Cephalochordate Nervous System
Abstract and Keywords
The central and peripheral nervous systems of amphioxus adults and larvae are characterized by morphofunctional features relevant to understanding the origins and evolutionary history of the vertebrate CNS. Classical neuroanatomical studies are mainly on adult amphioxus, but there has been a recent focus, both by TEM and molecular methods, on the larval CNS. The latter is small and remarkably simple, and new data on the localization of glutamatergic, GABAergic/glycinergic, cholinergic, dopaminergic, and serotonergic neurons within the larval CNS are now available. In consequence, it has been possible begin the process of identifying specific neuronal circuits, including those involved in controlling larval locomotion. This is especially useful for the insights it provides into the organization of comparable circuits in the midbrain and hindbrain of vertebrates. A much better understanding of basic chordate CNS organization will eventually be possible when further experimental data will emerge.
Amphioxus (amphis = both, oxys = sharp), also known as the lancelet, is a cephalochordate distributed in tropical and temperate seas. Adults live in shallow waters of tropical and temperate seas, generally near the coast, and are benthic organisms inhabiting normally loose sandy and shell-sand bottoms in various regions of the world (Fig. 1).
Adults spend most of their life as sessile organisms, protruding their rostral ends above the surface and filtering food particles, but are vigorous swimmers when required. The larvae, in contrast, are planktonic, and swim in part to maintain their position in the water column. Amphioxus was described for the first time by Pallas in 1774 who classified it as slug with the name Limax lanceolatus. Its close phylogenetic affinity to vertebrates was first recognized in 1834 by Costa, who named the genus Branchiostoma. At present three genera are known (Branchiostoma, Epigonichthys, and Asymmetron, see Kon et al., 2007) with ca. 30 known species. All living cephalochordates resemble each other closely. The name amphioxus was introduced by William Yarrell in 1836, who described for the first time the presence of the notochord, a morphological structure characteristic of chordates. In an interesting review on the evolution of chordates, Berrill (1987) defines amphioxus as “the riddle of the sands.” In fact, for a long time, amphioxus has been considered a vertebrate, but the molecular data now available shows them to be basal within the chordates, and is the sister group of the clade comprising urochordates and vertebrates (Bourlat et al., 2006; Delsuc et al., 2006; Holland et al., 2008).
Adult amphioxus are small, semitransparent fish-like animals 4–6 cm long. They have an elaborate segmented neuromuscular system and a notochord that extends from the tip of the rostrum to the end of the tail, but lack a typical vertebrate-type backbone and head. Nevertheless, amphioxus shares with vertebrates the same basic body plan, making it important for investigations of the evolutionary history of vertebrates. The recent sequencing of the amphioxus genome (Branchiostoma floridae) has confirmed the evolutionary conservation of numerous genes, including those expressed specifically in the nervous system (Holland et al., 2008; Putnam et al., 2008). Further, comparative studies in recent decades of both neuroanatomy and gene expression patterns reveal a vertebrate-like regional organization in the amphioxus CNS (Holland, 2009), which clarifies certain aspects of how chordate nervous systems have evolved (Holland et al., 2013).
The Adult Central Nervous System
The amphioxus nervous system consists of a hollow nerve cord, located dorsal to the notochord, and a peripheral nervous system (PNS) consisting of cranial nerves and segmental dorsal spinal nerves (Fig. 2), visceral plexi and numerous solitary sensory cells in the skin (Baatrup, 1981; Bone & Best, 1978).
The ventricular cavity of the nerve cord is approximately circular in cross-section in the most rostral region, that is, the anterior part of the cerebral vesicle, or anterior vesicle in some accounts, but is compressed to a vertical slit more caudally, including in the spinal cord.
Past studies have provided a basic outline of neural anatomy and histology for adult amphioxus (Boeke, 1913; Bone, 1960; Edinger, 1906; Franz, 1923; Joseph, 1904; Retzius, 1891; Rohde, 1887), and further details on the cytology and regional organization of the cerebral vesicle are reviewed by Ruppert (1997); Nieuwenhuys (1998); Wicht and Lacalli (2005); Castro et al. (2015); and Lacalli (2016). Ultrastructural, histochemical, and immunocytochemical studies on specific cell types include those of Meves (1973); Obermüller-Wilén and van Veen (1981); Holland and Holland (1993); Olsson, Yulis, and Rodriguez (1994); Anadón et al. (1998); Castro et al. (2003, 2004, 2006); Ekhart et al. (2003); Takeda et al. (2003); Moret et al. (2004); and Vopalensky et al. (2012). The neurotransmitters present in the adult CNS are incompletely known, but neurosecretory and monoaminergic neurons have been identified in the cerebral vesicle (Obermüller-Wilén & van Veen, 1981), as well as calcitonin, pancreatic polypeptide, and FMRFamide (Fang & Kobayashi, 1990; Van Noorden, 1984; Pestarino & Lucaroni, 1996), oxytocin (Vallet et al., 1985), methionine-enkephalin (Uemura et al., 1994), arginine vasopressin/vasotocin and oxytocin (Uemura et al., 1994), and pituitary transcription factor 1 (Pit-1) (Candiani & Pestarino, 1998a).
