Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in Aplysia.

Long-term memory (LTM) is believed to be stored in the brain as changes in synaptic connections. Here, we show that LTM storage and synaptic change can be dissociated. Cocultures of Aplysia sensory and motor neurons were trained with spaced pulses of serotonin, which induces long-term facilitation. Serotonin (5HT) triggered growth of new presynaptic varicosities, a synaptic mechanism of long-term sensitization. Following 5HT training, two antimnemonic treatments-reconsolidation blockade and inhibition of PKM--caused the number of presynaptic varicosities to revert to the original, pretraining value. Surprisingly, the final synaptic structure was not achieved by targeted retraction of the 5HT-induced varicosities but, rather, by an apparently arbitrary retraction of both 5HT-induced and original synapses. In addition, we find evidence that the LTM for sensitization persists covertly after its apparent elimination by the same antimnemonic treatments that erase learning-related synaptic growth. These results challenge the idea that stable synapses store long-term memories

Habituation of the C-start response in larval zebrafish exhibits several distinct phases and sensitivity to NMDA receptor blockade.

The zebrafish larva has been a valuable model system for genetic and molecular studies of development. More recently, biologists have begun to exploit the surprisingly rich behavioral repertoire of zebrafish larvae to investigate behavior. One prominent behavior exhibited by zebrafish early in development is a rapid escape reflex (the C-start). This reflex is mediated by a relatively simple neural circuit, and is therefore an attractive model behavior for neurobiological investigations of simple forms of learning and memory. Here, we describe two forms of short-lived habituation of the C-start in response to brief pulses of auditory stimuli. A rapid form, persisting for ≥1 min but <15 min, was induced by 120 pulses delivered at 0.5-2.0 Hz. A more extended form (termed "short-term habituation" here), which persisted for ≥25 min but <1 h, was induced by spaced training. The spaced training consisted of 10 blocks of auditory pulses delivered at 1 Hz (5 min interblock interval, 900 pulses per block). We found that these two temporally distinguishable forms of habituation are mediated by different cellular mechanisms. The short-term form depends on activation of N-methyl-d-aspartate receptors (NMDARs), whereas the rapid form does not

Long-term habituation of the gill-withdrawal reflex in aplysia requires gene transcription, calcineurin and L-type voltage-gated calcium channels.

Although habituation is possibly the simplest form of learning, we still do not fully understand the neurobiological basis of habituation in any organism. To advance the goal of a comprehensive understanding of habituation, we have studied long-term habituation (LTH) of the gill-withdrawal reflex (GWR) in the marine snail Aplysia californica. Previously, we showed that habituation of the GWR in a reduced preparation lasts for up to 12 h, and depends on protein synthesis, as well as activation of protein phosphatases 1 and 2A and postsynaptic glutamate receptors. Here, we have used the reduced preparation to further analyze the mechanisms of LTH in Aplysia. We found that LTH of the GWR depends on RNA synthesis because it was blocked by both the irreversible transcriptional inhibitor actinomycin-D and the reversible transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside (DRB). In addition, LTH requires activation of protein phosphatase 2B (calcineurin), because it was disrupted by ascomycin. Finally, LTH was blocked by nitrendipine, which indicates that activation of l-type voltage-gated Ca(2+) channels is required for this form of learning. Together with our previous results, the present results indicate that exclusively presynaptic mechanisms, although possibly sufficient for short-term habituation, are insufficient for LTH. Rather, LTH must involve postsynaptic, as well as presynaptic, mechanisms

Long-Term Habituation of the Gill-Withdrawal Reflex in Aplysia Requires Gene Transcription, Calcineurin and L-Type Voltage-Gated Calcium Channels

