György Kemenes

Role of delayed nonsynaptic neuronal plasticity in long-term associative memory.

BACKGROUND It is now well established that persistent nonsynaptic neuronal plasticity occurs after learning and, like synaptic plasticity, it can be the substrate for long-term memory. What still remains unclear, though, is how nonsynaptic plasticity contributes to the altered neural network properties on which memory depends. Understanding how nonsynaptic plasticity is translated into modified network and behavioral output therefore represents an important objective of current learning and memory research. RESULTS By using behavioral single-trial classical conditioning together with electrophysiological analysis and calcium imaging, we have explored the cellular mechanisms by which experience-induced nonsynaptic electrical changes in a neuronal soma remote from the synaptic region are translated into synaptic and circuit level effects. We show that after single-trial food-reward conditioning in the snail Lymnaea stagnalis, identified modulatory neurons that are extrinsic to the feeding network become persistently depolarized between 16 and 24 hr after training. This is delayed with respect to early memory formation but concomitant with the establishment and duration of long-term memory. The persistent nonsynaptic change is extrinsic to and maintained independently of synaptic effects occurring within the network directly responsible for the generation of feeding. Artificial membrane potential manipulation and calcium-imaging experiments suggest a novel mechanism whereby the somal depolarization of an extrinsic neuron recruits command-like intrinsic neurons of the circuit underlying the learned behavior. CONCLUSIONS We show that nonsynaptic plasticity in an extrinsic modulatory neuron encodes information that enables the expression of long-term associative memory, and we describe how this information can be translated into modified network and behavioral output.

A comparison of four techniques for mapping the distribution of serotonin and serotonin-containing neurons in fixed and living ganglia of the snail,Lymnaea

SummaryThe distribution of serotonin and serotonin-containing neurons was studied in the ganglia of the CNS of the snailLymnaea stagnalis. Results of the application of three different labelling techniques on wholemount preparations were compared with each other and with the serotonin content of the ganglia, measured by high-performance liquid chromatography. Serotonin immunocytochemistry resulted in the highest number of labelled neurons, but the more recently developedin vivo method of 5,6- or 5,7-dihydroxytryptamine-induced pigmentation also proved to be a reliable technique for the visualization of serotonin-containing cell bodies. In comparison with these two techniques, the glyoxylic acid fluorescence method appeared to be less sensitive. The distribution and number of serotonin-containing neurons and biochemically measured serotonin in specific ganglia showed a close correlation. By combining the results of the three labelling techniques, a detailed map of serotonin-containing neurons was constructed, and this was compared with maps of identified neurons prepared from earlier electrophysiological studies. Previously described serotonergic neurons were consistently found, as well as several new serotonin-containirig cell types in the cerebral, visceral and parietal ganglia. A network of serotonin-containing inter- and intraganglionic axon tracts, and thin serotonergic fibres in the perineurium were also demonstrated. Thisin vivo andin vitro identification of serotonin-containing neurons will facilitate further neurophysiological analysis of serotonergic neural mechanisms inLymnaea.

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.

Cyclic AMP response element binding (CREB)-like proteins in a molluscan brain: cellular localization and learning-induced phosphorylation

The phosphorylation and the binding to DNA of the nuclear transcription factor, cyclic adenosine 3′,5′‐monophosphate (cAMP) response element‐binding protein (CREB) are conserved key steps in the molecular cascade leading to the formation of long‐term memory (LTM). Here, we characterize, for the first time, a CREB1‐like protein in the central nervous system (CNS) of Lymnaea, a model system used widely for the study of the fundamental mechanisms of learning and memory. We demonstrate cAMP response element (CRE)‐binding activity in CNS protein extracts and show that one of the CRE‐binding proteins is recognized by a polyclonal antibody raised to mammalian (human) CREB1. The same antibody detects specific CREB1 immunoreactivity in CNS extracts and in the nuclei of most neurons in the brain. Moreover, phospho–CREB1‐specific immunoreactivity is increased significantly in protein extracts of the CNS by forskolin, an activator of adenylate cyclase. The forskolin‐induced increase in phospho–CREB1 immunoreactivity is localized to the nuclei of CNS neurons, some of which have an important role in the formation of LTM. Significantly, classical food–reward conditioning increases phospho–CREB1 immunoreactivity in Lymnaea CNS protein extracts. This increase in immunoreactivity is specific to the ganglia that contain the feeding circuitry, which undergoes cellular changes after classical conditioning. This work establishes the expression of a highly conserved functional CREB1‐like protein in the CNS of Lymnaea and opens the way for a detailed analysis of the role of CREB proteins in LTM formation in this model system.

