A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate

Similar to other invertebrate and vertebrate animals, cAMP dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using Matrix-Assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF) mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homologue of the vertebrate pituitary adenylate cyclase activating polypeptide (PACAP) and for the AC activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this strong food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a weak multi-trial food-reward conditioning paradigm, lip-touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multi-trial tactile conditioning accelerated the formation of transcription dependent memory.Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning

Reversal of age-related learning deficiency by the vertebrate PACAP and IGF-1 in a novel invertebrate model of aging: the pond snail (Lymnaea Stagnalis)

With the increase of life span, nonpathological age-related memory decline is affecting an increasing number of people. However, there is evidence that age-associated memory impairment only suspends, rather than irreversibly extinguishes, the intrinsic capacity of the aging nervous system for plasticity (1). Here, using a molluscan model system, we show that the age-related decline in memory performance can be reversed by administration of the pituitary adenylate cyclase activating polypeptide (PACAP). Our earlier findings showed that a homolog of the vertebrate PACAP38 and its receptors exist in the pond snail (Lymnaea stagnalis) brain (2), and it is both necessary and instructive for memory formation after reward conditioning in young animals (3). Here we show that exogenous PACAP38 boosts memory formation in aged Lymnaea, where endogenous PACAP38 levels are low in the brain. Treatment with insulin-like growth factor-1, which in vertebrates was shown to transactivate PACAP type I (PAC1) receptors (4) also boosts memory formation in aged pond snails. Due to the evolutionarily conserved nature of these polypeptides and their established role in memory and synaptic plasticity, there is a very high probability that they could also act as “memory rejuvenating” agents in humans

Delayed intrinsic activation of an NMDA-independent CaM-kinase II in a critical time window is necessary for late consolidation of an associative memory

Calcium/calmodulin-dependent kinases (CaM-kinases) are central to various forms of long-term memory (LTM) in a number of evolutionarily diverse organisms. However, it is still largely unknown what contributions specific CaM-kinases make to different phases of the same specific type of memory, such as acquisition, or early, intermediate, and late consolidation of associative LTM after classical conditioning. Here, we investigated the involvement of CaM-kinase II (CaMKII) in different phases of associative LTM induced by single-trial reward classical conditioning in Lymnaea, a well established invertebrate experimental system for studying molecular mechanisms of learning and memory. First, by using a general CaM-kinase inhibitor, KN-62, we found that CaM-kinase activation was necessary for acquisition and late consolidation, but not early or intermediate consolidation or retrieval of LTM. Then, we used Western blot-based phosphorylation assays and treatment with CaMKIINtide to identify CaMKII as the main CaM-kinase, the intrinsic activation of which, in a critical time window ( approximately 24 h after learning), is central to late consolidation of LTM. Additionally, using MK-801 and CaMKIINtide we found that acquisition was dependent on both NMDA receptor and CaMKII activation. However, unlike acquisition, CaMKII-dependent late memory consolidation does not require the activation of NMDA receptors. Our new findings support the notion that even apparently stable memory traces may undergo further molecular changes and identify NMDA-independent intrinsic activation of CaMKII as a mechanism underlying this "lingering consolidation." This process may facilitate the preservation of LTM in the face of protein turnover or active molecular processes that underlie forgetting

Critical time window for NO-cGMP-dependent long-term memory formation after one-trial appetitive conditioning

The nitric oxide (NO)-cGMP signaling pathway is implicated in an increasing number of experimental models of plasticity. Here, in a behavioral analysis using one-trial appetitive associative conditioning, we show that there is an obligatory requirement for this pathway in the formation of long-term memory (LTM). Moreover, we demonstrate that this requirement lasts for a critical period of ~5 hr after training. Specifically, we trained intact specimens of the snail Lymnaea stagnalis in a single conditioning trial using a conditioned stimulus, amyl-acetate, paired with a salient unconditioned stimulus, sucrose, for feeding. Long-term associative memory induced by a single associative trial was demonstrated at 24 hr and shown to last at least 14 d after training. Tests for LTM and its dependence on NO were performed routinely 24 hr after training. The critical period when NO was needed for memory formation was established by transiently depleting it from the animals at a series of time points after training by the injection of the NO-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO).By blocking the activity of NO synthase and soluble guanylyl cyclase enzymes after training, we provided further evidence that LTM formation depends on an intact NO-cGMP pathway. An electrophysiological correlate of LTM was also blocked by PTIO, showing that the dependence of LTM on NO is amenable to analysis at the cellular level in vitro. This represents the first demonstration that associative memory formation after single-trial appetitive classical conditioning is dependent on an intact NO-cGMP signaling pathway

Associative memory stored by functional novel pathway rather than modifications of preexisting neuronal pathways

