A neurochemical map of the developing amphioxus nervous system
© Candiani et al.; licensee BioMed Central Ltd. 2012
Received: 11 October 2011
Accepted: 27 April 2012
Published: 7 June 2012
Amphioxus, representing the most basal group of living chordates, is the best available proxy for the last invertebrate ancestor of the chordates. Although the central nervous system (CNS) of amphioxus comprises only about 20,000 neurons (as compared to billions in vertebrates), the developmental genetics and neuroanatomy of amphioxus are strikingly vertebrate-like. In the present study, we mapped the distribution of amphioxus CNS cells producing distinctive neurochemicals. To this end, we cloned genes encoding biosynthetic enzymes and/or transporters of the most common neurotransmitters and assayed their developmental expression in the embryo and early larva.
By single and double in situ hybridization experiments, we identified glutamatergic, GABAergic/glycinergic, serotonergic and cholinergic neurons in developing amphioxus. In addition to characterizing the distribution of excitatory and inhibitory neurons in the developing amphioxus CNS, we observed that cholinergic and GABAergic/glycinergic neurons are segmentally arranged in the hindbrain, whereas serotonergic, glutamatergic and dopaminergic neurons are restricted to specific regions of the cerebral vesicle and the hindbrain. We were further able to identify discrete groups of GABAergic and glutamatergic interneurons and cholinergic motoneurons at the level of the primary motor center (PMC), the major integrative center of sensory and motor stimuli of the amphioxus nerve cord.
In this study, we assessed neuronal differentiation in the developing amphioxus nervous system and compiled the first neurochemical map of the amphioxus CNS. This map is a first step towards a full characterization of the neurotransmitter signature of previously described nerve cell types in the amphioxus CNS, such as motoneurons and interneurons.
Although the genetic and developmental mechanisms of nervous system organization in vertebrates have attracted considerable attention, relatively little is known about the evolutionary origins of the vertebrate central nervous system (CNS). In all chordates, which, in addition to vertebrates, comprise the tunicates and cephalochordates (amphioxus), the anterior end of the dorsal, hollow nerve cord is enlarged to form a (at least diencephalic) forebrain, a possible midbrain and a hindbrain [1–5]. However, a definite midbrain and a midbrain–hindbrain organizer may have been vertebrate innovations [2, 6].
Here, we use neurotransmitter markers in amphioxus to reconstruct a neurochemical map of the developing cephalochordate nervous system. Using vesicular transporters and key enzymes for neurotransmitter biosynthesis as markers, we assessed the neuronal differentiation in the developing amphioxus nervous system focussing on the time of appearance of glutamatergic, GABAergic, glycinergic, cholinergic and serotonergic neurons. Although we are aware that physiological and pharmacological assays are required to definitively demonstrate the utilization of a specific neurotransmitter by a given amphioxus neuron, our work nonetheless revealed the distribution of neuronal cell bodies engaged in synthesizing particular neurochemicals in the developing amphioxus CNS and allowed us to construct the first neurochemical map of the amphioxus embryo.
List of the genes, encoding vesicular transporters and/or key enzymes for neutransmitter biosynthesis, studied in the present work
Vesicular Glutamate Transporter (VGLUT)
Serotonin Transporter (SERT)
Tryptophan Hydroxylase (TpH)
Vesicular GABA/Glycine Transporter (VGAT)
Glutamic Acid Decarboxylase (GAD)
Vesicular GABA/Glycine Transporter (VGAT)
Vesicular Acetylcholine Transporter (VAChT)
Amphioxus possesses at least one ortholog of the mammalian genes encoding tryptophan hydroxylase (TpH), the rate-limiting enzyme in serotonin synthesis and a serotonin transporter (SERT) acting as both symporter and antiporter in the presynaptic membrane (Additional file 1 and Additional file 2). Amphioxus TpH and SERT served as molecular markers for locating the serotonin-producing neurons in the developing amphioxus nervous system. Both markers show the same expression pattern and are likely expressed at very low levels, because, compared to the other neurotransmitter markers used in the present work, both required an extended development time with the alkaline phosphatase-mediated detection system. We were thus unable to obtain very strong signals in our double in situ hybridization experiments with these two markers.
To follow the differentiation of GABAergic neurons in amphioxus, we studied the expression of glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA biosynthesis. Moreover, we simultaneously assessed the developmental expression of the vesicular GABA/glycine transporter (VGAT) that is responsible for the uptake into the synaptic vesicle of both GABA and glycine . We found that amphioxus possesses single genes encoding GAD and VGAT (Additional file 1 and Additional file 2), both of which were cloned by PCR. However, since VGAT is expressed in both GABAergic and glycinergic neurons , in order to identify the glycinergic neurons in the amphioxus CNS, we needed to perform double in situ hybridization experiments with both GAD and VGAT. Thus, GABAergic neurons are VGAT-positive and GAD-positive, while glycinergic neurons are VGAT-positive and GAD-negative.
GABAergic and glycinergic neurons
Construction of a neurochemical map in amphioxus by double in situ hybridization
These expression patterns are maintained in later neurula stages (20 hrs to 24 hrs) (Figures 3, 9C-E, 10D-G and 11E,F), with the notable exception of the appearance of two groups of VGLUT-positive neurons: one located dorsally in the anterior cerebral vesicle (Figures 3, 9C and E, 10D,E,G) and one located posterior to the first pigment spot (Figures 3 and 10E-G). Moreover, we observed that the total number of neurons constituting the VGAT and VAChT clusters increased at these later stages (Figures 3, 9C-E, 10D-G). At larval stages, there is no substantial difference in the basic organization of neuronal cell types in the amphioxus CNS except for an additional increase in the total number of neurons constituting the VGAT and VAChT clusters (Figures 9F and G, 10H-K).
