Human spinal cord injury (SCI) results in persistent functional deficits as damaged axons in the mature central nervous system (CNS) fail to regenerate after injury. This is due to both growth-inhibiting compounds, e.g., the myelin-associated growth inhibitors and the chondroitin sulfate proteoglycans (CSPGs), in the extracellular environment, and growth-limiting intrinsic factors. Unlike mammals, the primitive sea lamprey robustly recovers swimming and other locomotor behaviors after complete spinal cord transection (TX), despite the presence of homologues of the mammalian growth-inhibiting molecules. This recovery is accompanied by heterogeneous anatomical regeneration of the reticulospinal (RS) system, which, in lampreys, is the dominant descending pathway for motor control. Within the RS system, there are 18 pairs of identifiable neurons that can be classified as “good” or “bad” regenerators based on the likelihood that their axons will regenerate beyond the TX site. Most bad regenerators undergo a delayed form of caspase-mediated cell death. Because both good and bad regenerators project through the same extracellular environment, investigating their divergent responses to axotomy has the potential to reveal the key intrinsic properties that regulate axon regeneration. And, since lampreys share much of the same CNS organization and signaling pathways with higher order mammals, regeneration mechanisms discovered in lampreys may be useful therapeutic targets in humans with SCI. Lampreys do not express myelin, so the CSPGs probably form the principal extracellular inhibitory component of the injured spinal cord. Mammalian in vitro and in vivo studies suggest that CSPGs bind the LAR-family receptor protein tyrosine phosphatases (RPTPs), PTPσ and LAR, leading to growth inhibiting cytoskeletal remodeling and reduced activity of pro-survival pathways via the small GTPAse, RhoA. Intriguingly, preliminary in situ hybridization experiments with antisense riboprobes revealed that PTPσ is preferentially expressed on bad regenerator neurons. Thus, we hypothesized that differential PTPσ expression may be a key signaling determinant of regeneration. Using antisense morpholino oligomers (MOs) applied to the proximal spinal cord stump immediately after TX, we inhibited PTPσ expression among lamprey RS neurons and assessed its effects on regeneration. Contrary to our hypothesis, PTPσ deletion did not promote supraspinal regeneration or enhance behavioral recovery. Most surprisingly, we observed reduced survival of RS neurons at long timepoints post-TX among the PTPσ knockdown cohort. Western blot analysis, using pan-LAR-family receptor antibodies, indicated that the PTPσ knockdown did not affect expression of other LAR-family receptors. Although these results are the opposite of what we expected, there are several potential biological explanations that may explain why the loss of PTPσ antagonizes survival. Notably, these include interactions with the pro-regenerative PTPσ ligands, heparin sulfate proteoglycans (HSPGS), exacerbation of inflammatory processes, reduced synaptogenesis leading to loss of trophic support, and potentially off-target toxicity. These explanations remain under investigation. Notably, pilot studies involving HSPG digestion using bacterial heperainase III did not recapitulate the knockdown phenotype. Following the surprising results of PTPσ knockdown, we stepped back and considered whether simpler factors between good and bad regenerators may contribute to their divergent response to axotomy. We had long noted that bad regenerators tended to be larger than good regenerators, but generally believed this was an epiphenomenon unrelated to axon regeneration. However, a careful reexamination of primary and historic data uncovered an even stronger inverse correlation between soma cross-sectional area and regenerative ability (r = -0.92) than we had suspected. Using a similar approach, we determined that RS neuron soma size is proportional to axon caliber. Because large axons may reseal more slowly following axotomy than smaller axons, we hypothesized that inefficient axolemmal resealing after axotomy may be a key driver of the degenerative processes observed among bad regenerators. Using dye exclusion assays with 10,000 MW