Ontogeny of gene expression of group IB phospholipase A2 isoforms in the red sea bream, Pagrus (Chrysophrys) major.

The red sea bream (Pagrus major) was previously found to express mRNAs for two group IB phospholipase A(2) (PLA(2)) isoforms, DE-1 and DE-2, in the digestive organs, including the hepatopancreas, pyloric caeca, and intestine. To characterize the ontogeny of the digestive function of these PLA(2)s, the present study investigated the localization and expression of DE-1 and DE-2 PLA(2) genes in red sea bream larvae/juveniles and immature adults, by in situ hybridization. In the adults, DE-1 PLA(2) mRNA was expressed in pancreatic acinar cells. By contrast, DE-2 PLA(2) mRNA was detected not only in digestive tissues, such as pancreatic acinar cells, gastric glands of the stomach, epithelial cells of the pyloric caeca, and intestinal epithelial cells, but also in non-digestive ones, including cardiac and lateral muscle fibers and the cytoplasm of the oocytes. In the larvae, both DE-1 and DE-2 PLA(2) mRNAs first appeared in pancreatic tissues at 3 days post-hatching (dph) and in intestinal tissue at 1 dph, and expression levels for both gradually increased after this point. In the juvenile stage at 32 dph, DE-1 PLA(2) mRNA was highly expressed in pancreatic tissue, and DE-2 PLA(2) mRNA was detected in almost all digestive tissues, including pancreatic tissue, gastric glands, pyloric caeca, and intestine, including the myomere of the lateral muscles. In conclusion, both DE-1 and DE-2 PLA(2) mRNAs are already expressed in the digestive organs of red sea bream larvae before first feeding, and larvae will synthesize both DE-1 and DE-2 PLA(2) proteins.

Artificial control of swimming in goldfish by brain stimulation: confirmation of the midbrain nuclei as the swimming center.

The midbrain locomotor region including the nucleus of the medial longitudinal fasciculus (Nflm) was electrically stimulated in free-moving goldfish. Stimulation of sites on the midline induced forward movement, whereas that of sites off midline induced turning toward the stimulated side. The closer the site of stimulation to Nflm, the lower the threshold stimulus intensity required to evoke locomotor movement. Using a wirelessly controlled two-channel microstimulator, the locomotion of goldfish in the horizontal plane could be controlled directly by stimulating Nflm for movements involving the trunk and tail. Forward and turning movements could be arbitrarily induced in goldfish equipped with the stimulation device and electrodes implanted in or near the right and left Nflm. Together with previous observations, these findings confirm the role of Nflm in driving spinal segmental rhythm generators initiating swimming, which involves rhythmic contractions of the trunk musculature.

Effects of partial ablation of the cerebellum on sustained swimming in goldfish.

To investigate the role of the corpus cerebelli in the control of sustained swimming or cruising in goldfish, Carassius auratus, we conducted experiments examining the effects of partial ablation of the corpus cerebelli (CC) on swimming performance against constant water flow at various speeds. Ten out of 15 CC-ablated fish successfully maintained sustained swimming against water flow even at the highest speed tested (3.0 body lengths per second). This result showed that the CC is not crucial for generating the simple swimming motor pattern, although some effects of the surgical operation itself on the capability of the sustained swimming were found in both sham-operated and CC-ablated fish. However, we found that both tail-beat amplitude and frequency in CC-ablated goldfish tended to be greater than that of control fish at the same swimming speeds. The thrust index (square of the value obtained by multiplying the tail beat frequency (Hz) by twice the tail beat amplitude (mm)) was significantly larger in CC-ablated fish than in control fish at higher swimming speeds (> or =2.0 body length per second). This result suggests that CC-ablated goldfish produced more thrust by tail beats than control fish to maintain sustained swimming at higher speeds. We concluded that in goldfish the CC plays no major role in the posture control and generation of simple forward swimming movement, although the integrity of the CC is important for execution of normal swim gait.

Central mechanisms underlying fish swimming.

