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The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling

  1. Kerrianne Ryan
  2. Zhiyuan Lu
  3. Ian A Meinertzhagen  Is a corresponding author
  1. Dalhousie University, Canada
Research Article
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Cite this article as: eLife 2016;5:e16962 doi: 10.7554/eLife.16962

Abstract

Left-right asymmetries in brains are usually minor or cryptic. We report brain asymmetries in the tiny, dorsal tubular nervous system of the ascidian tadpole larva, Ciona intestinalis. Chordate in body plan and development, the larva provides an outstanding example of brain asymmetry. Although early neural development is well studied, detailed cellular organization of the swimming larva’s CNS remains unreported. Using serial-section EM we document the synaptic connectome of the larva’s 177 CNS neurons. These formed 6618 synapses including 1772 neuromuscular junctions, augmented by 1206 gap junctions. Neurons are unipolar with at most a single dendrite, and few synapses. Some synapses are unpolarised, others form reciprocal or serial motifs; 922 were polyadic. Axo-axonal synapses predominate. Most neurons have ciliary organelles, and many features lack structural specialization. Despite equal cell numbers on both sides, neuron identities and pathways differ left/right. Brain vesicle asymmetries include a right ocellus and left coronet cells.

https://doi.org/10.7554/eLife.16962.001

eLife digest

Brains are made up of a network of nerve cells (neurons) that are connected to each other by junctions called synapses. The neurons on the left and right sides of the brain form different patterns of connections, but this asymmetry can be difficult to spot because the brain is large and complex. Understanding how the whole network operates is key to understanding how the brain works. However, a full map of all the connections between neurons – known as a connectome – has only been described for one species so far, a nematode worm called C. elegans.

The tadpole larva of the common sea squirt has a fairly simple brain distantly related to our own but made up of only about 330 cells. Ryan et al. used a technique called electron microscopy to study thin sections from the brains of sea squirt larvae to reveal this animal’s connectome and investigate left-right asymmetry in the brain.

The analysis revealed 177 neurons in this larval brain, just over half of its brain cells. These can be split into at least 25 types and each neuron has a simple, mostly unbranched shape with, on average, 49 synapses with other cells. This means that, even though it has such a small number of neurons, the neuron network is still relatively complex. The shortest sensory pathway to any muscle connects via three synapses, although most pathways involve more. The left and right sides of the brain differ in the types of neurons they contain and the connections these form, even though both sides have the same number of cells.

The findings of Ryan et al. reveal the second animal connectome and lay the groundwork for future studies on how each neuron in the network influences the behaviour of the sea squirt’s larva. Further work is also required to find out how the patterns of synapses in the brain change as the larva ages, and whether the connectome differs between siblings.

https://doi.org/10.7554/eLife.16962.002

Introduction

Animals exhibit various forms of left-right asymmetry (Ludwig, 1932; Neville, 1976; Brown and Wolpert, 1990; Palmer, 2009), and anatomical examples in brains are reported both in invertebrates (Hobert et al., 2002; Frasnelli et al., 2012), and vertebrates (Rogers and Andrew, 2002; Duboc et al., 2015) as differences in cell number (Blinkov and Glezer, 1968, their tables 184 and 185) and particularly in the lateralization of the mammalian cortex (Galaburda et al., 1978). Even predominantly symmetrical bilaterians exhibit various forms of sidedness, in both protostomes (Grande and Patel, 2009) and deuterostomes (Hamada et al., 2002; Duboc et al., 2005). In large, complex brains, these anatomical asymmetries are often structurally cryptic, and gene expression has been used as a proxy to examine the evolution of sidedness. As a sister group to vertebrates (Cameron et al., 2000; Satoh et al., 2014), ascidians have larvae that are chordate in body plan and mode of development, with a dorsal central nervous system (CNS). In the ~1 mm tadpole larva of Ciona intestinalis neurons are distributed rostrocaudally in three main centres, a brain vesicle, motor ganglion and caudal nerve cord (Katz, 1983; Nicol and Meinertzhagen, 1991; Meinertzhagen et al., 2004). The CNS forms from a neural tube, yet exhibits left/right differences, and so provides a useful model to study many aspects of brain asymmetry. This issue is important because brain laterality has been associated with increased fitness for animal life (Duboc et al., 2015).

The most studied tunicate species is Ciona intestinalis (Satoh, 1994). Not only does its development result from a fixed pattern of cell lineage and result in a mere ~ 2600 cells in the larva of Ciona intestinalis (Satoh, 1999), but the genome, first in Ciona intestinalis (Dehal et al., 2002) and now in nine other species (Brozovic et al., 2016), has been sequenced. Even though the events of early neural development and the nervous system’s subsequent metamorphosis have been identified, together with many of their underlying causal gene networks (Satoh, 2003; Sasakura et al., 2012), the detailed cellular organization of their product, the CNS of the swimming larva, still remains almost entirely unresolved.

Ciona releases 5000–10000 eggs per individual (Petersen and Svane, 1995), and its eggs are released either individually, or in a mucous string (Svane and Havenhand, 1993). Gametes undergo fertilization, cleavage, development, and then hatch into non-feeding lecithotrophic larvae in the water column. Initially after hatching, larvae swim up toward the surface of the water by negative geotaxis using the otolith cell (Tsuda et al., 2003) a behaviour retained in ocellus-ablated larvae. Later in larval life, larvae exhibit negative phototaxis, swimming down to find appropriate substrates for settlement (Tsuda et al., 2003). The swimming period exhibits three characterized behaviours: tail flicks (~10 Hz), ‘spontaneous’ swimming (~33 Hz), and shadow response (~32 Hz; Zega et al., 2006). Larvae swim more frequently and for longer periods earlier in life up to 2 hr post hatching (hph). Of the reported behaviors, the shadow response, in which a dimming of light results in symmetrical swimming, is the best studied, developing at 1.5 hph and increasing in tailbeat frequency after 2 hph (Zega et al., 2006). In addition to phototactic and geotactic behavior, there is evidence of chemotactic behavior just before settlement (Svane and Young, 1989) and of some mechanosensory responses in swimming larvae (Bone, 1992). Because larvae do not feed, their main biological imperative is survival and successful settlement to undergo metamorphosis into a sessile adult, in an environment with appropriate food and reproductive resources. Thus, entering the water current and avoiding predation by filter feeders may be the foundation for the larva’s many behavioral networks, especially in early life before settlement.

The substrate for these behaviours is the larva’s dorsal central nervous system, which is divided into the anterior sensory brain vesicle (BV), connected by a narrow neck to the motor ganglion (MG) within the larval trunk, and a caudal nerve cord (CNC) in the tail (Nicol and Meinertzhagen, 1991). Sensory neurons of the CNS and their interneurons reside in the BV, which has an expanded neural canal and the most complex neuropil. The relay neurons of the posterior brain vesicle extend axons through the neck to the motor ganglion, which overlies the anterior portion of the notochord, and contains neurons of the motor system. At the trunk-tail border, muscle cells of the tail flank the notochord and CNS, and these extend down through the tail alongside the narrow, simple CNC. In addition to the CNS several sensory epidermal neurons (ENs) of the peripheral nervous system (PNS) populate the dorsal and ventral axes of the larva in a rostrocaudal sequence, with axons running beneath the epidermis (Imai and Meinertzhagen, 2007b).

Many asymmetries have been uncovered by the developmental expression of Nodal and its signaling pathways (Hamada et al., 2002; Hudson, 2016). As in vertebrates, in ascidians, their sibling group (Satoh et al., 2014), Nodal expresses on the left hand side of the developing embryo (Boorman and Shimeld, 2002a, 2002b; Yoshida and Saiga, 2008). This is true neither of other deuterostomes (Duboc et al., 2005) nor lophotrochozoans (Grande and Patel, 2009), while ecdysozoans such as Drosophila and C. elegans lack Nodal (Schier, 2009), even though the brain in Drosophila is asymmetrical (Pascual et al., 2004). The development of brain asymmetry in the ascidian does however depend on the presence of an intact chorion in the embryo (Shimeld and Levin, 2006; Yoshida and Saiga, 2008Oonuma et al., 2016).

In contrast to the situation in most chordates, structural brain asymmetries, which include cell numbers, positions, and connections are externally visible in the tadpole larva of ascidians, for example from the pigment spots and right-sided ocellus in the head of Ciona intestinalis (Eakin and Kuda, 1971; Katz, 1983; Nicol and Meinertzhagen, 1991). Photoreceptor neurons associated with the ocellus are of the ciliary type, with outer segment lamellae orientated parallel to the cilium (Eakin and Kuda, 1971), in contrast to the perpendicular arrangement found in vertebrate rods (Lamb and Collin, 2007). The photoreceptor cells of the ocellus on the right of the brain vesicle, which number 20 cells (Horie et al., 2008), 17 and 18 of which had been reported by Nicol and Meinertzhagen (1991), are twinned with 17 to 19 structurally different coronet cells (previously claimed hydrostatic pressure receptor cells: Eakin and Kuda, 1971; Nicol and Meinertzhagen, 1991) on the left. This sidedness may also correspond to larval behavior because ascidian larvae pursue a helical trajectory when swimming (McHenry and Strother, 2003; McHenry, 2005) and ascidian larvae are thought to use klinotaxis to respond to visual cues by modulating the symmetry of tail kinematics (McHenry and Strother, 2003). The pattern of helical swimming arises from bilateral contractions of the tiered muscle bands on either side of the notochord. On each side, gap junctions connect all 18 uninucleate muscle cells, arranged in three rows, dorsal medial and ventral (Bone, 1992). Muscle activity along the tail is thus not segmental, and instead propagates posteriorly in a wave from innervated anterior dorsal and medial muscle cells at the trunk-tail border without the requirement for additional neuronal input along the tail (Bone, 1992).

In the past, the search for brain asymmetries has been frustrated by two features, the presumed relative rarity of such asymmetries, and the lack of structurally identified networks of neurons in which to recognise them. These obstacles are resolved in the CNS of the tadpole larva of Ciona, in which not only is the asymmetrical organization obvious, but the possibility exists to uncover the larva’s complete network of neuronal synaptic connections, or its connectome (Lichtman and Sanes, 2008). That possibility rests on the tiny size of the brain, the small number of its constituent cells, approximately 330 (Nicol and Meinertzhagen, 1991), and the morphological simplicity of its neurons (Imai and Meinertzhagen, 2007a). The apparent symmetry of the characteristic swimming pattern of chordate-like tail undulations (Bone, 1992; Video 1) stands in marked contrast to the asymmetries in the CNS that generates them. Here we present the connectome of the CNS of the tadpole larva in Ciona, and reveal its asymmetries in the left and right complements of neurons and their synaptic networks.

Video 1
Symmetrical undulations of the tail in a swimming Ciona larva.

The tail lacks segmentation and in the 2 hr hatchling larva oscillates at 20–30 Hz at the juncture with the rostral trunk (Bone, 1992).

https://doi.org/10.7554/eLife.16962.003

Results

A single larva was prepared for electron microscopy (EM) and a long continuous series of cross sections cut. A total of 3375 60 nm sections that started at the level of the otolith pigment and extended to the posterior motor ganglion, followed by 1360 70 nm sections cut into the anterior tail, and continued to its tip by a further 2193 100 nm sections (Figure 1; Figure 1—figure supplement 1) were collected and imaged at 3.85 nm per pixel. The entire imaged series thus included the anterior brain vesicle and motor ganglion (MG) cut at 60 nm, and the caudal nerve cord at 70, then 100 nm. These regions were additionally innervated by neurons of the PNS with cell bodies residing in the epidermis; these extend cilia into the tunic. Thus, unlike C. elegans, which lacks a distinction between a CNS and PNS, Ciona’s chordate nervous system has clear divisions between the two. We annotated mostly those PNS neurons that innervate the CNS (Figure 1A).

Figure 1 with 1 supplement see all
Ultrathin section series of Ciona intestinalis larva, its CNS and notochord.

(A) Diagram of whole larva with colour-coded cell types indicated by arrows. Types of relay neurons (RNs) are shown as colour-coded territories in the brain vesicle. Muscle cells align in dorsal (red), medial (orange), and ventral (yellow) tiers. (B) Larva from left side, illustrating major landmarks and indicating the number of sections of each thickness (60–100 nm) along the A-P axis. Diagrammatic profiles of representative sections at different levels along the A-P axis of the larva, shown to the same magnification. (C) Profile traces from Reconstruct of representative sections at six levels of the CNS. Nuclei are shown as small squares. Coloured cell types are shown in the key (Figure 1—source data 1). For labelled cell outlines of the sections see enlarged views in Figure 1—figure supplement 1. Abbreviations: Pap: papilla neuron: RTEN: rostral trunk epidermal neurons; ATENa: anterior apical trunk epidermal neurons; ATENp: posterior apical trunk epidermal neurons; Oc: ocellus; Ot: otolith; Ant: antenna neuron; Cor: coronet cell; PR: photoreceptor; ddN: descending decussating neuron; AMG/Ascending MG IN: ascending motor ganglion interneuron: MGIN: motor ganglion interneuron; MN: motor neuron; DCEN: dorsal caudal epidermal neuron; VCEN: ventral caudal epidermal neuron; BTN: bipolar tail neuron; Mu: muscle; pnsRNs: PNS relay neurons; PRRNs: photoreceptor relay neurons; AntRNs: antenna relay neurons; BV: brain vesicle; MG: motor ganglion; CNC: caudal nerve cord; Not: notochord; IN: interneuron; BVINs: brain vesicle interneurons. For descriptions and abbreviations of cell types also see key in Figure 1—source data 1 Scale bars in all panels: 10 µm.

https://doi.org/10.7554/eLife.16962.004

Synapses

Synapses were identified based on criteria established in other invertebrate species (White et al., 1986; Westfall, 1996; Meinertzhagen, 2016): primarily a cluster of vesicles at a presynaptic membrane. Small electron-lucent vesicles 30 to 60 nm in diameter were found exclusively at some presynaptic sites where they tended to cluster closely to form a tight cumulus (Figure 2A), accompanied at some synapses by larger electron-lucent vesicles (70—110 nm) (Figure 2B). The same vesicle types were found throughout the neuron, but their distribution was not quantified. Some synapses also had dense-core vesicles, large (110–140 nm; Figure 2C), medium (70–80 nm; Figure 2B), or small (40–60 nm) (Figure 2D; Figure 2—source data 1). The size of the internal density within these vesicles varied, some medium sized dense-core vesicles having small cores (Figure 2E). Other synapses had exclusively medium to large dense-core vesicles. Postsynaptic densities (Gray, 1959; Peters and Palay, 1996) were observed at some synapses (Figure 2C) but not at all, thus providing an unreliable criterion for a synaptic contact.

Synapses contain presynaptic vesicles of various sizes and types.

(A) Tightly packed cumulus of small (30–40 nm) vesicles at a single presynaptic site (arrow). (B) Mixed populations of small (30–50 nm) and large (70–110 nm) electron-lucent vesicles (arrow) as well as dense-core vesicles of medium size (arrowhead). (C) Large (100–110 nm) vesicles with dark cores (arrowheads). (D) Synapses containing electron-lucent vesicles (30–60 nm; arrow) as well as small (60 nm; small arrowhead) and medium (80 nm; large arrowhead) dense-core vesicles. See Figure 2—source data 1 for a list of those neurons with synapses having mixed vesicle populations. (E) Medium dense-core vesicles with small cores (arrows). (F) Membrane apposition (between arrows) interpreted as a gap junction, with membrane densities on both sides. Scale bars: 500 nm (A,B,C and E); 200 nm (D and F).

https://doi.org/10.7554/eLife.16962.007

Membrane appositions interpreted as putative gap junctions (Bennett and Goodenough, 1978) were annotated for contacts at which densities were present on the membranes of both sides (Figure 2F), except where such contacts formed directly adjacent to the neural canal, thus excluding junctions provisionally interpreted as desmosomes or adherens junctions.

Synapses and gap junctions varied in size, contributing profiles to between 1 and 29 sections (<66 sections for neuromuscular junctions) or 55 sections (gap junctions)(Figure 3A).

Figure 3 with 2 supplements see all
Synapse numbers (presynaptic sites) and sizes for all neurons (for complete list see Figure 3—source data 1).

(A) Most synaptic contacts extend over <10 60 nm sections. Those occupying >10 sections are neuromuscular junctions, inputs from relay neurons to MG neurons, and synapses from antenna cells. The frequency curve for chemical synapses reveals more large contacts than for gap junctions. (B) Plotted for all neurons, the total depth of presynaptic contact co-varies linearly with the total number of synapses (R2 = 0.91). Removing single-profile synapses eliminates 18% of all synaptic partnerships, and removing all 2-profile synapses would have eliminated a total of 35% of all synaptic partnerships. (C) The number of presynaptic sites co-varies with the number of postsynaptic partners according to a power function (R2 = 0.81). The number of synaptic partners is also referred to as the network statistic ‘degree’, and is mapped to the synaptic network (Figure 3—figure supplement 2). Five neurons lie well above the curve, having low degrees, with many synapses and few postsynaptic partners. These are: Antenna neuron 1 (Ant1) with many synapses onto seven relay neurons, and the two pairs of anterior-most motor neurons (MN1 and MN2) with many synapses onto muscle. (D) Cumulative distribution of the log number of presynaptic sites over the surfaces of neurons of major cell classes. TERM: terminal; AX: axon; CB: cell body; DEN: dendrite. Most synapses are located over axons and terminals (see Figure 3—figure supplement 1 for (D) averages per neuron and (E) postsynaptic site distribution). (E) Proportions of synapses and gap junctions in the connectome formed for particular partnerships. (F) Reciprocity of connections in the network given as the proportion of neuron partners that are reciprocally connected and the extent of their reciprocity (calculated as the cumulative depth of contacts in one direction divided by the sum of the depth of all contacts between the neuron pair). The total proportion of reciprocal synaptic connections between neuron pairs is 0.39.

https://doi.org/10.7554/eLife.16962.009

A total of 301 cells of the CNS were imaged, of which the CNS included 177 neurons with axons and presynaptic sites (Figure 1—source data 1; Figure 3—source data 1), and constituting the remainder, ependymal cells (those ciliated cells abutting the canal that lack an axon) and two cells of the CNS that are ambiguous, having presynaptic sites, but lacking a neuronal form (Figure 1—source data 1 and Figure 3—source data 1). Cells omitted from our EM series, those rostral to the otolith and caudal to the bipolar tail neurons (Imai and Meinertzhagen, 2007b; Stolfi et al., 2015), are presumed to account for the remainder of the >331 cells reported by Nicol and Meinertzhagen (1991) and thus to number at least 30. This assumes the constancy of cell number between different sibling batches of larvae. Between the CNS neurons (and the four bipolar tail neurons), we identified 8768 synapses (6618 >1 section), including 1772 neuromuscular synapses, and 2105 putative gap junctions (1206 >1 section). Each CNS neuron thus formed on average 49 (standard deviation, SD 61) presynaptic sites with a range of between 1 and 430 synapses and an average of 13 (SD 23) putative gap junctions, with a range between 0 and 166. Each postsynaptic neuron received an average of 39 (SD 42) synapses in total from all its presynaptic partners, with a range between 0 and 179.

