Ancient evolutionary origin of vertebrate enteric neurons from trunk-derived neural crest.

The enteric nervous system of jawed vertebrates arises primarily from vagal neural crest cells that migrate to the foregut and subsequently colonize and innervate the entire gastrointestinal tract. Here we examine development of the enteric nervous system in the basal jawless vertebrate the sea lamprey (Petromyzon marinus) to gain insight into its evolutionary origin. Surprisingly, we find no evidence for the existence of a vagally derived enteric neural crest population in the lamprey. Rather, labelling with the lipophilic dye DiI shows that late-migrating cells, originating from the trunk neural tube and associated with nerve fibres, differentiate into neurons within the gut wall and typhlosole. We propose that these trunk-derived neural crest cells may be homologous to Schwann cell precursors, recently shown in mammalian embryos to populate post-embryonic parasympathetic ganglia, including enteric ganglia. Our results suggest that neural-crest-derived Schwann cell precursors made an important contribution to the ancient enteric nervous system of early jawless vertebrates, a role that was largely subsumed by vagal neural crest cells in early gnathostomes.

An atlas of anterior hox gene expression in the embryonic sea lamprey head: Hox-code evolution in vertebrates.

In the hindbrain and the adjacent cranial neural crest (NC) cells of jawed vertebrates (gnathostomes), nested and segmentally-restricted domains of Hox gene expression provide a combinatorial Hox-code for specifying regional properties during head development. Extant jawless vertebrates, such as the sea lamprey (Petromyzon marinus), can provide insights into the evolution and diversification of this Hox-code in vertebrates. There is evidence for gnathostome-like spatial patterns of Hox expression in lamprey; however, the expression domains of the majority of lamprey hox genes from paralogy groups (PG) 1-4 are yet to be characterized, so it is unknown whether they are coupled to hindbrain segments (rhombomeres) and NC. In this study, we systematically describe the spatiotemporal expression of all 14 sea lamprey hox genes from PG1-PG4 in the developing hindbrain and pharynx to investigate the extent to which their expression conforms to the archetypal gnathostome hindbrain and pharyngeal hox-codes. We find many similarities in Hox expression between lamprey and gnathostome species, particularly in rhombomeric domains during hindbrain segmentation and in the cranial neural crest, enabling inference of aspects of Hox expression in the ancestral vertebrate embryonic head. These data are consistent with the idea that a Hox regulatory network underlying hindbrain segmentation is a pan vertebrate trait. We also reveal differences in hindbrain domains at later stages, as well as expression in the endostyle and in pharyngeal arch (PA) 1 mesoderm. Our analysis suggests that many Hox expression domains that are observed in extant gnathostomes were present in ancestral vertebrates but have been partitioned differently across Hox clusters in gnathostome and cyclostome lineages after duplication.

Characterization of Somatically-Eliminated Genes During Development of the Sea Lamprey (Petromyzon marinus).

The sea lamprey (Petromyzon marinus) is a basal vertebrate that undergoes developmentally programmed genome rearrangements (PGRs) during early development. These events facilitate the elimination of ∼20% of the genome from the somatic cell lineage, resulting in distinct somatic and germline genomes. Thus far only a handful of germline-specific genes have been definitively identified within the estimated 500 Mb of DNA that is deleted during PGR, although a few thousand germline-specific genes are thought to exist. To improve our understanding of the evolutionary/developmental logic of PGR, we generated computational predictions to identify candidate germline-specific genes within a new transcriptomic dataset derived from adult germline and the early embryonic stages during which PGR occurs. Follow-up validation studies identified 44 germline-specific genes and further characterized patterns of transcription and DNA loss during early embryogenesis. Expression analyses reveal that many of these genes are differentially expressed during early embryogenesis and presumably function in the early development of the germline. Ontology analyses indicate that many of these germline-specific genes play known roles in germline development, pluripotency, and oncogenesis (when misexpressed). These studies provide support for the theory that PGR serves to segregate molecular functions related to germline development/pluripotency in order to prevent their potential misexpression in somatic cells. This larger set of eliminated genes also allows us to extend the evolutionary/developmental breadth of this theory, as some deleted genes (or their gnathostome homologs) appear to be associated with the early development of somatic lineages, perhaps through the evolution of novel functions within gnathostome lineages.

