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Journal of Biological Rhythms Aug 2006In Aplysia californica, memory formation for long-term sensitization (LTS) and for a more complex type of associative learning, learning that food is inedible (LFI), is...
In Aplysia californica, memory formation for long-term sensitization (LTS) and for a more complex type of associative learning, learning that food is inedible (LFI), is modulated by a circadian clock. For both types of learning, formation of long-term memory occurs during the day and significantly less during the night. Aplysia eyes contain a well-characterized circadian oscillator that is strongly coupled to the locomotor activity rhythm. Thus, the authors hypothesized that the ocular circadian oscillator was responsible for the circadian modulation of LFI and LTS. To test this hypothesis, they investigated whether the eyes were necessary for circadian modulation of LFI and LTS. Eyeless animals trained during the subjective day and tested 24 h later demonstrated robust long-term memory for both LFI and LTS, while eyeless animals trained and tested during the subjective night showed little or no memory for LFI or LTS. The amplitude of the rhythm of modulation in eyeless animals was similar to that of intact Aplysia, suggesting that extraocular circadian oscillators were mainly responsible for the circadian rhythms in long-term memory formation. Next, the authors investigated whether the eyes played a role in photic entrainment for circadian regulation of long-term memory formation. Eyeless animals were exposed to a reversed LD cycle for 7 days and then trained and tested for long-term memory using the LFI paradigm. Eyeless Aplysia formed significant long-term memory when trained during the projected shifted day but not during the projected shifted night. Thus, the extraocular circadian oscillator responsible for the rhythmic modulation of long-term memory formation can be entrained by extraocular photoreceptors.
Topics: Animals; Aplysia; Behavior, Animal; Biological Clocks; Circadian Rhythm; Darkness; Learning; Light; Memory; Photoreceptor Cells, Invertebrate
PubMed: 16864645
DOI: 10.1177/0748730406289890 -
Journal of the American Society For... May 2011Mass spectrometry imaging (MSI) provides the ability to detect and identify a broad range of analytes and their spatial distributions from a variety of sample types,...
Mass spectrometry imaging (MSI) provides the ability to detect and identify a broad range of analytes and their spatial distributions from a variety of sample types, including tissue sections. Here we describe an approach for probing neuropeptides from sparse cell cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MSI--at single cell spatial resolution-in both MS and tandem MS modes. Cultures of Aplysia californica neurons are grown on an array of glass beads embedded in a stretchable layer of Parafilm M. As the membrane is stretched, the beads/neurons are separated physically and the separated beads/neurons analyzed via MALDI TOF MS. Compared with direct MS imaging of samples, the stretching procedure enhances analyte extraction and incorporation into the MALDI matrix, with negligible analyte spread between separated beads. MALDI tandem MSI using the stretched imaging approach yields localization maps of both parent and fragment ions from Aplysia pedal peptide, thereby confirming peptide identification. This methodology represents a flexible platform for MSI investigation of a variety of cell cultures, including functioning neuronal networks.
Topics: Animals; Aplysia; Cells, Cultured; Cytological Techniques; Image Processing, Computer-Assisted; Microscopy, Electron, Scanning; Molecular Imaging; Neurons; Neuropeptides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 21472517
DOI: 10.1007/s13361-011-0111-2 -
The Journal of Biological Chemistry Aug 2022Neuropeptides are a chemically diverse class of cell-to-cell signaling molecules that are widely expressed throughout the central nervous system, often in a...
Neuropeptides are a chemically diverse class of cell-to-cell signaling molecules that are widely expressed throughout the central nervous system, often in a cell-specific manner. While cell-to-cell differences in neuropeptides is expected, it is often unclear how exactly neuropeptide expression varies among neurons. Here we created a microscopy-guided, high-throughput single cell matrix-assisted laser desorption/ionization mass spectrometry approach to investigate the neuropeptide heterogeneity of individual neurons in the central nervous system of the neurobiological model Aplysia californica, the California sea hare. In all, we analyzed more than 26,000 neurons from 18 animals and assigned 866 peptides from 66 prohormones by mass matching against an in silico peptide library generated from known Aplysia prohormones retrieved from the UniProt database. Louvain-Jaccard (LJ) clustering of mass spectra from individual neurons revealed 40 unique neuronal populations, or LJ clusters, each with a distinct neuropeptide profile. Prohormones and their related peptides were generally found in single cells from ganglia consistent with the prohormones' previously known ganglion localizations. Several LJ clusters also revealed the cellular colocalization of behaviorally related prohormones, such as an LJ cluster exhibiting achatin and neuropeptide Y, which are involved in feeding, and another cluster characterized by urotensin II, small cardiac peptide, sensorin A, and FRFa, which have shown activity in the feeding network or are present in the feeding musculature. This mass spectrometry-based approach enables the robust categorization of large cell populations based on single cell neuropeptide content and is readily adaptable to the study of a range of animals and tissue types.
