Restricted co-localization of glutamate and dopamine in neurons of the adult sea lamprey brain.

Co-localization of dopamine with other classical neurotransmitters in the same neuron is a common phenomenon in the brain of vertebrates. In mammals, some dopaminergic neurons of the ventral tegmental area and the hypothalamus have a glutamatergic co-phenotype. However, information on the presence of this type of dopaminergic neurons in other vertebrate groups is very scant. Here, we aimed to provide new insights on the evolution of this neuronal co-phenotype by studying the presence of a dual dopaminergic/glutamatergic neuron phenotype in the central nervous system of lampreys. Double immunofluorescence experiments for dopamine and glutamate in adult sea lampreys revealed co-localization of both neurotransmitters in some neurons of the preoptic nucleus, the nucleus of the postoptic commissure, the dorsal hypothalamus and in cerebrospinal fluid-contacting cells of the caudal rhombencephalon and rostral spinal cord. Moreover, co-localization of dopamine and glutamate was found in dopaminergic fibres in a few brain regions including the lateral pallium, striatum, and the preoptic and postoptic areas but not in the brainstem. Our results suggest that the presence of neurons with a dopaminergic/glutamatergic co-phenotype is a primitive character shared by jawless and jawed vertebrates. However, important differences in the distribution of these neurons and fibres were noted among the few vertebrates investigated to date. This study offers an anatomical basis for further work on the role of glutamate in dopaminergic neurons.

Differential expression of somatostatin genes in the central nervous system of the sea lamprey.

The identification of three somatostatin (SST) genes (SSTa, SSTb, and SSTc) in lampreys (Tostivint et al. Gen Comp Endocrinol 237:89-97 https://doi.org/10.1016/j.ygcen.2016.08.006 , 2016) prompted us to study their expression in the brain and spinal cord of the sea lamprey by in situ hybridization. These three genes were only expressed in equivalent neuronal populations in the hypothalamus. In other regions, SST transcripts showed clear differential expression. In the telencephalon, SSTc-positive cells were observed in the medial pallium, ventral part of the lateral pallium, striatum, subhippocampal lobe, and preoptic region. In the diencephalon, SSTa-positive cells were observed in the thalamus and SSTc-positive cells in the prethalamus, posterior tubercle, pretectal area, and nucleus of the medial longitudinal fascicle. In the midbrain, SSTc-positive cells were observed in the torus semicircularis, lateral reticular area, and perioculomotor tegmentum. Different SSTa- and SSTc-positive populations were observed in the isthmus. SSTc neurons were also observed in the rostral octavolateralis area and caudal rhombencephalon. In the spinal cord, SSTa was expressed in cerebrospinal-fluid-contacting (CSF-c) neurons and SSTc in non-CSF-c interneurons. Comparison with previous immunohistochemical studies using anti-SST-14 antibodies strongly suggests that SST-14-like neurons correspond with the SSTa populations. Thus, the SSTc populations were not reported previously in immunohistochemical studies. Cluster-based analyses and alignments of mature peptides suggested that SSTa is an ortholog of SST1 and that SSTb is closely related to SST2 and SST6. These results provide important new insights into the evolution of the somatostatinergic system in vertebrates.

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.

High levels of mouse thymosin beta4 mRNA in differentiating P19 embryonic cells and during development of cardiovascular tissues.

The self assembly of actin and the large number of actin-binding proteins are important in the establishment of cell shape and function during embryogenesis. Thymosin beta4 (Tbeta4) is a small acidic peptide that participates in the regulation of actin polymerization in mammalian cells. In the present work, we report the presence of the mRNA encoding for Tbeta4 in mouse embryonic stem cells and its induction in P1 9 embryonal cells stimulated to differentiate into ectodermal-like (neurons and glia) or mesodermal-like cells (cardiac and skeletal muscle). The induction of Tbeta4, mRNA in P19 cells was confirmed by in situ hybridization analysis of early mouse postimplantation embryos. Noteworthy, we observed an important hybridization signal in several areas of the embryo specially in blood vessels and in heart tissues, suggesting a role for this peptide in angiogenesis. In conclusion, the results presented here demonstrate the expression of Tbeta4 gene during early embryogenesis which immediately suggests an important role for this peptide in developmental processes requiring actin-based functions such as the formation of cardiovascular system.