The Adult Cerebral Vesicle
The most anterior region of the nerve cord of adult amphioxus was initially described by Willey (1894). Its most anterior part, the anterior vesicle, or anterior cerebral vesicle (i.e., the region characterized by a circular central cavity, and ending with the infundibular cells), has been homologized to the diencephalon (Edinger, 1906; Franz, 1927; von Kupffer, 1906), but more recent molecular data (e.g., Albuixech-Crespo et al., 2017) indicates a closer relationship with the hypothalamus. The posterior cerebral vesicle, which begins at the infundibular cells and extends to the fourth myomere, has a central canal shaped as a vertical median cleft, and is considered homologous to the vertebrate midbrain plus hindbrain (Fritzsch, 1996; Holland & Garcia-Fernandez, 1996; von Kupffer, 1906). It includes a posterior zone referred to as the caudal brain region (Nieuwenhuys, 1998), or intercalated region (Ekhart et al., 2003), that is subdivided in three parts: anterior, intermediate, and posterior: adjacent respectively to the first, second, and third to fourth myomeres (Fig. 2). For the region as a whole, the molecular data indicates the point of transition from a zone homologous with midbrain to one homologous with hindbrain lies near the front of the second myomere (Holland & Garcia-Fernandez, 1996), but the diencephalon/mesencephalon subdivision has no obvious vertebrate counterpart (Albuixech-Crespo et al., 2017), and vertebrate-type rhombomeres are absent.
The Adult Spinal Cord and Spinal Nerves
The spinal cord has a triangular shape in cross-section with a central cavity shaped like an inverted keyhole. A bilateral series of dorsal sensory and visceromotor spinal nerves project from the dorsal part of the spinal cord, and travel between myomeres to the ventral part of the body, where they divide into dorsal ascending and ventral descending tracts (Bone, 1961) (Fig. 3).
The right and left dorsal spinal roots alternate as a consequence of corresponding alternation of the myomeres. In amphioxus, no vertebrate-like ventral nerve roots are present. Instead, the bundle of fibers extending from the myomere to the nerve cord, initially interpreted as ventral roots, are in fact slender muscle processes, and synapses to these are made across the basal lamina of the spinal cord from motoneurons whose fibers never leave the confines of the cord (Flood, 1996; Rohde, 1887).
Adult amphioxus of Branchiostoma lanceolatum has 64 myomeres and 65 pairs of spinal nerves. The two first pairs, conventionally numbered 1 and 2, emerge from the anterior cerebral vesicle and innervate the rostral region. Nerve 1 (in larvae, the rostral nerve) enters the nerve cord ventrally; nerve 2 (in larvae, the anterodorsal nerve) is dorsal. Nerves 3 to 6 arise from the intercalated region of the anterior nerve cord and connect two nerve plexi, the buccal plexus innervating the buccal cavity, cirri and associated muscles, and the velar plexus innervating tentacles and sphincter muscle of the velum, and send branches to the skin subepidermal nerve plexus. In conclusion, the first myomere is located between the dorsal nerves 2 and 3; the total number of spinal dorsal nerves is 64 (from nerve 2 to nerve 65) because the nerve 1 is ventral and the total number of myomeres is 64.
Cell Types of the Adult Amphioxus CNS
The Frontal Eye
In the tip of the anterior cerebral vesicle is located a cluster of pigment cells and uniciliate sensory cells, which together form the frontal eye (Fig. 2), considered to be a primitive homolog vertebrate paired eyes (Glardon et al., 1998; Lacalli, 1996a) though it is too small and simple in structure to form an image. The putative photoreceptors in this case lack any specialized rod- or cone-like structures, but coexpress vertebrate eye-specific regulatory genes (Rx, Otx, Pax4/6, and Mitf) and differentiation markers (c-opsins, inhibitory Gi-alpha subunit, melanin), which strongly supports the case for homology with the retinal pigment cells and photoreceptors of vertebrates (Vopalensky et al., 2012).
Circadian Clock Center
There are a number of small neurons located dorsally and behind the frontal eye and organized in the “dumbbell balls” that contain neurons expressing amphiPer and amphiBmal genes in a circadian fashion (Schomerus et al., 2008; Wicht et al., 2010). These are thought to be involved in the generation of circadian behaviors, and are reminiscent of the suprachiasmatic nucleus of mammals. Small GABAergic neurons and fibers have been observed in the caudodorsal region of the adult brain vesicle (Anadón et al., 1998), which suggests that this neurotransmitter may also be involved in the regulation of circadian circuitry in amphioxus, as GABA is a key neurotransmitter in the suprachiasmatic nucleus (SCN). The possibility here is that amphiPer- and amphiBmal-expressing cells in amphioxus coexpress GABA, but this is currently unproven.
