Catharine Rankin

Previous research on Caenorhabditis elegans by Giles (2012) and McEwan (2013) identified the goa-1 gene as a possible regulator of habituation of the tap withdrawal response because mutations in goa-1 caused abnormal habituation across multiple metrics. The original goal of this thesis was to use behavioural and genetic analyses to understand where and how goa-1 functions in habituation of the tap withdrawal response. I planned to use C. elegans with green fluorescent protein (GFP) inserted into wild-type goa-1 as well as tissue-specific degron strains to perform targeted degradation of the GOA-1 protein in subsets of cells. However, when I tested habituation the goa-1::GFP strain found that habituation for the probability of response looked similar to the probability of response habituation for the goa-1 null mutant, yet had a normal, wild-type phenotype for habituation of response duration. I compared our CRISPR goa-1::GFP strain to a second goa-1::GFP strain created by a different single copy insertion technique; the behaviour of this second goa-1::GFP was the same as our goa-1::GFP strain. I tested different mutant goa-1 strains to determine their effects on the habituation phenotypes, however, none of them matched the goa-1::GFP phenotype. Because the goa-1::GFP worms showed normal habituation of response duration I created three GOA-1::GFP degradation strains with pan-neuronal, ciliated neuron, and touch neuron specific degradation. The anatomical results showed that although goa-1::GFP degradation occurred, the behaviour results were inconclusive. Finally, I performed the habituation assay on worms with mutations in genes that were known interactors with goa-1 and found that mutations in dgk-1 led to similar habituation phenotypes as goa-1::GFP worms. The most likely explanation for the goa-1::GFP abnormal habituation phenotype is that the GFP interfered with a signalling pathway that activates the diacylglycerol kinase. These data suggest that the role of goa-1 in habituation of the probability of response is mediated through dgk-1.

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The goal of this thesis is to identify the potential contributions of two largely unstudied monoamines, tyramine and octopamine, in the context of tap habituation; a behavior who’s underlying circuitry heavily expresses genes encoding precursors and receptors for both these neurotransmitters. To pursue this goal I compared habituation of the tap withdrawal response in Caenorhabditis elegans - a model organism with a fully mapped connectome and proteome, and thoroughly characterized habituation - between wild type worms and worms that were null in tyramine and/or octopamine across different timepoints in young adulthood. I discovered that these gene products contribute to only isolated components of this behavior in an age-dependent manner. Although the impacts of these genes was interesting, the study of worms null in both of these precursors failed to find the striking differences in tap habituation that have been studied in animals expressing mutations in dopamine neurotransmission with much less representation in the neural circuitry responsible for this behavior. With some phenotypes identified by these mutations, their contributions to the biological underpinnings unique to each response-component can now be investigated in more detail.

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The simplest forms of learning are non-associative learning. In non-associative learning, both sensitization and dishabituation cause an increase in the behavioural response. A strong and/or surprising stimulus can enhance a naïve response above the baseline in sensitization, or facilitate a previously decremented response in dishabituation. While a lot has been done to unravel the cellular and molecular mechanisms for sensitization, the mechanism(s) for dishabituation remains elusive. Sensitization and dishabituation were considered as the same facilitatory process for decades, but recent evidence suggests that they may be two separate processes. The focus of this thesis was to investigate whether sensitization and dishabituation are the same or two different processes. In this research, sensitization and dishabituation of the response to optogenetic stimulation of ASH nociceptor neurons by a mechanosensory tap stimulus were characterized and compared in a series of behavioural paradigms in C. elegans. It was found that sensitization and dishabituation were produced in four ASH response components with different time-dependent dynamics. Using a candidate gene approach, the effects of several genes on sensitization and dishabituation of the ASH response were examined. It was found that genes played differential roles in mediating the two facilitatory processes; genes that were critical for sensitization did not affect dishabituation. Taken together, these findings strongly suggest that sensitization and dishabituation are two facilitatory processes mediated by distinct genetic and molecular pathways. This research deepens our understanding of the complex mechanisms of “simple” forms of learning.

