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Scientific Reports Jan 2022Neuropeptides, as pervasive intercellular signaling molecules in the CNS, modulate a variety of behavioral systems in both protostomes and deuterostomes. Allatostatins...
Neuropeptides, as pervasive intercellular signaling molecules in the CNS, modulate a variety of behavioral systems in both protostomes and deuterostomes. Allatostatins are neuropeptides in arthropods that inhibit the biosynthesis of juvenile hormones. Based on amino acid sequences, they are divided into three different types in arthropods: allatostatin A, allatostatin B, allatostatin C. Allatostatin C (AstC) was first isolated from Manduca sexta, and it has an important conserved feature of a disulfide bridge formed by two cysteine residues. Moreover, AstC appears to be the ortholog of mammalian somatostatin, and it has functions in common with somatostatin, such as modulating feeding behaviors. The AstC signaling system has been widely studied in arthropods, but minimally studied in molluscs. In this study, we seek to identify the AstC signaling system in the marine mollusc Aplysia californica. We cloned the AstC precursor from the cDNA of Aplysia. We predicted a 15-amino acid peptide with a disulfide bridge, i.e., AstC, using NeuroPred. We then cloned two putative allatostatin C-like receptors and through NCBI Conserved Domain Search we found that they belonged to the G protein-coupled receptor (GPCR) family. In addition, using an inositol monophosphate 1 (IP1) accumulation assay, we showed that Aplysia AstC could activate one of the putative receptors, i.e., the AstC-R, at the lowest EC, and AstC without the disulfide bridge (AstC') activated AstC-R with the highest EC. Moreover, four molluscan AstCs with variations of sequences from Aplysia AstC but with the disulfide bridge activated AstC-R at intermediate EC. In summary, our successful identification of the Aplysia AstC precursor and its receptor (AstC-R) represents the first example in molluscs, and provides an important basis for further studies of the AstC signaling system in Aplysia and other molluscs.
Topics: Amino Acid Sequence; Animals; Aplysia; CHO Cells; Cricetulus; Evolution, Molecular; Neuropeptides; Phylogeny
PubMed: 35075137
DOI: 10.1038/s41598-022-05071-8 -
Scientific Reports May 2023Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein...
Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein coupled receptors (GPCRs). Among them, the elevenin signaling system is now believed to be present primarily in protostomes. Although elevenin was first identified from the L11 neuron of the abdominal ganglion in mollusc Aplysia californica, no receptors have been described in Aplysia, nor in any other molluscs. Here, using two elevenin receptors in annelid Platynereis dumerilii, we found three putative elevenin GPCRs in Aplysia. We cloned the three receptors and tentatively named them apElevR1, apElevR2, and apElevR3. Using an inositol monophosphate (IP1) accumulation assay, we demonstrated that Aplysia elevenin with the disulfide bond activated the three putative receptors with low EC50 values (ranging from 1.2 to 25 nM), supporting that they are true receptors for elevenin. In contrast, elevenin without the disulfide bond could not activate the receptors, indicating that the disulfide bond is required for receptor activity. Using alanine substitution of individual conserved residues other than the two cysteines, we showed that these residues appear to be critical to receptor activity, and the three different receptors had different sensitivities to the single residue substitution. Finally, we examined the roles of those residues outside the disulfide bond ring by removing these residues and found that they also appeared to be important to receptor activity. Thus, our study provides an important basis for further study of the functions of elevenin and its receptors in Aplysia and other molluscs.
Topics: Animals; Amino Acid Sequence; Aplysia; Neuropeptides; Receptors, G-Protein-Coupled; Disulfides
PubMed: 37169790
DOI: 10.1038/s41598-023-34596-9 -
Learning & Memory (Cold Spring Harbor,... 2023Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been...
Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been hampered by the complexity of the nervous system. The marine mollusk , with a simpler nervous system and many large, identified neurons, presents several advantages for addressing this question and has been used to examine the roles of tens of peptides in behavior. To screen for other peptides that might also play roles in behavior, we observed immunoreactivity in individual neurons in the central nervous system of adult with antisera raised against the peptide FMRFamide and two mammalian peptides that are also found in , cholecystokinin (CCK) and neuropeptide Y (NPY), as well as serotonin (5HT). In addition, we observed staining of individual neurons with antisera raised against mammalian somatostatin (SOM) and peptide histidine isoleucine (PHI). However, genomic analysis has shown that these two peptides are not expressed in the nervous system, and we have therefore labeled the unknown peptides stained by these two antibodies as X and X There was an area at the anterior end of the cerebral ganglion that had staining by antisera raised against many different transmitters, suggesting that this may be a modulatory region of the nervous system. There was also staining for X and, in some cases, FMRFamide in the bag cell cluster of the abdominal ganglion. In addition, these and other studies have revealed a fairly high degree of colocalization of different neuropeptides in individual neurons, suggesting that the peptides do not just act independently but can also interact in different combinations to produce complex functions. The simple nervous system of is advantageous for further testing these ideas.
