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Abstract Background The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model. Results In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes. Conclusion This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Doyle and Croll.)

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 Ca 2+ 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- Ca 2+ 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stoyek, MacDonald, Mantifel, Baillie, Selig, Croll, Smith and Quinn.)

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-NH 2 -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-NH 2 and single copies of FIRF-NH 2 , FLRF-NH 2 , and pQFYRI-NH 2 . The second transcript encoded a precursor (Bgl-FaRP2; 347 amino acids) that comprised 14 copies of the heptapeptide GDPFLRF-NH 2 and 1 copy of SKPYMRF-NH 2 . The precursor encoded by the third transcript (Bgl-FaRP3; 287 amino acids) recapitulated Bgl-FaRP2 but lacked the full SKPYMRF-NH 2 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-NH 2 -like immunoreactive (FMRF-NH 2 -li) neurons were located in regions of the central nervous system associated with reproduction, feeding, and cardiorespiration. Antisera raised against non-FMRF-NH 2 peptides present in the tetrapeptide and heptapeptide precursors labeled independent subsets of the FMRF-NH 2 -li neurons. This study supports the participation of FMRF-NH 2 -related neuropeptides in the regulation of vital physiological and behavioral systems that are altered by parasitism in Biomphalaria., (© 2021 Wiley Periodicals LLC.)

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-NH 2 (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., (© 2020 Wiley Periodicals LLC.)

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., Competing Interests: Conflict of Interest Statement There are no conflicts of interest to declare., (Copyright © 2020. Published by Elsevier B.V.)

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.

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 (R o ) 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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© 2019 Wiley Periodicals, Inc.)

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., (© 2019 Wiley Periodicals, Inc.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Our understanding of the multiorgan pathology of cystic fibrosis (CF) has improved impressively during the last decades, but we still lack a full comprehension of the disease progression. Animal models have greatly contributed to the elucidation of specific mechanisms involved in CF pathophysiology and the development of new therapies. Soon after the cloning of the CF transmembrane conductance regulator ( CFTR ) gene in 1989, the first mouse model was generated and this model has dominated in vivo CF research ever since. Nonetheless, the failure of murine models to mirror human disease severity in the pancreas and lung has led to the generation of larger animal models such as pigs and ferrets. The following review presents and discusses data from the current animal models used in CF research.

Cephalopods are nontraditional but captivating models of invertebrate neurobiology, particularly in evolutionary comparisons. Cephalopod olfactory systems have striking similarities and fundamental differences with vertebrates, arthropods, and gastropods, raising questions about the ancestral origins of those systems. We describe here the organization and development of the olfactory system of the common cuttlefish, Sepia officinalis, using immunohistochemistry and in situ hybridization. FMRFamide and/or related peptides and histamine are putative neurotransmitters in olfactory sensory neurons. Other neurotransmitters, including serotonin and APGWamide within the olfactory and other brain lobes, suggest efferent control of olfactory input and/or roles in the processing of olfactory information. The distributions of neurotransmitters, along with staining patterns of phalloidin, anti-acetylated α-tubulin, and a synaptotagmin riboprobe, help to clarify the structure of the olfactory lobe. We discuss a key difference, the lack of identifiable olfactory glomeruli, in cuttlefish in comparison to other models, and suggest its implications for the evolution of olfaction.

The simpler nervous systems of certain invertebrates provide opportunities to examine colocalized classical neurotransmitters in the context of identified neurons and well defined neural circuits. This study examined the distribution of γ-aminobutyric acid-like immunoreactivity (GABAli) in the nervous system of the panpulmonates Biomphalaria glabrata and Biomphalaria alexandrina, major intermediate hosts for intestinal schistosomiasis. GABAli neurons were localized in the cerebral, pedal, and buccal ganglia of each species. With the exception of a projection to the base of the tentacle, GABAli fibers were confined to the CNS. As GABAli was previously reported to be colocalized with markers for dopamine (DA) in five neurons in the feeding network of the euopisthobranch gastropod Aplysia californica (Díaz-Ríos, Oyola, & Miller, 2002), double-labeling protocols were used to compare the distribution of GABAli with tyrosine hydroxylase immunoreactivity (THli). As in Aplysia, GABAli-THli colocalization was limited to five neurons, all of which were located in the buccal ganglion. Five GABAli-THli cells were also observed in the buccal ganglia of two other intensively studied panpulmonate species, Lymnaea stagnalis and Helisoma trivolvis. These findings indicate that colocalization of the classical neurotransmitters GABA and DA in feeding central pattern generator (CPG) interneurons preceded the divergence of euopisthobranch and panpulmonate taxa. These observations also support the hypothesis that heterogastropod feeding CPG networks exhibit a common universal design., (© 2018 Wiley Periodicals, Inc.)

