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The Journal of Biological Chemistry Oct 2022The protostome leucokinin (LK) signaling system, including LK peptides and their G protein-coupled receptors, has been characterized in several species. Despite the...
The protostome leucokinin (LK) signaling system, including LK peptides and their G protein-coupled receptors, has been characterized in several species. Despite the progress, molecular mechanisms governing LK peptide-receptor interactions remain to be elucidated. Previously, we identified a precursor protein for Aplysia leucokinin-like peptides (ALKs) that contains the greatest number of amidated peptides among LK precursors in all species identified so far. Here, we identified the first ALK receptor from Aplysia, ALKR. We used cell-based IP1 activation assays to demonstrate that two ALK peptides with the most copies, ALK1 and ALK2, activated ALKR with high potencies. Other endogenous ALK-derived peptides bearing the FXXWX-amide motif also activated ALKR to various degrees. Our examination of cross-species activity of ALKs with the Anopheles LK receptor was consistent with a critical role for the FXXWX-amide motif in receptor activity. Furthermore, we showed, through alanine substitution of ALK1, the highly conserved phenylalanine (F), tryptophan (W), and C-terminal amidation were each essential for receptor activation. Finally, we used an artificial intelligence-based protein structure prediction server (Robetta) and Autodock Vina to predict the ligand-bound conformation of ALKR. Our model predicted several interactions (i.e., hydrophobic interactions, hydrogen bonds, and amide-pi stacking) between ALK peptides and ALKR, and several of our substitution and mutagenesis experiments were consistent with the predicted model. In conclusion, our results provide important information defining possible interactions between ALK peptides and their receptors. The workflow utilized here may be useful for studying other ligand-receptor interactions for a neuropeptide signaling system, particularly in protostomes.
Topics: Animals; Amides; Aplysia; Artificial Intelligence; Ligands; Mutagenesis; Neuropeptides; Protein Conformation; Receptors, Neuropeptide
PubMed: 36049520
DOI: 10.1016/j.jbc.2022.102440 -
The Journal of Neuroscience : the... Jan 1988Until recently, dishabituation and sensitization have commonly been considered to reflect a unitary process: Sensitization refers to a general facilitation produced by...
Until recently, dishabituation and sensitization have commonly been considered to reflect a unitary process: Sensitization refers to a general facilitation produced by strong or noxious stimuli that enhances subsequent responding; dishabituation has been thought to represent a special instance of sensitization in which the facilitation is simply superimposed on a habituated response level. The unitary process hypothesis was based on the observation that both decremented and nondecremented responses are facilitated by a common noxious or strong stimulus. However, this observation does not rule out the possibility that dishabituation and sensitization could reflect separate processes that are activated in parallel by a strong stimulus. Recent cellular experiments by Hochner et al. (1986) suggest that this, in fact, occurs in the sensory neurons of the gill withdrawal reflex in Aplysia. A developmental analysis of learning in the marine mollusc Aplysia permits a direct behavioral test of this hypothesis. If dishabituation and sensitization reflect a unitary process then they should emerge at the same time ontogenetically. On the other hand, if they reflect different processes, then they might emerge according to different ontogenetic timetables. In the present study we examined the temporal emergence of dishabituation and sensitization in the defensive siphon withdrawal reflex in 3 stages of juvenile Aplysia: stage 11, early stage 12, and late stage 12. Animals received one of 2 kinds of training: Dishabituation training, in which the effect of strong tail shock on habituated responses were observed, and Sensitization training, in which the effect of strong tail shock on nondecremented responses was observed. We found that, while dishabituation was present in all stages examined, sensitization did not emerge until several weeks later, in late stage 12. These results were confirmed and extended in a group of animals that were tested twice: first in stage 11, when they showed no sensitization, and again 13 weeks later, in late stage 12, when they then showed significant sensitization. Our analysis of nondecremented responses prior to the emergence of sensitization also revealed an unexpected inhibitory component of tail shock that produces reflex depression. Moreover, there was a clear progression in the net effects of tail shock during development: reflex depression was produced in stages 11 and early stage 12, followed by a transition to reflex facilitation (sensitization) in late stage 12. Finally, when sensitization emerged in late stage 12, the process of dishabituation showed a significant increase compared with previous developmental stages.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Animals; Aplysia; Electric Stimulation; Habituation, Psychophysiologic; Learning; Physical Stimulation; Reflex; Tail; Touch
PubMed: 3339408
DOI: 10.1523/JNEUROSCI.08-01-00197.1988 -
PloS One 2012To what extent are motor networks underlying rhythmic behaviors rigidly hard-wired versus fluid and dynamic entities? Do the members of motor networks change from...