fluorescent dextran tracers, we assessed the rate of axolemma resealing for each of the identifiable neurons. Within 2 hours of TX, 75% of axons from small to medium sized neurons (≤ 20 x102 µm2; B5, I3, I5, mth’, M4, B6, I4, I6, M1, B2, I2) were impermeable to dye compared to only 5% of axons from the larger bad regenerator RS neurons (B1, M3, M2, B4, Mth, B3, I1). Indeed, many of these large bad regenerators remained permeable to dextran dye for more than 24 hours after injury. Importantly, approximately 65% of neurons with axons that remained dye permeable at 24 hours post-TX were positive for active caspases at +2 weeks, compared to only 10% of neurons with sealed axons (p<0.0001***). When axon resealing was artificially induced with the fusogen, polyethylene glycol (PEG), caspase activation was inhibited, suggesting that slow axolemma causatively promotes degeneration among lamprey RS neurons. Although this study did not investigate the underlying mechanisms, we suspect that prolonged influx of toxic mediators in the extracellular environment, particularly calcium, may drive the degenerative response. Together, these results demonstrate that axon regeneration and cell survival after spinal cord TX is a complex process strongly shaped by the intrinsic characteristics of the neurons themselves. Selective expression of putative inhibitory or pro-growth molecules may regulate the regeneration process in ways that can be difficult to predict a priori and with effects that vary among taxa. Because lampreys are one of the few vertebrates to recover after complete SCI, they remain an essential model organism to study true axon regeneration in the CNS. / Neuroscience

Pacific lamprey (Entosphenus tridentatus) is an endemic species to coastal watersheds along the Pacific Rim in North America and Asia. The species' geographic distribution is retracting and has not been mapped precisely, and the ability for the species to provide ecosystem services, such as water filtration, for supporting watershed management has not been tested. The San Luis Obispo Creek watershed, California, USA is at the southern end of Pacific lamprey’s retracting distribution, and the San Luis Obispo watershed is impacted by degraded water quality, making investigation of Pacific lamprey in the San Luis Obispo Creek watershed of direct local conservation importance. This thesis is presented as two manuscripts formatted for submission to scientific journals. The first manuscript features a study that estimates Pacific lamprey presence/absence throughout the San Luis Obispo Creek watershed using Environmental DNA. We compare our estimate to previous estimates of Pacific lamprey population distribution using established eFishing and redd survey methods. We found our estimate of distribution based on Environmental DNA to match the previous estimates throughout San Luis Obispo Creek, as well as indicate a location with Pacific lamprey presence that was previously unsurveyed. In this manuscript, we also discuss advantages and disadvantages to using Environmental DNA as a cost-effective and non-invasive method to survey Pacific lamprey in the wild. The second manuscript is a laboratory study focused on the larval (ammocoete) stage of Pacific lamprey. We tested the ability for an ammocoete population to reduce the concentration of harmful bacteria in water through filter-feeding. We found an increased decline of bacteria in experimental aquaria treated with ammocoetes than in control tanks or tanks containing non-filter feeding Pacific lamprey, indicating that ammocoetes can indeed significantly reduce bacterial loads in a contained environment. The laboratory study was conducted using water from San Luis Obispo Creek that was naturally inoculated with bacteria, generating broader applicability of the study in suggesting that Pacific lamprey have the potential to provide a water filtration ecosystem service in the wild for supporting high water quality watershed management objectives. These two studies contribute to knowledge on how to efficiently survey Pacific lamprey distribution in the wild and benefits the species may provide to ecosystems and human welfare. Collectively, they help us better understand the status and value of this often-forsaken anadromous species.

