Combining Stressors That Individually Impede Long-Term Memory Blocks All Memory Processes

The effects of stress on memory are typically assessed individually; however, in reality different stressors are often experienced simultaneously. Here we determined the effect that two environmentally relevant stressors, crowding and low calcium availability, have on memory and neural activity following operant conditioning of aerial respiration in the pond snail, Lymnaea stagnalis. We measured aerial breathing behaviour and activity of a neuron necessary for memory formation, right pedal dorsal 1 (RPeD1), in the central pattern generator (CPG) that drives aerial respiration in untrained animals, and assessed how these traits changed following training. In naïve animals both crowding and combined stressors significantly depressed burst activity in RPeD1 which correlated with a depression in aerial breathing behaviour, whereas low calcium availability had no effect on RPeD1 activity. Following training, changes in burst activity in RPeD1 correlated with behavioural changes, decreasing relative to their naïve state at 3 h and 24 h in control conditions when both intermediate-term memory (ITM: 3 h) and long-term memory (LTM: 24 h) are formed, at 3 h but not 24 h when exposed to individual stressors when only ITM is formed, and did not change in combined stressors (i.e. when no memory is formed). Additionally, we also found that Lymnaea formed short-term memory (STM: 10 min) in the presence of individual stressors or under control conditions, but failed to do so in the presence of combined stressors. Our data demonstrate that by combining stressors that individually block LTM only we can block all memory processes. Therefore the effects of two stressors with similar individual affects on memory phenotype may be additive when experienced in combination.     

Brain metabolic activity associated with long-term memory consolidation

The use of day-old chickens trained on a single-trial passive avoidance task provides a useful paradigm for investigations into cellular mechanisms underlying memory formation. Pharmacological intervention studies indicate that there are three temporally identifiable stages of memory processing leading to the consolidation of information for this task. These stages, designated as short-term (STM; up to 15 min), intermediate-term (ITM; 15-55 min), and long-term (LTM; more than 55 min) memory, have been found to be sequentially dependent (Ng and Gibbs, 1989). In addition, ITM appears to consist of two physiologically distinguishable phases, A and B. Evidence in this laboratory suggests that the crossover between these ITM phases (at 30 min after training) represents a critical time-point for the triggering of LTM.     

Persistence of long-term memory storage requires a late protein synthesis- and BDNF- dependent phase in the hippocampus

Persistence is the most characteristic attribute of long-term memory (LTM). To understand LTM, we must understand how memory traces persist over time despite the short-lived nature and rapid turnover of their molecular substrates. It is widely accepted that LTM formation is dependent upon hippocampal de novo protein synthesis and Brain-Derived Neurotrophic Factor (BDNF) signaling during or early after acquisition. Here we show that 12 hr after acquisition of a one-trial associative learning task, there is a novel protein synthesis and BDNF-dependent phase in the rat hippocampus that is critical for the persistence of LTM storage. Our findings indicate that a delayed stabilization phase is specifically required for maintenance, but not formation, of the memory trace. We propose that memory formation and memory persistence share some of the same molecular mechanisms and that recurrent rounds of consolidation-like events take place in the hippocampus for maintenance of the memory trace.     

