Lamprey Brain Research Articles

A lamprey neural cell type atlas illuminates the origins of the vertebrate brain

The vertebrate brain emerged more than ~500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey—a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits—based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of cell types, tissue structures and gene expression programs of the ancestral vertebrate brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral brain was probably devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors in the jawed vertebrate lineage. Altogether, our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.

No strict host specificity: Brain metacercariae Diplostomum petromyzifluviatilis Müller (Diesing, 1850) are conspecific with Diplostomum sp. Lineage 4 of Blasco-Costa et al. (2014).

Metacercariae of Diplostomum petromyzifluviatilis (Digenea, Diplostomidae) from the brain of European river lamprey Lampetra fluviatilis from the Baltic Sea basin and Arctic lamprey Lethenteron camtschaticum from the White Sea basin were studied with the use of genetic and morphological methods. Phylogenetic analysis based on cox1 marker showed that the parasites of both lamprey species were conspecific with Diplostomum sp. Lineage 4 of Blasco-Costa et al. (2014). The name Diplostomum petromyzifluviatilis Müller (Diesing, 1850) has historical precedence as a species described from the brain of lampreys and should be used in genus nomenclature. There were no morphological qualitative differences between the metacercariae from the two lamprey species but those from L. fluviatilis were larger than those from L. camtschaticum. We expanded the data on the second intermediate hosts and the localization of D. petromyzifluviatilis, showing that its metacercariae occur not only in the brain of lampreys but also in the brain and the retina of three-spined stickleback Gasterosteus aculeatus and the vitreous humour of the perch Perca fluviatilis across the European part of the Palearctic.

Neuromeric Distribution of Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase Activity in the Adult Lamprey Brain.

This study reports for the first time the distribution and morphological characterization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d; a reliable marker of nitric oxide synthase activity) positive elements in the central nervous system of the adult river lamprey (Lampetra fluviatilis) on the framework of the neuromeric model and compares their cytoarchitectonic organization with that of gnathostomes. Both NADPH-d exhibiting cells and fibers were observed in all major divisions of the lamprey brain as well as in the spinal cord. In the secondary prosencephalon, NADPH-d positive cells were observed in the mitral cell layer of the olfactory bulb, evaginated pallium, amygdala, dorsal striatum, septum, lateral preoptic nucleus, caudal paraventricular area, posterior entopeduncular nucleus, nucleus of the stria medullaris, hypothalamic periventricular organ and mamillary region sensu lato. In the lamprey diencephalon, NADPH-d labeled cells were observed in several nuclei of the prethalamus, epithalamus, pretectum, and the basal plate. Especially remarkable was the staining observed in the right habenula and several pretectal nuclei. NADPH-d positive cells were also observed in the following mesencephalic areas: optic tectum (two populations), torus semicircularis, nucleus M5 of Schöber, and a ventral tegmental periventricular nucleus. Five different cell populations were observed in the isthmic region, whereas the large sensory dorsal cells, some cells located in the interpeduncular nucleus, the motor nuclei of most cranial nerves, the solitary tract nucleus, some cells of the reticular nuclei, and small cerebrospinal fluid-contacting (CSF-c) cells were the most evident stained cells of the rhombencephalon proper. Finally, several NADPH-d positive cells were observed in the rostral part of the spinal cord, including the large sensory dorsal cells, numerous CSF-c cells, and some dorsal and lateral interneurons. NADPH-d positive fibers were observed in the olfactory pathways (primary olfactory fibers and stria medullaris), the fasciculus retroflexus, and the dorsal column tract. Our results on the distribution of NADPH-d positive elements in the brain of the adult lamprey L. fluviatilis are significantly different from those previously reported in larval lampreys and demonstrated that these animals possess a complex nitrergic system readily comparable to those of other vertebrates, although important specific differences also exist.

Inhibition of central axon regeneration: perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury.

Inhibition of central axon regeneration: perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury.

The neural bases of vertebrate motor behaviour through the lens of evolution.

