Focused ultrasound stimulation of an ex-vivo Aplysia abdominal ganglion preparation.

BACKGROUND: A growing body of research demonstrates that focused ultrasound stimulates activity in human and other mammalian nervous systems. However, there is no consensus on which sonication parameters are optimal. Furthermore, the mechanism of action behind ultrasound neurostimulation remains poorly understood. An invertebrate model greatly reduces biological complexity, permitting a systematic evaluation of sonication parameters suitable for ultrasound neurostimulation. NEW METHOD: Here, we describe the use of focused ultrasound stimulation with an ex-vivo abdominal ganglion preparation of the California sea hare, Aplysia californica, a long-standing model system in neurobiology. We developed a system for stimulating an isolated ganglion preparation while obtaining extracellular recordings from nerves. The focused ultrasound stimulation uses one of two single-element transducers, enabling stimulation at four distinct carrier frequencies (0.515 MHz, 1.l MHz, 1.61 MHz, 3.41 MHz). RESULTS: Using continuous wave ultrasound, we stimulated the ganglion at all four frequencies, and we present quantitative evaluation of elicited activation at four different sonication durations and three peak pressure levels, eliciting up to a 57-fold increase in spiking frequency. COMPARISON WITH ELECTRICAL STIMULATION: We demonstrated that ultrasound-induced activation is repeatable, and the response consistency is comparable to electrical stimulation. CONCLUSIONS: Due to the relative ease of long-term recordings for many hours, this ex-vivo ganglion preparation is suitable for investigating sonication parameters and the effects of focused ultrasound stimulation on neurons.

Sequences of Circadian Clock Proteins in the Nudibranch Molluscs Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea.

While much is known about the genes and proteins that make up the circadian clocks in vertebrates and several arthropod species, much less is known about the clock genes in many other invertebrates, including nudibranchs. The goal of this project was to identify the RNA and protein products of putative clock genes in the central nervous system of three nudibranchs, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea. Using previously published transcriptomes (Hermissenda and Tritonia) and a new transcriptome (Melibe), we identified nudibranch orthologs for the products of five canonical clock genes: brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1, circadian locomotor output cycles kaput, non-photoreceptive cryptochrome, period, and timeless. Additionally, orthologous sequences for the products of five related genes-aryl hydrocarbon receptor nuclear translocator like, photoreceptive cryptochrome, cryptochrome DASH, 6-4 photolyase, and timeout-were determined. Phylogenetic analyses confirmed that the nudibranch proteins were most closely related to known orthologs in related invertebrates, such as oysters and annelids. In general, the nudibranch clock proteins shared greater sequence similarity with Mus musculus orthologs than Drosophila melanogaster orthologs, which is consistent with the closer phylogenetic relationships recovered between lophotrochozoan and vertebrate orthologs. The suite of clock-related genes in nudibranchs includes both photoreceptive and non-photoreceptive cryptochromes, as well as timeout and possibly timeless. Therefore, the nudibranch clock may resemble the one exhibited in mammals, or possibly even in non-drosopholid insects and oysters. The latter would be evidence supporting this as the ancestral clock for bilaterians.

Localization and expression of putative circadian clock transcripts in the brain of the nudibranch Melibe leonina.

The nudibranch, Melibe leonina, expresses a circadian rhythm of locomotion, and we recently determined the sequences of multiple circadian clock transcripts that may play a role in controlling these daily patterns of behavior. In this study, we used these genomic data to help us: 1) identify putative clock neurons using fluorescent in situ hybridization (FISH); and 2) determine if there is a daily rhythm of expression of clock transcripts in the M. leonina brain, using quantitative PCR. FISH indicated the presence of the clock-related transcripts clock, period, and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry, respectively) in two bilateral neurons in each cerebropleural ganglion and a group of <10 neurons in the anterolateral region of each pedal ganglion. Double-label experiments confirmed colocalization of all four clock transcripts with each other. Quantitative PCR demonstrated that the genes clock, period, pcry and npcry exhibited significant differences in expression levels over 24 h. These data suggest that the putative circadian clock network in M. leonina consists of a small number of identifiable neurons that express circadian genes with a daily rhythm.

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome.

Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that less than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database--MirGeneDB ( http://mirgenedb.org )--to catalog this set of miRNAs, which complements the efforts of miRBase but differs from it by annotating the mature versus star products and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

The Mitochondrial Genomes of the Nudibranch Mollusks, Melibe leonina and Tritonia diomedea, and Their Impact on Gastropod Phylogeny.