Although the basic swimming rhythm is created by central pattern generators (CPGs) located in each spinal segment, command signals from the brain should be indispensable for the activation of CPGs to initiate swimming. We hypothesized that the nucleus of medial longitudinal fascicles (Nflm) is the midbrain locomotor region driving swimming rhythms in teleosts. To test this hypothesis, we recorded neuronal activities from Nflm neurons in swimming carp and analyzed the cytoarchitecture of the nucleus. We identified two types of Nflm neurons exhibiting electric activities closely related to swimming rhythms. Remarkably, tonic neurons that continued firing during swimming were found. The Nflm and neighboring oculomotor nucleus contain about 600 neurons in total, and among them as many as 500 were labeled retrogradely by an intraspinal tracer implantation and 400 neurons showed glutamatergic immunoreactivity. They are the most likely candidates for the descending neurons as the origin of driving signals that initiate swimming. Double-labeling experiments demonstrated direct connections of Nflm neurons to spinal neurons consisting of the CPG. These data imply that most Nflm neurons possibly exert an excitatory drive to the spinal CPGs through the descending axons with excitatory transmitter(s), probably glutamate. Furthermore, we confirmed that the caudal part of Nflm and the rostral part of the oculomotor nucleus overlap rostrocaudally by approximately 200 mum. In connection with the control of swimming by the brain, we carried out experiments to clarify the efferent system of the cerebellum of the goldfish. Cerebellar efferent fibers terminated in most brain regions except for the telencephalon. Importantly, the cerebellum projected also to the Nflm, suggesting the involvement of this brain region in the control of swimming. We have also determined that in the carp so-called eurydendroid cells are cerebellar efferent neurons.

Screening of freshwater fish species for their susceptibility to a betanodavirus.

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. Because the genomes are the smallest and simplest among viruses, betanodaviruses have been well studied using a reversed genetics system as model viruses. However, studies of virus-host interactions have progressed slowly because permissive hosts for betanodaviruses (basically larvae and juveniles of marine fish) are only available for limited periods of the year and are not suitable for the construction of a genetic engineering system. To obtain a model fish species that are not subject to these problems, 21 freshwater fish species were injected intramuscularly with a betanodavirus (redspotted grouper nervous necrosis virus) and tested for their susceptibility to the virus. Based on their responses, the tested fish were classified into 3 groups: 4 susceptible fish, 10 less susceptible fish, and 7 resistant fish. The susceptible fish, celebes rainbowfish Telmatherina ladigesi, threadfin rainbowfish Iriatherina werneri, dwarf rainbowfish Melanotaenia praecox, and medaka Oryzias latipes, exhibited erratic swimming and eventually died within 10 d post-inoculation. The virus was specifically localized in the brains, spinal cords, and retinas of the infected fish, similar to the pattern of infection in naturally infected marine fish. We believe that these susceptible freshwater fish species could act as good host models for betanodavirus-fish interaction studies.

Impact of dehydration on the forebrain preoptic recess walls in the mudskipper, Periophthalmus modestus: a possible locus for the center of thirst.

The forebrain lamina terminalis has not yet been examined for the role of osmosensing in teleosts, although the thirst center is well known to be present in this vascular permeable forebrain region in mammals. Here, we examined vascular permeability and neuronal responsiveness to dehydration in the lamina terminalis of the mudskipper, a euryhaline goby. Evans blue and N-hydroxysulfosuccinimide-biotin both bind to blood proteins, and are impermeable to the blood-brain barrier. Intraperitoneal injection of these probes stained the walls of the preoptic recess (PR) of the third ventricle, indicating increased vascular permeability in this region. When mudskippers kept in isotonic brackish water (ca. 11 psu) were challenged to seawater (ca. 34 psu) for 3 h, body water content showed a 1 % decrease, compared with mudskippers without hypertonic challenge. Simultaneously, the number of immunohistochemically identified cFos-expressing neurons in the anterior parvocellular preoptic nucleus (PPa) of the PR walls increased in a site-specific manner by approximately 1.6-fold compared with controls. Thus, these findings indicate that PPa neurons are activated, following dehydration in mudskippers. Taken together, the vascularly permeable PR walls may be involved in osmosensing, as in the mammalian thirst center.

Morphology and immunohistochemistry of efferent neurons of the goldfish corpus cerebelli.

In teleosts, cerebellar efferent neurons, known as eurydendroid cells, are dispersed within the cerebellar cortex rather than coalescing into deep cerebellar nuclei. To clarify their morphology, eurydendroid cells were labeled retrogradely by biotinylated dextran amine injection into the base of the corpus cerebelli. Labeling allowed the cells to be classified into three types-fusiform, polygonal, and monopolar-depending on their somal shapes and numbers of primary dendrites. The fusiform and polygonal type cells were distributed not only in the Purkinje cell layer but also in the molecular and granule cell layers. The monopolar type cells were distributed predominantly in the Purkinje cell layer of the ventrocaudal portion of the corpus cerebelli. These results suggest that there are some functional differences between these eurydendroid cell types. The eurydendroid cells were double-labeled by retrograde labeling and immunohistochemistry using specific antibodies against GABA, aspartate, and zebrin II. No GABA-like immunoreactivity was detected in the retrogradely labeled eurydendroid cells. About half of retrogradely labeled cells were immunoreactive to the anti-aspartate antibody, suggesting that some eurydendroid cells utilize aspartate as a neurotransmitter. Zebrin II reacts with cerebellar Purkinje cells but left all retrogradely labeled neurons nonreactive, although some of these were surrounded by immunopositive fibers. This relationship between the eurydendroid and Purkinje cells is similar to that between the deep cerebellar nuclei and Purkinje cells in mammals.

