Learning In Invertebrates Research Articles

NMDA‐like receptors in the nervous system of the crab Neohelice granulata: A neuroanatomical description

N-Methyl-D-aspartate receptors (NMDARs) are involved in learning and memory processes in vertebrates and invertebrates. In Neohelice granulata, NMDARs are involved in the storage of associative memories (see references in text). The aim of this work was to characterize this type of glutamate receptor in Neohelice and to describe its distribution in the central nervous system (CNS). As a first step, a detailed study of the CNS of N. granulata was performed at the neuropil level, with special focus on one of the main structures involved in this type of memory, the supraesophageal ganglion, called central brain. The characterization of the NMDAR was achieved by identifying the essential subunit of these receptors, the NR1-like subunit. The NR1-like signals were found via western blot and immunohistochemistry techniques in each of the major ganglia: the eyestalk ganglia, the central brain, and the thoracic ganglion. Western blots yielded two bands for the crab NR1-like subunit, at ∼88 and ∼84 kDa. This subunit is present in all the major ganglia, and shows a strong localization in synaptosomal membranes. NMDARs are distributed throughout the majority of each ganglion but show prominent signal intensity in some distinguishable neuropils and neurons. This is the first general description of the N. granulata nervous system as a whole and the first study of NMDARs in the CNS of decapods. The preferential localization of the receptor in some neuropils and neurons indicates the presence of possible new targets for memory processing and storage.

Effects of morphine on associative memory and locomotor activity in the honeybee (Apis mellifera)

Morphine can modulate the processes underlying memory in vertebrates. However, studies have shown various modulations by morphine: positive, negative and even neutral. The honeybee is a potential platform for evaluating the effects of drugs, especially addictive drugs, on the nervous system. However, the involvement of morphine in learning and memory in insects or other invertebrates is poorly understood. The current work evaluated whether morphine affects memory acquisition, consolidation and retrieval in honeybees, using the proboscis extension response (PER) paradigm. We demonstrated that morphine treatment (5 μg/bee) before training decreased the percentage of correct PERs and the response latency related to aversive rather than rewarding odors when tested after 1 or 24 h. Morphine treatment after training also caused a decrease in this latency when tested after 24 h. Meanwhile, morphine treatment reduced the ambulation distance when tested after 30 min. Our findings suggest that morphine impairs the acquisition of short- and long-term associative memory and slightly disrupts the consolidation of long-term memory in honeybees. These negative effects cannot be explained by reduced locomotion but by impaired memory associated with aversion.

Invertebrate learning and memory: Fifty years of olfactory conditioning of the proboscis extension response in honeybees

The honeybee Apis mellifera has emerged as a robust and influential model for the study of classical conditioning, thanks to the existence of a powerful Pavlovian conditioning protocol, the olfactory conditioning of the proboscis extension response (PER). In 2011, the olfactory PER conditioning protocol celebrates 50 years since it was first introduced by Kimihisa Takeda in 1961. Here, we review its origins, developments, and perspectives in order to define future research avenues and necessary methodological and conceptual evolutions. We show that olfactory PER conditioning has become a versatile tool for the study of questions in extremely diverse fields in addition to the study of learning and memory and that it has allowed behavioral characterizations, not only of honeybees, but also of other insect species, for which the protocol was adapted. We celebrate, therefore, Takeda's original work and prompt colleagues to conceive and establish further robust behavioral tools for an accurate characterization of insect learning and memory at multiple levels of analysis.

Octopus Conditioning: A Multi-Armed Approach to the LTP–Learning Question

A recent study shows that avoidance conditioning in the cephalopod Octopus vulgaris is mediated by long-term potentiation (LTP), a form of synaptic plasticity thought to be important in vertebrate associative learning. Thus, LTP appears to be an evolutionarily conserved learning mechanism.

Evolution of NMDA receptor cytoplasmic interaction domains: implications for organisation of synaptic signalling complexes.

