The glomerular network of the zebrafish olfactory bulb.

Each zebrafish olfactory bulb contains ~ 140 glomeruli that are distinguishable based on size, location, neurochemistry and function. Here we examine the mitral cell innervation of differently sized glomeruli in adult zebrafish. Type 1 glomeruli had diameters of 80.9 ± 8.1 µm and were innervated by 5.9 ± 0.9 mitral cells. The Type 1 mediodorsal glomeruli (mdG) were innervated by both uniglomerular (innervating only single glomeruli) and multiglomerular mitral cells (innervating two or more glomeruli). In contrast, the Type 1 ventroposterior (vpG) and lateral glomeruli (lG) were only innervated by uniglomerular mitral cells. Type 2 ventral glomeruli were 46 ± 5.1 µm in diameter and were innervated by 3.3 ± 0.2 mitral cells. Type 2 ventromedial glomeruli (vmG) were innervated exclusively by uniglomerular mitral cells. Type 3 glomeruli had diameters of 17 ± 2.5 µm and were innervated by 1.1 ± 0.6 multiglomerular mitral cells each. Finally, Type 4 glomeruli were small, with average diameters of 4.8 ± 3.9 µm and were restricted to the lateral plexus. These glomeruli were innervated mainly by multiglomerular mitral cells with extensively branching dendrites. This study provides the first specific associations between uni- and multiglomerular mitral cells with known zebrafish glomeruli. Our results suggest that glomeruli are distinguishable based on their postsynaptic compartment and that distinct input-output computations occur in different types of zebrafish glomeruli.

Biochemical and apoptotic changes in the nervous and ovotestis tissues of Biomphalaria alexandrina following infection with Schistosoma mansoni.

Infection with trematodes produces physiological and behavioural changes in intermediate snail hosts. One response to infection is parasitic castration, in which energy required for reproduction of the host is thought to be redirected to promote development and multiplication of the parasite. This study investigated some reproductive and biochemical parameters in the nervous (CNS) and ovotestis (OT) tissues of Biomphalaria alexandrina during the course of Schistosoma mansoni infection. Antioxidant and oxidative stress parameters including catalase (CAT), nitric oxide (NO) and lipid peroxidation (MDA) were measured. Levels of steroid hormones, including testosterone, progesterone and estradiol, were also assessed. Finally, flow cytometry was used to compare measures of apoptosis between control snails and those shedding cercariae by examining mitochondrial membrane potential with the stain 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) and poly(ADP-ribose) polymerase (PARP). Infection with S. mansoni caused a 47.7% reduction in the net reproductive rate (Ro) of B. alexandrina. CAT activity was increased in the CNS at 21 days post infection (dpi) but by 28 dpi it was reduced below control values. Also, CAT activity increased significantly in the OT at 14, 21 and 28 dpi. In CNS tissues, NO levels were reduced at 7 dpi, increased at 14 and 21 dpi, and reduced again at 28 dpi. The overall level of lipid peroxidation gradually increased during the course of infection to reach its highest levels at 28 dpi. Steroid hormone measurements showed that concentrations of testosterone and estradiol were reduced in the CNS tissues at 28 dpi, while those of progesterone were slightly increased in the CNS and OT tissues. The percentage of cells that positively stained with JC-1was significantly increased in CNS and OT tissues of infected snails while the percentage of cells positively stained with PARP was decreased compared to controls. Together, these findings indicate that infection initiates diverse biochemical and hormonal changes leading to loss of cells responsible for egg laying and reproduction in B. alexandrina.

Localization of keyhole limpet hemocyanin-like immunoreactivity in the nervous system of Biomphalaria alexandrina.