How stress alters memory in 'smart' snails

Cognitive ability varies within species, but whether this variation alters the manner in which memory formation is affected by environmental stress is unclear. The great pond snail, Lymnaea stagnalis, is commonly used as model species in studies of learning and memory. The majority of those studies used a single laboratory strain (i.e. the Dutch strain) originating from a wild population in the Netherlands. However, our recent work has identified natural populations that demonstrate significantly enhanced long-term memory (LTM) formation relative to the Dutch strain following operant conditioning of aerial respiratory behaviour. Here we assess how two populations with enhanced memory formation (i.e. 'smart' snails), one from Canada (Trans Canada 1: TC1) and one from the U.K. (Chilton Moor: CM) respond to ecologically relevant stressors. In control conditions the Dutch strain forms memory lasting 1-3 h following a single 0.5 h training session in our standard calcium pond water (80 mg/l [Ca(2+)]), whereas the TC1 and CM populations formed LTM lasting 5+ days following this training regime. Exposure to low environmental calcium pond water (20 mg/l [Ca(2+)]), which blocks LTM in the Dutch strain, reduced LTM retention to 24 h in the TC1 and CM populations. Crowding (20 snails in 100 ml) immediately prior to training blocks LTM in the Dutch strain, and also did so in TC1 and CM populations. Therefore, snails with enhanced cognitive ability respond to these ecologically relevant stressors in a similar manner to the Dutch strain, but are more robust at forming LTM in a low calcium environment. Despite the two populations (CM and TC1) originating from different continents, LTM formation was indistinguishable in both control and stressed conditions. This indicates that the underlying mechanisms controlling cognitive differences among populations may be highly conserved in L. stagnalis.     

Functional and evolutionary trade-offs co-occur between two consolidated memory phases in Drosophila melanogaster

Memory is a complex and dynamic process that is composed of different phases. Its evolution under natural selection probably depends on a balance between fitness benefits and costs. In Drosophila, two separate forms of consolidated memory phases can be generated experimentally: anaesthesia-resistant memory (ARM) and long-term memory (LTM). In recent years, several studies have focused on the differences between these long-lasting memory types and have found that, at the functional level, ARM and LTM are antagonistic. How this functional relationship will affect their evolutionary dynamics remains unknown. We selected for flies with either improved ARM or improved LTM over several generations, and found that flies selected specifically for improvement of one consolidated memory phase show reduced performance in the other memory phase. We also found that improved LTM was linked to decreased longevity in male flies but not in females. Conversely, males with improved ARM had increased longevity. We found no correlation between either improved ARM or LTM and other phenotypic traits. This is, to our knowledge, the first evidence of a symmetrical evolutionary trade-off between two memory phases for the same learning task. Such trade-offs may have an important impact on the evolution of cognitive capacities. On a neural level, these results support the hypothesis that mechanisms underlying these forms of consolidated memory are, to some degree, antagonistic.     

Repetitive noxious stimulus altered the shadow-induced withdrawal behavior in Lymnaea

Stress alters adaptive behaviors including vigilance behaviors. In Lymnaea one of these vigilance behavior is a heightened withdrawal response to a shadow. The shadow withdrawal response (SWR) is mediated by dermal photoreceptors located primarily on the foot, mantle cavity, and skin around the pneumostome area. Here we asked whether we could obtain a neural correlate of the heightened SWR and other essential behaviors following traumatic stress. We measured the electrophysiological properties of 'Right Pedal Dorsal 11 (RPeD11)', the interneuron that plays a major role in mediating the whole-body withdrawal response. In traumatized snails 24 hours after the trauma they responded not only to a shadow stimulus with an augmented withdrawal response, but suppressed in locomotive, feeding and respiratory behavior. Their behavioral change lasted at least one week. Accompanying the behavioral change in these 'traumatized' preparations there are a number of significant changes in the neuronal properties of RPeD11 compared to na?ve preparations. For example, RPeD11 is significantly more depolarized (～10 mV) has significantly larger input resistance, and the duration of the response elicited by the shadow persists longer. All these changes result in an increased RPeD11 response and seem to raise their defensive alert level.     

Molecular mechanisms underlying a unique intermediate phase of memory in aplysia

Short- and long-term synaptic facilitation induced by serotonin at Aplysia sensory-motor (SN-MN) synapses has been widely used as a cellular model of short- and long-term memory for sensitization. In recent years, a distinct intermediate phase of synaptic facilitation (ITF) has been described at SN-MN synapses. Here, we identify a novel intermediate phase of behavioral memory (ITM) for sensitization in Aplysia and demonstrate that it shares the temporal and mechanistic features of ITF in the intact CNS: (1) it declines completely prior to the onset of LTM, (2) its induction requires protein but not RNA synthesis, and (3) its expression requires the persistent activation of protein kinase A. Thus, in Aplysia, the same temporal and molecular characteristics that distinguish ITF from other phases of synaptic plasticity distinguish ITM from other phases of behavioral memory.     