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Although olfactory dysfunction is one of the hallmark symptoms in Alzheimer’s disease (AD) prior to the onset of cognitive impairments, little is known about the causes of this dysfunction. The nematode Caenorhabditis elegans is an ideal model for system-level genetic understanding of sensory neural circuits and behavior. Many cases of familial AD are linked to mutations of the presenilin (PS) genes. These genes are homologues with sel-12 genes in C. elegans. The purpose of this project was to examine the association between PS1 and olfactory deficits in order to investigate the cellular mechanism of these dementia-linked deficits. To gain a better understanding of the relationship between presenilin 1 (PS1) mutations in AD and olfactory deficits, chemotaxis experiments (with the attractant diacetyl, and the aversive octanol) were conducted on worms with a mutation in sel-12. I found that adult sel-12 mutant worms had a significantly decreased sensitivity to both odorants compared to wild-type worms. Extrachromosomal array expression of human wild-type PS1 into C. elegans rescued olfactory defects, confirming functional homology between the C. elegans and human gene. However, a PS1 mutant from an Alzheimer’s family was unable to rescue olfactory deficits. Moreover, C. elegans sel-12 mutant worms presented olfactory deficits throughout their lifespan, and the deficit increased with age, similar to the neurodegenerative progression of AD. Based on these data, I concluded that a mutation in the C. elegans homologue of PS1 is associated with decreased olfactory function, and this deficit was rescued by wild-type human PS1 gene. I suggest that altered functioning of the Notch pathway may be involved in these chemosensory deficits. Additionally, to localize the neuron(s) where wild-type sel-12 function is required for normal olfaction, sel-12 and PS1 rescues were conducted in specific sensory neurons, namely the ASH and the AWA neurons responsible for detecting octanol and diacetyl, respectively. Further, an examination of the morphology of the ASH neurons showed increased neurodegeneration over time in sel-12 mutant worms, demonstrating an association with the observed behavioral deficits.

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Habituation is a simple form of learning that manifests as a decrease in an innate response to a repeated stimulus that is not associated with important events. In this thesis, habituation-memory was studied using the tap-reversal response emitted by the roundworm C. elegans in response to a non-localized tap-stimulus. Delivery of 30 tap-stimuli produced a decline in the probability, duration, and speed of the reversal-response (i.e., learning). When stimulation was ceased for 10-minutes each of these response-components recovered partially but not completely, as evidenced by differences between training and test sessions (i.e., memory). Each of the components (probability, duration and speed) of the tap-reversal response showed different profiles of habituation-learning and habituation-memory in wild-type worms, with one not being predictive of the other. A genetic screen supported these findings and identified mutant strains that were deficient in habituation-memory for one component of the response but not the others. It was also shown that simply because a mutant strain showed more habituation-learning did not necessarily predict that it would show more habituation-memory. This suggested that different biological underpinnings likely underlie A) the persistence of habituation from stimulus-to-stimulus within a session of stimuli and B) the persistence of habituation from one session of stimuli to another. The genetic screen also identified mutant strains which supported a genetic dissociation of initial response-difference from overall average response-difference. This suggested that considering the phase of test-session is important for measuring memory. Finally, it was shown that the habituation-memory deficits shown by one mutant strain (pde-4) were selective to the frequency of training used and did not show deficits at all training frequencies. Together, these data suggest that habituation-memory is multidimensional and that considering both the components and time-scales of the response is important.

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Presenilins are well known as sites of mutations responsible for early-onset Alzheimer's disease. The normal functions of presenilins and the mechanisms by which presenilins cause Alzheimer disease are not yet known. Conservation of cellular and molecular functions between the C.elegans and human genes makes it a powerful experimental model organism to investigate cellular mechanisms of Alzheimer's disease and neurodegenerative disorders in general. Mutations in the C.elegans presenilin1homologue, sel-12, decrease Notch signaling activity, which results in an egg-laying deficit in these animals. It has been well established that pathogenic PS1 mutations impair Notch signaling; however, in the first part of this thesis we showed that a recently discovered PS1Δs₁₆₉ human mutation rescued the egg-laying deficit associated with Lin12/Notch pathway, suggesting that in this pathogenic PS1 mutation Notch processing remained intact. In the second part of this thesis the behavioural phenotypes of a mutation in the C.elegans presenilin homologue, sel-12, were studied. Our results revealed that a mutation in the sel-12 gene causes chemotaxis deficits toward volatile and water-soluble stimuli in sel-12 mutant animals. Reintroducing the sel-12 or the wild-type human presenilin gene decreased those behavioural phenotypes, indicating that the observed chemotaxis deficits were dependent on sel-12 activity. However, rescuing with the human PS1C₄₁₀Y mutation, which has a severe effect on Notch processing, did not ameliorate the chemotaxis deficit; in contrast, rescuing with PS1Δs₁₆₉ rescued both volatile and water-soluble chemotaxis impairments suggesting that the chemotaxis deficit causing by sel-12 mutation depends on the Notch pathway.

