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.

Figure 1:. Multineuron imaging and tracking in the head region of pan-neuronally labeled C. elegans (QW1217) over time. Images are of 3D volumetric data rendered in 2D by Maximum Intensity Projection along the Z-axis.

A) Image of C. elegans head region via light sheet imaging. B) As in (A), but nuclei tracked for automated signal extraction are displayed. Nuclei are tracked in all three dimensions, but shown here in 2D projection. C-H) Select frames from a single imaging trial, showing variation in GCaMP6s fluorescence (green), which reports neuronal activity.

Figure 2:. Neuronal Activity Distributions and Spectral Analysis.

A) Intensity trace of fluorescence activity of all 120 tracked neurons in an individual C. elegans at 0% (left), 4% (middle), and 8% (right) isoflurane. The color scale covers 0% to 75% of the dynamic range of the normalized intensities in order to remove rare high level activations and render lower level activity in a more discernable fashion. The time range covers 30 to 300 seconds, to exclude exuberant fluorescence caused by initial illumination. B) Distribution of all fluorescence measurements pooled across all neurons and worms at each level of isoflurane exposure. C) Mean cumulative power spectral densities of neuron fluorescence signals at each level of isoflurane exposure. Cumulative power spectral densities for bulk fluorescence measurements in dashed lines. The 95% spectral edge shown is shown with a dotted gray line. (Inset) Mean power spectral density of neuron fluorescence signals at each level of isoflurane.

Figure 3:. Neuronal Correlations

A) Correlation of fluorescence signals for each neuron-neuron pair from the intensity traces in Figure 2A at 0% (left), 4% (middle), 8% (right) isoflurane. The color scale is red when a correlation exists, blue when anti-correlated, and white when no correlation is found. The leading diagonal is red as each neuron is correlated to itself by definition. B) Distributions of all correlation values measured at each level of isoflurane. (Inset) Mean correlation values at each level of isoflurane. Sample variability reported as standard deviation. (_***, p<0.0001; ns, not significant)

Figure 4:. System-wide Activity States

A) 3D plots of the first three principal components (PCs) calculated from the 120 individual neuron signals shown in Figure 2A at 0% (left), 4% (middle), and 8% (right). Principal component analysis traces are colored by the fluorescence intensity of the neuron AVA, with the same color scale as in Figure 2A. B) Mean percentage of total variance captured by the first three principal components for all neurons and for all worms at each level of isoflurane. Sample variability reported as standard deviation. (_*, p<0.01; _**, p<0.001; _****, p<0.00001)