The primary driver of the evolution of the vertebrate nervous system has been the necessity to move, along with the requirement of controlling the plethora of motor behavioural repertoires seen among the vast and diverse vertebrate species. Understanding the neural basis of motor control through the perspective of evolution, mandates thorough examinations of the nervous systems of species in critical phylogenetic positions. We present here, a broad review of studies on the neural motor infrastructure of the lamprey, a basal and ancient vertebrate, which enjoys a unique phylogenetic position as being an extant representative of the earliest group of vertebrates. From the central pattern generators in the spinal cord to the microcircuits of the pallial cortex, work on the lamprey brain over the years, has provided detailed insights into the basic organization (a bauplan) of the ancestral vertebrate brain, and narrates a compelling account of common ancestry of fundamental aspects of the neural bases for motion control, maintained through half a billion years of vertebrate evolution.This article is part of the theme issue ‘Systems neuroscience through the lens of evolutionary theory’.

Expression of Kisspeptin 1 in the Brain of the Adult Sea Lamprey Petromyzon marinus.

Kisspeptin peptides play major roles in the regulation of reproduction and puberty onset in mammals. While most mammals only have one kisspeptin gene, other jawed vertebrates present two or three genes. Recent data also revealed the presence of two genes in lampreys (jawless vertebrates). However, apart from gene sequence data, there is almost no information on the kisspeptinergic system of lampreys. Here, we report phylogenetic and cluster-based analyses showing that the duplication of the ancestral kisspeptin gene occurred before the separation of jawless and jawed vertebrates. We also studied the expression of the kisspeptin transcripts in the brain of post-metamorphic juveniles and upstream migrating adult sea lampreys. Our in situ hybridization results revealed expression of kisspeptin 1 in hypothalamic neurons, which indicates that the hypothalamic expression of kisspeptins is an ancestral character in vertebrates. We also observed the presence of kisspeptin 1 expressing neurons in the paratubercular (posterior tubercle) nucleus of the diencephalon. This is the first description of the presence of kisspeptin 1 expressing neurons in this brain region in any vertebrate. We did not detect expression of kisspeptin 2 in the juvenile or adult sea lamprey brain with in situ hybridization. Our data provides an anatomical basis to study the role of kisspeptin 1 in the hypothalamic-pituitary system of lampreys and the contribution of diencephalic kisspeptinergic neurons to different circuits of the lamprey brain.

Expression of Urocortin 3 mRNA in the Central Nervous System of the Sea Lamprey Petromyzon marinus.

Simple SummaryNeuropeptides are small proteins produced and released by neurons. Neuropeptides are key mediators in the communication between neurons. Urocortin 3 is an important neuropeptide that is involved in stress-related behaviors. In this study, our aim was to determine in which regions of the lamprey brain urocortin 3 is expressed. The study of the distribution of neuroregulatory peptides in lamprey species helps in understanding their early evolution of neuropeptidergic systems in vertebrates because lampreys are one of the most ancient vertebrate groups. The sea lamprey life cycle lasts between five and nine years, and it includes a long larval stage and a transformation to the adult stage. We analyzed the organization of urocortin 3-expressing neuronal populations in the brain of larval and adult sea lampreys to establish whether this neuropeptide is expressed differentially in these two widely different phases of the sea lamprey life cycle. Our results revealed important differences between larval and adult stages. Moreover, comparing our results with the distribution of urocortin 3 reported in other vertebrate groups also revealed a poor evolutionary conservation of urocortin 3 expression in vertebrates. Our work sheds light on the evolution and role of urocortin 3 in vertebrates.In this study, we analyzed the organization of urocortin 3 (Ucn3)-expressing neuronal populations in the brain of the adult sea lamprey by means of in situ hybridization. We also studied the brain of larval sea lampreys to establish whether this prosocial neuropeptide is expressed differentially in two widely different phases of the sea lamprey life cycle. In adult sea lampreys, Ucn3 transcript expression was observed in neurons of the striatum, prethalamus, nucleus of the medial longitudinal fascicle, torus semicircularis, isthmic reticular formation, interpeduncular nucleus, posterior rhombencephalic reticular formation and nucleus of the solitary tract. Interestingly, in larval sea lampreys, only three regions showed Ucn3 expression, namely the prethalamus, the nucleus of the medial longitudinal fascicle and the posterior rhombencephalic reticular formation. A comparison with distributions of Ucn3 in other vertebrates revealed poor conservation of Ucn3 expression during vertebrate evolution. The large qualitative differences in Ucn3 expression observed between larval and adult phases suggest that the maturation of neuroregulatory circuits in the striatum, torus semicircularis and hindbrain chemosensory systems is closely related to profound life-style changes occurring after the transformation from larval to adult life.

