Urotensin II in Invertebrates: From Structure to Function in Aplysia californica

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.     

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.     

Studying the isolated central nervous system; a report on 35 years: more inquisitive than acquisitive

1. The CNS from invertebrate animals such as slugs, snails, leeches, and cockroaches, can be isolated and kept alive for many hours. 2. The electrical and pharmacological properties of invertebrate CNS neurons have many similarities and it is probable that the basic rules governing the CNS evolved more than 600 million years ago. 3. The nerve cells can show sodium action potentials, calcium action potentials, EPSP, IPSP, biphasic potentials, electrogenic sodium pump potentials, and a variety of potassium, sodium, calcium and chloride currents. 4. Invertebrate CNS ganglia contain identifiable individual nerve cells whose properties and responses to neurotransmitters and drugs are constant and repeatable from preparation to preparation. 5. It was possible to set up an isolated CNS-nerve trunk-muscle preparation and study the transport of radioactive material from the CNS to the muscle and from muscle to CNS. This has provided information about axoplasmic transport in both invertebrate and vertebrate preparations. 6. The methods developed from studies of invertebrate isolated CNS preparations have been applied to vertebrate isolated CNS preparations. 7. In addition to thin slices of the mammalian brain, it is possible to keep 5 cm lengths of the whole mammalian spinal cord and brain stem alive for many hours. 8. The isolated mammalian spinal cord has functional ipsilateral and contralateral reflexes, ascending and descending pathways, extensive sensory integrative local area networks, and inhibitory interneuron circuits. Much of the in vivo circuitry is functional in vitro. 9. The isolated mammalian spinal cord and brain stem can be developed to include functional higher brain circuits that will provide increased understanding of the control and integrative action of the mammalian central nervous system.     

Leptin does not directly affect CNS serotonin neurons to influence appetite

Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sert(cre)) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sert(cre) promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sert(cre)-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function.     

Oxytocinergic and serotonergic systems involvement in sodium intake regulation: satiety or hypertonicity markers?

Previous studies demonstrated the inhibitory participation of serotonergic (5-HT) and oxytocinergic (OT) neurons on sodium appetite induced by peritoneal dialysis (PD) in rats. The activity of 5-HT neurons increases after PD-induced 2% NaCl intake and decreases after sodium depletion; however, the activity of the OT neurons appears only after PD-induced 2% NaCl intake. To discriminate whether the differential activations of the 5-HT and OT neurons in this model are a consequence of the sodium satiation process or are the result of stimulation caused by the entry to the body of a hypertonic sodium solution during sodium access, we analyzed the number of Fos-5-HT- and Fos-OT-immunoreactive neurons in the dorsal raphe nucleus and the paraventricular nucleus of the hypothalamus-supraoptic nucleus, respectively, after isotonic vs. hypertonic NaCl intake induced by PD. We also studied the OT plasma levels after PD-induced isotonic or hypertonic NaCl intake. Sodium intake induced by PD significantly increased the number of Fos-5-HT cells, independently of the concentration of NaCl consumed. In contrast, the number of Fos-OT neurons increased after hypertonic NaCl intake, in both depleted and non-depleted animals. The OT plasma levels significantly increased only in the PD-induced 2% NaCl intake group in relation to others, showing a synergic effect of both factors. In summary, 5-HT neurons were activated after body sodium status was reestablished, suggesting that this system is activated under conditions of satiety. In terms of the OT system, both OT neural activity and OT plasma levels were increased by the entry of hypertonic NaCl solution during sodium consumption, suggesting that this system is involved in the processing of hyperosmotic signals.     

Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis

The swimming larvae of the chordate ascidians possess a dorsal hollowed central nervous system (CNS), which is homologous to that of vertebrates. Despite the homology, the ascidian CNS consists of a countable number of cells. The simple nervous system of ascidians provides an excellent experimental system to study the developmental mechanisms of the chordate nervous system. The neural fate of the cells consisting of the ascidian CNS is determined in both autonomous and non-autonomous fashion during the cleavage stage. The ascidian neural plate performs the morphogenetic movement of neural tube closure that resembles that in vertebrate neural tube formation. Following neurulation, the CNS is separated into five distinct regions, whose homology with the regions of vertebrate CNS has been discussed. Following their larval stage, ascidians undergo a metamorphosis and become sessile adults. The metamorphosis is completed quickly, and therefore the metamorphosis of ascidians is a good experimental system to observe the reorganization of the CNS during metamorphosis. A recent study has shown that the major parts of the larval CNS remain after the metamorphosis to form the adult CNS. In contrast to such a conserved manner of CNS reorganization, most larval neurons disappear during metamorphosis. The larval glial cells in the CNS are the major source for the formation of the adult CNS, and some of the glial cells produce adult neurons.     

