Enhanced Long-term Memory Formation Research Articles

No food for thought: An intermediate level of food deprivation enhances memory in Lymnaea.

Nutritional status plays an important role in cognitive functioning but there is disagreement on the role that food deprivation plays in learning and memory. In this study, we investigated the behavioral and transcriptional effects induced by different lengths of food deprivation: 1 day, which is a short-time period of food deprivation, and 3 days, which is an 'intermediate' level of food deprivation. Snails were subjected to different feeding regimens and then trained for operant conditioning of aerial respiration where they received a single 0.5h training session followed by a long-term memory (LTM) test 24h later. Immediately after the memory test, snails were sacrificed and the expression levels of key genes for neuroplasticity, energy balance, and stress response were measured in the central ring ganglia. We found that 1 day of food deprivation was not sufficient to enhance snails' LTM formation and subsequently did not result in any significant transcriptional effects. However, 3 days of food deprivation resulted in enhanced LTM formation and caused the upregulation of neuroplasticity and stress-related genes and the downregulation of serotonin-related genes. These data provide further insight into how nutritional status and related molecular mechanisms impact cognitive function.

CREBB repression of protein synthesis in mushroom body gates long-term memory formation in Drosophila

Learned experiences are not necessarily consolidated into long-term memory (LTM) unless they are periodic and meaningful. LTM depends on de novo protein synthesis mediated by cyclic AMP response element-binding protein (CREB) activity. In Drosophila, two creb genes (crebA, crebB) and multiple CREB isoforms have reported influences on aversive olfactory LTM in response to multiple cycles of spaced conditioning. How CREB isoforms regulate LTM effector genes in various neural elements of the memory circuit is unclear, especially in the mushroom body (MB), a prominent associative center in the fly brain that has been shown to participate in LTM formation. Here, we report that i) spaced training induces crebB expression in MB α-lobe neurons and ii) elevating specific CREBB isoform levels in the early α/βsubpopulation of MB neurons enhances LTM formation. By contrast, learning from weak training iii) induces 5-HT1A serotonin receptor synthesis, iv) activates 5-HT1A in early α/βneurons, and v) inhibits LTM formation. vi) LTM is enhanced when this inhibitory effect is relieved by down-regulating 5-HT1A or overexpressing CREBB. Our findings show that spaced training-induced CREBB antagonizes learning-induced 5-HT1A in early α/βMB neurons to modulate LTM consolidation.

CREBA and CREBB in two identified neurons gate long-term memory formation in Drosophila

Episodic events are frequently consolidated into labile memory but are not necessarily transferred to persistent long-term memory (LTM). Regulatory mechanisms leading to LTM formation are poorly understood, however, especially at the resolution of identified neurons. Here, we demonstrate enhanced LTM following aversive olfactory conditioning in Drosophila when the transcription factor cyclic AMP response element binding protein A (CREBA) is induced in just two dorsal-anterior-lateral (DAL) neurons. Our experiments show that this process is regulated by protein-gene interactions in DAL neurons: (1) crebA transcription is induced by training and repressed by crebB overexpression, (2) CREBA bidirectionally modulates LTM formation, (3) crebA overexpression enhances training-induced gene transcription, and (4) increasing membrane excitability enhances LTM formation and gene expression. These findings suggest that activity-dependent gene expression in DAL neurons during LTM formation is regulated by CREB proteins.

Black tea differs from green tea: it suppresses long-term memory formation in Lymnaea

ABSTRACTFoods, such as Green tea (GT), containing the flavonol, (-)-Epicatechin (Epi), enhance the formation of long-term memory (LTM) when snails are operantly conditioned in that substance. That is, a single 0.5 h training session results in LTM; whereas similar training in pond water does not result in LTM. It was of interest to determine if Black tea (BT), which is a more popular beverage than GT and which is derived from the same tea leaves, also enhances LTM formation. We found that BT, unlike GT, depressed homeostatic aerial respiratory behaviour and obstructed LTM formation. We used two different methods to determine if BT altered LTM formation and both procedures showed us that BT obstructed LTM formation. We conclude that BT obstructs LTM formation and depresses homeostatic aerial respiration

Heat stress enhances LTM formation in Lymnaea: role of HSPs and DNA methylation.