Taken together, our in situ hybridization experiments yielded a neurochemical map of the developing amphioxus CNS. Surprisingly, this map suggests that, at least in the developmental stages examined, neurotransmitters do not colocalize in the amphioxus CNS. This finding contrasts with some examples of neurochemical colocalization observed in mollusks and vertebrates [28–30].
Interpreting the organization of neuronal cell types in the developing amphioxus nervous system
Previous ultrastructural studies on the architecture and cellular organization of the larval amphioxus CNS have provided a detailed morphological map of different neuronal types [37–39]. The majority of these transmission electron microscope (TEM)-based neuroanatomical reconstructions in amphioxus have been carried out on 8-day and 12.5-day larvae and thus on developmental stages older than the material used in the present study. Comparisons of these morphological data with our neurochemical map will hence need to take these stage differences into account.
On the basis of morphological and topological features, three types of motoneurons have been identified in the ventrolateral nerve cord of amphious larvae: (i) dorsal compartment motoneurons (DC) innervating the dorsal fast fibers of the myotome, (ii) ventral compartment motoneurons (VC) innervating the ventral slow fibers of the myotome, (iii) visceral motoneurons (VM) innervating all of the remaining body musculature (Figure 12). The DC motoneurons are confined to the most anterior somites and are generally organized as pairs of cells. According to Lacalli and Kelly  at least one pair of DC motoneurons is slightly offset and is located at the level of the center of somite 2, whereas all the other pairs (at least five) are positioned at the junction of the somites. Expression of amphioxus islet and ERR genes coincides with prospective DC motoneurons [19, 20]. VC motoneurons have no evident periodical arrangement along the nerve cord, although a cluster of five VC neurons is found at the level of the posterior end of somite 1 [37, 39]. Finally, VM motoneurons seem to be very scarce in young larvae, but at least two have been described located posterior to the junction of somites 2 and 3 [37, 39].
Additionaly, different interneurons have been reported in the amphioxus ventrolateral nerve cord [37, 39]. At the ultrastructure level, four types of interneurons, described as large paired neurons (LPN1-4), are present in the amphioxus CNS (Figure 12). The first three at the level of the primary motor center (PMC) and the fourth at the level of the junction of somites 3 and 4 [37, 39]. Moreover, four ipsilateral projection interneurons (IPNs) have also been described, located predominantly in the most rostral part of the hindbrain posterior to the first pair of DC motoneurons [37, 39]. No information is available on the presence of other kinds of interneurons in more posterior regions of the amphioxus nerve cord.
In a previous report on the expression of the CGL (for cholinergic gene locus, i.e. ChAT/VAChT) , we have tentatively assigned a cholinergic neurotransmission to some of the ventrolateral neurons identified by TEM, most of which are motoneurons. This correlation of cell types identified by TEM and neurons expressing specific sets of neurotransmitters can now be expanded. We show, for instance, that cholinergic neurons are generally organized as discontinuous rows of cells starting anteriorly at the PMC and stretching posteriorly to the junction of somites 6 and 7 (Figure 3). Some of the most anterior cholinergic cells, located in proximity of the posterior end of somite 1, are probably VC motoneurons, whereas the pairs of cholinergic neurons at the junctions of the somites (from somites 2 and 3 to somites 5 and 6) are likely DC motoneurons, plus an additional pair of neurons located at the level of somite 2 (Figures 3 and 12). Additionally, the cholinergic neurons located just posterior to the junction of somites 2 and 3 might correspond to VM motoneurons (Figures 3 and 12).
One subset of cholinergic neurons identified in the CNS of developing amphioxus are most likely interneurons , which would correlate very well with the situation in vertebrates . For example, LPN3 located at the junction of somites 1 and 2 is a potential candidate for such a cholinergic amphioxus interneuron, although LPN3 could alternatively be interpreted as a pair of glutamatergic excitatory neurons located inside the PMC (Figures 12 and 13). In contrast, our data suggest that the anterior LPNs (LPN1 and 2) and IPNs (on the level of somite 2) are GABAergic and GABAergic/glycinergic, respectively (Figures 12 and 13).
In our neurochemical map, some neurons cannot be ascribed to one of the categories of motoneurons and interneurons identified by TEM . This is, for instance, the case for the cluster of cholinergic, glycinergic and glutamatergic neurons located just posterior to the first pigment spot as well as for the aggregation of a number of different neuronal types (expressing GABA, VGLUT or ChAT) at the level of the PMC (Figures 12 and 13). The PMC is a major integrative center of sensory and motor stimuli controlling the early locomotory activities of the larva, which could explain this concentration of different neuronal types. Moreover, the PMC interneurons (GABAergic and glutamatergic) probably connect to rostral sensory cells and project towards the posterior nerve cord, which likely contributes to locomotion and the startle response [41, 42].
GABAergic and glycinergic neurons in vertebrates and invertebrates
GABA is a major inhibitory neurotransmitter in both vertebrates and invertebrates [43–46]. In comparison to other animals, the distribution of GABA in the CNS of amphioxus embryos and larvae seems to be more discrete. In the CNS of the ascidian Ciona intestinalis, GABA is widely expressed and is associated with neural regions responsible for processing sensory information and motor integration, such as the sensory vesicle and the anterior visceral ganglion (Figure 13) . Interestingly, while a majority of neurons of the sensory vesicle are GABAergic, only very few cells of the anterior visceral ganglion contain GABA . In the C. intestinalis peripheral nervous system (PNS), there are only very few GABAergic cells, the majority of which are associated with the palps (Figure 13).
