Functional odor map heterogeneity is based on multifaceted glomerular connectivity in larval Xenopus olfactory bulb

Summary Glomeruli are the functional units of the vertebrate olfactory bulb (OB) connecting olfactory receptor neuron (ORN) axons and mitral/tufted cell (MTC) dendrites. In amphibians, these two circuit elements regularly branch and innervate multiple, spatially distinct glomeruli. Using functional multiphoton-microscopy and single-cell tracing, we investigate the impact of this wiring on glomerular module organization and odor representations on multiple levels of the Xenopus laevis OB network. The glomerular odor map to amino acid odorants is neither stereotypic between animals nor chemotopically organized. Among the morphologically heterogeneous group of uni- and multi-glomerular MTCs, MTCs can selectively innervate glomeruli formed by axonal branches of individual ORNs. We conclude that odor map heterogeneity is caused by the coexistence of different intermingled glomerular modules. This demonstrates that organization of the amphibian main olfactory system is not strictly based on uni-glomerular connectivity.


INTRODUCTION
The array of glomeruli in the vertebrate olfactory bulb (OB) represents the first relay station of olfactory information processing. A glomerulus consists of axon terminals of receptor neurons, dendritic processes of mitral/tufted cells (MTCs), and local interneurons. 1,2 Populations of receptor neurons expressing the same allele of a single odorant receptor gene coalesce to form distinct glomeruli. [3][4][5][6][7][8] Odor molecule detection at the peripheral sensory surface translates to spatiotemporal glomerular activity patterns integrated by the OB neuronal network. 9,10 At least two different modes of representing a chemosensory map on the glomerular array have been identified based on similarity of (1) odor molecule structure or (2) olfactory receptor protein. In the rodent and zebrafish main OB, glomeruli activated by structurally similar odor molecules are spatially close, and these molecular feature clusters are stereotypically arranged between animals. [11][12][13][14] Contrastingly, in the rodent accessory OB, glomeruli with similar odor tuning are spatially dispersed. 15 The sequence similarity of expressed receptor genes of vomeronasal receptor neurons is more indicative for the juxtaposition of connected glomeruli than molecular features of the detected odors. 15,16 Also, glomerular input convergence is less strict, and vomeronasal receptor neurons that express a specific receptor gene project into multiple, smaller glomeruli. 7,8,17 In a generalized olfactory system blueprint, glomerular circuit elements are primarily thought to be uniglomerular: Individual olfactory receptor neuron (ORN) axons and mitral cell dendrites coalesce into one functionally and morphologically distinct glomerulus. [3][4][5][6] In amphibians however, ORN axons and MTC dendrites regularly branch and innervate multiple, spatially distinct glomeruli. [18][19][20][21][22][23] Here, we report how this wiring layout affects the functional organization of the amphibian OB. The glomerular response map to amino acid odors was neither stereotypic between individual animals nor chemotopically organized. As a potential basis for this atypical map organization in a main OB, we show that different glomerular wiring paradigms functionally overlap in the OB of Xenopus laevis. This highlights that the main olfactory system is not necessarily based on strictly uni-glomerular connectivity and opens up new questions about the evolutionary position of amphibians during formation of distinct olfactory wiring strategies.

RESULTS
We focused on the ventro-lateral OB of larval X. laevis, since it is best characterized in terms of signal transduction pathways, potential odorant receptor families and suitable odorants. 21, [24][25][26][27][28][29] However, none of the latter studies has investigated the spatial glomerular organization and odor map transformation by OB circuitry on both the morphological and functional level. Amino acids were shown to evoke frequent and differential glomerular responses in the lateral glomerular cluster making those stimuli a powerful choice to investigate olfactory subsystem organization. 26 We performed fast volumetric calcium imaging experiments in the lateral OB to investigate the organization and transformation of odor representations. Application of structurally diverse amino acids to the peripheral olfactory organ led to robust fluorescence changes on the level of (1) glomerular input, (2) glomerular output, and (3) MTC somata ( Figures 1A and 1E). We recorded activity of glomerular input neuropil in transgenic tubb2b:GCaMP6s animals ( Figures 1E and 1F). The tubb2b promoter drives expression in peripheral ORNs and a subset of MTCs 30 ( Figure 1B). Postsynaptic glomerular output responses were measured in MTC tufts and juxtaglomerular cell (JGC) neurites via Fluo-4 AM injected into the mitral cell layer ( Figures 1E and 1F). Downstream somatic activity was quantified in a tubb2b + MTC subpopulation ( Figures 1E and 1F). Regions of interest with evoked calcium transients were identified in 3517 glomeruli on the input level (n = 17; tubb2b:GCaMP6s animals), 431 glomeruli and 299 JGCs on the output level (n = 10; Fluo-4 AM injected animals) and in 138 tubb2b + and 387 tubb2b À MTCs (n = 7; tubb2b:Katushka animals).

