Phosphoinositide-dependent Kinase Phosphorylation of Protein Kinase C Apl II Increases During Intermediate Facilitation in Aplysia

The Journal of Biological Chemistry. Oct, 2002  |  Pubmed ID: 12140280

Phosphorylation of protein kinase Cs (PKCs) by phosphoinositide-dependent kinase I (PDK) is critical for PKC activity. In the nervous system of the marine mollusk Aplysia, there are only two major PKC isoforms, the calcium-activated PKC Apl I and the calcium-independent PKC Apl II, and both PKCs are persistently activated during intermediate memory. We monitored the PDK-dependent phosphorylation of PKC Apl I and PKC Apl II using phosphopeptide antibodies. During persistent activation of PKCs in Aplysia neurons, there is a significant increase in the amount of PDK-phosphorylated PKC Apl II in the particulate fraction but no increase in the amount of PKC Apl I phosphorylated by PDK. PDK phosphorylation of PKCs was not sensitive to inhibitors of phosphatidylinositol 3-kinase, PKC, or expression of a kinase-inactive PDK. Localization of PDK-phosphorylated PKC Apl II using immunocytochemistry revealed an enrichment of phosphorylated PKC Apl II at the plasma membrane. These data suggest that increased PDK phosphorylation of PKC Apl II is important for persistent kinase activation.

Oxidation Induces Autonomous Activation of Protein Kinase C Apl I, but Not Protein Kinase C Apl II in Homogenates of Aplysia Neurons

Neuroscience Letters. Sep, 2002  |  Pubmed ID: 12183025

The Ca(2+)-independent protein kinase C (PKC) Apl II, but not the Ca(2+)-activated PKC Apl I, becomes autonomously active during intermediate periods of facilitation in Aplysia neurons. We examined the ability of superoxide formed by the enzymatic reaction of xanthine with xanthine oxidase (X/XO) to induce autonomous activity of PKCs in Aplysia. X/XO stimulated autonomous PKC activity in Aplysia nervous system homogenates, but this activity resulted solely from activation of PKC Apl I. PKC Apl I is also more sensitive to activation by X/XO when expressed in insect cells. Our results suggest that oxidation can autonomously activate PKC Apl I in the Aplysia nervous system, but that the activation of PKC Apl II during synaptic facilitation is not due to oxidation of the enzyme.

Protein Kinase C Isoforms Are Translocated to Microtubules in Neurons

The Journal of Biological Chemistry. Oct, 2002  |  Pubmed ID: 12183453

Activation of protein kinase C (PKC) increases microtubule (MT) growth lifetimes, resulting in extension of a nocodazole-sensitive population of MTs in Aplysia growth cones. We examined whether the two phorbol ester-activated PKCs in Aplysia, the Ca(2+)-activated PKC Apl I and the Ca(2+)-independent PKC Apl II, are associated with these MTs. Phorbol esters translocated PKC to the Triton X-100-insoluble fraction, and a significant portion of this translocated pool was sensitive to low concentrations of nocodazole. Low doses of nocodazole had no effect on the amount of PKC in the Triton X-100-insoluble fraction in the absence of phorbol esters, whereas higher doses of nocodazole reduced basal levels of PKC Apl II. The F-actin cytoskeletal disrupter, latrunculin A, removed both PKCs from the Triton X-100-insoluble fraction in both control and phorbol ester-treated nervous systems. PKC Apl II also directly interacted with purified MTs. In detergent-extracted cells, both PKCs immunolocalized predominantly with MTs. PKCs were associated with newly formed MTs invading the actin-rich peripheral growth cone domain after PKC activation. Our results are consistent with a central role for PKCs in regulating MT extension.

An Activity-dependent Switch to Cap-independent Translation Triggered by EIF4E Dephosphorylation

Nature Neuroscience. Mar, 2003  |  Pubmed ID: 12592407

In Vitro Autophosphorylation of Protein Kinase C Isozymes

Methods in Molecular Biology (Clifton, N.J.). 2003  |  Pubmed ID: 12840508

Phosphopeptide-specific Antibodies to Protein Kinase C

Methods in Molecular Biology (Clifton, N.J.). 2003  |  Pubmed ID: 12840511

Differential Regulation of Transmitter Release by Alternatively Spliced Forms of Synaptotagmin I

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2003  |  Pubmed ID: 12867508

We discovered a novel alternatively spliced form of synaptotagmin I (Syt I). This splicing event is conserved over evolution and, in Aplysia, results in a two amino acid insert in the juxtamembrane domain of Syt I (Syt IVQ). Both Syt I and Syt IVQ are localized to synaptic vesicles; however, we also observed punctae that contained one or the other spliced products. Both Syt I and Syt IVQ are phosphorylated at the adjacent PKC site. Overexpression of Syt IVQ, but not of Syt I, in Aplysia neurons blocked the ability of serotonin to reverse synaptic depression. This effect is upstream of PKC activation, because neither Syt IVQ nor Syt I blocked the effects of phorbol esters on reversing synaptic depression or the effects of serotonin on facilitating nondepressed synapses. Our results demonstrate a physiological role for splicing in the juxtamembrane domain of Syt I.

Activity-dependent Regulation of Neurohormone Synthesis and Its Impact on Reproductive Behavior in Aplysia

Biology of Reproduction. Feb, 2004  |  Pubmed ID: 14522824

The bag cell neurons (BCNs) of the mollusk Aplysia californica provide a simple model system for investigating cellular and molecular events regulating synthesis and secretion of a reproductive neuropeptide and their impact on physiology and behavior. The BCNs secrete a large amount of egg-laying hormone (ELH) in response to an electrical afterdischarge. The afterdischarge also triggers cellular and molecular events leading to upregulation of ELH biosynthesis to replenish the supply of releasable hormone that was lost because of secretion. In the present review, we discuss signal-transduction events that link membrane excitability to ELH biosynthesis. We present evidence that the afterdischarge stimulates ELH synthesis by upregulating translation of ELH mRNA rather than by activating ELH gene transcription. This increase in ELH synthesis is accompanied by a decrease in total protein synthesis, suggesting that the synthetic machinery is being funneled selectively toward ELH. We also discuss work showing that afterdischarge-induced ELH synthesis uses a novel mechanism of translation initiation, one involving a switch from cap-dependent to cap-independent translation initiation that activates an internal ribosome entry site (IRES) located in the 5'-untranslated region of ELH mRNA. The IRES-regulated translation provides a unique cellular mechanism to selectively upregulate synthesis of a critical reproductive hormone at the expense of nonessential proteins.