The lamellate cells, a type of putative ciliary photoreceptor, are found in the dorsal wall of the cerebral vesicle. They lie forward of the main group of Joseph cells, and are morphologically comparable to the cells of the pineal organ of lampreys and fish (Ekström & Meissl, 2003). Each lamellate cell has a single cilium that gives rise to parallel stacks of membranous lamellae (Eakin & Westfall, 1962; Ruiz & Anadón, 1991). Populations of small dorsal GABAergic (Anadón et al., 1998), serotonergic, and dopaminergic cells (Moret et al., 2004; Obermüller-Wilén & van Veen, 1981) have also been described from this region, but ultrastructural observations on adult amphioxus suggest that dopaminergic neurons and lamellate cells are not the same (Obermüller-Wilén & van Veen, 1981). From ultrastructural observations, the lamellate cells do not form a single “lamellar body” in the adult brain (Eakin & Westfall, 1962; Meves, 1973; Ruiz & Anadón, 1991), as they do in the larva (Lacalli, 1996a, 1996b).
Joseph cells are photoreceptors located in the dorsal roof of the posterior cerebral vesicle, extending caudally for some distance depending on species (Fig. 2). Ultrastructural studies of Joseph cells have demonstrated the presence of a rhabdomeric region characterized by an array of microvilli (Watanabe & Yoshida, 1986; Welsch, 1968), and the cells express amphioxus melanopsin, resembling invertebrate rhabdomeric photoreceptors in this respect (Gomez et al., 2009; Koyanagi et al., 2005). Arendt (2003) has proposed the homology of Joseph cells with the vertebrate retinal ganglion cells, which implies a link between ancestral rhabdomeric photoreceptors of prebilaterians and the circadian photoreceptors of higher vertebrates. Such a hypothesis could not be correct for amphioxus because the lamellar cells and Joseph cells (JCs) may overlap spatially, but they are entirely different cells types, and the role of retinal ganglion cells in determining day-length in higher vertebrates is probably secondary, evolving only when the skull became solid and opaque so the pineal could not receive light directly. Although some Joseph cells extend forward into a region homologous with forebrain, they extend posteriorly through regions considered homologous with both mid- and hindbrain (Shimeld & Holland, 2005).
Hesse eyecups, also known as dorsal ocelli, are photoreceptors consisting of a sensory cell and a cup-shaped pigment cell. They are arranged in segmental groups lying ventrally, to the right and left of the central canal of the spinal cord (Fig. 2). In the middle part of the spinal cord there is a region where the Hesse eyecups are rather sparse, and in this respect, they are somewhat similar to the Rohde cells. Electron microscopy of Hesse eyecups shows that the surface nearest the melanocyte is increased in area by microvilli (Eakin & Westfall, 1962), where the visual pigments are presumably located.
The infundibular cells are specialized secretory ependymal cells (Obermüller-Wilen, 1976; Olsson, 1955, 1956; Olsson et al., 1994), clustered to form an infundibular “organ” located in the caudal ventromedial region of the cerebral vesicle (Boeke, 1913; von Kupffer, 1906) (Fig. 2). The infundibular cells secrete the Reissner’s fibers, extruded into the ciliated ventral part of the central canal and passing caudally down it (Olsson et al., 1994; Sterba et al., 1983). In lower vertebrates, the ventral flexural organ can be considered homologous with the amphioxus infundibular organ (Olsson, 1956), whereas in higher vertebrates only the dorsal subcommisural organ is responsible for the secretion of Ressner’s fibers.
Retzius Bipolar Cells
Retzius bipolar cells were first reported from the spinal cord of amphioxus by Retzius (1891). Subsequently Edinger (1906) and Bone (1960) provided detailed descriptions. These cells are wedge-shaped with opposing longitudinal processes, one of which originates a collateral branch that exits the neural tube via a dorsal nerve. The series of Retzius bipolar begins just caudal to the point of entrance of nerve 2 into the nerve cord (Lacalli & Kelly, 2003a), with the first members of the series lying on either side of the lamellar body. The innervation of mouth structures by nerves 3–5 is notably asymmetric from early stages onward (Kaji et al., 2001, 2009). This suggests that the asymmetric arrangement of Retzius bipolar cells in the adult brain of amphioxus may be related to the exaggerated left–right asymmetry of nerves 3–5. Retzius bipolar cells of the spinal cord have been compared with the primary sensory neurons, or Rohon-Beard cells present at least transiently in the spinal cord of fishes and amphibians (Bone, 1960; Roberts, 2000). In lampreys, similar primary sensory cells are present in the caudal hindbrain and send peripheral processes in the trigeminal nerve and other branchiomeric cranial nerves (Anadón et al., 1989), which appears similar to those reported for amphioxus for the Retzius bipolar cells. Interestingly, such cells contain FMRFamide peptides (Pestarino & Lucaroni, 1996).
