Ph.d.

In Drosophila, eight insulin-like peptides (DILP1-8) are produced and secreted in different locations. They regulate many aspects of development and physiology, such as organism growth, metabolic homeostasis, reproduction, stress resistance and life span. DILP2, 3 and 5 are mainly produced by a cluster of median neurosecretory cells in the brain known as insulin producing cells (IPCs). Here we showed that IPCs are under tight regulation of two G-protein coupled receptors (GPCRs), serotonin receptor 5-HT1A and octopamine receptor OAMB. Genetic manipulations of these two receptors in IPCs affected transcription levels of DILPs, hence altered feeding, carbohydrate levels, and resistance to stress (Paper I and II). Moreover, we showed that the insulin receptor (dInR) is strongly expressed in leucokininergic neurons (LK neurons), and selectively regulates growth of around 300 neuropeptidergic neurons expressing the bHLH transcription factor DIMMED. Overexpression of dInR in DIMM-positive neurons led to substantial neuronal growth, including cell body size, golgi apparatus and nuclear size, while knockdown of dInR had the opposite effect (Paper III). Manipulations of components in the insulin signaling pathway in LK neurons resulted in the similar cell size phenotypes. Furthermore, dInR regulated size scaling of DIMM-postive neurons is nutrient-dependent and partially requires the presence of DIMM (Paper III). Finally, we investigated the roles of DILPs (2, 3, 5 and 7) and LK neurons in regulation of feeding and diuresis at the adult stage (Paper IV).  In summary, we have identified two more regulators for IPC activity and demonstrated developmental roles of  DILPs and dInR in regulating neuronal size. Moreover, DILPs regulate water homeostasis together with a diuretic hormone leucokinin and as a consequence affects feeding behavior. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press. Paper 4: Manuscript.</p>

While selective serotonin (5-HT) reuptake inhibitors (SSRIs) are utilized as the first-line strategy in treating depression, new approaches are still desired. Using in vivo electrophysiological techniques, the effects of co-administration of bupropion with the SSRI escitalopram on the firing rate of dorsal raphe 5-HT and locus coeruleus norepinephrine (NE) neurons were investigated. Escitalopram significantly decreased the firing of 5-HT and NE neurons at day 2. The 5-HT firing rate, unlike that of NE, recovered after the 14-day escitalopram regimen. Bupropion did not increase 5-HT firing but decreased that of NE after 2 days. Following 14-day bupropion, 5-HT firing was markedly enhanced, and NE firing was back to baseline. Co-administration of escitalopram and bupropion doubled 5-HT firing after 2 and 14 days, whereas NE neurons were inhibited after 2, but partially recovered after 14 days. Although sustained bupropion administration did not alter the sensitivity of 5-HT1A receptors in hippocampus, the tonic activation of postsynaptic 5-HT1A receptors was enhanced in 14-day bupropion-treated rats to a greater extent than in the 2-day and control rats. The function of terminal 5-HT1B autoreceptors was not changed. The inhibitory action of α2-adrenergic receptors on 5-HT terminals was, however, diminished. The function of terminal α2-adrenergic autoreceptors was also attenuated in rats given bupropion for 14 days. Administration of the antidepressant trazodone suppressed the 5-HT firing at day 2, which recovered to baseline following 14 days. Prolonged trazodone-administration enhanced the tonic activation of postsynaptic 5-HT1A receptors in hippocampus, and decreased the function of terminal 5-HT1B autoreceptors. Finally, a novel psychotropic agent asenapine showed potent antagonistic activity at 5-HT2A, D2, and α2-adrenoceptors. Asenapine, however, acted as a partial agonist at 5-HT1A receptors in dorsal raphe and hippocampus. Overall, the therapeutic effects of various antidepressants may be, at least in part, due to the enhancement of 5-HT and/or NE neurotransmission.

Although empirical evidence suggests that interactions occur between serotonin (5- HT) and the hypothalamic-pituitary-interrenal (HPI) axis teleost fish, the mechanisms of serotonergic regulation of the HPI axis require elucidation. The hypothesis that 5-HT regulates the stress response in rainbow trout (Oncorhynchus mykiss) by acting at multiple levels in the HPI axis through tissue-specific 5-HT receptor subtypes was examined. Messenger RNA for 5-HT1A, 5-HT2 and 5-HT4 receptor subtypes was expressed in all HPI axis tissues. Administration of 5-HT in vivo in cannulated trout caused significant increases in plasma cortisol and glucose concentrations. In vitro head kidney preparations revealed a stimulatory effect of 5- HT, acting through the 5-HT4 receptor, on cortisol production. Collectively, these data suggest that 5-HT plays a role in HPI axis activation in rainbow trout, and that at the head kidney level, these effects likely are mediated by the 5-HT4 receptor.