Genetic and Functional Characterization of Novel Brown-Like Adipocytes Around the Lamprey Brain

During the process of vertebrate evolution, many thermogenic organs and mechanisms have appeared. Mammalian brown adipose tissue (BAT) generates heat through the uncoupling oxidative phosphorylation of mitochondria, acts as a natural defense against hypothermia and inhibits the development of obesity. Although the existence, cellular origin and molecular identity of BAT in humans have been well studied, the genetic and functional characteristics of BAT from lampreys remain unknown. Here, we identified and characterized a novel, naturally existing brown-like adipocytes at the lamprey brain periphery. Similar to human BAT, the lamprey brain periphery contains brown-like adipocytes that maintain the same morphology as human brown adipocytes, containing multilocular lipid droplets and high mitochondrion numbers. Furthermore, we found that brown-like adipocytes in the periphery of lamprey brains responded to thermogenic reagent treatment and cold exposure and that lamprey UCP2 promoted precursor adipocyte differentiation. Molecular mapping by RNA-sequencing showed that inflammation in brown-like adipocytes treated with LPS and 25HC was enhanced compared to controls. The results of this study provide new evidence for human BAT research and demonstrate the multilocular adipose cell functions of lampreys, including: (1) providing material energy and protecting structure, (2) generating additional heat and contributing to adaptation to low-temperature environments, and (3) resisting external pathogens.

Differential expression of somatostatin genes in the central nervous system of the sea lamprey.

The identification of three somatostatin (SST) genes (SSTa, SSTb, and SSTc) in lampreys (Tostivint et al. Gen Comp Endocrinol 237:89-97 https://doi.org/10.1016/j.ygcen.2016.08.006 , 2016) prompted us to study their expression in the brain and spinal cord of the sea lamprey by in situ hybridization. These three genes were only expressed in equivalent neuronal populations in the hypothalamus. In other regions, SST transcripts showed clear differential expression. In the telencephalon, SSTc-positive cells were observed in the medial pallium, ventral part of the lateral pallium, striatum, subhippocampal lobe, and preoptic region. In the diencephalon, SSTa-positive cells were observed in the thalamus and SSTc-positive cells in the prethalamus, posterior tubercle, pretectal area, and nucleus of the medial longitudinal fascicle. In the midbrain, SSTc-positive cells were observed in the torus semicircularis, lateral reticular area, and perioculomotor tegmentum. Different SSTa- and SSTc-positive populations were observed in the isthmus. SSTc neurons were also observed in the rostral octavolateralis area and caudal rhombencephalon. In the spinal cord, SSTa was expressed in cerebrospinal-fluid-contacting (CSF-c) neurons and SSTc in non-CSF-c interneurons. Comparison with previous immunohistochemical studies using anti-SST-14 antibodies strongly suggests that SST-14-like neurons correspond with the SSTa populations. Thus, the SSTc populations were not reported previously in immunohistochemical studies. Cluster-based analyses and alignments of mature peptides suggested that SSTa is an ortholog of SST1 and that SSTb is closely related to SST2 and SST6. These results provide important new insights into the evolution of the somatostatinergic system in vertebrates.

Chromosome-level genome assembly of Lethenteron reissneri provides insights into lamprey evolution.