Unlike GABAergic cells, which are widespread in the vertebrate brain, most glycinergic neurons in the vertebrate CNS are restricted to the rhombencephalon and the spinal cord. For instance, in embryos of the frog Xenopus laevis the first glycinergic cells appear in the caudal hindbrain region and subsequently extend to the spinal cord , which was also observed in lamprey embryos [49, 50]. In C. intestinalis larvae, two pairs of glycinergic neurons have been reported in the tail  (Figure 13). These cells, called ACINs (for anterior caudal inhibitory neurons), are located in the anterior nerve cord, just posterior to the cholinergic motoneurons of the visceral ganglion, and act as a component of a neural circuit controlling alternative muscle contraction of the larva.
The distribution of glycinergic neurons in amphioxus is quite similar to that observed in the vertebrate CNS. Two pairs of glycinergic cells are visible in the hindbrain region at the neurula stage. At subsequent stages of development, two further clusters, intermingled with GABA cells, appear more anteriorly at the level of somites 2 and 3 (Figure 13). The present results thus reveal a dynamic developmental pattern of the glycinergic system in amphioxus, including early and late glycinergic neuron populations that might correlate with maturation changes occurring during brain differentiation.
Comparison of glutamatergic neurons in vertebrates and invertebrates
Glutamate is the predominant excitatory neurotransmitter in the CNS of invertebrates and vertebrates [52, 53]. Glutamate is also one of the major neurotransmitters used in the vertebrate retina pathway, with the three VGLUTs being differentially expressed in specific classes of retina neurons . Glutamate is further known to be present in the vestibular hair cells of different vertebrates [55–57] as well as in mechanosensory neurons of invertebrates . In C. intestinalis, one VGLUT gene has been identified, which is expressed in both the central and the peripheral nervous system (Figure 13) . More specifically, VGLUT is found in sensory organs (photoreceptor cells of ocellus and otolith) and interneurons of the posterior sensory vesicle. Moreover, most of the peripheral sensory neurons found in the larval head and tail are also glutamatergic.
In amphioxus, VGLUT is expressed at the neurula stage in a few cells of the nerve cord and in subpopulations of ectodermal cells. These ectodermal cells correspond to a subgroup of primary sensory cells expressing neural markers, such as Hu/Elav, ERR, β-tubulin, Delta, Brn3 (POU-IV) and synapsin [20, 59–63]. In the amphioxus CNS, glutamatergic neurons are detected in the most anterior and the central region of the cerebral vesicle (Figure 13). Moreover, whereas most of the amphioxus hindbrain is devoid of glutamatergic cells at least until 36-hr, a cluster of VGLUT cells is present in the PMC. The expression of VGLUT in some cells of the frontal eye complex of amphioxus suggests that, comparable to vertebrate retina cells, some of the photoreceptor cells and/or neurons in amphioxus are also glutamatergic. The posterior end of the amphioxus hindbrain, caudal to the first pigment spot, contains clusters of ventrolateral and dorsolateral VGLUT cells (Figure 13). The dorsolateral cluster has previously been homologized with Rohon Beard sensory neurons expressing the neural marker islet[19, 37]. Since several anamniote vertebrates exhibit Rohon Beard sensory neurons with a glutamatergic phenotype [64, 65], our data lend further support to this homology and suggest that glutamatergic transmission is likely to be a common feature of at least certain chordate sense organs.
The serotonergic and dopaminergic systems
Serotonin (5-HT) is one of the most widespread signaling molecules of metazoans [66–68] and probably even of single-celled eukaryotes, where it can modulate swimming behavior and growth . Dopamine (DA), a neurochemical molecule belonging to the catecholamines, has been identified in most metazoan phyla and hence represents another ancient neurotransmitter .
In tetrapod vertebrates, 5-HT neurons are located mainly in the raphe region of the hindbrain, whereas DA-synthesizing nuclei are located within the forebrain and the midbrain . However, in teleosts, for example, 5-HT neurons are also conspicuous in the hypothalamus, where serotonin can modulate neurohormone secretion [72–75]. It is well known that Shh and FGF8 signaling provide essential information to specify the fates and initial positions of DA and 5-HT neurons in the ventral midbrain and rostral hindbrain of vertebrates. These signaling cascades also seem to be important for the expression of early neural patterning genes, such as Pax2, Pax5, Wnt1 and Engrailed, which establish a fundamental organizing center at the boundary of the midbrain and the hindbrain . This midbrain-hindbrain boundary (MHB) acts as organizing center to direct the patterning of the adjacent neural territories and of the localization and size of the DA and 5-HT cell populations .
In the C. intestinalis larva, the TpH gene is expressed in cells of the visceral ganglion , whereas the TH gene (that encodes the tyrosine hydroxylase enzyme involved in DA synthesis) is expressed in the coronet cells of the ventral sensory vesicle, a region possibly homologous to the vertebrate hypothalamus (Figure 13) . In amphioxus, 5-HT neurons are mainly located in the cerebral vesicle, whereas TH-labeled cells were described in the lamellar organ and adjacent ventrolateral cells (Figure 13) . Taken together, since in amphioxus the first serotonergic and dopaminergic populations appear very close together in the cerebral vesicle, the ontogeny of the serotonergic and dopaminergic systems in amphioxus seem to differ substantially from the ontogeny of these systems in vertebrates. This difference between amphioxus and vertebrates might not be surprising given the absence in amphioxus of a vertebrate-like MHB organizer, which, in vertebrates, is required for defining the size and localization of the DA and 5-HT cell populations [78, 79].
