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Neuronal basis of olfactory imprinting and kin recognition in the zebrafish Danio rerio
Neuronal basis of olfactory imprinting and kin recognition in the zebrafish Danio rerio
Olfaction plays a fundamental role in detection and discrimination of the environment in all vertebrates, including teleosts, such as the zebrafish, Danio rerio. The zebrafish olfactory system is capable to detect a wide range of chemical compounds which trigger or contribute to behaviours crucial for survival such as foraging, migration, intraspecific communication, reproduction and predator avoidance. In contrast to terrestrial vertebrates, the teleost olfactory system lacks a separate vomeronasal organ (VNO), which is known to be involved in pheromone detection. However, although the zebrafish olfactory system consists only of one paired olfactory epithelium (OE), the containing olfactory sensory neurons express olfactory receptors related to those of the main OE and VNO of mammals. Thus, the fish olfactory system is capable to detect and process pheromones and show a context related behavioral response. Beside many olfactory driven social behaviors, kin recognition is of particular relevance to the field of neurobiology, because it depends on an imprinting paradigm which requires a two step learning process of olfactory and visual cues during a defined time window early in life. Zebrafish larvae imprint on the pigmentation pattern and olfactory cue of their kin on the 5th and 6th day of development. The created kin template allows discriminating between kin and non-kin and plays a fundamental role at early stages, as it was shown that zebrafish larvae prefer to group with their kin whereas sexually mature zebrafish use kin recognition to avoid inbreeding. Interestingly, larvae which are exposed to non-kin cues at the appropriate days show neither preference for kin nor for non-kin, suggesting a genetic predisposition for kin cues. However, the neuronal mechanisms underlying olfactory imprinting and kin recognition are unknown so far. Recent studies demonstrated that zebrafish recognize their kin based on Major Histocompatibility Complex (MHC) class II genotype similarity. Zebrafish which share MHC class II alleles show a similar pigmentation pattern (visual cue) as well as chemical signature (olfactory cue) and thus MHC class II genotype similarity may explain the genetic predisposition which prevents larvae to imprint on non-kin cues. Moreover, olfactory stimulation with MHC class II peptide ligands shows spatially overlapping activation of bulbar neurons compared to responses to kin odor, suggesting MHC peptides to be part of kin odor. Presently, the type of olfactory sensory neuron (OSN) which detects a kin odor related signal is unknown. The zebrafish OE bears four different types of OSNs, ciliated- and microvillous OSNs, kappe neurons and crypt cells; each type showing morphological- and immunohistochemical characteristics. Our study combines behavioral, genetic and neuroanatomical methods to investigate the neuronal mechanisms involved in the processes of olfactory imprinting and kin recognition in the zebrafish. The first aim of this study is to provide new insights to the anatomy of the larval and adult zebrafish olfactory system. Combinatorial immunohistological analysis of four different calcium binding proteins (CBPs), Parvalbumin, Calretinin, Calbindin and S100, reveals a differential expression pattern of OSNs and their axonal projections into the olfactory bulb (OB). Combinatorial double immunohistochemistry identifies at least eight subpopulations of OSNs. We report three subpopulations of ciliated OSNs - one major subpopulation expresses Parvalbumin, Calbindin and Calretinin, and two populations are either positive for Parvalbumin and Calbindin or Calretinin only. Furthermore, we identify four subpopulations of microvillous OSNs, one expresses only Parvalbumin, one minor population shows S100 and Parvalbumin positivity, one is positive for Parvalbumin and Calbindin and finally one subpopulation of microvillous OSNs which is immunoreactive for Parvalbumin, Calbindin and Calretinin. Crypt cells, absent in terrestrial vertebrates and only present in teleosts, express only S100 and are negative for all other CBPs. Consistent with other reports, axonal projections of ciliated OSNs terminate into dorso- and ventromedial bulbar fields whereas microvillous OSNs project their axon into the ventrolateral OB. Additionally, we newly describe axonal projections of likewise microvillous OSNs which only express Parvalbumin and