Kainic acid: neurophysiological and neurotoxic actions.

Kainic acid, an anthelmintic drug structurally related to glutamate, has excitatory electrophysiological actions on neurons in the vertebrate CNS and at the invertebrate neuromuscular junction. Recently, it has been shown to destroy neuronal cell bodies and dendrites in several regions of the vertebrate CNS, while sparing afferent fibers and fibers of passage. Kainic acid can be used to make lesions in experimental animals that closely resemble the pathology associated with certain neurological conditions in man. Its actions on vertebrate and invertebrate nervous systems are reviewed and possible neuroexcitatory and neurotoxic mechanisms are considered.     

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.     

Methylation of genomes and genes at the invertebrate-vertebrate boundary.

Patterns of DNA methylation in animal genomes are known to vary from an apparent absence of modified bases, via methylation of a minor fraction of the genome, to genome-wide methylation. Representative genomes from 10 invertebrate phyla comprise predominantly nonmethylated DNA and (usually but not always) a minor fraction of methylated DNA. In contrast, all 27 vertebrate genomes that have been examined display genome-wide methylation. Our studies of chordate genomes suggest that the transition from fractional to global methylation occurred close to the origin of vertebrates, as amphioxus has a typically invertebrate methylation pattern whereas primitive vertebrates (hagfish and lamprey) have patterns that are typical of vertebrates. Surprisingly, methylation of genes preceded this transition, as many invertebrate genes have turned out to be heavily methylated. Methylation does not preferentially affect genes whose expression is highly regulated, as several housekeeping genes are found in the heavily methylated fraction whereas several genes expressed in a tissue-specific manner are in the nonmethylated fraction.     

Chronic alterations in serotonin function: dynamic neurochemical properties in agonistic behavior of the crayfish, Orconectes rusticus

The biogenic amine serotonin [5-hydroxytryptamine (5-HT)] has received considerable attention for its role in behavioral phenomena throughout a broad range of invertebrate and vertebrate taxa. Acute 5-HT infusion decreases the likelihood of crayfish to retreat from dominant opponents. The present study reports the biochemical and behavioral effects resulting from chronic treatment with 5-HT-modifying compounds delivered for up to 5 weeks via silastic tube implants. High performance liquid chromatography with electrochemical detection (HPLC-ED) confirmed that 5,7-dihydroxytryptamine (5,7-DHT) effectively reduced 5-HT in all central nervous system (CNS) areas, except brain, while a concurrent accumulation of the compound was observed in all tissues analyzed. Unexpectedly, two different rates of chronic 5-HT treatment did not increase levels of the amine in the CNS. Behaviorally, 5,7-DHT treated crayfish exhibited no significant differences in measures of aggression. Although treatment with 5-HT did not elevate 5-HT content in the CNS, infusion at a slow rate caused animals to escalate more quickly while 5-HT treatment at a faster rate resulted in slower escalation. 5,7-DHT is commonly used in behavioral pharmacology and the present findings suggest its biochemical properties should be more thoroughly examined. Moreover, the apparent presence of powerful compensatory mechanisms indicates our need to adopt an increasingly dynamic view of the serotonergic bases of behavior like crayfish aggression.     

Molluscan neurons in culture: shedding light on synapse formation and plasticity

From genes to behaviour, the simple model system approach has played many pivotal roles in deciphering nervous system function in both invertebrates and vertebrates. However, with the advent of sophisticated imaging and recording techniques enabling the direct investigation of single vertebrate neurons, the utility of simple invertebrate organisms as model systems has been put to question. To address this subject meaningfully and comprehensively, we first review the contributions made by invertebrates in the field of neuroscience over the years, paving the way for similar breakthroughs in higher animals. In particular, we focus on molluscan (Lymnaea, Aplysia, and Helisoma) and leech (Hirudo) models and the pivotal roles they have played in elucidating mechanisms of synapse formation and plasticity. While the ultimate goal in neuroscience is to understand the workings of the human brain in both its normal and diseased states, the sheer complexity of most vertebrate models still makes it difficult to define the underlying principles of nervous system function. Investigators have thus turned to invertebrate models, which are unique with respect to their simple nervous systems that are endowed with a finite number of large, individually identifiable neurons of known function. We start off by discussing in vivo and semi-intact preparations, regarding their amenability to simple circuit analysis. Despite the 'simplicity' of invertebrate nervous systems however, it is still difficult to study individual synaptic connections in detail. We therefore emphasize in the next section, the utility of studying identified invertebrate neurons in vitro, to directly examine the development, specificity, and plasticity of synaptic connections in a well-defined environment, at a resolution that it is still unapproachable in the intact brain. We conclude with a discussion of the future of invertebrates in neuroscience in elucidating mechanisms of neurological disease and developing neuron-silicon interfaces.     

