Think small

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

Inter-individual stereotypy of the Platynereis larval visual connectome

This is the final version of the article. Available from the publisher via the DOI in this record.Developmental programs have the fidelity to form neural circuits with the same structure and function among individuals of the same species. It is less well understood, however, to what extent entire neural circuits of different individuals are similar. Previously, we reported the neuronal connectome of the visual eye circuit from the head of a Platynereis dumerilii larva (Randel et al., 2014). We now report a full-body serial section transmission electron microscopy (ssTEM) dataset of another larva of the same age, for which we describe the connectome of the visual eyes and the larval eyespots. Anatomical comparisons and quantitative analyses of the two circuits reveal a high inter-individual stereotypy of the cell complement, neuronal projections, and synaptic connectivity, including the left-right asymmetry in the connectivity of some neurons. Our work shows the extent to which the eye circuitry in Platynereis larvae is hard-wired.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013)/European Research Council Grant Agreement 260821.European Research Council (ERC): Grant Agreement 260821, Gaspar Jekel

Ciliomotor circuitry underlying whole-body coordination of ciliary activity in the Platynereis larva

This is the final version of the article. Available from the publisher via the DOI in this record.Ciliated surfaces harbouring synchronously beating cilia can generate fluid flow or drive locomotion. In ciliary swimmers, ciliary beating, arrests, and changes in beat frequency are often coordinated across extended or discontinuous surfaces. To understand how such coordination is achieved, we studied the ciliated larvae of Platynereis dumerilii, a marine annelid. Platynereis larvae have segmental multiciliated cells that regularly display spontaneous coordinated ciliary arrests. We used whole-body connectomics, activity imaging, transgenesis, and neuron ablation to characterize the ciliomotor circuitry. We identified cholinergic, serotonergic, and catecholaminergic ciliomotor neurons. The synchronous rhythmic activation of cholinergic cells drives the coordinated arrests of all cilia. The serotonergic cells are active when cilia are beating. Serotonin inhibits the cholinergic rhythm, and increases ciliary beat frequency. Based on their connectivity and alternating activity, the catecholaminergic cells may generate the rhythm. The ciliomotor circuitry thus constitutes a stop-and-go pacemaker system for the whole-body coordination of ciliary locomotion.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821. This project is supported by the Marie Curie ITN ‘Neptune’, GA 317172, funded under the FP7, PEOPLE Work Programme of the European Commission. This project is supported by the DFG - Deutsche Forschungsgemeinschaft (Reference no. JE 777/3–1).Deutsche Forschungsgemeinschaft 777/3-1 Gaspar JekelyMax-Planck-Gesellschaft Open-access funding Gaspar JekelyEuropean Commission GA 317172 Gaspar Jekel

A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes.

This is the final version of the article.Available from eLife Sciences Publications via the DOI in this record.Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821. This project is supported by the Marie Curie ITN "Neptune", GA 317172, funded under the FP7, PEOPLE Work Programme of the European Commission

Neuronal connectome of a sensory-motor circuit for visual navigation.

This is the final version of the article. Available from eLife Sciences Publications via the DOI in this record.Animals use spatial differences in environmental light levels for visual navigation; however, how light inputs are translated into coordinated motor outputs remains poorly understood. Here we reconstruct the neuronal connectome of a four-eye visual circuit in the larva of the annelid Platynereis using serial-section transmission electron microscopy. In this 71-neuron circuit, photoreceptors connect via three layers of interneurons to motorneurons, which innervate trunk muscles. By combining eye ablations with behavioral experiments, we show that the circuit compares light on either side of the body and stimulates body bending upon left-right light imbalance during visual phototaxis. We also identified an interneuron motif that enhances sensitivity to different light intensity contrasts. The Platynereis eye circuit has the hallmarks of a visual system, including spatial light detection and contrast modulation, illustrating how image-forming eyes may have evolved via intermediate stages contrasting only a light and a dark field during a simple visual task.The research leading to these results received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821

Ciliary and rhabdomeric photoreceptor-cell circuits form a spectral depth gauge in marine zooplankton.

This is the final version.Available from eLife Publications via the DOI in this record.All data generated or analysed during this study are included in the manuscript and supporting files.Source data files have been provided for Figures 1, 3 and 4 and Figure 2-figure supplement 2.Ciliary and rhabdomeric photoreceptor cells represent two main lines of photoreceptor-cell evolution in animals. The two cell types coexist in some animals, however how these cells functionally integrate is unknown. We used connectomics to map synaptic paths between ciliary and rhabdomeric photoreceptors in the planktonic larva of the annelid Platynereis and found that ciliary photoreceptors are presynaptic to the rhabdomeric circuit. The behaviors mediated by the ciliary and rhabdomeric cells also interact hierarchically. The ciliary photoreceptors are UV-sensitive and mediate downward swimming in non-directional UV light, a behavior absent in ciliary-opsin knockout larvae. UV avoidance overrides positive phototaxis mediated by the rhabdomeric eyes such that vertical swimming direction is determined by the ratio of blue/UV light. Since this ratio increases with depth, Platynereis larvae may use it as a depth gauge during vertical migration. Our results revealed a functional integration of ciliary and rhabdomeric photoreceptor cells in a zooplankton larva.The research was supported by a grant from the DFG - Deutsche Forschungsgemeinschaft (Reference no. JE 777/3–1). SY was supported by the National Institutes of Health (R01EY016400) and Emory University. KTR is supported by grants from the University of Vienna (research platform “Rhythms of Life”), the FWF (http://www.fwf.ac.at/en/) research project grant (#P28970), and the European Council under the European Community’s Seventh Framework Programme (FP7/2007-2013) ERC Grant Agreement 337011

Expression dynamics and protein localization of rhabdomeric opsins in Platynereis larvae.

The larval stages of polychaete annelids are often responsive to light and can possess one to six eyes. The early trochophore larvae of the errant annelid Platynereis dumerilii have a single pair of ventral eyespots, whereas older nectochaete larvae have an additional two pairs of dorsal eyes that will develop into the adult eyes. Early Platynereis trochophores show robust positive phototaxis starting on the first day of development. Even though the mechanism of phototaxis in Platynereis early trochophore larvae is well understood, no photopigment (opsin) expression has yet been described in this stage. In late trochophore larvae, a rhabdomeric-type opsin, r-opsin1, expressed in both the eyespots and the adult eyes has already been reported. Here, we identify another Platynereis rhabdomeric opsin, r-opsin3, that is expressed in a single photoreceptor in the eyespots in early trochophores, suggesting that it mediates early larval phototaxis. We also show that r-opsin1 and r-opsin3 are expressed in adjacent photoreceptor cells in the eyespots in later stages, indicating that a second eyespot-photoreceptor differentiates in late trochophore larvae. Using serial transmission electron microscopy (TEM), we identified and reconstructed both photoreceptors and a pigment cell in the late larval eyespot. We also characterized opsin expression in the adult eyes and found that the two opsins co-express there in several photoreceptor cells. Using antibodies recognizing r-opsin1 and r-opsin3 proteins, we demonstrate that both opsins localize to the rhabdomere in all six eyes. In addition, we found that r-opsin1 mRNA is localized to, and translated in, the projections of the adult eyes. The specific changes we describe in opsin transcription and translation and in the cellular complement suggest that the six larval eyes undergo spectral and functional maturation during the early planktonic phase of the Platynereis life cycle