Neural crest origin of sympathetic neurons at the dawn of vertebrates.

The neural crest is an embryonic stem cell population unique to vertebrates1 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 ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3-8. 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 ß-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.

Landmark discoveries in elucidating the origins of the hypothalamic-pituitary system from the perspective of a basal vertebrate, sea lamprey.

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.

Odorant organization in the olfactory bulb of the sea lamprey.

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.

Forebrain dopamine neurons project down to a brainstem region controlling locomotion.

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.

Anatomy of the lamprey ear: morphological evidence for occurrence of horizontal semicircular ducts in the labyrinth of Petromyzon marinus.

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.

A novel neural substrate for the transformation of olfactory inputs into motor output.

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.

Retinotopy of visual projections to the optic tectum and pretectum in larval sea lamprey.

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.

Functional re-organization of the gills of metamorphosing sea lamprey (Petromyzon marinus): preparation for a blood diet and the freshwater to seawater transition.

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 (Jamm) and urea excretion (Jurea) in FW, and following FW to artificial seawater (ASW) transfer. Rates of Jamm and Jurea 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.

Cholecystokinin in the central nervous system of the sea lamprey Petromyzon marinus: precursor identification and neuroanatomical relationships with other neuronal signalling systems.

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.

Sensory cutaneous papillae in the sea lamprey (Petromyzon marinus L.): I. Neuroanatomy and physiology.

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.

Development and organization of the descending serotonergic brainstem-spinal projections in the sea lamprey.

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.

Distribution of adrenomedullin-like immunoreactivity in the brain of the adult sea lamprey.

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.

Aspartate immunoreactivity in the telencephalon of the adult sea lamprey: comparison with GABA immunoreactivity.

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.

The early scaffold of axon tracts in the brain of a primitive vertebrate, the sea lamprey.

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.

Developmental changes of calretinin immunoreactivity in the lamprey spinal cord.

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.

Modulation of respiratory activity by locomotion in lampreys.

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.

Respiratory rhythms generated in the lamprey rhombencephalon.

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.

Olfactory sensory neurons in the sea lamprey display polymorphisms.

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.

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.

Ontogeny of 5-HT neurons in the brainstem of the lamprey, Petromyzon marinus.

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.