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The neural crest is an embryonic stem cell population unique to vertebrates 1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia 2 . Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates 3-8 . Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine β-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Sea lamprey (Petromyzon marinus) begin life as filter-feeding larvae (ammocoetes) before undergoing a complex metamorphosis into parasitic juveniles, which migrate to the sea where they feed on the blood of large-bodied fishes. The greater protein intake during this phase results in marked increases in the production of nitrogenous wastes (N-waste), which are excreted primarily via the gills. However, it is unknown how gill structure and function change during metamorphosis and how it is related to modes of ammonia excretion, nor do we have a good understanding of how the sea lamprey's transition from fresh water (FW) to sea water (SW) affects patterns and mechanisms of N-waste excretion in relation to ionoregulation. Using immunohistochemistry, we related changes in the gill structure of larval, metamorphosing, and juvenile sea lampreys to their patterns of ammonia excretion (J amm ) and urea excretion (J urea ) in FW, and following FW to artificial seawater (ASW) transfer. Rates of J amm and J urea were low in larval sea lamprey and increased in feeding juvenile, parasitic sea lamprey. In freshwater-dwelling ammocoetes, immunohistochemical analysis revealed that Rhesus glycoprotein C-like protein (Rhcg-like) was diffusely distributed on the lamellar epithelium, but following metamorphosis, Rhcg-like protein was restricted to SW mitochondrion-rich cells (MRCs; ionocytes) between the gill lamellae. Notably, these interlamellar Rhcg-like proteins co-localized with Na + /K + -ATPase (NKA), which increased in expression and activity by almost tenfold during metamorphosis. The distribution of V-type H + -ATPase (V-ATPase) on the lamellae decreased following metamorphosis, indicating it may have a more important role in acid-base regulation and Na + uptake in FW, compared to SW. We conclude that the re-organization of the sea lamprey gill during metamorphosis not only plays a critical role in allowing them to cope with greater salinity following the FW-SW transition, but that it simultaneously reflects fundamental changes in methods used to excrete ammonia.

Molecules present in an animal's environment can indicate the presence of predators, food, or sexual partners and consequently, induce migratory, reproductive, foraging, or escape behaviors. Three sensory systems, the olfactory, gustatory, and solitary chemosensory cell (SCC) systems detect chemical stimuli in vertebrates. While a great deal of research has focused on the olfactory and gustatory system over the years, it is only recently that significant attention has been devoted to the SCC system. The SCCs are microvillous cells that were first discovered on the skin of fish, and later in amphibians, reptiles, and mammals. Lampreys also possess SCCs that are particularly numerous on cutaneous papillae. However, little is known regarding their precise distribution, innervation, and function. Here, we show that sea lampreys (Petromyzon marinus L.) have cutaneous papillae located around the oral disk, nostril, gill pores, and on the dorsal fins and that SCCs are particularly numerous on these papillae. Tract-tracing experiments demonstrated that the oral and nasal papillae are innervated by the trigeminal nerve, the gill pore papillae are innervated by branchial nerves, and the dorsal fin papillae are innervated by spinal nerves. We also characterized the response profile of gill pore papillae to some chemicals and showed that trout-derived chemicals, amino acids, and a bile acid produced potent responses. Together with a companion study (Suntres et al., Journal of Comparative Neurology, this issue), our results provide new insights on the function and evolution of the SCC system in vertebrates., (© 2019 Wiley Periodicals, Inc.)

Cholecystokinin (CCK) is a neuropeptide that modulates processes such as digestion, satiety, and anxiety. CCK-type peptides have been characterized in jawed vertebrates and invertebrates, but little is known about CCK-type signalling in the most ancient group of vertebrates, the agnathans. Here, we have cloned and sequenced a cDNA encoding a sea lamprey (Petromyzon marinus L.) CCK-type precursor (PmCCK), which contains a CCK-type octapeptide sequence (PmCCK-8) that is highly similar to gnathostome CCKs. Using mRNA in situ hybridization, the distribution of PmCCK-expressing neurons was mapped in the CNS of P. marinus. This revealed PmCCK-expressing neurons in the hypothalamus, posterior tubercle, prethalamus, nucleus of the medial longitudinal fasciculus, midbrain tegmentum, isthmus, rhombencephalic reticular formation, and the putative nucleus of the solitary tract. Some PmCCK-expressing neuronal populations were only observed in adults, revealing important differences with larvae. We generated an antiserum to PmCCK-8 to enable immunohistochemical analysis of CCK expression, which revealed that GABA or glutamate, but not serotonin, tyrosine hydroxylase or neuropeptide Y, is co-expressed in some PmCCK-8-immunoreactive (ir) neurons. Importantly, this is the first demonstration of co-localization of GABA and CCK in neurons of a non-mammalian vertebrate. We also characterized extensive cholecystokinergic fibre systems of the CNS, including innervation of habenular subnuclei. A conspicuous PmCCK-8-ir tract ascending in the lateral rhombencephalon selectively innervates a glutamatergic population in the dorsal isthmic grey. Interestingly, this tract is reminiscent of the secondary gustatory/visceral tract of teleosts. In conclusion, this study provides important new information on the evolution of the cholecystokinergic system in vertebrates.