Structural selectivity and contrast increase from the glomerular input to output level
Due to a specific binding pocket, odorant receptors possess a molecular receptive range which imposes a tuning profile onto ORNs expressing them. 31,32 Differentially tuned glomeruli are associated with different odorant receptor types according to the elementary olfactory wiring principles in the vertebrate main 4-6 or accessory olfactory system. 7,8 Single amino acids have proven to be powerful odorants with both shared and highly dissimilar epitopes to study odor tuning and odor representations in the OB of aquatic animals. 14,25,26,33 Making assumptions about (dis-)similarities in odorant receptor binding and odor tunings based on measures of molecular structure (dis-)similarity between the odor ligands is difficult. Given the shared backbone of amino acids, we chose Tanimoto scores to particularly highlight amino acid residue differences, the main contributing steric factor in olfactory receptor binding. We used a selection of eight single amino acids with different side chains and thus different physical properties (basic, aromatic, small,  To investigate the relationship between odor-evoked signals and odor molecule structure, we calculated the correlation coefficient between the pooled response peak amplitudes of stimulus pairings ( Figure 1G). Amino acids were grouped by Tanimoto score similarity into (1) Figure 1G). Responses to structurally dissimilar amino acids R/W and K/H ( Figures 1C and 1D) had a weak positive correlation on the glomerular input level (0.11 and 0.22) and weak negative correlation on the glomerular output (À0.21 and À0.08) level ( Figure 1G). Hierarchical cluster analysis of response peak amplitude vectors on the glomerular input and output level revealed tight linkage between M, L, and I, K and R, and H, F, and W. Notably, on the glomerular input level, H is linked closer to K and R. This linkage was also apparent on the tubb2b + MTC level except for M clustering at a higher distance to L and I ( Figure 1H).
We next assessed if clustering of glomeruli according to response profile correlation was related to molecular similarity scores. A negative relation between both measures would imply that glomerular clustering according to activity similarity (low cluster distance) is correlated to the structural similarity of the stimulus pairings (Tanimoto scores). We found a significantly negative trend relation between cluster distances of stimulus pairings and the respective Tanimoto MCS and AP scores on the glomerular output level ( Figure 1I; OLS testing, H 0 : regression slope = 0). Higher structural similarities between stimulus pairings were connected to lower cluster distances. While the overall distributions of correlation coefficients between the pooled stimulus-response amplitudes showed no significant difference, the median correlation coefficient of the tubb2b + MTC soma level was more negative than of glomerular input and output level ( Figure 1J). The range of correlation coefficient values gradually increased on each level indicating enhanced capacity to show contrasting activity to structurally different odor molecules ( Figure 1J).
To characterize odor tuning across the three OB levels in a more general, stimulus-independent manner, we calculated the lifetime sparseness of each glomerulus/MTC (see STAR methods). A larger fraction of glomeruli of the output level exhibited higher lifetime sparseness values and mean lifetime sparseness of the glomerular output/MTC soma level were significantly increased compared to the glomerular input level ( Figure 1K). Mean lifetime sparseness between glomeruli of the output and tubb2b + MTC soma were not significantly different ( Figure 1K). The distribution of averaged response amplitude differences between different amino acid pairings also supported our findings ( Figure S2). Altogether, we observed an increase in structural and overall tuning selectivity from glomerular input to the output level. While the lifetime sparseness of MTC somata was comparable to the glomeruli of the output level, the negative relation between response profile similarity and molecular similarity of the stimuli was not conserved.
As a next step, we wanted to understand whether individual or groups of similarly tuned glomeruli occurred in all animals and whether they were comparable in number and distribution between animals.
Glomerular regions are dominantly tuned to one or a small set of structurally related amino acids Odor responses were complex, with graduated amplitude differences between amino acid stimuli. To identify preferred activation by specific odors, we classified responsive regions by amplitude thresholding (Figures 2A and 2B). We chose two different thresholds to correctly represent that amino acid response iScience Article profiles typically exhibited dominant response peaks to one or few amino acids in addition to more variable, lower amplitude responses ( Figures 2C and 2D). With increasing amplitude threshold, the number of regions tuned to one or few amino acid stimuli increased on both the glomerular input ( Figure 2C) and output level ( Figure 2D).

Identity and frequency of glomerular regions varies between specimen
The identity and frequency of glomeruli with similar default or dominant odor tuning (glomerular species; Figures 2E and 2F) varied strongly between individual animals. While certain glomerular species (W, R, KR, M) were present in the majority of animals on the glomerular output level, others were not found in more than half of the animals ( Figures 2E and 2F). Depending on tuning classification, we found an average of 1-4 G 1-2 glomeruli with similar tuning (Figures 2E and 2F; right panels). Lack of stereotypical glomerular numbers between animals was also observable on the glomerular input level ( Figures S3A and S3B). Even though glomerular species were variable in numbers in our findings, we wanted to test whether any of them showed positional consistency among the glomerular array.
We investigated the location of glomerular species within the lateral cluster along the medio-lateral, caudo-rostral and ventro-dorsal axis ( Figure 3). We found glomerular species (e.g., W or HW) with a strong caudal bias and an intermediate position along the medio-lateral axis ( Figure 3A). Glomerular species tuned to I were slightly biased toward the lateral part of the glomerular cluster, but spread along the medio-lateral axis ( Figure 3A). Overall, we observed high positioning variability between glomerular species independent of odor tuning classification ( Figures S4A-S4F).

