Kazumasa Uematsu

Effects of eicosapentaenoic and docosahexaenoic acids on growth, survival and brain development of larval Japanese flounder (Paralichthys olivaceus)

An investigation is reported on the effects of arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the growth and survival of larval Japanese flounder (Paralichthys olivaceus). The effects of dietary EPA and DHA on brain development in larvae were also investigated. Fifteen-day-old larvae were fed Artemia nauplii enriched with these fatty acids for 25 days. Larvae fed with Artemia enriched with oleic acid had low survival and poor growth. The survival and growth of larvae were improved by the addition of AA, EPA or DHA to their diet. However, AA was inferior to EPA and to DHA as an essential fatty acid (EFA). The relative volume of the cerebellum (volume of the cerebellum/total volume of the brain) of larvae fed EFA-deficient Artemia was significantly lower than that of larvae fed nauplii enriched with EPA or DHA. Other parts of the brain such as the telencephalon and the diencephalon revealed no significant difference among treatments. Thus, not only survival and growth, but also development of the brain, and of the cerebellum in particular, in larval Japanese flounder, was influenced by dietary EPA and DHA.

Comparison of behavioral responses to a novel environment between three teleosts, bluegill Lepomis macrochirus, crucian carp Carassius langsdorfii, and goldfish Carassius auratus

To investigate the emotional reactivity of fish in a novel environment, the swimway test was developed. The swimway apparatus consists of a shaded start chamber and an open, illuminated swimway. Fish were first introduced and habituated to the start chamber. A door partitioning the start chamber from the swimway was then opened, and behavioral responses of the fish in the apparatus were measured. By using the swimway test, behavioral responses to a novel environment of bluegill Lepomis macrochirus, crucian carp Carassius langsdorfii, and goldfish Carassius auratus were quantified and compared. The emotional reactivities in blue gill were found to be the lowest and crucian carp the highest, indicating bluegill are relatively active or ‘bold’, and that the crucian carp are relatively passive or ‘shy’, in a novel environment. It is suggested that the swimway test is applicable to examning inter-species differences in relative emotional reactivity or boldness in a simplified novel situation.

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.

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.

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. J. Comp. Neurol. 487:300–311, 2005.

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.

Morphogenesis of the Brain in Larval and Juvenile Japanese Eels, Anguilla japonica

We compared the morphology, including relative volumes (RV), of some brain regions in leptocephalus larvae (10-30 mm in total length), glass eels (elvers), young, and immature pre-adults of the Japanese eel Anguilla japonica. The external brain shape of the leptocephali gradually changes from a laterally compressed one to a depressed elongated one. These changes are fundamentally due to biased growth of the telencephalon and optic tectum. The dramatic brain transformation progresses until the juvenile stage and culminates in an adult-type brain arrangement, with a developed cerebellum and eminentia granularis and a much more flattened appearance. The RVs of brain regions closely related to somatic sensory functions are quite different in larvae and juveniles. The larvae possess larger optic tecta and smaller chemo- and mechanosensory regions than the juveniles. The RV of the olfactory bulb increases, and that of the optic tectum decreases, until the adult stage, unlike the condition in a pelagic fish, Pagrus major, suggesting that adult eels are inferior to pelagic fishes in visual perception. In contrast, the brain morphology of the larvae suggests that they are equipped with a well-developed visual system, while the functional significance of the system still remains a mystery. It might be important for feeding, orientation and diurnal vertical migration of the larvae.

Brain Morphogenesis of the Red Sea Bream, Pagrus major (Teleostei)

Post-embryonic morphogenesis and regional volumetric growth of the brain in larval and juvenile red sea bream, Pagrus major, were analyzed by employing a computer-aided three-dimensional reconstruction of serial sections of the head. The relative volume of the optic tectum rapidly increases after hatching until day 8, implying the importance of the visual sense for the first feeding, which begins on day 4. The differentiation and outgrowth of the primary gustatory center in the medulla after day 43 may reflect the increasing number of taste buds, suggesting a relationship to the onset of benthic feeding as juveniles. The rather rapid increases in the relative volumes of the corpus and valvula cerebelli after day 28 and their progressive morphogenesis may be correlated with improved motor performance. The relative volumes of the octaval-lateral line centers, including the eminentia granularis and the crista cerebellaris, and the relative volume of the torus longitudinalis, increase in parallel with those of the corpus and valvula cerebelli.

Physiological response of the fish, Cyprinus carpio, to formalin exposure—I. Effects of formalin on urine flow, heart rate, respiration

1. Carp were exposed to 280 ppm formalin. Haematocrit and plasma glucose and lactic acid increased. In moribund fish, blood pH was remarkably lower and plasma protein increased. 2. When exposed to formalin, heart rate (HR) and respiration increased briefly, and then decreased. 3. Some fish increased urine flow (UF) immediately and maintained higher values for a while, followed by gradual decrease, and others decreased UF consistently. UF stopped at 30 min or longer prior to fish death. Urine osmotic pressure was higher at the 1st to 2nd hour and immediately before UF stopped. 4. The relationship between UF, HR and respiration was also discussed.

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.