The overall aim of this project is to understand how neuromodulators influence the processing of chemosensory information in the olfactory bulb (OB) and ultimately animal behaviour in response to odours. Specifically, we proposed to determine how chemosensory information influences the activity of terminal nerve (TN) GnRH/NPY neurons and how TN GnRH/NPY neurons affect olfactory processing and odour-driven behaviours in zebrafish.
We have completed most of the work packages and achieved the scientific objectives in understanding the role of terminal nerve GnRH neurons in chemosensory processing and behaviour. However, through the project, it was difficult to generate NPY transgenic zebrafish lines. Thanks to our collaborators, we have the NPY transgenic zebrafish. Since all our methods/tools are optimized, we continue to work on achieving the remaining objectives.
For workpackage(WP)-1, we investigated how chemosensory information modulates the activity of terminal nerve GnRH/NPY neurons. To address this, first, we used genetic approach and established transgenic zebrafish line to label the TN GnRH neurons specifically with the genetically encoded calcium indicator. Thanks to our collaborator, we received transgenic line that marks all the NPY neurons in the zebrafish brain. Next, we established the method to image neural activity using two-photon microscopy in brain explant preparation. This allowed us to observe the activity of these neurons in response to natural odours. Finally, we developed the quantitative analytical tools to analyse the neural data. We found that GnRH secreting neurons are spontaneously active. They respond to natural odorants with complex dynamics and have strong preference for food related odours. This odour representation in the GnRH neurons are sexually dimorphic and strongly modulated by physiological states (satiety/hungry) of animals.
In WP-2, we had hypothesised that GnRH/NPY neurons modulate odour representation in the olfactory bulb. To test this hypothesis, we perturbed (downregulate/upregulate) the basal activity of GnRH/NPY neurons. This required establishing transgenic lines that label these neurons with optogenetic transgenes, halorhodopsin and channel rhodopsin, and chemogenetic gene, nitroreductase. Optogentic tools allowed temporal control over modulation of neural activity and chemogentic tool provided a means to study the acute effect of this modulation. We successfully down regulated or silenced the GnRH neuron activity using these methods and imaged neural activity in the olfactory bulb for any change in olfactory processing. Our initial findings suggested that modulation by GnRH neuron in olfactory bulb is quite subtle. Moreover, these neurons project to multiple brain regions including dorsomedial telencephalon (amygdala), dorsolateral telencephalon (hippocampus), ventral telencephalon, habenula and hypothalamus. Hence, we expanded the scope of our work to observe neural activity in these regions and modified our analytical tools for analysis of the new data set. Initial findings suggest that changes in odour representation likely involve network involved in aversion, such as habenula and amygdala. Moreover, we have established the framework to modulate the activity of NPY neuron and observe change in odour representation in the brain.
In WP-3, we investigated the impact of modulation of activity GnRH/NPY neuron on olfactory perception and odour driven feeding and reproductive behaviours. First, we established transgenic tools to modulate the activity of GnRH/NPY neurons in the olfactory bulb of freely behaving animals. Next, we developed and refined the behavioural assays to measure feeding rate, courtship intensity, reproductive success and olfactory behaviour assay. Finally, we optimized the quantitative analysis tools and parameters and analysed the behavioural data. Previously, hypothalamic GnRH has been shown to promote reproductive behaviour and suppress feeding behaviour. However, we found that TN GnRH neurons are essential for both feeding and reproduction. Thus, we found differential role of hypothalamic and terminal nerve GnRH in feeding behaviour.
As described in the proposal, we have presented our findings in seminars at our institute and at several conferences/meetings and venues(The 10th European Zebrafish Meeting 2017; The 11th FENS forum of Neurosciences 2018; The European Chemoreception Research Organization 2018; The XXXVI Annual conference of Indian Academy of Neurosciences 2018.). We are now preparing our first manuscript on the modulation of chemosensory representation in the zebrafish brain by GnRH neurons. It will be submitted to high quality peer-reviewed international scientific journal.