The hypothalamic-pituitary (HP) system, which is specific to vertebrates, is considered to be an evolutionary innovation that emerged prior to or during the differentiation of the ancestral jawless vertebrates (agnathans) leading to the neuroendocrine control of many complex functions. Along with hagfish, lampreys represent the oldest lineage of vertebrates, agnathans (jawless fish). This review will highlight our discoveries of the major components of the lamprey HP axis. Generally, gnathostomes (jawed vertebrates) have one or two hypothalamic gonadotropin-releasing hormones (GnRH) while lampreys have three hypothalamic GnRHs. GnRH(s) regulate reproduction in all vertebrates via the pituitary. In gnathostomes, there are three classical pituitary glycoprotein hormones (luteinizing hormone, LH; follicle stimulating hormone, FSH; and thyrotropin, TSH) interacting specifically with three receptors, LH-R, FSH-R, and TSH-R, respectively. In general, FSH and LH regulate gonadal activity and TSH regulates thyroidal activity. In contrast to gnathostomes, we propose that lampreys only have two heterodimeric pituitary glycoprotein hormones, lamprey glycoprotein hormone (lGpH) and thyrostimulin, and two lamprey glycoprotein hormone receptors (lGpH-R I and -R II). Our existing data also suggest the existence of a primitive, overlapping yet functional hypothalamic-pituitary-gonadal (HPG) and HP-thyroidal (HPT) endocrine systems in lampreys. The study of basal vertebrates provides promising models for understanding the evolution of the hypothalamic-pituitary-thyroidal and gonadal axes in vertebrates. We hypothesize that the glycoprotein hormone/glycoprotein hormone receptor systems emerged as a link between the neuroendocrine and peripheral control levels during the early stages of gnathostome divergence. Our discovery of a functional HPG axis in lamprey has provided important clues for understanding the forces that ensured a common organization of the hypothalamus and pituitary as essential regulatory systems in all vertebrates. This paper will provide a brief snapshot of my discoveries, collaborations and latest findings including phylogenomic analyses on the origins, co-evolution and divergence of ligand and receptor protein families from the perspective of the lamprey hypothalamic-pituitary system., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© 2017 Wiley Periodicals, Inc.)

Olfactory sensory neurons innervate the olfactory bulb, where responses to different odorants generate a chemotopic map of increased neural activity within different bulbar regions. In this study, insight into the basal pattern of neural organization of the vertebrate olfactory bulb was gained by investigating the lamprey. Retrograde labelling established that lateral and dorsal bulbar territories receive the axons of sensory neurons broadly distributed in the main olfactory epithelium and that the medial region receives sensory neuron input only from neurons projecting from the accessory olfactory organ. The response duration for local field potential recordings was similar in the lateral and dorsal regions, and both were longer than medial responses. All three regions responded to amino acid odorants. The dorsal and medial regions, but not the lateral region, responded to steroids. These findings show evidence for olfactory streams in the sea lamprey olfactory bulb: the lateral region responds to amino acids from sensory input in the main olfactory epithelium, the dorsal region responds to steroids (taurocholic acid and pheromones) and to amino acids from sensory input in the main olfactory epithelium, and the medial bulbar region responds to amino acids and steroids stimulating the accessory olfactory organ. These findings indicate that olfactory subsystems are present at the base of vertebrate evolution and that regionality in the lamprey olfactory bulb has some aspects previously seen in other vertebrate species., (© 2017. Published by The Company of Biologists Ltd.)

Background: The frontal lobe control of movement in mammals has been thought to be a specific function primarily related to the layered neocortex with its efferent connections. In contrast, we now show that the same basic organization is present even in one of the phylogenetically oldest vertebrates, the lamprey., Results: Stimulation of specific sites in the pallium/cortex evokes eye, trunk, locomotor, or oral movements. The pallial projection neurons target brainstem motor centers and basal ganglia subnuclei and have prominent dendrites extending into the outer molecular layer. They exhibit the characteristic features of pyramidal neurons and elicit monosynaptic glutamatergic excitatory postsynaptic potentials in output neurons of the optic tectum, reticulospinal neurons, and, as shown earlier, basal ganglia neurons., Conclusions: Our results demonstrate marked similarities in the efferent functional connectivity and control of motor behavior between the lamprey pallium and mammalian neocortex. Thus, the lamprey motor pallium/cortex represents an evolutionary blueprint of the corresponding mammalian system., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study hypothesizes the existence of three groups of sea lamprey Petromyzon marinus L. in Portugal (North/Central group, Tagus group, and Guadiana group), possibly promoted by seabed topography isolation during the oceanic phase of the life cycle. Within this context, our purpose was to analyze the existence of a stock structure on sea lamprey populations sampled in the major Portuguese river basins using both morphological characters and heart tissue fatty acid signature. In both cases, the multiple discriminant analysis revealed statistically significant differences among groups, and the overall corrected classification rate estimated from cross-validation procedure was particularly high for the cardiac muscle fatty acid profiles (i.e. 83.8%). Morphometric characters were much more useful than meristic ones to discriminate stocks, and the most important variables for group differentiation were eye length, second dorsal fin length and branchial length. Fatty acid analysis showed that all lampreys from the southern Guadiana group were correctly classified and not mixing with individuals from any other group, reflecting a typical heart fatty acid signature. Our results revealed that 89.5% and 72.2% of the individuals from the Tagus and North/Central groups, respectively, were also correctly classified, despite some degree of overlap between individuals from these groups. The fatty acids that contributed to the observed segregation were C16:0; C17:0; C18:1ω9; C20:3ω6 and C22:2ω6. Detected differences are probably related with environmental variables to which lampreys may have been exposed, which leaded to different patterns of gene expression. These results suggest the existence of three different sea lamprey stocks in Portugal, with implication in terms of management and conservation.

In jawed (gnathostome) vertebrates, the inner ears have three semicircular canals arranged orthogonally in the three Cartesian planes: one horizontal (lateral) and two vertical canals. They function as detectors for angular acceleration in their respective planes. Living jawless craniates, cyclostomes (hagfish and lamprey) and their fossil records seemingly lack a lateral horizontal canal. The jawless vertebrate hagfish inner ear is described as a torus or doughnut, having one vertical canal, and the jawless vertebrate lamprey having two. These observations on the anatomy of the cyclostome (jawless vertebrate) inner ear have been unchallenged for over a century, and the question of how these jawless vertebrates perceive angular acceleration in the yaw (horizontal) planes has remained open. To provide an answer to this open question we reevaluated the anatomy of the inner ear in the lamprey, using stereoscopic dissection and scanning electron microscopy. The present study reveals a novel observation: the lamprey has two horizontal semicircular ducts in each labyrinth. Furthermore, the horizontal ducts in the lamprey, in contrast to those of jawed vertebrates, are located on the medial surface in the labyrinth rather than on the lateral surface. Our data on the lamprey horizontal duct suggest that the appearance of the horizontal canal characteristic of gnathostomes (lateral) and lampreys (medial) are mutually exclusive and indicate a parallel evolution of both systems, one in cyclostomes and one in gnathostome ancestors., (© 2014 Anatomical Society.)

