The nervous system of the adult ascidian Ciona intestinalis Type A (Ciona robusta): Insights from transgenic animal models

The nervous system of ascidians is an excellent model system to provide insights into the evolutionary process of the chordate nervous system due to their phylogenetic positions as the sister group of vertebrates. However, the entire nervous system of adult ascidians has yet to be functionally and anatomically investigated. In this study, we have revealed the whole dorsal and siphon nervous system of the transgenic adult ascidian of Ciona intestinalis Type A (Ciona robusta) in which a Kaede reporter gene is expressed in a pan-neuronal fashion. The fluorescent signal of Kaede revealed the innervation patterns and distribution of neurons in the nervous system of Ciona. Precise microscopic observation demonstrated the clear innervation of the anterior and posterior main nerves to eight and six lobes of the oral and atrial siphons, respectively. Moreover, visceral nerves, previously identified as unpaired nerves, were found to be paired; one nerve was derived from the posterior end of the cerebral ganglion and the other from the right posterior nerve. This study further revealed the full trajectory of the dorsal strand plexus and paired visceral nerves on either side from the cerebral ganglion to the ovary, and precise innervation between the cerebral ganglion and the peripheral organs including the gonoduct, cupular organ, rectum and ovary. The differential innervation patterns of visceral nerves and the dorsal strand plexus indicate that the peripheral organs including the ovary undergo various neural regulations. Collectively, the present anatomical analysis revealed the major innervation of the dorsal and siphon nervous systems of adult Ciona.


Introduction
The biological functions and organization of the central nervous system are correlated with the evolutionary lineages of animals. Recent molecular phylogenetic analyses indicated that tunicates (ascidians, thaliaceans, and appendicuralians) constitute the sister group of vertebrates [1][2][3][4]. The structure and functions of the central and peripheral nervous system of the larva of a cosmopolitan ascidian, Ciona intestinalis, have been investigated including the number and types of neurons, axon projections, neuronal circuit, and development of the nervous were created by Minos-mediated transgenesis [38,39] and provided from the National BioResource Project. These lines were cultured using the inland culture system as described previously [40]. Adult transgenic animals were used in this study. The two transgenic lines, Tg

Tissue preparation
Animals were anesthetized using L-menthol based on the previous method [41]. In brief, 0.56% (weight per volume) L-menthol (Nacalai Tesque, Kyoto, Japan) in ethanol was prepared and used as a stock solution. The stock solution was further diluted by 1% (volume per volume) in artificial sea water before use. Animals were soaked into the diluted L-menthol solution for 10 min. Tissues were dissected under the fluorescence stereo microscope (Leica M205 FA; Leica Microsystems, Wetzlar, Germany) and fixed with 4% paraformaldehyde in PBS at 4 C˚overnight. The fixed tissues were soaked in 2 mg/ml glycine in PBS for quenching paraformaldehyde. The tissues were washed three times with PBS and used for morphological analyses.

Whole-mount observation of tissues
After removal of the tunic, fixed tissues were placed in a dish with a rubber sheet or a slide glass chamber filled with PBS (AGC TECHNO GLASS, Tokyo, Japan) and fixed with needles as appropriate. A fluorescence stereo microscope (Leica M205 FA; Leica Microsystems) and image acquisition software (Leica AF6000E; Leica Microsystems) were used for low magnification observation. A confocal microscope with a 10× or 20× objective (FLUOVIEW FV1000; Olympus, Tokyo, Japan) was used for high magnification observation and three-dimensional images acquisition. To construct three-dimensional images, a z-stack function was used and 200-300 cross section images of fixed tissues from top to bottom per sample were collected. The focus interval was 1μm for each section image. Names of the nerves were based on the previous study with partial modification [42]. The general morphological descriptions were based on the previous study [43]. The term "innervation" is used to explain the distribution of nerves but does not include their functions.

