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ATP and its ionotropic P2X receptors are components of the most ancient signaling system. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized the P2X receptors in the sea slug Aplysia californica—a prominent neuroscience model. AcP2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the AcP2X receptors than the suramin. AcP2X were uniquely expressed within the cerebral F-cluster, the multifunctional integrative neurosecretory center. AcP2X receptors were also detected in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering the evolution of neurotransmitters.

Long-term but not short-term memory and synaptic plasticity in many brain areas require neurotrophin signaling, transcription, and epigenetic mechanisms including DNA methylation. However, it has been difficult to relate these cellular mechanisms directly to behavior because of the immense complexity of the mammalian brain. To address that problem, we and others have examined numerically simpler systems such as the hermaphroditic marine mollusk Aplysia californica. As a further simplification, we have used a semi-intact preparation of the Aplysia siphon withdrawal reflex in which it is possible to relate cellular plasticity directly to behavioral learning. We find that inhibitors of neurotrophin signaling, transcription, and DNA methylation block sensitization and classical conditioning beginning ∼1 h after the start of training, which is in the time range of an intermediate-term stage of plasticity that combines elements of short- and long-term plasticity and may form a bridge between them. Injection of decitabine (an inhibitor of DNA methylation that may have other actions in these experiments) into an LE sensory neuron blocks the neural correlates of conditioning in the same time range. In addition, we found that both DNA and RNA methylation in the abdominal ganglion are correlated with learning in the same preparations. These results begin to suggest the functions and integration of these different molecular mechanisms during behavioral learning.

Transmitter signalling is the universal chemical language of any nervous system, but little is known about its early evolution. Here, we summarize data about the distribution and functions of neurotransmitter systems in basal metazoans as well as outline hypotheses of their origins. We explore the scenario that neurons arose from genetically different populations of secretory cells capable of volume chemical transmission and integration of behaviours without canonical synapses. The closest representation of this primordial organization is currently found in Placozoa, disk-like animals with the simplest known cell composition but complex behaviours. We propose that injury-related signalling was the evolutionary predecessor for integrative functions of early transmitters such as nitric oxide, ATP, protons, glutamate and small peptides. By contrast, acetylcholine, dopamine, noradrenaline, octopamine, serotonin and histamine were recruited as canonical neurotransmitters relatively later in animal evolution, only in bilaterians. Ligand-gated ion channels often preceded the establishment of novel neurotransmitter systems. Moreover, lineage-specific diversification of neurotransmitter receptors occurred in parallel within Cnidaria and several bilaterian lineages, including acoels. In summary, ancestral diversification of secretory signal molecules provides unique chemical microenvironments for behaviour-driven innovations that pave the way to complex brain functions and elementary cognition.This article is part of the theme issue ‘Basal cognition: multicellularity, neurons and the cognitive lens'.

Sponges and evolutionary origins Sponges represent our distant animal relatives. They do not have a nervous system but do have a simple body for filter feeding. Surveying the cell types in the freshwater sponge Spongilla lacustris , Musser et al . found that many genes important in synaptic communication are expressed in cells of the small digestive chambers. They found secretory machinery characteristic of the presynapse in small multipolar cells contacting all other cells and also the receptive apparatus of the postsynapse in the choanocytes that generate water flow and digest microbial food. These results suggest that the first directed communication in animals may have evolved to regulate feeding, serving as a starting point on the long path toward nervous system evolution. —BAP

The evolutionary origin of metazoan cell types such as neurons, muscles, digestive, and immune cells, remains unsolved. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identify 18 distinct cell types comprising four major families. This includes nitric-oxide sensitive contractile cells, digestive cells active in macropinocytosis, and a family of amoeboid-neuroid cells involved in innate immunity. We uncover ‘presynaptic’ genes in an amoeboid-neuroid cell type, and ‘postsynaptic’ genes in digestive choanocytes, suggesting asymmetric and targeted communication. Corroborating this, long neurite-like extensions from neuroid cells directly contact and enwrap choanocyte microvillar collars. Our data indicate a link between neuroid and immune functions in sponges, and suggest that a primordial neuro-immune system cleared intruders and controlled ciliary beating for feeding.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis., Biomphalaria glabrata is a fresh water snail that acts as a host for trematode Schistosoma mansoni that causes intestinal infection in human. This work describes the genome and transcriptome analyses from 12 different tissues of B glabrata, and identify genes for snail behavior and evolution.