Identification and Characterization of a Novel C2B Splice Variant of Synaptotagmin I

Journal of Neurochemistry. Apr, 2004  |  Pubmed ID: 15056279

We have identified an alternatively spliced form of synaptotagmin I in Aplysia neurons. This isoform, synaptotagmin I C2B-beta, is generated by alternative exon usage in the C2B domain leading to nine amino acid changes in the C2B sequence from the previously characterized synaptotagmin I, now designated as synaptotagmin I C2B-alpha. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that approximately 25% of mRNA encoding synaptotagmin I contained the C2B-beta exon in the nervous system. Synaptotagmin I C2B-beta showed greater resistance to digestion by chymotrypsin in the absence of calcium than did synaptotagmin I C2B-alpha, although both isoforms required the same amount of calcium to resist chymotrypsin digestion. The source of these changes in C2B properties was mapped to a single amino acid (threonine 358). We have also cloned SNAP 25 in Aplysia and show that it binds synaptotagmin I C2B-beta with a higher affinity than synaptotagmin I C2B-alpha. These results suggest that this splicing alters biochemical properties of the C2B domain, affecting a number of its important known interactions.

Characterization of a Novel Molluskan Tyrosine Kinase Receptor That Inhibits Neurite Regeneration

Journal of Neurobiology. Aug, 2004  |  Pubmed ID: 15266645

Receptor tyrosine kinases play many important roles in neuronal signaling including regulating neurite outgrowth. We have identified a novel receptor tyrosine kinase, neurite outgrowth regulating kinase (nork) from Aplysia californica. A fragment of this kinase was also identified in another mollusk, Lymnaea. The kinase domain is equally homologous to the Ret (rearranged during transformation) and fibroblast growth factor receptor families, but the extracellular domain is entirely novel, suggesting that it binds a nonconserved ligand. Overexpression of neurite outgrowth regulating kinase, but not a kinase dead form, causes a reduction in neurite outgrowth of Aplysia sensory neurons. Thus, we have identified a novel receptor tyrosine kinase implicated in regulating neurite outgrowth.

5-HT Stimulates EEF2 Dephosphorylation in a Rapamycin-sensitive Manner in Aplysia Neurites

Journal of Neurochemistry. Sep, 2004  |  Pubmed ID: 15341530

In Aplysia, serotonin mediates behavioral sensitization by increasing the strength of the synapse between sensory and motor neurons, a process known as facilitation. The retention of long-term facilitation is blocked by rapamycin, an inhibitor of a specific translational pathway. One possible rapamycin-sensitive target is the increased translation of 5'-terminal oligopyrimidine mRNAs. These transcripts encode components of the translational machinery and have been proposed to be important for retention of long-term facilitation. We have cloned the 5'-terminal oligopyrimidine mRNA encoding eukaryotic elongation factor 2 and shown that serotonin increased its translation in synaptosomes. Another possible rapamycin-sensitive target is the inactivation of eukaryotic elongation factor 2 kinase. Eukaryotic elongation factor 2 kinase phosphorylates and inactivates eukaryotic elongation factor 2, blocking translational elongation. Serotonin application decreased eukaryotic elongation factor 2 phosphorylation in synaptosomes and in isolated neurites, and this was blocked by rapamycin. We propose a role for the rapamycin-sensitive pathway in neurons. Stimulation blocks translation by inducing calcium entry and phosphorylation of eukaryotic elongation factor 2. This block is reversed through activation of the rapamycin-sensitive system and dephosphorylation of eukaryotic elongation factor 2.

ApTrkl, a Trk-like Receptor, Mediates Serotonin- Dependent ERK Activation and Long-term Facilitation in Aplysia Sensory Neurons

Neuron. Nov, 2004  |  Pubmed ID: 15541318

The Trk family of receptor tyrosine kinases plays a role in synaptic plasticity and in behavioral memory in mammals. Here, we report the discovery of a Trk-like receptor, ApTrkl, in Aplysia. We show that it is expressed in the sensory neurons, the locus for synaptic facilitation, which is a cellular model for memory formation. Serotonin, the facilitatory neurotransmitter, activates ApTrkl, which, in turn, leads to activation of ERK. Finally, inhibiting the activation of ApTrkl with the Trk inhibitor K252a or using dsRNA to inhibit ApTrkl blocks the serotonin-mediated activation of ERK in the cell body, as well as the cell-wide long-term facilitation induced by 5-HT application to the cell body. Thus, transactivation of the receptor tyrosine kinase ApTrkl by serotonin is an essential step in the biochemical events leading to long-term facilitation in Aplysia.

Ltrk is Differentially Expressed in Developing and Adult Neurons of the Lymnaea Central Nervous System

The Journal of Comparative Neurology. Jul, 2005  |  Pubmed ID: 15892101

The Trk receptor family plays diverse roles in both development and plasticity of the vertebrate nervous system. Ltrk is a related receptor that is expressed in the CNS of the mollusk Lymnaea, although little is known of its cellular distribution. This study provides three independent lines of evidence (based on RT-PCR, in situ hybridization, and immunohistochemistry) that Ltrk is universally expressed by neurons and dorsal body cells of both the juvenile and the adult Lymnaea CNS. The highest level of expression by neuronal somata occurs in the late juvenile stage, whereas axon collaterals express high levels throughout the animal's life span. Our data support multifunctional roles for Ltrk that parallel those of its mammalian counterparts.

