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1.
Proc Natl Acad Sci U S A ; 119(28): e2122301119, 2022 07 12.
Article in English | MEDLINE | ID: mdl-35867761

ABSTRACT

The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.


Subject(s)
Aplysia , Biological Evolution , Cognition , Synapses , Animals , Aplysia/genetics , Aplysia/metabolism , Neuronal Plasticity/genetics , Neurons/metabolism , Protein Isoforms/genetics , Proteome , Synapses/metabolism , Transcriptome
2.
Neuropharmacology ; 144: 1-8, 2019 01.
Article in English | MEDLINE | ID: mdl-30321611

ABSTRACT

Decades of work in Aplysia californica established the general rule that principles of synaptic plasticity and their molecular mechanisms are evolutionarily conserved from mollusks to mammals. However, an exquisitely sensitive, activity-dependent homosynaptic mechanism that protects against the depression of neurotransmitter release in Aplysia sensory neuron terminals has, to date, not been uncovered in other animals, including mammals. Here, we discover that depression at a mammalian synapse that is implicated in habit formation and habit learning acceleration by ethanol, the fast-spiking interneuron (FSI) to medium spiny principal projection neuron (MSN) synapse of the dorsolateral striatum, is subject to this type of synaptic protection. We show that this protection against synaptic depression is calcium- and PDZ domain interaction-dependent. These findings support activity dependent protection against synaptic depression as an Aplysia-like synaptic switch in mammals that may represent a leveraging point for treating alcohol use disorders.


Subject(s)
Central Nervous System Depressants/pharmacology , Corpus Striatum/physiology , Ethanol/pharmacology , Habits , Neuronal Plasticity/physiology , Synapses/physiology , Animals , Aplysia/physiology , Calcium/metabolism , Corpus Striatum/cytology , Corpus Striatum/drug effects , Female , Male , Mice, Transgenic , Neural Inhibition/drug effects , Neural Inhibition/physiology , Neuronal Plasticity/drug effects , Neurons/cytology , Neurons/drug effects , Neurons/physiology , PDZ Domains , Protein Kinase C/metabolism , Synapses/drug effects , Tissue Culture Techniques
3.
Curr Biol ; 27(18): R1020-R1023, 2017 09 25.
Article in English | MEDLINE | ID: mdl-28950086

ABSTRACT

New possibilities for treating posttraumatic stress disorder and anxiety disorders involving abnormal memories are emerging from analysis of persistent protein kinase activation and mechanisms of synapse-specific modification, known as synaptic tagging.


Subject(s)
Memory , Neuronal Plasticity , Neurons , Synapses
4.
Curr Biol ; 22(17): R705-11, 2012 Sep 11.
Article in English | MEDLINE | ID: mdl-22975001

ABSTRACT

For decades, the marine snail Aplysia has proven to be a powerful system for analyzing basic neurobiological mechanisms, particularly cellular and molecular mechanisms of neural plasticity. Three new findings on Aplysia may be relevant for the understanding and treatment of chronic human disorders. This research on this simple molluscan nervous system may lead to new therapeutic approaches for spinal cord injury, Fragile X syndrome, and genetic learning deficits more generally.


Subject(s)
Aplysia/physiology , Computer Simulation , Models, Neurological , Neurons/metabolism , Animals , Fragile X Syndrome/therapy , Humans , Learning Disabilities/genetics , Learning Disabilities/therapy , Nervous System Physiological Phenomena , Neuronal Plasticity , Neurons/physiology , Signal Transduction , Spinal Cord Injuries/therapy
5.
Nat Neurosci ; 15(8): 1144-52, 2012 Jul 08.
Article in English | MEDLINE | ID: mdl-22772333

ABSTRACT

Habituation of a behavioral response to a repetitive stimulus enables animals to ignore irrelevant stimuli and focus on behaviorally important events. In Aplysia, habituation is mediated by rapid depression of sensory synapses, which could leave an animal unresponsive to important repetitive stimuli, making it vulnerable to injury. We identified a form of plasticity that prevents synaptic depression depending on the precise stimulus strength. Burst-dependent protection from depression is initiated by trains of 2-4 action potentials and is distinct from previously described forms of synaptic enhancement. The blockade of depression is mediated by presynaptic Ca2+ influx and protein kinase C (PKC) and requires localization of PKC via a PDZ domain interaction with Aplysia PICK1. During protection from depression, PKC acts as a highly sensitive detector of the precise pattern of sensory neuron firing. Behaviorally, burst-dependent protection reduces habituation, enabling animals to maintain responsiveness to stimuli that are functionally important.