There is more than one way to develop neuronal complexity, and animals frequently use different molecular toolkits to achieve similar functional outcomes. Genomics and metabolomics data from basal metazoans suggest that neural signalling evolved independently in ctenophores and cnidarians/bilaterians. This polygenesis hypothesis explains the lack of pan-neuronal and pan-synaptic genes across metazoans, including remarkable examples of lineage-specific evolution of neurogenic and signalling molecules as well as synaptic components. Sponges and placozoans are two lineages without neural and muscular systems. The possibility of secondary loss of neurons and synapses in the Porifera/Placozoa clades is a highly unlikely and less parsimonious scenario. We conclude that acetylcholine, serotonin, histamine, dopamine, octopamine and gamma-aminobutyric acid (GABA) were recruited as transmitters in the neural systems in cnidarian and bilaterian lineages. By contrast, ctenophores independently evolved numerous secretory peptides, indicating extensive adaptations within the clade and suggesting that early neural systems might be peptidergic. Comparative analysis of glutamate signalling also shows numerous lineage-specific innovations, implying the extensive use of this ubiquitous metabolite and intercellular messenger over the course of convergent and parallel evolution of mechanisms of intercellular communication. Therefore: (i) we view a neuron as a functional character but not a genetic character, and (ii) any given neural system cannot be considered as a single character because it is composed of different cell lineages with distinct genealogies, origins and evolutionary histories. Thus, when reconstructing the evolution of nervous systems, we ought to start with the identification of particular cell lineages by establishing distant neural homologies or examples of convergent evolution. In a corollary of the hypothesis of the independent origins of neurons, our analyses suggest that both electrical and chemical synapses evolved more than once.

Biogenic amines are implicated in several mental disorders, many of which involve social interactions. Simple model systems, such as crustaceans, are often more amenable than vertebrates for studying mechanisms underlying behaviors. Although various cellular responses of biogenic amines have been characterized in crustaceans, the mechanisms linking these molecules to behavior remain largely unknown. Observed effects of serotonin receptor agonists and antagonists in abdomen posture, escape responses, and fighting have led to the suggestion that biogenic amine receptors may play a role in modulating interactive behaviors. As a first step in understanding this potential role of such receptors, we have cloned and fully sequenced two serotonin receptors, 5-HT(1Mac) and 5-HT(2Mac), from the CNS of the freshwater prawn Macrobrachium rosenbergii and have mapped their CNS immunohistochemical distribution. 5-HT(1Mac) was found primarily on the membranes of subsets of cells in all CNS ganglia, in fibers that traverse all CNS regions, and in the cytoplasm of a small number of cells in the brain and circum- and subesophageal ganglia (SEG), most of which also appear to contain dopamine. The pattern of 5-HT(2Mac) immunoreactivity was found to differ significantly; it was found mostly in the central neuropil area of all ganglia, in glomeruli of the brain's olfactory lobes, and in the cytoplasm of a small number of neurons in the SEG, thoracic, and some abdominal ganglia. The observed differences in terms of localization, distribution within cells, and intensity of immunoreactive staining throughout the prawn's CNS suggest that these receptors are likely to play different roles.

