Paul R. Benjamin

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 single time-window for protein synthesis-dependent long-term memory formation after one-trial appetitive conditioning.

Protein synthesis is generally held to be essential for long‐term memory formation. Often two periods of sensitivity to blockade of protein synthesis have been described, one immediately after training and another several hours later. We wished to relate the timing of protein synthesis‐dependence of behavioural long‐term memory (LTM) formation to an electrophysiological correlate of the LTM memory trace. We used the snail Lymnaea because one‐trial appetitive conditioning of feeding using a chemical conditioned stimulus leads to a stable LTM trace that can be monitored behaviourally and then electrophysiologically in preparations made from the same animals. Anisomycin (an inhibitor of translation) injected 10 min after training blocked behavioural LTM formation. Actinomycin D (an inhibitor of transcription) was also effective at 10 min. When anisomycin, at doses shown to be effective in blocking central nervous system protein synthesis, was injected at 1, 2, 3, 4, 5 and 6 h after training there was no effect on recall. These results indicate that there is a single period of sensitivity to protein synthesis inhibition in Lymnaea lasting for between 10 min and 1 h after training with no evidence for a second window of sensitivity. An electrophysiological correlate of LTM was found to be sensitive to anisomycin injected 10 min after training. It is unusual to find only one period of protein synthesis‐dependence in detailed time‐course studies of LTM, and this suggests that the consolidation processes involving protein synthesis are relatively rapid in one‐trial appetitive conditioning and complete within 1 h of training.

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.

Appetitive learning in snails shows characteristics of conditioning in vertebrates

The snail Lymnaea stagnalis can be rapidly conditioned to produce feeding response to a previously neutral tactile stimulus, repeatedly and specifically paired with positive reinforcement (food). The conditioned response has many of the same characteristics seen in appetitive learning in vertebrates. The relative simplicity of the molluscan CNS and the detailed knowledge of the neural circuitry underlying feeding in Lymnaea will facilitate the neurophysiological analysis of appetitive learning as well as stimulus generalization, discriminative learning and generalization of extinction all described in the present paper.

Persistent Sodium Current Is a Nonsynaptic Substrate for Long-Term Associative Memory

Although synaptic plasticity is widely regarded as the primary mechanism of memory [1], forms of nonsynaptic plasticity, such as increased somal or dendritic excitability or membrane potential depolarization, also have been implicated in learning in both vertebrate and invertebrate experimental systems [2-7]. Compared to synaptic plasticity, however, there is much less information available on the mechanisms of specific types of nonsynaptic plasticity involved in well-defined examples of behavioral memory. Recently, we have shown that learning-induced somal depolarization of an identified modulatory cell type (the cerebral giant cells, CGCs) of the snail Lymnaea stagnalis encodes information that enables the expression of long-term associative memory [8]. The Lymnaea CGCs therefore provide a highly suitable experimental system for investigating the ionic mechanisms of nonsynaptic plasticity that can be linked to behavioral learning. Based on a combined behavioral, electrophysiological, immunohistochemical, and computer simulation approach, here we show that an increase of a persistent sodium current of this neuron underlies its delayed and persistent depolarization after behavioral single-trial classical conditioning. Our findings provide new insights into how learning-induced membrane level changes are translated into a form of long-lasting neuronal plasticity already known to contribute to maintained adaptive modifications at the network and behavioral level [8].

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.

Neural Network Analysis in the Snail Brain

How can we hope to use the techniques of cellular neurobiology, which have been successfully used to analyze the properties of single neurons or small networks, to eventually understand a structure such as the snail brain which contains about 25,000 neurons? Our approach has been to carry out detailed analyses of specific neural networks underlying several types of function in the brain of the pond snail, Lymnaea, while collecting less specific information on more global aspects of brain organization and the larger scale of interactions within it. The concentration of the CNS into a compact brain and the distribution of cells of the same type over several ganglia (see, for instance, the neurosecretory cells of Fig. 1A) makes such a whole brain analysis almost inevitable even for simple mapping studies, but the global aspect of organization within the snail brain is also emphasized by our analysis of two interneurons that have follower cells in at least five of the ganglia of the CNS (Fig. 1C,D). Even neural systems underlying specific behavioral acts can be widely distributed and motoneurons responsible for whole body withdrawal responses occur in all nine ganglia of the central ganglionic ring (Fig. 1B).

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.

Gene expression and function of FMRFamide-related neuropeptides in the snail Lymnaea

FMRFamide and a large family of related peptides (FaRPs) have been identified in every major metazoan phylum examined, including chordates. In the pulmonate snail Lymnaea this family of neuropeptides is encoded by a five‐exon locus that is subject to alternative splicing. The two alternative mRNA transcripts are expressed in the CNS in a mutually exclusive manner at the single cell level, resulting in the differential distribution of the distinct sets of FaRPs that they encode in defined neuronal networks. Biochemical peptide purification, single‐cell analysis by mass spectroscopy, and immunocytochemistry have led to an understanding of the post‐translational processing patterns of the two alternative precursor proteins and identified at least 12 known and novel peptides contained in neuronal networks involved in cardiorespiration, penial control and withdrawal response. The pharmacological actions of single or co‐expressed peptides are beginning to emerge for the cardiorespiratory network and its central and peripheral targets. Peptides derived from protein precursor 1 and contained in the heart excitatory central motoneurons Ehe have distinct functions and also act in concert in cardiac regulation, based on their unique effects on heartbeat and their differential stimulatory effects on second messenger pathways. Precursor‐2 derived peptides, contained in the Visceral White Interneuron, a key neuron of the cardiorespiratory network, have mostly inhibitory effects on the VWIs central postsynaptic target neurons but with some of the peptides also exhibiting excitatory effects on the same cells. Microsc. Res. Tech. 49:547–556, 2000.

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.