Elsevier

Consciousness and Cognition

Volume 44, August 2016, Pages 72-88
Consciousness and Cognition

Review article
What is unconsciousness in a fly or a worm? A review of general anesthesia in different animal models

https://doi.org/10.1016/j.concog.2016.06.017Get rights and content

Highlights

  • All animals are immobilized by volatile general anesthetics.

  • Complex behaviors are more sensitive to volatile general anesthetics.

  • Loss of consciousness and total loss of behavioral responsiveness may represent distinct mechanisms of general anesthesia.

  • Loss of responsiveness may involve a presynaptic process.

Abstract

All animals are rendered unresponsive by general anesthetics. In humans, this is observed as a succession of endpoints from memory loss to unconsciousness to immobility. Across animals, anesthesia endpoints such as loss of responsiveness or immobility appear to require significantly different drug concentrations. A closer examination in key model organisms such as the mouse, fly, or the worm, uncovers a trend: more complex behaviors, either requiring several sub-behaviors, or multiple neural circuits working together, are more sensitive to volatile general anesthetics. This trend is also evident when measuring neural correlates of general anesthesia. Here, we review this complexity hypothesis in humans and model organisms, and attempt to reconcile these findings with the more recent view that general anesthetics potentiate endogenous sleep pathways in most animals. Finally, we propose a presynaptic mechanism, and thus an explanation for how these drugs might compromise a succession of brain functions of increasing complexity.

Introduction

General anesthesia is a drug-induced, reversible state of decreased responsiveness, capable of being induced in all animals, no matter how simple their nervous system. In humans, there are several key components of general anesthesia, termed anesthetic endpoints, which encompass the changes in perception or behavior that are essential to general anesthesia: immobility, amnesia, analgesia and loss of consciousness (Antognini & Carstens, 2002). These endpoints, particularly the perception of pain and loss of consciousness, are typically only associated with humans or higher mammals, so how comparable are these endpoints across the animal kingdom? This question is particularly relevant for simpler invertebrate model organisms, such as the fruit fly (fly, Drosophila melanogaster), which has a tiny brain, or the nematode (worm, Caenorhabditis elegans), which does not even have a brain. What does ‘unconsciousness’ look like in a fly, or a worm?

Here, we outline human general anesthesia endpoints and compare these with endpoints in three model organisms, with the aim to identify the likely analogues for human loss of consciousness in these different animal models. Our purpose is not to outline a molecular characterisation of anesthetic endpoints, which has been extensively discussed elsewhere (e.g., (Chau, 2010, Franks, 2006, Franks, 2008, Rudolph and Antkowiak, 2004)). Rather, we offer a hypothesis to explain the progression of behaviors that are reversibly abolished by volatile general anesthetics that relates to behavioral complexity: with increasing anesthetic dose, more complex behaviors, requiring coordination among multiple neuronal pathways, are lost first, and more simple behaviors require a higher anesthetic concentration to be attenuated. We propose that this pattern reflects a common presynaptic target of general anesthetics, and that different anesthetic endpoints reflect successive categories of synaptic coordination required for different behaviors. Our hypothesis does not imply a unitary theory of action of anesthetics, but rather offers an explanation for how different brain functions are affected in succession through disruption of synaptic coordination by general anesthetics. Consciousness is but one of these functions in humans, so should not be central to defining general anesthesia.

  • What is general anesthesia?

General anesthesia encompasses various behavioral and physiological traits, termed endpoints. These endpoints are quantitative measures of arousal, ranging from behaviors to physiological signals, which are attenuated and potentially abolished (reversibly) by general anesthetics. As such, numerous endpoints could be studied under general anesthesia, from heartbeat, to haemoglobin function to pain, but which endpoints are relevant for surgery to proceed? Moreover, some endpoints are not single behaviors but rather can encompass a whole set of functionally related behaviors. For example, in considering the immobility endpoint of general anesthesia in animals, in response to a painful tail-clamp stimulus, many investigators consider a pawing motion or movement of the head towards the stimulus as a positive response to the stimulus, meaning it is gross, purposeful movement. In contrast, increased breathing, coughing, swallowing, chewing, the stiffening or simple withdrawal of the limb is considered a negative response. General anesthetics can also produce some undesired endpoints, such as cardiovascular and respiratory depression, nausea, and in extreme cases, death. Thus there is a need to accurately define general anesthetic endpoints, and the behaviors these entail, especially in animal models used to understand mechanisms of general anesthesia.