Reward value determines memory consolidation in parasitic wasps

Animals can store learned information in their brains through a series of distinct memory forms. Short-lasting memory forms can be followed by longer-lasting, consolidated memory forms. However, the factors determining variation in memory consolidation encountered in nature have thus far not been fully elucidated. Here, we show that two parasitic wasp species belonging to different families, Cotesia glomerata (Hymenoptera: Braconidae) and Trichogramma evanescens (Hymenoptera; Trichogrammatidae), similarly adjust the memory form they consolidate to a fitness-determining reward: egg-laying into a host-insect that serves as food for their offspring. Protein synthesis-dependent long-term memory (LTM) was consolidated after single-trial conditioning with a high-value host. However, single-trial conditioning with a low-value host induced consolidation of a shorter-lasting memory form. For Cotesia glomerata, we subsequently identified this shorter-lasting memory form as anesthesia-resistant memory (ARM) because it was not sensitive to protein synthesis inhibitors or anesthesia. Associative conditioning using a single reward of different value thus induced a physiologically different mechanism of memory formation in this species. We conclude that the memory form that is consolidated does not only change in response to relatively large differences in conditioning, such as the number and type of conditioning trials, but is also sensitive to more subtle differences, such as reward value. Reward-dependent consolidation of exclusive ARM or LTM provides excellent opportunities for within-species comparison of mechanisms underlying memory consolidation.     

Dissociation of rugose‐dependent short‐term memory component from memory consolidation in Drosophila

Extensive investigations show several molecular and neuroanatomical mechanisms underlying short‐lived and long‐lasting memory in Drosophila. At the molecular level, the genetic pathway of memory formation, which was obtained through mutant research, seems to occur sequentially. So far, studies of Drosophila mutants appear to support the idea that mutants defective in short‐term memory (STM) are always associated with long‐term memory (LTM) impairment. At the neuroanatomical level, distinct memory traces are partially independently distributed. However, whether memory phase dissociation also exists at the molecular level remains unclear. Here, we report on molecular separation of STM and consolidated memory through genetic dissection of rugose mutants. Mutants in the rugose gene, which encodes an evolutionarily conserved A‐kinase anchor protein, show immediate memory defects as assayed through aversive olfactory conditioning. Intriguingly, two well‐defined consolidated memory components, anesthesia‐resistant memory and protein synthesis‐dependent LTM, are both normal in spite of the defective immediate memory after 10‐session massed and spaced training. Moreover, rugose genetically interacts with cyclic AMP‐protein kinase A signaling during STM formation. Considering our previous study that AKAP Yu specifically participates in LTM formation, these results suggest that there exists a molecular level of memory phase dissociation with distinct AKAPs in Drosophila.     

Roles of NO Signaling in Long-Term Memory Formation in Visual Learning in an Insect

Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.     

Parametric and genetic analysis of Drosophila appetitive long‐term memory and sugar motivation

Distinct forms of memory can be highlighted using different training protocols. In Drosophila olfactory aversive learning, one conditioning session triggers memory formation independently of protein synthesis, while five spaced conditioning sessions lead to the formation of long‐term memory (LTM), a long‐lasting memory dependent on de novo protein synthesis. In contrast, one session of odour–sugar association appeared sufficient for the fly to form LTM. We designed and tuned an apparatus that facilitates repeated discriminative conditioning by alternate presentations of two odours, one being associated with sugar, as well as a new paradigm to test sugar responsiveness (SR). Our results show that both SR and short‐term memory (STM) scores increase with starvation length before conditioning. The protein dependency of appetitive LTM is independent of the repetition and the spacing of training sessions, on the starvation duration and on the strength of the unconditioned stimulus. In contrast to a recent report, our test measures an abnormal SR of radish mutant flies, which might initiate their STM and LTM phenotypes. In addition, our work shows that crammer and tequila mutants, which are deficient for aversive LTM, present both an SR and an appetitive STM defect. Using the MB247‐P[switch] system, we further show that tequila is required in the adult mushroom bodies for normal sugar motivation.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

A sodium leak current regulates pacemaker activity of adult central pattern generator neurons in Lymnaea stagnalis

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na(+) (I(Leak-Na)). The I(Leak-Na) contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na(+) leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals.     