Our neurochemical map of the amphioxus CNS reveals that the developing amphioxus nervous system is characterized by a strict regionalization and segmented organization of discrete groups of neuronal cell types. At the neurula stage, GABAergic/glycinergic as well as cholinergic neurons show a segmented distribution in the hindbrain, while glutamatergic, serotonergic and dopaminergic neurons are detectable in very restricted groups of neurons with precise locations along the anteroposterior axis of the CNS.
Animal collection and RNA preparation
Amphioxus adults (B. floridae) were collected in Tampa Bay, Florida, USA, and electrostimulated to induce gamete release. Eggs were fertilized, and embryos were cultured and fixed according to the published methods . Embryos were collected at appropriate stages by low speed centrifugation and were frozen for RNA extraction or fixed for whole mount in situ hybridization. Following RNA extraction, the RNA was treated with RNAse-free DNAse I (Ambion Europe, UK) according to the manufacturer's recommendations to remove contaminating genomic DNA. First-strand cDNA was synthesized with 5 μg of RNA using the SuperScript first-strand synthesis system (Invitrogen, USA) and oligo(dT) primers.
Gene cloning and sequence analyses
The B. floridae genome sequence  was screened with vertebrate and invertebrate VGAT, GAD, SERT, TpH and VGLUT sequences using the TBLASTN algorithm  to identify candidate gene fragments. The automated gene annotation of the retained amphioxus sequences was verified and expanded by protein predictions obtained with the GenScan  and GenomeScan  programs. The resulting amino acid sequences of the amphioxus VGAT, GAD, SERT, TpH and VGLUT candidates were aligned with different vertebrate and invertebrate VGAT, GAD, SERT, TpH and VGLUT sequences using Clustal W v.1.83  and subjected to phylogenetic tree reconstruction analyses. The phylogenetic trees were reconstructed using the Neighbor Joining (NJ) and Maximum Likelihood (ML) methods with distance estimated by JTT amino acid matrix implemented in the program MEGA 5 . Robustness of the resulting trees was calculated by bootstrap analyses in 1,000 replicates. The sequences used for calculating the phylogenies are provided in Additional file 1 and the resulting trees are presented in Additional file 2.
In sum, the in silico analyses yielded single amphioxus VGAT, GAD, SERT, TpH and VGLUT sequences that were used to design specific primers for PCR amplification. The following primers were used to amplify partial sequences of: VGAT, 5' primer GGTCCAGTGTTTGTACGAGGA and 3' primer GATCCCACTTCAGCTTCATGT; GAD, 5' primer ACATCCCCGCTTTTTCAAC and 3' primer GGAGATAGAACGGCTGGGA; SERT, 5' primer TGCGTTCCTTGTCCCTTATTTCA and 3' primer CCCGCGGGTACTCGTCACTCAG; TpH 5’ primer AAGCCGACAAGACCCGAATGAAC and 3’ primer TCTAAGGCGTGGCTAATGGTGTCC; VGLUT 5’ primer TGGGGATACATCGTCACTCA and 3’ primer GGGAGCAATGTCAAGATGGT. An embryonic cDNA library was used to isolate amphioxus amplicons. In some cases, the amplicons were identified by RT-PCR on RNA samples from amphioxus adults. PCR experiments were carried out in a 50 μl reaction mixture using the Hot Master mix according to the manufacturer's instructions (Eppendorf, Italy). The PCR products were directly cloned using a TOPO TA cloning kit (Invitrogen, USA) and subsequently sequenced using a 377 PerkinElmer sequencer (PerkinElmer, USA).
In situ hybridization and histological analysis
The cDNA sequences corresponding to the clones of amphioxus VGAT, GAD, SERT, TpH and VGLUT isolated by PCR were used as templates for in vitro transcription using a DIG RNA labeling kit according to the supplier's instructions (Roche, Italy). Cholinergic neurons were studied using a riboprobe against VAChT previously isolated in the laboratory . In situ hybridization analysis on amphioxus embryos was carried out following the published protocol . Labeled whole mount embryos were photographed using an Olympus IX71 microscope (Olympus, Italy), and subsequently counterstained with 1% Ponceau S in 1% acetic acid, dehydrated in ethanol, embedded in Spurr's resin and serially sectioned at 3–4 μm. Negative control experiments were performed using sense riboprobes and no specific signal was obtained.
For double in situ hybridization assays, embryos were hybridized simultaneously with two probes labeled with fluorescein and digoxigenin and developed with the appropriate antibody conjugated to alkaline phosphatase (Boehringer, Germany). Double-staining in situ hybridization was performed as previously described .
Confocal images (1024 X 1024 X 8 bit) were acquired on a Leica TCS SP5 AOBS confocal laser scanning microscope (Leica Microsystems, Germany). The NBT/BCIP signal was acquired with a pinhole of 2 airy units and a 633 nm gas laser with the detection window set to 630–640 nm. The red fluorescence of Fast Red (Ex Max: 553 nm, Em Max: 619 nm) was excited at 514 nm and collected with a 590 nm longpass filter. Serial optical sections were taken at a z-step of 0.5 mm. The Leica Confocal Software program was used for image acquisition, storage and analysis (Leica Microsystems, Germany).
The authors would like to thank Skip Pierce, John M. Lawrence, Ray Martinez and Marilyn Wetzel (Department of Biology, University of South Florida, Tampa, FL, USA) for providing laboratory space, equipment and logistic support during the amphioxus spawning season and Jim Langeland (Department of Biology, Kalamazoo College, Kalamazoo, MI, USA) for providing the cDNA library. Moreover, we are indebted to Nicholas D. Holland for critical reading of the manuscript (Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA).
