An odor-reward, associative learning task was used to investigate the differential effects of physiological manipulation on long-term and short-term memory.
Robust and simple behavioral paradigms of appetitive, associative memory are crucial for researchers interested in cellular and molecular mechanisms of memory. In this paper, an effective and low-cost mouse behavioral protocol is described for examining the effects of physiological manipulation (such as the infusion of pharmacological agents) on the learning rate and duration of odor-reward memory. Representative results are provided from a study examining the differential role of tyrosine kinase receptor activity in short-term (STM) and long-term memory (LTM). Male mice were conditioned to associate a reward (sugar pellet) with one of the two odors, and their memory for the association was tested 2 or 48 h later. Immediately prior to the training, a tyrosine kinase (Trk) receptor inhibitor or vehicle infusions were delivered into the olfactory bulb (OB). Although there was no effect of the infusion on the learning rate, blockade of the Trk receptors in the OB selectively impaired LTM (48 h), and not short-term memory (STM; 2 h). The LTM impairment was attributed to the diminished odor selectivity as measured by the length of the digging time. The culmination of the results of this experiment showed that Trk receptor activation in the OB is the key in olfactory memory consolidation.
The mechanisms of associative memory formation have previously been investigated predominantly based on one-trial fear conditioning studies. However, many mundane tasks typically have more complex acquisition patterns and rely on repeated encounters. The goal of this protocol is to provide a cost-effective rodent behavioral paradigm that is used to understand the cellular and molecular mechanisms of multi-trial appetitive learning and memory.
Odor learning that is dependent on the main olfactory bulb (OB) provides several advantages for the study of multi-trial appetitive memory. First, OB-dependent memories have varying durations (STM, LTM, and intermediate-term memory1) and rely on the same molecular2,3 and structural mechanism as elsewhere in the brain, including neuromodulation4, long-term potentiation5, and adult neurogenesis6,7,8. Second, in contrast to the higher-order regions, like the hippocampus, OB-dependent memories allow for observations of a more direct correspondence between experimenter manipulations of the perceptual environment and changes to the neural circuitry responsible for learning8,9,10,11. In this paper, an OB-dependent associative learning and memory paradigm, that can be used to study general molecular and structural mechanisms, is detailed. It was developed to allow researchers to access the advantages of olfactory learning for the study of cellular and molecular mechanisms of memory.
In our recent publication3, the protocol described here was used to demonstrate that the consolidation of appetitive odor learning is dependent upon Trk receptor activation within the OB. In the protocol below, areas where the behavioral paradigm can be adjusted for different experimental needs, are also discussed.
A total of 27 adult male CD-1 mice, 8 weeks old at the time of cannulation, were used in this study. For the precise group distributions and odor set use, see the methods section of our previous publication3. Male mice were used to avoid large fluctuations in estrogen levels because previous research12 showed that olfactory memory is enhanced by increased estrogen levels. These mice were always kept on a 12:12 h reverse light/dark cycle and had access to water. During the behavioral experiments, the diets of the mice were restricted to maintain them at ~90% of their free feeding weight. Diet restriction began 3 days before the commencement of the behavioral experiment. As will be described below, the same set of mice are presented with different odor sets in order to reach the appropriate levels of statistical power while minimizing the animal use. The statistical analysis section shows how to account for the random variance that may be introduced by this.
One-trial fear conditioning is a powerful behavioral protocol for studying molecular and cellular dynamics of memory, but much of natural learning is incremental and best modeled through a paradigm like the one described above. The inhibition of Trk receptors in the OB prevented the consolidation of olfactory memory in a multi-trial, appetitive learning paradigm as shown previously by our group3. The finding opens new avenues for research into the differential timing of molecular mechanisms, like neurotrophins, in appetitive and aversive learning.
This experiment consisted of two critical parts: (1) the cannulation and (2) the associative discrimination task (subdivided into shaping, training, and testing). Experimenters can adapt this protocol to their specific research question. For example, we were primarily interested in the OB and this established protocol can be easily applied to other OB studies. For experimenters with other regions of interest, it will be important to validate the infusion sites in a pilot study. Experimenters may also need to consider the diffusion rate, spatial penetration, and bio-activity duration of what they are infusing.
The shaping steps outlined in the protocol have been used extensively by the authors of this protocol. Adhering to them as described appears to be important to have the mice learn the task in a timely manner. Using other timelines, the authors observed more variance amongst the mice in their familiarity with the task and this meant additional training to get all mice to a criterion for testing with experimental odors. For training and testing, the researcher has flexibility, depending on their research interests, with the number of trials, the concentration of the experimental odors, and the similarity of the odors to one another. We recommend the use of multiple odor sets when possible, as we’ve described, in order to reduce the number of animals used for the experiment. See the Statistical Analysis section for the guidance on how to account for the use of multiple order sets in the final analysis. In principle, the similarity of the odor pair can be varied to adjust the difficulty of the discrimination. In the previously published study from our group3, the odor pairs consist of two odorants of the same functional group but differed from each other by one carbon length. These discriminations are more difficult than pairs that differ by two or more carbon lengths, but easier than enantiomers (e.g., (+)-limonene and (-)-limonene). Odorants from different functional groups are highly different perceptually. Cleland et al13 discusses more stimulus variations and their effect on specific learning parameters.
One major limitation of this protocol is that it takes a much longer time to carry out compared to automated associative learning tasks where multiple animals could be tested in parallel. For a given test, it would take one researcher at least 20 minutes to complete 20 trials for one mouse. However, it is this lack of automation that means the protocol is more financially accessible, a priority for many institutions. Importantly, in the case of this experiment, it has been found that this protocol is highly tractable and effective for training undergraduate researchers with interests in behavioral neuroscience. In particular, these students develop strong animal handling skills in addition to the usual benefits of research participation.
