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After spinal cord injury (SCI) in mammals, the loss of serotonin coming from the brainstem reduces the excitability of motor neurons and leads to a compensatory overexpression of serotonin receptors. Despite the key role of the serotonin receptor 1a in the control of locomotion, little attention has been put in the study of this receptor after SCI. In contrast to mammals, lampreys recover locomotion after a complete SCI, so, studies in this specie could help to understand events that lead to recovery of function. Here, we showed that in lampreys there is an acute increase in the expression of the serotonin 1A receptor transcript (5-ht1a) after SCI and a few weeks later expression levels go back to normal rostrally and caudally to the lesion. Overexpression of the 5-ht1a in rostral levels after SCI has not been reported in mammals, suggesting that this could be part of the plastic events that lead to the recovery of function in lampreys. The analysis of changes in 5-ht1a expression by zones (periventricular region and horizontally extended grey matter) showed that they followed the same pattern of changes detected in the spinal cord as a whole, with the exception of the caudal periventricular layer, where no significant differences were observed between control and experimental animals at any time post lesion. This suggests that different molecular signals act on the periventricular cells of the rostral and caudal regions to injury site and thus affecting their response to the injury in terms of expression of the 5-ht1a.

Spinal muscular atrophy (SMA) is characterized by the selective loss of spinal motor neurons owing to reduced levels of survival motor neuron (Smn) protein. In addition to its well-established role in assembling constituents of the spliceosome, diverse cellular functions have been proposed for Smn, but the reason why low levels of this widely expressed protein result in selective motor neuron pathology is still debated. In longitudinal studies of exon-level changes in SMA mouse model tissues, designed to determine the contribution of splicing dysfunction to the disease, we have previously shown that a generalized defect in splicing is unlikely to play a causative role in SMA. Nevertheless, we identified a small subset of genes that were alternatively spliced in the spinal cord compared with control mice before symptom onset, indicating a possible mechanistic role in disease. Here, we have performed functional studies of one of these genes, chondrolectin (Chodl), known to be highly expressed in motor neurons and important for correct motor axon outgrowth in zebrafish. Using in vitro and in vivo models of SMA, we demonstrate altered expression of Chodl in SMA mouse spinal motor neurons, show that Chodl has distinct effects on cell survival and neurite outgrowth and that increasing the expression of chodl can rescue motor neuron outgrowth defects in Smn-depleted zebrafish. Our findings thus link the dysregulation of Chodl to the pathophysiology of motor neuron degeneration in SMA.

Spinal cord injury (SCI) causes permanent functional deficits in mammals, and therefore it constitutes a public health and economic problem. Changes in the different spinal neurotransmitters systems have been observed after the injury. In this chapter, we describe a very useful method to quantify the changes in the number of fibers in the spinal neurotransmitters systems. This method combines confocal microscopy together with the use of appropriate free computer software. Standardizing this type of studies will be very useful to compare results between studies. This method can also be used with bright-field microscopy and be adapted to other experimental conditions and animal models. © 2014 Springer Science+Business Media New York.

Spinal cord injuries are an important sanitary and economical problem for the society. In mammals, including humans, a traumatic injury to the spinal cord leads to a loss of motor and sensorial function, which is irreversible due to the low regenerative ability of the central nervous system. In contrast to mammals, functional recovery occurs spontaneously after a complete spinal cord transection in lampreys. Functional recovery occurs because in these animals about 50% of the reticulospinal axons regenerate after injury and also because of the occurrence of processes of reorganization and plasticity of the spinal circuits. In this review, we first analyze the characteristics and regeneration ability of lampreys as compared to mammals. Then, we compile the knowledge about the process of recovery after a spinal cord injury acquired in studies using the lampreys as animal model and finally we provide some general perspectives about the molecular processes implicated in regeneration that can be investigated in a very advantageous way in this animal model and which knowledge could allow to develop new therapies for patients suffering spinal cord injury.