The contribution of dopamine (DA) to locomotor control is traditionally attributed to ascending dopaminergic projections from the substantia nigra pars compacta and the ventral tegmental area to the basal ganglia, which in turn project down to the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion in vertebrates. However, a dopaminergic innervation of the pedunculopontine nucleus, considered part of the MLR, was recently identified in the monkey. The origin and role of this dopaminergic input are unknown. We addressed these questions in a basal vertebrate, the lamprey. Here we report a functional descending dopaminergic pathway from the posterior tuberculum (PT; homologous to the substantia nigra pars compacta and/or ventral tegmental area of mammals) to the MLR. By using triple labeling, we found that dopaminergic cells from the PT not only project an ascending pathway to the striatum, but send a descending projection to the MLR. In an isolated brain preparation, PT stimulation elicited excitatory synaptic inputs into patch-clamped MLR cells, accompanied by activity in reticulospinal cells. By using voltammetry coupled with electrophysiological recordings, we demonstrate that PT stimulation evoked DA release in the MLR, together with the activation of reticulospinal cells. In a semi-intact preparation, stimulation of the PT elicited reticulospinal activity together with locomotor movements. Microinjections of a D1 antagonist in the MLR decreased the locomotor output elicited by PT stimulation, whereas injection of DA had an opposite effect. It appears that this descending dopaminergic pathway has a modulatory role on MLR cells that are known to receive glutamatergic projections and promotes locomotor output., Competing Interests: The authors declare no conflict of interest.

Although there is abundant evidence for segregated processing in the olfactory system across vertebrate taxa, the spatial relationship between the second order projection neurons (PNs) of olfactory subsystems connecting sensory input to higher brain structures is less clear. In the sea lamprey, there is tight coupling between olfaction and locomotion via PNs extending to the posterior tuberculum from the medial region of the olfactory bulb. This medial region receives peripheral input predominantly from the accessory olfactory organ. However, the axons from olfactory sensory neurons residing in the main olfactory epithelium extend to non-medial regions of the olfactory bulb, and the non-medial bulbar PNs extend their axons to the lateral pallium. It is not known if the receptive fields of the PNs in the two output pathways overlap; nor has the morphology of these PNs been investigated. In this study, retrograde labelling was utilized to investigate the PNs belonging to medial and non-medial projections. The dendrites and somata of the medial PNs were confined to medial glomerular neuropil, and dendrites of non-medial PNs did not enter this territory. The cell bodies and dendrites of the non-medial PNs were predominantly located below the glomeruli (frequently deeper in the olfactory bulb). While PNs in both locations contained single or multiple primary dendrites, the somal size was greater for medial than for non-medial PNs. When considered with the evidence-to-date, this study shows different neuroanatomical organization for medial olfactory bulb PNs extending to locomotor control centers and non-medial PNs extending to the lateral pallium in this vertebrate.

Lampreys are jawless vertebrates, the most basal group of extant vertebrates. This phylogenetic position makes them invaluable models in comparative studies of the vertebrate central nervous system. Lampreys have been used as vertebrate models to study the neuronal circuits underlying locomotion control and axonal regeneration after spinal cord injury. Inhibitory inputs are key elements in the networks controlling locomotor behaviour, but very little is known about the descending inhibitory projections in lampreys. The aim of this study was to investigate the presence of brain-spinal descending inhibitory pathways in larval stages of the sea lamprey Petromyzon marinus by means of tract-tracing with neurobiotin, combined with immunofluorescence triple-labeling methods. Neurobiotin was applied in the rostral spinal cord at the level of the third gill, and inhibitory populations were identified by the use of cocktails of antibodies raised against glycine and GABA. Glycine-immunoreactive (-ir) neurons that project to the spinal cord were observed in three rhombencephalic reticular nuclei: anterior, middle and posterior. Spinal-projecting GABA-ir neurons were observed in the anterior and posterior reticular nuclei. Double glycine-ir/GABA-ir spinal cord-projecting neurons were only observed in the posterior reticular nucleus, and most glycine-ir neurons did not display GABA immunoreactivity. The present results reveal the existence of inhibitory descending projections from brainstem reticular neurons to the spinal cord, which were analyzed in comparative and functional contexts. Further studies should investigate which spinal cord circuits are affected by these descending inhibitory projections., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Despite the importance of glutamate as a major excitatory neurotransmitter in the brain, the distribution of glutamatergic populations in the brain of most vertebrates is still unknown. Here, we studied for the first time the distribution of glutamatergic neurons in the forebrain of the sea lamprey (Petromyzon marinus), belonging to the most ancient group of vertebrates (agnathans). For this, we used in situ hybridization with probes for a lamprey vesicular glutamate transporter (VGLUT) in larvae and immunofluorescence with antiglutamate antibodies in both larvae and adults. We also compared glutamate and γ-aminobutyric acid (GABA) immunoreactivities in sections using double-immunofluorescence methods. VGLUT-expressing neurons were observed in the olfactory bulb, pallium, septum, subhippocampal lobe, preoptic region, thalamic eminence, prethalamus, thalamus, epithalamus, pretectum, hypothalamus, posterior tubercle, and nucleus of the medial longitudinal fascicle. Comparison of VGLUT signal and glutamate immunoreactivity in larval forebrain revealed a consistent distribution of positive cells, which were numerous in most regions. Glutamate-immunoreactive cell populations were also found in similar regions of the adult forebrain. These include mitral-like cells of the olfactory bulbs and abundant cells in the lateral pallium, septum, and various diencephalic regions, mainly in the prethalamus, thalamus, habenula, pineal complex, and pretectum. Only a small portion of the glutamate-immunoreactive cells showed colocalization with GABA, which was observed mainly in the olfactory bulb, telencephalon, hypothalamus, ventral thalamus, and pretectum. Comparison with glutamatergic cells observed in rodent forebrains suggests that the regional distribution of glutamatergic cells does not differ greatly in lampreys and mammals., (Copyright © 2011 Wiley-Liss, Inc.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In larval lamprey, spinal locomotor activity can be initiated by pharmacological microstimulation from the following higher order brain locomotor areas [Paggett et al. (2004) Neuroscience 125:25-33; Jackson et al. (2007) J Neurophysiol 97:3229-3241]: rostrolateral rhombencephalon (RLR); ventromedial diencephalon (VMD); or dorsolateral mesencephalon (DLM). In the present study, pharmacological microstimulation with excitatory amino acids (EAAs) or their agonists in the brains of in vitro brain/spinal cord preparations was used to determine the sizes, pharmacology, and organization of these locomotor areas. First, the RLR, DLM and VMD locomotor areas were confined to relatively small areas of the brain, and stimulation as little as 50 μm outside these areas was ineffective or elicited tonic or uncoordinated motor activity. Second, pharmacological stimulation with NMDA, kainate, or AMPA in the VMD or DLM reliably initiated well-coordinated spinal locomotor activity. In the RLR, stimulation with all three ionotropic EAA receptor agonists could initiate spinal locomotor activity, but NMDA or AMPA was more reliable than kainate. Third, with synaptic transmission blocked only in the brain, stimulation in the RLR, VMD, or DLM no longer initiated spinal locomotor activity, suggesting that these locomotor areas do not directly activate spinal locomotor networks. Fourth, following a complete transection at the mesencephalon-rhombencephalon border, stimulation in the RLR no longer initiated spinal motor activity. Thus, the RLR locomotor area does not appear able to initiate spinal locomotor activity by neural circuits confined entirely within the rhombencephalon but requires more rostral neural centers, such as those in the VMD and DLM, as previously proposed [Paggett et al. (2004) Neuroscience 125:25-33]., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