Recent years have led to increased effort to describe and understand the peripheral nervous system and its influence on central mechanisms and behavior in gastropod molluscs. This study revealed that an antibody raised against keyhole limpet hemocyanin (KLH) cross-reacts with an antigen(s) found extensively in both the central and the peripheral nervous systems of Biomphalaria alexandrina. The results revealed KLH-like immunoreactive (LIR) neurons in the cerebral, pedal, buccal, left pleural, right parietal, and visceral ganglion within the CNS with fibers projecting throughout all the peripheral nerves. Numerous KLH-LIR peripheral sensory neurons located in the foot, lips, tentacles, mantle, esophagus, and penis exhibited a bipolar morphology with long tortuous dendrites. KLH-LIR cells were also present in the eye and statocyst, thus suggesting the labeling of multiple sensory modalities/cell types. KLH-LIR cells did not co-localize with tyrosine hydroxylase (TH)-LIR cells, which have previously been described in this and other gastropods. The results thus provide descriptions of thousands of peripheral sensory neurons, not previously described in detail. Future research should seek to pair sensory modalities with peripheral cell type and attempt to further elucidate the nature of KLH-like reactivity. These findings also emphasize the need for caution when analyzing results obtained through use of antibodies raised against haptens conjugated to carrier proteins, suggesting the need for stringent controls to help limit potential confounds caused by cross-reactivity. In addition, this study is the first to describe neuronal cross-reactivity with KLH in Biomphalaria, which could provide a substrate for host-parasite interactions with a parasitic trematode, Schistosoma.

An immunohistochemical analysis of peptidergic neurons apparently associated with reproduction and growth in Biomphalaria alexandrina.

Peptide hormones and neurotransmitters involved in reproduction and growth have been studied extensively in certain gastropod molluscs, such as Lymnaea stagnalis and Aplysia californica. The present study employs antisera that have been used to study peptidergic neurons in those species to probe the central nervous system of another gastropod, Biomphalaria alexandrina, an intermediate host of the parasitic trematode that causes schistosomiasis in humans. Whole mount preparations of central ganglia were stained immunohistochemically, and several populations of neurons appeared to be homologous to those forming the neuroendocrine axis that has been previously described in L. stagnalis. These cells include the caudodorsal cells and the light green and canopy cells, which produce hormones that regulate ovulation and growth, respectively. Other populations of cells containing APGWamide, FMRFamide and/or related peptides are consistent with ones that innervate the penis in L. stagnalis and other gastropods. Identification of neurons that might be responsible for the control of reproduction and growth in Biomphalaria provides an important initial step toward the development of novel methods of disease control and pest management directed toward reducing snail populations.

The in vitro zebrafish heart as a model to investigate the chronotropic effects of vapor anesthetics.

In addition to their intended clinical actions, all general anesthetic agents in common use have detrimental intrasurgical and postsurgical side effects on organs and systems, including the heart. The major cardiac side effect of anesthesia is bradycardia, which increases the probability of insufficient systemic perfusion during surgery. These side effects also occur in all vertebrate species so far examined, but the underlying mechanisms are not clear. The zebrafish heart is a powerful model for studying cardiac electrophysiology, employing the same pacemaker system and neural control as do mammalian hearts. In this study, isolated zebrafish hearts were significantly bradycardic during exposure to the vapor anesthetics sevoflurane (SEVO), desflurane (DES), and isoflurane (ISO). Bradycardia induced by DES and ISO continued during pharmacological blockade of the intracardiac portion of the autonomic nervous system, but the chronotropic effect of SEVO was eliminated during blockade. Bradycardia evoked by vagosympathetic nerve stimulation was augmented during DES and ISO exposure; nerve stimulation during SEVO exposure had no effect. Together, these results support the hypothesis that the cardiac chronotropic effect of SEVO occurs via a neurally mediated mechanism, while DES and ISO act directly upon cardiac pacemaker cells via an as yet unknown mechanism.

Skeletal stiffening in an amphibious fish out of water is a response to increased body weight.

Terrestrial animals must support their bodies against gravity, while aquatic animals are effectively weightless because of buoyant support from water. Given this evolutionary history of minimal gravitational loading of fishes in water, it has been hypothesized that weight-responsive musculoskeletal systems evolved during the tetrapod invasion of land and are thus absent in fishes. Amphibious fishes, however, experience increased effective weight when out of water - are these fishes responsive to gravitational loading? Contrary to the tetrapod-origin hypothesis, we found that terrestrial acclimation reversibly increased gill arch stiffness (∼60% increase) in the amphibious fish Kryptolebias marmoratus when loaded normally by gravity, but not under simulated microgravity. Quantitative proteomics analysis revealed that this change in mechanical properties occurred via increased abundance of proteins responsible for bone mineralization in other fishes as well as in tetrapods. Type X collagen, associated with endochondral bone growth, increased in abundance almost ninefold after terrestrial acclimation. Collagen isoforms known to promote extracellular matrix cross-linking and cause tissue stiffening, such as types IX and XII collagen, also increased in abundance. Finally, more densely packed collagen fibrils in both gill arches and filaments were observed microscopically in terrestrially acclimated fish. Our results demonstrate that the mechanical properties of the fish musculoskeletal system can be fine-tuned in response to changes in effective body weight using biochemical pathways similar to those in mammals, suggesting that weight sensing is an ancestral vertebrate trait rather than a tetrapod innovation.