Glutamate is the major excitatory neurotransmitter in vertebrates, and glutamatergic cells probably represent a majority of neurons in the brain. Physiological studies have demonstrated a wide presence of excitatory (glutamatergic) neurons in lampreys. The present in situ hybridization study with probes for the lamprey vesicular glutamate transporter (VGLUT) provides an anatomical basis for the general distribution and precise localization of glutamatergic neurons in the sea lamprey brainstem. Most glutamatergic neurons were found within the periventricular gray layer throughout the brainstem, with the following regions being of particular interest: the optic tectum, torus semicircularis, isthmus, dorsal and medial nuclei of the octavolateral area, dorsal column nucleus, solitary tract nucleus, motoneurons, and reticular formation. The reticular population revealed a high degree of cellular heterogeneity including small, medium-sized, large, and giant glutamatergic neurons. We also combined glutamate immunohistochemistry with neuronal tract-tracing methods or γ-aminobutyric acid (GABA) immunohistochemistry to better characterize the glutamatergic populations. Injection of Neurobiotin into the spinal cord revealed that retrogradely labeled small and medium-sized cells of some reticulospinal-projecting groups were often glutamate-immunoreactive, mostly in the hindbrain. In contrast, the large and giant glutamatergic reticulospinal perikarya mostly lacked glutamate immunoreactivity. These results indicate that glutamate immunoreactivity did not reveal the entire set of glutamatergic populations. Some spinal-projecting octaval populations lacked both VGLUT and glutamate. As regards GABA and glutamate, their distribution was largely complementary, but colocalization of glutamate and GABA was observed in some small neurons, suggesting that glutamate immunohistochemistry might also detect non-glutamatergic cells or neurons that co-release both GABA and glutamate., Grant sponsor: Spanish Ministry of Science and Innovation; Grant numbers: BFU2007-61056 and BFU2010-17174 (to M.C.R.); Grant sponsor: Xunta de Galicia; Grant number: INCITE09ENA200036ES (to R.A.).

UNC5 receptors mediate repulsive signaling of netrin on neurons. Although only one UNC5 receptor has been identified in invertebrates, four members of the UNC5 family have been identified in gnathostomes. Lampreys, together with mixynes, belong to the oldest branch of extant vertebrates, and their phylogenetic position near to the vertebrate root makes them an interesting model for understanding molecular evolution. Here, we cloned three sea lamprey UNC5 (UNC5L) receptors, and phylogenetic analyses indicated that the first two duplications of the ancestral UNC5 gene occurred before the separation of jawless and jawed vertebrates. UNC5 receptors play important roles during early development, but expression studies have also suggested that UNC5 receptors play roles in the mature nervous system. Here, we report the expression of the different UNC5L receptor transcripts in identified reticulospinal neurons of mature larval or adult sea lampreys detected by in situ hybridization in wholemounted brain preparations. In addition, an extensive expression of the UNC5 receptors was also observed in most brain regions of the adult lamprey. An increase in the types of identifiable reticulospinal neurons expressing the UNC5L receptors was observed in adults compared with larvae. Expression of UNC5 receptors at late developmental stages appears to be a shared characteristic of lampreys and mammals. In larvae, expression of UNC5L receptors was observed in reticulospinal neurons that when axotomized are known to be “bad regenerators.” Results in lampreys and mammals suggest that the UNC5-Netrin axonal guidance system may play a role in limiting axonal regeneration after spinal cord injury. J. Comp. Neurol. 520:4141–4156, 2012. © 2012 Wiley Periodicals, Inc.

Apoptosis is a major feature in neural development and important in traumatic diseases. The presence of active caspases is a widely accepted marker of apoptosis. We report here the development of a method to study neuronal apoptotic death in whole-mounted brain preparations using fluorochrome-labeled inhibitors of caspases (FLICA). As a model we used axotomy-induced retrograde neuronal death in the CNS of larval sea lampreys. Once inside the cell, the FLICA reagents bind covalently to active caspases causing apoptotic cells to fluoresce, whereas nonapoptotic cells remain unstained. The fluorescent probe, the poly caspase inhibitor FAM-VAD-FMK, was applied to whole-mounted brain preparations of larval sea lampreys 2 weeks after a complete spinal cord (SC) transection. Specific labeling occurred only in identifiable spinal-projecting neurons of the brainstem previously shown to undergo apoptotic neuronal death at later times after SC transection. These neurons also exhibited intense labeling 2 weeks after a complete SC transection when a specific caspase-8 inhibitor (FAM-LETD-FMK) served as the probe. In this study we show that FLICA reagents can be used to detect specific activated caspases in identified neurons of the whole-mounted lamprey brain. Our results suggest that axotomy may cause neuronal apoptosis by activation of the extrinsic apoptotic pathway.