Intestinal HCO3- secretion and the excretion of resultant CaCO3 precipitates have become a recognized characteristic of seawater osmoregulation in teleosts; however, this is the first report of this osmoregulatory strategy outside of teleosts and also includes evidence for its use in green turtles, Chelonia mydas. Furthermore, the effects of feeding on intestinal HCO3- secretion were newly investigated in teleosts. Intestinal base secretion via apical Cl-/HCO3- exchange was found to increase following feeding, at a magnitude sufficient to offset the "alkaline tide" commonly associated with digestion. Intestinal HCO3- secretion in marine teleosts draws HCO3- from both endogenous (via hydration of intracellular CO2) and serosal (blood) sources, of which serosal HCO3- was found to contribute a greater proportion to the elevated postprandial intestinal base secretion measured in gulf toadfish, Opsanus beta. The mechanism by which this serosal HCO3- crosses the basolateral membrane for subsequent secretion into the intestinal lumen was confirmed in toadfish to be a basolateral Na+/HCO3- co-transporter, tfNBCe1. Furthermore, the isolated intestinal tissue was found to have a high metabolic rate in both control and postprandial toadfish, with respect to that of the whole animal, and shows a considerable specific dynamic action (SDA) response to feeding. Overall, this dissertation provides evidence for the widespread use of intestinal HCO3- secretion as a strategy of marine osmoregulation across aquatic taxa, and also for its newly recognized involvement in postprandial acid-base balance.

Synucleins represent a conserved family of small proteins that include α-, β-, and γ- isoforms, which are highly expressed in neurons of the vertebrate nervous system. The normal function of these proteins is not well understood. However, in humans α- synuclein dysfunction is causatively linked to Parkinson’s Disease (PD), where it abnormally accumulates in neuronal cell bodies as protein aggregates that are associated with neuronal death. Although the associations between synuclein accumulation and cellular death are established in PD, the extent to which this occurs in other contexts, such as neuronal injury, is unknown. Furthermore, the effects of synuclein aggregation on the function of synapses, where synuclein is normally localized, are not well understood. To address these questions I took advantage of the experimentally accessible nervous system of the sea lamprey (Petromyzon marinus). I used molecular cloning and phylogenetic analyses to characterize three lamprey synuclein orthologues, one of which is highly expressed within a class of neurons called the giant reticulospinal (RS) neurons. Spinal cord injury induces the accumulation of synuclein protein only within a population of poor surviving RS neurons, and this accumulation is correlated with cellular death. Thus, similar to PD, the abundance of synuclein protein is associated with neuronal toxicity. In a related project, I demonstrated that elevating synuclein levels at synapses, such as occurs in PD, is deleterious to synaptic function through an inhibition of synaptic vesicle (SV) recycling. By injecting excess synuclein protein directly into the axons of giant RS neurons, and analyzing the ultrastructural morphology of synapses, I have shown that clathrin-mediated synaptic vesicle endocytosis was greatly inhibited. The conserved N-terminal domain was sufficient to inhibit vesicle recycling, and injecting synuclein mutants with disrupted N-terminal α-helices caused reduced defects in SV recycling. Therefore the α-helical structure of the N-terminus is necessary to inhibit SV recycling at early stages of clathrin-mediated endocytosis. Binding interactions with clathrin-mediated endocytosis components, such as the phosphoinositide lipid PI(4)P support this hypothesis. These studies provide a better understanding of the mechanisms by which synuclein dysfunction leads to neuronal death after injury and synaptic dysfunction in PD and other synuclein-associated diseases. / text

Calcified teeth comprise the hardest vertebrate tissues (enamel and dentine) and they undoubtedly represent a key evolutionary advantage for their users. Nevertheless, teeth have been lost many times and they were often replaced by keratinised structures, such as rhamphoteca of birds or of turtle beaks, or labial tooth structures and scratching laminae of many aquatic vertebrates. This thesis is intended to analyse levels of similarities among keratinised teeth and structures of three phylogenetically distinct lineages of amniotes and to compare them to morphogenesis of true teeth. Detailed developmental and structural characteristics of oral apparatus possessing keratinous teeth of lampreys, Loricarid cathfishes and frog tadpoles were identified and distinguished. Keratinous structures of cathfishes include only horny projections from single cell surface (unculi), whereas in frog tadpoles both horny jaw sheet and unicellular labial teeth composed by cell columns were found; the lamprey oral apparatus consists of multicellular teeth and laminae instead. Disparate phenotypes of these keratinous structures can, however, developmentally derive from a general process of keratinization: morphological disparities arise by distinct gradual accretion of keratinous layers and collumns during development. My...