Associative conditioning involves changes in the processing pathways activated by sensory information to link the conditioned stimulus (CS) to the conditioned behavior. Thus, conditioning can recruit neuronal elements to form new pathways for the processing of the CS and/or can change the strength of existing pathways. Using a behavioral and systems level electrophysiological approach on a tractable invertebrate circuit generating feeding in the mollusk Lymnaea stagnalis, we identified three independent pathways for the processing of the CS amyl acetate used in appetitive conditioning. Two of these pathways, one suppressing and the other stimulating feeding, mediate responses to the CS in naive animals. The effects ofthese two pathways on feeding behavior are unaltered by conditioning. In contrast, the CS response ofa third stimulatory pathway is significantly enhanced after conditioning, becoming an importantcontributor to the overall CS response. This is unusual because, in most of the previous examples in which naive animals already respond to the CS, memory formation results from changes in the strength of pathways that mediate the existing response. Here, we show that, in the molluscan feeding system, both modified and unmodified pathways are activated in parallel by the CS after conditioning, and it is their integration that results in the conditioned respons

Phase-dependent molecular requirements for memory reconsolidation: differential roles for protein synthesis and protein kinase A activity

After consolidation, a process that requires gene expression and protein synthesis, memories are stable and highly resistant to disruption by amnestic influences. Recently, consolidated memory has been shown to become labile again after retrieval and to require a phase of reconsolidation to be preserved. New findings, showing that the dependence of reconsolidation on protein synthesis decreases with the age of memory, point to changing molecular requirements for reconsolidation during memory maturation. We examined this possibility by comparing the roles of protein synthesis (a general molecular requirement for memory consolidation) and the activation of protein kinase A (PKA) (a specific molecular requirement for memory consolidation), in memory reconsolidation at two time points after training. Using associative learning in Lymnaea, we show that reconsolidation after the retrieval of consolidated memory at both 6 and 24 h requires protein synthesis. In contrast, only reconsolidation at 6 h after training, but not at 24 h, requires PKA activity, which is in agreement with the measured retrieval-induced PKA activation at 6 h. This phase-dependent differential molecular requirement for reconsolidation supports the notion that even seemingly consolidated memories undergo further selective molecular maturation processes, which may only be detected by analyzing the role of specific pathways in memory reconsolidation after retrieval

Role of tonic inhibition in associative reward condiitoning in Lymnaea

Changes in the strength of excitatory synaptic connections are known to underlie associative memory formation in the molluscan nervous system but less is known about the role of synaptic inhibition. Tonic or maintained synaptic inhibition has an important function in controlling the Lymnaea feeding system and is known to suppress feeding in the absence of food or in satiated animals. Tonic inhibition to the feeding network is provided by the N3t interneuron that has inhibitory monosynaptic connection with the central pattern generator interneuron, the N1M. Here we asked whether a reduction in the level of tonic inhibition provided by the N3t cell could play a role in reward conditioning? Semi-intact preparations made from hungry snails were conditioned using a previously-developed one-trail chemical conditioning paradigm. We recorded electrical activity in a feeding motoneuron, the B3, at various time-points after conditioning. This allowed us to measure the frequency of spike activity in the N3t interneuron and monitor fictive feeding patterns that generate the rhythmic movements involved in food ingestion. We show that there is a reduction in N3t spiking at 1, 2, 3 and 4 hours after conditioning but not at 10 minutes and 30 minutes and the reduction in N3t firing inversely correlates with an increase in the conditioned fictive feeding response. Computer simulation of N3t-N1M interactions suggests that changes in N3t firing are sufficient to explain the increase in the fictive feeding activity produced by conditioning. A network model is presented that summarizes evidence suggesting that reward conditioning in Lymnaea is due to the combined effects of reduced tonic inhibition and enhanced excitatory synaptic connections between the CS pathway and feeding command neurons

Timed and targeted differential regulation of nitric oxide synthase (NOS) and anti-NOS genes by reward conditioning leading to long-term memory formation

In a number of neuronal models of learning, signaling by the neurotransmitter nitric oxide (NO), synthesized by the enzyme neuronal NO synthase (nNOS), is essential for the formation of long-term memory (LTM). Using the molluscan model system Lymnaea, we investigate here whether LTM formation is associated with specific changes in the activity of members of the NOS gene family: Lym-nNOS1, Lym-nNOS2, and the antisense RNA-producing pseudogene (anti-NOS). We show that expression of the Lym-nNOS1 gene is transiently upregulated in cerebral ganglia after conditioning. The activation of the gene is precisely timed and occurs at the end of a critical period during which NO is required for memory consolidation. Moreover, we demonstrate that this induction of the Lym-nNOS1 gene is targeted to an identified modulatory neuron called the cerebral giant cell (CGC). This neuron gates the conditioned feeding response and is an essential part of the neural network involved in LTM formation. We also show that the expression of the anti-NOS gene, which functions as a negative regulator of nNOS expression, is downregulated in the CGC by training at 4 h after conditioning, during the critical period of NO requirement. This appears to be the first report of the timed and targeted differential regulation of the activity of a group of related genes involved in the production of a neurotransmitter that is necessary for learning, measured in an identified neuron of known function. We also provide the first example of the behavioral regulation of a pseudogene

Synaptic metaplasticity underlies tetanic potentiation in Lymnaea: a novel paradigm

We present a mathematical model which explains and interprets a novel form of short-term potentiation, which was found to be use-, but not time-dependent, in experiments done on Lymnaea neurons. The high degree of potentiation is explained using a model of synaptic metaplasticity, while the use-dependence (which is critically reliant on the presence of kinase in the experiment) is explained using a model of a stochastic and bistable biological switch.Comment: 12 pages, 7 figures, to appear in PLoS One (2013