BackgroundGlutamate gated postsynaptic receptors in the central nervous system (CNS) are essential for environmentally stimulated behaviours including learning and memory in both invertebrates and vertebrates. Though their genetics, biochemistry, physiology, and role in behaviour have been intensely studied in vitro and in vivo, their molecular evolution and structural aspects remain poorly understood. To understand how these receptors have evolved different physiological requirements we have investigated the molecular evolution of glutamate gated receptors and ion channels, in particular the N-methyl-D-aspartate (NMDA) receptor, which is essential for higher cognitive function. Studies of rodent NMDA receptors show that the C-terminal intracellular domain forms a signalling complex with enzymes and scaffold proteins, which is important for neuronal and behavioural plasticityResultsThe vertebrate NMDA receptor was found to have subunits with C-terminal domains up to 500 amino acids longer than invertebrates. This extension was specific to the NR2 subunit and occurred before the duplication and subsequent divergence of NR2 in the vertebrate lineage. The shorter invertebrate C-terminus lacked vertebrate protein interaction motifs involved with forming a signaling complex although the terminal PDZ interaction domain was conserved. The vertebrate NR2 C-terminal domain was predicted to be intrinsically disordered but with a conserved secondary structure.ConclusionWe highlight an evolutionary adaptation specific to vertebrate NMDA receptor NR2 subunits. Using in silico methods we find that evolution has shaped the NMDA receptor C-terminus into an unstructured but modular intracellular domain that parallels the expansion in complexity of an NMDA receptor signalling complex in the vertebrate lineage. We propose the NR2 C-terminus has evolved to be a natively unstructured yet flexible hub organising postsynaptic signalling. The evolution of the NR2 C-terminus and its associated signalling complex may contribute to species differences in behaviour and in particular cognitive function.

The Role of Rapid, Local, Postsynaptic Protein Synthesis in Learning-Related Synaptic Facilitation in Aplysia

The discovery that dendrites of neurons in the mammalian brain possess the capacity for protein synthesis stimulated interest in the potential role of local, postsynaptic protein synthesis in learning-related synaptic plasticity. But it remains unclear how local, postsynaptic protein synthesis actually mediates learning and memory in mammals. Accordingly, we examined whether learning in an invertebrate, the marine snail Aplysia, involves local, postsynaptic protein synthesis. Previously, we showed that the dishabituation and sensitization of the defensive withdrawal reflex in Aplysia require elevated postsynaptic Ca(2+), postsynaptic exocytosis, and functional upregulation of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Here, we tested whether the synaptic facilitation that underlies dishabituation and sensitization in Aplysia requires local, postsynaptic protein synthesis. We found that the facilitatory transmitter, serotonin (5-HT), enhanced the response of the motor neuron to glutamate, the sensory neuron transmitter, and this enhancement depended on rapid protein synthesis. By using individual motor neurites surgically isolated from their cell bodies, we showed that the 5-HT-dependent protein synthesis occurred locally. Finally, by blocking postsynaptic protein synthesis, we disrupted the facilitation of the sensorimotor synapse. By demonstrating its critical role in a synaptic change that underlies learning and memory in a major model invertebrate system, our study suggests that local, postsynaptic protein synthesis is of fundamental importance to the cell biology of learning.

Automated analysis of behavior: A computer‐controlled system for drug screening and the investigation of learning

Efforts to understand cognition will be greatly facilitated by computerized systems that enable the automated analysis of animal behavior. A number of controversies in the invertebrate learning field have resulted from difficulties inherent in manual experiments. Driven by the necessity to overcome these problems during investigation of neural function in planarian flatworms and frog larvae, we designed and developed a prototype for an inexpensive, flexible system that enables automated control and analysis of behavior and learning. Applicable to a variety of small animals such as flatworms and zebrafish, this system allows automated analysis of innate behavior, as well as of learning and memory in a plethora of conditioning paradigms. We present here the schematics of a basic prototype, which overcomes experimenter effects and operator tedium, enabling a large number of animals to be analyzed with transparent on-line access to primary data. A scaled-up version of this technology represents an efficient methodology to screen pharmacological and genetic libraries for novel neuroactive reagents of basic and biomedical relevance.

