The Olfactory Hole Board test is an adaptation of the classic Hole Board Test that is often used in the assessment of unconditioned behavior, cognition, and social interaction. The Olfactory Hole Board apparatus combines the advantages of the conventional Hole Board Test with that of the Open-Field Test apparatus. The task adds the element of odor to evaluate their effect on the rodent’s behavioral responses.

Odors play a crucial role in molding basic survival behaviors. In the wild, odors help animals identify and distinguish threats and communicate information such as gender, reproductive state, social status, and identity. Pheromones are odors that are associated with individuals of the same species and accompany mediation of different social behaviors within the species. On the other hand, kairomones are odors that are usually associated with predators. Kairomones evoke defensive behaviors in preys and thus are beneficial to the preys while being disadvantageous to the animals releasing them (Brennan and Kendrick, 2006; Fortes-Marco et al., 2013).

The apparatus is similar in construction to the classic Hole Board Test apparatus. It comprises a transparent box with a perforated floor. The holes can be closed with lids to create different configurations of open and closed holes. Other similar tests and apparatuses include the Attentional Set Shifting (IDED) Chamber, the Odor Span Test, the Dig Task, and the Fear Conditioning Chamber.

The Olfactory Hole Board test consists of three testing transparent acrylic boxes of dimension 51.5 x 51.5 x 41 cm and a removable grid-patterned hole board. The holes of the grid are 3 cm in diameter and come equipped with lids to allow closing of holes to create variations in the grid configuration.

Prior to the experiment clean the apparatus thoroughly. Since the task deals with odor, apparatus should be cleaned between trials or animals. Appropriately illuminate the apparatus. Tracking and video recording can be done using a tracking system such as the Noldus EthoVision XT. Infrared detectors can be used to monitor head-dipping behavior.

Olfactory Hole Board Task

Prior to introducing the subject into the apparatus, collect sample odors and place them in small containers under the holes of the hole board floor. Ensure that the samples are placed such that the subject can only smell it but not access it. Use the lids to close the holes that are not required in the experiment. Place the subject in the center of the apparatus and observe its response to the odor(s) for at least 20 minutes. Repeat the trial as required.

Investigation of behavioral responses to the simultaneous presentation of attractive and aversive odors

Male Sprague-Dawley rats were evaluated for their responses to being presented with attractive odors and aversive odors. Urine samples of fox, bobcat and female rat along with water were used as the odors in the experiment. Each sample was kept under one of the four corner holes, thus creating an environment wherein all the odors existed simultaneously. As expected, the subjects preferred the hole containing the female urine significantly more than the other three samples. The subjects showed avoidance behavior towards the holes containing the aversive stimuli. (Wernecke and Fendt, 2015)

Investigation of behavioral responses to predator versus non-predator odors

Male Sprague-Dawley rats were tested for their ability to distinguish between odors associated with a predator from harmless species. For this experiment, urine samples of a carnivorous puma, an omnivorous mona monkey, and a herbivorous elk were used with water as the control odor. The odors were simultaneously used in the trial. Percentage hole visit showed that the subjects avoided the holes containing the puma or the mona monkey urine while the elk urine evoked a neutral response similar to the water sample. (Wernecke and Fendt, 2015)

Evaluation of anxiolytic treatment on aversive odor avoidance behavior

Male Sprague Dawley rats received intraperitoneal injections of either saline or 5-HT1A receptor agonist buspirone (0.1, 1 mg/kg), 20 minutes before being tested in the Olfactory Hole Board test. Fox urine, coyote urine, female rat urine, and water as control odor were placed under one of the four corner holes. Based on the data obtained from the trials it was concluded that buspirone led to decreased avoidance response towards the aversive stimuli at both doses. However, at 1 mg/kg buspirone, the percentage of hole visits for different corner holes did not differ much from the water odor hole. (Wernecke and Fendt, 2015)

The following parameters are usually recorded during the Olfactory Hole Board Task.

• Number of head dips (nose pokes)
• Frequency of visits
• Percentage of total hole visits
• Total hole visits
• Hole preference

Strengths

The Olfactory Hole Board apparatus combines the advantages of the conventional Hole Board with that of the Open Field task. The apparatus is simple to construct and easy to use. The apparatus is also highly modifiable to enable different modes of investigation. The removable hole board allows the use of different configurations of open and closed holes by using lids. Placing different odors simultaneously during the task creates a more naturalistic olfactory environment. Testing protocols can be varied as per needs of the investigation such as using a single odor per task or multiple odors. The task can be used to access the effects of pharmacological manipulations and lesions on olfactory-based behavioral responses.

Limitations

In the Olfactory Hole Board task, placing odors too close to one another can lead to an infusion of the odors consequently influencing performances. The subjects also tend to have a preference for corner holes over the central holes (Wernecke and Fendt, 2015). Subjects that tend to be inactive or highly active are not recommended for the Olfactory Hole Board task. Presence of unwanted olfactory, auditory and visual cues can impact task performance. Handling and habituation processes can also affect the performance. When evaluating drug effects care must be taken to consider their effect on the olfactory and motor abilities of the subject.

• Olfactory Hole Board Test is used in the assessment of behavioral responses to odors.
• Olfactory Hole Board task combines the advantages of the Open Field Test and the classic Hole Board test.
• Animals tend to have a preference for corner holes than the central holes.
• The configuration of the hole grid can be changed by covering holes with lids.
• Simultaneous use of different odors in the Olfactory Hole Board test creates a natural olfactory environment.
• Attractive odors result in frequent hole visits and head dips. The converse is true for aversive odors.
• Certain drugs and pharmacological compounds can dull the olfactory and motor abilities of the subject.

1. Brennan P. A., Kendrick K. M. (2006). Mammalian social odours: attraction and individual recognition. Trans. R. Soc. B 361 2061–2078. 10.1098/rstb.2006.1931
2. Fortes-Marco L., Lanuza E., Martinez-Garcia F. (2013). Of pheromones and kairomones: what receptors mediate innate emotional responses? Rec. 296 1346–1363. 10.1002/ar.22745
3. Wernecke KE, Fendt M (2015). The olfactory hole-board test in rats: a new paradigm to study aversion and preferences to odors. Front Behav Neurosci. 9:223. doi: 10.3389/fnbeh.2015.00223.