The neurotransmitter dopamine has an important role in the vertebrate brain, modulating behaviours that include locomotion, aggression, feeding and reward. Dysregulation of dopamine signalling is also linked several human diseases including Parkinson’s disease, ADHD and schizophrenia. Furthermore, mutations in the dopamine transporter, the protein that reuptakes dopamine in the synapse causes dopamine transporter deficiency syndrome (DTDS), an early-onset form of Parkinson’s disease in children. Our laboratory has recently established fast-scan cyclic voltammetry (FSCV) in adult zebrafish, a technique that permits the release- and reuptake of neurotransmitters at the synapse to be measured. In this project we will establish a protocol to perform FSCV in the intact larval zebrafish brain and couple this to virtual reality simulation of behaviour. These experiments will permit us to investigate neurotransmitter release in awake freely behaving animals.
Research in our laboratory investigates the genetic, neurological and pharmacological basis of zebrafish behaviour. Zebrafish are an ideal model for behavioural neuroscience since their genes and neurotransmitter signalling pathways are highly conserved with other vertebrates1. We combine behavioural measurements with techniques to examine the brain such as high precision liquid chromatography (HPLC –basal levels of neurotransmitters), fast-scan cyclic voltammetry (FSCV – release of neurotransmitters), in situ hybridisation (ISH – localisation of gene expression) and immunohistochemistry. For example, our laboratory has shown that fibroblast growth factor receptor 1a mutants exhibit increased aggression and a malformation of the hypothalamus demonstrating the power of this model to connect behaviour and neurobiology2.
In a recent project, we used FSCV to show that electrical stimulation of the adult telencephalon triggers the release of dopamine and histamine coupled to a change in pH3. These experiments, the first demonstration of FSCV in zebrafish, were performed on sagittal sections of the brain cultured in artificial cerebrospinal fluid. In this project we will now develop a setup to measure neurotransmitter release in awake behaving animals. Importantly, we have access to several zebrafish lines that will enable us to complete this research: - Tg(VMAT2:GFP) transgenic fish that label monoaminergic neurons in the brain with GFP. - slc6a4a/dat mutant fish that lack dopamine transporter function in the brain and so are hyperdopaminergic.
We will investigate 3 objectives: 1) Establish an FSCV protocol to measure dopamine release in the intact larval (6 day-old) forebrain. We will use Tg(VMAT2:GFP) to accurately target DA neurons in the intact larval brain.
2) Build an optic flow setup to stimulate behaviour in larval fish. The setup will use a flat screen computer to play films to larvae. Fish will be restrained in agarose using a preparation that permits the eyes and tail to move freely. Films played on the screen will stimulate Behaviour: for example, a looming shadow will elicit an escape response, whereas small bite-sized stimuli will stimulate hunting and feeding.
3) Measure the release of neurotransmitters in real-time by combining FSCV with optic flow. If dopamine release or reuptake correlates with the behavioural reaction to optical stimulation, we will then manipulate dopaminergic signalling. We will either genetically alter dopamine levels using the slc6a4a/dat mutant line that has heightened dopamine levels in the brain, or use drugs that selectively target dopaminergic receptors.
In summary, this project combines state-of-the-art techniques to investigate the contribution of dopaminergic signalling to behaviour by measuring the release of neurotransmitters in the intact vertebrate brain. It also has the potential to improve our understanding of DTDS, with a view to developing novel drug treatments for this disease.