Demand-feeding rhythms and feeding-entrainment of locomotor activity rhythms in tench (Tinca tinca)

Tench (Tinca tinca) has been described as a strictly nocturnal species whose locomotor activity rhythms, albeit strongly synchronised by light, have an endogenous nature. Aside from light, a number of other environmental factors, such as mealtime, can act as circadian system synchronisers in fish; however, there is a scarcity of information on tench feeding rhythms. This study describes daily self-feeding rhythms in tench, and analyses the role of feeding time on synchronisation of locomotor activity rhythms. Tench were able to operate string sensor-activated self-feeders, and they displayed a strictly nocturnal behavior, both under indoor and outdoor conditions. Locomotor activity remained strictly nocturnal irrespective of whether tench were fed only during the scotophase (D-feeding) or the photophase (L-feeding). However, no statistically significant differences were detected between both groups in terms of food intake or growth performance. Furthermore, unlike L-feeding, D-feeding elicited a clear anticipatory activity (FAA). When tench were given the possibility of feeding at both times of the day, they showed a clear preference for D-feeding. Finally, in fish exposed to constant darkness (DD), feeding time acted as a true zeitgeber and FAA was observed. When animals were fasted under DD conditions, locomotor activity free-run and 6 out of 12 individuals yielded significant results in the periodogram analysis. Under DD, fish resynchronised when regular food was resumed, with some tench displaying FAA. The obtained results indicated the existence of a feeding-entrainable oscillator (FEO) in tench.     

Morphology of the Mauthner axon inhibitory system in tench (Tinca tinca L.) spinal cord

Using ultrastructural features as a natural tracer we identified a commissural interneuron in the tench (Tinca tinca L.) spinal cord with the following characteristics: (1) the unipolar cell soma is located 100-150 micron dorsal from the central canal, the frequency of its occurrence being one cell on each side per spinal cord segment; (2) the first node of Ranvier of its axon is connected to a presynaptic branch of the ipsilateral Mauthner axon via gap junctions; (3) its postsynaptic targets are contralateral moto- and interneurons; (4) its terminal boutons contain f-type vesicles and form Gray-type-2 synapses abut on the initial segment or node of Ranvier of these target neurons distal to the input site of monosynaptic excitatory links from the contralateral Mauthner axon. Thus, it appears that this segmental interneuron may provide the structural basis for the well-known mutual crossed inhibition within the spinal circuit of Mauthner axons.     

Comparative study on the accumulation of heavy metals in different organs of tench (Tinca tinca L. 1758) and plerocercoids of its endoparasite Ligula intestinalis

Concentrations of heavy metals (Cu, Fe, Zn, Mn, Cr, Pb and Cd) in some organs of tench and tissues of its parasite found in Kovada Lake (Turkey) were analyzed using atomic absorption spectrophotometry and subsequently compared with the data from sediments and water. Only Cu, Fe, Zn and Mn were detected in water, sediment, plerocercoids of Ligula intestinalis and fish samples, while levels of Cr, Pb and Cd were below the detection limits. Four elements were found at higher concentrations in the plerocercoid than in different fish tissues (muscle, liver and gill), being 1.6–37.4 times higher than that measured in muscle, liver and gill. Significant positive (for Cu) and negative (for Fe, Zn and Mn) correlations were found between the quantity of heavy metals in water and tissues of L. intestinalis plerocercoids while there were significant positive (for Cu and Zn) and negative (for Fe and Mn) correlations between the quantity of heavy metals in bottom sediment and tissues of L. intestinalis plerocercoids. Cestodes were found suitable to reflect the amount heavy metals in sediments, providing more reliable information about the actual pollution of the reservoirs.     

Further morphological (freeze-fracture) evidence for an impulse generating function of Mauthner axon collaterals in the tench (Tinca tinca L.) spinal cord

The freeze-etching technique was applied to the tench spinal cord. Replicas of Mauthner axons revealed that the non-junctional axolemma at the tip of axon collaterals alone exhibits intramembranous E-face particles whose size, distribution and overall density are comparable with those at Ranvier nodes. Since typical Ranvier nodes are lacking along the stem of the heavily myelinated Mauthner giant axon, this finding contributes further evidence to our earlier observations suggesting that the spiny Mauthner axon collaterals represent true nodal equivalents and most likely the long-sought substrate of impulse propagation.     