Evolutionarily conserved role for SoxC genes in neural crest specification and neuronal differentiation.

Members of the Sox family of transcription factors play a variety of critical developmental roles in both vertebrates and invertebrates. Whereas SoxBs and SoxEs are involved in neural and neural crest development, respectively, far less is known about members of the SoxC subfamily. To address this from an evolutionary perspective, we compare expression and function of SoxC genes in neural crest cells and their derivatives in lamprey (Petromyzon marinus), a basal vertebrate, to frog (Xenopus laevis). Analysis of transcript distribution reveals conservation of lamprey and X. laevis SoxC expression in premigratory neural crest, branchial arches, and cranial ganglia. Moreover, morpholino-mediated loss-of-function of selected SoxC family members demonstrates essential roles in aspects of neural crest development in both organisms. The results suggest important and conserved functions of SoxC genes during vertebrate evolution and a particularly critical, previously unrecognized role in early neural crest specification.

A fate-map for cranial sensory ganglia in the sea lamprey.

Cranial neurogenic placodes and the neural crest make essential contributions to key adult characteristics of all vertebrates, including the paired peripheral sense organs and craniofacial skeleton. Neurogenic placode development has been extensively characterized in representative jawed vertebrates (gnathostomes) but not in jawless fishes (agnathans). Here, we use in vivo lineage tracing with DiI, together with neuronal differentiation markers, to establish the first detailed fate-map for placode-derived sensory neurons in a jawless fish, the sea lamprey Petromyzon marinus, and to confirm that neural crest cells in the lamprey contribute to the cranial sensory ganglia. We also show that a pan-Pax3/7 antibody labels ophthalmic trigeminal (opV, profundal) placode-derived but not maxillomandibular trigeminal (mmV) placode-derived neurons, mirroring the expression of gnathostome Pax3 and suggesting that Pax3 (and its single Pax3/7 lamprey ortholog) is a pan-vertebrate marker for opV placode-derived neurons. Unexpectedly, however, our data reveal that mmV neuron precursors are located in two separate domains at neurula stages, with opV neuron precursors sandwiched between them. The different branches of the mmV nerve are not comparable between lampreys and gnatho-stomes, and spatial segregation of mmV neuron precursor territories may be a derived feature of lampreys. Nevertheless, maxillary and mandibular neurons are spatially segregated within gnathostome mmV ganglia, suggesting that a more detailed investigation of gnathostome mmV placode development would be worthwhile. Overall, however, our results highlight the conservation of cranial peripheral sensory nervous system development across vertebrates, yielding insight into ancestral vertebrate traits.

Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis.

BACKGROUND: Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics. RESULTS: We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection. CONCLUSIONS: HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.

Importance of SoxE in neural crest development and the evolution of the pharynx.

The neural crest, a defining character of vertebrates, is of prime importance to their evolutionary origin. To understand neural crest evolution, we explored molecular mechanisms underlying craniofacial development in the basal jawless vertebrate, sea lamprey (Petromyzon marinus), focusing on the SoxE (Sox8, Sox9 and Sox10) gene family. In jawed vertebrates, these are important transcriptional regulators of the neural crest, and the loss of Sox9 causes abnormal craniofacial development. Here we report that two lamprey SoxE genes are expressed in migrating neural crest and crest-derived prechondrocytes in posterior branchial arches, whereas a third paralogue is expressed later in the perichondrium and mandibular arch. Morpholino knock-down of SoxE1 reveals that it is essential for posterior branchial arch development, although the mandibular arch is unaffected. The results show that chondrogenic function of SoxE regulators can be traced to the lamprey-gnathostome common ancestor and indicate that lamprey SoxE genes might have undergone independent duplication to have distinct functions in mandibular versus caudal branchial arches. This work sheds light on the homology of vertebrate branchial arches and supports their common origin at the base of vertebrates.

An ancient mechanism of hindbrain patterning has been conserved in vertebrate evolution.

