Neurobiology of social attachment: A comparative approach to behavioral, neuroanatomical, and neurochemical studies

Abstracts

Over the past few decades there have been extensive studies on metal uptake in marine animals (mainly the invertebrates). Many of these studies are guided by chemical speciation principle (model) and thus metal environmental chemistry is the focus of these studies. There have been relatively fewer comparative studies on metal uptake and accumulation, yet such study can yield important understanding of the processes controlling metal uptake in marine animals. One of such comparative approaches is the use of different marine species which exhibit contrasting patterns of metal handling and sequestration (even within the taxonomically similar species). In this talk I will try to illustrate and highlight how the comparative approach in examining different species can help our understanding of metal uptake in aquatic environments. Specifically, I will present a few examples of the differences of metal handling among different species of invertebrates (such as barnacles, mussels, and scallops), or between the freshwater and marine fish. Such difference in metal handling can lead to their different uptake and body concentrations. The ecotoxicological significance of these body concentrations is also highly variable for different species. In addition, there are contrasting patterns of metal accumulation among different metals, and a comparative approach can also help in understanding the process of metal uptake. Clearly, study of metal uptake needs to consider the great difference among different marine species and metals. Comparative approach certainly needs to be further appreciated by the scientific community. doi:10.1016/j.cbpc.2008.10.070

DOWN-REGULATION OF CAVEOLIN-1 LEADS TO EARLY TRANSFORMATION IN MAMMARY EPITHELIAL CELLS: DEREGULATION ESTROGEN RECEPTOR ALPHA (ERΑ) SIGNALING Wang Xi, Zhang Hong, Feng Shuang, Zhao Chun-Hui, Zou Wei College of Life Science, Liaoning Normal University, Dalian 116029, China E-mail: [email protected] Caveolin, a 21–24 kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Caveolin-1 can associate with a variety of molecules, involved in signal transduction, endocytosis and transcytosis, cytoskeletal arrangement and also with mammary cell transformation and oncogenesis. Through gene trapping, we have obtained a cell line, named MCF10A-ST1, which only expresses 50% Cav-1 compared with parental cells MCF 10A (human mammary epithelial cell line). The decreased expression of Cav-1 is sufficient for phenotypic transformation of MCF10A cells, which involved in the loss of anchorage-dependent growth and morigenicity in nude mice with the existence of E2. However, the mechanism of Cav-1 down-regulation in the early transformation and signaling transduction of mammary epithelial cells is unclear. Here, we report that down-regulation of caveolin-1 protein expression leads to deregulated estrogen receptor α (ERα) signaling and consequently early transformation in mammary epithelia. Previous study in our lab on microarray assay showed that the expression of Cyclin D1 (cell cycle protein) were up-regulated in ST1 cell line. Here, we not only confirmed the results by Western Blot but also demonstrated that Cav-1 down-regulation accelerate the progression of mammary cells from G1 phrase into S phrase by Flow Cytometry (FCM). This proposed that the Cav-1 down-regulation could change the progress of cell cycle in the mammary cells. Otherwise, microarray assay also showed that the transcription factor (c-Jun) was up-regulated. But, the original carcinoma gene (c-Fos) and transcription factor (AP-1) have no obviously changed compared with MCF10A. ST1 cells obtained the morigenicity in nude mice with the existence of E2, and immunoprecipitation showed the interactions of Cav-1 with ERα in both MCF10A and ST1. ERα expression were increased as further downregulation of Cav-1. So, we hypothesize that Cav-1 down-regulation could induce the activation of ERα-associated signaling pathway, in order to adjust the development and proliferation. By siRNA technology, the down regulation of Cav-1 could activate MAP kinase and Akt signaling pathway, including the phosphorylation of ERK1/2 and AKT. These results above suggest that Cav-1 plays an important role in the transformation in mammary epithelial cells and could be responsible for the initiation of breast cancer as a tumor suppressor. doi:10.1016/j.cbpc.2008.10.071

