Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30–80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.

The field of optogenetics uses channelrhodopsins (ChRs) for light-induced neuronal activation. However, optimized tools for cellular inhibition at moderate light levels are lacking. We found that replacement of E90 in the central gate of ChR with positively charged residues produces chloride-conducting ChRs (ChloCs) with only negligible cation conductance. Molecular dynamics modeling unveiled that a high-affinity Cl(-)-binding site had been generated near the gate. Stabilizing the open state dramatically increased the operational light sensitivity of expressing cells (slow ChloC). In CA1 pyramidal cells, ChloCs completely inhibited action potentials triggered by depolarizing current injections or synaptic stimulation. Thus, by inverting the charge of the selectivity filter, we have created a class of directly light-gated anion channels that can be used to block neuronal output in a fully reversible fashion.

Channelrhodopsins (ChRs) are directly light-gated ion channels that function as sensory photoreceptors in flagellated green algae, allowing these algae to identify optimal light conditions for growth. In neuroscience, ChRs constitute the most versatile tools for the light-induced activation of selected cells or cell types with unprecedented precision in time and space. In recent years, many ChR variants have been discovered or engineered, and countless electrical and spectroscopic studies of these ChRs have been carried out, both in host cells and on purified recombinant proteins. With significant support from a high-resolution 3D structure and from molecular dynamics calculations, scientists are now able to develop models that conclusively explain ChR activation and ion conductance on the basis of chromophore isomerization, structural changes, proton transfer reactions, and water rearrangement on timescales ranging from femtoseconds to minutes.

Background: Dual-color activation of two cell types with channelrhodopsins is a major challenge because all available well expressing variants absorb blue light. Results: We engineered channelrhodopsin hybrids with color-shifted spectra, as well as altered kinetics and selectivity. Conclusion:The results provide deeper insight into channelrhodopsin function. Significance: The combination of novel and established channelrhodopsins can activate distinct cell populations by dual-color excitation.

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.