Both chronic psychosocial stress and chronic administration of corticosterone have been shown to alter serotonergic type 2A (5-HT2A) receptor activity. A non-invasive behavioural index of 5-HT2A receptor activity is the frequency of "wet dog shakes" (WDS) or serotonergic stereotypy. In addition to WDS, 5-HT2A receptors mediate effects on sexual behaviour in the rat, in particular, inhibition in the male and stimulation in the female. In the present series of experiments, the potential involvement of stress and corticosterone in the regulation of WDS and sexual behaviour in the male and female rat was investigated. In Experiments 1-4, chronic exposure to several different forms of psychosocial stress was found to facilitate female and inhibit male rat sexual behaviour while concurrently increasing the display of WDS in both sexes. In Experiment 5, nefazodone, an antidepressant with 5-HT2A antagonistic properties, blocked the effects of stress on WDS but not sexual behaviour in female rats. In Experiments 6-7, the corticosterone synthesis inhibitor, metyrapone, blocked the effects of stress on sexual proceptivity and WDS in female rats. Metyrapone blocked the effects of stress on WDS but not sexual behaviour in male rats. In Experiments 8-9, high doses of corticosterone administered chronically facilitated female and inhibited male rat sexual behaviour while concurrently increasing WDS in both sexes. In Experiments 10-11, the 5-HT2A antagonist ketanserin was found to completely attenuate the effects of corticosterone on sexual behaviour and WDS in both male and female rats. In Experiments 12-13, the acute administration of corticosterone was found to exert no effect on either sexual behaviour or WDS in male or female rats. The present results indicate that both chronic corticosterone treatment and exposure to chronic stress inhibit male and facilitate female sexual behaviour while concurrently increasing WDS behaviour. The stress-induced facilitation of WDS appears to be related to elevated corticosterone levels and is suggestive of increased 5-HT2A activity. Both corticosterone and stress exerted effects on sexual behaviour in the direction that would be predicted by increased 5-HT2A activity. While the effects of corticosterone on sexual behaviour appear to be mediated by 5-HT2A activity, the effects of stress on sexual behaviour do not appear to be related to either elevations in corticosterone levels or alterations in 5-HT2A activity. / Arts, Faculty of / Psychology, Department of / Graduate

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.

No description available.

Methionine sulfoxide reductase (MSR) is an important antioxidant to help mitigate oxidative stress that contributes to age-associated neurodegenerative diseases, such as Alzheimer’s Disease and Parkinson’s Disease. In MSR deficient Drosophila melanogaster (fruit flies), larvae show a developmental delay like that seen when wild-type larvae are reared on nutrient deficit culture medium. These investigators further showed that serotonin levels were depressed in these nutrient deficient larvae. The overarching aim of this study was to better understand the role of serotonin in MSR regulated physiology. Supplementing food with serotonin partially rescued the slower mouth hook movements (MHM) observed in the MSR-deficient flies. However, supplementation with serotonin altering drugs that cross the blood brain barrier (5-hydroxytryptophan, fluoxetine, or paravi chlorophenylalanine) did not rescue MHM and caused impairments to the growth of larvae during development. This study indicates that serotonin regulates feeding behavior partially through the regulation of MSR production but acts independently to regulate development. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

No description available.

Major depression is a complex disease involving genetic and environmental factors. Previous studies suggest that functional genetic polymorphisms that alter the serotonin (5-HT) system in combination with psychosocial stress can synergistically increase the strength of these associations. In addition, depression is associated with several neurological disorders involving neuronal injury, including stroke (i.e. post-stroke depression, PSD). Both forms of depression are treated with 5-HT-selective antidepressants like fluoxetine, but remission rates do not exceed 50%. Evidence showed that alterations affecting the 5-HT system, directly or indirectly, lead to anxiety or depression phenotypes and may elucidate determinants of response to antidepressants. To better understand common and unique alterations in both genetic- and injury-related depression, I have generated and investigated two novel mouse models that exemplify a serotonin-related genetic risk (Flx-Freud-1 mice) and an injury model (ischemic lesion), to identify similarities and differences in their behavioral phenotypes, and in response to fluoxetine treatment. In the Flx-Freud-1 mouse model, 5-HT neuron-specific adult knockout of Freud-1, a key repressor of the 5-HT1A receptor gene, led to overexpression of 5-HT1A autoreceptors thought to negatively regulate the 5-HT system. These mice showed increased 5-HT1A autoreceptor responses, reduced 5-HT levels and a robust anxiety and depression phenotype that was resistant to chronic fluoxetine treatment. These behaviors were dependent on increased 5-HT1A autoreceptors since they were not seen in mice lacking 5-HT1A autoreceptors in adult Freud-1 knockout background. Instead an opposite anti-depressed phenotype emerged, suggesting that Freud-1 might have additional functions in 5-HT cells. In the PSD model, the vasoconstrictor endothelin-1 was injected to induce transient ischemia in the left medial prefrontal cortex, iv part of the circuitry thought to be damaged in PSD in humans. This stroke resulted in a persistent anxiety, depression and cognitive impairment. Chronic fluoxetine treatment alone or combined with voluntary exercise was effective to reverse the behavioral and cognitive phenotypes in this PSD mouse model.The results of genetic and SSRI treated stroke models show that changes in 5-HT system contribute to widespread dysregulation of the neuronal circuitry implicated in depression, anxiety. Genetic alteration of the 5-HT system conferred fluoxetine-resistance, while cortical stroke which indirectly altered the 5-HT system remained responsive to fluoxetine. Following unilateral stroke, there was increased activity of the contralateral hemisphere, including the prefrontal cortex and limbic areas involved in anxiety and depression, and activation of the 5-HT system. Effective treatment with chronic fluoxetine alone or combined with exercise significantly reduced and balanced the contralesional neuronal activation in affected regions that correlated with improvements in phenotypes. In conclusion, this work implicates genetic changes that directly alter the 5-HT system in resistance to chronic fluoxetine treatment. Therefore, the Flx-Freud-1-induced 5-HT1A autoreceptor overexpression mouse model may provide a useful pre-clinical model of antidepressant resistance. In contrast, in the PSD model, in which expression of 5-HT1A autoreceptors remained intact, chronic fluoxetine treatment reversed depression and anxiety phenotypes. This model may provide insight into changes in neuronal activity that allows antidepressants to mediate behavioral and cognitive improvement.