems can occur with this technique. First, in common cases where the sample contains extreme variations in brightness, for example between large somas and much finer dendritic processes, it is often impossible to capture the full range of signals without either saturating the detector when scanning over bright regions, or losing signal when scanning over dim regions. Second, when imaging time-varying signals from functional reporters such as GCaMP, large brightness variations occur that cannot be predicted in advance, forcing the user to use a low illumination to minimize the possibility of detector saturation, thus potentially compromising SNR. Third, when performing volumetric scans through an extended range of depths, a single laser power becomes either too weak at large depths or too strong at shallow depths. We propose a simple solution to solve all these problems. The solution involves actively regulating the laser power pixel-by-pixel using feedback electronics. We have demonstrated that our technique can improve the dynamic range of two-photon microscopes by several orders of magnitude for moderately fast pixel times of 20?s, achieving an unprecedentedly high dynamic range (HDR) of 1011:1. Our goals for this project are the: 1) Development of ultrafast feedback electronics for video-rate HDR imaging. 2) Development of switched multiplexing technique for large-scale multi-region HDR imaging. 3) Application of multiphoton HDR imaging to anatomical and functional mouse brain imaging. Our goal is to enable comprehensive large-scale multiphoton imaging with unprecedented dynamic range in a simple manner that can be readily implementable by many labs at reasonable cost and with minimal hardware modifications.

Miniature head-mounted fluorescence microscopes allow neuroscientists to record from populations of neurons longitudinally at cellular resolution in freely moving animals. However, off-the-shelf devices currently lack a number of desirable features such as easy modification, wireless interfacing, and flexible real-time analysis software. This project will disseminate an open-source miniature microscope (?miniscope?) that meets these needs. New features realized in the miniscope include a 3D-printed housing for easy microscope reconfiguration, wireless telemetry, and color CMOS sensors for simultaneous recording of multiple fluorescence indicators. In addition to the microscope, this project disseminates open-source software for controlling the microscope. This software is capable of real-time image processing and feedback for closed-loop experiments that trigger stimulation or other events in response to patterns of recorded neural activity. The strategy for dissemination is two-fold. First, the project provides fully functioning miniscopes and associated components and training for 14 collaborating labs. These end-users will use the microscopes to address a wide range of questions in surface and deep brain nuclei, focused on many distinct cell types. These groups will study learning in normal brain functioning and pathological activity patterns in multiple disease models. Second, the project will create a public web repository containing resources and documentation necessary to reproduce the microscope in other laboratories. Finally, the project will implement a number of design variations requested by end-users. These design variations include changes in the field of view, specialized microscope housings, adaptations for simultaneous electrophysiology, new color imaging strategies, and user-defined changes to the real-time software. These design variations will also be described in the public web resources. Ultimately, the goal of the project is to provide a platform for innovation in the use of customized miniature microscopes.

ABSTRACT Scale is a fundamental obstacle in linking neural activity to behavior. While perception and cognition arise from interactions between diverse brain areas separated by long distances, neural codes and computations are implemented at the scale of individual neurons. An integrative understanding of brain dynamics thus requires cellular-resolution measurements across sensory, motor, and executive areas spanning more than a centimeter. While such comprehensive sampling has recently been achieved in head-fixed animals, comparable data during complex behaviors in freely moving animals is not possible with current technology. Head-mounted ?miniscopes? provide powerful tools for long-term monitoring of neural population activity in behaving mice. While new surgical methods now offer optical access to most of neocortex, current miniscope optics limit their field of view (FOV) to less than 1 mm2, precluding imaging across multiple brain regions. Large- scale imaging also requires mouse models with widely expressed Ca2+ sensors, severely constraining size and weight. Fundamental physical principles dictate that conventional optics cannot meet the joint requirements of large FOV, high resolution, and efficient light collection in a compact and lightweight device that can be carried by small animal models. Here we propose to develop a next-generation miniscope for cellular-scale imaging across the majority of cortex in freely moving mice, based on novel computational imaging framework that jointly design optics and algorithms. Our Computational Miniature Mesoscope (CM2) uses parallel sampling with single lightweight microlens array to dramatically simplify the optical path, increasing FOV by ~2 orders of magnitude while maximizing light-throughput, resolution and signal-to-noise ratio. The CM2 system will provide resolution of approximately 10 µm across distances of centimeter or more, enabling brain-wide recording of multiscale neural dynamics inaccessible with existing technology. Our goals are to: 1) Develop novel optical designs for ?wearable? cellular-resolution Ca2+ imaging throughout neocortex. 2) Validate and optimize the CM2 for cortex-wide functional imaging in behaving mice. To this end, our interdisciplinary team brings together highly complementary expertise in computational imaging, neurophotonics innovation, and neural recordings in behaving rodents. Overall, this work will establish powerful enabling technology that greatly expands the scale of activity measurements possible in behaving animals, providing access to a wide range of questions about distributed cortical function.

Project Summary Innate social behaviors such as aggression, mating, and parenting offer a powerful window on the brain networks that map sensory input onto appropriate motor outputs. The compact architecture of the rodent vomeronasal system is ideal for characterizing the circuits that translate identified, sex-specific chemosignals into responses matched to the characteristics of different conspecifics. Here we address two seemingly contradictory roles of this system: the need to mediate stereotyped interactions with same- or opposite-sex partners, while still providing flexibility in responses to different individuals based on established social relationships. We characterize the learning mechanisms that mediate changes in aggression, a prototypical and highly robust male behavior that is powerfully altered by dominance relationships. Using powerful genetic tools to access functionally distinct sensory pathways in the accessory olfactory bulb, our experiments will address (1) the cellular plasticity processes that adapt aggression levels to reflect social experience, (2) the way that familiarity shapes the sensory representations of conspecifics during active social interactions, and (3) the circuit anatomy that organizes behaviorally relevant sensory signals and routes them to downstream limbic centers. A deeper understanding of the neural mechanisms for regulating aggression may help design strategies for mitigating pathological behaviors in human communities. More broadly, a deeper knowledge of the brain networks for social behaviors is highly relevant for understanding autism spectrum disorders.