Evidence of Active-Forgetting Mechanisms? Blocking Arachidonic Acid Release May Slow Forgetting of Sensitization in Aplysia.

Long-term sensitization in Aplysia is accompanied by a persistent up-regulation of mRNA encoding the peptide neurotransmitter Phe-Met-Arg-Phe-amide (FMRFa), a neuromodulator that opposes the expression of sensitization through activation of the arachidonic acid second-messenger pathway. We completed a preregistered test of the hypothesis that FMRFa plays a critical role in the forgetting of sensitization. Aplysia received long-term sensitization training and were then given whole-body injections of vehicle (N = 27), FMRFa (N = 26), or 4-bromophenacylbromide (4-BPB; N = 31), a phospholipase inhibitor that prevents the release of arachidonic acid. FMRFa produced no changes in forgetting. 4-BPB decreased forgetting measured 6 d after training [d s = 0.55 95% CI(0.01, 1.09)], though the estimated effect size is uncertain. Our results provide preliminary evidence that forgetting of sensitization may be a regulated, active process in Aplysia, but could also indicate a role for arachidonic acid in stabilizing the induction of sensitization.

Registered Report: Transcriptional Analysis of Savings Memory Suggests Forgetting is Due to Retrieval Failure.

There is fundamental debate about the nature of forgetting: some have argued that it represents the decay of the memory trace, others that the memory trace persists but becomes inaccessible because of retrieval failure. These different accounts of forgetting lead to different predictions about savings memory, the rapid re-learning of seemingly forgotten information. If forgetting is because of decay, then savings requires re-encoding and should thus involve the same mechanisms as initial learning. If forgetting is because of retrieval failure, then savings should be mechanistically distinct from encoding. In this registered report, we conducted a preregistered and rigorous test between these accounts of forgetting. Specifically, we used microarray to characterize the transcriptional correlates of a new memory (1 d after training), a forgotten memory (8 d after training), and a savings memory (8 d after training but with a reminder on day 7 to evoke a long-term savings memory) for sensitization in Aplysia californica (n = 8 samples/group). We found that the reactivation of sensitization during savings does not involve a substantial transcriptional response. Thus, savings is transcriptionally distinct relative to a newer (1-d-old) memory, with no coregulated transcripts, negligible similarity in regulation-ranked ordering of transcripts, and a negligible correlation in training-induced changes in gene expression (r = 0.04 95% confidence interval (CI) [-0.12, 0.20]). Overall, our results suggest that forgetting of sensitization memory represents retrieval failure.

Transcriptional changes before and after forgetting of a long-term sensitization memory in Aplysia californica.

Most long-term memories are forgotten, becoming progressively less likely to be recalled. Still, some memory fragments may persist, as savings memory (easier relearning) can be detected long after recall has become impossible. What happens to a memory trace during forgetting that makes it inaccessible for recall and yet still effective to spark easier re-learning? We are addressing this question by tracking the transcriptional changes that accompany learning and then forgetting of a long-term sensitization memory in the tail-elicited siphon withdrawal reflex of Aplysia californica. First, we tracked savings memory. We found that even though recall of sensitization fades completely within 1 week of training, savings memory is still detectable at 2 weeks post training. Next, we tracked the time-course of regulation of 11 transcripts we previously identified as potentially being regulated after recall has become impossible. Remarkably, 3 transcripts still show strong regulation 2 weeks after training and an additional 4 are regulated for at least 1 week. These long-lasting changes in gene expression always begin early in the memory process, within 1 day of training. We present a synthesis of our results tracking gene expression changes accompanying sensitization and provide a testable model of how sensitization memory is forgotten.

Savings memory is accompanied by transcriptional changes that persist beyond the decay of recall.

Most long-term memories are forgotten. What happens, then, to the changes in neuronal gene expression that were initially required to encode and maintain the memory? Here we show that the decay of recall for long-term sensitization memory in Aplysia is accompanied both by a form of savings memory (easier relearning) and by persistent transcriptional regulation. A behavioral experiment (N = 14) shows that sensitization training produces a robust long-term sensitization memory, but that recall fades completely within 1 wk. This apparent forgetting, though, is belied by persistent savings memory, as we found that a weak reminder protocol reinstates a long-term sensitization memory only on the previously trained side of the body. Using microarray (N = 8 biological replicates), we found that transcriptional regulation largely decays along with recall. Of the transcripts known to be regulated 1 d after training, 98% (1172/1198) are no longer significantly regulated 7 d after training. Still, there is a small set of transcripts which remain strongly regulated even when recall is absent. Using qPCR (N = 11 additional biological replicates) we confirmed that these include the peptide transmitter FMRFamide, a transcript encoding a putative homolog of spectrin beta chain (Genbank: EB255259) , a transcript encoding a protein with a predicted EF-hand calcium-binding domain (Genbank: EB257711), and eight uncharacterized transcripts. To our knowledge, this is the first work to show that transcriptional changes evoked by learning can outlast recall. The small set of transcriptional changes that persist could mediate the rapid relearning of the memory (savings), or the decay of recall, or both, or neither.

Transcriptional correlates of memory maintenance following long-term sensitization of Aplysia californica.

We characterized the transcriptional response accompanying maintenance of long-term sensitization (LTS) memory in the pleural ganglia of Aplysia californica using microarray (N = 8) and qPCR (N = 11 additional samples). We found that 24 h after memory induction there is strong regulation of 1198 transcripts (748 up and 450 down) in a pattern that is almost completely distinct from what is observed during memory encoding (1 h after training). There is widespread up-regulation of transcripts related to all levels of protein production, from transcription (e.g., subunits of transcription initiation factors) to translation (e.g., subunits of eIF1, eIF2, eIF3, eIF4, eIF5, and eIF2B) to activation of components of the unfolded protein response (e.g., CREB3/Luman, BiP, AATF). In addition, there are widespread changes in transcripts related to cytoskeleton function, synaptic targeting, synaptic function, neurotransmitter regulation, and neuronal signaling. Many of the transcripts identified have previously been linked to memory and plasticity (e.g., Egr, menin, TOB1, IGF2 mRNA binding protein 1/ZBP-1), though the majority are novel and/or uncharacterized. Interestingly, there is regulation that could contribute to metaplasticity potentially opposing or even eroding LTS memory (down-regulation of adenylate cyclase and a putative serotonin receptor, up-regulation of FMRFa and a FMRFa receptor). This study reveals that maintenance of a "simple" nonassociative memory is accompanied by an astonishingly complex transcriptional response.

Transcriptional analysis of a whole-body form of long-term habituation in Aplysia californica.

Habituation is the simplest form of learning, but we know little about the transcriptional mechanisms that encode long-term habituation memory. A key obstacle is that habituation is relatively stimulus-specific and is thus encoded in small sets of neurons, providing poor signal/noise ratios for transcriptional analysis. To overcome this obstacle, we have developed a protocol for producing whole-body long-term habituation of the siphon-withdrawal reflex (SWR) of Aplysia californica. Specifically, we constructed a computer-controlled brushing apparatus to apply low-intensity tactile stimulation over the entire dorsal surface of Aplysia at regular intervals. We found that 3 d of training (10 rounds of stimulation/day; each round = 15 min brushing at a 10-sec ISI; 15-min rest between rounds) produces habituation with several characteristics favorable for mechanistic investigation. First, habituation is widespread, with SWR durations reduced whether the reflex is evoked by tactile stimulation to the head, tail, or the siphon. Second, long-term habituation is sensitive to the pattern of training, occurring only when brushing sessions are spaced out over 3 d rather than massed into a single session. Using a custom-designed microarray and quantitative PCR, we show that long-term habituation produces long-term up-regulation of an apparent Aplysia homolog of cornichon, a protein important for glutamate receptor trafficking. Our training paradigm provides a promising starting point for characterizing the transcriptional mechanisms of long-term habituation memory.

Immediate and persistent transcriptional correlates of long-term sensitization training at different CNS loci in Aplysia californica.

