Morphological and functional aspects of the epidermis of the sea lamprey Petromyzon marinus throughout development.

The development of the epidermis of sea lamprey Petromyzon marinus along the whole life cycle was studied using conventional staining techniques and lectin histochemistry. The epidermis undergoes variations in morphology and thickness throughout development. The simple cuboidal epithelium found in the epidermis of prolarvae becomes stratified cubic in the adult by increasing the number of cell layers. The cuticle thickness undergoes a steady increase during the larval period. There are changes in the glycoconjugate composition of the three main cell types of the P. marinus epidermis, mucous, granular and skein cells, which are more pronounced after metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method shows the presence of both acidic and neutral glycoconjugates in the mucous cells, indicating their secretory function. Moreover, lectin analysis reveals a mucous secretion containing glycoconjugates such as sulphated glycosaminoglycans (N-acetylglucosamine and N-acetylgalactosamine) and N-glycoproteins rich in mannose. Although granular cells are AB-PAS negative, they exhibit a similar glycoconjugate composition to the mucous cells. Moreover, granular cells show sialic acid positivity in larvae but this monosaccharide residue is not detected after metamorphosis. The skein cells, a unique cell of lampreys, are negative to AB-PAS staining but they mostly contain l-fucose and sialic acid residues, which also disappear after metamorphosis. The function of the granular and skein cells is still unknown but the role of their glycoconjugate composition is discussed. In addition, a different cellular origin is suggested for these two types of cells.

Distal-less-like protein distribution in the larval lamprey forebrain.

A polyclonal antibody against the Drosophila distal-less (DLL) protein, cross-reactive with cognate vertebrate proteins, was employed to map DLL-like expression in the midlarval lamprey forebrain. This work aimed to characterize in detail the separate diencephalic and telencephalic DLL expression domains, in order to test our previous modified definition of the lamprey prethalamus [Pombal and Puelles (1999) J Comp Neurol 414:391-422], adapt our earlier schema of prosomeric subdivisions in the lamprey forebrain to more recent versions of this model [Pombal et al. (2009) Brain Behav Evol 74:7-19] and reexamine the pallio-subpallial regionalization of the lamprey telencephalon. We observed a large-scale conservation of the topologic distribution of the DLL protein, in consonance with patterns of Dlx expression present in other vertebrates studied. Moreover, evidence was obtained of substantial numbers of DLL-positive neurons in the olfactory bulb and the cerebral hemispheres, in a pattern consistent with possible tangential migration out of the subpallium into the overlying pallium, as occurs in mammals, birds, frogs and teleost fishes.

Developmental changes of the GABA-immunoreactive fibers in the lamprey spinal cord.

The changes in distribution and number of GABA immunoreactive (GABA-ir) fibers from postembryonic stages to adulthood in the lamprey spinal cord white matter were studied by using immunocytochemical techniques. From prolarvae to adult spawning animals there was an increase of the number of GABA-ir fibers. Three phases can be distinguished: (a) from prolarvae to middle size larvae (around 50 mm in body length) an increase in the number of GABA-ir fibers per section is observed. Furthermore, an adult-like pattern of GABA-ir fibers distribution is established during this phase. (b) Then, the number of GABA-ir fibers remains stable until metamorphosis, the end of the larval period. (c) Finally, in young postmetamorphic and adult animals the number of GABA-ir fibers is higher than in larvae. These observations, joined to the changes previously reported in the GABA-ir neurons, indicate that at least parts of the GABA inhibitory component of the spinal locomotor network is reorganized during the lamprey life cycle and it may indicate different inhibitory requirements in the locomotor network.

Development of GABA-immunoreactive cells in the spinal cord of the sea lamprey, P. marinus.

