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.

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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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At high concentrations, glutamate (Glu) exerts potent neurotoxic properties, leading to irreversible brain damages found in numerous neurological disorders. The accepted notion that Glu homeostasis in brain interstitial fluid is maintained primarily through the activity of Glu transporters present on glial cells does not take into account the possible contribution of endothelial cells constituting the blood-brain barrier (BBB) to this process. Here, we present evidence for the presence of the Glu transporters, excitatory amino-acid transporters (EAATs) 1 to 3, in porcine brain endothelial cells (PBECs) and show their participation in Glu uptake into PBECs. Moreover, transport of Glu across three in vitro models of the BBB is investigated for the first time, and evidence for Glu transport across the BBB in both directions is presented. Our results provide evidence that the BBB can function in the efflux mode to selectively remove Glu, via specific transporters, from the abluminal side (brain) into the luminal compartment (blood). Furthermore, we found that glial cells lining the BBB have an active role in the efflux process by taking up Glu and releasing it, through hemichannels, anion channels, and possibly the reversal of its EAATs, in close proximity to ECs, which in turn take up Glu and release it to the blood.

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Adaptation is typically associated with attenuation of the neuronal response during sustained or repetitive sensory stimulation, followed by a gradual recovery of the response to its baseline level thereafter. Here, we examined the process of recovery from sensory adaptation in layer IV cells of the rat barrel cortex using in vivo intracellular recordings. Surprisingly, in approximately one-third of the cells, the response to a test stimulus delivered a few hundred milliseconds after the adapting stimulation was significantly facilitated. Recordings under different holding potentials revealed that the enhanced response was the result of an imbalance between excitation and inhibition, where a faster recovery of excitation compared with inhibition facilitated the response. Hence, our data provide the first mechanistic explanation of sensory facilitation after adaptation and suggest that adaptation increases the sensitivity of cortical neurons to sensory stimulation by altering the balance between excitation and inhibition.

Thalamic inputs of cells in sensory cortices are outnumbered by local connections. Thus, it was suggested that robust sensory response in layer 4 emerges due to synchronized thalamic activity. To investigate the role of both inputs in the generation of correlated cortical activities, we isolated the thalamic excitatory inputs of cortical cells by optogenetically silencing cortical firing. In anaesthetized mice, we measured the correlation between isolated thalamic synaptic inputs of simultaneously patched nearby layer 4 cells of the barrel cortex. Here we report that in contrast to correlated activity of excitatory synaptic inputs in the intact cortex, isolated thalamic inputs exhibit lower variability and asynchronous spontaneous and sensory-evoked inputs. These results are further supported in awake mice when we recorded the excitatory inputs of individual cortical cells simultaneously with the local field potential in a nearby site. Our results therefore indicate that cortical synchronization emerges by intracortical coupling.