Translumenal Neurons and Rohde Cells
Populations of translumenal neurons, characterized by apical processes that cross the neural canal, occur in amphioxus CNS (Anadón et al., 1998; Edinger, 1906; Ekhart et al., 2003; Franz, 1923; Rohde, 1887). The anterior series of large translumenals are Rohde cells and start at level of the left sixth dorsal nerve according to Rohde (1887) and marks, approximately, the neuroanatomical boundary between the cerebral vesicle as defined for the adult, and the spinal cord (Franz, 1923). An anterior group of Rohde cells is located behind the cerebral vesicle and is separated from a most caudal posterior group by a region devoid of such cell type. On the other hand, molecular studies indicate that, at early stages of development, the boundary between the cerebral vesicle and spinal cord may be located more caudally to the first Rohde cell, at least at the level of the third or fourth myomeres (Shimeld & Holland, 2005). The giant axons of Rohde cells cross the ventral midline and project contralaterally (Rohde, 1887) (Fig. 2). Rohde cells probably play a role in coordinating and possibly in initiating locomotory responses (e.g., forward and backward swimming, Guthrie, 1975), where large axon diameter, and hence rapid transmission speed, would be advantageous.
Classical studies focus only on the largest examples, that is, the Rohde cells and other giant translumenals. Of these, five examples with descending ipsilateral axons are reported rostral to the first Rohde neuron in the larval brain (Bone, 1959), possibly corresponding with those described in the adult by Ekhart et al. (2003), whose axons are shown to project to the spinal cord. Putative transmitters for translumenal neurons include GABA (Anadón et al., 1998), FMRFamide, urotensin 1, NPY, arginine vasotocin (Castro et al., 2003; Kubokawa et al., 2010; Uemura et al., 1994), prolactin and calcitonin (Obermüller-Wilén, 1984) (Fig. 4).
In addition, translumenal neurons with supraspinal projections have been shown to contain high levels of glutamate and aspartate (D’Aniello & Garcia-Fernandez, 2007). Translumenal neurons, in short, appear to be neurochemically very heterogeneous.
This population of neurons located ventral to the central canal of the anterior nerve cord was first reported by Rohde (1887) and it has been suggested that the Rohde nucleus might have a neuroendocrine function (Ekhart et al., 2003). The distribution and shape of cells of this nucleus are visible at level of the ventrolateral “infundibulum,” a structure related to Hatschek’s pit (Fig. 2). Nozaki et al. (1999) described a protrusion of the nerve cord that extends to the Hatschek’s pit along the right side of the notochord, and Gorbman (1999) describes endocrine cells in Hatchek’s pit that may be comparable to those in the vertebrate adenohypophysis (Candiani et al., 1998a; Candiani & Pestarino, 2008a; Castro et al., 2003; Gorbman et al., 1999). The amphioxus “infundibulum” has been compared to the vertebrate neurohypophysis (Gorbman, 1999; Gorbman et al., 1999). Moreover, the presence of Pit1 (one of the master genes involved in the differentiation of the vertebrate adenohypophysis), has been demonstrated at the level of the Hatschek’s pit in adults and larvae of amphioxus (Candiani et al., 2008; Candiani & Pestarino, 1998a, 1998b).
Edinger cells of amphioxus (Fig. 2) are thought to be comparable to cerebrospinal fluid-contacting neurons reported from the spinal cord of various vertebrates, most of which appear to be GABAergic and sensory in function (Dale et al., 1987; Martin et al., 1998; Melendez-Ferro et al., 2003). A sensory role for the Edinger cells is possible, but has not yet been confirmed.
Various types of glial cells have been described in the amphioxus nerve cord (Bone, 1960): Müller’s glia, which are small cells arrayed near the dorsal nerve roots; Schneider’s glia (Schneider, 1879), and radial (or ependymal) glia (Bone, 1960). Further glial cells have been observed in the larval nerve cord where they have a role in axon guidance (Wicht & Lacalli, 2005), but the fate of these cells is not known. Myelin sheaths are absent in both the CNS and PNS.
Sensory and Motor Systems
The nerve cord of amphioxus contains both somatomotor and visceromotor neurons. All are ventral in terms of their position in the ependymal layer, but the latter are more ventral. None are translumenal, and all have only ipsilateral projections (Castro et al., 2004, 2015), as with most vertebrate motoneurons. In adult amphioxus, the motoneurons are slender pyramidal cells with axons that travel caudally along the basal lamina, synapsing with muscle processes in transit, that is, none of their axons leave the nerve cord. The somatomotor system is a major constituent of the CNS, reflecting the fact that the coordination of swimming is crucial to amphioxus survival. The somatic muscles contract out-of-phase to produce an undulatory swimming motion, and amphioxus is able to swim either forward or backward with equal facility, either fast or slow depending on the stimulus, and in sand, that is, to form burrows. Relative to body length, it is as fast and efficient a swimmer as most fish (Guthrie, 1975).