their serotonergic system aft er injury in the central nervous system (CNS), I would like to introduce the concept of ‘evorego’. In this case, ‘evorego’ refers to the idea of how we could also try to understand evolution by studying the diff erences and similarities in the regenerative ability between extant species. In mammals, including humans, injury to the adult CNS usually causes an irreversible loss of function because of the inability of the mature CNS to regenerate. One of the main causes for the absence of recovery is the inability of injured axons to regenerate and reconnect with their appropriate targets. By contrast, teleost fi shes and lampreys are able to regain functionality aft er injury in the mature CNS. For example, both zebrafi sh and lampreys are able to recover locomotion that appears normal aft er a complete spinal cord transection [Becker and Becker, 2008; Barreiro-Iglesias and Rodicio, 2011]. Recent studies have shown that serotonergic neurons have an impressive regenerative ability aft er spinal cord injury in both zebrafi sh [Kuscha et al., 2011] and lampreys [Cornide-Petronio et al., 2011]. Using a double labeling tract-tracing tech‘Evodevo’ is an acronym from the words ‘evolution and development’ that is used to name the fi eld of life sciences focused on the study of diff erences and similarities in the developmental process of extant species with the aim of understanding how they have evolved from common ancestors. Th e ‘evodevo’ research fi eld has expanded greatly over the last century thanks to an increase in the number of research groups focusing their eff orts in this discipline and the appearance of science journals specifi cally devoted to publishing new advances in the fi eld (e.g. Evolution and Development ) or journals from a variety of disciplines that publish a considerable percentage of studies that use the ‘evodevo’ approach (e.g. Brain, Behavior and Evolution ). ‘Evodevo’ researchers use a comparative approach to study a specifi c developmental process in diff erent but related species to understand the evolutionary process and try to identify primitive and derived characters, homologies, parallelisms or phenomena of convergent evolution. Here, using as an example recent data [CornidePetronio et al., 2011; Hawthorne et al., 2011; Kuscha et al., 2011] on the ability of diff erent vertebrate species to regenerate Published online: December 6, 2011

Recent research has shown that at least two tryptophan hydroxylase (Tph) genes are present in gnathostome vertebrates, but it is not known when the duplication of the ancestral Tph gene took place during evolution. By their position as an out-group of gnathostomes, lampreys (agnathans) are key models to understand molecular evolution in vertebrates. Here, we report the cloning of a Tph cDNA of the sea lamprey and the pattern of Tph mRNA expression in larval and postmetamorphic (young adult) sea lampreys using in situ hybridization. Phylogenetic analysis indicated that the lamprey Tph is an orthologue of Tphs of other vertebrates and suggested that the duplication of the ancestral Tph gene occurred before the separation of agnathans and gnathostomes, although alternative hypothesis are also discussed in the present study. In the sea lamprey brain, the Tph transcript was expressed in perikarya of the pineal organ, the retina, the diencephalic and rhombencephalic nuclei reported previously with serotonin immunohistochemistry and in small cells of the spinal cord, with a pattern similar to that observed with anti-serotonin antibodies. This suggests that expression of this Tph gene is shared by all lamprey serotonergic brain populations, unlike that reported in zebrafish and mammals for their different Tph genes. However, no Tph expression was observed in peripheral serotonergic cells, which, unlike in other vertebrates, are widely distributed in lampreys. Our results suggest that the selection of Tph2 to be expressed in raphe neurons may have occurred along the line leading to gnathostomes.

The sea lamprey has a complex life cycle with very different larval and adult stages. The eyes of larvae are subcutaneous, lack a differentiated lens and probably work only as an ocellus-like photoreceptor organ, while the well-developed adult eyes are capable of forming images. The larval retina differs greatly from the adult retina and presents a central region with differentiated photoreceptors and a lateral, largely undifferentiated part that grows in the second half of larval life. In the present study, we examined the retinotopy of projections from larval ganglion cells to the optic tectum and pretectum in sea lamprey by using retrograde tract-tracing techniques. In most regions of the tectum, application of the tracer neurobiotin (NB) resulted in labelled ganglion cells in the lateral retina, mostly in the contralateral eye. Ganglion cells of the lateral retina showed a very simple dendritic tree, possibly because of the lack of differentiation of most retinal layers in this region. The retinotectal projection is already retinotopically organized in larvae and follows a pattern similar to that observed in adult lampreys and other vertebrates. Application of NB to the central region of the tectum also led to labelling of a few ganglion cells in the central retina, which were clearly more complex than those in the lateral region, as they had dendrites that branched both in the outer and inner plexiform layers. Application of NB to the medial pretectum led to labelling of ganglion cells in the contralateral central retina. Occasional cells were also labelled in the lateral retina. The differential organization of larval retinal projections to the pretectum and tectum suggests a different role for these projections, which is consistent with the different involvement of these centres in visual behaviour, as determined in adult lampreys. The observations in larvae also reveal very different developmental timetables for these putative functions.

