Respiratory rhythm generation: A modeling study of the respiratory central pattern generator and the phrenic motor neuron

The respiratory central pattern generator is a collection of medullary neurons that generates the rhythm of respiration. The respiratory central pattern generator feeds phrenic motor neurons, which, in turn, drive the main muscle of respiration, the diaphragm. The purpose of this thesis is to understand the neural control of respiration through mathematical models of the respiratory central pattern generator and phrenic motor neurons. ^ We first designed and validated a Hodgkin-Huxley type model that mimics the behavior of phrenic motor neurons under a wide range of electrical and pharmacological perturbations. This model was constrained physiological data from the literature. Next, we designed and validated a model of the respiratory central pattern generator by connecting four Hodgkin-Huxley type models of medullary respiratory neurons in a mutually inhibitory network. This network was in turn driven by a simple model of an endogenously bursting neuron, which acted as the pacemaker for the respiratory central pattern generator. Finally, the respiratory central pattern generator and phrenic motor neuron models were connected and their interactions studied. ^ Our study of the models has provided a number of insights into the behavior of the respiratory central pattern generator and phrenic motor neurons. These include the suggestion of a role for the T-type and N-type calcium channels during single spikes and repetitive firing in phrenic motor neurons, as well as a better understanding of network properties underlying respiratory rhythm generation. We also utilized an existing model of lung mechanics to study the interactions between the respiratory central pattern generator and ventilation. ^

The role of deregulated E2F1 expression in brain tumors

Mutations disabling the retinoblastoma (Rb) pathway are among the most common in human cancers, including brain cancer. These mutations promote tumor development through deregulated control of the E2F family of transcription factors. E2F1 belongs to a class of E2F's identified as transcriptional activators and involved in the G1/S phase transition of the cell. However, E2F-1 presents with a paradox as it is considered to have membership in two gene classes, functioning as both an oncogene and a tumor suppressor. This unusual trait generates a degree of uncertainty on the role that E2F1 plays in the development or maintenance of any given tumor. Here we show that E2F1 functions as an oncogene in brain tumors through the generation of mice engineered to overexpress E2F1 specifically within glial cells and neuronal progenitors as directed by the GFAP promoter. Mice carrying the transgene develop with high penetrance a phenotype characterized by neurological deficits including paresia, ataxia, head tilt and seizures. MRI imagining of the tgE2F1 mice reveals a low incidence of mild hydrocephalus, and most notably, histological analysis demonstrates that 25% of tgE2F1 mice present with the spontaneous formation of malignant brain tumors. Overall these neoplasms show histological features from a wide range of aggressive brain cancers including medulloblastoma, choroid plexus carcinoma, primary neuroectodermic tumor and malignant gliomas. Isolation and characterization of astrocytes from the tgE2F1 animal reveals a highly proliferative population of cells with 55% ± 2.5 of the tgE2F1astrocytes, 35% ± 3.4 normal mouse astrocytes in S-phase and the acquired capacity to grow in anchorage independent conditions. Additionally tgE2F1 astrocytes show an aberrant phenotype with random chromosomal fusions and nearly all cells demonstrating polyploidy. Taken together, this model forces a comparison to human brain tumor formation. Mouse age as related to tumoral mimics the human scenario with juvenile tgE2F1 mice presenting embryonal tumors typically identified in children, and older tgE2F1 mice demonstrating gliomas. In this regard, this study suggests a global role for E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. ^

Metastatic lung cancer in a new mouse model inheriting p53 and latent K-ras mutations