It is widely recognized that animals respond to odors by generating or modulating specific motor behaviors. These reactions are important for daily activities, reproduction, and survival. In the sea lamprey, mating occurs after ovulated females are attracted to spawning sites by male sex pheromones. The ubiquity and reliability of olfactory-motor behavioral responses in vertebrates suggest tight coupling between the olfactory system and brain areas controlling movements. However, the circuitry and the underlying cellular neural mechanisms remain largely unknown. Using lamprey brain preparations, and electrophysiology, calcium imaging, and tract tracing experiments, we describe the neural substrate responsible for transforming an olfactory input into a locomotor output. We found that olfactory stimulation with naturally occurring odors and pheromones induced large excitatory responses in reticulospinal cells, the command neurons for locomotion. We have also identified the anatomy and physiology of this circuit. The olfactory input was relayed in the medial part of the olfactory bulb, in the posterior tuberculum, in the mesencephalic locomotor region, to finally reach reticulospinal cells in the hindbrain. Activation of this olfactory-motor pathway generated rhythmic ventral root discharges and swimming movements. Our study bridges the gap between behavior and cellular neural mechanisms in vertebrates, identifying a specific subsystem within the CNS, dedicated to producing motor responses to olfactory inputs., Competing Interests: The authors have declared that no competing interests exist.

The bodies of fish change shape over propulsive, behavioral, developmental, and evolutionary time scales, a general phenomenon that we call "reconfiguration". Undulatory, postural, and form-reconfiguration can be distinguished, studied independently, and examined in terms of mechanical interactions and evolutionary importance. Using a combination of live, swimming fishes and digital robotic fish that are autonomous and self-propelled, we examined the functional relation between undulatory and postural reconfiguration in forward swimming, backward swimming, and yaw turning. To probe how postural and form reconfiguration interact, the yaw turning of leopard sharks was examined using morphometric and kinematic analyses. To test how undulatory reconfiguration might evolve, the digital robotic fish were subjected to selection for enhanced performance in a simulated ecology in which each individual had to detect and move towards a food source. In addition to the general issue of reconfiguration, these investigations are united by the fact that the dynamics of undulatory and postural reconfigurations are predicted to be determined, in part, by the structural stiffness of the fish's body. Our method defines undulatory reconfiguration as the combined, point-by-point periodic motion of the body, leaving postural reconfiguration as the combined deviations from undulatory reconfiguration. While undulatory reconfiguration appears to be the sole or primary propulsive driver, postural reconfiguration may contribute to propulsion in hagfish and it is correlated with differences in forward, and backward, swimming in lamprey. Form reconfigures over developmental time in leopard sharks in a manner that is consistent with an allometric scaling theory in which structural stiffness of the body is held constant. However, correlation of a form proxy for structural stiffness of the body suggests that body stiffness may scale in order to limit maximum postural reconfiguration during routine yaw turns. When structural stiffness and undulatory frequency are modeled as determining the tail's undulatory wave speed, both factors evolve under selection for enhanced foraging behavior in the digital fish-like robots. The methods used in making these distinctions between kinds of reconfiguration have broad applicability in fish biology, especially for quantifying complex motor behaviors in the wild and for simulating selection on behavior that leads to directional evolution of functional phenotypes.

The amino acid D-serine is an endogenous coagonist of N-methyl-D-aspartate (NMDA) receptors in mammals that has been shown to play an important role in synaptic function, behavior, learning, and memory. The distribution and cellular location of D-serine in the brain of the sea lamprey was investigated by using immunofluorescence methods. One major finding of our study, unlike early studies of mammals, was the localization of D-serine immunoreactivity in perikarya and dendrites of neurons, whereas D-serine immunoreactivity was not generally observed in the lamprey glia. D-serine-immunoreactive neurons were observed in different brain regions, including the olfactory bulb, medial pallium, thalamus, torus semicircularis, isthmus, and reticular formation. The colocalization of D-serine with gamma-aminobutyric acid (GABA) was also studied with a double-immunofluorescence technique. The relationship between D-serine and glycine immunoreactivities was studied in alternate parallel series of sections stained for either D-serine/GABA or glycine/GABA. Colocalization with GABA was observed in various D-serine-immunoreactive populations, and codistribution and possible colocalization with glycine was also observed in some populations, mainly in the dorsal isthmic gray, medial octavolateral nucleus, dorsal column nucleus, interpeduncular nucleus, and reticular formation. Although numerous fibers were strongly GABA- and glycine-immunoreactive, D-serine immunoreactivity was observed mostly in cell perikarya and dendrites. The present results indicate that the D-serine immunoreactive cells are small to medium-sized neurons, some exhibiting classical inhibitory neurotransmitters, in which D-serine might be acting as a modulator. The neuronal distribution of D-serine and its frequent colocalization and/or codistribution with the two main inhibitory neurotransmitters appeared early in vertebrates., ((c) 2009 Wiley-Liss, Inc.)

