William F. Gilly - US grants

In skeletal muscle, the mechanisms coupling the action potential across the sarcolemma and transverse tubular (T-) membranes to initiation of calcium release from the sarcoplasmic reticulum (SR) remain unknown. This represents the major gap in our knowledge of the regulation of muscle contraction. The morphological pathway for this coupling is thought to be the anatomically well-defined T:SR junctions (e.g. diads and triads), but these junctions represent only a small part of the total muscle cell membrane area and generally are buried deep within a structurally and electrically complicated T-system. The chief aim of this proposal is to study these junctions in the most direct way technically possible. An extracellular patch-clamp electrode (lMu dia,) will be employed to make electrophysiological measurements from small regions of muscle cell membranes naturally enriched in these junctions. This technique will be utilized with certain vertebrate and invertebrate preparations in which T:SR junctions are in close physical and electrical proximity to the cell surface. This approach will provide the most direct electrical recordings to date from these poorly understood junctions. Success in electrical measurements from a single T-tubule from scorpion muscle or from a single surface membrane:SR junction in frog slow muscle will give a direct indication of what electrical activity underlying T:SR Coupling looks like and localize this activity to junctional membrane. These experiments will also enable characterization of the activity in order to determine whether the currents are capacitive, ionic, or both and to determine whether extracellular calcium plays an important role in their generation. These answers are basic to discovering the mechanisms involved in T:SR Coupling. They are also fundamental to understanding the role this process might play in both cause and treatment of muscle disorders.

Nerve cell activity depends on various ions crossing the cell membrane through channels that are sometimes very specific. These channels are formed by protein molecules in the membrane. Nerve cells often have a long process, the axon which extends from the cell body known as the soma. Often channels in the axon are distributed differently from those in the soma. This research addresses the question of how different proteins, which are continuously synthesized in the soma of nerve cells, are spatially distributed. This project will examine the molecular mechanisms of sorting sodium channel proteins in nerve cells. Sodium channel proteins are synthesized in the soma, but are found in the membrane of the axon and are absent in the soma. In the squid, an invertebrate, mRNA coding from rat sodium channels will be injected into the soma. As a result the cells will synthesize two different types of sodium channels making it possible to compare the spatial distribution of the two protein molecules. The part of the molecule responsible for its peculiar spatial distribution can be changed by changing the coding and thus is able to be identified. This study will advance our understanding of long-term regulation of how nerve cells function.

The training program, "Molecular Approaches to Ion Channels", will provide advanced graduate students and professional scientists with in-depth exposure to a modern, multi-disciplinary approach to the study of ion channels in cell membranes at the molecular level. Methods covered include patch clamp applied to native cells and transformed cells expressing cloned gene products, and biochemical and molecular analysis of channel expression. Training provided by this course will directly have an impact on the scientific community by guiding the efforts of students and established investigators who are seeking to explore new aspects of current research problems.

Specificity of neuronal function at the cellular level is dictated by the exact functional properties of channels and receptors and the precise spatial distribution of these molecules. This specificity is vital to systems-level function. For example, severe loss of motor ability occurs in response to disorganization of Na channel distributions in demyelinating diseases such as multiple sclerosis and as a result of mutated Na channel genes that perturb specific functional properties in hyperkalemic periodic paralysis and related myotonias. Other neuromuscular and central disorders are likely to involve related mechanisms, but the molecular and cell biological dynamics that control function and distribution of channels and receptors are not well understood. This is true even for the Na and K channels responsible for a fundamental function like action potential conduction, especially in light of the fact that both of these "channels" represent multi-gene families. At least 7 Na channel mRNA species are expressed in skeletal muscle. Four distinct sub- families of K channel genes exist; at least 10 human members of just the Shaker-related subfamily 1 have been reported. Expression of cloned channels in heterologous systems reveal a broad array of functional differences, even between closely related subtypes. How much of this complexity exists in vivo at the level of a single neuron, and to what degree can we understand the complexity of a single cell given available technology? These fundamental questions demand answering. This proposal describes a systematic approach to this end by focusing on the Na and Shaker-like K channels responsible for impulse conduction in a single neuron, the squid giant axon and its cell bodies. Unique features of this system, coupled with the enormous data base on functional properties of squid Na and K channels, will lead to a deep understanding of the processes acting to control neuronal function in a biologically relevant context. Specific aims will answer: What channel subtypes are expressed and how do their native functional properties compare to those for their cloned counterparts? What is the spatial distribution of each channel isotype? How do post-translational modifications and interactions with other proteins act to control these spatial distributions and functional attributes? Finally, how are these control processes involved in long-term modification of expression patterns for specific channel isotypes that occur during development or in acquisition of new firing patterns? Answers to these questions will significantly advance our understanding of maintenance and modulation of neuronal function in living systems.

