The neurotoxic effects of manganese on the dopaminergic innervation of the gill of the bivalve mollusc, Crassostrea virginica

We examined effects of manganese on the nervous system and innervation of lateral cilia of Crassostrea virginica. While essential in trace amounts, tissue manganese accumulation is neurotoxic, inducing Manganism, a Parkinson's-like disease in humans. Lateral cilia of the gill of C. virginica are controlled by a reciprocal serotonergic-dopaminergic innervation from their ganglia. Oysters were incubated 3 days in the presence of up to 1 mM manganese, followed by superfusion of the cerebral ganglia, visceral ganglia or gill with dopamine or serotonin. Beating rates of cilia were measured by stroboscopic microscopy of isolated gill preparations or gill preparations with the ipsilateral cerebral and/or visceral ganglia attached. Acute manganese treatments impaired the dopaminergic, cilio-inhibitory system, while having no effect on the serotonergic, cilio-excitatory system, which is in agreement with the proposed mechanism of manganese toxicity in humans. Manganese treatments also decreased endogenous dopamine levels in the cerebral and visceral ganglia, and gills, but not serotonin levels. We demonstrated that manganese disrupts the animal's dopaminergic system, and also that this preparation can be used to investigate mechanisms that underlie manganese neurotoxicity. It also may serve as a model in pharmacological studies of drugs to treat or prevent Manganism and other dopaminergic cell disorders.     

合浦珠母贝鳃的显微与超微结构

合浦珠母贝(Pinctada fucata)是典型的滤食性瓣鳃类动物,也是我国重要的海水珍珠养殖贝类。本研究用光学显微镜、扫描电镜和透射电镜观察了合浦珠母贝鳃的显微和超微结构。结果表明,合浦珠母贝鳃结构属于异丝鳃型,左右两侧各2个鳃瓣,每个鳃瓣由内鳃瓣和外鳃瓣组成。鳃瓣由主鳃丝和普通鳃丝构成,主鳃丝在鳃瓣中主要起支架作用,每2根主鳃丝之间的9～12根普通鳃丝由"簇内连接"(intrabunchial junction)相连成簇。普通鳃丝之间通过"丝间连接"(interfilament junction)相连,丝间连接的上皮细胞与普通鳃丝的扁平细胞结构一样,为鳃的呼吸上皮。丝间连接的存在扩大了鳃的表面积,这种结构有助于进行气体交换。主鳃丝和普通鳃丝表面有前纤毛和侧纤毛,与食物运送和气体交换有关。普通鳃丝表面的纤毛为典型的"9+2"型微管结构。     

The nervous system control of lateral ciliary activity of the gill of the bivalve mollusc, Crassostrea virginica

Lateral cilia of the gill of Mytilus edulis are controlled by a reciprocal serotonergic-dopaminergic innervation from their ganglia. Other bivalves have been studied to lesser degrees and lateral cilia of most respond to serotonin and dopamine when applied directly to the gill indicating a possible neuro or endocrine mechanism. Lateral cilia in Crassostrea virginica are affected by serotonin and dopamine, but little work has been done regarding ganglionic control of their cilia. We examined the role of the cerebral and visceral ganglia in innervating the lateral ciliated cells of the gill epithelium of C. virginica. Ciliary beating rates were measured in preparations which had the ipsilateral cerebral or visceral ganglia attached. Superfusion of the cerebral or visceral ganglia with serotonin increased ciliary beating rates which was antagonized by pretreating with methysergide. Superfusion with dopamine decreased beating rates and was antagonized by ergonovine. This study demonstrates there is a reciprocal serotonergic-dopaminergic innervation of the lateral ciliated cells, similar to that of M. edulis, originating in the cerebral and visceral ganglia of the animal and this preparation is a useful model to study regulatory mechanisms of ciliary activity as well as the pharmacology of drugs affecting biogenic amines in nervous systems.     