Organization of glomeruli is not chemotopic and juxtaposition does not indicate odor tuning similarity
Coarse chemotopical maps and molecular feature clusters have been described especially on the macroscopic level of the OB. [11][12][13]25 However, there is also the notion of fractured domains of glomeruli mapped according to tuning similarity, chemical groups or structural features on the microscopic glomerular level. 14,[34][35][36] We wanted to find general consistencies and principles in arrangement of glomeruli among the lateral cluster related to their tuning.
To check for organization according to a molecular receptive range, we analyzed spatial territories of glomerular species on the output level dominantly tuned to structurally similar amino acids ( Figures 3A,  S4G, and S4H). The territories of those individual groups were largely overlapping. We found no differences in distances between centroids of glomerular species tuned to similar (three groups: H, F and W; R and K; M, L and I) vs. dissimilar (all other stimulus pairings) amino acids ( Figure 3B). To assess the possibility of gradual, structure-based transitions of odor representations across the glomerular cluster, we analyzed whether the centroids of glomerular species with compound profiles (e.g., HFW) were located near the hypothetical centroid defined by the glomerular species tuned to the single stimuli only (e.g., H, F, and W). We did not find significant differences in the hypothetical centroid deviation ( Figure 3C), indicating that trajectories across the glomerular cluster do not imply gradual changes of odor representations between reference points.
Next, we explored how glomerular tuning was related to that of other adjacent glomeruli. We found no linear relation (OLS regression analysis, R 2 = 0.025) between the inter-glomerular Euclidean distance and the correlation coefficient between raw fluorescence traces of glomerular pairings ( Figure 3D). Also, the number of common stimuli in dominant odor tunings of glomeruli showed no obvious dependence on inter-glomerular distance ( Figure 3E). We frequently found glomeruli tuned to structurally dissimilar amino acids (e.g., W vs. R or K) close to each other in multiple animals ( Figures 3F-3H). In conclusion, juxtaposition of glomeruli was a poor predictor of similarity in odor tuning. iScience Article Bifurcating ORN axons can be associated with amino acid-responsive glomeruli As a next step, we examined how a seemingly inconsistent glomerular organization is connected to the wiring of the OB inputs. In amphibians, ORN axons frequently bifurcate to innervate distinct glomerular targets. We labeled individual ORNs and recorded amino acid-evoked responses to determine whether glomerular target regions innervated by the same ORN are tuned to amino acids ( Figure 4). We labeled 18 multi-glomerular ORNs and found six to be associated with glomerular regions responsive to amino acids. Labeled ORNs were variable in axon terminal segregation, complexity, and size. Four glomeruli innervated by single ORNs were associated with basic amino acids K and R, but also glomerular reactivity to aromatic amino acids as F and W occurred. While in our experiments, axon endings of multi-glomerular ORNs projected to similarly tuned glomeruli, the sample size did not allow conclusions about general properties of glomeruli innervated by multi-glomerular ORNs. Especially, since a fraction of them seemed to be amino acid insensitive. In one ORN, we found axon terminals branching into bipartite glomerular regions with differential tuning to amino acids ( Figure 4, ORN3). We could not exclude a functional synaptic connection into these regions. This supports a divergence of olfactory information into glomeruli with the same or different odor tuning.

MTC innervate distinct glomerular units in a coarse topological manner
This raises the question of how the postsynaptic glomerular network is organized to process a divergence of odor input. We assessed this first on the population level, by comparing the distribution of glomerular species on the output level to the positioning of MTC somata we recorded in tubb2b:Katushka animals according to their dominant odor tunings. Similar to the glomerular output level ( Figure Figure S5I) biased dendritic projection fields among the cluster, respectively. We analyzed the spatial distribution of pixels in the glomerular and MTC layer that showed at least a 2:1 ratio of color intensity to the other respective dyes (classified by the dominant color into GL or MCL blue, green and red color category). In all five animals analyzed, we saw a clear topological link of MTC somata and the glomeruli of similar color category ( Figure S6, two animals displayed: A-D and E-H). Overall, we observed a topological arrangement of glomerular modules in the lateral OB despite the lack of chemotopy, stereotypy, and consistency in glomerular wiring.