Results and discussions
Overview of the Kaede-positive innervation of the transgenic adult Ciona The transgenic Ciona strongly expressed Kaede fluorescent protein in the nervous system throughout the body. The Kaede-positive nerve connections were visualized among the cerebral ganglion, siphons and body muscles (Fig 1A, 1B and 1C). Furthermore, Kaede-positive neural structure lied between the cerebral ganglion and the ovary via the orange-pigmented organ (OPO), gonoduct and rectum ( Fig 1A, 1B and 1C). These anatomical views prompted us to investigate the innervation in a whole-mount preparation of the transgenic adult Ciona in greater detail. We focused on the innervation of the dorsal and siphon nervous system, which had been poorly understood.

Innervation of anterior and posterior nerves to the siphons
Several early studies demonstrated the innervation of anterior nerves to the oral siphon and posterior nerves to the atrial siphon in ascidians including Ciona [24,44]. However, the precise number of nerves and their innervation patterns remained to be elucidated. The present study clearly showed that eight main anterior nerves and ten main posterior nerves are derived from the cerebral ganglion and innervate the oral siphon, atrial siphon, and periphery branching into thin nerves (Fig 2A and 2B). These nerves innervated the edge of the siphon lobes and pigment organs (Fig 2C), suggesting that they regulate the movement and/or sensory functions of siphons. Interestingly, the eight lobes of the oral siphon were found to be innervated by four  different anterior nerves. Three ventral lobes (numbered by 4 to 6 in Fig 2D) and two left-lateral lobes (numbered by 2 and 3 in Fig 2D) were innervated by the anterior lateral nerve 1a (ALN1a) and anterior medial nerve 1 (AMN1), respectively, derived from the left region of the cerebral ganglion (Fig 2A and 2D). In contrast, one dorsal lobe (numbered by 1 in Fig 2D) and two right-lateral lobes (numbered by 7 and 8 in Fig 2D) were innervated by the anterior medial nerve 2 (AMN2) and anterior lateral nerve 2a (ALN2a), respectively, derived from the right region of the cerebral ganglion (Fig 2A and 2D). The body walls were innervated by the other four anterior main nerves, ALN 1b, 1c, 2b, and 2c (Fig 2A and 2D). Minor neural connections were seen between these nerves and the oral siphon and between these nerves and tentacles (Fig 2A). These innervation patterns to the oral siphon suggest that the lobes of the oral siphon are grouped into four histological units according to the projected nerves: three ventral lobes, left two and right two lateral lobes, and one dorsal lobe. Each of six lobes of the atrial siphon was innervated by a single nerve of six posterior main nerves, posterior lateral nerve 1a (PLN1a), PLN2a, posterior medial nerve 1a, (PMN1a), PMN1b, PMN2a and PMN2b, and peripheral body walls were innervated by the other four posterior main nerves, PLN1b, 1c, 2b and 2c (Fig 2B and 2D). These results revealed an outstanding difference in the innervation patterns between the oral and atrial siphons. The aforementioned innervation of major nerves was observed in all transgenic lines, while the number and shape of fiber projection was found to vary among individuals (S1 Fig). Thus, the innervation of major nerves is conserved as a basal nervous system in Ciona.
We observed efferent peptidergic innervations from the cerebral ganglion to the siphons in the previous immunohistochemical study [45]. In addition, afferent synapses were dominantly found in the tentacles of siphons in Ciona [31]. Afferent innervations of the peptidergic neurons in the siphons were also reported in other ascidian species Corella inflata [46]. These findings will contribute to identification of major efferent and afferent nerves in the Kaedepositive nervous system.
Ciona individuals have eight siphon lobes and pigment organs located between lobes [47]. All of the oral siphon lobes and pigment organs were regenerated in their original forms when the distal tip or middle part of the oral siphon was removed [48]. On the other hand, the number of regenerated lobes and pigmented organ was disordered when the siphon was removed at the base part below the tentacle row, suggesting that the proximal-distal axis determines the regeneration pattern of the oral siphon [48]. Notably, this study showed that the corresponding base part is innervated by ALN1a, ALN2a, AMN1, and AMN2 that are divided into eight branches above the tentacle row and further projects numerous nerves to the oral lobes and pigment organs (Fig 2A, 2C and 2D). Our findings allow us to hypothesize that these nerves participate in the proximal-distal axis-directed normal regeneration of the oral siphon lobes and pigment organs.