Researchers who are interested in adopting this paradigm can vary several parameters. Most apparently, pharmacological manipulations of mechanisms are diverse, and this behavioral protocol can be used with chemogenetic techniques or various other ways of manipulating molecular and cellular pathways (e.g. optogenetics). The paradigm itself can be adjusted to vary the kind of learning and memory tested. For example, researchers can adjust the similarity of the two odors presented in order to control the learning rate. In our study3, the odor pairs consist of two odorants of the same functional group but differed from each other by one carbon length. These discriminations are more difficult than pairs that differ by two or more carbon lengths, but easier than enantiomers (e.g. (+)-limonene and (-)-limonene). Odorants from different functional groups are highly different perceptually. Cleland et al13 discusses more stimulus variations and their effect on specific learning parameters, concluding that more similar odors are more difficult to discriminate and therefore take longer to learn14. These manipulations would also affect the strength of memories. Along this vein, researchers may be interested in testing memory at different timepoints after learning. For example, two studies15,16 examined the role of BDNF in LTM persistence for a one-trial aversive learning task. BDNF is a ligand of TrkB. The studies showed that anti-BDNF antisense oligonucleotide infusion in the hippocampus 12 hours after learning blocked LTM 7 days later, but not 2 days later. This study shows that the timescale of molecular mechanisms after initial learning play interesting, and yet-to-be-understood, roles in LTM. This paper describes a behavioral protocol that would allow for the investigation of these timescales. Other parameters of interest for future application include the mouse model used. For example, it would be interesting to replace male mice (who have a significantly better studied neurobiology)17 with female mice in future studies to examine variations in the learning rate, STM, and LTM, as female mammals have a higher sensitivity and selectivity to odors than male mammals18. Of course, rodent models of diseases can also be used effectively with this protocol.
The authors have nothing to disclose.
The project was supported by the Scantland Summer Collaborative Research Gift and the Stephen and Sylvia Tregidga Burges Endowed Research Fund. The authors would like to acknowledge colleagues in the Department of Psychology and the Neuroscience Program at Earlham College for their support and guidance.
Double guide cannula | PlasticsOne | C235GS-5-1.5/SPC | Custom order |
(-)-limonene | Sigma-Aldrich | 218367-50G | |
(+)-limonene | Sigma-Aldrich | 183164-100ML | |
2-hetanone | Sigma-Aldrich | 537683 | |
2-octanone | Sigma-Aldrich | O4709 | |
5mg sucrose pellets | Test Diet | 1811560 | Custom size. Used for rewards |
Butanoic acid | Sigma-Aldrich | B103500 | |
butyl acetate | Sigma-Aldrich | 402842 | |
Dental Cement Powder (Coral) | A-M Systems | 525000 | |
Dental Cement Solvent | A-M Systems | 526000 | |
Double connector assembly | PlasticsOne | C232C | |
Double dummy cannula | PlasticsOne | C235DCS-5/SPC dummy dbl | Custom order |
Double injector | PlasticsOne | C235IS-5/SPC | Custom order |
Drill | Kopf Instruments | Model 1474 High Speed Stereotaxic Drill | This drill requires an additional "adaptor" piece in order to fit certain drill bits. We get by this problem by wrapping the drill bit with lab tape to increase the circumference of the drill it to fit. This may not be an option for surgeries requiring sterile technique. |
Eye Ointment | Purchase from local pharmacy | ||
Figure 1 illustration software | BioRender | ||
heptanal | Sigma-Aldrich | W254002 | |
hexanal | Sigma-Aldrich | 115606 | |
hexanol | Sigma-Aldrich | H13303 | |
Infusion pump model 11 | Harvard Apparatus | 4169D | Used pumps available via American Instrument Exchange |
Isoflurane | Santa Cruz Animal Health | sc-363629Rx | Vet prescription needed for order |
K252a | Sigma-Aldrich | K2015 | Mixed to 50uM in DMSO (5%) |
Ketoprofen | Allivet | 25920 | Vet prescription needed for order |
Lidocaine | Aspercreme | Purchased from Amazon | |
Mounting Screws | PlasticsOne | 00-96 X 3/32 | |
Mouse Anesthesia Mask | Kopf Instruments | Model 907 Mouse Anesthesia Mask | Used with the stereotaxic to allow oxygen and anesthesia while mouse in stereotax |
Mouse Nose Adaptor | Kopf Instruments | Model 926 Mouse Adaptor | Used with the stereotaxic to allow for head of mouse to be secured. |
Novalsan | Jeffers | 41375 | |
Pentanoic acid | Sigma-Aldrich | 240370 | |
pentanol | Sigma-Aldrich | 138975 | |
Petri dish glass bottoms | VWR | 10754-804 | |
Polycarbonate Café bottoms | Ancare | N10PCSEC | Use normal housing cages and custom fit a track in the middle to act as the track for an opaque plexiglass divider |
propyl acetate | Sigma-Aldrich | 537438 | |
Quikrete Premium Play Sand | Purchase from local hardware store | ||
Saline | Insight Needles | N/A | Sterile saline for drug mixing |
Stereotaxic apparatus | Kopf Instruments | Model 902 Small Animal Stereotaxic Instrument | |
Testing chamber | Ancare | N10PCSEC | Our testing chambers are modified using the regular mouse housing cage. The manuscript details what was done. |
Vetbond Tissue Adhesive | 3M | Purchased from Amazon |
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