FSES

Fast and slow endocytosis at the synapse

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 Obiettivo del progetto (Objective)

'The mechanism of neurotransmitter release is a fundamental aspect of nervous system function, but it is still highly controversial. There is overwhelming evidence that vesicles can release their contents by fully collapsing into the cell surface, after which they are retrieved slowly (10-20 s) by formation of a clathrin cage around the membrane. But it has also been suggested that there is a second mode of exocytosis in which vesicular content is released through a transitory fusion pore and then quickly retrieved (“kiss-and-run”). Several lines of evidence indicate that a fast mechanism of endocytosis (~1 s) does indeed operate at some synapses, but the idea that the vesicle does not merge with the surface membane remains controversial. We will investigate synaptic vesicle endocytosis in two types of retinal synapse (conventional and ribbon-type), using genetically modified zebrafish expressing fluorescent fusion proteins that report synaptic vesicle fusion and retrieval. Neurons will be isolated and total internal reflection fluorescence microscopy (TIRFM) used to monitor the synaptic vesicle cycle in real-time at the level of individual vesicles. A key aim of this project will be to establish the role of fast and slow endocytosis during both spontaneous vesicle fusion and ongoing synaptic activity and identify proteins recruited to the membrane during these processes (including clathrin, dynamin and endophilin).'

Descrizione progetto (Article)

Sensory systems continually adjust their sensitivity to transmit stimuli under a wide variety of natural conditions. For the visual system, this process starts in the retina where neurons adapt to changes in visual scene contrast and ambient light level.

The bipolar cells (BCs) represent the sole direct excitatory connection between photoreceptors and ganglion cells. However, specific role of neuropeptides mediating calcium signalling within the BC microcircuits is still poorly understood. The EU-funded 'Fast and slow endocytosis at the synapse' (FSES) Marie Curie Fellowship aimed to study the impact of the neuromodulatory regulation at the level of bipolar terminals (BPs) of BCs.

Most of the experiments were performed using transgenic zebrafish. Through pharmacological manipulations, researchers observed that disinhibition of calcium signalling at BPs increases response to visual stimulus such as luminance and frequency. The enhancement of the synaptic calcium responses was clear across the activation BPs, but not through the inhibiting pathway. An increase in the spike rate could amplify visual signal at BPs and increase the sensitivity to luminance and frequency.

Interestingly, inhibiting terminals presented a faster and transient response to light offset and a shift in the frequency with no changes in the spike rate. Luminance and frequency modulations were eliminated when pharmacological manipulations inhibited depolarisation of activating terminals at light on. This finding suggests that inhibiting terminals are being modulated by the increased excitability at the activating pathway.

Overall, the project demonstrated that changes in the intrinsic properties of BPs through disinhibition of calcium signalling can alter the sensitivity of the inner retina.