For each neuron, the number of presynaptic sites varies with the number of its postsynaptic partners, plotted for all neurons and their synapses but excluding the neuromuscular junctions (Figure 3C). This relationship indicates that with each additional partner, the number of synapses made by a presynaptic neuron increases, thus reflecting a postsynaptic drive to the total synapse load. There was no overall relationship for each neuron between the volume of its soma and the number of its synapses (r2 = 0.4), nor between soma volume and axon/terminal surface area (r2 = 0.4; Figure 3—figure supplement 1). However, axon/terminal surface area and number of synapses were weakly correlated (r2 = 0.7) (Figure 3—figure supplement 1C).

Synaptic structure is sometimes unpolarised (Figure 4A), with synaptic vesicles situated on either side of the synaptic cleft; neurons also frequently connected to form both reciprocal (Figure 4B) and serial synapses (Figure 4C; Table 1). Of the 8617 synapses, 922 were polyadic, having multiple postsynaptic elements (Figure 4C; Figure 4—source data 1; Table 1). The most common of these were dyads, which constituted 93% of all polyadic synapses, and were common especially in antenna neurons (see below).

Unpolarized, reciprocal, and serial synapses.

(A) Unpolarized mixed synapse between cell 115 and cell 23 with dense-core (arrowhead) and electron-lucent (arrow) vesicles on both sides of the synaptic cleft. (B) Single section with synapse from cell 102 to cell 120 (black arrow) and a reciprocal partner synapse from cell 120 to cell 102 (red arrow). Arrowhead: membrane apposition marking a putative gap junction. (C) Serial dyad synapse (black arrow) from single neuron onto two postsynaptic targets (157 and 74), one of which is presynaptic in the same section (red arrow) at a dyad synapse onto two neurites (AMG1 and AMG2). (D) Serial monad synapse (black arrow) onto a single postsynaptic target (126) that is presynaptic at an adjacent synapse (red arrow) to a single postsynaptic target (116). Scale bars: 1 µm. (E) Series of four 60 nm sections through a single synapse. The pre- and postsynaptic cell are labelled in the top image. A clear cumulus of presynaptic vesicles is visible all images, and a clear postsynaptic density in the penultimate image (arrowhead). Scale bar: 500 nm.

https://doi.org/10.7554/eLife.16962.013
Table 1

Numbers of synapses and gap junctions.

https://doi.org/10.7554/eLife.16962.015
Total no.Total no.
sections
Mean no. sections
/contact
No. synapses>
1 section
Mean no. sections
/contact >1 section
% Unpolarized% Polyad% Dcv

Synapses

8617

30163

3.5

6618

4.3

5.2

10.7

8

Gap junctions

3205

5765

1.8

1206

3.1

?

3

N/A

  1. Percentage (%) refers to the percentage of all synapses that are unpolarized (presynaptic vesicles on either side of the cleft between both neuron partners); polyadic (having >1 postsynaptic neurite); or containing dense-core vesicles (dcv) at the presynaptic site.

The network forms a single connected component, with all cells (nodes) being connected by a synapse (edge) to another node in the network (Figure 3—figure supplement 2). The network statistics (Table 2) reveal that the characteristic path length between two neurons is 2.7 (from one neuron, through one other to its target), with neurons having an average of 20 neighbors (synaptic partners) and an overall average network clustering coefficient (existing edges between neighbors of a neuron/possible edges between neighbors of a neuron) of 0.333.

Table 2

Network statistics for networks of chemical synapses and putative gap junctions.

https://doi.org/10.7554/eLife.16962.016

Statistic

Synaptic network

Gap junction network

Full network

CNS neurons only

Full network

CNS neurons only

(>0.06 μm)

Clustering co-efficient

0.333

0.335

0.25

0.305

Connected component

1

1

7

1

Network diameter

9

7

8

8

Radius

1

4

1

4

Shortest paths

90% [41001]

95% [29759]

85% [31536]

100% [16770]

Characteristic path length

2.684

2.541

3.078

2.775

Average number of neighbours

20.169

20.689

8.674

10.369

Number of nodes

213

177

193

130

Network density

0

0

0.045

0.08

Network heterogeneity

-

-

0.935

0.76

Number of self-loops

19

16

13

9

Multi-edge node pairs

826

699

30

22

Network centralisation

-

-

0.191

0.257

  1. Network statistics calculated using the Cytoscape Network Analyzer for network of chemical synapses (Synaptic network) and putative gap junctions (Gap Junction network) for both the full network thus including PNS neurons, muscle, ambiguous cells, and synapses onto basal lamina, as well as CNS neurons; and the network for CNS neurons only (CNS neurons). Note that the 'CNS neurons only' network excludes one additional isolated profile of a single branch of one photoreceptor terminal, probably pr10.

Neurons

Within the CNS the distribution of presynaptic sites over the surface of the neuron varies by cell type. Most neurons are monopolar, <25% only having dendrites, and their axons usually form a clear terminal. Axons fasciculate in bundles but braid their positions within their bundle or sometimes defasciculate. Brain vesicle (BV) intrinsic interneurons have approximately equal numbers of presynaptic sites over their axons as their terminals, each constituting approximately 40% of their total. In contrast, axo-axonal synapses are the predominant synapses involving relay and motor ganglion (MG) interneurons, comprising >50% for both ascending and descending MG interneurons and >35% for BV relay neurons. The abundance of en passant synaptic contacts in the larval CNS of Ciona is particularly apparent when examining the proportional distribution of postsynaptic sites. Synapses formed by BV relay neurons at their terminals form en passant onto the axons and terminals of both relay and MG neuron targets, which together constitute >20% of all BV relay neurons’ presynaptic contacts. Likewise, >50% of BV intrinsic interneuron synapses form en passant from their axons or terminals onto the axons or terminals of their targets. Synapses from terminal to terminal also constitute the greatest proportion of photoreceptor synapses (43%), and en passant synapses constitute over 60% of their contacts.

Overall, within the CNS and among the axons of the dorsal PNS, 68% of all neuron-neuron synapses terminate on axons or terminals. More synaptic contacts are made upon axons than upon terminals, the latter constituting only 23%–26% of all synapses, whereas those onto axons comprise 42%–44%. These numbers support the impression that presynaptic sites are formed at various places over the cell surface, and that each cell type has a preferred location to form them, but that this location is neither absolute nor exclusive. For further information see Figure 3 and Figure 3—figure supplement 1D

Asymmetry in cellular composition

The overall cell complement including neurons, ependymal and accessory cells, is closely similar on the two sides (left: 125; right: 129; midline 46). The brain vesicle has unequal numbers of neurons and pigment cells, however, with more sensory neurons and pigment cells on the right side and more interneurons on the left (Table 3). Many of these left-side interneurons were previously identified as ciliated ependymal cells (Nicol and Meinertzhagen, 1991), but have been seen here to possess axons and synapses that were not visible by light microscopy, and are thus to be considered neurons. Excluding ependymal cells, each brain region has approximately equal numbers of cells on both sides (Table 3). Despite this near equal distribution in their overall numbers, however, examples of asymmetries in the numbers of identified neuron types, or numbers of neurons of each type, were nevertheless also found amongst specific interneuron classes in the brain vesicle. In the ventral motor ganglion most neurons are paired, including five pairs of motor neurons and four pairs of interneurons, whereas caudally among the ascending contralateral interneurons, ACINs, there were two representatives on the left and one on the right, as well as two descending posterior motor ganglion neurons (PMGNs) found only on the right (Table 3).

Table 3

Numbers of cells in the left, right and centre of the CNS and PNS.

https://doi.org/10.7554/eLife.16962.017

Left

Centre

Right

Lens cells

3

Pigment cells

2

Total: Pigment and lens cells

5

Coronet

13*

2

1

Photoreceptors

37*

Antenna neurons

1

1

Photoreceptor tract interneurons

3*

Anterior BV neurons

29

1

BV peripheral interneurons

4

4

1

Bipolar neurons

2

Anaxonal arborizing neurons

1

2

Posterior BV peripheral interneurons

2

1

1

Photoreceptor relay neurons

6

Photoreceptor-peripheral relay neurons

2

2

6

Photoreceptor-coronet relay neurons

2

1

Antenna-coronet relay neuron

1

Antenna relay neurons

7

2

Peripheral relay neurons

2

1

Relay interneurons

5

Total: BV neurons

72

14

57

Neck neurons

1

1

Total: Neck neurons

1

1

Ascending MG peripheral interneurons

3

1

3

Descending decussating neurons

1

1

MG interneurons

3

3

Motor neurons

5

5

Total: MG neurons

12

1

12

Ascending contralateral inhibitory neurons (ACINs)

2

1

Posterior MG interneurons

2

Mid-tail neurons**

2

2

Total: CNC neurons

4

5

All CNS neurons

88

15

75

Peripheral nervous system

Bipolar tail neurons

2

2

Peripheral neurons (RTENa)

6

6

anterior ATENs

2

2

posterior ATENs

4

DCENs

4

Total: PNS neurons

8

6

14

  1. Neurons of the left side of the nervous system outnumber those of the right, which in turn outnumber those of the centre. All CNS neurons include known neurons that lack synapses (*).

  2. **Additional mid-tail neurons which lay beyond the analysed region of the EM series are excluded from the totals.

Sidedness in the brain vesicle

Neurons are distributed unequally with respect to cell type. The most obvious case for sidedness in the CNS has been long recognized from the composition and placement of the ocellus and coronet cells (Meinertzhagen and Okamura, 2001; Meinertzhagen et al., 2004).

On the left side, we found the 17 enigmatic coronet cells (Figure 5A–B), each structurally distinguished by a single bulbous protrusion and expressing immunoreactivity to dopamine (Moret et al., 2005). These have short axons that terminate against the basal lamina where they form synapses with almost exclusively dense-core vesicles 80–110 nm in diameter (Figure 6A). Additional synapses are formed at close range onto other interneurons, including relay neurons (pr-corRN and ant-corRN), or onto neighbouring coronet cells (Figure 6B). Alongside the coronet cells lie ciliated neurons, some of which have cilia that project towards the bulbous protrusions (Figure 6C).

Sensory neurons and associated cells have sided distributions.

Reconstructed coronet cells (Cor) with their bulbous protrusions (BP, one with a black arrow) and -- in their correct relative position -- six layers of photoreceptor neurons, excluding their terminals, together with otolith (Ot) and ocellar (Oc) pigment cells. Reconstructions shown from a dorsal (A), or frontal (B) view. (C) Reconstruction of visual system components (including photoreceptor tract (trIN) and vacuolated sensory (vacIN) interneurons, shown from the right side, anterior to the right. (D) Sensory neurons (spheroids) and their modified cilia reconstructed within the outline of the CNS from left lateral, dorsal, and frontal views. Cells coloured as in panels A-C, PR-I outer segments in yellow, Pr-II outer segments in teal, coronet bulbous protrusions in green.

https://doi.org/10.7554/eLife.16962.018
Synapses of coronet cells.

(A) Synapse, containing exclusively dense-core vesicles (arrow), from a coronet cell onto the basal lamina (BL). (B) Unpolarized synapse between two coronet cells, with dense-core vesicles (arrows) on both sides of a synaptic cleft. (C) Reconstruction of coronet cells each with a bulbous protrusion (arrow) alongside coronet-cell associated somata (grey) of ciliated neurons, with cilia reconstructed in black. Scale bars: 1 µm (in A and B).

https://doi.org/10.7554/eLife.16962.019

On the right side, five rows of photoreceptors which, like those of vertebrates, and as previously reported (Dilly, 1962, 1969; Eakin and Kuda, 1971), extend a stalk that contains a basal body and expands to form a ciliary outer segment in the ocellus pigment cup. In addition to the Group I photoreceptors of the ocellus, two anterior rows of Group II photoreceptors adjacent to the ocellus have ciliary outer segments that extend into the neural canal, with smaller terminals extending a short distance into the posterior brain vesicle neuropil. The number and arrangement of photoreceptor neurons confirms the presence of the additional class of photoreceptors identified using Ci-opsin immunolabeling, with outer segments outside the ocellus pigment (Horie et al., 2005, 2008). These photoreceptors, along with those of the ocellus, total 30, as reported previously (Horie et al., 2005, 2008). We identify 23 with outer segments that projected into the ocellus pigment, and 7 into the neural canal (Video 2; Video 3; Video 4). The 17–18 photoreceptors reported by Nicol and Meinertzhagen (1991) appear to represent just some of the rows of nuclei from the five fans of ocellus photoreceptors, those that were visible in semithin sections and that lay close to the opaque pigment. In addition to the 30 photoreceptors, three anterior sensory interneurons, unique in having axons within the sensory axon tract, lie on the right side in the anterior brain vesicle (Figure 5C). We also identify a further group of seven Group III (Horie et al., 2008) right-side photoreceptors which lie posterior and ventral to the 30 (Figure 5C), are vacuolated, and have outer segments that are less well organized.

Video 2
Rotation of reconstructed sensory structures.

Reconstructed pigment cells (black) with otolith associated ciliated cells (yellow) and vacuoles observed in a variety of cell types (lime green). Outer segments reconstructed as spheres for group I photoreceptors projecting into the ocellus (darker purple) and group II photoreceptors projecting into the canal (lighter purple) and group III modified outer segments (blue), as well as coronet bulbous protrusions (orange).

https://doi.org/10.7554/eLife.16962.020
Video 3
Rotation of reconstructed sensory neurons.

Reconstruction including transparent cell bodies illustrating pigment cells (black), group I (dark purple), group II (light purple) and group III (blue) photoreceptors with their outer segments reconstructed as spheres, lens cells (white) with their vacuoles reconstructed (bright green); also shown are vacuolated photoreceptor tract interneurons (lime), antenna cells (green), otolith associated ciliated cells (yellow), and coronet bulbous protrusions. Terminals of antenna and photoreceptor neurons are truncated in this view.

https://doi.org/10.7554/eLife.16962.021
Video 4
Reconstruction of spheroids representing the cell body positions of sensory structures.

Pigment, photoreceptor and coronet cells with the bulbous protrusions (green) and photoreceptor outer segments (type I and type II: yellow; and type III: purple).

https://doi.org/10.7554/eLife.16962.022

Sidedness in CNS pathways

In addition to receptor neurons, interneurons of the brain vesicle also exhibit sidedness in the position of their somata (Figure 7).

Figure 7 with 1 supplement see all
Representation and relative sizes of cell bodies and their positions along the neuraxis, with corresponding axon tracts.

(A) Cell bodies of CNS neurons, dorsal view. Colours denote cell types (key). (B) Corresponding axon tracts, shown as skeleton reconstruction, dorsal view, colours as in (A) (for a network graph of synaptic connectome formed by corresponding neurons sorted by connectivity see Figure 7—figure supplement 1). (C) Cell bodies of CNS neurons and axon tracts, corresponding to (A) and (B), left lateral view. Pigment oc/ot: ocellus and otolith pigment cells; PR (oc): type I photoreceptor; PR (can): type II photoreceptor; PNIN: peripheral interneuron; PNIN (cilia) peripheral interneuron with cilium; vac IN: vacuolated photoreceptor-associated interneuron; Antenna: antenna cell; Coronet: coronet cell; aaIN: anaxonal arborizing interneuron; BVlN (cilia): ciliated brain vesicle interneuron: pr-AMG RN: photoreceptor-AMG relay neuron; trIN: photoreceptor tract interneuron; cor-ass BVIN: ciliated coronet associated brain vesicle interneuron; prRN: photoreceptor relay neuron; BVIN (no cilia): brain vesicle interneuron lacking cilium; non-sensory relay neuron (RN); antRN: antenna relay neuron; ant-corRN: antenna-coronet relay neuron: Eminens: eminens neuron: PNRN: peripheral relay neuron: PBV PNIN: posterior brain vesicle peripheral interneuron; MGINs 1–2: motor ganglion paired interneurons 1 and 2; MGINs 3: motor ganglion paired interneurons 3; ddN: descending decussating neuron pair: AMG: ascending motor ganglion neuron; MN: motor neuron: ACIN: ascending contralateral inhibitory neuron; PMGN: posterior motor ganglion neuron; Midtail neurons: short descending neurons of the caudal nerve cord. Scale bar 10 µm.

https://doi.org/10.7554/eLife.16962.023

Right-side interneurons can be identified by various morphological features not resolved in previous light microscopy studies. Two classes of intrinsic interneurons are:

  1. two bipolar neurons (cells 90 and 92), both photoreceptor interneurons (Figure 8A).

  2. three anaxonal arborizing interneurons (aaINs), which receive inputs from antenna and photoreceptor neurons, with highly branched terminals adjacent to their cell bodies (Figure 8B).

Posterior to these intrinsic neurons are three additional classes of interneuron:

  1. Teardrop-shaped neurons (cells 108, 116, 127, 157, 124, and 140) in the dorsal right brain vesicle that project axons ventrally to terminate in the anterior motor ganglion, one type with forked terminals (Figure 8C). This entire group also forms a connectivity class (pr-AMG relay neurons) that integrates input from photoreceptors and ascending peripheral interneurons (AMGs).