MTCs are classified by the number of dendritic tufts
After investigating MTCs of the lateral cluster on the population level, we aimed to dissect the MTC population further in terms of morphological and functional subtypes contributing to the glomerular module circuitry. We reconstructed the morphology of MTCs of the lateral OB labeled via single/sparse cell electroporation (n = 61, Figure 5A). MTCs with single (42.6%) or two (37.7%) dendritic tufts were most common, while three (13.1%), four (4.9%) and five (1.6%) tufts were considerably less abundant. The analyzed MTC population was generally heterogeneous and clear subgroups could not be detected based on morphological features using principal component analysis (PCA, Figure 5B)  iScience Article multi-tufted MTCs had 605 G 812 mm 3 (n = 41 tufts). In terms of number of secondary dendrites and primary/secondary basal neurites, the population was very heterogeneous, and we found no correlation between the number of tufts and the number of neurites. Number of primary and secondary basal neurites across the population averaged around 1.6 G 1.2 and 3.4 G 2.8, respectively. However, the number of basal neurites and secondary dendrites were anticorrelated.
Multi-glomerular MTCs integrate broadly tuned glomerular inputs with archetypical, low amplitude reactivity The high abundance of multi-glomerular MTCs in the lateral OB implied a potentially pivotal role in glomerular input integration. To investigate which odor information they integrate, we labeled individual multi-tufted MTCs (n = 16 MTCs) and measured amino acid evoked responses in associated glomerular input regions (n = 42; Figure 6). We found a weak, non-significant negative relation between (1) mean interglomerular distance and glomerular cross-sectional area and a significant, negative relation between iScience Article (2) interglomerular distance and response profile similarity ( Figure 6B). Especially the latter observation must be seen in context of the observed dependency between the signal-to-background ratio (SBR) of individual glomeruli and their (1) cross-sectional area and (2) mean correlation coefficient to associated glomerular response profiles ( Figure 6B). We found three main functional types of multi-glomerular MTCs. First, MTCs with highly correlated response profiles and similar response amplitude differences ( Figure 6C). Second, MTCs with similar dominant odor tuning, but differences in the lower amplitude responses ( Figure 6D). Third, MTCs exhibiting low amplitude or no responses above noise level ( Figure 6E).
We pooled the fluorescent time courses of all glomeruli confined by dendrites of multi-glomerular MTCs for further analysis ( Figure 7A). Independent of noise levels, all glomeruli were broadly responsive to amino acid stimuli. This was particularly evident when compared to the mixed population of previously recorded glomerular inputs with more selective tuning ( Figure 7B, see also Figure 1). Glomeruli innervated by multiglomerular MTCs had a broader tuning curve compared to glomeruli of the input level with unknown connectivity, but similar signal-to-background ratio ( Figures 7C-7E). They are characterized by an underlying, broad, but characteristic amino acid response profile and consequently feature a highly significant lower lifetime sparseness ( Figures 7C-7E).
As an indicator of tuning similarity within glomerular populations, we computed correlations of all possible pairings of response peak amplitude vectors. The ranked distribution of correlation coefficients between glomeruli innervated by the same multi-glomerular MTC (MTC-linked) was generally positive ( Figure 7F). All glomeruli innervated by multi-glomerular MTCs (MULTI) featured also negative correlation coefficients, but generally had a strong bias toward positive values. Overall, tuning within this population was more similar than its similarity to the tuning of glomerular input, output, and tubb2b + MTC populations. It is important to note that glomeruli of the MULTI group were compared to glomeruli that could be innervated either by uni-or multiglomerular MTCs, since the respective wiring was unknown. The fraction of response peak amplitude vectors with high similarity to MULTI (>0.7) decreased from the input over the output to the MTC level ( Figure 7F).
Glomeruli innervated by multi-glomerular MTCs also clustered tightly in our response amplitude-based analysis display of glomeruli and MTCs in odor space, where output level glomeruli and tubb2b + MTCs occurred less frequently ( Figure S7, see also Figures S2 and S3C). Outliers were glomeruli innervated by MTCs with a bias toward W on the R-W amplitude difference axis ( Figure 7G, also shown in Figures 6C-6E).

Individual MTCs can innervate glomeruli associated with bifurcating ORN axons
The morphology of ORN axonal arborizations 23 and MTC tufted dendritic configurations seemed to follow similar patterns. Our functional recording of presynaptic glomerular responses associated with the tufted dendrites of multi-glomerular MTCs as well as functional recordings of postsynaptic glomerular responses associated with the axon terminals of bifurcating ORNs supported this idea: Diverging ORN axon terminals and converging MTC tufts can independently innervate glomerular neuropil with similar response profiles (Figures 4, 5, and 6).
We wanted to test the hypothesis, whether glomeruli innervated by bifurcating ORN axons could also be innervated by a specific set of MTCs. They could reconverge the information of both glomeruli with their glomerular dendrites or could integrate between different glomerular channels. To find connected pairs of diverging/converging neurons, we sparsely co-labeled ORNs and MTCs (Figure 8). We found sister MTCs that innervated the same pair of glomeruli with both their tufted dendrites (Figures 8D and 8E). Furthermore, we found one bifurcating ORN projecting into two distinct glomeruli that were convergently innervated by dendrites of a single MTC (pseudo-multi-glomerular wiring; Figures 8A-8C).

