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First published online May 30, 2008
Journal of Experimental Biology 211, 1841-1849 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018028

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Redundancy of olfactory sensory pathways for odor-aversion memory in the terrestrial slug Limax valentianus

Miki Yamagishi, Etsuro Ito and Ryota Matsuo^*

Laboratory of Functional Biology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Shido, Sanuki, Kagawa 769-2193, Japan

View larger version (23K): [in this window] [in a new window]   Fig. 1. Apparatus and procedure used in odor-aversion conditioning. (A) A view of a slug's head and tentacles. (B) The experimental apparatus. Concentric circles with radii of 45 mm and 15 mm were drawn on black paper, and a glass plate was superimposed on the paper. This was lit from above. (C) Procedure for odor-aversion conditioning. (i) Carrot juice (1 ml) was laid on the glass plate in the shape of the larger circle with a radius of 45 mm. A slug was placed on the center of the circle. (ii) In the paired conditioning, 1 ml of quinidine sulfate solution was applied to the mouth of the slug when it was just about to touch the carrot juice.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Odor-aversion learning was not affected by the control treatment, but was completely abolished by amputation of both stets of tentacles, the STs and ITs. (Ai) Experimental schedule of the pre-conditioning control treatment. (Aii) The avoidance rate (%) of the slugs with the pre-conditioning control treatment. (Bi) Experimental schedule of the post-conditioning control treatment. (Bii) The avoidance rate (%) of the slugs with the post-conditioning control treatment. (Ci,Cii) Odor-aversion learning was affected by the amputation of both STs and ITs. (Ci) Experimental schedule of the post-conditioning ST+IT amputation. (Cii) The avoidance rate (%) of the slugs with the post-conditioning ST+IT amputation. The conditioning, the control treatment (or ST+IT amputation), and the retention test are indicated by •, and , respectively. The numbers above the columns indicate the number of slugs used for the retention tests. *P<0.05 and **P<0.01 by 2 test. n.s., not significantly different.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Odor-aversion learning was not affected by ST amputation. (Ai) Experimental schedule of the pre-conditioning ST amputation. (Aii) The avoidance rate (%) of the slugs with the pre-conditioning ST amputation. (Bi) Experimental schedule of the post-conditioning ST amputation. (Bii) The avoidance rate (%) of the slugs with the post-conditioning ST amputation. The numbers above the columns indicate the number of slugs used for the retention tests. **P<0.01 by 2 test.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Odor-aversion learning of the slugs with IT amputation. (Ai) Experimental schedule of the pre-conditioning IT amputation. (Aii) The avoidance rate (%) of the slugs with the pre-conditioning IT amputation. (Bi) Experimental schedule of the post-conditioning IT amputation. (Bii) The avoidance rate (%) of the slugs with the post-conditioning IT amputation. The numbers above the columns indicate the number of slugs used for the retention tests. **P<0.01 by 2 test (pre-conditioning amputation) or by Fisher's exact probability test (post-conditioning amputation).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Tracings of the paired and the unpaired conditioned slugs with IT amputation in the odor sensibility test. The humidified powder mixture of everyday food was placed on the upper half of each circle, and garlic homogenate was placed on the lower half. The time (in seconds) when the slugs reached either odor source is indicated. All slugs chose everyday food.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Apparatus and procedure of odor-aversion conditioning with a shading box. (A) The experimental apparatus with a shading box. (B) During the conditioning, 1 ml of carrot juice was laid in the shape of a half circle with a radius of 60 mm. (C) The conditioning procedure. (i) The slugs were placed just behind the center of the start line. (ii) For the slugs in the paired conditioned group, 1 ml of saturated (approximately 1% w/v) quinidine sulfate solution was applied to the mouth of the slug when it was just about to touch the carrot juice. At the same time, we recorded the time it took to reach the carrot juice after the middle of the slug's body passed the start line. If the slug did not reach the carrot juice within 3 min, it was eliminated from the experiments. (D) In the retention test, the carrot juice was laid in the shape of a half circle with a radius of 90 mm, and we tested whether they avoided the carrot juice for at least 3 min.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Odor-aversion learning of the slugs with IT amputation, using a shading box. (Ai) Experimental schedule of the pre-conditioning control treatment or IT amputation. (Aii) The avoidance rate (%) of the slugs with the pre-conditioning control treatment. (Aiii) The avoidance rate (%) of the slugs with the pre-conditioning IT amputation. (Bi) Experimental schedule of the post-conditioning control treatment or IT amputation. (Bii) The avoidance rate (%) of the slugs with the post-conditioning control treatment. (Biii) The avoidance rate (%) of the slugs with the post-conditioning IT amputation. The numbers above the columns indicate the number of slugs used for the retention tests. **P<0.01 by 2 test.

View larger version (32K): [in this window] [in a new window]   Fig. 8. The mobility test with cucumber juice. The average (± s.e.m.) time to reach the cucumber juice is shown as columns: ST, the superior tentacle-amputated group; IT, the inferior tentacle-amputated group. The numbers in or above the columns indicate the number of slugs used for that test. (A) Times taken in the lit environment (see Fig. 1). A significant difference was observed only in the pre-conditioning IT amputation group between the paired and unpaired-conditioned slugs. *P<0.05 by Student's t-test. (B) Times taken under a shading box (Fig. 6). (C) Average time taken by slugs that underwent different surgical treatments to reach the cucumber juice in the lit or shaded environments. **P<0.01, *P<0.05 by Student's t-test. (D) The avoidance rates in the mobility tests with cucumber juice (>3 min). **P<0.01 by 2 test.

View larger version (117K): [in this window] [in a new window]   Fig. 9. Histological analysis of the tentacles of the slugs. Toluidine Blue-stained sections of the tentacles. (Ai) The brain and tentacles of an intact slug. (Aii,Aiii) Longitudinal sections of tentacles of intact slugs. (Bi) The brain and tentacles of a slug after an 8 day recovery period following ST amputation. (Bii–v) Longitudinal sections of the tentacles of slugs after an 8 day recovery period from ST amputation. Scale bars, 1 mm.

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