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Dopaminergic neurons and the reward system in fish: a functional neuroanatomical multidisciplinary study

Periodic Reporting for period 1 - FISHDOPA (Dopaminergic neurons and the reward system in fish: a functional neuroanatomical multidisciplinary study)

Reporting period: 2016-03-01 to 2018-02-28

The current project aims at elucidating the role of the dopaminergic (DA) system in regulation and control of reward systems in fish. DA has been known to regulate reward, as well as other vital neurological functions, but the underlying molecular mechanisms are still not fully understood. The DA system has also been associated with several neuropsychological illnesses and therefore, the study of DA regulation is of great importance. In mammals, DA neurons originate in the midbrain and reward stimuli activation and processing has been linked to activation of the midbrain DA neural populations, which in turn activate forebrain networks, such as the amygdala, hippocampus and prefrontal cortex. In this way, the DA system helps animals react, process and integrate reward stimuli with internal physiological cues in order to display an adaptive behavioral response. In this context, recent studies point to the DA system as also the main signaling center for reward in fish. However, a thorough functional characterization of DA neurons to reward stimuli in fishes is still lacking.
The zebrafish (Danio rerio) as a model species possess several advantages, such as, a sequenced and annotated genome, a comparatively small and easy to map nervous system (particularly in larvae), ease of maintenance, a substantial use by the scientific community and that a great deal of its biology has already been studied. Notably, the transparent zebrafish larvae have proven to be an ideal candidate for the study of large neuronal networks and populations in vivo by the use of novel imaging technologies, such as calcium imaging and optogenetics. Therefore, we have used the zebrafish model to study the function of DA neurons in reward processing. That is, we wished to establish which forebrain DA neuronal populations, brain targets and signaling molecules are active during reward conditions, as well as establish a causal relationship between these areas and the control of reward-motivated behavior in fish.
We started by developing a behavioral protocol to study reward in adult zebrafish and larvae. We then subjected adult fish to reward and sampled them consecutively in order to study brain activity and signaling molecules regulating reward behavior throughout reward learning. We found that there was a consistent activation of DA neuronal populations in two forebrain areas, called the dorsal (Vd) and the ventral (Vv) part of the ventral telencephalon. Following these results, extraction of these areas was done by means of laser microdissections and samples were sequenced in order to discover which signaling molecules were being regulated before and after reward learning. Results from sequencing showed that while the Vd shows an upregulation of genes associated with dopamine signaling before reward learning, the Vv shows upregulation of genes associated with learning after reward learning. This implies that the Vd is important for reward processing and the Vv for reward learning. Furthermore, we are using our new behavioral reward paradigm for fish larvae in order to conduct optogenetic studies. That is, we are currently conducting experiments in which genetically modified fish larvae are exposed to a light wavelength via a laser in order to activate or inhibit the activity of specific neurons. In this case, we are activating and inhibiting DA neurons during reward learning in order to confirm the importance of DA activity to process and learn about reward. These results have and will be presented at international scientific conferences. Furthermore, once all data has been collected and analyzed, it will be submitted for publication as one manuscript encompassing all aspects of the project (in order to publish in an open access high impact factor journal). Once publication is achieved, we will actively contact journalists in order to publish popular science articles in different news outlets (e.g. newspapers, magazines, popular science websites, social media) in order to reach the general public. In addition, part of these results were presented as part of an interactive activity for school children of ages between 10-12 at the Annual Science Festival of Gothenburg.
The results obtained from this project are very important since, the dysregulation of the DA system is associated with several psychiatric and neurological diseases, such as Parkinson’s, Alzheimer’s, Huntington’s, schizophrenia and restless leg syndrome and with the pathophysiology of stress-related illnesses, such as depression. In addition, DA has a pivotal role in addiction. In this context, the zebrafish has in recent years gained popularity as a model for the study of central nervous system function, particularly in the study of psychiatric and neurological diseases, including those associated with DA systems. However, as mentioned above, even though several studies have linked the DA system to have the same function in fish as in mammals, there is a lack of functional studies that have concentrated on the normal function (i.e. in healthy functioning animals) of DA in fish. For example, even though several studies have mapped the location of different DA neuronal populations, these studies have not included functional analysis of DA neurons to reward and have therefore only speculated on which populations may be important reward centers in the zebrafish brain. Notably, a recent study pinpoints to forebrain DA populations being more important than midbrain populations in the processing of reward stimuli in fish and this is in agreement with our results. That is, we mapped activity of specific DA neuronal populations to reward and found that forebrain DA neurons of the Vd and Vv are important centers for reward processing. This is the first time that this has been achieved and importantly, it helps validate the use of fish models for the study of DA related illnesses, since it confirms that DA neuronal networks work in the same way in fish as it does in humans. These results will aid in the understanding of neural mechanisms and development of treatments for neurological diseases associated with DA function. With the execution of this project the fellow learned valuable skills in visual imaging techniques, optogenetics, microscopy, fish husbandry, RNA sequencing and laser capture microdissections, which was used in the study of functional neuroanatomy in fish, an area in which the fellow wishes to specialize in. In addition, this experience gave the fellow the possibility of running a project in an independent manner but with the possibility of having a senior colleague at hand when required. All these skills have helped the fellow mature in his professional academic career. Thanks to the skills and execution of the current project the fellow has been able to procure financing for the execution of another functional neuroanatomy project and is in the process of applying for financing of several other projects in which the skills learned during the FISHDOPA project will be utilized. Importantly, the innovation activities carried out during this project include the development of new behavioral assays that will benefit the scientific community.