Differential Impacts of Repeated Sampling on Odor Representations

Research Report: Regular Manuscript Differential impacts of repeated sampling on odor representations by genetically-defined mitral and tufted cell subpopulations in the mouse olfactory bulb https://doi.org/10.1523/JNEUROSCI.0258-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0258-20.2020 Received: 3 February 2020 Revised: 16 June 2020 Accepted: 18 June 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 Title 2 Differential impacts of repeated sampling on odor representations by genetically-defined mitral 3 and tufted cell subpopulations in the mouse olfactory bulb 4 5 Abbreviated title 6 Reformatting of odor representations by sniffing 7 8 Journal Section 9 Systems/Circuits 10 11 Authors 12 Thomas P. Eiting, Matt Wachowiak 13 14 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 15 16 Corresponding author 17 Thomas P. Eiting 18 Department of Neurobiology and Anatomy 19 University of Utah 20 Salt Lake City, UT 84103 21 [email protected] 22 23 Number of pages: 36 24 Number of figures: 6 25 Abstract: 250 words 26 Introduction: 590 words 27 Discussion: 1335 words 28 29 Conflicts of interest 30 The authors declare no competing financial interests 31 32 Acknowledgments 33 We thank S. Burton, A. Moran, S. Short, and I. Youngstrom for helpful comments, G. Vasquez 34 for help with virus injections, and J. Ball and R. Kummer for help with animal husbandry and 35 histological processing. We also thank two anonymous reviewers, whose constructive feedback 36 improved this paper substantially. This work was supported by funding from NIH 37 (F32DC015389 to TPE and DC006441 to MW). 38 1 39 Abstract 40 Sniffing—the active control of breathing beyond passive respiration—is used by 41 mammals to modulate olfactory sampling. Sniffing allows animals to make odor-guided 42 decisions within ~200 ms, but animals routinely engage in bouts of high-frequency sniffing 43 spanning several seconds; the impact of such repeated odorant sampling on odor representations 44 remains unclear. We investigated this question in the mouse olfactory bulb, where mitral and 45 tufted cells (MTCs) form parallel output streams of odor information processing. To test the 46 impact of repeated odorant sampling on MTC responses, we used two-photon imaging in 47 anesthetized male and female mice to record activation of MTCs while precisely varying 48 inhalation frequency. A combination of genetic targeting and viral expression of GCaMP6 49 reporters allowed us to access mitral (MC) and superficial tufted cell (sTC) subpopulations 50 separately. We found that repeated odorant sampling differentially affected responses in MCs 51 and sTCs, with MCs showing more diversity than sTCs over the same time period. Impacts of 52 repeated sampling among MCs included both increases and decreases in excitation, as well as 53 changes in response polarity. Response patterns across simultaneously-imaged MCs reformatted 54 over time, with representations of different odorants becoming more distinct. Individual MCs 55 responded differentially to changes in inhalation frequency, whereas sTC responses were more 56 uniform over time and across frequency. Our novel results support the idea that MCs and TCs 57 comprise functionally distinct pathways for odor information processing, and we suggest that the 58 reformatting of MC odor representations by high-frequency sniffing may serve to enhance the 59 discrimination of similar odors. 60 61 2 62 Significance Statement 63 Repeated sampling of odorants during high-frequency respiration (sniffing) is a hallmark 64 of active odorant sampling by mammals, however the adaptive function of this behavior remains 65 unclear. We found distinct effects of repeated sampling on odor representations carried by the 66 two main output channels from the mouse olfactory bulb, mitral and tufted cells. Mitral cells 67 showed more diverse changes in response patterns over time as compared to tufted cells, leading 68 to odorant representations that were more distinct after repeated sampling. These results support 69 the idea that mitral and tufted cells contribute different aspects to encoding odor information, and 70 they indicate that mitral cells (but not tufted cells) may play a primary role in the modulation of 71 olfactory processing by sampling behavior. 