Marine cone snails of the genus Conus are predators that paralyze their prey by injecting potent neurotoxins. These toxins are peptides (11-45 amino acids long) that target ion channels crucial for normal electrochemical activity in nerve and muscle cells. A novel toxin has been discovered in one species, Conus californicus, which is a non-selective predator on worms, snails and fish. This toxins blocks nerve transmission by targeting voltage-gated sodium channels. This project uses biochemical, biophysical, and molecular approaches to elucidate the chemical structure of this peptide toxin, its mechanism of action, and its specificity profile, and to localize and clarify the basic biology of the venom production in this animal.
Results will be important in developing a new experimental reagent for basic research on channels, and in understanding how different toxic peptides are made and used by these snails in a biologically relevant context. This lab also will continue important multi-disciplinary training of undergraduates in neuroscience.

Inshore and open-ocean (pelagic) squid are the most athletic of all invertebrates. They are highly active, jet-propelled swimmers, have high metabolic rates and grow at a prodigious rate throughout their short life spans of one to two years. Such squid are abundant in all the world's oceans where they play important ecological roles as major predators. Adult squid serve as essential prey for many top predators, including sharks, tuna, billfish and marine mammals. In addition, squid are becoming increasingly important in commercial fisheries worldwide as they replace slow-growing fish, particularly where these stocks are being depleted. Dosidicus gigas, also known as the jumbo or Humboldt squid, is a true giant, reaching 2-3 m in overall length and over 50 kg in mass. It is widely distributed over the eastern Pacific, ranging from Chile to Canada and nearly to Hawaii at the equator. It forms the basis of a major commercial fishery, presently the third largest in Mexico. Despite the ecological and economic importance of D. gigas, little is known about its life history, behavior or physiology. Its large size and open-ocean habitat complicate traditional field and laboratory studies. This project focuses on integrative field and laboratory studies of D. gigas in the Gulf of California using recently developed techniques that facilitate such studies. Pilot tagging studies have revealed that D. gigas spends the daytime in cold, deep, oxygen-depleted water (~10 deg C at 300 meters) and migrates at night to shallow, aerated surface waters that can reach 30 deg C. Frequent rapid dives at night to daytime depths cover several hundred meters in minutes. It is a mystery how these large, metabolically active squid can tolerate the stress of chronic daytime hypoxia at depth. Conversely, warm surface waters also may present a stress that limits the time squid can spend in this zone. This proposal will employ electronic tagging to track vertical migrations of this pelagic predator and to monitor natural jetting and respiration at different depths. Oxygen consumption determined from these data, with calibrations provided by laboratory swim-tunnel experiments and biochemical indices of anaerobic metabolism, will provide a measure of the true energetic costs to the squid itself. Extreme low-light video methods will reveal natural behaviors over the range of a typical vertical migration, both day and night. Thus, this project will reveal what this remarkable squid is doing in its oceanic habitat, why it is doing it, and what physiological and biochemical adaptations permit these behaviors at some depths and preclude them at others.

This study will greatly advance our understanding of the biology of D. gigas and provide a model for an integrated approach to studying the ecological physiology of other pelagic predators. It will also establish a life-history framework that will ultimately be necessary to manage this fishery at the ecosystem level in Mexico and elsewhere. It includes training of a postdoctoral fellow and two graduate students, and collaboration with two Mexican scientists and their students. Cephalopods, particularly large squid, are charismatic and appealing to the public, and this project has significant potential for mass public outreach through a variety of media.