terminate into the mediodorsal OB. Moreover, we show S100 positive crypt cells to terminate into one single mediodorsal glomerulus, the mdg2, but also show additional axonal input into this glomerulus from S100 and Parvalbumin expressing microvillous OSNs. To investigate the type(s) of OSNs which detect a kin odor related signal, we focused on finding a reliable marker for neuronal activity in response to olfactory stimulation. The Extracellular signal Regulated Kinase (ERK) is a member of the ERK / Mitogen Activated Protein Kinase (MAPK) signaling pathway. Activation, for instance by binding of a ligand to an olfactory receptor leads to phosphorylation and therefore activation of ERK (pERK) which in turn translocates into the cell nucleus to modulate gene expression. In mammals, pERK is a common marker for neuronal activity and was previously used in the field of olfaction. Before starting to approach the identity of the OSN type involved in kin recognition, we validated pERK as a reliable marker for neuronal activation in the larval zebrafish after odor exposure. To this aim, we stimulated group raised larvae at the 9th day of development with different odors and analyzed neuronal activation visualized by pERK immunopositivity in the larval zebrafish OE. With the use of accepted morphological criteria, we identified the four differenttypes of OSNs of the zebrafish OE. Additionally, we used the CBP S100 to mark specifically crypt cells. For the first time in larval zebrafish, we performed a timescale experiment to test best odor exposure duration with the result that detectable pERK levels are recognizable already after 3 minutes of odor exposure. However, prolonging the exposure duration does not lead to better pERK signals in OSNs. Furthermore, we demonstrate that olfactory stimulation with food and non-kin odor (conspecific odor) clearly results in a differential activation pattern of OSNs. Consistent with other studies, we identify activated ciliated and microvillous OSNs after exposure to food odor, whereas only microvillous cells show responses to conspecific odor. Crypt cells show activation neither to food odor nor to non-kin odor. Upon validation of pERK to mark neuronal activation in the larval zebrafish OE, we stimulated imprinted and non-imprinted 9 day old larvae with kin odor and analyzed activated OSNs. In two rounds of stimulation experiments, each with slightly different raising conditions, we provide the first direct evidence for crypt cells as well as a small subpopulation of microvillous cells to be involved in detection of a kin odor related signal. Interestingly, only larvae which were successfully imprinted show activated crypt cells in response to kin odor, whereas crypt cells of non-imprinted larvae show no increase in pERK levels. A difference in crypt cell number does not account for this difference in activation pattern as a comparison of crypt cell quantity reveals no significant difference between imprinted and non-imprinted larvae. Furthermore, we analyzed neuronal activation of bulbar neurons after exposure to kin odor. Consistent with our results on activation at the level of the larval OE, bulbar neurons of imprinted larvae show increased neuronal activation compared to non-imprinted larvae especially around the mediodorsal glomerulus that receives crypt cell input (mdG2) after kin odor exposure. The final aim of this study is to identify the existence of an accessory olfactory pathway in teleosts. In tetrapods, vomeronasal information is mainly transferred from the VNO to the accessory olfactory bulb (AOB) and from there to the medial amygdala. The medial amygdala is a part of the subpallium and initiates via amygdalo-hypothalamic pathways behavioral and also hormonal responses to incoming signals. Moreover, besides an involvement in fear and associative learning, the medial amygdala is also known to be involved in processing of conspecific odors in rodents. Although a separate VNO is absent in teleosts, we newly identify an accessory olfactory pathway in the zebrafish. By injection of DiI tracer into the mediodorsal OB, which is the target region of crypt cells and some microvillous OSNs, we demonstrate a neuronal circuit running from the mediodorsal OB to the medial amygdala and from there to the tuberal hypothalamus. Interestingly, non-imprinted zebrafish larvae show increased activity of neurons in the medial amygdala compared to imprinted larvae. Finally, we demonstrate for the first time the OSN type which is involved in the detection as well as processing targets of a kin odor related signal in larval zebrafish.
