Several studies show that short-term memory content is integrated into cortical long-term memory during slow-wave sleep and that this process can be selectively influenced by a combined application of odor cues during encoding and during slow-wave sleep at night (targeted memory reactivation, TMR). In the present study, we replicated this beneficial effect of an odor cue. In particular, we found that (1) odor cues not only work in a lab environment, but also with students in a regular school setting; that (2) odor cues also take effect if presented continuously during the complete sleep period at night, rather than selectively during slow-wave sleep stages. (3) We observed some indirect indication for a further performance benefit with additional cueing during the recall test.

Real life settings and effect sizes

Leaving the experimental lab environments and doing field research in real life situations is a priori challenging. In a lab, most of the potentially confounding parameters can be controlled and their influence can be minimized. This is not the case in real life situations. Confounding influences typically increase the noise factors of the statistical terms and ultimately reduce the effect sizes. The following constraints need to be kept in mind, with respect to the present results:

Due to organisational reasons, the embedding of the current study in the daily school routine did neither allow a new allocations of the students to either test and control group for each experimental condition, nor a randomization of the order of conditions between students within the two groups. It is thus possible that at least part of the reported effects may reflect an order effect.

The vocabulary learning units as well as the vocabulary tests in school had been conducted by two different teachers in two different school classes and with slightly different vocabulary tests. Therefore, there might be small differences in the instructions given to the students and other realization details.

We had no control over how often the students repeated the learning material at home within the seven days before the final test.

We had to rely on the students’ reports on their use of the odor cues while studying at home and during their sleep in the seven nights between the initial encoding of the vocabulary material and the final test.

We had no control over the spatial distance between the odor cue and the students (i.e. individual odor intensity) while they were studying at home, during sleep, and during the test itself.

Several studies reported about reduction of sleep time and changes in sleep pattern (reduction in EEG amplitudes and a linear increase in peak spectral frequency of EEG sleep spindles) during adolescence within an age range between 11 and 17 24,25 . We did not record sleep EEG and had no control about the students’ absolute sleep time or their sleep quality. However, if a reduction of sleep duration and/or a change in sleep pattern did affect consolidation of our student participants negatively, the control group must have been affected more strongly than the test group, given the current results. Also, the test group must have been selectively more affected in the N (no odor cue) and LT (odor cues during learning and the test but not during sleep) conditions. Of course, in principle, it might be possible that the students in the test group slept longer only and selectively in the conditions containing an odor cue during sleep (LS and LST). But it is not very probable. Future (lab and field) studies may investigate the relation between adolescent changes in sleep pattern and duration and the efficacy of odor cueing during sleep.

The different experimental conditions were based on different vocabulary material. Within-group comparisons across experimental conditions may thus be confounded by variations in the difficulty of the vocabulary material and/or final tests. Comparisons between test and control groups within conditions are unaffected by this factor. However, comparisons between conditions and interaction tests may have been affected by this.

Time-on-test, potential verbal and non-verbal interactions between students during tests had not been perfectly controlled.

Given that all of these factors (except the first) could have introduced additional “experimental noise” into our data, it is remarkable that our effect sizes with Cohen’s d between 0.6 and 1.2 are in the same range as, or larger than the effect size (d ≈ 0.6) in Rasch et al.’s study15 (we have estimated this value from the leftmost graph of their Fig. 2A).

Control conditions

Odor cueing paradigms typically include test conditions in which a vehicle contains an odor, which is then distributed at different steps during the learning process. These conditions are contrasted with control conditions in which the vehicle is either presented without the odor or with varying odors (e.g.18). Unfortunately, no vehicle had been distributed to the participants of the control conditions in the present study. This may in principle make our results vulnerable to psychological expectancy effects. However, the following argumentation makes this an unlikely explanation for our findings.

The odor cue effect is only present if the odor cue had been given during night. No difference is found between test and control groups in the LT condition (odor during learning and during test – but not during sleep). In contrast, for both the LS (odor during learning and during sleep) and LST (odor during learning, sleep and final test) conditions, we found significant differences between the test and control groups (keep in mind that the results from the LS condition is based on data from the two classes, whereas the LST condition is restricted to data from Class 1). It is very unlikely that a psychological expectancy is selectively coupled to receiving the odor device at night (LS and LST conditions), but not for receiving it during the learning period (LT condition). Further, a recent study, applying auditory word cues in the context of a vocabulary-learning paradigm, found that memory performance of those words not having been cued at night is similar to the memory performance of words in a condition without any cue during night26. If psychological expectancy would completely explain the effect of memory cueing during sleep, it should not be restricted to a subset of words being cued at night.

Odor as contextual cue for learning

Odor has already been used as a cue for context-dependent memory formation prior to the findings established by Rasch and his colleagues in their seminal study17,27,28. Typically, odor cues had only been presented during encoding and during retrieval, but not during sleep. It was shown that “context” is not necessarily a spatial parameter, but that fragrances can also establish an olfactory context, which can improve later retrieval (e.g.27,28).