Partial cloning and expression of mRNA coding choline acetyltransferase in the spinal cord of the goldfish, Carassius auratus.

Choline acetyltransferase (ChAT, EC 2.3.1.6) synthesizes a neurotransmitter, acetylcholine in cholinergic neurons. ChAT is considered to be the most specific marker for cholinergic neurons. To obtain a better marker of the neurons, as the first step, we isolated a partial ChAT cDNA from the goldfish (Carassius auratus) brain by RT-PCR methods. The partial cDNA of the goldfish ChAT was composed of 718 nucleotides. The amino acid sequence of the goldfish ChAT is approximately 70% identical to those of mammalian and chicken ChAT. Northern blot analysis demonstrated that ChAT mRNA was expressed in the brain and the spinal cord of the goldfish, and much abundant in the spinal cord. In the spinal cord of the goldfish, ChAT-positive neurons were detected mainly in the ventral horn by in situ hybridization. In addition, fluorescence in situ hybridization combined with a retrograde labeling by using True Blue demonstrated ChAT mRNA positive neurons were exactly motoneurons. In the cord, putative presynaptic sympathetic neurons were also labeled.

Involvement of the cerebellum in classical fear conditioning in goldfish.

To investigate the emotional role of the cerebellum of fish, we conducted experiments examining effects of cerebellar manipulations on fear-related classical heart rate conditioning in goldfish. We performed total ablation of the corpus cerebelli to examine the effect of irreversible effects. We also performed localized cooling of the corpus cerebelli, in place of the ablation, for reversible inactivation of the cerebellar function. Both the cardiac arousal response to the first presentation of the conditioned stimulus and the cardiac reflex to the aversive unconditioned stimulus were not impaired by the ablation or cooling of the corpus cerebelli. On the other hand, inactivation of cerebellar function severely impaired the acquisition of a conditioned cardiac response in the fear-related conditioning. In addition, localized cooling of the corpus cerebelli reversibly suppressed the expression of established conditioned response. We suggest that the cerebellum of fish is not only being a motor coordination center but also is involved in emotional learning.

Betanodavirus as a novel transneuronal tracer for fish.

In order to obtain a potential new tool to analyze networks of the central nervous system of teleost fishes, we tested a fish-pathogenic betanodavirus, sevenband grouper nervous necrosis virus (SGNNV), as a transneuronal tracer using the freshwater angelfish Pterophyllum scalare as a test animal. Intravitreous injections of SGNNV into the right eye resulted first in the labelings of neuronal cell bodies in the ganglion cell layers of the retina and then those in the inner and outer nuclear layers in sequence. For the first time, labeled neurons were found also in the stratum periventriculare of the contralateral optic tectum, the ventrolateral and ventromedial thalamic nuclei, and the periventricular nucleus of posterior tuberculum in the brain, then the periventricular pretectal nucleus pars dorsalis and pars ventralis. In contrast, by injections of biotinylated dextran amine into the eye no labeled cell bodies were observed in these brain areas, but axons and terminals were labeled anterogradely. These results suggest that the virus could be transported in both directions in axons of the first order neuron and transfected the second and third order neurons by passing across synaptic clefts, and that this technique is practically applicable to the study of neurobiology in teleost.

Post-hatching development of the brain in Octopus ocellatus.

To investigate the post-hatching development of the brain in a benthic octopod, Octopus ocellatus, we performed volumetric analyses of the brain. The brain consisting of the supra- and subesophageal masses was divided into 5 regions according to the functions suggested for the brain of another benthic octopod Octopus vulgaris, and the volume of each region was estimated at three post-hatching ages. We found that the inferior frontal lobe system and the brachial lobe increased in relative volume as the animals grew, while the basal lobe system decreased in relative volume. This result suggests that increasing demand for processing tactile information after hatching is reflected in the higher developmental rate in the centers devoted for tactile sense and related learning. We also found that the inner neuropile layer mainly consisting of dendrites, synapses and axons showed great increases in volume compared with the outer neural-cell-body layer. Although the increase in volume of the inner layer was marked during 1 month after hatching in all brain regions examined, the extent of the increase varied among brain regions. Developmental changes in cell densities in the outer layer also varied among the regions. The present results suggest that the post-hatching development of the brain in O. ocellatus is not homogeneous but varies among brain regions depending on different roles in controlling the behavior.