The visceromotor neurons are located at or near the ventral midline, and there are both large segmentally repeated cells and more numerous small ones. The axons in each case exit through the nearest dorsal nerve and travel to the visceral organs and atrium. Bone (1958) has suggested that the dendrites of visceromotor neurons may receive synapses directly from visceral afferents, that is, neurons in the peripheral plexi, but how such circuits work in practice is poorly understood.
The Adult Peripheral Nervous System
The PNS of amphioxus consists of a set of peripheral plexi derived from neurons located in the skin and probably including the peripheral neurites of the intramedullary processes from Retzius bipolar cells and dorsal root cells (Bone, 1960, 1961; Wicht & Lacalli, 2005).
The term atrial nervous system (ANS) is used for the part of the PNS that innervates the buccal and velar region, the gut, gonads, and the pterygial muscle, and it is also responsible for controlling the ciliated epithelium lining the gill bars. It has both sensory and motor components. The former includes a variety of sensory cell types, for example, unipolar sensory neurons located on the surface of the pterygial muscle and the parietal walls of the atrium, and multipolar sensory neurons associated with the foregut and its diverticulum. The ANS is evidently mainly involved in the control of feeding and spawning, but very little is known of the cells themselves or their transmitters. The latter appear to differ from those used by autonomic neurons in vertebrates and, in fact, only FMRFamide has so far been identified in amphioxus as a putative transmitter (Bone et al., 1996). There is little evidence at this point for any significant degree of homology between the amphioxus ANS and the visceral innervation of vertebrates.
Amphioxus Larval Nervous System
The nervous system of the amphioxus larva is essentially similar to that of the adult, but much smaller and simpler in organization, with fewer specialized cell types and much less neuropiles. There is also a considerable difference in complexity between young larvae, which begin to feed at a little over a millimeter in length, and late-stage larvae, 10 times that size. Embryos and larvae up to about 3 days in age are used widely for studies of developmental gene expression, and an unexpected level of complexity has been revealed in the nerve cord both in its regional subdivisions and the diversity of cell types (Albuixech-Crespo et al., 2017; Holland & Holland, 1999; Wicht & Lacalli, 2005).
The neural tube of amphioxus larva is wider at the front, a region known as the cerebral vesicle, but its relation to the adult cerebral vesicle seems to be different (in the larva, cerebral vesicle stop at somite 2) while in an adult at the fourth myomere. Therefore, the term posterior cerebral vesicle has a different meaning for larva and adult, depending on how well developed the animal is. The larval cerebral vesicle, in turn, is divided into an anterior portion, whose circular central canal opens anteriorly to the outside through the neuropore, and a gradually narrowing posterior region that extends to about the end of somite 1, where appearance, both in size and organization, becomes essentially indistinguishable from the rest of the nerve cord. The anterior cerebral vesicle includes structures of the frontal eye, a photoreceptor organ, and a balance organ probably involved in perceiving of movement and/or gravity (Lacalli et al., 1994; Lacalli & Kelly, 2000) (Fig. 5).
The posterior region cerebral vesicle has a laterally compressed central canal with a ventral floor plate, a characteristic of the rest of the nerve cord, and contains the dorsal lamellar body (a photoreceptor organ) and a zone of ventral neuropile that includes a commissural fiber bundle derived from the cells of the lamellar body (Lacalli et al., 1994). The junction between the anterior and posterior part of cerebral vesicle is marked ventrally by a cluster of infundibular cells, responsible for producing Reissner’s fiber, which in vertebrates is formed by the subcommisural organ (Lacalli et al., 1994). Other distinctive features of the posterior cerebral vesicle include the primary motor center (PMC), which is a cluster of large, paired neurons involved in locomotory control located near the junction between somites 1 and 2, and the paired anterior-most group of Retzius bipolar cells, which lie on either side of the lamellar body. The region just below the bipolar cells is of special interest, as the major site of synaptic input to the locomotory complex from both the rostral sensory cells and the bipolar cells themselves, referred to by Lacalli and Candiani (2017) as the primary synaptic zone. The caudal limit of the posterior cerebral vesicle is marked by the end of the lamellar body. In older larvae, this corresponds approximately with the junction between somites 1 and 2, also the location of the PMC.
