This is the final version of the article. Available from the publisher via the DOI in this record.The tadpole larva of a sea squirt is only the second animal to have its entire nervous system mapped out, and the results confirm that there is still much to learn from the smallest brains

Bezares-Calderón, LA

Jékely, G

eLife

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CONNECTOMICSThink smallThe tadpole larva of a sea squirt is only the second animal to have itsentire nervous system mapped out, and the results confirm that there isstill much to learn from the smallest brains.LUIS A BEZARES-CALDERO´N AND GA´SPA´R JE´KELYBrains come in different sizes. Spermwhales have the largest, weighing in atover 7 kilograms, whereas the dwarfmales of a worm called Dinophilus gyrociliatushave the smallest, containing just 66 neurons(Windoffer and Westheide, 1988). Whetherlarge or small, one feature unites all brains morethan anything else: their neurons are connectedto form intricate networks. This property is notseen in other organs, like the liver, kidneys orskin. Mapping networks of neurons has nowbecome a central theme in neuroscience, as evi-denced by the rise of the field of connectomics.Producing wiring diagrams of neural networks– or connectomes – that show the specific con-nections between cells requires use of electronmicroscopy to image ultrathin slices of neural tis-sue. This approach has led to spectacular prog-ress in recent years, but it can only analyserelatively small volumes. For example, a mousebrain is approximately 1 cm3, which is about100,000 times larger than the volume that cancurrently be imaged and analysed. This is whysize matters in connectomics. To date, a com-plete connectome has been produced for justone species, the nematode worm Caenorhabditiselegans. This wiring diagram, which waspublished 30 years ago, contains 302 neuronsand took over a decade of effort (White et al.,1986).Now, in eLife, Kerrianne Ryan, Zhiyuan Lu andIan Meinertzhagen from Dalhousie Universityreport that they have mapped the connectomeof a tadpole larva of the ascidian Ciona intestina-lis (Ryan et al., 2016). Adult ascidians are quiteunremarkable, water-filled sacs that squirt whenremoved from the sea – which is why they arecommonly called “sea squirts” (Figure 1A).However, their tadpole-like larvae have attractedinterest ever since the Russian embryologistAlexander Kowalewsky discovered in 1867 thatthey have the same basic body plan as the verte-brates (i.e. animals with backbones, rangingfrom fish to humans; Mikhailov and Gilbert,2002). Later molecular studies confirmed thatthey were indeed our closest “invertebrate” rel-atives (Delsuc et al., 2006), and Ciona has nowbecome an important laboratory animal in manyareas of biology.With fewer than 200 neurons, the nervoussystem of a Ciona larva is small and comes closeto the lower limits of brain size. Nevertheless,imaging and reconstructing most of the neuronsin the nervous system of a single specimenrequired a gargantuan effort. First, Ryan et al.sliced a single larva into 7,000 sections, just tensof nanometres thick, and imaged the sectionsusing electron microscopy (Figure 1B). Thehigh-resolution images were then aligned andthe neurons were painstakingly traced by hand.Finally, the connections for each and every neu-ron were identified and annotated. This resultedin the near-complete wiring diagram of theCiona larva, representing the second connec-tome of any animal.Copyright Bezares-Caldero´n andJe´kely. This article is distributedunder the terms of the CreativeCommons Attribution License, whichpermits unrestricted use andredistribution provided that theoriginal author and source arecredited.Related research article Ryan K, Lu Z,Meinertzhagen IA. 2016. The CNSconnectome of a tadpole larva of Cionaintestinalis (L.) highlights sidedness in thebrain of a chordate sibling. eLife 5:e16962.doi: 10.7554/eLife.16962Bezares-Caldero´n and Je´kely. eLife 2016;5:e22497. DOI: 10.7554/eLife.22497 1 of 4INSIGHTSo, what can we learn from the connectomeof Ciona? First, it is what is called a “small-world” network, meaning that it has highly con-nected local sub-networks linked together by asmaller number of long-range connections. Thisis similar to how the C. elegans connectome,social networks and power grids are organised(Watts and Strogatz, 1998). Overall, the 177neurons are connected such that, on average, itonly takes 2.7 steps to go from one node in thenetwork to another.The connectome also shows that a Cionalarva has clearly separate peripheral and centralnervous systems. The peripheral nervous systemcontains a few sensory neurons that are in directcontact with the environment. The centralFigure 1. Connectome of the Ciona intestinalis tadpole larva. (A) As adults, sea squirts like Ciona intestinalis areimmobile, filter-feeding animals. The scale bar is 10 mm. (B) The tadpole-like larva of Ciona, however, can swimand has a vertebrate-like body plan with a flexible rod that runs along its back called a notochord. Ryan et al. cut asingle larva into ~7,000 ultrathin slices and used these to reconstruct the larval connectome. (C) The larva also hassensory organs, including a single eyespot on the right side and an organ involved in sensing gravity (calledotolith) on the left. The scale bars in B and C are 100 mm. (D) The Ciona connectome shows that the larval nervoussystem consists of a peripheral and a central nervous system; the latter includes a brain vesicle and a motorganglion. The brain vesicle contains the photoreceptors for the eyespot, the coronet cells (which are thought todetect pressure), antenna cells (which are involved in sensing the position of the otolith), and relay neurons. Themotor ganglion contains neurons that directly control the contractions of the muscles. The brain shows manyasymmetries in its cellular anatomy and wiring. For example, there are more relay neurons on the left of the larvae,and the motorneurons in the motor ganglion (numbered 1–5) are connected differently on the left and right sidesof the larvae (red arrows show asymmetric connections). Photographs in A, B and C are courtesy of AntonioPalladino.Bezares-Caldero´n and Je´kely. eLife 2016;5:e22497. DOI: 10.7554/eLife.22497 2 of 4Insight Connectomics Think smallnervous system consists of sensory systems con-taining a single eye on the right side of thehead, an organ that is sensitive to gravity, andcells that are thought to sense pressure(Figure 1C). These sensory systems connect tohighly interconnected relay neurons found at theback of the brain. These relay neurons in turnproject to interneurons and motorneurons thatare also part of the central nervous system andthat eventually innervate into the larva’smuscles.How can the connectome help us understandhow the Ciona larva does what it does? Well,almost everything these creatures do involvestheir muscles. Even before hatching, asymmetricmuscle contractions cause the larva’s tail to flick10 times per second and help it to hatch from itsegg. Tail flicks then persist after hatching andmay be important for changing direction whenswimming (Mast, 1921). Moreover, as a larvaages, its movements change and become drivenby symmetric tail contractions that occur about20–30 times per second. The larvae also respondto different stimuli – including light, gravity andshadows – by altering the activity of theirmuscles (Zega et al., 2006; McHenry andStrother, 2003); for example, young larvae willswim up against the direction of gravity(Tsuda et al., 2003). Ryan et al. have now fullymapped neuronal pathways responsible forthese relatively simple behaviours, from sensoryorgans to muscles (Figure 1D).Simple as these behaviours may be, they arenot independent. Instead, the network allowscrosstalk at different levels. This suggests thatthe brain may indeed integrate different stimulibut only trigger one pattern of movements at atime – which is consistent with how these larvaeactually swim (Zega et al., 2006). These findingsshow that, even though each larva is small andanatomically simple, the underlying neural net-works in its nervous system are complex andintegrated.Ryan et al. noted another intriguing featureof