Targeting light-gated chloride channels to neuronal somatodendritic domain reduces their excitatory effect in the axon — TEI-tagged view

MessierpersName, ChenpersName, CaipersName, XuepersName (2018date). “Targeting light-gated chloride channels to neuronal somatodendritic domain reduces their excitatory effect in the axon.” eLifetitle·j.

Object of study — what a light-gated chlorideterm channelterm actually does to a neuronterm, given that its effect depends on a gradientterm that differs between compartmentsterm

Research question — whether an actuator adopted for inhibitionterm inhibits everywhere in the cell, or only where the gradientterm happens to point the right way

Contribution — the same channelterm inhibits the somaterm and EXCITES the axonterm and terminals, causing transmitter release - which explains published paradoxical results - and a targeting motifterm fixes it by changing where the protein goes

Full text — tagged

Targeting light-gated chlorideterm channelsterm to neuronal somatodendritic domain reduces their excitatoryterm effect in the axonterm eLife 7 :e38506.

https://doi.org/10.7554/eLife.38506 Copy to clipboard Download BibTeX Download .RIS Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Article and author information Metrics Light-gated chlorideterm channelsterm are emerging as promising optogeneticterm·mth tools for inhibitionterm of neural activity. However, their effects depend on the transmembrane chlorideterm electrochemical gradientterm and may be complex due to the heterogeneity of this gradientterm in different developmental stages, neuronal types, and subcellular compartmentsterm. Here we characterized a light-gated chlorideterm channelterm, GtACR2term, in mouse cortical neuronsterm. We found that GtACR2term activation inhibited the somaterm, but unexpectedly depolarized the presynapticterm terminals resulting in neurotransmitterterm release. Other light-gated chlorideterm channelsterm had similar effects. Reducing the chlorideterm concentrations in the axonterm and presynapticterm terminals diminished the GtACR2-induced neurotransmitterterm release, indicating an excitatoryterm effect of chlorideterm channelsterm in these compartmentsterm. A novel hybrid somatodendritic targeting motifterm reduced the GtACR2-induced neurotransmitterterm release while enhancing the somatic photocurrentsterm. Our results highlight the necessity of precisely determining the effects of light-gated chlorideterm channelsterm under specific experimental conditions and provide a much-improved light-gated chlorideterm channelterm for optogeneticterm·mth inhibitionterm.

https://doi.org/10.7554/eLife.38506.001 One way to study the role of a specific neuronterm is to activate or inhibit the cell and then observe the consequences. This can be achieved by using optogeneticsterm·mth, a technique that involves introducing ‘light-gated’ ion channelsterm in the outer membrane of a target neuronterm. When light is shone on the cell, these pore-like proteins open their channelsterm: this allows ions to move into or out of the neuronterm.

Ions flow from high concentration to low concentration areas. Typically, when a neuronterm is at rest, there are fewer chlorideterm ions inside the cell than outside. Activating a light-gated chlorideterm channelterm should thus cause these negatively charged ions to enter the neuronterm. The charge inside of the cell would become more negative relative to the outside: this would inhibit the neuronterm, making it less likely to fire.

Here, MessierpersName et al. looked into using a light-gated chlorine channelterm called GtACR2term to inhibit the activity of neuronsterm in mouse brain slices, but the results were not as expected. Activating the chlorideterm channelterm did inhibit the cell body, the area of the neuronterm that contains the nucleus. Yet, it had the opposite effect in the axonterm, the structure that carries electrical signals away from the cell body.

There, activating GtACR2term caused chlorideterm ions to leave the axonterm, which resulted in the neuronterm firing. Testing other types of optogeneticterm·mth chlorideterm channelsterm produced the same result. Further experiments revealed that the concentration of chlorideterm ions is higher inside the axonterm than the cell body, explaining the observed effects.

MessierpersName et al. then tried to redistribute the channelsterm from the axonterm to the cell body, where the proteins are inhibitory. This was accomplished by fitting GtACR2term with a molecular tag that acts like an address label, with the cell body as the target destination. Overall, when these modified channelsterm were activated, the neuronterm was more strongly inhibited.

