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Dorsal raphe nucleus (DRN) neurons projects to widespread areas throughout the brain and are involved in many physiological functions and neuropsychiatric disorders. In particular, DRN serotonin (5-HT) neurons are thought to be implicated in major depressive disorder (MDD) as are steroid hormones. Therefore, the aim of this thesis was to assess the effects of some neurosteroids on DRN neurons in non-anaesthetized rats. Initially, we examined electrophysiological properties of dorsal raphe cells across the sleep---wake cycle in non-anaethetized rats. In this first study we characterized six distinct neuronal populations in the DRN based on spike waveform and firing pattern. We then examined the effects of DHEA-S and testosterone (T) on the firing properties of DRN neuronal populations previously characterized. We observed that most populations exhibited an initial decrease in firing activity following one week of treatment. However, there was a great variability in responses across the populations.

Breathing is an essential homeostatic function and its disruption leads to disability, brain damage, and death. Serotonin (5-hydroxytryptamine; 5-HT) neurons in the brainstem play an important role in control of breathing. Medullary 5-HT neurons are stimulated by increased CO₂ and subsequently stimulate respiratory nuclei to increase ventilation and maintain normal blood gas levels. Anesthetic-induced breathing dysfunction is a serious concern in healthcare settings. In research settings, experiments are often performed under anesthesia, and therefore it is important to understand how these drugs affect animal physiology. Unfortunately, little is known about how anesthetics modulate 5-HT neurons, breathing, and CO₂ chemoreception in mice, as many of the previous studies have been performed in different species. Characterizing how anesthetics commonly used in both research and clinical settings affect 5-HT neurons, breathing and CO₂ chemoreception is valuable to the broader field of neuroscience since these drugs are so ubiquitously used in research. Breathing dysfunction and defects in the serotonergic system have been implicated in disorders, such as sudden unexpected death in epilepsy (SUDEP) and sudden infant death syndrome (SIDS), which means better characterizing the role of 5-HT neurons in breathing has translational impact as well. Here I examine whether halogenated inhalational anesthetics, which potentiate TWIK-related acid-sensitive K⁺ (TASK) currents and GABAA receptors, could mask an effect of CO₂ on 5-HT neurons. During in vivo plethysmography in mice, a therapeutic level of isoflurane (1%) markedly reduced the hypercapnic ventilatory response (HCVR) in all mouse strains tested. In dissociated cell cultures, isoflurane (1%) hyperpolarized 5-HT neurons and inhibited spontaneous firing. A subsequent decrease in pH from 7.4 to 7.2 depolarized 5-HT neurons, but that was insufficient to reach threshold for firing. Depolarizing current restored baseline firing and the firing frequency response to acidosis, indicating that isoflurane did not block the underlying mechanisms mediating chemosensitivity. These results demonstrate that isoflurane masks 5-HT neuron chemosensitivity in vitro, and markedly decreases the HCVR in vivo. Next, I demonstrate that ketamine-xylazine or urethane anesthesia also significantly reduced the HCVR in mice at both therapeutic and sub-therapeutic doses. However, mice treated with a sub-therapeutic dose of anesthesia decreased their O₂ consumption in parallel, and thus matched their ventilation to metabolic demands. Mice that were anesthetized with the therapeutic dose did not sufficiently match their breathing and metabolic demands, and thus anesthesia induced hypoventilation. Recordings from 5-HT neurons in culture indicated that neither ketamine nor urethane affected 5-HT neuron chemosensitivity. These data demonstrate that anesthetics with different molecular targets similarly reduce the HCVR in mice, but not all of their effects are mediated via 5-HT neurons. Moreover, both ketamine-xylazine and urethane anesthesia altered baseline breathing in different ways, suggesting they targeted different parts of the respiratory network. Finally I show that isoflurane anesthesia in neonatal mice caused depression of resting ventilation, which was different from isoflurane-anesthetized adults. This effect was more pronounced in wildtype mice compared to littermates with genetic deletion of 5-HT neurons. Isoflurane-induced breathing depression decreased and mice fully recovered following washout of isoflurane at P8. I observed that genetic deletion of 5-HT neurons in mice with a congenic C57Bl/6 background led to a more severe phenotype than previously described in mixed genetic background strains. These mice had decreased survival, severe growth retardation, and reduced baseline ventilation. These results indicate that 5-HT neurons have a different role during the neonatal period and that some mouse strains are more sensitive to genetic deletion of 5-HT neurons; thus, background genetics play an important role in phenotype presentation. In summary, different classes of anesthetics each strongly depress chemoreception. Isoflurane seems to affect breathing, in part, by hyperpolarizing 5-HT neurons and masking their chemosensitivity, whereas ketamine and urethane have less effect on 5-HT neurons. However, both ketamine-xylazine and urethane anesthesia alter baseline breathing. Isoflurane anesthesia decreases baseline ventilation in neonates, but this effect is absent in adults, which suggests that the effects of isoflurane on breathing changes as mice age. These data are important for the field of respiratory physiology because they highlight the sensitivity of breathing to the effects of anesthetics. These results are valuable to the broader field of neuroscience, because anesthetics are widely used during in vivo research. Additionally, some transgenic mouse strains are more sensitive to 5-HT neuron deletion depending on their genetic background. In the future it will be critical to characterize the molecular mechanisms that underlie these phenomena.