Molecular Characterization of NMDA-Like Receptors inAplysiaandLymnaea:Relevance to Memory Mechanisms

The N-methyl-D-aspartate (NMDA) receptor belongs to the group of ionotropic glutamate receptors and has been implicated in synaptic plasticity, memory acquisition, and learning in both vertebrates and invertebrates, including molluscs. However, the molecular identity of NMDA-type receptors in molluscs remains unknown. Here, we cloned two NMDA-type receptors from the sea slug Aplysia californica, AcNR1-1 and AcNR1-2, as well as their homologs from the freshwater pulmonate snail Lymnaea stagnalis, LsNR1-1 and LsNR1-2. The cloned receptors contain a signal peptide, two extracellular segments with predicted binding sites for glycine and glutamate, three recognized transmembrane regions, and a fourth hydrophobic domain that makes a hairpin turn to form a pore-like structure. Phylogenetic analysis suggests that both the AcNR1s and LsNR1s belong to the NR1 subgroup of ionotrophic glutamate receptors. Our in situ hybridization data indicate highly abundant, but predominantly neuron-specific expression of molluscan NR1-type receptors in all central ganglia, including identified motor neurons in the buccal and abdominal ganglia as well as groups of mechanosensory cells. AcNR1 transcripts were detected extrasynaptically in the neurites of metacerebral cells of Aplysia. The widespread distribution of AcNR1 and LsNR1 transcripts also implies diverse functions, including their involvement in the organization of feeding, locomotory, and defensive behaviors.

Potentiation and prolongation of long-term odor memory in neonate rats using a phosphodiesterase inhibitor

Cyclic AMP has been shown to have a critical role in learning and memory in invertebrates. Here we use the rat pup odor preference learning model in which odor acts as a conditioned stimulus and β-adrenoceptor stimulation acts as an unconditioned stimulus to test the role of cyclic AMP in an associative mammalian paradigm. A phosphodiesterase inhibitor that prevents cyclic AMP breakdown (cilomilast) makes a low, learning-ineffective dose of a β-adrenoceptor agonist (isoproterenol, 1mg/kg) an effective unconditioned stimulus in pup odor preference learning. A dose of the phosphodiesterase inhibitor (cilomilast, 1mg/kg) that induces learning with a weak unconditioned stimulus interferes with learning using a normally optimal unconditioned stimulus (isoproterenol, 2mg/kg). Cilomilast (3mg/kg) paired with peppermint odor during learning, prolonged memory at least four times longer than without the drug (24 h vs. 96 h). These data demonstrate a causal role for cyclic AMP in the acquisition and duration of odor preference learning in the rat pup.

Invertebrate learning: what can't a worm learn?

The nematode worm Caenorhabditis elegans can learn and remember the stimuli it encounters, the environment it is in, and its own physiological state. Analyses of mutations in C. elegans that affect different aspects of experience are beginning to address the nature of learning.

Calexcitin‐like immunoreactivity in the pond snail Lymnaea stagnalis

AbstractCalexcitin (CE) is a low molecular weight Ca2+‐ and guanosine triphosphate‐ binding protein, which is phosphorylated during associative learning in both vertebrates and invertebrates. The purpose of this study was to determine the presence of CE in the central nervous system (CNS) of the pond snail Lymnaea stagnalis, which can acquire classical and operant conditioning. Immunoblotting of CE showed that the anti‐CE antibody prepared from squid can detect Lymnaea CE. In the cerebral ganglia, CE‐like immunoreactivity was exhibited in two pairs of cell clusters that receive taste signals from the superior or median lip nerves. In both pedal ganglia, CE‐like immunoreactivity was detected in 1‐4 cell of the PeA clusters, which are involved in the withdrawal response. Our results therefore showed that CE is involved in the feeding and withdrawal neural networks, suggesting that CE may function in associative learning of feeding and withdrawal behavior in L. stagnalis.

