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.

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.

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.

The sea lamprey tyrosine hydroxylase: cDNA cloning and in situ hybridization study in the brain.

Lampreys belong to the oldest group of extant vertebrates, the agnathans or cyclostomes. Thus, they occupy a key phylogenetic position near the root of the vertebrate tree, which makes them important to the study of nervous system evolution. Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis and is considered a marker of catecholaminergic neurons. In the present study, we report partial cloning of the sea lamprey tyrosine hydroxylase (TH) cDNA and the pattern of TH transcript expression in the adult brain by means of in situ hybridization. Sea lamprey TH mRNA is characterized by the presence of a large untranslated sequence in the 3' end that contains a typical polyadenylation signal (ATTAAA). The deduced partial TH protein sequence presents a conserved domain with two His residues coordinating Fe(2+) binding and a conserved cofactor binding site. Neurons expressing the TH transcript were observed in the preoptic, postoptic commissure, dorsal hypothalamic, ventral hypothalamic, mammillary and paratubercular nuclei of the prosencephalon. In situ hybridization experiments also confirmed the existence of a catecholaminergic (dopaminergic) striatal population in the brain of the adult sea lamprey. A few granule-like cells in the olfactory bulbs also showed weak TH transcript expression. No cells showing TH transcript expression were observed in the rostral rhombencephalon, which suggests the absence of a locus coeruleus in the sea lamprey. Comparison of the pattern of TH mRNA expression in the prosencephalon between lampreys and teleost fishes revealed both similarities and differences. Our results suggest that the duplication of the TH gene might have occurred before the separation of agnathans and gnathostomes.

New insights on the neuropeptide Y system in the larval lamprey brain: neuropeptide Y immunoreactive neurons, descending spinal projections and comparison with tyrosine hydroxylase and GABA immunoreactivities.

Lampreys are useful models for studying the evolution of the nervous system of vertebrates. Here we used immunofluorescence and tract-tracing methods to study new aspects of the neuropeptide Y-immunoreactive (NPY-ir) system in larval sea lampreys. NPY-ir neurons were observed in brain nuclei that contain NPY-ir cells in other lamprey species. Moreover, a group of NPY-ir cells that migrated away the periventricular layer was observed in the lateral part of the dorsal hypothalamus, which suggests a role for NPY in feeding behavior in lampreys. We also report NPY-ir cells in the dorsal column nucleus, which appears to be unique among vertebrates, and in the habenula. A combination of tract-tracing and immunohistochemical labeling demonstrated the presence of spinal projecting NPY-ir reticular cells in the anterior rhombencephalic reticular formation, and the relationships between the NPY-ir system and the reticulospinal nuclei and some afferent systems. The colocalization of catecholamines and GABA in lamprey NPY-ir neurons was investigated by double immunofluorescence methods. Colocalization of tyrosine hydroxylase (TH) and NPY immunoreactivities was not observed in any brain neuron, although reported in amphibians and mammals. The frequent presence of NPY-ir terminals on TH-ir cells suggests that NPY modulates the activity of some dopaminergic nuclei in lampreys. Colocalization of NPY and GABA immunoreactivities was frequently observed in neurons of different rhombencephalic and diencephalic NPY-ir populations. These results in lampreys suggest that the coexpression of NPY and GABA in neurons appeared early on in the brains of vertebrates.

Pineal projections in the zebrafish (Danio rerio): overlap with retinal and cerebellar projections.

The pineal organ in fishes is a photoreceptive organ with dual outputs, neuroendocrine and neural. The neural projections of the zebrafish pineal were experimentally studied by means of tract-tracing with carbocyanine dyes (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO)). Double-labeling experiments were also performed in order to investigate the degree of overlapping of pineal, retinal or cerebellar projections in zebrafish. The pineal organ sends efferent fibers bilaterally to the rostral hypothalamus, thalamus, pretectum, posterior tubercle and the mesencephalic tegmentum. A few pinealofugal fibers could also be traced to the optic tectum. Most of the targets of the zebrafish pineal also receive retinal and/or cerebellar afferents, indicating a high degree of overlap with these projections. Since most of the targets of the pineal projections of zebrafish appear to be premotor and precerebellar centers, the neural output of the pineal organ is probably, because of its photoreceptive and circadian function, involved in photic and circadian modulation of these centers.