Since their introduction into medical practice in the mid-nineteenth century, there was a need for an accurate measure to compare anesthetic potencies to ensure accurate dosing. Indeed, some of first recorded anesthetic procedures performed with the inhalational anesthetics ether and chloroform resulted in patient deaths, which most likely stemmed from over-dosing (Jacob, Kopp, Bacon, & Smith, 2013). Early measures assessed anesthetic depth based on reflexes, such as eye-blinking, and changes in breathing and muscle movement and tone (Guedel, 1937, Woodbridge, 1957). These measures proved to be unreliable between different inhalational compounds, limiting their clinical utility. In the early 1960s, Eger and colleagues in their research on both dogs and human patients introduced the concept of minimum alveolar concentration or MAC as a measure of general anesthesia depth (Merkel & Eger, 1963). MAC is defined as the minimum alveolar concentration of an inhalational anesthetic at which 50% of subjects do not respond to surgical incision. Importantly, Eger and colleagues showed that MAC was remarkably consistent, with anesthesia induced at a very similar dose in the dogs. Also, increasing the intensity of the stimulus did not change MAC. That is, administering two painful stimuli to the dogs, an electric shock and tail-clamp, did not increase anesthetic requirements. This suggests MAC is a reliable measure of general anesthetic potency.

Skin-incision remains the standard stimulus in humans for determining MAC, asking: “When this noxious stimulus is applied, is there any movement?” Movement is often defined as gross, purposeful movement, and thus simple reflexes are not considered. The definition of MAC closely ties this measure with immobility, a cardinal feature of general anesthesia. Since MAC represents the concentration at which 50% of subjects no longer respond to the surgical incision, MAC therefore reflects another common measure in anesthesia research: it is a general anesthetic 50% effective concentration, or EC50.

How are these EC50s calculated? In the case for human patients, MAC is measured with a quantal study design (Sonner, 2002). Patients are exposed to an anesthetic dose for a set-period of time, after which a skin incision is administered. The resulting response is assigned a quantal, or categorical response of “move” or “no-move”. Plotting the percentage of patients responding as a function of increasing anesthetic concentration creates a dose-response curve (Eger et al., 1965, Saidman et al., 1967). MAC represents a single concentration on this dose-response curve at which 50% of patients no longer respond to the noxious stimulus (Fig. 1). MAC is 1.17 vol% for isoflurane in humans (Mapleson, 1996). In humans, the standard deviation of MAC is around 10% (de Jong & Eger, 1975), meaning that at concentrations 2 standard deviations around MAC, 95% of patients will not respond to the noxious stimulus. This narrow concentration range (0.8–1.2 MAC) reflects the high reproducibility of anesthetic effects between individuals. Since MAC measures are highly reproducible within subjects, this highlights the clinical value of this anesthetic endpoint.

Before discussing general anesthesia endpoints across animal species, it is worth noting an important distinction between anesthesia studies in humans and non-humans. Whereas human studies use the quantal design mentioned above, animal experiments usually implement a bracketed design, where MAC is calculated for each individual animal. The response or lack of response to an anesthetic concentration is noted, as with the quantal design. However, additional measures are made. If the animal moved at the initial concentration, the dose is increased in step-wise fashion until no response is observed. MAC for an individual reflects the average of the highest concentration that permitted movement, and the lowest concentration that prevented movement. For a group of animals, the MAC of individual animals is averaged. Importantly, Sonner (2002) showed that both the quantal and bracketed designs give the same MAC (response to tail-clamp) in mice, highlighting the utility of comparisons between human and non-human anesthesia studies.

What physiological effects has MAC been associated with? There is a large body of research showing that inhaled anesthetics achieve immobility, which is closely associated with MAC, through their actions on the spinal cord. For example, the activity of spinal sensory (Yamauchi, Sekiyama, Shimada, & Collins, 2002), and motor neurons (Rampil & King, 1996), and also central pattern generators are depressed by inhaled anesthetics (Jinks, Atherley, Dominguez, Sigvardt, & Antognini, 2005). Even when brain activity is in burst suppression induced by isoflurane (Rampil & Laster, 1992), or if the cortex and thalamus are removed (Rampil, Mason, & Singh, 1993), movement in response to a noxious tail-clamp stimulus in mice is still possible. These studies indicate that the spinal cord is important in generating movement in response to a noxious stimulus, yet the spinal cord is obviously less relevant for mediating loss of consciousness.