We have previously cloned and characterized a novel 14-3-3 gene from the euryhaline telost Fundulus heteroclitus, Fh14-3-3a (Kultz et al., 2001). The corresponding gene product is osmoregulated and most highly expressed in gill epithelium of this fish. In the present study we have expressed Fh14-3-3a cRNA in Xenopus laevis oocytes and investigated the survival and electrophysiological parameters of Xenopus oocytes in isosmotic and various hyperosmotic media. Xenopus oocytes expressing Fh14-3-3a show no mortality after a 16 hour exposure to hyperosmolality in the form of elevating medium K+, Na+, polyethylene glycol, or sorbitol concentrations up to 444 mosmol/kg. In contrast, 16 hours of the same hyperosmolality caused 100% mortality in control Xenopus oocytes injected with water. As a result of hyperosmolality the Xenopus oocyte membrane potential decreased between 10 and 70% in oocytes expressing Fh14-3-3a whereas it was completely abolished in control oocytes. We report that one potential cause for the osmoprotective effect of Fh14-3-3a on Xenopus oocytes could be its inhibition of an endogenous chloride current. Hyperosmotic urea was not as harmful to Xenopus oocytes as hypertonicity and maybe acting through a different mechanism. Coexpression of Fh14-3-3a with a human calcium channel in Xenopus oocytes did not affect the electrophysiological properties of this exogenous channel. Thus, the osmoprotective effect of Fh14-3-3a may prove a valuable tool for the characterization of exogenous ion channels in Xenopus oocytes exposed to hyperosmotic conditions. J. Exp. Zool. 299A:103–109, 2003. © 2003 Wiley-Liss, Inc.

Nature Communications 8: Article number: 15451 (2017); Published 16 May 2017; Updated 23 August 2017 The original version of this Article contained an error in the spelling of the author Leon Di Stefano, which was incorrectly given as Leon di Stephano. This has now been corrected in both the PDF andHTML versions of the Article.

Schistosomes are parasitic flatworms that cause schistosomiasis, a major tropical disease. The current drug of choice against schistosomiasis is praziquantel (PZQ), which has minimal side effects and is potent against all schistosome species. The mode of action of PZQ is unknown, though the drug clearly affects Ca(2+) homeostasis in worms, and there is indirect evidence for interaction of PZQ with schistosome voltage-gated Ca(2+) channels. We have cloned and expressed two Ca(2+) channel beta subunits, one from Schistosoma mansoni and one from Schistosoma japonicum. These two subunits (SmCa(v)beta A and SjCa(v)beta) have structural motifs that differ from those found in other known beta subunits. Surprisingly, coexpression of either SmCa(v)beta A or SjCa(v)beta with a cnidarian (CyCa(v)1) or mammalian (Ca(v)2.3) Ca(2+) channel alpha(1) subunit results in a striking reduction in current amplitude. In the case of Ca(v)2.3, this current reduction can be partially reversed by addition of 100 nm PZQ, which results in a significant increase in current amplitude. Thus, these unusual schistosome beta subunits can confer PZQ sensitivity to an otherwise PZQ-insensitive mammalian Ca(2+) channel, indicating that a possible target for PZQ action is the interaction between beta subunits and pore-forming alpha(1) subunits in schistosomes.

Prohormone substrates are required for investigation of the proteolytic processing of prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant prohormones in Escherichia coli. High-level expression of milligrams of prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases "prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different prohormones was demonstrated. PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for prohormone substrates illustrates the importance of expressing recombinant prohormones for in vitro processing studies.

RNA-seq or transcriptome analysis of individual cells and small-cell populations is essential for virtually any biomedical field. It is especially critical for developmental, aging, and cancer biology as well as neuroscience where the enormous heterogeneity of cells present a significant methodological and conceptual challenge. Here we present two methods that allow for fast and cost-efficient transcriptome sequencing from ultra-small amounts of tissue or even from individual cells using semiconductor sequencing technology (Ion Torrent, Life Technologies). The first method is a reduced representation sequencing which maximizes capture of RNAs and preserves transcripts' directionality. The second, a template-switch protocol, is designed for small mammalian neurons. Both protocols, from cell/tissue isolation to final sequence data, take up to 4 days. The efficiency of these protocols has been validated with single hippocampal neurons and various invertebrate tissues including individually identified neurons within a simpler memory-forming circuit of Aplysia californica and early (1-, 2-, 4-, 8-cells) embryonic and developmental stages from basal metazoans.

