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Nature Chemical Biology Oct 2023Phe-Met-Arg-Phe-amide (FMRFamide)-activated sodium channels (FaNaCs) are a family of channels activated by the neuropeptide FMRFamide, and, to date, the underlying...
Phe-Met-Arg-Phe-amide (FMRFamide)-activated sodium channels (FaNaCs) are a family of channels activated by the neuropeptide FMRFamide, and, to date, the underlying ligand gating mechanism remains unknown. Here we present the high-resolution cryo-electron microscopy structures of Aplysia californica FaNaC in both apo and FMRFamide-bound states. AcFaNaC forms a chalice-shaped trimer and possesses several notable features, including two FaNaC-specific insertion regions, a distinct finger domain and non-domain-swapped transmembrane helix 2 in the transmembrane domain (TMD). One FMRFamide binds to each subunit in a cleft located in the top-most region of the extracellular domain, with participation of residues from the neighboring subunit. Bound FMRFamide adopts an extended conformation. FMRFamide binds tightly to A. californica FaNaC in an N terminus-in manner, which causes collapse of the binding cleft and induces large local conformational rearrangements. Such conformational changes are propagated downward toward the TMD via the palm domain, possibly resulting in outward movement of the TMD and dilation of the ion conduction pore.
Topics: FMRFamide; Cryoelectron Microscopy; Ion Channel Gating; Neuropeptides; Sodium Channels
PubMed: 37550431
DOI: 10.1038/s41589-023-01401-7 -
Journal of Neuroscience Methods Aug 2023The analyses of neuronal circuits require high-throughput technologies for stimulating and recording many neurons simultaneously with single-neuron precision....
BACKGROUND
The analyses of neuronal circuits require high-throughput technologies for stimulating and recording many neurons simultaneously with single-neuron precision. Voltage-sensitive dyes (VSDs) have enabled the monitoring of membrane potentials of many (10-100 s) neurons simultaneously. Carbon fiber electrode (CFE) arrays allow for stimulation and recording of many neurons simultaneously, including intracellularly.
NEW METHOD
Combining CFE with VSD leverages the advantages of both technologies, allowing for stimulation of single neurons while recording the activity of the entire network. 3-D printing technology was used to develop a chamber to simultaneously perform VSD imaging, CFE array recording, and extracellular recording from individual glass electrodes.
RESULTS
Aplysia buccal ganglia were stained with VSD and imaged while also recording using a CFE array and extracellular nerve electrodes. Coincident spiking activity was recorded by VSD, CFE, and extracellular nerve electrodes. Current injection with CFE electrodes could activate and inhibit individual neurons as detected by VSD and nerve recordings.
COMPARISON TO EXISTING METHODS
The large size of traditional manipulators limits the number of electrodes used and the number of neurons recorded during an experiment. Here we present a method to build a 3-D printed recording chamber that includes a 3-axis micromanipulator to position a CFE array and eight 2-axis manipulators to position eight extracellular electrodes.
CONCLUSIONS
3-D printing technology can be used to build a custom recording chamber and micromanipulators. Combining these technologies allows for the direct modulation of the activity of neurons while recording the activity of 100 s of neurons simultaneously.
Topics: Carbon Fiber; Action Potentials; Neurons; Electrodes; Fluorescent Dyes
PubMed: 37524249
DOI: 10.1016/j.jneumeth.2023.109935 -
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 -
The Science of the Total Environment Oct 2023The presence of plastic debris in the marine environment has reached massive levels in the past decades. In marine environments, microplastics can exist for hundreds of...
The presence of plastic debris in the marine environment has reached massive levels in the past decades. In marine environments, microplastics can exist for hundreds of years and the presence of microplastics in this environment has been reported since 1970 and since then has been considered ubiquitous. Mollusks are being used as microplastic pollution indicators, especially in coastal areas and bivalves are more often used in microplastic-monitoring studies. On the other hand, gastropods are poorly used as indicators for microplastic pollution, even though they are the most diverse group of mollusks. The sea hares of the genus Aplysia are herbivorous gastropods, important model organisms commonly used in neuroscience studies, isolating the compounds in their defensive ink. Until today, there is no previous record of the presence of MPs in Aplysia gastropods. Therefore, this study aims to investigate the presence of microplastics in tissues of A. brasiliana found in southeastern Brazil. We collected seven individuals of A. brasiliana from a beach in southeastern Brazil, dissected them to isolate the digestive tract and the gills, and digested the tissues with a solution of 10 % NaOH. In the end, 1021 microplastic particles were found, 940 in the digestive tissue, and 81 in the gills. These results represent the first record of the presence of microplastics in the Brazilian sea hare A. brasiliana.
Topics: Humans; Animals; Aplysia; Microplastics; Plastics; Brazil; Hares; Water Pollutants, Chemical; Environmental Monitoring; Bivalvia
PubMed: 37385493
DOI: 10.1016/j.scitotenv.2023.165156 -
Chemistry & Biodiversity Aug 2023A new irieane-type diterpene, 12-hydroxypinnaterpene C (1), and 21 known compounds, angasiol acetate (2), angasiol (3), 11-deacetylpinnaterpene C (4), palisadin A (5),...
