Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.
In many neurodegenerative diseases and particularly in Parkinson’s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson’s disease as well as in testing potential disease-modifying therapies.
Olfactory dysfunction is linked to a number of neurodegenerative disorders including Parkinson’s disease (PD), Alzheimer’s disease, and Huntington’s disease1. In PD, olfactory impairments include deficits in odor identification, detection, and discrimination and are found in up to 70–95% of patients2-5. These deficits can precede the cardinal motor symptoms of PD by up to 4 years, indicating that olfactory dysfunction may signal the early stages of PD6-10. The early occurrence of olfactory deficits in PD has led to a keen interest in olfactory dysfunction and the underlying mechanisms involved. In preclinical studies in rodents, olfactory dysfunction could be a sensitive outcome measure to predict the therapeutic potential of novel therapeutic strategies.
Many tests have been designed and extensively used to characterize sensorimotor impairments in rodent models of PD and to test the therapeutic potential of novel treatments11-15. Even though olfactory deficits are well documented in PD, olfactory function has not been routinely measured in many models. This view is changing though with the discovery of genetic forms of PD and the more accepted notion that PD is a systemic disorder affecting more than just sensorimotor function. Currently, there are numerous studies in genetic mouse models of PD and other neurodegenerative disorders that now include analysis of olfaction in the characterization16-24. Given the growing interest of olfactory dysfunction in neurodegenerative disorders, we sought to assemble a battery of olfactory tests that can be used to characterize novel models of neurodegeneration as well as test potential disease-modifying therapies in preclinical studies. The tests described in the present study have been used in both characterization and preclinical studies18,25.
The tests highlighted in this study have been shown to be sensitive in detecting olfactory dysfunction in a frequently utilized alpha-synuclein overexpressing mouse model of Parkinson’s disease18. They include the buried pellet test26,27, an adapted version of the block test23,24,28,29, and the habituation/dishabituation test30. It is important to note that there are several adaptations of the tests described in this study that are sensitive measures of olfactory function in mice, the ones highlighted in this study are the tests our laboratory has the most experience with and routinely use.
All steps of the protocol follow the animal care and use guidelines and regulations set by the IACUC of the University of Cincinnati.
1. General Considerations
2. Buried Pellet Test
3. Block Test
4. Habituation/Dishabituation
5. Analysis of Videotapes
6. Statistics
The buried pellet, block, and habituation/dishabituation tests are all highly advantageous evaluations of olfaction in mice. Using these tests, we have found significant changes in olfaction in multiple genetic mouse models of Parkinson’s disease, including alpha-synuclein overexpressing (Thy1-aSyn)18 and Atp13a2 knockout mice. Both Thy1-aSyn and Atp13a2 knockout mice take longer to find the buried pellet than controls18. Figures 1-3 show data collected from wildtype and Atp13a2 knockout mice. In the buried pellet test, Atp13a2 knockout mice show increased latency to find the buried pellet compared to wildtype control mice (Figure 1). In the block test both wildtype and Atp13a2 knockout mice sniff the novel block from another mouse’s cage compared to blocks from their own home cage (Figure 2). In the habituation/dishabituation test wildtype mice show habituation to one odor and then increased sniffing when a novel odor is introduced (Figure 3).
Figure 1. Buried pellet test. Latency to find the pellet in wildtype (n = 10) and Atp13a2 KO (n = 14) at 20-27 m of age. * p < 0.05, Mann-Whitney U.
Figure 2. Block test. Time sniffing the novel block (E) in the block test in wildtype (n = 10) and Atp13a2 KO (n = 12) female mice at 20 – 27m of age. ΔΔ represents p < 0.01 compared to blocks A, B, and C from the same genotype. Mann-Whitney U.
Figure 3. Habituation/dishabituation test. Mean time sniffing a scented cartridge in wildtype mice (age 9 m, n = 7) across 6 trials and the introduction of a cartridge with a novel scent on trial 7.
Each of the tests described in this study measure different aspects of olfactory function in mice. The buried pellet test measures the food motivation aspect of olfaction, testing the ability of hungry (food restricted, not food deprived) mice to detect a palatable piece of sweetened cereal buried under bedding. The block test measures more of the social aspect of olfactory function, testing the ability of mice to discriminate between their own scent and that of a conspecific. The habituation/dishabituation test assesses the ability of the mouse to discriminate between familiar and novel, innocuous scents. Using multiple tests is important when characterizing a new model because anomalies in food motivation or fear could contribute to differences observed in mutant mice compared to controls. For example, if only the block test is performed and decreased sniffing of the block that has the odor of another mouse is observed, it is unclear whether there is an olfactory deficit or an enhanced fear response to the conspecific that may lead to avoidance of that block. When multiple tests are performed and deficits are observed in all tests, it strongly supports an interpretation of olfactory impairment. However, if differences are observed in only one test but not the others then there may be a more subtle olfactory impairment but it will be essential to rule out other explanations (i.e., enhanced fear or reduced food motivation). Additional important factors to keep in mind when testing olfactory function mice include the background strain and sex of the mice. Different genetic background strains (i.e., C57BL/6, DBA, etc.) can have profound effects on mouse behavior therefore, it is recommended to adapt each protocol in wildtype mice of the same background before performing the entire experiment. It is also recommended that male and female mice be tested separately from each other because the male olfactory system is highly sensitive to females in estrous and olfactory detection of a female in estrous could interfere with performance in all three tests.
Some steps are absolutely critical to follow when testing olfaction in mice. It is important to be cognizant of the odors the experimenter is introducing to the animals. Wearing gloves for the procedures is essential and frequent changing of gloves between animals is required. It is always a good practice for the experimenter not to wear cologne or perfume on olfaction testing days. While some parts of the procedure are inflexible, there are other parts that can be modified and adapted without reducing the validity or sensitivity of the test. For example, the number of habituation trials can be increased or decreased depending on the strain of mice being tested and how quickly they habituate to the stimuli. The major limitation of these tests is that they are all driven by some form of motivation, be it food or social, making it difficult to completely rule out anomalies in motivation as an explanation when an altered response observed. This can be minimized by analyzing additional parameters, such as activity during testing and time to approach stimuli.
Once mastered, the tests can be easily used in examining the phenotype of novel mouse models of neurodegenerative disease. In addition, the tests that have the highest power can be included in preclinical studies testing potential therapeutics (for an example see ref. 25).
The authors have nothing to disclose.
This protocol follows any and all animal care and use guidelines and regulations set by IACUC at the University of Cincinnati. This work is funded by NIH/NINDS NS07722 and the Gardner Family Center for Parkinson’s Disease and Movement Disorders.
Cap'n Crunch | Quaker Oats | 3E+10 | |
Wooden Blocks | Lara’s Crafts | 10144 | |
Flavor Extracts | Kroger | Almond: 011110664716 | |
Anise: 011110615619 | |||
Banana: 011110669919 | |||
Coconut: 011110669889 | |||
Lemon: 011110669957 | |||
Orange: 0011110669964 | |||
Tissue Cartridges | Sigma-Aldrich | Z672122-500EA | Manufactured by Simport no: M490-2 |
Cotton Balls | Kroger | 1.1111E+10 | |
Mouse Cages | Ancare | 19 x 29 x 12.7 cm | |
Camcorder | Sony | HDR-HC9 | |
MiniDV Tapes | Sony DVC premium | 2.7243E+10 | 60 minute long play |