The lamprey spinal cord increases in length and size during all its life cycle; thus, it is expected that new cells will be generated. This expectation suggests that the locomotor circuits must be continuously remodeled. Key elements in the cellular network controlling locomotor behavior are inhibitory cells. Here, we studied the gamma-aminobutyric acid-immunoreactive (GABA-ir) cells in the lamprey spinal cord during postembryonic development. Three major populations of GABA-ir cells were identified according to their distribution: those located in the gray matter, those contacting the cerebrospinal liquid (LC cells), and those located in the white matter. The results show (1). the number of GABA-ir cells per segment increase from prolarvae (<10 mm) to adulthood; (2). the lower number of GABA-ir cells in 100 microm of spinal cord is 66 +/- 7, found in premetamorphic larvae, and the highest is 107 +/- 6, found in postmetamorphic animals; (3). the gray matter and LC GABA-ir cells show different variations in number depending on the developmental period. Thus, in the 10-mm larvae, the gray matter GABA-ir cells are more abundant than LC cells, whereas in the young postmetamorphic specimens, the contrary occurs. Most of the GABA-ir cells located in the white matter were classified as edge cells. They increase in number from the beginning of the prolarval period, where there are not white matter-positive cells, to the middle larval period, where there are 9 +/- 4 GABA-ir edge cells per segment. This value was unaltered in later periods, where GABA-ir edge cells represent 20-30% of the total number of edge cells per segment. The increase in number of GABA-ir cells in these populations during a specific point of the lamprey life cycle may indicate different inhibitory requirements of the locomotor circuit at different developmental periods.

Immunocytochemical localization of calretinin in the olfactory system of the adult lamprey, Lampetra fluviatilis.

The distribution of calretinin immunoreactive (CR-ir) structures in the adult lamprey (Lampetra fluviatilis) olfactory system was studied by using immunocytochemical techniques. In the olfactory epithelium, a subpopulation of olfactory receptor cells was CR-ir. In the olfactory bulbs, three different cell populations were observed. Large CR-ir cells (mitral cells) were located medially to the olfactory glomeruli and occasionally between them. In the inner cellular layer, two types of CR-ir perikarya were found: numerous small CR-ir cells (granule cells) and some medium-sized CR-ir cells (putative displaced periglomerular cells). In addition, different intensities of CR-ir fibers were present in particular rootlets of the olfactory nerves, as well as in particular glomeruli. The presence of CR-ir cells and fibers in all layers of the lamprey olfactory bulbs supports the idea that this protein is present in pathways underlying the processing of sensory information throughout evolution.

Distribution of choline acetyltransferase-immunoreactive structures in the lamprey brain.

The distribution of cholinergic neurons and fibers was studied immunohistochemically in the brain of two species of lampreys (Petromyzon marinus and Lampetra fluviatilis), by using an antiserum against choline acetyltransferase (ChAT). The results obtained in both species were similar, but there appeared some interspecies differences. In the forebrain, cholinergic cells were present in the striatum, preoptic region, paraventricular nucleus, pineal and parapineal organs, habenula, and pretectum. The cranial nerve motoneurons (III, IV, V, VI, VII, IX, and X), the first and second spino-occipital nerves (so), and the ventral horn of the spinal cord showed a strong ChAT immunoreactivity. Additional cholinergic neurons were observed: the mesencephalic M5 nucleus of Schober, two different cell populations in the isthmic region, the efferent component of the eighth nerve, putative preganglionic parasympathetic cells, cells in the solitary tract nucleus, and the rhombencephalic reticular formation. Cholinergic fibers were widely distributed in the brain. Comparison with previous studies in other vertebrates suggests that major cholinergic pathways, like tectal innervation from the isthmic region, are also present in lampreys. Of particular interest was the prominent projection to the neurohypophysis from cholinergic neurons in the preoptic region and paraventricular nucleus. Present data were analyzed within the segmental paradigm, as was previously done in other vertebrates. Our results reveal that the organization of many cholinergic systems in the lamprey as, for example, in the striatal, preoptic, and isthmic regions, comprises features of the anamniote brain that remain common to all living amniotes studied so far, thus being conservative to a surprisingly high degree. Therefore, the distribution of ChAT-immunoreactive structures in the lamprey brain is, in general, comparable to that previously described in other vertebrate species.

GABA-immunoreactive internuclear neurons in the ocular motor system of lampreys.

