The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.

[Truncated abstract] Formation and consolidation of the retinotectal projection during optic nerve regeneration has been associated with two major interlinked processes. Initially, retinal ganglion cell (RGC) axons are guided by molecular guidance cues, such as the Eph receptor tyrosine kinases and their ligands, the ephrins, to their approximately correct location and form a coarse topographic map in the optic tecum. Such axon guidance occurs in the absence of neural activity and is considered to be activity-independent. The second process involves glutamatergic excitation, whereby correctly located connections are strengthened by correlated neural activity, whilst removing inappropriately located ones thereby sharpening the topography.The second process is considered to be activitydependent. Here, a number of experiments were undertaken to further examine the interrelationships of activity-dependent and independent processes with respect to functional outcomes. Two models of optic nerve regeneration were studied. In goldfish, following optic nerve crush, regeneration is successful. … In goldfish, guidance along the medio-lateral tectal axis may occur through preordering of axons prior to entering the tectum via the appropriate medial and lateral brachium, with EphA/ephrin-A then guiding axons over the rostral-caudal axis establishing gross topography. The increase in involvement of NMDA-mediated transmission during the period of activity-dependent refinement consolidated the role of this receptor in synapse plasticity. However what triggers NMDA-mediated activity to increase is still largely unresolved, although as the factors governing receptor trafficking during development and synaptic plasticity become better understood, these can be applied to the period of plasticity associated with regenerating axons. And finally, as GABAergic inhibition appears to suppress activity-dependent refinement, means of overcoming this inhibition through 4 such methods as visual training or pharmacological intervention may have significance for mammalian regeneration.

Horizontal cells (HCs) are retinal interneurons which provide feedback to photoreceptors to produce visual contrast. They are depolarized by glutamate released from photoreceptors, leading to a constant influx of Ca2+ which would be fatal to most neurons. In addition, HCs present spontaneous Ca2+-based action potentials, which are poorly understood and whose function is unknown. Given these unique Ca2+ dynamics, the present thesis sought to define action potentials (APs) and mechanisms of Ca2+ homeostasis in HCs. APs were observed in isolated goldfish HCs with electrophysiology, Ca2+ imaging, and voltage-sensitive dye imaging. Pharmacological inhibition of ion channels suggests APs required extracellular Ca2+ entry via L-type Ca2+ channels, followed by Ca2+-induced Ca2+ release from ryanodine receptors. Next, we developed a novel system to classify all four HC subtypes in vitro, and validated it with immunocytochemistry for a subtype-specific biomarker. All subtypes presented APs, although frequency and duration varied by subtype. APs were also found in HCs of tissue slices prepared from whole retina, where similar trends were found between subtype, frequency, and duration. This highlights subtype-specific differences in Ca2+ dynamics. Lastly, [Ca2+]i was monitored throughout hypoxia in HCs of the hypoxia-tolerant goldfish and the hypoxia-sensitive rainbow trout. In Ca2+ imaging experiments, hypoxia destabilized [Ca2+]i in HCs of trout; but in goldfish, HCs were resistant to the effects of hypoxia. However, when mitochondrial ATP-dependent K+ (mKATP) channels were inhibited, goldfish HCs lost the ability to maintain [Ca2+]i homeostasis during hypoxia. By contrast, in trout HCs, opening of mKATP stabilized [Ca2+]i during hypoxia. Furthermore, in goldfish, hypoxia protected against increases in [Ca2+]i caused by inhibiting glycolysis, showing that hypoxia is not just tolerated, but is actively protective in goldfish HCs. The present thesis includes the first comprehensive description of spontaneous Ca2+-based APs in HCs, and introduces the first cellular model of intrinsic hypoxic neuroprotection in the vertebrate retina.

No description available.