"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)."

This project concerns the ecological physiology of Dosidicus gigas, a large squid endemic to the eastern Pacific where it inhabits both open ocean and continental shelf environments. Questions to be addressed include: 1) How does utilization of the OML by D. gigas vary on both a daily and seasonal basis, and how do the vertical distributions of the OML and its associated fauna vary? 2)What behaviors of squid are impaired by conditions found in the OML, and how are impairments compensated to minimize costs of utilizing this environment? and 3)What are the physiological and biochemical processes by which squid maintain swimming activity at such remarkable levels under low oxygen conditions? The investigators will use an integrated approach involving oceanographic, acoustic, electronic tagging, physiological and biochemical methods. D. gigas provides a trophic connection between small, midwater organisms and top vertebrate predators, and daily vertical migrations between near-surface waters and a deep, low-oxygen environment (OML) characterize normal behavior of adult squid. Electronic tagging has shown that this squid can remain active for extended periods in the cold, hypoxic conditions of the upper OML. Laboratory studies have demonstrated suppression of aerobic metabolism during a cold, hypoxic challenge, but anaerobic metabolism does not appear to account for the level of activity maintained. Utilization of the OML in the wild may permit daytime foraging on midwater organisms. Foraging also occurs near the surface at night, and Dosidicus may thus be able to feed continuously. D. gigas is present in different regions of the Guaymas Basin on a predicable year-round basis, allowing changes in squid distribution to be related to changing oceanographic features on a variety time scales.

This research is of broad interest because Dosidicus gigas has substantially extended its range over the last decade, and foraging on commercially important finfish in invaded areas off California and Chile has been reported. In addition, the OML has expanded during the last several decades, mostly vertically by shoaling, including in the Gulf of Alaska, the Southern California Bight and several productive regions of tropical oceans, and a variety of ecological impacts will almost certainly accompany changes in the OML. Moreover, D. gigas currently supports the world's largest squid fishery, and this study will provide acoustic methods for reliable biomass estimates, with implications for fisheries management in Mexico and elsewhere. A related goal is to work with colleagues in Mexico on a squid fishery management plan. Previous work involved collaboration with Mexican colleagues, including training and research opportunities for undergraduate, graduate and postdoctoral students from both nations. It also involved public outreach efforts through television, print and web media. This charismatic species provides an excellent means to connect climate change with ecological effects, and outreach activities will continue with this theme. A new international effort will establish a laboratory for research on squid at the site of field work in Santa Rosalia, Baja California Sur, Mexico. The facility will involve Mexican college students in marine research and implement local educational programs.

Stanford University has been awarded a grant to purchase a confocal scanning laser microscope and establish a related multi-user facility at Hopkins Marine Station (HMS) to serve multiple marine laboratories in the Monterey Bay Peninsula, California. High-resolution imaging is now an essential part of the contemporary tool kit of cell and developmental biological processes. Confocal microscopy, along with advances in fluorescent labeling of protein and nucleic acids, has resulted in unprecedented insights into the 3D architecture and function of cells and tissues of many marine organisms. An important application of this technology is visualization of cellular elements and processes related to early development, physiology, and neurobiology. This technical ability also has broader implications for elucidating the interactions of marine animals with their environment. Ultimately, the environment acts on organisms through its effects on cells and tissues. Ocean acidification, deoxygenation and other processes will alter ecosystems and fisheries through effects on individual organisms. This microscope will help address how the various facets of climate change are impacting critical aspects of the cell/developmental biology and physiology of a variety of marine species.

