Real-time place preference – RTPP

Original Research Paper

Impairments in laterodorsal tegmentum to VTA projections underlie glucocorticoid-triggered reward deficits

Protocol

Real time place preference in rats

Bárbara Coimbra^{1, 2}, Ana João Rodrigues^{1, 2}

¹ Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal;

² ICVS/3B’s–PT Government Associate Laboratory, Braga/Guimarães, Portugal

[Abstract] 

The real time place preference (RTPP) is a standard behavioral test used to evaluate preference or avoidance behaviors associated with optogenetic or electrical stimulation in rodents. This behavioral test is a modified version of the classical conditioned place preference (CPP) test. Using this protocol, preference and avoidance behaviors can be easily assessed within the same apparatus with high spatial and temporal selectivity. This task involves the use of a two chamber apparatus, in which one side is associated with a particular stimulus (optogenetic or electric stimulation), and the other side with the absence of the stimulus. Here, we provide a detailed protocol for assessing the rewarding or aversive properties of optogenetic stimulation of laterodorsal tegmentum (LDT) terminals the ventral tegmental area (VTA).

[Background] 

Optogenetic tools coupled with behavioral testing is a valuable tool to study the role of distinct brain circuits, or discrete neuronal populations, in particular types of behavior. In the context of reward/aversion-related behaviors optogenetics allows spatio-temporally dissection of how brain circuits or specific populations of cells affect behavior. 

One classical method to evaluate the rewarding or aversion properties of drugs of abuse, and more lately, the effects of specific neuronal stimulation, is the Conditioned Place Preference (CPP). The CPP is based on the premise of Pavlovian association, in which a particular environment is associated with a rewarding or aversive experience (Tzschentke, 1998). The pairing of a stimulus (drugs, stimulation) with a neutral environment can later lead to approach or avoidance after the association, even in the absence of the rewarding/aversive stimulus, respectively. The CPP is usually performed in an apparatus containing three distinct compartments, such as floor texture or wall patterns. During the conditioning phase, the subject is exposed to the drug (or optogenetic stimulus) and is restricted to one of the compartments (drug/stimulus side), and in the next session, the individual is exposed to vehicle (or mock-stimulation) and restricted to the other side. The reinforcing properties of the drug are assessed in a drug-free session where the subject is allowed to freely explore the whole apparatus. If the animal spends more time in the drug-paired compartment, the drug has induced preference - rewarding effects. If the drug is perceived as aversive, the animal will avoid the drug-paired compartment and spend more time in the saline-paired compartment, indicating conditioned place aversion (CPA).

The real-time place preference (RT-PP) box has two chambers that are visually similar (Stamatakis & Stuber, 2012) and one chamber is randomly assigned to deliver optical stimulation whenever the animal is located in this chamber, while in the other chamber no stimulation is performed. Contrary to the classical CPP paradigm, in which the subject is conditioned to one of the sides (non-contingent conditioning), in the RT-PP, the subject is allowed to move freely in the entire apparatus and behavior is recorded throughout the session (contingent). Entry into one of the compartments is paired with intracranial light-stimulation. If the light-stimulation is perceived as rewarding, the rat will remain in the light-paired compartment, while if the light-stimulation is perceived as aversive, the rat will exit the compartment to escape and avoid the stimulation. In this configuration, the RT-PP paradigm measures if optogenetically-induced neuronal modulation is rewarding or aversive. 

Equipment

1. Custom made transparent RTPP arena (60x60x40cm) with middle division between chambers
2. Rotary joint (Doric lenses, FRJ_1x1_FC-FC)
3. Digital camera with tripod to support, placed directly above the arena
4. Master-8 pulse generator (Microprobes, Master-8 Pulse stimulator)
5. Optic patch cable fiber (made in house, materials from Thorlabs)
6. Laser 100 mW 473nm Laser System (Laser2000; CL-473-100) and FC cable connector for Master-8
7. Power meter with sensor (Thorlabs; PM100D)
8. Ferrule mating sleeve (Thorlabs; ADAF1)

Software

1. EthoVision XT tracking system (Noldus Information Technology, Inc. Leesburg, Virginia)
2. Microsoft Excel (Microsoft)
3. Statistical Software (i.e., SPSS, GraphPad Prism, R)

Procedure

This particular protocol has been carried out using Wistar han rats , aged >8 weeks, unilaterally injected with 0.5μl of AAV5–EF1a–WGA–Cre–mCherry in the VTA and 0.5 μl AAV5–EF1a–DIO–hChR2–YFP in the LDT (Coimbra et al., 2017). An optic fiber ferrule (200μm core fiber optic; 2.5 mm stainless steel ferrule; Thorlabs) was implanted in the VTA and and fixed in the skull, together with 2 screws (2.4mm screws, Bilaney, with dental cement (C&B kit, Sun Medical). The RTPP protocol was performed 4 weeks after surgery. Animals were previously handled by the researcher for several days (at least 5) to reduce experimenter-related anxious behavior.

