Rodent Olfactory Exploration Box

Rodents, like all other mammals, highly depend on olfactory cues (or biological odors) for their survival. Olfaction plays a significant role in rodent behavior such as developing social hierarchies, mating, feeding, and for defense against potential predators (Wernecke and Fendt, 2015).

During olfaction, olfactory receptor neurons present in the olfactory epithelium at the back of the nasal cavity detect the chemical stimulus. These signals are sent to the main olfactory bulb (MOB) connected to the brain. The brain processes these signals, identifies and discriminates various odors in this way (Branigan and Tadi, 2022). Neural dysfunction or interneuron turnover rate can cause alterations in MOB neurogenesis thereby affecting the olfactory response (Perez-Villalba et al., 2015). The olfactory exploration box is a behavioral paradigm used for conducting habituation-dishabituation and threshold detection experiments for analyzing olfactory responses in rodents.

The olfactory exploration box fabricated from acrylic material comprises solid green walls. One wall has a hole drilled within it. The box is covered with a transparent lid to avoid odor dissipation during the test. The exploration paradigm contains a video recorder mounted at the top at a 30-40cm distance from the transparent cover. The video camera with high resolution records the subjects during the behavioral assay without interruption. 

Rodent location can be tracked using a video tracking software package such as Noldus EthoVision, ANY-Maze, or BehaviorCloud.

Olfactory Threshold Detection

A threshold detection test determines odorant sensitivity i.e., the lowest concentration of an odorant that can be perceived by the olfactory system. You can follow the underlying protocol for the olfactory threshold detection test (Perez-Villalba et al., 2015).

1.   Take male mice of desired strain (e.g., CD1 and C57BL/6J) and house them in groups of 4-5 per cage at a temperature between 21-25oC and 12:12 light: dark cycle. Provide the animals with food and water ad libitum.
2. Extract the subject from its cage under a hood and place it in a clean empty box for transportation to the test area.
3. Cover the box ground with a 1cm thick uniform layer of bedding (wooden chips).
4. For general habituation to the testing environment, place the mouse in the olfactory exploration box and allow it to move freely for about 3 minutes for context processing.
5.  Pipette out 20ꭒl mineral oil and deposit it directly into the head of the cotton swab.
6.  Introduce the cotton stick soaked in mineral oil into the box via the hole located in one of its walls. Protrude the stick approximately 3 cm and hold it there for 1 minute.
7.  Repeat steps 5 and 6 four times using a new stick soaked freshly in mineral oil each time to habituate the subject to the swab’s movement in and out of the box.
8.   Use a video recording system to monitor the animal’s behavior throughout the experiment.
9.  After completing five trials with “non-odorant stimulus,” soak the cotton swab in different dilutions of a novel test scent prepared in mineral oil (1:10, 1:20, 1:40, 1:80, and 1:160) to determine the lowest concentration that stimulates olfactory exploration in the subject.
10. Starting with the lowest concentration, dip a cotton swab into the scent, introduce it into the box through the hole, and hold it there for 1 minute.
11. Repeat step 10 separately for each concentration of the test stimulus and record the animal’s behavior.

The “inter-trial interval” (ITI) described as “the time between exposures to olfactory cues” allows the subject to show an olfactory response towards each swab impregnated in scent without losing interest in the task. An ITI of 1-2 minutes is advised for this task.

Olfactory Habituation-Dishabituation Assay (Perez-Villalba et al., 2015)

In the cotton swab-based habituation/dishabituation test, the subject is exposed to two different odors to assess “olfactory discrimination.” During the experiment, the researcher sequentially presents the subject with different odors using cotton swabs. The following protocol can be followed for this test.

Note: Inter-trial interval (ITI) for this task is 1-2 minutes.

1. Follow steps 2-8 in the above protocol for habituating the animal to testing conditions.
2. After completing five trials with “non-odorant stimulus,” expose the subject to five presentations of one odor followed by five presentations of a second odor.
3. Take a micropipette and fill it with 20ꭒl geraniol at concentration 1:20 and impregnate a swab head with it.
4. Introduce the cotton swab into the box and hold it in place for 1 minute.
5. Then introduce a cotton stick soaked in mineral oil into the box in the same way.
6. Repeat steps 2-5 four times.
7. Following this, dip a cotton swab in 20ꭒl citralva at 1:20, protrude it approximately 3 cm into the box, and hold it in place for 60 seconds.
8.  Then introduce a cotton stick soaked in mineral oil into the box in the same way.
9.  Repeat steps 7 and 8 four more times.
10. Use a video camera to record the animal’s olfactory behavior throughout the behavioral paradigm.
11. After completing the experiment, shift the animal to its cage and thoroughly wipe the olfactory exploration box with 5% alcohol and let it dry.

The following parameters can be observed using the Rodent Olfactory Exploration Box:

• Odor detection
• Olfactory discrimination
• Olfactory perception

Analysis of the Role of α-SYN in Parkinson’s disease

Perez-Villalba et al. (2018) studied the role of a synaptic protein α-Synuclein (α-SYN) in neurotransmission modulation and its aggregation in the cytoplasm of the degenerating neurons in a neurodegenerative disorder called Parkinson’s disease. The α-SYN is also responsible for olfactory neurogenesis in adult male and female mice. The researchers utilized an olfactory exploration box for their experiments. They took adult male and female mice and injected them with α-SYN to generate Snca mutant mice. The animals were gender group-housed and provided with food and water ad libitum.