The presence of internuclear neurons in the abducens and oculomotor nuclei of lampreys [González, M.J., Pombal, M.A., Rodicio, M.C. and Anadón, R., Internuclear neurons of the ocular motor system of the larval sea lamprey, J. Comp. Neurol. 401 (1998) 1-15] indicates that coordination of eye movements by internuclear neurons appeared early during the evolution of vertebrates. In order to investigate the possible involvement of inhibitory neurotransmitters in internuclear circuits, the distribution of gamma-aminobutyric acid (GABA) in the extraocular motor nuclei of the lamprey was studied using immunocytochemical techniques. Small GABA-immunoreactive (GABAir) neurons were observed in the three ocular motor nuclei. Numerous GABAir neurons were observed in the group of internuclear neurons of the dorsal rectus oculomotor subnucleus. A second group of GABAir neurons was observed among and below the trochlear motoneurons. Two further groups of GABAir interneurons, periventricular and lateral, were located in the abducens nucleus among the cells of the caudal rectus and the ventral rectus motor subnuclei, respectively. In addition to the presence of GABAir neurons, in all the ocular motor nuclei the motoneurons were contacted by numerous GABAir boutons. Taken together, these results suggest that GABA is involved as a neurotransmitter in internuclear pathways of the ocular motor system of lampreys.

Prosomeric map of the lamprey forebrain based on calretinin immunocytochemistry, Nissl stain, and ancillary markers.

The structural organization of the lamprey extratelencephalic forebrain is re-examined from the perspective of the prosomeric segmental paradigm. The question asked was whether the prosomeric forebrain model used for gnathostomes is of material advantage for interpreting subdivisions in the lamprey forebrain. To this aim, the main longitudinal and transverse landmarks recognized by the prosomeric model in other vertebrates were identified in Nissl-stained lamprey material. Lines of cytoarchitectural discontinuity and contours of migrated neuronal groups were mapped in a two-dimensional sagittal representation and were also classified according to their radial position. Immunocytochemical mapping of calretinin expression in adjacent sections served to define particular structural units better, in particular, the dorsal thalamus. These data were complemented by numerous other chemoarchitectonic observations obtained with ancillary markers, which identified additional specific formations, subdivisions, or boundaries. Emphasis was placed on studying whether such chemically defined neuronal groups showed boundaries aligned with the postulated inter- or intraprosomeric boundaries. The course of diverse axonal tracts was studied also with regard to their prosomeric topography. This analysis showed that the full prosomeric model applies straightforwardly to the lamprey forebrain. This finding implies that a common segmental and longitudinal organization of the neural tube may be primitive for all vertebrates. Interesting novel aspects appear in the interpretation of the lamprey pretectum, the dorsal and ventral thalami, and the hypothalamus. The topologic continuity of the prosomeric forebrain regions with evaginated or non-evaginated portions of the telencephalon was also examined.

Choline acetyltransferase immunoreactivity in the hypothalamoneurohypophysial system of the lamprey.

The cholinergic innervation of the neurohypophysis of the lampreys Petromyzon marinus and Lampetra fluviatilis was studied by means of immunocytochemical techniques with antibodies directed against the enzyme choline acetyltransferase (ChAT). The results obtained in both species were basically similar. A rich innervation by ChAT-immunoreactive fibres was found throughout the neurohypophysis. These fibres originate from cholinergic neurons located in the preoptic region and the paraventricular nucleus. Some of these cholinergic neurons are in contact with the cerebrospinal fluid. Numerous axonal swellings were evident in the tuberal region of the sea lamprey, but not in the river lamprey. The possible pathways of cholinergic release in the lamprey hypophysis are discussed.

Cholinergic and GABAergic neuronal elements in the pineal organ of lampreys, and tract-tracing observations of differential connections of pinealofugal neurons.

The putative cholinergic and GABAergic elements of the pineal organ of lampreys were investigated with immunocytochemistry to choline acetyltransferase (ChAT) and gamma-aminobutyric acid (GABA), and by acetylcholinesterase (AChE) histochemistry. For comparison we also carried out immunocytochemistry to serotonin (5-HT) and a tract-tracing investigation of the two types of projecting cells, i. e., ganglion cells and long-axon photoreceptors. Most photoreceptors were ChAT-immunoreactive (ChAT-ir) and AChE-positive, while ganglion cells and the pineal tract were ChAT-negative and AChE-negative or only faintly positive. These results strongly suggest the presence of a cholinergic system of photoreceptors in the lamprey pineal organ. GABA-ir fibers that appear to originate from faintly to moderately stained ganglion cells were observed in the pineal stalk. Immunocytochemistry to 5-HT indicated the presence of two types of 5-HT-ir cells, bipolar cells and ganglion-like cells. The connections of the ganglion cells and long-axon photoreceptors were also studied by application of DiI to the pineal stalk in fixed brains or of biotinylated dextran amine (BDA) to one of the main targets of pinealofugal fibers (optic tectum or mesencephalic tegmentum) in isolated brains in vitro. Some long-axon photoreceptors and ganglion cells were labeled from the optic tectum. However, BDA application to the tegmentum exclusively labeled ganglion cells in the pineal organ. These results indicate that the two morphological types of afferent pineal neuron have different projections. No labeled cells were observed in the parapineal organ in BDA experiments, indicating that this organ and the pineal organ are involved in different neural circuits.