Experimental study of the connections of the preglomerular nuclei and corpus mamillare in the rainbow trout, Oncorhynchusmykiss.

The preglomerular complex of trout consists of the anterior (aPGN) and medial (mPGN) preglomerular nuclei and the corpus mamillare (CM). In order to improve knowledge on this complex, we applied a lipophilic neuronal tracer (DiI) to the three nuclei. These nuclei received afferents from the medial part of the dorsal telencephalic area (Dm), the ventral part of the ventral telencephalic area (Vv), the preoptic nucleus, the periventricular layer of the rostral optic tectum and the central posterior thalamic nucleus. The aPGN also received numerous toral projections and, sent efferents to the anterior tuberal nucleus. In addition, both the aPGN and the mPGN nuclei gave rise to efferents to the dorsal region of the dorsal telencephalic area (Dd), whereas the medial preglomerular nucleus and the CM sent fibers to the torus lateralis and the diffuse nucleus, as confirmed by reciprocal labeling. A small mPGN/CM subgroup projected to the optic tectum. These results suggest close functional inter-relationship between the trout preglomerular complex and two telencephalic regions (Dm and Vv). In addition, all nuclei of the complex receive preoptic, tectal and dorsal thalamic afferents, whereas the aPGN and mPGN are related with acoustic-lateral ascending pathways, and the mPGN and CM with the central region of the dorsal telencephalic area and visceral/gustatory pathways.

Thymosin beta10 mRNA expression during early postimplantation mouse development.

The beta-thymosins are a family of monomeric actin sequestering peptides that regulate actin dynamics within the cells. During embryogenesis the control of actin polymerization is essential in processes such as cell migration, angiogenesis and neurogenesis. Here we report that the levels of thymosin beta10 (Tbeta10) mRNA strongly increase during early postimplantation mouse embryogenesis as well as during in vitro P19 cell differentiation, indicating that this peptide plays an important role in early development. Moreover, analysis of the spatial distribution of Tbeta10 mRNA in 9.5-12.5 days postcoitum mouse embryos showed a remarkable presence of this transcript in mesenchymal structures as well as in the mantle layer of spinal cord. Interestingly, we observed differences in the distribution of the mRNAs encoding Tbeta10 and Tbeta4, another member of the beta-thymosin family, suggesting different roles for these peptides during mouse embryogenesis.

Afferent and efferent connections of the habenula in the rainbow trout (Oncorhynchus mykiss): an indocarbocyanine dye (DiI) study.

The habenula is a conserved structure in the brain of vertebrates. With the aim of further understanding of the evolution of the habenular system in vertebrates, we studied the afferent and efferent connections of the habenula of the rainbow trout. Experiments included application of the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the habenula, telencephalon, pineal organ, posterior tubercle, and interpeduncular nucleus (IPN). The results obtained reveal a consistent pattern of habenular connections. Most afferents originate from three nuclei, one extending from the preoptic region to the rostral thalamus (the entopeduncular nucleus), the second located in the region of the hypothalamus-posterior tubercle and consisting of large bipolar cells (tuberculohabenular nucleus), and the third in the preoptic region (preoptic nucleus). A few large neurons of the locus coeruleus appeared to be labeled in some cases. The trout habenula also receives pineal and parapineal projections. Small labeled glial cells were observed in the thalamus around the fasciculus retroflexus and, sometimes, around the IPN. The most conspicuous efferents coursed in the fasciculus retroflexus to the IPN, the isthmal raphe, and the central gray. The existence of olfactohabenular or habenulotelencephalic projections is discussed.

Afferent and efferent connections of the habenula in the larval sea lamprey (Petromyzon marinus L.): an experimental study.