CHARGE syndrome is linked to autosomal-dominant mutations in the CHD7 gene and results in a number of physiological and structural abnormalities, including heart defects, hearing and vision loss, and gastrointestinal (GI) problems. Of these challenges, GI problems have a profound impact throughout an individual's life, resulting in increased morbidity and mortality. A homolog of CHD7 has been identified in the zebrafish, the loss of which recapitulates many of the features of the human disease. Using a morpholino chd7 knockdown model complemented by a chd7 null mutant zebrafish line, we examined GI structure, innervation, and motility in larval zebrafish. Loss of chd7 resulted in physically smaller GI tracts with normal epithelial and muscular histology, but decreased and disorganized vagal projections, particularly in the foregut. chd7 morphant larvae had significantly less ability to empty their GI tract of gavaged fluorescent beads, and this condition was only minimally improved by the prokinetic agents, domperidone and erythromycin, in keeping with mixed responses to these agents in patients with CHARGE syndrome. The conserved genetics and transparency of the zebrafish have provided new insights into the consequences of chd7 gene dysfunction on the GI system and cranial nerve patterning. These findings highlight the opportunity of the zebrafish to serve as a preclinical model for studying compounds that may improve GI motility in individuals with CHARGE syndrome., (© 2018 Federation of European Biochemical Societies.)

Objective: Globally, more than 200 million people live at risk of the neglected tropical disease schistosomiasis (or snail fever). Larval schistosomes require the presence of specific snail species that act as intermediate hosts, supporting their multiplication and transformation into forms that can infect humans. This project was designed to generate a transcriptome from the central nervous system (CNS) of Biomphalaria alexandrina, the major intermediate host for Schistosoma mansoni in Egypt., Results: A transcriptome was generated from five pooled central nervous systems dissected from uninfected specimens of B. alexandrina. Raw Illumina RNA-seq data (~ 20.3 million paired end reads of 150 base pairs length each) generated a transcriptome consisting of 144,213 transcript elements with an N50 contig size of 716 base pairs. Orthologs of 15,246 transcripts and homologs for an additional 16,810 transcripts were identified in the UniProtKB/Swiss-Prot database. The B. alexandrina CNS transcriptome provides a resource for future research exploring parasite-host interactions in a simpler nervous system. Moreover, increased understanding of the neural signaling mechanisms involved in the response of B. alexandrina to infection by S. mansoni larvae could lead to novel and highly specific strategies for the control of snail populations.

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., (Copyright © 2017 the American Physiological Society.)

