Neuroscience Letters 555 (2013) 149–153 Contents lists available at ScienceDirect Neuroscience Letters jou rn al hom epage: www.elsevier.com/locate/neulet High voltage with little current as an unconditional stimulus for taste avoidance conditioning in Lymnaea stagnalis Satoshi Takigami a,b , Hiroshi Sunada c , Ken Lukowiak c , Manabu Sakakibara a,b,d,∗ a Graduate School of High-Technology for Human Welfare, Tokai University, 410-0321 Numazu, Shizuoka, Japan b Graduate School of Bioscience, Tokai University, 410-0321 Numazu, Shizuoka, Japan c Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, AB, Canada T2N 4N1 d School of High-Technology for Human Welfare, Tokai University, 410-0321 Numazu, Shizuoka, Japan h i g h l i g h t s • A new and better taste avoidance conditioning paradigm for Lymnaea has been developed. • 15 pairs of CS (sucrose) and UC (high voltage) resulted in LTM lasting 1 week. • 5 pairs of CS–US resulted in STM. a r t i c l e i n f o Article history: Received 5 August 2013 Received in revised form 12 September 2013 Accepted 14 September 2013 Keywords: Taste avoidance conditioning Lymnaea stagnalis High-voltage stimulus Short-term memory Long-term memory a b s t r a c t A new and better taste avoidance conditioning paradigm for Lymnaea has been developed that replaces the previously used tactile unconditional stimulus (US) with an brief electrical stimulus (1000 V, 80 A), while continuing to use a sucrose application to the lips as the conditional stimulus (CS). With 15 paired CS–US presentations on a single day, we were able to elicit both short-term memory (STM) and long-term memory (LTM). The LTM persisted for at least one week. While STM was elicited with 5, 8, or 10 paired presentations of the CS–US on a single day, LTM was not. The new US used here was more consistent than the previously used US, and this stimulus consistency may explain why 15 paired CS–US presentations now result in LTM formation. © 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Our understanding of the causal neuronal mechanisms that underlie learning and the subsequent formation of long-lasting memory has been greatly advanced by studies of simpler inver- tebrate model systems [2,5]. Previously, we developed taste avoidance conditioning (TAC) in the pond snail Lymnaea to study the neuronal basis of associative learning and memory formation. To accomplish this, we applied Abbreviations: ANOVA, analysis of variance; CGC, cerebral giant cell; CS, condi- tional stimulus; IPSP, inhibitory post-synaptic potential; ITM, intermediate-term memory; LTM, long-term memory; N1M, neuron 1 medial cell; N3t, neuron 3 transient cell; STM, short-term memory; TAC, taste avoidance conditioning; US, unconditional stimulus. ∗ Corresponding author at: Laboratory of Neurobiological Engineering, School of High-Technology for Human Welfare, Tokai University, 410-0321 Numazu, Shizuoka, Japan. Tel.: +81 55 968 1111x4504; fax: +81 55 968 1156. E-mail addresses: manabu@tokai.ac.jp, msakaki@chime.ocn.ne.jp (M. Sakakibara). a sucrose solution to the lips, which elicits feeding, as the condi- tional stimulus (CS) and we applied a tactile stimulus to the head, which stops feeding, as the unconditional stimulus (US) [6]. After paired CS–US presentations, the snail learns and forms memory for TAC. Importantly, however, this method of conditioning requires manual skill to consistently apply the US to the head of the ani- mal. If the US is too weak, feeding does not cease, and if the US is too strong, the snails respond with whole-body-withdrawal behav- ior, from which recovery took ten minutes to hours. To establish a more consistently effective conditioning procedure, we developed new methodology for use as the US. We used a high electric voltage field around the animal, which we refer to as US2, while the tactile stimulus is referred to as US1. US2 in our new procedure serves as an aversive stimulus that causes the snail to start withdrawing, which is incompatible with feeding. Thus, the feeding response elicited by the CS is immedi- ately terminated. However, possibly because there is little current flow (i.e., micro-ampere order), the snails quickly cease with- drawal. Thus, paired presentations of the CS–US2 can occur, and with successful conditioning, the CS no longer elicits feeding. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.09.042