Review | Integrative Systems Integrative Neuroscience of Paramecium, a “Swimming Neuron” https://doi.org/10.1523/ENEURO.0018-21.2021 Cite as: eNeuro 2021; 10.1523/ENEURO.0018-21.2021 Received: 15 January 2021 Revised: 17 March 2021 Accepted: 18 March 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 Brette This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 1. Manuscript title: Integrative neuroscience of Paramecium, a “swimming neuron” 2 3 2. Abbreviated title: Integrative neuroscience of Paramecium 4 5 3. Author Names and Affiliations 6 Romain Brette 7 Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 8 75012 Paris, France 9 10 4. Author Contributions 11 RB wrote the paper, designed and performed research. 12 13 5. Correspondence should be addressed to Romain Brette, Institut de la Vision, 17, rue Moreau, 14 75012 Paris, France 15 [email protected] 16 17 6. Number of figures: 14 18 7. Number of Tables: 0 19 8. Number of Multimedia: 0 20 9. Number of words for Abstract : 67 21 10. Number of words for Significance Statement : 67 22 11. Number of words for Introduction : 916 23 12. Number of words for Discussion : 1035 24 25 13. Acknowledgements 26 None. 27 28 14. Conflict of Interest 29 The author reports no conflict of interest. 30 31 15. Funding sources 32 This work was supported by Agence Nationale de la Recherche (ANR-20-CE30-0025-01), by the 33 Programme Investissements d’Avenir IHU FOReSIGHT (ANR-18-IAHU-01), by Sorbonne Université 34 (Programme Emergence, grant NEUROSWIM) and by Fondation Pour l’Audition (FPA RD-2017-2). 35 1 36 37 Integrative neuroscience of Paramecium, a “swimming neuron” 38 39 Abstract 40 Paramecium is a unicellular organism that swims in fresh water by beating thousands of cilia. When 41 it is stimulated (mechanically, chemically, optically, thermally…), it often swims backward then turns 42 and swims forward again. This “avoiding reaction” is triggered by a calcium-based action potential. 43 For this reason, some authors have called Paramecium a “swimming neuron”. This review 44 summarizes current knowledge about the physiological basis of behavior of Paramecium. 45 46 Significance statement 47 Paramecium is a unicellular organism that swims in fresh water by beating thousands of cilia. When 48 it is stimulated (mechanically, chemically, optically, thermally…), it often swims backward then turns 49 and swims forward again. This “avoiding reaction” is triggered by a calcium-based action potential. 50 For this reason, some authors have called Paramecium a “swimming neuron”. This review 51 summarizes current knowledge about the physiological basis of behavior of Paramecium. 52 53 Introduction 54 Even the simplest behavior must engage at least a sensory organ, a large part of the nervous system, 55 the body (muscles, skeleton), and the environment. Thus, understanding the biological basis of 56 behavior requires an integrative approach, which remains highly challenging given the complexity of 57 both the nervous and musculoskeletal systems of vertebrates. A fruitful research strategy is to study 58 model organisms that are structurally simpler and have experimental advantages. For example, the 59 biophysical basis of excitability was studied in the giant axon of the squid (Hodgkin, 1964); the 60 molecular basis of learning and memory was studied in Aplysia (Kandel, 2009). A recent model 61 organism to develop integrative approaches to behavior is C. Elegans, with its 302 neurons and a 62 known connectome (Schafer, 2018). In C. Elegans, modeling the entire organism and its interaction 63 with the body and environment seems more feasible in principle (Cohen and Denham, 2019; Cohen 64 and Sanders, 2014). Nevertheless, even in this more favorable situation, developing functional and 65 empirically valid neuromechanical models of C. Elegans remains very challenging. Two other recently 66 introduced model organisms for this type of integrative work are Hydra, which has a few thousand 67 neurons and the advantage of being transparent (Dupre and Yuste, 2017; Wang et al., 2020), and 68 jellyfish Aurelia aurita (Pallasdies et al., 2019). Here I will present a model organism that is 69 significantly simpler as it consists of a single “neuron”. 70 71 Figure 1. Paramecium morphology. A, Scanning electron microscopy image of Paramecium tetraurelia (scale 72 bar: 10 µm) (Valentine et al., 2012). B, Paramecium caudatum (Herbert S. Jennings, 1899), a large species 2 73 (about 200 µm) with a pointed posterior end. a, anterior end; p, posterior end; g, oral groove; m, mouth; o, 74 oral side; ab, aboral side; cv, contractile vacuole. The drawing also shows food vacuoles and cilia. 