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Parkinson’s disease (PD) is a neurodegenerative disorder of central nervous system affecting more than 10 million people globally. This disease has a strong genetic component with 14% of individuals with PD reporting a first-degree relative with the disease. While genes like PARK2 and PINK1 have been associated with familial early onset PD, LRRK2, DNAJC13, and VPS35 have been linked to idopathic PD. These genes are largely involved in the maintenance of mitochondria or in the quality control via the cellular machinery that degrades unneeded proteins. Mutations in these genes affect the function and survival of particular neurons in parts of the brain that regulate normal movement, balance, and coordination. One endophenotype of PD is abnormal habituation; habituation is a simple form of learning in which repeated stimulation causes a decrement in responding over time. By investigating how these PD associated genes are involved in habituation, we hope to better understand the underlying pathophysiology induced by these genetic differences. In this study mechanosensory habituation was examined in Caenorhabditis elegans (C.elegans; microscopic transparent roundworms) in both wild-type worms and in worms with mutations in homologues of genes implicated in PD. Each strain of mutant worms showed a unique combination of basal characteristics and habituation phenotypes that distinguished them from wild-type worms. The integrity of dopamine neurotransmission was also investigated using the SWIP assay and ON/OFF Food Habituation assay. The C. elegans homologue of the PD gene VPS35, vps-35, an essential component of the retromer complex, was found to display the characteristics of abnormal dopamine signaling and was chosen for additional testing. Experiments using channelrhodopsin confirmed a lower dopamine signaling phenotype, with overexpression and rescue strains of this mutant being created to further explore how this protein affects behavior. These experiments set the groundwork for using C. elegans as a genetic model of PD that can help us better understand this complicated neurological disorder.

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Despite its apparent simplicity, the soil-dwelling nematode Caenorhabditis elegans has a surprisingly large capacity to learn and remember. Previous characterization of C. elegans genome and neuronal circuit makes this worm an ideal choice for studying behavior and the mechanisms that underlie it. Through careful behavioral and genetic studies, nematodes have been shown to form many different types of memory, including short-term non-associative memory called habituation. Habituation is the defined as the decrement in response after repeated, irrelevant stimuli. In the first part of this thesis, detailed analyses showed that as one response type, reversals, decreased other responses, accelerations, decelerations and pauses became more prevalent. An earlier large-scale screen of mutant strains of C.elegans showed that several genes related to heterotrimeric G-protein family of signaling pathways exhibited striking defects in habituation. To follow-up on those findings, in the second part of my thesis I investigated the role of heterotrimeric G-protein signaling pathway and showed that Gαi and Gαq signaling pathways share a broad role regulating habituation whereas the Gαs pathway modulates the rate of habituation. The analyses of all the behaviours nematodes preform in response to habituation training showed that heterotrimeric G-protein signaling pathways play a role in regulating the shift in behaviour during habituation training. Together these data add to our understanding of the mechanisms underlying habituation of the tap response in C. elegans.

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These studies were designed to investigate how environmental cues are associated during a non-associative learning process by studying chemosensory context conditioning for habituation in the nematode Caenorhabditis elegans. In chemosensory context conditioning for habituation animals that are trained and tested in the presence of either a taste or smell context cue show greater retention of habituation to tap stimuli when compared to animals trained and tested in different environments. This thesis is based on the work of Rankin (2000), in which taste (sodium acetate) context conditioning of habituation, extinction and latent inhibition of the cue were demonstrated. Here, I have shown context conditioning for an olfactory chemosensory cue (diacetyl) and dissociated the taste and smell pathways for this form of learning. odr-7 worms, with non-functional AWA olfactory chemosensory neuron (that detects diacetyl), showed short-term context conditioning to the taste but not to smell; the reverse was true for osm-3 worms with non-functional taste chemosensory neurons. This dissociation allows me to distinguish learning genes from genes involved in the detection of taste or smell. I also demonstrated long-term associative memory (24h) for context conditioning; context conditioning did not enhance normal long-term habituation, however, it produced memory in a training procedure that normally does not produce memory. My results showed that glr-1 (an AMPA-type ionotropic glutamate receptor subunit) and nmr-1 (an NMDA-type ionotropic glutamate receptor subunit) mutant worms did not show either short- or long-term context conditioning. To identify one site of plasticity, I showed that NMR-1 in the RIM interneurons was critical to produce short-term olfactory context conditioning. These studies lay the foundation to elucidate the cellular mechanisms of non-associative and associative learning for both short- and long-term memory, and may provide insights into how interneurons integrate information from multiple sensory systems.

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