Although habituation is possibly the simplest form of learning, we still do not fully understand the neurobiological basis of habituation in any organism. To advance the goal of a comprehensive understanding of habituation, we have studied long-term habituation (LTH) of the gill-withdrawal reflex (GWR) in the marine snail Aplysia californica. Previously, we showed that habituation of the GWR in a reduced preparation lasts for up to 12 h, and depends on protein synthesis, as well as activation of protein phosphatases 1 and 2A and postsynaptic glutamate receptors. Here, we have used the reduced preparation to further analyze the mechanisms of LTH in Aplysia. We found that LTH of the GWR depends on RNA synthesis because it was blocked by both the irreversible transcriptional inhibitor actinomycin-D and the reversible transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside (DRB). In addition, LTH requires activation of protein phosphatase 2B (calcineurin), because it was disrupted by ascomycin. Finally, LTH was blocked by nitrendipine, which indicates that activation of l-type voltage-gated Ca2+ channels is required for this form of learning. Together with our previous results, the present results indicate that exclusively presynaptic mechanisms, although possibly sufficient for short-term habituation, are insufficient for LTH. Rather, LTH must involve postsynaptic, as well as presynaptic, mechanisms

Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia.

Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report that LTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and maintenance of LTM depend on DNA methylation

RNA from Trained Aplysia

The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it is reported that RNA extracted from the central nervous system of Aplysia given long-term sensitization (LTS) training induced sensitization when injected into untrained animals; furthermore, the RNA-induced sensitization, like training-induced sensitization, required DNA methylation. In cellular experiments, treatment with RNA extracted from trained animals was found to increase excitability in sensory neurons, but not in motor neurons, dissociated from naïve animals. Thus, the behavioral, and a subset of the cellular, modifications characteristic of a form of nonassociative long-term memory (LTM) in Aplysia can be transferred by RNA. These results indicate that RNA is sufficient to generate an engram for LTS in Aplysia and are consistent with the hypothesis that RNA-induced epigenetic changes underlie memory storage in Aplysia

The Potential Role of Postsynaptic Phospholipase C Activity in Synaptic Facilitation and Behavioral Sensitization in Aplysia

Previous findings indicate that synaptic facilitation, a cellular mechanism underlying sensitization of the siphon withdrawal response (SWR) in Aplysia, depends on a cascade of postsynaptic events, including activation of inositol triphosphate (IP3) receptors and release of Ca2+ from postsynaptic intracellular stores. These findings suggest that phospholipase C (PLC), the enzyme that catalyzes IP3 formation, may play an important role in postsynaptic signaling during facilitation and learning in Aplysia. Using the PLC inhibitor U73122, we found that PLC activity is required for synaptic facilitation following a 10-min treatment with 5-HT, as measured at 20 min after 5-HT washout. Prior work has indicated that facilitation at this time is supported primarily by postsynaptic processes. To determine whether postsynaptic PLC activity is involved in 5-HT–mediated facilitatory actions, we examined the effect of U73122 on enhancement of the response of motor neurons isolated in cell culture to glutamate, the sensory neuron transmitter. A 10-min application of 5-HT induced persistent (>40 min) enhancement of glutamate-evoked potentials (Glu-EPs) recorded from isolated motor neurons, and this enhancement was blocked by U73122. Finally, we showed that injecting U73122 into intact animals before behavioral training impaired intermediate-term sensitization, indicating that PLC activity contributes to this form of nonassociative learning

RNA from Trained Aplysia Can Induce an Epigenetic Engram for Long-Term Sensitization in Untrained Aplysia.

The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it is reported that RNA extracted from the central nervous system of Aplysia given long-term sensitization (LTS) training induced sensitization when injected into untrained animals; furthermore, the RNA-induced sensitization, like training-induced sensitization, required DNA methylation. In cellular experiments, treatment with RNA extracted from trained animals was found to increase excitability in sensory neurons, but not in motor neurons, dissociated from naïve animals. Thus, the behavioral, and a subset of the cellular, modifications characteristic of a form of nonassociative long-term memory (LTM) in Aplysia can be transferred by RNA. These results indicate that RNA is sufficient to generate an engram for LTS in Aplysia and are consistent with the hypothesis that RNA-induced epigenetic changes underlie memory storage in Aplysia

Isoform Specificity of PKMs during Long-Term Facilitation in Aplysia Is Mediated through Stabilization by KIBRA

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