DISCUSSION
Basic elements required for olfactory glomerular map formation are orderly converging axons of receptor neurons expressing the same individual odorant receptor gene. 37 The molecular receptive range of each iScience Article odorant receptor is associated with a characteristic tuning to certain structural features of odor molecules. 32,38 In the main OB, spatial grouping of glomeruli with similar receptive ranges forms the basis for a stereotypical and coarsely topographical functional odor representation. 12,13,31 In the Xenopus lateral OB, the input or output level of glomeruli featured dominant responses to structural characteristics of mostly one or two individual amino acids, but numerous smaller responses were apparent. Even with a restricted set of odors, this led to a high diversity of response profiles in glomeruli as previously shown in ORN soma responses from OE slice preparations. 39,40 Like zebrafish, glomerular responses in larval X. laevis have been shown to functionally cluster into distinct classes according to their tuning to amino acid structures. 14,26,33,41 We observed a significant increase in tuning selectivity, lifetime sparseness and contrast for structural features from the glomerular input to the output level. This implied the existence of a feature-based sculpting of glomerular odor representations by the OB network. 26,29,42 No stereotypic or chemotopic arrangement among glomerular clusters Non-delineated and often small glomeruli of similar tuning were distributed over the lateral OB and varied in number, identity and position between different animals, notwithstanding some topological bias. iScience Article Non-selectively tuned glomerular species showed no clear topological arrangement at all. Overall, we did not find any hints for chemotopy or molecular feature clusters with gradual transitions or odor tuning similarity based on juxtaposition within the lateral cluster. However, chemotopy across the OB surface is also controversially discussed within the mammalian olfactory research and despite findings of glomerular arrangements based on functional groups or molecular features 12,13,31,36,43 other studies have shown, that especially on the anatomically finer scale this arrangement can follow different principles or collapse entirely. 34,35 This raises the question of how our findings compare to the OB organization of other aquatic vertebrates. The lateral olfactory subsystems of Xenopus and zebrafish have often been compared due to their shared cAMPindependent signal transduction cascade, amino acid sensitivity and association with V2R type odorant receptors and microvillous ORNs. 25,[44][45][46][47] In the lateral chain glomeruli of zebrafish, stereotypic, fractured spatial maps of amino acid-sensitive regions occur. 14,41,48 Environmental enrichment with amino acids in young zebrafish alters the number and size of certain sets of glomeruli, whereas others are unaffected. 49 This hints toward a certain flexibility in aquatic OB organization, at least in some glomerular modules. Our work focused mainly on the spatial component, but it is important to note that odor representations are highly transient and OB activity is shaped over time. On the MTC level, chemotopic odor representations are dynamically reorganized in the range of hundreds of milliseconds after stimulus application. 33,50,51 Coexistence of uni-and multi-glomerular modules as a source of odor map heterogeneity The axonal and dendritic connectivity of a single glomerulus forms the basis of the functional OB organization and can be uni-or multi-glomerular. 18,20 In zebrafish, ORNs and the majority of MTCs are only connected to a single glomerulus and thus OB organization is based on uni-glomerular modules. 18,52-55 In iScience Article Xenopus, ORN axons and MTCs regularly split and connect to more than a single glomerulus. 18,21, 23 In addition to these multi-glomerular modules, also uni-glomerular connectivity is found on both levels of the Xenopus OB. 23,24 The organization of the olfactory network in amphibians of all developmental stages is primarily based on the coexistence of uni-and multi-glomerular connectivity with different hypothetical, overlapping wiring schemes. 18,19,23,56 Uni-glomerular MTCs connecting to the same glomerulus exhibit highly correlated activity patterns, 24,57 whereas multi-glomerular MTCs show lower correlation between their glomerular input activity patterns. Glomeruli selectively tuned to amino acids might be carried by uni-glomerular modules and arrange in a topological manner among the lateral cluster. The multi-glomerular modules possibly increase the variability in glomerular numbers and obscure the topology, due to the influence of a broad, archetypic amino acid response signature associated with glomeruli innervated by these MTCs. It is unclear how these information channels interact and multiple potential wiring strategies within and across the groups of uni-and multi-glomerular modules might exist.
We found that multi-tufted MTCs can convergingly connect to anatomically distinct glomeruli that are innervated by the bifurcating axonal branches of an individual receptor neuron. In this multi-glomerular wiring logic, incoming olfactory information is spatially split into two glomerular fractions and could still form an isolated transmission channel comparable to canonical uni-glomerular wiring strategies as in the rodent main OB. 4,58 Nonetheless, this pseudo-multiglomerular wiring might potentially facilitate differential modulation of olfactory information in spatially segregated glomerular regions for downstream integration. This organization remotely resembles the rodent AOB with lack of stereotypy in glomerular number and position between animals. 7,8 Multi-glomerular accessory mitral cells selectively integrate glomerular input in a heterotypic or homotypic manner resulting in a non-chemotopic but odorant receptor sequence and biological relevance-based odor map. 15,16,59 We conclude that odor map heterogeneity in Xenopus arises from the variability of uni-and multi-glomerular innervation on both input and output level.

Potential odorant receptor co-expression associated with multi-glomerular modules
An archetypical, low amplitude response profile in glomeruli innervated by multi-glomerular MTCs supports an underlying co-expression of a broadly amino acid-sensitive odorant receptor. Expression of more than one olfactory receptor could explain highly variable odor tuning classes observed in ORNs. 39 Expression patterns of ancestral v2r genes and the broadly expressed v2r-C overlap in the lateral MOE of larval Xenopus and are associated with cAMP-independent, trpc2-positive, microvillous amino acid sensitive ORNs. 25,46,47,60 In zebrafish, a similar lateral subsystem of microvillous ORNs expressing trpc2 and V2R-type receptors is also amino acid sensitive. 14,44 Amino acid responses are severely impaired in knockouts of a broadly expressed OlfC receptor, a v2r-C equivalent, supporting its role as co-receptor or chaperone. 61 OlfCc1 is required for detection of basic, aromatic, and neutral amino acids, while tuned to hydrophobic amino acids at a low sensitivity. 61 In larval Xenopus, an ORN population could express a co-receptor leading to a baseline amino acid sensitivity that can be superimposed by the response profile of the main receptor. V2Rs are primary suspects for the glomerular cAMP-independent, amino acid sensitivity we observe. However, we cannot conclude a clear association of multi-glomerular innervation and V2R/co-receptor expression, since multi-glomerular wiring also exists in the cAMP-dependent, OR receptor expressing medial glomerular cluster. 25 In fact, the general co-expression of multiple odorant receptors (OR, V1R, taars) might have led to the occurrence of multi-glomerular wiring strategies, possibly coinciding with the expansion of V1R/V2R and OR-type genes in amphibians. 19,37 Multi-glomerular modules might be a prevalent phenomenon linked to ORN axon bifurcation in amphibians, but not necessarily exclusive to them. 17,18 They could represent an intermediary type of neuronal module between fast, highly correlated uni-glomerular modules (latency coding of odor identity and concentration 24,57 ) and slower, integrative multi-glomerular modules (detection of biologically relevant blends 15,62 ). It is conceivable that a multitude of wiring logics emerges from the combination of singular and co-expression of odorant receptor types, increasing the coding capability of the olfactory system already at the OB level.