Cerebellar efferent neurons in teleost fish.

In tetrapods, cerebellar efferent systems are mainly mediated via the cerebellar nuclei. In teleosts, the cerebellum lacks cerebellar nuclei. Instead, the cerebellar efferent neurons, termed eurydendroid cells, are arrayed within and below the ganglionic layer. Tracer injections outside of the cerebellum, which retrogradely label eurydendroid cells demonstrate that most eurydendroid cells possess two or more primary dendrites which extend broadly into the molecular layer. Some eurydendroid cells mostly situated in caudal portions of the cerebellum have only one primary dendrite. The eurydendroid cells receive inputs from the Purkinje cells and parallel fibers, but apparently do not receive inputs from the climbing fibers. Eurydendroid cells of the corpus cerebelli and medial valvula project to many brain regions, from the diencephalon to the caudal medulla. A few eurydendroid cells in the valvula project directly to the telencephalon. About half of the eurydendroid cells are aspartate immunopositive. Anti-GABA and anti-zebrin II antibodies that are known as markers for the Purkinje cells in mammals also recognize the Purkinje cells in the teleost cerebellum, but do not recognize the eurydendroid cells. These results suggest that the eurydendroid cells receive GABAergic inputs from the Purkinje cells. This relationship between the eurydendroid and Purkinje cells is similar to that between the cerebellar nuclei and Purkinje cells in mammals. The eurydendroid cells of teleost have both dissimilar as well as similar features compared to neurons of the cerebellar nuclei in tetrapods.

Molecular cloning and effect of c-fos mRNA on pharmacological stimuli in the goldfish brain.

c-fos is an immediate early gene, and is rapidly and transiently induced in neurons of the central nervous system according to their activities. To investigate neuronal activities in the brain of the goldfish (Carassius auratus), we considered that expression of c-fos mRNA would be an available marker for the neuronal activities. Therefore, we firstly isolated a cDNA clone encoding c-Fos from the goldfish brain by RT-PCR and RACE methods. A full length cDNA of the goldfish c-fos was composed of 1044 bp open reading frame. The amino acid sequence of the goldfish c-Fos was approximately 56-90% identical to those of other teleostean fish c-Fos. Northern blot analysis showed that the expression of c-fos mRNA was rapidly and transiently induced in the brain of the goldfish by the intraperitoneal administration of kainic acid. We also showed that the identification of the c-fos mRNA expression site by in situ hybridization will be able to be used as an anatomical marker for the identification of the activated neuronal region in the goldfish brain.

Efferent connections of the cerebellum of the goldfish, Carassius auratus.

Efferent fiber connections of the corpus and valvula cerebelli in the goldfish, Carassius auratus, were studied using an anterograde neural fiber tracing technique. Efferent targets of the corpus cerebelli are the posterior parvocellular preoptic nucleus, the ventromedial and ventrolateral thalamic nucleus, dorsal posterior thalamic nucleus, periventricular nucleus of posterior tuberculum, dorsal periventricular pretectal nucleus, inferior lobe, optic tectum, torus semicircularis, nucleus of the medial longitudinal fascicle, nucleus ruber, dorsal tegmental nucleus, nucleus lateralis valvulae, reticular formation, torus longitudinalis, and the medial and lateral lobe of the valvula cerebelli. Projections to the posterior parvocellular preoptic nucleus and the periventricular nucleus of posterior tuberculum are not reported in previous studies. Efferent targets of the medial lobe of the valvula cerebelli are similar to that of the corpus cerebelli except for lacking a projection to the inferior lobe and torus longitudinalis, but showing one to the corpus cerebelli. On the other hand, the lateral lobe of the valvula cerebelli projects only to the dorsal zone of the periventricular hypothalamus, the diffuse nucleus of the inferior lobe, corpus mamillare, vagal lobe and the corpus cerebelli. There are topographical projections from the lateral valvula to the inferior lobe. These results suggest that the function of the corpus and medial lobe of the valvula cerebelli include not only motor control but also functions similar to the mammalian higher cerebellum. This study also suggests that there are obvious functional divisions between the medial and lateral lobes of the valvula cerebelli.

Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major.

We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major. Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides. Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence. The alpha-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy. The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens. The three peptides were also hemolytic. Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills. Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.