Made available in DSpace on 2016-06-02T20:18:07Z (GMT). No. of bitstreams: 1 TeseACPF.pdf: 1993727 bytes, checksum: 42770c78b76a4d455bc87403cef8c453 (MD5) Previous issue date: 2006-06-02 / The aim of this study was to verify the effects of histamine on the process of functional recovery after hemilabyrinthectomy in goldfish, Carassius auratus. To this end, the fish were submitted to unilateral vestibular lesion and treated with a histamine precursor (L-histidine) and antagonists of the receptors H1, H2 and H3 (respectively chlorpheniramine, zolantidine and thioperamide). The success of the surgery was evidenced by the ataxia they suffered during approximately thirty minutes of observation and the presence of these signs was used as criterion for the inclusion of the animal in the experiment. The subjects were treated during fifteen consecutive days with L-histidine (150 mg/kg), chlorpheniramine (24 mg/kg), zolantidine (18 mg/kg) and thioperamide (15 mg/kg). All substances were administrated in a volume of 1.5 ml/ kg of body weight and sterile saline (1.5 ml/kg) was used with the experimental control group. Another group, the sham lesion group, was submitted to the same surgical procedure, but in this case the vestibular lesion was not carried out. The subjects were submitted to seven tests, the first test being carried out on the first day after the lesion, and then five more at three day intervals. The seventh test was carried out three days after the conclusion of treatment. In each test, the subject was placed inside a translucent tube inside a black aquarium which was illuminated on the ipsilateral side for five minutes. The fish behavior was recorded with a video camera located in front of a small opening in the front wall of the aquarium. Body tilt was measured by comparing a line traced between the subject s eyes with a horizontal line traced on the television screen. The mean of the tilt in degrees obtained every thirty seconds was considered. The data were subjected to variance analysis (Two-way ANOVA), followed by the Student Newman Keuls multiple comparisons test. / O objetivo deste estudo foi verificar os efeitos da histamina sobre o processo de recuperação funcional, após hemilabirintectomia em peixes da espécie Carassius auratus. Para tanto, foram utilizados a L-histidina, um precursor histaminérgico e antagonistas dos receptores H1, H2 e H3, sendo a clorfeniramina, antagonista H1, a zolantidina antagonista H2 e a tioperamida, antagonista do receptor histaminérgico H3. Os peixes foram anestesiados e submetidos à lesão vestibular unilateral. O sucesso da cirurgia foi evidenciado pela ataxia, caracterizada pelo rolamento do corpo e nado incoordenado durante aproximadamente trinta minutos, e a presença desses sinais foi utilizada como critério de inclusão do animal no experimento. Os sujeitos foram tratados durante quinze dias consecutivos com L-histidina (150 mg/kg), clorfeniramina (24 mg/kg), zolantidina (18 mg/kg) e tioperamida (15 mg/kg). Todas as substâncias foram administradas em um volume de 1.5 ml/kg, e a salina estéril (1.5 ml/kg) foi utilizada como controle experimental. Um outro grupo foi submetido ao procedimento cirúrgico, mas a lesão vestibular não foi realizada (Lesão Fictícia). Os sujeitos foram submetidos a sete testes. O primeiro teste foi realizado no primeiro dia após a lesão, e os demais, a cada três dias, totalizando seis testes. O sétimo teste foi realizado três dias após o término do tratamento. Cada teste consistiu em posicionar o sujeito dentro de um cilindro, no interior de um aquário revestido com uma película preta e iluminar esse aquário no lado ipsilateral à lesão durante cinco minutos. O comportamento animal foi registrado através de uma câmera de vídeo posicionada em frente a uma pequena abertura presente na parede anterior do aquário. O ângulo de inclinação corporal foi formado por uma linha correspondente ao posicionamento dos olhos e uma linha horizontal e medido com um transferidor na tela do televisor. A média desses ângulos obtidos a cada trinta segundos foi considerada. Os dados foram analisados através da Análise de Variância (ANOVA) de duas vias, seguida do teste de comparações múltiplas de Student Newman Keuls.

Caffeine is one of the most popular psychostimulant drugs worldwide. Its effects are exerted through a variety of complex mechanisms, apparently primarily via interactions with adenosine A1 and A2A receptors. This drug has also been proven to elicit neuroprotective responses in a number of different brain disorders of the Central Nervous System (CNS), as well as provide enhancement of cognitive abilities. Moreover, a biphasic set of functional and structural neurological changes are often found in these receptors among diverse vertebrates. I investigated the effects of chronic caffeine exposure on functional recovery of the dorsal light reflex (DLR) in hemilabyrinthectomized common goldfish, Carassius auratus. In this lesion model the unilateral removal of vestibular organs results in the temporary loss of gravitational regulated postural control, which over time corrects itself by a vestibular compensation (VC) mechanism and can be quantified via the DLR. We compared the functional recovery over 24 post -surgery days in goldfish continuously held in a caffeine solution of 2.5mg/L (n=10), 5.0mg/L (n=10), 10.0mg/L (n=11) or a control 0.0mg/L (n=9). Compared to a sham surgery group (n=11), statistically significant changes in the DLR of all hemilabyrinthectomized goldfish was observed on day 1. The control group recovered over the study period by approaching but not entirely reaching sham surgery DLR. The 2.5mg/L and 5.0mg/L groups initiated postural recovery similar to the controls, but then regressed to a stronger DLR. Beginning on day 10 the caffeine groups deviated from the control and all three experimental caffeine groups were statistically different from the control group on days 15-24. Results suggest early caffeine exposure may be innocuous; however, chronic exposure inhibits the functional recovery process.