Limitations of the study
Our stimulation approach aimed for a maximal activation of the glomerular array ( Figure S8). Stimulation with high concentrations of odor molecules might obscure possible topologies of glomeruli very sensitive to individual odorants. 36  iScience Article the set of labeled cells random and limited in numbers, but also the finite set of stimuli limits the analysis of glomerular tuning. This limitation extends to the classification of homotypic or heterotypic innervation. It would be interesting to check if uni-glomerular modules show more spatial organization and chemotopy. Due to the lack of a specific marker, we were not able to only label uni-glomerular modules to differentiate population activity between uni-and multi-glomerular MTCs. Observations of the pseudo-multi-glomerular wiring pattern and sister MTCs innervating the same set of glomeruli were low in numbers due to the stochastic labeling approach (Figure 8). These observations should be seen as 'proof of principle' of an existing, unique wiring strategy and not as the generalized blueprint of glomerular modules. We investigated functional glomerular organization in larval animals, and it would be interesting to test how the OB is organized in post-metamorphic frogs.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Thomas Hassenklö ver (thomas.hassenkloever@physzool.bio.uni-giessen.de).

Materials availability
This study did not create any new unique reagents.
Data and code availability d Raw, processed, and intermediate multiphoton imaging data and morphological reconstructions reported in this paper will be shared by the lead contact upon request.
d All code is available as of the date of publication upon request.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Ethical statement
All experiments followed the guidelines of laboratory animal research and were approved by the regional board (RP Giessen; Az: V54-19c2015h01 GI 15/7) and the Niedersä chsisches Landesamt fü r

Background and developmental stages
In this work we used larval Xenopus laevis albino (wild-type; NASCO strain) or transgenic tadpoles all of developmental stages 50-53. 65 Health status and lack of developmental deformations was assessed beforehand. The sex of the animals was not determined.

Anesthesia and whole-mount preparation
We anesthetized larval Xenopus laevis in 0.02% tricaine/MS-222 solution (pH 7.6 in tap water) until complete irresponsiveness. 66 We killed anesthetized animals by severing the nervous system at the brainstem. A rectangular tissue block from the upper jaw, including the peripheral olfactory system plus olfactory bulbs, was excised for whole-mount preparations. Connective tissue occluding the nostrils and superincumbent palatial tissue on the ventral olfactory bulb was removed using forceps and fine scissors. For imaging experiments, the whole mount preparation was placed in a recessed recording chamber and stabilized by a nylon-stringed platinum grid. 66,67 Wheat germ agglutinin dye-based glomerular labeling We removed residual water in the nostrils of anesthetized tadpoles with microfiber tissue and gradually applied $1 mL of 250 ng/mL WGA-Alexa Fluor 594 (Thermo Fisher, dissolved in frog ringer) using micro loader pipette tips (Eppendorf, Microloader 25 mL). Gently pipetting the dye solution up and down assured homogeneous distribution in the nasal cavities ( Figure S1E). After being kept in a moistened chamber for 10 min, we washed off excess dye solution and put the anesthetized tadpoles back to their aquaria to recover from anesthesia.

Dextran dye based labeling of glomerular cluster
Prior to sparse-cell electroporations of MTCs, we labeled ORNs via bulk electroporation of fluorophorecoupled dextrans. 68 After anesthesia, dextran-dye crystals (Oregon green BAPTA dextran, MW 10,000; 3 mM in frog saline) were dissolved in residual water in the nostrils. We positioned 0.2 mm platinum wire electrodes and applied four/six (stage 50-51/52-53) sets of three square-pulses, alternating the polarity after each set (15/20 V, 500 ms duration and 25 ms delay; NPI Electronics; 3 mF capacitor connected in parallel). We performed the procedure on both nostrils with one electrode touching the animal laterally of the ON. Anesthetized animals were transferred to aquaria for recovery and used for experiments after 1-3 days. We labeled sparse sets of ORNs via electroporation 69 ( Figure S1F). Micropipettes with small tapers (8)(9)(10)(11)(12)(13)(14)(15) were pulled (P-1000, Sutter Instruments) from borosilicate glass capillaries (outer diameter 1.5 mm, inner diameter 0.86 mm, length 100 mm, with filament; Warner Instruments). Micropipettes were filled with 3-4 mL of fluorophore-coupled dextran solution (Alexa Fluor 594/568 dextran; 10 kD, 3 mM in frog ringer, Thermo Fisher) and mounted to the head stage of a single cell electroporator (Axoporator 800A, Axon Instruments/Molecular Devices). We positioned the anesthetized animal in a Petri dish, covered with a moistened microfiber tissue and in contact with the reference electrode. Under a microscope (Olympus SZX16; light source: X-Cite Series 120 Q, Excellitas technologies), the micropipette was penetrated into the lateral part of the main olfactory epithelium using a micromanipulator (Scientifica, PatchStar). A train of voltage pulses was applied (50 V, 300 ms pulse length, 500 ms train duration, 300 Hz). Fluorescent cell bodies were visible a few seconds after successful electroporation. Animals were transferred to aquaria for recovery from anesthesia and used for experiments after one to three days.

Sparse cell labeling of MTCs using dextran dyes
Whole-mount preparations were placed under a microscope with fluorescent illumination (Olympus BX51WI; light source AMH-200-F6S; Andor Technology-Oxford Instruments). Micropipettes filled with dextran-coupled fluorophores (see above; 3 mM in frog ringer, Alexa Fluor 488/555/594 and cascade blue dextran, 10 kD, Thermo Scientific) were mounted to the head stage of a single cell electroporator. We lowered the pipette tip toward the OB surface using a micromanipulator before penetrating it into the mitral cell layer and applying a voltage pulse train (50 V, 300 ms pulse length, 500 ms train duration, 300 Hz; details see. 68 We used prior bulk electroporation with fluorophore-coupled dextrans, WGA application, or the Katushka fluorescence of the tubb2b transgenic reporter line 30 to identify the mitral cell layer.

Sparse ORN labeling in transgenic brainbow tadpoles
Fluorescent protein expression was induced in multiple ORNs by injection of CRE recombinase mRNA into the MOE of transgenic CMV:Brainbow tadpoles with subsequent bulk electroporation (Figures S1H-S1I). We fabricated microinjection pipettes for CRE mRNA injection from borosilicate glass capillaries (outer diameter 1.0 mm, inner diameter 0.58 mm, length 100 mm; Warner instruments) that we then sharpened at an angle of 20-30 (Micropipette Beveler 48000; World Precision Instruments). CleanCap NLS-Cre Recombinase mRNA solution (100 ng/mL; TriLink Biotechnologies) was mixed 10:1 with cascade blue or Alexa Fluor 594 dextran (3 mM in frog ringer, 10 kDa, Thermo Fisher) and stored on ice. We mounted micropipettes filled with mRNA solution to a micromanipulator connected to a pressurized microinjector (FemtoJet, Eppendorf). Brainbow tadpoles were anesthetized and transferred to a moistened silicone rubber-filled Petri dish. We penetrated the pipette into the mucosa and increased the supply pressure until dye extrusion was visible. The pressure was increased by small increments (100-1000 hPa) until the dye slowly spread in the surrounding tissue. This was repeated at up to five injection sites in the main olfactory epithelium. Immediately after injection, we performed bulk electroporation (as described above). Tadpoles were transferred back to aquaria and left for recovery. Recombination-mediated cyan/yellow fluorescent protein expression was detectable in ORNs after a few days, axonal staining in the OB after three to five weeks.

Loading of MTCs with AM-dyes/calcium indicators
For functional experiments, we injected calcium indicator solution into the ventral OB ( Figure S1G, Offner et al., 2020). Fluo-4 AM (50 mg, Thermo Fisher) was dissolved in DMSO (Sigma-Aldrich) and mixed with 10 mL Pluronic F-127 (Biotium) and 35 mL frog ringer. After centrifugation at 16100g for 60 s, we mixed the supernatant with 0.3 mL cascade-blue dextran (3 mM in frog ringer; 10 kDa) and 3 mL MK571 (50 mM in frog ringer; Alexis Biochemicals). Micropipettes (see above) were filled with 3 mL calcium indicator solution and mounted to a head stage with a manual pressure supply system (syringe with three-way valve). The micropipette was pierced into the lateral mitral cell layer using a micromanipulator under a microscope with fluorescent illumination (Olympus BX51WI; light source AMH-200-F6S; Andor Technology-Oxford Instruments). The pressure was built up in the syringe by manual compression and released by opening the valve. Dye extrusion into the OB was observed, and the applied pressure was adjusted if necessary to gradually load the tissue surrounding the injection site for a maximum of 5 s. We performed three injections at different sites in the lateral mitral cell layer to achieve visible Fluo-4 fluorescence in neurons of interest.  Figures 6A, 6E, S1D, and S1E).

Multiphoton microscopy and imaging settings
We obtained 3D-image stacks of the OB with a multiphoton microscope (Nikon A1R MP Odor stimuli and odorant application system We applied odorants to the olfactory epithelia of the whole-mount preparations for 5 s via two gravity-fed eight-channel perfusion systems (ALA-VM-8 Series; VC3-8xP Series; ALA Scientific) connected via silicone tubing to a shared outflow (Milli Manifold; 16 inlet ports; ALA Scientific) positioned in front of the nostril. Without stimulations, constant perfusion of frog ringer (98 mM NaCl, 2 mM KCl, 1 mM CaCl 2 , 2 mM MgCl, 5 mM Na-pyruvate, 5 mM glucose, 10 mM HEPES, pH 7.8, osmolarity of 230 mOsmol/l) was present, and excess solution drained by a peristaltic pump (Cyclo II, Roth). We recorded for 15 s before each stimulus application. We chose inter-stimulus intervals of 1 min to avoid desensitization effects. Each experiment consisted of at least two pairs of stimulus sequences to eight stimuli. There was one repetition of the respective amino acid/odorant mix in each sequence at the beginning and end of the recording. We used all four sequences in MTC somatic recordings and only one pair of sequences in glomerular recordings for CaImAn analysis. Detailed schematics of the different experimental protocols are depicted in Figures S1A-S1C.

QUANTIFICATION AND STATISTICAL ANALYSIS
Imaging data pre-processing We corrected 5D calcium imaging data (two color channels, x, y, z, and time dimension) for a line shift originating from bilinear scanning. During the 16/32 min recordings under perfusion, x, y, and z drift occurred in all measurements. We merged multiple subsequent z-planes (4/6 planes, interplane distances 4-6 mm) to one virtual plane (20-30 mm) by maximum intensity projection to compensate z drift. While losing z-resolution with this method, spatial footprints were not easily lost due to z-or x-y drift later. We used a piecewise-rigid motion correction algorithm (NoRMCorre) to obtain lateral drift-corrected image stacks and subsequent plane-wise denoising, deconvolution, and de-mixing of the data by CaImAn. [70][71][72][73]

