Behavioral paradigms
This protocol is extracted from research article:
A central control circuit for encoding perceived food value
Sci Adv, Nov 21, 2018; DOI: 10.1126/sciadv.aau9180

Behavioral paradigm 1. Lymnaea’s behavior was observed by placing them in a custom-built behavioral chamber filled with Cu2+-free water. The chamber held the animal on the surface of the water, allowing for the application of sensory stimuli to the mouth of the snail while being able to fully observe movements of the feeding structures. All behavioral experiments were videoed and analyzed using ImageJ software. Animals were left to acclimatize for 10 min before testing. An ingestion bite was triggered by brief application of an appetitive stimulus (lettuce) to the lips of the animal, eliciting a bite response in all animals tested, regardless of hunger state (148 animals). Upon opening of the mouth, either a tactile probe was placed inside or 50 μl of 0.008% AA was applied to the mouth/lips of the animal. The tactile probe consisted of a 1-ml syringe whose tip had been heated and pulled into a fine point. In both conditions, a radula motor program was induced in response to the stimulus. All animals were videoed, and these responses were later analyzed (see below section for details of analysis).

Behavioral paradigm 2. To test whether tactile stimulation of the esophagus elicited ingestion or egestion, an incision was made under the mantle cavity to expose a region of the esophagus, allowing for the mechanical stimulation of the structure with a pair or forceps. Esophageal stimulation was sufficient to elicit an egestion bite even when presented during a period of quiescence. The elicited bite was videoed and analyzed (see below).

Behavioral paradigm 3. To determine the effects of drug injection on animal’s locomotion, we tracked animals in a novel environment for 30 min. Briefly, the animals were placed in a 14-cm-diameter petri dish filled with 100 ml of Cu2+-free water. Recording started as soon as they were placed in the arena so as to monitor their initial behavior. Animals were recorded at one frame/s for 30 min. Videos were analyzed using idTracker (54), and total distance was traversed compared between groups.

Behavioral paradigm 4. The effects of drug treatment on the animal’s food searching behavior in a novel environment were tested by counting the number of appetitive bites during the first 10 min from being placed in a petri dish filled with 100 ml of Cu2+-free water.

Behavioral paradigm 5. The effects of drug treatment on the animal’s responsiveness to sucrose were tested by placing the animal in a petri dish filled with 90 ml of Cu2+-free water. Animals were allowed to acclimatize for 10 min, and then 5 ml of water was added to the dish. The number of bites performed was counted for 2 min, and then 5 ml of sucrose (0.33% final solution) was added, and the number of bites performed was counted for 2 min. A feeding score was obtained by subtracting the number of bites in response to water from the number performed in response to sucrose.

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