The habenula is an integrative center between the striatum and the limbic and motor systems. With the aim of achieving further understanding of the evolution of this structure in vertebrates, we carried out an experimental study of the afferent and efferent connections of the habenula of larval sea lamprey. Experimental procedures included in vivo and in vitro transport after injections of horseradish peroxidase (HRP) into the habenula, telencephalon, pineal organ, dorsal thalamus, and posterior tubercle as well as carbocyanine dye tracing (DiI). The combined results of these experiments showed that the pattern of habenular connections is very simple. Most afferents appear to originate from the lobus subhippocampalis and neighboring area, whereas the only efferents found coursed in the fasciculus retroflexus to the neuropil of the nucleus interpeduncularis. This neuropil comprises a commissural region in the rostral mesencephalon, two long bilateral areas extending in the basal mesencephalon and medulla oblongata to the trigeminal level, and, finally, a caudal commissural zone. The conspicuous habenular commissure contains interhemispheric fibers that appear to form occasional contacts within the habenulae. The lamprey habenula also receives a few immunocytochemically identified fibers (somatostatinergic, catecholaminergic, and serotoninergic fibers) from other sources.

Neural connections of the pineal organ in the primitive bony fish Acipenser baeri: a carbocyanine dye tract-tracing study.

The pineal organ of the sturgeon (Acipenser baeri; Chondrostei) consists of an elongated terminal vesicle and a long stalk that runs to the region of the posterior commissure. In order to investigate the neural connections of the pineal organ of the surgeon, a fluorescent indocarbocyanine (DiI) was applied to the pineal stalk of paraformaldehyde-fixed brains. This application strongly labeled the pineal tracts (right and left), which form an extensive bilateral projection to the brain. The fibers of these tracts could be followed to their different targets in the brain, namely the preoptic region, the rostral habenulae, the medial subhabenular and retrohabenular (post- and subcommissural) region, the medial thalamus, the dorsal hypothalamus, the pretectal area, and medial and dorsolateral tegmental mesencephalic regions (interstitial nucleus of Cajal, oculomotor nucleus, mesencephalic reticular area, nucleus profundus mesencephali, and central gray). A few fibers reach the interpeduncular nucleus and the rostral optic tectum. Pinealofugal fibers branched profusely, and some of them crossed the midline ventrally in the optic chiasma, the postoptic commissure, the conspicuous preinfundibular commissure or, occasionally, the mesencephalic tegmentum. In addition to pinealofugal fibers, this type of DiI application labeled a few cells, here interpreted as pinealopetal neurons, in the posthabenular-dorsomedial thalamic region and the central gray of the mesencephalon. The wide distribution of pinealofugal projections in sturgeon is reminiscent of that in the lamprey. Comparison of the present findings with those of other authors in teleosts, amphibians, and reptiles also suggests that there has been a reduction in the number of pineal projections in these lines.

The optic tectum of the dogfish Scyliorhinus canicula L.: a Golgi study.

The optic tectum of the dogfish Scyliorhinus canicula L. was studied by using the methods of Nissl, reduced silver nitrate, Golgi-aldehyde, and Golgi-Cox. Six layers and eight types of neurons were recognized. These cell types are not restricted to one layer; in fact some are found in all six tectal layers. The types of cells found are A) monopolar, B) triangular, C) radial bipolar, D) horizontal fusiform, E) large tectal, F) small tectal, G) pyriform, and H) stellate cells. In at least six of the cell types a series of dendritic specializations can be observed, such as spines in the form of "drumsticks" and thin varicose appendages, similar to those reported previously in the optic tecta of amphibians and teleosts. The optic tectum of the dogfish shows a degree of complexity comparable to that of amphibians and teleosts.

Golgi study of the telencephalon of the small-spotted dogfish Scyliorhinus canicula L.

The telencephalon of the small-spotted dogfish, Scyliorhinus canicula L., was examined by Nissl and Golgi-aldehyde techniques. On the basis of differences in perikaryal and dendritic morphology and size, several cell types were distinguished in pallial and subpallial regions, most of them reported here for the first time in elasmobranchs. In the pallium, the pallium dorsalis is the richest in cell types (eight types of neurons), whereas the neuron population of the pallium medialis is the most homogeneous. Dendrites of most neuron types in the pallium are smooth or sparsely thorny. Interestingly, the pallium dorsalis and pallium lateralis contain a type of primitive pyramidal cell characterized by the dense appearance of its thorny dendrites. In the subpallium, the area superficialis basalis contains a heterogeneous population (six types of neurons): large radial cells are the most characteristic cell type. Dendrites of these cell types are smooth or sparsely thorny. The cell populations of the nucleus N are roughly similar to those of the area superficialis basalis, but they lack the large radial cells characteristic of this area. The area centralis subpallialis and striatum consist of populations of small neurons. The regio septalis contains a rather homogeneous population of small cells. The populations in the nucleus entopeduncularis and the nucleus interstitialis of the basal forebrain bundle are the least varied and consist of large radial cells and bipolar cells similar to those of the area superficialis basalis. This investigation reveals important differences in cytoarchitecture that should be useful in the interpretation of immunocytochemical, tracing, and electrophysiological studies of the telencephalon of elasmobranchs.