Roles of Calcium/Calmodulin-Dependent Kinase II in Long-Term Memory Formation in Crickets

Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca²⁺/CaM and cAMP signaling participates in long-term memory (LTM) formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM). Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca²⁺ influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC) activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca²⁺ signaling and cAMP signaling for LTM formation, a new role of CaMKII in learning and memory.     

Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila

Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7) on sleep and long-term memory (LTM) formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation "window" that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state.     

Molecular pharmacological dissection of short- and long-term memory

1. It has been discussed for over 100 years whether short-term memory (STM) is separate from, or just an early phase of, long-term memory (LTM). The only way to solve this dilemma is to find out at least one treatment that blocks STM while keeping LTM intact for the same task in the same animal.2. The effect of a large number of treatments infused into the hippocampus, amygdala, and entorhinal, posterior parietal or prefrontal cortex on STM and LTM of a one-trial step-down inhibitory avoidance task was studied. The animals were tested at 1.5 h for STM, and again at 24 h for LTM. The treatments were given after training.3. Eleven different treatments blocked STM without affecting LTM. Eighteen treatments affected the two memory types differentially, either blocking or enhancing LTM alone. Thus, STM is separate from, and parallel to the first hours of processing of, LTM of that task.4. The mechanisms of STM are different from those of LTM. The former do not include gene expression or protein synthesis; the latter include a double peak of cAMP-dependent protein kinase activity, accompanied by the phosphorylation of CREB, and both gene expression and protein synthesis.5. Possible cellular and molecular events that do not require mRNA or protein synthesis should account for STM. These might include a hyperactivation of glutamate AMPA receptors, ribosome changes, or the exocytosis of glycoproteins that participate in cell addition.     

Cellular and systems reconsolidation in the hippocampus

Cellular theories of memory consolidation posit that new memories require new protein synthesis in order to be stored. Systems consolidation theories posit that the hippocampus has a time-limited role in memory storage, after which the memory is independent of the hippocampus. Here, we show that intra-hippocampal infusions of the protein synthesis inhibitor anisomycin caused amnesia for a consolidated hippocampal-dependent contextual fear memory, but only if the memory was reactivated prior to infusion. The effect occurred even if reactivation was delayed for 45 days after training, a time when contextual memory is independent of the hippocampus. Indeed, reactivation of a hippocampus-independent memory caused the trace to again become hippocampus dependent, but only for 2 days rather than for weeks. Thus, hippocampal memories can undergo reconsolidation at both the cellular and systems levels.     

Ventromedial prefrontal cortex activation is associated with memory formation for predictable rewards

During reinforcement learning, dopamine release shifts from the moment of reward consumption to the time point when the reward can be predicted. Previous studies provide consistent evidence that reward-predicting cues enhance long-term memory (LTM) formation of these items via dopaminergic projections to the ventral striatum. However, it is less clear whether memory for items that do not precede a reward but are directly associated with reward consumption is also facilitated. Here, we investigated this question in an fMRI paradigm in which LTM for reward-predicting and neutral cues was compared to LTM for items presented during consumption of reliably predictable as compared to less predictable rewards. We observed activation of the ventral striatum and enhanced memory formation during reward anticipation. During processing of less predictable as compared to reliably predictable rewards, the ventral striatum was activated as well, but items associated with less predictable outcomes were remembered worse than items associated with reliably predictable outcomes. Processing of reliably predictable rewards activated the ventromedial prefrontal cortex (vmPFC), and vmPFC BOLD responses were associated with successful memory formation of these items. Taken together, these findings show that consumption of reliably predictable rewards facilitates LTM formation and is associated with activation of the vmPFC.