Hermissenda crassicornis is a model for studying the molecular and cellular basis for classical conditioning, based on its ability to associate light with vestibular stimulation. We used confocal microscopy to map histamine (HA), FMRF-amide, and γ-aminobutyric acid (GABA) immunoreactivity in the central nervous system (CNS), eyes, optic ganglia and statocysts of the nudibranchs. For HA immunoreactivity, we documented both consistently and variably labeled CNS structures across individuals. We also noted minor differences in GABA immunoreactivity in the CNS compared to previous work on Hermissenda. Contrary to expectations, we found no evidence for GABA inside the visual or vestibular systems. Instead, we found only FMRFamide- and HA immunoreactivity (FMRFamide: 4 optic ganglion cells, 4-5 hair cells; HA: 3 optic ganglion cells, 8 hair cells). Overall, our results can act as basis for comparisons of nervous systems across nudibranchs, and suggest further exploration of intraspecific plasticity versus evolutionary changes in gastropod nervous systems., (© 2017 Wiley Periodicals, Inc.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Within a common body plan, echinoid planktotrophic larvae are morphologically diverse, with variations in overall size, the length, and number of arms and the presence or absence of epidermal structures. In this report, we are interested in variation in larval arm-flexing behavior and correlated differences in larval musculature. Larvae of the cidaroid Eucidaris tribuloides exhibit conspicuous and regular arm-flexing behavior. In contrast, Lytechinus variegatus, a representative of the euechinoid clade, does not exhibit this behavior. We hypothesized that there were differences in musculature that correlated with this behavioral contrast and compared the development and structure of larval muscles between these species. We report substantial differences in some aspects of larval musculature. In addition to previously described oral musculature, both larvae possessed polygon-shaped musculature at the basal end of the larva. However, larval musculature in E. tribuloides was larger and contained additional muscles not observed in larvae of L. variegatus. Therefore, a conspicuous larval behavior consisting of repeated flexing of the postoral and posterodorsal larval arms was correlated with a larger, more complex musculature. This simple contrast indicates that larval musculature not associated with endoderm evolves in a manner that relates to differences in larval behavior and that additional comparisons are warranted.

Several lines of evidence suggest that serotonin (5-HT) has a regulatory role in cardiovascular function from embryogenesis through adulthood. However, the reported actions of 5-HT are often contradictory and include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction. Clarifying such cardiac effects requires further research and may benefit from utilizing a model simpler than the mammalian hearts traditionally used in these studies. In the present study, we describe the cardiac distribution and chronotropic responses of 5-HT in the zebrafish heart. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the involvement of 5-HT pathways, and to compare neural and direct myocardial pathways of biological action. Immunohistochemical methods revealed 5-HT in endocardial cells, glial-like cells, and intracardiac neurons in the atrium. Electrocardiogram (ECG) recordings combined with the administration of pharmacological agents demonstrated that 5-HT acted predominantly through direct myocardial pathways resulting in a reduction of heart rate. Overall, the results of this study contribute significant advances in the establishment of the zebrafish as a new model for studies of the role of 5-HT in autonomic cardiac control., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. Arduino microprocessors were used to control the delivery of auditory or visual stimuli to groups of adult or juvenile zebrafish in their home tanks in a conventional zebrafish facility. An automatic feeder dispensed precise amounts of food immediately after the conditioned stimuli, or at variable delays for controls. Responses were recorded using inexpensive cameras, with the video sequences analysed with ImageJ or Matlab. Fish showed significant conditioned responses in as few as 5 trials, learning that the conditioned stimulus was a predictor of food presentation at the water surface and at the end of the tank where the food was dispensed. Memories of these conditioned associations persisted for at least 2days after training when fish were tested either as groups or as individuals. Control fish, for which the auditory or visual stimuli were specifically unpaired with food, showed no comparable responses. This simple, low-cost, automated system permits scalable conditioning of zebrafish with minimal human intervention, greatly reducing both variability and labour-intensiveness. It will be useful for studies of the neural basis of learning and memory, and for high-throughput screening of compounds modifying those processes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In many marine invertebrates, larval metamorphosis is induced by environmental cues that activate sensory receptors and signalling pathways. Nitric oxide (NO) is a gaseous signalling molecule that regulates metamorphosis in diverse bilaterians. In most cases NO inhibits or represses this process, although it functions as an activator in some species. Here we demonstrate that NO positively regulates metamorphosis in the poriferan Amphimedon queenslandica. High rates of A. queenslandica metamorphosis normally induced by a coralline alga are inhibited by an inhibitor of nitric oxide synthase (NOS) and by a NO scavenger. Consistent with this, an artificial donor of NO induces metamorphosis even in the absence of the alga. Inhibition of the ERK signalling pathway prevents metamorphosis in concert with, or downstream of, NO signalling; a NO donor cannot override the ERK inhibitor. NOS gene expression is activated late in embryogenesis and in larvae, and is enriched in specific epithelial and subepithelial cell types, including a putative sensory cell, the globular cell; DAF-FM staining supports these cells being primary sources of NO. Together, these results are consistent with NO playing an activating role in induction of A. queenslandica metamorphosis, evidence of its highly conserved regulatory role in metamorphosis throughout the Metazoa.

