Florman, Jeremy T. et al. (2021) International Worm Meeting "Chemical in vivo activation of C. elegans neurons using a histamine-gated cation channel"

Francis, Michael M., Florman, Jeremy T., Araya Budnik, Antonia, Alkema, Mark J.

[International Worm Meeting, 2021]

The ability to non-invasively activate and inactivate neurons in intact behaving animals provides powerful methods to study the functions of neural circuits. Recently, transgenic expression of a Drosophila melanogaster histamine-gated chloride channel (HisCl1) in C. elegans has enabled hyperpolarization of specific neurons in response to histamine (Pokala et al., 2014). The small molecule histamine is used as a transmitter in many species however C. elegans lack endogenous histamine signaling. We will present data to show that modification of the pore domain of this histamine-gated channel renders it selective to cations rather than anions. We find that cell specific expression of this modified channel, which we named HisCat1, enables histamine induced activation of neurons and can elicit specific behaviors. Pan-neuronal expression of HisCat1 induced uncoordinated seizure like behavior, whereas expression in cholinergic neurons caused hypercontraction and inhibited locomotion in animals exposed to histamine. When we expressed HisCat1 in serotonergic neurons, histamine exposure induced persistent pharyngeal pumping in the absence of food, consistent with the serotonin release. Finally, we demonstrate that HisCat1 can be used to depolarize non-neuronal tissues as expression in body-wall muscle induces calcium influx in response to histamine. Chemical activation using the HisCat1 channel provides a useful complement to existing optogenetic tools. Neuronal activation using HisCat1 appears to be stable over long durations, allowing prolonged activation that is more difficult to achieve with existing optogenetic methods. Furthermore, the HisCat1 channel can be combined with Channelrhodopsin and optical sensors such as GCaMP, so light and histamine can be used to activate and monitor several neurons independently in the same experiment. Therefore, HisCat1 is an effective means to non-invasively activate neurons in C. elegans and increases the existing toolkit of light induced sensors and ion channels. Pokala, N., Liu, Q., Gordus, A., Bargmann, C.I., 2014. Inducible and titratable silencing of Caenorhabditis elegans neurons in vivo with histamine-gated chloride channels. Proc. Natl. Acad. Sci. U. S. A. 111, 2770-5.

Wirak, Gregory et al. (2021) International Worm Meeting "Age-related decline of neuronal function is linked to a loss of inhibitory signaling in C. elegans"

Wirak, Gregory, Gabel, Christopher, Alkema, Mark, Connor, Christopher, Florman, Jeremy

[International Worm Meeting, 2021]

Neuronal function declines with age resulting in behavioral deficits. How this age-related decline is related to changes in neuronal connectivity and circuit function on the cellular level is largely unknown. Here we employ multi-neuronal fluorescence imaging in C. elegans to measure changes in neuronal activity and connectivity with age across the animal's nervous system. Employing transgenic expression of the fluorescent calcium indicator GCaMP6s in specific neurons, we identify changes in the activity of individual neurons and circuits linked to locomotory behaviors that are modulated by age. We further perform multi-neuronal imaging, employing light sheet microscopy to capture neuronal activity at single-cell resolution across the organism's entire head region. We identify substantial age-related alterations in the activity dynamics of neurons as well as changes in connectivity and functional organization. As the animals age, we observe a loss of system-wide organization and a corresponding shift in individual neuron activity toward higher frequencies. We also observe a specific loss of anti-correlative (i.e. inhibitory) signaling between neurons, resulting in an overall shift in the excitatory-inhibitory (E/I) balance of the system. In support of this, we find that application of the GABAA agonist muscimol diminishes certain aspects of nervous system decline in aged animals. Interestingly, age-related effects are recapitulated in young animals bearing a gain-of-function mutation within the unc-2 gene, encoding a pre-synaptic voltage-gated calcium channel. Conversely, aging effects are greatly diminished by loss-of-function mutations in either unc-2 or in ced-4. Ced-4 is a key mediator of the conserved apoptotic cell-death pathway and is known to play a role in synaptic elimination across multiple species. During development, UNC-2/CaV2 activity is known to trigger the removal of inhibitory GABAergic synapses in motor neurons through a CED-4 dependent pathway. Our results suggest that a similar process is occurring as the animals age, resulting in a loss of inhibitory signaling and disruption of the system dynamics. Through comprehensive multi-neuronal imaging, we are able to measure the progressive breakdown of neuronal activity and system dynamics with age and begin to identify the cellular processes and changes in synaptic signaling that contribute to this decline.