Studies on the gill ciliation of the American oyster Crassostrea virginica (Gmelin)

Latero-frontal, para-latero-frontal, and frontal ciliary tracts on the gill filaments of Crassostrea virginica (Gmelin) were studied with light microscopy and scanning electron microscopy. Latero-frontal cirri are complex structures composed of varying numbers of paired cilia. The multiple pairs of cilia which constitute a single cirrus are closely appressed for a portion of their length; they then branch laterally from the central axis in a plume-like fashion. Latero-frontal cirri of adjacent gill filaments create a filtration sieve which should be capable of retaining particles smaller than 1 μm in diameter. Para-latero-frontal cilia are short, closely spaced cilia arranged as a staggered row along the frontal side of each tract of latero-frontal cirri. Latero-frontal cirri and para-latero-frontal cilia occur on ordinary, principal, and transitional gill filaments. Frontal ciliary tracts of ordinary filaments are divided into a central, ventrally directed coarse tract, flanked on either side by a dorsally directed fine ciliary tract. The coarse tract is covered by cirri which are comprised of five to eight cilia, while the fine frontal tracts are made up of individually functioning cilia. The frontal ciliary tracts of principal and transitional filaments bear only dorsally directed fine cilia. The unique direction of effective beat of the coarse frontal cirri of ordinary filaments, in combination with the action of fine frontal cilia and the strategic location of mucus producing cells, is used to describe a possible mechanism for the sorting of filtered particles.     

STUDIES ON CILIA : The Fixation of the Metachronal Wave

Upon excision into spring water, the lateral cilia of the gill of the freshwater mussel Elliptio complanatus (Solander) stop beating, but 0.04 M potassium ion can activate the gill so that these cilia again beat with metachronal rhythm. One per cent osmium tetroxide quickly pipetted onto a fully activated gill fixes the lateral cilia in a pattern that preserves the form and arrangement of the metachronal wave, and permits the cilia to be studied with the electron microscope in all stages of their beat cycle. Changes are seen in the fixed active preparation that are not present in the inactive control, i.e., in the packing of the cilia, the position of the axis of the ciliary cross-section, and the diameter of the ring of peripheral filaments. Analysis of these parameters may lead to new correlations between ciliary fine structure and function.     

An endogenous SCP-related peptide modulates ciliary beating in the gills of a venerid clam, Mercenaria mercenaria

The activities of both the lateral and frontal cilia of Mercenaria mercenaria were unaffected, either by the two endogenous SCP-related peptides AMSFYFPRMamide and YFAFPRQamide, or by FMRFamide (all at 10(-6) M). Dopamine (DA) inhibited the lateral cilia; the mean EC50 was 2 x 10(-6) M. The peptide YFAFPRQamide--but neither AMSFYFPRMamide nor FMRFamide--antagonized the inhibition induced by DA; this effect was dependent on both time and dose. At a DA concentration of 5 x 10(-7) M, the effect of YFAFPRQamide appeared within 20 min and became maximal within 40-60 min; the mean EC50 at these times was 4.7 x 10(-11) M. If the concentration of DA was increased to 10(-6) M, the maximal effect of the peptide was delayed to 50 min, and the mean EC50 increased to 1.1 x 10(-7) M. Particle transport by the frontal cilia was inhibited by 5-hydroxytryptamine (5HT); the mean EC50 was 5.7 x 10(-7) M. Again, only YFAFPRQamide had an antagonistic effect on the 5HT-induced inhibition. At a 5HT concentration of 10(-6) M, the effects of YFAFPRQamide did not appear until 45 min; the mean EC50 was 10(-6) M. When radioimmunoassayed with an SCP antiserum, the elution profile of a gill extract overlapped those of the SCP-related peptides that had previously been identified in extracts of whole animals. These data suggest that all three SCP analogs occur in the gill. Immunohistochemistry of the gill, carried out with a monoclonal antibody raised to SCPB, stained many varicose neuronal fibers. Most of these were associated with the gill musculature, but a sparse innervation of the filaments underlying the cilia was also observed. Some fluorescent nerve cell bodies were also seen in the gill tissue. Our results are consistent with the hypothesis that YFAFPRQamide modulates branchial activities--muscular as well as ciliary--that are associated with feeding.     