The visceral nerves and dorsal strand plexus around the cerebral ganglion
Little has been known about innervation patterns between the cerebral ganglion and the ovary through the dorsal strand plexus and the visceral nerves [15,24,[49][50][51]. In the previous studies, the visceral nerve was identified as an unpaired nerve in Ciona [13,[52][53][54]. In contrast, we identified "paired" visceral nerves. The visceral nerve 1 (VN1) originated from the cerebral ganglion adjacent to the right posterior nerve 2 (PN2), and visceral nerve 2 (VN2) branched from PN2, both of which extended to the periphery along with either side of the dorsal strand plexus (Fig 3A-3D). The observation from the serial sections also confirmed the presence of the two visceral nerves arising from the posterior cerebral ganglion and PN2 (S2 Fig). VN1 and VN2 were derived from the right part of the posterior cerebral ganglion, suggesting that the right half of the cerebral ganglion regulates VN1 and VN2. VN1 that is derived from the posterior cerebral ganglion corresponds to the previously reported visceral nerve [13,[52][53][54], and VN2 that is derived from PN2 is newly reported in this study. In the immunohistochemical studies of gonadotropin-releasing hormone (GnRH), only a single visceral nerve was observed [52,53]. In other studies using transgenic lines CiPhox2::YFP [11] or E[MiTSAdT-POG]15 line [54] that expresses a fluorescent protein in nervous tissues also reported a single visceral nerve. It is highly likely that all nerves were not visualized and VN2 might have not been observed by immunohistochemistry or morphological analyses of transgenic ascidians in these studies, particularly, due to the difficulty in distinguishing VN2 from other nerves that also branch from PN2 as shown in Fig 3B. In addition, fibrous tissues lie above the branching point of VN2 and surgical removal of this tissues was quite difficult (S3 Fig). Altogether, the present study provides evidence that Ciona is endowed with paired visceral nerves.
This study also revealed entire trajectories of the visceral nerves and the dorsal strand plexus from the cerebral ganglion to the peripheral organs. Furthermore, we identified the origin of the dorsal strand plexus in detail with spatial information in a three-dimensional view. The neurons constituting the dorsal strand plexus emerged between the cerebral ganglion and neural gland, and passed above PN2 and curved around the dorsal blood sinus, and built the dorsal strand plexus (Fig 3B and 3C). The neural gland is not a nervous tissue [24,34] and Kaede fluorescent signal was not seen in the neural gland (Fig 3B), suggesting that the neurons innervating to the dorsal strand plexus are derived from the ventral region of the cerebral ganglion.

Innervation of the dorsal strand plexus and visceral nerves to the orange-pigmented organ
Neurons of the dorsal strand plexus built a broad band-like structure and passed near the orange-pigmented organ (OPO) located at the end of spermiduct (Fig 4A). Two visceral nerves, VN1 and VN2, were observed on either side of the dorsal strand plexus (Fig 4A). Interestingly, numerous nerves from VN2 innervated OPO, compared with only a few VN1 nerves (Fig 4B and 4C). Kaede-positive neurons and their axons were also observed on top of OPO (Fig 4A and 4B). These results are largely consistent with the previous study using other transgenic lines [55]. Interestingly, the axons from OPO neurons mainly projected to VN2 (Fig 4B  and 4C). Together, these innervation patterns suggest both afferent and efferent trajectories between OPO and VN2. In addition, OPO is known as a duct for sperm release [55], suggesting the regulation of the sperm release by VN2. Some neurons in the dorsal strand plexus also projected to OPO (Fig 4C), suggesting their neural communication. These observations are in part consistent with the previous studies on the distribution of GnRH immunoreactive neurons in the dorsal strand plexus [15,50,52,56]. In the present study, however, a greater number of neurons were found in the dorsal strand plexus than the GnRH-immunoreactive neurons, suggesting other neuropeptidergic neurons in the dorsal strand plexus.
Subsequently, we observed innervation of neurons to the site located posterior to OPO. The dorsal strand plexus, VN1, and VN2 run along with the dorsal blood sinus located adjacent to the oviduct and spermiduct (Fig 5A). Moreover, Fig 5B-5D demonstrates the distribution of numerous monopolar, bipolar, and multipolar neurons in the dorsal strand plexus, neurons in the cupular organ, and nerves of VN1 and VN2. Furthermore, neurons in the cupular organ were found to project their axons to the neurons in the dorsal strand plexus, VN1, and VN2 ( Fig 5C). The cupular organ is a putative hydrodynamic sensor consisting of neurons and sensory cells in Ciona [55,57]. Combined with these findings, the present observation suggests that the dorsal strand plexus and visceral nerves are involved in the transmission of sensory information from cupular organs to the cerebral ganglion.