This article provides supporting data for the research article "A simple automated system for appetitive conditioning of zebrafish in their home tanks" (J.M. Doyle, N. Merovitch, R.C. Wyeth, M.R. Stoyek, M. Schmidt, F. Wilfart, A. Fine, R.P. Croll, 2016) [1]. In that article, we described overall movements of zebrafish toward a food source as a response to auditory or visual cues as conditioned stimuli in a novel learning paradigm. Here, we describe separate analyses of the vertical and horizontal components of the learned response. These data provide evidence that the conditioning might result from both classical conditioning of an innate response of zebrafish to move to the surface in response to food cues and secondary conditioning of the fish to associate a food presentation with a specific location in the tank. Movement data from the twenty trial acquisition period and probe trials from 2-32 days post conditioning are included.

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 β2 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., (Copyright © 2016 the American Physiological Society.)

Heavy metals are common environmental pollutants to the aquatic ecosystems. Several aquatic species have been used as bioindicators and biomonitoring subjects for heavy metals pollution. In the present study, the effects of cadmium (Cd) and manganese (Mn) on the survival, attachment, locomotion, and feeding behaviours of the gastropod snail Biomphalaria alexandrina were determined. The short-term (96 h) LC50 for Cd and Mn were found to be 0.219 and 154.2 mg/l, respectively. Long-term exposures (16-20 days) to ascending concentrations of Cd (0.01-1 mg/l) and Mn (50-500 mg/l) also caused gradual decreases in the survival rate of B. alexandrina in a dose-dependent manner. Attachment, locomotion and feeding behaviours of snails exposed to lethal and sublethal concentrations of Cd and Mn at acute (96 h) and chronic exposure (24 days) intervals, respectively, were also recorded. Compared to controls, a significant decrease (p ≤ 0.05) was recorded in the different behaviours of exposed snails. These changes in behaviour would potentially impact the snail's ability to survive in the wild. Although Cd caused a more severe decline in snail survivorship than Mn, the behavioural effects of Mn were much more severe than Cd when the metals were roughly matched for lethality. In sum, the present study demonstrates B. alexandrina to be a sensitive bioindicator and model organism to assess heavy metals risk factors for severe toxicity in freshwater ecosystems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The siphon of Aplysia californica has several functions, including involvement in respiration, excretion, and defensive inking. It also provides sensory input for defensive withdrawals that have been studied extensively to examine mechanisms that underlie learning. To better understand the neuronal bases of these functions, we used immunohistochemistry to catalogue peripheral cell types and innervation of the siphon in stage 12 juveniles (chosen to allow observation of tissues in whole-mounts). We found that the siphon nerve splits into three major branches, leading ultimately to a two-part FMRFamide-immunoreactive plexus and an apparently separate tyrosine hydroxylase-immunoreactive plexus. Putative sensory neurons included four distinct types of tubulin-immunoreactive bipolar cells (one likely also tyrosine hydroxylase immunoreactive) that bore ciliated dendrites penetrating the epithelium. A fifth bipolar neuron type (tubulin- and FMRFamide-immunoreactive) occurred deeper in the tissue, associated with part of the FMRFamide-immunoreactive plexus. Our observations emphasize the structural complexity of the peripheral nervous system of the siphon, and the importance of direct tests of the various components to better understand the functioning of the entire organ, including its role in defensive withdrawal responses., (© 2015 Wiley Periodicals, Inc.)