Histopathological Changes in Tissues of Bithynia siamensis goniomphalos Incubated in Crude Extracts of Camellia Seed and Mangosteen Pericarp

The present study was performed to observe histopathological changes in tissues of Bithynia siamensis goniomphalos (Gastropoda, Bithyniidae) incubated in crude extract solutions of camellia (Camellia oleifera) seed and mangosteen (Garcinia mangostana) pericarp, and furthermore to estimate the molluscicidal effects of 2 plant substances. Substantial numbers of bithyniid snails were incubated in various concentrations of 2 plant solution for 24 hr. As the positive control, snails incubated in various concentrations of niclosamide, a chemical molluscicide, were used. The histopathological findings were observed in sectioned snail specimens of each experimental and control groups. The results showed that both camellia and mangosteen extracts had molluscicidal effects at 24 hr with 50% lethal concentration (LC₅₀) at concentrations of 0.003 and 0.002 g/ml, respectively, while niclosamide had LC₅₀ at concentrations 0.599 ppm. B. siamensis goniomphalos snail tissues (foot, gill, and digestive system) showed disruption of columnar muscle fibers of the foot, reduction of the length and number of gill cilia, numerous mucous vacuoles, and irregularly shaped of epithelial cells. Irregular apical and calciferous cells, dilatation of the digestive gland tubule, and large hemolymphatic spaces, and irregular apical surfaces, detachment of cilia, and enlargement of lysosomal vacuoles of epidermis were also shown in all groups. By the present study, it is confirmed that 2 plants, camellia and mangosteen, are keeping some substance having molluscicidal effects, and histopathological findings obtained in this study will provide some clues in further studies on their action mechanisms to use them as natural molluscicides.     

Dopamine produces muscle contractions and modulates motoneuron-induced contractions inAplysia gill

1. Dopamine has been reported to exist in unusually large quantities in Aplysia gill. The physiological role of this neurotransmitter in this organ was examined. 2. The addition of dopamine to a gill perfusate results in the contractions of the lateral and medial external pinnule muscles, the circular and longitudinal muscles of the afferent vessel, and the circular muscles of the efferent vessel. 3. Dopamine-induced contractions persist after chemical synaptic transmission is eliminated in the gill. This suggests that excitatory dopamine receptors are present on gill smooth muscle fibers themselves. 4. Dopamine also potentiates the gill response to action potentials in single identified gill motoneurons. Evidence presented suggests that muscle contractions and modulation of motoneuron contractions are independent phenomena. 5. While modulation may in part be mediated by increases in excitatory junction potential (EJP) amplitude, in many cases large increases in muscle contractions occur while the enhancement of EJPs is disproportionately small. 6. Dopamine's ability to produce muscle contractions suggests that there may be dopaminergic motoneuron innervation of the gill. We suggest that dopamine's modulatory actions may be mediated via modification of excitation-contraction coupling in smooth muscle fibers.     

The calcium-dependent neuronal release of serotonin and its antagonism by lithium

The cilio-excitatory serotonergic innervation of lateral gill cilia of Mytilus edulis was studied in vivo. Peripheral serotonin release was dependent on the external calcium concentration. Serotonin release was inhibited by autodialyzing calcium from the tissue or by increasing the calcium concentration in the medium, as determined by measuring ciliary activity stroboscopically and by biochemical and radioassays of serotonin. Lithium also inhibited serotonin release when added to the external bathing medium. Concomitantly, altering calcium concentrations altered the degree of inhibition of serotonin release caused by lithium. The study demonstrates that the terminal release of the monoamine serotonin is a calcium-dependent mechanism. The pharmacological effects of lithium in this system appear to be interrelated with the calcium-dependent releasing mechanism.     

NEUROID TRANSMISSION IN CILIATED EPITHELIUM

Evidence from several sources indicates that there is present in certain types of ciliated tissue a primitive form of conduction regulating the rate of beat of the cilia. (Kraft, 1890; Engelmann, 1898). A similar type of conduction has been observed by Parker (1910) in the sponge Stylotella. In the foregoing experiments a study was made of such conduction in the ciliated tissue of the gills of the clam Unio. Observations were made on the effects of temperature changes on the rate of beat of cilia adjacent to areas not themselves directly influenced. The following results were obtained. 1. The transmission through the gill of the effects of warmth applied locally is apparent through increased rate of ciliary beat on adjacent gill tissue in all directions from the region of application. Effects are not observed laterally at a distance greater than 9 to 11 mm. from the nearest edge of the stimulated area. The narrowness of the gill makes it impossible to determine the vertical limits of the transmission. 2. Effects of low temperatures are not observable beyond the limits of the region of direct application. These results differ from those of Kraft in tissue from the frog''s pharynx, where conduction was shown to take place only down a row of beating cilia. On the other hand they agree with his results in indicating that the effects of warmth only are transmitted. The phenomenon might be explained by the stimulating effect of the action current of the directly excited cilia on the neighboring relatively quiet cilia. A similar explanation has been offered by Lillie for waves of coordinated beating in the rows of swimming plates of ctenophores. Such an explanation, though in accord with the work on Unio, is inconsistent with certain of the observations of Kraft on the tissue from the frog''s pharynx.     