La détection de molécules chimiques par l'odorat est importante pour guider le comportement des animaux. Chez la lamproie marine, Petromyzon marinus, l'olfaction est vitale pour plusieurs fonctions telles que l’alimentation, l’évitement des prédateurs et la reproduction. Les différents comportements olfactifs de la lamproie sont les mieux caractérisés parmi tous les vertébrés aquatiques et ils font l’objet du premier chapitre de l’introduction. Les circuits du cerveau responsables des mouvements produits lors de la détection de stimuli olfactifs ont été examinés chez la lamproie. Des études récentes révèlent qu’il existe deux organes olfactifs périphériques ayant des projections parallèles qui innervent des parties distinctes du bulbe olfactif (BO). Dans les deux cas, le signal olfactif atteint éventuellement les cellules réticulospinales (RS), qui activent les réseaux locomoteurs spinaux. La littérature portant sur ces circuits neuronaux est décrite dans le deuxième chapitre introductif. Le substrat neuronal par lequel le signal olfactif est transmis aux cellules RS n'est pas complètement caractérisé mais des données du laboratoire Dubuc suggèrent que le tubercule postérieur (TP) serait une cible importante des projections du BO. Puisque cette région contient des neurones dopaminergiques (DA) impliqués dans le contrôle moteur, l’objectif principal de cette thèse était de déterminer leur rôle dans le traitement du signal olfactif et la production de locomotion. Nos résultats ont permis de caractériser l'innervation DA du BO de la lamproie et d’observer que les neurones DA du TP projettent à la partie médiane du BO chez les animaux de stade larvaire et adulte. De plus, l’activation de récepteurs D2 dans cette région diminue la transmission du signal olfactif aux cellules RS. Dans le reste du BO, des neurones DA apparaissent au stade adulte. Ces observations sont rapportées dans le premier chapitre des résultats. Puisque les neurones DA du TP peuvent moduler la transmission olfactomotrice au niveau du BO, ils pourraient aussi jouer un rôle via leurs projections connues vers le tronc cérébral. Le deuxième chapitre des résultats se penche donc sur l’implication du TP dans le relai de l’information olfactive au système moteur. L’étude des projections du BO montre que les neurones DA sont ciblés, incluant ceux qui projettent à la région locomotrice mésencéphalique (RLM), responsable de l’initiation et du contrôle de la locomotion. Aussi, la stimulation olfactive active des neurones du TP qui projettent à la RLM. Dans une préparation dont la tête est fixée mais le corps peut se déplacer, la stimulation olfactive induit de la nage en recrutant simultanément le TP et les cellules RS. Nous montrons aussi que le TP est recruté durant la nage survenant spontanément, ce qui indique que cette région joue un rôle important dans le contrôle locomoteur. Cette thèse révèle que les neurones DA du TP peuvent 1) être activés par la détection d’odeurs et ensuite 2) moduler la transmission au niveau du BO ainsi que 3) recruter la RLM pour produire un épisode de nage. Ces données suggèrent qu’ils occupent une position-clé dans l’intégration sensorimotrice des stimuli olfactifs puisqu’ils encodent à la fois de l’information sensorielle et motrice. / The detection of chemical molecules by smell is important in guiding the behavior of animals. In the sea lamprey, Petromyzon marinus, olfaction is vital for several functions such as feeding, predator avoidance and reproduction. The various olfactory behaviors of the lamprey are the best characterized among all aquatic vertebrates and they were reviewed in the first chapter of the introduction. The brain circuitry responsible for producing movement upon sensing olfactory stimuli has been examined in lamprey. Recent studies revealed that there are two peripheral olfactory epithelia with parallel projections that reach distinct parts of the olfactory bulb (OB). In both cases, the olfactory signal eventually reaches reticulospinal (RS) cells, which activate the locomotor networks of the spinal cord. The literature describing these neural circuits is thoroughly reviewed in the second chapter of the introduction. The neuronal substrate by which the olfactory