This piece of equipment will directly benefit 5 research labs at HMS with an immediate potential benefit to 10 graduate students, and provide outstanding undergraduate training opportunities during research training apprenticeships. The microscope will be housed at HMS but be available to researchers and their students at other research institutions in the local area, including the Monterey Bay Aquarium, Monterey Bay Aquarium Research Institute at Moss Landing and California State University Monterey Bay (CSUMB). There are many faculty at these institutions with a wide range of research interests who plan to use this microscope upon availability. HMS will partner with CSUMB Bay to create training opportunities on this microscope for the McNair Scholars program for gifted minority students to prepare them for doctoral studies. The image data generated from this equipment will be highlighted in the regular public lectures and open houses at HMS, and on a web-based resource dedicated to early embryogenesis and larval development of marine invertebrates. An imaging collaboration between HMS and the Monterey Bay Aquarium (MBA) will develop a greater public awareness of the diversity of embryos and larvae of marine invertebrates by spotlighting image data using the extensive established outreach mechanisms of the MBA both in formal public exhibits and Facebook postings. For more information about Hopkins Marine Station, visit the website at http://www-marine.stanford.edu/.

This project will examine the response of Dosidicus gigas (Humboldt squid) to an El Niño event in 2009-2010 that was accompanied by a collapse of the commercial fishery for this squid in the Guaymas Basin within the Gulf of California. This large squid is a major predator of great ecological and economic importance in the Gulf of California, the California Current, and Peru Current systems. In early 2010, these squid abandoned their normal coastal-shelf habitats in the Guaymas Basin and instead were found in the Salsipuedes Basin to the north, an area buffered from the effects of El Niño by the upwelling of colder water. The commercial fishery also relocated to this region and large squid were not found in the Guaymas Basin from 2010-2012, instead animals that matured at an unusually small size and young age were abundant. A return to the large size-at-maturity condition has still not occurred, despite the apparent return of normal oceanographic conditions.

The El Niño of 2009-2010 presented an unforeseen opportunity to reveal an important feature of adaptability of Dosidicus gigas to an acute climatic anomaly, namely a large decrease in size and age at maturity. Now these investigators will have the opportunity to document recovery to the normal large size-at-maturity condition. The specific aims of this project are: 1) continue a program of acoustic surveys and direct sampling of squid that has already been established in the Gulf of California in order to assess distribution, biomass, life history strategy diet, and migratory and foraging behaviors relative to pre-El Niño conditions and 2) conduct analogous surveys in Monterey Bay, California in conjunction with long-term remote operated vehicle surveys of squid abundance. The data from these studies will provide a comparison of recovery in the two different squid populations and yield valuable insights into what ecological effects an area is expected to experience with an invasion of either small or large Humboldt squid. As long-term climate change progresses, squid of both forms may expand northward into the California Current System.

Training will be provided for participating graduate and undergraduate students and an established collaboration will be continued with a technical college in Mexico that involves Mexican undergraduates in local sampling and developing public outreach aimed at the local squid fishing community. Squid abundance (biomass) and foraging (diet) data will be incorporated into NOAA fishery-management models being developed for Humboldt squid. Findings of the project concerning El Niño, climate change, and squid fisheries will be incorporated into an established outreach program with NOAA (Squids4Kids), the Google Science Fair Science Hangouts program, and a NEH Summer Institute on John Steinbeck at Hopkins Marine Station. The investigators will continue to contribute exhibits being developed on squid at both the Monterey Bay Aquarium and Hatfield Marine Science Center.

Complex color-changing (chromogenic) behaviors in squid provide camouflage by mimicking benthic features such as rocks, coral or algae, and also serve in intra-specific signaling. However, few studies have been carried out under natural lighting conditions in the field, or without the presence of human observers or noisy remotely operated vehicles. This project will extend field studies of natural chromogenic behaviors to the family Ommastrephidae as exemplified by Dosidicus gigas (Humboldt squid). To accomplish the research under natural lighting conditions, the project will develop an improved low-light Crittercam and an improved Driftcam that can operate effectively at greater depths. These improved video platforms will lead to new understanding of the generation and regulation of camouflage and communication in the open-ocean environment. Little is known of predatory behavior in midwater region occupied by Humboldt squid, which in the eastern Pacific Ocean is associated with a vast volume of water that is naturally hypoxic called the oxygen minimum zone (OMZ). This OMZ is currently expanding and moving toward the surface, largely due to climate change. Observations of predatory activity in the upper OMZ will increase our understanding of how pelagic ecosystems might change as climate change progresses, and how these changes could impact economically and ecologically important species like the Humboldt squid.