In the vertebrate heart the intracardiac nervous system is the final common pathway for autonomic control of cardiac output, but the neuroanatomy of this system is not well understood. In this study we investigated the innervation of the heart in a model vertebrate, the zebrafish. We used antibodies against acetylated tubulin, human neuronal protein C/D, choline acetyltransferase, tyrosine hydroxylase, neuronal nitric oxide synthase, and vasoactive intestinal polypeptide to visualize neural elements and their neurotransmitter content. Most neurons were located at the venous pole in a plexus around the sinoatrial valve; mean total number of cells was 197 ± 23, and 92% were choline acetyltransferase positive, implying a cholinergic role. The plexus contained cholinergic, adrenergic, and nitrergic axons and vasoactive intestinal polypeptide-positive terminals, some innervating somata. Putative pacemaker cells near the plexus showed immunoreactivity for hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and the transcription factor Islet-1 (Isl1). The neurotracer neurobiotin showed that extrinsic axons from the left and right vagosympathetic trunks innervated the sinoatrial plexus proximal to their entry into the heart; some extrinsic axons from each trunk also projected into the medial dorsal plexus region. Extrinsic axons also innervated the atrial and ventricular walls. An extracardiac nerve trunk innervated the bulbus arteriosus and entered the arterial pole of the heart to innervate the proximal ventricle. We have shown that the intracardiac nervous system in the zebrafish is anatomically and neurochemically complex, providing a substrate for autonomic control of cardiac effectors in all chambers., (© 2015 Wiley Periodicals, Inc.)

Histamine appears to be an important transmitter throughout the Animal Kingdom. Gastropods, in particular, have been used in numerous studies establishing potential roles for this biogenic amine in the nervous system and showing its involvement in the generation of diverse behaviours. And yet, the distribution of histamine has only previously been described in a small number of molluscan species. The present study examined the localization of histamine-like immunoreactivity in the central and peripheral nervous systems of pulmonate snails of the genus Biomphalaria. This investigation demonstrates immunoreactive cells throughout the buccal, cerebral, pedal, left parietal and visceral ganglia, indicative of diverse regulatory functions in Biomphalaria. Immunoreactivity was also present in statocyst hair cells, supporting a role for histamine in graviception. In the periphery, dense innervation by immunoreactive fibers was observed in the anterior foot, perioral zone, and other regions of the body wall. This study thus shows that histamine is an abundant transmitter in these snails and its distribution suggest involvement in numerous neural circuits. In addition to providing novel subjects for comparative studies of histaminegic neurons in gastropods, Biomphalaria is also the major intermediate host for the digenetic trematode parasite, which causes human schistosomiasis. The study therefore provides a foundation for understanding potential roles for histamine in interactions between the snail hosts and their trematode parasites.

Embryonic cuttlefish can first respond to a variety of sensory stimuli during early development in the egg capsule. To examine the neural basis of this ability, we investigated the emergence of sensory structures within the developing epidermis. We show that the skin facing the outer environment (not the skin lining the mantle cavity, for example) is derived from embryonic domains expressing the Sepia officinalis ortholog of pax3/7, a gene involved in epidermis specification in vertebrates. On the head, they are confined to discrete brachial regions referred to as "arm pillars" that expand and cover Sof-pax3/7-negative head ectodermal tissues. As revealed by the expression of the S. officinalis ortholog of elav1, an early marker of neural differentiation, the olfactory organs first differentiate at about stage 16 within Sof-pax3/7-negative ectodermal regions before they are covered by the definitive Sof-pax3/7-positive outer epithelium. In contrast, the eight mechanosensory lateral lines running over the head surface and the numerous other putative sensory cells in the epidermis, differentiate in the Sof-pax3/7-positive tissues at stages ∼24-25, after they have extended over the entire outer surfaces of the head and arms. Locations and morphologies of the various sensory cells in the olfactory organs and skin were examined using antibodies against acetylated tubulin during the development of S. officinalis and were compared with those in hatchlings of two other cephalopod species. The early differentiation of olfactory structures and the peculiar development of the epidermis with its sensory cells provide new perspectives for comparisons of developmental processes among molluscs., (© 2014 Wiley Periodicals, Inc.)