The phylogenetic relationships among certain groups of gastropods have remained unresolved in recent studies, especially in the diverse subclass Opisthobranchia, where nudibranchs have been poorly represented. Here we present the complete mitochondrial genomes of Melibe leonina and Tritonia diomedea (more recently named T. tetraquetra), two nudibranchs from the unrepresented Cladobranchia group, and report on the resulting phylogenetic analyses. Both genomes coded for the typical thirteen protein-coding genes, twenty-two transfer RNAs, and two ribosomal RNAs seen in other species. The twelve-nucleotide deletion previously reported for the cytochrome oxidase 1 gene in several other Melibe species was further clarified as three separate deletion events. These deletions were not present in any opisthobranchs examined in our study, including the newly sequenced M. leonina or T. diomedea, suggesting that these previously reported deletions may represent more recently divergent taxa. Analysis of the secondary structures for all twenty-two tRNAs of both M. leonina and T. diomedea indicated truncated d arms for the two serine tRNAs, as seen in some other heterobranchs. In addition, the serine 1 tRNA in T. diomedea contained an anticodon not yet reported in any other gastropod. For phylogenetic analysis, we used the thirteen protein-coding genes from the mitochondrial genomes of M. leonina, T. diomedea, and seventy-one other gastropods. Phylogenetic analyses were performed for both the class Gastropoda and the subclass Opisthobranchia. Both Bayesian and maximum likelihood analyses resulted in similar tree topologies. In the Opisthobranchia, the five orders represented in our study were monophyletic (Anaspidea, Cephalaspidea, Notaspidea, Nudibranchia, Sacoglossa). In Gastropoda, two of the three traditional subclasses, Opisthobranchia and Pulmonata, were not monophyletic. In contrast, four of the more recently named gastropod clades (Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia) were all monophyletic, and thus appear to be better classifications for this diverse group.

Circadian rhythms of crawling and swimming in the nudibranch mollusc Melibe leonina.

Daily rhythms of activity driven by circadian clocks are expressed by many organisms, including molluscs. We initiated this study, with the nudibranch Melibe leonina, with four goals in mind: (1) determine which behaviors are expressed with a daily rhythm; (2) investigate which of these rhythmic behaviors are controlled by a circadian clock; (3) determine if a circadian clock is associated with the eyes or optic ganglia of Melibe, as it is in several other gastropods; and (4) test the hypothesis that Melibe can use extraocular photoreceptors to synchronize its daily rhythms to natural light-dark cycles. To address these goals, we analyzed the behavior of 55 animals exposed to either artificial or natural light-dark cycles, followed by constant darkness. We also repeated this experiment using 10 animals that had their eyes removed. Individuals did not express daily rhythms of feeding, but they swam and crawled more at night. This pattern of locomotion persisted in constant darkness, indicating the presence of a circadian clock. Eyeless animals also expressed a daily rhythm of locomotion, with more locomotion at night. The fact that eyeless animals synchronized their locomotion to the light-dark cycle suggests that they can detect light using extraocular photoreceptors. However, in constant darkness, these rhythms deteriorated, suggesting that the clock neurons that influence locomotion may be located in, or near, the eyes. Thus, locomotion in Melibe appears to be influenced by both ocular and extraocular photoreceptors, although the former appear to have a greater influence on the expression of circadian rhythms.

Homology and homoplasy of swimming behaviors and neural circuits in the Nudipleura (Mollusca, Gastropoda, Opisthobranchia).

How neural circuit evolution relates to behavioral evolution is not well understood. Here the relationship between neural circuits and behavior is explored with respect to the swimming behaviors of the Nudipleura (Mollusca, Gastropoda, Opithobranchia). Nudipleura is a diverse monophyletic clade of sea slugs among which only a small percentage of species can swim. Swimming falls into a limited number of categories, the most prevalent of which are rhythmic left-right body flexions (LR) and rhythmic dorsal-ventral body flexions (DV). The phylogenetic distribution of these behaviors suggests a high degree of homoplasy. The central pattern generator (CPG) underlying DV swimming has been well characterized in Tritonia diomedea and in Pleurobranchaea californica. The CPG for LR swimming has been elucidated in Melibe leonina and Dendronotus iris, which are more closely related. The CPGs for the categorically distinct DV and LR swimming behaviors consist of nonoverlapping sets of homologous identified neurons, whereas the categorically similar behaviors share some homologous identified neurons, although the exact composition of neurons and synapses in the neural circuits differ. The roles played by homologous identified neurons in categorically distinct behaviors differ. However, homologous identified neurons also play different roles even in the swim CPGs of the two LR swimming species. Individual neurons can be multifunctional within a species. Some of those functions are shared across species, whereas others are not. The pattern of use and reuse of homologous neurons in various forms of swimming and other behaviors further demonstrates that the composition of neural circuits influences the evolution of behaviors.

Serotonin influences locomotion in the nudibranch mollusc Melibe leonina.

