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Final Activity Report Summary - NEURAL CIRCUIT (Combining genetic, physiological and viral tracing methods to understand the structure and function of neural circuits)

Our scientific goal was to combine different disciplines including mouse genetics, viral tracing, molecular biology, electrophysiology and two-photon imaging to understand the structure and function of local circuits in the mammalian retina and to use this knowledge to restore visual function in retinal degeneration. Firstly, we identified an approach-sensitive ganglion cell type in the mouse retina (Nature Neuroscience, 2009a), resolved elements of its afferent neural circuit, and described how these confer approach sensitivity on the ganglion cell. The key point was that a neuronal type can perform very different computations depending on the state of the circuit. Another circuit we investigated was that of the intrinsically photosensitive, melanopsin-containing retinal ganglion cells (ipRGCs), which control important physiological processes including the circadian rhythm, pupillary reflex and suppression of locomotor behaviour.

With the use of transsynaptic pseudorabies viruses (PRVs), we traced the local circuit of different ipRGC subtypes in the mouse retina (Current Biology, 2007). Our discovery that dopaminergic amacrine cells are synaptically connected to ipRGCs provided a circuitry link between the control of light-dark circuit-adaptation and ipRGC function. We then investigated how circuit computations develop in the retina. We followed the spatial distribution of synaptic strengths between starburst and directional selective cells during early postnatal development (Nature, accepted for publication, 2010). We showed that asymmetry develops rapidly over a 2-day period through an intermediate state in which random or symmetric synaptic connections have been established. The development of asymmetry involves the spatially selective reorganization of inhibitory synaptic inputs.

Our work demonstrated a rapid developmental switch from a symmetric to asymmetric input distribution for inhibition in the neural circuit of a principal cell. Secondly, in order to restore visual function in animal models of retinitis pigmentosa we expressed light-sensitive genes in identified retinal cell types. We showed that expression of archaebacterial halorhodopsin in light-insensitive cones can substitute for the native phototransduction cascade and restore their light sensitivity in mouse models of retinitis pigmentosa (Science, 2010). Resensitized photoreceptors activate all retinal cone pathways, drive sophisticated retinal circuit functions including directional selectivity, activate cortical circuits, and mediate visually guided behaviours. Using human ex vivo retinas, we showed that halorhodopsin can reactivate light-insensitive human photoreceptors.

As another approach, we genetically targeted a light-activated cation channel, channelrhodopsin-2, to second-order neurons, ON bipolar cells (Nature Neuroscience, 2008). In the absence of 'classical' photoreceptors, we found that ON bipolar cells that were engineered to be photosensitive induced light-evoked spiking activity in ganglion cells. The rescue of light sensitivity was selective to the ON circuits that would naturally respond to increases in brightness. Thirdly, our unbiased large-scale screen for GFP-labelled retinal cell types resulted in finding ~100 mouse lines with cell type- or retinal stratum-specific GFP expression, bright enough to perform two-photon laser targeted electrophysiological recordings. As a result (Nature Neuroscience, 2009b), we can now specifically target different cell types for recordings. We also described a set of PRV viruses (Nature Methods, 2009) with different molecular tools, which are helpful for tracing circuits across many synapses. Finally, we studied the changes in gene expression in identified cell types during light and dark adaptation, and analysed the functional consequences of such changes. We have identified microRNAs regulated by different light levels in the mouse retina, independent of the circadian time (Cell, 2010).

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