Determining the role of activity-dependent bulk endocytosis in brain function via VAMP4

Efficient and accurate synaptic communication is essential for the maintenance of normal brain function and neuronal health across the human lifespan. Synaptic function is critically dependent on efficient release of neurotransmitter during action potential stimulation at central nerve terminals and the subsequent reformation of synaptic vesicles (SVs) by endocytosis. SVs are retrieved by a series of different endocytosis modes which are triggered by specific patterns of stimuli. During intense neuronal activity, the dominant SV endocytosis mode is activity-dependent bulk endocytosis (ADBE). ADBE is exclusively triggered during high frequency stimulation and forms endosomes (called bulk endosomes) direct from the plasma membrane from which SVs subsequently bud. Since ADBE is the dominant mode of SV generation during intense activity, it should contribute towards key biological events such as learning and memory and potentially pathological events such as epileptic seizure.

Decoding the role of ADBE in brain function has been hampered by a lack of detailed molecular information, since all currently identified ADBE molecules have multiple functions at the synapse. Our recent discovery that VAMP4 is a unique ADBE molecule has provided an exciting opportunity to determine its physiological role, since now the function of ADBE can be examined in isolation from all other synaptic events for the first time.

This project will determine the role of ADBE at the synapse, circuit and behavioural level by taking advantage of a conditional VAMP4 knockout mouse which is currently being generated. In doing so it will provide training in cell / molecular Neuroscience, electrophysiology, in vivo skills, mouse colony maintenance and behavioural tasks.

Synaptic role – VAMP4 is essential for ADBE, however the molecular interactions that mediate its role are still undetermined. VAMP4 interaction partners will be identified using a series of standard biochemical techniques such as recombinant protein expression, GST-pull-downs and Western blotting. VAMP4 mutants that do not interact with identified partners will be generated via standard molecular biology techniques and expressed in primary cultures of VAMP4 knockout neurones. The role of these interactions in ADBE will be assessed using fluorescent genetically-encoded reporters and wide-field microscopy.

Circuit role – The role of ADBE in synaptic transmission will be determined using acute brain slices from VAMP4 knockout mice and monitoring a series of key parameters via patch clamp electrophysiology. Circuit activity will be monitored using multi-electrode arrays. To determine how VAMP4 interactions control synaptic transmission, modified adeno-associated virus expressing VAMP4 mutants will be delivered into knockout mice via stereotaxic injection.

Behavioural role – Learning and memory are dependent on high frequency firing, suggesting ADBE will be critical for these events. To test this, VAMP4 knockout mice will perform learning / behaviour tasks to test spatial learning and memory, context-dependent fear conditioning and spontaneous object exploration that assess spatial and episodic memory.

This project will therefore provide a multi-disciplinary training in a series of state of the art technologies by exploiting a unique ADBE-deficient mouse model.