Innervation of the dorsal strand plexus and visceral nerves to the rectum
Nerves from VN1 innervated the rectum and Kaede-positive cells at the edge of the rectum, suggesting VN1 regulate the rectum (Fig 6A).
Nerves from VN2 and/or the dorsal strand plexus also projected to the rectum (Fig 6A). Nerves from VN2 extended to the periphery along with the dorsal strand plexus (Fig 6A and  6D). The dorsal strand plexus became thinner near the rectum (Fig 6A, 6C and 6D). 20-30 neurons were laterally distributed in the dorsal strand plexus above the rectum (Fig 6B), while less than ten neurons were distributed in the thinner dorsal strand plexus (Fig 6C). Unlike the upper part of the dorsal strand plexus located above the rectum (Fig 6B), the cupular organs were not seen in the lower part of the dorsal strand plexus (Fig 6C). The absence of the cupular organs suggests that the lower part of the dorsal strand plexus is not involved in sensory transmission, although the possibility cannot be excluded that other sensory neurons, not contained in the cupular organs, exist in these regions (e.g. proprioceptive neurons [46]).
Numerous Kaede-positive small cells were also found in the rectum, and these cells were widely distributed to the dorsal region of the rectum (Fig 6A). Although innervation to other regions of the rectum than the posterior edge awaits further study, such localization of Kaedepositive small cells suggests some neuronal regulations of the rectum. Compared with the previous study [24], the present study revealed the structural organization of the nervous system around the rectum. GnRH-immunoreactive axons were found to poorly innervate the rectum [52]. Therefore, other neuropeptides and neurotransmitters than GnRH are likely to regulate the functions of the rectum via the dorsal strand plexus and visceral nerves.

Innervation of the dorsal strand plexus to the ovary
We observed that the dorsal strand plexus reaches the junction between the oviduct and the ovary (Fig 7A). This observation is in part consistent with the previous immunohistochemistry using several anti-GnRH antibodies [15,50,52,56]. Furthermore, the innervation was observed not only at the junction but also around the whole ovary (Fig 7B and 7C). Some neuron-like cells were also detected on the surface of the ovary (Fig 7C). The detailed innervation of the dorsal strand plexus to the ovary indicates that the ovary is regulated by the materials secreted from the neurons of the dorsal strand plexus in Ciona. In our previous study, more than thirty neuropeptides were identified from the central nervous system of adult Ciona [16,21]. Among these peptides, Ciona tachykinin-I (Ci-TK-I) enhanced oocyte growth by inducing expression of proteases including cathepsin D, chymotrypsin, and carboxy-peptidase B1 in the ovary [26,27]. Most of the identified neuropeptides including Ci-TK-I were expressed exclusively in the cerebral ganglion but not in the ovary, whereas many of their receptors were expressed in the ovary [16,21,26,27]. Combined with these findings, the present study suggests that the neuropeptides produced in the cerebral ganglion are secreted to the ovary via the dorsal strand plexus and/or visceral nerves, and regulate the oocyte growth and maturation.
In conclusion, we have identified a wide range of innervation patterns of the siphon and dorsal nervous system of adult Ciona between the cerebral ganglion and the periphery. Investigation of innervation to other tissues, such as stomach, heart, gill, and endostyle by the ventral nervous system is currently in progress. Identification of afferent and efferent nerves will reveal more rigorous neural network in adult Ciona. In addition, the immunohistochemistry and photoconverting of Kaede in the transgenic animal will allow us to further understand the peptidergic regulatory network of Ciona. These studies will also provide insights into the evolutionary process of neuroendocrine system in chordates.