DOI: 10.5061/dryad.tb2rbp098
title: Independent operations of appetitive and aversive conditioning systems lead to simultaneous production of conflicting memories in an insect

description: Pavlovian conditioning is a ubiquitous form of associative learning that
 enables animals to remember appetitive and aversive experiences. Animals
 possess appetitive and aversive conditioning systems that memorize and
 retrieve appetitive and aversive experiences. Here we addressed the
 question of whether integration of competing appetitive and aversive
 information takes place during the encoding of the experience or during
 memory retrieval. We developed novel experimental procedures to address
 this question using crickets (Gryllus bimaculatus), which allowed
 selective blockade of the expression of appetitive and aversive memories
 by injecting octopamine and dopamine receptor antagonists. We conditioned
 an odor (conditioned stimulus 1, CS1) with water and then with sodium
 chloride solution. We developed datasets of relative preference between
 conditioned odor and control odor before training and at each step of the
 conditioning trials for statistical evaluation of the conditioning effect.
 At 24 hours after conditioning, crickets retained both appetitive and
 aversive memories, and the memories were integrated to produce a
 conditioned response (CR). Importantly, when a visual pattern (CS2) was
 conditioned with CS1, appetitive and aversive memories formed
 simultaneously. This indicates that appetitive and aversive second-order
 conditionings are achieved at the same time. The memories were integrated
 for producing a CR. We conclude that appetitive and aversive conditioning
 systems operate independently to form parallel appetitive and aversive
 memories, which compete to produce learned behavior in crickets.
description: # Independent operations of appetitive and aversive conditioning systems
 lead to simultaneous production of conflicting memories in an insect
 [https://doi.org/10.5061/dryad.tb2rbp098](https://doi.org/10.5061/dryad.tb2rbp098) The PI (preference index) for the vanilla odor of individual crickets is described for each experiment. ## Description of the data and file structure We developed datasets to evaluate the effect of various types of conditioning in crickets for addressing a question of whether integration of competing appetitive and aversive information takes place during the encoding of the experience or during memory retrieval [1, 2]. This study is based on our accumulated knowledge on the roles of octopamine neurons and dopamine neurons in formational and retrieval of memory in Pavlovian conditioning and second-order conditioning in crickets. [3-10]. Relative preference between the conditioned odor or visual pattern and the control odor or pattern was collected using test apparatuses [3, 4] at each step of conditioning trainings in crickets. Relative preference was defined as the preference index (PI) for the conditioned odor or visual pattern, defined as Tc/ (Tc+Tn), where Tc was the time spent exploring the odor or pattern used in conditioning and Tn was the time spent exploring the odor or pattern not used in conditioning in the test. The name of each dataset (Data Figs. 2a, 2b, 2c, 2d, 3a, 3b, 4a, 4b, 4c, S2a, S2b) is the name of the figure of our associated paper. The experiments for each data are as follows: Data Fig. 2a-d. We investigated retention of appetitive and aversive memories after conditioning of an odor with water and then with sodium chloride solution as evaluated by injection of epinastine (OA receptor antagonist) or flupentixol (DA receptor antagonist). The time schedules of the experiments are shown above the graphs. (a, b) Two groups of crickets received appetitive and aversive conditioning for the same odor (CS). Odor preference was tested before (Pre-test), 30 min after appetitive conditioning (Post-test 1) and 30 min and 24 hours after aversive conditioning (Post-tests 2 and 3). Each test took 85-90 min. Crickets in each group were then injected with epinastine (a) or flupentixol (b) and received the final test 30 min later (Post-test 4). (c, d) Two groups of crickets received appetitive and aversive conditioning. Odor preference was tested before (Pre-test), 30 min after appetitive conditioning (Post-test 1) and 30 min after aversive conditioning (Post-test 2). Then the crickets in each group were injected with epinastine (c) or flupentixol (d) and received the final test 30 min later (Post-test 3). The result of this experiment demonstrates formation and expression of parallel memories after sequential appetitive and aversive conditioning of a CS.  In addition, the experiment demonstrates that expression of previously formed appetitive memory is transiently inhibited after subsequent aversive conditioning of the same CS. Data Fig. 3a, b. We investigated formation of appetitive and aversive memories by pairing a visual pattern (CS2) with an odor (CS1) that was paired with water and then with sodium chloride solution, evaluated by injection of epinastine (a) or flupentixol (b). The time schedules of the experiments are shown above the graphs. Four groups of crickets received pairing of an odor (CS1) with water and then sodium chloride solution, and they received pairing of a pattern (CS2) with the odor (CS1) 24 hours later. Then the crickets in each group were injected with either saline (a, b) or saline containing (a) epinastine or flupentixol (b). Pattern preference was tested before training (Pre-test), 1 hour after CS2-CS1 pairing (Post-test 1) and 30 min after injection (Post-test 2). The results of this study indicate that appetitive and aversive memories were formed simultaneously for CS2 by pairing CS2 with CS1. In other words, appetitive and aversive SOCs are achieved at the same time by this pairing. Data Fig. 4a, b. We investigated reduction of the expression of appetitive memory by US (water) devaluation after pairing of an odor (CS1) with water and then with sodium chloride solution (a, b) and after pairing of a visual pattern (CS2) with the odor (CS1) (c), evaluated by injection of epinastine or flupentixol. The time schedules of the experiments are shown above the graphs. (a, b) Crickets in four groups received an odor preference test 2 hours before training (Pre-test), 30 min after providing water until satiation (Post-test 1), and 30 min after injection of saline (a, b) or saline containing epinastine (a) or flupentixol (b) (Post-test 2). (c). Crickets in another two groups received a pattern preference test before training (Pre-test), 1 hour after pairing of a pattern (CS2) with the odor (CS1) paired with water and sodium chloride solution (Post-test 1), 1 hour after providing water until satiation (Post-test 2), and 30 min after injection of saline or flupentixol (Post-test 3). The results of this experiment indicate that expression of appetitive memory is inhibited in water-satiated crickets and hence aversive memory governs the response to the CS2. Data Fig. S2a, b. We investigated retention of aversive and appetitive memories after aversive-to-appetitive counterconditioning (a, b) Crickets in two groups received pairing of an odor with sodium chloride solution and then with water. Odor preference was tested before (Pre-test), 30 min after aversive conditioning (Post-test 1) and 30 min and 24 hours after appetitive conditioning (Post-tests 2 and 3). The crickets were then injected with epinastine (a) or flupentixol (b) and received the final test 30 min later (Post-test 4). The results of this experiment demonstrate that aversive-to-appetitive counterconditioning is achieved. By evaluating the dataset, we conclude that that appetitive and aversive conditioning systems operate independently to form parallel appetitive and aversive memories, which compete to produce learned behavior in crickets. 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