Raj, Vishnu et al. (2021) International Worm Meeting "Dopaminergic neurons are critical for encoding and retrieval of adaptive memory in Caenorhabditis elegans"

Raj, Vishnu, Thekkuveettil, Anoopkumar

[International Worm Meeting, 2021]

The neuronal basis of learning and memory has not been well elucidated. Alterations in neurotransmitter release and synaptic plasticity allow connectome alterations to play a critical role in learning and memory formation. Dopamine (DA) is one of the neurotransmitters involved in motivation and motor function. In the present study, we used Caenorhabditis elegans to verify the role of DA and dopamine neurons in olfactory adaptive learning. Both wild-type (N2) and DA deficient (cat-2) mutant showed learning and memory formation when worms were trained towards butanone (conditional stimulus) in association with food (unconditional stimulus). However, the transgenic worms (dat-1::ICE) with loss of dopamine neurons due to interleukin-1 beta converting enzyme (ICE) expressing under dat1 promoter showed significant loss of memory retention. A similar loss of memory retention was observed in UA44 worms expressing human alpha-synuclein, a protein involved in dopaminergic neurodegeneration. The worms treated with exogenous DA could partially rescue memory retention. When the dat-1::ICE worms were rescued from dopamine neuron degeneration with siRNA targeting ICE, the memory retention was regained comparable to that of control worms. Transgenic worms expressing GCaMP in dopaminergic neurons (CEP and ADE) were used to measure the calcium dynamics during memory recall. An increased firing pattern of dopamine neurons was observed during memory recall, confirming these neuronal connectomes are crucial in olfactory adaptive learning in worms. Furthermore, worms with dopamine neuron degeneration have been found to have low acetylcholine release. These results suggest the critical role of DA in memory recall and might be playing a significant role in cognitive function. Key words: Dopamine; Learning and Memory; C.elegans; Neurodegeneration; Calcium imaging.

Varghese, Amal et al. (2021) International Worm Meeting "Monoaminergic molecular pathways in modulating memory and behaviour"

Thekkuveettil, Anoopkumar, Varghese, Amal

[International Worm Meeting, 2021]

To exercise information about its immediate surroundings C.elegans receive a variety of external stimuli, which establishes behavioral modifications in animals. Monoamines seem to mediate a wide range such functions along with physiological and homeostatic functions in animals. Recent evidences suggest Tyramine (TA), one of the precursors for monoamines like dopamine and serotonin, could act as a neurotransmitter. However, the molecular mechanisms underlying the tyramine pathway is poorly understood. Tyraminergic neurons, expressing SER-2 receptors, could act as neuromodulator and facilitate escape response in C. elegans. Yet another receptor TYRA-2 acts as determining factor in shut down of aversive behaviours in unharmonious situations for Risk-Reward pathway.Tyramine secretion being extrasynaptic from Motor interneuron RIM is likely to affect the head neurons, presenting receptors towards tyramine.This mechanism might be credited in establishing essential behavioral modifications and short lived neuronal alterations in the animals. In this study, we verified the role of monoamines by c.elegans behaviour using olfactory learning and memory generation. A significant increase in memory was found in worms on providing tyramine extra-synaptically. Mechano-stimulation assay in presence of tyramine also correlates with behavioral alterations via omega turns and reversal of worm, suggesting escape response activity coordinated with motor neuronal stimulation (RIM). These results suggest that the monoaminergic molecular modulation in associated neurons facilitates alterations in memory formations. The top down approach of multisensory decision involving ASH and AWA is advocated by tyramine, this can also possibly alter preferences based on learning and memory in worms. Key words- Tyramine, SER-2, TYRA-2, Mechano-stimulation, Associative memory

A, Aswathy et al. (2021) International Worm Meeting "Neuronal circuits and molecular pathways involved in olfactory imprinting in Caenorhabditis elegans"

Thekkuveettil, Anoopkumar, A, Aswathy

[International Worm Meeting, 2021]