Planorbid snails of the genus Biomphalaria are major intermediate hosts for the digenetic trematode parasite Schistosoma mansoni. Evidence suggests that levels of the neurotransmitter dopamine (DA) are reduced during the course of S. mansoni multiplication and transformation within the snail. This investigation used immunohistochemical methods to localize tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, in the nervous system of Biomphalaria. The two species examined, Biomphalaria glabrata and Biomphalaria alexandrina, are the major intermediate hosts for S. mansoni in sub-Saharan Africa, where more than 90% of global cases of human intestinal schistosomiasis occur. TH-like immunoreactive (THli) neurons were distributed throughout the central nervous system (CNS) and labeled fibers were present in all commissures, connectives, and nerves. Some asymmetries were observed, including a large distinctive neuron (LPeD1) in the pedal ganglion described previously in several pulmonates. The majority of TH-like immunoreactive neurons were detected in the peripheral nervous system (PNS), especially in lip and foot regions of the anterior integument. Independent observations supporting the dopaminergic phenotype of THli neurons included 1) block of LPeD1 synaptic signaling by the D2/3 antagonist sulpiride, and 2) the similar localization of aqueous aldehyde (FaGlu)-induced fluorescence. The distribution of THli neurons indicates that, as in other gastropods, dopamine functions as a sensory neurotransmitter and in the regulation of feeding and reproductive behaviors in Biomphalaria. It is hypothesized that infection could stimulate transmitter release from dopaminergic sensory neurons and that dopaminergic signaling could contribute to modifications of both host and parasite behavior., (© 2014 Wiley Periodicals, Inc.)

In this study, we present a morphological description of the fine structure of the tissues composing the caudal tip of the adult zebrafish swim bladder and an immunochemical survey of the innervation at this site. The internal aspect of the caudal tip is lined by an epithelium specialized to secrete surfactant into the lumen as evinced by the exocytosis of lamellar bodies. The sole innervation to this region consists of a neural plexus, present on the external surface, of nitric oxide synthase-positive (nNOS) neuronal cell bodies that are contacted by axon terminals, some containing neuropeptide Y and vasoactive intestinal polypeptide. As the specialized epithelium and neural plexus are coincident and of common extent, we suggest that the morphological relationship between the two elements allows the nervous system to affect surfactant processing, possibly through a paracrine mechanism., (© 2014 Wiley Periodicals, Inc.)

The intracardiac nervous system represents the final common pathway for autonomic control of the vertebrate heart in maintaining cardiovascular homeostasis. In teleost fishes, details of the organization of this system are not well understood. Here we investigated innervation patterns in the heart of the goldfish, a species representative of a large group of cyprinids. We used antibodies against the neuronal markers zn-12, acetylated tubulin, and human neuronal protein C/D, as well as choline acetyltransferase, tyrosine hydroxylase, nitric oxide synthetase, and vasoactive intestinal polypeptide (VIP) to detect neural elements and their transmitter contents in wholemounts and sections of cardiac tissue. All chambers of the heart were innervated by choline acetyltransferase-positive axons, implying cholinergic regulation; and by tyrosine hydroxylase-containing axons, implying adrenergic regulation. The mean total number of intracardiac neurons was 713 ± 78 (SE), nearly half of which were cholinergic. Neuronal somata were mainly located in a ganglionated plexus around the sinoatrial valves. Somata were contacted by cholinergic, adrenergic, nitrergic, and VIP-positive terminals. Putative pacemaker cells, identified by immunoreactivity for hyperpolarization activated, cyclic nucleotide-gated channel 4, were located in the base of the sinoatrial valves, and this region was densely innervated by cholinergic and adrenergic terminals. We have shown that the goldfish heart possesses the necessary neuroanatomical substrate for fine, region-by-region autonomic control of the myocardial effectors that are involved in determining cardiac output., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