Olfactory projections in a chondrostean fish, Acipenser baeri: an experimental study.

The presence of extrabulbar primary olfactory projections has been well established in teleosts. In order to investigate the phylogeny of these projections and to compare their targets with those of the secondary olfactory projections, the connections of the olfactory epithelium and olfactory bulb were studied by means of tract-tracing methods in a chondrostean, Acipenser baeri. Primary olfactory projections mainly extend to the glomerular layer of the olfactory bulb, but a significant number of extrabulbar efferent fibers course to various telencephalic and diencephalic regions. Both extrabulbar primary and olfactory bulb projections course in diffuse pathways. Extensive overlap was observed between the targets of these extrabulbar primary olfactory fibers and those of the secondary efferent projections of the olfactory bulb, though the latter were more numerous and reached additional targets. Tracer application to the olfactory bulb also revealed a number of bulbopetal neurons in the ipsilateral and contralateral telencephalic areas, as well as crossed interbulbar projections. The presence in a chondrostean of important extrabulbar primary projections and their extensive overlap with secondary olfactory projections suggest that such projections are a derived characteristic of bony fishes.

Calretinin expression in specific neuronal systems in the brain of an advanced teleost, the grey mullet (Chelon labrosus).

The distribution of calretinin (CR) in the brain of an "advanced" teleost, the grey mullet, was studied by using immunoblotting and immunocytochemical techniques. In immunoblots of protein extracts of rat and mullet brains, the CR antibody stained a single band of about 29 kDa. CR immunoreactivity was observed in specific neuronal populations of all brain regions. The primary olfactory system, the optic nerve fibers, and some sensory fibers of other cranial nerves exhibited strong CR immunoreactivity. In the forebrain, the CR-immunoreactive (CR-ir) populations were scarce in the telencephalon and hypophysiotrofic hypothalamus, but numerous in many specialized nuclei of the diencephalon (preglomerulosus complex, nucleus glomerulosus, anterior glomerular nucleus, nucleus diffusus) and pretectum (parvocellular and magnocellular superficial pretectal nuclei, central pretectal nucleus), which are related to sensory systems. The two main forebrain bundles, medial and lateral, contained numerous CR-ir fibers. The midbrain sensory centers (optic tectum and torus semicircularis) exhibited numerous CR-ir cells and fibers. Likewise, the secondary gustatory nucleus of the isthmus is one of the nuclei exhibiting more intense CR immunoreactivity. Characteristically, the efferent cerebellar system (eurydendroid cells and brachium conjunctivum) and some afferent cerebellar fibers were CR-ir. In the medulla oblongata, a number of reticular cells, the inferior olive, and the magnocellular octaval nucleus exhibited CR immunoreactivity. CR-ir motoneurons were also observed in the spinal cord and in the oculomotor nucleus. Together with results obtained in other vertebrates, present results suggest that neural systems using calretinin to maintain intracellular calcium concentration have been rather well conserved during vertebrate evolution.

Fine structure of the medullary lateral line area of Chelon labrosus (order perciformes), a nonelectroreceptive teleost.