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METABOLIC AND OSMOTIC RESPONSES OF LAKE QINGHAI NAKED CARP GYMNOCYPRIS PRZEWALSKII TO ACUTE HYPOXIA DURING SPAWNING MIGRATION Yuxiang Wanga,b, X.-Q. Chena, J.-Z. Dua, C.J. Braunerc, J. Bahramib, R. Daviesb, C.M. Woodd, J. Rogersd, J.W. Semplee, B. Murrayf, J.G. Richardsc a Department of Biotechnology, College of Life Science, Zhejiang University, Hangzhou P.R. China b Department of Biology, Queen's University, Kingston, Ontario, Canada c Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada d Department of Biology, McMaster University, Hamilton, Canada e Department of Biology, University of Waterloo, Waterloo, Canada f Department of Biology, University of Northern British Columbia, Prince George, British Columbia, Canada E-mail: [email protected] Members of carp family are well known for their sluggish lifestyle and their extreme hypoxia tolerance. The endemic naked carp of Lake Qinghai is a relatively active fish, undergoing an annual spawning migration from the saline and alkaline lake water to freshwater rivers, but little is known about its metabolic and osmotic responses to hypoxia. Are there lifestyle tradeoffs between high performance and hypoxia tolerance? We investigated 1) the metabolic responses of this high altitude-living fish to 24 h acute hypoxic exposure (~ 0.3 mg O2/L) and an additional 12 hr recovery in normoxic water; 2) if this high altitude-living fish can cope with osmotic balance under acute hypoxic exposure. Fish were caught in freshwater during their spawning migration. Unlike typical hypoxia tolerant animals, muscle ATP was not defended during hypoxia exposure, but fell by 80% after 24 h exposure to hypoxia. Muscle lactate accumulation points to an activation of glycolysis for energy supply during hypoxia; however, the lactate accumulated could not be accounted for by muscle glycogen depletion. Instead, large decreases in liver glycogen suggest that glycolytic flux in muscle may be supported by hepatic glucose production. In addition, there was a 36% increase in the length and 40% decrease in the thickness of gills secondary lamellae within 12 h exposure, gill Na/K-ATPase, Ca-ATPase and Mg-ATPase activities increased more than 50%, while blood plasma osmolality remained unchanged. Other key metabolic enzymes and metabolites were measured in ionic regulatory sites such as kidney and intestine. We conclude that, when challenged with hypoxia, the migrating fish can enhance their gills gas exchange surface area, ionic transport capability and metabolic activity without compromising their osmotic homeostasis in a hypoosmotic environment. (Funded by NSF China and NSERC Canada) doi:10.1016/j.cbpc.2008.10.072

NEUROBIOLOGY OF SOCIAL ATTACHMENT: A COMPARATIVE APPROACH TO BEHAVIORAL, NEUROANATOMICAL, AND NEUROCHEMICAL STUDIES Zuoxin Wang Department of Psychology and Program in Neuroscience, Florida State University, Tallahassee, FL 32306, USA E-mail: [email protected] Voles (Microtus) are closely related rodent species that show remarkable differences in life strategy and social behavior, and thus provide an excellent opportunity for comparative studies. We compared monogamous with promiscuous voles to examine their social behaviors and underlying neurochemical mechanisms. Monogamous prairie voles (M. ochrogaster), but not promiscuous meadow (M. pennsylvanicus) or montane voles (M. montanus), show biparental care and mating-induced pair bonding. To examine the neurochemcial mechanism of pair bonding, we focused on the neuropeptide vasopressin (AVP), neurotransmitter dopamine (DA), and their interactions. We found species differences in the distribution pattern of AVP receptors, indicating species differences in brain responsiveness to released AVP. In addition, the AVP pathway, particularly from the bed nucleus of the stria terminalis (BST), lateral septum (LS), and ventral pallidum (VP), is involved in pair bonding. Site-specific administration of AVP induced pair bonding in monogamous, but not promiscuous, voles, whereas administration of the AVP V1a receptor antagonist blocked mating-induced pair bonding in monogamous voles. The neurotrans-