The reissner lamprey Lethenteron reissneri, belonging to the class Cyclostomata, serves as a bridge between invertebrates and jawed vertebrates, and is considered the sister group of jawed vertebrates. However, despite this evolutionary significance, the genetic mechanisms underlying the adaptive evolution of the lamprey lineage remain unclear. Here, we assembled a 1.06Gb chromosome-level draft genome of L. reissneri, with 72 chromosomes (ranging in length from 4.5Mb to 25.9Mb) and a scaffold N50 length of 13.23Mb. Genome quality comparisons revealed that the reissner lamprey genome has higher completeness and contiguity than the previously published sea lamprey and Japanese lamprey genomes. Moreover, reissner lamprey, sea lamprey, and Japanese lamprey species share similar transposable element profiles and Hox gene cluster compositions, suggesting that a burst of transposable element activity and whole genome duplication occurred before their divergence. Additionally, the Lip gene copy numbers, which have been studied for their functions in the host defence system, were found to be expanded uniquely in lamprey lineages, suggesting key roles for these genes in lamprey evolution and adaptation. We also identified two neural-related genes, Nrn1 and Unc13a, with copy number expansions in jawed vertebrates, which may be functionally relevant to the origin of lamprey brains. Hence, this study not only provides the first chromosome-level reference genome for Cyclostomata, but also highlights features of the unique biology and adaptive evolution of the lamprey lineage.

Waterborne pheromones modulate gonadotropin-inhibitory hormone levels in sea lamprey (Petromyzon marinus)

The relationships between pheromone stimuli and neuropeptides are not well established in vertebrates due to the limited number of unequivocally identified pheromone molecules. The sea lamprey (Petromyzon marinus) is an advantageous vertebrate model to study the effects of pheromone exposure on neuropeptides since many pheromone molecules and neuropeptides have been identified in this species. Sexually mature male sea lamprey release pheromones 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate, 3kPZS) and 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3-keto allocholic acid, 3kACA) that differentially regulate gonadotropin-releasing hormone (lGnRH) and steroid levels in sexually immature sea lamprey. However, the effects of these pheromones on gonadotropin-inhibitory hormones (GnIHs), hypothalamic neuropeptides that regulate lGnRH release, are still elusive. In this report, we sought to examine the effects of waterborne pheromones on lamprey GnIH-related neuropeptide levels in sexually immature sea lamprey. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analyses revealed sex differences in GnIH-related neuropeptide levels in the brain and plasma of immature sea lamprey. Exposure to 3kPZS and 3kACA exerted differential effects on GnIH-related neuropeptide levels in both sexes, but the effects were more prominent in female brains. We conclude that sea lamprey pheromones regulate GnIH-related neuropeptide levels in a sexually dimorphic manner.

Galanin in an Agnathan: Precursor Identification and Localisation of Expression in the Brain of the Sea Lamprey Petromyzon marinus.

Galanin is a neuropeptide that is widely expressed in the mammalian brain, where it regulates many physiological processes, including feeding and nociception. Galanin has been characterized extensively in jawed vertebrates (gnathostomes), but little is known about the galanin system in the most ancient extant vertebrate class, the jawless vertebrates or agnathans. Here, we identified and cloned a cDNA encoding the sea lamprey (Petromyzon marinus) galanin precursor (PmGalP). Sequence analysis revealed that PmGalP gives rise to two neuropeptides that are similar to gnathostome galanins and galanin message-associated peptides. Using mRNA in situ hybridization, the distribution of PmGalP-expressing neurons was mapped in the brain of larval and adult sea lampreys. This revealed PmGalP-expressing neurons in the septum, preoptic region, striatum, hypothalamus, prethalamus, and displaced cells in lateral areas of the telencephalon and diencephalon. In adults, the laterally migrated PmGalP-expressing neurons are observed in an area that extends from the ventral pallium to the lateral hypothalamus and prethalamus. The striatal and laterally migrated PmGalP-expressing cells of the telencephalon were not observed in larvae. Comparison with studies on jawed vertebrates reveals that the presence of septal and hypothalamic galanin-expressing neuronal populations is highly conserved in vertebrates. However, compared to mammals, there is a more restricted pattern of expression of the galanin transcript in the brain of lampreys. This work provides important new information on the early evolution of the galanin system in vertebrates and provides a genetic and neuroanatomical basis for functional analyses of the galanin system in lampreys.