Lung cancer is a devastating disease with very poor prognosis. The design of better treatments for patients would be greatly aided by mouse models that closely resemble the human disease. The most common type of human lung cancer is adenocarcinoma with frequent metastasis. Unfortunately, current models for this tumor are inadequate due to the absence of metastasis. Based on the molecular findings in human lung cancer and metastatic potential of osteosarcomas in mutant p53 mouse models, I hypothesized that mice with both K-ras and p53 missense mutations might develop metastatic lung adenocarcinomas. Therefore, I incorporated both K-rasLA1 and p53RI72HΔg alleles into mouse lung cells to establish a more faithful model for human lung adenocarcinoma and for translational and mechanistic studies. Mice with both mutations ( K-rasLA1/+ p53R172HΔg/+) developed advanced lung adenocarcinomas with similar histopathology to human tumors. These lung adenocarcinomas were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that seen in lung cancer patients. This mouse model also showed gender differences in cancer related death and developed pleural mesotheliomas in 23.2% of them. In a preclinical study, the new drug Erlotinib (Tarceva) decreased the number and size of lung lesions in this model. These data demonstrate that this mouse model most closely mimics human metastatic lung adenocarcinoma and provides an invaluable system for translational studies. ^ To screen for important genes for metastasis, gene expression profiles of primary lung adenocarcinomas and metastases were analyzed. Microarray data showed that these two groups were segregated in gene expression and had 79 highly differentially expressed genes (more than 2.5 fold changes and p<0.001). Microarray data of Bub1b, Vimentin and CCAM1 were validated in tumors by quantitative real-time PCR (QPCR). Bub1b , a mitotic checkpoint gene, was overexpressed in metastases and this correlated with more chromosomal abnormalities in metastatic cells. Vimentin, a marker of epithelial-mesenchymal transition (EMT), was also highly expressed in metastases. Interestingly, Twist, a key EMT inducer, was also highly upregulated in metastases by QPCR, and this significantly correlated with the overexpression of Vimentin in the same tumors. These data suggest EMT occurs in lung adenocarcinomas and is a key mechanism for the development of metastasis in K-ras LA1/+ p53R172HΔg/+ mice. Thus, this mouse model provides a unique system to further probe the molecular basis of metastatic lung cancer.^

Forebrain-cerebellum interactions revealed by trace eyelid conditioning

While it is commonly assumed that brain systems receive and process information from other brain systems, there are few examples of tractable behaviors that allow such interactions to be studied. With the experiments presented in this dissertation we provide evidence that trace eyelid conditioning, a simple form of associative learning, is mediated by cerebellar learning in response to the output of persistent neural activity in the prefrontal cortex (PFC) and thus may be useful in analyses of PFC-cerebellar interactions. In a series of stimulation and reversible inactivation experiments we provide evidence that trace eyelid conditioning is mediated by cerebellar learning in response to a learned forebrain-driven input. Specifically, we provide evidence that this input is driven by the medial PFC and persists through the stimulus free trace interval of trace eyelid conditioning. In the next set of experiments we show that directly presenting the cerebellum with a pattern of input that mimics the classic persistent activity of PFC neurons reconstitutes trace eyelid conditioning, as assessed by a number of stringent tests. Finally, in set of reversible inactivation experiments, we provide evidence that bidirectional learning during trace eyelid conditioning involves the omission of the persistent, PFC-driven input that the cerebellum learns and responds to during trace eyelid conditioning. Given that persistent activity in PFC is often associated with working memory, these experiments suggest that trace eyelid conditioning may be useful in analyses of working memory mechanisms, cerebellar information processing and their interaction. To facilitate future analyses, we conclude with a working hypothesis of forebrain-cerebellum interactions during trace eyelid conditioning that addresses how persistent activity in PFC is induced and how the cerebellum decodes and uses PFC-driven input. ^

Minimal DNA sequences that control the cell lineage-specific expression of the pro$\alpha$2(I) collagen promoter in transgenic mice

The pattern of expression of the pro$\alpha$2(I) collagen gene is highly tissue-specific in adult mice and shows its strongest expression in bones, tendons, and skin. Transgenic mice were generated harboring promoter fragments of the mouse pro$\alpha$2(I) collagen gene linked to the Escherichia coli $\beta$-galactosidase or firefly luciferase genes to examine the activity of these promoters during development. A region of the mouse pro$\alpha$2(I) collagen promoter between $-$2000 and +54 exhibited a pattern of $\beta$-galactosidase activity during embryonic development that corresponded to the expression pattern of the endogenous pro$\alpha$2(I) collagen gene as determined by in situ hybridization. A similar pattern of activity was also observed with much smaller promoter fragments containing either 500 or 350 bp of upstream sequence relative to the start of transcription. Embryonic regions expressing high levels of $\beta$-galactosidase activity included the valves of the developing heart, sclerotomes, meninges, limb buds, connective tissue fascia between muscle fibers, osteoblasts, tendon, periosteum, dermis, and peritoneal membranes. The pattern of $\beta$-galactosidase activity was similar to the extracellular immunohistochemical localization of transforming growth factor-$\beta$1 (TGF-$\beta$1). The $-$315 to $-$284 region of the pro$\alpha$2(I) collagen promoter was previously shown to mediate the stimulatory effects of TGF-$\beta$1 on the pro$\alpha$2(I) collagen promoter in DNA transfection experiments with cultured fibroblasts. A construct containing this sequence tandemly repeated 5$\sp\prime$ to both a very short $\alpha$2(I) collagen promoter ($-$40 to +54) and a heterologous minimal promoter showed preferential activity in tail and skin of 4-week old transgenic mice. The pattern of expression mimics that of the $-$350 to +54 pro$\alpha$2(I) collagen promoter linked to a luciferase reporter gene in transgenic mice. ^