Characterization of an RNA Granule from Developing Brain

Molecular & Cellular Proteomics : MCP. Apr, 2006  |  Pubmed ID: 16352523

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.

Mnk is a Negative Regulator of Cap-dependent Translation in Aplysia Neurons

Journal of Neurochemistry. Apr, 2006  |  Pubmed ID: 16515558

To investigate the mechanisms underlying regulation of eukaryotic initiation factor 4E (eIF4E) phosphorylation in Aplysia neurons, we have cloned the Aplysia homolog of the vertebrate eIF4E kinases, Mnk1 and -2. Aplysia Mnk shares many conserved regions with vertebrate Mnk, including putative eukaryotic initiation factor 4G binding regions, activation loop phosphorylation sites, and a carboxy-terminal anchoring site for MAP kinases. As expected, purified Aplysia Mnk phosphorylated Aplysia eIF4E at a conserved carboxy-terminal serine and over-expression of Aplysia Mnk in sensory neurons led to increased phosphorylation of endogenous eIF4E. Over-expression of Aplysia Mnk led to strong decreases in cap-dependent translation, while generally sparing internal ribosomal entry site (IRES)-dependent translation. However, decreases in cap-dependent translation seen after expression of Aplysia Mnk could only be partly explained by increases in eIF4E phosphorylation. In Aplysia sensory neurons, phosphorylation of eIF4E is reduced during intermediate memory formation. However, we found that this physiological regulation of eIF4E phosphorylation was independent of changes in Aplysia Mnk phosphorylation. We propose that changes in eIF4E phosphorylation in Aplysia neurons are a consequence of changes in cap-dependent translation that are independent of regulation of Aplysia Mnk.

Tracing the Evolution and Function of the Trk Superfamily of Receptor Tyrosine Kinases

Brain, Behavior and Evolution. 2006  |  Pubmed ID: 16912468

Most growth factors and their receptors have been strongly conserved during evolution. In contrast, Trks (Tropomyosin-related kinases) and related receptors in the Trk superfamily, Rors (receptor tyrosine kinase-like orphan receptors), Musks (muscle specific kinases) and Ddrs (discoidin domain receptor family), appear to be ancient, but their function has been lost in multiple lineages and the roles for the receptors have been modified over time. We will trace the evolution of the Trk superfamily and discuss possible conserved functional roles, including a unifying theme of target recognition by growing axons. We present an analogy between the evolution of G-protein-coupled receptors and receptor tyrosine kinases (RTKs), proposing that an important driving force for the divergence of receptors is the ease of divergence of their ligands.

Isoform Specificity of PKC Translocation in Living Aplysia Sensory Neurons and a Role for Ca2+-dependent PKC APL I in the Induction of Intermediate-term Facilitation

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2006  |  Pubmed ID: 16928874

Protein kinase Cs (PKCs) are important effectors of synaptic plasticity. In Aplysia, there are two major phorbol ester-activated PKCs, Ca2+-activated PKC Apl I and Ca2+-independent PKC Apl II. Functional Apl II, but not Apl I, in sensory neurons is required for a form of short-term facilitation induced at sensorimotor synapses by the facilitatory transmitter serotonin (5-HT). Because PKCs are activated by translocating from the cytoplasm to the membrane, we used fluorescently tagged PKCs to determine the isoform and cell-type specificity of translocation in living Aplysia neurons. In Sf9 cells, low levels of diacylglycerol translocate Apl II, but not Apl I, which requires calcium for translocation at low concentrations of diacylglycerol. Accordingly, application of 5-HT to Aplysia sensory neurons in the absence of neuronal firing translocates Apl II, but not Apl I, consistent with the role of Apl II in short-term facilitation. This translocation is observed in sensory neurons, but not in motor neurons. Apl I translocates only if 5-HT is coupled to firing in the sensory neuron; firing alone is ineffective. Because combined 5-HT and firing are required for the induction of one type of intermediate-term facilitation at these synapses, we asked whether this form of synaptic plasticity involves activation of Apl I. We report here that dominant-negative Apl I, but not Apl II, blocks intermediate-term facilitation. Thus, different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity: Ca2+-independent Apl II is involved in short-term facilitation, and Ca2+-dependent Apl I contributes to intermediate-term facilitation.

Serotonin Increases Phosphorylation of Synaptic 4EBP Through TOR, but Eukaryotic Initiation Factor 4E Levels Do Not Limit Somatic Cap-dependent Translation in Aplysia Neurons

Molecular and Cellular Biology. Nov, 2006  |  Pubmed ID: 16982686

The target of rapamycin (TOR) plays an important role in memory formation in Aplysia californica. Here, we characterize one of the downstream targets of TOR, the eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP) from Aplysia. Aplysia 4EBP contains the four critical phosphorylation sites regulated by TOR as well as an N-terminal RAIP motif and a C-terminal TOS site. Aplysia 4EBP was hypophosphorylated in synaptosomes, and serotonin addition caused a rapamycin-sensitive increase in 4EBP phosphorylation both in synaptosomes and in isolated neurites. Aplysia 4EBP was regulated in a fashion similar to that of mammalian 4EBPs, binding to eIF4E when dephosphorylated and releasing eIF4E after phosphorylation. Overexpression of 4EBP in the soma of Aplysia neurons caused a specific decrease in cap-dependent translation that was rescued by concomitant overexpression of eIF4E. However, eIF4E overexpression by itself did not increase cap-dependent translation, suggesting that increasing levels of free eIF4E by phosphorylating 4EBP is not important in regulating cap-dependent translation in the cell soma. Total levels of eIF4E were also regulated by 4EBP, suggesting that 4EBP can also act as an eIF4E chaperone. These studies demonstrate the conserved nature of 4EBP regulation and its role in cap-dependent translation and suggest differential roles of 4EBP phosphorylation in the soma and synapse.

