Aversive Olfactory Imprinting in Caenorhabditis Elegans

Early memories are especially robust and enduring, among which the most evocative example is imprinting. Imprinting was first described in newly hatched geese that form a lasting attachment to the first moving object they see. As observed in many animal species, imprinting is a process in which a sensory cue presented early in animal’s life – a critical period – subsequently gains unique access to ecologically relevant behaviors. Little is known about the molecular and neural underpinnings of imprinting. I have used C. elegans as a model organism to study imprinting because of its compact and well characterized nervous system, an armory of available genetic tools, and a versatile behavioral repertoire. Using a ethologically relevant training regime, I found that exposing newly hatched larvae C. elegans to pathogenic bacteria can generate an aversive memory of bacterial odors that is sustained into adulthood (4 days), in contrast to training of adults that results in a medium-term memory that lasts for less than a day. This long lasting aversive memory is specific to the experienced pathogen and has a critical period in the first larval stage (L1), and is defined as a form of aversive imprinting. Through chemical-genetic silencing of candidate neurons, I identified neurons essential for memory formation but not for memory retrieval (interneurons AIB and RIM), and complementary neurons essential for memory retrieval but not for memory formation (interneurons AIY and RIA) (Chapter 2). The RIM memory formation neurons synthesize the neuromodulator tyramine, which is required in the L1 stage for learning. This learning signal is transmitted to the AIY memory retrieval neurons by the tyramine receptor SER-2, which is required for imprinted aversion but not for adult learned aversion (Chapter 3). Tyramine modulation bridges the two subcircuits by linking tyramine production during learning with memory retrieval days later. Functional calcium imaging indicates that early imprinting experience modifies neuronal activity and output of the memory circuit. Among several neurons examined, changes in RIA best express the context and specificity of the imprinted memory (Chapter 4). Combining classical neuroethology, molecular genetics, and functional imaging, I have mapped distinct groups of neurons required for the formation and retrieval of an imprinted memory, defined neuromodulation that enables this critical period learning (tyramine and SER-2), and identified neuronal activity changes associated with memory. These findings provide insight into neuronal substrates of different forms of learning and memory, and lay a foundation for further understanding of early plasticity

A cellular defense memory imprinted by early life toxic stress

Stress exposure early in life is implicated in various behavioural and somatic diseases. Experiences during the critical perinatal period form permanent, imprinted memories promoting adult survival. Although imprinting is widely recognized to dictate behaviour, whether it actuates specific transcriptional responses at the cellular level is unknown. Here we report that in response to early life stresses, Caenorhabditis elegans nematodes form an imprinted cellular defense memory. We show that exposing newly-born worms to toxic antimycin A and paraquat, respectively, stimulates the expression of toxin-specific cytoprotective reporters. Toxin exposure also induces avoidance of the toxin-containing bacterial lawn. In contrast, adult worms do not exhibit aversive behaviour towards stress-associated bacterial sensory cues. However, the mere re-encounter with the same cues reactivates the previously induced cytoprotective reporters. Learned adult defenses require memory formation during the L1 larval stage and do not appear to confer increased protection against the toxin. Thus, exposure of C. elegans to toxic stresses in the critical period elicits adaptive behavioural and cytoprotective responses, which do not form imprinted aversive behaviour, but imprint a cytoprotective memory. Our findings identify a novel form of imprinting and suggest that imprinted molecular defenses might underlie various pathophysiological alterations related to early life stress. © 2019, The Author(s)

Physical exercise impact on aging-related pathways across generations in C. elegans

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, 2023. Tutor/a: Christian Griñán FerréAging has been defined as a gradual functional decline with a progressive physiological integrity loss, increasing the organism’s vulnerability to death. Otherwise, this described deterioration is the major risk factor for most current human pathologies, including neurodegenerative diseases, cancer, cardiovascular disorders, and diabetes. However, in the last decades, non-pharmacological treatments like physical exercise have provided general health benefits against aging decline. In this study, we aim to analyze how lifestyle factors such as physical exercise can modify the molecular expression of aging-related pathways and observe if this gene expression modification pattern is inherited transgenerationally throughout the following generations. Among all hallmarks of aging process, in this study we highlight epigenetic alterations. Even though extensive studies of transgenerational epigenetic inheritance have been reported in Caenorhabditis elegans, there is still a gap regarding how physical exercise might benefit organisms through epigenetic pathways. Then, we implement a physical exercise treatment in the first C. elegans generation, and we analyze the subsequent generations to study transgenerational heritable changes in gene expression involved in aging-related pathways. We emphasized on CREB transcription factor pathway, early growth response 1 (EGR1) transcription factor, superoxide dismutase 1 (Sod1) as a powerful antioxidant, and disintegrin and metalloproteinase domain-containing protein (ADAM10) pathway since its role in correct neurological development. Key words: exercise, C. elegans, aging, transgenerational epigeneti

Improvements in optical techniques to investigate the behavior and neuronal network dynamics over long timescales

Developments in optical technology have produced an important shift in experimental neuroscience from electrophysiological methods for observation and stimulation to all-optical solutions. One expects this trend to continue as future developments continue to deliver, and improve upon, the original promises of the technology: 1) minimally invasive actuation and recording of neurons, and 2) a drastic increase in targets that can be treated simultaneously. Moreover, as the high costs of the technology are reduced, one may expect its larger-scale adoption in the neuroscience community. In this thesis, I describe the development and implementation of two alloptical solutions for the analysis of behavior, neuronal signaling, and stimulation, which improve on previous state-of-the-art: (1) A minimally-invasive, high signal-to-noise twophoton microscopy setup capable of simultaneous, live-imaging of a large subset of sensory neurons post activation, and (2) a low-cost tracking solution to stimulate and record behavior. I begin this thesis with a review of recent advances in optical neuroscience techniques for the study of neuronal networks with the focus on work done in Caenorhabditis elegans. Then, in chapter 2, I describe my implementation of a two-photon temporal focusing microscopy setup and show significant improvements through the use of a high power/ high pulse repetition rate excitation system, enabling live imaging with high resolution for extended periods of time. I model temperature increase during a physiological imaging scenario for different repetition rates at fixed peak intensities and find range centered around 1 MHz to be optimal. Lastly, I describe the low-cost tracking setup with the ability to stimulate and record behavior over the course of hours. The setup is capable of two-color stimulation of optogenetic proteins over the area of the behavioral arena in combination with volatile chemicals. To showcase the utility of the system, I demonstrate behavioral analysis of integration of contradictory cues. In summary, I present a set of techniques for the interrogation of neural networks from animal behavior to neuronal activity, over timescales of potentially hours and days. These techniques can be used to address a new dimension of scientific questions.Okinawa Institute of Science and Technology Graduate Universit

Use of the Model Organism Caenorhabditis elegans to Elucidate Neurotoxic and Behavioral Effects of Commercial Fungicides

Fungicides are widely used in agriculture and medicine and there are several different types of fungicides that are distributed globally in the soil and water through water runoff and drift from spraying as well as other avenues of distribution. Understanding the biological effects of fungicide contaminants in the environment on non-target organisms including humans is critical. This chapter considers the use of the model organism and key beneficial soil nematode, Caenorhabditis elegans, as an effective strategy for examining fungicide effects on growth, reproduction, nervous system and behavior. We present novel evidence of the effects of a general use fungicide, mancozeb, on behavior and neuronal structure