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

A major goal of chemotherapy is to selectively kill cancer cells while minimizing toxicity to normal cells. Identifying biological differences between cancer and normal cells is essential in designing new strategies to improve therapeutic selectivity. Superoxide dismutases (SOD) are crucial antioxidant enzymes required for the elimination of superoxide (O2·− ), a free radical produced during normal cellular metabolism. Previous studies in our laboratory demonstrated that 2-methoxyestradiol (2-ME), an estradiol derivative, inhibits the function of SOD and selectively kills human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The present work was initiated to examine the biochemical basis for the selective anticancer activity of 2-ME. Investigations using two-parameter flow cytometric analyses and ROS scavengers established that O2·− is a primary and essential mediator of 2-ME-induced apoptosis in cancer cells. In addition, experiments using SOD overexpression vectors and SOD knockout cells found that SOD is a critical target of 2-ME. Importantly, the administration of 2-ME resulted in the selective accumulation of O 2·− and apoptosis in leukemia and ovarian cancer cells. The preferential activity of 2-ME was found to be due to increased intrinsic oxidative stress in these cancer cells versus their normal counterparts. This intrinsic oxidative stress was associated with the upregulation of the antioxidant enzymes SOD and catalase as a mechanism to cope with the increase in ROS. Furthermore, oxygen consumption experiments revealed that normal lymphocytes decrease their respiration rate in response to 2-ME-induced oxidative stress, while human leukemia cells seem to lack this regulatory mechanism. This leads to an uncontrolled production of O2·−, severe accumulation of ROS, and ultimately ROS-mediated apoptosis in leukemia cells treated with 2-ME. The biochemical differences between cancer and normal cells identified here provide a basis for the development of drug combination strategies using 2-ME with other ROS-generating agents to enhance anticancer activity. The effectiveness of such a combination strategy in killing cancer cells was demonstrated by the use of 2-ME with agents/modalities such as ionizing radiation and doxorubicin. Collectively, the data presented here strongly suggests that 2-ME may have important clinical implications for the selective killing of cancer cells. ^

Sea ice cover, and bottom water and sediment characteristics at stations in the Amundsen Gulf, Canada

Seasonal dynamics in the activity of Arctic shelf benthos have been the subject of few local studies, and the pronounced among-site variability characterizing their results makes it difficult to upscale and generalize their conclusions. In a regional study encompassing five sites at 100-595 m water depth in the southeastern Beaufort Sea, we found that total pigment concentrations in surficial sediments, used as proxies of general food supply to the benthos, rose significantly after the transition from ice-covered conditions in spring (March-June 2008) to open-water conditions in summer (June-August 2008), whereas sediment Chl a concentrations, typical markers of fresh food input, did not. Macrobenthic biomass (including agglutinated foraminifera >500 µm) varied significantly among sites (1.2-6.4 g C/m**2 in spring, 1.1-12.6 g C/m**2 in summer), whereas a general spring-to-summer increase was not detected. Benthic carbon remineralisation also ranged significantly among sites (11.9-33.2 mg C/m**2/day in spring, 11.6-44.4 mg C/m**2/day in summer) and did in addition exhibit a general significant increase from spring-to-summer. Multiple regression analysis suggests that in both spring and summer, sediment Chl a concentration is the prime determinant of benthic carbon remineralisation, but other factors have a significant secondary influence, such as foraminiferan biomass (negative in both seasons), water depth (in spring) and infaunal biomass (in summer). Our findings indicate the importance of the combined and dynamic effects of food supply and benthic community patterns on the carbon remineralisation of the polar shelf benthos in seasonally ice-covered seas.

