Regeneration of the gill filaments and replacement of serotonergic neuroepithelial cells in adult zebrafish (Danio rerio).

Respiratory epithelia and chemoreceptors of the gills and mammalian lung derive from the same embryonic structures. While the lung is limited to facultative regeneration, the regenerative capacity of the gill has not been adequately explored. We report regeneration of gill filaments and respiratory lamellae in adult zebrafish (Danio rerio). Gill filaments retained a constitutive population of mitotic cells identified by the proliferating cell nuclear antigen (PCNA). Within 24 h of resection, a new mass of PCNA-positive cells appeared at the filament tip. At 40 days post-resection, approximately half of resected tissue was replaced; and at 160 days post-resection, regeneration was nearly complete. Chemoreceptive neuroepithelial cells, identified by serotonin immunohistochemistry, were present in regenerates and established innervation by nerve fibres. Use of the transgenic zebrafish line Tg(fli1a:EGFP), in which the gill vasculature was labelled with enhanced green fluorescent protein, indicated that angiogenesis occurred during the regenerative process. Thus, the zebrafish is capable of substantive gill regeneration and replacement of respiratory chemoreceptors.

Sensory cutaneous papillae in the sea lamprey (Petromyzon marinus L.): II. Ontogeny and immunocytochemical characterization of solitary chemosensory cells.

Solitary chemosensory cells (SCCs) and their innervating fibers are located in the respiratory system of many vertebrates, including papillae on lamprey gill pores. In order to gain stronger insight for the role of these chemosensory cells, we examined immunocytochemical and innervation characteristics, as well as abundance at the different stages of the lamprey life cycle. The SCCs were distinguished from the surrounding epithelial cells by calretinin and phospholipase C140 immunoreactivity. Nerve fibers extended into the gill pore papillae, as far as the SCCs and serotonergic fibers extended from the underlying dermis into the papillar base. Gill pore papillae were absent and SCCs were sparse during the larval stage and in newly transformed lamprey. Few SCCs were located on small nub-like papillae during the parasitic juvenile stage, but SCCs were abundant on prominent papillae in migrating and in spawning adults. These findings show similarities between the SCCs in lampreys and other vertebrates and suggest that gill SCC function may be important during the feeding juvenile and the adult stages of the lamprey life cycle.

Ventilatory responses of the clown knifefish, Chitala ornata, to arterial hypercapnia remain after gill denervation.

The aim of this study was to corroborate the presence of CO2/H+-sensitive arterial chemoreceptors involved in producing air-breathing responses to aquatic hypercarbia in the facultative air-breathing clown knifefish (Chitala ornata) and to explore their possible location. Progressively increasing levels of CO2 mixed with air were injected into the air-breathing organ (ABO) of one group of intact fish to elevate internal PCO2 and decrease blood pH. Another group of fish in which the gills were totally denervated was exposed to aquatic hypercarbia (pH ~ 6) or arterial hypercapnia in aquatic normocarbia (by injection of acetazolamide to increase arterial PCO2 and decrease blood pH). Air-breathing frequency, gill ventilation frequency, heart rate and arterial PCO2 and pH were recorded during all treatments. The CO2 injections into the ABO induced progressive increases in air-breathing frequency, but did not alter gill ventilation or heart rate. Exposure to both hypercarbia and acetazolamide post-denervation of the gills also produced significant air-breathing responses, but no changes in gill ventilation. While all treatments produced increases in arterial PCO2 and decreases in blood pH, the modest changes in arterial PCO2/pH in the acetazolamide treatment produced the greatest increases in air-breathing frequency. These results strengthen the evidence that internal CO2/H+ sensing is involved in the stimulation of air breathing in clown knifefish and suggest that it involves extra-branchial chemoreceptors possibly situated either centrally or in the air-breathing organ.

Fgf- and Bmp-signaling regulate gill regeneration in Ambystoma mexicanum.