Afferent and efferent connections of the parapineal organ in lampreys: a tract tracing and immunocytochemical study.

The neural connections of the parapineal organ of two species of lampreys were studied with the fluorescent dye 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) and with immunocytochemistry. The lamprey parapineal organ consists of a vesicle and a ganglion that are connected to the left habenula. Labeling experiments included the application of DiI to the parapineal organ, left and right fasciculus retroflexus, left habenula, and the left pretectal region. Afferent parapineal fibers run in the left fasciculus retroflexus to the interpeduncular nucleus. The parapineal fibers of this fascicle arose from parapineal ganglion cells, whereas DiI application to the left habenula labeled both neurons of this ganglion and bipolar cells in the parapineal vesicle. Efferent neurons were observed in the left habenula, and bilaterally in the subhippocampal nucleus and the dorsal pretectum. Labeling with DiI also revealed a hippocampal projection. Immunocytochemical study of the parapineal vesicle revealed serotonin-immunoreactive cells in both species of lamprey, as well as substance P-immunoreactive (SP-ir) cells in sea lamprey and choline acetyltransferase-immunoreactive (ChAT-ir) cells in the river lamprey. The SP-ir cells and ChAT-ir cells formed a rich neuropil in the parapineal ganglion. Calretinin-ir cells were numerous in the ganglion. Neuropeptide Y-immunoreactive and gamma-aminobutyric acid-immunoreactive efferent fibers were observed in the parapineal organ. Neuropeptide Y-immunoreactive fibers originate in the subhippocampal nucleus, whereas gamma-aminobutyric acid-immunoreactive fibers might also arise in the pretectal nucleus. A few galanin-ir fibers were observed. These results indicate that the parapineal connections are completely different from those of the pineal organ. The possible homology between parapineal organs of vertebrates is discussed.

Internuclear neurons of the ocular motor system of the larval sea lamprey.

The internuclear neurons of the ocular motor system of lampreys are characterized here for the first time. Horseradish peroxidase (HRP), fluorescein-, or Texas red-(TRDA) coupled dextran-amine applied into the oculomotor nucleus of larval lamprey (Petromyzon marinus) retrogradely labeled two populations of contralateral abducens interneurons, one lateral and the other periventricular. Tracer application to the abducens nucleus anterogradely labeled thick contralateral fibers that specifically contact the medial rectus motor subnucleus by means of large boutons. Local application of TRDA to this subnucleus allowed identification of the lateral abducens interneurons as the origin of this projection. Electron microscopy of the medial rectus motor subnucleus showed large boutons bearing round synaptic vesicles that contact on the perikarya, as well as small boutons with pleomorphic vesicles. This lateral rectus (abducens) -- medial rectus (oculomotor) internuclear projection of lampreys appears to be similar to those involved in the coordination of horizontal eye movements in mammals. The periventricular abducens interneurons projected bilaterally to other oculomotor subnuclei. Tracer application to the abducens nucleus labeled a group of small interneurons in the ipsilateral dorsal rectus motor subnucleus. Anterograde labeling indicates that oculomotor interneurons project ipsilaterally to the ventral rectus abducens subnucleus, thus, corresponding to oculomotor interneurons found in mammals and frogs. The interneurons of the dorsal rectus and ventral rectus motor subnuclei are probably involved in the control of conjugate vertical eye movements. The present results strongly suggest that the internuclear coordination of conjugate eye movements appeared in the earliest vertebrates. The homologies of extraocular muscles of lampreys and gnathostomes were reexamined.

Centrifugal fibers are the only GABAergic structures of the retina of the larval sea lamprey: an immunocytochemical study.