ROI extraction and neuronal activity visualization
We used CaImAn's constrained non-negative matrix factorization (CNMF) to extract spatial footprints and their temporal components. 72 To visualize stimulus-induced neuronal activity, we created fluorescence intensity difference maps. Therefore, we subtracted the mean of the maximum fluorescence values of the calcium transients and the two neighboring frames (8 s after stimulation onset) from the mean baseline fluorescence (5 time frames: 5 s prior to stimulus; see also Figure S1A). We used the deconvolved and gaussian-filtered data for intensity difference maps to visualize only ROIs detected by CaImAn. In this approach aiming to display glomerular/neuronal tuning in a qualitative manner, we used the resulting individual (response peak) intensity difference image (grayscale) for each individual stimulus and merged those images in Fiji with individual stimulus specific primary colors as multicolor stacks, mostly the ll

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All remaining raw fluorescence time traces were baseline corrected by asymmetric least squares smoothing, 74 absolute fluorescence changes transformed into DF/F, 26

Essential parameters for odor tuning analysis
We analyzed the calcium responses of glomeruli and neurons on the glomerular input, output, and MTC soma level according to their tuning to the eight single amino acid stimuli (M, L, I, H, K, R, F, W). We used the stimulus-response peak amplitude of the normalized DF/F fluorescent time traces (in the interval 15 s after stimulus application start) as a measure of odor-evoked response intensity. For all stimuli that were applied multiple times, the maximum response amplitudes were averaged.

Odor tuning
We analyzed all glomeruli and neurons according to a 'default' and a 'dominant odor tuning'. We defined the default/dominant odor tuning as the set of stimuli triggering calcium responses (peak amplitude) above 2x/3x the standard deviation of averaged baseline fluorescence of the entire stimulation sequence (15 s intervals, 25 s after stimulus onset). For assignment of these tunings to each glomerulus/neuron, we normalized each fluorescent time trace by its maximum fluorescence intensity value (as described above). The default/dominant odor tuning of glomeruli/neurons is presented as a sequence of the single-letter amino acid codes. A region with dominant odor tuning HFW exhibits suprathreshold calcium responses to H, F, and W, and all potential peaks below 3x SD of the baseline fluorescence are neglected (Figure 2A). We linked 21 of the most frequent default/dominant odor tunings with particular colors (Figures 2E, 2F, and S4A).

Correlation and response amplitude (difference) based analysis
We calculated the pairwise differences (ranging from À1 to 1) between the normalized response peak amplitudes to amino acid stimuli. Among others, those amplitude differences were used as x, y, and z coordinates of individual ROIs in a simplified 3D odor space representation (for details see Figure S2).
We normalized the response peak amplitudes for odor tuning analysis to the maximum response evoked by the selected eight stimuli (M, L, I, H, K, R, F, W; range 0-1 iScience Article method, using correlation distances), and (lifetime) sparseness calculation. 75 Lifetime sparseness equals 0, when a region responds equally strongly to all stimuli and 1, when exclusively responding to one. N represents the length, and r n the nth response of the amplitude response vector. sparseness = 1 À P r n N 2 ∕ P r 2 n N 1 À 1 N We calculated the normalized distances between the columns of the hierarchical cluster diagram (cluster distances; 0-1) as a measure of how individual stimuli were linked on the level of their amplitude response vectors.
Signal to background/noise ratio Normalized spatial territories of glomerular species We normalized glomerular/neuronal positions according to their relative positioning along the mediolateral, caudo-rostral, and dorsoventral axis. Therefore x, y, and z coordinates were normalized to manually defined coordinates at the respective boundaries of the glomerular cluster/mitral cell layer. For the glomerular output layer, we used WGA-Alexa Fluor 594 to label all glomeruli in vivo and high z-resolutions during image acquisition to ensure full coverage of the lateral cluster and its glomeruli in all animals. On the glomerular input layer, faint green GCaMP6s expression marked the outlines of the lateral cluster. Centroids of the positioning of individual glomerular species (glomeruli of a specific default/dominant odor tuning) were calculated from the mean x, y, and z coordinates along each of these normalized axes. Ellipsoids of 1x SD along each axis with the centroid coordinate as origin served to estimate the territories of each glomerular species.

Measures of juxtaposition and chemotopy
We calculated the Euclidean distances between the positional centroids of different glomerular species as a measure of juxtaposition. For chemotopy analysis, centroids of glomerular species dominantly tuned to two or three amino acid stimuli were used (compound profiles, e.g., KR or HFW). We calculated their positional deviation from the centroid of the two or three glomerular species dominantly tuned to the individual stimuli of the compound profile only (e.g., K and R or H, F and W) by Euclidean distance. The smaller this distance, the more centered were the glomerular species with a compound profile between the glomerular species tuned to the individual stimuli only.
We defined glomeruli tuned to (combinations of) H, F, and W; M, L and I, or K and R as glomeruli tuned to structurally similar amino acids (similar molecular receptive range).

In silico structural similarity scoring
We used the online tool CheMineTools to calculate Tanimoto scores of maximum common substructure (MCS) and atom pair distance (AP) as a quantitative descriptor of structural similarity between amino acid pairings. 76 Tanimoto scores were calculated using the reference numbers of the amino acids: H: 6274, W: 6305, F: 6140, R: 6322, K: 5962, M: 6137, I: 6106, and L: 6306.

MTC morphological analysis
MTCs labeled via sparse/single cell electroporation in the MOB of larval Xenopus laevis were semi-automatically reconstructed from two-photon image stacks using Vaa3D. 56, 77 The reconstructions were translated into a hierarchical tree-structure by defining branching and endpoints. The cell soma was defined as root segment of the tree, with each segment connecting to exactly one parent segment. The DBSCAN algorithm (Density-Based Spatial Clustering of Applications with Noise) implemented in the ll OPEN ACCESS