Spinal cord injury (SCI) is a debilitating and devastating condition, and there are approximately 12,000 new cases in the USA each year and an estimated number of sufferers reaching 250,000–300,000 in the USA alone. Although important advances have been made in the medical treatment of SCI

The present is a review of the gustatory system of lampreys, which are representative of the earliest vertebrates. They are the oldest extant vertebrates that possess taste buds. Because of the phylogenetic position of lampreys, the study of their gustatory system will provide important information to help understand the early evolution of this system in vertebrates. The taste buds of larval lampreys, which are papillae located on the first six pairs of gill arches facing the water current, respond to classical taste substances. They consist of two types of tall differentiated cells, serotonergic biciliated taste receptors (‘light’ cells) and microvillous sustentacular cells (‘dark cells’). The taste buds also contain basal proliferative cells. Afferent gustatory fibers of the glossopharyngeal and vagal nerves innervate the taste buds of lampreys and contact the basal surface of the biciliated cells without entering the bud. Central processes of the glossopharyngeal and vagal cranial nerves terminate in a caudal rhombencephalic region that may correspond to the nucleus of the solitary tract of gnathostomes. To date, most studies in lampreys have focused on characterizing taste buds; future research should focus on the central processing of the gustatory information. Here we will review the current knowledge about the gustatory system of lampreys to provide a basis for establishing the direction of further studies of this chemosensory system.

Lampreys belong to the oldest group of extant vertebrates, the agnathans or cyclostomes. Thus, they occupy a key phylogenetic position near the root of the vertebrate tree, which makes them important to the study of nervous system evolution. Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis and is considered a marker of catecholaminergic neurons. In the present study, we report partial cloning of the sea lamprey tyrosine hydroxylase (TH) cDNA and the pattern of TH transcript expression in the adult brain by means of in situ hybridization. Sea lamprey TH mRNA is characterized by the presence of a large untranslated sequence in the 3' end that contains a typical polyadenylation signal (ATTAAA). The deduced partial TH protein sequence presents a conserved domain with two His residues coordinating Fe(2+) binding and a conserved cofactor binding site. Neurons expressing the TH transcript were observed in the preoptic, postoptic commissure, dorsal hypothalamic, ventral hypothalamic, mammillary and paratubercular nuclei of the prosencephalon. In situ hybridization experiments also confirmed the existence of a catecholaminergic (dopaminergic) striatal population in the brain of the adult sea lamprey. A few granule-like cells in the olfactory bulbs also showed weak TH transcript expression. No cells showing TH transcript expression were observed in the rostral rhombencephalon, which suggests the absence of a locus coeruleus in the sea lamprey. Comparison of the pattern of TH mRNA expression in the prosencephalon between lampreys and teleost fishes revealed both similarities and differences. Our results suggest that the duplication of the TH gene might have occurred before the separation of agnathans and gnathostomes.

Lampreys are useful models for studying the evolution of the nervous system of vertebrates. Here we used immunofluorescence and tract-tracing methods to study new aspects of the neuropeptide Y-immunoreactive (NPY-ir) system in larval sea lampreys. NPY-ir neurons were observed in brain nuclei that contain NPY-ir cells in other lamprey species. Moreover, a group of NPY-ir cells that migrated away the periventricular layer was observed in the lateral part of the dorsal hypothalamus, which suggests a role for NPY in feeding behavior in lampreys. We also report NPY-ir cells in the dorsal column nucleus, which appears to be unique among vertebrates, and in the habenula. A combination of tract-tracing and immunohistochemical labeling demonstrated the presence of spinal projecting NPY-ir reticular cells in the anterior rhombencephalic reticular formation, and the relationships between the NPY-ir system and the reticulospinal nuclei and some afferent systems. The colocalization of catecholamines and GABA in lamprey NPY-ir neurons was investigated by double immunofluorescence methods. Colocalization of tyrosine hydroxylase (TH) and NPY immunoreactivities was not observed in any brain neuron, although reported in amphibians and mammals. The frequent presence of NPY-ir terminals on TH-ir cells suggests that NPY modulates the activity of some dopaminergic nuclei in lampreys. Colocalization of NPY and GABA immunoreactivities was frequently observed in neurons of different rhombencephalic and diencephalic NPY-ir populations. These results in lampreys suggest that the coexpression of NPY and GABA in neurons appeared early on in the brains of vertebrates.