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.

Distribution and development of FMRFamide-like immunoreactive neuronal systems in the brain of the brown trout, Salmo trutta fario.

The distribution of Phe-Met-Arg-Phe-amide (FMRFamide) peptide-immunoreactive (FMRF-ir) cells and fibers in the terminal nerve and central nervous system was investigated in developing stages and adults of the brown trout, Salmo trutta fario. The first FMRF-ir neurons appeared in the terminal nerve system of 8-mm embryos in and below the olfactory placode. In the brain, FMRF-ir neurons were first observed in the rostral hypothalamus, primordial hypothalamic lobe, mesencephalic laminar nucleus, and locus coeruleus of 12- to 13 -m embryos. After hatching, FMRF-ir cells appeared in the lateral part of the ventral telencephalic area and the anterior tuberal nucleus. In adult trout, FMRF-ir cells were observed in all these areas. The number of FMRF-ir neurons increased markedly in some of these populations during development. Dense innervation by FMRF-ir fibers was observed in the dorsal and lateral parts of the dorsal telencephalic area, and in the ventral telencephalic area, the lateral preoptic area, the medial hypothalamic and posterior tubercle regions, midbrain tegmentum and rhombencephalic reticular areas, the central gray, the superior raphe nucleus, the secondary visceral nucleus, the vagal nuclei, and the area postrema. Fairly rich FMRF-ir innervation was also observed in the optic tectum and some parts of the torus semicircularis. The saccus vasculosus and hypophysis received a moderate amount of FMRF-ir fibers. Innervation of most of these regions appeared either in late alevins or fry, although FMRF-ir fibers in the preoptic area, hypothalamus, and reticular areas appeared in embryos. Comparative analysis of the complex innervation pattern observed in the brain of trout suggests that FMRF is involved in a variety of functions, like the FMRF family of peptides in mammals.

A DiI-tracing study of the neural connections of the pineal organ in two elasmobranchs (Scyliorhinus canicula and Raja montagui) suggests a pineal projection to the midbrain GnRH-immunoreactive nucleus.

The pineal organ of elasmobranchs is an elongated photoreceptive organ. In order to investigate the afferent and efferent connections of the pineal organ of two elasmobranchs, the skate (Raja montagui) and the dogfish (Scyliorhinus canicula), a fluorescent carbocyanine (DiI) was applied to the pineal organ of paraformaldehyde-fixed brains. This application strongly labeled the pineal tract, which formed extensive bilateral projections. In both species, the pinealofugal fibers coursed to the dorsomedial thalamus, the medial pretectal area, the posterior tubercle, and the medial mesencephalic tegmentum and branched profusely in these areas. Application of DiI to the pineal organ also labeled occasional perikarya in the dorsomedial thalamus, posterior commissural region, posterior tubercle, and mesencephalic tegmentum. A comparison of these results with those of immunocytochemical analyses of the dogfish brain with an anti-salmon gonadotropin-releasing hormone (sGnRH) antiserum revealed a close topographical relation between the pineal projections and the midbrain sGnRH-immunoreactive (ir) nucleus, the only structure in the dogfish brain that contained sGnRHir neurons. This and the widespread distribution of sGnRHir fibers in the brain suggest that the midbrain sGnRHir nucleus is a part of the secondary pineal pathways and may be involved in light-mediated pineal regulation of brain function. Although GnRH distribution has not been studied in the skate, a midbrain GnRHir nucleus has been identified in three other elasmobranchs, including a skate relative. The probable existence of direct pineal projections to the GnRHir midbrain nucleus in elasmobranchs and other anamniotes is discussed.

Differential expression of thymosins beta(4) and beta(10) during rat cerebellum postnatal development.