Cell Types of the Most Anterior Larval CNS
The anterior cerebral vesicle in young amphioxus is ca. 75–80 μm long and thickened ventrally, which is where the main groups of neurons reside at this stage. The neurons are generally flask-shaped and include the following:
1. Cells of the frontal eye, consisting of a pigment cup, two transverse rows of photoreceptor cells, and several more of associated neurons, which together act as a low-sensitivity directional photoreceptor. The location of the frontal eye in comparison with the unpaired medial eye rudiment of vertebrate embryos suggests homology (Lacalli, 1996a; Lacalli et al., 1994; Fig. 5), and this is supported by molecular data (e.g., Vopalensky et al., 2012). Its function as a photoreceptor has been confirmed by observations on four-day old larvae (Vopalensky et al., 2012).
2. A balance organ, located just forward of the infundibular cells, consisting of a transverse cluster of cells (typically 10) with unusually large, club-shaped cilia. The cells, referred to by Lacalli (1996a, 1996b) as ciliary bulb cells (Fig. 5), are probably involved in the perception of movement or gravity.
3. A preinfundibular region containing an assortment of flask-shaped neurons, typically with mixed clear and dense-cored vesicles, and basal axons that project to varying degrees to more caudal regions of the nerve cord. Three specific subclasses have been identified and traced in some detail (Lacalli, 2002a; Lacalli & Kelly, 2000, 2003b): two classes of type 1 preinfundibular projection neurons (PPN1 and 2), and one class of type 2 preinfundibular projection neurons (PPN2s) (Fig. 5). The latter are involved in locomotory control (Lacalli, 2002b), but the morphology for all these cells implies they function as intramedullary sensory cells, possibly with a role in homeostasis. This is supported by molecular evidence that the region in question is homologous with vertebrate hypothalamus (Albuixech-Crespo et al., 2017).
The posterior cerebral vesicle includes the following:
1. The initial part of the floor plate, which begins immediately behind the infundibular cells and continues posteriorly along the neural tube.
2. The dorsal lamellar body, a probable homolog of the pineal organ of vertebrates (Lacalli et al., 1994), which is known both from TEM (Lacalli et al., 1994) and immunohistochemistry (Bozzo et al., 2017). The lamellar body itself is a mass of parallel stacks of lamellae arising from the cilium of each of a bilateral series of dorsal-positioned lamellar cells (lc, see Fig. 5). Each lamellar cell has a single large axon that projects ventrally and across the developing post-infundibular neuropile. Lamellar cells are reported also in the adult, but are scattered rather than combined together to form a unitary structure. This may be due to the forward growth of the Joseph cells during the later phase of larval development, which may disrupt and displace other dorsal structures.
3. The region occupied by the anterior group of Retzius bipolar, referred to by Lacalli (1996a), as a “tectum” in recognition of the dorsal positioning of the bipolar (or tectal) cells (tc in Fig. 5) over the synaptic zone just beneath. A better term of the latter is the primary synaptic zone (psz), and this is clearly a region of great functional significance in the early larval brain. It remains an open question whether there is any homology between this region and the tectum of vertebrates, or of any of the constituent cells, but the region in question in amphioxus does appear to lie within the domain whose closest homologous vertebrate counterpart lies within the midbrain.
4. The primary motor center (PMC), a ventral cluster of motoneurons and large neurons predominantly arranged in pairs. Particularly notable are three pairs of interneurons, which are located at the junction between the first and second somite and consist of one pair of especially large (i.e., “giant”) cells and two pairs of smaller cells of similar type (all are glutamatergic, Lacalli & Candiani, 2017) (Fig. 5). The PMC appears to be the control center for initiating rapid bursts of larval swimming in response to mechanosensory stimuli, that is, the larval escape response (Lacalli, 1996a; Lacalli & Candiani, 2017).
Amphioxus Larval PNS
The sensory component of the amphioxus PNS includes a number of different types of sensory receptors (Benito-Gutiêrrez et al., 2005; Holland & Yu, 2002; Lacalli & Hou, 1999; Stokes & Holland, 1995) and peripheral nerves (Yasui et al., 1998). Most are individual sensory neurons and are widely distributed on the body. These have been generally classed as type I receptors (primary sensory neurons having axons projecting to CNS, of which there are diverse types; e.g., see Lacalli & Hou, 1999) and type II receptors (secondary sensory neurons, lacking axons, with synaptic terminals close to the cell body) of which the principal type so far described has a peculiar microvillar collar around an erect cilium). Type I receptors are the first to differentiate and appear at neurula stage, while the type II receptors are seen mainly in older larvae. One category of the former develops as a ventral population that secondarily disperses in the epithelium and moves dorsally (Holland & Yu, 2002), and there is evidence that some of these cells may cross to basal lamina and reposition themselves in the dermal layer (Benito-Gutiêrrez et al., 2005; Kaltenbach, Yu, & Holland, 2009). Peripheral sensory neurons in amphioxus expresses a suit of pan-neuronal markers (AmphiTrk, AmphiHu/Elav, AmphiCoe, Amphi-POU-IV, SoxB, AmphiTlx, AmphiSyn, amphioxus Ash (Candiani et al., 2006, 2010; Kaltenbach et al., 2009; Lu et al., 2012; Mazet et al., 2004; Meulemans & Bronner-Fraser, 2007; Satoh, Wang, Zhang, & Satoh, 2011), and their migration may be controlled by signals originating from mesoderm, and possibly from somite boundaries (Candiani et al., 2010; Kaltenbach et al., 2009). Finally, molecular data support the idea that these cells are specified by a combinatorial code of genes influenced by both RA and Hox genes (Schubert et al., 2005). In addition, Delta/Notch signaling is involved in the specification of individual epidermal sensory neurons from neighboring general epidermal cells, and BMP signaling positively regulate the induction of a progenitor field in the ventral ectoderm for generating epidermal sensory neurons in amphioxus embryos (Lu et al., 2012)
Other type I and II sensory cells are found around mouth and preoral ectoderm, along with the peripheral nerve cells supplying the circumoral nerve (Lacalli et al., 1999). The latter include intrinsic neurons, which are embedded in the oral nerve itself, and extrinsic ones that lie outside it, the largest of which develops near the preoral pit and containing GABA (Zieger et al., 2017). An additional type of secondary sensory cell, clustered in small groups, forms the oral spines around the edges of the mouth, which trigger a cough response used to expel food debris trapped in the mouth.