Since its discovery, the possible corelease of classic neurotransmitters from neurons has received much attention. Colocalization of monoamines and amino acidergic neurotransmitters [mainly glutamate and dopamine (DA) or serotonin] in mammalian neurons has been reported. However, few studies have dealt with the colocalization of DA and gamma-aminobutyric acid (GABA) in neurons. With the aim of providing some insight into the colocalization of neurotransmitters during early vertebrate phylogeny, we studied GABA expression in dopaminergic neurons in the sea lamprey brain by using double-immunofluorescence methods with anti-DA and anti-GABA antibodies. Different degrees of colocalization of DA and GABA were observed in different dopaminergic brain nuclei. A high degree of colocalization (GABA in at least 25% of DA-immunoreactive neurons) was observed in populations of the caudal rhombencephalon, ventral isthmus, postoptic commissure nucleus, preoptic nucleus and in granule-like cells of the olfactory bulb. A new DA-immunoreactive striatal population that showed colocalization with GABA in about a quarter of its neurons was observed. In the periventricular hypothalamus, colocalization was observed in only a few cells, despite the abundance of DA- and GABA-immunoreactive neurons, and no double-labelled cells were observed in the paratubercular nucleus. The frequent colocalization of DA and GABA reveals that the dopaminergic populations of lampreys are more complex than previously reported. Double-labelled fibres or terminals were observed in different brain regions, suggesting possible corelease of DA and GABA by these lamprey neurons. The present results suggest that colocalization of DA and GABA in neurons appeared early in vertebrate evolution.

Lampreys are vertebrate animal models in spinal cord regeneration studies. In order to gain knowledge on the mechanisms that provide to the lamprey spinal cord its capacity of regeneration we decided to compare the expression patterns of the growth-associated protein 43 (GAP-43) in the CNS of the sea lamprey before and after a complete spinal transection by immunocytochemical methods using an anti-GAP-43 antibody. Surprisingly, in the brain/spinal cord of both normal and injured animals, anti-GAP-43-like labeling was only observed in the subcommissural organ (SCO) and Reissner's fibre (RF). In injured larvae, a dotted labeling was also observed in the meninges and in the blood the vessels of the neighbouring tissues at the site of lesion. The experiments in injured animals showed that after complete spinal cord transection the SCO seems to continue to produce the Reissner's substance (RS), which is accumulated at the proximal site of spinal transection. The dotted labeling observed in the neighbouring tissues could correspond to RS that was released from the site of injury. In Western blot experiments done using protein extracts of the lamprey brain, the anti-GAP-43 antibody did not recognize any protein band of the expected GAP-43 molecular weight, indicating that the secreted material is not this protein. An anti-serotonin antibody was also used as a marker of some brain structures. Serotonergic afferent fibres innervated the SCO. Here we show a new tool that can be used as a highly specific marker in further studies of the SCO/RF system of lampreys.