Inhibition of neogenin promotes neuronal survival and improved behavior recovery after spinal cord injury

Following spinal cord trauma, axonal regeneration in the mammalian spinal cord does not occur and functional recovery may be further impeded by retrograde neuronal death. By contrast, lampreys recover after spinal cord injury (SCI) and axons re-connected to their targets in spinal cord. However, the identified reticulospinal (RS) neurons located in the lamprey brain differ in their regenerative capacities – some are good regenerators, and others are bad regenerators – despite the fact that they have analogous projection pathways. Previously, we reported that axonal guidance receptor Neogenin involved in regulation of axonal regeneration after SCI and downregulation of Neogenin synthesis by morpholino oligonucleotides (MO) enhanced the regeneration of RS neurons. Incidentally, the bad regenerating RS neurons often undergo a late retrograde apoptosis after SCI. Here we report that, after SCI, expression of RGMa mRNA was upregulated around the transection site, while its receptor Neogenin continued to be synthesized almost inclusively in the “bad-regenerating” RS neurons. Inhibition of Neogenin by MO prohibited activation of caspases and improved the survival of RS neurons at 10 weeks after SCI. These data provide new evidence in vivo that Neogenin is involved in retrograde neuronal death and failure of axonal regeneration after SCI.

GABAergic modulation of olfactomotor transmission in lampreys.

Odor-guided behaviors, including homing, predator avoidance, or food and mate searching, are ubiquitous in animals. It is only recently that the neural substrate underlying olfactomotor behaviors in vertebrates was uncovered in lampreys. It consists of a neural pathway extending from the medial part of the olfactory bulb (medOB) to locomotor control centers in the brainstem via a single relay in the caudal diencephalon. This hardwired olfactomotor pathway is present throughout life and may be responsible for the olfactory-induced motor behaviors seen at all life stages. We investigated modulatory mechanisms acting on this pathway by conducting anatomical (tract tracing and immunohistochemistry) and physiological (intracellular recordings and calcium imaging) experiments on lamprey brain preparations. We show that the GABAergic circuitry of the olfactory bulb (OB) acts as a gatekeeper of this hardwired sensorimotor pathway. We also demonstrate the presence of a novel olfactomotor pathway that originates in the non-medOB and consists of a projection to the lateral pallium (LPal) that, in turn, projects to the caudal diencephalon and to the mesencephalic locomotor region (MLR). Our results indicate that olfactory inputs can induce behavioral responses by activating brain locomotor centers via two distinct pathways that are strongly modulated by GABA in the OB. The existence of segregated olfactory subsystems in lampreys suggests that the organization of the olfactory system in functional clusters may be a common ancestral trait of vertebrates.

Molecular Cloning of Synucleins in River Lamprey Lampetra fluviatilis

Synaptic proteins synucleins are found in pathologic aggregates in human brain during neurodegenerative diseases and in some tumors. Normal functions of these proteins in synapses are still unclear. In the present study, we used cDNA cloning to determine amino acid sequences of synucleins in the central nervous system of river lamprey (Lampetra fluviatilis), which is used as a model organism to study molecular mechanisms of synaptic transmission. Three genes are identified. High similarity in amino acid sequences as compared to other vertebrate species is revealed. The bioinformatic analysis predicts that the river lamprey synucleins relate to the group of gamma-synucleins. High homology with human alpha-synuclein is reported. The hydrophobic region required for the formation of alpha-synuclein amyloid fibers is also present in the river lamprey synucleins. The latter suggests that this region appeared at early stages of evolution. The obtained amino acid sequences of synucleins in the river lamprey brain will allow generating novel molecular tools for dissecting physiological functions of these proteins.