To what extent are motor networks underlying rhythmic behaviors rigidly hard-wired versus fluid and dynamic entities? Do the members of motor networks change from moment-to-moment or from motor program episode-to-episode? These are questions that can only be addressed in systems where it is possible to monitor the spiking activity of networks of neurons during the production of motor programs. We used large-scale voltage-sensitive dye (VSD) imaging followed by Independent Component Analysis spike-sorting to examine the extent to which the neuronal network underlying the escape swim behavior of Tritonia diomedea is hard-wired versus fluid from a moment-to-moment perspective. We found that while most neurons were dedicated to the swim network, a small but significant proportion of neurons participated in a surprisingly variable manner. These neurons joined the swim motor program late, left early, burst only on some cycles or skipped cycles of the motor program. We confirmed that this variable neuronal participation was not due to effects of the VSD by finding such neurons with intracellular recording in dye-free saline. Further, these neurons markedly varied their level of participation in the network from swim episode-to-episode. The generality of such unreliably bursting neurons was confirmed by their presence in the rhythmic escape networks of two other molluscan species, Tritonia festiva and Aplysia californica. Our observations support a view that neuronal networks, even those underlying rhythmic and stereotyped motor programs, may be more variable in structure than widely appreciated.
Topics: Animals; Aplysia; Behavior, Animal; Motor Activity; Nerve Net; Neurons; Optical Imaging; Swimming
PubMed: 22815768
DOI: 10.1371/journal.pone.0040579 -
PloS One 2019The simplified nervous system of Aplysia californica (Aplysia) allows for detailed studies of physiological and molecular changes in small sets of neurons. Sensory...
The simplified nervous system of Aplysia californica (Aplysia) allows for detailed studies of physiological and molecular changes in small sets of neurons. Sensory neurons of the biting and tail withdrawal reflexes are glutamatergic and show reduced L-Glutamate current density in aged animals, making them a good candidate to study age-related changes in glutamatergic responses. To examine if changes in ionotropic L-Glu receptor (iGluR) transcription underlie reduced physiology, mRNA expression of iGluR was quantified in two sensory neuron clusters of two cohorts of Aplysia at both sexual maturity (~8 months) and advanced age (~12 months). Sensory neuron aging resulted in a significant overall decrease in expression of iGluR subunits in both sensory neuron clusters and cohorts. Although the individual subunits differentially expressed varied between sensory neuron clusters and different cohorts of animals, all differentially expressed subunits were downregulated, with no subunits showing significantly increased expression with age. Overall declines in transcript expression suggest that age-related declines in L-Glu responsiveness in Aplysia sensory neurons could be linked to overall declines in iGluR expression, rather than dysregulation of specific subunits. In both sensory neuron clusters tested the N-methyl-D-aspartate receptor subtype was expressed at significantly greater levels than other iGluR subtypes, suggesting an in vivo role for NMDAR-like receptors in Aplysia sensory neurons.
Topics: Aging; Animals; Aplysia; Protein Subunits; RNA, Messenger; Receptors, Ionotropic Glutamate; Sensory Receptor Cells; Transcription, Genetic
PubMed: 31120976
DOI: 10.1371/journal.pone.0217300 -
Proceedings of the National Academy of... May 2004The induction of different phases of memory depends on the amount and patterning of training, raising the question of whether specific training patterns engage different...
The induction of different phases of memory depends on the amount and patterning of training, raising the question of whether specific training patterns engage different cellular mechanisms and whether these mechanisms operate in series or in parallel. We examined these questions by using a cellular model of memory formation: facilitation of the tail sensory neuron-motor neuron synapses by serotonin (5-hydroxytryptamine, 5-HT) in the CNS of Aplysia. We studied facilitation in two temporal domains: intermediate-term facilitation (1.5-3 h) and long-term facilitation (LTF, >24 h). Both forms can be induced by using several different temporal and spatial patterns of 5-HT, including (i) repeated, temporally spaced pulses of 5-HT to both the sensory neuron soma and the sensory neuron-motor neuron synapse, and (ii) temporally asymmetric exposure of 5-HT to the soma and synapse under conditions in which neither exposure alone induces LTF. We first examined the protein and RNA synthesis requirements for LTF induced by these two patterns and found that asymmetric (but not repeated) 5-HT application induced LTF that required postsynaptic protein and RNA synthesis. We next focused on the patterning and protein synthesis requirements for intermediate-term facilitation. We found that intermediate-term facilitation (i) is induced locally at the synapse, (ii) requires multiple pulses of 5-HT, and (iii) requires synaptic protein synthesis. Our findings show that different temporal and spatial patterns of 5-HT induce specific temporal phases of long-lasting facilitation in parallel by engaging different cellular and molecular mechanisms.
Topics: Animals; Aplysia; Neuronal Plasticity; Serotonin; Synapses
PubMed: 15123836
DOI: 10.1073/pnas.0402163101 -
ELife Nov 2014Long-term memory (LTM) is believed to be stored in the brain as changes in synaptic connections. Here, we show that LTM storage and synaptic change can be dissociated....
Long-term memory (LTM) is believed to be stored in the brain as changes in synaptic connections. Here, we show that LTM storage and synaptic change can be dissociated. Cocultures of Aplysia sensory and motor neurons were trained with spaced pulses of serotonin, which induces long-term facilitation. Serotonin (5HT) triggered growth of new presynaptic varicosities, a synaptic mechanism of long-term sensitization. Following 5HT training, two antimnemonic treatments-reconsolidation blockade and inhibition of PKM--caused the number of presynaptic varicosities to revert to the original, pretraining value. Surprisingly, the final synaptic structure was not achieved by targeted retraction of the 5HT-induced varicosities but, rather, by an apparently arbitrary retraction of both 5HT-induced and original synapses. In addition, we find evidence that the LTM for sensitization persists covertly after its apparent elimination by the same antimnemonic treatments that erase learning-related synaptic growth. These results challenge the idea that stable synapses store long-term memories.