The structures of the retina immunoreactive to GABA are described in larval lamprey. Although GABAergic cells develop early in the retinas of vertebrates, no GABA-immunoreactive perikarya were observed in the retina of lamprey larvae. The only GABA-immunoreactive structures were beaded fibers of the centrifugal system, which produced a dense plexus at the level of the optic fiber/inner plexiform layer in both the central (photoreceptor-bearing) and lateral (no-photoreceptor) parts of the retina. These fibers do not ascend toward the outer plexiform layer. Nerve fibers in the optic nerve and neuronal perikarya of the M5 nucleus of the mesencephalon, which is known to project to the retina, were also GABA-immunoreactive. The distribution of centrifugal fibers closely matches that of ganglion cells revealed by retrograde labelling with fluorescein-coupled dextran-amine, and the presence of biplexiform ganglion cells in larvae is confirmed. That the ganglion cells and the centrifugal fibers appears to be the only structures differentiated in the lateral retina of the larva suggests that the GABAergic centrifugal fibers may have a role, perhaps the neurotrophic maintenance of retinal ganglion cells, during the very long larval phase of lampreys.

Afferents of the lamprey striatum with special reference to the dopaminergic system: a combined tracing and immunohistochemical study.

The origin of afferents to the striatum in lamprey (Lampetra fluviatilis) was studied by using fluorescein-coupled dextran-amines (FDA). Injection of FDA into the striatum retrogradely labeled several cell populations in the forebrain and the rostral rhombencephalon. No retrograde labeled cells were seen in the mesencephalon. A dopamine-specific antiserum was used to determined the distribution of dopaminergic perikarya and fibers. Many dopamine-immunoreactive (DA-ir) fibers were present throughout the brain, but the highest density of labeled fibers was in the mediobasal prosencephalon, especially in the striatum, the lateral hypothalamic area, and the neurohypophysis. Most DA-ir cells were located in the mediobasal diencephalon (preoptic region, nucleus commissurae postopticae, hypothalamus, and nucleus tuberculi posterioris). In the mesencephalon, only a few immunopositive cells were observed in the tectum opticum. In the rhombencephalon, DA-ir cells were observed in the isthmic region, dorsally to the descending trigeminal tract, and caudally to the posterior rhombencephalic reticular nucleus. The rostralmost spinal cord received many descending DA-ir fibers from the brainstem. Along the spinal cord, DA-ir neurons were also found, some of which projected to the medioventral surface, forming a prominent plexus. On the basis of double-labeling experiments, it is shown that the dopaminergic input to the striatum originates from the nucleus tuberculi posterioris. Thus, the striatum receives inputs from different structures, including a strong dopaminergic innervation from the diencephalon. Much of the dopaminergic system in Lampetra fluviatilis is basically similar to that seen in some teleosts, but it presents differences with other anamniote (elasmobranch) as well as amniote groups.

Organization of the lamprey striatum - transmitters and projections.

The purpose of the present study is to characterize the striatum of the lamprey by immunohistochemical and tracing techniques. Cells immunoreactive for GABA and substance P (SP), and positive for acetylcholinesterase, are present in the lamprey striatum. Immunoreactive (ir) fibers were detected by antisera raised against SP, dopamine, enkephalin and serotonin. These immunoreactive fibers were mainly located in the periventricular neuropil that borders the striatum and in which GABAergic striatal neurons distributed their dendritic arbors. Putative connections between the striatum, the ventral part of the lateral pallium, and the diencephalic motor centers involved in the control of locomotion were studied by using fluorescein-coupled dextran amines (FDA) as a tracer. The striatum projects to the ventral part of the lateral pallium (lpv), where GABA-ir cells and SP-ir fibers were also present. The lpv in turn projects to the ventral thalamus, which has descending connections to the reticulospinal cells involved in the control of locomotion. These results, together with previous findings of histaminergic and neurotensin projections, suggest that the lamprey striatum and its inputs with regard to neurotransmitters/modulators are very similar to those of modem amniotes, including primates, and are thus conserved to a high degree.

An immunocytochemical study of encephalic photoreceptors in three species of lamprey.

