Binding Studies of Neuronal Nicotinic Acetylcholine Receptors Expressing Unnatural Amino Acids

Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels mediating fast synaptic transmission throughout the peripheral and central nervous systems. They have been implicated in various processes related to cognitive functions, learning and memory, arousal, reward, motor control and analgesia. Therefore, these receptors present alluring potential therapeutic targets for the treatment of pain, epilepsy, Alzheimer’s disease, Parkinson’s disease, Tourette’s syndrome, schizophrenia, anxiety, depression and nicotine addiction. The work detailed in this thesis focuses on binding studies of neuronal nicotinic receptors and aims to further our knowledge of subtype specific functional and structural information.

Chapter 1 is an introductory chapter describing the structure and function of nicotinic acetylcholine receptors as well as the methodologies used for the dissertation work described herein. There are several different subtypes of nicotinic acetylcholine receptors known to date and the subtle variations in their structure and function present a challenging area of study. The work presented in this thesis deals specifically with the α4β2 subtype of nicotinic acetylcholine receptor. This subtype assembles into 2 closely related stoichiometries, termed throughout this thesis as A3B2 and A2B3 after their respective subunit composition. Chapter 2 describes binding studies of select nicotinic agonists on A3B2 and A2B3 receptors determined by whole-cell recording. Three key binding interactions, a cation-π and two hydrogen bonds, were probed for four nicotinic agonists, acetylcholine, nicotine, smoking cessation drug varenicline (Chantix®) and the related natural product cytisine.

Results from the binding studies presented in Chapter 2 show that the major difference in binding of these four agonists to A3B2 and A2B3 receptors lies in one of the two hydrogen bond interactions where the agonist acts as the hydrogen bond acceptor and the backbone NH of a conserved leucine residue in the receptor acts as the hydrogen bond donor. Chapter 3 focuses on studying the effect of modulating the hydrogen bond acceptor ability of nicotine and epibatidine on A3B2 receptor function determined by whole-cell recording. Finally, Chapter 4 describes single-channel recording studies of varenicline binding to A2B3 and A3B2 receptors.

Investigations on the vertical migration of planktonic Crustacea in the Bodensee-Obersee. [Translation from: Schriften des Vereins fur Geschichte des Bodensees und seiner Umgebung, 87, 177-187, 1969.]

The vertical zoning of the planktonic Crustacea in a lake is the expression of a complex set of different factors. Besides the measurable, external influences such as light, temperature, acid and C02 stratification, a particularly large part is played by internal factors, which co-ordinate a specific reaction in each species depending on state of development, age and sex. Supporting this extensive, predictable, annual course of diurnal depths and the daily vertical migrations, whose extent is again dependent on external conditions, primarily of course on the amount of light. The individual factors mentioned, however, are here also of great significance. Within the scope of a long-term study of the planktonic Copepoda of Lake Constance, some day and night series were in 1963 also carried out in the Obersee, in order to obtain at least volumetric data on the extent of the daily migrations of these creatures.

On drift and diel vertical migration of bottom invertebrates of the Amur. [Translation from: Zoologicheskie Zhurnal 42 1601-1612, 1963.]

Planktobenthos was sampled in 1957-58 in the river Amur. A determination of the kind of organisms drifting in the mass of water of the Amur was carried out. Of special interest for the authors was the activity of drifting of benthic larvae.

Bird migration and the spread of Crustacea. [Translation from: Verhandlungen der Deutschen Zoologischen Gesellschaft 27, 311-316, 1963.]

The passive spread of a high percentage of freshwater organisms is one of the most important requirements in short-lived and insular communities for species to attai n and survive - and consequently to balance the lack of a topographical continuity of most inland waters. Unfortunately hardly anything is known about the amounts of seed material typical for any lake into which it is carried. The causes of passive dissemination - wind, water and animals as well as man - are confirmed by many examples. It has been assumed now for at least a hundered years that , among animals, birds play a prominent role, although also disappointingly few facts are at hand. The passage and spread through birds' intestines has up to now been supported only by some limited data. This paper reports on experimental research where the eggs of Daphnia magna, Triops cancriformis, Artemia salina, Diaptomus spinosus and Cypris pubera were introduced by means of gelatine capsules into the oesophagus of a drake. The bird's excrements were inspected under a microscope for eggs and resting stages, and these were transferred into corresponding cultures.

Biologie de Penaeus duorarum (Burkenroad) en Côte d'Ivoire: 2 - ponte et migration

The pink shrimp Penaeus duorarum spawns from 25 to 60m, mostly in summer (October to June). Size at first sexual maturity is 31 mm (carapace length). The observed difference with the Caribbean pink shrimp is analysed. Immature shrimps migrate all year round but a peak migration occurs from January to March (in summer) and is associated with maximum salinities. A secondary peak migration occurs in October corresponding to minimum salinity and maximum river discharge. The action of salinity on migration is discussed and a preponderant action of currents in the process is also suggested. Migration is also related to moon phase, tide and day-night cycles. Migration intensity as expressed by catch per unit of effort is maximum at night, during ebb tide, on new and full moon. Seasonal variation of mean migration size and abundance are related by a negative linear correlation on a logarithmic plot (R = 0.776). This phenomenon is perhaps related to competition for food.