Olfactory imprinting is a type of behavioural alteration produced during the early developmental stages of an animal. Like imprinting, associative learning can also alter the behavioural pattern. Caenorhabditis elegans is an excellent model to study alterations in neuronal connectomes because of their precise mapping and well-defined neurotransmitter pathways. C. elegans has been found to adapt to several learning paradigms, including olfactory, temperature, and salt chemotaxis, as well as olfactory imprinting. This study tried to elucidate the connectomes and neuro-modulators involved in olfactory imprinting and associative learning and memory. Expression of sra-11 receptors is critical for imprinted memory in worms. The difference in associative learning and memory after imprinting also suggests that AIY interneurons play a critical role in both these memory formations. We used a set of mutants with connectome defects to elucidate the neuronal circuitries involved in memory formation. Our results show a significant overlap between imprinting and associative learning; both sharing common neuronal circuits such as AWC, AIY, and RIA neurons in C. elegans. These results suggest that animals with imprinted olfactory memory have a better ability to learn and form both long-term and short-term memory. Key words - Olfactory imprinting, connectome, neuromodulator, associative learning

Santhosh Kanakalatha, Rasitha et al. (2021) International Worm Meeting "Role of Insulin and Insulin-like pathway in Learning and Memory of Caenorhabditis elegans"

Thekkuveettil, Anoopkumar, Santhosh Kanakalatha, Rasitha

[International Worm Meeting, 2021]

Insulin is implicated in various neurodegenerative diseases including Alzheimer's Disease that primarily result in memory dysfunctions. Hence, it has been a topic of growing interest among scientists across the globe. We used the model organism Caenorhabditis elegans to elucidate the role of insulin pathway in the learning and memory processes. In C. elegans, the insulin pathway is mediated by a number of insulin peptides and a receptor, daf-2. Our approach included an appetitive memory paradigm associated with an odor to analyse the learning and memory alterations in this model system. Our study involving both short- and long-term trainings showed that insulin affects learning and memory in a differential manner. Post- dauers with a developmental history of downregulated insulin pathway showed a defective memory recall. However, some mutants in the initial pathway of insulin, like the ligand ins-1 and the receptor daf-2, show better retention of memory. We were also able to show that this altered memory is not through the known Akt-1 pathway. In our study, we also found that excess insulin antagonises the pathway and shows extended memory. Our studies also point towards the implications of dopamine in the process. The calcium imaging data also correlates with the role of dopamine neurons in this memory. This work establishes the role of the insulin pathway in learning and memory and that insulin pathway modifies the learning and memory of the worms. Key Words: insulin, Learning and memory, ins-1, daf-2, dauers

Jetto, Cuckoo et al. (2017) International Worm Meeting "Spontaneous nucleolysis occurs during organismal death in Caenorhabditis elegans."

Jetto, Cuckoo, Raj, Vishnu, Nair, Agrima, Vijayan, Bejoy, Thekkuveettil, Anoopkumar

[International Worm Meeting, 2017]

Necrotic cell death has been extensively studied for its involvement in many human diseases. In C elegance change in pH or temperature induces organismal death and is marked by a characteristic wave of necrotic cell death of worm's digestive track; a phenomenon also marked with a blue fluorescence (called death fluorescence or DF) due to anthranilate release from stored vesicles. One of the critical events in this pathway is the increase in cytoplasmic calcium levels in the digestive track, which is essential for both for the development of DF as well as for the organismal death. But whether the organismal death, being a spontaneous event, follows the necrotic cell death pathway involving calcium-triggered activation of calpain and cathepsin is not clear. Here we report a uniquely different mechanism in organismal death through calcium-regulated calpain proteases, bypassing the cathepsin/ aspartyl protease pathway. Our findings also show that nucleolysis is an immediate intracellular change occurs during organismal death and is a calcium-dependent process. These cellular responses during organismal death are unique, compare to necrotic neurodegeneration in C. elegans and could have significance in the onset of organismal death.

Mangalath, Aswathy et al. (2017) International Worm Meeting "Imprinting enhances learning and memory in C. elegans."

Raj, Vishnu, Santhosh, Rasitha, Mangalath, Aswathy, Nair, Agrima, Thekkuveettil, Anoopkumar

[International Worm Meeting, 2017]

Imprinted memory, thought to be an epigenetic phenomenon, develops during a critical period in the early development. This form of memory is an unconditional learning and can last even in adulthood. The role of imprinting in normal learning and memory process is however not known. We addressed this question by inducing imprinted olfactory memory in C. elegans and assayed for its impact in olfactory learning and memory. Imprinted wild type N2 worms showed a better learning and memory compared to naive. Sra-11 mutants, a known imprinting defective strain, however, showed significantly better learning. However, cima-1 mutants, defective in AIY presynaptic G-protein coupled receptor showed both imprinting and learning defect. These results suggested that though both imprinting and learning share same neuronal circuits, the molecular pathways could be unique in both. Besides, results suggest that imprinting involves both sra-11 and cima-1 receptors localized at post- and pre-synaptic terminal of AIY interneuron, respectively, connecting AWC and RIA.