The ultrastructure and synaptic organization of the nucleus medialis and cerebellar crest of the teleost Chelon labrosus have been investigated. The nucleus medialis receives projections from the anterior and posterior lateral line nerves. This nucleus consists of oval neurons and large crest cells ("Purkinje-like" cells) whose apical dendrites branch in the overlying molecular layer, the cerebellar crest. In the dorsal region of the nucleus medialis, the perikarya and smooth primary dendrites of the crest cells are interspersed among myelinated fibers and nerve boutons. The ventral layer of the nucleus medialis contains crest cell perikarya and dendrites as well as oval neurons. The cerebellar crest lacks neuronal bodies, but the apical dendrites of crest cells receive synapses from unmyelinated and myelinated fibers. In the cerebellar crest, two types of terminals are presynaptic to the crest cell dendrites: boutons with spherical vesicles that form asymmetric synapses with dendritic spines and boutons containing pleomorphic vesicles that form symmetric synapses directly on the dendritic shaft. Most axon terminals found on the somata and primary dendrites of crest cells in the nucleus medialis have pleomorphic vesicles and form symmetric contacts, though asymmetric synapses with spherical vesicles and mixed synapses can be observed; these mixed synapses exhibit gap junctions and contain spherical vesicles. Unlike crest cells, the oval neuron perikarya receive three types of contacts (symmetric, asymmetric, and mixed). The origins and functions of these different bouton types in the nucleus medialis are discussed.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the central nervous system of a chondrostean, the siberian sturgeon (Acipenser baeri).

All studies to date of cholinergic systems of bony fishes have been done in teleosts. To gain further insight into the evolution of the cholinergic systems of bony fishes, we have studied the brain of a chondrostean fish, the Siberian sturgeon (Acipenser baeri, Brandt), by using an antibody against choline acetyltransferase (ChAT). This study showed the presence of ChAT-immunoreactive (ChAT-ir) neurons in the preoptic region (parvocellular and magnocellular preoptic nuclei and suprachiasmatic nucleus), the periventricular and tuberal hypothalamus, the saccus vasculosus, the dorsal thalamus, and the habenula. The mesencephalic tegmentum contained ChAT-ir cells in the torus semicircularis and torus lateralis. The isthmus contained several cholinergic populations: the nucleus isthmi, the lateral nucleus of the valvula, the secondary visceral nucleus, and the dorsal tegmental nucleus. The motor neurons of the cranial nerves and the spinal motor column were strongly immunoreactive. The medial (sensory) trigeminal nucleus also contained a ChAT-ir neuronal population. The distribution of ChAT-ir neurons in the sturgeon brain showed some notable differences with that observed in teleosts, such as the absence of cholinergic cells in the telencephalon and the optic tectum. Several brain regions were richly innervated by ChAT-ir fibers, particularly the telencephalon, optic tectum, thalamus, posterior tubercle, and interpeduncular nucleus. The hypothalamo-hypophyseal tract, the tract of the saccus vasculosus, the fasciculus retroflexus, and an isthmo-mesencephalo-thalamic tract were the most conspicuous cholinergic bundles. Comparative analysis of these results suggests that teleosts have conserved most traits of the cholinergic system of the sturgeon, having acquired new cholinergic populations during evolution.

Organization of the visual system in larval lampreys: an HRP study.

The organization of the visual system of larval lampreys was studied by anterograde and retrograde transport of HRP injected into the eye. The retinofugal system has two different patterns of organization during the larval period. In small larvae (less than 60-70 mm in length) only a single contralateral tract, the axial optic tract, is differentiated. This tract projects to regions in the diencephalon, pretectum, and mesencephalic tegmentum. In larvae longer than 70-80 mm, there is an additional contralateral tract, the lateral optic tract, which extends to the whole tectal surface. In addition, ipsilateral retinal fibers are found in both small and large larvae. Initially, the ipsilateral projection is restricted to the thalamus-pretectum, but it reaches the optic tectum in late larvae. Changes in the organization of the optic tracts coincide with the formation of the late-developing retina and consequently, the origin of the optic tracts can be related to specific retinal regions. The retinopetal system is well developed in all larvae. Most retinopetal neurons are labeled contralaterally and are located in the M2-M5 nucleus of the mesencephalic tegmentum, in the caudolateral mesencephalic reticular area and adjacent ventrolateral portions of the optic tectum. Dendrites of these cells are apparent, especially those directed dorsally, which in large larvae extend to the optic tectum overlapping with the retino-tectal projection. These results indicate that in lampreys, visual projections organize mainly during the blind larval period before the metamorphosis, their development being largely independent of visual function.

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.

Octavolateral neurons projecting to the middle and posterior rhombencephalic reticular nuclei of larval lamprey: a retrograde horseradish peroxidase labeling study.