  2. Two relay neurons (cells 123 and 130) that lack unique morphological features, but share aspects of their connectivity, both postsynaptic to photoreceptor and bipolar tail neurons.

  3. Two antenna relay neurons (cells 142 and 152), one having an axon that splits to form long collateral bifid axons terminating at different depths in the motor ganglion, and the other having a long dendrite extending anteriorly from its soma (Figure 8D,E).

Right-side interneurons reconstructed from the brain vesicle, left lateral views, anterior to the left.

(A) Intrinsic bipolar interneuron with two axons (arrows). (B) Anaxonal arborizing interneuron with large branched terminal (arrow). (C) Photoreceptor-ascending motor ganglion (pr-AMGRN) relay neuron with forked terminal (arrow). (D) Antenna relay neuron with bifid axon (arrows). (E) Antenna relay neuron with single axon, terminal and soma dendrite (arrow). Scale bar 10 µm.

https://doi.org/10.7554/eLife.16962.025

Most interneurons of the brain vesicle are however located on the left side and many are structurally anonymous. Aside from those mentioned above (a-c), photoreceptor and antenna relay neurons are left-sided. In addition, other relay interneurons that lack direct input from sensory neurons are also left-sided (Table 3).

The only apparent difference in photoreceptor input to left and right subclasses of relay neurons is that Type II canal photoreceptors are presynaptic only to right-side relay neurons of the pr-AMG class (Figure 9). Unlike their right-side counterparts PR-AMGRN(R) (cells 108, 116, 127, 157, 123, and 130), the left-side photoreceptor relay neurons PRRN(L) (cells 74, 94, 80, 86, 96, 100, 121, and 126) receive photoreceptor input exclusively from Type I ocellus photoreceptors (Figure 9). Two ventral antenna neurons (Ant1 and Ant2), which are proposed to signal input from otolith position (Torrence, 1986; Tsuda et al., 2003), lack obvious sidedness in their cell body positions, and extend collateral axons toward the posterior brain vesicle (BV). The terminals of these antenna cells, however, differ in their synaptic input to sides of the posterior BV: Antenna cell 1 to both sides, and Antenna cell 2 predominantly to right-side relay neurons (AntRN). One right-side antenna relay neuron, located more ventrally (Ant-corRN; Figure 1—source data 1), is also postsynaptic to coronet cells.

Figure 9 with 1 supplement see all
Asymmetrical sensory input to the two sides of the motor ganglion MG(L) and MG(R) via relay neurons.

Sensory input arises from coronet cells (Cor); antenna cells Ant1 and Ant2 (combined as Ant); and photoreceptors (PR) of two types: ocelli (oc: PR I) and neural canal (can: PR II). Signals are relayed through respective interneuron classes: photoreceptor relay neurons on the left, PRRN(L); photoreceptor-ascending motor ganglion (PR-AMGRN(R)) relay neurons on the right (Figure 8c); and antenna relay neurons (AntRN) of the left and right sides. PR-AMG relay neurons of the right side receive input from ascending motor ganglion neurons that is reciprocated. Pathways with weak connections are shown with dashed lines. Details of pathway strength appear in Figure 9—figure supplement 1

https://doi.org/10.7554/eLife.16962.026

Compared with the brain vesicle, the motor ganglion has paired neurons and thus appears more bilaterally symmetrical (Figure 7; Figure 10—figure supplement 1), but nevertheless receives asymmetrical inputs from the relay neurons of the brain vesicle (Figure 10). These project mostly to the motor ganglion’s interneurons, however, although some sparse connections are made directly to the motor neurons themselves (Figure 10—figure supplement 1). The shortest sensory pathway to any motor neuron connects via a brain vesicle interneuron, and is thus disynaptic, although most direct pathways involve two interneurons and are thus trisynaptic (Figure 11). However, these shortest paths fail to depict the complexity of integration revealed in the total network (Figure 7—figure supplement 1; Figure 9—figure supplement 1).

Figure 10 with 2 supplements see all
Classes of relay neurons (presynaptic) in the CNS of Ciona and the inputs these provide to cells on the left and right sides of the motor ganglion (for details of relay inputs see Figure 10—figure supplement 1 and for antenna pathway see Figure 10—figure supplement 2).

For relay neuron class names see the key in Figure 1—source data 1). Each circle represents the input synapses to the first (column 1), second (column 2) or both (column 3) paired MG interneurons. Inputs to the left (blue) or right (red) partners are shown as an angular subtense of a circle the area of which represents the overall synaptic strength. Most sensory inputs are predominantly one-sided, some entirely so. Total synaptic input varies widely (see Figure 10—source data 1 for actual values and proportions).

https://doi.org/10.7554/eLife.16962.028
The shortest CNS pathways between sensory neurons and motor neurons for different sensory modalities are three-synapse arcs.

Four modalities are indicated, from top to bottom: light, gravity, coronet cells (possibly hydrostatic pressure) and PNS mechano/chemosensory. Members of the same cell types are assigned the same colour. Each pathway originates in the particular class of sensory neuron (photoreceptor: PR; antenna neuron: Ant; coronet cell, Cor; and PNS sensory neuron, pns) connecting via relay neurons (RN) and interneurons (IN) of the brain vesicle, to interneurons (IN) of the motor ganglion, to motor neurons (MNs). AMG: addition relay neurons of the motor ganglion receive input from the PNS. These pathways are all interconnected, overlapping networks for all sensory modalities thus underlying the complex reality of the actual behavioral network (Figure 9—figure supplement 1).

https://doi.org/10.7554/eLife.16962.032

The relay neurons of the photoreceptor and antennal pathways project asymmetrically (Figure 10—source data 1; Figure 10—figure supplement 2), photoreceptor interneurons connecting 70% to left-side interneurons, and antenna interneurons connecting 70% to right-side interneurons, of the motor ganglion (Figure 10; Figure 10—source data 1). The asymmetry is greatest among relay interneurons that receive additional input from coronet cells. Those that receive photoreceptor combined with coronet cell input project entirely to the left, and those that receive antennal and coronet cell input project entirely to the right. The asymmetry in pathways to motor ganglion interneurons parallels that of the direct input to motor neurons (photoreceptor pathways connecting to the left and antenna to the right) (Figure 10; Figure 10—source data 1).

The motor ganglion

The five pairs of motor neurons (MN1-MN5) form morphologically distinct neuromuscular junctions. Two anterior primary motor neurons (MN1 and MN2) each form 200–360 large synapses with many vesicles and postsynaptic densities on the muscle (Figure 12A), whereas the three more posterior motor neurons (MN3-MN5) each form 15–50 smaller neuromuscular junctions, often with fewer vesicles and less obvious muscle postsynaptic specializations (Figure 12B). The input to left and right muscle at these junctions is asymmetrical from all motor neuron pairs except the MN2 neuron pair, which provides symmetrical synaptic input in both number (46% to left and 54% to right) and total size (49% to left and 51% to right). Asymmetries in synapses observed between left and right muscle are not great, however, falling within a left:right ratio range between 60:40 and 40:60, MN1 having greater input to the right, and MN3-MN5 greater to the left (Table 4).

Ciona intestinalis larval motor neuron terminals and neuromuscular junctions.

(A) Neuromuscular junction (arrow) of MN1, with a postsynaptic specialization on the muscle (arrowheads). A basal lamina (red arrow) extends in the cleft between neuron and muscle. (B) Two adjacent neuromuscular synapses (arrows) with postsynaptic cisternae (arrowheads), but lacking postsynaptic membrane densities. (C,D) Enlarged views of anterior tail with reconstructed puncta representing colour-coded neuromuscular junctions of each motor neuron pair. (D) Top: Dorsal view. Bottom: Left lateral view. Scale bars: 1 µm (A, B); 10 µm (C, D).

https://doi.org/10.7554/eLife.16962.033
Table 4

Input to left and right dorsal and medial muscle bands from motor neuron pairs at their neuromuscular junctions.

https://doi.org/10.7554/eLife.16962.034

Motor neuron pair

Left muscle band

Right muscle band

Ratios

Dorsal

Medial

Dorsal

Medial

Left: Right

No. syn

No. sec

No. syn

No. sec

No. syn

No. sec

No. syn

No. sec

No. syn

No. sec

MN1

192

969

47

145

230

1181

130

558

40: 60

39: 61

MN2

224

1583

258

1636

46: 54

49: 51

MN3

42

156

28

101

60: 40

61: 39

MN4

45

189

30

116

60: 40

62: 38

MN5

21

128

15

55

58: 42

70: 30

  1. Number of synapses (No. syn) and number of synaptic profiles (No. sec) provided for each motor neuron and left:right ratios expressed as percentages of neuromuscular junction input from left and right partners for each motor neuron pair.

Motor neurons also form synapses with each other, with some further asymmetries between connections on the left and right sides (Figure 13A). On the right side only, MN1 is presynaptic to MN4 and postsynaptic to MN3. MN2 also shows asymmetries, receiving input from MN3 on the right, but providing input to MN3 of the left, and receiving feedback input from MN5 just on the left side. These synaptic asymmetries contrast with the obvious symmetry of the network of gap junctions between the motor neurons (Figure 13A–D).

The networks of motor neurons MN1-MN5 and descending ipsilateral neurons (MG1-MG3) of the left and right side of the motor ganglion (MG).

(A) Synaptic network of motor neurons 1–5 on the left (MN1-5L) and right (MN1-5R) sides. (B) Network of putative gap junctions between motor neurons of the MG. (C) Summary diagrams of motor neuron synaptic networks of left and right sides. (D) Summary diagrams of putative gap junction network of motor neurons of the left and right sides. Dotted lines represent tentative connections, dashed lines minimal contacts, and solid line connections with many large contact sites. (E) Synaptic network of descending ipsilateral interneurons (MGINs) of the motor ganglion. (F) Network of putative gap junctions between descending ipsilateral interneurons of the motor ganglion. For a, b, e and f: Arrows illustrate polarity of synapse, line thickness show the cumulative depth of synaptic contact in µm (see Materials and methods). Red lines illustrate synaptic contacts that differ between left and right sides of the MG.

https://doi.org/10.7554/eLife.16962.035

The synaptic strengths (see Materials and methods) of the interneuron network on left and right sides are also asymmetrical (Figure 13E). In particular, the strength of the synaptic input from the first interneuron (cell MGIN1) to the second (cell MGIN2) is far greater on the left side. This asymmetry is paralleled by the gap junction network, in which the second interneuron is connected to the first only on the left side. In addition, this second left interneuron (MGIN2L) is also connected to the contralateral first right interneuron (cell MGIN1R) by gap junctions (Figure 13F).

Sidedness in the neural network of the motor ganglion is also evident in the synaptic connections between interneurons and motor neurons (Figure 14). The first motor neuron receives more inputs on the right side from both the first and third interneuron (cells MGIN1 and MGIN3) than it does on the left (synaptic depths 1.08 and 1.2 µm versus 0.42 and 0.72 µm). The second motor neuron (MN2) likewise receives more inputs from the second interneuron on the right (MGIN2R) than the left (MGIN2L) side (synaptic depths 4.69 versus 1.24 µm). Other asymmetries are apparent in connections between cells that are reciprocal on only one side: MN1L to MGIN3L, MN3L to MGIN2L, on the left, and MN5R to MGIN1R, MN1R to MGIN2R, and MGIN2R to MN4R on the right (Figure 14A). These pathways refer to chemical synapses (Figure 14A), relative to which those exhibited by gap junctions (Figure 14B) are more left/right symmetrical and involve more connected partners across the midline, especially for the anterior components.

Left-right asymmetries in the overall synaptic pathways of the motor ganglion.

Pathways shown are between motor neurons (MNs), descending ipsilateral interneurons (MGINs) and descending mid-tail neurons. (A) Synaptic network with arrows indicating polarity of synaptic contacts. (B) Summary network of gap junctions for descending motor ganglion neurons illustrated in (A). Blue lines represent gap junction inputs from motor neurons, green represent gap junction inputs from interneurons, and pale blue are gap junction inputs from mid-tail neurons. Pathway strength varies over a wide (>25 times) range and is more left-right symmetrical than the synaptic network in (A). In both (A) and (B) pathways shown by orange lines are left/right asymmetrical, and those in pink are present only on one side (key). Thickness of lines indicates cumulative depth of synaptic contacts (see Materials and methods) (scale).

https://doi.org/10.7554/eLife.16962.036

Aside from the obvious asymmetry in their number, ACINs are also asymmetric in their projections and connections between left and right sides of the posterior motor ganglion (Video 5). On the right, although the ACIN crosses the midline, it does not pass the ependymal cell to extend into the right neuropil, and so forms no contralateral synapses. On the left, however, both ACINs decussate fully, crossing to the contralateral neuropil and forming synapses there. These synapses are as a result not formed directly onto contralateral motor neurons, as previously proposed (Horie et al., 2010), but instead onto interneurons (cells MGIN1R, MGIN2R and MGIN3R) of the right side (Figure 15A). Both left and right ACINs are presynaptic to their ipsilateral motor neurons (Figure 15B) and first two pairs of interneurons. All three ACINs are also presynaptic to a right-side bipolar tail neuron (cell BTN2, reported in Stolfi et al., 2015), and additionally form multiple synaptic contacts directly onto the ventral basal lamina (Figure 15C). That input resembles the synaptic input from the motor neuron terminals, which form onto the basal lamina that ensheathes the muscle (Sanes et al., 1978).

ACIN synapses and network.

(A) Presynaptic site (arrow) from the left ACIN onto contralateral MGIN interneurons at a dyad synapse. BTN2: bipolar tail neuron profile. (B) Dyad synapse (arrow) onto ipsilateral motor neuron MN3R and an unpaired tail interneuron (PMGN2) on the right side. (C) Synapse (arrow) from ACIN1L onto the ventral basal lamina (BL) opposite the notochord. Scale bar (a-c): 1 µm. (D) Network diagram of ACIN pathways. Layout plotted as an edge-weighted spring embedded network (Cytoscape 3.1.0: NRNB.org) based on synapse pathway strengths (see Materials and methods). Right and left neuropiles are each enclosed in a dashed line. Pathway strengths are shown as the line thickness sorted by the cumulative depth of synaptic profiles (key). The right side includes two sided PMGN interneurons and their partners. Note reciprocity of connections for ipsilateral but not contralateral partners. Thus ACINs are presynaptic to contralateral partners but not postsynaptic. Cell types abbreviated as in Figure 1—source data 1 (E) Dorsal view of reconstructed ACINs. Scale bar 10 µm.

https://doi.org/10.7554/eLife.16962.037
Video 5
Rotations of reconstructed ACINs decorated with their presynaptic sites.

Reconstructed ACINs with presynaptic sites colour-coded by postsynaptic cell type: basal lamina (black); motor neuron (blue), descending MG interneuron (green), bipolar tail neuron (red), and posterior MG descending neuron (brown)

https://doi.org/10.7554/eLife.16962.038

Presynaptic input to ACINs is not only asymmetric, but differs between each of the three ACIN cells. All are postsynaptic, however, to at least one ipsilateral interneuron and one ipsilateral motor neuron (Figure 15D). Both left ACINs receive input from MN3L and MGIN2L, but ACIN1L receives additional input from MN1L and a descending decussating neuron (cell ddR), while ACIN2L receives input from MGIN1L and the third a contralateral interneuron (MGIN3R). On the right, the only symmetrical input partner is the ipsilateral first interneuron, MGIN1(R), with asymmetrical input from MN2R as well as right-side bipolar tail neurons.

Summarizing: the connectivity matrix of all neurons reveals the following features (Figure 16): (a) Most cell types form synapses among members of the same class of neuron. (b) Many synapses also form on the basal lamina and from motor neurons onto muscle cells. (c) A high degree of reciprocity is manifest between members of different neuron classes, especially those that are interneurons. (d) Neurons are both pre- and postsynaptic, those that have many presynaptic sites generally also have many that are postsynaptic, except for sensory neurons in which presynaptic sites predominate. (e) The pathway strength is in general greater in the motor ganglion but also high for the relay neurons and among neurons of the pathway from the peripheral neurons. (f) Cell classes with fewer representative neurons tend to have greater pathway strengths. Features of the sidedness of the matrix are reported in Figure 16—figure supplement 1

Figure 16 with 2 supplements see all
Entire connectivity matrix for the complete brain of a larva of Ciona intestinalis.

Shown for all synapses are the pre- (rows) and post- (columns) synaptic cells, colour-coded by cell type (see Figure 1—source data 1) and arranged in their rostro-caudal sequence along the longitudinal axis (presented in Figure 16—figure supplement 1 is the same matrix with cells sorted into left and right sides). Each intercept is colour-coded for the cumulative depth of presynaptic contacts made by that neuron upon its postsynaptic partner (key, bottom). In the case of dyads or triads, all connections are plotted. Also included are muscle cells, and the basal lamina of the CNS, both of which are exclusively postsynaptic. Other cell types, particularly ependymal cells lacking axons, are excluded. Muscle cells of the dorsal and medial bands are pooled on each side, because these are connected via gap junctions (Bone, 1992). The matrix is bounded by nested boxes between specific cell types. The smaller boxes enclosed by dashed lines indicate the connections between neurons of the same subtype. These are enclosed within boxes bounded by coloured lines, which indicate connections between neurons of the same brain region. Neurons of the brain vesicle are segregated into sensory neurons (orange lines, Sensory), intrinsic interneurons (pink lines, BVIN) and relay neurons (green lines, RN). Remaining boxes are as follows, neurons of the: PNS (PNS); motor ganglion (MG); and tail neurons of the caudal nerve cord (CNC). For matrix file see (Figure 16—source data 1).

https://doi.org/10.7554/eLife.16962.039

Discussion

We report the full synaptic connectome of a single tadpole larva of a model chordate species, the ascidian Ciona intestinalis, and use this to identify the complete inventory of the many asymmetrical features in its CNS. Some connectomic analyses rely on symmetry of the connections between left and right sides to validate the synaptic connections of neurons that are paired (Durbin, 1987; Randel et al., 2014; Ohyama et al., 2015). This approach has not been possible in Ciona, because many cells are not bilaterally paired in the brain vesicle, while in the motor ganglion where cells are paired, presynaptic inputs from the brain vesicle are similarly asymmetrical. Additionally, we cannot exclude the possibility that minor left/right asymmetries might be the product of developmental noise or imprecision, insofar as only a small number of larvae need to survive and eventually become adults.

