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.

Colocalization of dopamine and GABA in spinal cord neurones in the sea lamprey.

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.

Neurochemical differentiation of horizontal and amacrine cells during transformation of the sea lamprey retina.

The sea lamprey is a modern representative of the earliest vertebrates (the agnathans) in which development of the eye and retina shows unique patterns. In larval stages the retina is poorly developed, and although a small central region has developed glutamatergic vertical pathways, there is no evidence of chemical differentiation of amacrine and horizontal cells in the central or lateral larval retina [Villar-Cerviño, V., Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Holstein, G.R., Martinelli, G.P., Rodicio, M.C., Anadón, R., 2006. Presence of glutamate, glycine, and gamma-aminobutyric acid in the retina of the larval sea lamprey: comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas. J. Comp. Neurol. 499, 810-827.]. However, in adults all the retina was differentiated and both amacrine and horizontal cells are well developed. Present immunocytochemical results show that the horizontal and amacrine cells of the retina begin their neurochemical differentiation during metamorphosis, when they start to express GABA, glycine, serotonin and dopamine; this occurs several years after the onset of development. Immunoreactivity for GABA, glycine and serotonin was found at early metamorphic stages, while expression of the markers of catecholaminergic amacrine cells, dopamine and tyrosine hydroxylase, was found to be delayed until intermediate metamorphic stages. GABA, which is found in some amacrine and horizontal cells of adults, was first observed in amacrine cells during early stages of transformation and then in horizontal cells during middle stages. All cells immunoreactive to serotonin or tyrosine hydroxylase/dopamine were amacrine cells. Interestingly, all these markers began expression before the appearance of opsin-immunoreactive photoreceptors in the lateral retina. The pattern of chemical differentiation of amacrine and horizontal cells was compared with that of other vertebrates and their significance was discussed.

The tegmental proliferation region in the sea lamprey.

The diencephalic/midbrain tegmental domain of the developing lamprey was characterized by the special features of the ventricular zone and distribution of some postmitotic neuronal populations, using proliferating cell nuclear antigen (PCNA) and HNK-1 immunocytochemistry. In late prolarvae and early larvae, the tegmental ventricular zone differentiated a striking arrangement of thin longitudinal crests with strongly PCNA-immunoreactive cells protruding toward the ventricle and separated by shallow valleys whose cells were faintly or moderately PCNA-immunoreactive. The tegmental ventricular zone was composed of a large caudal region located ventral to the pretectum and the midbrain tectum, and of a smaller rostral wedge-shaped region that extended dorsally between the dorsal and the ventral thalamus, in the last one, ventricular crests coursing in the zona limitans intrathalamica. In other diencephalic and midbrain regions, the ventricular zone lacked this crest-like organization and exhibited different smooth-surfaced domains rather homogeneous in thickness and appearance. The distribution of HNK-1-immunoreactive cell populations in the tegmental domain appears to be closely related with the pattern of proliferation crests. Together, our results reveal a shared basic organization in the tegmental domains of the diencephalon and midbrain of developing lamprey, indicating early appearance of the domain in vertebrate phylogeny.

Chemoarchitecture of the dorsal column nucleus of the larval sea lamprey.

We studied the organization of the dorsal column nucleus (DCN) of larval sea lamprey with immunohistochemical and tract-tracing techniques. Texas red-coupled dextran amine was injected into the spinal cord, which allowed tracing the dorsal column fibers and characterizing the DCN. The dorsal column fibers formed a dense tract coursing adjacent to the dorsal midline of the spinal cord to the caudal rhombencephalon alar plate. In larvae, most spinal cord dorsal cells and spinal ganglion perikarya, and many dorsal column fibers, were calretinin-immunoreactive. We delineated the DCN in the dorsomedial portion of the obex and preobecular alar plate. It consists of a periventricular neuronal cell layer and neurons scattered in the lateral neuropil and receives dorsal column fibers. After immunohistochemistry with antibodies against glutamate, glycine, and GABA numerous immunoreactive perikarya were observed in the DCN. In addition to glutamate-, glycine-, and GABA-immunoreactive processes, serotonin- and dopamine-immunoreactive fibers coursed in the neuropil of this nucleus. A few small calretinin-immunoreactive perikarya were also observed in the DCN. Our results reveal the presence of inhibitory and excitatory transmitters in neurons of the DCN, and suggest that dopamine and serotonin modulate the activity of this nucleus.

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.

Reelin immunoreactivity in the larval sea lamprey brain.

In order to analyze the presence of a reelin-like protein in the brain of a primitive vertebrate with a laminar-type brain, such as the sea lamprey, Western blot and immunohistochemical approaches were employed by using the G10 and 142 reelin-specific monoclonal antibodies. Western blots of lamprey brain extracts showed bands of about 400 kDa, 180 kDa and others below 100 kDa; similar bands were observed in samples from rat cerebellum. In different larval stages there was a prominent reelin immunolabeling associated with the olfactory bulb, pallial regions, habenula, hypothalamus and optic tectum. In addition, the olfactory and optic tracts, as well as the afferent and efferent (fasciculus retroflexus) tracts of the habenular ganglion, also showed immunopositivity in these stages. Interestingly, the highest level of labeling was observed in premetamorphic larvae, just prior to entering the metamorphic stage. These data indicate that reelin expression is also prominent in brains of primitive vertebrates without layered cortical regions, suggesting that some physiological roles of reelin not related to the regulation of neuronal migration in layered cortical regions (i.e. involvement in axon pathfinding, synaptogenesis, dendritic arborization and neuronal plasticity) might have appeared earlier in evolution.