Antognini and colleagues have demonstrated that the brain alone has greater anesthetic requirements than the brain and spinal cord together. These studies were done in goats, due to their unique circulatory system that allows cerebral vascular isolation (Reimann, Lluch, & Glick, 1972). The amount of isoflurane required to anesthetize goats, and prevent movement to a painful forelimb clamp stimulus is 1.2 vol% isoflurane (MAC). When isoflurane is preferentially delivered to the brain alone, MAC increased to around 3 vol%. This is in stark contrast to only 0.3 vol% isoflurane being required to suppress movement if isoflurane is delivered to the body, and hence spinal cord, alone (Antognini & Schwartz, 1993). Studies in rats lend further support to the differential anesthetic requirements of the brain and spinal cord. Precollicular decerebration (Rampil et al., 1993) or complete severing of the upper thoracic spinal cord (Rampil, 1994) has minimal effect on MAC. These studies suggest the immobility endpoint depends primarily on spinal cord activity, and highlights the need to separate endpoints that will involve the brain, such as consciousness-related endpoints, from those that just involve movement.

Having a reliable, repeatable measure of general anesthesia is essential for comparison both within and between subjects. MAC provides such a measure, and remains the benchmark in measuring anesthetic depth, particularly for immobility. But what about measuring general anesthesia in organisms that do not have lungs, a spinal cord or even a brain?

Section snippets

Volatile anesthetics produce immobility at different concentrations across the animal kingdom

Since general anesthetics affect all animal species (Humphrey, Sedensky, & Morgan, 2002), we now highlight what concentrations of volatile anesthetics are required for MAC, a reliable measure of anesthetic potency, across the animal kingdom. For invertebrate species in our survey, we consider the endpoint of immobility, to be as similar to human MAC as possible. We restrict our survey to two volatile anesthetics: isoflurane and halothane. Isoflurane and halothane EC50 concentrations required

General anesthesia endpoints depend on behavioral complexity across animal species

The concentration of volatile anesthetic required to produce immobility is but a snapshot of the endpoints that describe general anesthesia. There are actually several endpoints of general anesthesia, which are more sensitive than immobility, which can be defined across all species. We now canvass what is known in three animal species that have greatly advanced our understanding of general anesthesia: the rodent model, the fly and the worm, comparing endpoints in these animals to what has been

Electrophysiological measures of general anesthesia endpoints also show a progression of effects under general anesthesia

In mammals, general anesthetics affect many electrophysiological measures of arousal, and this has been amply discussed (Cimenser et al., 2011, John and Prichep, 2005, Ku et al., 2011, Lee et al., 2009, Purdon et al., 2013). For example, brain activity in the first stages of anesthesia strongly resembles those seen in sleep (Brown, Lydic, & Schiff, 2010), supporting the thesis that anesthetics first activate sleep pathways. As anesthesia deepens, there is reduced functional connectivity between

Conclusion

General anesthesia is a reversible state of decreased behavioral responsiveness that can be defined in all animals. Here, we have compared general anesthesia in four animal species: humans, and three model organisms: mice, flies and nematodes. We propose that behavioral or neuronal circuit complexity can explain the order in which various endpoints are reversibly abolished by general anesthetics. However, the initial entry into the behaviorally anesthetized state seems to require activation of

Acknowledgment

This work was funded by the Australian Research Council (FT100100725), the National Health and Medical Research Council (APP1103921), and The Queensland Brain Institute.

References (153)

  • B. Kottler et al.

    A sleep/wake circuit controls isoflurane sensitivity in Drosophila

    Current Biology

    (2013)
  • T. Lebestky et al.

    Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits

    Neuron

    (2009)
  • U. Lee et al.

    The directionality and functional organization of frontoparietal connectivity during consciousness and anesthesia in humans

    Consciousness and Cognition

    (2009)
  • B.W. Levinson

    States of awareness during general anaesthesia. Preliminary communication

    British Journal of Anaesthesia

    (1965)
  • J.S. Lin et al.

    A critical role of the posterior hypothalamus in the mechanisms of wakefulness determined by microinjection of muscimol in freely moving cats

    Brain Research

    (1989)
  • J.E. MacKenzie

    Determination of MAC for halothane, cyclopropane and ether in the rabbit

    British Journal of Anaesthesia

    (1977)
  • W.W. Mapleson

    Effect of age on MAC in humans: A meta-analysis

    British Journal of Anaesthesia

    (1996)
  • J.T. Moore et al.

    Direct activation of sleep-promoting VLPO neurons by volatile anesthetics contributes to anesthetic hypnosis

    Current Biology

    (2012)
  • D.E. Newton et al.

    Levels of consciousness in volunteers breathing sub-MAC concentrations of isoflurane

    British Journal of Anaesthesia

    (1990)
  • D.A. Nitz et al.

    Electrophysiological correlates of rest and activity in Drosophila melanogaster

    Current Biology

    (2002)
  • T.M. Ramage et al.