The preference of the ‘prohormone thiol protease’ (PTP), a candidate prohormone processing enzyme, for different peptide precursors was assessed in vitro with recombinant prohormones near estimated in vivo levels. Pro-neuropeptide Y (pro-NPY), prooplomelanocortin (POMC), and proenkephalin (PE) were expressed at high levels in E. coli. Purification of prohormones utilized a combination of DEAE-Sepharose, Mono Q, and preparative electrophoresis. PTP cleaved PE most readily, and also cleaved pro-NPY. The processing of POMC by PTP was minimal. These results demonstrate PTP's preference for certain prohormone substrates.

Sulfate conjugation by sulfotransferases is important for the metabolism of drugs, xenobiotics, monoamines, and neuropeptides (1-3). In this study, molecular cloning resulted in the isolation of a 1.2-kb human brain hippocampal cDNA encoding a member of the phenol sulfotransferase (PST) family of enzymes. The hippocampal phenol sulfotransferase (H-PST) cDNA possesses differences in its deduced primary sequence compared to human liver monoamine (M form) (4,5) and phenol (P form) (6,7) isoforms of PST. The hippocampal PST contrasts with previous studies that identified two human brain PST cDNAs whose coding sequences are identical to the human liver M and P forms of PST. Importantly, this study provides evidence for a human hippocampal form Of PST.

Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands.

There is ample evidence linking octopamine (OA) and tyramine (TA) to several neurophysiological functions in arthropods. In our laboratory we use the freshwater prawn Macrobrachium rosenbergii to study the neural basis of aggressive behavior. As a first step towards understanding the possible role of these amines and their receptors in the modulation of interactive behaviors, we have cloned a putative octopamine/tyramine receptor. The predicted sequence of the cloned OA/TA Mac receptor consists of 1,579 base pairs (bp), with an open reading frame of 1,350 bp that encodes a 450 amino acid protein. This putative protein displays sequence identities of 70% to an Aedes aegypti mosquito TA receptor, followed by 60% to a Stegomyia aegypti mosquito OA receptor, 59% and 58% to the migratory locust TA-1 and -2 receptors respectively, and 57% with the silkworm OA receptor. We also mapped the OA/TA Mac receptor distribution by in-situ hybridization to the receptor's mRNA, and by immunohistochemistry to its protein. We observed stained cell bodies for the receptor's mRNA, mainly in the midline region of the thoracic and in the abdominal ganglia, as well as diffuse staining in the brain ganglia. For the receptor's protein, we observed extensive punctate staining within the neuropil and on the membrane of specific groups of neurons in all ganglia throughout the CNS, including the brain, the midline region and neuropiles of the thoracic ganglia, and ventral part and neuropiles of the abdominal ganglia. The same pattern of stained cells was observed on the thoracic and abdominal ganglia in both in-situ hybridization and immunohistochemistry experiments. Diffuse staining observed with in-situ hybridization also coincides with punctate staining observed in brain, SEG, thoracic, and abdominal ganglia in immunohistochemical preparations. This work provides the first step towards characterizing the neural networks that mediate octopaminergic signaling in prawn.

Nitric oxide (NO) is one of the smallest and most diffusible signal molecules known. It can be synthesized in virtually any cell of our body and found in nearly every major group of organisms on our planet. Here, we will discuss several enzymatic and nonenzymatic pathways of NO synthesis in both prokaryotes and eukaryotes including protists, plants and animals. Many of these synthetic mechanisms can coexist within the same cell or cell population. We will also briefly review comparative aspects of NO signaling with a focus on the diversity of NO synthases in invertebrate animals and nonanimal groups. NO-related regulatory mechanisms may be as old as cellular organization itself, so that “ancestral” functions of NO in prokaryotes and basal eukaryotes are likely well preserved across billions of years of biological evolution and can be essential for biomedical studies and clinical applications. On the other hand, NO synthetic pathways might represent examples of parallel evolution in different lineages of organisms.

