The partial segregation of the LC into ventromedial and dorsolateral lobe as a reflection of adjacent V2R and V1R expression domains
The injections of WGA-coupled fluorophores into the ventral OB strengthened the idea of the bimodal olfactory streams in larval Xenopus laevis (Gliem et al., 2013). Moreover, the staining revealed glomerular morphologies in a punctuate and less fiber-like manner than in dextran bulk electroporation (Figure 1; A). I described a dorsolateral lobe of the LC that is directly adjacent to the more prominent ventromedial lobe. The coarse boundaries between the lobes were distinguishable in stages 52-54. These two lobes are very likely the equivalent to the projection fields named PF4 and PF 5 by Gaudin and Gascuel (Gaudin and Gascuel, 2005).
The segregation of the two projection fields / lobes were described, especially during ongoing development (Gaudin and Gascuel, 2005). I did not observe responses to amino acids in the dorsolateral lobe, at least within the limitations of the methods I used. While there is no concrete proof yet, ORN responses to amino acids in Xenopus laevis are highly correlated to V2R odorant receptor class expression (Syed et al., 2013, 2017). The retrogradely traced population of ORNs that innervate the two lobes of the LC might consist of both V2R and V1R-expressing neurons. One could speculate that the ventromedial lobe of the LC characterized in this work is dominated by V2R-expressing ORNs (amino-acid sensitive) while the dorsolateral lobe could be a target of V1R-expressing ORNs. In the rodent AOB, the two VR domains are dichotomous (Belluscio et al., 1999; Rodriguez et al., 1999).
However, the individual neuronal networks processing the caudal and rostral domains' information interact on the bulb level (Larriva-Sahd, 2008). It might very well be that in larval Xenopus, two subsystems exist within the LC similar to the rodent AOB but less anatomically distinct.
93 Amino acids and sulfated steroids – Suitable odorants for different sets of V2Rs The calcium imaging experiments I performed in the LC of larval Xenopus MOB revealed that sulfated steroids induced odor responses on the glomerular and MTC level (Figure 22 published in (Sansone et al., 2015). Both, rodent V1Rs and V2Rs can potentially be activated by sulfated steroids (Hammen et al., 2014; Isogai et al., 2012). However, to the best of my knowledge, there are no reports of rodent V1Rs or ORAs in fish that are tuned to single amino acids. In rodents, a small but consistent population of sulfated steroid-responsive glomeruli was characterized in the posterior AOB domain (Hammen et al., 2014), which is assumed to be exclusively innervated by V2R-expressing VRNs (Belluscio et al., 1999; Rodriguez et al., 1999). The authors interpreted this surprising observation as either ‘displaced' V1R glomeruli or a subset of V2Rs that respond to sulfated steroids. Xenopus laevis possesses an extensive V2R repertoire, and V2Rs are broadly expressed in its MOE (Hagino-Yamagishi et al., 2004;
Syed et al., 2013). Consequently, it seems relatively realistic that a set of V2Rs could carry sensitivity to sulfated steroids in Xenopus. Different contributions of V1Rs and V2Rs might explain differences in detection thresholds of sulfated steroids between the VNO and MOE (Sansone et al., 2015) could be explained by different contributions of V1Rs and V2Rs. The AOS of Xenopus laevis harbors phylogenetically younger, mammalian-like V2Rs and lacks the more ancestral ‘fish-like’ V2Rs (Hagino-Yamagishi et al., 2004; Syed et al., 2013). The glomeruli and AMCs I measured in larval Xenopus AOB, did not respond to amino acids, but to both sulfated steroid mixes (E- and P-mix). If one assumes these response profiles to be representative for sets of later-diverging V2Rs of the AOS, the glomerular responses to sulfated steroids in the larval MOS are more likely a result of the ancestral V2Rs or V1Rs expressed (Date-Ito et al., 2008; Hagino-Yamagishi et al., 2004; Syed et al., 2013).