Veuthey, Tania et al. (2021) International Worm Meeting "Tyramine modulates the systemic stress response by stimulating the release of intestinal insulin like-peptides (ILPs)"

Rayes, Diego, Alkema, Mark, Giunti, Sebastian, Veuthey, Tania, De Rosa, Maria Jose, Florman, Jeremy

[International Worm Meeting, 2021]

Multicellular organisms trigger a complex and coordinated response against systemic stress. We have recently shown that in C. elegans, the neural stress-hormone tyramine supplies a state-dependent neural switch between acute flight- and long-term environmental-stress responses (De Rosa et al, 2019). Tyramine release during the flight response, stimulates the DAF-2/Insulin/IGF-1 signaling (IIS) pathway and precludes the nuclear translocation of the DAF-16/FOXO transcription factor through the activation of an adrenergic-like receptor TYRA-3 in the intestine. We hypothesize that tyramine stimulates the release of agonist ILPs from the intestine which acts as an autocrine and/or paracrine signal to systemically activate the DAF-2/IIS pathway. To test this hypothesis we are screening ILPs mutants for their resistance to environmental stressors (oxidative and thermal stress). The C. elegans genome encodes 40 ILPs, 28 of which expressed in the intestine. We performed a screening of intestinal peptides described as strong DAF-2 agonist, by silencing individual intestinal ILPs and testing worm resistance to environmental stressors (oxidative and thermal stress). Thus, so far we found that ins-3 and ins-7 mutants are resistant to environmental stress, like tyramine-deficient and tyra-3 mutants. We are further testing whether tyramine directly stimulates the release of these ILPs from the intestine through a Gq-protein mediated signaling pathway. These studies will provide insight into how a neurohormone coordinates systemic cellular stress responses.

Rayes, D. et al. (2019) International Worm Meeting "The flight response impairs cytoprotective mechanisms through neural inhibition of the insulin pathway."

Veuthey, Tania, Blanco, Gabriela, Alkema, Mark, Florman, Jeremy, Andersen, Natalia, Grant, Jeff, Rayes, D., De Rosa, Maria Jose

[International Worm Meeting, 2019]

An animal uses different survival strategies to cope with life-threatening situations. For instance, it can engage in a rapid and energy-demanding "fight-or-flight" response when encountering a predator, or it can induce the gradual and long-lasting activation of highly conserved cytoprotective processes in response to environmental stressors such as hypoxia, heat, oxidative stress, or food shortage. In animals across the evolutionary spectrum the continued activation of the fight-or-flight response weakens the animal's resistance to environmental challenges. In humans, for instance, the recurrent experience of stress in patients that suffer from post-traumatic stress disorder (PTSD) has been associated with decreased antioxidant capacity, accelerated aging and increased susceptibility to metabolic, cardiovascular and infectious diseases. However, the molecular and cellular mechanisms that regulate the trade-off between flight response and long-term stressors are poorly understood. Here we show that repeated induction of the C. elegans flight response shortens lifespan and inhibits conserved cytoprotective mechanisms. The flight response activates neurons that release tyramine, the invertebrate analog of adrenaline/noradrenaline. Tyramine stimulates the DAF-2/Insulin/IGF-1 pathway and precludes the induction of stress response genes by activating an adrenergic-like receptor in the intestine. In contrast, long-term environmental stressors, such as heat or oxidative stress, reduce tyramine release allowing the induction of cytoprotective genes. These findings demonstrate that a neural stress-hormone supplies a state-dependent neural switch between acute flight and long-term environmental stress responses and provides mechanistic insights into how the flight response impairs cellular defense systems and accelerates aging.