The extraretinal and extrapineal photoreceptors of three species of adult lamprey, sea lamprey (Petromyzon marinus), river lamprey (Lampetra fluviatilis) and silver lamprey (Ichthyomyzon unicuspis) were studied using antibodies raised against photoreceptor rod and cone opsins, alpha-transducin and arrestin. In all three species cells in the pineal organ (P), parapineal organ (PP), nucleus preopticus (T5), nucleus commissurae postopticae (D8), nucleus ventralis hypothalami (D10) and nucleus dorsalis hypothalami (D11) were labelled by one or more of the anti-opsin antibodies. In addition, anti-arrestin antibodies labelled cells within the D8 and anti-alpha-transducin antibodies labelled cells within the pineal complex and hypothalamus (primarily D8 and/or D10). A more variable and species dependent pattern of opsin, arrestin and alpha-transducin labelling was observed within the nucleus commissurae postinfundibularis (D12) in an area comprising the nucleus dorsalis thalami pars subhabenularis (D4sh) and nucleus dorsalis thalami pars caudalis/nucleus commissurae posterioris (D4c/M1), and in the proximity of the second Müller cells in the ventrocaudal diencephalon (2.MZ/M6). The majority of the neurons labelled within the pineal and parapineal organs and hypothalamus were periventricular with clear cerebrospinal fluid contacts (CSF-contacting neurons). Labelled neurons in the epithalamic (D4sh and D4c/M1) and caudal diencephalon (2.MZ/M6) had no obvious ventricular contacts. We speculate that the "primitive" vertebrate brain of lampreys represents an ancestral condition in which different populations of encephalic photoreceptors are associated with different behavioural and physiological responses. Image-forming vision needs an eye, but irradiance detection does not require a specialised organ. Rather the photoreceptors could be closely associated with their effector systems within the brain.

A tract-tracing study of the central projections of the mesencephalic nucleus of the trigeminus in the guppy (Lebistes reticulatus, teleostei), with some observations on the descending trigeminal tract.

We studied the central projections of the mesencephalic nucleus of the trigeminal nerve (MesV) in the guppy (Lebistes reticulatus), after application of horseradish peroxidase or fluorescein dextran amine into the eye orbit. A small number (1 to 13) of large mesencephalic trigeminal neurons were solid labeled in the ipsilateral rostral mesencephalon. At the level of the trigeminal nerve entrance, the united process of each mesencephalic trigeminal cell bifurcates, giving rise to a peripheral branch that exits in the trigeminal nerve and a descending branch that runs caudally in a medial bundle separated from the descending trigeminal tract. This bundle passes close to the visceromotor nuclei of the medulla oblongata. Descending processes give rise to short collaterals to the descending nucleus of the trigeminus and the ventrolateral reticular area. Most MesV descending fibres terminate in this ventrolateral field at the transition of the medulla to the spinal cord, but one or two fibres could be followed to the C6 level, where they give rise to collaterals to the dorsal funicular nucleus. No collaterals directed to the trigeminal motor nucleus, the cerebellum, or the mesencephalic tegmentum were observed. These projections were also compared with those of the descending trigeminal tract.

Diencephalic projection to reticulospinal neurons involved in the initiation of locomotion in adult lampreys Lampetra fluviatilis.

Morphological and electrophysiological techniques were used to characterize a diencephalic projection from the ventral thalamus to reticulospinal neurons and its role in initiating rhythmic locomotor activity in the spinal cord of adult lampreys (Lampetra fluviatilis). Injection of fluorescein-coupled dextran amine (FDA) into the rhombencephalic reticular nuclei labeled neurons in the ventral thalamus region on both the ipsilateral side and the contralateral side. Injection of FDA into the ventral thalamus labeled axonal projections in all reticular nuclei, but no direct projections were found to the spinal cord. Extracellular stimulation of the ventral thalamus elicited monosynaptic excitatory postsynaptic potentials (EPSPs), polysynaptic EPSPs, and inhibitory postsynaptic potentials (IPSPs) in reticulospinal neurons in the posterior (prrn) and middle (mrrn) rhombencephalic reticular nuclei. The monosynaptic EPSPs were blocked by the glutamate antagonist kynurenic acid and can be considered glutamatergic. The monosynaptic EPSPs were potentiated (up to 12 minutes) following a brief high-frequency stimulation. Stimulation of the ventral thalamus induced rhythmic firing of reticulospinal neurons and elicited rhythmic burst activity in the spinal ventral roots. The projections from the ventral thalamus to the reticulospinal neurons in the prrn and mrrn thus provide excitatory inputs to the reticulospinal neurons, which, in turn, can activate the spinal circuits underlying locomotion. Also, the input nuclei to the ventral thalamus were labeled following injection of FDA into this nucleus. Labeled cells were found in the olfactory bulb, pallial areas, striatum, preoptic nucleus, hypothalamus, dorsal thalamus, optic tectum, and dorsal isthmic gray. The ventral thalamus, therefore, receives inputs from several different regions in the brain and controls the level of excitability in reticulospinal neurons.