Zebrafish heart as a model to study the integrative autonomic control of pacemaker function.

The cardiac pacemaker sets the heart's primary rate, with pacemaker discharge controlled by the autonomic nervous system through intracardiac ganglia. A fundamental issue in understanding the relationship between neural activity and cardiac chronotropy is the identification of neuronal populations that control pacemaker cells. To date, most studies of neurocardiac control have been done in mammalian species, where neurons are embedded in and distributed throughout the heart, so they are largely inaccessible for whole-organ, integrative studies. Here, we establish the isolated, innervated zebrafish heart as a novel alternative model for studies of autonomic control of heart rate. Stimulation of individual cardiac vagosympathetic nerve trunks evoked bradycardia (parasympathetic activation) and tachycardia (sympathetic activation). Simultaneous stimulation of both vagosympathetic nerve trunks evoked a summative effect. Effects of nerve stimulation were mimicked by direct application of cholinergic and adrenergic agents. Optical mapping of electrical activity confirmed the sinoatrial region as the site of origin of normal pacemaker activity and identified a secondary pacemaker in the atrioventricular region. Strong vagosympathetic nerve stimulation resulted in a shift in the origin of initial excitation from the sinoatrial pacemaker to the atrioventricular pacemaker. Putative pacemaker cells in the sinoatrial and atrioventricular regions expressed adrenergic ß2 and cholinergic muscarinic type 2 receptors. Collectively, we have demonstrated that the zebrafish heart contains the accepted hallmarks of vertebrate cardiac control, establishing this preparation as a viable model for studies of integrative physiological control of cardiac function by intracardiac neurons.

Experience-dependent versus experience-independent postembryonic development of distinct groups of zebrafish olfactory glomeruli.

Olfactory glomeruli are innervated with great precision by the axons of different olfactory sensory neuron types and act as functional units in odor information processing. Approximately 140 glomeruli are present in each olfactory bulb of adult zebrafish; these units consist of either highly stereotypic large glomeruli or smaller anatomically indistinguishable glomeruli. In the present study, we investigated developmental differences among these types of glomeruli. We observed that 10 large and individually identifiable glomeruli already developed before hatching, at 72 h after fertilization, in configurations that resembled their mature organization. However, the cross-sectional area of these glomeruli increased throughout larval development, and they eventually comprised the largest units in postlarval olfactory bulbs. In contrast, small and anatomically indistinguishable glomeruli formed only after hatching, apparently by segregating from five larger precursors that were identifiable during embryonic development. The differentiation of these small glomeruli proceeded with conspicuous variation in number and arrangement, both among larvae and between olfactory bulbs of the same individuals. To determine factors that might contribute to this variability, we investigated the effects of olfactory enrichment on the development of amino acid-responsive lateral glomeruli, which include both large and small units. Larvae reared in an amino acid-enriched environment had normal large lateral glomeruli, but the small lateral glomeruli were more numerous and displayed reduced cross-sectional areas compared with glomeruli in control animals. Our results suggest that large and small glomeruli mature via distinct developmental processes that may be differentially influenced by sensory experience.

Use of axonal projection patterns for the homologisation of cerebral nerves in Opisthobranchia, Mollusca and Gastropoda.

