MODULATORY ACTIONS OF SEROTONERGIC SYSTEM IN CARDIAC FUNCTION, BEHAVIOR, AND SENSORIMOTOR CIRCUIT ACTIVITY IN DROSOPHILA MELANOGASTER

In this dissertation, I have focused on the role of serotonin (5-HT) as a modulator in heart rate, feeding and locomotion behaviors as well as sensorimotor circuit activity in Drosophila melanogaster. A general overview in the actions of the serotonergic (5-HTergic) system on the larval heart and nervous system in larvae and adults is reviewed in Chapter One. I sought to further study the actions of serotonergic system to provide additional insights into cellular and molecular underpinnings in the actions of 5-HT.In Chapter two, I present studies on mechanisms of action by 5-HT in larvae cardiac system. For this purpose, genetic and pharmacological approaches were used. The transgenic flies used expressed hM4Di receptors (designer receptors exclusively activated by designer drugs (DREADDs)) which were employed to manipulate the activity of Gαi heterotrimeric protein through activation of engineered G-protein coupled receptors hM4Di DREADD. The activation of hM4Di DREADD receptors by clozapine-N-oxide (CNO) arrested the heart beat; however, pharmacological manipulation of adenylyl cyclase activity and cAMP levels had no significant effect on heart rate. In Chapter Three the role of various 5-HT receptor subtypes that mediate 5-HT action in larval cardiac tissue is addressed. In this study, various 5-HT agonists and antagonists were employed. The pharmacological results demonstrate that a 5-HT2 agonist significantly increases the heart rate. Furthermore, 5-HT2 antagonist, markedly reduces the effect of 5-HT. In addition, I employed genetic approaches to corroborate the pharmacological results. In addition, I investigated the role of the 5-HTergic system in locomotion and feeding behaviors as well as in modulation of sensorimotor circuits. This study is delineated in Chapter Four. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae various pharmacological agents. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or induced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotor activity and feeding behavior in larvae. In addition, acute activation of 5-HT neurons disrupts normal locomotor activity in adult flies. In Chapter Five, I addressed direct actions of fluoxetine on synaptic transmission at neuromuscular junctions (NMJs), neural properties, and cardiac function unrelated to fluoxetine’s action as a selective 5-HT reuptake inhibitor using Drosophila, crayfish and primary neurons in mouse model system. Fluoxetine application blocked action potentials in crayfish axons, enhanced occurrences of spontaneous synaptic vesicle fusion events at NMJs of both Drosophila and crayfish. In rodent primary neurons, fluoxetine application resulted in increase of cytoplasmic Ca2+. I also developed teaching modules, which are presented in Chapter Seven, to guide students how to exploit a vast array of genetic tools, such as optogenetics in Drosophila to manipulate various neural circuits and to observe their effects on behavior and sensorimotor circuit activity. I also developed a module to teach college level students a hands-on experiment regarding proprioception and tension receptors in crab limb, which is detailed in Chapter Eight

Optogenetic Stimulation of Drosophila Heart Rate at Different Temperatures and Ca\u3csup\u3e2+\u3c/sup\u3e Concentrations

Optogenetics is a revolutionary technique that enables noninvasive activation of electrically excitable cells. In mammals, heart rate has traditionally been modulated with pharmacological agents or direct stimulation of cardiac tissue with electrodes. However, implanted wires have been known to cause physical damage and damage from electrical currents. Here, we describe a proof of concept to optically drive cardiac function in a model organism, Drosophila melanogaster. We expressed the light sensitive channelrhodopsin protein ChR2.XXL in larval Drosophila hearts and examined light‐induced activation of cardiac tissue. After demonstrating optical stimulation of larval heart rate, the approach was tested at low temperature and low calcium levels to simulate mammalian heart transplant conditions. Optical activation of ChR2.XXL substantially increased heart rate in all conditions. We have developed a system that can be instrumental in characterizing the physiology of optogenetically controlled cardiac function with an intact heart

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, \u3cem\u3eHirudo medicinalis\u3c/em\u3e

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism

A Novel Educational Module to Teach Neural Circuits for College and High School Students: NGSS-Neurons, Genetics, and Selective Stimulations