The hindbrain is a vertebrate-specific embryonic structure of the central nervous system formed by iterative transitory units called rhombomeres (r). Rhombomeric cells are segregated by interhombomeric boundaries which are prefigured by sharp gene expression borders. The positioning of the first molecular boundary within the hindbrain (the prospective r4/r5 boundary) responds to the expression of an Iroquois (Irx) gene in the anterior (r4) and the gene vHnf1 at the posterior (r5). However, while Irx3 is expressed anteriorly in amniotes, a novel Irx gene, iro7, acts in teleosts. To assess the evolutionary history of the genes responsible for the positioning of the r4/r5 boundary in vertebrates, we have stepped outside the gnathostomes to investigate these genes in the agnathans Lethenteron japonicum and Petromyzon marinus. We identified one representative of the Hnf1 family in agnathans. Its expression pattern recapitulates that of vHnf1 and Hnf1 in higher vertebrates. Our phylogenetic analysis places this gene basal to gnathostome Hnf1 and vHnf1 genes. We propose that the duplication of an ancestral hnf1 gene present in the common ancestor of agnathans and gnathostomes gave rise to the two genes found in gnathostomes. We have also amplified 3 Irx genes in L. japonicum: LjIrxA, LjIrxC, LjIrxD. The expression pattern of LjIrxA (the agnathan Irx1/3 ortholog) resembles those of Irx3 or iro7 in gnathostomes. We propose that an Irx/hnf1 pair already present in early vertebrates positioned the r4/r5 boundary and that gene duplications occurred in these gene families after the divergence of the agnathans.

Dissecting early regulatory relationships in the lamprey neural crest gene network.

The neural crest, a multipotent embryonic cell type, originates at the border between neural and nonneural ectoderm. After neural tube closure, these cells undergo an epithelial-mesenchymal transition, migrate to precise, often distant locations, and differentiate into diverse derivatives. Analyses of expression and function of signaling and transcription factors in higher vertebrates has led to the proposal that a neural crest gene regulatory network (NC-GRN) orchestrates neural crest formation. Here, we interrogate the NC-GRN in the lamprey, taking advantage of its slow development and basal phylogenetic position to resolve early inductive events, 1 regulatory step at the time. To establish regulatory relationships at the neural plate border, we assess relative expression of 6 neural crest network genes and effects of individually perturbing each on the remaining 5. The results refine an upstream portion of the NC-GRN and reveal unexpected order and linkages therein; e.g., lamprey AP-2 appears to function early as a neural plate border rather than a neural crest specifier and in a pathway linked to MsxA but independent of ZicA. These findings provide an ancestral framework for performing comparative tests in higher vertebrates in which network linkages may be more difficult to resolve because of their rapid development.

Development of the viscerocranial skeleton during embryogenesis of the sea lamprey, Petromyzon Marinus.

Evolution of the skeleton was a key transition in early vertebrates. Lampreys lack a mineralized skeleton but possess cartilaginous neurocranial and viscerocranial elements. In lampreys, the visceral skeleton develops as a fused branchial basket supporting the pharynx. Here, we have adapted Alcian blue staining of lamprey cartilage and show this method results in cartilage fluorescence that we used to describe development of the branchial skeleton in Petromyzon marinus between 17 and 63 days of development. We show that skeletal rods develop from condensations of flattened discoidal chondrocytes and may involve cellular intercalation. Lamprey trabecular, parachordal, and subchordal cartilages consist of aggregations of polygonal chondrocytes positioned on the ventral and lateral surfaces of the notochord. We speculate that morphological differences relate to functional differences in the cartilage. We show that differentiated skeletal rods are derived from neural crest. Finally, we show how branchial muscles intercalate with skeletal rods of the branchial basket.

Epicardial development in lamprey supports an evolutionary origin of the vertebrate epicardium from an ancestral pronephric external glomerulus.

The epicardium is the outer layer of the vertebrate heart. Both the embryonic epicardium and its derived mesenchyme are critical to heart development, contributing to the coronary vasculature and modulating the proliferation of the ventricular myocardium. The embryonic epicardium arises from an extracardiac, originally paired progenitor tissue called the proepicardium, a proliferation of coelomic cells found at the limit between the liver and the sinus venosus. Proepicardial cells attach to and spread over the cardiac surface giving rise to the epicardium. Invertebrate hearts always lack of epicardium, and no hypothesis has been proposed about the origin of this tissue and its proepicardial progenitor in vertebrates. We herein describe the epicardial development in a representative of the most basal living lineage of vertebrates, the agnathan Petromyzon marinus (lamprey). The epicardium in lampreys develops by migration of coelomic cells clustered in a paired structure at the roof of the coelomic cavity, between the pronephros and the gut. Later on, these outgrowths differentiate into the pronephric external glomerulus (PEG), a structure composed of capillary networks, mesangial cells, and podocytes. This observation is consistent with the conclusion that the primordia of the most anterior pair of PEG in agnathans have been retained and transformed into the proepicardium in gnathostomes. Glomerular progenitor cells are highly vasculogenic and probably allowed for the vascularization of a cardiac tube primarily devoid of coronary vessels. This new hypothesis accounts for the striking epicardial expression of Wt1 and Pod1, two transcription factors essential for development of the excretory system.

