Materials and Reagents

Anti-drip bottles with a capacity of 150 ml (Classic Drinker de Luxe; Zooplus) (see Figure 1)

Figure 1. Anti-drip bottles
Animals: Adult male Wistar rats (Envigo Laboratories, Barcelona, Spain) with 11 weeks of age and weighing 281.2 (±2.1) g were used
Filtered and autoclaved water
Sucrose (white sugar suitable for human consumption. Azucarera Española S.A.)
32% sucrose solution (see Recipes)
4% sucrose solution (see Recipes)

Equipment

Timer and chronometer (Digital Onstart 100) (Figure 2A)
Consumption chamber
Rectangular (30 x 30 x 15 cm) Plexiglas boxes (Figure 2B)
Weighing scale (WLC 1/A2 Precision Balance; Radwag^©) (Figure 2C)
Personal protective equipment (Laboratory coat, gloves, masks, etc.) (Figure 2D)

Figure 2. Equipment. A. Timer and chronometer. B. Consumption chamber. C. Weighing scale. D. Personal protective equipment.

Procedure

See Figure 3 for the Timeline of daily experimental procedure.

Figure 3. Timeline of daily experimental procedure. *High/low value sucrose depending on each experimental group.

Individualize each animal in polycarbonate standard cages with free access to water and maintain under controlled standard conditions (12 h dark/light cycles starting at 08:00 h, 24 °C temperature, and 50%-60% humidity). Deprive animals of food to 82%-85% of their ad lib weights throughout the experiment. Once animals reach the required weight, feed daily with 9 g (±1.5 g) of rat chow to maintain body weight. All manipulations must be carried out at the same time every day and preferably by the same person. Efforts must be made to minimize suffering and reduce the number of animals used in the experiments. To maintain the animal’s body weight as assessed before the start of the experiment, it is important to weigh each specimen every day and provide supplementary food no less than 30 min after the end of each session. Moreover, sucrose solution must be prepared fresh daily to avoid contamination.
cSNC training lasts 19 daily sessions: 16 preshift sessions followed by 3 postshift sessions.
In the cSNC task, at least two experimental groups are required. During the preshift sessions, one of them has access to a large reward (e.g., 32% sucrose solution) and the other to a small reward (e.g., 4% sucrose solution). During postshift sessions, both groups are exposed to the small reward (4% sucrose solution).
Before each session, animal racks must be moved into a different room where the experiment will be held, allowing the rats 15 min in their home cages to settle down, before placing them the consumption chambers.
The contrast session lasts 5 min from the first contact with the sipper tube. The position of the bottle is counterbalanced daily to minimize the effects of side preference. The animal is placed back in its home cage at the end of the session.
Session duration for each box can be measured manually with digital clocks (Digital Onstart 100).
Sucrose consumption is recorded for each animal weighing the bottles before and after the session, and then transforming the weight to milliliters (having accounted for the density of each solution) using the formula: Grams of solution consumed/density (grams per milliliter). All animals receive training every day in groups of 4, with the order of groups counterbalanced across days. At the end of every session, contrast chambers must be wiped with a wet paper towel and feces removed as needed.
Animals must be fed with the proper amount of rat chow to maintain body weight no less than 30 min after the end of each session.

Data analysis

In the cSNC situation, the dependent variable was the total amount of sucrose consumed in each session (in milliliters; Figure 4).

Figure 4. Mean (±SEM) sucrose consumption (ml) during the last preshift session (16) and the three postshift sessions (17-19). 32-4: animals exposed to reward devaluation from 32% to 4% sucrose during postshift sessions. 4-4: animals exposed to an unshifted reward condition, always receiving access to 4% sucrose throughout the experiment. The asterisk reflects a significant difference between the downshifted and unshifted groups. (Figure modified from Jiménez-García et al., 2016.)

Notes

The consumption chamber should be cleaned with sterile water to remove any animal odor. It’s necessary to cover the boxes’ floor with standard home cage sawdust. No access to food or water should be allowed.
For the sucrose presentation, a graduated cylinder (in 0.01 ml units) with a metallic sipper tube can be used.
cSNC training can be reduced to 15 daily sessions: 10 preshift sessions followed by 5 postshift sessions. This procedure is the typical protocol in cSNC experiments and, for that reason, it is advantageous too. Some experiments have reported a cSNC effect with minimal preshift experience (one session: Flaherty et al., 1983), but less than 10 preshift sessions is infrequently used. The final protocol chosen will depend on other requirements associated with the problem of interest.
A recording system (i.e., camera) could be used optionally. Thus, place the recording system on the side of the chamber. Ensure that the oral region of the animal is clearly visible.
A magnetic stirrer may be useful to properly dissolve sucrose.

Recipes

32% sucrose solution
32 g sucrose (commercial white sugar)
68 g distilled water
4% sucrose solution
4 g sucrose (commercial white sugar)
96 g distilled water

Acknowledgments

This paper was partially supported by the Spanish Ministry of Economy and Competitiveness (grant PSI-2013-44945-P), the Junta de Andalucía (grant CTS109), FEDER funds and the Master’s program in Basic and Applied Neuroscience and Pain, University of Granada, Spain. MRP’s participation was supported by a Fulbright US Scholar Award. Animals were maintained according to the EU Directive 2010/63/EU for animal experiments. We thank the CTS430 group, especially M. Á. Zafra, for their help. This protocol and figures are adapted from our previous work (Jiménez-García et al., 2016).

Competing interests

The authors declare no conflicts of interest or competing interests.

Ethics

The experimental protocols were approved by the University of Granada Research Ethics Committee. Reference: SSA/SIS/MD/jv (valid from 31/03/2016 to 31/03/2021).

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