A Persistent Cellular Change in a Single Modulatory Neuron Contributes to Associative Long-Term Memory

Most neuronal models of learning assume that changes in synaptic strength are the main mechanism underlying long-term memory (LTM) formation. However, we show here that a persistent depolarization of membrane potential, a type of cellular change that increases neuronal responsiveness, contributes significantly to a long-lasting associative memory trace. The use of a model invertebrate network with identified neurons and known synaptic connectivity had the advantage that the contribution of this cellular change to memory could be evaluated in a neuron with a known function in the learning circuit. Specifically, we used the well-understood motor circuit underlying molluscan feeding and showed that a key modulatory neuron involved in the initiation of feeding ingestive movements underwent a long-term depolarization following behavioral associative conditioning. This depolarization led to an enhanced single cell and network responsiveness to a previously neutral tactile conditioned stimulus, and the persistence of both matched the time course of behavioral associative memory. The change in the membrane potential of a key modulatory neuron is both sufficient and necessary to initiate a conditioned response in a reduced preparation and underscores its importance for associative LTM.

Voltage-gated ionic currents in an identified modulatory cell type controlling molluscan feeding

An important modulatory cell type, found in all molluscan feeding networks, was investigated using two‐electrode voltage‐ and current‐clamp methods. In the cerebral giant cells of Lymnaea, a transient inward Na+ current was identified with activation at −58 ± 2 mV. It was sensitive to tetrodotoxin only in high concentrations (≈ 50% block at 100 µm), a characteristic of Na+ channels in many molluscan neurons. A much smaller low‐threshold persistent Na+ current (activation at < −90 mV) was also identified. Two purely voltage‐sensitive outward K+ currents were also found: (i) a transient A‐current type which was activated at −59 ± 4 mV and blocked by 4‐aminopyridine; (ii) a sustained tetraethylammonium‐sensitive delayed rectifier current which was activated at −47 ± 2 mV. There was also evidence that a third, Ca2+‐activated, K+ channel made a contribution to the total outward current. No inwardly rectifying currents were found. Two Ca2+ currents were characterized: (i) a transient low‐voltage (−65 ± 2 mV) activated T‐type current, which was blocked in NiCl2 (2 mm) and was completely inactivated at ≈ −50 mV; (ii) A sustained high voltage (−40 ± 1 mV) activated current, which was blocked in CdCl2 (100 µm) but not in ω‐conotoxin GVIA (10 µm), ω‐agatoxin IVA (500 nm) or nifedipine (10 µm). This current was enhanced in Ba2+ saline. Current‐clamp experiments revealed how these different current types could define the membrane potential and firing properties of the cerebral giant cells, which are important in shaping the wide‐acting modulatory influence of this neuron on the rest of the feeding network.

Training in a novel environment improves the appetitive learning performance of the snail, Lymnaea stagnalis

The effect of novelty, an environmental background variable affecting feeding and appetitive learning performance, was examined in a behavioral study of the pond snail Lymnaea stagnalis. Transfer of snails into a novel aquatic environment (clean water) evoked exploratory behavior which manifested itself in an increased number of spontaneous rasping movements of the mouth over the second to fifth minute after exposure to the novel environment. The intensity of this behavior was much weaker in a familiar environment (used water from the home tank). Similarly, sucrose-induced feeding rates were highest when the snails were given the sucrose stimulus in a novel environment. The effectiveness of appetitive conditioning using tactile stimulus paired with food (Kemenes & Benjamin, 1989a) improved when the snails were subjected to conditioning in a novel environment. Satiety, an internal variable, suppressed the stimulating effects of the novel environment on the spontaneous, unconditioned, and conditioned feeding alike. After training in the novel environment, the conditioned response was retained for up to 12 days and thus provided a robust behavioral paradigm for the extension of the analysis to the neurophysiological mechanisms of factors affecting appetitive learning in molluscs.

Dynamic control of a central pattern generator circuit : a computational model of the snail feeding network

Central pattern generators (CPGs) are networks underlying rhythmic motor behaviours and they are dynamically regulated by neuronal elements that are extrinsic or intrinsic to the rhythmogenic circuit. In the feeding system of the pond snail, Lymnaea stagnalis, the extrinsic slow oscillator (SO) interneuron controls the frequency of the feeding rhythm and the N3t (tonic) has a dual role; it is an intrinsic CPG interneuron, but it also suppresses CPG activity in the absence of food, acting as a decision‐making element in the feeding circuit. The firing patterns of the SO and N3t neurons and their synaptic connections with the rest of the CPG are known, but how these regulate network function is not well understood. This was investigated by building a computer model of the feeding network based on a minimum number of cells (N1M, N2v and N3t) required to generate the three‐phase motor rhythm together with the SO that was used to activate the system. The intrinsic properties of individual neurons were represented using two‐compartment models containing currents of the Hodgkin–Huxley type. Manipulations of neuronal activity in the N3t and SO neurons in the model produced similar quantitative effects to food and electrical stimulation in the biological network indicating that the model is a useful tool for studying the dynamic properties of the feeding circuit. The model also predicted novel effects of electrical stimulation of two CPG interneurons (N1M and N2v). When tested experimentally, similar effects were found in the biological system providing further validation of our model.

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 (∼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.

Dynamic clamp with StdpC software

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording.