Breakdown of Neural Function under Isoflurane Anesthesia: In Vivo, Multineuronal Imaging in Caenorhabditis elegans

Abstract

What we already know about this topic: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Previous work on the action of volatile anesthetics has focused at either the molecular level or bulk neuronal measurement such as electroencephalography or functional magnetic resonance imaging. There is a distinct gulf in resolution at the level of cellular signaling within neuronal systems. The authors hypothesize that anesthesia is caused by induced dyssynchrony in cellular signaling rather than suppression of individual neuron activity.

Methods: Employing confocal microscopy and Caenorhabditis elegans expressing the calcium-sensitive fluorophore GCaMP6s in specific command neurons, the authors measure neuronal activity noninvasively and in parallel within the behavioral circuit controlling forward and reverse crawling. The authors compare neuronal dynamics and coordination in a total of 31 animals under atmospheres of 0, 4, and 8% isoflurane.

Results: When not anesthetized, the interneurons controlling forward or reverse crawling occupy two possible states, with the activity of the "reversal" neurons AVA, AVD, AVE, and RIM strongly intercorrelated, and the "forward" neuron AVB anticorrelated. With exposure to 4% isoflurane and onset of physical quiescence, neuron activity wanders rapidly and erratically through indeterminate states. Neuron dynamics shift toward higher frequencies, and neuron pair correlations within the system are reduced. At 8% isoflurane, physical quiescence continues as neuronal signals show diminished amplitude with little correlation between neurons. Neuronal activity was further studied using statistical tools from information theory to quantify the type of disruption caused by isoflurane. Neuronal signals become noisier and more disordered, as measured by an increase in the randomness of their activity (Shannon entropy). The coordination of the system, measured by whether information exhibited in one neuron is also exhibited in other neurons (multiinformation), decreases significantly at 4% isoflurane (P = 0.00015) and 8% isoflurane (P = 0.0028).

Conclusions: The onset of anesthesia corresponds with high-frequency randomization of individual neuron activity coupled with induced dyssynchrony and loss of coordination between neurons that disrupts functional signaling.

Figure 1. Gross and functional neuroanatomy of the C. elegans model

A. C. elegans, standard bright field microscopy. B. Confocal fluorescent image of the C. elegans head, demonstrating the expression of a pan-neuronal calcium-sensitive green marker (GCaMP) and a red nuclear marker (RFP). Representative of the region of interest marked in Figure 1A. C. Neurons AVA, AVB, AVD, AVE and RIM identified under selective expression of GCaMP in the QW1574 worm. D. Schematic diagram of the C. elegans neuronal circuit controlling forward/backward crawling.

Figure 2. Disruption of C. elegans behavior under isoflurane anesthesia

A. Average crawling speed at indicated levels of isoflurane. B. Examples of crawling tracks with and without exposure to isoflurane. C. Touch avoidance response measured as % of animals responding (crawling reversal) to a mechanical buzz at indicated levels of isoflurane.

Figure 3. Functional imaging of the command interneurons AVA, AVD, AVE and RIM, showing spontaneous activity at varying levels of anesthesia. Example traces of individual animals are displayed (left), along with a histogram (right) of the normalized neuronal signals across all animals measured under that condition. The oppositional activity of AVB is shown in the unanesthetized state

A. Control, unanesthetized (0%vol isoflurane) B. At moderate levels of anesthesia (4%vol isoflurane) C. At deep levels of anesthesia (8%vol isoflurane)

Figure 4. Correlation heatmaps displaying the correlation coefficient (SD) between neurons AVA, AVD, AVE and RIM at varying levels of anesthesia. AVB is shown only in the unanesthetized state

A. Control, unanesthetized (0%vol isoflurane) B. At moderate levels of anesthesia (4%vol isoflurane) C. At deep levels of anesthesia (8%vol isoflurane) D. The averaged absolute correlation value between neuron pairs AVA, AVD, AVE and RIM across all worms measured at each condition. (Separable with statistical significance as shown.)

Figure 5. Mean power spectra of neuronal dynamics at varying levels of anesthesia

Mean power spectra were measured across all neurons imaged at 0%vol, 4%vol, 8%vol isoflurane. In addition, measurements from animals expressing GFP in the imaged neurons are displayed as controls. Insert: The cumulative normalized power spectra measured at 0%vol and 4%vol isoflurane.