Although four different primary olfactory pathways have been described in tetrapod vertebrates, polymorphic olfactory sensory neurons comingle in the olfactory epithelium and project axons into separate bulbar regions in teleost fish. However, spatially segregated neurons may exist in the peripheral olfactory organ of lampreys, extant representatives of ancestral jawless vertebrates. In lampreys, the caudoventral portion of the peripheral olfactory organ contains tubular diverticula, named the accessory olfactory organ (AOO). Short, ciliated AOO cells were retrogradely labelled following application of biocytin or carbocyanine dyes to the medial region of the olfactory bulb. Tracer application to eight radial locations within the layer of glomeruli with mitral cells, of the olfactory bulb, showed that AOO projections were restricted to the medial region of the olfactory bulb. The outer boundary of the AOO projection extended to the ventromedial region of glomerular neuropil in 43% of the specimens. The olfactory sensory neurons in the main olfactory epithelium projected to glomerular neuropil throughout the olfactory bulb, including sparse projections to the medial region of the olfactory bulb. This study shows that these AOO neurons and their projections in the medial region of the olfactory bulb are anatomically distinct regions of the primary olfactory pathway in the sea lamprey., (Copyright 2009 Wiley-Liss, Inc.)

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.

Light avoidance in larval lampreys is mediated by dermal photoreceptors located in the tail. These photoreceptors continue to function in adults, but they seem redundant because post-metamorphic lampreys possess well-developed eyes. This study examined the role of dermal photoreceptors in adult sea lampreys by testing whether temperature-induced changes in refuge-seeking behavior are mediated by a reduction in dermal photosensitivity. In a lighted arena containing a single shaded refuge platform, lampreys at 22 degrees C displayed five times less search activity and were less likely to attach beneath the refuge platform than lampreys at either 7 or 15 degrees C. A behavioral assay for tail photosensitivity (locomotor response to tail illumination) revealed a corresponding reduction in dermal photosensitivity at 22 degrees C. Moreover, the responses to head illumination (eyes and pineal) did not correspond with the observed light avoidance behaviors. The head was less responsive to light than the tail and was not influenced by temperature. These results provide strong evidence that the dermal photoreceptors continue to mediate light avoidance in adult lampreys, even though adults possess fully functional eyes. The fact that the eyes apparently do not take on this role suggests that there is functional specialization between these two light sensing systems.

The organization and development of the descending spinal projections from serotonergic rhombencephalic neurons in the larval sea lamprey were investigated by double labeling, tract-tracing methods and immunocytochemistry against serotonin. The results showed that two serotonergic populations of the isthmic and vagal reticular regions present reticulospinal neurons from the beginning of the larval period. Of the three serotonergic subpopulations recognized in the isthmic reticular group [Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Anadón, R., Rodicio, M.C., 2007. Development of the serotonergic system in the central nervous system of the sea lamprey. J. Chem. Neuroanat. 34, 29-46], only two - the medial and ventral subpopulations - project to the spinal cord, with most of the projecting cells in the caudal part of the medial isthmic subpopulation. Occasional cells projecting to the spinal cord were observed in the ventral subpopulation. The vagal reticular serotonergic nucleus situated in the caudal rhombencephalon also presents cells with descending projections. The early development of the brainstem serotonergic projections to the spinal cord appears to be a conserved trait in all vertebrates studied. Although a serotonergic hindbrain-spinal projection system appears to have been present before the divergence of agnathans and gnathostomes, no serotonergic cells were observed in the raphe region in lamprey. Moreover, proportionally more rostral hindbrain serotonergic cells contribute to the spinal serotonergic projections in the sea lamprey than in jawed vertebrates.

In this study, double immunofluorescence methods were used to investigate possible colocalization of the neurotransmitters dopamine [DA] and GABA in rostral spinal cord neurones in the upstream migrating adult sea lamprey (Petromyzon marinus). Double immunofluorescence revealed that all the DA-immunoreactive (ir) cerebrospinal fluid-contacting (CSF-c) cells, approximately 30% of the medioventral DA-ir cells, and most of the DA-ir cells located in the grey lateral to the central canal were also GABA-ir. The results also revealed some DA-ir cells located dorsally to the central canal, which increases the number of dopaminergic cell types known in lamprey. Double-labelled fibres were mainly distributed in the ventral column, and double-labelled boutons contacted some dorsal GABA-ir CSF-c cells, as well as some non-CSF-c GABA-ir cells and ventromedial dendrites of motoneurones. The findings reveal colocalization of dopamine and GABA in some cells and fibres, which suggests co-release of these substances in some synaptic terminals. Although dopaminergic/GABAergic CSF-c cells have been reported in some other vertebrates, the other double-labelled spinal populations appear exclusive to lampreys.