Intracellular Trafficking of RNA in Neurons

Traffic (Copenhagen, Denmark). Dec, 2006  |  Pubmed ID: 17054760

The transport of messenger RNAs (mRNAs) in neurons serves many purposes. During development, trafficking of mRNAs to both axonal and dendritic growth cones regulates neuronal growth. After synapse formation, mRNAs continue to be transported to dendrites both as a mechanism for the localization of proteins to specific compartments and as a substrate for local translational regulation of synaptic plasticity. Finally, activity-dependent mRNAs are transported quickly to dendrites after transcription. Determining how mRNAs are transported and specifically translated in these different paradigms is a major unanswered question. Addressing this question is also complicated by the presence of many other RNA processing and storage centers that may not be involved in transport but share components with the transport structures. In the present review, we will discuss several recent studies addressing mechanisms of mRNA transport in neurons, as well as proteomic characterization of mRNA transporting structures in neurons. We define two types of RNA transport structures in neurons, transport particles and RNA granules and distinguish them by the presence or absence of ribosomes. We will present a number of different molecular models for how mRNAs are repressed during transport, and how these may affect the regulation of local translation in neurons.

Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

Learning & Memory (Cold Spring Harbor, N.Y.). Apr, 2007  |  Pubmed ID: 17404386

Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used Aplysia as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented physiological roles for PKC in synaptic plasticity in this system. In particular, we have shown that distinct isoforms mediate distinct types of synaptic plasticity induced by the same neurotransmitter: The novel calcium-independent PKC Apl II is required for actions mediated by serotonin (5-HT) alone, while the classical calcium-dependent PKC Apl I is required for actions mediated when 5-HT is coupled to activity. We will discuss the reasons for PKC isoform specificity, assess the tools used to uncover isoform specificity, and discuss the implications of isoform specificity for understanding the roles of PKC in regulating synaptic plasticity.

Something Old, Something New: BDNF-induced Neuron Survival Requires TRPC Channel Function

Nature Neuroscience. May, 2007  |  Pubmed ID: 17453053

PKC Modulation of Transmitter Release by SNAP-25 at Sensory-to-motor Synapses in Aplysia

Journal of Neurophysiology. Jan, 2007  |  Pubmed ID: 16971689

Activation of phosphokinase C (PKC) can increase transmitter release at sensory-motor neuron synapses in Aplysia, but the target of PKC phosphorylation has not been determined. One putative target of PKC at synapses is the synaptosomal-associated protein of 25 kDa (SNAP-25), a member of the SNARE protein complex implicated in synaptic vesicle docking and fusion. To determine whether PKC regulated transmitter release through phosphorylation of SNAP-25, we cloned Aplysia SNAP-25 and expressed enhanced green fluorescent protein (EGFP)-coupled SNAP-25 constructs mutated at the PKC phosphorylation site Ser198 in Aplysia sensory neurons. We found several distinct effects of expression of EGFP-SNAP-25 constructs. First, the rates of synaptic depression were slowed when cells contained SNAP-25 with phosphomimetic residues Glu or Asp. Second, PDBu-mediated increases in transmitter release at naïve synapses were blocked in cells expressing nonphosphorylated-state SNAP-25. Finally, expression of EGFP-coupled SNAP-25 but not uncoupled SNAP-25 inhibited 5-HT-mediated reversal of depression and the ability of EGFP-coupled SNAP-25 to inhibit the reversal of depression was affected by changes at Ser198. These results suggest SNAP-25 and phosphorylation of SNAP-25 by PKC can regulate transmitter release at Aplysia sensory-motor neuron synapses by a number of distinct processes.

The Significance of Organellar Proteomics for the Nervous System

Proteomics. Clinical Applications. Nov, 2007  |  Pubmed ID: 21136641

Organellar proteomics is a useful tool for gaining biological insights about structures in the cell. Here, we discuss the tools used in organellar proteomics and the impact of this technique in understanding nervous system function. We will review insights gained from the proteomes of nervous system-specific organelles such as synaptic vesicles and the postsynaptic density. Moreover, we will show how comparison of proteomes between organelles isolated from the nervous system and from other tissues highlight nervous system-specific functions using the examples of clathrin-coated vesicles and RNA granules.

Defining Memories by Their Distinct Molecular Traces

Trends in Neurosciences. Apr, 2008  |  Pubmed ID: 18329733

It is often stated that short-term memory is consolidated in a protein-synthesis-dependent manner into long-term memory. Alternatively, memories might consist of distinct molecular traces that last for different periods of time. These traces can be graded by their 'volatility'; traces encoded by activation of protein kinases are more volatile than traces encoded by morphological changes at preexisting synapses. The least volatile ('static') traces are due to the generation and stabilization of new synapses. Importantly, whereas at the cellular level these traces are generated independently of each other, they might be linked at the network level where volatile memory traces are required to set up a cellular network that is in turn required to induce the static memory trace.

The Essence of Memory. Preface

Progress in Brain Research. 2008  |  Pubmed ID: 18394464

Molecular Memory Traces

Progress in Brain Research. 2008  |  Pubmed ID: 18394465

To understand the essence of memory, one must examine the working of the brain on many levels. It is important to find the appropriate level to study the particular aspect of memory under investigation. In this review, I will focus on insights gained from examining memory at the molecular level. I will illustrate these insights with specific examples from examining the molecular and cellular mechanisms underlying long-term facilitation in the marine mollusk Aplysia and long-term potentiation, studied mainly in rodents. In particular, I will discuss how molecular memory traces are formed and focus in detail on what role increasing the level of proteins through protein synthesis and gene expression plays in memory formation. I will point out three important constraints from molecular work that should impact on cognitive modeling of the nervous system: (i) the induction of plasticity depends on the 'state' of the synapse; (ii) there are multiple independent molecular traces formed after experience with different half-lives; and (iii) the requirement for the conjunction of synaptic activation and new protein synthesis implies that new conjunctions are required to induce long-term memory formation.

