Learning-induced lateralized activation of the MAPK/ERK cascade in identified neurons of the food-aversion network in the mollusk Helix lucorum

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Abstract

The MAPK/ERK pathway plays an important role in the regulation of gene expression during memory formation both in vertebrates and invertebrates. In the mollusk Helix lucorum, serotonin induces activation of MAPK/ERK in the central nervous system (CNS) upon food aversion learning. Such learning depends on a neuronal network in which specialized neurons play distinct roles so that they may exhibit different activation levels of the MAPK/ERK pathway. Here we performed a comparative analysis of MAPK/ERK activation in single neurons of the food-aversion network, focusing both on command neurons, which mediate withdrawal behavior and process information pertaining to the unconditioned stimulus, and on neurons of the procerebrum, the mollusk’s olfactory center, which process information from the conditioned stimulus. By means of Western blots designed to detect micro amounts of proteins, we determined MAPK/ERK activation in these neurons and found that after food aversion learning phospho-ERK levels increased significantly in RPa(2/3) command neurons of the right parietal ganglia and in the procerebrum. Such an increase was prevented by injection of PD98095, an inhibitor of the ERK upstream kinase (MEK-1). In contrast, no activation of MAPK/ERK was detected in similar conditions in the corresponding neurons of the left parietal ganglia LPa(2/3). This asymmetry was verified after serotonin application to the CNS in order to mimic learning. Our results thus show that learning involves synchronous and asymmetric serotonin-dependent MAPK/ERK activation. Such an asymmetry may reflect lateralization of memory processes in the mollusk brain.

Introduction

Intracellular regulatory systems, including the signal transduction pathway mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), play a fundamental role in adaptive processes in the central nervous system. Their activation patterns determine survival or apoptosis, effectiveness of preexisting synapses or growth of new synaptic connections (Kaplan and Miller, 2000, Thomas and Huganir, 2004). Activation of the MAPK/ERK cascade is also an essential step for the formation of long-term memory (Atkins et al., 1998, Feld et al., 2005, Martin et al., 1997, Sananbenesi et al., 2003). Furthermore, its dysfunction can lead to neurodegenerative disorders accompanied by memory loss (Einat et al., 2003, Kyosseva, 2004).

The MAPK/ERK cascade influences genome-dependent plastic changes in the central nervous system through the direct phosphorylation of transcription factors (TF’s) such as Elk-1, a member of ternary complex factors (TCFs), the calcium/cAMP response element binding protein (CREB) and the serum response factor (SRF) (Orban et al., 1999, Thomas and Huganir, 2004). Although the role of the MAPK/ERK cascade in cellular processes related to memory formation has been studied in some vertebrates (Davis, Vanhoutte, Pages, Caboche, & Laroche, 2000), research on various mollusks species such as Aplysia, Hermissenda, Helix and Lymnaea stagnalis has been crucial to demonstrate its involvement in cellular and molecular mechanisms of long-term memory formation (Balaban, 2002, Crow et al., 2001, Kandel, 2001, Martin et al., 1997, Ribeiro et al., 2005, Sharma and Carew, 2004). Most of these studies used neurons in culture (Martin et al., 1997), or focused on entire mollusk ganglia or sensory organs (Crow et al., 2001, Feld et al., 2005, Ribeiro et al., 2005), thus overlooking the potential different contributions of distinct cell populations and/or functionally different neurons existing in such structures. Until now, studies on MAPK/ERK activation in single neurons of neuronal networks underlying learning and memory formation have never been reported. It has been, therefore, impossible to answer the question of whether neurons with different functions within an associative network exhibit different levels of MAPK/ERK activation upon memory formation.

Here we aimed at answering this question using the terrestrial mollusk Helix lucorum, in which several forms of conditioned avoidance reflex have been reported (Balaban, 2002, Stepanov et al., 1988). Basically, snails can be trained to avoid a piece of food (the conditioned stimulus; e.g. carrot) if it is appropriately paired with an electric shock (the unconditioned stimulus) delivered to the snails’ head. Neuronal networks underlying feeding behavior and withdrawal in Helix have been determined and neural correlates of withdrawal behavior have been described in detail (Balaban, 2002).