Our findings reveal not only those features with possible counterparts in the vertebrate CNS, but also the many features of all nervous systems. These include the wide range and combination of cells that share synaptic contacts, the poorly segregated distribution of synapses over the neuron surface; synapses onto non-neuronal cells and basal lamina; unpolarized and mixed vesicle synapses resembling those in cnidarians (Westfall, 1996); and the apparent general redundancy in the connectome. Unpolarized synapses have been previously reported between coelenterate (Horridge and Mackay, 1962) and pulmonate mollusc (McCarrager and Chase, 1985) neurons, while synapses onto the basal lamina have been reported in muscle after removing the underlying myofibres (Sanes et al., 1978). Synaptic sites that occur onto the basal lamina, and that therefore lack postsynaptic partners, resemble other presynaptic sites in the CNS, and resemble neuromuscular junctions, where neurotransmitter must cross the basal lamina to act on adjacent muscle cells. Some presumed sites of synaptic release across the basal lamina lie opposite non-neuronal cells, including epidermal cells or cells of the notochord. Neurotransmitter receptor expression studies are critical to our understanding of the roles these special sites of synaptic vesicle release may play. Sites that lack adjacent cells may be sites of neuromodulator release. This is particularly likely in the case of coronet cells, which express reporters for dopamine and have exclusively dense-core vesicles at their synapses onto the basal lamina. Synaptic reciprocity and serial networks are commonplace in many nervous systems (e.g. Dowling and Boycott, 1966). Aided by the numerical simplicity of the Ciona CNS, we are also able to detect features such as cilia that may lie undetected in highly populated vertebrate brains, and confirm that many neurons in Ciona are ciliated (Figure 3—figure supplement 1).

An obvious point of comparison is set by the nervous system of C. elegans, the sole precedent for a completely reported connectome, with which the Ciona larva is dimensionally comparable and with which it has a numerically similar network -- 302 identified neurons in the nervous system of a single hermaphrodite C. elegans (White et al., 1986; Varshney et al., 2011) compared with 177 neurons in the larval CNS of Ciona. Neurons form comparable numbers of synapses in both these model nervous systems -- an average of 37 presynaptic sites and 7 putative gap junctions (>1 section) compared with 28 synapses and 3.2 gap junctions per non-pharyngeal neuron in C. elegans (calculated from data at http://www.wormatlas.org/hermaphrodite/nervous/Neuroframeset.html). These numbers are smaller than in Drosophila, in which optic lobe neurons may have in excess of 100 presynaptic sites (Meinertzhagen and Sorra, 2001; Takemura et al., 2008, 2015) and clearly less than typical vertebrate neurons (for example a mouse somatosensory cortex neuron has about 8200 synapses: Schüz and Palm, 1989). Further discussion of this topic is provided elsewhere (Meinertzhagen, 2010).

For brain asymmetry to appear in the hatched larva, the expression of Nodal and ciliary action are both required during embryonic development (Nishide et al., 2012; Thompson et al., 2012). These events are perturbed during the process of dechorionation that has been used for many experimental interventions, especially electroporation of genetic reagents (Shimeld and Levin, 2006), so that many asymmetries may have escaped detection in previous reports that are revealed in our larva reared with an intact chorion. Furthermore, within the chorion the developing embryo invariably curls around itself along the left side of the trunk (Katsumoto et al., 2013), and this may further influence sidedness in the larval brain, in ways that are lost after dechorionation. In addition to pigment cell displacement, arrestin expression indicating photoreceptor cell fate, is significantly altered, often expanding to the left brain in dechorionated embryos (Oonuma et al., 2016).

The overall asymmetry of the larval ascidian CNS finds deep parallels with asymmetries in other chordates, including vertebrates (Boorman and Shimeld, 2002b), and clear differences from those of other deuterostomes, such as echinoderms (Duboc et al., 2005). Most difficulties in comparing between ascidian larval and vertebrate nervous systems come however from differences in their respective cell numbers. The extreme miniaturization of the former exposes sidedness in the ascidian larval brain that can actually exist in both chordate clades, such as in the vertebrate epithalamus (Concha and Wilson, 2001; Hamada et al., 2002). Structurally most obvious in the ascidian larva is sidedness in the position of the ocellus, which is driven by the same left-side action of Nodal (Yoshida and Saiga, 2011) as drives asymmetry in all chordate brains, including those of vertebrates (Halpern, 2003; Carl et al., 2007).

Migration of the ocellus pigment driven in Ciona by Nodal (Yoshida and Saiga, 2011) calls to mind the vertebrate pineal, which is also lateralized based on Nodal’s action (Carl et al., 2007). Nodal acts on the left side of the CNS in both, but in Ciona the pigment cells migrate to the right, whereas vertebrate parapineal cells migrate to the left. Moreover, based on the projection of their outer segments, which is inward, not toward the outside world, Ciona’s larval eyes are more akin to lateral eyes than to a single pineal (Lamb, 2013). The photoreceptor cells of the ocellus likely share a common lineage, blastomeres A9.14 and A9.16 on the right side (Oonuma et al., 2016), with the coronet cells of the left side (Cole and Meinertzhagen, 2004). Ascidian coronet cells are likewise components of a morphologically homologous structure that is bilateral in vertebrates, the saccus vasculosus (Smeets et al., 1983). In vertebrates, the saccus vasculosus comprises coronet cells in addition to neurons contacting the cerebrospinal fluid. In Ciona we report new ciliated coronet-associated neurons, with cilia that project into the neural canal toward the coronet cells’ bulbous protrusions. In both vertebrate and ascidian cases, it is these neurons rather than the coronet cells that give rise to an axonal pathway, forming the axon tract emanating from the saccus vasculosus in vertebrates (Rodríguez-Moldes and Anadón, 1988) and the coronet complex in Ciona, further strengthening the similarities between coronet complex and saccus vasculosus. Both ocellus and coronet complex structures are lateralized in Ciona, unlike the single medial structures reported for cyclopic mutants (Belloni et al., 1996; Chiang et al., 1996) or fused, unpaired saccus vasculosus phenotypes (Nieuwenhuys, 1998).

Given that ascidian larvae swim in a helical pattern (McHenry, 2005) and have a single-sided ocellus, their phototactic behaviour follows a helical, not visual pattern, as defined by Randel and Jékely (2016). Unlike simple helical phototaxis, however, the complement of cell types and complexity of component connections involved in Ciona’s visual circuit (Video 6) compare much more closely to circuits responsible for visual phototaxis than those for simple helical phototaxis (Randel and Jékely, 2016: their Figure 2f,g). Helical swimming of the ascidian larva provides a mechanism by which a preexisting bilateral visual phototaxis circuit could have been co-opted into a complex hybrid helical phototaxis circuit, still allowing mechanisms such as delay, sensory integration, and modulation to take place, and unlike the more direct helical phototaxis mechanisms in ciliated forms, such as protists and trochophore larvae (Randel and Jékely, 2016).

Video 6
Animated reconstruction of the photoreceptor pathway.

Cell bodies, shown as spheroids, from photoreceptor through relay neurons to the motor ganglion. Cells are colour-coded as in Figure 1—source data 1

https://doi.org/10.7554/eLife.16962.044

The presence of unilateral systems in the larval CNS of Ciona that show homology with bilateral structures in the vertebrate brain can be interpreted as a reduction in one side, and as an outcome of the small cell numbers in ascidian larvae. A defining feature of larval Ciona and its CNS is indeed the miniaturization of both. The larva’s small cell numbers can be seen as a direct outcome of the few embryonic cleavage generations, no more than 14 for the entire CNS (Cole and Meinertzhagen, 2004), the lack of metamerism (Garstang, 1928; Crowther and Whittaker, 1994), the lack of feeding in this lecithotrophic larvae, and consequently its short life. The small number of its component neurons is reflected in turn in the relatively small numbers of synapses formed by each, conforming to an overall relationship between neuron number and synapse number per neuron seen in nervous systems generally. In contrast to both is the richness of cell types. Among the 177 larval neurons we can distinguish at least 25 different types and 52 subtypes identified on the basis of morphological and connectivity differences. C. elegans has a numerically comparable richness, with 118 cell types among its 302 neurons (White et al., 1986).

The asymmetries we observe start to appear with the failure of cells to pair left and right, at around 75% of embryonic development (Cole and Meinertzhagen, 2004). After this the brain vesicle pushes to the right between 75% and 85% of embryonic development, and cell positions begin to shift, followed by the loss of strict bilateral symmetry among the cells of the motor ganglion. The pattern of cell lineage is left/right symmetrical until the 11th cleavage, with ventral divisions becoming desynchronized after the 10th cleavage (Cole and Meinertzhagen, 2004). The photoreceptor pigment cell begins its migration at the 11th generation. The ACINs appear after the 11th embryonic cleavage (Nishitsuji et al., 2012) and fate choices select cells as either neurons or ependymal cells at around this stage too (Cole and Meinertzhagen, 2004). The asymmetry in position within the motor ganglion and between the cell numbers of the two sides of the caudal nerve cord is apparent by the final stages of embryonic development.

Total cell numbers are mostly left-right symmetrical throughout the CNS, and sidedness is mostly a question of cell fate. Thus the fate choice, for example between neuron and ependymal cell type, generates the sidedness in the numbers of cells comprising the same class. A striking example is provided by the ACIN neurons in the rostral caudal nerve cord. Thus A11.116 undergoes two divisions, first to yield A12.231 and A12.232 (Nishitsuji et al., 2012: their Figure 5B), and a further round of divisions, yielding a total of four 13th-generation cells that become two ACIN and two ependymal cells. The fate decision to generate ependymal and ACIN cells is then proposed to occur after this final division, based on the expression of extracellular signals and transcription factors. The total number of ACINs varies between larvae (Nishino et al., 2010), and in the larva we report, this variation manifests itself as an asymmetry, in which we observe two progeny of the left side as ACINs, with only a single ACIN present on the right, the other site being occupied by ependymal cell tail 4. Thus variation in cell fate decision is, we propose, the basis for the left/right asymmetry in ACIN neurons. We also find neurons on the right side (PMGN1 and PMGN2) of a hitherto unreported type that are located anterior to the single ACIN (ACIN2L) of that side, but posterior to motor neuron pair MN5. Their asymmetrical location suggests they may represent similar late choices between neuronal and ependymal cell fate.

Together with these asymmetries in cell types, there are clear asymmetries in connectivity. These are most obvious in inputs to, and connections within, the motor ganglion. Similar asymmetries may exist in other motor systems but are revealed only from comparisons between the left and right sides of a complete network, and so have rarely been revealed. In the mouse, however, an imbalance index for the motor innervation of interscutularis muscles reveals an asymmetry in the morphological features of motor innervation (Lu et al., 2009), while in the polychaete Platynereis there is also an asymmetric connectivity pattern for one class of motor neuron, both for the inputs it provides to the muscle it innervates and the inputs it provides to contralateral neuron partners (Randel et al., 2014, 2015). These instances compare to both the asymmetrical input of, for example, MN1 to muscle and other network asymmetries we find in Ciona in which, like Platynereis, an input from two ACINs on the left side of the brain to interneurons on the right side is not matched by a contralateral input from the ACIN on the right side.

Our conclusions are based on a single larva, although four unrelated sibling larvae have been reported with closely similar overall cell complements (Nicol and Meinertzhagen, 1991). Data are still lacking on the constancy of neuron cell types and their connections between individuals. The consistency of asymmetrical differences we see in this larva will only be resolved when its sibling larvae are examined, as we now undertake.

Mechanosensory drive of symmetrical swimming was initially dismissed because tails can retain swimming independent of the head, and no known input from mechanosensory cells to motor neurons was known. However, we find multiple inputs from mechanosensory tail neurons’ interneurons to motor neurons, both from AMGs in the dorsal MG, and as reported from the bipolar tail neurons (Stolfi et al., 2015). It is not yet clear whether the cilia of DCENs or VCENs may alternatively have thermoreceptive, electroreceptive, or chemoreceptive properties, although there is evidence for chemotactic behavior during larval settlement, mediated by epidermal sensory neurons.

A proposed central pattern generator (CPG) for Ciona has been compared (Horie et al., 2010) to the vertebrate swimming CPG of the lamprey (Grillner and Wallén, 1999) but in Ciona omits the role of excitatory interneurons in general and ipsilateral connections within the motor network in particular. Moreover the depicted network is left-right symmetrical and with proposed direct contralateral input to motor neurons, features we now show to be lacking in Ciona’s CNS connectome. This difference highlights the power of a complete connectome to reveal the actual connections between identified neurons. We find the left-right difference of the ACIN connections, in particular, to be most surprising, and endorsed by the failure of the right ACIN to send a neurite into the left neuropile even though it crosses the midline. Additional features of the network’s right side also support this asymmetry: the lack of ACIN1 on the right side, and the two additional right-side interneurons with neurites that remain ipsilateral, and the lack of inhibitory feedback to the first interneuron on the right from the ipsilateral ACIN. An additional left-right asymmetry is provided by the input from the first interneurons MG1 L and R to the second interneurons MG2 L and R, which is far greater on the left than on the right side, and supported by an asymmetrical distribution of gap junctions. The latter is endorsing, and unusual because putative pathways formed by gap junctions are in general more symmetrical than those formed by chemical synapses.

Larvae swim in a helical pattern (McHenry, 2005) from simple bilateral flexions of the tail (Bone, 1992; Nishino et al., 2010). The helical pattern may reflect the asymmetry of motor pathways, but varying the direction of incident light relative to the ocellus could also generate helical klinotaxis, enablng phototaxis in response to cyclical changes in light intensity as the shadow of the ocellus pigment sweeps across the outer segments (McHenry and Strother, 2003; McHenry, 2005; Randel and Jékely, 2016). Based on gravitaxis in young larvae (Tsuda et al., 2003) we also predict that the antenna neurons’ circuits underlie a directional response to gravity. It is interesting that the visual pathway originating in the right-sided ocellus and the gravity pathway from the antennal cells, both converge asymmetrically in the motor ganglion, the visual pathway stronger to the left of the ganglion and the gravity pathway to the right. These asymmetries in sensory input to the MG, particularly for the antenna pathway, suggest that a sensory input ought to generate a sided swimming movement, such as a large unilateral contraction (Video 7). We predict from the network that this should predominantly be on a fixed side of each animal, as video recordings indeed suggest (Video 8; Ryan, unpublished observations).

Video 7
Unilateral tail flick.

A larva exhibits a unilateral tail flick.

https://doi.org/10.7554/eLife.16962.045
Video 8
Asymmetrical tail flicks.

A larva exhibits repeated tail flicks to the same side of the trunk.

https://doi.org/10.7554/eLife.16962.046

Despite its shared evolutionary ancestry with chordates, the synaptic network we report incorporates many pathways that are manifestly left-right asymmetrical but that compare to pathways in the vertebrate brain so far reported to be bilaterally symmetrical in form and function. We interpret these asymmetries as an ascidian specialization brought about by the small cell numbers and rapid development of the larval stage, and compatible with the larva’s sensorimotor behaviour and helical swimming pattern.

Materials and methods

Animals

Adult sea squirts, Ciona intestinalis (L.), were collected by Mr. Peter Darnell from Mahone Bay, Nova Scotia. Adults were kept in tanks under constant illumination at the Aquatron facility of Dalhousie University, with flowing sea-water (5–6 L/min) at ~18°C. Adults kept for 1–5 days were removed from the tank and dissected to expose the oviduct and sperm duct. Eggs were collected from the oviduct using a pipette and placed in Petri dishes containing sea-water filtered through a Nalgene 0.2 µm syringe filter. The animals were then washed with sea-water and the sperm duct pierced with a pipette and sperm sucked directly into the pipette and placed in a microcentrifuge tube. Sperm from one adult was added two drops at a time into a Petri dish containing eggs from a different adult and the dish gently swirled to distribute the sperm. Eggs and sperm were left for 15 min for fertilization to occur, then eggs were rinsed several times with filtered sea-water and placed in a Petri dish with filtered sea-water, wrapped in aluminium foil, and placed in an incubator at 18°C.

Electron microscopy

Dark-reared larvae were removed from the 18°C incubator after 20 hr, and larvae (21 hr post fertilization/2 hr post hatching) were fixed at 4°C for 2 hr in 1% OsO4 in 0.2M Na2PO4 (phosphate buffer) adjusted to pH 7.2 with HCl. The animals were then post-fixed in 0.2M phosphate buffer containing 2% glutaraldehyde for 1 hr at 4°C. Prior to fixation newly hatched larvae received light from a dissecting microscope fibre optic illuminator only briefly to check that they were swimming, and to pipette them into the fixative. Fixed specimens were dehydrated in an ethanol series (30%, 50%, 70%, for 10 min each, followed by 90% 95%, 100% then propylene oxide for 5 min each). After dehydration, specimens were placed overnight at 23°C in a dish containing propylene oxide and Poly/Bed 812 resin 1:1. Sibling larvae, the products of a single cross, were next transferred to 100% resin for 3 hr and then placed in 100% resin and polymerized at 60°C for 48 hr. Larvae were sectioned and checked for acceptable fixation. Nervous tissue from marine animals presents special problems for good EM fixation (Cobb and Pentreath, 1978) and a single larva selected for its clear synaptic vesicle profiles and intact cell membranes was cut in a series at 60, 70 or 100 nm, as reported in Results (Figure 1A). All sections were post-stained for 5–6 min in freshly prepared aqueous uranyl acetate followed by 2–3 min in lead citrate. Sections were viewed using an FEI Tecnai 12 electron microscope operated at 80kV and images captured initially using a Kodak Megaview II camera with software (AnalySIS: SIS GmbH, Münster, Germany), or a later Gatan 832 Orius SC1000 CCD camera using Gatan DigitalMicrograph software (Gatan Inc., Pleasanton, CA).

Imaging

High magnification 3.85 nm per pixel images were collected for the neuropil region of each section. The profile area ranged from five 5 × 5 montages per section in the posterior brain vesicle to single 2 × 2 montages in the tail. Independent lower magnification 13.9 nm per pixel images of the entire CNS and overlying epidermis were collected for every section in the anterior BV, and for every fourth section through the posterior brain vesicle, neck, motor ganglion and anterior tail.

The montages were compiled automatically with the Gatan DigitalMicrograph software or with AnalySIS. Given a limitation of the Gatan software-generated montages, final montages were also manually montaged in Adobe Photoshop. Images were imported into either a high magnification or low magnification series in Reconstruct (Fiala, 2005), and sections manually aligned using this software. All profiles in every third section, somata in the low-magnification series and all profiles in the high-magnification series, were then traced completely. Fewer than five neurites were candidate orphans that lacked synapses, and thus did not contribute to the CNS connectome. Skeletonization of neurite projections was accomplished using the function Z-Trace in Reconstruct, which connects the mid-point of each traced profile. In the high magnification series, traces were hidden and all sections then blindly annotated for synapses, putative gap junctions, dense-core vesicles and cilia. After each block of sections was annotated, the traces were then made visible and annotated elements assigned to specific pre- and postsynaptic elements. Blind annotation was duplicated in 100 section blocks by an independent annotator (Ms Carlie Langille). Most annotations of each viewer duplicated existing synaptic contacts seen by the other, but neither viewer annotated a synapse between two partners that did not replicate a synapse formed elsewhere by the same two cells. Of the differences observed, 95% were simply those between the numbers of sections in which a synapse was observed.

Synapse analysis

Each neuron was classified from structural criteria, mostly from the identity of its presynaptic partner(s) (see key in Figure 1—source data 1). Synapses were identified based on the criteria established in C. elegans (White et al., 1986) of a cluster of vesicles at a presynaptic membrane. Although postsynaptic densities were observed at some synapses (Peters and Palay, 1996), these were either not clear or not present at all sites with a presynaptic vesicle cluster. Putative gap junctions were annotated at sites with juxtaposed membranes and densities on the membranes of both sides, except where such contacts were directly adjacent to the neural canal, which are candidate desmosomes and were not studied further. The numbers of synapses and the numbers of sections in which a synaptic profile, a single imaged cross-section of a synapse, was observed from each were both measures used to quantify synaptic strength, as was their product, the total depth of synaptic profiles. The cumulative depth of synaptic contact was calculated by multiplying the number of sections in which a synapse was observed by the section thickness. These values for each presynaptic neuron were linearly proportional (Figure 3B); the relationship remained unchanged for the numbers of synapses per neuron when small synapses – those containing only a single section -- were removed. This depth in µm was used for network diagrams as a putative proxy for synaptic strength.

Nucleus location was used to determine neuron sidedness. A line through the midline of the CNS drawn from the ventral to dorsal surfaces through the neural canal was used to classify nuclei as left or right. Those with nuclei intersected by the midline were classified as midline neurons. Nuclear x/y positions are reported in Figure 1—source data 1 Partitioning the parts of each neuron (cell body/soma, axon, terminal, or dendrite) was determined by examining the reconstructed neuron and identifying the sections at the boundaries between these parts. Axons were distinguished from their somata of origin by the reduction in profile diameter, and terminals were distinguished from axons where we observed branching or expansions with presynaptic sites near their final termination. Many terminals had but small swellings, and their synapses occurred onto collateral terminals or axons, so were considered en passant. Dendrites were those regions proximal to the cell body in which short neurites extended, with or without branching. Some relay neurons contain expanded branched regions near their axon hillock, which we classified as ‘BVterm’ and these were included in the analysis of dendritic synapse. Exclusively postsynaptic dendrites were lacking in the CNS. Despite our attempts to partition them in this way, neurons were characterized by a lack of segregation of pre- and postsynaptic sites along their length and a general absence of defining features for each region.

Video microscopy

For Video 1 and Video 7, larval swimming was recorded using a Motionscope CCD camera by Redlake Imaging (Model PCI 2000 S #1108–0009) mounted on a Leica MZFLIII dissecting microscope equipped with a Plan Apo 1/0x objective and recording these on Sterling computer using MIDAS software. Clips were selected and converted to. mov format using iMovie. For Video 8, hatched larvae were recorded using a Leica MZFLIII dissecting microscope equipped with a Plan Apo 1/0x objective, capturing their images with a high resolution CCD camera (Elmo TSM 41OH) on VHS video tapes, and then transforming these to digitized image sequences at up to 60 fps by software (QuickTime Pro: Apple Inc.).

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Decision letter

  1. Eve Marder
    Reviewing Editor; Brandeis University, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Sidedness in the brain of a chordate sibling, the tadpole larva of Ciona intestinalis" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jason Pipkin (Reviewer #1); Scott Emmons (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Your manuscript provides an important report of the connectome of the larva of Ciona intestinalis, a chordate with a very small number of neurons. You present the result of a tremendous effort reconstructing the connectome of larval Ciona intestinalis from a series of electron micrographs. This represents the second whole-organism connectome ever fully reconstructed and is a finding of major significance.

The reviewers were universally impressed with the potential significance of this work, and were enthusiastic about is eventual publication. Nonetheless, because of its unusual and rare nature, and therefore its potential value to the field, the reviewers were very concerned that the data be presented as clearly and transparently as possible and that the manuscript itself be brought to the same high quality as the work itself. We all understand the inherent difficulty of figuring out how to present this amount of data in an accessible form, and we also understand that after a project that must have taken years of work, that you must be ready to see it in print. But all three of the reviewers found the presentation less than ideal. I am loath to tell you exactly how to reorganize it. I am taking the unusual step for eLife and including the full reviews so that you can both appreciate the enthusiasm of the reviewers and also see first-hand the myriad issues that arose during the discussion and the reviews. I understand that addressing some of the issues raised below may lengthen the paper. That is ok, as long as you use appropriate headings to orient the reader and to provide signposts to the work. Obviously, the ideal would be to achieve greater transparency and clarity without too much additional length.

Most importantly, please read and think about all of these comments, and then do your best to prepare a revision that deals with these issues. You will note that some issues arise in all three reviews, such as a clearer description of the system and its behavior. Please pay especial attention to those issues that are brought up in multiple reviews, as they are crucial to address. Many of the comments can be addressed with a few words, while others will require more thought.

Important: eLife would like you to release in an appropriate location the source data and reconstruction data and identifying criteria for use by the community. The major image dataset and reconstructions together seem an ideal candidate for sharing via the Open Connectome Project. Ideally, as much of the original data and the reconstructions should be openly available so that the connectome becomes a working data set for whomever is interested. Please let us know what your plans are in this regard.

Reviewer #1:

Ryan et al. present the result of a tremendous effort reconstructing the connectome of larval Ciona intestinalis from a series of electron micrographs. This represents the second whole-organism connectome ever fully reconstructed and is a finding of major significance. The authors relate their findings within the context of structural and connectivity asymmetries, particularly emphasizing relevant comparisons to vertebrates. As many readers are unlikely to be familiar with C. intestinalis, the Introduction should discuss the life cycle of the larva and its behavior. My understanding is that younger larvae swim upwards and older larvae swim downwards to eventually settle based in part on sensors for gravity and light. The authors should emphasize throughout how the Ciona connectome relates to its behaviors.

The generalizability of the conclusions drawn about the asymmetry here are tempered by the single-organism sample size. There is space for even more discussion about how variable these left-right asymmetries are from animal-to-animal. What percentage of larvae eventually settle? Perhaps the larval connectome can be "noisy" in terms of cell number and connection strength if only a small number of larvae need to survive and eventually become adults.

Some relevant comparisons to other connectomics work are missing. For instance, there are several recent papers from Cardona's group on larval Drosophila that briefly touch on issues of right-left symmetry (sometimes only in supplemental material). Their work, along with Randel et al. (2015), rely on an assumption of symmetry to determine the confidence that a given synaptic connection is real. This work challenges that assumption, and it would be good to address this contrast.

The manuscript as currently written lacks adequate elaboration of its methods. Neurons are divided into types, soma position is defined as left, right, or medial, and neuronal arbors are divided into different parts (axon, terminal, etc.) without any explicit and rigorous description of the criteria by which these decisions were made.

The authors report a measure of synaptic strength that depends on the number of sections containing a presynaptic profile. However, section thickness varied and it is not clear that the necessary compensations were done. Inevitably, all anatomically-based hypotheses of synaptic strength fall short of direct physiological measurement, so the authors must make absolutely clear at all times what exactly is being measured. The authors describe synapses "onto" basal lamina, a non-cellular structure. Because the concept of a synapse is ingrained the minds of many readers as involving at least two cells (excepting autapses), these should be described differently, perhaps as "putative sites of neurotransmitter release." Could these be sites of neuromodulator release?

There are at least two discrepancies in the presented results. First, there are missing connections in Figure 12C that are present in 12A. Second, the number of polyadic synapses is variously reported as 920, 921, and 2150 (25% of 8601) in the Abstract, Results, and Table 1 respectively.

Finally, the manuscript would greatly benefit from a key that relates neuron type and abbreviation.

Reviewer #2:

Ryan, Lu, and Meinertzhagen present the complete connectome of the larval ascidian Ciona intestinalis. This is a paper of potentially historic importance. Not only is this the second whole-animal connectome deciphered, it is the first connectome to be obtained from a single individual. Moreover, Ciona is a chordate, having a notochord and dorsal nerve cord. Therefore, the features of the connectome are of particular interest as a point of comparison to the previously available connectome of C. elegans. When there was only a single connectome available, and that of a tiny, potentially highly-derived invertebrate, it was difficult to know what to make of it and whether one could generalize its characteristics to other animals. Even if C. elegans were representative of invertebrates, how similar would the nervous system of an animal from the other great branch of metazoans be?

In fact, interestingly, Ryan et al. find they are very similar! Similar features include similar overall cell numbers but a rather large number of cell types, largely unbranched neurons making en passant synapses, similar numbers of chemical and gap junction synapses, many polyadic synapses, and little segregation of neurons into separated axonal and dendritic regions, indicating graded potential neurons. In the beginning of the Discussion the authors claim that many features of the connectome are basal. These may indeed be, but there really is little support for this claim. As there are no other connectomes to compare to apart from C. elegans, how can we know, for example, that "the apparent general lack of precision" is basal? The only support mentioned for one feature being basal is that unpolarized synapses are seen in coelenterates. But they are also seen in a pulmonate mollusc. Is the pulmomate mollusc primitive or basal?

Summary of substantive concerns

An important feature of the C. elegans connectome is that it comprises a neural network making a single giant component. That is, everything is connected to everything, eventually. One gathers this is true for the Ciona connectome as well (Discussion, first paragraph: features include the "wide range and combination of cells that share synaptic contacts"), but it is not discussed. In general, the authors should pay more attention to the overall graph properties of the network. The degree distributions are particularly important and should be given.

The authors have chosen to focus their manuscript on left/right asymmetries in the connectome. This is an over-emphasis at the expense of the other interesting features in the opinion of this reviewer. The Introduction does not tell us why this is such an important issue. It begins by suggesting such asymmetries are commonly known. Then later on (last paragraph) it is said they are rare and difficult to recognize. Apparently the issue of l/r asymmetry has been well-studied in ascidians and results from asymmetric nodal expression during embryogenesis as it is in vertebrates. Most of the Discussion is devoted to this issue. Since we know many morphological features of the body are asymmetric, we can expect this will be true for the nervous system as well; it's not clear why so much is made of this here.

Representing as it does a descriptive presentation of a very large number of observations, it is important for the manuscript to give the reader a framework that is easy to grasp and keep in mind into which the more detailed observations may be placed. This is particularly so in this instance, as very few readers will have any prior knowledge of the Ciona larva. Figure 1 is not clear and sufficient in this regard. It should have an excellent diagram of the entire larva, showing the locations of sensory structures, ganglia, nerve tracts, and muscles. Since the neurons are unbranched, do they run in bundles with a constant set of neighbors as they do in C. elegans? There should be a description of the larva's behavior. One finds out in the Discussion (fifth paragraph) that it does not feed! That's going to make a big difference in the complexity of the nervous system. What does it do besides swim? Does it chemotax, thermotax, and so forth? What sensory-behavioral pathways are revealed in the connectome? What circuits governing swimming behavior and its control?

Here are some additional questions or comments that occurred to this reviewer:

1) What constitutes the CNS and the PNS? CNS is referred to early in the paper without definition. Figure 15 shows that there are also a small number of PNS neurons. How are these distinguished in such a small nervous system? There is an error in the second paragraph of the Discussion, where it is stated there are 302 neurons in "most of the CNS" of C. elegans. There are 302 neurons in the entire nervous system and there is no clear division in C. elegans between CNS and PNS; these terms are not used.

2) Neuron types: this concept is used throughout, but how neuron "type" is defined is not described. Do the neurons really fall into a set of discreet, unambiguous types? In C. elegans, the broad classifications sensory neuron, interneuron, motorneuron are not unambiguous. Many neurons generally considered to be interneurons because of the large number of synapses they make with other neurons also make nmj's, while many neurons considered to be motor neurons also have lots of synapses onto other neurons. "Motor neurons" can have stretch receptors, and "sensory neurons" can have synapses onto muscle. Careful examination reveals that where the cut-offs should in fact be among these broadest classes is unclear and may be somewhat arbitrary. When the manuscript first refers to different neuron types (subsection “Neurons”: brain vesicle intrinsic interneurons, MG interneurons, BV relay interneurons), the reader does not know what these neuron types are. So the descriptions of their various properties are hard to appreciate. Diagrams of the various types, what defines them, where their cell bodies lie, and where their processes go, are needed.

3) In the first paragraph of the subsection “Neurons”, how can a "terminal" make an en passant synapse?

4) In the third paragraph of the subsection “Synapses”; junctions <2 sections were omitted from consideration. What is the justification for this cutoff? Why not junctions <3? How many junctions does this exclude and what fraction of the "load" do they carry?

5) In the fourth paragraph of the subsection “Synapses”, and throughout the paper: average is used as a statistic (average 37 presynaptic sites per CNS neuron). This doesn't seem to be a meaningful statistic. The distributions need to be given. In this regard, Figure 3A should probably be a semi-log survival curve. The curve shown doesn't tell us much as most of the points either don't move much along the X-axis or much along the Y axis.

6) Figure 3B is described in the text (subsection “Synapses”, fifth paragraph) as showing the number of presynaptic sites versus the number of postsynaptic partners, but the axes in the figure are labeled number of synapse profiles vs number of synapses. What are synapse profiles? And why is this ratio of interest? What's the point being made here, that all neurons have the same density of synapses?

7) In the second paragraph of the subsection “Sidedness in the brain vesicle”: there are said to be 17 enigmatic coronet cells on the left side, and Figure 5A-B is referred to. But I cannot see 17 enigmatic coronet cells in this figure. In fact, it's hard to distinguish any cells in the figures produced by Reconstruct (I presume). There need to be drawn diagrams to accompany them. And the drawings should be shown in the context of the entire larva, like the drawings of C. elegans neurons in WormAtlas (e.g. http://wormatlas.org/neurons/Individual%20Neurons/AVAframeset.html).

8) Figure 12. Can't read the labels in A and B. How do we know these left/right differences aren't just developmental noise?

9)In the sixth paragraph of the Discussion: number of neuron types. This seems to me to be Results, not Discussion.

10) I can't relate the cells listed (or cell types?) in the legend to Figure 7A to the cell types (?) listed in Table 2. I can't relate the cells listed in the master cell list Figure 3—figure supplement 1 with the cells e.g. in Figure 3D. The master cell list Figure 3—figure supplement 1 is said to be an excel file, but mine's a PDF. Excel file would be much easier to work with (e.g. the heading row can be kept visible as one scrolls down the list).

11) I don't have a Figure 9—figure supplement 1.

12) Table 1 lists 3 polyadic gap junctions, but polyads are not defined for gap junctions because there is no defined pre- and post-synaptic cell.

13) Figure 15. This is the main presentation, along with individual neuron maps (missing), of the findings of this reconstruction. This is presumably the directed graph of chemical connections. The corresponding undirected graph of the gap junction connections needs to be included. I don't understand why the weights of the matrix are given as "percentile synaptic strength." In fact, I don't know what this means, rank order percentile? or percent of the highest value? Is a matrix with numbers (number of EM sections, or physical size in microns, since section thickness varied across this reconstruction) available rather than these colors? Modelers or theoreticians who would like to study the properties of the network (there will be many!) will need this.

Reviewer #3:

This is a beautiful and very important work describing the entire neuronal connectome of the Ciona tadpole larva. Ciona early development and neuronal development are very well understood and the full larval connectome is a milestone addition to the resources available for this important chordate model organism. Importantly, urochordates are the sister group to the vertebrates, and have been studied intensively to understand the evolution of key vertebrate features. The connectome also provides a very important reference point for broader circuit comparisons across animals.

Given that this is a full connectome paper, I think the authors should not feel constrained by the 5000 word limit, but aim to describe the neuron complement and the full circuits in more detail. They should also think about how they can relate the connectivity to known larval behaviors (e.g., phototaxis, gravitaxis, larval settlement).

First, larval behaviors:

There is one important mistake that the authors need to correct first to be able to better interpret the connectome.

They state in the paper twice that the tadpole swims in a bilateral fashion with tail undulations: "The apparent symmetry of the characteristic swimming pattern of chordate-like tail undulations" and "Larvae exhibit a simple bilaterally symmetrical swimming response to light "

This is not true, Ciona and other ascidian larvae swim in a helical pattern. This was already known to Eakin and Kuda (1971) who wrote "When an ascidian larva is actively swimming the body rotates about its long axis." More importantly, a detailed kinematic analysis of swimming of the Ciona tadpole revealed how the larva turns towards light during helical swimming.

The authors should consult and cite "The kinematics of phototaxis in larvae of the ascidian Aplidium constellatum" by McHenry and Strother (Marine Biology, 2003) and "The morphology, behavior, and biomechanics of swimming in ascidian larvae" by McHenry (Can. J. Zool. 83: 62-74 2005). Appreciating the correct motor pattern is important to correctly interpret how the circuits work. The helical pattern emerges due to the asymmetry in tail bending. This is likely one of the main reasons for the high degree of asymmetry in this animal.

