Behavioral and neuronal mechanisms of olfactory imprinting in zebrafish
Final Report Abstract
Towards the end of the first project period we concluded that the phosphorylated extracellular signal kinase pERK (and not its downstream immediate early genes such as egr1) is the most promising activity marker in behavioral olfactory tests for zebrafish larvae. Initial positive tests using an immunohistochemical assay for pERK after stimulation with general food and conspecific odors yielded clearly different activity response profiles of olfactory sensory cells (OSNs). Thus, we used raising protocols to generate imprinted and non-imprinted zebrafish larvae which were then stimulated more specifically for kin recognition using kin odor. Olfactory imprinting did not change quantitative expression of specific olfactory receptor genes. But our results showed highly significantly that crypt cells and a fraction of (S100-negative) microvillous OSNs respond to kin odor in imprinted - but not in non-imprinted - larvae. Furthermore, enhanced activity was also seen in the dorsomedial area of the olfactory bulb receiving crypt cell projections. In addition, combination of neuroanatomical tracing and neurochemical assays allowed for describing a synaptic chain leading from the olfactory epithelium via the dorsomedial the olfactory bulb to a telencephalic nucleus (presumably the medial amygdala) and from there into the tuberal hypothalamus. This multisynaptic pathway (which includes the crypt cell information) is identical to the accessory olfactory pathway seen in tetrapods. Unexpected developments which led to modification of the original research plan resulted partially from insights through collaborations on projects outside of the SPP tandem (e.g., use of activity marker pERK or transcription factor otp as marker for medial amygdala; see Final Progress Report for details). In parallel, we mapped neuronal activity evoked by MHC peptides, conspecific odor and food odor throughout the olfactory bulb (OB) and parts of the telencephalon of zebrafish larvae by multiphoton calcium imaging. The results show that MHC peptides are potent odorants that evoke complex, distributed patterns of activity in the OB. Attempts to further correlate representations of MHC peptides with behavioral imprinting were unsuccessful because genotypes that imprint on defined MHC peptides could not be bred to homozygosity, which may be a direct consequence of imprinting. Nevertheless, these experiments provide the first detailed activity maps of MHC odorants in the central nervous system. In addition, we established an efficient procedure for olfactory discrimination learning that will be used in future projects.
Publications
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(2012) Correlated basal expression of immediate early gene egr1 and tyrosine hydroxylase in zebrafish brain and downregulation in olfactory bulb after transitory olfactory deprivation J Chem Neuroanat 46: 51-66
Kress S, Wullimann MF
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(2012) Influence of Kinship and MHC Class II Genotype on Visual Traits in Zebrafish Larvae (Danio rerio). Plos One 7:e5118210.5118137
Hinz C, Gebhardt K, Hartmann AK, Sigman L, Gerlach G
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(2013) Kin recognition in zebrafish, Danio rerio, is based on imprinting on olfactory and visual stimuli. Anim Behav 85:925-930
Hinz C, Kobbenbring S, Kress S, Sigman L, Müller A, Gerlach G
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(2013) Olfactory imprinting is triggered by MHC peptide ligands. Sci Rep 3: 2800
Hinz C, Namekawa I, Behrmann-Godel J, Oppelt C, Jaeschke A, Müller A, Friedrich RW, Gerlach G
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(2015) Combinatorial analysis of calcium-binding proteins in larval and adult zebrafish primary olfactory system identifies differential olfactory bulb glomerular projection fields. Brain Struct Funct 220: 1951-70
Kress S, Biechl D, Wullimann MF
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(2016) Crypt cells are involved in kin recognition in larval zebrafish. Sci Rep 6:24590
Biechl D, Tietje K, Gerlach G, Wullimann MF
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(2017) Identification of accessory olfactory system and medial amygdala in the zebrafish. Sci Rep 7:44295
Biechl D, Tietje K, Ryu S, Grothe B, Gerlach G, Wullimann MF