Gill regeneration has not been well studied compared to regeneration of other appendages, such as limb and tail regeneration. Here, we focused on axolotl gill regeneration and found that Fgf- and Bmp-signaling are involved in their gill regeneration mechanism. Axolotls have three pairs of gill rami, and each gill ramus has multiple gill filaments. The gills consist of mesenchyme rich in extracellular matrix and epidermis. The gill nerves are supplied from the trigeminal ganglia located in the head. Denervation resulted in no gill regeneration responses. Nerves and gills express Bmp and Fgf genes, and treating animals with Fgf- and Bmp-signaling inhibitors results in phenotypes similar to those seen in denervated gills. Inducing an accessory appendage is a standard assay in amphibian regeneration research. In our study, an accessory gill could be induced by lateral wounding, suggesting that thin axon fibers and mesenchymal Fgfs and Bmps contributed to the induction of the accessory structure. Such accessory gill induction was inhibited by the denervation. Exogenous Fgf2+Fgf8+Bmp7, which have been determined to function as a regeneration inducer in urodele amphibians, could compensate for the effects denervation has on accessory blastema formation. Our findings suggest that regeneration of appendages in axolotls is regulated by common Fgf- and Bmp-signaling cascades.

Manganese toxicity is targeting an early step in the dopamine signal transduction pathway that controls lateral cilia activity in the bivalve mollusc Crassostrea virginica.

Manganese is a neurotoxin causing manganism, a Parkinson-like clinical disorder. Manganese has been shown to interfere with dopaminergic neurotransmission, but the neurotoxic mechanism involved is not fully resolved. In the bivalve mollusc Crassostrea virginica also known as the eastern oyster, beating rates of lateral cilia of the gill are controlled by dopaminergic-serotonergic innervation originating from their cerebral and visceral ganglia. Terminal release of dopamine activates D2-like receptors on these gill cells inhibiting adenylyl cyclase and slowing cilia beating rates. In C. virginica, manganese treatment disrupts this dopaminergic innervation of the gill, preventing the normal cilio-inhibitory response of lateral cells to dopamine. In this study an adenylyl cyclase activator (forskolin) and two different inhibitors (MDL-12,330A and SQ 22,536) were used to determine if manganese had any effects on the adenylyl cyclase step of the dopamine D2 receptor signal transduction pathway. The results showed that neither the adenylyl cyclase activator nor the inhibitors were affected by manganese in the control of lateral ciliary activity. This suggests that in C. virginica the mechanism of manganese toxicity on the dopaminergic control of lateral ciliary activity is targeting an early step in the D2R signal transduction pathway, which may involve interference with D2 receptor activation or alternatively some other downstream signaling activity that does not affect adenylyl cyclase.

Extrinsic nerves are not involved in branchial 5-HT dynamics or pulsatile urea excretion in Gulf toadfish, Opsanus beta.

Gulf toadfish (Opsanus beta) can switch from continuously excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses. Previous studies have shown that the neurotransmitter serotonin (5-HT) is involved in controlling this process, but it is unknown if 5-HT availability is under central nervous control or if the 5-HT signal originates from a peripheral source. Following up on a previous study, cranial nerves IX (glossopharyngeal) and X (vagus) were sectioned to further characterize their role in controlling pulsatile urea excretion and 5-HT release within the gill. In contrast to an earlier study, nerve sectioning did not result in a change in urea pulse frequency. Total urea excretion, average pulse size, total nitrogen excretion, and percent ureotely were reduced the first day post-surgery in nerve-sectioned fish but recovered by 72h post-surgery. Nerve sectioning also had no effect on toadfish urea transporter (tUT), 5-HT transporter (SERT), or 5-HT2A receptor mRNA expression or 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) abundance in the gill, all of which were found consistently across the three gill arches except 5-HIAA, which was undetectable in the first gill arch. Our findings indicate that the central nervous system does not directly control pulsatile urea excretion or local changes in gill 5-HT and 5-HIAA abundance.

Osmotic versus adrenergic control of ion transport by ionocytes of Fundulus heteroclitus in the cold.

In eurythermic vertebrates, acclimation to the cold may produce changes in physiological control systems. We hypothesize that relatively direct osmosensitive control will operate better than adrenergic receptor mediated control of ion transport in cold vs. warm conditions. Fish were acclimated to full strength seawater (SW) at 21°C and 5°C for four weeks, gill samples and blood were taken and opercular epithelia mounted in Ussing style chambers. Short-circuit current (Isc) at 21°C and 5°C (measured at acclimation temperature), was significantly inhibited by the α2-adrenergic agonist clonidine but the ED50 dose was significantly higher in cold conditions (93.8±16.4nM) than in warm epithelia (47.8±8.1nM) and the maximum inhibition was significantly lower in cold (-66.1±2.2%) vs. warm conditions (-85.6±1.3%), indicating lower sensitivity in the cold. ß-Adrenergic responses were unchanged. Hypotonic inhibition of Isc, was higher in warm acclimated (-95%), compared to cold acclimated fish (-75%), while hypertonic stimulations were the same, indicating equal responsiveness to hyperosmotic stimuli. Plasma osmolality was significantly elevated in cold acclimated fish and, by TEM, gill ionocytes from cold acclimated fish had significantly shorter mitochondria. These data are consistent with a shift in these eurythermic animals from complex adrenergic control to relatively simple biomechanical osmotic control of ion secretion in the cold.

Gill denervation eliminates the barostatic reflex in a neotropical teleost, the tambaqui (Colossoma macropomum).

The baroreflex is one of the most important regulators of cardiovascular homeostasis in vertebrates. It begins with the monitoring of arterial pressure by baroreceptors, which constantly provide the central nervous system with afferent information about the status of this variable. Any change in arterial pressure relative to its normal state triggers autonomic responses, which are characterized by an inversely proportional change in heart rate and systemic vascular resistance and which tend to restore pressure normality. Although the baroreceptors have been located in mammals and other terrestrial vertebrates, their location in fish is still not completely clear and remains quite controversial. Thus, the objective of this study was to locate the baroreceptors in a teleost, the Colossoma macropomum. To do so, the occurrence and efficiency of the baroreflex were both analyzed when this mechanism was induced by pressure imbalancements in intact fish (IN), first-gill-denervated fish (G1), and total-gill-denervated fish (G4). The pressure imbalances were initiated through the administration of the α1-adrenergic agonist phenylephrine (100 µg kg(-1)) and the α1-adrenergic antagonist prazosin (1 mg kg(-1)). The baroreflex responses were then analyzed using an electrocardiogram that allowed for the measurement of the heart rate, the relationship between pre- and post-pharmacological manipulation heart rates, the time required for maximum chronotropic baroreflex response, and total heart rate variability. The results revealed that the barostatic reflex was attenuated in the G1 group and nonexistent in G4 group, findings which indicate that baroreceptors are exclusively located in the gill arches of C. macropomum.

A time differential staining technique coupled with full bilateral gill denervation to study ionocytes in fish.

Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the gill where they transport ions such as Na+, Cl- and Ca2+ via a variety of ion channels, pumps and exchangers. The teleost gill is extrinsically innervated by the facial (VI), glossopharyngeal (IX) and vagus (X) nerves. The IX and X nerves are also the extrinsic source of branchial IC innervation. Here, two techniques used to study the innervation, proliferation and distribution of ICs are described: a time differential staining technique and a full bilateral gill denervation technique. Briefly, goldfish are exposed to a vital mitochondrion-specific dye (e.g., MitoTracker Red) which labels (red fluorescence) pre-existing ICs. Fish were either allowed to recover for 3-5 days or immediately underwent a full bilateral gill denervation. After 3-5 days of recovery, the gills are harvested and fixed for immunohistochemistry. The tissue is then stained with an α-5 primary antibody (targets Na+/K+ ATPase containing cells) in conjunction with a secondary antibody that labels all (both new and pre-existing) ICs green. Using confocal imaging, it was demonstrated that pre-existing ICs appear yellow (labelled with both a viable mitochondrion-specific dye and α-5) and new ICs appear green (labelled with α-5 only). Both techniques used in tandem can be applied to study the innervation, proliferation and distribution of ICs on the gill filament when fish are exposed to environmental challenges.

Heme oxygenase-1 (HO-1) mediated respiratory responses to hypoxia in the goldfish, Carassius auratus.

In this study we investigated the role of heme oxygenase-1 (HO-1) in modulating the hypoxic and hyperoxic ventilatory responses of goldfish (Carassius auratus) acclimated to 7 and 25°C. HO-1 was present in the neuroepithelial cells (NECs; putative branchial O2 chemoreceptors) of fish acclimated to 7°C only. Hypoxia exposure increased gill HO-1 activity in 7°C fish (14.0±1.4 to 42.5±3.2pmolbilirubinmin(-1)mgprotein(-1)). Inhibition of HO-1 activity with zinc protophorphyrin IX (ZnPPIX) increased the ventilation frequency response to acute hypoxia (30mmHg); frequency increased from 48.3±5.1 to 137.4±16.0 breaths per min (BPM) in hypoxic 7°C fish treated with ZnPPIX compared to 46.2±4.2 to 77.9±5.3 BPM in control fish. Unlike in the control (untreated) 7°C fish exposed to hyperoxia, fish injected with ZnPPIX did not significantly decrease breathing frequency. Inhibiting HO-1 activity was without effect on the hypoxic or hyperoxic ventilatory responses of fish acclimated to 25°C. Based on these observations, we suggest that HO-1 plays an inhibitory role in regulating breathing frequency but only in goldfish acclimated to 7°C.

Effects of hypoxia-induced gill remodelling on the innervation and distribution of ionocytes in the gill of goldfish, Carassius auratus.

The presence of an interlamellar cell mass (ILCM) on the gills of goldfish acclimated to 7°C leads to preferential distribution of branchial ionocytes to the distal edges of the ILCM, where they are likely to remain in contact with the water and hence remain functional. Upon exposure to hypoxia, the ILCM retracts, and the ionocytes become localized to the lamellar surfaces and on the filament epithelium, owing to their migration and the differentiation of new ionocytes from progenitor cells. Here we demonstrate that the majority of the ionocytes receive neuronal innervation, which led us to assess the consequences of ionocyte migration and differentiation during hypoxic gill remodelling on the pattern and extent of ionocyte neuronal innervation. Normoxic 7°C goldfish (ILCM present) possessed significantly greater numbers of ionocytes/mm(2) (951.2 ± 94.3) than their 25°C conspecifics (ILCM absent; 363.1 ± 49.6) but a statistically lower percentage of innervated ionocytes (83.1% ± 1.0% compared with 87.8% ± 1.3%). After 1 week of exposure of goldfish to hypoxia, the pool of branchial ionocytes was composed largely of pre-existing migrating cells (555.6 ± 38.1/mm(2)) and to a lesser extent newly formed ionocytes (226.7 ± 15.1/mm(2)). The percentage of new (relative to pre-existing) ionocytes remained relatively constant (at ∼30%) after 1 or 2 weeks of normoxic recovery. After hypoxia, pre-existing ionocytes expressed a greater percentage of innervation than newly formed ionocytes in all treatment groups; however, their percentage innervation steadily decreased over 2 weeks of normoxic recovery.

A cellular model of memory reconsolidation involves reactivation-induced destabilization and restabilization at the sensorimotor synapse in Aplysia.

The memory reconsolidation hypothesis suggests that a memory trace becomes labile after retrieval and needs to be reconsolidated before it can be stabilized. However, it is unclear from earlier studies whether the same synapses involved in encoding the memory trace are those that are destabilized and restabilized after the synaptic reactivation that accompanies memory retrieval, or whether new and different synapses are recruited. To address this issue, we studied a simple nonassociative form of memory, long-term sensitization of the gill- and siphon-withdrawal reflex in Aplysia, and its cellular analog, long-term facilitation at the sensory-to-motor neuron synapse. We found that after memory retrieval, behavioral long-term sensitization in Aplysia becomes labile via ubiquitin/proteasome-dependent protein degradation and is reconsolidated by means of de novo protein synthesis. In parallel, we found that on the cellular level, long-term facilitation at the sensory-to-motor neuron synapse that mediates long-term sensitization is also destabilized by protein degradation and is restabilized by protein synthesis after synaptic reactivation, a procedure that parallels memory retrieval or retraining evident on the behavioral level. These results provide direct evidence that the same synapses that store the long-term memory trace encoded by changes in the strength of synaptic connections critical for sensitization are disrupted and reconstructed after signal retrieval.

Confocal imaging of Merkel-like basal cells in the taste buds of zebrafish.