Forskolin Induction of Late-LTP and Up-regulation of 5' TOP MRNAs Translation Via MTOR, ERK, and PI3K in Hippocampal Pyramidal Cells

Journal of Neurochemistry. Aug, 2008  |  Pubmed ID: 18466337

The late phase of long-term potentiation (LTP) requires activation of the mammalian target of rapamycin (mTOR) pathway and synthesis of new proteins. mTOR regulates protein synthesis via phosphorylation of 4E-binding proteins (4E-BPs) and S6K, and via selective up-regulation of 5' terminal oligopyrimidine (5' TOP) mRNAs that encode components of the translational machinery. In this study, we explored the regulation of 5' TOP mRNAs during late-LTP (L-LTP). Synaptic plasticity was studied at Schaffer collateral--CA1 pyramidal cell synapses in rat organotypic hippocampal slices. Forskolin, an adenylate cyclase activator, induced L-LTP in organotypic slices that was mTOR-dependent. To determine if 5' TOP mRNAs are specifically up-regulated during L-LTP, we generated a 5' TOP-myr-dYFP reporter to selectively monitor 5' TOP translation. Confocal imaging experiments in cultured slices revealed an increase in somatic and dendritic fluorescence after forskolin treatment. This up-regulation was dependent on an intact TOP sequence and was mTOR, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-dependent. Our findings indicate that forskolin induces L-LTP in hippocampal neurons and up-regulates 5' TOP mRNAs translation via mTOR, suggesting that up-regulation of the translational machinery is a candidate mechanism for the stabilization of LTP.

Physiological Role for Phosphatidic Acid in the Translocation of the Novel Protein Kinase C Apl II in Aplysia Neurons

Molecular and Cellular Biology. Aug, 2008  |  Pubmed ID: 18505819

In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKCepsilon, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.

Mechanisms Regulating ApTrkl, a Trk-like Receptor in Aplysia Sensory Neurons

Journal of Neuroscience Research. Oct, 2008  |  Pubmed ID: 18521934

An Aplysia Trk-like receptor (ApTrkl) was previously shown to be involved in cell wide long-term facilitation (LTF) and activation of ERK when serotonin (5-HT) is applied to the cell soma. The current study investigated the regulation of ApTrkl by overexpressing the receptor and several variants in Aplysia sensory neuron cultures. Kinase activity-dependent constitutive activation of ApTrkl was observed mainly on the plasma membrane. These studies revealed two modes of receptor internalization: (1) kinase activity-dependent internalization and (2) 5-HT-dependent, kinase activity-independent internalization. Both modes of internalization were ligand independent, and the action of 5-HT was mediated through G-protein-coupled receptors (GPCRs). On the other hand, methiothepin, an inverse agonist of 5-HT GPCRs activated endogenous ApTrkl to the same extent as 5-HT, suggesting a transactivation mechanism due to a novel coupling of GPCRs to receptor tyrosine kinase (RTK) activation that is also activated through inverse agonist binding. The neuropeptide sensorin could transiently activate ApTrkl but was not required for 5-HT-induced ApTrkl activation.

Translational Control of Long-lasting Synaptic Plasticity and Memory

Neuron. Jan, 2009  |  Pubmed ID: 19146809

Long-lasting forms of synaptic plasticity and memory are dependent on new protein synthesis. Recent advances obtained from genetic, physiological, pharmacological, and biochemical studies provide strong evidence that translational control plays a key role in regulating long-term changes in neural circuits and thus long-term modifications in behavior. Translational control is important for regulating both general protein synthesis and synthesis of specific proteins in response to neuronal activity. In this review, we summarize and discuss recent progress in the field and highlight the prospects for better understanding of long-lasting changes in synaptic strength, learning, and memory and implications for neurological diseases.

The Atypical Protein Kinase C in Aplysia Can Form a Protein Kinase M by Cleavage

Journal of Neurochemistry. May, 2009  |  Pubmed ID: 19302474

In vertebrates, a brain-specific transcript from the atypical protein kinase C (PKC) zeta gene encodes protein kinase M (PKM) zeta, a constitutively active kinase implicated in the maintenance of synaptic plasticity and memory. We have cloned the atypical PKC from Aplysia, PKC Apl III. We did not find a transcript in Aplysia encoding PKMzeta, and evolutionary analysis of atypical PKCs suggests formation of this transcript is restricted to vertebrates. Instead, over-expression of PKC Apl III in Aplysia sensory neurons leads to production of a PKM fragment of PKC Apl III. This cleavage was induced by calcium and blocked by calpain inhibitors. Moreover, nervous system enriched spliced forms of PKC Apl III show enhanced cleavage. PKC Apl III could also be activated through phosphorylation downstream of phosphoinositide 3-kinase. We suggest that PKM forms of atypical PKCs play a conserved role in memory formation, but the mechanism of formation of these kinases has changed over evolution.

Combinations of DEAD Box Proteins Distinguish Distinct Types of RNA: Protein Complexes in Neurons

Molecular and Cellular Neurosciences. Apr, 2009  |  Pubmed ID: 19340935

Transport of mRNAs to axons and dendrites in neurons is important for growth, polarization and plasticity. Recent proteomic studies in neurons have identified a number of DEAD box proteins as components of RNA granules. Using DEAD box proteins as markers, we have defined classes of RNA:protein structures present in neurons. In particular, we demonstrate that the conjunction of DEAD box 1 and DEAD box 3 identifies a motile ribosome-containing RNA granule present in both axons and dendrites that is similar to the biochemically isolated RNA granule. Conjunction of DEAD box 1 and the novel protein CGI-99 defines a distinct complex in neurons. Attempts to define a P-body like structure with expression of DEAD box 6 and decapping enzymes suggest that this structure may be more complex in neuronal processes than in other compartments. These studies hint at a great complexity in RNA transport and storage in neuronal processes.

