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.

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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The medial prefrontal cortex (mPFC) mediates a variety of complex cognitive functions via its vast and diverse connections with cortical and subcortical structures. Understanding the patterns of synaptic connectivity that comprise the mPFC local network is crucial for deciphering how this circuit processes information and relays it to downstream structures. To elucidate the synaptic organization of the mPFC, we developed a high-throughput optogenetic method for mapping large-scale functional synaptic connectivity. We show that mPFC neurons that project to the basolateral amygdala display unique spatial patterns of local-circuit synaptic connectivity within the mPFC, which distinguish them from the general mPFC cell population. Moreover, the intrinsic properties of the postsynaptic mPFC cell and anatomical position of both cells jointly account for ~7.5% of the variation in probability of connection between mPFC neurons, with anatomical distance and laminar position explaining most of this fraction in variation. Our findings demonstrate a functional segregation of mPFC excitatory neuron subnetworks, and reveal the factors determining connectivity in the mPFC.

Retinal direction-selectivity originates in starburst amacrine cells (SACs), which display a centrifugal preference, responding with greater depolarization to a stimulus expanding from soma to dendrites than to a collapsing stimulus. Various mechanisms were hypothesized to underlie SAC centrifugal preference, but dissociating them is experimentally challenging and the mechanisms remain debatable. To address this issue, we developed the Retinal Stimulation Modeling Environment (RSME), a multifaceted data-driven retinal model that encompasses detailed neuronal morphology and biophysical properties, retina-tailored connectivity scheme and visual input. Using a genetic algorithm, we demonstrated that spatiotemporally diverse excitatory inputs–sustained in the proximal and transient in the distal processes–are sufficient to generate experimentally validated centrifugal preference in a single SAC. Reversing these input kinetics did not produce any centrifugal-preferring SAC. We then explored the contribution of SAC-SAC inhibitory connections in establishing the centrifugal preference. SAC inhibitory network enhanced the centrifugal preference, but failed to generate it in its absence. Embedding a direction selective ganglion cell (DSGC) in a SAC network showed that the known SAC-DSGC asymmetric connectivity by itself produces direction selectivity. Still, this selectivity is sharpened in a centrifugal-preferring SAC network. Finally, we use RSME to demonstrate the contribution of SAC-SAC inhibitory connections in mediating direction selectivity and recapitulate recent experimental findings. Thus, using RSME, we obtained a mechanistic understanding of SACs’ centrifugal preference and its contribution to direction selectivity.

The medial prefrontal cortex (mPFC) mediates a variety of complex cognitive functions via its vast and diverse connections with cortical and subcortical structures. Understanding the patterns of synaptic connectivity that comprise the mPFC local network is crucial for deciphering how this circuit processes information and relays it to downstream structures. To elucidate the synaptic organization of the mPFC, we developed a high-throughput optogenetic method for mapping large-scale functional synaptic connectivity in acute brain slices. We show that in male mice, mPFC neurons that project to the basolateral amygdala (BLA) display unique spatial patterns of local-circuit synaptic connectivity, which distinguish them from the general mPFC cell population. When considering synaptic connections between pairs of mPFC neurons, the intrinsic properties of the postsynaptic cell and the anatomical positions of both cells jointly account for ~7.5% of the variation in the probability of connection. Moreover, anatomical distance and laminar position explain most of this fraction in variation. Our findings reveal the factors determining connectivity in the mPFC and delineate the architecture of synaptic connections in the BLA-projecting subnetwork.

Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways in cognitive and behavioral processes requires temporally precise manipulation of their activity in vivo. However, existing optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homologue of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro as well as in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents leads to an ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at synaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

Neurons in neocortical layer 1 (L1) are thought to regulate attentional processes through integration of longrange inputs and disinhibitory effects on the underlying cortex. A new study combines genetically targeted voltage imaging and optogenetics to elucidate the input-output transformations of the L1 network in the mouse somatosensory cortex, revealing unique features of sensory-evoked dynamics in L1 neurons.