Though the evidence is sparse, the majority of sensory cells mentioned above appear to be mechanoreceptors, though chemoreception is also a possibility (Baatrup et al., 1981; Bone & Best, 1978). Gene expression data on a G protein-coupled receptor related to the olfactory receptor family genes, support the idea that amphioxus has a chemoreceptive and possibly an olfactory sense (Churcher & Taylor, 2010; Satoh, 2005), though the cells involved have yet to be identified.
The peripheral nerves of the amphioxus PNS first appeared on the anterior dorsal surface of the nerve cord at neurula stage (Yasui et al., 1998). The first nerve, arising from the anterior tip of the nerve cord, is thought to be formed exclusively of sensory elements and may be related to the preoptic nerve of vertebrates. Later in development, axons from the corpuscles of de Quatrefages are incorporated into this nerve. This is an encapsulated sense organ peculiar to amphioxus that is probably involved in sensing mechanical stress to the rostral region (Baatrup, 1981; Fritzsch, 1996). The second nerve appears during early larval development, and innervates the oral region. The oral region is also innervated by the third and fourth nerves. Innervation patterns change during metamorphosis, when the entire oral region is rapidly remodeled, and subsequently, so the oral region in the adult is innervated by the third through seventh nerves (Ayers, 1921). Unlike the situation in vertebrates, no clear distinction can be made between cranial and spinal nerves in amphioxus, implying the latter represents a more primitive condition for the chordate PNS.
Molecular Homology of Amphioxus Larva and Chordate Brains
The anterior nerve cord in amphioxus larvae has been shown by both TEM analysis and gene expression data to be subdivided and structured along similar lines to vertebrate brain. From TEM, structures have been identified that can be assigned to the optic rudiment (frontal eye complex), hypothalamus (the ventral preinfundibular region), diencephalon (the lamellar body), and midbrain (the tegmental neuropile and PMC, the latter being a putative homolog of the mesencephalic locomotor center), but there is no obvious counterpart of the telencephalon (Lacalli, 1996a, 1996b, 2016; Lacalli et al., 1994; Wicht & Lacalli, 2005). Except for the pineal, it is mainly ventral structures/regions of the vertebrate brain that are best represented in the comparisons to the amphioxus anterior nerve chord. Gene expression patterns support the morphological evidence: Otx is expressed in the cerebral vesicle, as in vertebrate forebrain and midbrain, while markers for vertebrate hindbrain, for example, Hox, Gbx and islet genes, are expressed caudal to this region in amphioxus (Castro et al., 2006; Holland & Holland, 1999; Jackman et al., 2000; Jackman & Kimmel, 2002; see Fig. 6), which places the beginning of the amphioxus hindbrain homolog at a level somewhere near the front of somite 2.
The amphioxus nerve cord lacks anatomical subdivisions comparable to vertebrate rhombomeres, but several genes are expressed in the relevant region in a segmentally repeating fashion, which may indicate an underlying segmental organization related to that of vertebrate hindbrain (Bardet et al., 2005; Candiani et al., 2008, 2010; Ferrier, Brooke, Panopoulou, & Holland, 2011; Jackman & Kimmel, 2002; Mazet & Shimeld, 2002). The presence of a midbrain homolog in amphioxus has been somewhat controversial in the past, along with the midbrain-hindbrain boundary (MHB), a major organizing center of the vertebrate brain (Castro et al., 2006; Holland & Holland, 1999). Recent evidence has shown that the amphioxus homologs of the caudal forebrain (i.e., the diencephalon) and mesencephalon are specified as a single unit, the diencephalon-mesencephalon primordium (the DiMes; see Albuixech-Crespo et al., 2017) (Fig. 6), which may explain why the axial extent of a putative diencephalic structure, the lamellar body, overlaps in amphioxus larvae with that of the tegmental neuropile, a basal mesencephalic structure in vertebrates. A further addition to the molecular toolkit for comparative analysis of CNS regions and cell types are the microRNAs (Candiani et al., 2011), and these show some promise for identify specific neural cell types (for example: miR-124 in differentiating and mature neurons, miR-9 in differentiated neurons, miR-183 in cells of sensory organs as well as miR-137 and miR-184 in restricted CNS regions).
