Zoological Science

Four components of stimulation-induced changes in neurotransmitter release are known as the synaptic plasticity at the frog neuromuscular junction. These are: fast and slow facilitation, augmentation and potentiation, classified by their decay time constants after repetitive nerve stimulation. Most experiments support the view that fast facilitation is caused by residual Ca²⁺. However, the causes of the other three components are not clear. We have studied electrophysiologically the effect of Mg²⁺ on these three components. Transmitter release was estimated by the amplitudes of endplate potential (EPP) and by the frequencies of miniature endplate potential (MEPP). The increase in the transmitter release by nerve stimulation is described as the product of four components. The magnitude of potentiation of MEPP frequencies after a tetanic nerve stimulation (100 Hz, 5000 times) increased markedly in high Mg²⁺ concentrations. Conversely, the magnitude of augmentation (MEPP frequencies and EPP amplitude) decreased in the higher Mg²⁺ Ringer solution.

The efficiency in O₂ transport of an O₂ carrier is best expressed by the slope of O₂ equilibrium curve (dS/dP), where S and P denote the fractional O₂ saturation of the carrier and the partial O₂ pressure, respectively. The aim of this study is to characterize the O₂ binding to arthropod hemocyanins (Hcs) in term of the O₂ transport efficiency, and for that purpose we have reexamined the previously published O₂ equilibrium data and their mathematical interpretations. Examination of the data for 6- and 12-meric Hcs from lobsters (Panulirus japonicus, Procambarus clarkii) revealed the relationship P _dmax < P ₅₀ ≈ P _nmax, where P _dmax, P ₅₀ and P _nmax denote the P at which dS/dP is maximized, P at half-saturation and P at which the Hill coefficient is maximized, respectively. On the other hand, 48-meric Hcs from horseshoe crabs (Tachypleus gigas, and Limulus polyphemus) gave the relationship P _dmax ≤ P ₅₀ < P _nmax, when the O₂ binding is cooperative. The results indicate that Hc functions most effectively at P values lower than that expected from the maximal degree of cooperativity, similarly to the previous results obtained for human hemoglobin (Hb) [6]. The profile of dS/dP vs. P curves for lobster Hcs was similar to that of Hb, showing a single maximum and a relatively symmetric shape. On the other hand, the curves for horseshoe crab Hcs showed multiple maxima tailing in the low P range, indicating that the O₂ transport efficiency is high even under hypoxic circumstances. As far as the O₂ transport efficiency is concerned, horseshoe crab Hcs are regarded as unique, and the features seem to reflect their uniqueness in the molecular architecture and the mode of allosteric transition.

Adrenocorticotropic hormone (ACTH) and insulin negatively influenced the phagocytic activity of Tetrahymena. The two hormones had diverse effects after 4 hr of treatments on no-test-particle containing, “0-cells”. At this time the number of “0 cells” was significantly lower in the ACTH-treated groups, while in the insulin-treated groups there was an increase of “0-cells” compared to the control and to the results of the starting experiment. Considering previous results, when small molecular weight hormones, if did at all, positively influenced phagocytosis in Tetrahymena, the experiments call the attention to the differences caused by the size of the signal molecules. In the light of the literary data on hormone effects to phagocytosis in mammals and men, the similarity of the effects in species being very far from each other in evolution, could be concluded.

Lipofuscin is the autofluorescent material, which accumulates with aging in the cells of various tissues. However, its autofluorescence characteristics are different among tissues. In the present study, the autofluorescence features of lipofuscin in the brain and adrenal were compared. In 18–21-month-old rats, the brain lipofuscin was granular and its autofluorescence was bright whitish-yellow to bright orange. On the contrary, the adrenal lipofuscin was not demarkated as granules, and its autofluorescence was subdued orange. The emission maximum of the bright whitish-yellow brain lipofuscin was 540 nm to 570 nm and that of the adrenal lipofuscin was 640 nm to 660 nm, when excited at 330 nm to 380 nm. When the spectra were drawn after correcting the wavelength-dependent bias of microspectrofluorometer, the autofluorescence spectra were consistent with microscopically observable tint. To conclude, the present results showed that the autofluorescence features of the bright whitish-yellow brain lipofuscin and the adrenal lipofuscin were quite different.

Coelomocytes (blood cells) of the solitary ascidian Halocynthia roretzi were observed in culture by phase-contrast, time-lapse video and scanning electron microscopy. Seven types and one sub-type of cells were distinguished with respect to their morphology and behavior. These cell types are discussed in relation to those appearing in the literature. A table of correlation between cells reported under various names in the literature is presented in the ^Appendix.