The development and cellular distribution of the inhibitory neurotransmitter glycine in the spinal cord of the sea lamprey were studied by immunocytochemistry and double immunofluorescence and compared with the distribution of gamma-aminobutyric acid (GABA). Results in lamprey embryos and prolarvae reveal that the appearance of glycine-immunoreactive (-ir) spinal neurons precedes that of GABA-ir neurons. Throughout development, glycine-ir cells in the lateral and dorsomedial gray matter of the spinal cord are more numerous than the GABA-ir cells. Only a subset of these neurons shows colocalization of GABA and glycine, suggesting that they are primarily disparate neuronal populations. In contrast, most cerebrospinal fluid (CSF)-contacting neurons of the central canal walls are strongly GABA-ir, and only a portion of them are faintly glycine-ir. Some edge cells (lamprey intraspinal mechanoreceptors) were glycine-ir in larvae and adults. The glycine-ir and GABA-ir neuronal populations observed in the adult spinal cord were similar to those found in larvae. Comparison of glycine-ir and GABA-ir fibers coursing longitudinally in the spinal cord of adult lamprey revealed large differences in diameter between these two types of fiber. Commissural glycine-ir fibers appear in prolarvae and become numerous at larval stages, whereas crossed GABA-ir are scarce. Taken together, results in this primitive vertebrate indicate that the spinal glycinergic cells do not arise by biochemical shift of preexisting GABAergic cells but instead suggest that glycine is present in the earliest circuitry of the developing lamprey spinal cord, where it might act transiently as an excitatory transmitter., ((c) 2008 Wiley-Liss, Inc.)

We studied the distribution of calretinin immunoreactivity (CR-ir) in the rostral and intermediate levels of the spinal cord of lampreys from embryonic to adult periods. CR-ir was first observed at hatching in motoneurons and primary sensory neurons of the spinal cord, the dorsal cells. During the prolarval period two new cell types showed CR-ir: ganglion cells and interneurons. Motoneurons, dorsal cells, and ganglion cells were strongly positive, whereas interneurons were weakly stained in late prolarvae. The intensity of CR-ir in these four types of cells changed during the larval period. Increase of CR-expression was found in interneurons but a decrease in dorsal cells and in ganglion cells. These changes were more evident in premetamorphic larvae. Postmetamorphic lampreys showed almost no CR-ir in dorsal cells. In adult lampreys, the interneurons showed the highest CR-ir, whereas motoneurons were more weakly stained than in earlier stages of development. Moreover, in adults the dorsal cells and the ganglion cells showed no CR-ir. The present study shows that CR-ir changes during lamprey spinal cord development in different types of neurons, sometimes in opposite ways. This plasticity of CR-expression may indicate different needs from subsets of lamprey spinal cord cells involved in the different locomotor behaviors along its life cycle.

The excitatory amino acid l-aspartate (Asp) plays a number of roles in neuronal function. We studied the distribution of Asp-immunoreactive (ir) cells in the telencephalon of young and upstream migrating adult sea lamprey, Petromyzon marinus, and compared it with the distribution of gamma-aminobutyric acid (GABA) immunoreactivity, by using double immunofluorescence methods. Our results reveal for the first time the existence of Asp-ir neuronal populations in the lamprey forebrain. In the olfactory bulbs, Asp-ir neurons were observed in the mitral cell layer and in the inner cellular layer. Many granule-like cells were both Asp-ir and GABA-ir. In the pallium, Asp-ir cells were abundant in the lateral pallium and most of them were also GABA-ir. In the septum/terminal lamina nucleus, some cerebrospinal fluid-contacting type (CSF-c) cells were either Asp-ir or GABA-ir, and a few were double-labeled. Some non-CSF-c septal cells were both Asp-ir and GABA-ir. In the striatum, Asp-ir and/or GABA-ir cells were either subependymal or located in the characteristic arched cell row. In the lateral preoptic region, a few small Asp-ir/GABA-ir neurons were observed. In the caudal preoptic recess nucleus, numerous CSF-c cells were Asp-ir and/or GABA-ir. This study also reveals that colocalization of GABA and Asp immunoreactivities in telencephalic neurons is partial. Further investigation is required to establish whether Asp is a neurotransmitter and/or an intermediate in GABA synthesis in lamprey telencephalon.

Adrenomedullin (AM) is a neuropeptide widely distributed in vertebrates. In jawed vertebrates it has been localized in distinct regions of the central nervous system by means of antisera against human AM because the molecule seems to be well conserved across species. In this study, we have analyzed the localization of AM-like immunoreactive (AM-ir) cell bodies and fibers throughout the brain of the adult sea lamprey Petromyzon marinus, by using immunohistochemistry. Several AM-ir cell populations were found in the basal plate of the secondary prosencephalon, being more numerous in the hypothalamus, as well as two in the diencephalon and one in the mesencephalon; in addition two cell populations were found in the rhombencephalic alar plate, one in the isthmic region and other in the nucleus of the solitary tract. Immunolabeled fibers were widespread throughout the lamprey brain, but were more abundant in the basal plate. Of particular interest was the conspicuous innervation of the striatum by AM-ir fibers. In addition, our results indicate that AM-ir cells and fibers are present in the lamprey hypothalamo-neurohypophyseal system, suggesting that AM plays some important role in the control of pituitary gland function.

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.

Adrenocorticotropin (ACTH), melanotropins (MSHs), growth hormone (GH) and gonadotropin (GTH) have been identified or cloned from the pituitary gland of sea lampreys (Petromyzon marinus). The present study was designed to gain insights into the functional significance of these hormones through a description of changes in the occurrence and distribution of cells immunoreactive to their antibodies at several different stages of the sea lamprey life cycle. ACTH-like cells and MSH-like cells were distributed in the rostral pars distalis and the pars intermedia, respectively, throughout the life cycle from ammocoetes (larvae) to pre-spawning adults. A large number of ACTH-like cells were observed during the pre-spawning period when animals may experience the highest stressful conditions. On the other hand, the number of MSH-like cells increased markedly during metamorphosis, in accordance with the completion of eye development. A small number of GH-like cells were present in the proximal pars distalis during the larval and metamorphic phases, but the number of cells increased markedly during the parasitic period, which corresponded well with the rapid somatic growth. GTH-like cells were not observed in the pituitary during the larval and metamorphic phases, but were present in the proximal pars distalis of immediately post-metamorphosed animals. Since there was a high accumulation of GTH-like cells in pre-spawning adults, these cells appeared to be involved in gonadotropic functions. The results of changing immunoreactivity during the lamprey life cycle suggest that lamprey adenohypophysial hormones, ACTH, MSH, GH and GTH, may possess biological functions similar to those of more advanced gnathostome vertebrates. Given that lampreys represent the most ancient group of vertebrates, it is most likely that these hormones have been conserved for their functions throughout vertebrate evolution.