Sediment metabolism in samples off Northwest Africa

Sediments off northwest Africa were assayed for activities of the respiratory electron transport system (ETS) and for primary amino nitrogen. ETS activities were used to compute respiratory oxygen consuption, carbon oxidation, and nitrate reduction rates. Activities were correlated with depth of the water column, and their longshore distribution resembled that of euphotic zone phytoplankton productivity. Protein concentrations were closely correlated with ETS activities. Carbon biomass was calculated from protein and compared with other computed values. The carbon oxidation rate accounted for 13 % of the region's primary production.

Data from PhD thesis

The ocean quahog, Arctica islandica is the longest-lived non-colonial animal known to science. A maximum individual age of this bivalve of 405 years has been found in a population off the north western coast of Iceland. Conspicuously shorter maximum lifespan potentials (MLSPs) were recorded from other populations of A. islandica in European waters (e.g. Kiel Bay: 30 years, German Bight: 150 years) which experience wider temperature and salinity fluctuations than the clams from Iceland. The aim of my thesis was to identify possible life-prolonging physiological strategies in A. islandica and to examine the modulating effects of extrinsic factors (e.g. seawater temperature, food availability) and intrinsic factors (e.g. species-specific behavior) on these strategies. Burrowing behavior and metabolic rate depression (MRD), tissue-specific antioxidant and anaerobic capacities as well as cell-turnover (= apoptosis and proliferation) rates were investigated in A. islandica from Iceland and the German Bight. An inter-species comparison of the quahog with the epibenthic scallop Aequipecten opercularis (MLSP = 8-10 years) was carried out in order to determine whether bivalves with short lifespans and different lifestyles also feature a different pattern in cellular maintenance and repair. The combined effects of a low-metabolic lifestyle, low oxidative damage accumulation, and constant investment into cellular protection and tissue maintenance, appear to slow-down the process of physiological aging in A. islandica and to afford the extraordinarily long MLSP in this species. Standard metabolic rates were lower in A. islandica when compared to the shorter-lived A. opercularis. Furthermore, A. islandica regulate mantle cavity water PO2 to mean values < 5 kPa, a PO2 at which the formation of reactive oxygen species (ROS) in isolated gill tissues of the clams was found to be 10 times lower than at normoxic conditions (21 kPa). Burrowing and metabolic rate depression (MRD) in Icelandic specimens were more pronounced in winter, possibly supported by low seawater temperature and food availability, and seem to be key energy-saving and life-prolonging parameters in A. islandica. The signaling molecule nitric oxide (NO) may play an important role during the onset of MRD in the ocean quahog by directly inhibiting cytochome-c-oxidase at low internal oxygenation upon shell closure. In laboratory experiments, respiration of isolated A. islandica gills was completely inhibited by chemically produced NO at low experimental PO2 <= 10 kPa. During shell closure, mantle cavity water PO2 decreased to 0 kPa for longer than 24 h, a state in which ROS production is supposed to subside. Compared to other mollusk species, onset of anaerobic metabolism is late in A. islandica in the metabolically reduced state. Increased accumulation of the anaerobic metabolite succinate was initially detected in the adductor muscle of the clams after 3.5 days under anoxic incubation or in burrowed specimens. A ROS-burst was absent in isolated gill tissue of the clams following hypoxia (5 kPa)-reoxygenation (21 kPa). Accordingly, neither the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), nor the specific content of the ROS-scavenger glutathione (GSH) was enhanced in different tissues of the ocean quahog after 3.5 days of self-induced or forced hypoxia/anoxia to prepare for an oxidative burst. While reduced ROS formation compared to routine levels lowers oxidative stress during MRD and also during surfacing, the general preservation of high cellular defense and the efficient removal and replacement of damaged cells over lifetime seem to be of crucial importance in decelerating the senescent decline in tissues of A. islandica. Along with stable antioxidant protection over 200 years of age, proliferation rates and apoptosis intensities in most investigated tissues of the ocean quahog were low, but constant over 140 years of age. Accordingly, age-dependent accumulations of protein and lipid oxidation products are lower in A. islandica tissues when compared to the shorter-lived bivalve A. opercularis. The short-lived swimming scallop is a model bivalve species representing the opposite life and aging strategy to A. islandica. In this species permanently high energy throughput, reduced investment into antioxidant defense with age, and higher accumulation of oxidation products are met by higher cell turnover rates than in the ocean quahog. The only symptoms of physiological change over age ever found in A. islandica were decreasing cell turnover rates in the heart muscle over a lifetime of 140 years. This may either indicate higher damage levels and possibly ongoing loss of functioning in the heart of aging clams, or, the opposite, lower rates of cell damage and a reduced need for cell renewal in the heart tissue of A. islandica over lifetime. Basic physiological capacities of different A. islandica populations, measured at controlled laboratory conditions, could not explain considerable discrepancies in population specific MLSPs. For example, levels of tissue-specific antioxidant capacities and cell turnover rates were similarly high in individuals from the German Bight and from Iceland. Rather than genetic differences, the local impacts of environmental conditions on behavioral and physiological traits in the ocean quahog seem to be responsible for differences in population-specific MLSPs.