INTRODUCTION: Gastropoda are guided by several sensory organs in the head region, referred to as cephalic sensory organs (CSOs). These CSOs are innervated by distinct nerves. This study proposes a unified terminology for the cerebral nerves and the categories of CSOs and then investigates the neuroanatomy and cellular innervation patterns of these cerebral nerves, in order to homologise them. The homologisation of the cerebral nerves in conjunction with other data, e.g. ontogenetic development or functional morphology, may then provide insights into the homology of the CSOs themselves. RESULTS: Nickel-lysine axonal tracing ("backfilling") was used to stain the somata projecting into specific nerves in representatives of opisthobranch Gastropoda. Tracing patterns revealed the occurrence, size and relative position of somata and their axons and enabled these somata to be mapped to specific cell clusters. Assignment of cells to clusters followed a conservative approach based primarily on relative location of the cells. Each of the four investigated cerebral nerves could be uniquely identified due to a characteristic set of soma clusters projecting into the respective nerves via their axonal pathways. CONCLUSIONS: As the described tracing patterns are highly conserved morphological characters, they can be used to homologise nerves within the investigated group of gastropods. The combination of adequate number of replicates and a comparative approach allows us to provide preliminary hypotheses on homologies for the cerebral nerves. Based on the hypotheses regarding cerebral nerve homology together with further data on ultrastructure and immunohistochemistry of CSOs published elsewhere, we can propose preliminary hypotheses regarding homology for the CSOs of the Opisthobranchia themselves.

Superficial neuromasts facilitate non-visual feeding by larval striped bass (Morone saxatilis).

To investigate whether mechanoreception is used in non-visual feeding in larval striped bass (Morone saxatilis), the ontogeny of superficial neuromasts along the lateral line was described using the vital stain FM1-43FX and fluorescent microscopy. The number of neuromasts visible along one flank increased from 11 at first feeding [5 to 7 days post-hatch (dph)] to >150 by the juvenile stage (27 dph). A neomycin dose response (0, 1, 2 and 5 mmol l(-1)) was evaluated for neuromast ablation of bass aged 10, 13, 17 and 20 dph. Using these same age groups, the ability of bass to catch Artemia salina prey in both dark and light tank-based feeding trials was compared between larvae with neuromasts ablated using neomycin (5 mmol l(-1)) and controls. Neomycin significantly reduced the incidence of feeding in the light and dark. Among larvae that fed, those in the dark treated with neomycin caught fewer Artemia (~5 prey h(-1); P<0.05) than controls (16 prey h(-1) at 10 dph; 72 prey h(-1) at 20 dph). In the light, by contrast, neomycin treatment had no significant effect on prey capture by larvae age 13 to 20 dph, but did inhibit feeding of 10 dph larvae. Verification that neomycin was specifically ablating the hair cells of superficial neuromasts and not affecting either neuromast innervation, olfactory pits, or taste cells was achieved by a combination of staining with FM1-43FX and immunocytochemistry for tubulin and the calcium binding proteins, S100 and calretinin.

A Critical Review of Zebrafish Models of Parkinson's Disease.

A wide variety of human diseases have been modelled in zebrafish, including various types of cancer, cardiovascular diseases and neurodegenerative diseases like Alzheimer's and Parkinson's. Recent reviews have summarized the currently available zebrafish models of Parkinson's Disease, which include gene-based, chemically induced and chemogenetic ablation models. The present review updates the literature, critically evaluates each of the available models of Parkinson's Disease in zebrafish and compares them with similar models in invertebrates and mammals to determine their advantages and disadvantages. We examine gene-based models, including ones linked to Early-Onset Parkinson's Disease: PARKIN, PINK1, DJ-1, and SNCA; but we also examine LRRK2, which is linked to Late-Onset Parkinson's Disease. We evaluate chemically induced models like MPTP, 6-OHDA, rotenone and paraquat, as well as chemogenetic ablation models like metronidazole-nitroreductase. The article also reviews the unique advantages of zebrafish, including the abundance of behavioural assays available to researchers and the efficiency of high-throughput screens. This offers a rare opportunity for assessing the potential therapeutic efficacy of pharmacological interventions. Zebrafish also are very amenable to genetic manipulation using a wide variety of techniques, which can be combined with an array of advanced microscopic imaging methods to enable in vivo visualization of cells and tissue. Taken together, these factors place zebrafish on the forefront of research as a versatile model for investigating disease states. The end goal of this review is to determine the benefits of using zebrafish in comparison to utilising other animals and to consider the limitations of zebrafish for investigating human disease.

Drivers of Sinoatrial Node Automaticity in Zebrafish: Comparison With Mechanisms of Mammalian Pacemaker Function.