This report introduces various approaches to target defined neural pathways for stimulation and to address the effect of particular neural circuits on behavior in a model animal, the fruit fly (Drosophila melanogaster). The objective of this novel educational module described can be used to explain and address principle concepts in neurobiology for high school and college level students. A goal of neurobiology is to show how neural circuit activity controls corresponding behavior in animals. The fruit fly model system provides powerful genetic tools, such as the UAS-Gal4 system, to manipulate expression of non-native proteins in various populations of defined neurons: glutamergic, serotonergic, GABAergic, and cholinergic. The exhibited behaviors in the examples we provide allows teachers and students to address questions from behaviors to details at a cellular level. We provided example sets of data, obtained in a research lab, as well as ideas on ways to present data for participants and instructors. The optogenetic tool, channelrhodpsin 2 (ChR2), is employed to increase the activity of each population of neurons in a spatiotemporal controlled manner in behaving larvae and adult flies. Various behavioral assays are used to observe the effect of a specific neuron population activation on crawling behavior in larvae and climbing behavior in adult flies. Participants using this module become acquainted with the actions of different neurotransmitters in the nervous system. A pre- and post- assessment survey on the content is provided for teachers, as templates, to address learning of content and concepts

Modulatory Action by the Serotonergic System: Behavior and Neurophysiology in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

Serotonin modulates various physiological processes and behaviors. This study investigates the role of 5-HT in locomotion and feeding behaviors as well as in modulation of sensory-motor circuits. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae 5-HT, a 5-HT precursor, or an inhibitor of tryptophan hydroxylase during early stages of development. The effects of feeding fluoxetine, a selective serotonin reuptake inhibitor, during early second instars were also examined. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or enhanced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotion and feeding behaviors in larvae. Acute activation of 5-HT neurons disrupts normal locomotion activity in adult flies. To determine which 5-HT receptor subtype modulates the evoked sensory-motor activity, pharmacological agents were used. In addition, the activity of 5-HT neurons was enhanced by expressing and activating TrpA1 channels or channelrhodopsin-2 while recording the evoked excitatory postsynaptic potentials (EPSPs) in muscle fibers. 5-HT2 receptor activation mediates a modulatory role in a sensory-motor circuit, and the activation of 5-HT neurons can suppress the neural circuit activity, while fluoxetine can significantly decrease the sensory-motor activity

DREADDs in Drosophila: A Pharmacogenetic Approach for Controlling Behavior, Neuronal Signaling, and Physiology in the Fly

SummaryWe have translated a powerful genetic tool, designer receptors exclusively activated by designer drugs (DREADDs), from mammalian systems to Drosophila melanogaster to selectively, rapidly, reversibly, and dose-dependently control behaviors and physiological processes in the fly. DREADDs are muscarinic acetylcholine G protein-coupled receptors evolved for loss of affinity to acetylcholine and for the ability to be fully activated by an otherwise biologically inert chemical, clozapine-N-oxide. We demonstrate its ability to control a variety of behaviors and processes in larvae and adults, including heart rate, sensory processing, diurnal behavior, learning and memory, and courtship. The advantages of this particular technology include the dose-responsive control of behaviors, the lack of a need for specialized equipment, and the capacity to remotely control signaling in essentially all neuronal and nonneuronal fly tissues

A Cross-sectional Study of Clinical Characteristics and Outcomes among Adults with Laboratory-confirmed SARS-CoV-2 Infection with Omicron Variant

The emergence of the SARS-CoV-2 Omicron variant has raised concerns due to its increased transmissibility and potential implications on clinical characteristics and outcomes in infected individuals. The aims of this report were to study the profile of SARS-CoV-2 infection with omicron variant, investigate the infection outcome, reinfection rates with associated factors, antibody levels, and explore the associations between biochemical markers and disease severity. This prospective cohort study was conducted in Duhok city in the Northern of Iraq. All volunteers with confirmed SARS-CoV-2 RT–PCR and confirmed Omicron infection who were older than 18 years old and agreed to participate were recruited for this study. The study was carried out from January to April 2022. There were 234 cases of confirmed SARS-CoV-2 RT–PCR Omicron infection. The mean age was 48.12±17.3 years, 43.2% were vaccinated, and 40.2% were male. Among the recruited patients, 99.1% recovered and did not need hospitalization. In this study, (38.9%) had a history of previously confirmed COVID-19 infection. Reinfection was significantly higher in females than males (p=0.04; OR= 0.56). It was found that the IgG antibody levels were higher in patients who received Pfizer-BioNTech than in those who received other vaccines (p=0.001). The levels of IgG were also significantly higher in patients with mild infection (p=0.046), whereas the levels of D-dimer were significantly higher in patients with severe cases of the infection compared to those with mild or moderate cases (p=0.001). Additionally, the levels of C-reactive protein (CRP) were observed to be higher in individuals with moderate cases of infection than in mild and severe cases (0.001). Individuals who contracted the Omicron strain generally had positive outcomes. Reinfection with the Omicron variant was relatively high. IgG levels were higher in patients with mild disease, implying that they were associated with decreased disease severity. We found significant associations between D-dimer levels and the severity of the disease. Additional research is required to investigate the long-term effects of Omicron infection