Allelopathic Potential of Invasive Plantago virginica on Four Lawn Species

Plantago virginica L. has invaded many lawn ecosystems in the Eastern part of China. The invasion has incurred an economic cost to remove them. In order to prevent the invasion, it is critical to understand the invasive mechanisms of this species. However, few studies have been conducted on the allelopathic mechanisms of its invasion. In this study, we examined allelopathic effects of P. virginica on germination of seeds and growth of seedlings of four widely used lawn species. We found extensive allelopathic potential of P. virginica on other lawn species, which varied with species and developmental stage. While most effects of the extracts of P. virginica were inhibitory, some variables in some species were promoted by the addition of the extracts. The extracts of P. virginica significantly inhibited seed germination of Agrostis matsumurae. While the overall differences in seed germination rate of Poa annua were significant among treatments, difference between control and any of the treatments was not significant. The height of seedlings of A. matsumurae and Cynodon dactylon was significantly lower under the treatments of adding extracts of P. virginica. In contrast, growth of seedlings of Festuca elata and P. annua did not show significant differences among treatments. The root length of A. matsumurae, C. dactylon and P. annua was suppressed by the extracts of P. virginica whereas root length of F. elata was not affected. Aboveground biomass of A. matsumurae and F. elata was significantly higher than control, except for F. elata at the concentration of 50mg/mL, whereas aboveground biomass of C. dactylon and P. annua was reduced at higher concentrations of the extracts. Except for A. matsumurae, root biomass of the other three lawn species declined under the treatments with the extracts of P. virginica. Our results revealed that P. virginica had allelopathic potential on four lawn species and supported the theory of “novel weapons hypothesis”. Invasion by P. virginica in lawn can be moderated by selecting those species that are not affected or promotionally affected by it.     

Control of ciliary beat frequency in the gill of Mytilus--II. Effects of saponin and Brij-58 on the lateral cilia

The effects of saponin and Brij-58 on the beat activation of the lateral cilia on the gill of Mytilus edulis were investigated. The ciliary activation by 5-hydroxytryptamine (5HT) decreased as the saponin-induced permeabilization progressed, increasing the reactivation of the ciliary beat by extracellularly applied ATP (1 mM). The cilia were activated by 5HT even after the treatment with saponin (0.01 and 0.02% w/v) or Brij-58 (0.07%) rendered the preparation capable of the reactivation by ATP. The saponin treatment itself stimulated the beat of the cilia. Theophylline (1 mM) augmented the saponin-induced activation of the cilia.     

Short-term manganese pretreatment partially protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that induces parkinsonism in human and non-human primates. Its mechanism of action is not fully elucidated.Recently, the participation of trace metals, such as manganese, on its neurotoxic action has been postulatted. In this work, we studied the effect of manganese administration on the neurochemical consequences of MPTP neurotoxic action. Male Swiss albino mice were treated with manganese chloride (MnCl₂ ·4H₂O; 0.5 mg/ml or 1.0 mg/ml of drinking water) for 7 days, followed by three MPTP administrations (30 mg/Kg, intraperitoneally). Seven days after the last MPTP administration, mice were sacrificed and dopamine and homovanillic acid contents in corpus striatum were analyzed. Striatal concentration of dopamine was found increased by 60% in mice pretreated with 0.5 mg/ml and 52% in the group treated of 1.0 mg/ml as compared versus animals treated with MPTP only. Hornovanillic acid content in both groups treated with manganese was the same as those in control animals. The results indicate that manganese may interact with MPTP, producing an enhancement of striatal dopamine turnover, as the protective effect of manganese was more pronounced in the metabolite than in the neurotransmitter.     