Cardiac excitation originates in the sinoatrial node (SAN), due to the automaticity of this distinct region of the heart. SAN automaticity is the result of a gradual depolarisation of the membrane potential in diastole, driven by a coupled system of transarcolemmal ion currents and intracellular Ca2+ cycling. The frequency of SAN excitation determines heart rate and is under the control of extra- and intracardiac (extrinsic and intrinsic) factors, including neural inputs and responses to tissue stretch. While the structure, function, and control of the SAN have been extensively studied in mammals, and some critical aspects have been shown to be similar in zebrafish, the specific drivers of zebrafish SAN automaticity and the response of its excitation to vagal nerve stimulation and mechanical preload remain incompletely understood. As the zebrafish represents an important alternative experimental model for the study of cardiac (patho-) physiology, we sought to determine its drivers of SAN automaticity and the response to nerve stimulation and baseline stretch. Using a pharmacological approach mirroring classic mammalian experiments, along with electrical stimulation of intact cardiac vagal nerves and the application of mechanical preload to the SAN, we demonstrate that the principal components of the coupled membrane- Ca2+ pacemaker system that drives automaticity in mammals are also active in the zebrafish, and that the effects of extra- and intracardiac control of heart rate seen in mammals are also present. Overall, these results, combined with previously published work, support the utility of the zebrafish as a novel experimental model for studies of SAN (patho-) physiological function.

Effects of altered ambient pressure on the volume and distribution of gas within the swimbladder of the adult zebrafish, Danio rerio.

Many teleosts use gas-filled swimbladders to control buoyancy and influence three-dimensional orientation (pitch and roll). However, swimbladder volume, and its contributions to these functions, varies with depth-related pressure according to Boyle's law. Moreover, the swimbladder volume at a given depth also depends on the compliance of the swimbladder wall, but this latter factor has been investigated in only a limited number of species. In this study, changes in the volume of the zebrafish swimbladder were observed both in vitro and in situ in pressure chambers that allowed simulations of movements within the water column to and from depths of >300 cm. Results show the anterior chamber to be highly compliant, varying ±38% from its initial volume over the range of simulated depths. This large volume change was accomplished, at least in part, by a series of regular corrugations running along the ventral aspect of the chamber wall and another set of pleats radiating from around the communicating duct in the caudal aspect of the chamber wall. The posterior chamber, in contrast, was found to be minimally compliant, varying only a fraction of a percent from its initial volume over the same pressure range. The different volumetric responses of the chambers caused a significant shift in the distribution of gas within the swimbladder system, but only resulted in a change in the whole-body pitch angle of ±2 deg over the range of pressures tested. Together, our findings provide new insights into the control of buoyancy and trim within teleosts and suggest novel mechanisms that may contribute to swimbladder performance.

Identification and localization of a gonadotropin-releasing hormone-related neuropeptide in Biomphalaria, an intermediate host for schistosomiasis.

Freshwater snails of the genus Biomphalaria serve as obligatory hosts for the digenetic trematode Schistosoma mansoni, the causative agent for the most widespread form of intestinal schistosomiasis. Within Biomphalaria, S. mansoni larvae multiply and transform into the cercariae form that can infect humans. Trematode development and proliferation is thought to be facilitated by modifications of host behavior and physiological processes, including a reduction of reproduction known as "parasitic castration." As neuropeptides participate in the control of reproduction across phylogeny, a neural transcriptomics approach was undertaken to identify peptides that could regulate Biomphalaria reproductive physiology. The present study identified a transcript in Biomphalaria alexandrina that encodes a peptide belonging to the gonadotropin-releasing hormone (GnRH) superfamily. The precursor and the predicted mature peptide, pQIHFTPDWGNN-NH2 (designated Biom-GnRH), share features with peptides identified in other molluscan species, including panpulmonates, opisthobranchs, and cephalopods. An antibody generated against Biom-GnRH labeled neurons in the cerebral, pedal, and visceral ganglia of Biomphalaria glabrata. GnRH-like immunoreactive fiber systems projected to all central ganglia. In the periphery, immunoreactive material was detected in the ovotestis, oviduct, albumen gland, and nidamental gland. As these structures serve crucial roles in the production, transport, nourishment, and encapsulation of eggs, disruption of the GnRH system of Biomphalaria could contribute to reduced reproductive activity in infected snails.

Disrupted local innervation results in less VIP expression in CF mice tissues.