Topics: Animals; Aplysia; Behavior, Animal; Benzophenanthridines; Coculture Techniques; Epigenesis, Genetic; Histone Deacetylases; Memory, Long-Term; Microscopy, Confocal; Protein Kinase C; Serotonin; Synapses
PubMed: 25402831
DOI: 10.7554/eLife.03896 -
Journal of Neurophysiology Nov 2005
Topics: Animals; Aplysia; Body Temperature Regulation; Feedback; Models, Neurological; Motor Neurons; Nerve Net; Neuromuscular Junction; Neuronal Plasticity; Temperature
PubMed: 16222070
DOI: 10.1152/jn.00636.2005 -
Comparative Biochemistry and... Jan 2014Endo-β-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly...
Endo-β-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly degrades laminaribiose. For the complete depolymerization of laminarin, other enzymes that can hydrolyze laminaribiose appear to be necessary. In the present study, we successfully isolated a laminaribiose-hydrolyzing enzyme from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatographies. This enzyme, AkLab, named after the scientific name of this animal and substrate specificity toward laminaribiose, shows an approximate molecular mass of 110kDa on SDS-PAGE, and optimum pH and temperature at around pH5.5 and 50°C, respectively. AkLab rapidly hydrolyzes laminaribiose and p-nitrophenyl-β-D-glucoside, and slowly cellobiose, gentiobiose and lactose, but not sucrose and maltose. AkLab shows high transglycosylation activity and can produce a series of laminarioligosaccharides larger than laminaritetraose from laminaribiose (a donor substrate) and laminaritriose (an acceptor substrate). This enzyme is suggested to be a member of glycosyl hydrolase family 1 by the analysis of partial amino-acid sequences.
Topics: Amino Acid Sequence; Animals; Aplysia; Disaccharides; Glycoside Hydrolases; Glycosylation; Hydrolysis; Molecular Sequence Data; Substrate Specificity
PubMed: 23912026
DOI: 10.1016/j.cbpb.2013.07.008 -
The Journal of Neuroscience : the... Sep 1989The transport of neuropeptides between central ganglia was studied in Aplysia. Peptide transport was determined by incubating ganglia with 35S-methionine and measuring...
The transport of neuropeptides between central ganglia was studied in Aplysia. Peptide transport was determined by incubating ganglia with 35S-methionine and measuring the appearance of labeled peptides in connected ganglia. Selected interganglionic connectives were left intact and passed through a diffusion barrier separating the ganglia. Labeled peptides transported between ganglia included FMRFamide, myomodulin, and pedal peptide. Each of these peptides has been shown to be physiologically active in Aplysia. In addition to these previously characterized neuropeptides, a number of other as yet uncharacterized labeled peptides were also transported. All the peptides were transported by fast axonal transport as judged by the distance transported and/or the sensitivity to colchicine. Overall, FMRFamide and several unidentified peptides were the predominant transported peptides. However, the nature and amount of the peptides transported differed for each ganglia. These results support the proposition that the labeled peptides have transmitterlike actions and suggest that there are a number of neuropeptides that are likely to have central actions that have not yet been characterized in Aplysia.
Topics: Abdomen; Animals; Aplysia; Axons; Biological Transport; Brain; Cheek; Ganglia; Neuropeptides; Pleura
PubMed: 2795162
DOI: 10.1523/JNEUROSCI.09-09-03243.1989 -
ENeuro 2020As they interact with their environment and encounter challenges, animals adjust their behavior on a moment-to-moment basis to maintain task fitness. This dynamic...
As they interact with their environment and encounter challenges, animals adjust their behavior on a moment-to-moment basis to maintain task fitness. This dynamic process of adaptive motor control occurs in the nervous system, but an understanding of the biomechanics of the body is essential to properly interpret the behavioral outcomes. To study how animals respond to changing task conditions, we used a model system in which the functional roles of identified neurons and the relevant biomechanics are well understood and can be studied in intact behaving animals: feeding in the marine mollusc We monitored the motor neuronal output of the feeding circuitry as intact animals fed on uniform food stimuli under unloaded and loaded conditions, and we measured the force of retraction during loaded swallows. We observed a previously undescribed pattern of force generation, which can be explained within the appropriate biomechanical context by the activity of just a few key, identified motor neurons. We show that, when encountering load, animals recruit identified retractor muscle motor neurons for longer and at higher frequency to increase retraction force duration. Our results identify a mode by which animals robustly adjust behavior to their environment, which is experimentally tractable to further mechanistic investigation.
Topics: Animals; Aplysia; Biomechanical Phenomena; Feeding Behavior; Models, Biological; Motor Neurons; Muscles
PubMed: 32332081
DOI: 10.1523/ENEURO.0016-20.2020