- Holland ND: Early central nervous system evolution: an era of skin brains?. Nat Rev Neurosci. 2003, 4: 617-627. 10.1038/nrn1175.PubMedView ArticleGoogle Scholar
- Holland LZ: Chordate roots of the vertebrate nervous system: expanding the molecular toolkit. Nat Rev Neurosci. 2009, 10: 736-746.PubMedView ArticleGoogle Scholar
- Lacalli TC: New perspectives on the evolution of protochordate sensory and locomotory systems, and the origin of brains and heads. Philos Trans R Soc Lond B Biol Sci. 2001, 356: 1565-1572. 10.1098/rstb.2001.0974.PubMed CentralPubMedView ArticleGoogle Scholar
- Wada H, Saiga H, Satoh N, Holland PW: Tripartite organization of the ancestral chordate brain and the antiquity of placodes: insights from ascidian Pax-2/5/8, Hox and Otx genes. Development. 1998, 125: 1113-1122.PubMedGoogle Scholar
- Wada H, Satoh N: Patterning the protochordate neural tube. Curr Opin Neurobiology. 2001, 11: 16-21. 10.1016/S0959-4388(00)00168-9.View ArticleGoogle Scholar
- Takahashi T, Holland PW: Amphioxus and ascidian Dmbx homeobox genes give clues to the vertebrate origins of midbrain development. Development. 2004, 131: 3285-3294. 10.1242/dev.01201.PubMedView ArticleGoogle Scholar
- Putnam NH: The amphioxus genome and the evolution of the chordate karyotype. Nature. 2008, 453: 1064-1071. 10.1038/nature06967.PubMedView ArticleGoogle Scholar
- Holland LZ: The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Res. 2008, 18: 1100-1111. 10.1101/gr.073676.107.PubMed CentralPubMedView ArticleGoogle Scholar
- Guthrie DM: The physiology and structure of the nervous system of amphioxus, Branchiostoma lanceolatum. Symposia of the Zoological Society of London. 1975, 36: 43-80.Google Scholar
- Bone Q: The central nervous system in amphioxus. J Comp Neurol. 1960, 115: 27-51. 10.1002/cne.901150105.View ArticleGoogle Scholar
- Lacalli TC, Holland ND, West JE: Landmarks in the Anterior Central Nervous System of Amphioxus Larvae. Philos Trans R Soc Lond Biol. 1994, 344: 165-185. 10.1098/rstb.1994.0059.View ArticleGoogle Scholar
- Wicht H, Lacalli TC: The nervous system of amphioxus: structure, development, and evolutionary significance. Can J Zool. 2005, 83: 122-150. 10.1139/z04-163.View ArticleGoogle Scholar
- Holland LZ, Holland ND: Chordate origins of the vertebrate central nervous system. Curr Opin Neurobiol. 1999, 9: 596-602. 10.1016/S0959-4388(99)00003-3.PubMedView ArticleGoogle Scholar
- Williams NA, Holland PWH: Old head on young shoulders. Nature. 1996, 383: 490-10.1038/383490a0.View ArticleGoogle Scholar
- Holland LZ, Holland ND: Expression of AmphiHox-1 and AmphiPax-1 in amphioxus embryos treated with retinoic acid: insights into evolution and patterning of the chordate nerve cord and pharynx. Development. 1996, 122: 1829-1838.PubMedGoogle Scholar
- Wada H, Garcia-Fernàndez J, Holland PWH: Colinear and segmental expression of amphioxus hox genes. Dev Biol. 1999, 213: 131-141. 10.1006/dbio.1999.9369.PubMedView ArticleGoogle Scholar
- Schubert M, Holland ND, Laudet V, Holland LZ: A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus. Dev Biol. 2006, 296: 190-202. 10.1016/j.ydbio.2006.04.457.PubMedView ArticleGoogle Scholar
- Holland PWH, Holland LZ, Williams NA, Holland ND: An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution. Development. 1992, 116: 653-661.PubMedGoogle Scholar
- Jackman WR, Langeland JA, Kimmel CB: islet reveals segmentation in the Amphioxus hindbrain homolog. Dev Biol. 2000, 220: 16-26. 10.1006/dbio.2000.9630.PubMedView ArticleGoogle Scholar
- Bardet PL, Schubert M, Horard B, Holland LZ, Laudet V, Holland ND, Vanacker JM: Expression of estrogen-receptor related receptors in amphioxus and zebrafish: implications for the evolution of posterior brain segmentation at the invertebrate-to-vertebrate transition. Evol Dev. 2005, 7: 223-233. 10.1111/j.1525-142X.2005.05025.x.PubMedView ArticleGoogle Scholar
- Candiani S, Oliveri D, Parodi M, Castagnola P, Pestarino M: AmphiD1/β, a dopamine D1/β-adrenergic receptor from the amphioxus Branchiostoma floridae: evolutionary aspects of the catecholaminergic system during development. Dev Genes Evol. 2005, 215: 631-638. 10.1007/s00427-005-0019-6.PubMedView ArticleGoogle Scholar
- Candiani S, Lacalli TC, Parodi M, Oliveri D, Pestarino M: The cholinergic gene locus in amphioxus: molecular characterization and developmental expression patterns. Dev Dynamics. 2008, 237: 1399-1411. 10.1002/dvdy.21541.View ArticleGoogle Scholar
- Holland ND, Holland LZ: Serotonin-containing cells in the nervous system and other tissues during ontogeny of a lancelet, Branchiostoma floridae. Acta Zool. 1993, 74: 195-204. 10.1111/j.1463-6395.1993.tb01234.x.View ArticleGoogle Scholar