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The class Bacillariophyceae, encompasses all diatoms that possess a raphe. Members of the orders Bacillariales, Rhopalodiales and Surirellales have a canal raphe system. Historically, the possession of this character was considered support for a close evolutionary relationship. To investigate phylogenetic relationships, a three gene dataset was constructed for 49 strains representing 9 of the 11 orders within the Bacillariophyceae. We provide the first formal tests of homology of the canal raphe system and the first demonstration that the Rhopalodiales are nested within the Surirellales. Results strongly reject the monophyly of Bacillariales, Rhopalodiales, and Surirellales thereby discounting the canal raphe as a homologous character. The Surirellales include three families: Surirellaceae, Entomoneideaceae, and Auriculaceae, while Rhopalodiales has only three genera in one family, the Rhopalodiaceae. In order to test familial and generic concepts, I expanded taxon representation and collected DNA sequence data for 125 strains. Taken together, only 5 of the 12 genera (Entomoneis, Stenopterobia, Cymatopleura, Petrodictyon and Epithemia) were found to be monophyletic. Our current concepts of the two most species-rich genera, Surirella and Campylodiscus, are too broad as the analyses resolved taxa from these two genera into multiple independent lineages. The “Robustoid” lineage, comprised of Surirella Robustae, Campylodiscus Robusti, and Stenopterobia, exhibits a high degree of endemism within ancient Lake Ohrid, with 17 species considered endemic or relict taxa. A dataset of three molecular markers and 71 Robustoid taxa from Lake Ohrid, Europe, and North America was used to construct a preliminary phylogeny. The aim was to investigate phylogenetic relationships and test hypotheses of speciation and morphological evolution. The recovered paraphyly of Lake Ohrid taxa eliminates the possibility that the Ohrid Robustoids as a whole are a product of intralacustrine speciation. However, sister relationships between putative Ohrid endemics and unexplored morphological diversity within some species complexes (e.g. Campylodiscus marginatus, Scoliodiscus spp.) leave open the possibility that these lineages may be the result of intralacustrine speciation. / text

Stroke is the third leading cause of death and the primary cause of adult long-term disability in Canada. Despite advances in rehabilitation research, stroke survivors experience an unusually high incidence of depressive symptoms which undermine recovery outcomes by reducing patient motivation levels. Human and animal studies have linked the incidence of post-stroke depression and the extent of prefrontal cortex (PFC) damage. The PFC and the lateral habenula (LHb) are limbic structures that are strongly connected to the serotonergic dorsal raphe nucleus (DRN), a key neuronal hub for mood regulation. We hypothesized that PFC stroke produces a depressive phenotype by triggering maladaptive reorganization in mood-related networks. We used viral and optogenetic strategies to functionally characterize PFC and LHb projections to DRN. Moreover, we found that PFC stroke causes a time-dependent remodeling of LHb inputs to DRN 5-HT neurons which results in altered postsynaptic glutamate receptor number and subunit composition. This remodeling likely reflects a homeostatic upregulation of LHb-DRN synapses in response to stroke-induced challenge to network activity. Since these synapses encode stress and aversion, potentiation of this pathway could contribute to depressive symptoms following stroke. However, more work will be needed to identify the behavioral and network-level consequences of altered LHb-DRN dynamics. Thus, a deeper understanding of circuit mechanisms implicated in post-stroke depression will provide insights into this disease and open new treatment avenues to improve recovery.

