We investigated the efficacy of optogenetic inhibition at presynaptic terminals using halorhodopsin, archaerhodopsin and chloride-conducting channelrhodopsins. Precisely timed activation of both archaerhodopsin and halorhodpsin at presynaptic terminals attenuated evoked release. However, sustained archaerhodopsin activation was paradoxically associated with increased spontaneous release. Activation of chloride-conducting channelrhodopsins triggered neurotransmitter release upon light onset. Our results indicate that the biophysical properties of presynaptic terminals dictate unique boundary conditions for optogenetic manipulation.

Reversible silencing of neuronal activity is a powerful approach for isolating the roles of specific neuronal populations in circuit dynamics and behavior. In contrast with neuronal excitation, for which the majority of studies have used a limited number of optogenetic and chemogenetic tools, the number of genetically encoded tools used for inhibition of neuronal activity has vastly expanded. Silencing strategies vary widely in their mechanism of action and in their spatial and temporal scales. Although such manipulations are commonly applied, the design and interpretation of neuronal silencing experiments present unique challenges, both technically and conceptually. Here, we review the most commonly used tools for silencing neuronal activity and provide an in-depth analysis of their mechanism of action and utility for particular experimental applications. We further discuss the considerations that need to be given to experimental design, analysis, and interpretation of collected data. Finally, we discuss future directions for the development of new silencing approaches in neuroscience.

SummarySensory information is encoded within the brain in distributed spatiotemporal patterns of neuronal activity. Understanding how these patterns influence behavior requires a method to measure and to bidirectionally perturb with high spatial resolution the activity of the multiple neuronal cell types engaged in sensory processing. Here, we combined two-photon holography to stimulate neurons expressing blue light-sensitive opsins (ChR2 and GtACR2) with two-photon imaging of the red-shifted indicator jRCaMP1a in the mouse neocortex in vivo. We demonstrate efficient control of neural excitability across cell types and layers with holographic stimulation and improved spatial resolution by opsin somatic targeting. Moreover, we performed simultaneous two-photon imaging of jRCaMP1a and bidirectional two-photon manipulation of cellular activity with negligible effect of the imaging beam on opsin excitation. This all-optical approach represents a powerful tool to causally dissect how activity patterns in specified ensembles of neurons determine brain function and animal behavior.

Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. However, the limitations of inhibitory optogenetic tools impose stringent constraints on experimental paradigms. The high light power requirement of light-driven ion pumps and their effects on intracellular ion homeostasis pose unique challenges, particularly in experiments that demand inhibition of a widespread neuronal population in vivo. Guillardia theta anion-conducting channelrhodopsins (GtACRs) are promising in this regard, due to their high single-channel conductance and favorable photon-ion stoichiometry. However, GtACRs show poor membrane targeting in mammalian cells, and the activity of such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential in this compartment. Here, we address these problems by enhancing membrane targeting and subcellular compartmentalization of GtACRs. The resulting soma-targeted GtACRs show improved photocurrents, reduced axonal excitation and high light sensitivity, allowing highly efficient inhibition of neuronal activity in the mammalian brain.

Social encounters are associated with varying degrees of emotional arousal and stress. The mechanisms underlying adequate socioemotional balance are unknown. The medial amygdala (MeA) is a brain region associated with social behavior in mice. Corticotropin-releasing factor receptor type-2 (CRF-R2) and its specific ligand urocortin-3 (Ucn3), known components of the behavioral stress response system, are highly expressed in the MeA. Here we show that mice deficient in CRF-R2 or Ucn3 exhibit abnormally low preference for novel conspecifics. MeA-specific knockdown of Crfr2 (Crhr2) in adulthood recapitulated this phenotype. In contrast, pharmacological activation of MeA CRF-R2 or optogenetic activation of MeA Ucn3 neurons increased preference for novel mice. Furthermore, chemogenetic inhibition of MeA Ucn3 neurons elicited pro-social behavior in freely behaving groups of mice without affecting their hierarchal structure. These findings collectively suggest that the MeA Ucn3-CRF-R2 system modulates the ability of mice to cope with social challenges.

24Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. 25However, the limitations of inhibitory optogenetic tools impose stringent constraints on experimental 26 paradigms. The high light power requirement of light-driven ion pumps and their effects on intracellular 27 ion homeostasis pose unique challenges, particularly in experiments that demand inhibition of a 28 widespread neuronal population in vivo. Guillardia theta anion-conducting channelrhodopsins (GtACRs) 29 are promising in this regard, due to their high single-channel conductance and favorable photon-ion 30 stoichiometry. However, GtACRs show poor membrane targeting in mammalian cells, and the activity of 31 such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential 32 in this compartment. Here we address both problems by enhancing membrane targeting and subcellular 33 compartmentalization of GtACRs. The resulting GtACR-based optogenetic tools show improved 34 photocurrents, greatly reduced axonal excitation, high light sensitivity and rapid kinetics, allowing highly 35 efficient inhibition of neuronal activity in the mammalian brain. 36 37 amplitudes of up to 90% within a minute of illumination, leading to reduced silencing efficacy over time 54 12,8,13 . Because of their insensitivity to electrochemical gradients, ion-pumping microbial rhodopsins can 55 shift the concentrations of intracellular ions to non-physiological levels. In the case of halorhodopsin, 56 this can lead to accumulation of chloride in the neuron, inducing changes in the reversal potential of 57 GABAergic synapses 14 . While in the case of archaerhodopsin this can increase the intracellular pH, 58 inducing action potential-independent Ca 2+ influx and elevated spontaneous vesicle release 13 . 59Furthermore, the hyperpolarization mediated by ion-pumping activity together with the fast off kinetics 60 can lead to an increased firing rate upon termination of the illumination 6,13 . 61Anion-conducting channelrhodopsins (ACRs), a newly established set of optogenetic tools 15,16,17 , are 62 distinct from ion-pumping rhodopsins in two major aspects: first, they can conduct multiple ions during 63 each photoreaction cycle. This increased photocurrent yield per photon makes channelrhodopsins 64 superior in terms of their operational light-sensitivity. Second, conducting ions according to the reversal 65 potential, ACRs are more likely to avoid non-physiological changes in ion concentration gradients. A 66 light-gated chloride conductance will shunt membrane depolarization, which can be used to effectively 67 clamp the neuronal membrane potential to the reversal potential of chloride, given that the ion 68 permeability is sufficiently high. Anion-conducting channelrhodopsins could therefore relieve constrains 69 imposed by ion-pumping rhodopsins. The naturally-occurring anion-conducting channelrhodopsins 70 (nACRs) from the cryptophyte alga Guillardia theta 16 are particularly interesting in this ...

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Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers, and specific targeting sequences to the employed photoreceptorencoding DNA. This powerful approach allows precise characterization and manipulation of cellular