RhoA activation in axotomy-induced neuronal death

After spinal cord injury (SCI) in mammals, severed axons fail to regenerate, due to both extrinsic inhibitory factors, e.g., the chondroitin sulfate proteoglycans (CSPGs) and myelin-associated growth inhibitors (MAIs), and a developmental loss of intrinsic growth capacity. The latter is suggested by findings in lamprey that the 18 pairs of individually identified reticulospinal neurons vary greatly in their ability to regenerate their axons through the same spinal cord environment. Moreover, those neurons that are poor regenerators undergo very delayed apoptosis, and express common molecular markers after SCI. Thus the signaling pathways for retrograde cell death might converge with those inhibiting axon regeneration. Many extrinsic growth-inhibitory molecules activate RhoA, whereas inhibiting RhoA enhances axon growth. Whether RhoA also is involved in retrograde neuronal death after axotomy is less clear. Therefore, we cloned lamprey RhoA and correlated its mRNA expression and activation state with apoptosis signaling in identified reticulospinal neurons. RhoA mRNA was expressed widely in normal lamprey brain, and only slightly more in poorly-regenerating neurons than in good regenerators. However, within a day after spinal cord transection, RhoA mRNA was found in severed axon tips. Beginning at 5 days post-SCI RhoA mRNA was upregulated selectively in pre-apoptotic neuronal perikarya, as indicated by labelling with fluorescently labeled inhibitors of caspase activation (FLICA). After 2 weeks post-transection, RhoA expression decreased in the perikarya, and was translocated anterogradely into the axons. More striking than changes in RhoA mRNA levels, RhoA was continuously active selectively in FLICA-positive neurons through 9 weeks post-SCI. At that time, almost no neurons whose axons had regenerated were FLICA-positive. These findings are consistent with a role for RhoA activation in triggering retrograde neuronal death after SCI, and suggest that RhoA may be a point of convergence for inhibition of both axon regeneration and neuronal survival after axotomy.

Organization of alpha-transducin immunoreactive system in the brain and retina of larval and young adult Sea Lamprey (Petromyzon marinus), and their relationship with other neural systems.

We employed an anti-transducin antibody (Gαt-S), in combination with other markers, to characterize the Gαt-S-immunoreactive (ir) system in the CNS of the sea lamprey, Petromyzon marinus. Gαt-S immunoreactivity was observed in some neuronal populations and numerous fibers distributed throughout the brain. Double Gαt-S- and opsin-ir neurons (putative photoreceptors) are distributed in the hypothalamus (postoptic commissure nucleus, dorsal and ventral hypothalamus) and caudal diencephalon, confirming results of García-Fernández et al. (Cell and Tissue Research, 288, 267-278, 1997). Singly Gαt-S-ir cells were observed in the midbrain and hindbrain, increasing the known populations. Our results reveal for the first time in vertebrates the extensive innervation of many brain regions and the spinal cord by Gαt-S-ir fibers. The Gαt-S innervation of the habenula is very selective, fibers densely innervating the lamprey homologue of the mammalian medial nucleus (Stephenson-Jones et al., Proceedings of the National Academy of Sciences of the United States of America, 109, E164-E173, 2012), but not the lateral nucleus homologue. The lamprey neurohypophysis was not innervated by Gαt-S-ir fibers. We also analyzed by double immunofluorescence the relation of this system with other systems. A dopaminergic marker (TH), serotonin (5-HT) or GABA do not co-localize with Gαt-S-ir neurons although codistribution of fibers was observed. Codistribution of Gαt-S-ir fibers and isolectin-labeled extrabulbar primary olfactory fibers was observed in the striatum and hypothalamus. Neurobiotin retrograde transport from the spinal cord combined with immunofluorescence revealed spinal-projecting Gαt-S-ir reticular neurons in the caudal hindbrain. Present results in an ancient vertebrate reveal for the first time a collection of brain targets of Gαt-S-ir neurons, suggesting they might mediate non-visual modulation by light in many systems.

Restricted co-localization of glutamate and dopamine in neurons of the adult sea lamprey brain.

Co-localization of dopamine with other classical neurotransmitters in the same neuron is a common phenomenon in the brain of vertebrates. In mammals, some dopaminergic neurons of the ventral tegmental area and the hypothalamus have a glutamatergic co-phenotype. However, information on the presence of this type of dopaminergic neurons in other vertebrate groups is very scant. Here, we aimed to provide new insights on the evolution of this neuronal co-phenotype by studying the presence of a dual dopaminergic/glutamatergic neuron phenotype in the central nervous system of lampreys. Double immunofluorescence experiments for dopamine and glutamate in adult sea lampreys revealed co-localization of both neurotransmitters in some neurons of the preoptic nucleus, the nucleus of the postoptic commissure, the dorsal hypothalamus and in cerebrospinal fluid-contacting cells of the caudal rhombencephalon and rostral spinal cord. Moreover, co-localization of dopamine and glutamate was found in dopaminergic fibres in a few brain regions including the lateral pallium, striatum, and the preoptic and postoptic areas but not in the brainstem. Our results suggest that the presence of neurons with a dopaminergic/glutamatergic co-phenotype is a primitive character shared by jawless and jawed vertebrates. However, important differences in the distribution of these neurons and fibres were noted among the few vertebrates investigated to date. This study offers an anatomical basis for further work on the role of glutamate in dopaminergic neurons.