Serotonin (5-HT) influences locomotion in many animals, from flatworms to mammals. This study examined the effects of 5-HT on locomotion in the nudibranch mollusc Melibe leonina (Gould, 1852). M. leonina exhibits two modes of locomotion, crawling and swimming. Animals were bath-immersed in a range of concentrations of 5-HT or injected with various 5-HT solutions into the hemolymph and then monitored for locomotor activity. In contrast to other gastropods studied, M. leonina showed no significant effect of 5-HT on the distance crawled or the speed of crawling. However, the highest concentration (10(-3) mol l(-1) for bath immersion and 10(-5) mol l(-1) for injection) significantly increased the time spent swimming and the swimming speed. The 5-HT receptor antagonist methysergide inhibited the influence of 5-HT on the overall amount of swimming but not on swimming speed. These results suggest that 5-HT influences locomotion at the behavioral level in M. leonina. In conjunction with previous studies on the neural basis of locomotion in M. leonina, these results also suggest that this species is an excellent model system for investigating the 5-HT modulation of locomotion.

Different roles for homologous interneurons in species exhibiting similar rhythmic behaviors.

It is often assumed that similar behaviors in related species are produced by similar neural mechanisms. To test this, we examined the neuronal basis of a simple swimming behavior in two nudibranchs (Mollusca, Opisthobranchia), Melibe leonina and Dendronotus iris. The side-to-side swimming movements of Dendronotus [1] strongly resemble those of Melibe [2, 3]. In Melibe, it was previously shown that the central pattern generator (CPG) for swimming is composed of two bilaterally symmetric pairs of identified interneurons, swim interneuron 1 (Si1) and swim interneuron 2 (Si2), which are electrically coupled ipsilaterally and mutually inhibit both contralateral counterparts [2, 4]. We identified homologs of Si1 and Si2 in Dendronotus. (Henceforth, homologous neurons in each species will be distinguished by the subscripts (Den) and (Mel).) We found that Si2(Den) and Si2(Mel) play similar roles in generating the swim motor pattern. However, unlike Si1(Mel), Si1(Den) was not part of the swim CPG, was not strongly coupled to the ipsilateral Si2(Den), and did not inhibit the contralateral neurons. Thus, species differences exist in the neuronal organization of the swim CPGs despite the similarity of the behaviors. Therefore, similarity in species-typical behavior is not necessarily predictive of common neural mechanisms, even for homologous neurons in closely related species.

Different functions for homologous serotonergic interneurons and serotonin in species-specific rhythmic behaviours.

Closely related species can exhibit different behaviours despite homologous neural substrates. The nudibranch molluscs Tritonia diomedea and Melibe leonina swim differently, yet their nervous systems contain homologous serotonergic neurons. In Tritonia, the dorsal swim interneurons (DSIs) are members of the swim central pattern generator (CPG) and their neurotransmitter serotonin is both necessary and sufficient to elicit a swim motor pattern. Here it is shown that the DSI homologues in Melibe, the cerebral serotonergic posterior-A neurons (CeSP-As), are extrinsic to the swim CPG, and that neither the CeSP-As nor their neurotransmitter serotonin is necessary for swim motor pattern initiation, which occurred when the CeSP-As were inactive. Furthermore, the serotonin antagonist methysergide blocked the effects of both the serotonin and CeSP-As but did not prevent the production of a swim motor pattern. However, the CeSP-As and serotonin could influence the Melibe swim circuit; depolarization of a cerebral serotonergic posterior-A was sufficient to initiate a swim motor pattern and hyperpolarization of a CeSP-A temporarily halted an ongoing swim motor pattern. Serotonin itself was sufficient to initiate a swim motor pattern or make an ongoing swim motor pattern more regular. Thus, evolution of species-specific behaviour involved alterations in the functions of identified homologous neurons and their neurotransmitter.

Homologues of serotonergic central pattern generator neurons in related nudibranch molluscs with divergent behaviors.

Homologues of a neuron that contributes to a species-specific behavior were identified and characterized in species lacking that behavior. The nudibranch Tritonia diomedea swims by flexing its body dorsally and ventrally. The dorsal swim interneurons (DSIs) are components of the central pattern generator (CPG) underlying this rhythmic motor pattern and also activate crawling. Homologues of the DSIs were identified in six nudibranchs that do not exhibit dorsal-ventral swimming: Tochuina tetraquetra, Melibe leonina, Dendronotus iris, D. frondosus, Armina californica, and Triopha catalinae. Homology was based upon shared features that distinguish the DSIs from all other neurons: (1) serotonin immunoreactivity, (2) location in the Cerebral serotonergic posterior (CeSP) cluster, and (3) axon projection to the contralateral pedal ganglion. The DSI homologues, named CeSP-A neurons, share additional features with the DSIs: irregular basal firing, synchronous inputs, electrical coupling, and reciprocal inhibition. Unlike the DSIs, the CeSP-A neurons were not rhythmically active in response to nerve stimulation. The CeSP-A neurons in Tochuina and Triopha also excited homologues of the Tritonia Pd5 neuron, a crawling efferent. Thus, the CeSP-A neurons and the DSIs may be part of a conserved network related to crawling that may have been co-opted into a rhythmic swim CPG in Tritonia.