Prediction and confirmation of seasonal migration of Pacific sardine (Sardinops sagax) in the California Current Ecosystem

During the last century, the population of Pacific sardine (Sardinops sagax) in the California Current Ecosystem has exhibited large fluctuations in abundance and migration behavior. From approximately 1900 to 1940, the abundance of sardine reached 3.6 million metric tons and the “northern stock” migrated from offshore of California in the spring to the coastal areas near Oregon, Washington, and Vancouver Island in the summer. In the 1940s, the sardine stock collapsed and the few remaining sardine schools concentrated in the coastal region off southern California, year-round, for the next 50 years. The stock gradually recovered in the late 1980s and resumed its seasonal migration between regions off southern California and Canada. Recently, a model was developed which predicts the potential habitat for the northern stock of Pacific sardine and its seasonal dynamics. The habitat predictions were successfully validated using data from sardine surveys using the daily egg production method; scientific trawl surveys off the Columbia River mouth; and commercial sardine landings off Oregon, Washington, and Vancouver Island. Here, the predictions of the potential habitat and seasonal migration of the northern stock of sardine are validated using data from “acoustic–trawl” surveys of the entire west coast of the United States during the spring and summer of 2008. The estimates of sardine biomass and lengths from the two surveys are not significantly different between spring and summer, indicating that they are representative of the entire stock. The results also confirm that the model of potential sardine habitat can be used to optimally apply survey effort and thus minimize random and systematic sampling error in the biomass estimates. Furthermore, the acoustic–trawl survey data are useful to estimate concurrently the distributions and abundances of other pelagic fishes.

Defining plausible migration rates based on historical tagging data: a Bayesian mark-recapture model applied to English sole (Parophrys vetulus)

A generalized Bayesian population dynamics model was developed for analysis of historical mark-recapture studies. The Bayesian approach builds upon existing maximum likelihood methods and is useful when substantial uncertainties exist in the data or little information is available about auxiliary parameters such as tag loss and reporting rates. Movement rates are obtained through Markov-chain Monte-Carlo (MCMC) simulation, which are suitable for use as input in subsequent stock assessment analysis. The mark-recapture model was applied to English sole (Parophrys vetulus) off the west coast of the United States and Canada and migration rates were estimated to be 2% per month to the north and 4% per month to the south. These posterior parameter distributions and the Bayesian framework for comparing hypotheses can guide fishery scientists in structuring the spatial and temporal complexity of future analyses of this kind. This approach could be easily generalized for application to other species and more data-rich fishery analyses.

Oceanic migration rates of Upper Chesapeake Bay striped bass (Morone saxatilis), determined by otolith microchemical analysis*

Oceanic incidence and spawning frequency of Chesapeake Bay striped bass (Morone saxatilis) were estimated by using microchemical analysis of strontium in otoliths. Otoliths from 40 males and 82 females sampled from Maryland’s portion of the Chesapeake Bay were analyzed for seasonal and age-specific patterns in strontium and calcium levels. The proportion of oceanic females increased from 50% to 75% between ages seven to 13; the proportion of oceanic males increased from 20% to ~50% between ages four to 13. Contrary to an earliermodel of Chesapeake Bay striped bass migration, results indicated that a substantial number of males undertook oceanic migrations. Further, we observed no mass emigration of females from three to four years of age from the Chesapeake Bay. Seasonal patterns of estuarine habitat use were consistent with annual spawning runs by striped bass of mature age classes, but with noteworthy exceptions for newly mature females. Evidence of an early oceanic presence indicated that Chesapeake Bay yearlings move into coastal regions—a pattern observed also for Hudson River striped bass. Otolith microchemical analyses revealed two types of behaviors (estuarine and oceanic) that confirm migratory behaviors recently determined for other populations of striped bass and diadromous species (e.g., American eels [Anguilla rostrata] American shad [Alosa sapidissima] and white perch [Morone Americana]).

Variation in the distribution of walleye pollock (Theragra chalcogramma) with temperature and implications for seasonal migration

Aspects of the feeding migration of walleye pollock (Theragra chalcogramma) in the eastern Bering Sea (EBS) were investigated by examining the relationship between temperatures and densities of fish encountered during acoustic and bottom trawl surveys conducted in spring and summer between 1982 and 2001. Bottom temperature was used as an indicator of spring and summer warming of the EBS. Clusters of survey stations were identified where the density of walleye pollock generally increased or decreased with increasing water temperature. Inferences about the direction and magnitude of the spring and summer feeding migration were made for five length categories of walleye pollock. Generally, feeding migrations appeared to be northward and shoreward, and the magnitude of this migration appeared to increase with walleye pollock size up to 50 cm. Pollock larger then 50 cm showed limited migratory behavior. Pollock may benefit from northward feeding migrations because of the changes in temperature, zooplankton production, and light conditions. Ongoing climate changes may affect pollock distribution and create new challenges for pollock management in the EBS.