Co-localization of three gonadotropin-releasing hormone transcripts in larval, parasitic, and adult sea lamprey brains

RNA expression of lamprey gonadotropin-releasing hormone (lGnRH)-I, -II, and -III was demonstrated in the brains of larval, parasitic phase and adult sea lampreys, Petromyzon marinus, using a highly sensitive triple-label in situ hybridization technique. In female larval lampreys, lGnRH-I and-II were co-expressed in the same neurons throughout the olfactory bulbs, preoptic area (POA), and rhombencephalon (hindbrain); lGnRH-I, -II and -III were triple co-expressed in the hypothalamus and in the paranuclear region of neuronal somas in the rhombencephalon. In female parasitic phase lampreys, lGnRH-I and -II were co-expressed in the POA, thalamus, and preoptico-neurohypophyseal tract (PNT); lGnRH-III was minimally triple co-expressed with lGnRH-I and -II in the hypothalamus. In adult female lampreys, lGnRH-I and -III were co-expressed in the hypothalamus; lGnRH-I was also expressed in the neurohypophysis (NH). In adult male lampreys, lGnRH-I and-III were co-expressed in the primordial hippocampus, POA, thalamus, hypothalamus, NH, and PNT; lGnRH-I was also expressed in the epithalamus. In summary, we provide the first study using in situ hybridization of all three lGnRHs (lGnRH-I, -II, and -III) at three major life stages (larval, parasitic, and adult) of lampreys, which strongly supports previous immunohistological studies and suggests that lGnRH-I and -II are the predominant lGnRHs in larval and parasitic phase lampreys, and that lGnRH-I and -III are the predominant lGnRHs in adult female and male lampreys. Therefore, our results show that lGnRH-I, -II, and -III have different localization and co-expression in the development and sexual maturation of lampreys, which may suggest unique physiological roles at each life stage and sex in the developing and mature lamprey brain.

The expression of chemorepulsive guidance receptors and the regenerative abilities of spinal-projecting neurons after spinal cord injury

Spinal cord injury (SCI) in mammals leads to permanent loss of function because axons do not regenerate in the central nervous system (CNS). To date, treatments based on neutralizing inhibitory environmental cues, such as the myelin-associated growth inhibitors and chondroitin sulfate proteoglycans, or on adding neurotrophic factors, have had limited success in enhancing regeneration. Published studies suggested that multiple axon guidance cues (repulsive guidance molecule (RGM) family, semaphorins, ephrins, and netrins) persist in adult animals, and that their expression is upregulated after CNS injury. Moreover, many adult CNS neurons continue to express axon guidance receptors. We used the advantages of the lamprey CNS to test the hypotheses that the regenerative abilities of spinal-projecting neurons depend upon their expression of chemorepulsive guidance receptors. After complete spinal transection, lampreys recover behaviorally, and injured axons grow selectively in their correct paths. However, the large identified reticulospinal (RS) neurons in the lamprey brain are heterogeneous in their regenerative abilities – some are high regeneration capacity neurons (probability of axon regeneration >50%), others are low regeneration capacity neurons (<30%). Here we report that the RGM receptor Neogenin is expressed preferentially in the low regeneration capacity RS neurons that regenerate poorly, and that downregulation of Neogenin by morpholino antisense oligonucleotides enhances regeneration of RS axons after SCI. Moreover, lamprey CNS neurons co-express multiple guidance receptors (Neogenin, UNC5 and PlexinA), suggesting that the regenerative abilities of spinal-projecting neurons might reflect the summed influences of the chemorepulsive guidance receptors that they express.