Topics: Animals; Aplysia; Central Nervous System; Neurons; Neuropeptides; Single-Cell Analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 35835221
DOI: 10.1016/j.jbc.2022.102254 -
Journal of Neurophysiology Jun 2022These experiments focus on an interneuron (B63) that is part of the feeding central pattern generator (CPG) in . Previous work has established that B63 is critical for...
These experiments focus on an interneuron (B63) that is part of the feeding central pattern generator (CPG) in . Previous work has established that B63 is critical for program initiation regardless of the type of evoked activity. B63 receives input from a number of different elements of the feeding circuit. Program initiation occurs reliably when some are activated, but we show that it does not occur reliably with activation of others. When program initiation is reliable, modulatory neuropeptides are released. For example, previous work has established that an ingestive input to the feeding CPG, cerebral buccal interneuron 2 (CBI-2), releases feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP-2). Afferents with processes in the esophageal nerve (EN) that trigger egestive motor programs release small cardioactive peptide (SCP). Previous studies have described divergent cellular and molecular effects of FCAP/CP-2 and SCP on the feeding circuit that specify motor activity. Here, we show that FCAP/CP-2 and SCP additionally increase the B63 excitability. Thus, we show that peptides that have well-characterized divergent effects on the feeding circuit additionally act convergently at the level of a single neuron. Since convergent effects of FCAP/CP-2 and SCP are not necessary for specifying the type of network output, we ask why they might be important. Our data suggest that they have an impact during a task switch, i.e., when there is a switch from egestive to ingestive activity. The activity of multifunctional central pattern generators (CPGs) is often configured by neuromodulators that exert divergent effects that are necessary to specify motor output. We demonstrate that ingestive and egestive inputs to the feeding CPG in act convergently (as well as divergently). We ask why this convergence may be important and suggest that it may be a mechanism for a type of arousal that occurs during task switching.
Topics: Animals; Aplysia; Central Pattern Generators; Feeding Behavior; Ganglia, Invertebrate; Interneurons; Neuropeptides
PubMed: 35507477
DOI: 10.1152/jn.00025.2022 -
PloS One 2013How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified...
How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.
Topics: Acetylcholine; Animals; Aplysia; Base Sequence; Cellular Senescence; Cholinergic Agonists; Molecular Sequence Data; Neurons; Receptors, Cholinergic
PubMed: 24386417
DOI: 10.1371/journal.pone.0084793 -
Fish & Shellfish Immunology Jun 2023The internal defense system of mollusks represents an efficient protection against pathogens and parasites, involving several biological immune processes, such as...
The internal defense system of mollusks represents an efficient protection against pathogens and parasites, involving several biological immune processes, such as phagocytosis, encapsulation, cytotoxicity, and antigenic recognition of self/non-self. Mollusks possess professional, migratory, and circulating cells that play a key role in the defense of the organism, the hemocytes. Several studies have been performed on hemocytes from different mollusks, but, to date, these cells are still scarcely explored. Different hemocyte populations have been found, according to the presence or absence of granules, size, and the species of mollusks studied. Our study aims to deepen the knowledge of the hemocytes of the gastropod Aplysia depilans using morphological techniques and light and confocal microscopy, testing Toll-like receptor 2, inducible nitric oxide synthetase, and nicotinic acetylcholine receptor alpha 7 subunit. Our results show two hemocyte populations distinguishable by size, and presence/absence of granules in the cytoplasm, strongly positive for the antibodies tested, suggesting for the first time the presence of these receptors on the surface of sea hare hemocytes by immunohistochemistry. These data help in the understanding of the immune system of this gastropod, providing additional data for comprehending the evolution of the defense response in metazoan phylogenesis.
Topics: Animals; Aplysia; Gastropoda; Hemocytes; Mollusca; Phagocytosis
PubMed: 37146849
DOI: 10.1016/j.fsi.2023.108791 -
Journal of Visualized Experiments : JoVE Apr 2011Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and...
Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca(2+) and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca(2+)-independent. The atypical PKCs are activated by neither Ca(2+) nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation (1-2). Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail (3). In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article (4), expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.