Effects of the cilioexcitatory neurohumors dopamine and 5-hydroxytryptamine on cyclic AMP levels in the gill of the mussel Mytilus edulis.

Cyclic 3′, 5′-adenosine monophosphate (cAMP) has been identified in the ciliated gill epithelium of the marine mussel $\text{Mytilus}$$\text{edulis}$. In concentrations which stimulate the rate of particle transport by frontal gill cilia, DA and 5HT stimulate levels of cAMP within the gill. The stimulation occurs in as early as 15 sec and is graded from 10^?6M to 10^?4M. DA plus 5HT is not additive at maximal effective concentrations of both amines. ACH does not mimic the DA or 5HT stimulation of cAMP. Theophylline alone has a weak effect on cAMP levels; however, the effect of theophylline is potentiated in the presence of DA or 5HT. Dibutyryl cAMP produces a gradual stimulation in the rate of particle transport. It is suggested that the dopaminergic and serotonergic excitatory control of particle transport by frontal gill cilia of $\text{Mytilus}$$\text{edulis}$ is mediated through a cAMP second messenger system.     

Alteration of dopaminergic innervation and voluntary movements after long period of thirst in a semi-desert rodent, Meriones shawi: Behavioral and immunohistochemical studies

Dehydration is a powerful stimulus causing disequilibrium in homeostasis of water and electrolytes resulting from depletion in total body water. Most studies have focused on domestic and laboratory animals; however, the study of desert animals allows improved understanding about water balance and resistance to dehydration and associated behavioral changes, including those related to voluntary movements. Meriones shawi (Shaw's Jird) is a desert rodent characterized by its resistance to long periods of thirst that can extend for several months. In the present study, M. shawi were subjected to water deprivation for 1 month. We used tyrosine hydroxylase immunohistochemistry (TH: the key enzyme of catecholamine biosynthesis) to evaluate the effects of prolonged dehydration on the dopaminergic system in both substancia nigra pars compacta and ventral tegmental area (SNpc and VTA), which are the main sources of dopamine input to several brain areas; the immunolabelling was performed also in both the medial forebrain bundle and the caudate putamen (striatum). In addition, the open-field test was used to evaluate the effect of dehydration on locomotor activity in M. shawi. The results showed an increase in TH immunolabelling in both SNpc and VTA following 1 month of dehydration compared to control levels. The same results were obtained with fibers in both MFB and striatum. This augmentation of TH immunoreactivity was accompanied by noticeable changes in locomotor activity behavior of Meriones, the recording test shows the hyperactivity of animals which is probably caused by dehydration. Overall, the results indicate that dehydration is able to increase dopaminergic neurotransmission, which might be involved in generating hyperactivity in this desert animal.     

Lack of Dopaminergic Inputs Elongates the Primary Cilia of Striatal Neurons

In the rodent brain, certain G protein-coupled receptors and adenylyl cyclase type 3 are known to localize to the neuronal primary cilium, a primitive sensory organelle protruding singly from almost all neurons. A recent chemical screening study demonstrated that many compounds targeting dopamine receptors regulate the assembly of Chlamydomonas reinhardtii flagella, structures which are analogous to vertebrate cilia. Here we investigated the effects of dopaminergic inputs loss on the architecture of neuronal primary cilia in the rodent striatum, a brain region that receives major dopaminergic projections from the midbrain. We first analyzed the lengths of neuronal cilia in the dorsolateral striatum of hemi-parkinsonian rats with unilateral lesions of the nigrostriatal dopamine pathway. In these rats, the striatal neuronal cilia were significantly longer on the lesioned side than on the non-lesioned side. In mice, the repeated injection of reserpine, a dopamine-depleting agent, elongated neuronal cilia in the striatum. The combined administration of agonists for dopamine receptor type 2 (D2) with reserpine attenuated the elongation of striatal neuronal cilia. Repeated treatment with an antagonist of D2, but not of dopamine receptor type 1 (D1), elongated the striatal neuronal cilia. In addition, D2-null mice displayed longer neuronal cilia in the striatum compared to wild-type controls. Reserpine treatment elongated the striatal neuronal cilia in D1-null mice but not in D2-null mice. Repeated treatment with a D2 agonist suppressed the elongation of striatal neuronal cilia on the lesioned side of hemi-parkinsonian rats. These results suggest that the elongation of striatal neuronal cilia following the lack of dopaminergic inputs is attributable to the absence of dopaminergic transmission via D2 receptors. Our results provide the first evidence that the length of neuronal cilia can be modified by the lack of a neurotransmitter''s input.     

