Our comprehensive examination of the molecular and functional characteristics of the DA cells of the sensory vesicle of Ciona
intestinalis revealed unanticipated common features with the DA-producing amacrine cells of the vertebrate retina. Because DA cells of the sensory vesicle of C. intestinalis also share traits with the DA neurons of the vertebrate hypothalamus, a major implication of our work is that these types very likely derive from DA-synthesizing cells that were already present in a primitive proto-hypothalamo-retinal territory at the anterior neural tube of ancestral chordates. A second conclusion of these findings is that the multiple cell types found in the vertebrate retina and involved in photoreception, including PRCs, ganglion cells and amacrine cells, existed before the formation of the retina as an organ, as suggested from other studies conducted in Drosophila . Thus, the absence of amacrine and other accessory cell types in the retina of hagfish, an animal that effectively lives in the dark, would represent a secondary loss, in agreement with the monophyly of cyclostomes  and with the presence of monoaminergic amacrine cells in lampreys .
The evidence supporting homology between vertebrate DA amacrine cells and the DA cells of the sensory vesicle of Ciona
intestinalis are based both on shared neurochemical and molecular signatures and on functional characteristics. Beside the fact they express the whole set of molecules required to synthesize, store and release DA (TH, AADC and VMAT), the DA cells in Ciona display the unusual ability to take up and store serotonin (5HT). Serotonin-accumulating amacrine cells have been described in several vertebrate species [17, 31, 43] and, similarly, DA cells of the C. intestinalis larva are able to take up 5-HT via the SERT present in these cells. We postulate that this CiSERT should be able to transport DA in addition to 5HT, since no DAT (absent from the genome) is present in DA cells of the sensory vesicle, although this is an unusual property for SERT [30, 44]. Supporting this contention, the blockage of CiSERT by Fluoxetine, a widely used SERT inhibitor, has a robust inhibitory effect on the swimming activity induced by shadowing, in Ciona larvae. This inhibitory effect is probably mediated by the action of DA on ADRα2-like receptors, since no DA receptors are present in the Ciona genome . The action of DA through ADRα2-a receptors has also been described in the vertebrate hypothalamus [39, 40]. The affinity of ADRα2-like receptors for dopamine is only about 10-fold lower than that of noradrenaline in most vertebrates [39, 40], allowing them to be contingently used as DA receptors in some tissues and some species.
In vertebrates, DA amacrine cells modulate light responses of the retinal network through lateral inhibition [1, 2] and the ascidian DA cells modulate the swimming behavior elicited by PRC activation. In the two cases, this effect in mediated, at least in part, by ADRα2-like receptors. In C. intestinalis, ADRα2-like receptors are located on DA cells, as in vertebrate amacrine cells where they act as autoreceptors , and also on glutamatergic photoreceptors and GABAergic interneurons, the probable targets of secreted DA. A reminiscent situation is found in vertebrates where the three paralogous ADRα2A/B/C receptor subtypes are expressed in PRCs (glutamatergic), RGCs (glutamatergic) and amacrine cells (mostly GABAergic), with some species differences . A role of this DA-induced inhibition in ascidian larvae could be to promote the onset of metamorphosis, which is associated with the immobilization of the larvae and attachment to a substrate. In contrast, it was shown that blocking 5-HT receptors delayed the onset in P. mammillata larvae . It is thus very likely that 5HT and DA have opposing effects on motor behavior in ascidians, this effect depending on the integrity of the connection between the sensory vesicle and the motor pattern generator in the trunk (see Additional file 1 Figure S3).
Incidentally, our data also highlight the large molecular flexibility and evolutionary adaptability of these DA and 5HT systems, with the loss in ascidian genomes of monoamine transporter genes except SERT , the loss of D1-like and D2-like receptors encoding genes . Yet the systems remain functional by using SERT and ADRα2-like receptors for DA uptake and effect on target cells.
The similarity between DA cells of the ascidian sensory vesicle and DA amacrine cells of the vertebrate retina is reinforced by the shared expression of Ptf1a, bHLH transcription factor [25, 26]. Ptf1a is mandatory for the formation of horizontal and amacrine cells, especially GABAergic amacrine cells . The DA cells of Ciona do not contain GABA in addition to DA as in vertebrates , but Ptf1a can be expressed in all types of amacrine cells in vertebrates , and GABA and DA cells can be separate cell types. Other transcription factors, such as CiRx, CiSix3/6, CiPax6 and CiMeis, which contribute to retina development [20, 21, 47], are also localized in the anterior medial and lateral part of the sensory vesicle, where DA cells are located in Ciona [18, 19, 47, 48]. Overall, these observations clearly show that DA cells are present in a photoreceptive territory in the ascidian sensory vesicle, as the amacrine cells are in the vertebrate retina.
In addition to their similarity to amacrine cells, the DA cells of the sensory vesicle of C. intestinalis also share traits with the CSF-contacting dopamine neurons of the vertebrate hypothalamus . In Ciona, the ventral location of the DA cells and their protrusions (coronets) in the lumen of the sensory vesicle make them resemble the DA coronet cells of the saccus vasculosus of cartilaginous and teleost fishes, a paraventricular organ of the hypothalamus, secondarily lost in tetrapods, as well as to the dopamine-synthesizing cells of the caudal hypothalamus in teleosts [reviewed in ].
Based on commonalities with the hypothalamus of vertebrates, we previously hypothesized that the area of the ascidian sensory vesicle where DA cells are located was homologous as a field to the hypothalamus of the vertebrate brain . Based on our present results, it seems now more likely that the sensory vesicle of ascidians on the one hand, and, on the other hand, the hypothalamus and retina together, ancestrally derive from a photoreceptive neuroepithelium folded into the anterior neural tube of ancestral chordates, as initially proposed by F.K. Studnicka in 1898, based on embryological data and a remarkable intuition [reviewed in ].
This hypothesis fits with the observation that deep brain photoreceptors and photoreceptive organs (retina and epiphysis) are all derived from the ventricle-lining prosencephalon [51, 52]. In this respect, the ascidian PRCs would be equally related to the PRCs of the retina, the epiphysis and the hypothalamus of vertebrates. Furthermore, in the amphioxus Branchiostoma floridae, a non-craniate chordate which lacks a true retina, some cilliary PRCs express Pax6 , and these cells are part of the cerebrospinal fluid (CSF)-contacting neurons sitting in the wall of the cerebral vesicle [51, 52], a region proposed to be homologous to the vertebrate prosencephalon [52, 53]. In addition, DA cells of the anterior cerebral vesicle of the amphioxus are located close to these photoreceptors [54, 55], a situation somewhat reminiscent of that of Ciona.