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

Multiple types of control by identified interneurons in a sensory-activated rhythmic motor pattern.

Modulatory interneurons that can drive central pattern generators (CPGs) are considered as good candidates for decision-making roles in rhythmic behaviors. Although the mechanisms by which such neurons activate their target CPGs are known in detail in many systems, their role in the sensory activation of CPG-driven behaviors is poorly understood. In the feeding system of the mollusc Lymnaea, one of the best-studied rhythmical networks, intracellular stimulation of either of two types of neuron, the cerebral ventral 1a (CV1a) and the slow oscillator (SO) cells, leads to robust CPG-driven fictive feeding patterns, suggesting that they might make an important contribution to natural food-activated behavior. In this paper we investigated this contribution using a lip-CNS preparation in which feeding was elicited with a natural chemostimulant rather than intracellular stimulation. We found that despite their CPG-driving capabilities, neither CV1a nor SO were involved in the initial activation of sucrose-evoked fictive feeding, whereas a CPG interneuron, N1M, was active first in almost all preparations. Instead, the two interneurons play important and distinct roles in determining the characteristics of the rhythmic motor output; CV1a by modulating motoneuron burst duration and SO by setting the frequency of the ongoing rhythm. This is an example of a distributed system in which (1) interneurons that drive similar motor patterns when activated artificially contribute differently to the shaping of the motor output when it is evoked by the relevant sensory input, and (2) a CPG rather than a modulatory interneuron type plays the most critical role in initiation of sensory-evoked rhythmic activity

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

Pattern generating role for motoneurons in a rythmically active neuronal network

The role of motoneurons in central motor pattern generation was investigated in the feeding system of the pond snail Lymnaea stagnalis, an important invertebrate model of behavioral rhythm generation. The neuronal network responsible for the three-phase feeding motor program (fictive feeding) has been characterized extensively and divided into populations of central pattern generator (CPG) interneurons, modulatory interneurons, and motoneurons. A previous model of the feeding system considered that the motoneurons were passive followers of CPG interneuronal activity. Here we present new, detailed physiological evidence that motoneurons that innervate the musculature of the feeding apparatus have significant electrotonic motoneuron¿interneuron connections, mainly confined to cells active in the same phase of the feeding cycle (protraction, rasp, or swallow). This suggested that the motoneurons participate in rhythm generation. This was assessed by manipulating firing activity in the motoneurons during maintained fictive feeding rhythms. Experiments showed that motoneurons contribute to the maintenance and phase setting of the feeding rhythm and provide an efficient system for phase-locking muscle activity with central neural activity. These data indicate that the distinction between motoneurons and interneurons in a complex CNS network like that involved in snail feeding is no longer justified and that both cell types are important in motor pattern generation. This is a distributed type of organization likely to be a general characteristic of CNS circuitries that produce rhythmic motor behavior

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

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

A central control circuit for encoding perceived food value

Hunger state can substantially alter the perceived value of a stimulus, even to the extent that the same sensory cue can trigger antagonistic behaviors. How the nervous system uses these graded perceptual shifts to select between opposed motor patterns remains enigmatic. Here, we challenged food-deprived and satiated Lymnaea to choose between two mutually exclusive behaviors, ingestion or egestion, produced by the same feeding central pattern generator. Decoding the underlying neural circuit reveals that the activity of central dopaminergic interneurons defines hunger state and drives network reconfiguration, biasing satiated animals toward the rejection of stimuli deemed palatable by food-deprived ones. By blocking the action of these neurons, satiated animals can be reconfigured to exhibit a hungry animal phenotype. This centralized mechanism occurs in the complete absence of sensory retuning and generalizes across different sensory modalities, allowing food-deprived animals to increase their perception of food value in a stimulus-independent manner to maximize potential calorific intake

Suppression of nitric oxide (NO)-dependent behavior by double-stranded RNA-mediated silencing of a neuronal NO synthase gene

We have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to disrupt neuronal nitric oxide (NO) synthase (nNOS) gene function in the snail Lymnaea stagnalis and have detected a specific behavioral phenotype. The injection of whole animals with synthetic dsRNA molecules targeted to the nNOS-encoding mRNA reduces feeding behavior in vivo and fictive feeding in vitro and interferes with NO synthesis by the CNS. By showing that synthetic dsRNA targeted to the nNOS mRNA causes a significant and long-lasting reduction in the levels of Lym-nNOS mRNA, we verify that specific RNAi has occurred. Importantly, our results establish that the expression of nNOS gene is essential for normal feeding behavior. They also show that dsRNA can be used in the investigation of functional gene expression in the context of whole animal behavior, regardless of the availability of targeted mutation technologies

Computational model of a modulatory cell type in the feeding network of the snail, Lymnaea stagnalis

Realistic mathematical models of single neurons are significant in assessing the contribution of specific ionic conductances to neuronal excitability. This study presents a detailed computational model of the Cerebral Giant Cells (CGCs), a pair of serotonergic neurons in the feeding network of Lymnaea stagnalis, which are critical for the expression of motor behaviour (feeding) and the formation of long-term memory

A CREB2-targeting microRNA is required for long-term memory after single-trial learning

Although single-trial induced long-term memories (LTM) have been of major interest in neuroscience, how LTM can form after a single episode of learning remains largely unknown. We hypothesized that the removal of molecular inhibitory constraints by microRNAs (miRNAs) plays an important role in this process. To test this hypothesis, first we constructed small non-coding RNA (sncRNA) cDNA libraries from the CNS of Lymnaea stagnalis subjected to a single conditioning trial. Then, by next generation sequencing of these libraries, we identified a specific pool of miRNAs regulated by training. Of these miRNAs, we focussed on Lym-miR-137 whose seed region shows perfect complementarity to a target sequence in the 3’ UTR of the mRNA for CREB2, a well-known memory repressor. We found that Lym-miR-137 was transiently up-regulated 1 h after single-trial conditioning, preceding a down-regulation of Lym-CREB2 mRNA. Furthermore, we discovered that Lym-miR-137 is co-expressed with Lym-CREB2 mRNA in an identified neuron with an established role in LTM. Finally, using an in vivo loss-of-function approach we demonstrated that Lym-miR-137 is required for single-trial induced LTM