Environmentally relevant stressors alter the memory-forming process in Lymnaea following operant conditioning of aerial respiration. One such stressor is heat. Previously, we found that following a 1 h heat shock, long-term memory (LTM) formation was enhanced. We also had shown that the heat stressor activates at least two heat shock proteins (HSPs): HSP40 and HSP70. Here, we tested two hypotheses: (1) the production of HSPs is necessary for enhanced LTM formation; and (2) blocking DNA methylation prevents the heat stressor-induced enhancement of LTM formation. We show here that the enhancing effect of the heat stressor on LTM formation occurs even if snails experienced the stressor 3 days previously. We further show that a flavonoid, quercetin, which inhibits HSP activation, blocks the enhancing effect of the heat stressor on LTM formation. Finally, we show that injection of a DNA methylation blocker, 5-AZA, before snails experience the heat stressor prevents enhancement of memory formation.

Training Lymnaea in the presence of a predator scent results in a long-lasting ability to form enhanced long-term memory.

Lymnaea exposed to crayfish effluent (CE) gain an enhanced ability to form long-term memory (LTM). We test the hypothesis that a single CE exposure and operant conditioning training leads to long lasting changes in the capability of snails to form LTM when tested in pond water fourweeks later. We trained both juvenile and adult snails with a single 0.5h training session in CE and show that LTM was present 24h later. Snails trained in a similar manner in just pond water show no LTM. We then asked if such training in CE conferred enhanced memory forming capabilities on these snails fourweeks later. That is, would LTM be formed in these snails fourweeks later following a single 0.5h training session in pond water? We found that both adult and juvenile snails previously trained in CE onemonth previously had enhanced LTM formation abilities. The injection of a DNA methylation blocker, 5-AZA, prior to training in adult snails blocked enhanced LTM formation fourweeks later. Finally, this enhanced LTM forming ability was not passed on to the next generation of snails.

Spaced taste avoidance conditioning in Lymnaea

We succeeded in taste avoidance conditioning with sucrose as the conditional stimulus (CS) and an electrical stimulus (∼1000V, 80μA) as the unconditional stimulus (US). With 15 paired CS–US presentations on a single day, we were able to elicit both short-term memory (STM) and long-term memory (LTM) persisting for at least one week. However, while STM was elicited with 5, 8, 10, and 20 paired presentations of the CS–US on a single day, LTM was not. We found, however, that if we inserted a 3h interval between a first and a second set of CS–US pairings that both 8 and 20 paired CS–US presentations on a single day was now sufficient to cause LTM formation. Exposing snails to bryostatin before or during training enhanced LTM formation such that 8 paired presentations of the CS–US resulted in LTM.

Interaction between environmental stressors mediated via the same sensory pathway

The great pond snail, Lymnaea stagnalis, is a calciphile, requiring approximately 20 mg/l dissolved calcium for natural populations to live long and prosper. However, despite population survival we have previously demonstrated that acute exposure for 1 week to low environmental calcium (20 mg/l) acts as a stressor on the snail, blocking long-term memory (LTM) formation. This response to calcium availability is mediated by the snail directly sensing the calcium concentration in its environment using a sensory structure called the osphradium. In addition to sensing the calcium environment, the osphradium also mediates the response to predator kairomones which has an opposite effect on memory i.e. kairomone exposure during training enhances LTM formation. Here we demonstrate how these two stressors (low calcium availability and predator kairomones), that alter memory formation in opposing directions via the same sensory system, interact when experienced simultaneously.

Upregulation of CREB-Mediated Transcription Enhances Both Short- and Long-Term Memory

Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.

Intracellular Trafficking of Histone Deacetylase 4 Regulates Long‐Term Memory Formation

Histone acetylation is important for gene transcription, which is controlled by the balance between two kinds of opposing enzymes: histone acetyltransferases and histone deacetylases (HDACs). HDACs repress gene transcription by decreasing histone acetylation levels. Our hypothesis was that shuttling of Class II HDACs, such as HDAC4, between the nucleus and cytoplasm is critical for its function. We constructed mutants of mammalian HDAC4 that had different cellular locations and checked their function during memory formation using Caenorhabditis elegans as a model. The deletion of hda4, a homolog of HDAC4, was able to enhance learning and long-term memory (LTM) in a thermotaxis model. Transgenic experiments showed that mammalian wild-type HDAC4 rescued the phenotype of hda4-deleted worms but impaired LTM formation in wild-type worms. The cytosol-localized HDAC4 mutant was not able to alter the phenotype of knock-out worms but led to enhanced LTM formation in wild-type worms similar to hda4-deletion mutants. Constitutive nuclear localization of HDAC4 rescued the phenotype of deletion worms similar to wild-type HDAC4 but had no effect on wild-type worms. These results support our hypothesis that HDAC4's biological function is regulated by its intracellular distribution.

The role of serotonin in the enhancement of long-term memory resulting from predator detection in Lymnaea

Serotonergic systems play important roles in modulating stress-induced arousal and vigilance behaviours. The pond snail, Lymnaea, shows multiple defensive vigilance behaviours in response to the stress associated with predator detection. Predator detection elicited by crayfish effluent (CE), increases the time to re-emerge from the shell and enhances the shadow withdrawal response. More importantly, in Lymnaea, CE enhances the ability to form long-term memory (LTM). We investigated the role of the serotonergic system in these anti-predator responses in Lymnaea. Using a serotonin-receptor antagonist, mianserin, we found that two defensive vigilance behaviours (e.g. increasing the time to re-emerge from their shell and shadow response) elicited by CE were not observed when the serotonergic system was disrupted. Also, methysergide, another serotonin antagonist, blocked the enhanced LTM formation after training in CE. Importantly, mianserin did not alter LTM formation in pond water (PW). These data suggest that a serotonergic system is activated only when Lymnaea detect a predator. When snails were trained in CE using a training procedure that in PW produces a 24-h LTM, a more persistent form of LTM (5 days) occurred. This more persistent form of LTM was abolished after mianserin treatment. Increasing 5-HT levels in the snail by the injection of 5-HT was also associated with enhanced LTM formation. Lastly, we tested whether the osphradium is implicated in CE detection and subsequent enhanced formation of LTM. Cutting the osphradial nerve to the CNS resulted in the loss of the ability to form enhanced LTM in CE. Together, these findings support the hypothesis that the serotonergic system plays a key role in modulating the predator-induced stress responses in Lymnaea.

Increase in excitability of RPeD11 results in memory enhancement of juvenile and adult Lymnaea stagnalis by predator-induced stress

Memory consolidation following learning is a dynamic process. Thus, long-term memory (LTM) formation can be modulated by many factors, including stress. We examined how predator-induced stress enhances LTM formation in the pond snail Lymnaea stagnalis at both the behavioral and electrophysiological levels. Training snails in crayfish effluent (CE; i.e., water from an aquarium containing crayfish) significantly enhanced LTM. That is, while memory persists for only 3 h in adult control experiments following a single 0.5-h training session in pond water in which the pneumostome receives a contingent tactile stimulus to the pneumostome; when the snails are trained in CE, the memory persists for at least 24 h. In juveniles, the data are more dramatic. Juveniles are unable to form LTM in pond water, but form LTM when trained in CE. Here we examined whether juvenile snails form LTM following a one-trial training procedure (1TT). Following the 1TT procedure (a single-trial aversive operant conditioning training procedure), juveniles do not form LTM, unless trained in CE. Concomitantly, we observe changes in the excitability of RPeD11, a key neuron mediating the whole snail withdrawal response, which may be a neural correlate of enhanced memory formation.