Subject(s)
Aplysia/enzymology , Isoenzymes/physiology , Protein Kinase C/physiology , Sensory Gating/physiology , Sensory Receptor Cells/enzymology , Action Potentials/physiology , Animals , Behavior, Animal/physiology , Calcium/physiology , Calcium Channels/physiology , Habituation, Psychophysiologic/physiology , Neuronal Plasticity/physiology , Synaptic Transmission/physiology
6.
J Pharmacol Toxicol Methods ; 65(3): 122-5, 2012.
Article in English | MEDLINE | ID: mdl-22504007

ABSTRACT

INTRODUCTION: Lead exposure can cause learning disabilities, memory loss and severe damage to the nervous system. However, the exact mechanism by which lead causes learning disabilities is not fully understood. The effects of lead on calcium-regulated signaling pathways are difficult to study biochemically; with the traditional method of controlling the free calcium concentration with EGTA, the exact concentrations of free lead and calcium ions in solution are interdependent and prone to error because EGTA also buffers lead. METHODS AND RESULTS: In our approach, we first reduced the free calcium concentration in the solution using calcium-binding resins before adding lead to buffers. The solution was sequentially treated with Chelex-100 ion exchange resin, followed by immobilized BAPTA resin. The final concentration of free calcium in the solution was measured with Fluo-3 indicator. Our protocol successfully produced buffers with free calcium levels below 15 nM, which is substantially below threshold for activation of calcium-dependent enzymes in signaling pathways (which is typically a few hundred nanomolar calcium, when determined in vitro). CONCLUSION: This method provides an improved approach to study the effect of heavy metals on calcium-stimulated signaling pathways.


Subject(s)
Calcium Signaling/drug effects , Calcium/chemistry , Metals, Heavy/adverse effects , Metals, Heavy/chemistry , Animals , Buffers , Egtazic Acid/chemistry , Lead/chemistry , Lead/toxicity , Male , Rats , Solutions/chemistry
7.
Proc Natl Acad Sci U S A ; 107(35): 15607-12, 2010 Aug 31.
Article in English | MEDLINE | ID: mdl-20702764

ABSTRACT

Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neurons (SNs) in Aplysia has been proposed as a molecular coincidence detector during conditioning. We identified four putative ACs in Aplysia CNS. CaM binds to a sequence in the C1b region of AC-AplA that resembles the CaM-binding sequence in the C1b region of AC1 in mammals. Recombinant AC-AplA was stimulated by Ca(2+)/CaM. AC-AplC is most similar to the Ca(2+)-inhibited AC5 and AC6 in mammals. Recombinant AC-AplC was directly inhibited by Ca(2+), independent of CaM. AC-AplA and AC-AplC are expressed in SNs, whereas AC-AplB and AC-AplD are not. Knockdown of AC-AplA demonstrated that serotonin stimulation of cAMP-dependent plasticity in SNs is predominantly mediated by this CaM-sensitive AC. We propose that the coexpression of a Ca(2+)-inhibited AC in SNs, together with a Ca(2+)/CaM-stimulated AC, would enhance the associative requirement for coincident Ca(2+) influx and serotonin for effective stimulation of cAMP levels and initiation of plasticity mediated by AC-AplA.


Subject(s)
Adenylyl Cyclases/metabolism , Neuronal Plasticity/drug effects , Sensory Receptor Cells/drug effects , Serotonin/pharmacology , Action Potentials/drug effects , Adenylyl Cyclases/classification , Adenylyl Cyclases/genetics , Amino Acid Sequence , Animals , Aplysia/cytology , Aplysia/genetics , Aplysia/metabolism , Calcium/metabolism , Calcium/pharmacology , Calmodulin/metabolism , Calmodulin/pharmacology , Cloning, Molecular , Cyclic AMP/metabolism , Cyclic AMP/pharmacology , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Knockdown Techniques , Immunoblotting , Molecular Sequence Data , Phylogeny , Sensory Receptor Cells/cytology , Sensory Receptor Cells/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Serotonin Agents/pharmacology
8.
Brain Behav Evol ; 74(3): 191-205, 2009.
Article in English | MEDLINE | ID: mdl-20029183

ABSTRACT

The protein kinase C (PKC) and the cAMP-dependent kinase (protein kinase A; PKA) pathways are known to play important roles in behavioral plasticity and learning in the nervous systems of a wide variety of species across phyla. We briefly review the members of the PKC and PKA family and focus on the evolution of the immediate upstream activators of PKC and PKA i.e., phospholipase C (PLC) and adenylyl cyclase (AC), and their conservation in gastropod mollusks, taking advantage of the recent assembly of the Aplysiacalifornica and Lottia gigantea genomes. The diversity of PLC and AC family members present in mollusks suggests a multitude of possible mechanisms to activate PKA and PKC; we briefly discuss the relevance of these pathways to the known physiological activation of these kinases in Aplysia neurons during plasticity and learning. These multiple mechanisms of activation provide the gastropod nervous system with tremendous flexibility for implementing neuromodulatory responses to both neuronal activity and extracellular signals.