The octavolateral area of lampreys, which receives primary fibers from the octaval and lateral line nerves, is involved in the premotor organization of body movements through secondary projections to the reticular formation. Here, the typology of neurons of the three octavolateral nuclei (ventral, medial, and dorsal) that putatively project to the middle and posterior rhombencephalic reticular nuclei were studied by retrograde transport of horseradish peroxidase (HRP) applied to these reticular nuclei. Several types of neurons were labeled in the ventral nucleus, both ipsilateral and contralateral to the site of HRP application. Some of these neurons showed a rather simple morphology (octavomotor neurons, monopolar cells), but most had more- or less-branched dendrites that were associated with one, or several, fields of terminal fibers in the octavolateral area. Unlike those of the ventral nucleus, labeled neurons of the medial nucleus were homogeneous in appearance (mostly pear-shaped). The dorsal nucleus was scarcely developed in larvae, as judged from the very simple and small labeled cells. The presence of terminal or "en-passant" boutons of secondary octavolateral fibers in the reticular area and the commissural nature of these fibers were also investigated by means of application of HRP or indocarbocyanine dye to the octavolateral nuclei. In addition, neurons of other alar plate nuclei that were labeled by the HRP application to the reticular nuclei (trigeminal descending root nucleus and solitary nucleus) were also characterized. The functional significance of these results is discussed.

Development and organization of the ocular motor nuclei in the larval sea lamprey, Petromyzon marinus L.: an HRP study.

Retrograde transport of horseradish peroxidase (HRP) after its application into the orbit was used to investigate the development of the different ocular motor nuclei in larvae of the sea lamprey (Petromyzon marinus) and to identify their regions of origin. In the smallest larvae studied (10-19 mm in length), the oculomotor and abducens neurons were ipsilateral to the site of HRP application, whilst trochlear neurons were contralateral. These motoneurons did not have dendritic processes. In larvae more than 19 mm in length, both ipsilateral and contralateral components were found in the oculomotor and trochlear nuclei; dendrites were present, and their length and branching increased with larval age. An adult-like pattern of topographic organization and dendritic arborization was reached in larvae of about 45-60 mm in length. In oculomotor neurons, medial dendrites appear first, then dorsolateral dendrites, and finally ventral dendrites. Similarly, in trochlear neurons ventral and ventrolateral dendrites develop first, followed by dorsal dendrites that course either to the caudal optic tectum or to the terminal fields of the octaval and lateral line nerves in the cerebellar plate. Dorsal and ventral dendrites of the abducens neurons arise at the same time, but dorsal dendrites attain an adult-like morphology earlier. A few motoneurons showed ventricular attachments in larvae longer than 40 mm. The significance of these processes and their possible usefulness as a marker for the regions of origin of the ocular motor nuclei are discussed. Finally, the results presented here indicate that differentiation of the ocular motor nuclei in larval lampreys precedes and is independent of the maturation of the eye at transformation.

Distribution of GABA immunoreactivity in the central and peripheral nervous system of amphioxus (Branchiostoma lanceolatum Pallas).

On the basis of labeling with an anti-gamma-aminobutyric acid (GABA) antibody, we report for the first time the presence and distribution of GABA-immunoreactive cells in the central and peripheral nervous system of amphioxus. In the nerve cord, there is a large dorsorostral group of cerebrospinal-fluid-contacting (CSFc) cells at the caudal end of the brain vesicle that gives rise to a large ventral commissure and neuropilar region. In the middle and caudal region of the brain, numerous commissural and CSFc neurons are situated below the region of large dorsal cells. In the spinal cord, several types of GABA-immunoreactive neurons of different size, appearance, and distribution were observed. In the dorsalmost region, very small commissural cells are scattered regularly along the cord. More ventrally in the cord, GABAergic neurons, both of commissural and CSFc cell types, form segmental groups, but scattered cells are observed throughout. These cells give rise to dense longitudinal fascicles of GABAergic fibers and to scattered commissural fibers. The caudal ampulla lacks GABAergic cells and fibers. Some of the fibers of the most rostral and caudal peripheral (sensory) nerves, as well as some sensory cells of the rostral and caudal epidermis, are GABA immunoreactive. The significance of these results for the understanding of the evolution of GABAergic systems of vertebrates is discussed.