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.

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.

Dynamic expression of the LIM-homeodomain gene Lhx15 through larval brain development of the sea lamprey (Petromyzon marinus).

LIM-homeodomain genes encode a family of transcription factors with highly conserved roles in the patterning and regionalisation of the vertebrate brain. The expression of one of those genes, Lhx15, in the embryonic lamprey brain, characterises precise functional subdivisions. In order to analyse the non-embryonic development of the lamprey brain, we chose this gene to perform in situ hybridisations in Petromyzon marinus larvae of different ages. We demonstrate the usefulness of Lhx15 to follow the development and morphogenesis of brain structures and show the dynamical expression of this gene through time. Furthermore, we provide evidence for the evolutionary conservation of the expression of this gene in the spinal cord, notochord and urogenital system.

Evolution of the vertebrate claudin gene family: insights from a basal vertebrate, the sea lamprey.

Claudins are major constituents of tight junctions, contributing both to their intercellular sealing and selective permeability properties. While claudins and claudin-like molecules are present in some invertebrates, the association of claudins with tight junctions has been conclusively documented only in vertebrates. Here we report the sequencing, phylogenetic analysis and comprehensive spatiotemporal expression analysis of the entire claudin gene family in the basal extant vertebrate, the sea lamprey. Our results demonstrate that clear orthologues to about half of all mammalian claudins are present in the lamprey, suggesting that at least one round of whole genome duplication contributed to the diversification of this gene family. Expression analysis revealed that claudins are expressed in discrete and specific domains, many of which represent vertebrate-specific innovations, such as in cranial ectodermal placodes and the neural crest; whereas others represent structures characteristic of chordates, e.g. pronephros, notochord, somites, endostyle and pharyngeal arches. By comparing the embryonic expression of claudins in the lamprey to that of other vertebrates, we found that ancestral expression patterns were often preserved in higher vertebrates. Morpholino mediated loss of Cldn3b demonstrated a functional role for this protein in placode and pharyngeal arch morphogenesis. Taken together, our data provide novel insights into the origins and evolution of the claudin gene family and the significance of claudin proteins in the evolution of vertebrates.

Development of the dopamine-immunoreactive system in the central nervous system of the sea lamprey.

The development of dopamine-immunoreactive (DAir) populations in the central nervous system of the sea lamprey, a modern representative of the earliest vertebrates, was studied to achieve further understanding of dopaminergic systems in vertebrates. The first DAir cell groups appeared in the spinal cord, the posterior tubercle nucleus and the dorsal hypothalamic nucleus of prolarval stages. In larvae, new DAir cell groups were observed in the caudal preoptic nucleus, the postoptic commissure nucleus, the postinfundibular commissure nucleus and the caudal rhombencephalon. All these DAir cell groups observed in larvae were also DA-positive in adults, which showed only one new DAir cell group found in the ventral hypothalamic nucleus. Occasional DAir cells were observed in the telencephalon, the ventral thalamus and/or the isthmus of large larvae and adults. From medium-sized larvae to adults, the regions most richly innervated by DAir fibers were the neurohypophysis, the striatum, the pretectum and the midbrain tegmentum. Striking differences are observed between lampreys and other vertebrates in regard to the relative time of appearance of DAir cells, which is probably related with the complex life cycle of the sea lamprey.

RNA interference technology to control pest sea lampreys--a proof-of-concept.

The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

The sea lamprey tryptophan hydroxylase: new insight into the evolution of the serotonergic system of vertebrates.

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

Inhibitory descending rhombencephalic projections in larval sea lamprey.

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.

Localization, pharmacology, and organization of brain locomotor areas in larval lamprey.

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].