75 Paramecium is a single-cell eukaryote, 100-300 µm long depending on species (Fokin, 2010) (Fig. 1), 76 which has long been a model organism for many aspects of eukaryotic biology (Görtz, 1988; 77 Wichterman, 1986). It is a ciliate that has been living in ponds and lakes all over the world for 78 hundreds of millions of years (Parfrey et al., 2011) - a fossil has been discovered in a 200 million 79 years old piece of amber (Schönborn et al., 1999). Its abundance and large size made it a popular 80 subject of behavioral study in the late 19th century; Jennings described his culture method as follows 81 (Jennings, 1897): “A handful of hay or grass is placed in a jar and covered with hydrant water. In a few 82 weeks the solution of decaying vegetable matter swarms with Paramecia.”. 83 Paramecium swims in fresh water by beating its thousands of cilia, and feeds on smaller 84 microorganisms such as bacteria and algae. It is a prey for other microorganisms such as Didinium. 85 As beautifully described by Jennings more than a century ago (1906), Paramecium lives in a rich 86 sensory environment: it finds food by detecting and following chemicals produced by decaying plants 87 and fellow paramecia; it moves towards the water surface by gravitaxis; it avoids obstacles thanks to 88 its mechanosensitivity; it resists water currents by rheotaxis; it avoids bright light; it avoids hot and 89 cold waters; it even communicates chemically. It typically swims in helicoidal paths interrupted by 90 abrupt changes in direction called avoiding reactions, which form the “trial-and-error” basis of its 91 behavior. When an unfavorable condition is met (obstacle, unwanted chemical), the avoiding 92 reaction is triggered (Fig. 2A): Paramecium swims backward for a brief time, then turns and swims 93 forward in a new direction. By this simple mechanism, Paramecium can navigate in crowded 94 multisensory environments. 95 96 Figure 2. The avoiding reaction triggered by an action potential. A, Avoiding reaction against an obstacle, as 97 illustrated by Jennings (Jennings, 1906). B, Action potential in response to a 2 ms current pulse (top), 98 recorded with the hanging droplet method (bottom) (Naitoh et al., 1972). 99 This avoiding reaction is triggered by an action potential produced by voltage-gated calcium 100 channels located in the cilia (Fig. 2B) (Eckert, 1972). These are L-type calcium channels related to the 101 CaV1 family found in neurons, heart and muscles of mammals (Lodh et al., 2016). A number of other 102 ionic currents have been identified (Eckert and Brehm, 1979), and genes for many more ionic 103 channels have been found in the genome, often homologs of mammalian channels (Martinac et al., 104 2008). Sensitivity to various sensory signals is provided by transduction into ionic currents, which 105 may then trigger action potentials. Piezo channels, which convey mechanosensitivity in many species 106 including mammals (Coste et al., 2010) have been identified in the genome. A rhodopsin-like protein 107 has been identified in Paramecium bursaria, a photosensitive species (Nakaoka et al., 1991). In fact, 108 many signaling pathways of neurons have been found in Paramecium, in particular calcium signaling 109 pathways (Plattner and Verkhratsky, 2018) – calcium release channels, pumps, calmodulin, centrin, 110 calcineurin, SNARE proteins, cAMP and cGMP-dependent kinases, etc. For this reason, some authors 111 have called Paramecium a “swimming neuron” (Kung and Saimi, 1985). 112 Many other motile unicellular organisms have rich behavior (Wan and Jékely, 2020) and produce 113 action potentials, including microalgae (Eckert and Sibaoka, 1968; Harz and Hegemann, 1991; 114 Taylor, 2009), other ciliates such as Stentor (Wood, 1991), other protists such as Actinocoryne 115 contractilis (Febvre-Chevalier et al., 1986) and even bacteria (Kralj et al., 2011; Masi et al., 2015). One 116 advantage of Paramecium is its large size, allowing relatively simple electrophysiological recordings 3 117 (Kulkarni et al., 2020; Naitoh and Eckert, 1972). For this reason, there is a rich literature on 118 Paramecium electrophysiology, mostly from the 1960-80s (Eckert, 1972; Eckert and Brehm, 1979). 119 In addition, Paramecium is still an active model organism in genetics, and benefits from many tools 120 such as RNA interference (Galvani and Sperling, 2002); its genome has also been sequenced (Aury et 121 al., 2006; McGrath et al., 2014). 122 I will first give an overview of the behavior of Paramecium, then I will explain how it moves with its 123 body and cilia, and finally I will describe the physiological basis of behavior, with a special focus on 124 the avoiding reaction.