Subject(s)
Adenylyl Cyclases/metabolism , Biological Evolution , Cyclic AMP-Dependent Protein Kinases/metabolism , Gastropoda/physiology , Protein Kinase C/metabolism , Type C Phospholipases/metabolism , Adenylyl Cyclases/genetics , Animals , Intracellular Signaling Peptides and Proteins/metabolism , Neuronal Plasticity/physiology , Protein Kinase C/genetics
9.
Neurobiol Learn Mem ; 92(2): 155-65, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19345275

ABSTRACT

This review focuses on synaptic depression at sensory neuron-to-motor neuron synapses in the defensive withdrawal circuit of Aplysia as a model system for analysis of molecular mechanisms of sensory gating and habituation. We address the following topics: 1. Of various possible mechanisms that might underlie depression at these sensory neuron-to-motor neuron synapses in Aplysia, historically the most widely-accepted explanation has been depletion of the readily releasable pool of vesicles. Depletion is also believed to account for synaptic depression at long interstimulus intervals in a variety of other systems. 2. Multiple lines of evidence now indicate that vesicle depletion is not an important contributing mechanism to synaptic depression at Aplysia sensory neuron-to-motor neuron synapses. More generally, it appears that vesicle depletion does not contribute substantially to depression that occurs with those stimulus patterns that are typically used in studying behavioral habituation. 3. Recent evidence suggests that at these sensory neuron-to-motor neuron synapses in Aplysia, synaptic depression is mediated by an activity-dependent, but release-independent, switching of individual release sites to a silent state. This switching off of release sites is initiated by Ca2+ influx during individual action potentials. We discuss signaling proteins that may be regulated by Ca2+ during the silencing of release sites that underlies synaptic depression. 4. Bursts of 2-4 action potentials in presynaptic sensory neurons in Aplysia prevent the switching off of release sites via a mechanism called "burst-dependent protection" from synaptic depression. 5. This molecular switch may explain the sensory gating that allows animals to discriminate which stimuli are innocuous and appropriate to ignore and which stimuli are more important and should continue to elicit responses.


Subject(s)
Habituation, Psychophysiologic/physiology , Motor Neurons/physiology , Sensory Receptor Cells/physiology , Synapses/physiology , Action Potentials/physiology , Animals , Aplysia , Calcium/metabolism , Calcium Channels/metabolism , Models, Animal , Models, Neurological , Reflex/physiology , Synaptic Transmission/physiology , Synaptic Vesicles/physiology
10.
Curr Biol ; 18(5): R220-3, 2008 Mar 11.
Article in English | MEDLINE | ID: mdl-18334201

ABSTRACT

A novel mechanism of persistent facilitation induced by serotonin at Aplysia synapses depends upon rapid postsynaptic protein synthesis and increased responsiveness to glutamate; whereas the memory for this synaptic change is postsynaptic, the initiating signal may be an increase in spontaneous release of glutamate from the presynaptic terminals.


Subject(s)
Aplysia/metabolism , Glutamic Acid/metabolism , Neuronal Plasticity/physiology , Serotonin/metabolism , Synapses/metabolism , Animals , Motor Neurons/metabolism
11.
J Neurophysiol ; 95(4): 2713-20, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16236785

ABSTRACT

Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+ currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 microM 5-HT was unaffected by 100 microM spiperone, whereas it was effectively blocked by 100 microM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 microM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.


Subject(s)
Aplysia/physiology , Cyclic AMP-Dependent Protein Kinases/physiology , Neuronal Plasticity/drug effects , Neurons, Afferent/physiology , Protein Kinase C/physiology , Serotonin Antagonists/pharmacology , Action Potentials/drug effects , Action Potentials/physiology , Adenylyl Cyclases/physiology , Animals , Cyclic AMP/physiology , Electrophysiology , Long-Term Synaptic Depression/drug effects , Long-Term Synaptic Depression/physiology , Methiothepin/pharmacology , Neuronal Plasticity/physiology , Neurons, Afferent/drug effects , Neurons, Afferent/enzymology , Nifedipine/pharmacology , Receptors, Serotonin/drug effects , Receptors, Serotonin/physiology , Serotonin/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Spiperone/pharmacology , Synapses/drug effects , Synapses/physiology , Type C Phospholipases/physiology
12.
J Neurophysiol ; 89(3): 1440-55, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12611939