Alzheimer´s disease (AD) is an age-related, irreversible and progressive neurodegenerative pathology that deteriorates cognitive function including learning and memory. AD is characterised neuropathologically by the presence of neuritic plaques (Aβ), neurofibrillary tangles (NFTs), synaptic loss and neuronal death. AD affects specific brain regions involved in mnestic function such as the neocortex and the hippocampus. The dorsal (DR) and the median raphe (MR) nuclei give rise to serotonergic (5-HT) projections that innervate multiple brain regions including the cortex and the hippocampus, playing an important role in learning and memory processes. For a long time the degeneration of cholinergic (ACh) system was considered as the main neurochemical changes in AD brains, however, more recent studies highlight the involvement of other neurotransmitter systems including 5-HT. This thesis entitled “Altered serotonergic neurotransmission as a main player in the pathophysiology of Alzheimer’s disease: structural and ultrastructural studies in a triple transgenic mouse model of the disease” demonstrates that there exist specific alterations in the serotonergic projections of the hippocampus during the progression of AD using the triple transgenic (3xTg-AD) mouse model of the disease, which closely resemble human AD. Mr. Harun N. Noristani is submitting this thesis to the University of Manchester for the degree of PhD in the Faculty of Life Science. The results obtained in this thesis show for the first time a biphasic increase in serotonergic fibre sprouting in the 3xTg-AD mouse model of AD that occurs in parallel with evident intraneuronal/extracellular Aβ deposition in the hippocampus (Chapter 3). In addition, serotonergic fibre sprouting correlated with reduced perforated synapses in the hippocampus, suggesting a structural remodeling process to maintain hippocampal connectivity (Chapter 4). Increased 5-HT neurotransmission (via high dietary intake of tryptophan, 5-HT precursor) reduced intraneuronal Aβ accumulation in the hippocampus, suggesting a direct role of 5-HT neurotransmission in modifying AD neuropathology (Chapter 5). Given the protective role of increased 5-HT neurotransmission, treatment with 5-HT enhancing drugs may be beneficial in reducing the underlying pathology as well as improving the behavioural and cognitive abnormalities associated with AD. Nevertheless, the role of specific 5-HT receptors responsible for such neuro-protective effect of 5-HT in AD awaits further research.

Indiana University-Purdue University Indianapolis (IUPUI) / Serotonin (5-HT) is heavily implicated in severe anxiety and trauma-related disor-ders, such as panic and post-traumatic stress disorders. Overall, site-specific pharmacolog-ical manipulations show that while 5-HT enhances anxiety-associated/avoidance behaviors in the amygdala, 5-HT inhibits panic-associated escape behaviors in the perifornical hypo-thalamus region (PeFR). Yet, our understanding of how specific serotonergic networks and co-transmitters regulate these conditions, but also other aspects of innate panic (e.g., car-dioexcitation or thermal response that occur during a flight or escape response) or condi-tioned fear behaviors is still elusive. Therefore, utilizing circuit-based gain- and loss-of-function approaches to selectively manipulate amygdala- and PeFR-projecting sero-tonergic systems, we hypothesize that specific serotonergic networks projecting to the amygdala and PeFR respectively enhance conditioned fear responses and attenuate innate panic-associated behaviors and physiological responses. There are two main chapters in this dissertation. In Chapter III, retrograde tracing revealed that the amygdala-projecting neurons from dorsal Raphe (DR) were almost exclusively serotonergic (92-95%) concen-trated in the dorsal/ventral (DRD/DRV) DR, with few non-serotonergic neurons. While selective lesioning of this network with saporin toxin (SAP) facilitated the extinction of conditioned fear behavior, selective optogenetic activation of amygdala-projecting DRD/DRV cell bodies using intersectional genetics reduced extinction of conditioned fear behavior and enhanced anxiety avoidance. In Chapter IV, retrograde tracing showed that the PeFR was innervated by equally selective serotonergic networks concentrated in the lateral wings DR (lwDR) and median Raphe (MR). Contrasting with the results from the amygdala-innervating 5-HT system, lesioning the PeFR-projecting serotonergic network from lwDR/MR was accompanied by reduced extinction of conditioned fear behavior, in-creased anxiety avoidance, and increased CO2-induced panic (elevated escape responses and enhanced cardioexcitation). Conversely, selective activation of lwDR/MR serotonergic terminals in the PeFR decreased anxiety-associated behaviors; inhibited CO2-induced panic, and induced unconditioned and conditioned place preferences. The circuit-based ap-proach data presented here show that amygdala- and PeFR-projecting 5-HT neurons com-prise distinct circuits underlying opposite roles enhancing anxiety/fear responses in the amygdala and dampening fear/panic responses in the PeFR. The identification of distinct circuits controlling anxiety, fear, and panic responses is a fundamental step towards the development of more effective therapies for psychiatric conditions such as anxiety and trauma-related disorders. / 2021-11-04

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