The development of glycine immunoreactivity in the brain of the sea lamprey was studied by use of immunofluorescence techniques at embryonic to larval stages. Glycine distribution was also compared with that of gamma-aminobutyric acid (GABA) by use of double immunofluorescence. The first glycine-immunoreactive (ir) cells appeared in the caudal rhombencephalon of late embryos, diencephalon of early prolarvae, and mesencephalon of late prolarvae, in which glycine-ir cells were observed in several prosencephalic regions (preoptic nucleus, hypothalamus, ventral thalamus, dorsal thalamus, pretectum, and nucleus of the medial longitudinal fascicle), mesencephalon (M5), isthmus, and rhombencephalon. In larvae, glycine-ir populations were observed in the olfactory bulbs, preoptic nucleus and thalamus (prosencephalon), M5 and oculomotor nucleus (mesencephalon), dorsal isthmic gray, isthmic reticular formation, and various alar and basal plate rhombencephalic populations. No glycine-ir cells were observed in the larval optic tectum or torus semicircularis, which contain glycine-ir populations in adults. A wide distribution of glycine-ir fibers was observed, which suggests involvement of glycine in the function of most lamprey brain regions. Colocalization of GABA and glycine in prolarvae was found mainly in cell groups of the diencephalon, in the ventral isthmic group, and in trigeminal populations. In larvae, colocalization of GABA and glycine was principally observed in the M5 nucleus, the reticular formation, and the dorsal column nucleus. The present results reveal for the first time the complex developmental pattern of the glycinergic system in lamprey, including early glycine-ir populations, populations transiently expressing glycine, and late-appearing populations, in relation to maturation changes that occur during metamorphosis.

We used Neurobiotin as a retrograde tract tracer in both larval and adult sea lampreys and observed a number of neuronal brainstem populations (mainly reticular and octaval populations and some diencephalic nuclei) that project to the spinal cord, in agreement with the results of previous tracer studies. We also observed small labeled neurons in the ventral hypothalamus, the mammillary region, and the paratubercular nucleus, nuclei that were not reported as spinal projecting. Notably, most of the labeled cells of the mammillary region and some of the ventral hypothalamus were cerebrospinal fluid-contacting (CSF-c) neurons. Combined tract tracing and immunocytochemistry showed that some of the labeled neurons of the mammillary and paratubercular nuclei were dopamine immunoreactive. In addition, some CSF-c cells were labeled in the caudal rhombencephalon and rostral spinal cord, and many were also dopamine immunoreactive. Results with other tracers (biotinylated dextran amines, horseradish peroxidase, and the carbocyanine dye DiI) also demonstrated that the molecular weight or the molecular nature of the tracer was determinant in revealing diencephalic cells with very thin axons. The results show that descending systems afferent to the spinal cord in lampreys are more varied than previously reported, and reveal a descending projection from CSF-c cells, which is unknown in vertebrates. The present results also reveal the existence of large differences between agnathans and gnathostomes in the organization of the dopaminergic cells that project to the spinal cord. J. Comp. Neurol. 511:711–723, 2008. © 2008 Wiley-Liss, Inc.

Neuroactive substances such as serotonin and other monoamines have been suggested to be involved in the transmission of gustatory signals from taste bud cells to afferent fibers. Lampreys are the earliest vertebrates that possess taste buds, although these differ in structure from taste buds in jawed vertebrates, and their neurochemistry remains unknown. We used immunofluorescence methods with antibodies raised against serotonin, tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), glutamate, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), calretinin, and acetylated alpha-tubulin to characterize the neurochemistry and innervation of taste buds in the sea lamprey, Petromyzon marinus L. For localization of proliferative cells in taste buds we used bromodeoxyuridine labeling and proliferating cell nuclear antigen immunohistochemistry. Results with both markers indicate that proliferating cells are restricted to a few basal cells and that almost all cells in taste buds are nonproliferating. A large number of serotonin-, calretinin-, and CGRP-immunoreactive bi-ciliated cells were revealed in lamprey taste buds. This suggests that serotonin participates in the transmission of gustatory signals and indicates that this substance appeared early on in vertebrate evolution. The basal surface of the bi-ciliated taste bud cells was contacted by tubulin-immunoreactive fibers. Some of the fibers surrounding the taste bud were calretinin immunoreactive. Lamprey taste bud cells or afferent fibers did not exhibit TH, GABA, glutamate, or NPY immunoreactivity, which suggests that expression of these substances evolved in taste buds of some gnathostomes lines after the separation of gnathostomes and lampreys.