Rostrocaudal distribution of 5-HT innervation in the lamprey spinal cord and differential effects of 5-HT on fictive locomotion.

5-hydroxytryptamine (5-HT) is known to modulate the locomotion generator network in the lamprey spinal cord, but little is known about the pattern of 5-HT innervation along the spinal cord. The distribution of 5-HT-immunoreactive (5-HT-ir) cells and fibers, as well as the effects of 5-HT on the locomotor network in the rostral and caudal parts of the spinal cord were compared in two lamprey species, Lampetra fluviatilis and Petromyzon marinus. Intraspinal 5-HT cells form a very dense ventromedial plexus in which the dendrites of neurons forming the locomotor network are distributed. The number of 5-HT cells and varicosities in this plexus decreases in the fin area (segments 70-90), and then increases somewhat in the most caudal segments. The descending 5-HT fibers from the rhombencephalon are located in the lateral and ventral columns, and their numbers gradually decrease to around 50% in the tail part of the spinal cord. In contrast, the number of 5-HT-ir axons in the dorsal column remains the same along the spinal cord. Bath application of both N-methyl-D-aspartic acid (NMDA, 20-250 microM) and D-glutamate (250-1000 microM) was used to induce fictive locomotion in the isolated spinal cord. Bath application of 5-HT (1 microM) reduced the burst frequency in the presence of NMDA. The 5-HT effect was, however, significantly greater in the rostral as compared to the caudal part. With D-glutamate, the 5-HT effects was instead more pronounced in the caudal spinal cord. To account for this difference in 5-HT effects on NMDA- and D-glutamate-induced fictive locomotion, the cellular effect of D-glutamate was further investigated. It activates not only NMDA, but also alpha amino-3-hydroxy-5-methyl-4-isoxyl propionate (AMPA)/kainate and metabotropic glutamate receptors. In contrast to NMDA, D-glutamate did not elicit tetrodotoxin (TTX)-resistant membrane potential oscillations. This difference in action between NMDA (selective NMDA receptor agonist) and D-glutamate (mixed agonist) may partially account for the differences in effect of 5-HT on the locomotor pattern.

Secondary vestibulo-oculomotor projections in larval sea lamprey: anterior octavomotor nucleus.

The ventral octavolateral area of lampreys contains three nuclei: the anterior, intermediate and posterior octavomotor nuclei, formed of large neurons that are contacted by thick primary vestibular fibres. We used horseradish peroxidase (HRP) or fluorescein-dextran-amine (FDA) labelling to study the projections of the anterior octavomotor nucleus (AON) in the larval sea lamprey, Petromyzon marinus. The tracers were injected either in the AON, the oculomotor nucleus or the rostralmost spinal cord. HRP injection in the AON labelled thick axons that coursed to the basal mesencephalic tegmentum, where most decussate and project to the oculomotor nucleus and the third Müller cell. Electron microscopy confirmed that AON axons contact with the contralateral third Müller cell and with oculomotor neurons. Some AON axons run in the mesencephalic tegmentum and the ventral diencephalon. An AON axon was observed to run close to the axon of the contralateral third Müller cell, establishing what appeared to be en passant contacts. HRP injection in the AON also revealed commissural fibres projecting to the contralateral octavolateral area. HRP or FDA injections in the oculomotor nucleus labelled both large and small neurons of the AON, mostly contralateral to the injection site, as well as of cells in the intermediate octavomotor nucleus, mainly ipsilateral. HRP injection in the AON or in the rostral spinal cord did not reveal any projections from the AON to the spinal cord. Our results indicate that the pattern of octavo-oculomotor connections in the lamprey is different from that observed in other vertebrates.