Evolution of vertebrate central nervous system is accompanied by novel expression changes of duplicate genes

The evolution of the central nervous system (CNS) is one of the most striking changes during the transition from invertebrates to vertebrates. As a major source of genetic novelties, gene duplication might play an important role in the functional innovation of vertebrate CNS. In this study, we focused on a group of CNS-biased genes that duplicated during early vertebrate evolution. We investigated the tempo-spatial expression patterns of 33 duplicate gene families and their orthologs during the embryonic development of the vertebrate Xenopus laevis and the cephalochordate Brachiostoma belcheri. Almost all the identified duplicate genes are differentially expressed in the CNS in Xenopus embryos, and more than 50% and 30% duplicate genes are expressed in the telencephalon and mid-hindbrain boundary, respectively, which are mostly considered as two innovations in the vertebrate CNS. Interestingly, more than 50% of the amphioxus orthologs do not show apparent expression in the CNS in amphioxus embryos as detected by in situ hybridization, indicating that some of the vertebrate CNS-biased duplicate genes might arise from non-CNS genes in invertebrates. Our data accentuate the functional contribution of gene duplication in the CNS evolution of vertebrate and uncover an invertebrate non-CNS history for some vertebrate CNS-biased duplicate genes.     

Retinoic acid induces neurite outgrowth and growth cone turning in invertebrate neurons

Identification of molecules involved in neurite outgrowth during development and/or regeneration is a major goal in the field of neuroscience. Retinoic acid (RA) is a biologically important metabolite of vitamin A that acts as a trophic factor and has been implicated in neurite outgrowth and regeneration in many vertebrate species. Although abundant in the CNS of many vertebrates, the precise role of RA in neural regeneration has yet to be determined. Moreover, very little information is available regarding the role of RA in invertebrate nervous systems. Here, we demonstrate for the first time that RA induces neurite outgrowth from invertebrate neurons. Using individually identified neurons isolated from the CNS of Lymnaea stagnalis, we demonstrated that a significantly greater proportion of cells produced neurite outgrowth in RA. RA also extended the duration of time that cells remained electrically excitable in vitro, and we showed that exogenously applied RA acted as a chemoattractive factor and induced growth cone turning toward the source of RA. This is the first demonstration that RA can induce turning of an individual growth cone. These data strongly suggest that the actions of RA on neurite outgrowth and cell survival are highly conserved across species.     

Neuromodulatory Control of a Goal-Directed Decision

Many cost-benefit decisions reduce to simple choices between approach or avoidance (or active disregard) to salient stimuli. Physiologically, critical factors in such decisions are modulators of the homeostatic neural networks that bias decision processes from moment to moment. For the predatory sea-slug Pleurobranchaea, serotonin (5-HT) is an intrinsic modulatory promoter of general arousal and feeding. We correlated 5-HT actions on appetitive state with its effects on the approach-avoidance decision in Pleurobranchaea. 5-HT and its precursor 5-hydroxytryptophan (5-HTP) augmented general arousal state and reduced feeding thresholds in intact animals. Moreover, 5-HT switched the turn response to chemosensory stimulation from avoidance to orienting in many animals. In isolated CNSs, bath application of 5-HT both stimulated activity in the feeding motor network and switched the fictive turn response to unilateral sensory nerve stimulation from avoidance to orienting. Previously, it was shown that increasing excitation state of the feeding network reversibly switched the turn motor network response from avoidance to orienting, and that 5-HT levels vary inversely with nutritional state. A simple model posits a critical role for 5-HT in control of the turn network response by corollary output of the feeding network. In it, 5-HT acts as an intrinsic neuromodulatory factor coupled to nutritional status and regulates approach-avoidance via the excitation state of the feeding network. Thus, the neuromodulator is a key organizing element in behavioral choice of approach or avoidance through its actions in promoting appetitive state, in large part via the homeostatic feeding network.     

Structure and function of invertebrate 5-HT receptors: a review

Over the last decade, knowledge of invertebrate serotonin receptors has expanded greatly. The first 5-HT receptor from Drosophila was cloned 10 years ago, and subsequently, 11 additional receptor genes have been cloned from Drosophila, molluscs (Lymnaea and Aplysia) and nematodes (Caenorhabditis and Ascaris). Information has also accumulated from physiological and biochemical studies that have used vertebrate serotonergic ligands to characterize endogenous invertebrate receptors. Although the endogenous receptors are often classified according to mammalian-based categories, in many cases the pharmacological properties of vertebrate and invertebrate receptors differ significantly and the actual identity of the latter is questionable. By providing information on the gene structure and amino acid sequence, molecular cloning studies offer a more definitive way to identify and classify invertebrate 5-HT receptors. This review summarizes information on the pharmacological and transductional properties of cloned invertebrate 5-HT receptors, and considers recent studies of endogenous receptors in the light of this new data.     