Brainstem networks generating the respiratory rhythm in lampreys are still not fully characterized. In this study, we described the patterns of respiratory activities and we identified the general location of underlying neural networks. In a semi-intact preparation including the brain and gills, rhythmic discharges were recorded bilaterally with surface electrodes placed over the vagal motoneurons. The main respiratory output driving rhythmic gill movements consisted of short bursts (40.9+/-15.6 ms) of discharge occurring at a frequency of 1.0+/-0.3 Hz. This fast pattern was interrupted by long bursts (506.3+/-174.6 ms) recurring with an average period of 37.4+/-24.9 s. After isolating the brainstem by cutting all cranial nerves, the frequency of the short respiratory bursts did not change significantly, but the slow pattern was less frequent. Local injections of a glutamate agonist (AMPA) and antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D,L-amino-5-phosphonopentanoic acid (AP5)) were made over different brainstem regions to influence respiratory output. The results were similar in the semi-intact and isolated-brainstem preparations. Unilateral injection of AP5 or CNQX over a rostral rhombencephalic region, lateral to the rostral pole of the trigeminal motor nucleus, decreased the frequency of the fast respiratory rhythm bilaterally or stopped it altogether. Injection of AMPA at the same site increased the rate of the fast respiratory rhythm and decreased the frequency of the slow pattern. The activity recorded in this area was synchronous with that recorded over the vagal motoneurons. After a complete transverse lesion of the brainstem caudal to the trigeminal motor nucleus, the fast rhythm was confined to the rostral area, while only the slow activity persisted in the vagal motoneurons. Our results support the hypothesis that normal breathing depends on the activity of neurons located in the rostral rhombencephalon in lampreys, whereas the caudal rhombencephalon generates the slow pattern.

The sea lamprey (Petromyzon marinus) is an ancient jawless fish phyletically removed from modern (teleost) fishes. It is an excellent organism in the study of olfaction due to its accessible olfactory pathway, which is susceptible to manipulation, and its important location in the evolution of vertebrates. There are many similarities in the olfactory systems of all fishes, and they also share characteristics with the olfactory system of mammals. Teleost fishes lack the distinctive vomeronasal organ of mammals; rather all odours are processed initially by olfactory sensory neurons (OSNs) of three morphotypes within the olfactory epithelium. We sought to identify olfactory sensory neuron polymorphisms in the sea lamprey. Using retrograde tracing with dyes injected into the olfactory bulb, we identified three morphotypes which are highly similar to those found in teleosts. This study provides the first evidence of morphotypes in the sea lamprey peripheral olfactory organ, and indicates that olfactory sensory neuron polymorphism may be a trait highly conserved throughout vertebrate evolution.

In vertebrates, locomotion is associated with changes in respiratory activity, but the neural mechanisms by which this occurs remain unknown. We began examining this in lampreys using a semi-intact preparation of young adult Petromyzon marinus, in which respiratory and locomotor behaviors can be recorded simultaneously with the activity of the underlying neural control systems. Spontaneous fictive respiration was recorded with suction electrodes positioned over the glossopharyngeal or the rostral vagal motor nucleus. In this preparation, locomotor activity, characterized by symmetrical tail movements (electromyogram recordings), was evoked by mechanical stimulation of the skin. During locomotion, the mean respiratory frequency and the mean area of the motor bursts were significantly increased (81.6+/-28.6% and 62.8+/-25.4%, respectively; P<0.05). The frequency returned to normal 92+/-51 s after the end of locomotion. There were fluctuations in the instantaneous respiratory and locomotor frequencies that were rhythmical but antiphasic for the two rhythmic activities. The changes in respiratory activity were also examined during bouts of locomotion occurring spontaneously, and it was found that a modification in respiratory activity preceded the onset of spontaneous locomotion by 3.5+/-2.6 s. This suggests that the early respiratory changes are anticipatory and are not caused by feedback generated by locomotion. The increase in respiratory frequency during locomotion induced by sensory stimulation persisted after removal of the mesencephalon. When both the mesencephalon and spinal cord were removed, resulting in the isolation of the rhombencephalon, changes in the respiratory activity were also present following skin stimulations that would have normally induced locomotion. Altogether, the results suggest that respiratory changes are programmed to adjust ventilation prior to motor activity, and that a central rhombencephalic mechanism is involved.

Variability is increasingly recognized as a characteristic feature of cellular, synaptic, and network properties. While studies have traditionally focused on mean values, significant effects can result from changes in variance. This study has examined cellular and synaptic variability in the lamprey spinal cord and its modulation by the neuropeptide substance P. Cellular and synaptic variability differed in different types of cell and synapse. Substance P reduced the variability of subthreshold locomotor-related depolarizations and spiking in motor neurons during network activity. These effects were associated with a reduction in the variability of spiking in glutamatergic excitatory network interneurons and with a reduction in the variance of excitatory interneuron-evoked excitatory postsynaptic potentials (EPSPs). Substance P also reduced the variance of postsynpatic potentials (PSPs) from crossing inhibitory and excitatory interneurons, but it increased the variance of inhibitory postsynpatic potentials (IPSPs) from ipsilateral inhibitory interneurons. The effects on the variance of different PSPs could occur with or without changes in the PSP amplitude. The reduction in the variance of excitatory interneuron-evoked EPSPs was protein kinase A, calcium, and N-methyl-d-aspartate (NMDA) dependent. The NMDA dependence suggested that substance P was acting postsynaptically. This was supported by the reduced variability of postsynaptic responses to glutamate by substance P. However, ultrastructural analyses suggested that there may also be a presynaptic component to the modulation, because substance P reduced the variability of synaptic vesicle diameters in putative glutamatergic terminals. These results suggest that cellular and synaptic variability can be targeted for modulation, making it an additional source of spinal cord plasticity.