We characterized the expression of sall4, a gene encoding a zinc finger transcription factor involved in the maintenance of embryonic stem cells, in taste buds of zebrafish (Danio rerio). Using an enhancer trap line (ET5), we detected enhanced green fluorescent protein (EGFP) in developing and adult transgenic zebrafish in regions containing taste buds: the lips, branchial arches, and the nasal and maxillary barbels. Localization of EGFP to taste cells of the branchial arches and lips was confirmed by co-immunolabeling with antibodies against calretinin and serotonin, and a zebrafish-derived neuronal marker (zn-12). Transgenic insertion of the ET construct into the zebrafish genome was evaluated and mapped to chromosome 23 in proximity (i.e. 23 kb) to the sall4 gene. In situ hybridization and expression analysis between 24 and 96 h post-fertilization (hpf) demonstrated that transgenic egfp expression in ET5 zebrafish was correlated with the spatial and temporal pattern of expression of sall4 in the wild-type. Expression was first observed in the central nervous system and branchial arches at 24 hpf. At 48 hpf, sall4 and egfp expression was observed in taste bud primordia surrounding the mouth and branchial arches. At 72 and 96 hpf, expression was detected in the upper and lower lips and branchial arches. Double fluorescence in situ hybridization at 3 and 10 dpf confirmed colocalization of sall4 and egfp in the lips and branchial arches. These studies reveal sall4 expression in chemosensory cells and implicate this transcription factor in the development and renewal of taste epithelia in zebrafish., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

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.

Odor molecules are transduced by thousands of olfactory sensory neurons (OSNs) located in the nasal cavity. Each OSN expresses a single functional odorant receptor protein and projects an axon from the sensory epithelia to an olfactory bulb glomerulus, which is selectively innervated by only one or a few OSN types. We used whole-mount immunocytochemistry to study the neurochemistry and anatomical organization of glomeruli in the zebrafish olfactory system. By employing combinations of antibodies against G-protein α subunits, calcium-binding proteins, and general neuronal markers, we selectively labeled various OSN types, their axonal projections to glomeruli, and the detailed anatomical distributions of individual glomeruli in different regions of the olfactory bulb. In this way we identified ≈140 glomeruli in each olfactory bulb of mature zebrafish. A small subset (27) of these glomeruli was unambiguously identifiable in nearly all animals examined. These units were large and, located mainly in the medial olfactory bulbs. Most glomeruli, however, were comparatively small, anatomically indistinguishable, and located in coarsely circumscribed regions; almost all of these latter glomeruli were innervated by OSNs that were labeled with anti-G(α s/olf) and/or anti-calretinin antibodies. Collectively, our results provide a uniquely detailed description of a vertebrate olfactory system and highlight anatomically distinct parallel neural pathways that mediate early aspects of olfactory processing in the zebrafish., (Copyright © 2012 Wiley Periodicals, Inc.)

The swimbladder of teleost fishes is the primary organ for controlling whole-body density, and thus buoyancy. The volume of gas in the swimbladder is adjusted to bring the organism to near neutral buoyancy at a particular depth. Swimbladder morphology varies widely among teleosts, but all species are capable of inflating and deflating this organ under reflex control by the autonomic nervous system, to achieve neutral buoyancy. Here we review the control of effectors within the swimbladder, including acid-secreting cells, vasculature and musculature, that are involved in determining gas volume. This control system is complex. It incorporates the "classical" efferent elements of the autonomic nervous system, the spinal autonomic and cranial autonomic limbs and their neurotransmitters (typically noradrenaline (NA)/adrenaline (ADR), and acetylcholine, respectively), but also non-adrenergic, non-cholinergic neurotransmitters such as peptides, purines and nitric oxide. The detailed patterns of autonomic innervation of swimbladder effectors are not well understood, nor are the relationships of terminals releasing non-adrenergic, non-cholinergic neurotransmitters onto these effectors. Furthermore, in most cases the complement of postjunctional receptor subtypes activated by adrenergic, cholinergic and other neurotransmitters, and the biological effects of these neurochemicals, have not been completely established. In order to clarify some of these issues and to provide insight into basic principles underlying autonomic control of swimbladder function, we propose the zebrafish as a potentially useful model teleost., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