zebrafish, olfaction, kin recognition, imprinting
Biechl, Daniela
2017
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Biechl, Daniela (2017): Neuronal basis of olfactory imprinting and kin recognition in the zebrafish Danio rerio. Dissertation, LMU München: Faculty of Biology
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Abstract

Olfaction plays a fundamental role in detection and discrimination of the environment in all vertebrates, including teleosts, such as the zebrafish, Danio rerio. The zebrafish olfactory system is capable to detect a wide range of chemical compounds which trigger or contribute to behaviours crucial for survival such as foraging, migration, intraspecific communication, reproduction and predator avoidance. In contrast to terrestrial vertebrates, the teleost olfactory system lacks a separate vomeronasal organ (VNO), which is known to be involved in pheromone detection. However, although the zebrafish olfactory system consists only of one paired olfactory epithelium (OE), the containing olfactory sensory neurons express olfactory receptors related to those of the main OE and VNO of mammals. Thus, the fish olfactory system is capable to detect and process pheromones and show a context related behavioral response. Beside many olfactory driven social behaviors, kin recognition is of particular relevance to the field of neurobiology, because it depends on an imprinting paradigm which requires a two step learning process of olfactory and visual cues during a defined time window early in life. Zebrafish larvae imprint on the pigmentation pattern and olfactory cue of their kin on the 5th and 6th day of development. The created kin template allows discriminating between kin and non-kin and plays a fundamental role at early stages, as it was shown that zebrafish larvae prefer to group with their kin whereas sexually mature zebrafish use kin recognition to avoid inbreeding. Interestingly, larvae which are exposed to non-kin cues at the appropriate days show neither preference for kin nor for non-kin, suggesting a genetic predisposition for kin cues. However, the neuronal mechanisms underlying olfactory imprinting and kin recognition are unknown so far. Recent studies demonstrated that zebrafish recognize their kin based on Major Histocompatibility Complex (MHC) class II genotype similarity. Zebrafish which share MHC class II alleles show a similar pigmentation pattern (visual cue) as well as chemical signature (olfactory cue) and thus MHC class II genotype similarity may explain the genetic predisposition which prevents larvae to imprint on non-kin cues. Moreover, olfactory stimulation with MHC class II peptide ligands shows spatially overlapping activation of bulbar neurons compared to responses to kin odor, suggesting MHC peptides to be part of kin odor. Presently, the type of olfactory sensory neuron (OSN) which detects a kin odor related signal is unknown. The zebrafish OE bears four different types of OSNs, ciliated- and microvillous OSNs, kappe neurons and crypt cells; each type showing morphological- and immunohistochemical characteristics. Our study combines behavioral, genetic and neuroanatomical methods to investigate the neuronal mechanisms involved in the processes of olfactory imprinting and kin recognition in the zebrafish. The first aim of this study is to provide new insights to the anatomy of the larval and adult zebrafish olfactory system. Combinatorial immunohistological analysis of four different calcium binding proteins (CBPs), Parvalbumin, Calretinin, Calbindin and S100, reveals a differential expression pattern of OSNs and their axonal projections into the olfactory bulb (OB). Combinatorial double immunohistochemistry identifies at least eight subpopulations of OSNs. We report three subpopulations of ciliated OSNs - one major subpopulation expresses Parvalbumin, Calbindin and Calretinin, and two populations are either positive for Parvalbumin and Calbindin or Calretinin only. Furthermore, we identify four subpopulations of microvillous OSNs, one expresses only Parvalbumin, one minor population shows S100 and Parvalbumin positivity, one is positive for Parvalbumin and Calbindin and finally one subpopulation of microvillous OSNs which is immunoreactive for Parvalbumin, Calbindin and Calretinin. Crypt cells, absent in terrestrial vertebrates and only present in teleosts, express only S100 and are negative for all other CBPs. Consistent with other reports, axonal projections of ciliated OSNs terminate into dorso- and ventromedial bulbar fields whereas microvillous OSNs project their axon into the ventrolateral OB. Additionally, we newly describe axonal projections of