Tectal afferents were studied in adult lampreys of three species (Ichthyomyzon unicuspis, Lampetra fluviatilis, and Petromyzon marinus) following unilateral BDA injections into the optic tectum (OT). In the secondary prosencephalon, neurons projecting to the OT were observed in the pallium, the subhipoccampal lobe, the striatum, the preoptic area and the hypothalamus. Following tectal injections, backfilled diencephalic cells were found bilaterally in: prethalamic eminence, ventral geniculate nucleus, periventricular prethalamic nucleus, periventricular pretectal nucleus, precommissural nucleus, magnocellular and parvocellular nuclei of the posterior commissure and pretectal nucleus; and ipsilaterally in: nucleus of Bellonci, periventricular thalamic nucleus, nucleus of the tuberculum posterior, and the subpretectal tegmentum, as well as in the pineal organ. At midbrain levels, retrogradely labeled cells were seen in the ipsilateral torus semicircularis, the contralateral OT, and bilaterally in the mesencephalic reticular formation and inside the limits of the retinopetal nuclei. In the hindbrain, tectal projecting cells were also bilaterally labeled in the dorsal and lateral isthmic nuclei, the octavolateral area, the sensory nucleus of the descending trigeminal tract, the dorsal column nucleus and the reticular formation. The rostral spinal cord also exhibited a few labeled cells. These results demonstrate a complex pattern of connections in the lamprey OT, most of which have been reported in other vertebrates. Hence, the lamprey OT receives a large number of nonvisual afferents from all major brain areas, and so is involved in information processing from different somatic sensory modalities., (Copyright (c) 2007 S. Karger AG, Basel.)

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, gamma-aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter-synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin-immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin-immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA-immunolabeled perikarya were observed. GABA-immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate-immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter-expressing cells of the larval retina appear to participate only in the vertical processing pathway.

This study examined the spatial and temporal distribution of serotonin-immunoreactive (5-HT-ir) neurons in the brainstem of Petromyzon marinus at three developmental stages, larval, postmetamorphic, and reproductive. Computer-assisted 3-D reconstructions were made of the three main 5-HT-ir neuron groups. The rostralmost brainstem group was located near the posterior commissure, the second group at the isthmus, and the third group in the bulbar area. For each of those groups, the distribution of the 5-HT-ir neurons was very similar in the three developmental stages examined, suggesting that the 5-HT system is relatively mature early in larval animals. The soma of 5-HT-ir neurons increased in size and their dendritic fields increased in complexity with development. Furthermore, the number of 5-HT-ir neurons in each group increased significantly from the larval to the reproductive stage. To determine whether this was due to the genesis of 5-HT neurons, bromodeoxyuridine (BrdU) was injected into larval, metamorphosing, and postmetamorphic lampreys. These experiments revealed a few neurons colocalizing BrdU and 5-HT in metamorphosing animals. Taken together, the present results suggest that 5-HT neurons increase in number during maturation and that neurogenesis could, at least partially, contribute to the appearance of new 5-HT cells at different developmental stages.

The distribution of lamprey gonadotropin-releasing hormone (GnRH)-I and -III has been extensively characterized by immunocytochemistry in the forebrain of the sea lamprey, Petromyzon marinus. However, the cellular location of lamprey GnRH-III mRNA expression by in situ hybridization in the lamprey brain has not been determined. We show for the first time the location of expression of lamprey GnRH-III, as well as provide a more comprehensive in situ study of lamprey GnRH-I and glutamic acid decarboxylase (GAD; GABA-synthesizing enzyme) mRNA expression in the brain of the lamprey in different reproductive life stages. Colorimetric and dual-label fluorescent amplification methods of in situ hybridization were used on brain tissue sections of adult, juvenile, and larval sea lamprey. In each life stage of the lamprey, expression of lamprey GnRH-I was shown in the preoptic area (POA) and the hypothalamus forming the characteristic arc-like cell population extending from the preoptic nucleus (NPO) to the neurohypophysis. Lamprey GnRH-III expression was also seen in the POA of each life stage in close proximity to lamprey GnRH-I mRNA containing neurons. GAD expression was shown in distinct cell clusters in and around the POA, in the olfactory bulb, in the dorsal thalamus beneath the habenular region, and also in the ventral-medial hypothalamus stretching from the periventricular region to the anterior portion of the rhombencephalon. Using dual-label in situ hybridization, we have shown that lamprey GnRH-I and -III mRNA are colocalized in the same cells in the POA in adult lampreys. Dual-label in situ hybridization also showed close proximity of GAD mRNA containing neurons and GnRH containing neurons in the POA. These data suggest that gamma-aminobutyric acid (GABA) may directly affect GnRH release in the brain of the sea lamprey.

The presence of tachykinins in the CNS of vertebrates has been known for many decades, and numerous studies have described their distribution in mammals. Tachykinins were also reported in the CNS of lampreys using immunohistochemistry, chromatography, and radioimmunoassay methods, but the use of substance P (SP)-specific antibodies to reveal those tachykinins could have led to an underestimation of their number in this genus. Therefore, we carried out a new immunohistochemical study on Petromyzon marinus using a commercial polyclonal antibody that binds not only to mammalian SP, but also to other neurokinins. This antibody labeled all previously described lamprey tachykinin-containing neuronal populations, but more important, labeled new populations in several parts of the brain. These include the dorsal gray of the rostral spinal cord, the dorsal column nuclei, the octavolateral area, the nucleus of the solitary tract, the medial rhombencephalic reticular formation, the lateral tegmentum of the rostral rhombencephalon, the torus semicircularis, the optic tectum, the habenula, the mammillary area, the dorsal thalamic area, the lateral hypothalamus, and the septum area. Preabsorption experiments confirmed the binding of the antibody to neurokinins and allowed us to propose that the CNS of P. marinus contains at least two different tachykinins., (Copyright 2004 Wiley-Liss, Inc.)