A. Experiment setup

1. Place the apparatus in a room dedicated to behavioral research, devoid of extraneous odors or sounds. The room's lights will be used to be able to track rats in the videos during testing.
2. Use a shelf or rack close to the apparatus and place all equipment: Laser with connecting fiber patch cords coupled with rotary joint, Master-8. The camera should be placed directly pointing to apparatus and tested in a non-test animal to confirm that the position is suitable. 
3. Connect the laser source to the optic fiber and rotary joint. Connect a patch cord to the rotary joint. Confirm that fiber patch cord is long enough to allow animal to freely explore the entire apparatus and stabilize rotary joint above the apparatus.
4. Use a power meter with a sensor to set the laser power to 10 mW.
5. Set stimulation parameters in Master-8 pulse stimulator (for this study: frequency 20 Hz, pulse duration 10ms).
6. Set the stimulation-paired chamber in a randomly-assigned counterbalanced manner.

B. Experiment

1. Start trial by turning on recording camera.
2. Gently take the rat out of its cage and connect the fiber-optic implant to the fiber-optic patch cord using a ceramic sleeve.
3. Place the animal in the non-stimulation side of the chamber.
4. Determine total time of 15min using a timer.
5. Allow animal to freely explore the apparatus without any disturbances (noise, smells).
6. Use camera screen to monitor animal and turn on laser (if using manual stimulation) in Master-8, whenever the animal crosses to stimulation-paired side and keep it turned on while it stays on stimulation-paired side. See NOTE 1 for automatic procedure.
7. Monitoring the animal in the camera screen, check whenever the animal exits the stimulation-paired compartment, and turn laser off in Master-8.
8. After 15min, turn recording off and gently take animal out of the apparatus and unclouple fiber-optic patch cord.
9. Place animal back in its cage.
10. Clean arena very well with 10% ethanol between subjects, let it dry before next subject. At the end of the protocol, clean arena with 70% ethanol.

Data analysis

Behavior is analyzed by plotting the time spent on either chamber of the RTPP apparatus by manually or automatically visualizing the videos. Researcher should be blind to experimental group that is being analyzed. In addition, the number of entries to either side of the chamber is also collected. Number of entries is collected when half of the animal crosses the board of each arena.

Other option to analyze the behavioral videos is to use the EthoVision XT tracking software. The arena setup should be calibrated and a ruler used to measure a specific part of the physical apparatus, drawing a line corresponding to the part measured on the image within the software under the Draw Scale to Calibrate tab and enter the known value. After designing the arena, create zones assigned to stimulation-paired chamber or non-stimulation-paired chamber. Set trial time for a 15min RTPP session and confirm that animals are correctly detected in all videos. In addition to time spent in each chamber, in this way, the distances traveled may also be collected

For statistical analysis please refer to original publication in statistical analysis (Coimbra et al., 2017). In short, if testing 2 or more groups (after assessing normality distribution of data), compare means with a repeated measures 2way-ANOVA considering comparisons within subjects (stimulation-paired vs non stimulation-paired chamber) and between subjects.

Note 1

1. In addition to manual stimulation using the Master-8 pulse stimulator, this equipment can be used together with EthoVision XT tracking software in real time during the behavior task. In this case, a computer running EthoVision XT connected to a USB IO-box is able to control when stimulation is delivered during the task; EthoVision XT sends out signals via the USB IO-box that is connected to the Master-8, whenever it tracks the animal crossing zones. This automatic method is preferred over the manual.

References

Coimbra, B., Soares-Cunha, C., Borges, S., Vasconcelos, N. A., Sousa, N., & Rodrigues, A. J. (2017). Impairments in laterodorsal tegmentum to VTA projections underlie glucocorticoid-triggered reward deficits. ELife, 6. https://doi.org/10.7554/eLife.25843

Stamatakis, A. M., & Stuber, G. D. (2012). Activation of lateral habenula inputs to the ventral midbrain promotes behavioral avoidance. Nature Neuroscience, 15(8), 1105–1107. https://doi.org/10.1038/nn.3145

Tzschentke, T. M. (1998). Measuring reward with the conditioned place preference paradigm: A comprehensive review of drug effects, recent progress and new issues. Progress in Neurobiology, 56(6), 613–672. https://doi.org/10.1016/s0301-0082(98)00060-4

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