This study aimed to develop a behavioral test of olfactory acuity in rats that could serve as a translational biomarker for early detection of cognitive decline in aging. The researchers trained rats on an odor identification task using operant conditioning chambers. Rats were tested on their ability to discriminate target odors from similar odor mixtures in probe trials without reinforcement. Reaction times on the probe trials provided a sensitive measure of odor perception abilities. The results showed that rats could distinguish odor mixtures that differed from the target by as little as 1%, and their reaction times increased systematically with harder mixtures. However, the steepness of generalization gradients depended on the target odor. This behavioral test shows promise as a sensitive olfactory biomarker across the lifespan in rats

1. Background and significance
Characterizing olfactory generalization in Fischer 344 rats using behavioral reaction times
Wendy M Yoder1 Ÿ Olivia Munizza1 Ÿ Leslie Gaynor2,4 Ÿ Ethan Windham3 Ÿ Michelle Lyman4 Ÿ Barry Setlow4,5,7 Ÿ Jennifer L Bizon4,5,6,7 Ÿ David W Smith4,6
1Program in Interdisciplinary Studies, Neurobiological Sciences Ÿ 2Department of English Ÿ 3Health Science Program Ÿ 4Program in Behavioral and Cognitive Neuroscience, Department of Psychology Ÿ 5Department of Psychiatry Ÿ 6Center for Smell and Taste Ÿ 7Department of Neuroscience, University of Florida College of Medicine, Gainesville, Florida
5 5
Behavioral methodology: Operant Chamber
Acknowledgments
References
Modified Operant
Chamber
Odor Port
Odor Stream
Stimulus Behavior Response Measure Observation
Figure 2: Psychophysical principle of reaction time. Reaction times can be used to measure stimulus
intensity. In the illustration above, the hotter the flame, the faster the hand will withdrawal. Similar behavioral
methodology may be applied to odor-guided tasks to create a generalization gradient and increase difficulty.
Stimulus Behavior Response Measure Observation
Figure 4: Procedure for using non-reinforced probe trials. (A) If the rat identifies the mixture (S+ : S-) as the
target (S+), the rat will lick. If the animal decides the mixture (S+ : S-) is NOT the target (S+), the animal will not
lick. (B) How fast the rat licks in response to the mixture is a measure of how certain the rat is that the mixture
(S+ : S-) is the same as the target (S+).
=
Mixture
S+ : S-
Rat Sniffs
S+ or S- ?
Decision: S+
Rat Licks
Delay to
Decide / Lick
Probe smells
like S+
Figure 3: Operant chamber.
Licking in the presence of the
target odorant (S+) results in 5 µl
of liquid reinforcement (Ensure).
Conversely, incorrectly licking in
the presence of the control
odorant (S-) results in a 5 second
time-out; the rat cannot initiate
new trials during this interval.
Reaction times are defined as the
interval from the time of stimulus
onset until the rat makes contact
with the lick tube.4
Behavioral
Biomarker
Several
Limitations
Need Better
Translational
Measures
Odor
Identification
Target
Choices
A) Can rats be trained to “say” POPCORN?
B) Can the odor-guided task be more
difficult to minimize ceiling effects?
• Olfactory changes are an early sign of many forms of neural and cognitive dysfunction.1
• To thoroughly investigate the underlying
mechanisms involved in these processes,
cross-species assessments are needed
that are both sensitive and specific.
Figure 1: Odor identification schematic.
Rodents are frequently used in olfactory research,
but current measures are often not comparable to
olfactory tests used with human subjects.2
Cross-species
Comparisons
Step 1: Obtain thresholds for individual rats
Step 2: Reinforcement (100%) on S+ trials
Step 3: Partial reinforcement (50%)
Step 4: Non-reinforced probe trials
S+ : S- S+ : S- S+ : S-S+ S-
+ + +
Criterion:
100% Accuracyvs.
S+ S-
Criterion:
100% Accuracyvs.
S+ S-
Figure 3: Stimuli Presentation. Each regular block
contains 20 trials (10 S+ and 10 S-). Probe trials (2
per block) are pseudorandomized and presented
during 5 separate sessions (n=10 per mixture ratio).