Repeated noxious stimulation produces long-term sensitization of defensive withdrawal reflexes in Aplysia californica, a form of long-term memory that requires changes in both transcription and translation. Previous work has identified 10 transcripts which are rapidly up-regulated after long-term sensitization training in the pleural ganglia. Here we use quantitative PCR to begin examining how these transcriptional changes are expressed in different CNS loci related to defensive withdrawal reflexes at 1 and 24 hours after long-term sensitization training. Specifically, we sample from a) the sensory wedge of the pleural ganglia, which exclusively contains the VC nociceptor cell bodies that help mediate input to defensive withdrawal circuits, b) the remaining pleural ganglia, which contain withdrawal interneurons, and c) the pedal ganglia, which contain many motor neurons. Results from the VC cluster show different temporal patterns of regulation: 1) rapid but transient up-regulation of Aplysia homologs of C/EBP, C/EBPγ, and CREB1, 2) delayed but sustained up-regulation of BiP, Tolloid/BMP-1, and sensorin, 3) rapid and sustained up-regulation of Egr, GlyT2, VPS36, and an uncharacterized protein (LOC101862095), and 4) an unexpected lack of regulation of Aplysia homologs of calmodulin (CaM) and reductase-related protein (RRP). Changes in the remaining pleural ganglia mirror those found in the VC cluster at 1 hour but with an attenuated level of regulation. Because these samples had almost no expression of the VC-specific transcript sensorin, our data suggests that sensitization training likely induces transcriptional changes in either defensive withdrawal interneurons or neurons unrelated to defensive withdrawal. In the pedal ganglia, we observed only a rapid but transient increase in Egr expression, indicating that long-term sensitization training is likely to induce transcriptional changes in motor neurons but raising the possibility of different transcriptional endpoints in this cell type.

Characterization of the rapid transcriptional response to long-term sensitization training in Aplysia californica.

We used a custom-designed microarray and quantitative PCR to characterize the rapid transcriptional response to long-term sensitization training in the marine mollusk Aplysia californica. Aplysia were exposed to repeated noxious shocks to one side of the body, a procedure known to induce a long-lasting, transcription-dependent increase in reflex responsiveness that is restricted to the side of training. One hour after training, pleural ganglia from the trained and untrained sides of the body were harvested; these ganglia contain the sensory nociceptors which help mediate the expression of long-term sensitization memory. Microarray analysis from 8 biological replicates suggests that long-term sensitization training rapidly regulates at least 81 transcripts. We used qPCR to test a subset of these transcripts and found that 83% were confirmed in the same samples, and 86% of these were again confirmed in an independent sample. Thus, our new microarray design shows strong convergent and predictive validity for analyzing the transcriptional correlates of memory in Aplysia. Fully validated transcripts include some previously identified as regulated in this paradigm (ApC/EBP and ApEgr) but also include novel findings. Specifically, we show that long-term sensitization training rapidly up-regulates the expression of transcripts which may encode Aplysia homologs of a C/EBPγ transcription factor, a glycine transporter (GlyT2), and a vacuolar-protein-sorting-associated protein (VPS36).

Harmonin enhances voltage-dependent facilitation of Cav1.3 channels and synchronous exocytosis in mouse inner hair cells.

Cav1.3 channels mediate Ca(2+) influx that triggers exocytosis of glutamate at cochlear inner hair cell (IHC) synapses. Harmonin is a PDZ-domain-containing protein that interacts with the C-terminus of the Cav1.3 α1 subunit (α11.3) and controls cell surface Cav1.3 levels by promoting ubiquitin-dependent proteosomal degradation. However, PDZ-domain-containing proteins have diverse functions and regulate other Cav1.3 properties, which could collectively influence presynaptic transmitter release. Here, we report that harmonin binding to the α11.3 distal C-terminus (dCT) enhances voltage-dependent facilitation (VDF) of Cav1.3 currents both in transfected HEK293T cells and in mouse inner hair cells. In HEK293T cells, this effect of harmonin was greater for Cav1.3 channels containing the auxiliary Cav ß1 than with the ß2 auxiliary subunit. Cav1.3 channels lacking the α11.3 dCT were insensitive to harmonin modulation. Moreover, the 'deaf-circler' dfcr mutant form of harmonin, which does not interact with the α11.3 dCT, did not promote VDF. In mature IHCs from mice expressing the dfcr harmonin mutant, Cav1.3 VDF was less than in control IHCs. This difference was not observed between control and dfcr IHCs prior to hearing onset. Membrane capacitance recordings from dfcr IHCs revealed a role for harmonin in synchronous exocytosis and in increasing the efficiency of Ca(2+) influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Cav1.3 Ca(2+) channels and exocytosis.

