bioRxiv preprint doi: https://doi.org/10.1101/2020.05.04.076604; this version posted May 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 4 The effect of odor enrichment on olfactory acuity: Olfactometric testing in 5 mice using two mirror-molecular pairs. 6 7 Alyson Blount1 and David M. Coppola1,* 8 9 10 1Department of Biology, Randolph-Macon College, Ashland, Virginia, United States of America 11 12 13 14 *Corresponding Author 15 Email: [email protected] (DMC) 16 17 18 19 20 Short Title: The effect of odor enrichment on olfactory acuity. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.04.076604; this version posted May 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 21 Abstract 22 Intelligent systems in nature like the mammalian nervous system benefit from adaptable inputs 23 that can tailor response profiles to their environment that varies in time and space. Study of such 24 plasticity, in all its manifestations, forms a pillar of classical and modern neuroscience. This 25 study is concerned with a novel form of plasticity in the olfactory system referred to as 26 induction. In this process, subjects unable to smell a particular odor, or unable to differentiate 27 similar odors, gain these abilities through mere exposure to the odor(s) over time without the 28 need for attention or feedback (reward or punishment). However, few studies of induction have 29 rigorously documented changes in olfactory threshold for the odor(s) used for “enrichment.” We 30 trained 36 CD-1 mice in an operant-olfactometer (go/no go task) to discriminate a mixture of 31 stereoisomers from a lone stereoisomer using two enantiomeric pairs: limonene and carvone. 32 We also measured each subject’s ability to detect one of the stereoisomers of each odor. In order 33 to assess the effect of odor enrichment on enantiomer discrimination and detection, mice were 34 exposed to both stereoisomers of limonene or carvone for 2 to 12 weeks. Enrichment was 35 effected by adulterating the subject’s food (passive enrichment) with one pair of enantiomers or 36 by exposing them to the enantiomers in daily operant discrimination testing (active enrichment). 37 We found that neither form of enrichment altered discrimination nor detection. And this result 38 pertained using either within-subject or between-subject experimental designs. Unexpectedly, 39 our threshold measurements were among the lowest ever recorded for any species, which we 40 attributed to the relatively greater amount of practice (task replication) we allowed our mice 41 compared to other reports. Interestingly, discrimination thresholds were no greater (limonene) or 42 only modestly greater (carvone) from detection thresholds suggesting chiral-specific olfactory 43 receptors determine thresholds for these compounds. The super-sensitivity of mice, shown in 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.04.076604; this version posted May 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 44 this study, to the limonene and carvone enantiomers, compared to the much lesser acuity of 45 humans for these compounds, reported elsewhere, may resolve the mystery of why the former 46 group with four-fold more olfactory receptors have tended, in previous studies, to have similar 47 thresholds to the latter group. Finally, our results are consistent with the conclusion that 48 supervised-perceptual learning i.e. that involving repeated feedback for correct and incorrect 49 decisions, rather than induction, is the form of plasticity that allows animals to fully realize the 50 capabilities of their olfactory system. 51 52 53 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.04.076604; this version posted May 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 54 Introduction 55 The task of natural intelligence, like artificial intelligence, is to correctly interpret environmental 56 information in pursuit of certain goals. This requires sensory systems that can capture mission- 57 critical data in a world teeming with stimuli that are irrelevant, ambiguous or under-specified. 58 Universal and enduring sensory statistics, the knowledge of which can promote survival, are 59 often “hardwired” into neural systems. For example, natural scene statistics which have a 60 preponderance of cardinal contours are matched by a mammalian visual cortex with an innate 61 preponderance of neurons responsive to vertical and horizontal edges [1-3]. 62 However, it is impractical to program all needed sensory information into intelligent 63 systems, pointing to the survival advantage of learning or, more broadly, neural plasticity. One 64 of the most studied forms of plasticity, that involving “critical periods” during development, 65 enjoys the advantages of both adaptability to a unstable environment and resistance to alteration 66 after formation. Mammalian vision, again, provides a classic example in ocular dominance 67 columns of visual cortex, which are only mutable during a prescribed period of early life [4, 5]. 68 Adult plasticity, including the various forms of memory, round out the modes of information 69 processing and storage with these later forms representing the most evanescent. 70 Here we are concerned with “induction” a mode of information processing in the 71 olfactory system that has been likened to perceptual learning, a form of implicit memory by 72 which abilities in a sensory task are improved upon with or without the necessity of feedback [6- 73 8]. The classic form of induction involves anosmias. In these deficits, which are widespread in 74 humans, there is an inability to smell a particular odor in a subject that otherwise has a normal 75 sense of smell [9]. However, in certain cases non-smellers can be converted, over time, to 76 smellers by repeated exposure to the odor for which they were initially anosmic [10]. In 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.04.076604; this version posted May 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 77 animals, a related phenomenon has been reported in which subjects that initially are unable to 78 discriminate like-pairs of odors develop the ability to do so without feedback after several days 79 of enrichment [11]. These unsupervised forms of olfactory learning that have been associated 80 with peripheral or, at least, low-level plastic changes in the nervous system will be referred to as 81 induction in keeping with Wysocki and colleagues [10]. The modes of odor exposure that have 82 typically precipitated induction will be referred to as “enrichment.” Usually, these regimens have 83 involved one or a small number of purified odorants delivered to subjects at relatively high 84 concentrations with exposures lasting from several days to several months [10, 11]. 85 Relatively few studies have rigorously compared the effects of passive (unsupervised) 86 enrichment and active (supervised) enrichment on behavioral acuity for the enriched odor. Here 87 we combine operant olfactometric testing with extensive replication to ask if enrichment with the 88 enantiomers (mirror-molecules) of limonene and carvone cause improved behavioral acuity 89 through either heightened ability to discriminate the stereoisomers of these odors or to detect one 90 stereoisomer. Though we found no evidence of induction---no change in discrimination or 91 detection thresholds--after either active or passive enrichment, we did obtain among of the 92 lowest thresholds ever measured for any species. The implications of these data for interpreting 93 thresholds in other studies including the comparison of mice and humans and for assessing the 94 role of perceptual learning in olfactory information processing are discussed. 95 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.04.076604; this version posted May 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 96 Methods 97 Animals 98 All animal procedures were approved and supervised by the Randolph-Macon College 99 Institutional Animal Care and Use Committee and comply with the “Guide for the Care and Use 100 of Laboratory Animals” (8th Edition, National Academies Press USA). Mice were kept in an 101 approved animal room maintained on a 12hr/12hr light cycle with mouse chow available ad lib. 102 Subjects were females of the CD-1 outbred strain obtained from Charles River Laboratories 103 (Wilmington MA, USA) at 56-60 days of age and used in the study up to 7 months of age. This 104 strain was used because it has been the most common target of olfactometric studies, in general, 105 and thresholds have previously been obtained from the strain using similar stimuli and 106 procedures [12]. 107 Thirty-six mice completed threshold testing.