There is evidence from in situ hybridization studies, that Xenopus V2Rs and the V2R-C receptor could selectively be co-expressed (Syed et al., 2013) similar as observed in fish (DeMaria et al., 2013) and rodent V2Rs (Ishii and Mombaerts, 2011; Silvotti et al., 2007). In zebrafish, the OlfCc1 receptor serves as amino acid sensor and co-receptor for other OlfC receptors. Loss of OlfCc1 results in general loss of sensitivity to polar, basic, and acidic amino acids (DeMaria et al., 2013) and thus might be essential to detect food stimuli in zebrafish (Koide et al., 2009). Amino acid responses in the ventromedial lobe could be explained by similar combinations of Xenopus ancestral V2Rs and the broadly expressed v2r-C receptors (Syed et al., 2013). V2Rs tuned to hydrophobic, hetero-cyclic features in amino acid residues like tryptophan might potentially also be activated by similar features found in the sulfated steroids as a result of combinatorial receptor coding (Malnic et al., 1999; Sansone et al., 2015).
The calcium imaging experiments in the LC of the larval MOB revealed MTCs exclusively responding to either sulfated steroids or amino acids but also subsets that responded to both groups (Figure 22). Amino acid-insensitive glomeruli that reacted to sulfated steroids might
94 carry information from V2Rs tuned to particular features of sulfated steroids that are not present in amino acids. Another possibility is that the amino acid-insensitive fraction of sulfated steroid-responsive glomeruli in Xenopus receives input from V1Rs. In rodents, V1Rs can be activated by sulfated steroids already at concentrations of 1µM (Hammen et al., 2014).
The apparent sensitivity of the larval Xenopus MOS to sulfated steroids could be composed of a more sensitive V1R part and a less sensitive V2R part (Sansone et al., 2015). The V2R-based responses to sulfated steroids at higher odorant concentrations (Sansone et al., 2015) could be due to less specific odorant receptor-binding This could explain the lower average sensitivity of ORNs in the MOE to sulfated steroids (both V1R and V2R components) in comparison to the VRNs (V2R components only; (Date-Ito et al., 2008; Sansone et al., 2015).
If the dorsolateral lobe (PF5) should indeed represent the amino acid-insensitive V1R expression zone, its small size could be a reflection of the low number of V1R genes in Xenopus laevis (Date-Ito et al., 2008; Shi and Zhang, 2007). However, quantification of this observation will be necessary. To sum it up, the described ventromedial lobe of the LC could represent a non-exclusive V2R expression domain with amino acids and sulfated steroids as potent stimuli. Its responsiveness to amino acids might be based on ORNs co-expressing ancestral V2Rs and V2R-Cs. The responses to sulfated steroids in Xenopus MOB could be a result of low-affinity binding to those ORNs' odorant receptors at high odorant concentrations. Alternatively, V2R-expressing ORN species tuned to sulfated steroids might account for the observed responses. The dorsolateral lobe might represent the amino-acid insensitive V1R expression zone, possibly responsive to sulfated steroids. No evidence for cAMP-dependent olfactory signal transduction was found in the LC of larval Xenopus (Gliem et al., 2013). The G-protein Gi2 seems to be associated with the signal transduction cascade of V1Rs in Xenopus (Date-Ito et al., 2008). Immunohistochemical stainings against Gi2 or calcium imaging using V1R specific ligands would undoubtedly help to deduce odorant receptor contributions to the observed ventromedial and dorsolateral lobe. Overall, the first part of my results confirms but at the same time, expands the existing idea of the bimodal olfactory processing streams in larval Xenopus laevis. I could reveal the full extent of the ventrolateral ORN population projecting to the LC. MTCs associated with particular glomerular clusters reside in proximity to those. Their somatic position can be predictive for their dendritic glomerular innervation among the LC. However, there are subsets of MTCs that can project to distal or even multiple glomeruli scattered across the glomerular array. In addition to amino acids, I established sulfated steroids as suitable stimuli for the postsynaptic neuronal network associated with the LC. The observed glomerular response patterns to those odorant groups, rule in the possibility of two distinct, but partially overlapping subsystems within the LC. The medioventral and dorsolateral lobes of the LC could be reflections of different VR-based subsystems
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Schematic 1
Schematic 18 Distribution of glomerular reactivity to different odorant groups among the LC lobes and the AOB