Scanning electron microscopic study of the splenic vascular casts in common tree shrew (Tupaia glis)

Summary Splenic vascular casts of the common tree shrew,Tupaia glis, were constructed with Batson's No. 17 plastic mixture and studied with the scanning electron microscope (SEM). Fifteen adult animals of both sexes, weighing between 120 and 180 g were used. Under ether anaesthesia, each animal was injected with 0.05 ml heparin intracardially; the right atrium was cut open and then 250 ml of 0.9% NaCl, followed by 50 ml of 10% neutral formalin, (in four animals) was injected through the left ventricle. Plastic mixture was injected through the same opening. After complete polymerization of the plastic, the spleen and surrounding tissues were removed and macerated in 40% KOH. The air-dried casts were then coated with carbon and gold before viewing and photographing under SEM at 15 kV. It was found that the splenic arteries penetrated deep into the organ before they divided into trabecular arteries and divided again into central arterioles. Each central arteriole sent out 15 to 30 radiating arterioles, called penicillar arterioles, and further divided into smaller vessels entering the marginal zone and red pulp. In this area each arteriole continued directly into either marginal or red pulp sinusoids. The sinusoids emptied into pulp venules which joined to form trabecular veins. Most of the trabecular veins travelled to the cortical area underneath the splenic capsule before approaching the hilum, where they finally drained into splenic and short gastric veins. It is likely that the spleen of the common tree shrew has a closed circulation.This paper is dedicated to Professor Fred Walberg on the occasion of his 70th birthday.     

Impairment of the splenic immune system in P2X(2)/P2X(3) knockout mice

The isolated spleens from male and female mice lacking P2X(2) and P2X(3) receptors (P2X(2)/P2X(3) knockout (KO) mice) and those from wild-type (WT) mice were investigated by flow cytometry, immunohistochemistry and functionally by organ-bath pharmacology. The spleens from the P2X(2)/P2X(3) KO mice weighed significantly more than the corresponding WT mice. Flow cytometry was used to isolate the mononuclear cells, which were then phenotyped. T-lymphocytes, B-lymphocytes and macrophages were identified and counted. It was found that the increase in size of the spleens from the KO animals corresponded to an increase in the numbers of mononuclear cells present and that all three cell types (T-lymphocytes, B-lymphocytes and macrophages) increased in much the same proportion as those from the WT animals. Immunohistochemical localisation of P2Y(1), P2Y(2) and P2X(1) receptors revealed their presence on the spleen capsule and trabeculae. P2X(1) receptors were also present on blood vessels. There was no difference in the expression of these receptors between the WT and P2X(2)/P2X(3) KO spleens. Functional studies revealed the presence of multiple P2 receptors inducing the contraction of the spleen capsule, from both WT and KO mice. There was no difference in the contractions induced by adenosine 5'-triphosphate (ATP), alpha,beta-methylene ATP, 2-methylthio ADP or uridine triphosphate from WT and KO mice. It is concluded that mice lacking both P2X(2) and P2X(3) receptors have enlarged spleens and that this is correlated with an increase in the number of immune cells, perhaps as a consequence of a compromised immune system and chronic infection.     

Cytochemistry and morphology of granulocytes of the caecilian <Emphasis Type="Italic">Siphonops annulatus</Emphasis> (Amphibia,Gymnophiona)

The caecilians (Amphibia, Gymnophiona) constitute one of the least known groups of terrestrial vertebrates because most species live underground in quite inaccessible environments. Siphonops annulatus is an exclusively fossorial species and is the most extensively distributed caecilian in South America. Little is known of this order concerning circulating granulocytes, including their morphological and cytochemical structure and ultrastructure. This paper is part of a project covering the study of granulocytes in representative species of the order Amphibia. Blood extensions were carried out and submitted to Leishman, Toluidine Blue, Periodic acid Schiff, Sirius Red and hydrogen o-toluidine peroxide methods. Part of the samples was prepared for conventional transmission electron microscopy. Among granular leukocytes, mature and immature neutrophils and eosinophils were identified, plus basophils. The most frequent granulocyte encountered in S. annulatus peripheral blood is the neutrophil. This is a cell with a hyper-segmented nucleus and with a very clear cytoplasm when compared to the eosinophil, which presents large cytoplasmic acidophilic granules. On the other hand, the basophils present basophilic and metachromatic granules. Glycogen was detected in the cytoplasm of the neutrophils and eosinophils, while basic protein rich in amino acids was observed in the eosinophil’s granules. Myeloperoxidase activity was detected in the cytoplasm of the neutrophils and eosinophils. Neutrophils were ultrastructurally detected with three types of small granules: eosinophils with large and small spherical granules and basophils with large spherical granules with lamellate structures.     