A new irieane-type diterpene, 12-hydroxypinnaterpene C (1), and 21 known compounds, angasiol acetate (2), angasiol (3), 11-deacetylpinnaterpene C (4), palisadin A (5), 12-acetoxypalisadin B (6), 12-hydroxypalisadin B (7), aplysistatin (8), luzodiol (9), 5-acetoxy-2-bromo-3-chloro-chamigra-7(14),9-dien-8-one (10), neoirietriol (11), neoirietetraol (12), (3Z)-laurenyne (13), cupalaurenol (14), cupalaurenol acetate (15), (3Z)-venustinene (16), 10-hydroxykahukuene B (17), aplysiol B (18), (3Z)-13-epipinnatifidenyne (19), 3Z,6R,7R,12S,13S-obtusenyne (20), (3Z,9Z)-7-chloro-6-hydroxy-12-oxo-pentadeca-3,9-dien-1-yne (21), and cholest-7-en-3,5,7-triol (22) were isolated from the digestive diverticula of Aplysia argus from the Ikei Island in Okinawa, Japan. The structures of these compounds were determined using spectroscopic methods such as NMR and HR-ESI-MS. These compounds were tested for their antibacterial activity against the phytopathogen Ralstonia solanacearum. Compounds 11 and 21 exhibited antibacterial activity at 30 μg/disc. In this study, we also discuss the types of red algae that A. argus feeds on in the shallow waters of Okinawa Prefecture.
Topics: Animals; Anti-Bacterial Agents; Aplysia; Magnetic Resonance Spectroscopy; Molecular Structure; Rhodophyta; Diterpenes
PubMed: 37350187
DOI: 10.1002/cbdv.202300791 -
ACS Chemical Neuroscience Jul 2023Neuropeptides with the C-terminal Wamide (Trp-NH) are one of the last common ancestors of peptide families of eumetazoans and play various physiological roles. In this...
Neuropeptides with the C-terminal Wamide (Trp-NH) are one of the last common ancestors of peptide families of eumetazoans and play various physiological roles. In this study, we sought to characterize the ancient Wamide peptides signaling systems in the marine mollusk , i.e., APGWamide (APGWa) and myoinhibitory peptide (MIP)/Allatostatin B (AST-B) signaling systems. A common feature of protostome APGWa and MIP/AST-B peptides is the presence of a conserved Wamide motif in the C-terminus. Although orthologs of the APGWa and MIP signaling systems have been studied to various extents in annelids or other protostomes, no complete signaling systems have yet been characterized in mollusks. Here, through bioinformatics, molecular and cellular biology, we identified three receptors for APGWa, namely, APGWa-R1, APGWa-R2, and APGWa-R3. The EC values for APGWa-R1, APGWa-R2, and APGWa-R3 are 45, 2100, and 2600 nM, respectively. For the MIP signaling system, we predicted 13 forms of peptides, i.e., MIP1-13 that could be generated from the precursor identified in our study, with MIP5 (WKQMAVWa) having the largest number of copies (4 copies). Then, a complete MIP receptor (MIPR) was identified and the MIP1-13 peptides activated the MIPR in a dose-dependent manner, with EC values ranging from 40 to 3000 nM. Peptide analogs with alanine substitution experiments demonstrated that the Wamide motif at the C-terminus is necessary for receptor activity in both the APGWa and MIP systems. Moreover, cross-activity between the two signaling systems showed that MIP1, 4, 7, and 8 ligands could activate APGWa-R1 with a low potency (EC values: 2800-22,000 nM), which further supported that the APGWa and MIP signaling systems are somewhat related. In summary, our successful characterization of APGWa and MIP signaling systems represents the first example in mollusks and provides an important basis for further functional studies in this and other protostome species. Moreover, this study may be useful for elucidating and clarifying the evolutionary relationship between the two Wamide signaling systems (i.e., APGWa and MIP systems) and their other extended neuropeptide signaling systems.
Topics: Animals; Aplysia; Amino Acid Sequence; Neuropeptides; Mollusca; Peptides
PubMed: 37339428
DOI: 10.1021/acschemneuro.3c00158 -
Neuroscience Research Nov 2023Anorexia is a behavioral change caused by functional brain disorders in patients with Alzheimer's disease (AD). Amyloid-β (1-42) oligomers (o-Aβ) are possible...
Administration of amyloid-β oligomer to the buccal ganglia may reduce food intake and cholinergic synaptic responses within the feeding neural circuit in Aplysia kurodai.