Topics: Animals; Aplysia; Cytological Techniques; Fluorescent Dyes; Isoenzymes; Microscopy, Confocal; Neurons, Afferent; Protein Kinase C; Spodoptera
PubMed: 21505406
DOI: 10.3791/2516 -
Learning & Memory (Cold Spring Harbor,... 2002In Aplysia, three distinct phases of memory for sensitization can be dissociated based on their temporal and molecular features. A single training trial induces...
In Aplysia, three distinct phases of memory for sensitization can be dissociated based on their temporal and molecular features. A single training trial induces short-term memory (STM, lasting <30 min), whereas five trials delivered at 15-min intervals induces both intermediate-term memory (ITM, lasting >90 min) and long-term memory (LTM, lasting >24 h). Here, we explore the interaction of amount and pattern of training in establishing ITM and LTM by examining memory for sensitization after different numbers of trials (each trial = one tail shock) and different patterns of training (massed vs. spaced). Under spaced training patterns, two trials produced STM exclusively, whereas four or five trials each produced both ITM and LTM. Three spaced trials failed to induce LTM but did produce an early decaying form of ITM (E-ITM) that was significantly shorter and weaker in magnitude than the late-decaying ITM (L-ITM) observed after four to five trials. In addition, E-ITM was induced after three trials with both massed and spaced patterns of training. However, L-ITM and LTM after four to five trials require spaced training: Four or five massed trials failed to induce LTM and produced only E-ITM. Collectively, our results indicate that in addition to three identified phases of memory for sensitization--STM, ITM, and LTM--a unique temporal profile of memory, E-ITM, is revealed by varying either the amount or pattern of training.
Topics: Animals; Aplysia; Learning; Memory
PubMed: 11917004
DOI: 10.1101/lm.44802 -
Journal of the American Chemical Society Jun 2018Acetylcholine, the first neurotransmitter identified more than a century ago, plays critical roles in human activities and health; however, its synaptic concentration...
Acetylcholine, the first neurotransmitter identified more than a century ago, plays critical roles in human activities and health; however, its synaptic concentration dynamics have remained unknown. Here, we demonstrate the in situ simultaneous measurements of synaptic cholinergic transmitter concentration and release dynamics. We used nanoscale electroanalytical methods: nanoITIES electrode of 15 nm in radius and nanoresolved scanning electrochemical microscopy (SECM). Time-resolved in situ measurements unveiled information on synaptic acetylcholine concentration and release dynamics of living Aplysia neurons. The measuring technique enabled the quantitative sensing of acetylcholine with negligible interference of other ionic and redox-active species. We measured cholinergic transmitter concentrations very close to the synapse, with values as high as 2.4 mM. We observed diverse synaptic transmitter concentration dynamics consisting of singlet, doublet and multiplet events with a signal-to-noise ratio of 6 to 130. The unprecedented details about synaptic neurotransmission unveiled are instrumental for understanding brain communication and diseases in a way distinctive from extra-synaptic studies.
Topics: Animals; Aplysia; Cholinergic Agents; Microscopy, Electrochemical, Scanning; Neurons; Synaptic Transmission
PubMed: 29883110
DOI: 10.1021/jacs.8b01989 -
Neuro-Signals 2004Aplysia feeding is striking in that it is executed with a great deal of plasticity. At least in part, this flexibility is a result of the organization of the feeding... (Review)
Review
Aplysia feeding is striking in that it is executed with a great deal of plasticity. At least in part, this flexibility is a result of the organization of the feeding neural network. To illustrate this, we primarily discuss motor programs triggered via stimulation of the command-like cerebral-buccal interneuron 2 (CBI-2). CBI-2 is interesting in that it can generate motor programs that serve opposing functions, i.e., programs can be ingestive or egestive. When programs are egestive, radula-closing motor neurons are activated during the protraction phase of the motor program. When programs are ingestive, radula-closing motor neurons are activated during retraction. When motor programs change in nature, activity in the radula-closing circuitry is altered. Thus, CBI-2 stimulation stereotypically activates the protraction and retraction circuitry, with protraction being generated first, and retraction immediately thereafter. In contrast, radula-closing motor neurons can be activated during either protraction or retraction. Which will occur is determined by whether other cerebral and buccal neurons are recruited, e.g. radula-closing motor neurons tend to be activated during retraction if a second CBI, CBI-3, is recruited. Fundamentally different motor programs are, therefore, generated because CBI-2 activates some interneurons in a stereotypic manner and other interneurons in a variable manner.
Topics: Action Potentials; Animals; Aplysia; Behavior, Animal; Digestive System Physiological Phenomena; Feedback; Feeding Behavior; Ganglia, Invertebrate; Motor Activity; Nerve Net; Neural Inhibition; Neural Networks, Computer; Neurons
PubMed: 15004426
DOI: 10.1159/000076159