In vitro degradation of endocytosed protein in pronephros cells of the char (Salmo alpinus L.). The effects of temperature and inhibitors

In vitro degradation of 125I-formaldehyde treated human serum albumin (fHSA) in char (Salmo alpinus L.) pronephros cells was studied. The labelled protein was injected intravenously and after various intervals of time pronephros cells were isolated and degradation of internalized protein was measured. No degradation could be observed in cells isolated 30 min after injection. The degradation was very effective in cells isolated at later time points (60-90 min); as much as 65% of the initial cell associated labelled protein was degraded during 90 min incubation at 15 degrees C. The effect of temperature on degradation showed a linear course in the temperature range 0-20 degrees C when plotted in an Arrhenius plot. Monensin and ammonium ions inhibited degradation while colchicine had no effect when pronephros cells were isolated 75 min after the injection.     

Topography and ultrastructure of commissural interneurons that may establish reciprocal inhibitory connections of the Mauthner axons in the spinal cord of the tench,Tinca tinca L.

Summary This study was made to identify the inhibitory interneurons belonging to the spinal circuitry activated by the Mauthner axons in the tench (Tinea tinea L.). The histological investigations were focused on a segmentai pair of commissural interneurons that were reconstructedin toto from their distinguishing topographical and ultrastructural features. These features are: (a) the adendritic soma located 100–150 m dorsal to the central canal; (b) the first node of Ranvier which is precommissural and connected to the ipsilateral Mauthner axon via gap junction; (c) the second node of Ranvier, from which two first-order branches arise postcommissurally each supplying roughly the rostral and caudal half of the contralateral spinal cord segment; (d) their second-order branches, which arise at intervals that correspond closely to those of the Mauthner axon collaterals; (e) the postsynaptic targets of the second-order branches, which are exclusively all the motoneurons and interneurons innervated by the contralateral Mauthner axon; (f) the axon terminals of these branches, which contain F-type vesicles, form Gray type-2 synapses, and abut either on the initial segment or on the first node of Ranvier of the target neurons. Thus, it appears that this segmental interneuron has all the appropriate features that could provide the structural basis for the reciprocal fast-acting inhibitory coupling underlying the startle reflex elicited by the Mauthner neurons in response to auditory stimuli.     

Stromal cells, macrophages and lymphoid cells in the head-kidney of sea bass (Dicentrarchus labrax L.). An ultrastructural study

The ultrastructure of the stromal cells, macrophages and lymphoid cells in the head-kidney of the sea bass (Dicentrarchus labrax L.) was studied. Like mammals, stroma cell types here include endothelial and adventitial cells comprising the sinusoidal wall, fibroblast-like reticular cells related to scarce reticular fibres, and macrophage-type reticulum cells, the last probably corresponding to the resident macrophage population of higher vertebrates. Their possible role in the haemopoietic microenvironment is considered. Monocyte-macrophages, macrophages and melano-macrophages, probably corresponding to ontogenic or functional stages of the same cell type were identified and their functional significances are discussed. Scarce, free lymphoid cells or small clusters of lymphocytes but no lymphopoietic islets were recognizable. Large lymphocytes, small lymphocytes and very scarce developing and mature plasma cells were identified. The lymphoid function and defensive role of the head-kidney were analyzed.     