Avoidance learning in the crayfish ( Procambarus clarkii) depends on the predatory imminence of the unconditioned stimulus: a behavior systems approach to learning in invertebrates

Signaled avoidance learning in crayfish ( Procambarus clarkii) was investigated when the crayfish were not confined, by indexing two types of locomotive movement to the escape compartment. Mild shocks, which induced tail flipping in the crayfish, and light illumination were used as unconditioned and conditioned stimuli, respectively. In Experiment 1, two groups of crayfish were trained in a one-way shuttle box. The crayfish in Group Forward were placed in the start compartment facing the escape compartment and they were able to escape/avoid shocks by walking forward, while the crayfish in Group Backward were placed in the compartment facing the opposite direction and they were able to escape by tail flipping. Avoidance learning was achieved only by walking, and not by tail flipping despite the fact that consistent tail flipping allowed the crayfish to avoid shocks. In Experiment 2, the experimental conditions were switched by using the ABA design. In this experiment, we confirmed that avoidance behavior was restricted to walking. These results are readily explained by the behavior systems approach.

A learning and memory area in the octopus brain manifests a vertebrate-like long-term potentiation.

Cellular mechanisms underlying learning and memory were investigated in the octopus using a brain slice preparation of the vertical lobe, an area of the octopus brain involved in learning and memory. Field potential recordings revealed long-term potentiation (LTP) of glutamatergic synaptic field potentials similar to that in vertebrates. These findings suggest that convergent evolution has led to the selection of similar activity-dependent synaptic processes that mediate complex forms of learning and memory in vertebrates and invertebrates.

Calcium-regulated GTPase activity in the calcium-binding protein calexcitin

Calexcitin (CE) is a calcium-binding protein, closely related to sarcoplasmic calcium-binding proteins, that is involved in invertebrate learning and memory. Early reports indicated that both Hermissenda and squid CE also could bind GTP; however, the biochemical significance of GTP-binding and its relationship to calcium binding have remained unclear. Here, we report that the GTPase activity of CE is strongly regulated by calcium. CE possessed a P-loop-like structure near the C-terminal similar to the phosphate-binding regions in other GTP-binding proteins. Site-directed mutagenesis of this region showed that Gly 182, Phe 186 and Gly 187 are required for maximum affinity, suggesting that the GTP-binding motif is G-N-x-x-[FM]-G. CE cloned from Drosophila CNS possessed a similar C-terminal sequence and also bound and hydrolyzed GTP. GTPase activity in Drosophila CE was also strongly regulated by Ca 2+, exhibiting over 23-fold higher activity in the presence of 0.3 μM calcium. Analysis of the conserved protein motifs defines a new family of Ca 2+-binding proteins representing the first example of proteins endowed with both EF-hand calcium binding domains and a C-terminal, P-loop-like GTP-binding motif. These results establish that, in the absence of calcium, both squid and Drosophila CE bind GTP at near-physiological concentrations and hydrolyze GTP at rates comparable to unactivated ras. Calcium functions to increase GTP-binding and GTPase activity in CE, similar to the effect of GTPase activating proteins in other low-MW GTP-binding proteins. CE may, therefore, act as a molecular interface between Ca 2+ cytosolic oscillations and the G protein-coupled signal transduction.

Honey Bees (<em>Apis mellifera</em>) and the Solution of Practical Problems

Experiments on the use of honey bees ( Apis mellifera ) to solve practical problems are described. These problems include studies of alcoholism, influence of consumer products on Africanized honey bees, the effect of pesticides considered “harmless,” and the detection of adulterated hive products. The results of these experiments indicate that the study of invertebrate learning conducted within a comparative-psychological paradigm can provide much useful data to the solution of practical problems of significance to psychologists.