72 3 73 Introduction 74 Olfaction, a key sensory modality for mammals, is an important model system for 75 understanding how active sensation contributes to sensory processing (Uchida et al., 2006; 76 Wachowiak, 2011; Jordan et al., 2018b). Sniffing—the active control of breathing beyond 77 passive respiration—is used by mammals to modulate olfactory sampling. Sniffing provides 78 temporal control over olfactory sensation and can increase access to odor information by 79 increasing the flow of odorants through the nasal cavity, and also by allowing multiple samples 80 of odorant in a shorter time (Zhao et al., 2006; Verhagen et al., 2007; Courtiol et al., 2011; Rygg 81 et al., 2017). Repeated high-frequency (3 - 10 Hz) inhalations are a hallmark of sniffing behavior 82 in most mammals, including rodents (Youngentob et al., 1987; Laska, 1990; Thesen et al., 1993; 83 Wesson et al., 2008a). While mice and rats can discriminate odors in as little as one sniff (150- 84 200 ms (Uchida and Mainen, 2003; Abraham et al., 2004), they frequently exhibit longer sniffing 85 bouts lasting several seconds (Welker, 1964; Wesson et al., 2008a). How repeated sampling 86 influences olfactory processing by neural circuits in the olfactory bulb (OB) or elsewhere is not 87 well understood. 88 Previous work has shown that rapid sniffing induces adaptation of olfactory sensory 89 inputs to the OB (Verhagen et al., 2007); more recently, we and others have found that inhalation 90 frequency modulates subthreshold membrane potential and spiking patterns in mitral and tufted 91 cells (MCs and TCs), the two major subclasses of olfactory bulb output neurons (Bathellier et al., 92 2008; Carey and Wachowiak, 2011; Díaz-Quesada et al., 2018; Jordan et al., 2018b). Multiple 93 functions of this frequency-dependent modulation have been proposed, including enhancing 94 olfactory sensitivity, increasing the salience of odorants against a background, and improving 95 fine-scale odor discrimination. 4 96 There is substantial evidence that mitral cells (MCs) and tufted cells (TCs) have distinct 97 odorant response properties and likely contribute differentially to encoding odor information 98 (Nagayama et al., 2004; Griff et al., 2008; Fukunaga et al., 2012; Jordan et al., 2018a; Short and 99 Wachowiak, 2019). Putative mitral and tufted cells also respond to odorants differently during 100 brief bouts of high-frequency sniffing (Jordan et al., 2018b). However, how - or whether - these 101 populations differentially represent odorants during repeated sampling of odorants remains 102 unclear. Ultimately, because odor coding involves combinatorial activity patterns across many 103 cells, understanding how sniffing impacts odor representations requires recording from multiple 104 cells simultaneously, ideally using genetic and strict anatomical criteria to define subpopulations 105 independent of their functional properties. 106 Here, we addressed these knowledge gaps by imaging from genetically- and 107 anatomically-defined subsets of MCs and TCs during repeated odorant sampling at different 108 inhalation frequencies. We use an artificial inhalation paradigm in anesthetized mice to allow for 109 precise reproduction of odorant sampling regimes and comparison of responses across cell type 110 subpopulations, with minimal impact of centrifugal inputs or other behavioral state-dependent 111 factors. We found substantial population-level differences between MCs and a subset of TCs 112 defined by their superficial location and their expression of the peptide transmitter 113 cholecystokinin (CCK). The CCK+ superficial TC (sTC) population showed stronger coupling to 114 individual inhalations, few suppressive responses, and little change in its population response 115 pattern during repeated odorant sampling. In contrast, MCs showed diverse changes in excitation 116 and suppression with repeated odorant sampling, such that the MC population response pattern 117 reformatted substantially from the beginning to the end of the odorant presentation, and did so in 118 a frequency-dependent manner. These effects point to an important role for inhalation 5 119 frequency-dependence in odor perception and differential contributions by MC and sTC 120 populations in encoding odor information over repeated samples. 121 Materials and Methods 122 Animals. Adult transgenic mice (ages 6-16 weeks) of both sexes (n = 12 males and 13 females) 123 were used in all experiments. Imaging in mice was carried out in Cck-IRES-cre (Jackson 124 Laboratory (Jax) stock #012706) (Taniguchi et al., 2011) mice for imaging in sTCs or in either 125 Pcdh21-cre (Mutant Mouse Resource and Research Center stock #030952-UCD) (Gong et al., 126 2003) mice or Tbet-cre (Jax stock #024507) (Haddad et al., 2013) mice for imaging in MCs. 127 Mice were either crossed with Ai95(RCL-GCaMP6f)-D (Jax stock #024105)

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