In vitro and in vivo study of the clastogenicity of the flavone cirsitakaoside extracted from Scoparia dulcis L. (Scrophulariaceae)

The mutagenic effect of the flavone cirsitakaoside extracted from the medicinal herb Scoparia dulcis was evaluated in vitro by using human peripheral blood cultures treated with doses of 5, 10, and 15 microg of the flavone/ml culture medium for 48 h. The compound proved to be mutagenic at the highest concentration tested (15 microg/ml). Furthermore, the proliferative index was significantly reduced in all cultures treated with the flavone, although the mitotic index was not reduced. However, the clastogenic activity of the flavone cirsitakaoside was not observed when Swiss mice were treated orally with doses of 10, 20, and 30 mg/animal for 24 h.     

In vitro and in vivo responses of murine granulocytes to human complement-derived, haemolytically inactive C5b67 (iC5b67).

Haemolytically inactive C5b67 (iC5b67), which was made from purified human components and decayed to a haemolytically inactive form, was evaluated as an agonist for murine leucocytes both in vitro and in vivo. In an in vitro assay, iC5b67 stimulated chemotaxis for both neutrophils purified from mouse bone marrow and splenic eosinophils of IL-5 transgenic mice. The stimulation was dose-dependent, with high dose inhibition. As with human neutrophils, iC5b67 also failed to up-regulate CR3 (CD11b/CD18) expression and to stimulate superoxide generation in murine bone marrow neutrophils, in vitro. In vivo, iC5b67 elicited an inflammatory response in a mouse model of pleuritis. A marked infiltration of neutrophils, which peaked at 4 h, was followed by an infiltration of eosinophils and mononuclear leucocytes. This inflammatory response was dose- and time-dependent. However, the protein concentration in the pleural wash fluid did not increase, indicating that iC5b67 did not induce a capillary leak. Although the infiltration of neutrophils could not be reproduced by pure C7 or human serum albumin (HSA), C5b6 did induce an influx of neutrophils. We were able to document the existence of C7, both antigenically and functionally, in pleural washes of normal mice, making it likely that the activity of C5b6 resulted from the in situ formation of C5b67 and iC5b67. The mouse model of pleuritis promises to be a useful in vivo system in which to evaluate the pro- and anti-inflammatory effects of iC5b67 that have been noted in vitro.     

Hyperplastic and hypertrophic growth of lateral muscle in Dicentrarchus labrax (L.). An ultrastructural and morphometric study

Summary In this EM study of lateral muscle in Dicentrarchus labrax, we observed that during the larval period, growth of the presumptive red and white muscle layers occurs both by hypertrophy (as fibres already present at hatching complete their maturation) and by production of new fibres in germinal zones specific to the two muscle layers.In the first half of larval life the presumptive white muscle increases in thickness by the addition, superficially, of new fibres derived from a germinal zone of presumptive myoblasts lying beneath the red muscle layer. In the second half of larval life new fibres produced in this same zone form the intermediate (or pink) muscle layer. Dorsoventrally the myotome grows throughout larval life, largely by addition of new fibres from germinal zones at the hypo- and epi-axial extremities. Towards the end of larval life all these germinal zones are becoming exhausted, but another source of fibres arises as satellite cells, associated with large-diameter presumptive white muscle fibres, are activated to produce new fibres. The addition of small, new fibres gives the white muscle its mosaic appearance.Morphometric analysis of fibre diameters in the white muscle confirms that whereas these hyperplastic processes are important during the larval and juvenile periods, when growth is very rapid, they have ceased by the time the adult stage is attained. By contrast, fibre hypertrophy continues through into adult life.The presumptive red muscle consists initially of a monolayer of fibres present only near the lateral line, and during larval life it grows hypo- and epi-axially by addition of fibres derived from myoblasts already present in these areas at hatching. Lying superficially to the presumptive red muscle monolayer there is a near-continuous layer of external cells with a flattened profile. During the second half of larval life, differentiation of these external cells into myoblasts provides the source of new fibres which are added to the red muscle layer. This process, which occurs initially in the region around the lateral line and later spreads outwards, is responsible for the increase in thickness of the red muscle.     