Influencia del estilo de vida y la funcionalidad sobre la calidad de vida relacionada con la salud en población mexicana con salud comprometida

Este estudio tiene como objetivo estudiar la influencia de los factores del estilo de vida y funcionalidad sobre la calidad de vida relacionada con la salud (CVRS) en población con salud comprometida. Método: 139 sujetos formaron parte de este estudio divididos en cuatro grupos de enfermedades co-morbidas (sobrepeso-obesidad, diabetes, hipertensión e hiperlipidemias). El estilo de vida se determinó mediante la encuesta FANTASTIC, calidad de vida relacionada con la salud mediante el SF-36 y la funcionalidad con los Test Sentar y Lenvantar (TSL) e Ir y Venir (TIV). Los resultados nos muestras un estilo de vida excelente influye positivamente sobre la Compuesta Salud Física y Compuesta Salud Mental (p < 0.05) en relación estilo de vida inferiores (bueno, regular y bajos) y no influye sobre la capacidad funcional (TSL y TIV). Además, diferentes condiciones de enfermedades co-morbidas (SO vs SOD vs SODT vs ODTH) parecen no afectar la CVRS, y si la capacidad funcional (p < 0.05). Conclusiones: niveles excelentes de estilo de vida influyen positivamente sobre la CVRS específicamente sobre los aspectos físicos y mentales. Las pruebas funcionales de TSL y TIV parecen ser útiles para diferencias los niveles funcionales en población con salud comprometida o población con enfermedades co-morbidas

Benthic oxygen flixes on the Washington shelf and slope

Benthic oxygen fluxes calculated from in situ microelectrode profiles arc compared with benthic flux chamber O2 uptake measurements on a transect of eight stations across the continental shelf and three stations on the slope of Washington State. Station depths ranged from 40 to 630 m and bottom-water oxygen concentrations were 127-38 µM. The fluxes measured by the two methods were similar on the slope, but on the shelf, the chamber flux exceeded the microelectrode flux by as much as a factor of 3-4. We attribute this difference to pore-water irrigation, a process which apparently accounts for the oxidation of a significant amount of organic C in the continental shelf sediments. Combining our diffusive flux data with other data demonstrates clearly that the bottomwater oxygen concentration must play some significant role in determining the sedimentary oxygen consumption rate. Numerical simulation of the microelectrode 0, profiles suggests that roughly half the diffusive 0, flux must be consumed within - 1 mm of the sediment surface. If this conclusion is correct, then the magnitude of the diffusive flux depends both on the bottom-water oxygen concentration and on the supply rate of labile C to the sediment surf'ace.