Topics: Animals; Aplysia; FMRFamide; Central Nervous System; Neuropeptides; Ganglia; Mammals
PubMed: 37442624
DOI: 10.1101/lm.053758.123 -
Journal of Experimental Psychology.... Oct 2019Defensive responses to threatening events in the environment are displayed by a vast number of animals, both vertebrate and invertebrate. These defensive responses can...
Defensive responses to threatening events in the environment are displayed by a vast number of animals, both vertebrate and invertebrate. These defensive responses can be associated with salient neutral stimuli that are present along with the threatening stimulus. This is referred to as aversive conditioning. Animals with more simple nervous systems, such as Aplysia, C elegans, and Drosophila, have facilitated identification of some the physiological processes that support aversive conditioning. Perhaps even more basic information regarding the neurobiology of learning and memory may be gleaned from animals that have special characteristics not found in other species. Tardigrades, also known as "water bears," are microscopic eight-legged animals that live in various aquatic and terrestrial environments. They are known for their resilience to extreme conditions because of their ability to enter a cryptobiotic "tun" state during which they turn off their metabolism. Thus, tardigrades present an ideal model to study the metabolic requirements for memory storage. However, there is no prior research on tardigrade learning and memory. The purpose of this study was to demonstrate aversive conditioning in a tardigrade species, Dactylobiotus dispar. Associative learning was confirmed by numerous control conditions (unconditioned stimulus [US] only, conditional stimulus [CS] only, backward pairing, random pairing). Short-term memories were formed after a single pairing of the CS and US. This research introduces an important new animal model to the study of the neurobiology of aversive conditioning with important ramifications for understanding the metabolic influences on learning and memory. (PsycINFO Database Record (c) 2019 APA, all rights reserved).
Topics: Animals; Behavior, Animal; Conditioning, Classical; Memory, Short-Term; Models, Animal; Tardigrada
PubMed: 31368766
DOI: 10.1037/xan0000218 -
HardwareX Dec 2023The California sea hare () provides a powerful biomedical model system for studying aspects of neurological development and damage, behavior, aging, and hypoxia....
Untangling the Gordian Knot of sea hare egg masses: An integrated open-hardware system for standardized egg strand sizing and packaging for cryopreservation research and application.
The California sea hare () provides a powerful biomedical model system for studying aspects of neurological development and damage, behavior, aging, and hypoxia. encapsulate their zygotes within strands that result in tangled egg masses that greatly complicate culture and experimentation. The historical and current importance of for biomedical research and the mounting climate crisis necessitates protection of genetic resources. The goal of this work was to prototype open-hardware sizing, processing, and packaging devices for early life stages suitable for integration into a cryopreservation pathway. The Strand Centi-Sizer was a low-cost, fused filament fabrication 3-D printable device that increased experiment preparation efficiency and standardized the cutting of egg strands customizable to user needs. A downstream system of 3-D printed devices was also prototyped to address inefficiencies in handling of egg strand sections for processing and packaging into existing cryopreservation straw platforms. Time studies were conducted comparing manual methods (i.e., no specialized equipment) with open hardware to demonstrate utility of the devices and to encourage community members to design and prototype new devices to address recurrent and novel problems in other aquatic animals that produce egg strands. Improvements in design could further increase efficiency, standardization, and reproducibility, and extend the application of these devices to other research communities, such as shrimp or salamander spermatophores, sea anemone body part (e.g., pedal lacerate) cryopreservation, or study areas such as vitrification.
PubMed: 38020540
DOI: 10.1016/j.ohx.2023.e00476 -
Journal of Visualized Experiments : JoVE Apr 2022Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS)....
Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described-single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)-to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.
Topics: Animals; Aplysia; Chromatography, Liquid; Mass Spectrometry; Neurons; RNA; RNA Processing, Post-Transcriptional
PubMed: 35532275
DOI: 10.3791/63940 -
The Journal of Neuroscience : the... Nov 2023Many neurons exhibit regular firing that is limited to the duration and intensity of depolarizing stimuli. However, some neurons exhibit all-or-nothing plateau...