Santhosh, Rasitha et al. (2017) International Worm Meeting "Insulin pathway differentially alters learning and memory in Caenorhabditis elegans."

Suresh, Swathy, Mangalath, Aswathy, Raj, Vishnu, Nair, Agrima, Santhosh, Rasitha, Thekkuveettil, Anoopkumar

[International Worm Meeting, 2017]

Dauer constitutive (Daf-c) mutants of C. elegans have been extensively studied as a model to understand the regulatory molecules involved in organismal development and longivity. Recently one of the longevity extending factor, insulin/IGF-1 receptor mutant Daf-2 have been reported to have enhanced learning and memory. To elucidate this further, we tested butanone-associated appetitive learning in insulin mutant ins-1 and found enhanced learning in this strain similar to that of Daf-2. However, daf-16, akt-1 and age-1 mutants did not show a significant reduction in learning, suggesting the limited involvement of Daf-2 downstream pathways in learning and memory. Surprisingly, associative-learning in post-dauer N2 strains showed a significant reduction in learning and memory paradigm compared to the control wild-type adults. Furthermore, 24-hour starved worms also showed significantly low learning and memory. These results suggest that the learning deficiency could be linked to the environment or epigenetic factors during early development of an organism. Besides, AWC sensory neuron, which is known to express the insulin receptor Daf-2 might have a regulatory role in integrating various cues to determine the learning and memory capabilities of the organism.

Nair, Agrima et al. (2017) International Worm Meeting "Connectome specific storage of short-term and long-term memory in Caenorhabditis elegans."

Thekkuveettil, Anoopkumar, Nair, Agrima, Wilson, Amal, Mangalath, Aswathy, Raj, Vishnu, Santhosh, Rasitha

[International Worm Meeting, 2017]

Molecular pathways involved in memory formation both long-term and short-term are still unknown, while the involvement of synaptic plasticity in these pathways has been documented. C.elegans show distinct short-term and long-term associated memory (STAM and LTAM) towards butanone. Here we use this two forms of memory and assay the connectomes involved in the pathways. Blocking translation using actinomycin-D affected the STAM and LTAM, while blocking transcription using cyclohexamide blocked LTAM and maintained STAM. The role of NMDA receptors was probed using nmr-1 and -2 mutants as well as using AP-5, a NMDA receptor antagonist. All these mutants and the AP-5 treated N2 worms showed a selective loss of LTAM. AMPA receptor glr-2 mutants, however, showed significantly low STAM and LTAM as compare to the control. Interestingly AMPA receptor glr-1 mutants showed STAM. This suggests the existence of selective neuronal circuits in C. elegans for short term and long term memory formation. To test this possibility, we assayed sra-11 mutant (AIY neuron-specific protein) and tdc-1 mutant (RIM neuron-specific protein). LTAM showed significant impairment in both these strains while STAM was similar to that of the controls, confirming the involvement of connectome in memory storage.

Raj, Vishnu et al. (2017) International Worm Meeting "Degeneration of dopamine neurons affects memory formation in Caenorhabditis elegans."

Mangalath, Aswathy, Raj, Vishnu, Santhosh, Rasitha, Nair, Agrima, Thekkuveettil, Anoopkumar, Vijayan, Bejoy

[International Worm Meeting, 2017]

Degeneration of dopamine neurons often found to develop dementia as a comorbidity. It is not deciphered, however, the involvement of the dopamine connectome in memory formation as well as in dementia. Caenorhabditis elegans is an excellent model system to study the connectome variations because of its elementary and well-mapped nervous system. The four CEP neurons in the head region of the organism use dopamine as their neurotransmitter and play a critical role in the movement. To understand the role of dopamine circuit in learning, we used adaptive learning paradigm of Caenorhabditis elegans to olfactory cues. Significantly low associative learning towards butanone was observed in mutant DAT-1::ICE, which develops dopamine neuron degeneration in their late larval stages. These results were comparable to that of mutants of str-2, a G-protein coupled receptor in AWC neurons, having a significantly low associative learning. A similar pattern of learning defects were observed in UA-44 strain with age-associated dopamine neurodegeneration. However, Cat-2 and Tph-1 mutants, which are defective in dopamine and serotonin synthesis respectively, showed no significant changes in learning pattern. These findings suggest dopamine connectome have a critical role in memory formation.