Influencia del estilo de vida y la funcionalidad sobre la calidad de vida relacionada con la salud en población mexicana con salud comprometida

Este estudio tiene como objetivo estudiar la influencia de los factores del estilo de vida y funcionalidad sobre la calidad de vida relacionada con la salud (CVRS) en población con salud comprometida. Método: 139 sujetos formaron parte de este estudio divididos en cuatro grupos de enfermedades co-morbidas (sobrepeso-obesidad, diabetes, hipertensión e hiperlipidemias). El estilo de vida se determinó mediante la encuesta FANTASTIC, calidad de vida relacionada con la salud mediante el SF-36 y la funcionalidad con los Test Sentar y Lenvantar (TSL) e Ir y Venir (TIV). Los resultados nos muestras un estilo de vida excelente influye positivamente sobre la Compuesta Salud Física y Compuesta Salud Mental (p < 0.05) en relación estilo de vida inferiores (bueno, regular y bajos) y no influye sobre la capacidad funcional (TSL y TIV). Además, diferentes condiciones de enfermedades co-morbidas (SO vs SOD vs SODT vs ODTH) parecen no afectar la CVRS, y si la capacidad funcional (p < 0.05). Conclusiones: niveles excelentes de estilo de vida influyen positivamente sobre la CVRS específicamente sobre los aspectos físicos y mentales. Las pruebas funcionales de TSL y TIV parecen ser útiles para diferencias los niveles funcionales en población con salud comprometida o población con enfermedades co-morbidas

Influencia del estilo de vida y la funcionalidad sobre la calidad de vida relacionada con la salud en población mexicana con salud comprometida

Este estudio tiene como objetivo estudiar la influencia de los factores del estilo de vida y funcionalidad sobre la calidad de vida relacionada con la salud (CVRS) en población con salud comprometida. Método: 139 sujetos formaron parte de este estudio divididos en cuatro grupos de enfermedades co-morbidas (sobrepeso-obesidad, diabetes, hipertensión e hiperlipidemias). El estilo de vida se determinó mediante la encuesta FANTASTIC, calidad de vida relacionada con la salud mediante el SF-36 y la funcionalidad con los Test Sentar y Lenvantar (TSL) e Ir y Venir (TIV). Los resultados nos muestras un estilo de vida excelente influye positivamente sobre la Compuesta Salud Física y Compuesta Salud Mental (p < 0.05) en relación estilo de vida inferiores (bueno, regular y bajos) y no influye sobre la capacidad funcional (TSL y TIV). Además, diferentes condiciones de enfermedades co-morbidas (SO vs SOD vs SODT vs ODTH) parecen no afectar la CVRS, y si la capacidad funcional (p < 0.05). Conclusiones: niveles excelentes de estilo de vida influyen positivamente sobre la CVRS específicamente sobre los aspectos físicos y mentales. Las pruebas funcionales de TSL y TIV parecen ser útiles para diferencias los niveles funcionales en población con salud comprometida o población con enfermedades co-morbidas

Influencia del estilo de vida y la funcionalidad sobre la calidad de vida relacionada con la salud en población mexicana con salud comprometida

Este estudio tiene como objetivo estudiar la influencia de los factores del estilo de vida y funcionalidad sobre la calidad de vida relacionada con la salud (CVRS) en población con salud comprometida. Método: 139 sujetos formaron parte de este estudio divididos en cuatro grupos de enfermedades co-morbidas (sobrepeso-obesidad, diabetes, hipertensión e hiperlipidemias). El estilo de vida se determinó mediante la encuesta FANTASTIC, calidad de vida relacionada con la salud mediante el SF-36 y la funcionalidad con los Test Sentar y Lenvantar (TSL) e Ir y Venir (TIV). Los resultados nos muestras un estilo de vida excelente influye positivamente sobre la Compuesta Salud Física y Compuesta Salud Mental (p < 0.05) en relación estilo de vida inferiores (bueno, regular y bajos) y no influye sobre la capacidad funcional (TSL y TIV). Además, diferentes condiciones de enfermedades co-morbidas (SO vs SOD vs SODT vs ODTH) parecen no afectar la CVRS, y si la capacidad funcional (p < 0.05). Conclusiones: niveles excelentes de estilo de vida influyen positivamente sobre la CVRS específicamente sobre los aspectos físicos y mentales. Las pruebas funcionales de TSL y TIV parecen ser útiles para diferencias los niveles funcionales en población con salud comprometida o población con enfermedades co-morbidas

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.