Serotonin activation of the ERK pathway in Hermissenda: contribution of calcium-dependent protein kinase C.

The mitogen-activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal-regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one-trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5-hydroxytryptamine (5-HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by 'upstream' signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5-HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA-dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5-HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA-AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca(2+)-dependent PKC activation contributes to ERK phosphorylation, although a PKC-independent pathway is also involved in 5-HT-dependent ERK phosphorylation and activation.

Cellular analysis of appetitive learning in invertebrates

In recent years significant progress has been made in the analysis of the cellular mechanisms underlying appetitive learning in two invertebrate species, the pond snail Lymnaea stagnalis and the honeybee Apis mellifera. In Lymnaea, both chemical (taste) and tactile appetitive conditioning paradigms were used and cellular traces of behavioural classical conditioning were recorded at several specific sites in the nervous system. These sites included sensory pathways, central pattern generator and modulatory interneurones as well as motoneurones of the feeding network. In the honeybee, a chemical (odour) appetitive conditioning paradigm resulted in cellular changes at different sites in the nervous system. In both the pond snail and the honeybee the activation of identified modulatory interneurones could substitute for the use of the chemical unconditioned stimulus, making these paradigms even more amenable to more detailed cellular and molecular analysis.

Second Messenger Systems Underlying Plasticity at the Neuromuscular Junction

Publisher Summary The Drosophila neuromuscular junction provides elegant significant experimental model to study signal transduction pathways that mediate synaptic plasticity because of its accessibility to electrophysiological techniques, morphological analyses, and pharmacological and genetic manipulations at identifiable synapses. Much of what is known concerning synaptic transmission and plasticity in Drosophila learning and memory mutants has been analyzed using the larval neuromuscular junction preparation, because the Drosophila central nervous system (CNS) is relatively inaccessible to electrophysiological recordings. Although the neuromuscular junction itself is not involved in learning and memory, these studies have yielded profound insights into the mechanisms underlying both functional and structural plasticity. Many of the second messenger molecules found at the larval neuromuscular junction are involved in learning and memory in both invertebrates and vertebrates. This chapter examines the role that second messenger systems play in both structural and functional plasticity at the Drosophila neuromuscular junction. It is believed that further analysis of various mutants allow determining the relationship between structural and functional change.

Biochemistry of molluscan learning and memory

AbstractStudies of learning in marine invertebrates have yielded new information, implicating protein kinase C and calmodulin‐dependent protein kinase as critical components in pathways for learning and memory that are shared with higher vertebrates. Recent advances correlating in vitro biochemical and biophysical measurements with in vivo learning have begun to elaborate the roles in memory storage for these two kinases, their substrates, and signaling proteins such as calexcitin and calmodulin. Other studies have implicated transcription factors associated with kinases such as the cyclic AMP response element binding protein (CREB). These experiments have resulted in a greater understanding of the role of second messengers, principally calcium and cyclic AMP (cAMP), as vehicles for transforming memory‐specific effects occurring at the membrane into permanent DNA‐mediated cellular and structural changes.

Mediation of classical conditioning in Aplysia californica by long-term potentiation of sensorimotor synapses.

Long-term potentiation (LTP) is considered an important neuronal mechanism of learning and memory. Currently, however, there is no direct experimental link between LTP of an identified synapse and learning. A cellular analog of classical conditioning in Aplysia was used to determine whether this form of invertebrate learning involves N-methyl-D-aspartate (NMDA)-type LTP. The NMDA receptor-antagonist dl-2-amino-5-phosphonovalerate significantly disrupted synaptic enhancement after associative training but did not disrupt synaptic enhancement after nonassociative training. Thus, classical conditioning in Aplysia appears to be mediated, in part, by LTP due to activation of NMDA-related receptors.