Vasoactive Intestinal Peptide (VIP) is the major physiological agonist of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride channel activity. VIP functions as a neuromodulator and neurotransmitter secreted by neurons innervating all exocrine glands. VIP is also a potent vasodilator and bronchodilator that regulates exocrine gland secretions, contributing to local innate defense by stimulating the movement of water and chloride transport across intestinal and tracheobronchial epithelia. Previous human studies have shown that the rich intrinsic neuronal networks for VIP secretion around exocrine glands could be lost in tissues from patients with cystic fibrosis. Our research has since confirmed, in vitro and in vivo, the need for chronic VIP exposure to maintain functional CFTR chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissues morphology [1]. The goal of the present study was to examine changes in VIP in the lung, duodenum and sweat glands of 8- and 17-weeks old F508del/F508del mice and to investigate VIPergic innervation in the small intestine of CF mice, before important signs of the disease development. Our data show that a low amount of VIP is found in CF tissues prior to tissue damage. Moreover, we found a specific reduction in VIPergic and cholinergic innervation of the small intestine. The general innervation of the primary and secondary myenteric plexus was lost in CF tissues, with the presence of enlarged ganglionic cells in the tertiary layer. We propose that low amount of VIP in CF tissues is due to a reduction in VIPergic and cholinergic innervation and represents an early defect that constitutes an aggravating factor for CF disease progression.

FMRF-NH2 -related neuropeptides in Biomphalaria spp., intermediate hosts for schistosomiasis: Precursor organization and immunohistochemical localization.

Freshwater snails of the genus Biomphalaria serve as intermediate hosts for the digenetic trematode Schistosoma mansoni, the etiological agent for the most widespread form of intestinal schistosomiasis. As neuropeptide signaling in host snails can be altered by trematode infection, a neural transcriptomics approach was undertaken to identify peptide precursors in Biomphalaria glabrata, the major intermediate host for S. mansoni in the Western Hemisphere. Three transcripts that encode peptides belonging to the FMRF-NH2 -related peptide (FaRP) family were identified in B. glabrata. One transcript encoded a precursor polypeptide (Bgl-FaRP1; 292 amino acids) that included eight copies of the tetrapeptide FMRF-NH2 and single copies of FIRF-NH2 , FLRF-NH2 , and pQFYRI-NH2 . The second transcript encoded a precursor (Bgl-FaRP2; 347 amino acids) that comprised 14 copies of the heptapeptide GDPFLRF-NH2 and 1 copy of SKPYMRF-NH2 . The precursor encoded by the third transcript (Bgl-FaRP3; 287 amino acids) recapitulated Bgl-FaRP2 but lacked the full SKPYMRF-NH2 peptide. The three precursors shared a common signal peptide, suggesting a genomic organization described previously in gastropods. Immunohistochemical studies were performed on the nervous systems of B. glabrata and B. alexandrina, a major intermediate host for S. mansoni in Egypt. FMRF-NH2 -like immunoreactive (FMRF-NH2 -li) neurons were located in regions of the central nervous system associated with reproduction, feeding, and cardiorespiration. Antisera raised against non-FMRF-NH2 peptides present in the tetrapeptide and heptapeptide precursors labeled independent subsets of the FMRF-NH2 -li neurons. This study supports the participation of FMRF-NH2 -related neuropeptides in the regulation of vital physiological and behavioral systems that are altered by parasitism in Biomphalaria.

Adrenergic control of swimbladder deflation in the zebrafish (Danio rerio).

Many teleosts actively regulate buoyancy by adjusting gas volume in the swimbladder. In physostomous fishes such as the zebrafish, a connection is maintained between the swimbladder and the oesophagus via the pneumatic duct for the inflation and deflation of this organ. Here we investigated the role of adrenergic stimulation of swimbladder wall musculature in deflation of the swimbladder. Noradrenaline (NA), the sympathetic neurotransmitter (dosage 10(-6) to 10(-5) mol l(-1)), doubled the force of smooth muscle contraction in isolated tissue rings from the anterior chamber, caused a doubling of pressure in this chamber in situ, and evoked gas expulsion through the pneumatic duct, deflating the swimbladder to approximately 85% of the pre-NA volume. These effects were mediated by beta-adrenergic receptors, representing a novel role for these receptors in vertebrates. No effects of adrenergic stimulation were detected in the posterior chamber. In a detailed examination of the musculature and innervation of the swimbladder to determine the anatomical substrate for these functional results, we found that the anterior chamber contained an extensive ventral band of smooth muscle with fibres organized into putative motor units, richly innervated by tyrosine hydroxylase-positive axons. Additionally, a novel arrangement of folds in the lumenal connective tissue in the wall of the anterior chamber was described that may permit small changes in muscle length to cause large changes in effective wall distensibility and hence chamber volume. Taken together, these data strongly suggest that deflation of the zebrafish swimbladder occurs primarily by beta-adrenergically mediated contraction of smooth muscle in the anterior chamber and is under the control of the sympathetic limb of the autonomic nervous system.