We previously found that transcription factors controlling gene expression via CRE, SRE and AP-1 elements are involved in the regulation of food aversion learning in Helix and that their activation depends both on the МАРK/ERK cascade and on serotonin (5-HT) (Grinkevich et al., 2003, Grinkevich and Vasil’ev, 2000). Moreover, we have shown that the neurotoxin 5,7-DHT, which induces dysfunction of serotoninergic terminals and reduces conditioned food aversion learning, abolishes МАРK/ERK activation (Grinkevich et al., 2007, Grinkevich et al., 2008). Immature mechanisms of sensitization and impaired conditioned avoidance responses are found in juvenile Helix snails, which differ from adults in a spectrum of transcription factors binding to regulatory elements SRE and АР-1 (Grinkevich and Vasil’ev, 2000, Grinkevich et al., 2003) and which do not exhibit MAPK/ERK activation after training in contrast to adults (Grinkevich et al., 2008).

We have also demonstrated that МАРK/ERK activation differs between different regions of the central nervous system (visceral complex of ganglia, cerebral and pedal ganglia), which play different roles in conditioned food aversion learning (Grinkevich et al., 2007). However a comparative analysis of MAPK/ERK activation in identified neurons of the food-aversion network has never been performed. Here we focused on procerebral neurons (the mollusk’s olfactory center processing information pertaining to the conditioned stimuli (CS)) and on command neurons controlling withdrawal behavior as both kinds of neurons establish the basic associative network underlying conditioned food aversion learning. Command neurons are located symmetrically in the right and left parietal ganglia so that besides comparing MAPK/ERK activation between neuronal populations (procerebral versus command neurons), a lateralized analysis of MAPK/ERK activation is possible.

Our results show that phospho-ERK level increased significantly after conditioned food aversion learning in the procerebrum and in command neurons of the right parietal ganglia RPa(2/3) but not in symmetrical command neurons of the left parietal ganglia LPa(2/3), where no activation was visible. Mimicking learning by means of serotonin application to the nervous system resulted in the same asymmetric distribution of phospho-ERK within the food-aversion network. Thus, conditioned food aversion learning involves synchronous serotonin-dependent MAPK/ERK activation both in procerebral neurons processing information from the conditioned stimulus and in command neurons processing information from the unconditioned stimulus (US) and controlling withdrawal behavior. Command neurons exhibit a functional asymmetry in MAPK/ERK activation, which could reflect lateralization of memory processes in mollusks.

Section snippets

Molecular cloning of Helix MAPK/ERK2 kinase

Total RNA was purified from the nervous tissue of Helix lucorum and RT-PCR analysis was performed using standard procedures. ERK kinase product was amplified using primers designed from two regions of the ERK kinase of Aplysia californica U40484: 5′-CCGTT TGAAC ATCAG ACCTA for the 5′ region, and forward 5′-AACAT CTCTG CCAGG ATACA T for the 3′ region. PCR fragment was purified and sequenced using the Sanger method.

Animals and behavioral studies

Experiments were carried out on adult (20–25 g) snails Helix lucorum. Animals were

Sequencing of Helix Lucorum ERK kinase

First, we verified that a MAP kinase isoform previously reported in the CNS of Helix lucorum does indeed correspond to the ERK2 kinase of vertebrates. The high similarity between kinases has been confirmed by several biological and computational methods such as NCBI-Blast Analysis of MAP-kinase sequences of diverse species using Genome bank databases, peptide competition control for the specificity of ERK antibodies of Human origin to Helix, and pretreatment with the ERK upstream inhibitor

Discussion

Our work shows that the MAPK/ERK pathway is selectively activated in different neurons of the central nervous system of Helix lucorum upon food aversion learning. Specifically, MAPK/ERK activation was found in procerebral neurons and in RPa(2/3) command neurons of the right parietal ganglia. While the former process odors and thus information pertaining to the CS, the latter control withdrawal behavior upon electric shock stimulation and process therefore information pertaining to the US.

Acknowledgments

This work was supported by the Governmental Program No. 0120.0408876, Grant RFBR No. 08-04-01325. We would like to thank Eugene Zabarovski and Vladimir Kashuba from Karolinska Institutet Stockholm for the help in ERK kinase sequencing. We would like to thank Gennady Vasil’ev for the performing a phylogenetic analysis. We are also grateful to Tom Abrams for helpful advices and discussions.

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