Video 1 does not demonstrate bilateral swimming, it is actually impossible to tell from this video what the locomotor pattern is (bilateral or helical) because the larva moves out of focus and the frame rate is too low. This video may be removed or just used as an illustration.

Neuron types and connectivity:

Figure 3——figure supplement 1 lists all cells and their important features. Since it is a comprehensive paper, the morphology and position of at least one example from each cell type should be shown in a supplementary figure (or figures) and where each cell type is located in the body (show body outline).

Likewise, some of the anatomical diagrams are difficult to interpret because the cells are not shown in the context of the whole body or together with other cells from the same circuit.

Same for the videos: Videos 2 and 3 – the detail of the ocellus should be embedded into the larger anatomical context (e.g., by showing the body outline).

It is in general difficult to follow the description of connectivity of the different subsystems (e.g., eyes, otolith), because the relevant details are embedded in the summery diagrams and are not explained in full. I would really encourage the authors to describe the different sensory-motor systems in more detail. This should include the reconstructed neurons in the whole-body context (anatomical diagrams) and the corresponding circuit diagrams. For example, what is the full cell complement and wiring diagram of the PRC circuit? Show type I, II, III PRCs and their downstream circuitry, to the muscles. Likewise, show the otolith and coronet circuits. What are the PNS neurons and their circuitry? How about the tail circuitry that probably tunes the left-right motor pattern?

The integration can then be described following the details of the subsystems (as is now e.g., in Figure 9).

It would also be very interesting to see the strength of all reciprocal connections (e.g., represented as the geometric mean of 2-way synapse numbers) at the level of the entire connectome. This can easily be computed from the whole matrix.

It is hard to read Figure 7—figure supplement 1. The network should be shown in higher magnification. An alternative representation that would be useful would be to merge cells of the same type and connectivity into one node and show the average synaptic strength.

Figure 11. Also show cell morphology of MN classes. Please also show a schematic of the different muscle types and their position relative to the MN axons. It is not clear how an MN projecting on the left side can synapse on muscles both on the left and right sides of the body.

Figure 15 is hard to read – please label neuron categories also on the presynaptic side. Provide list of abbreviations. Label major categories in the figure (corresponding to the boxes e.g., sensory neurons).

Discussion:

The functional and comparative implications of the Ciona connectome should be spelled out in more detail. For example, how does the connectome explain negative phototactic behavior? The strategy of ascidian phototaxis is helical (not visual, for an explanation see Randel Philos Trans R Soc Lond B Biol Sci. 2016 doi: 10.1098/rstb.2015.0042.). It is known that upon light off, the tail begins to vibrate (Grave, 1921; Dilly, 1964). This would mean that when the helically swimming tadpole ocellus turns away from the light source, it initiates tail movement. What can one deduce from the connectome? How does it compare to other phototactic systems (fish, lamprey, annelid)?

The authors interpret the one sidedness of the eye as a reduction in one side, which is likely correct, and may have been possible as the larva lost visual phototaxis (which requires two eyes) and evolved helical phototaxis.

Gravitaxis has also been studied by otolith ablations. Is the motor pattern known? Does the connectome explain how gravitaxis works?

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for submitting your article "Sidedness in the brain of a chordate sibling, the tadpole larva of Ciona intestinalis" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The major issue is that all three reviewers feel that this paper has the potential to be a classic, and therefore they have strong feelings about further improving it for the benefit of readers for years to come. Everyone involved in the review understands that the wealth of information in the raw data makes deciding how to organize the paper difficult. That said, we believe that one last round of revision will benefit the paper and its utility to future readers. We fully appreciate that this paper is yours, and that honest differences of opinions might arise between well-intentioned reviewers and authors. That said, the reviewers are reading the paper without benefit of all of the authors' working knowledge of the data, and therefore, their input provides crucial information of how other readers may confront the paper.

Reviewer #2:

This paper is improved after the first revision. However, it remains difficult for the general reader to follow. The difficulty lies in the fact that it is basically descriptive with many different neuron types referred to.

For the general reader, there needs to be a more careful and complete description of the work. First of all, the paper does not present an "entire synaptic connectome" (Abstract) or "full synaptic connectome" (first sentence of Discussion). It presents only a partial connectome, that of the CNS. This is just the beginning of how the presentation gets the reader off the track. Subsequently, CNS, PNS (also described as "epidermal"), and non-neuronal ependymal and support cells are all mixed in together in the descriptions. While connectivity, network properties and structure, and neuronal pathways are described, much more of the manuscript is devoted to individual neuron descriptions. Indeed, in the subsection “Sidedness in CNS pathways”, referring to some of their pathway diagrams, they state "However, these shortest paths fail to depict the complexity of integration revealed in the total network." The real emphasis can be seen in the subsection “Asymmetry in cellular composition”, where it out with the sentence "The overall cell complement is closely similar on the two sides (left: 125; right: 129; midline 46)." Total: 300, not 177 as we read in the Abstract. It is really a paper on the cellular structure of the Ciona larval nervous system, with emphasis on its left-right asymmetry, as properly presented in the title. Most of the Discussion is devoted to the developmental origin of the asymmetry.

For what they're worth, I offer the following further comments and suggestions for the authors' consideration.

1) I still don't understand the significance of the issue of left-right asymmetry. In the first paragraph of the Introduction, after the sentence that ends "and so provide a useful model to study many aspects of brain asymmetry," I need a next sentence that begins: "This issue is important because…" In the Abstract: "Chordate in body plan and development, the larva provides by contrast an outstanding example of brain asymmetry." By contrast to what?

2) The sentences that begin the Results section belong in Methods. Similarly, the second paragraph of the subsection “Synapses”. Results need to begin: "The nervous system of the Ciona larva consists of.…" We need a general description of the larva and its nervous system so that when the EM series (and everything else) are described we know where we are. For example, for the general reader, "starting at the level of the otolith pigment.…" is meaningless as we don't know where that is. The major nervous system partitions given in the first paragraph ("posterior motor ganglion," "anterior brain vesicle," "motor ganglion," need to be shown in Figure 1A.

3) What does it mean in the subsection “Synapses”, that "A total of 301 cells of the CNS were imaged,"? Wasn't every cell in an EM cross section "imaged"? Do you mean reconstructed or traced? The issue of the ependymal cells should not be taken so lightly. Indeed, the authors feel the need to define these as "those ciliated cells abutting the canal that lack an axon," but they only do so in passing in the subsection “Asymmetry in cellular composition”. And apparently this work shows some cells previously thought to be ependymal are in fact neuronal. In the subsection “Synapses” it is explained that there are at least 30 CNS cells that were not studied. As we don't know whether these are neurons, ependymal, or other support cells, we don't really know that "the entire CNS included 177 neurons". Maybe it includes 207.

4) Relay neurons. In such a highly cross-connected connectome as this can the authors really distinguish "relay" interneurons from other types? Figure 11 labels some neurons "interneurons" and some "relay" neurons. This distinction needs to be discussed.

5) Explain and emphasize at the top that PNS neurons are often referred to as "epidermal."

6) In the second paragraph of the Discussion: delete "most of".

7) The authors say they have determined the connectivity of 177 neurons. But their table of network statistics, Table 2, says there are 213 nodes in the chemical network and 193 in the gap junction network. Please explain this discrepancy.

https://doi.org/10.7554/eLife.16962.047

Author response

[…]

Reviewer #1:

Ryan et al. present the result of a tremendous effort reconstructing the connectome of larval Ciona intestinalis from a series of electron micrographs. This represents the second whole-organism connectome ever fully reconstructed and is a finding of major significance. The authors relate their findings within the context of structural and connectivity asymmetries, particularly emphasizing relevant comparisons to vertebrates. As many readers are unlikely to be familiar with C. intestinalis, the Introduction should discuss the life cycle of the larva and its behavior. My understanding is that younger larvae swim upwards and older larvae swim downwards to eventually settle based in part on sensors for gravity and light. The authors should emphasize throughout how the Ciona connectome relates to its behaviors.

We now added the following text to the Introduction:

Ciona releases 5000-10000 eggs per individual (Peterson and Svane, 1995) and its eggs are released either individually, or in a mucous string (Svane and Havenhand, 1993). […] Of the reported behaviors, the shadow response, in which a dimming of light results in symmetrical swimming, is the best studied, developing at 1.5 hph and increasing in frequency after 2 hph (Zega et al., 2006).

In addition, we address the issue of how the Ciona connectome relates to its behaviors, later, in the Discussion.

The generalizability of the conclusions drawn about the asymmetry here are tempered by the single-organism sample size. There is space for even more discussion about how variable these left-right asymmetries are from animal-to-animal. What percentage of larvae eventually settle? Perhaps the larval connectome can be "noisy" in terms of cell number and connection strength if only a small number of larvae need to survive and eventually become adults.

Of course we would love to address the issue of the constancy of the connectome in sibling larvae, and we address these rather general points on how generalizable our conclusions are with the following minor additions:

“Additionally, we cannot exclude the possibility that minor left/right asymmetries might be the product of developmental noise, insofar as only a small number of larvae need to survive and eventually become adults.” (Discussion) and:

“Our conclusions are based on a single larva, although four sibling larvae have been reported with closely similar overall cell complements (Nicol and Meinertzhgen, 1991). Data are still lacking on the constancy of neuron cell types and their connections between individuals. The consistency of asymmetrical differences will only be resolved when additional sibling larvae are examined, as we now undertake.” (Discussion).

Some relevant comparisons to other connectomics work are missing. For instance, there are several recent papers from Cardona's group on larval Drosophila that briefly touch on issues of right-left symmetry (sometimes only in supplemental material). Their work, along with Randel et al. (2015), rely on an assumption of symmetry to determine the confidence that a given synaptic connection is real. This work challenges that assumption, and it would be good to address this contrast.

We now added the following text:

“Some connectomic analyses rely on symmetry of the connections between left and right sides to validate the synaptic connections of neurons that are paired (Durbin, 1987; Randel et al., 2014; Ohyama et al., 2015). This approach has not been possible in Ciona, because many cells are not bilaterally paired in the brain vesicle, while in the motor ganglion where cells are paired, presynaptic inputs from the brain vesicle are similarly asymmetrical.”

The manuscript as currently written lacks adequate elaboration of its methods. Neurons are divided into types, soma position is defined as left, right, or medial, and neuronal arbors are divided into different parts (axon, terminal, etc.) without any explicit and rigorous description of the criteria by which these decisions were made.

We now added the following text:

Nucleus location was used to determine neuron sidedness. A line through the midline of the CNS drawn from the ventral to dorsal surfaces through the neural canal was used to classify nuclei as left or right. […] Despite our attempts to partition them in this way, neurons were characterized by a lack of segregation of pre- and postsynaptic sites along their length and a general absence of defining features for each region.”

The authors report a measure of synaptic strength that depends on the number of sections containing a presynaptic profile. However, section thickness varied and it is not clear that the necessary compensations were done.

We are grateful for this comment, and compiled depth determinations for each synapse were made as suggested, and we have now added these in the text. As it turns out, the corrections made little difference to the overall analysis. To clarify the issue, however, we added the following text:

The depth of synaptic contact was calculated by multiplying the number of sections in which a synapse was observed by the section thickness. […] This depth in µm was used for network diagrams as a proxy for synaptic strength.”

We also changed the chart and R2 value in the legend to Figure 3B.

Inevitably, all anatomically-based hypotheses of synaptic strength fall short of direct physiological measurement, so the authors must make absolutely clear at all times what exactly is being measured. The authors describe synapses "onto" basal lamina, a non-cellular structure. Because the concept of a synapse is ingrained the minds of many readers as involving at least two cells (excepting autapses), these should be described differently, perhaps as "putative sites of neurotransmitter release." Could these be sites of neuromodulator release?

We acknowledge this issue, and exclude these sites of synaptic vesicle release from network analysis, and we provide comparisons of summary data with and without “sites of putatative release” onto the basal lamina in Table 1 (see below). On the other hand, synaptic sites formed onto the basement membrane, and thus without obvious postsynaptic partners, have a presynaptic ultrastructure that resembles other synapses in the CNS, and also resemble neuromuscular junctions where a basal lamina ensheathes the muscle cell that neurotransmitter must cross to act postsynaptically. Other cases where synapses form onto a basal lamina may lack an adjacent muscle cell, and indeed may be sites of neuromodulator release. This possibility seems particularly likely in the case of coronet cells, which express reporters for dopamine and which have exclusively dense-core vesicles at their “synapses”.

We therefore now add the following text:

“Synaptic sites that occur onto the basal lamina, and that therefore lack postsynaptic partners, resemble other presynaptic sites in the CNS, and resemble neuromuscular junctions, where neurotransmitter must cross the basal lamina to act on adjacent muscle cells. […] This is particularly likely in the case of coronet cells, which express reporters for dopamine and have exclusively dense-core vesicles at their synapses onto the basal lamina.”

There are at least two discrepancies in the presented results. First, there are missing connections in Figure 12C that are present in 12A. Second, the number of polyadic synapses is variously reported as 920, 921, and 2150 (25% of 8601) in the Abstract, Results, and Table 1 respectively.

These results were based on different subsets of neurons, and have been corrected in the text and tables, and the following comparative data have been added as Table 1. Subsumed by data in the Table, the numbers of synapses are now as follows:

Of ALL synapses recorded: Total 8617;% Reciprocal 5.2%;% Polyad 10.7%

Of ALL without bm, Ep, Mu, space: Total 6661; Reciprocal 6.7%; Polyad 13.8%

Of synapses >1 section without bm, Ep, space:

Without Mu: Total 4913/1124.4; Reciprocal 6.7%/7.8%; Polyad 14%/12.3%

With Mu Number of synapses: Total 6083; Reciprocal 330 (5.4%); Polyad 692 (11.4%); Mu 1170 (19.2%)

With Mu Depth of total synapses: Total 1559.7; Reciprocal 88.28 (5.7%); Polyad 138.8 (8.9%); Mu 435.364 (27.9%)

Finally, the manuscript would greatly benefit from a key that relates neuron type and abbreviation.

We see now that the key requested by the reviewer was in fact not appended to the original submission, and is now attached as a supplementary summary data table (Figure 1—figure supplement 2). This was our mistake. An additional, enlarged version of this key with annotated cartoons of neuron shape will be deposited in Open Connectome.

Reviewer #2:

Ryan, Lu, and Meinertzhagen present the complete connectome of the larval ascidian Ciona intestinalis. This is a paper of potentially historic importance. Not only is this the second whole-animal connectome deciphered, it is the first connectome to be obtained from a single individual. Moreover, Ciona is a chordate, having a notochord and dorsal nerve cord. Therefore, the features of the connectome are of particular interest as a point of comparison to the previously available connectome of C. elegans. When there was only a single connectome available, and that of a tiny, potentially highly-derived invertebrate, it was difficult to know what to make of it and whether one could generalize its characteristics to other animals. Even if C. elegans were representative of invertebrates, how similar would the nervous system of an animal from the other great branch of metazoans be?

In fact, interestingly, Ryan et al. find they are very similar! Similar features include similar overall cell numbers but a rather large number of cell types, largely unbranched neurons making en passant synapses, similar numbers of chemical and gap junction synapses, many polyadic synapses, and little segregation of neurons into separated axonal and dendritic regions, indicating graded potential neurons. In the beginning of the Discussion the authors claim that many features of the connectome are basal. These may indeed be, but there really is little support for this claim. As there are no other connectomes to compare to apart from C. elegans, how can we know, for example, that "the apparent general lack of precision" is basal? The only support mentioned for one feature being basal is that unpolarized synapses are seen in coelenterates. But they are also seen in a pulmonate mollusc. Is the pulmomate mollusc primitive or basal?

This has been an object of discussion between the authors and internal reviewers and on balance we are persuaded to remove the term ‘basal’ from the text, as being indefensible. We really mean ancestral, but all features of known nervous systems derive from extant species that don’t allow observation of ancestral forms. We have simply removed the word “basal” or, in the Abstract, and substituted “lack structural specialization(s)” in the text.See also the same point raised by Reviewers 1 and 3.

Summary of substantive concerns

An important feature of the C. elegans connectome is that it comprises a neural network making a single giant component. That is, everything is connected to everything, eventually. One gathers this is true for the Ciona connectome as well (Discussion, first paragraph: features include the "wide range and combination of cells that share synaptic contacts"), but it is not discussed. In general, the authors should pay more attention to the overall graph properties of the network. The degree distributions are particularly important and should be given.

We have added the following text:

“The network forms a single connected component, with all cells (nodes) being connected by a synapse (edge) to another node in the network (Figure 3—figure supplement 2). The network statistics (Table 2) reveal that the characteristic path length between two neurons is 2.7 (from one neuron, through one other to its target), with neurons having an average of 20 neighbors (synaptic partners) and an overall average network clustering coefficient (existing edges between neighbors of a neuron/possible edges between neighbors of a neuron) of 0.333.”

The authors have chosen to focus their manuscript on left/right asymmetries in the connectome. This is an over-emphasis at the expense of the other interesting features in the opinion of this reviewer. The Introduction does not tell us why this is such an important issue. It begins by suggesting such asymmetries are commonly known. Then later on (last paragraph) it is said they are rare and difficult to recognize. Apparently the issue of l/r asymmetry has been well-studied in ascidians and results from asymmetric nodal expression during embryogenesis as it is in vertebrates. Most of the Discussion is devoted to this issue. Since we know many morphological features of the body are asymmetric, we can expect this will be true for the nervous system as well; it's not clear why so much is made of this here.

We chose left/right asymmetries as the most distinctive feature of ascidian larval brains, of greatest interest to a general readership, and think it represents one outcome of reducing cell number, allowing each side of the CNS to specialize in a particular function, especially a sensory function, and for the opposite side to lack the cells for that function. But this perspective is, we think, too speculative and insufficiently warranted to present in a concise revision of our manuscript.