likewise microvillous OSNs which only express Parvalbumin and terminate into the mediodorsal OB. Moreover, we show S100 positive crypt cells to terminate into one single mediodorsal glomerulus, the mdg2, but also show additional axonal input into this glomerulus from S100 and Parvalbumin expressing microvillous OSNs. To investigate the type(s) of OSNs which detect a kin odor related signal, we focused on finding a reliable marker for neuronal activity in response to olfactory stimulation. The Extracellular signal Regulated Kinase (ERK) is a member of the ERK / Mitogen Activated Protein Kinase (MAPK) signaling pathway. Activation, for instance by binding of a ligand to an olfactory receptor leads to phosphorylation and therefore activation of ERK (pERK) which in turn translocates into the cell nucleus to modulate gene expression. In mammals, pERK is a common marker for neuronal activity and was previously used in the field of olfaction. Before starting to approach the identity of the OSN type involved in kin recognition, we validated pERK as a reliable marker for neuronal activation in the larval zebrafish after odor exposure. To this aim, we stimulated group raised larvae at the 9th day of development with different odors and analyzed neuronal activation visualized by pERK immunopositivity in the larval zebrafish OE. With the use of accepted morphological criteria, we identified the four differenttypes of OSNs of the zebrafish OE. Additionally, we used the CBP S100 to mark specifically crypt cells. For the first time in larval zebrafish, we performed a timescale experiment to test best odor exposure duration with the result that detectable pERK levels are recognizable already after 3 minutes of odor exposure. However, prolonging the exposure duration does not lead to better pERK signals in OSNs. Furthermore, we demonstrate that olfactory stimulation with food and non-kin odor (conspecific odor) clearly results in a differential activation pattern of OSNs. Consistent with other studies, we identify activated ciliated and microvillous OSNs after exposure to food odor, whereas only microvillous cells show responses to conspecific odor. Crypt cells show activation neither to food odor nor to non-kin odor. Upon validation of pERK to mark neuronal activation in the larval zebrafish OE, we stimulated imprinted and non-imprinted 9 day old larvae with kin odor and analyzed activated OSNs. In two rounds of stimulation experiments, each with slightly different raising conditions, we provide the first direct evidence for crypt cells as well as a small subpopulation of microvillous cells to be involved in detection of a kin odor related signal. Interestingly, only larvae which were successfully imprinted show activated crypt cells in response to kin odor, whereas crypt cells of non-imprinted larvae show no increase in pERK levels. A difference in crypt cell number does not account for this difference in activation pattern as a comparison of crypt cell quantity reveals no significant difference between imprinted and non-imprinted larvae. Furthermore, we analyzed neuronal activation of bulbar neurons after exposure to kin odor. Consistent with our results on activation at the level of the larval OE, bulbar neurons of imprinted larvae show increased neuronal activation compared to non-imprinted larvae especially around the mediodorsal glomerulus that receives crypt cell input (mdG2) after kin odor exposure. The final aim of this study is to identify the existence of an accessory olfactory pathway in teleosts. In tetrapods, vomeronasal information is mainly transferred from the VNO to the accessory olfactory bulb (AOB) and from there to the medial amygdala. The medial amygdala is a part of the subpallium and initiates via amygdalo-hypothalamic pathways behavioral and also hormonal responses to incoming signals. Moreover, besides an involvement in fear and associative learning, the medial amygdala is also known to be involved in processing of conspecific odors in rodents. Although a separate VNO is absent in teleosts, we newly identify an accessory olfactory pathway in the zebrafish. By injection of DiI tracer into the mediodorsal OB, which is the target region of crypt cells and some microvillous OSNs, we demonstrate a neuronal circuit running from the mediodorsal OB to the medial amygdala and from there to the tuberal hypothalamus. Interestingly, non-imprinted zebrafish larvae show increased activity of neurons in the medial amygdala compared to imprinted larvae. Finally, we demonstrate for the first time the OSN type which is involved in the detection as well as processing targets of a kin odor related signal in larval zebrafish.