Anorexia is a behavioral change caused by functional brain disorders in patients with Alzheimer's disease (AD). Amyloid-β (1-42) oligomers (o-Aβ) are possible causative agents of AD that impair signaling via synaptic dysfunction. In this study, we used Aplysia kurodai to study functional disorders of the brain through o-Aβ. Administration of o-Aβ to the buccal ganglia (feeding brain for oral movements) by surgical treatment significantly reduced food intake for at least five days. Furthermore, we explored the effects of o-Aβ on the synaptic function in the feeding neural circuit, focusing on a specific inhibitory synaptic response in jaw-closing motor neurons produced by cholinergic buccal multi-action neurons because we recently found that this cholinergic response decreases with aging, which is consistent with the cholinergic hypothesis for aging. Administration of o-Aβ to the buccal ganglia significantly reduced the synaptic response within minutes, whereas administration of amyloid-β (1-42) monomers did not. These results suggest that o-Aβ may impair the cholinergic synapses, even in Aplysia, which is consistent with the cholinergic hypothesis for AD.
Topics: Animals; Humans; Amyloid beta-Peptides; Aplysia; Alzheimer Disease; Ganglia; Synapses; Cholinergic Agents; Eating
PubMed: 37328111
DOI: 10.1016/j.neures.2023.06.004 -
Journal of the American Society For... Sep 2023Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility...
Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and / proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from . We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited / proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and / proline isomerization.
Topics: Amino Acid Sequence; Aspartic Acid; Isoaspartic Acid; Tandem Mass Spectrometry; Peptides; Proline; Isomerism
PubMed: 37102735
DOI: 10.1021/jasms.3c00092 -
Journal of Proteome Research Oct 2023Protein database search engines are an integral component of mass spectrometry-based peptidomic analyses. Given the unique computational challenges of peptidomics, many...
Protein database search engines are an integral component of mass spectrometry-based peptidomic analyses. Given the unique computational challenges of peptidomics, many factors must be taken into consideration when optimizing search engine selection, as each platform has different algorithms by which tandem mass spectra are scored for subsequent peptide identifications. In this study, four different database search engines, PEAKS, MS-GF+, OMSSA, and X! Tandem, were compared with and peptidomics data sets, and various metrics were assessed such as the number of unique peptide and neuropeptide identifications, and peptide length distributions. Given the tested conditions, PEAKS was found to have the highest number of peptide and neuropeptide identifications out of the four search engines in both data sets. Furthermore, principal component analysis and multivariate logistic regression were employed to determine whether specific spectral features contribute to false C-terminal amidation assignments by each search engine. From this analysis, it was found that the primary features influencing incorrect peptide assignments were the precursor and fragment ion / errors. Finally, an assessment employing a mixed species protein database was performed to evaluate search engine precision and sensitivity when searched against an enlarged search space containing human proteins.
Topics: Humans; Animals; Rats; Search Engine; Peptides; Algorithms; Tandem Mass Spectrometry; Neuropeptides; Databases, Protein; Software
PubMed: 36809008
DOI: 10.1021/acs.jproteome.2c00307 -
Neuromodulation : Journal of the... Dec 2023Small-diameter afferent axons carry various sensory signals that are critical for vital physiological conditions but sometimes contribute to pathologies. Infrared (IR)...
OBJECTIVES
Small-diameter afferent axons carry various sensory signals that are critical for vital physiological conditions but sometimes contribute to pathologies. Infrared (IR) neural inhibition (INI) can induce selective heat block of small-diameter axons, which holds potential for translational applications such as pain management. Previous research suggested that IR-heating-induced acceleration of voltage-gated potassium channel kinetics is the mechanism for INI. Therefore, we hypothesized that other heating methods, such as resistive heating (RH) in a cuff, could reproduce the selective inhibition observed in INI.
MATERIALS AND METHODS
We conducted ex vivo nerve-heating experiments on pleural-abdominal connective nerves of Aplysia californica using both IR and RH. We fabricated a transparent silicone nerve cuff for simultaneous IR heating, RH, and temperature measurements. Temperature elevations (ΔT) on the nerve surface were recorded for both heating modalities, which were tested over a range of power levels that cover a similar ΔT range. We recorded electrically evoked compound action potentials (CAPs) and segmented them into fast and slow subcomponents on the basis of conduction velocity differences between the large and small-diameter axonal subpopulations. We calculated the normalized inhibition strength and inhibition selectivity index on the basis of the rectified area under the curve of each subpopulation.
RESULTS
INI and RH showed a similar selective inhibition effect on CAP subcomponents for slow-conducting axons, confirmed by the inhibition probability vs ΔT dose-response curve based on approximately 2000 CAP measurements. The inhibition selectivity indexes of the two heating modalities were similar across six nerves. RH only required half the total electrical power required by INI to achieve a similar ΔT.
SIGNIFICANCE
We show that selective INI can be reproduced by other heating modalities such as RH. RH, because of its high energy efficiency and simple design, can be a good candidate for future implantable neural interface designs.
Topics: Humans; Neural Conduction; Heating; Neural Inhibition; Action Potentials; Axons
PubMed: 36707292
DOI: 10.1016/j.neurom.2022.12.004