Chromogenic behavior in squid and other cephalopods is generated by muscular organs called chromatophores that are controlled by neural activity in the brain, and this direct, descending motor-control for chromatophores is unique to cephalopods. Pilot studies with the National Geographic Remote Imaging (NGRI) "Crittercam" video-package deployed on free-swimming Humboldt under natural lighting have revealed static patterning-type displays, like those seen in loliginid species, as well as a unique "flashing" behavior, with the entire body rapidly alternating between pale white and deep red. Flashing appeared to occur only during interactions with conspecifics, strongly suggesting a communication role. These data also revealed a more subtle wave-like "flickering" of chromatophores that may emulate fluctuations of light in the water column and provide dynamic countershading or crypsis. Neither type of behavior has been described in the scientific literature. The generation and control of the flashing and flickering chromatophore signals is therefore of great interest. This project will increase our knowledge of chromatophore signals through in situ video observations and anatomic analyses of Humboldt squid that include electrophysiology assessments, and immunohistochemical analyses of mantle tissue. An improved version of an autonomous low-light drift-camera package ("Driftcam") that can operate at midwater depths to observe natural behaviors of Humboldt squid that are too small to carry a Crittercam will be developed during the course of the project. Stanford graduate and undergraduate students will benefit from direct involvement in all of the research activities, and the researchers will also participate in wider public science dissemination efforts through regional K-12 programs, international educational outreach programs, and collaboration with major public media outlets.

Squid and their relatives (other cephalopods such as octopuses) have the ability to change skin color with chromatophores, microscopic muscular organs that are under control of the nervous system. All work on the cellular mechanisms of chromatophore control in squid has focused on three related species that inhabit relatively shallow coastal areas that have prominent features like seaweed, rocks and coral on the ocean floor. Skin-color changes in these species are associated with camouflage, signaling between individuals of the same species and threat displays with other species. The deeper open ocean presents a radically different environment that is also inhabited by many squids, primarily of different taxonomic families from the one commonly inhabiting coastal waters. An important open-ocean family includes the Humboldt squid (Dosidicus gigas). There is little light in the ocean at depths inhabited by these squid during daytime, and visual features such as coral and rocks are non-existent. Novel color-change behaviors in Dosidicus include repetitive whole-body "flashing," used for signaling between individuals of this species, and chaotic "flickering" that may underlie camouflage in the open ocean. Although these dynamic behaviors contrast with the more static patterns typical of coastal species, squids of both families employ temporal and spatial patterning to varying degrees. It is therefore likely that basic mechanisms for controlling the chromatophore network are the same in most, if not all, squids. "Vertical" control from the brain to the chromatophore muscles is known in the coastal squids, and may account for most chromatophore-based behaviors in those species, but behaviors like flickering in deeper-water species may be more influenced by processes within the skin itself that permit changes in chromatophores to spread from one to another without directly involving the nervous system. This hypothetical pathway would define a "horizontal" or distributed control system in the periphery that would permit autonomous behavior within the chromatophore network. This issue is the primary significance of the project. Understanding the fundamentals of horizontal control of chromatophores has the potential of being transformative to the field, because the current paradigm is that all control is directly exerted by the brain. Horizontal control is relevant to blood delivery to local tissues by circulatory systems, gut function and nervous system micro-circuits in vertebrates. Therefore, results from this project would also influence understanding of local control more broadly. From a wider perspective, results of this project will provide insight into the interactions of distributed (horizontal) and top-down (vertical) control mechanisms, a subject relevant to the general ability of complex systems to generate non-predictable, emergent phenomena. This concept is of fundamental interest to a broad sector of society, ranging from engineering to economics to politics.