Comparative mapping of serotonin-immunoreactive neurons in the central nervous systems of nudibranch molluscs.

The serotonergic systems in nudibranch molluscs were compared by mapping the locations of serotonin-immunoreactive (5-HT-ir) neurons in 11 species representing all four suborders of the nudibranch clade: Dendronotoidea (Tritonia diomedea, Tochuina tetraquetra, Dendronotus iris, Dendronotus frondosus, and Melibe leonina), Aeolidoidea (Hermissenda crassicornis and Flabellina trophina), Arminoidea (Dirona albolineata, Janolus fuscus, and Armina californica), and Doridoidea (Triopha catalinae). A nomenclature is proposed to standardize reports of cell location in species with differing brain morphologies. Certain patterns of 5-HT immunoreactivity were found to be consistent for all species, such as the presence of 5-HT-ir neurons in the pedal and cerebral ganglia. Also, particular clusters of 5-HT-ir neurons in the anterior and posterior regions of the dorsal surface of the cerebral ganglion were always present. However, there were interspecies differences in the number of 5-HT-ir neurons in each cluster, and some clusters even exhibited strong intraspecies variability that was only weakly correlated with brain size. Phylogenetic analysis suggests that the presence of particular classes of 5-HT-ir neurons exhibits a great deal of homoplasy. The conserved features of the nudibranch serotonergic system presumably represent the shared ancestral structure, whereas the derived characters suggest substantial independent evolutionary changes in the number and presence of serotonergic neurons. Although a number of studies have demonstrated phylogenetic variability of peptidergic systems, this study suggests that serotonergic systems may also exhibit a high degree of homoplasy in some groups of organisms.

Neural correlates of swimming behavior in Melibe leonina.

The nudibranch Melibe leonina swims by rhythmically bending from side to side at a frequency of 1 cycle every 2-4 s. The objective of this study was to locate putative swim motoneurons (pSMNs) that drive these lateral flexions and determine if swimming in this species is produced by a swim central pattern generator (sCPG). In the first set of experiments, intracellular recordings were obtained from pSMNs in semi-intact, swimming animals. About 10-14 pSMNs were identified on the dorsal surface of each pedal ganglion and 4-7 on the ventral side. In general, the pSMNs in a given pedal ganglion fired synchronously and caused the animal to flex in that direction, whereas the pSMNs in the opposite pedal ganglion fired in anti-phase. When swimming stopped, so did rhythmic pSMN bursting; when swimming commenced, pSMNs resumed bursting. In the second series of experiments, intracellular recordings were obtained from pSMNs in isolated brains that spontaneously expressed the swim motor program. The pattern of activity recorded from pSMNs in isolated brains was very similar to the bursting pattern obtained from the same pSMNs in semi-intact animals, indicating that the sCPG can produce the swim rhythm in the absence of sensory feedback. Exposing the brain to light or cutting the pedal-pedal connectives inhibited fictive swimming in the isolated brain. The pSMNs do not appear to participate in the sCPG. Rather, they received rhythmic excitatory and inhibitory synaptic input from interneurons that probably comprise the sCPG circuit.

Modulation of swimming in the gastropod Melibe leonina by nitric oxide.

Nitric oxide (NO) is a gaseous intercellular messenger produced by the enzyme nitric oxide synthase. It has been implicated as a neuromodulator in several groups of animals, including gastropods, crustaceans and mammals. In this study, we investigated the effects of NO on the swim motor program produced by isolated brains and by semi-intact preparations of the nudibranch Melibe leonina. The NO donors sodium nitroprusside (SNP, 1 mmol x l(-1)) and S-nitroso-N-acetylpenicillamine (SNAP, 1 mmol x l(-1)) both had a marked effect on the swim motor program expressed in isolated brains, causing an increase in the period of the swim cycle and a more erratic swim rhythm. In semi-intact preparations, the effect of NO donors was manifested as a significant decrease in the rate of actual swimming. An NO scavenger, reduced oxyhemoglobin, eliminated the effects of NO donors on isolated brains, supporting the assumption that the changes in swimming induced by donors were actually due to NO. The cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (1 mmol x l(-1)) produced effects that mimicked those of NO donors, suggesting that NO is working via a cGMP-dependent mechanism. These results, in combination with previous histological studies indicating the endogenous presence of nitric oxide synthase, suggest that NO is used in the central nervous system of Melibe leonina to modulate swimming.