Despite being integral to the practice of surgery, the mechanisms by which general anesthetics mediate their effects remain unknown. For this reason, it is difficult to predict adverse side-effects and to determine how treatment should be modified for specific patient populations. Recent clinical studies have reported post-operative neuropsychological and behavioral abnormalities in children and protracted periods of post-operative cognitive decline in elderly patients. Definitively linking these post-operative consequences to the agents used to induce anesthesia has been difficult, due to a lack of proper clinical controls and an abundance of confounding health factors. Animal studies, have repeatedly shown that general anesthetics can be neurotoxic and lead to lasting impairments in learning and memory acquisition in both the very young and old. However, the scope and causes of these post-exposure impairments and the reasons why age seems to measurably affect outcomes remain unclear. Here we employ multi-neuronal fluorescence imaging in the nematode Caenorhabditis elegans to measure changes in neuronal activity and connectivity across the animal’s nervous system, following exposure to the volatile anesthetic isoflurane during neurodevelopment and senescence. Employing transgenic expression of the fluorescent calcium indicator GCaMP6s, we measure neuronal activity of specific command interneurons as well as across the majority of the nervous system with single cell resolution. Isoflurane exposure during developing, results changes in the transition rate between neuronal activity states and an overall increase in excitatory connectivity. Importantly these effects are dependent on cellular stress pathways involved mTOR and daf-16 but not on apoptotic cell death (medatied by ced-3). Measuring neuronal activity across the animals lifespan, we identify substantial age-related alterations to neural activity, connectivity and functional organization of the system. These include a progressive 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 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. We further identify genes that either hasten or delay the progression toward senescent neural activity patterns, including the presynaptic voltage-gated Ca2+ channel UNC-2/CaV2, and also CED-4, a key mediator of the conserved cell-death pathway. Finally, imaging post-exposure consequences of isoflurane during senescence reveals long term effects on neuronal signaling that involved a decrease in excitatory connectivity, the opposite of what is observed during development. We conclude that anesthetic exposure during development cause permanent alteration in neuronal activity and signaling which involves cellular stress pathways but that these effects are distinct from long-term effect of anesthetic exposure we observe in age animals. Our studies also begin to define the changes in neuronal dynamics with age and demonstrate the importance of excitatory/inhibitory balance in this processes. Through comprehensive multi-neuronal imaging in C. elegans, we are able to measure the progressive breakdown of neuronal activity and system dynamics with age and isoflurane exposure and begin to identify the cellular processes and changes in synaptic signaling that contribute to these declines. Moreover, we leverage this platform to gain insight into the age-dependency of isoflurane-induced insult to neural systems.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/45520 |
| Date | 25 January 2023 |
| Creators | Wirak, Gregory Scott |
| Contributors | Gabel, Christopher V. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-NonCommercial-ShareAlike 4.0 International, http://creativecommons.org/licenses/by-nc-sa/4.0/ |
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