the Ciona connectome. Most animals studiedto date display a left-right symmetry, so that foreach neuron on the left, there is a counterparton the right. However, most of the nervous sys-tem of Ciona lacks this symmetry (Figure 1D).Moreover, even those groups of neurons thatare symmetric – such as the motorneurons – areconnected to other cells in an asymmetric way. Itis not known why the nervous system of Ciona isso strongly asymmetric, but it may have some-thing to do with the streamlining of the ascidianbody plan. Asymmetries can be found in allbrains, including our own, but to a lesser extent;so Ciona may provide clues on the developmentand function of asymmetry in brains.The Ciona connectome holds lessons forthose trying to understand how the brain works.It clearly shows that we are not done with thesmallest brains yet; despite having few neurons,the Ciona network is complex and we are still farfrom fully understanding it. More generally, itsuggests that it would be wise to solve someother small brains before we tackle more com-plex ones. This suggestion also makes economicsense, when you consider that a single mouseconnectome would roughly cost the same as100,000 small brain connectomes. Perhapsothers working in the connectomics field will beinspired by the beautiful work of Ryan, Lu andMeinertzhagen, and turn their attention to thesmallest of brains first.Luis A Bezares-Caldero´n is in the Max-Planck-Institute for Developmental Biology, Tu¨bingen,Germanyhttp://orcid.org/0000-0001-6678-6876Ga´spa´r Je´kely is in the Max-Planck-Institute forDevelopmental Biology, Tu¨bingen, Germanygaspar.jekely@tuebingen.mpg.dehttp://orcid.org/0000-0001-8496-9836Competing interests: The authors declare thatno competing interests exist.Published 06 December 2016ReferencesDelsuc F, Brinkmann H, Chourrout D, Philippe H.2006. Tunicates and not cephalochordates are theclosest living relatives of vertebrates. Nature 439:965–968. doi: 10.1038/nature04336, PMID: 16495997Mast SO. 1921. Reactions to light in the larvae of theascidians, Amaroucium constellatum and Amarouciumpellucidum with special reference to photicorientation. Journal of Experimental Zoology 34:148–187. doi: 10.1002/jez.1400340204McHenry M, Strother J. 2003. The kinematics ofphototaxis in larvae of the ascidian Aplidiumconstellatum. Marine Biology 142:173–184. doi: 10.1007/s00227-002-0929-zMikhailov AT, Gilbert SF. 2002. From development toevolution: the re-establishment of the "AlexanderKowalevsky Medal". The International Journal ofDevelopmental Biology 46:693–698. PMID: 12216980Ryan K, Lu Z, Meinertzhagen I. 2016. The CNSconnectome of a tadpole larva of Ciona intestinalis (L.)highlights sidedness in the brain of a chordate sibling.eLife 5:e16962. doi: 10.7554/eLife.16962Tsuda M, Sakurai D, Goda M. 2003. Direct evidencefor the role of pigment cells in the brain of ascidianlarvae by laser ablation. Journal of ExperimentalBiology 206:1409–1417. doi: 10.1242/jeb.00235,PMID: 12624175Bezares-Caldero´n and Je´kely. eLife 2016;5:e22497. DOI: 10.7554/eLife.22497 3 of 4Insight Connectomics Think smallWatts DJ, Strogatz SH. 1998. Collective dynamics of’small-world’ networks. Nature 393:440–442. doi: 10.1038/30918, PMID: 9623998White JG, Southgate E, Thomson JN, Brenner S. 1986.The structure of the nervous system of the nematodeCaenorhabditis elegans. Philosophical Transactions ofthe Royal Society B: Biological Sciences 314:1–340.doi: 10.1098/rstb.1986.0056Windoffer R, Westheide W. 1988. The nervous systemof the male Dinophilus gyrociliatus (Polychaeta,Dinophilidae): II. Electron microscopical reconstructionof nervous anatomy and effector cells. The Journal ofComparative Neurology 272:475–488. doi: 10.1002/cne.902720403, PMID: 3417897Zega G, Thorndyke MC, Brown ER. 2006.Development of swimming behaviour in the larva ofthe ascidian Ciona intestinalis. Journal of ExperimentalBiology 209:3405–3412. doi: 10.1242/jeb.02421,PMID: 16916975Bezares-Caldero´n and Je´kely. eLife 2016;5:e22497. DOI: 10.7554/eLife.22497 4 of 4Insight Connectomics Think small

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