The oropharyngeal cavity in fish supports a range of sensory modalities, including detection of chemical and mechanical stimuli. Taste buds are found throughout this tissue and may participate in both processes. We used confocal microscopy and immunohistochemistry to characterize the morphology of Merkel-like cells and their association with other cell types and nerve fibers of the taste bud in the vertebrate model, the zebrafish. In addition, we document procedures for the observation of these structures in whole-tissue preparations from larvae and adults using zebrafish-specific and monoclonal antibodies. A single microvillus Merkel-like cell was found in each taste bud regardless of age or location. Merkel-like cells were neurosecretory, as indicated by labelling with the styryl dye, FM1-43, and the synaptic vesicle marker, SV2. Merkel-like cells were associated with SV2- and calretinin-positive taste receptor cells, received innervation from discoid aggregations of nerve fibers, and retained serotonin-filled synaptic vesicles oriented within the cytoplasm toward adjacent innervation. Moreover, a ring-like formation of nerve endings was identified with the neuronal marker, zn-12 that circumscribed the taste receptor area, surrounding calretinin-immunoreactive taste cell microvilli, and appeared to associate with the nerve plexus adjacent to Merkel-like cells. We suggest that these nerve fibers are somatosensory, perhaps associated with mechanoreception or the common chemical sense.

Hypercarbic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): the role of branchial CO2 chemoreceptors.

The aim of the present study was to determine the roles that externally versus internally oriented CO(2)/H(+)-sensitive chemoreceptors might play in promoting cardiorespiratory responses to environmental hypercarbia in the air-breathing fish, Hoplerythrinus unitaeniatus (jeju). Fish were exposed to graded hypercarbia (1, 2.5, 5, 10 and 20% CO(2)) and also to graded levels of environmental acidosis (pH approximately 7.0, 6.0, 5.8, 5.6, 5.3 and 4.7) equal to the pH levels of the hypercarbic water to distinguish the relative roles of CO(2) versus H(+). We also injected boluses of CO(2)-equilibrated solutions (5, 10 and 20% CO(2)) and acid solutions equilibrated to the same pH as the CO(2) boluses into the caudal vein (internal) and buccal cavity (external) to distinguish between internal and external stimuli. The putative location of the chemoreceptors was determined by bilateral denervation of branches of cranial nerves IX (glossopharyngeal) and X (vagus) to the gills. The data indicate that the chemoreceptors eliciting bradycardia, hypertension and gill ventilatory responses (increased frequency and amplitude) to hypercarbia are exclusively branchial, externally oriented and respond specifically to changes in CO(2) and not H(+). Those involved in producing the cardiovascular responses appeared to be distributed across all gill arches while those involved in the gill ventilatory responses were located primarily on the first gill arch. Higher levels of aquatic CO(2) depressed gill ventilation and stimulated air breathing. The chemoreceptors involved in producing air breathing in response to hypercarbia also appeared to be branchial, distributed across all gill arches and responded specifically to changes in aquatic CO(2). This would suggest that chemoreceptor groups with different orientations (blood versus water) are involved in eliciting air-breathing responses to hypercarbia in jeju.

Effects of p-Aminosalicylic acid on the neurotoxicity of manganese on the dopaminergic innervation of the cilia of the lateral cells of the gill of the bivalve mollusc, Crassostrea virginica.

The lateral cilia of the gill of Crassostrea virginica are controlled by a dopaminergic-serotonergic innervation. Dopamine is the neurotransmitter causing cilio-inhibition. High levels of manganese are neurotoxic to people, causing Manganism, a Parkinson-like disease. Clinical interventions for Manganism have not been very successful. Recently, p-Aminosalicylic acid (PAS) was reported as an effective treatment of severe Manganism in humans; however, its mechanism of action is unknown. Previously, we reported that manganese treatments caused disruption of the dopaminergic innervation of gill of C. virginica. Here we compared the effects of manganese on gill innervation in the presence of PAS, EDTA or Acetylsalicylic acid (ASA), and examined whether co-treating animals with PAS could block the deleterious effects of manganese on the oyster's dopaminergic innervation of the gill. Beating rates of the lateral cilia of the gill were measured by stroboscopic microscopy. Pre-treating gill preparations with PAS or EDTA blocked the neurotoxic effects of manganese, while ASA did not. In other experiments, animals exposed to three day treatments with manganese produced a dose dependent impairment of the dopaminergic, cilio-inhibitory system, which was decreased by co-treatment with PAS. The study shows that PAS protects the animal against neurotoxic effects of manganese and the mechanism of action of PAS in alleviating Manganism is more likely related to its chelating abilities than its anti-inflammatory actions.

A neural analysis of avoidance conditioning with the feeding attractant glycine in Pleurobranchaea japonica.