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Abstracts

mitter DA in the reward circuit is also involved in pair bonding in a receptorspecific manner in monogamous prairie voles. The vole's nucleus accumbens (NAcc) contains both DA terminals and receptors, and mating induced DA release. Intra-NAcc administration of a D2-type receptor agonist induced pair bond formation whereas administration of the D2-type receptor antagonist blocked this behavior induced by mating, suggesting the importance of D2-type receptor activation in pair bond formation. Interestingly, pair-bonded prairie voles showed an increased level of D1-type receptor expression in the NAcc, which was associated with pair bond maintenance. Finally, we also found interactions between AVP and DA in the regulation of pair bonding behavior. Together, these data illustrate a neural circuit important in pair bonding; the AVP-mediated circuit for individual recognition, learning and memory and DAmediated circuit for reward associated with mating. This research was supported by grants from NIMH, NIDA, and NICHD. doi:10.1016/j.cbpc.2008.10.073

UNEXPECTED INTERACTIONS BETWEEN PB, CD, AND THE GILLS OF RAINBOW TROUT IN MODERATELY ACIDIC, SOFT WATERS: IMPLICATIONS FOR THE BIOTIC LIGAND MODEL (BLM) AND PREDICTIONS OF TOXICITY M.P. Wilkiea, O. Birceanua, P. Gillisb, Y. Karaa, J. Chowdhuryb, J. McGeera, C.M. Woodb a Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada N2L 3C5 b Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 E-mail: [email protected] The Biotic Ligand Model (BLM) predicts how natural organic matter (NOM) and competing ions (e.g. Ca2+, H+, Na+) affect metal bioavailability and metal toxicity in aquatic organisms. However, the BLM mainly focuses upon individual, rather than metal mixtures, which are more common. Two such metals are Pb and Cd, which may be co-released into aquatic ecosystems due to mining and smelting operations, from industrial sources, and coal combustion. Our goal was to determine how Pb and Cd interacted with the gill of rainbow trout (Oncorhynchus mykiss) exposed to biologically relevant concentrations of Cd (< 100 nmol L− 1) plus Pb (< 500 nmol L− 1). Because both metals act on gill Ca2+ channels, we predicted that Pb- and Cdgill binding would be additive, and exacerbate disturbances to Ca2+ influx when fish were exposed to both metals. Since Pb would mainly be in its ionized Pb2+ form in soft (< 100 μmol Ca2+ L− 1), acidic (pH 6.0) water, we also predicted that Pb toxicity would be greater than previously reported. The 96 h LC50 for Pb was 482 nmol L− 1, indicating that Pb was approximately one-order of magnitude more toxic in soft, acidic water than in harder, circumneutral pH waters. The LC50 for Cd alone was also low, approximately 6.7 nmol L− 1. Surprisingly, fish acclimated to soft water had a greater number of Pb-gill binding sites (Bmax = 18.2 nmol g− 1 gill tissue) than Cdgill binding sites (Bmax = 13.5 nmol g− 1 gill binding sites), suggesting that Pb was binding to additional sites on or within the gill. We suggest that this greater than anticipated Pb binding resulted from Pb2+ trapping within the more alkaline gill cytosol due to Pb2+ conversion to Pb(OH)2 and PbOH+, and/or non-specific binding to cytosolic proteins. This explanation was supported by reductions in Na+ influx at relatively low Pb concentrations (~ 100 nmol L− 1), which likely arose from Pb-induced inhibition of intracellular carbonic anhydrase and/or basolateral Na+/K+-ATPases. Cd-gill and Pb-gill binding were additive at relatively low concentrations of both metals. However, at higher Cd concentrations, there was less than predicted Pb-gill binding suggesting that Cd was out-competing Pb for gill binding sites. This was supported by observations that Cd alone, but not Pb alone, inhibited Ca2+ influx. In mixtures, however, the presence of Pb exacerbated Cd-induced disturbances to Ca2+ influx. We conclude that the binding and physiological disturbances caused by Cd and Pb at the gill are additive. Moreover, the toxicity of both metals is much greater in soft, moderately acidic waters that characterize the Canadian Shield, Scandinavia and other sensitive freshwaters. The authors gratefully acknowledge the support of the NSERC MITHE-Research Network. doi:10.1016/j.cbpc.2008.10.074

