MRC Doctoral Training Partnership: Using zebrafish to understand how genetic defects affect neuronal circuits

The overall goal of this PhD project is to develop an experimental paradigm for investigating differences in the development and function of neuronal circuits in health and disease, using a novel zebrafish model of epilepsy as an exemplar.

Molecular mechanisms of neurodevelopmental disorders such as epilepsy are extensively studied. Much less is known about how these neurological conditions affect neuronal circuit structure and function. To begin to address this question, it is necessary to compare the activities of large numbers of neurons (ideally the entire brain) in healthy and diseased brains. In the proposed project, we will use deep learning and automatic clustering on large neuronal activity datasets from zebrafish larvae expressing the neuronal activity reporter transgene NBT:GCaMP3 (Bergmann et al., 2018), to establish such a methodological approach.

First, the PhD student will establish research protocols involving light-sheet microscope imaging to detect neuronal activity in immobilized NBT:GCaMP3 transgenic zebrafish larvae exposed to visual, auditory or olfactory stimuli. These stimuli will include distinct colours and patterns of light, sounds of distinct frequency and amplitude, and a variety of potent water-soluble attractive and aversive chemicals. The student will then employ an unsupervised clustering approach to define stereotypic populations of neurons responsible for processing of different forms of sensory stimuli and map them on a template brain (Year 1).

Once developed, this approach will then be used to determine how genes implicated in neurodevelopmental disorders, such as as epilepsy, affect the neuronal circuits. Epilepsy, in particular infantile epileptic encephalopathy, is often associated with mutations in a range of genes including syntaxin-binding protein 1 (STXBP1). The role of STXBP1 in development of neuronal circuits is not well understood. We have created novel loss-of-function mutations in the zebrafish orthologues of STXBP1, stxbp1a and stxbp1b. We and others (Grone et al., 2016) have observed that such stxbp1a mutants are homozygous lethal, whereas stxbp1b mutants are homozygous viable. Our unpublished studies further indicate that stxbp1b mutants exhibit heightened sensitivity to sensory stimuli compared to wild-type larvae, which suggests that this mutant may be a tractable model for elucidating the molecular and cellular basis of epilepsy due to loss of STXBP1 function.
Using this experimental approach, the responses of neurons in the brains of wild-type and stxbp1b mutant zebrafish that also express the NBT:GCaMP3 transgene, will be characterised. The student will test whether the stxbp1b mutation selectively affects the activity of specific subpopulations of neurons with distinct activation profiles in response to the sensory stimuli described above. Thus, the neuronal ensembles whose activities are most clearly dependent on stxbp1b function will be identified and located on a template brain. We will then use fluorescent whole-mount in situ hybridisation and light-sheet imaging to generate high resolution, three-dimensional images of the expression domains of genes encoding receptors for neurotransmitters and neuromodulators such as glutamate, GABA, glycine, catecholamines, and acetylcholine. These maps will then be computationally aligned with the stxbp1b¬-dependent activity map generated this project, in order to determine whether the stimulus-sensitive stxbp1b¬¬-dependent neuronal ensembles exhibit specific neurotransmitter sensitivities.