Neurotransmitter Phenotypes of the Amphioxus Nervous System
Data on the localization of biosynthetic enzymes and vesicular transporters have been used to construct a neurochemical map of the early larval nervous system in amphioxus (Candiani et al., 2012), confirming in general terms the TEM data obtained by Lacalli and Kelly (2003a) on older larvae. Of special interest are the glutamatergic neurons, which are key excitatory components in both vertebrate and invertebrate nervous systems. In amphioxus, glutamate occurs in epidermal sensory neurons in the rostrum and elsewhere, and in the anterior CNS, in both the anterior group of Retzius bipolar cells and the large paired neurons of the PMC (Lacalli & Candiani, 2017). The latter are central components of the locomotory control circuit that, when activated by sensory inputs, initiates the larval escape response. Glutamate appears therefore to be a major excitatory transmitter, acting both at a sensory and an interneuronal level (Fig. 7).
The remaining ventral neurons of the anterior cord include cells containing acetylcholine, GABA, glycine, and acetylcholine (Fig. 7). Acetylcholine is found in motoneurons, of which there are two types, responsible for the fast and slow mode swimming respectively (Lacalli, 2002b; Lacalli & Kelly, 1999), and in one class of interneurons, the ipsilateral projection neurons that, like motoneurons, have exclusively ipsilateral projections. There are also segmentally repeating groups of neurons containing GABA and a number of glycinergic neurons, which appear to correspond, respectively, with the commissural neurons and multipolar neurons identified by TEM (Fig. 7). Both are presumed to be inhibitory, but the exact role of these cells in locomotory control is not known, nor do we know how the frequency and phase of contraction are controlled so as to generate alternating contractions on the two sides of the body. The large paired neurons (LPN3) may play a role here as discussed by Lacalli and Candiani (2017) (Fig. 7), but the source of pacemaker activity in amphioxus remains uncertain, as does the question of how similar the amphioxus system is in this respect to vertebrates. GABAergic neurons also occur in the PNS, the most noteworthy being the first of the extrinsic oral neurons to develop (Lu et al., 2012; Zieger et al., 2017).
Amphioxus has a dopaminergic system, located in the anterior part of the diencephalic homolog (i.e., the anterior DiMes), described for the adult by Moret et al. (2004) and for larvae by Candiani et al. (2005) and Zieger et al. (2017). The first of the putative (TH-containing) dopaminergic neurons to develop correspond to the type 1 parainfundibular neurons (PIN1s) described by Lacalli and Kelly (2003a) and appear to act to positively reinforce the activity of the early-developing circuits controlling the escape response. Positive reinforcement of this kind parallels, in a sense, the role dopamine plays in vertebrate brain, and implies that dopamine may provide a similar kind of positive feedback throughout the development of the anterior CNS in amphioxus as new circuits are formed and elaborated. Finally, serotonergic neurons have been identified in the frontal eye complex, localized to the Row2 receptor cells (Candiani et al., 2010; Vopalensky et al., 2012).
Moreover, recent data provide evidences of the involvement of RA signaling in the patterning of amphioxus GABAergic and dopaminergic cells (Zieger et al., 2017).
From a comparative standpoint, amphioxus neuroanatomy provides a valuable window on the origin of vertebrates and vertebrate CNS from invertebrate antecedents. Amphioxus CNS is much simpler than that of vertebrates, but is subdivided and organized along similar lines. The forebrain homolog, that is, the cerebral vesicle, has only been recognized in the last two decades on the basis of detailed molecular and EM-level studies of early development. There is so far no evidence for an amphioxus counterpart to the telencephalon, and generally most of the dorsal centers, receiving and integrating input from the major paired sense organs, are either absent or present as no more than small rudiments in amphioxus. The PNS of amphioxus is diffuse and organized along quite different lines than that of vertebrates, presumably in part a consequence of the absence of a neural crest cells. Much remains to be learned about neuronal cell types, tract organization, and circuitry in amphioxus, all of which will contribute to a better understanding of CNS organization and evolution in vertebrates.
We thank Nick and Linda Holland for helping to obtain Branchiostoma floridae adults and embryos, Hector Escrivà and Stéphanie Bertrand for providing access to amphioxus embryos of Branchiostoma lanceolatum, and Thurston Lacalli for critical reading and helpful comments on the manuscript.
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