signal is transmitted to RS cells is not fully characterized, but data from the Dubuc laboratory suggest that the posterior tubercle (PT) may be an important target for OB projections. Since this region contains dopaminergic (DA) neurons involved in motor control, the main objective of this thesis was to determine their role in olfactory signal processing and the production of locomotion. Our results have allowed to characterize the DA innervation of the lamprey OB and show that DA neurons of the PT send projections to the medial part of the OB in larval and adult animals. In addition, the activation of D2 receptors in this region decreases the transmission of the olfactory signal to RS cells. In the rest of the OB, DA neurons appear in adult animals. These observations are reported in the first chapter of the Results. Since DA neurons of the PT can modulate olfactory-motor transmission at the level of the OB, they could also play a role through existing descending projections to the brainstem. Thus, in the second chapter of the Results, we studied the involvement of the PT in the relay of olfactory information to the motor system. The analysis of OB projections shows that DA neurons are targeted, including those that project to the mesencephalic locomotor region (MLR), which is responsible for initiating and controlling locomotion. Moreover, olfactory stimulation activates PT neurons that project to the MLR. In a head-fixed preparation in which the body moves, olfactory stimulation induces swimming simultaneously with PT and RS cell activity. We also show that the PT is recruited during spontaneously occurring swimming, which indicates that this region plays an important role in locomotor control. This thesis reveals that DA neurons in the PT can 1) be activated following odorant detection and then 2) modulate the transmission at the level of the OB as well as 3) recruit the MLR to produce a swimming episode. These data suggest that they occupy a key position in the sensorimotor integration of olfactory stimuli since they encode both sensory and motor information.

Lampreys are primitive jawless fishes that diverged over 550 million years ago. As adults, they are either parasitic or non-parasitic. In non-parasitic species, sexual differentiation and oocyte development generally occur earlier than in parasitic species; fecundity is reduced and sexual maturation is accelerated following metamorphosis. The genes controlling ovarian differentiation and maturation in lampreys are poorly understood. This study used RNA-Seq data in the parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey Ichthyomyzon fossor to identify suites of genes expressed during different stages of ovarian development that show different developmental trajectories with respect to ovarian differentiation and sexual maturation. For this, reference-guided and de novo assembly pipelines were designed for studying a non-model species. To test and explore the relative advantages of the pipelines, expression of insulin superfamily genes was used. This research helps to identify genes involved in lamprey ovarian development and provides insight into evolution of the insulin superfamily in vertebrates. / May 2017

The aim of this thesis is to evaluate the impact of agricultural activity on hydrochemical parameters of surface waters in connection with the occurence of specially protected species within the surface waters of a monitored area. A model was chosen for the evaluation of the situation - Bedřichovský potok, which is located in Novohradské hory. The river consists of lower and upper sub-waters. Forest management is applied within upper sub-basin, while agricultural management on arable land, meadows and pastures is used in the lower sub-basin. The monitored parameters were indicators of eutrophication of surface waters: Nitrate nitrogen (N-NO3-), Phosphate phosphorus (P-PO43-), and conductivity of undissolved material (NL105). The results showed that the agriculture management (especially on arable land) burdens the soil with nutrients and consequently erosion enters the substances, bringing them to the surface and ground water as a result. There was an increase of substances in the water after rain.