Glycine (Gly) is one of the amino acids that most strongly provoke feeding behavior in the carnivorous opisthobranch sea slug Pleurobranchaea japonica. Placing of an aliquot of a Gly solution in front of the anterior end of this animal induced feeding responses such as orientation to the origin of the stimulus and extrusion of the proboscis. In contrast, light stimulation of the body of the animal with a glass-fiber light guide evoked aversive responses involving the gill withdrawal response. Animals were trained by pairing a Gly solution stimulus (conditioned) and a light stimulus (unconditioned). After training with repetitive paired stimuli, we found that animals exhibited aversive responses to the Gly solution. We confirmed achievement of a conditioned Gly-aversive reflex in intact animals by recording of motor impulses with an electrode implanted on the branchial nerve responsible for the gill withdrawal response, the most reliable index of the reflex. Motor discharge of the branchial nerve associated with the conditioned Gly-aversive reflex persisted even in a preparation isolated from an animal which had previously acquired the reflex. This study provides an experimental model for neural analysis with in vivo long-term nerve recording using an electrode implanted in a nerve of an intact animal for neuroethological training.

The relationship between O2 chemoreceptors, cardio-respiratory reflex and hypoxia tolerance in the neotropical fish Hoplias lacerdae.

The localization, distribution and orientation of O(2) chemoreceptors associated with the control of cardio-respiratory responses were investigated in the neotropical, Hoplias lacerdae. Selective denervation of the cranial nerves (IX and X) was combined with chemical stimulation (NaCN) to characterize the gill O(2) chemoreceptors, and the fish were then exposed to gradual hypoxia to examine the extent of each cardio-respiratory response. Changes in heart rate (f(H)) and ventilation amplitude (V(amp)) were allied with chemoreceptors distributed on both internal and external surfaces of all gill arches, while ventilation rate (f) was allied to the O(2) chemoreceptors located only in the internal surface of the first gill arch. H. lacerdae exposed to gradual hypoxia produced a marked bradycardia (45%) and 50% increase in V(amp), but only a relatively small change in f (32%). Thus, the low f(R) response yet high V(amp) were in accord with the characterization of the O(2) chemoreceptors. Comparing these results from H. lacerdae with hypoxia-tolerant species revealed a relationship existent between general oxygenation of the individual species environment, its cardio-respiratory response to hypoxia and the characterization of O(2) chemoreceptors.

Nervous control of the gills.

The fish gill is a highly complex organ that performs a wide variety of physiological processes and receives extensive nervous innervation from both afferent (sensory) and efferent (motor) fibres. Innervation from the latter source includes autonomic nerve fibres of spinal (sympathetic) and cranial (parasympathetic) origin whose primary role is to induce vasomotor changes within the respiratory or nonrespiratory pathways of the gill vasculature. Autonomic control of the gill occurs by nerve fibres identified as adrenergic, cholinergic, and more recent evidence indicates that nonadrenergic-noncholinergic (NANC) nerve fibres, such as those that express amines, peptides, or nitric oxide, may also play an important role. The distribution and physiological function of NANC nerve fibres, however, is less clear. This review primarily discusses histochemical studies that have characterized the nervous innervation and autonomic control of the gill vasculature. In addition, supporting evidence from recent studies for the efferent control, or modulation, of other homeostatic processes in the gill is examined.

New developments on gill innervation: insights from a model vertebrate.

The fish gill is a highly specialized and complex organ that performs a variety of important physiological functions. In this article, we briefly review the innervation of important structures of the branchial region, such as the gill filaments, respiratory lamellae and pseudobranch, and discuss the physiological significance of this innervation within the context of homeostatic functions of the gill, such as oxygen sensing and ion regulation. Studies in zebrafish utilizing techniques of confocal microscopy and immunolabelling, with specific antibodies against neuronal markers, have recently led to the characterization of innervation patterns in the gills not attained with traditional techniques of histochemistry and electron microscopy. We will discuss the association of putative sensory nerve fibres with O2-chemoreceptive neuroepithelial cells and the implications of dual sensory pathways for cardiorespiratory and vascular control. In addition, the idea of the neural control of ion regulation in the gill based on the apparent innervation of mitochondria-rich cells, and the role of innervation in the pseudobranch, will be presented.

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.