Summary Molecular analyses of Aplysia, a well-established model organism for cellular and systems neural science, have been seriously handicapped by a lack of adequate genomic information. By sequencing cDNA libraries from the central nervous system (CNS), we have identified over 175,000 expressed sequence tags (ESTs), of which 19,814 are unique neuronal gene products and represent 50%–70% of the total Aplysia neuronal transcriptome. We have characterized the transcriptome at three levels: (1) the central nervous system, (2) the elementary components of a simple behavior: the gill-withdrawal reflex—by analyzing sensory, motor, and serotonergic modulatory neurons, and (3) processes of individual neurons. In addition to increasing the amount of available gene sequences of Aplysia by two orders of magnitude, this collection represents the largest database available for any member of the Lophotrochozoa and therefore provides additional insights into evolutionary strategies used by this highly successful diversified lineage, one of the three proposed superclades of bilateral animals.

Nitric oxide (NO) is synthesized enzymatically by nitric oxide synthase (NOS). Several groups have previously presented evidence for NOS activity and immunoreactivity in several parasitic platyhelminths, including schistosomes. Here, we use 4,5-diaminofluorescein-2 diacetate (DAF-2 DA), a fluorescent indicator of NO, to detect NO in living schistosomes. In adult worms, DAF-2 fluorescence is found selectively in epithelial-like cells. Fluorescence increases when worms are incubated in L-arginine, the precursor of NO synthesis, and decreases dramatically in the presence of the NOS inhibitor N(G)-nitro-L-arginine methyl ester, indicating that predicted NO release may be NOS-dependent, and that enzymatic NO signaling pathways may play an important role in schistosome physiology.

The distribution of nitric oxide synthase (NOS) immunoreactivity and putative NOS activity in adult Schistosoma mansoni was analysed using 3 different types of NOS antibodies and NADPH-diaphorase histochemistry. Although potential involvement of the gaseous radical nitric oxide (NO) in host response to infection by schistosomes has been suggested, there is little or no information available regarding the role, or even the presence, of the NO pathway in schistosomes themselves. Here, we demonstrate that antibodies against neuronal NOS (nNOS) and inducible NOS (iNOS) isoforms stain adult worms with distinctive patterns; anti-endothelial NOS (eNOS) shows no selective labelling. nNOS-like immunoreactivity is found in the main nerve cords and the peripheral nervous system. Putative sensory neurons with apical neuronal processes leading to the tegument of male worms are also immunoreactive for nNOS. Anti-iNOS labels a variety of predominantly non-neuronal tissues, showing intense labelling at or near the surface of the worm and in components of the gastrointestinal tract. The distribution of NADPH-diaphorase reactivity (a histochemical marker of NOS), is generally similar to the pattern of NOS immunoreactivity, including labelling of neuronal-like cells as well as developing eggs. These results suggest that an NOS-like enzyme is present in S. mansoni, and indicate potential roles for the different NOS isoforms in neuronal signalling, reproduction and development.

Molecular cloning of bovine adrenal medulla (AM) and pituitary (Pit)α 1 -antichymotrypsin cDNAs indicated novel isoforms of ACT. The deduced primary sequences indicated that the AM ACT and Pit ACT possess COOH-terminal reactive-site domains that are characteristic of serpins ( ser ineprotease in hibitors). Of high interest was the finding of unique reactive sites within AM ACT and Pit ACT which are predicted to possess Arg as P 1 residue. Arginine as P 1 residue parallels the cleavage specificity of neuroendocrine prohormone processing enzymes cleaving at basic residues. Furthermore, RT-PCR indicated tissue-specific expression of AM and Pit ACT mRNAs. The AM and Pit isoforms of ACT may regulate novel target proteases involved in neuroendocrine function.