Les mécanismes neuronaux contrôlant la respiration sont présentement explorés à l’aide de plusieurs modèles animaux incluant le rat et la grenouille. Nous avons utilisé la lamproie comme modèle animal nous permettant de caractériser les réseaux de neurones du tronc cérébral qui génèrent et modulent le rythme respiratoire. Nous avons d’abord caractérisé une nouvelle population de neurones, dans le groupe respiratoire paratrigéminal (pTRG), une région du tronc cérébral essentielle à la genèse du rythme respiratoire chez la lamproie. Les neurones de cette région sont actifs en phase avec le rythme respiratoire. Nous avons montré que ces neurones possèdent une arborisation axonale complexe, incluant des projections bilatérales vers les groupes de motoneurones du tronc cérébral qui activent les branchies ainsi que des connexions reliant les pTRG de chaque côté du tronc cérébral. Ces résultats montrent que le pTRG contient un groupe de cellules qui active les motoneurones respiratoires des deux côtés et qui pourrait être impliqué dans la synchronisation bilatérale du rythme respiratoire. Nous avons ensuite étudié les mécanismes neuronaux par lesquels le rythme respiratoire est augmenté en lien avec l’effort physique. Nous avons montré que la région locomotrice du mésencéphale (MLR), en plus de son rôle dans la locomotion, active les centres respiratoires pendant la nage, et même en anticipation. Les neurones de la MLR projetant vers les centres locomoteurs et respiratoires sont ségrégés anatomiquement, les neurones localisés plus dorsalement étant ceux qui possèdent des projections vers les centres respiratoires. Nous avons aboli la contribution de la partie dorsale de la MLR aux changements respiratoires en injectant des bloqueurs des récepteurs glutamatergiques localement, sur des préparations semi-intactes. Nous avons montré que lors d’épisodes de nage, une majeure partie de l’effet respiratoire est abolie par ces injections, suggérant un rôle prépondérant des neurones de cette région dans l’augmentation respiratoire pendant la locomotion. Nos résultats confirment que le rythme respiratoire est généré par une région rostrolatérale du pons de la lamproie et montrent que des connexions des centres locomoteurs arrivent directement à cette région et pourraient être impliquées dans l’augmentation respiratoire reliée à l’effort physique. / The neural control of breathing is currently investigated on multiple animal models such as frogs and rats. We have used the lamprey as an experimental model to characterize the brainstem neural networks involved in the genesis and modulation of the respiratory rhythm. We have first characterized a new population of respiratory neurons in the paratrigeminal respiratory group (pTRG). The pTRG is a region that was shown to be essential to respiratory rhythmogenesis in lampreys. We have shown that the pTRG contains a group of neurons with complex axonal arborisations, including bilateral projections to the motoneuron pools of the brainstem that activate gills, as well as bilateral projections connecting the pTRGs on the two sides of the brainstem. These results suggest that pTRG neurons could participate in the descending control of respiratory motoneurons as well as the bilateral synchrony of the respiratory rhythm. We have then studied the neural mechanisms by which respiration is increased during locomotion. We have shown that the mesencephalic locomotor region (MLR), in addition to its role in controlling locomotion, also increases breathing during locomotion. Neurons in the MLR are anatomically segregated, those projecting to the respiratory centers being located more dorsally. We have abolished the contribution of the dorsal part of the MLR to respiratory changes by injecting glutamate receptor blockers locally in semi-intact preparations. We have shown that during swimming episodes, a major part of the respiratory effect is dependent on the dorsal part of the MLR. Our results confirm that the respiratory rhythm is generated by a rostrolateral region in the pons of lampreys and show that connections from locomotor centers can directly activate this region. These connections could be implicated in the increase of breathing activity related to locomotion.

Pacific lamprey (Entosphenus tridentatus) are experiencing population declines throughout their range. Xenobiotics could be an important risk factor for lamprey populations. Our goal was to establish if common herbicides, as used in forest management, could affect reproductive fitness. We determined that atrazine was a likely compound of greatest concern to lamprey populations. Using an odorant response behavioral assay we were able to demonstrate that environmentally relevant concentrations of atrazine caused a depressed response to adult lamprey holding tank effluent, likely pheromones. Atrazine also depressed their activity level; the number of times they crossed into the effluent arm after being treated with atrazine was significantly lower than controls. In addition, activity level post exposure to atrazine differed between adult life history stages, something which was not significantly different during control trials. Using an odorant detection assay, based on evaluating ventilation rate, we were able to show that environmentally relevant concentrations of atrazine caused a significant increase in ventilatory response to a repulsive odorant, a conspecific necromone. Through the detection study we also showed that lamprey,exposed to atrazine, had a slight increase in ventilatory response to odor from adult lamprey. If we are concerned about the decline in Pacific lamprey populations, then we should logically be concerned with their exposure to atrazine in the environment. / Graduation date: 2012