- Candiani S, Augello A, Oliveri D, Passalacqua M, Pennati R, De Bernardi F, Pestarino M: Immunocytochemical localization of serotonin in embryos, larvae and adults of the lancelet, Branchiostoma floridae. Histochem J. 2001, 33: 413-420. 10.1023/A:1013775927978.PubMedView ArticleGoogle Scholar
- Chaudhry FA, Reimer RJ, Bellocchio EE, Danbolt NC, Osen KK, Edwards RH, Storm-Mathisen J: The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons. J Neurosci. 1998, 18: 9733-9750.PubMedGoogle Scholar
- Mazet F, Masood S, Luke GN, Holland ND, Shimeld SM: Expression of AmphiCoe, an amphioxus COE/EBF gene, in the developing central nervous system and epidermal sensory neurons. Genesis. 2004, 38: 58-65. 10.1002/gene.20006.PubMedView ArticleGoogle Scholar
- Kaltenbach SL, Yu JK, Holland ND: The origin and migration of the earliest-developing sensory neurons in the peripheral nervous system of amphioxus. Evol Dev. 2009, 11 (2): 142-151. 10.1111/j.1525-142X.2009.00315.x.PubMedView ArticleGoogle Scholar
- Kupfermann I: Functional studies of cotransmission. Physiol Revs. 1991, 71: 683-732.Google Scholar
- Boulland JL, Qureshi T, Seal RP, Rafiki A, Gundersen V, Bergersen LH, Fremeau RT, Edwards RH, Storm-Mathisen J, Chaudhry FA: Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters. J Comp Neurol. 2004, 480 (3): 264-280. 10.1002/cne.20354.PubMedView ArticleGoogle Scholar
- Barreiro-Iglesias A, Cornide-Petronio ME, Anadón R, Rodicio MC: Serotonin and GABA are colocalized in restricted groups of neurons in the larval sea lamprey brain: insights into the early evolution of neurotransmitter colocalization in vertebrates. J Anat. 2009, 215 (4): 435-443. 10.1111/j.1469-7580.2009.01119.x.PubMed CentralPubMedView ArticleGoogle Scholar
- Uemura H, Tezuka Y, Hasegawa C, Kobayashi H: Immunohistochemical investigation of neuropeptides in the central nervous system of the amphioxus, Branchiostoma belcheri. Cell Tissue Res. 1994, 277: 279-287. 10.1007/BF00327775.View ArticleGoogle Scholar
- Pestarino M, Lucaroni B: FMRFamide-like immunoreactivity in the central nervous system of the lancelet Branchiostoma lanceolatum. Isr J Zool. 1996, 42: 227-234.Google Scholar
- Moret F, Guilland JC, Coudouel S, Rochette L, Vernier P: Distribution of tyrosine hydroxylase, dopamine, and serotonin in the central nervous system of amphioxus (Branchiostoma lanceolatum): implications for the evolution of catecholamine systems in vertebrates. J Comp Neurol. 2004, 468: 135-150. 10.1002/cne.10965.PubMedView ArticleGoogle Scholar
- Anadón R, Adrio F, Rodríguez-Moldes I: Distribution of GABA immunoreactivity in the central and peripheral nervous system of amphioxus (Branchiostoma lanceolatum Pallas). J Comp Neurol. 1998, 401: 293-307. 10.1002/(SICI)1096-9861(19981123)401:3<293::AID-CNE1>3.0.CO;2-F.PubMedView ArticleGoogle Scholar
- Pascual-Anaya J, D'Aniello S: Free amino acids in the nervous system of the amphioxus Branchiostoma lanceolatum. A comparative study. Int J Biol Sci. 2006, 2 (2): 87-92.PubMed CentralPubMedView ArticleGoogle Scholar
- Meinertzhagen IA, Lemaire P, Okamura Y: The neurobiology of the ascidian larva: recent developments in an ancient chordate. Annu Rev Neurosci. 2004, 27: 453-485. 10.1146/annurev.neuro.27.070203.144255.PubMedView ArticleGoogle Scholar
- Lacalli TC, Kelly SJ: Ventral neurons in the anterior nerve cord of amphioxus larvae. I. An inventory of cell types and synaptic patterns. J Morphol. 2003, 257: 190-211. 10.1002/jmor.10114.PubMedView ArticleGoogle Scholar
- Lacalli TC: The dorsal compartment locomotory control system in amphioxus larvae. J Morphol. 2002, 252: 227-237. 10.1002/jmor.1101.PubMedView ArticleGoogle Scholar
- Lacalli TC: Ventral neurons in the anterior nerve cord of amphioxus larvae. II. Further data on the pacemaker circuit. J Morphol. 2003, 257: 212-218. 10.1002/jmor.10133.PubMedView ArticleGoogle Scholar
- Woolf NJ: Cholinergic systems in mammalian brain and spinal cord. Prog Neurobiol. 1991, 37: 475-524. 10.1016/0301-0082(91)90006-M.PubMedView ArticleGoogle Scholar
- Lacalli TC: Frontal eye circuitry, rostral sensory pathways and brain organization in amphioxus larvae: evidence from 3D reconstructions. Phil Trans R Soc Lond B. 1996, 351: 243-263. 10.1098/rstb.1996.0022.View ArticleGoogle Scholar
- Lacalli TC: Sensory pathways in amphioxus larvae I. Constituent fibres of the rostral and anterodorsal nerves, their targets and evolutionary significance. Acta Zool (Stock). 2002, 83: 149-166. 10.1046/j.1463-6395.2002.00109.x.View ArticleGoogle Scholar