THE METACHRONAL WAVE OF LATERAL CILIA OF MYTILUS EDULIS

The form of beat of cilia and the structure of the metachronal wave on the lateral gill epithelium of Mytulus edulis have been studied on living material by interference-contrast microscopy and stroboscopic illumination, and compared with the same features in rapid-fixed preparations studied by light microscopy and with the scanning electron microscope. The most striking finding is that the beat of the cilia is not planar, as previously assumed, but involves a sideways movement in the recovery stroke Previous reports on nonplanar ciliary beating from protozoan examples describe a planar effective stroke and a counterclockwise rotation in the recovery stroke; in this molluscan example there is a clockwise rotation in the recovery stroke The lateral inclination of the cilia in the recovery stroke is in the same direction as the propagation of the waves, and the orientation of cilia in the recovery stroke is thought to determine whether the waves move to the left or right of the direction of the effective stroke     

Effect of temperature and salinity on acetylcholinesterase activity, a common pollution biomarker, in Mytilus sp. from the south-western Baltic Sea

Acetylcholinesterase (AChE) activity is a well established biomarker to monitor environmental pollution caused by neurotoxic compounds, such as organophosphorus and carbamate pesticides. The presence of these compounds results in a measurable inhibition of the enzyme. This has been shown for different marine species including blue mussels. Besides pollution, environmental variables may also have a direct or indirect effect on AChE activity, particularly in estuarine and brackish water environments. To assess the impact of abiotic factors on the AChE activity the seasonal course of gill AChE activity was followed in relation to temperature and salinity in Mytilus sp. collected from the south-western Baltic Sea. In addition, the effect of salinity on AChE activity was investigated in an experimental study. The AChE activity showed significant seasonal differences with maximum activities during the summer period and minimum activities in winter. These changes correlate significantly (p<0.001) and positively with water temperature. The experimental exposure of Mytilus sp. to different salinities (5, 7, 16 or 20 psu) resulted in changes in the gill AChE activity. Empirical orthogonal function (EOF) analysis revealed that AChE activity was significantly and negatively correlated with salinity (p<0.01). These results clearly demonstrate the need to consider the potential influence of temperature and salinity on AChE activity when applying this biomarker to monitor exposure to and effect of neurotoxic substances in estuarine and brackish water blue mussels.     

The mussel filter–pump – present understanding,with a re‐examination of gill preparations

Filter feeding in mussels is a secondary adaptation where the gills have become W‐shaped and greatly enlarged, acting as the mussel filter–pump. Water pumping and particle capture in the blue mussel, Mytilus edulis, have been studied over many years. Here, we give a short status of the present understanding of ciliary structure and function of the mussel filter–pump, supplemented with new photo‐microscope and scanning electron microscopy (SEM) pictures of gill preparations. Pumping rate (filtration) and pressure to maintain flow have been extensively studied so the power delivered by the mussel pump to the water flow is known (1.1% of total respiratory power), but the actual cost based on gill respiration is much higher (19%), implying that the cost of maintaining of the large gill pump is considerable and that only relatively little energy can be saved by stopping or reducing the activity of the water‐pumping cilia so that continuous feeding with a ‘minimal scaled’ pump is cheaper than discontinuous feeding with a correspondingly larger pump. According to the present view, the pump proper is the beating lateral cilia (lc) on the gill filaments and particle capture is accomplished by the action of laterofrontal cirri (lfc) transferring particles from the main water current to the frontal gill filament currents driven by frontal cilia (fc). Unexplained aspects include retention efficiency according to particle size and the role of pro‐laterofrontal cilia (p‐lfc) placed between the lfc and fc. The structure of cilia and the mode of ciliary beating have been re‐examined in this study by new high‐resolution light and scanning electron microscopy of isolated gill preparations exposed to serotonin (5‐HT) stimulation which can activate the lc and lfc at low concentrations (10^?6 M), but removes the lfc from the interfilament canals at higher concentrations (10^?5 M).