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A more thorough description of the changes in synaptic strength underlying synaptic plasticity may be achieved with quantal resolution measurements at individual synaptic sites. Here, we demonstrate that by using a membrane targeted genetic calcium sensor, we can measure quantal synaptic events at the individual synaptic sites of "Aplysia" sensory neuron to motor neuron synaptic connections. These results show that synaptic strength is not evenly distributed between all contacts in these cultures, but dominated by multiquantal sites of synaptic contact, likely clusters of individual synaptic sites. Surprisingly, most synaptic contacts were not found opposite presynaptic varicosities, but instead at areas of pre- and postsynaptic contact with no visible thickening of membranes. The release probability, quantal size, and quantal content can be measured over days at individual synaptic contacts using this technique. Homosynaptic depression was accompanied by a reduction in release site probability, with no evidence of individual synaptic site silencing over the course of depression. This technique shows promise in being able to address outstanding questions in this system, including determining the synaptic changes that maintain long-term alterations in synaptic strength that underlie memory.

Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems, including the hermaphrodite marine mollusk, Aplysia californica Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform is required, the kidney and brain expressed adaptor protein (KIBRA) is also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform-specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant-negative-PKMs to erase LTF is correlated with isoform-specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an α-helix "handle" in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in Aplysia SIGNIFICANCE STATEMENT Long-lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs are not isoform-specific, isoform specificity is defined by a "handle" helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.

In neurons, mRNAs can be repressed postinitiation and assembled into granules enabling the transport and later, regulated reactivation of the paused mRNAs. It has been suggested that a large percentage of transcripts in neuronal processes are stored in these stalled polysomes. Given this, it is predicted that nascent peptides should be abundant in these granules. Nascent peptides can be visualized in real time by the SunTag system. Using this system, we observe nascent peptides in neuronal processes that are resistant to runoff with the initiation inhibitor homoharringtonin (HHT) and to release by puromycin, properties expected from RNA granules consisting of stalled polysomes. In contrast, nascent peptides in nonneuronal cells and neuronal cell bodies were not resistant to HHT or puromycin. Stalled polysomes can also be visualized after runoff with ribopuromycylation and the RNA granules imaged with ribopuromycylation were the same as those with SunTag visualized nascent peptides. Accordingly, the ribopuromycylated puncta in neuronal dendrites were also resistant to puromycin. Thus, the SunTag technique corroborates in situ evidence of stalled polysomes and will allow for the live examination of these translational structures as a mechanism for mRNA transport and regulated protein synthesis.

The integrated stress response (ISR), a pivotal protein homeostasis network, plays a critical role in the formation of long-term memory (LTM). The precise mechanism by which the ISR controls LTM is not well understood. Here, we report insights into how the ISR modulates the mnemonic process by using targeted deletion of the activating transcription factor 4 (ATF4), a key downstream effector of the ISR, in various neuronal and non-neuronal cell types. We found that the removal of ATF4 from forebrain excitatory neurons (but not from inhibitory neurons, cholinergic neurons, or astrocytes) enhances LTM formation. Furthermore, the deletion of ATF4 in excitatory neurons lowers the threshold for the induction of long-term potentiation, a cellular model for LTM. Transcriptomic and proteomic analyses revealed that ATF4 deletion in excitatory neurons leads to upregulation of components of oxidative phosphorylation pathways, which are critical for ATP production. Thus, we conclude that ATF4 functions as a memory repressor selectively within excitatory neurons. [ABSTRACT FROM AUTHOR]

Atypical PKM, a persistently active form of atypical PKC, is proposed to be a molecular memory trace, but there have been few examinations of the role of PKMs generated from other PKCs. We demonstrate that inhibitors used to inhibit PKMs generated from atypical PKCs are also effective inhibitors of other PKMs. In contrast, we demonstrate that dominant-negative PKMs show isoform-specificity. A dominant-negative PKM from the classical PKC Apl I blocks activity-dependent intermediate-term facilitation (a-ITF) when expressed in the sensory neuron, while a dominant-negative PKM from the atypical PKC Apl III does not. Consistent with a specific role for PKM Apl I in activity-dependent facilitation, live imaging FRET-based cleavage assays reveal that activity leads to cleavage of the classical PKC Apl I, but not the atypical PKC Apl III in the sensory neuron varicosities of "Aplysia." In contrast, massed intermediate facilitation (m-ITF) induced by 10 min of 5HT is sufficient for cleavage of the atypical PKC Apl III in the motor neuron. Interestingly, both cleavage of PKC Apl I in the sensory neuron during a-ITF and cleavage of PKC Apl III in the motor neuron during m-ITF are inhibited by a dominant-negative form of a penta-EF hand containing classical calpain cloned from "Aplysia." Consistent with a role for PKMs in plasticity, this dominant-negative calpain also blocks both a-ITF when expressed in the sensory neuron and m-ITF when expressed in the motor neuron. This study broadens the role of PKMs in synaptic plasticity in two significant ways: (i) PKMs generated from multiple isoforms of PKC, including classical isoforms, maintain memory traces; (ii) PKMs play roles in the presynaptic neuron.

The presynaptic release apparatus can be specialized to enable specific synaptic functions. Habituation is the diminishing of a physiological response to a frequently repeated stimulus and in Aplysia, habituation to touch is mediated by a decrease in transmitter release from the sensory neurons that respond to touch even after modest rates of action potential firing. This synaptic depression is not common among Aplysia synaptic connections suggesting the presence of a release apparatus specialized for this depression. We found that specific splice forms of ApCaV2, the calcium channel required for transmitter release, are preferentially used in sensory neurons, consistent with a specialized release apparatus. However, we were not able to find a specific ApCaV2 splice uniquely required for synaptic depression. The C-terminus of ApCaV2 alpha1 subunit retains conserved binding to Aplysia rab-3 interacting molecule (ApRIM) and ApRIM-binding protein (ApRBP) and the C-terminus is required for full synaptic expression of ApCaV2. We also identified a splice form of ApRIM that did not interact with the ApCav2 alpha 1 subunit, but it was not preferentially used in sensory neurons. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamcyin (mTOR) is a central player in cell growth throughout the organism. However, mTOR takes on an additional, more specialized role in the developed neuron, where it regulates the protein synthesis-dependent, plastic changes underlying learning and memory. mTOR is sequestered in two multiprotein complexes (mTORC1 and mTORC2) that have different substrate specificities, thus allowing for distinct functions at synapses. We will examine how learning activates the mTOR complexes, survey the critical effectors of this pathway in the context of synaptic plasticity, and assess whether mTOR plays an instructive or permissive role in generating molecular memory traces.

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.

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.

A study was conducted on ability of fusion proteins containing different domains of the protein kinase C (PKC) Apl II regulatory region to bind phorbol ester. The findings suggest that the presence of the C2 domain lowers the affinity of protein kinase C activators for the C1 domains. Moreover, phosphatidic acid is more potent than phosphatidylserine in reducing C2 domain-mediated inhibition, implying the phosphatidic acid may be an essential cofactor for the activation of PKC Apl II.