This dissertation examines theatre and film in Ireland between 1988 and 2005, focusing on the plays of Sebastian Barry and Marina Carr, as well as a select group of films from this period. Employing a method of analysis that couples close-readings with attention to socio-cultural context, aesthetic form, and issues of representation, the dissertation demonstrates how theatre and film work to complicate conventional Irish historical narratives and thereby encourages a reassessment of contemporary constructs of Irish identity. The introduction provides a contextual framework for significant contemporaneous social, cultural and economic changes in Ireland, and includes a case study of ‘The Spire,’ a monument unveiled on Dublin’s central boulevard in 2003, which I argue is the architectural metonym for the transitional nature of Celtic Tiger Ireland. The case study explores the aesthetics of the monument, as well as the politicised public debate that ensued, and thereby provides a snapshot of issues relevant to the readings pursued in dissertation’s remaining chapters. The discussion of Sebastian Barry’s ‘family plays’ reveals the playwright’s effort to refuse traditional binary conceptions of identity and to proffer, instead, a dramatic landscape that similarly refuses to allow conflict to dominate. Barry’s use of a non-conflictual dramatic form supports his narrative interest in compassion and peaceful resolution, and provides a model for living with otherness that could prove useful in an increasingly diverse and globalised Ireland. Marina Carr’s plays share Barry’s desire to represent aspects of Irish character anew, but they also dramatise how cultural transitions are difficult and never linear, and how the conventional pull of memory and the past has a residual presence in the ‘new’ Ireland. Taken together, these chapters reveal Barry’s hopefulness as an antidote to Carr’s tragic endings. The final chapter provides close readings of several ‘Celtic Tiger’ films, arguing that the representation of landscape is the key lens through which Irish film communicates shifting images of Irish identity. A cycle of films from the first years of the new millennium ekes out a space for new modes of representation through a critical dialogue with major tropes in Irish film history.

This dissertation examines theatre and film in Ireland between 1988 and 2005, focusing on the plays of Sebastian Barry and Marina Carr, as well as a select group of films from this period. Employing a method of analysis that couples close-readings with attention to socio-cultural context, aesthetic form, and issues of representation, the dissertation demonstrates how theatre and film work to complicate conventional Irish historical narratives and thereby encourages a reassessment of contemporary constructs of Irish identity. The introduction provides a contextual framework for significant contemporaneous social, cultural and economic changes in Ireland, and includes a case study of ‘The Spire,’ a monument unveiled on Dublin’s central boulevard in 2003, which I argue is the architectural metonym for the transitional nature of Celtic Tiger Ireland. The case study explores the aesthetics of the monument, as well as the politicised public debate that ensued, and thereby provides a snapshot of issues relevant to the readings pursued in dissertation’s remaining chapters. The discussion of Sebastian Barry’s ‘family plays’ reveals the playwright’s effort to refuse traditional binary conceptions of identity and to proffer, instead, a dramatic landscape that similarly refuses to allow conflict to dominate. Barry’s use of a non-conflictual dramatic form supports his narrative interest in compassion and peaceful resolution, and provides a model for living with otherness that could prove useful in an increasingly diverse and globalised Ireland. Marina Carr’s plays share Barry’s desire to represent aspects of Irish character anew, but they also dramatise how cultural transitions are difficult and never linear, and how the conventional pull of memory and the past has a residual presence in the ‘new’ Ireland. Taken together, these chapters reveal Barry’s hopefulness as an antidote to Carr’s tragic endings. The final chapter provides close readings of several ‘Celtic Tiger’ films, arguing that the representation of landscape is the key lens through which Irish film communicates shifting images of Irish identity. A cycle of films from the first years of the new millennium ekes out a space for new modes of representation through a critical dialogue with major tropes in Irish film history.