THE ROLE OF THE NMDA RECEPTOR (NMDAR) IN THE HIGH ANOXIA-TOLERANCE AND AMMONIA-TOLERANCE OF THE GOLDFISH (CARASSIUS AURATUS) M.P. Wilkiea,b, M.E. Pamenterb, D.S. Shinb, G. Skeltona, S. Alkabiea, L.T. Buckb Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada N2L 3C5 b Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3G5 E-mail: [email protected]

a

Recently, evidence has begun to accumulate suggesting that fish that can withstand long periods of oxygen deprivation (hypoxia/anoxia) are generally ammonia tolerant (Walsh et al., 2007. Comp. Biochem. Physiol. 147A:332). One such fish is the goldfish, which is one of the most anoxia tolerant vertebrates known. However, we report that the goldfish is also ammonia tolerant, with a 96 h LC50 of 270–290 μmol NH3 L− 1, making them one of the most ammonia-tolerant, exclusively aquatic fishes. Our most recent experiments have focused upon the NMDAr because oxygen starvation and ammonia toxicity are each associated with over-activation of this glutamate receptor. This over-activation is referred to as excitotoxicity, and it is associated with a massive depolarization of neurons in the CNS due to the uncontrolled entry of Ca2+ via continuously opening of NMDAr's. We used electrophysiological, biochemical and pharmacological approaches to test the hypothesis that the NMDAr of the goldfish was resistant to excitotoxicity caused by both anoxia and increased ammonia. Whole cell patch-clamping of goldfish brain (telencephalon) slices demonstrated that goldfish lowered NMDAr currents by 60% during oxygen starvation. We concluded that such “channel arrest” likely prevented excitotoxicity, as observed in the anoxiatolerant western painted turtle (Chrysemys picta: Shin and Buck. 2002. Physiol. Biochem. Zool. 76:41). Moreover, our observations of reduced brain Na+/K+ ATPase activity in the anoxic goldfish suggested that a reduced permeability of goldfish neurons to Na+, may have also had a protective effect against anoxia. In contrast to anoxia, exposure of brain slices to NH4Cl (5 and 10 mmol L− 1) led to 2.0- to 2.5-fold increases in NMDAr currents. However, this effect was reversible suggesting that the goldfish NMDAr can withstand acute build-ups of ammonia. The conclusion was substantiated by the lack of protective effect that an NMDAr channel blocker, MK801, had when fish were exposed to lethal concentrations of ammonia. These finding suggest ammonia may directly activate the NMDAr, but that ammonia toxicity is not related to excitotoxicity in the goldfish. In more sensitive species, such as the rainbow trout (Oncorhynchus mykiss) ammonia toxicity was likely related to excitoxicity of the NMDAr because MK801 increased survival during exposure to high environmental ammonia. Finally, the resting membrane potential of goldfish neurons was unaltered by high environmental ammonia, suggesting that depolarization of the neurons by NH4+ is not involved in the initiation of ammonia-induced excitoxicity in neurons as suggested in mammalian models of ammonia toxicity. We conclude that the high anoxia and ammonia tolerance of the goldfish is related to the ability of their NMDAr's to withstand these normally neurotoxic insults compared to more sensitive fishes and mammals. Funded by NSERC Discovery Grants to LTB and MPW. doi:10.1016/j.cbpc.2008.10.075

THE BIOTIC LIGAND MODEL: USING PHYSIOLOGY, GEOCHEMISTRY, AND MODELING TO PREDICT METAL TOXICITY Chris M. Wood McMaster University, Hamilton, Ontario, Canada and Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Florida, U.S.A. E-mail: [email protected] Historically, ambient water quality criteria (AWQC) for trace metals have been based simply on total or dissolved metal levels in the water. However a wealth of physiological and toxicological research has demonstrated that the bioavailability and toxicity of metals to aquatic organisms is dependent on site-specific water chemistry factors such as hardness, salinity, specific ion levels, pH, alkalinity, and dissolved organic matter. The Biotic Ligand Model (BLM) is a relatively new computational approach for the generation of site-