The beta-thymosins are a family of actin monomer-sequestering proteins widely distributed among vertebrate classes. The most abundant beta-thymosins in mammalian species are thymosin beta(4) (Tbeta(4)) and thymosin beta(10) (Tbeta(10)), two small peptides (43 amino acids) sharing a high degree of sequence homology. In the present work, we have analyzed the distribution of Tbeta(4) and Tbeta(10) in the developing and adult rat cerebellum using in situ hybridization and immunohistochemistry techniques. Our results show that the temporal and cellular patterns of expression of both beta-thymosins are different. In the young (7 and 18 postnatal days) and adult (1 and 4 months old) rat cerebellum, Tbeta(4) was mainly expressed in the glia (microglia, Golgi epithelial cells and oligodendrocytes), neurons (granule cells and Purkinje cells), and in the capillaries. In 14-month-old rats, the Tbeta(4) immunoreactivity was only detected in some microglia cells. In young and adult animals, most of the Tbeta(10) immunoreactivity was localized in several types of neuronal cells including granule cells, Golgi neurons and Purkinje cells. In old animals, a faint Tbeta(10) signal could be detected in a few Purkinje cells. Our results suggest that each beta-thymosin could play a different function in the control of actin dynamics.

GABA immunoreactivity in the olfactory bulbs of the adult sea lamprey Petromyzon marinus L.

The distribution of gamma-aminobutyric acid (GABA) immunoreactivity in the olfactory bulbs of the adult sea lamprey was studied using an antibody against this transmitter. Five types of GABA-immunoreactive (GABAir) cells were observed. Medium-sized GABAir cells (periglomerular cells) were located around the olfactory glomeruli and occasionally within them. In the inner cellular layer of the bulbs and around the olfactory ventricles, two types of GABAir perikarya were present: some medium-sized GABAir cells and numerous small GABAir cells (granules). In the walls of the olfactory ventricle, some medium-sized GABAir cells of cerebrospinal fluid-contacting type were observed. At the entrance of the olfactory nerves, medium-sized GABAir bipolar cells were present, mostly located between the olfactory nerve and the glomerular layer or close to the meninges, but some in the intracranial portion of the olfactory nerve. GABAir processes were present in all layers of the olfactory bulb. In addition there were also GABAir cells in the dorsal interbulbar commissure. The distribution of GABA observed in the olfactory system of lampreys indicates that this transmitter plays a major role in the modulation of bulbar circuits. The presence of granular and periglomerular cells in lampreys indicates that these two intrinsic GABAergic neurons of the olfactory bulbs are shared by most vertebrates, although lampreys have additional GABAir cell types.

Presence and development of thyrotropin-releasing hormone-immunoreactive amacrine cells in the retina of a teleost, the brown trout (Salmo trutta fario).

The presence of thyrotropin-releasing hormone-immunoreactive (TRHir) amacrine cells is described for the first time in the retina of a teleost. These amacrine cells were mostly located in the inner nuclear layer, with occasional perikarya in the ganglion cell layer. Their processes formed a conspicuous plexus at the level of the ganglion cell perikarya. The TRHir amacrine cells appeared in posthatching stages, with the total number in retinas of juveniles approximately four times the number of cells in adults. Two types of TRHir cells, large and small, can be distinguished in developing stages, small cells outnumbering large cells. The TRHir cells of adults appears mainly to correspond to large, multistratified amacrine cells of developing stages. The possibility of transient expression of TRH in small amacrine cells during development is discussed.

Development of thyrotropin-releasing hormone immunoreactivity in the brain of the brown trout Salmo trutta fario.