The researchers conducted an olfactory habituation-dishabituation test using two synthetic scents geraniol and citralva. The mice were placed in the olfactory exploration box consisting of solid green walls with ground having 1cm thick bedding of wooden chips. They introduced the olfactory cue into the box via a hole of 1 cm diameter in one wall. For this, they impregnated a cotton swab soaked in mineral oil, protruded it approximately 3 cm into the hole, and held it there for 60 seconds. This step was followed by 5 consecutive trials with the same non-odorant stimulus. After completing 6 trials with mineral oil, the experimenters conducted six subsequent trials of geraniol and citralva at 1:20, both diluted in mineral oil. They used a new stick every time and kept the used sticks in a sealed container. In addition, they used a video camera to record the subjects’ olfactory responses like sniffing, smelling, or heading their nose toward the swab either by physical contact or through a close distance of 2-3 cm. After the experiment, they thoroughly cleaned the olfactory exploration box with 5% alcohol and dried it.

The scientists concluded that α-SYN is responsible for sustaining neurogenic potential in adult neural stem cells (NSCs) and plays a significant role in neurogenic defects.

Evaluation of the role of NT-3 in neuronal survival

Delgado et al. (2014) analyzed the role of neurotrophin (NT)-3 in the quiescence and long-term maintenance of neural stem cells (NSCs) for promoting neuronal survival. The experimenters used an olfactory exploration box for threshold detection test and habituation-dishabituation assay. In a threshold detection test, they took mice strains and injected them with recombinant human NT-3 using the suggested protocol. Then they placed mice in an olfactory exploration box having solid green walls and 1 cm thick wooden chips bedding on its floor. Following 3 minutes of free exploration, the subjects were exposed to a cotton swab soaked in mineral oil via the hole in one wall of the box located 8 cm above the box ground and 11.2 cm from the corners. They held the stick in place for one minute and repeated the same procedure for 5 subsequent trials of non-odorant stimulus. New sticks soaked in fresh mineral oil were used every time. Then, the experimenters exposed mice to six trials of increasing concentrations of citralva diluted in mineral oil (from 1:160 to 1:10). They also conducted a habituation-dishabituation test (six trials of geraniol and citralva at 1:20 diluted in mineral oil) using the same protocol as described above. They recorded the animals’ olfactory behavior using a video camera. In the end, they thoroughly cleaned the equipment with 5% alcohol.

They concluded that stem cells could control their quiescence by NT-3 in response to endothelium-secreted molecules.

Strengths 

The olfactory exploration box has numerous advantages. The foremost advantage is that it is easy to use. In addition, it allows the performance of multiple behavioral tests associated with olfaction. The equipment allows direct visual recording of the subject’s olfactory responses minimizing the chance of errors during data collection.

Limitations

A potential disadvantage is that a slight exposure to any odorant other than the one required for the experiment can negatively affect the results. However, discarding the used cotton swabs in a sealed container and covering the box with a transparent lid can help overcome this issue.

Precautions

 To hold the stick in place without movement, pass the stick through the hole and fix the other hand using adhesive tape.

• Do not let the cotton swab touch the inner walls of the box.
•  Use a lab timer to measure the exact time of exposure to the olfactory cue.
• Use a new stick for each trial and discard the used cotton swab in a sealed container to avoid odor dissipation.
• Thoroughly wipe the equipment with 5% alcohol after use and let it dry.

 An olfactory exploration box is employed to evaluate animals’ ability to identify and discriminate between different odors.

• It is fabricated from acrylic material and comprises solid green walls. One wall has a hole of 1 cm diameter through which odorant is introduced using a cotton stick.
• The olfactory exploration box is used to conduct threshold detection tests and habituation-dishabituation assay associated with olfactory responses in rodents.
• It is frequently used to study animal models of various human disorders resulting from olfactory dysfunction like schizophrenia, post-traumatic stress disorder (PTSD), Alzheimer’s disease, Parkinson’s disease, bipolar disorder, and so on.

Branigan, B., & Tadi, P. (2022). Physiology, Olfactory. In StatPearls [Internet]. StatPearls Publishing.

Delgado, A. C., Ferrón, S. R., Vicente, D., Porlan, E., Perez-Villalba, A., Trujillo, C. M., … & Fariñas, I. (2014). Endothelial NT-3 delivered by vasculature and CSF promotes the quiescence of subependymal neural stem cells through nitric oxide induction. Neuron, 83(3), 572-585.

Perez-Villalba, A., Palop, M. J., Pérez-Sánchez, F., & Fariñas, I. (2015). Assessment of olfactory behavior in mice: odorant detection and habituation-dishabituation tests. Bio-protocol, 5(13), e1518-e1518.

Perez-Villalba, A., Sirerol-Piquer, M. S., Belenguer, G., Soriano-Cantón, R., Muñoz-Manchado, A. B., Villadiego, J., … & Fariñas, I. (2018). Synaptic regulator α-synuclein in dopaminergic fibers is essentially required for the maintenance of subependymal neural stem cells. Journal of Neuroscience, 38(4), 814-825.

Wernecke, K. E., & Fendt, M. (2015). The olfactory hole-board test in rats: a new paradigm to study aversion and preferences to odors. Frontiers in Behavioral Neuroscience, 9, 223.