- Jackson FR, Newby LM, Kulkarni SJ: Drosophila GABAergic systems: sequence and expression of glutamic acid decarboxlyase. J Neurochem. 1990, 54: 1068-1078. 10.1111/j.1471-4159.1990.tb02359.x.PubMedView ArticleGoogle Scholar
- McIntire S, Jorgensen EM, Horvitz HR: Genes required for GABA function in the nematode Caenorhabditis elegans. Nature. 1993, 364: 334-337. 10.1038/364334a0.PubMedView ArticleGoogle Scholar
- Mueller T, Vernier P, Wullimann MF: A phylotypic stage in vertebrate brain development: GABA cell patterns in zebrafish compared with mouse. J Comp Neurol. 2006, 494: 620-634. 10.1002/cne.20824.PubMedView ArticleGoogle Scholar
- Martyniuk CJ, Awad R, Hurley R, Finger TE, Trudeau VL: Glutamic acid decarboxylase 65, 67, and GABA-transaminase mRNA expression and total enzyme activity in the goldfish (Carassius auratus) brain. Brain Res. 2007, 1147: 154-166.PubMedView ArticleGoogle Scholar
- Zega G, Biggiogero M, Groppelli S, Candiani S, Oliveri D, Parodi M, Pestarino M, Bernardi F, Pennati R: Developmental expression of glutamic acid decarboxylase andof γ-aminnobutyric acid type B Receptors in the ascidian Ciona intestinalis. J Comp Neurol. 2008, 506: 489-505. 10.1002/cne.21565.PubMedView ArticleGoogle Scholar
- Roberts A, Dale N, Ottersen OP, Storm-Mathisen J: Development and characterization of commissural interneurones in the spinal cord of Xenopus laevis embryos revealed by antibodies to glycine. Development. 1988, 103 (3): 447-461.PubMedGoogle Scholar
- Villar-Cerviño V, Barreiro-Iglesias A, Anadón R, Rodicio MC: Development of glycine immunoreactivity in the brain of the sea lamprey: comparison with gamma-aminobutyric acid immunoreactivity. J Comp Neurol. 2009, 512: 747-767. 10.1002/cne.21916.PubMedView ArticleGoogle Scholar
- Villar-Cerviño V, Barreiro-Iglesias A, Rodicio MC, Anadón R: D-serine is distributed in neurons in the brain of the sea lamprey. J Comp Neurol. 2010, 518: 1688-1710. 10.1002/cne.22296.PubMedView ArticleGoogle Scholar
- Horie T, Nakagawa M, Sasakura Y, Kusakabe TG: Cell type and function of neurons in the ascidian nervous system. Dev Growth Differ. 2009, 51: 207-220. 10.1111/j.1440-169X.2009.01105.x.PubMedView ArticleGoogle Scholar
- Antzoulatos EG, Byrne JH: Learning insights transmitted by glutamate. Trends Neurosci. 2004, 27: 555-560. 10.1016/j.tins.2004.06.009.PubMedView ArticleGoogle Scholar
- Collingridge GL, Lester RAJ: Excitatory amino acid receptors in the vertebrate central nervous system. Pharmacol Rev. 1989, 40: 143-210.Google Scholar
- Fyk-Kolodziej B, Dzhagaryan A, Qin P, Pourcho RG: Immunocytochemical localization of three vesicular glutamate transporters in the cat retina. J Comp Neurol. 2004, 475: 518-530. 10.1002/cne.20199.PubMedView ArticleGoogle Scholar
- Panzanelli P, Valli P, Cantino D, Fasolo A: Glutamate and carnosine in the vestibular system of the frog. Brain Res. 1994, 662: 293-296. 10.1016/0006-8993(94)90829-X.PubMedView ArticleGoogle Scholar
- Demêmes D, Wenthold RJ, Moniot B, Sans A: Glutamate-like immunoreactivity in the peripheral vestibular system of mammals. Hear Res. 1990, 46: 261-269. 10.1016/0378-5955(90)90007-C.PubMedView ArticleGoogle Scholar
- Harper A, Blythe WR, Grossman G, Petrusz P, Prazma J, Pillsbury HC: Immunocytochemical localization of aspartate and glutamate in the peripheral vestibular system. Hear Res. 1995, 86: 171-182. 10.1016/0378-5955(95)00068-F.PubMedView ArticleGoogle Scholar
- Horie T, Kusakabe T, Tsuda M: Glutamatergic networks in theCiona intestinalislarva. J Comp Neurol. 2008, 508: 249-263. 10.1002/cne.21678.PubMedView ArticleGoogle Scholar
- Satoh G, Wang Y, Zhang P, Satoh N: Early development of amphioxus nervous system with special reference to segmental cell organization and putative sensory cell precursors: a study based on the expression of pan-neuronal marker gene Hu/elav. J Exp Zool. 2001, 291: 354-364. 10.1002/jez.1134.PubMedView ArticleGoogle Scholar
- Yasui K, Tabata S, Ueki T, Uemura M, Zhang SC: Early development of the peripheral nervous system in a lancelet species. J Comp Neurol. 1998, 393: 415-425. 10.1002/(SICI)1096-9861(19980420)393:4<415::AID-CNE2>3.0.CO;2-3.PubMedView ArticleGoogle Scholar
- Candiani S, Oliveri D, Parodi M, Bertini E, Pestarino M: Expression of AmphiPOU-IV in the developing neural tube and epidermal sensory neural precursors in amphioxus supports a conserved role of class IV POU genes in the sensory cells development. Dev Genes Evol. 2006, 216: 623-633. 10.1007/s00427-006-0083-6.PubMedView ArticleGoogle Scholar