The peripheral nervous system in gastropods plays a key role in the neural control of behaviors, but is poorly studied in comparison with the central nervous system. Peripheral sensory neurons, although known to be widespread, have been studied in a patchwork fashion across several species, with no comprehensive treatment in any one species. We attempted to remedy this limitation by cataloging peripheral sensory cells in the cephalic sensory organs of Lymnaea stagnalis employing backfills, vital stains, histochemistry, and immunohistochemistry. By using at least two independent methods to corroborate observations, we mapped four different cell types. We have found two different populations of bipolar sensory cells that appear to contain catecholamines(s) and histamine, respectively. Each cell had a peripheral soma, an epithelial process bearing cilia, and a second process projecting to the central nervous system. We also found evidence for two populations of nitric oxide-producing sensory cells, one bipolar, probably projecting centrally, and the second unipolar, with only a single epithelial process and no axon. The various cell types are presumably either mechanosensory or chemosensory, but the complexity of their distributions does not allow formation of hypotheses regarding modality. In addition, our observations indicate that yet more peripheral sensory cell types are present in the cephalic sensory organs of L. stagnalis. These results are an important step toward linking sensory cell morphology to modality. Moreover, our observations emphasize the size of the peripheral nervous system in gastropods, and we suggest that greater emphasis be placed on understanding its role in gastropod neuroethology., (Copyright © 2011 Wiley-Liss, Inc.)

FMRFamide-related peptides (FaRPs) are involved in numerous neural functions across the animal kingdom and serve as important models for understanding the evolution of neuropeptides. Gastropod molluscs have proved to be particularly useful foci for such studies, but the developmental expression of FaRPs and the evolution of specific transcripts for different peptides are unclear within the molluscs. Here we show that FaRPs are encoded by two transcripts that appear to be splice variants of a single gene in the abalone, Haliotis asinina, which represents the basal vetigastropods. Has-FMRF1 comprises 1,438 nucleotides and encodes a precursor protein of 329 amino acids that can potentially produce two copies of FLRFamide, one copy each of TLAGDSFLRFamide, QFYRIamide, SDPDLDDVIRASLLAYSLDDSPNN, and SVATAPVEAKAVEAGNKDIE, and 13 copies of FMRFamide. The second 1,241-nucleotide transcript, Has-FMRF2, encodes a 206-amino acid precursor protein with single copies of FLRFamide and FMRFamide along with such extended forms as NFGEPFLRFamide, FDSYEDKALRFamide, and NGWLHFamide, in addition to SDPGEDMLKSILLRGAPSNNGLQY and DTUDETTUNDNAHSRQ. Both transcripts are present early in life and are expressed in different but overlapping patterns within the developing larval nervous system. Mass spectrometry and immunocytochemistry demonstrate that FaRPs are cleaved from larger precursors and localize to the developing nervous system. Our results confirm previous evidence that FaRPs are expressed early and potentially play many roles during molluscan development and suggest that the last common ancestor to living gastropods used alternative splicing of an FMRFamide gene to generate a diversity of FaRPs in spatially restricted patterns in the nervous system., (Copyright © 2011 Wiley-Liss, Inc.)

The gas-filled swimbladder of teleost fishes provides hydrodynamic lift which counteracts the high density of other body tissues, and thereby allows the fish to achieve neutral buoyancy with minimal energy expenditure. In this study, we examined whether the absence of a constant direction gravitational vector affects the ontogeny of the swimbladder and buoyancy control in zebrafish (Danio rerio). We exposed fertilized eggs to simulated microgravity (SMG) in a closed rotating wall vessel with control eggs placed in a similar but nonrotating container. All eggs hatched in both groups. At 96 hr of postfertilization (hpf), all larvae were removed from the experimental and control vessels. At this point, 62% of the control larvae, but only 14% of SMG-exposed larvae, were observed to have inflated their swimbladder. In addition, the mean volume of the inflated swimbladders was significantly greater in the control larvae compared with larvae raised in SMG. After transfer to open stationary observation tanks, larvae with uninflated swimbladders in both groups swam to the surface to complete inflation, but this process was significantly delayed in larvae exposed to SMG. Initial differences in swimbladder inflation and volume between groups disappeared by 144 hpf. Furthermore, there were no apparent changes in patterns of development and maturation of swimbladder musculature, vasculature, or innervation resulting from SMG exposure at later stages of ontogeny. These data indicate that, despite a transient delay in swimbladder inflation in zebrafish larvae exposed to SMG, subsequent swimbladder development in these animals proceeded similarly to that in normal larvae., (Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.)