Representing as it does a descriptive presentation of a very large number of observations, it is important for the manuscript to give the reader a framework that is easy to grasp and keep in mind into which the more detailed observations may be placed. This is particularly so in this instance, as very few readers will have any prior knowledge of the Ciona larva. Figure 1 is not clear and sufficient in this regard. It should have an excellent diagram of the entire larva, showing the locations of sensory structures, ganglia, nerve tracts, and muscles.

We agree, and now present a new version of the entire larva, with most colour-coded cell types to which the account refers, in a new panel (Figure 1A). Reference to existing panels has been revised in the text. We hope this addition makes the general anatomy of the larva more clear.

Since the neurons are unbranched, do they run in bundles with a constant set of neighbors as they do in C. elegans?

Although there are axon bundles containing neurons of specific types, this bundling is not strict and neurons braid within axon bundles, with many exiting their bundle and change neighbours along their lengths, unlike the case in C. elegans. We have added the following text in Results:

“Axons fasciculate in bundles but braid their positions within their bundle or sometimes defasiculate.”

There should be a description of the larva's behavior. One finds out in the Discussion (fifth paragraph) that it does not feed! That's going to make a big difference in the complexity of the nervous system. What does it do besides swim? Does it chemotax, thermotax, and so forth?

These are global questions to which we can respond only in brief in our text. We regret the lack of a comprehensive review to which we could refer. See also Reviewer 1 comment 6. We indicate that larvae do not feed in this comment (non-feeding lecithotrophic larvae). We add the following text in the Introduction: “In addition to phototactic and geotactic behavior, there is evidence of chemotactic behavior just before settlement (Svane and Young, 1989) and of some mechanosensory responses in swimming larvae (Bone, 1992).”

The main goal of swimming is thought to be that of dispersal, yet field studies and models also reveal that many animals have a dispersal distance of less than 6km/generation from adult colonies (Svane and Havenhand, 1993; Petersen and Svane, 1995; Kanary et al., 2011). Some eggs and hatched larvae also remain in a mucous string secreted by the adult (only 40-60% secreted in such a string actually escape the mucous, further limiting their dispersal.

In fact, active swimming was not considered in a recent dispersal model for ascidians despite their sometimes long swimming period (3-6 days) because their mean speeds are only 0.6-4mm/s at bursts of 5-20 seconds (Tsuda et al. 2001; Tsuda et al. 2003; McHenry and Patek 2004; Zega et al. 2006), which are negligible compared with the local current velocity of 27mm/s (Kanary et al., 2011). Thus, rather than retaining networks for active dispersal by swimming, retention of a complex network underlying larval behaviours may be selected for by survival pressure. We add the following text in the Introduction: “Because larvae do not feed, their main biological imperative is survival and successful settlement to undergo metamorphosis into a sessile adult, in an environment with appropriate food and reproductive resources. Thus, entering the water current and avoiding predation by filter feeders may be the foundation for the larva’s many behavioral networks, especially in early life before settlement.”

What sensory-behavioral pathways are revealed in the connectome? What circuits governing swimming behavior and its control?

We add the following text to the Discussion to address this comment: “Mechanosensory drive of symmetrical swimming was initially dismissed because tails can retain swimming independent of the head, and no known input from mechanosensory cells to motor neurons was known. However, we find multiple inputs from mechanosensory tail neurons’ interneurons to motor neurons, both from AMGs in the dorsal MG, and as reported from the bipolar tail neurons (Stolfi et al., 2015). It is not yet clear whether the cilia of DCENs or VCENs may alternatively have thermoreceptive, electroreceptive, or chemoreceptive properties, although there is evidence for chemotactic behavior during larval settlement, mediated by epidermal sensory neurons.”

We also added a summary figure to the following text to relate connectome pathways to specific sensory modalities.

“The shortest sensory pathway to any motor neuron connects via a brain vesicle interneuron, and is thus disynaptic, although most direct pathways involve two interneurons and are thus trisynaptic (Figure 11). However, these shortest paths fail to depict the complexity of integration revealed in the total network (Figure 7—figure supplement 1; Figure 9—figure supplement 1).”

Figure 11. The shortest CNS pathways between sensory neurons and motor neurons for different sensory modalities are three-synapse arcs. […] These pathways are all interconnected, overlapping networks for all sensory modalities thus underlying the complex reality of the actual behavioral network (Figure 9—figure supplement 1).”

Here are some additional questions or comments that occurred to this reviewer:

1) What constitutes the CNS and the PNS? CNS is referred to early in the paper without definition. Figure 15 shows that there are also a small number of PNS neurons. How are these distinguished in such a small nervous system? There is an error in the second paragraph of the Discussion, where it is stated there are 302 neurons in "most of the CNS" of C. elegans. There are 302 neurons in the entire nervous system and there is no clear division in C. elegans between CNS and PNS; these terms are not used.

We acknowledge this distinction by indicating that the 302 neurons refers to most of the nervous system of C. elegans whereas 177 neurons refers explicitly to the CNS of Ciona. In addition, we have added the following text in the Results:

“These regions were additionally innervated by peripheral neurons with cell bodies residing in the epidermis that extend cilia into the tunic. Thus, unlike C. elegans, which lacks a distinction between a CNS and peripheral nervous system (PNS), Ciona’s chordate nervous system has clear divisions between the CNS and PNS. We annotated only those PNS neurons that innervate the CNS (Figure 1A).”

2) Neuron types: this concept is used throughout, but how neuron "type" is defined is not described. Do the neurons really fall into a set of discreet, unambiguous types? In C. elegans, the broad classifications sensory neuron, interneuron, motorneuron are not unambiguous. Many neurons generally considered to be interneurons because of the large number of synapses they make with other neurons also make nmj's, while many neurons considered to be motor neurons also have lots of synapses onto other neurons. "Motor neurons" can have stretch receptors, and "sensory neurons" can have synapses onto muscle. Careful examination reveals that where the cut-offs should in fact be among these broadest classes is unclear and may be somewhat arbitrary. When the manuscript first refers to different neuron types (subsection “Neurons”: brain vesicle intrinsic interneurons, MG interneurons, BV relay interneurons), the reader does not know what these neuron types are. So the descriptions of their various properties are hard to appreciate. Diagrams of the various types, what defines them, where their cell bodies lie, and where their processes go, are needed.

We agree with the reviewer on this point. The primary criteria for each cell type are: a) the location of the soma along a rostrocaudal axis, whether it falls into the brain vesicle, neck, motor ganglion or caudal nerve cord; its termination in these same regions; and its connectivity. Some neurons have specialized structural features, as for example whether the neurons have cilia or sensory neurons that have specific structures. The revised key (Figure 1—figure supplement 2) helps to enumerate and clarify these features.

3) In the first paragraph of the subsection “Neurons”, how can a "terminal" make an en passant synapse?

See our response to reviewer 1, response 4, as follows:

“Many terminals had but small swellings, and their synapses occurred onto collateral terminals or axons, so were considered en passant.”

4) In the third paragraph of the subsection “Synapses”; junctions <2 sections were omitted from consideration. What is the justification for this cutoff? Why not junctions <3? How many junctions does this exclude and what fraction of the "load" do they carry?

The justification was of course mostly to ensure greater accuracy in our connectome, by requiring connections to be validated by >2 synaptic profiles between the same partner neurons. By removing single-profile synapses we eliminated 18% of all synaptic partnerships, and by removing all 2-profile synapses we would have eliminated a total of 35% of all synaptic partnerships. We now add the following text to the legend of Figure 3:

“Removing single-profile synapses eliminates 18% of all synaptic partnerships, and removing all 2-profile synapses would have eliminated a total of 35% of all synaptic partnerships.”

5) In the fourth paragraph of the subsection “Synapses”, and throughout the paper: average is used as a statistic (average 37 presynaptic sites per CNS neuron). This doesn't seem to be a meaningful statistic. The distributions need to be given. In this regard, Figure 3A should probably be a semi-log survival curve. The curve shown doesn't tell us much as most of the points either don't move much along the X-axis or much along the Y axis.

On reflection we agree with this point and now give the number of synapses per neuron as a mean and standard deviation, with the corresponding range, and have added/amended the following text:

“Each CNS neuron thus formed on average 49+61 (SD) presynaptic sites with a range of between 1 and 430 synapses and an average of 13+23 (SD) putative gap junctions, with a range between 0 and 166. Each postsynaptic neuron received an average of 39+42 (SD) synapses from each presynaptic partner, with a range between 0 and 179.”

6) Figure 3B is described in the text (subsection “Synapses”, fifth paragraph) as showing the number of presynaptic sites versus the number of postsynaptic partners, but the axes in the figure are labeled number of synapse profiles vs number of synapses. What are synapse profiles?

We agree, and have exchanged the original Figure 3B for a plot that shows depth of synaptic contacts, as explained in response 5 to reviewer 1. We now add a new Figure 3C which plots the numbers of synapses as a function of the numbers of synaptic partnerships. We now cite this Figure in the text in the place of the former Figure 3B, and trivially revise the text to read:

“For each neuron, the number of presynaptic sites varies with the number of its postsynaptic partners, plotted for all neurons and their synapses, but excluding the neuromuscular junctions (Figure 3C).”

And why is this ratio of interest? What's the point being made here, that all neurons have the same density of synapses?

This is related to the previous point. The ratio is of interest because it indicates that each of its partners increases the number of synapses made by a presynaptic neuron, and thus reflects a postsynaptic drive to the total synapse load. We now add this in the following short text:

“This relationship indicates that each of its partners increases the number of synapses made by a presynaptic neuron, and thus reflects a postsynaptic drive to the total synapse load.”

7) In the second paragraph of the subsection “Sidedness in the brain vesicle”: there are said to be 17 enigmatic coronet cells on the left side, and Figure 5A-B is referred to. But I cannot see 17 enigmatic coronet cells in this figure. In fact, it's hard to distinguish any cells in the figures produced by Reconstruct (I presume). There need to be drawn diagrams to accompany them. And the drawings should be shown in the context of the entire larva, like the drawings of C. elegans neurons in WormAtlas (e.g. http://wormatlas.org/neurons/Individual%20Neurons/AVAframeset.html).

This is a difficult point to address. We agree about Reconstruct and could wish that our study would have been commenced later, when superior reconstruction software would have been available to us. Figure 5 is an honest presentation of what a real reconstruction looks like without editing. The figures in Wormatlas are beautiful drawings, but that is an extensive document. We do now illustrate the coronet cells in our new Figure 1A and in Figure 7C, which is also highly rendered, where their position is illustrated by orange profiles. To reveal the coronet cells in particular we therefore now render them from a dorsal view in a new panel, Figure 5D, to address the reviewer’s point.

8) Figure 12. Can't read the labels in A and B. How do we know these left/right differences aren't just developmental noise?

Labels in A and B of what is now Figure 13, have been changed to white on blue, to enhance their chromatic contrast. We don’t in fact know that the left/right differences aren't just developmental noise, which would require the reconstruction of multiple larvae at different stages. We allow the possibility of developmental noise in the following addition (Discussion):

“Additionally, we cannot exclude the possibility that minor left/right asymmetries might be the product of developmental noise.”

9)In the sixth paragraph of the Discussion: number of neuron types. This seems to me to be Results, not Discussion.

It could be either Results or Discussion. These types are now provided in the key (Figure 1—figure supplement 2) referred to within the Results. We put it in the Discussion chiefly to compare with C. elegans.

10) I can't relate the cells listed (or cell types?) in the legend to Figure 7A to the cell types (?) listed in Table 2. I can't relate the cells listed in the master cell list Figure 3—figure supplement 1 with the cells e.g. in Figure 3D. The master cell list Figure 3—figure supplement 1 is said to be an excel file, but mine's a PDF. Excel file would be much easier to work with (e.g. the heading row can be kept visible as one scrolls down the list).

This was our fault. The abbreviations now appear in the key (Figure 1—figure supplement 2), and we have changed the figures so that abbreviations and colours for the cell types are now cross-referred to the key. Additionally, we have now included small thumbnails of individual neuron reconstructions (whole cells, or terminals for photoreceptors) in Figure 3—figure supplement 1.

11) I don't have a Figure 9—figure supplement 1.

Likewise, this was our fault, the supplement was omitted from the submission, and is now Figure 9—figure supplement 1 (see response to comment 8 of reviewer 1).

12) Table 1 lists 3 polyadic gap junctions, but polyads are not defined for gap junctions because there is no defined pre- and post-synaptic cell.

We could not find a more suitable descriptive term for membrane appositions (GJs) that link more than two elements at a single site. These number 3% of the total number of such appositions. Given the uncertainty anyway in identifying GJs from membrane appositions we prefer not to remake the table for this point.

13) Figure 15. This is the main presentation, along with individual neuron maps (missing), of the findings of this reconstruction. This is presumably the directed graph of chemical connections. The corresponding undirected graph of the gap junction connections needs to be included.

We have now included small thumbnails of individual neuron reconstructions (whole cells, or terminals for photoreceptors) in Figure 3—figure supplement 1. We have also now included the corresponding undirected graph of putative GJs as Figure 16—figure supplement 2.

I don't understand why the weights of the matrix are given as "percentile synaptic strength." In fact, I don't know what this means, rank order percentile? or percent of the highest value? Is a matrix with numbers (number of EM sections, or physical size in microns, since section thickness varied across this reconstruction) available rather than these colors? Modelers or theoreticians who would like to study the properties of the network (there will be many!) will need this.

We have now remade both matrices of chemical and electrical synapses, using the color-coded depth by cumulative depth of synaptic contacts rather than percentile synaptic strengths. Colors represent ranges as illustrated in the new key.

Reviewer #3:

This is a beautiful and very important work describing the entire neuronal connectome of the Ciona tadpole larva. Ciona early development and neuronal development are very well understood and the full larval connectome is a milestone addition to the resources available for this important chordate model organism. Importantly, urochordates are the sister group to the vertebrates, and have been studied intensively to understand the evolution of key vertebrate features. The connectome also provides a very important reference point for broader circuit comparisons across animals.

Given that this is a full connectome paper, I think the authors should not feel constrained by the 5000 word limit, but aim to describe the neuron complement and the full circuits in more detail. They should also think about how they can relate the connectivity to known larval behaviors (e.g., phototaxis, gravitaxis, larval settlement).

We have now extended the length of the text, albeit conservatively, and are glad to have this reviewer’s and the editor’s sanction to do so.

First, larval behaviors:

There is one important mistake that the authors need to correct first to be able to better interpret the connectome.

They state in the paper twice that the tadpole swims in a bilateral fashion with tail undulations: "The apparent symmetry of the characteristic swimming pattern of chordate-like tail undulations" and "Larvae exhibit a simple bilaterally symmetrical swimming response to light "

This is not true, Ciona and other ascidian larvae swim in a helical pattern. This was already known to Eakin and Kuda (1971) who wrote "When an ascidian larva is actively swimming the body rotates about its long axis." More importantly, a detailed kinematic analysis of swimming of the Ciona tadpole revealed how the larva turns towards light during helical swimming.

The authors should consult and cite "The kinematics of phototaxis in larvae of the ascidian Aplidium constellatum" by McHenry and Strother (Marine Biology, 2003) and "The morphology, behavior, and biomechanics of swimming in ascidian larvae" by McHenry (Can. J. Zool. 83: 62-74 2005). Appreciating the correct motor pattern is important to correctly interpret how the circuits work. The helical pattern emerges due to the asymmetry in tail bending. This is likely one of the main reasons for the high degree of asymmetry in this animal.

This is an essential point. Our reference to bilateral swimming was to the pattern of muscular contraction as recorded, for example, in Nishino et al. (2010) their Figure 2B, rather than to larval trajectory, as reported by Eakin and Kuda and in the papers of McHenry and colleagues. We should acknowledge that we were of course aware of the latters’ reports, but these were cut from the final version in view of the word limit. Now that we include more detail on larval behavior, it makes sense to relate our circuits to the helical pattern of larval swimming. We do this in the following text:

In the Introduction we write: “This sidedness may also correspond to larval behavior because ascidian larvae pursue a helical trajectory when swimming (McHenry and Strother, 2003; McHenry, 2005) and ascidian larvae are thought to use klinotaxis to respond to visual cues by modulating the symmetry of tail kinematics (McHenry and Strother, 2003).”

We continue in the Discussion with the following amended sentence: “Larvae swim in a helical pattern (McHenry, 2005) from simple bilateral flexions of the tail (Video 1: Bone, 1992) responding to light and a directional response to gravity (Tsuda et al., 2003).”

On the other hand, we do not entirely agree that the “Appreciating the correct motor pattern is important to correctly interpret how the circuits work” because asymmetry may be introduced by the tail muscle itself, which may even be more likely in muscle cells that are held in a curved posture, and may not reside entirely, or even at all, in the motor circuits that innervate the muscle. We don’t believe the last proviso, but think it sufficient simply to refer to the pattern of helical swimming, leaving any functional outcome hanging for the moment. On the other hand, we think helical movement bears importantly on the pattern of illumination it provides to the ocelli, when the shadow of the pigment crosses the outer segments. We cover these points in the following revised of the Discussion:

“Larvae swim in a helical pattern (McHenry, 2005) from simple bilateral flexions of the tail (Bone, 1992; Nishino et al., 2010). […] Based on gravitaxis in young larvae (Tsuda et al., 2003) we also predict that the antenna neurons’ circuits underlie a directional response to gravity.”

Video 1 does not demonstrate bilateral swimming, it is actually impossible to tell from this video what the locomotor pattern is (bilateral or helical) because the larva moves out of focus and the frame rate is too low. This video may be removed or just used as an illustration.

We agree and have substituted a high-speed video, which is used simply as an illustration for readers who may never have seen larval swimming. Reference to this video is now omitted from the Discussion.

Neuron types and connectivity:

Figure 3—figure supplement 1 lists all cells and their important features. Since it is a comprehensive paper, the morphology and position of at least one example from each cell type should be shown in a supplementary figure (or figures) and where each cell type is located in the body (show body outline).

Likewise, some of the anatomical diagrams are difficult to interpret because the cells are not shown in the context of the whole body or together with other cells from the same circuit.

Same for the videos: Videos 2 and 3 – the detail of the ocellus should be embedded into the larger anatomical context (e.g., by showing the body outline).