The development and adult distribution of thyrotropin-releasing hormone-immunoreactive (TRHir) neurons in the brain of the brown trout, Salmo trutta fario, was studied with the streptavidin-biotin immunohistochemical method. Study of embryos, alevin, and juveniles revealed groups of TRHir neurons in the mesencephalon and rhombencephalon that have not been noted previously in adult teleosts. The earliest TRHir cells observed were those of the trigeminal motor nucleus, which expressed this substance only in embryos and alevins. In the forebrain, early-arising TRH populations were observed in the supra- and postcommissural region of the ventral telencephalic area, the anterior parvocellular preoptic nucleus, the organon vasculosum laminae terminalis, the magnocellular preoptic nucleus, the suprachiasmatic nucleus, and the posterior tuberal nucleus. TRHir cells of the olfactory bulb, abundant in the adult, appeared later. A small TRHir neuronal population was transiently observed in the habenula of alevins and juveniles. The laminar nucleus of the mesencephalon contained a small population of TRH cells in alevins and juveniles. In the isthmus, TRH was observed in cells of the interpeduncular nucleus, the nucleus isthmi, the dorsolateral tegmental nucleus, the superior reticular nucleus, and the central gray, although perikarya were TRHir only in alevin and/or juvenile stages. Some vagal motoneurons were TRHir from the late embryo stage onward. TRHir fibers were abundant in several forebrain regions of alevins and juveniles, including the medial region of the dorsal telencephalic area, the ventral telencephalic area and commissural region, the preoptic neuropil, the posterior tubercle, the anterior tuberal nucleus, and the posterior hypothalamic lobe. TRHir fibers invaded the neurohypophysis in early alevins, and their number increased subsequently to adulthood. The parvocellular superficial pretectal nucleus and the optic tectum received a rich TRHir innervation from juvenile stages onward. The interpeduncular nucleus and the secondary gustatory nucleus contained many TRHir fibers. In the rhombencephalon, TRHir fibers were scarce, except in the ventrolateral regions and the inferior olive. The distribution of TRHir fibers suggests that they were mainly related to hypophysiotropic and visceral centers, although the presence of TRH in centers related to the visual system indicates that TRH also plays other roles in the brain. We discuss the possibility that the strong expression of TRH in the embryonic trigeminal motoneurons plays a role in head morphogenesis.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the adult trout and tract-tracing observations on the connections of the nuclei of the isthmus.

The distribution of cholinergic neurons and fibers was studied in the brain and rostral spinal cord of the brown trout and rainbow trout by using an antiserum against the enzyme choline acetyltransferase (ChAT). Cholinergic neurons were observed in the ventral telencephalon, preoptic region, habenula, thalamus, hypothalamus, magnocellular superficial pretectal nucleus, optic tectum, isthmus, cranial nerve motor nuclei, and spinal cord. In addition, new cholinergic groups were detected in the vascular organ of the lamina terminalis, the parvocellular and magnocellular parts of the preoptic nucleus, the anterior tuberal nucleus, and a mesencephalic tegmental nucleus. The presence of ChAT in the magnocellular neurosecretory system of trout suggests that acetylcholine is involved in control of hormone release by neurosecretory terminals. In order to characterize the several cholinergic nuclei observed in the isthmus of trout, their projections were studied by application of 1,1;-dioctadecyl-3,3,3;, 3;-tetramethylindocarbocyanine perchlorate (DiI) to selected structures of the brain. The secondary gustatory nucleus projected mainly to the lateral hypothalamic lobes, whereas the nucleus isthmi projected to the optic tectum and parvocellular superficial pretectal nucleus, as previously described in other teleost groups. In addition, other isthmic cholinergic nuclei of trout may be homologs of the mesopontine system of mammals. We conclude that the cholinergic systems of teleosts show many primitive features that have been preserved during evolution, together with characteristics exclusive to the group.

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.

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.

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.

Ingestion, faecal pellet and egg production rates of Calanus helgolandicus feeding coccolithophorid versus non-coccolithophorid diets.

Ingestion rates, faecal pellet and egg production were obtained in laboratory experiments with females of the copepod Calanus helgolandicus collected from the English Channel in November 1994. Five different algal monocultures were used as food: Prorocentrum micans (30 µm ESD), Thalassiosira weissflogii (13 µm ESD), Dunaliella tertiolecta (7 µm ESD), Emiliania huxleyi (5 µm ESD) and Coccolithus pelagicus (14 µm ESD). Results obtained suggest the low ingestion efficiency of the copepod when feeding on coccolithophorids during late autumn-early winter. From the five species offered, only the largest non coccolithophorid Prorocentrum micans and Thalassiosira weissflogii supported efficient feeding and calculated respiratory demand for C. helgolandicus. Both coccolithophorids, irrespective of cell size, were ingested at very low rates even when offered at high concentrations (233-468 µg C l(-1)). Besides low ingestion, no egg production was found in the copepods fed with Emiliania huxleyi, although unusual high gross efficiency (reaching 72%) was obtained in experiments performed with Coccolithus pelagicus. The late seasonal timing of the experiments (November) could explain the low ingestion and egg production rates.

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.