Cellular identity and Ca2+ signaling activity of the non-reproductive GnRH system in the Ciona larva

Tunicate larvae have a non-reproductive GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. In the Ciona larva, one of the gnrh genes, gnrh2, is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord, respectively, of vertebrates. The gnrh2 gene is also expressed in the proto-placodal sensory neurons, which are the proposed homologue of vertebrate olfactory neurons. The tunicate larvae occupy a non-reproductive dispersal stage, yet the roles of their GnRH system remain elusive. In this study, we investigated neuronal types of gnrh2-expressing cells in the Ciona larva and visualized activity of these cells by fluorescence imaging using a calcium sensor protein. Some cholinergic neurons as well as dopaminergic cells express gnrh2, suggesting that a role of GnRH in the control of swimming behavior. By contrast, none of the gnrh2-expressing cells overlap with glycinergic or GABAergic neurons. A role in the motor control is also suggested by correlation between activity of some gnrh2-expressing cells and tail movements. Interestingly, gnrh2-positive ependymal cells in the nerve cord, known as a kind of glia cells, actively produced Ca2+ transients, suggesting that neuroendocrine signaling occurs in glia cells of the nerve cord.


Introduction
Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive functions in vertebrates (Okubo and Nagahama 2008;Oka 2009). Non-reproductive roles of GnRH have been suggested in the nervous system and during development (Dolan et al. 2003;Albertson et al. 2008;Sherwood and Wu 2005;Wu et al. 2006;Abraham et al. 2008;Kanaho et al. 2009;Ramakrishnan et al. 2010). Compared to its reproductive roles, the non-reproductive roles of GnRH are less well understood.
Tunicates are the sister group of vertebrates (Delsuc et al. 2006;Putnam et al. 2008). A conspicuous non-reproductive GnRH system has been reported in the larva of the sessile tunicate Ciona Kamiya et al. 2014). Six GnRH peptides and four receptors are encoded by the Ciona genome (Adams et al. 2003;Kusakabe et al. 2003;Tello et al. 2005;Sakai et al. 2010Sakai et al. , 2012. In the Ciona larva, the GnRH genes are strikingly expressed in the central nervous system (CNS) through the entire anteroposterior body axis . Correspondingly, the GnRH receptor genes are specifically expressed in the tissues and organs located along the CNS, namely the notochord, the tail muscle, and epidermal sensory neurons ).
One of the Ciona gnrh genes, gnrh2, is conspicuously expressed in the motor ganglion and nerve cord of the larva, which are homologous structures to the hindbrain and spinal cord, respectively, of vertebrates. The gnrh2 gene is also expressed in the proto-placodal sensory neurons, which are the proposed homologue of vertebrate olfactory neurons (Abitua et al. 2015).
Ciona GnRH has been implicated to play a pivotal role in the control of metamorphosis (Kamiya et al. 2014). Considering the complex and well-developed nature of the larval GnRH system in Ciona, GnRH may play diverse and important roles in developmental and physiological processes in the Ciona larva. To date, however, the roles of the Ciona GnRH system remain elusive.
In this study, we investigated neuronal types of gnrh2expressing cells in the Ciona larva and visualized activity of these cells by fluorescence imaging using a calcium sensor protein. Some cholinergic motor neurons as well as unique cholinergic cells along the nerve cord express gnrh2, suggesting that a role of GnRH in the control of swimming behavior. By contrast, none of the gnrh2expressing cells overlap with glycinergic or GABAergic neurons. A role in the motor control is also suggested by simultaneous activation of some gnrh2-expressing cells with tail movements.
Interestingly, gnrh2-positive ependymal cells in the nerve cord, known as a kind of glia cells, produced Ca 2+ transients, suggesting that active signaling, presumably involving GnRH, occurs in the nerve cord.

Results
Gnrh2 is expressed in proto placode-derived sensory neurons and caudal glial ependymal cells First, we examined whether gnrh2-expressing cells include glutamatergic neurons. In the Ciona larva, glutamate is a major neurotransmitter in the peripheral sensory neurons and photoreceptor cells (Horie et al. 2008). Some interneurons in the posterior brain is also glutamatergic (Horie et al. 2008). As previously reported (Abitua et al. 2015), proto-placode derived gnrh2-expressing epidermal neurons (aATENs) are glutamatergic ( Fig. 2A). aATENs are the only glutamatergic neurons that express Cellular retinaldehyde binding protein (CRALBP) is specifically localized in the glial ependymal cells in the brain vesicle and the motor ganglion (Tsuda et al. 2003;Kusakabe et al. 2009).
CRALBP-positive cells were never overlapped with gnrh2epxressing cells (Fig. 2B). Thus, in contrast to the conspicuous gnrh2 expression in the ependymal cells of the nerve cord, gnrh2 is not expressed in the ependymal cells in the brain vesicle and the motor ganglion.