An Aplysia Egr homolog is rapidly and persistently regulated by long-term sensitization training.

The Egr family of transcription factors plays a key role in long-term plasticity and memory in a number of vertebrate species. Here we identify and characterize ApEgr (GenBank: KC608221), an Egr homolog in the marine mollusk Aplysia californica. ApEgr codes for a predicted 593-amino acid protein with the highly conserved trio of zinc-fingered domains in the C-terminus that characterizes the Egr family of transcription factors. Promoter analysis shows that the ApEgr protein selectively recognizes the GSG motif recognized by vertebrate Egrs. Like mammalian Egrs, ApEgr is constitutively expressed in a range of tissues, including the CNS. Moreover, expression of ApEgr is bi-directionally regulated by changes in neural activity. Of most interest, the association between ApEgr function and memory may be conserved in Aplysia, as we observe rapid and long-lasting up-regulation of expression after long-term sensitization training. Taken together, our results suggest that Egrs may have memory functions that are conserved from mammals to mollusks.

Transcriptional changes following long-term sensitization training and in vivo serotonin exposure in Aplysia californica.

We used Aplysia californica to compare the transcriptional changes evoked by long-term sensitization training and by a treatment meant to mimic this training, in vivo exposure to serotonin. We focused on 5 candidate plasticity genes which are rapidly up-regulated in the Aplysia genus by in vivo serotonin treatment, but which have not yet been tested for regulation during sensitization: CREB1, matrilin, antistasin, eIF3e, and BAT1 homolog. CREB1 was rapidly up-regulated by both treatments, but the regulation following training was transient, falling back to control levels 24 hours after training. This suggests some caution in interpreting the proposed role of CREB1 in consolidating long-term sensitization memory. Both matrilin and eIF3e were up-regulated by in vivo serotonin but not by long-term sensitization training. This suggests that in vivo serotonin may produce generalized transcriptional effects that are not specific to long-term sensitization learning. Finally, neither treatment produced regulation of antistasin or BAT1 homolog, transcripts regulated by in vivo serotonin in the closely related Aplysia kurodai. This suggests either that these transcripts are not regulated by experience, or that transcriptional mechanisms of memory may vary within the Aplysia genus.

Harmonin inhibits presynaptic Cav1.3 Ca²âº channels in mouse inner hair cells.

Harmonin is a scaffolding protein that is required for normal mechanosensory function in hair cells. We found a presynaptic association of harmonin and Ca(v)1.3 Ca(2+) channels at the mouse inner hair cell synapse, which limits channel availability through a ubiquitin-dependent pathway.

Regulation of the preprotachykinin-I gene promoter through a protein kinase A-dependent, cyclic AMP response element-binding protein-independent mechanism.

Preprotachykinin-I (PPT) gene expression is regulated by a number of stimuli that signal through cyclic AMP (cAMP)-mediated pathways. In the present study, forskolin, an adenylyl cyclase stimulator, significantly increased PPT mRNA levels in PPT-expressing RINm5F cells, an effect paralleled by an increase in PPT promoter-luciferase reporter construct activity. The forskolin-induced stimulation of PPT transcription was protein kinase A dependent (PKA), as shown by blockade with the PKA inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide. We found that the activation protein 1/cAMP response element (AP1/CRE) site centered at -196 relative to the transcription start site was important for basal and forskolin-induced PPT promoter activity. Because of the involvement of PKA and the similarity of the AP1/CRE element to consensus CRE sequences, we investigated the role of CRE-binding protein (CREB) in the regulation of the PPT promoter. Surprisingly, overexpression of a dominant-negative CREB (i.e. CREB-A) did not affect basal or forskolin-induced PPT promoter activity. Furthermore, binding of CREB to the PPT promoter AP1/CRE site was not demonstrable in electrophoretic mobility shift assays. Rather, our experiments suggested that c-Jun is a member of the complex that binds to this site. We conclude that, at least in RINm5F cells, cAMP-mediated up-regulation of PPT gene expression does not involve CREB or CREB-related transcription factor recruitment to the AP1/CRE site.