When 10 μl of silicone oil was injected into the hemocoel of a 5-day-old 5th-instar larva of Samia cynthia ricini, the globular oil was encapsulated by hemocytes. The initial process of encapsulation was examined with scanning electron microscopy (SEM). Within 2 min after injection, granulocytes attached to the oil surface, and a thin membrane of amorphous substance occurred around the granulocytes. The cells immediately discharged numerous granules. A reticular network appeared around the degranulated cells, resulting in the formation of the innermost layer of the developing capsule. Compaction of capsule-forming hemocytes occurred after completion of the innermost layer of the developing capsule. These results suggest that a localized coagulation of hemolymph on the oil surface, which resembles ordinary hemolymph coagulation, constitutes the initial phase of encapsulation.

The distribution of the urotensin I (UI)- and II (UII)-like immunoreactants in the central nervous system of the primitive bony fish Polypterus senegalus was studied by means of immunohistochemistry. Noticeably, UI-positive perikarya were present chiefly in the brain stem and the entire spinal cord with the exception of the caudalmost spinal cord segments, and their axons terminated on the wall of the adjacent blood vessels. In the cranial portion, the positive cells were found in the nucleus preopticus, the caudal diencephalon and the medulla oblongata. In contrast, the UII-immunoreactive fibers were distributed in the terminal part of the spinal cord, anterior to the 15th-to-the last vertebra; and a few UII-immunopositive perikarya occurred only in the caudalmost 5 to 6 segments. The most caudal region of the spinal cord showed a simplified or reduced neurohemal structure, where only UII-immunoreactive fibers were found. Accordingly, this area may represent the caudal neurosecretory system of the bichir, irrespective of a lack of UI-immunoreactivity. In the midregion of the spinal cord, UII-immunoreactive cerebrospinal fluid (CSF)-contacting neurons were sporadically encountered.

Specific polyclonal antibodies against the two salmonid gonadotropins (GTHs) were developed using two synthetic oligopeptides corresponding to GTH Iβ-(96–113) and GTH IIβ-(107–119), respectively. By Western blot analysis under reducing conditions, anti-GTH Iβ-(96–113) detected one specific band corresponding to GTH Iβ (17 kDa) and anti-GTH IIβ-(107–119) detected one specific band corresponding to GTH IIβ (20 kDa). Neither antibody recognized bands under nonreducing conditions. By immunohistochemical analysis, immunoreactive (ir-) Iβ-(96–113)-and ir-IIβ-(107–119)-cells had different regional distributions among the aldehyde fuchsin positive cells and were not stained with an anti-human thyrotropin β antibody. In salmonid fishes (chum salmon, rainbow trout, brook trout, whitespotted char, dolly varden and huchen), each antibody reacted with distinctly separate hypophyseal cells. However, in nonsalmonid species, and even in the salmoniform fish, the ayu (Plecoglossus altivelis), no pituitary cell was stained with these antibodies. These data indicate that these antibodies are useful tools for studying the change and the function of GTHs in salmonid fishes.

Axonal regeneration in the central nervous system (CNS) was investigated in the fine structural and histochemical aspects using carp spinal cord, which was completely transected at the level of the dorsal fin. Fusion of the transection region and the regeneration of axons already began to be recognized 26 days after operation by electron microscopy. At 115 days after operation, the rostral and caudal parts of the transected spinal cord were completely connected by the regenerating nervous tissue, which contained numerous axons among the ependymal and glial processes.

Horseradish peroxidase (HRP), which was injected in the spinal cord at the portion caudal to the transection site was detected in the cytoplasm of large neurons located in the reticular formation of midbrain. This demonstrates that these long axons were regenerated passing through the ablation gap 151–204 days after operation. These findings indicate that regenerating axons in the carp spinal cord can pass through the glial scar formed in the transected portion, which is considered to be the main obstacle for the prolongation of axons in the mammalian CNS.

Many regenerating axons, both unmyelinated and myelinated, were observed being in contact directly with the cell membrane of the ependymal as well as astroglial cells. This indicates that neither ependymal nor glial cells play a role as an obstacle for elongation of axons in the carp spinal cord. Numerous GFAP (glial fibrillary acidic protein) positive intermediate filaments were observed in the cell bodies and cytoplasmic processes of both ependymal cells and astroglia. Their morphological similarity and similar property for axonal elongation indicate that the cells of both types in the carp spinal cord are intimately related with each other in origin, morphology and function.