Predator detection enables juvenileLymnaeato form long-term memory

Learning and memory provide the flexibility an organism requires to respond to changing social and ecological conditions. Juvenile Lymnaea have previously been shown to have a diminished capacity to form long-term memory (LTM) following operant conditioning of aerial respiratory behavior. Juvenile Lymnaea, however, can form LTM following classical conditioning of appetitive behaviors. Here, we demonstrate that laboratory-reared juvenile Lymnaea have the ability to detect the presence of a sympatric predator (i.e. crayfish) and respond to the predator by altering their aerial respiratory behavior. In addition to increasing their total breathing time, predator detection confers on juvenile Lymnaea an enhanced capability to form LTM following operant conditioning of aerial respiratory behavior. That is, these juveniles now have the ability to form long-lasting memory. These data support the hypothesis that biologically relevant levels of stress associated with predator detection induce behavioral phenotypic alterations (i.e. enhanced LTM formation) in juveniles, which may increase their fitness. These data also support the notion that learning and memory formation in conjunction with predator detection is a form of inducible defense.

`Different strokes for different folks': geographically isolated strains ofLymnaea stagnalisonly respond to sympatric predators and have different memory forming capabilities

Gaining insight into how natural trait variation is manifest in populations shaped by differential environmental factors is crucial to understanding the evolution, ecology and sensory biology of natural populations. We have demonstrated that lab-reared Lymnaea detect and respond to the scent of a crayfish predator with specific, appropriate anti-predator behavioral responses, including enhanced long-term memory (LTM) formation, and that such predator detection significantly alters the electrophysiological activity of RPeD1, a neuron that is a necessary site for LTM formation. Here we ask: (1) do distinct populations of wild Lymnaea stagnalis respond only to sympatric predators and if so, can these traits be quantified at both the behavioral and neurophysiological levels, and (2) does the presence of a non-sympatric predator elicit anti-predator behaviors including augmentation of LTM? We tested three different populations of wild (i.e. not lab-reared) snails freshly collected from their natural habitat: (1) polders near Utrecht in The Netherlands, (2) six seasonally isolated ponds in the Belly River drainage in southern Alberta, Canada and (3) a 20-year-old human-made dugout pond in southern Alberta. We found strain-specific variations in the ability to form LTM and that only a sympatric predator evoked anti-predatory behaviors, including enhanced LTM formation and changes in RPeD1 activity.

Electrophysiological and Behavioral Evidence Demonstrating That Predator Detection Alters Adaptive Behaviors in the SnailLymnaea

Stress has been shown to both impair and enhance learning, long-term memory (LTM) formation, and/or its recall. The pond snail, Lymnaea stagnalis, both detects and responds to the scent of a crayfish predator with multiple stress-related behavioral responses. Using both behavioral and electrophysiological evidence, this investigation is a first attempt to characterize how an environmentally relevant stressor (scent of a predator) enhances LTM formation in Lymnaea. Using a training procedure that, in "standard" pond water (PW), results in an intermediate-term memory that persists for only 3 h, we found that training snails in "crayfish effluent" (CE) induces a memory that persists for 48 h (i.e., its now an LTM). In addition, if we use a training procedure that in PW produces an LTM that persists for 1 d, we find that snails trained in CE have an LTM that persists for at least 8 d. Furthermore, we describe how a single neuron (RPeD1), which has been shown to be a necessary site for LTM formation, reflects the behavioral changes in its firing properties that persist for the duration of the LTM. Finally, Lymnaea exhibit context-specific memory, that is, when a memory is formed in a specific context (food odorant), it is only recalled in that context. Here, we found that snails trained in CE demonstrate context generalization, that is, memory is recalled in multiple contexts. All data are consistent with the hypothesis that learning in a stressful, yet biologically relevant, environment enhances LTM and prolongs its retention.

Notch signaling in Drosophila long-term memory formation.

Notch (N) is a cell surface receptor that mediates an evolutionarily ancient signaling pathway to control an extraordinarily broad spectrum of cell fates and developmental processes. To gain insights into the functions of N signaling in the adult brain, we examined the involvement of N in Drosophila olfactory learning and memory. Long-term memory (LTM) was disrupted by blocking N signaling in conditional mutants or by acutely induced expression of a dominant-negative N transgene. In contrast, neither learning nor early memory were affected. Furthermore, induced overexpression of a wild-type (normal) N transgene specifically enhanced LTM formation. These experiments demonstrate that N signaling contributes to LTM formation in the Drosophila adult brain.