The distribution of glycinergic cells in the brain of nonmammalian vertebrates is still unknown. Lampreys are the most primitive extant vertebrates, and they may provide important data on the phylogeny of this system. Here, we studied for the first time the distribution of glycine immunoreactivity in the sea lamprey brain and compared it with γ-aminobutyric acid (GABA)-ergic populations. Most glycine-immunoreactive neurons were found at midbrain and hindbrain levels, and most of these cells did not exhibit GABA immunoreactivity. We describe glycine-immunoreactive cell populations in the olfactory bulbs, the preoptic nucleus, and the thalamus of the sea lamprey, which is in striking contrast to their lack in the mammalian forebrain. We also observed glycine-immunoreactive populations in the optic tectum, the torus semicircularis and the midbrain tegmentum, the isthmus, the octavolateral area, the dorsal column nucleus, the abducens nucleus, the trigeminal motor nucleus, the facial motor nucleus, and the rhombencephalic reticular formation. In these populations, colocalization with GABA was observed in only some cells of the tegmental M5 nucleus, ventral isthmus, medial octavolateral nucleus, dorsal column nucleus, and lateral reticular region. The present results allow us to conclude that the distribution of glycine-immunoreactive cells changed notably from lamprey to mammals, with a decrease in glycinergic populations in the forebrain and a specialization of brainstem cell groups. Although knowledge of the glycinergic populations in lampreys is important for understanding the early evolution of this system, there is a notable gap of information regarding its organization in brains of other nonmammalian vertebrates. J. Comp. Neurol. 507:1441–1463, 2008. © 2008 Wiley-Liss, Inc.

Lamprey eyes exhibit dual retinal development, with highly different larval and adult phases. Here, cell proliferation and photoreceptor differentiation was investigated in late larvae and during transformation (occurring several years after egg hatching) by using immunohistochemistry against the proliferating cell nuclear antigen (PCNA) and opsins. In large larvae proliferating cells are mainly located in the lateral retina, a wide undifferentiated region, whereas opsin immunoreactivity revealed only a single type of photoreceptors in the very small central retina. In premetamorphic larvae, retinal cell proliferation increases considerably, but at metamorphosis it becomes progressively restricted to the periphery of the lateral retina. Proliferating (PCNA-immunoreactive) cells were mainly observed in the inner nuclear layer but also in the outer plexiform layer and outer nuclear layer, suggesting that the latter proliferating cells migrate to the outer nuclear layer and differentiate into photoreceptors. In the lateral retina, first photoreceptors expressing opsins were observed at middle metamorphic stages, and outer and inner segments were present at latter stages. Some immature photoreceptors were also observed in postmetamorphic retina. Unlike teleost and amphibian retinas, no proliferating cells were observed in the retina after metamorphosis, indicating that the retinal growth after this period is due to cellular reorganization and increase in cell size.

The development of the early axonal scaffold formed by early-differentiating neurons was studied in a primitive vertebrate (the sea lamprey), by immunohistochemistry against acetylated alpha-tubulin and a cell surface marker (HNK-1 antibodies), to determine the degree of conservation of this process in vertebrate evolution. The medial and dorsolateral longitudinal fascicles were the first longitudinal axonal bundles observed to develop in the neural tube, followed by the tract of the postoptic commissure and the supraoptic tract. Establishment of the first dorso-ventral tracts occurs after the appearance of the tract of the postoptic commissure and the medial longitudinal fascicle, the basal plate longitudinal axonal system. The dorsolateral longitudinal fascicle appears to be equivalent to the "descending tract" of the mesencephalic nucleus of the trigeminal nerve of mouse and birds; the possible homologies between other early scaffold tracts of the sea lamprey and those of other vertebrates are also discussed. In addition, present results suggest the presence of highly conserved brain regions that would allow for early neuronal differentiation and axonal pathfinding in vertebrates, which were probably defined before the divergence of Agnathans and Gnathostomes.

Spinal cord injuries are an important sanitary and economical problem for the society. In mammals, including humans, a traumatic injury to the spinal cord leads to a loss of motor and sensorial function, which is irreversible due to the low regenerative ability of the central nervous system. In contrast to mammals, functional recovery occurs spontaneously after a complete spinal cord transection in lampreys. Functional recovery occurs because in these animals about 50% of the reticulospinal axons regenerate after injury and also because of the occurrence of processes of reorganization and plasticity of the spinal circuits. In this review, we first analyze the characteristics and regeneration ability of lampreys as compared to mammals. Then, we compile the knowledge about the process of recovery after a spinal cord injury acquired in studies using the lampreys as animal model and finally we provide some general perspectives about the molecular processes implicated in regeneration that can be investigated in a very advantageous way in this animal model and which knowledge could allow to develop new therapies for patients suffering spinal cord injury.