Pretreatment with Resveratrol Prevents Neuronal Injury and Cognitive Deficits Induced by Perinatal Hypoxia-Ischemia in Rats

Despite advances in neonatal care, hypoxic-ischemic brain injury is still a serious clinical problem, which is responsible for many cases of perinatal mortality, cerebral palsy, motor impairment and cognitive deficits. Resveratrol, a natural polyphenol with important anti-oxidant and anti-inflammatory properties, is present in grapevines, peanuts and pomegranates. The aim of the present work was to evaluate the possible neuroprotective effect of resveratrol when administered before or immediately after a hypoxic-ischemic brain event in neonatal rats by analyzing brain damage, the mitochondrial status and long-term cognitive impairment. Our results indicate that pretreatment with resveratrol protects against brain damage, reducing infarct volume, preserving myelination and minimizing the astroglial reactive response. Moreover its neuroprotective effect was found to be long lasting, as behavioral outcomes were significantly improved at adulthood. We speculate that one of the mechanisms for this neuroprotection may be related to the maintenance of the mitochondrial inner membrane integrity and potential, and to the reduction of reactive oxygen species. Curiously, none of these protective features was observed when resveratrol was administered immediately after hypoxia-ischemia.

Forty Years of Winter: Cetaceans Observed During the Southbound Migration of Gray Whales, Eschrichtius robustus, Near Granite Canyon, Central California

From December to February in most years from 1967 to 2007, observers counted gray whales, Eschrichtius robustus, from shore sites south of Carmel in central California. In addition to gray whales, other cetacean species were also recorded. These observations were summarized and compared among survey platforms and to ocean conditions. Eleven cetacean species were identified including eight odontocete species (killer whale, Orcinus orca; Pacific white-sided dolphin, Lagenorhynchus obliquidens; common dolphin, Delphinus spp.; bottlenose dolphin, Tursiops truncatus, northern right whale dolphin, Lissodelphis borealis; Risso’s dolphin, Grampus griseus; Dall’s porpoise, Phocoenoides dalli; and harbor porpoise, Phocoena phocoena) and three mysticete species (humpback whale, Megaptera novaeangliae; minke whale, Balaenoptera acutorostrata; and blue whale, Balaenoptera musculus). As expected, the detection of certain species among survey platforms (shore-based census watches, 25-power “Big Eye” binocular watches, and aerial surveys) was limited by species surfacing behavior and/or bathymetric preference. Comparisons among the shore-based census efforts showed a significant difference in sightings rates from 1967–84 (n = 14, mean = 0.11, SD = 0.11) to 1985–2007 (n = 11, mean = 1.48, SD = 0.47; t-Test: p < 0.001, df = 23). The warm period observed during the 1990’s may partially explain the increase in sighting rates and diversity of species observed at the census site compared to the much cooler temperatures of the 1970’s.

Length-weight Relationships of Dolphinfish, Coryphaena hippurus, and Wahoo, Acanthocybium solandri: Seasonal Effects of Spawning and Possible Migration in the Central North Pacific

Weight-on-length (W-L) relationships for 2,482 dolphinfish, Coryphaena hippurus, and 1,161 wahoo, Acanthocybium solandri, were examined. Data on fork length, whole (round) weight, and sex were collected for dolphinfish at the Honolulu fish auction from March 1988 through November 1989. Unsexed weight and length data for wahoo were collected at the auction from July 1988 through November 1989. We also used sex specific weight and length data of 171 wahoo collected during 1977–1985 research cruises for analysis. Coefficients of W-L regressions were significantly different between the sexes for dolphinfish. Coefficients did not significantly differ between the sexes for wahoo based on research cruise data. In a general linear model evaluating month as a categorical factor, month was significant for female dolphinfish, male dolphinfish, and wahoo with sexes pooled. W-L and length-on-weight (L-W) relationships were fitted by nonlinear regression for all dolphinfish, female dolphinfish, male dolphinfish, and all wahoo sexes pooled. W-L relationships for monthly samples of female dolphinfish, male dolphinfish, and all wahoo with sexes pooled were also fitted by nonlinear regression. Predicted mean weight at length for wahoo was highest at the beginning of the spawning season in June and lowest after the spawning season in September. Maximum and minimum predicted mean weight at length for both sexes of dolphinfish did not correspond with the peak spawning period (March–May). Plausible migration models in conjunction with reproductive behavior were examined to explain the variability in monthly predicted mean weight at length for dolphinfish.

The assessment of the flow requirements for upstream migration of salmonids in some rivers of North West England

This is the assessment of the flow requirements for upstream migration of salmonids in some rivers of North West England produced by the North West Water Authority in 1985. This report focuses on the automatic fish counters operating on the resistivity principle used for several years in North West England. This report aims to investigate the flow requirements for upstream migration of salmon and migratory trout. The data obtained confirmed that during summer months most fish movement occurs in the higher range of the available flows, but the migration flow range varied from year to year, depending on prevailing river levels. Of the other environmental variables measured, only water temperature and incident light intensity appear to have any direct association with fish movement. Information on migration flow ranges were used in conjunction with computer simulations of the effects of abstraction proposals on historical flows to assess the implications of these proposals for migratory fish.