An integrated approach will permit testing the hypothesis that control of the chromatophore network in squid involves peripheral mechanisms that are distinct from the neuronal motor-control pathway that descends from the brain. Spontaneous chromatophore activity that is independent of canonical neural control will be isolated by experimental manipulations in coastal loliginid squid (Doryteuthis opalescens), including chronic denervation and pharmacological block of neuronal activity with tetrodotoxin. In addition, a comparative approach will take advantage of an oceanic ommastrephid species, Dosidicus gigas, in which spontaneous, tetrodotoxin-resistant chromatophore activity is extremely prominent. Relevant methods involve molecular transcriptomics, cellular electrophysiology, immunohistochemistry with confocal microscopy and high-resolution electron microscopy. Specific aims are: 1) identify molecular and physiological properties of relevant ion channels and receptors that control excitability in the radial muscle fibers that operate individual chromatophore organs; 2) define structural, molecular and physiological features of coupling mechanisms between muscle fibers of neighboring chromatophores that define an excitatory transmission pathway within the skin; 3) elucidate the inhibitory role in controlling spontaneous chromatophore activity played by serotonin; 4) carry out parallel experiments in Dosidicus, a member of a family of ecologically important squid in which cellular studies of chromatophores have never been carried out. This project will support undergraduate and graduate student training, and includes significant efforts to involve students from groups underrepresented in STEM.

Coastal marine ecosystems in the US and the economies that depend on them are experiencing the brunt of an increasingly variable climate, characterized by increasing frequency and intensity of heat waves, strong El Niño events, storms, low-oxygen episodes and gradual ocean acidification. These changes in the environment have cumulative biological effects, and a great deal of research shows how each one separately affects many marine species. What is needed next is a set of experiments on how these environmental factors act when they act in combination with each other. This project will establish a new multi-user experimental facility at Hopkins Marine Station that will provide independent control of seawater temperature, dissolved oxygen concentration and acidification. These units will allow researchers to expose marine species to the wide range of physical conditions found naturally in upwelling ecosystems, and to alter those conditions to match future predictions. The proposed facility will enable training in experimental biology and climate change science for postdoctoral researchers, graduate and undergraduate students at all Monterey Bay institutions, K-12 outreach programs, and a training program for graduate students from the University of Puerto Rico. A major component of the proposed training activities is through collaboration with faculty at California State University Monterey Bay in supporting broad participation of underrepresented minorities, women and first generation college students in independent research and course-based projects.

The impact of multiple environmental stressors acting together on marine species has been hypothesized to be more than the sum of the impacts of single stressors alone. Yet this hypothesis has not often been tested across a range of species that naturally experience multi-stressor environmental impacts. The proposed facility will enable researchers at marine institutions in the Monterey Bay area to conduct the next generation of multi-stressor experiments with a suite of benthic and pelagic marine invertebrates and fishes that are relevant to local ecosystems and environmental variability. This project will address this issue by establishing a new multi-user experimental facility at Hopkins Marine Station (https://hopkinsmarinestation.stanford.edu) that will allow independent control of seawater temperature, dissolved oxygen concentration and pH over a range of parameter values that regularly occur in Monterey Bay and the offshore waters of the California Current System. The facility will include 40 independently controlled 189-liter aquaria and is based on successful construction and experimental use of four fully working prototypes. Thirty-six units will be added as part of this project. Four 500-liter round tanks to facilitate the study of small pelagic fishes and squid, and a small-tank system designed to allow the rapid change of multiple physical parameters through mixing of controlled water volumes for experiments on pelagic squid and fishes that involve equipment-intensive electrophysiological and biomechanical approaches will be components of the system to be employed. Experiments conducted in this facility will be analyzed through a wide range of modern tools including advanced genomics, neurophysiology, analysis of species interaction strengths, and ecosystem modeling. Overall, these facilities will provide an unprecedented ability to conduct collaborative joint experiments on a wide range of ecologically and economically relevant species and will support a creative and comprehensive research program on the effects of changing physical parameters on biodiversity and ecosystem functioning of the future oceans.