    Distinct long-term neurocognitive outcomes after equipotent sevoflurane or isoflurane anaesthesia in immature rats

    British Journal of Anaesthesia

    (2013)
  • M.T. Alkire et al.

    Relative amnesic potency of five inhalational anesthetics follows the Meyer-Overton rule

    Anesthesiology

    (2004)
  • M.T. Alkire et al.

    Consciousness and anesthesia

    Science

    (2008)
  • R. Allada et al.

    Drosophila melanogaster as a model for study of general anesthesia: The quantitative response to clinical anesthetics and alkanes

    Anesthesia and Analgesia

    (1993)
  • J.F. Antognini et al.

    Exaggerated anesthetic requirements in the preferentially anesthetized brain

    Anesthesiology

    (1993)
  • M. Bali et al.

    Defining the propofol binding site location on the GABAA receptor

    Molecular Pharmacology

    (2004)
  • A.B. Barron et al.

    What insects can tell us about the origins of consciousness

    Proceedings of the National Academy of Sciences of the United States of America

    (2016)
  • J.P. Baumgart et al.

    Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling

    Proceedings of the National Academy of Sciences of the United States of America

    (2015)
  • R.J. Blanchard et al.

    Crouching as an index of fear

    Journal of Comparative and Physiological Psychology

    (1969)
  • K. Bonath et al.

    Experimental studies on the clinical use and control of inhalation anesthesia in reptiles

    Zentralblatt für Veterinärmedizin. Reihe A

    (1979)
  • V. Bonhomme et al.

    Neural correlates of consciousness during general anesthesia using functional magnetic resonance imaging (fMRI)

    Archives Italiennes de Biologie

    (2012)
  • V. Bonnet et al.

    Analyse oscillographique des depressions fonctionnelles de la substance grise spinale

    International Archives of Physiology and Biochemistry

    (1948)
  • F. Bremer et al.

    Action particuliere des barbituriques sur la transmission synaptique centrale

    International Archives of Physiology and Biochemistry

    (1948)
  • B.R. Brown et al.

    A comparative study of the effects of five general anesthetics on myocardial contractility. I. Isometric conditions

    Anesthesiology

    (1971)
  • E.N. Brown et al.

    General anesthesia, sleep, and coma

    New England Journal of Medicine

    (2010)
  • E.N. Brown et al.

    General anesthesia and altered states of arousal: A systems neuroscience analysis

    Annual Review of Neuroscience

    (2011)
  • D.B. Campbell et al.

    Use of Drosophila mutants to distinguish among volatile general anesthetics

    Proceedings of the National Academy of Sciences of the United States of America

    (1994)
  • J.L. Campbell et al.

    The visually-induced jump response of Drosophila melanogaster is sensitive to volatile anesthetics

    Journal of Neurogenetics

    (1998)
  • M. Chalfie et al.

    The neural circuit for touch sensitivity in Caenorhabditis elegans

    Journal of Neuroscience

    (1985)
  • J.P. Changeux

    Conscious processing: Implications for general anesthesia

    Current Opinion in Anesthesiology

    (2012)
  • P.L. Chau

    New insights into the molecular mechanisms of general anaesthetics

    British Journal of Pharmacology

    (2010)
  • S. Chennu et al.

    Brain connectivity dissociates responsiveness from drug exposure during propofol-induced transitions of consciousness

    PLoS Computational Biology

    (2016)
  • B.S. Chortkoff et al.

    Subanesthetic concentrations of isoflurane suppress learning as defined by the category-example task

    Anesthesiology

    (1993)
  • A. Cimenser et al.

    Tracking brain states under general anesthesia by using global coherence analysis

    Proceedings of the National Academy of Sciences of the United States of America

    (2011)
  • C.M. Crowder et al.

    Behavioral effects of volatile anesthetics in Caenorhabditis elegans

    Anesthesiology

    (1996)
  • R.H. de Jong et al.

    MAC expanded: AD50 and AD95 values of common inhalation anesthetics in man

    Anesthesiology

    (1975)
  • J.E. Deady et al.

    Anesthetic potencies and the unitary theory of narcosis

    Anesthesia and Analgesia

    (1981)
  • J.M. Donlea et al.

    Inducing sleep by remote control facilitates memory consolidation in Drosophila

    Science

    (2011)
  • J.C. Drummond

    MAC for halothane, enflurane, and isoflurane in the New Zealand white rabbit: And a test for the validity of MAC determinations

    Anesthesiology

    (1985)
  • S.L. Dubovsky et al.

    Absence of recall after general anesthesia: Implications for theory and practice

    Anesthesia and Analgesia

    (1976)
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