- Candiani S, Moronti L, Pennati R, De Bernardi F, Benfenati F, Pestarino M: The synapsin gene family in basal chordates: evolutionary perspectives in metazoans. BMC Evol Biol. 2010, 10: 32-10.1186/1471-2148-10-32.PubMed CentralPubMedView ArticleGoogle Scholar
- Rasmussen SL, Holland LZ, Schubert M, Beaster-Jones L, Holland ND: Amphioxus AmphiDelta: evolution of Delta protein structure, segmentation, and neurogenesis. Genesis. 2007, 45: 113-22. 10.1002/dvg.20278.PubMedView ArticleGoogle Scholar
- Marek KW, Kurtz LM, Spitzer NC: cJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification. Nat Neurosci. 2010, 13: 944-950. 10.1038/nn.2582.PubMed CentralPubMedView ArticleGoogle Scholar
- Drapeau P, Saint-Amant L, Buss RR, Chong M, McDearmid JR, Brustein E: Development of the locomotor network in zebrafish. Prog Neurobiol. 2002, 68: 85-111. 10.1016/S0301-0082(02)00075-8.PubMedView ArticleGoogle Scholar
- Zega G, Pennati R, Fanzago A, De Bernardi F: Serotonin involvement in the metamorphosis of the hydroid Eudendrium racemosum. Int J Dev Biol. 2007, 51: 307-313. 10.1387/ijdb.062195gz.PubMedView ArticleGoogle Scholar
- Makrosova TG, Zaitseva OV, Smirnov RV: Monoamine- and peptide-containing elements in the nemertine digestive tract. Zh Evol Biokhim Fiziol. 2007, 43: 60-68.PubMedGoogle Scholar
- Pires A, Woollacott RM: Serotonin and dopamine have opposite effects on phototaxis in larvae of the bryozoan Bugula neritina. Biol Bull. 1997, 192: 399-409. 10.2307/1542749.View ArticleGoogle Scholar
- Csaba G: Presence in and effects of pineal indoleamines at very low level of phylogeny. Experientia. 1993, 49: 627-634. 10.1007/BF01923943.PubMedView ArticleGoogle Scholar
- Anctil M, Hurtubise P, Gillis MA: Tyrosine hydroxylase and dopamine-beta-hydroxylase immunoreactivities in the cnidarian Renilla koellikeri. Cell Tissue Res. 2002, 310 (1): 109-117. 10.1007/s00441-002-0601-4.PubMedView ArticleGoogle Scholar
- Goridis C, Rohrer H: Specification of catecholaminergic and serotonergic neurons. Nat Rev Neurosci. 2002, 3 (7): 531-541.PubMedView ArticleGoogle Scholar
- Kah O, Chambolle P: Serotonin in the brain of the goldfish, Carassius auratus. An immunocytochemical study. Cell Tissue Res. 1983, 234: 319-333. 10.1007/BF00213771.PubMedView ArticleGoogle Scholar
- Jacobs BL, Azmitia EC: Structure and function of the brain serotonin system. Physiol Rev. 1992, 72: 165-229.PubMedGoogle Scholar
- Khan IA, Thomas P: Immunocitochemical localization of serotonin and gonadotropin-releasing hormone in the brain and pituitary gland of the Atlantic croaker Micropogonias undulates. Gen Comp Endocrinol. 1993, 91: 167-180. 10.1006/gcen.1993.1116.PubMedView ArticleGoogle Scholar
- Oliveri D, Candiani S, Parodi M, Bertini E, Pestarino M: A serotonergic system in the brain of the Antarctic fish, Trematomus bernacchii. Polar Biol. 2005, 28: 366-371. 10.1007/s00300-004-0706-1.View ArticleGoogle Scholar
- Wurst W, Bally-Cuif L: Neural plate patterning: upstream and downstream of the isthmic organizer. Nat Rev Neurosci. 2001, 2: 99-108. 10.1038/35053516.PubMedView ArticleGoogle Scholar
- Pennati R, Candiani S, Biggiogero M, Zega G, Groppelli S, Oliveri D, Parodi M, De Bernardi F, Pestarino M: Developmental expression of tryptophan hydroxylase gene in Ciona intestinalis. Dev Genes Evol. 2007, 217: 307-313. 10.1007/s00427-007-0138-3.PubMedView ArticleGoogle Scholar
- Holland LZ, Kene M, Williams N, Holland ND: Sequence and embryonic expression of the amphioxus engrailed gene (AmphiEn): the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development. 1997, 124: 1723-1732.PubMedGoogle Scholar
- Kozmik Z, Holland ND, Kalousova A, Paces J, Schubert M, Holland LZ: Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region. Development. 1999, 126: 1295-1304.PubMedGoogle Scholar
- Holland LZ, Holland PWH, Holland ND, Ferraris JD, Palumbi SR: Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: amphioxus versus vertebrates. In Molecular Zoology: Advances, Strategies, and Protocols. 1996, New York: Wiley-Liss, 267-282.Google Scholar
- JGI (Joint Genome Institute). [http://genome.jgi-psf.org/].
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410.PubMedView ArticleGoogle Scholar
- GenScan. [http://genes.mit.edu/GENSCAN.html].
- GenomeScan. [http://genes.mit.edu/genomescan.html].
- Higgins DG, Thompson JD, Gibson TJ: Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 1996, 226: 383-402.View ArticleGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol. 2011, 28 (10): 2731-2739. 10.1093/molbev/msr121.PubMed CentralPubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.