Bioaccumulation and biotransformation of pyrene and 1-hydroxypyrene by the marine whelk Buccinum undatum.

The fates of a phenolic contaminant and its hydrocarbon precursor have rarely been compared, especially in an invertebrate species. Two groups of Buccinum undatum were exposed to equimolar amounts of pyrene and 1-hydroxypyrene over 15 d through their diets. Tissue extracts from the muscle and visceral mass were analyzed by liquid chromatography with fluorescence and mass spectrometry detection. Nine biotransformation products were detected in animals from both exposures. These included 1-hydroxypyrene, pyrene-1-sulfate, pyrene-1-glucuronide, pyrene glucose sulfate, two isomers each of pyrenediol sulfate and pyrenediol disulfate, and one isomer of pyrenediol glucuronide sulfate. These compounds represent a more complex metabolic pathway for pyrene than is typically reported. Diconjugated metabolites were as important in animals exposed to pyrene as in those exposed to 1-hydroxypyrene. Biotransformation products represented >90% of the material detected in the animals and highlight the importance of analyzing metabolites when assessing exposure. A mean of only 2 to 3% of the body burden was present in muscle compared with the visceral mass of both groups. The analytical methods were sufficiently sensitive to detect biotransformation products both in laboratory control whelks and in those sampled offshore. The tissue distribution of [(14)C]pyrene was also studied by autoradiography. Radioactivity was present primarily in the digestive and excretory system of the whelks and not in the gonads or muscle tissue.

Taste bud development and patterning in sighted and blind morphs of Astyanax mexicanus.

In the blind cave-dwelling morph of A. mexicanus, the eye degenerates while other sensory systems, such as gustation, are expanded compared to their sighted (surface-dwelling) ancestor. This study compares the development of taste buds along the jaws of each morph. To determine whether cavefish have an altered onset or rate of taste bud development, we fluorescently labeled basal and receptor cells within taste buds over a developmental series. Our results show that taste bud number increases during development in both morphs. The rate of development is, however, accelerated in cavefish; a small difference in taste bud number exists at 5 dpf reaching threefold by 22 dpf. The expansion of taste buds in cavefish is, therefore, detectable after the onset of eye degeneration. This study provides important insights into the timing of taste bud expansion in cavefish as well as enhances our understanding of taste bud development in teleosts in general.

Histamine and histidine decarboxylase in the olfactory system and brain of the common cuttlefish Sepia officinalis (Linnaeus, 1758).

Cephalopods are radically different from any other invertebrate. Their molluscan heritage, innovative nervous system, and specialized behaviors create a unique blend of characteristics that are sometimes reminiscent of vertebrate features. For example, despite differences in the organization and development of their nervous systems, both vertebrates and cephalopods use many of the same neurotransmitters. One neurotransmitter, histamine (HA), has been well studied in both vertebrates and invertebrates, including molluscs. While HA was previously suggested to be present in the cephalopod central nervous system (CNS), Scaros, Croll, and Baratte only recently described the localization of HA in the olfactory system of the cuttlefish Sepia officinalis. Here, we describe the location of HA using an anti-HA antibody and a probe for histidine decarboxylase (HDC), a synthetic enzyme for HA. We extended previous descriptions of HA in the olfactory organ, nerve, and lobe, and describe HDC staining in the same regions. We found HDC-positive cell populations throughout the CNS, including the optic gland and the peduncle, optic, dorso-lateral, basal, subvertical, frontal, magnocellular, and buccal lobes. The distribution of HA in the olfactory system of S. officinalis is similar to the presence of HA in the chemosensory organs of gastropods but is different than the sensory systems in vertebrates or arthropods. However, HA's widespread abundance throughout the rest of the CNS of Sepia is a similarity shared with gastropods, vertebrates, and arthropods. Its widespread use with differing functions across Animalia provokes questions regarding the evolutionary history and adaptability of HA as a transmitter.