Some cholinergic and dopaminergic neurons express gnrh2
Acetylcholine is a major neurotransmitter at the neuromuscular junctions of the Ciona larva (Yoshida et al. 2004;Horie et al. 2010). Cholinergic neurons were visualized by a fluorescence protein expressed under the control of cis-regulatory region of the vacht gene (Yoshida et al. 2004). Gnrh2-expressing neurons were shown to be cholinergic both in the brain vesicle and the motor ganglion (Fig. 3A).
The caudal part of the CNS (nerve cord) mainly consists of non-neuronal ependymal cells (Katz 1983). The nerve cord also contains two types of neurons: ACINs and bilateral pairs of cholinergic caudal neurons (Horie et al. 2010). Some of these cholinergic caudal neurons seem to express gnrh2 (Fig. 3B).

Correlation between tail movements and Ca 2+ transients in the motor ganglion and the anterior nerve cord
A neural circuit in the motor ganglion and the anterior nerve cord is thought to control muscle contraction of the tail (Nishino et al. 2010;Horie et al. 2010;Kusakabe 2017). Because active Ca 2+ transients in gnrh2-expressing cells were observed in these regions, we examined temporal correlation between tail movement and the activity of gnrh2-expressing cells. Ca 2+ transients were frequently observed when the tail stopped movement, whereas the Ca 2+ transients were seemingly suppressed during the term when the tail was moving (Fig. 7). This finding suggests possible involvement of gnrh2-expressing cells in the control of swimming behavior.

Discussion
In this study, we identified cell types of gnrh2-expressing Periodic Ca 2+ transients observed in the motor ganglion of young larvae are reminiscent of spontaneous rhythmic activities observed in developing nervous systems of vertebrates (Gu et al. 1994;Wong et al. 1995;Feller et al. 1996;Zhou 1999;Kerr et al. 2005;Chang and Spitzer 2009). These periodic neuronal activities are thought to be important for the development of neural circuits in the central nervous system and the retina (Zhou 1999;Spitzer et al. 2000;Feller 2006). Similar rhythmic oscillation of Ca 2+ transients were reported in the developing motor ganglion of the Ciona embryo (Akahoshi et al. 2017). By contrast, we observed rhythmic Ca 2+ transients only in young larvae. Swimming behavior of Ciona larvae reveals ontogenic changes and their photo-responsiveness appears a few hours after hatching (Nakagawa et al. 1999;Tsuda et al. 2003;Zega et al. 2006). Thus, the spontaneous rhythmic Ca 2+ transients may play an important role in the neural circuit development of Ciona larvae.
We observed correlation between the tail movements and Ca 2+ transients in the motor ganglion and the nerve cord. This suggests that gnrh2-expressing cells are involved in the control of swimming locomotion. Ca 2+ transients appeared when the tail stops movement and Ca 2+ signal was low when the tail was moving (Fig.   7B). In other words, the tail movement precedes the Ca 2+ spike.

Preparation of reporter constructs and electroporation
Construction of the vglut>kaede was described previously (Horie et al., 2011, Razy-Krajka et al. 2012). The vacht>cfp plasmid was made by inserting the 3.8-kb upstream region of  (Hozumi et al. 2010). The gnrh2 upstream region was also used to generate the gnrh2>g-camp8 construct. The Kaede coding sequence of pSP-Kaede was replaced with a DNA fragment coding for G-CaMP8 (Ohkura et al. 2012) using NotI/EcoRI sites. The gnrh2 upstream region was amplified from the gnrh2>kaede plasmid using a pair of nucleotide primers (5'-

TGACGCGGCCGCTGTTACGTTATCTCTCTAGAAG-3'), digested with
BamHI and NotI, and then inserted into the BamHI/NotI sites upstream of the G-CaMP8 in the pSP vector. Plasmid DNA constructs were electroporated into Ciona fertilized eggs as described by Corbo et al. (1997).

Immunofluorescent staining
Immunofluorescent staining was carried out according to the method described by Nishitsuji et al. (2012). Fluorescent images were obtained by using a laser scanning confocal microscope (FV1200 IX83; Olympus, Tokyo, Japan).