Individual thresholds are used to generate binary mixtures
Figure 6. Comparison of
individual, threshold
estimates for octanol and
citral. Columns refer to
descending concentrations
(% v/v) of the dilutions.
Each panel/symbol
represents discrimination
accuracy for the target
odorant (octanol or citral) vs.
the odorless, control
odorant (diethyl phthalate).
Lines represent accuracy
across three consecutive
blocks for each
concentration. The lowest
concentration at which the
rat receives 85% or greater
on at least one of the three
blocks is recorded as
threshold. These are used
in subsequent steps to
match intensity levels
between odorants.
Threshold:
Lowest concentration
the rat receives ≥ 85%
vs.
S+ S-
vs.
S-S+
vs.
individual Mixture Probe Trials
Figure 8: Groupmean reaction times (ms) for binary
mixture ratio probe trials. Circles represent mean reaction
times for non-reinforced probe trials containing different
concentrations of citral and octanol. For visual simplification,
only the two highest ratios recorded as S- are shown here.
Figure 9: Individualreaction times for non-reinforced,
probe trials. Each mixture ratio contains 10 probe trials (2
per block) randomized across sessions. Ratios not shown
indicate the rats did not lick – responded to the mixture as S-.
Figure 7: Comparison of mean reaction times (ms) for
reinforced target (S+) trials and non-reinforced, probe
trials of equal concentration (threshold level). Mean
values are not statistically different (i.e., reinforcement
contingency does not affectreaction times).
Funding for this research was provided by NIH R01 AG024671
and the McKnight Brain Research Foundation.
Asymmetrical Response
Aging is a complex process and a key
risk factor for cognitive and neural
dysfunction, but declines are rapid
without early intervention. Researchers
are scrambling to identify biomarkers.
Is olfactory dysfunction
a promising behavioral
biomarker?
This measure could be
included in a battery with
other olfactory tests.
Importantly, our laboratory has expertise
assessing olfactory acuity in both
humans subjects and animal models.
Fischer 344 rats may be an important
piece of the puzzle. As these rats age,
some show cognitive declines that
correlate with olfactory impairments.
Olfactory changes may be predictive.4
For both citral and octanol, reaction times were rapid (~150-200 ms) when the
probe trials consisted of a single, monomolecular component.
Binary mixtures that differed from the target odorant by as little as 1% v/v were
different enough to increase reaction times -- rats hesitated before responding.
Reaction times changed systematically as a function of task difficulty for both
odorants, but the steepness of the generalization gradients depended on the
target odorant presented.
95:5 84:16
Figure 10. Asymmetrical responses.5 Less
citral was required to “suppress” the presence of
octanol than vice versa. On average, animals
ceased responding to the probe trials as the target
at the ratio 95:5 for octanol and 85:15 for citral.
• Probe trials “ask” the rat to respond without the
contingency of reinforcement.
• This measure captures subtle, behavioral
changes and minimizes ceiling effects.
• Mixtures can be more or less complex
depending on the experimental objectives.
Disadvantages
Advantages
• Threshold matching is labor intensive.
• Stimuli interaction effects should be
considered.
1. Atanasova, B., Graux, J., El Hage, W., Hommet, C., Camus, V., Belzung, C. 2008. Olfaction: a potential cognitive marker of psychiatric disorders.
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2. Doty R, Frye R, Agrawal U. 1989. Internal consistency reliability of the fractionated and whole University of Pennsylvania Smell Identification Test.
Perception & Psychophysics 45: 381-384.
3. LaSarge, C., Montgomery, K.S., Tucker, C., Slaton, S., Griffith, W., Setlow, B., Bizon, J. 2007. Deficits across multiple cognitive domains in a subset of aged
Fischer 344 rats. Neurobiology of aging 28(6), 928-36.
4. Slotnick, B. 2007. Odor-Sampling Time of Mice under Different Conditions. Chemical Senses 32(5), 445-54.
5. Sokolic L, Laing D, McGregor I. 2007. Asymmetric suppression of components in binary aldehyde mixtures: behavioral studies in the laboratory rat.
Chemical senses 32: 191-199.