ABSTRACT

We attempted to identify compounds that are effective in blocking the serotonin (5-hydroxytryptamine, 5-HT) receptor(s) that activate adenylyl cyclase (AC) in Aplysia CNS. We call this class of receptor 5-HT(apAC). Eight of the 14 antagonists tested were effective against 5-HT(apAC) in CNS membranes with the following rank order of potency: methiothepin > metergoline approximately fluphenazine > clozapine > cyproheptadine approximately risperidone approximately ritanserin > NAN-190. GR-113808, olanzapine, Ro-04-6790, RS-102221, SB-204070, and spiperone were inactive. Methiothepin completely blocked 5-HT stimulation of AC with a K(b) of 18 nM. Comparison of the pharmacological profile of the 5-HT(apAC) receptor with those of mammalian 5-HT receptor subtypes suggested it most closely resembles the 5-HT(6) receptor. AC stimulation in Aplysia sensory neuron (SN) membranes was also blocked by methiothepin. Methiothepin substantially inhibited two effects of 5-HT on SN firing properties that are mediated by a cAMP-dependent reduction in S-K(+) current: spike broadening in tetraethylammonium/nifedipine and increased excitability. Consistent with cyproheptadine blocking 5-HT stimulation of AC, cyproheptadine also blocked the 5-HT-induced increase in SN excitability. Methiothepin was less effective in blocking AC-mediated modulatory effects of 5-HT in electrophysiological experiments on SNs than in blocking AC stimulation in CNS or SN membranes. This reduction in potency appears to be due to effects of the high ionic strength of physiological saline on the binding of this antagonist to the receptor. Methiothepin also antagonized AC-coupled dopamine receptors but not AC-coupled small cardioactive peptide receptors. In conjunction with other pharmacological probes, this antagonist should be useful in analyzing the role of 5-HT in various forms of neuromodulation in Aplysia.


Subject(s)
Adenylyl Cyclases/metabolism , Methiothepin/pharmacology , Receptors, Serotonin/metabolism , Serotonin Antagonists/pharmacology , Animals , Aplysia , Cyproheptadine/pharmacology , Electrophysiology , Ganglia, Invertebrate/cytology , Ganglia, Invertebrate/physiology , Mammals , Neural Inhibition/drug effects , Neurons, Afferent/drug effects , Neurons, Afferent/physiology , Neurotransmitter Agents/metabolism , Radioligand Assay , Serotonin/pharmacology , Sodium Chloride/pharmacology
13.
J Neurosci ; 22(5): 1942-55, 2002 Mar 01.
Article in English | MEDLINE | ID: mdl-11880525

ABSTRACT

The synaptic connections of Aplysia sensory neurons (SNs) undergo dramatic homosynaptic depression (HSD) with only a few low-frequency stimuli. Strong and weak SN synapses, although differing in their probabilities of release, undergo HSD at the same rate; this suggests that the major mechanism underlying HSD in these SNs may not be depletion of the releasable pool of vesicles. In computational models, we evaluated alternative mechanisms of HSD, including vesicle depletion, to determine which mechanisms enable strong and weak synapses to depress with identical time courses. Of five mechanisms tested, only release-independent, stimulus-dependent switching off of release sites resulted in HSD that was independent of initial synaptic strength. This conclusion that HSD is a release-independent phenomenon was supported by empirical results: an increase in Ca2+ influx caused by spike broadening with a K+ channel blocker did not alter HSD. Once induced, HSD persisted during 40 min of rest with no detectable recovery; thus, release does not recover automatically with rest, contrary to what would be expected if HSD represented an exhaustion of the exocytosis mechanism. The hypothesis that short-term HSD involves primarily a stepwise silencing of release sites, rather than vesicle depletion, is consistent with our earlier observation that HSD is accompanied by only a modest decrease in release probability, as indicated by little change in the paired-pulse ratio. In contrast, we found that there was a dramatic decrease in the paired-pulse ratio during serotonin-induced facilitation; this suggests that heterosynaptic facilitation primarily involves an increase in release probability, rather than a change in the number of functional release sites.


Subject(s)
Exocytosis/physiology , Neural Inhibition/physiology , Neurons, Afferent/physiology , Synapses/physiology , Synaptic Transmission/physiology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Aplysia , Computer Simulation , Electric Stimulation , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Ganglia, Invertebrate/cytology , Ganglia, Invertebrate/drug effects , Ganglia, Invertebrate/physiology , In Vitro Techniques , Models, Neurological , Monte Carlo Method , Neurons, Afferent/drug effects , Probability , Serotonin/pharmacology , Synapses/drug effects , Synaptic Transmission/drug effects , Synaptic Vesicles/metabolism
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