We have addressed this concern also raised by reviewer 1 in our response to that reviewer: “An additional, very large version of this key with annotated cartoons of neuron shape will be deposited in Open Connectome” and by reviewer 2, in our response to that reviewer. “The body outline is also now shown (for the BV, neck and MG), along with a rendered version of cell bodies for Videos 2 and 3)”.

It is in general difficult to follow the description of connectivity of the different subsystems (e.g., eyes, otolith), because the relevant details are embedded in the summery diagrams and are not explained in full. I would really encourage the authors to describe the different sensory-motor systems in more detail.

This comment relates to others from reviewers 1 and 2. We have summarized pathways pictorially in a new figure (Figure 11). We do not agree that text is more clear than figures, and think instead that this new figure goes a long way to making the existing text more readable, as indicated in response to Reviewer 2’s comment given above. Further text could we think only increase the length and opacity of our already long paper. The primary problem is simply that the full CNS connectome is very complex, but that each sensorimotor network can ultimately be reduced to a trisynaptic pathway, and this we have now tried to present more clearly. One of the chief issues in presenting a more complete network for Ciona is that, unlike the connectome for C. elegans, we still lack essentially all functional information on each type of interneuron and its particular neurotransmitter identity.

This should include the reconstructed neurons in the whole-body context (anatomical diagrams) and the corresponding circuit diagrams. For example, what is the full cell complement and wiring diagram of the PRC circuit? Show type I, II, III PRCs and their downstream circuitry, to the muscles. Likewise, show the otolith and coronet circuits. What are the PNS neurons and their circuitry? How about the tail circuitry that probably tunes the left-right motor pattern?

We agree with the reviewer, and Figure 11 now has the simplified summary of these pathways, and we do now also include a new video (4) that shows an animation of the visual pathway with cell bodies as spheroids within the outline of the CNS. We also report the antenna cell (otolith) pathways in Figure 10—figure supplement 2. But a more comprehensive treatment would require an addition equal in length to the existing text, and really lies beyond the scope of this first submission.

The integration can then be described following the details of the subsystems (as is now e.g., in Figure 9).

This, we think, is simply beyond the scope of a single paper, much as we would like to take an entire issue of eLife to accommodate the request.

It would also be very interesting to see the strength of all reciprocal connections (e.g., represented as the geometric mean of 2-way synapse numbers) at the level of the entire connectome. This can easily be computed from the whole matrix.

Could calculate

It is hard to read Figure 7—figure supplement 1. The network should be shown in higher magnification. An alternative representation that would be useful would be to merge cells of the same type and connectivity into one node and show the average synaptic strength.

We have now added a further network diagram (Figure 9—figure supplement 1) in which we pool neurons of the same class into a single node and summed their synapse numbers. This comment was not quite clear to us. Given that many cells of the same class converge upon a single postsynaptic target, it makes more sense to us to refer to the pooled synaptic strength than to show the average synaptic strength. See also our response to Reviewer 2.

Figure 11. Also show cell morphology of MN classes. Please also show a schematic of the different muscle types and their position relative to the MN axons. It is not clear how an MN projecting on the left side can synapse on muscles both on the left and right sides of the body.

We have now included a figure showing the pairs of motor neurons reconstructed (Figure 12—figure supplement 1) as requested. The general pattern of innervation for different motor neurons and the arrangement of the three different muscle bands is we think sufficiently reported in the literature (see for example: Nishino et al. 2010, their supplementary figures). We do not understand how our text could have been misinterpreted to indicate that a motor neuron projecting on the left side can synapse on muscles both on the left and right sides of the body, but must accept that it was unclear to the reviewer. We inserted the word “pairs” in the subsection “The motor ganglion”, in case this was the cause for the misunderstanding. All motor neurons project unilaterally, of course.

Figure 15 is hard to read – please label neuron categories also on the presynaptic side. Provide list of abbreviations. Label major categories in the figure (corresponding to the boxes e.g., sensory neurons).

We understand this problem. There are many cells and if all are to be presented in a single-page matrix, each necessarily will be small. We have added labels on the left-hand presynaptic side in a revised version of the figure and the abbreviations used are now provided in Figure 1—figure supplement 2, with colour coding of the cells that corresponds to the colours in the network diagrams. We have however retained the numerical labelling of the boxes in this figure, because to add text would have obscured the information in the matrix intercepts, which we needed to convey.

Discussion:

The functional and comparative implications of the Ciona connectome should be spelled out in more detail. For example, how does the connectome explain negative phototactic behavior? The strategy of ascidian phototaxis is helical (not visual, for an explanation see Randel Philos Trans R Soc Lond B Biol Sci. 2016 doi: 10.1098/rstb.2015.0042.). It is known that upon light off, the tail begins to vibrate (Grave, 1921; Dilly, 1964). This would mean that when the helically swimming tadpole ocellus turns away from the light source, it initiates tail movement.

We now cite the important paper by Randel and Jékely, the publication of which overlapped the period of preparation for our own manuscript. We cannot of course respond to the question of how does the connectome explain negative phototactic behavior, because our data are entirely anatomical and to seek such an explanation immediately leads us into undue speculation. We share the reviewer’s curiosity, of course, and even went so far as to generate the following text, which we have since removed, but which illustrates the uncertainties in all such speculations from anatomical connectome data:

“helical phototaxis could be explained by a simple circuit from hyperpolarizing glutamatergic photoreceptors onto possibly excitatory brain vesicle relay neurons, which in turn are presynaptic to cholinergic motor ganglion interneurons on the left and right sides, with anatomically greater synaptic drive on the left side than the right (ocellus) side that are themselves presynaptic to cholinergic motor neurons; thus allowing shading to differentially drive muscle contraction on both sides. What is unclear, however, is what role anatomical circuit asymmetries actually play in phototaxis as periodic shading activates and deactivates photoreceptors, given the differences between neurotransmitter phenotypes of different photoreceptors and the complexity of their downstream pathways.”

We are however encouraged by the thought that the identification of visual interneurons we now provide, can be combined with genetic intervention studies to generate appropriate functional studies.

What can one deduce from the connectome? How does it compare to other phototactic systems (fish, lamprey, annelid)?

The authors interpret the one sidedness of the eye as a reduction in one side, which is likely correct, and may have been possible as the larva lost visual phototaxis (which requires two eyes) and evolved helical phototaxis.

Gravitaxis has also been studied by otolith ablations. Is the motor pattern known? Does the connectome explain how gravitaxis works?

Our response to these important issues raised by the reviewer is essentially the same as we give above. The complement of cells and their pathways in the visual network of larval Ciona is most similar to the level of complexity approaching visual phototaxis as illustrated by stage (f) or (g) in Figure 2 of Randel and Jékely’s paper. In relation to this figure, the complexity of pathways found in Ciona suggests to us that the unilateral ascidian larval ocellus may have arisen by loss of a contralateral ocellus in ancestral forms. We have added this idea more explicitly to the Discussion as a new paragraph, as follows:

“Given that ascidian larvae swim in a helical pattern (McHenry, 2005) and have a single-sided ocellus, their phototactic behaviour is of a helical, not visual nature, as defined by Randel and Jékely (2016). […] Helical swimming of the ascidian larva provides a mechanism by which a preexisting bilateral visual phototaxis circuit could have been co-opted into a complex hybrid helical phototaxis circuit, still allowing mechanisms such as delay, sensory integration, and modulation to take place, and unlike the more direct helical phototaxis mechanisms in ciliated forms, such as protists and trochophore larvae (Randel and Jékely, 2016).”

[Editors' note: further revisions were requested prior to acceptance, as described below.]

[…]

Reviewer #2:

This paper is improved after the first revision. However, it remains difficult for the general reader to follow. The difficulty lies in the fact that it is basically descriptive with many different neuron types referred to.

For the general reader, there needs to be a more careful and complete description of the work. First of all, the paper does not present an "entire synaptic connectome" (Abstract) or "full synaptic connectome" (first sentence of Discussion). It presents only a partial connectome, that of the CNS.

We do appreciate that our text will not be an easy read for many readers, because the system is unfamiliar to most, even to most workers in the tunicate field. However, most readers would, we think, understand connectome as CNS connectome. We should also point out that our connectome provides most of the PNS connectome, because it identifies PNS neurons that provide input to CNS cells and that overlie the trunk and tail epithelium. To address the reviewer’s concern, however, we have nevertheless inserted ‘CNS’ as a qualifier to ‘connectome’ in the Impact Statement and in two later places, in the Abstract and in the thirteenth paragraph of the Discussion, where we refer to a ‘connectome’.

We also revised the composition of Table 2, adding two columns to provide segregated data for the Full network and for CNS neurons only.

Reflecting this reviewer’s concern, and our own interest, we have also now somewhat revised the title of our submission to become “The CNS connectome of a tadpole larva of Ciona intestinalis highlights sidedness in the brain of a chordate sibling”.

This is just the beginning of how the presentation gets the reader off the track. Subsequently, CNS, PNS (also described as "epidermal"), and non-neuronal ependymal and support cells are all mixed in together in the descriptions.

We now define and describe PNS earlier in the text.

The mixture of PNS, and designations as "epidermal", non-neuronal ependymal and support cells the reviewer finds is mostly a problem that cell types are not always segregated from each other in the larval CNS, as they would be in other nervous systems. For example, epidermal neurons are so identified based on previous accounts (Takamura, Imai and Meinertzhagen) that we chose not to revise. A subset of these epidermal PNS cannot be overlooked because they have axons that terminate and make synaptic connections in the CNS.

Ependymal cells are defined in the subsection “Synapses” (“those ciliated cells abutting the canal that lack an axon”).

The confusion between 300 and 177 is that not all cells are neurons, of course, as we remind the reader with the following change: “and constituting the remainder, ependymal cells (those ciliated cells abutting the canal that lack an axon) and three cells of the CNS that are ambiguous, having presynaptic sites, but lacking a neuronal form”. The discussion of ependymal cells is important for the question of asymmetry because the total cell numbers exhibit symmetry that is not upheld when examining neurons only.

While connectivity, network properties and structure, and neuronal pathways are described, much more of the manuscript is devoted to individual neuron descriptions.

The many newly identified cell types in this nervous system and many general readers’ unfamiliarity with the larval tunicate nervous necessitate this description. While the network may be valuable, its biological significance relies on what and how components are connected.

Indeed, in the subsection “Sidedness in CNS pathways”, referring to some of their pathway diagrams, they state "However, these shortest paths fail to depict the complexity of integration revealed in the total network." The real emphasis can be seen in the subsection “Asymmetry in cellular composition”, where it out with the sentence "The overall cell complement is closely similar on the two sides (left: 125; right: 129; midline 46)." Total: 300, not 177 as we read in the Abstract. It is really a paper on the cellular structure of the Ciona larval nervous system, with emphasis on its left-right asymmetry, as properly presented in the title. Most of the Discussion is devoted to the developmental origin of the asymmetry.

We have clarified the book-keeping on our cell numbers, which we confess may have been complex and confusing, and was written with concision in view of the eLife word limit. We now indicate the left/right and middle cell numbers on as “including neurons. ependymal and accessory cells”.

For what they're worth, I offer the following further comments and suggestions for the authors' consideration.

1) I still don't understand the significance of the issue of left-right asymmetry. In the first paragraph of the Introduction, after the sentence that ends "and so provide a useful model to study many aspects of brain asymmetry," I need a next sentence that begins: "This issue is important because…" In the Abstract: "Chordate in body plan and development, the larva provides by contrast an outstanding example of brain asymmetry." By contrast to what?

We added the following sentence and reference to Duboc et al. (2015), as follows: “This issue is important because brain laterality has been associated with increased fitness for animal life” We split the paragraph at this point to make this the concluding short sentence, before resuming discussion of Ciona.

We gave now removed “by contrast” from the Abstract, and we write later in the Introduction “In contrast to the situation in most chordates….”

2) The sentences that begin the Results section belong in Methods. Similarly, the second paragraph of the subsection “Synapses”. Results need to begin: "The nervous system of the Ciona larva consists of.…" We need a general description of the larva and its nervous system so that when the EM series (and everything else) are described we know where we are. For example, for the general reader, "starting at the level of the otolith pigment.…" is meaningless as we don't know where that is. The major nervous system partitions given in the first paragraph ("posterior motor ganglion," "anterior brain vesicle," "motor ganglion," need to be shown in Figure 1A.

3) What does it mean in the subsection “Synapses”, that "A total of 301 cells of the CNS were imaged,"? Wasn't every cell in an EM cross section "imaged"? Do you mean reconstructed or traced?

We meant that all 301 cells were imaged and we go on in the text to say: “Cells omitted from our EM series, those rostral to the otolith and caudal to the bipolar tail neurons (Imai and Meinertzhagen, 2007b; Stolfi et al., 2015), are presumed to account for the remainder of the >331 cells reported by Nicol and Meinertzhagen (1991) and thus to number at least 30”.

The issue of the ependymal cells should not be taken so lightly. Indeed, the authors feel the need to define these as "those ciliated cells abutting the canal that lack an axon," but they only do so in passing in the subsection “Asymmetry in cellular composition”. And apparently this work shows some cells previously thought to be ependymal are in fact neuronal. In the subsection “Synapses” it is explained that there are at least 30 CNS cells that were not studied. As we don't know whether these are neurons, ependymal, or other support cells, we don't really know that "the entire CNS included 177 neurons". Maybe it includes 207.

We now introduce ependymal cells earlier in the fourth paragraph of the subsection “Synapses”, which we think clarifies the account. We do not think the identification of ependymal cells is in doubt at EM level, only that a previous account at LM level could not resolve axons.

For the question of cell numbers raised by the reviewer, it is correct that our analysis does not include the possible tail neurons that lay beyond the caudal extent of our analysed series. In a rostral direction there are only two axons, which are included in the totals given in Table 3, and which come from somata beyond our section series. They are reported separately in Figure 3—source data 1 and belong to BVIN class of neurons.

We make the following revisions in our paper to take account of these further bookkeeping issues: Impact statement: “Serial-section EM analysis uncovers the CNS connectome of a Ciona larva, the second of any entire nervous system, and exposes left-right asymmetries in its synaptic circuits.” Additionally we removed the word “entire” when used to qualify reference to the connectome.

4) Relay neurons. In such a highly cross-connected connectome as this can the authors really distinguish "relay" interneurons from other types? Figure 11 labels some neurons "interneurons" and some "relay" neurons. This distinction needs to be discussed.

We think this is justifiable, and use relay to identify neurons that extend from one brain region to another, as they would be identified in, for example, the vertebrate brain. This is now clarified in the following sentence of the new paragraph added to the Introduction as follows:

“The relay neurons of the posterior brain vesicle extend axons through the neck to the motor ganglion, which overlies the anterior portion of the notochord, and contains neurons of the motor system.”.

5) Explain and emphasize at the top that PNS neurons are often referred to as "epidermal."

We included the following sentence to address this point, as follows:

“In addition to the CNS several sensory epidermal neurons (ENs) of the peripheral nervous system (PNS) populate the dorsal and ventral axis of the larva in a rostrocaudal sequence, with axons running beneath the epidermis (Imai and Meinertzhagen, 2007b).”.

6) In the second paragraph of the Discussion: delete "most of".

We deleted “most of”.

7) The authors say they have determined the connectivity of 177 neurons. But their table of network statistics, Table 2, says there are 213 nodes in the chemical network and 193 in the gap junction network. Please explain this discrepancy.

As explained above: “muscle was indeed included (4 nodes, dorsal left, dorsal right, medial left and medial right), as were PNS neurons and basal lamina, one orphan photoreceptor terminal profile, and two ambiguous cells which lack axons but have presynaptic sites. To cover these additional cells we added the following: “and two cells of the CNS that are ambiguous, having presynaptic sites, but lacking a neuronal form (Figure 1—figure supplement 2 and Figure 3—source data 1)”. We made a new network with just the CNS neurons and analysis of this network is now added in Table 2 for both gap junctions and chemical synapses.”

https://doi.org/10.7554/eLife.16962.048

Article and author information

Author details

  1. Kerrianne Ryan

    1. Department of Biology, Life Sciences Centre, Dalhousie University, Halifax, Canada
    2. Department of Psychology and Neuroscience, Life Sciences Centre, Dalhousie University, Halifax, Canada
    Contribution
    KR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Zhiyuan Lu

    1. Department of Biology, Life Sciences Centre, Dalhousie University, Halifax, Canada
    2. Department of Psychology and Neuroscience, Life Sciences Centre, Dalhousie University, Halifax, Canada
    Contribution
    ZL, Cut the entire ultrathin section series upon which this study is based and provided essential expertise on serial section electron microscopy, Acquisition of data
    Competing interests
    The authors declare that no competing interests exist.
  3. Ian A Meinertzhagen

    1. Department of Biology, Life Sciences Centre, Dalhousie University, Halifax, Canada
    2. Department of Psychology and Neuroscience, Life Sciences Centre, Dalhousie University, Halifax, Canada
    Contribution
    IAM, Conception and design, Drafting or revising the article
    For correspondence
    iam@dal.ca
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6578-4526

Funding

Natural Sciences and Engineering Research Council of Canada (DIS0000065)

  • Kerrianne Ryan
  • Zhiyuan Lu
  • Ian A Meinertzhagen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work has been supported by grant DIS-0000065 (to IAM) from the Natural Sciences and Engineering Council of Canada. We thank Ms Carlie Langille for assistance in proofreading neurite profiles and annotating synapses, Drs. Dianne Nicol (Tasmania), Janice Imai (and Ayami Matsushima (Fukuoka) for their video images of larval swimming, and Ms Jane Anne Horne for assistance with computer techniques. We also acknowledge Dr. Scott W Emmons (Albert Einstein College of Medicine, New York) for help in comparing our findings with the C. elegans connectome.

Ethics

Animal experimentation: This study was approved by Dalhousie University protocol I9-015.

Reviewing Editor

  1. Eve Marder, Brandeis University, United States

Publication history

  1. Received: April 26, 2016
  2. Accepted: October 17, 2016
  3. Version of Record published: December 6, 2016 (version 1)

Copyright

© 2016, Ryan et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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