Effect of prolactin, in vivo and in vitro, upon heterophil phagocytic function in the ring dove (Streptopelia risoria)

The purpose of this study was to investigate the effects of prolactin, both in vitro and in vivo, upon heterophil phagocytic activity of the ring dove, Streptopelia risoria. In vitro incubation of heterophils with either 0.1 or 100 μg/mL of ovine prolactin for 2 h significantly increased both latex bead phagocytosis and Nitroblue tetrazolium (NBT) reduction, an index of phagocytic metabolic activity. Ring doves given antigen demonstrated an increase in latex bead phagocytosis and NBT reduction. The greatest increase in both of these parameters was observed in those birds which possessed elevated plasma prolactin concentrations, either through exogenous administration or naturally through being in the later stages of incubation. These results suggest that during the incubation period of the avian breeding cycle there is an increase in phagocytic activity which is a direct consequence of the elevation shown by these birds in the concentration of plasma prolactin.     

In vitro anti-inflammatory,cytotoxic and antioxidant activities of boesenbergin A,a chalcone isolated from Boesenbergia rotunda (L.) (fingerroot)

The current in vitro study was designed to investigate the anti-inflammatory, cytotoxic and antioxidant activities of boesenbergin A (BA), a chalcone derivative of known structure isolated from Boesenbergia rotunda. Human hepatocellular carcinoma (HepG2), colon adenocarcinoma (HT-29), non-small cell lung cancer (A549), prostate adenocarcinoma (PC3), and normal hepatic cells (WRL-68) were used to evaluate the cytotoxicity of BA using the MTT assay. The antioxidant activity of BA was assessed by the ORAC assay and compared to quercetin as a standard reference antioxidant. ORAC results are reported as the equivalent concentration of Trolox that produces the same level of antioxidant activity as the sample tested at 20 µg/mL. The toxic effect of BA on different cell types, reported as IC₅₀, yielded 20.22 ± 3.15, 10.69 ± 2.64, 20.31 ± 1.34, 94.10 ± 1.19, and 9.324 ± 0.24 µg/mL for A549, PC3, HepG2, HT-29, and WRL-68, respectively. BA displayed considerable antioxidant activity, when the results of ORAC assay were reported as Trolox equivalents. BA (20 µg/mL) and quercetin (5 µg/mL) were equivalent to a Trolox concentration of 11.91 ± 0.23 and 160.32 ± 2.75 µM, respectively. Moreover, the anti-inflammatory activity of BA was significant at 12.5 to 50 µg/mL and without any significant cytotoxicity for the murine macrophage cell line RAW 264.7 at 50 µg/mL. The significant biological activities observed in this study indicated that BA may be one of the agents responsible for the reported biological activities of B. rotunda crude extract.     

脑胶质瘤间质内化疗联合用药的敏感性研究

目的 观察卡铂（CBP）、威猛（Vm-26）、甲氨喋呤（MTX）及尼莫地平（NIM）联合应用对脑腔质瘤的抑制作用，探讨脑间质内联合用药与服质瘤细胞敏感性的关系，为临床脑胶质瘤间质内化疗提供有效的化疗依据。方法 40倒脑胶质瘤标母体外细胞培养，采用噻唑兰（MTT）比色法测定体外用药对脑肢质瘤的增殖抑制作用。光镜和电镜下观察药物作用的细胞形态学改变，并采用流式细胞术（FCM）测定药物在脑腔质瘤细胞周期中的分布厦细胞凋亡情况，比较脑胶质瘤细胞对CBP、Vm-26、MTX及NIM单独使用、联合使用的敏感性。结果CBP、Vm-26、MTX、NIM联合用药对肿瘤细胞的抑制率可连06．64％；明显高于CBP＋NIM（69.03％）、Vm-26＋NIM（71.53％）、MTX＋NIM（52.75％）及CBP＋Vm-26＋MTX（78．59％）的抑制效果（P〈0．01），同时CBP、Vm-26、MTX的浓度降低为单独用药的1／10～I／100；光镜和电镜检查发现，在药物作用下肢质瘤细胞形态发生明显改变；FCM检查结果表明，MTX＋CBP＋Vm-26＋NIM联合应用能显著诱导细胞发生凋亡，且主要作用于G2期、S期细胞。结论 联合应用CBP、Vm～26、MTX及NIM对肢质瘤细胞的敏感性明显优于单用药，具有抑制率高、用药浓度低的特点。药物对胶质瘤细胞各个周期均有杀伤力，以G2期和S细胞为主。