Many neurons exhibit regular firing that is limited to the duration and intensity of depolarizing stimuli. However, some neurons exhibit all-or-nothing plateau potentials that, once elicited, can lead to prolonged activity that is independent of stimulus intensity or duration. To better understand this diversity of information processing, we compared the voltage-gated and Ca-gated currents of three identified neurons from hermaphroditic Two of these neurons, B51 and B64, generated plateau potentials and a third neuron, B8, exhibited regular firing and was incapable of generating a plateau potential. With the exception of the Ca-gated potassium current ( ), all three neuron types expressed a similar array of outward and inward currents, but with distinct voltage-dependent properties for each neuron type. Inhibiting voltage-gated Ca channels with Ni prolonged the plateau potential, indicating is important for plateau potential termination. In contrast, inhibiting persistent Na ( ) blocked plateau potentials, empirically and in simulations. Surprisingly, the properties and level of expression of were similar in all three neurons, indicating that the presence of does not distinguish between regular-firing neurons and neurons capable of generating plateau potentials. Rather, the key distinguishing factor is the relationship between and outward currents such as the delayed outward current ( ), and We then demonstrated a technique for predicting complex physiological properties such as plateau duration, plateau amplitude, and action potential duration as a function of parameter values, by fitting a curve in parameter space and projecting the curve beyond the tested values. Plateau potentials are intrinsic properties of neurons that are important for information processing in a wide variety of nervous systems. We examined three identified neurons in with different propensities to generate a plateau potential. No single conductance was found to distinguish plateau generating neurons. Instead, plateau generation depended on the ratio between persistent Na current ( ), which favored plateaus, and outward currents such as , which facilitated plateau termination. Computational models revealed a relationship between the individual currents that predicted the features of simulated plateau potentials. These results provide a more solid understanding of the conductances that mediate plateau generation.
Topics: Calcium; Neurons; Action Potentials
PubMed: 37699717
DOI: 10.1523/JNEUROSCI.0789-23.2023 -
The Journal of Experimental Biology Aug 2019Grasping soft, irregular material is challenging both for animals and robots. The feeding systems of many animals have adapted to this challenge. In particular, the...
Grasping soft, irregular material is challenging both for animals and robots. The feeding systems of many animals have adapted to this challenge. In particular, the feeding system of the marine mollusk , a generalist herbivore, allows it to grasp and ingest seaweeds of varying shape, texture and toughness. On the surface of the grasper of is a structure known as the radula, a thin flexible cartilaginous sheet with fine teeth. Previous studies suggested that intrinsic muscles, I7, are responsible for opening the radula. Lesioning I7 does not prevent animals from grasping and ingesting food. New studies demonstrate that a set of fine muscle fibers on the ventral surface of the radula - the sub-radular fibers (SRFs) - mediate opening movements even if the I7 muscles are absent. Both and lesions demonstrate that removing the SRFs leads to profound deficits in radular opening, and significantly reduces feeding efficiency. A theoretical biomechanical analysis of the actions of the SRFs suggests that they induce the radular surface to open around a central crease in the radular surface and to arch the radular surface, allowing it to softly conform to irregular material. A three-dimensional model of the radular surface, based on observations and magnetic resonance imaging of intact animals, provides support for the biomechanical analysis. These results suggest how a soft grasper can work during feeding, and suggest novel designs for artificial soft graspers.
Topics: Animals; Aplysia; Biomechanical Phenomena; Feeding Behavior; Mouth
PubMed: 31350299
DOI: 10.1242/jeb.191254 -
International Journal of Molecular... May 2023Amyloids are fibrillar protein aggregates with a cross-β structure. More than two hundred different proteins with amyloid or amyloid-like properties are already known....
Amyloids are fibrillar protein aggregates with a cross-β structure. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Functional amyloids with conservative amyloidogenic regions were found in different organisms. Protein aggregation appears to be beneficial for the organism in these cases. Therefore, this property might be conservative for orthologous proteins. The amyloid aggregates of the CPEB protein were suggested to play an important role in the long-term memory formation in , , and . Moreover, the FXR1 protein demonstrates amyloid properties among the Vertebrates. A few nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human Nup153 and Nup58), are supposed or proved to form amyloid fibrils. In this study, we performed wide-scale bioinformatic analysis of nucleoporins with FG-repeats (phenylalanine-glycine repeats). We demonstrated that most of the barrier nucleoporins possess potential amyloidogenic properties. Furthermore, the aggregation-prone properties of several Nsp1 and Nup100 orthologs in bacteria and yeast cells were analyzed. Only two new nucleoporins, Nup98 and Nup98, aggregated in different experiments. At the same time, Nup58 only formed amyloids in bacterial cells. These results rather contradict the hypothesis about the functional aggregation of nucleoporins.
Topics: Mice; Animals; Humans; Nuclear Pore Complex Proteins; Saccharomyces cerevisiae; Amyloid; Drosophila melanogaster; Saccharomyces cerevisiae Proteins; Amyloidogenic Proteins; RNA-Binding Proteins; Nuclear Proteins
PubMed: 37239918
DOI: 10.3390/ijms24108571 -
Bioorganic & Medicinal Chemistry Dec 2022A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety...
A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N-2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N-2'-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N-2'-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release [T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686-26691]. 2'-O-propargyl-cADPR was recognized as an agonist which elicited Ca release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N-2'-O-propargyl cyclic ADP-ribose did not elicit Ca release at low concentration or impact cyclic ADP-ribose mediated Ca release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.
Topics: Humans; Cyclic ADP-Ribose; NAD; Adenosine
PubMed: 36446271
DOI: 10.1016/j.bmc.2022.117099