It is possible that the odor-context-dependent memory effects and the effects of odor cueing during learning and during sleep are based on the same underlying mechanisms. It is also possible that the impact of an odor on the retrieval of information is independent of its impact on consolidation. These earlier studies may have shown retrieval effects, and odor-during-sleep studies may have shown consolidation effects. Our study shows significant consolidation effects and some indication for beneficial retrieval with odor cues (twice as large effect sizes in condition LST than in condition LS but no significant ANOVA interaction). The question remains, why we did not see any pattern of cueing effects in the LT condition? One possible explanation is the larger inherent noise level of the present field study compared to better-controlled laboratory studies.

Do odor cues during sleep improve both memory consolidation and memory retrieval?

The formation of memories is typically divided into three major steps: encoding, consolidation and retrieval29. If we are not able to recall a certain past event or fact, the relevant information may not have been consolidated or, alternatively, it has been consolidated, i.e. the information is somewhere in our memories, but we cannot retrieve it. So far, several studies have shown that cueing during learning and during SWS increases memory performance. Given the state of the art in memory research, this improvement was interpreted as an improvement of the memory consolidation processes (e.g.19). However, memory performance tests, conducted several days after the cueing during SWS, always test both consolidation and retrieval success. In the current study, we added cueing during retrieval as another experimental parameter in order to disentangle both effects and to test for further memory improvement.

Testing the effects of odor cues on memory retrieval was unfortunately restricted to the data sets from Class 1, as no data from Class 2 was available for the LST condition. A significant interaction between the factors GROUP (test vs. control) and CONDITION (LS vs LST) in the second ANOVA would have indicated an effect of odor cueing on memory retrieval during test. However, the ANOVA did not reveal a significant interaction. Despite the absence of a significant ANOVA interaction we calculated post-hoc tests for the conditions LS and LST, comparing test performance of test and control groups. In our view, this is reasonable because the comparison between conditions – as part of the interaction test – can be confounded by the different vocabulary materials used in the different conditions, as already discussed above. The post-hoc tests revealed significantly better memory performance of the test group compared to the control group with a considerably large effect size (d = 1.23) for the condition LST and with half the effect size for the condition LS (d = 0.6). No significant difference between groups was found for the conditions LT and N (see also Table 1 for an overview).

An effect size (d = 1.22) twice as large for condition LST than for condition LS (d = 0.6), indicates some additional influence of the odor cue for retrieval. However with regard of the results of the ANOVA this point remains controversial. Our pattern of results motivates at least a second look on combined cueing during learning, sleep and retrieval in a future repetition of the current study.

One and the same odor cue for different learning materials

One and the same odor cue was used with the same participants in different experimental conditions and thus with different sets of English vocabulary. Thus, one and the same odor fragrance had to serve as cue for 60 (Class 1) or even 90 (Class 2) different words, which could theoretically have led to memory interference effects. Furthermore, former studies used one and the same odor cue for different items to learn.

Having this in mind, the efficacy of odor cues for consolidation and retrieval is surprising. It may be interesting to check whether it is possible to further increase odor cuing efficacy by using more odor cues.

Timing of memory cues during sleep

Current theories about memory consolidation are based on numerous empirical findings10,11,12,13,30 and assume that memory consolidation implies a reactivation of hippocampal short-term memory traces and an integration into pre-existing cortical memory networks during slow wave sleep (SWS) at night17,19,20,31. Most studies indicate the necessity of controlling the sleep stages with EEG in order to be able to selectively apply the odor cues during the critical SWS stages. However, monitoring sleep stages and presenting cues only during SWS is a considerable operational expense with related difficulties for a potential practical application. Further, sleeping in the lab, wearing an EEG cap with many electrodes and cables associated, may influence sleep quality in a negative way. Our results are highly interesting in this context, as they show comparable memory benefits with continuous odor stimulation during the whole night. Furthermore, recent evidence indicates beneficial effects from whole-night odor cueing in a creativity task32. Thus, temporally selective cueing during sleep is apparently not a necessary precondition in order to achieve a beneficial effect of odor cueing at night, which is an important finding for practical application perspectives. However, we cannot rule out that a more specific cueing during certain sleep periods may have further increased the effectiveness of cueing.

Interestingly, memory cueing during night is not restricted to the olfactory modality, as auditory cues, even the auditory presentation of previously learned words during SWS, also work13,19,20. However, continuous stimulation with auditory cues can be problematic because of a certain refractoriness pattern: Auditory stimulus presentation needs to be below a certain temporal duration33 and spaced in time within the SWS stage in order to prevent the disappearance of the beneficial effect of cueing. The latter strongly reminds of well-known spacing effects15,34,35,36 or retroactive interference effects during learning16. Our continuous odor stimulation during night shows that the refractoriness problem is at least not that severe in the olfactory domain. Further auditory cueing may affect sleep quality and in the worst case wake the participants (e.g.37). So far, there is some evidence that sleep quality is not affected by odor cueing38. However, further studies are necessary to better clarify this important point.