Collapse of Global Neuronal States in Caenorhabditis elegans under Isoflurane Anesthesia

Abstract

Background A comprehensive understanding of how anesthetics facilitate a reversible collapse of system-wide neuronal function requires measurement of neuronal activity with single-cell resolution. Multineuron recording was performed in Caenorhabditis elegans to measure neuronal activity at varying depths of anesthesia. The authors hypothesized that anesthesia is characterized by dyssynchrony between neurons resulting in a collapse of organized system states. Methods Using light-sheet microscopy and transgenic expression of the calcium-sensitive fluorophore GCaMP6s, a majority of neurons (n = 120) in the C. elegans head were simultaneously imaged in vivo and neuronal activity was measured. Neural activity and system-wide dynamics were compared in 10 animals, progressively dosed at 0%, 4%, and 8% isoflurane. System-wide neuronal activity was analyzed using principal component analysis. Results Unanesthetized animals display distinct global neuronal states that are reflected in a high degree of correlation (R = 0.196 ± 0.070) between neurons and low-frequency, large-amplitude neuronal dynamics. At 4% isoflurane, the average correlation between neurons is significantly diminished (R = 0.026 ± 0.010; P < 0.0001 vs. unanesthetized) and neuron dynamics shift toward higher frequencies but with smaller dynamic range. At 8% isoflurane, interneuronal correlations indicate that neuronal activity remains uncoordinated (R = 0.053 ± 0.029; P < 0.0001 vs. unanesthetized) with high-frequency dynamics that are even further restricted. Principal component analysis of unanesthetized neuronal activity reveals distinct structure corresponding to known behavioral states. At 4% and 8% isoflurane this structure is lost and replaced with randomized dynamics, as quantified by the percentage of total ensemble variance captured by the first three principal components. In unanesthetized worms, this captured variance is high (88.9 ± 5.4%), reflecting a highly organized system, falling significantly at 4% and 8% isoflurane (57.9 ± 11.2%, P < 0.0001 vs. unanesthetized, and 76.0 ± 7.9%, P < 0.001 vs. unanesthetized, respectively) and corresponding to increased randomization and collapse of system-wide organization. Conclusions Anesthesia with isoflurane in C. elegans corresponds to high-frequency randomization of individual neuron activity, loss of coordination between neurons, and a collapse of system-wide functional organization. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

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“…off transitions) were manually identified by clear rises and falls in GCaMP6s fluorescence intensity. As previously described (Wirak et al, 2020), nonlinear fitting of hyperbolic tangent functions to these on and off transitions facilitated the unbiased analysis of activity state transition properties and eliminated noise. To determine the Rise Time of a given on transition, its fitted function was used to calculate the time required to rise from 5% of the maximum fluorescence above baseline to 95%.…”

“…Confocal fluorescence microscopy was performed as previously described (Wirak et al, 2020) to capture spontaneous in vivo activity of the premotor interneuron AVA. In short, worms were briefly paralyzed in 5 mM tetramisole immediately prior to encasement in a transparent and permeable polyethylene hydrogel (Burnett et al, 2018).…”

“…This is made visually apparent by overlaying and averaging the individual calcium transient onsets (Fig1F). Transient rise times were calculated as the time required to shift from 5% to 95% of the maximum fluorescence (as previously described (Wirak et al, 2020), see Statistical Methods). A progressive increase in AVA Rise Time is observed through days 1-12 of nematode adulthood (Fig1G).…”

“…Recent advances in multi-neuron fluorescence imaging present an unprecedented ability to measure and understand the dynamics, function, and breakdown of small neuronal systems. Applied to the microscopic nematode Caenorhabditis elegans , such techniques enable measurement of neuronal activity across the majority of the nervous system with single cell resolution, revealing spontaneous behavioral state-dynamics (Kato et al, 2015) (Nguyen et al, 2016) (Venkatachalam et al, 2016) that are lost under unique circumstances, such as developmental quiescence or exposure to volatile anesthetics (Nichols et al, 2017) (Awal et al, 2020). Moreover, C. elegans is also a powerful system for the study of aging with longevity studies contributing much to our understanding of conserved molecular pathways affecting organismal aging (Mack et al, 2018).…”

Metabolomics and Whole-Exome Sequencing in Patients with Differential Sensitivity to Sevoflurane: A Protocol for a Prospective Observational Trial

“…Mutations of several genes are associated with altered sensitivity to volatile anesthetics in fruit flies, nematodes and mice ( Campbell et al, 2009 ; Nagasaka et al, 2017 ; Awal et al, 2020 ). The product of Gas-1 gene is a subunit of mitochondrial complex I ( Kayser et al, 2004 ).…”