Final Report Summary - FSES (Fast and slow endocytosis at the synapse) Sensory systems continually adjust their sensitivity to encode stimuli under a wide variety of natural conditions. For visual system, this process starts in the retina where neurons adapt through different mechanisms including adaptation to changes in visual scene contrast and ambient light level. In the retina, bipolar cells (BCs) represent the sole direct excitatory connection between photoreceptors and ganglion cells. With their axons ramifying at different layers of the inner plexiform layer (IPL), BCs receive reciprocal and nonreciprocal synapses from the inhibitory interneuron amacrine (ACs). Reciprocal synapses take place when BCs receive inputs from ACs activated directly by the same BC {Dowling and Boycott 1996}. In this synapse, diverse neuromodulatory inputs shape the visual signal within milliseconds of precision and modulate the output of ganglion cell to downstream the brain {Euler 2000}. At nonreciprocal synapses, BCs receive inputs from ACs generated from the opposite visual stream: amacrine cells carry ON inhibition to the OFF cells and carry OFF inhibition to the ON cells {Molnar 2007}. This mechanism is known as “crossover inhibition” and helps the retina to compensate for nonlinearities generated by rectifying synaptic currents. Recent works indicate that crossover inhibition is predominantly glycinergic and occurs mostly from ON to OFF system. Crossover inhibition is the dominant form of inhibition in almost all OFF bipolar cell, but was found to dominate in less than half of ON bipolar cell {Roska 2006; Molnar 2007; Cafaro 2013}.On top of the fast GABAergic and glycinergic inhibitory synapses from ACs, BCs receive extensive synaptic feedback from neuropeptides such as dopamine, somatostatin and substance P {Eggers 2011}. Substance P released by amacrine cells upon light stimulation is the preferred ligand of a Gi/o-protein coupled receptor (GPCR), named NK1 (NK1-R) {Casini 2005}. Functionally, substance P has been reported to reduce voltage-dependent calcium currents in bipolar cells {Ayoub 1992}, which may modulate the release of neurotransmitters from these cells altering their excitability and shaping the responses to the visual inputs. The vast and rich synaptic connections within the IPL represent a place of signal integration shaping the BC output to ganglion cells; however little is know about the specific role of neuropeptides mediating Ca2+ signalling within the microcircuits of the IPL. The project I carried on during my Marie Curie Fellowship aimed to understand the potential impact of the neuromodulatory regulation at the level of bipolar terminals. For this purpose, the majority of the experiments were performed using transgenic zebrafish (Danio Renio) expressing either ribeye::SyGCaMP2 (Dreosti et al., 2009) for in vivo imaging of presynaptic calcium signals across the population of bipolar cells projecting to all layers of the IPL or eno2::GCaMP3 to record calcium signals across ganglion cell population (Nikolaev 2013). Using pharmacological manipulations (intravitreous injection) we observed that disinhibition of calcium signalling at level of bipolar terminals by a specific NK1-R blocker (L-077,60) increases response to visual stimulus such as luminance and frequency. The enhancement of the synaptic calcium responses was clear across the ON bipolar terminals, but not through the OFF pathway. Additionally, calcium disinhibition increased the spike rate by 4.3-fold in ON terminals. The increase in the spike rate is expected since substance P (the endogenous agonist of NK1-R) decreases the gating of voltage-dependent calcium channels, changing in about 5 mV the threshold for regenerative responses. Likely, an increase in the spike rate would amplify visual signal at bipolar terminals and together with graded signal would increase the sensitivity to luminance and frequency. The specific effects at ON pathway support the presence of NK1-R at ON bipolar cells {Casini 2002; Catalani 2006}. On the other hand, OFF terminals presented a faster and transient response to light offset and a shift in the frequency preference towards high frequency, with no changes in the spike rate. Luminance and frequency modulations were eliminated when an agonist of mGluR6 receptors L-AP4 was used to inhibit depolarization of ON terminals at light on. This finding suggests that OFF terminals are being modulated by the increased excitability at ON pathway after the inhibition of NK1-R. Overall, we show that changes in the intrinsic properties of bipolar terminals through disinhibition of calcium signalling can alter the sensitivity of the inner retina via (at least) two cellular mechanisms: increase in the spike rate at ON bipolar terminals and modulation of the OFF pathway through a crossover inhibition.