Evolution of motor innervation to vertebrate fins and limbs

The evolution and diversification of vertebrate behaviors associated with locomotion depend highly on the functional transformation of paired appendages. Although the evolution of fins into limbs has long been a focus of interest to scientists, the evolution of neural control during this transition has not received much attention. Recent studies have provided significant progress in the understanding of the genetic and developmental bases of the evolution of fin/limb motor circuitry in vertebrates. Here we compare the organization of the motor neurons in the spinal cord of various vertebrates. We also discuss recent advances in our understanding of these events and how they can provide a mechanistic explanation for the evolution of fin/limb motor circuitry in vertebrates.     

Gastrointestinal hormones regulating appetite

The role of gastrointestinal hormones in the regulation of appetite is reviewed. The gastrointestinal tract is the largest endocrine organ in the body. Gut hormones function to optimize the process of digestion and absorption of nutrients by the gut. In this capacity, their local effects on gastrointestinal motility and secretion have been well characterized. By altering the rate at which nutrients are delivered to compartments of the alimentary canal, the control of food intake arguably constitutes another point at which intervention may promote efficient digestion and nutrient uptake. In recent decades, gut hormones have come to occupy a central place in the complex neuroendocrine interactions that underlie the regulation of energy balance. Many gut peptides have been shown to influence energy intake. The most well studied in this regard are cholecystokinin (CCK), pancreatic polypeptide, peptide YY, glucagon-like peptide-1 (GLP-1), oxyntomodulin and ghrelin. With the exception of ghrelin, these hormones act to increase satiety and decrease food intake. The mechanisms by which gut hormones modify feeding are the subject of ongoing investigation. Local effects such as the inhibition of gastric emptying might contribute to the decrease in energy intake. Activation of mechanoreceptors as a result of gastric distension may inhibit further food intake via neural reflex arcs. Circulating gut hormones have also been shown to act directly on neurons in hypothalamic and brainstem centres of appetite control. The median eminence and area postrema are characterized by a deficiency of the blood-brain barrier. Some investigators argue that this renders neighbouring structures, such as the arcuate nucleus of the hypothalamus and the nucleus of the tractus solitarius in the brainstem, susceptible to influence by circulating factors. Extensive reciprocal connections exist between these areas and the hypothalamic paraventricular nucleus and other energy-regulating centres of the central nervous system. In this way, hormonal signals from the gut may be translated into the subjective sensation of satiety. Moreover, the importance of the brain-gut axis in the control of food intake is reflected in the dual role exhibited by many gut peptides as both hormones and neurotransmitters. Peptides such as CCK and GLP-1 are expressed in neurons projecting both into and out of areas of the central nervous system critical to energy balance. The global increase in the incidence of obesity and the associated burden of morbidity has imparted greater urgency to understanding the processes of appetite control. Appetite regulation offers an integrated model of a brain-gut axis comprising both endocrine and neurological systems. As physiological mediators of satiety, gut hormones offer an attractive therapeutic target in the treatment of obesity.     

Revisiting the origin of the vertebrate Hox14 by including its relict sarcopterygian members

Bilaterian Hox genes play pivotal roles in the specification of positional identities along the anteroposterior axis. Particularly in vertebrates, their regulation is tightly coordinated by tandem arrays of genes [paralogy groups (PGs)] in four gene clusters (HoxA-D). Traditionally, the uninterrupted Hox cluster (Hox1-14) of the invertebrate chordate amphioxus was regarded as an archetype of the vertebrate Hox clusters. In contrast to Hox1-13 that are globally regulated by the "Hox code" and are often phylogenetically conserved, vertebrate Hox14 members were only recently revealed to be present in an African lungfish, a coelacanth, chondrichthyans and a lamprey, and decoupled from the Hox code. In this study we performed a PCR-based search of Hox14 members from diverse vertebrates, and identified one in the Australian lungfish, Neoceratodus forsteri. Based on a molecular phylogenetic analysis, this gene was designated NfHoxA14. Our real-time RT-PCR suggested its hindgut-associated expression, previously observed also in cloudy catshark HoxD14 and lamprey Hox14α. It is likely that this altered expression scheme was established before the Hox cluster quadruplication, probably at the base of extant vertebrates. To investigate the origin of vertebrate Hox14, by including this sarcopterygian Hox14 member, we performed focused phylogenetic analyses on its relationship with other vertebrate posterior Hox PGs (Hox9-13) as well as amphioxus posterior Hox genes. Our results confirmed the hypotheses previously proposed by other studies that vertebrate Hox14 does not have any amphioxus ortholog, and that none of 1-to-1 pairs of vertebrate and amphioxus posterior Hox genes, based on their relative location in the clusters, is orthologous.