Role of Protein Kinase C in the Induction and Maintenance of Serotonin-dependent Enhancement of the Glutamate Response in Isolated Siphon Motor Neurons of Aplysia Californica

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2009  |  Pubmed ID: 19386905

Serotonin (5-HT) mediates learning-related facilitation of sensorimotor synapses in Aplysia californica. Under some circumstances 5-HT-dependent facilitation requires the activity of protein kinase C (PKC). One critical site of PKC's contribution to 5-HT-dependent synaptic facilitation is the presynaptic sensory neuron. Here, we provide evidence that postsynaptic PKC also contributes to synaptic facilitation. We investigated the contribution of PKC to enhancement of the glutamate-evoked potential (Glu-EP) in isolated siphon motor neurons in cell culture. A 10 min application of either 5-HT or phorbol ester, which activates PKC, produced persistent (> 50 min) enhancement of the Glu-EP. Chelerythrine and bisindolylmaleimide-1 (Bis), two inhibitors of PKC, both blocked the induction of 5-HT-dependent enhancement. An inhibitor of calpain, a calcium-dependent protease, also blocked 5-HT's effect. Interestingly, whereas chelerythrine blocked maintenance of the enhancement, Bis did not. Because Bis has greater selectivity for conventional and novel isoforms of PKC than for atypical isoforms, this result implicates an atypical isoform in the maintenance of 5-HT's effect. Although induction of enhancement of the Glu-EP requires protein synthesis (Villareal et al., 2007), we found that maintenance of the enhancement does not. Maintenance of 5-HT-dependent enhancement appears to be mediated by a PKM-type fragment generated by calpain-dependent proteolysis of atypical PKC. Together, our results suggest that 5-HT treatment triggers two phases of PKC activity within the motor neuron, an early phase that may involve conventional, novel or atypical isoforms of PKC, and a later phase that selectively involves an atypical isoform.

Synapse- and Stimulus-specific Local Translation During Long-term Neuronal Plasticity

Science (New York, N.Y.). Jun, 2009  |  Pubmed ID: 19443737

Long-term memory and synaptic plasticity require changes in gene expression and yet can occur in a synapse-specific manner. Messenger RNA localization and regulated translation at synapses are thus critical for establishing synapse specificity. Using live-cell microscopy of photoconvertible fluorescent protein translational reporters, we directly visualized local translation at synapses during long-term facilitation of Aplysia sensory-motor synapses. Translation of the reporter required multiple applications of serotonin, was spatially restricted to stimulated synapses, was transcript- and stimulus-specific, and occurred during long-term facilitation but not during long-term depression of sensory-motor synapses. Translational regulation only occurred in the presence of a chemical synapse and required calcium signaling in the postsynaptic motor neuron. Thus, highly regulated local translation occurs at synapses during long-term plasticity and requires trans-synaptic signals.

PKC Differentially Translocates During Spaced and Massed Training in Aplysia

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2009  |  Pubmed ID: 19692602

Learning is highly regulated by the pattern of training. In Aplysia, an important organism for the development of cellular and molecular models of learning, spaced versus massed application of the same stimulus leads to different forms of memory. A critical molecular step underlying memory is the serotonin (5HT)-mediated activation of the novel PKC Apl II. Here, we demonstrate that activation of PKC Apl II is highly sensitive to the pattern of 5HT application. Spaced applications downregulate PKC translocation through PKA signaling, whereas massed applications lead to persistent translocation of PKC. Differential regulation of PKC translocation is mediated by competing feedback mechanisms that act through protein synthesis. These studies elucidate a fundamental molecular difference between spaced and massed training protocols.

Evolutionary Conservation of the Signaling Proteins Upstream of Cyclic AMP-dependent Kinase and Protein Kinase C in Gastropod Mollusks

Brain, Behavior and Evolution. 2009  |  Pubmed ID: 20029183

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.

Ribosomal Protein S6 Kinase is a Critical Downstream Effector of the Target of Rapamycin Complex 1 for Long-term Facilitation in Aplysia

The Journal of Biological Chemistry. Apr, 2010  |  Pubmed ID: 20177060

Long-term facilitation (LTF) in Aplysia is a leading cellular model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. In Aplysia, LTF requires translational control downstream of the target of rapamycin (TOR) complex 1 (TORC1). The major known downstream targets of TORC1 are 4E binding protein (4E-BP) and S6 kinase (S6K). By removing the site within these regulators required for their interaction with TORC1, we have generated dominant negative proteins that disrupt specific pathways downstream of TORC1. Expression of dominant negative S6K, but not dominant negative 4E-BP, in Aplysia sensory neurons (SNs) blocked 24-h LTF. TORC1 is directly activated by the small GTP-binding protein, Ras homologue enriched in brain (Rheb). To determine the effects of TORC1 activation on translation in Aplysia neurons, we have examined the effects of expressing a constitutively active form of the Aplysia orthologue of Rheb, ApRheb (ApRheb(Q63L)). Expression of ApRheb(Q63L) increased 4E-BP phosphorylation and the level of general, cap-dependent translation within the SN cell soma in a rapamycin-sensitive manner. This increase in cap-dependent translation was blocked neither by dominant negative 4E-BP nor dominant negative S6K. Thus, we demonstrate that S6K is an important downstream target of TORC1 in Aplysia and that it is necessary for 24-h LTF, but not for TORC1-mediated increases in somatic cap-dependent translation.

