The procerebrum is necessary for odor-aversion learning in the terrestrial slug Limax valentianus

Abstract

The terrestrial slug Limax has a highly developed ability to associate the odor of some foods (e.g., carrot juice) with aversive stimuli such as the bitter taste of quinidine solution. The procerebrum (PC) is a part of the slug's brain thought to be involved in odor-aversion learning, but direct evidence is still lacking. Here we present evidence showing that the PC is essential for odor-aversion learning. Unlike sham-operated slugs, PC ablation 7 d prior to conditioning showed that most slugs did not avoid carrot juice in the memory retention test conducted 24 h after the conditioning. Slugs with the PC ablated 3 h, 1 d, 3 d, or 7 d after conditioning and examined by the memory retention test at 3 d after the PC ablation were also less likely to avoid carrot juice than sham-operated slugs. The PC ablation did not damage the ability of the slugs to sense attractive odor (everyday food) or innately aversive odor (onion or garlic). These results demonstrate that the PC is a necessary component in the retention and/or retrieval of odor-aversion memory.

Figure 1.

Photograph of the PC ablation surgery. (A) An anesthetized slug was laid on a paper towel, and the side of the head was cut. (B) Magnified view of the cut site. The arrow indicates the left side of the PC.

Figure 2.

Experiment I: Pre-conditioning PC ablation impaired odor-aversion learning. (A) Experimental schedule. (B) The avoidance rate (%) was substantially reduced in the PC-ablated group in comparison to the sham-operated groups on the day after the conditioning (P < 0.001 by χ² test). The numerals above the columns indicate the number of slugs used for the conditioning.

Figure 3.

Tracings of the PC-ablated and sham-operated slugs 8 d after surgery. The humidified everyday food was placed on the upper half of each circle, and repellent odorant ([A] onion or [B] garlic homogenates) was placed on the lower half. The time the slug reached either odor source is indicated.

Figure 4.

Histological analysis of the PC-ablated brain of the slugs. Toluidine blue-stained sections of the brains of (A) 11 randomly chosen slugs that had not avoided the conditioned odor of the carrot juice and (B) five randomly chosen slugs that avoided the conditioned odor of the carrot juice. (C) A section of the brain from a naive slug. The arrow indicates the PC. Scale bar, 500 μm.

Figure 5.

There was no significant difference in the residual area of the PC between the slugs that reached the carrot juice within 3 min (n = 5, left dots) and those that did not (n = 11, right dots). The average areas (1.42 ± 0.92 × 10⁻² mm² and 1.07 ± 0.89 × 10⁻² mm²) are indicated as broken and dotted lines, respectively.

Figure 6.

Experiment II: The post-conditioning PC-ablation impaired the odor-aversion learning. (A) The experimental schedule. The conditioning, the surgery, and the retention test are indicated by ●, ▲, and ■, respectively. (B) The avoidance rate (%) was substantially reduced in the PC-ablated group than in the sham-operated groups irrespective of the interval between the conditioning and the surgery. The numerals above the columns indicate the number of slugs used for the conditioning. *P < 0.05 and **P < 0.01 by χ² test. Solid bars: PC-ablated slugs; open bars: sham-operated slugs; shaded bars: naive slugs.