We studied expression of the epidermis-specific gene, designated ep37, by means of Northern blotting in various tissues and at different developmental stages of the Japanese newt, Cynops pyrrhogaster. Using the coding region of ep37 cDNA as a probe, we detected five RNA species. Of these, two were found before metamorphosis and the other three were detected in adult tissues. Amounts of the pre-metamorphic products markedly decreased and two of the three adult products promptly appeared at metamorphosis. These observations along with the difference in affinity of each RNA species to the cDNA probe suggest that the ep37 gene is a member of a novel epidermis-specific gene family, the expression of which is under metamorphic regulation.

This study examined the effect of light pulses on the free-running rhythm of locomotor activity of the hagfish, Eptatretus burgeri, reared for prolonged periods under constant darkness. The free-running period (τ) of circadian locomotor activity of 55 hagfishes followed a normal distribution with mean 24.1 hr (min.: 22.8 hr; max.: 25.1 hr). After establishing the τ in constant darkness for each specimen, we examined the effect of single light pulses (intensity; 670 lx at the bottom of the aquarium, duration; 15 min) on the free-running rhythm. Light pulses in the early subjective night lengthened τ and caused a slight phase delay, whereas those in the late subjective night shortened τ and provoked a slight phase advance. Thus, the hagfish showed a distinct “period response” to short light pulse, but the “phase response” was not so clear as other organisms.

The incidence of diapause and details of body coloration were examined in adults of the bean bug, Riptortus clavatus (Thunberg) (Heteroptera, Alydidae) under various photoperiodic and temperature conditions. The insects showed a long-day photoperiodic response for the induction of diapause at 20, 25 and 30°C. However, some adults at 30°C failed to enter diapause even under short-day conditions. A photoperiodic response that produced light-colored adults under long-day conditions was common among 20, 25 and 30°C, although high temperature increased the proportion of light-colored adults. The photoperiodic response curve for the induction of diapause and that for in the determination of the coloration of the lateral metathorax exhibited a high degree of similarity at 20, 25 and 30°C, supporting a common time-measurement system for these two responses. With respect to the coloration of the ventral metathorax, temperature rather than photoperiod was the major determinant, although an effect of photoperiod was also detectable. There was no significant correlation between the lateral and ventral coloration in the threshold zone.

Differential-interference-contrast microscopy with video enhancement displayed the movement of particles for the first time in the isolated axonic process of cone-type photoreceptor cells of Rana catesbiana. This movement was observed under visible light which visual pigments could absorb. The number of retrograde moving particles in an arbitrary area on the axonic process was twice that of those moving in the anterograde direction. The mean velocities were 1.03 ± 0.55 μm/sec for anterograde particles and 0.41 ± 0.30 μm/sec for retrograde particles, which are of the same order as those found in isolated neurons.

Paramecium primaurelia stock 90 clones were examined in their mating immaturity and maturity stages, under starving conditions triggering mating reactivity in mature cells. Living samples were labelled by cycloheptaamilosedansyl chloride (CDC) complex, which has been proved to be an effective fluorescent marker of outer membrane proteins in living ciliates, as well as a sensitive probe towards microenvironment hydrophobicity. The analysis of CDC-labelled surface membrane region, usually engaged in conjugation, of immature and unpaired mature cells of mating types I and II, reveals differences in emission spectra, which can be referred to different hydrophobicity conditions. These findings lead us to assume that variations in surface membrane chemico-physical properties, differently affecting CDC fluorescence spectra, would be mating type-dependent and related to the functional states of cell immaturity or maturity, thus supporting the suggested role of hydrophobic interactions in Paramecium pair formation.

The transforming growth factor-β (TGF-β) family has been implicated in the regulation of cell differentiation during early development. Bone morphogenetic proteins (BMPs) are thought to play particularly important roles in the formation of the dorso-ventral axis of Xenopus laevis. We found by means of in situ hybridization that BMP transcripts were localized during early Xenopus embryogenesis. BMP-4 transcripts were localized first to neural crest cells, presumptive heart mesoderm and around the yolk plug at the neurula stage and to the eye, auditory vesicles, the heart and mesenchymal cells of fin at the tailbud stage. In sharp contrast, BMP-2 transcripts were strictly localized to olfactory placodes at the tailbud stage. These results showed temporal and spatial regulation of the genes and thus support the notion that BMP proteins are important for directing cell growth and differentiation during organogenesis in the amphibian embryo.