Postnatal Deamidation of 4E-BP2 in Brain Enhances Its Association with Raptor and Alters Kinetics of Excitatory Synaptic Transmission

Molecular Cell. Mar, 2010  |  Pubmed ID: 20347422

The eIF4E-binding proteins (4E-BPs) repress translation initiation by preventing eIF4F complex formation. Of the three mammalian 4E-BPs, only 4E-BP2 is enriched in the mammalian brain and plays an important role in synaptic plasticity and learning and memory formation. Here we describe asparagine deamidation as a brain-specific posttranslational modification of 4E-BP2. Deamidation is the spontaneous conversion of asparagines to aspartates. Two deamidation sites were mapped to an asparagine-rich sequence unique to 4E-BP2. Deamidated 4E-BP2 exhibits increased binding to the mammalian target of rapamycin (mTOR)-binding protein raptor, which effects its reduced association with eIF4E. 4E-BP2 deamidation occurs during postnatal development, concomitant with the attenuation of the activity of the PI3K-Akt-mTOR signaling pathway. Expression of deamidated 4E-BP2 in 4E-BP2(-/-) neurons yielded mEPSCs exhibiting increased charge transfer with slower rise and decay kinetics relative to the wild-type form. 4E-BP2 deamidation may represent a compensatory mechanism for the developmental reduction of PI3K-Akt-mTOR signaling.

Mechanisms of Translational Regulation in Synaptic Plasticity

Current Opinion in Neurobiology. Aug, 2010  |  Pubmed ID: 20430610

The plasticity of the nervous system is due to the ability of neurons to change their properties by altering the function of their proteome. A major mechanism for this is through altering the amount of proteins by regulating their translation. In this review, we focus on recent advances in the elucidation of the mechanisms by which neurons regulate translation during synaptic plasticity. Particular focus will be on the different transduction mechanisms that selectively target distinct elements of the mRNA in the regulation of translation during plasticity.

Aplysia Cell Adhesion Molecule and a Novel Protein Kinase C Activity in the Postsynaptic Neuron Are Required for Presynaptic Growth and Initial Formation of Specific Synapses

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2010  |  Pubmed ID: 20573882

To explore the role of both Aplysia cell adhesion molecule (ApCAM) and activity of specific protein kinase C (PKC) isoforms in the initial formation of sensory neuron synapses with specific postsynaptic targets (L7 but not L11), we examined presynaptic growth, initial synapse formation, and the expression of the presynaptic neuropeptide sensorin following cell-specific reduction of ApCAM or of a novel PKC activity. Synapse formation between sensory neurons and L7 begins by 3 h after plating and is accompanied by a rapid accumulation of a novel PKC to sites of synaptic interaction. Reducing ApCAM expression specifically from the surface of L7 blocks presynaptic growth and initial synapse formation, target-induced increase of sensorin in sensory neuron cell bodies and the rapid accumulation of the novel PKC to sites of interaction. Selective blockade of the novel PKC activity in L7, but not in sensory neurons, with injection of a dominant negative construct that interferes with the novel PKC activity, produces the same actions as downregulating ApCAM; blockade of presynaptic growth and initial synapse formation, and the target-induced increase of sensorin in sensory neuron cell bodies. The results indicate that signals initiated by postsynaptic cell adhesion molecule ApCAM coupled with the activation of a novel PKC in the appropriate postsynaptic neuron produce the retrograde signals required for presynaptic growth associated with initial synapse formation, and the target-induced expression of a presynaptic neuropeptide critical for synapse maturation.

Regulation of Protein Kinase C Apl II by Serotonin Receptors in Aplysia

Journal of Neurochemistry. Nov, 2010  |  Pubmed ID: 20964689

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.

Translation of 5' Terminal Oligopyrimidine Tract (5'TOP) MRNAs in Aplysia Californica is Regulated by the Target of Rapamycin (TOR)

Biochemical and Biophysical Research Communications. Jan, 2011  |  Pubmed ID: 21172307

Aplysia californica is a model organism for determining the molecular basis of memory. In this system identified synaptic changes have been closely linked to behavioral memories. Long-term sensitization and long-term synaptic changes between sensory neurons and motor neurons require both gene expression followed by translational control of the newly expressed mRNAs. One important mechanism for translational control is mediated through the target of rapamycin (TOR) and one mechanism downstream of TOR is the translational control of mRNAs containing a 5' terminal oligopyrimidine tract (5'TOP) sequence in their mRNA transcript. These include all ribosomal proteins, elongation factors and a few other translational regulators. TOR regulation of 5'TOP mRNAs in vertebrates is thought to be due to TOR dependent removal of the translational repression mediated by the 5'TOP sequence. Here, we show that this mechanism is similar in Aplysia, whereby Aplysia 5'TOP mRNAs are repressed under basal conditions and this repression is removed by serotonin in a rapamycin-sensitive manner.

Compartment-specific, Differential Regulation of Eukaryotic Elongation Factor 2 and Its Kinase Within Aplysia Sensory Neurons

Journal of Neurochemistry. Jun, 2011  |  Pubmed ID: 21426346

Long-term facilitation (LTF) in Aplysia is a leading model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. LTF requires translational control downstream of target of rapamycin complex 1. Our lab has previously shown that treatment with the facilitating neurotransmitter, 5-hydroxytryptamine (5-HT), causes a target of rapamycin complex 1-mediated decrease in phosphorylation of eukaryotic elongation factor 2 (eEF2) within the neurites of sensory neurons involved in LTF. Here, we characterize the Aplysia orthologue of eEF2 kinase (eEF2K). We show that the Aplysia eEF2K orthologue contains an S6 kinase phosphorylation site and that a serine-to-alanine mutation at this site blocks the ability of 5-HT to decrease eEF2 phosphorylation in neurites. We also show that within the soma, 5-HT has the opposite effect, decreasing eEF2K phosphorylation at the S6 kinase site and, concomitantly, increasing eEF2 phosphorylation. Surprisingly, while eEF2K over-expression inhibits translation of a marker for internal ribosome entry site-dependent translation, it stimulates the translation of a marker for cap-dependent translation. This study demonstrates that eEF2 is differentially regulated in separate compartments and contributes to a growing body of evidence that inhibition of elongation can stimulate the translation of certain transcripts.

