Classical conditioning of feeding in Aplysia: I. Behavioral analysis

Abstract

A training protocol was developed to classically condition feeding behavior in Aplysia californica using tactile stimulation of the lips as the conditional stimulus (CS) and food as the unconditional stimulus (US). Paired training induced a greater increase in the number of bites to the CS than unpaired training or US-only stimulation. Memory for classical conditioning was retained for at least 24 hr. The organization of the reinforcement pathway that supports classical conditioning was analyzed in additional behavioral experiments. No evidence was found for the contribution to appetitive reinforcement of US-mediating pathways originating in the lips of the animals. Bilateral lesions of the anterior branch of the esophageal nerve, which innervates parts of the foregut, however, were found to attenuate classical conditioning. Thus, it appears likely that reinforcement during appetitive classical conditioning of feeding was mediated by afferent pathways that originate in the foregut. The companion paper () describes two neurophysiological correlates of the classical conditioning.

Fig. 1.

Protocol for classical conditioning of feeding behavior. A, Animals were restrained in clear plastic tubes, which facilitated stimulation of the mouth and lips (see Materials and Methods for details). Tactile stimulation of the lips with a paintbrush served as the conditional stimulus (CS) and food as the unconditional stimulus (US). B, A delayed pairing procedure was used in which the CS onset preceded the US onset by 3 sec. CS and US overlapped for 5 sec, after which the CS was terminated. The US was presented until the animal ingested it, but no longer than 60 sec. Unpaired training consisted of CS and US presentations at an ISI of 120 sec. An additional control group received US presentations only. The ITI was 4 min in all experiments. C, Animals were tested for feeding behavior (biting) by offering them a piece of seaweed (US) before the experiment. Immediately before training, animals received four CS presentations (pretest). The number of bites during this stimulation period was counted. Subsequently, animals received 10 trials of paired, unpaired, or US-only training. After training, the number of bites elicited by four CSs (test) was determined and compared to the score obtained during the pretest. Finally, animals were tested once again for feeding behavior by offering a piece of food (US).

Fig. 2.

One hour retention for classical conditioning of feeding behavior. Conditioning was measured by a change in the number of bites elicited by the CS before (pretest) and after (test) training. Paired training resulted in a greater increase than unpaired training and US presentations only. In this and subsequent illustrations, data are displayed as means + SEM.

Fig. 3.

Long-term memory for classical conditioning of feeding. Associative memory for the CS, induced by paired training was retained for a minimum of 24 hr. Paired training resulted in a significantly greater increase in the number of bites to the CS than unpaired training. Before testing, animals were fed a piece of seaweed to mimic the conditions of the pretest. This procedure may account for the higher scores in both groups, compared to those reported in other experiments.

Fig. 4.

Organization of US-mediating afferents. In the training protocol outlined above, the CS is limited to the tactile stimulation of the lips. In contrast, the US (food) comes in contact with the sensory epithelia of the lips and the foregut. The lips and other regions of the head are innervated by nerves of the cerebral ganglion. The foregut is exclusively innervated by nerves of the buccal ganglia. Although food-induced activation of cerebral afferents reliably elicits biting behavior, it is not known whether these afferents also mediate reinforcement during classical conditioning. An alternative hypothesis is that buccal afferents mediate this reinforcement instead or contribute to reinforcement.

Fig. 5.

Behavioral dissection of the reinforcing pathways. Groups of animals were conditioned by pairing the CS with food that could be ingested as in previous experiments (normal US) or food that was brought in contact with the lips, but was not made available for ingestion (lips-only US). Unpaired controls received the same treatment at the longer ISI. Only paired training with the normal US resulted in associative learning, as measured by the number of bites during CS presentation before and after training. Thus, cerebral afferents originating in the lips were not sufficient to mediate reinforcement during classical conditioning, despite the fact that they reliably elicit biting behavior. US presentation to only the lips resulted in a decrease in the number of bites to the CS 1 hr after training. This decrease was not pairing-specific and was examined in more detail in the experiment below (Fig. 6).

Fig. 6.

Time-dependent decrements in CS-evoked biting. To determine whether food presented only to the lips caused an inhibition of feeding behavior, the effect of US presentation to the lips only was compared to the time-dependent decrement in food arousal over an interval equivalent to training and 1 hr retention. Ten trials of food stimulation of only the lips resulted in a decrease in responsiveness to the CS 1 hr after the last food presentation. This decrease, which was similar in magnitude to the decrease seen after paired training with US presentation to the lips only, however, was not distinguishable from the time-dependent decrease in biting responses in control animals that did not receive any stimulation after the pretest. This result suggests that food stimulation of the lips only does not have an inhibitory effect on feeding, but rather fails to support food arousal over the duration of the retention interval.

Fig. 7.

Effect of esophageal nerve lesions on classical conditioning. Animals with bilateral lesions of the anterior branch of the esophageal nerve (En₂) did not show a significant increase in the number of bites to the CS 1 hr after training when compared with unpaired control animals. In contrast, sham-operated animals that were trained concurrently showed normal conditioning.

Fig. 8.

Model circuit for classical conditioning of feeding behavior in Aplysia. Information about the CS is mediated by cerebral mechanosensory afferents that originate in the lips. These afferents make weak synaptic connections to a response system consisting of command neurons, located in the cerebral ganglion, and central pattern generators in the cerebral and buccal ganglia that produce consummatory feeding behavior (bites). As a result of classical conditioning, these weak connections and/or the response system itself undergo modulation that leads to an increased probability for feeding behavior to occur in response to mechanosensory stimulation of the lips. The modulatory system is activated by sensory afferents mediating the US (food). Although US-mediating cerebral afferents from the lips reliably activate the response system, no evidence was found that they activate the modulatory system (dashed lines), and thus do not contribute to appetitive reinforcement. In contrast, experimental evidence is consistent with the idea that afferents from the foregut, including the En₂, mediate the reinforcing component of the US by activating the modulatory system during conditioning.