Staufen 2 Regulates MGluR Long-term Depression and Map1b MRNA Distribution in Hippocampal Neurons

Learning & Memory (Cold Spring Harbor, N.Y.). 2011  |  Pubmed ID: 21508097

The two members of the Staufen family of RNA-binding proteins, Stau1 and Stau2, are present in distinct ribonucleoprotein complexes and associate with different mRNAs. Stau1 is required for protein synthesis-dependent long-term potentiation (L-LTP) in hippocampal pyramidal cells. However, the role of Stau2 in synaptic plasticity remains unexplored. We found that unlike Stau1, Stau2 is not required for L-LTP. In contrast, Stau2, but not Stau1, is necessary for DHPG-induced protein synthesis-dependent long-term depression (mGluR-LTD). While Stau2 is involved in early development of spines, its down-regulation does not alter spine morphology or spontaneous miniature synaptic activity in older cultures where LTD occurs. In addition, Stau2, but not Stau1, knockdown reduces the dendritic localization of Map1b mRNA, a specific transcript involved in mGluR-LTD. Moreover, mGluR stimulation with DHPG induces Map1b, but not Map2, mRNA dissociation from mRNA granules containing Stau2 and the ribosomal protein P0. This dissociation was not observed in cells in which Stau2 was depleted. Finally, Stau2 knockdown reduces basal Map1b protein expression in dendrites and prevents DHPG-induced increases in dendritic Map1b protein level. We suggest a role for Stau2 in the generation and regulation of Map1b mRNA containing granules that are required for mGluR-LTD.

A New Mechanism of Action of a C2 Domain-derived Novel PKC Inhibitor Peptide

Neuroscience Letters. Oct, 2011  |  Pubmed ID: 21982802

Novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. We have previously shown that the Aplysia novel PKC Apl II's C2 domain inhibits binding of diacylglycerol (DAG) to the C1 domain and that this inhibition is removed by phosphatidic acid (PA) binding to the C1b domain. Another model for C2 domain regulation of nPKCs suggests that the C2 domain binds to receptors for activated C kinase (RACKs) to assist in kinase translocation and activation. In the present study, we examined how a pharmacological peptide derived from RACK-binding site in the vertebrate novel PKCɛ regulates translocation of PKC Apl II from the cytosol to the plasma membrane. We found that a C2 domain-derived inhibitor peptide inhibited PKC Apl II translocation. This inhibition was removed by R273H mutation in the C1b domain and by phosphatidic acid, which can both remove C2-domain mediated inhibition suggesting that the peptide can regulate C1-C2 domain interactions.

The Rates of Protein Synthesis and Degradation Account for the Differential Response of Neurons to Spaced and Massed Training Protocols

PLoS Computational Biology. Dec, 2011  |  Pubmed ID: 22219722

The sensory-motor neuron synapse of Aplysia is an excellent model system for investigating the biochemical changes underlying memory formation. In this system, training that is separated by rest periods (spaced training) leads to persistent changes in synaptic strength that depend on biochemical pathways that are different from those that occur when the training lacks rest periods (massed training). Recently, we have shown that in isolated sensory neurons, applications of serotonin, the neurotransmitter implicated in inducing these synaptic changes during memory formation, lead to desensitization of the PKC Apl II response, in a manner that depends on the method of application (spaced versus massed). Here, we develop a mathematical model of this response in order to gain insight into how neurons sense these different training protocols. The model was developed incrementally, and each component was experimentally validated, leading to two novel findings: First, the increased desensitization due to PKA-mediated heterologous desensitization is coupled to a faster recovery than the homologous desensitization that occurs in the absence of PKA activity. Second, the model suggests that increased spacing leads to greater desensitization due to the short half-life of a hypothetical protein, whose production prevents homologous desensitization. Thus, we predict that the effects of differential spacing are largely driven by the rates of production and degradation of proteins. This prediction suggests a powerful mechanism by which information about time is incorporated into neuronal processing.

Inhibitory Responses in Aplysia Pleural Sensory Neurons Act to Block Excitability, Transmitter Release, and PKC Apl II Activation

Journal of Neurophysiology. Jan, 2012  |  Pubmed ID: 21994260

Expression of the 5-HT(1Apl(a)) receptor in Aplysia pleural sensory neurons inhibited 5-HT-mediated translocation of the novel PKC Apl II in sensory neurons and prevented PKC-dependent synaptic facilitation at sensory to motoneuron synapses (Nagakura et al. 2010). We now demonstrate that the ability of inhibitory receptors to block PKC activation is a general feature of inhibitory receptors and is found after expression of the 5-HT(1Apl(b)) receptor and with activation of endogenous dopamine and FMRFamide receptors in sensory neurons. Pleural sensory neurons are heterogeneous for their inhibitory response to endogenous transmitters, with dopamine being the most prevalent, followed by FMRFamide, and only a small number of neurons with inhibitory responses to 5-HT. The inhibitory response is dominant, reduces membrane excitability and synaptic efficacy, and can reverse 5-HT facilitation at both naive and depressed synapses. Indeed, dopamine can reverse PKC translocation during the continued application of 5-HT. Reversal of translocation can also be seen after translocation mediated by an analog of diacylglycerol, suggesting inhibition is not through blockade of diacylglycerol production. The effects of inhibition on PKC translocation can be rescued by phosphatidic acid, consistent with the inhibitory response involving a reduction or block of production of this lipid. However, phosphatidic acid could not recover PKC-dependent synaptic facilitation due to an additional inhibitory effect on the non-L-type calcium flux linked to synaptic transmission. In summary, we find a novel mechanism downstream of inhibitory receptors linked to inhibition of PKC activation in Aplysia sensory neurons.