Ultimately, the GtACR2term channelterm designed by MessierpersName et al. is a powerful new inhibitory optogeneticterm·mth tool. In addition, this tool could be used to study chlorideterm gradientsterm in brain regions, cell types and areas of cells that are otherwise difficult to access.

https://doi.org/10.7554/eLife.38506.002 Targeted manipulation of neural activity is a powerful approach in neuroscience that has provided fundamental insights into the roles of specific neuronsterm in nervous system functions. Genetically encoded actuators such as light-gated ion channelsterm or pumpsterm enable control of neural activity with unprecedented spatiotemporal specificity and are transforming neuroscience research ( Boyden et al., 2005 ; Han and Boyden, 2007 ; Li et al., 2005 ; Nagel et al., 2003 ; Zhang et al., 2007 ). Actuators that enable neuronal activation are frequently used, but inhibitory optogeneticterm·mth tools are increasingly crucial because reversible and temporally precise suppression of neuronal activity is key to revealing the causal roles of specific neuronsterm in network dynamics and behavior. The widely used light-driven inward chlorideterm pumpsterm and outward proton pumpsterm, such as Natronomonas pharaonis halorhodopsin (NpHR) and Halorubrum sodomense archaerhodopsin (Arch), can hyperpolarize membrane potentialsterm, independent of the electrochemical gradientsterm, to inhibit action potentials with millisecond precision ( Chow et al., 2010 ; Chuong et al., 2014 ; Han and Boyden, 2007 ; Han et al., 2011 ; Zhang et al., 2007 ). However, their efficacies are limited because only one ion is transported per absorbed photon, and their activation does not decrease membrane resistance. Light-gated chlorideterm channelsterm, such as Guillardia theta anion channelrhodopsin 1 and 2 (GtACR1 and GtACR2term), iC++, and iChloC, overcome these limitations ( Berndt et al., 2014 ; Govorunova et al., 2015 , 2017b ; Wietek et al., 2014 ). They are highly sensitive to light, allow multiple ions to cross the membrane per photocycle, and reduce membrane resistance, thereby potently inhibiting action potentials. Thus, light-gated chlorideterm channelsterm are emerging as promising optogeneticterm·mth tools for suppressing neuronal activity ( Govorunova et al., 2017a ).

Determining the precise effects of light-gated ion channelsterm or pumpsterm under defined conditions is a prerequisite to use them for interrogating the functions of specific neuronsterm and circuits. This is due to the possibility that these channelsterm or pumpsterm not only modulate membrane potentialsterm but also may affect other processes such as ion homeostasis or neurotransmitterterm release. For example, activation of a light-driven inward chlorideterm pumpterm, eNpHR3.0, can transiently change the reversal potentialterm of GABA A receptors and alter the inhibitory synaptic inputs ( Raimondo et al., 2012 ). Prolonged activation of a light-driven outward proton pumpterm, eArch3.0, can increase presynapticterm calcium concentrations and spontaneous neurotransmitterterm release ( Mahn et al., 2016 ). Despite these potential confounds, these inhibitory optogeneticterm·mth molecules become increasingly important tools for targeted silencing of neuronal populations, as long as these confounds are understood and controlled ( Allen et al., 2015 ). Therefore, it is also crucial to thoroughly characterize light-gated chlorideterm channelsterm, as their effect depends on the difference between the membrane potentialterm and the reversal potentialterm for chlorideterm, both of which can vary in different neuronal types and subcellular compartmentsterm ( Marty and Llano, 2005 ; Trigo et al., 2008 ).

To this end, we investigated the effects of activating GtACR2term in mouse cortical excitatoryterm and inhibitory neuronsterm. Much to our surprise, wide-field light activation of GtACR2term not only inhibited the somaterm, but also caused neurotransmitterterm release onto neighboring neuronsterm. A similar phenomenon was observed with GtACR1, iC++, and iChloC. We further showed that GtACR2term activation in the axonterm and presynapticterm terminals directly depolarized the membrane to induce neurotransmitterterm release due to high chlorideterm concentrations in these compartmentsterm. These data explain the recent observations that photostimulation of neuronsterm expressing GtACR1 or GtACR2term can paradoxically release neurotransmittersterm or generate action potentials ( Mahn et al., 2016 ; Malyshev et al., 2017 ). To reduce the excitatoryterm effect of GtACR2term, we screened a panel of somatodendritic targeting motifsterm to reduce the trafficking of GtACR2term to the axonterm and presynapticterm terminals. We created a hybrid motifterm·def (Kv2.1C-linker-TlcnC) that is most effective in concentrating GtACR2term in the somatodendritic domain. Activation of somatodendritically targeted GtACR2term resulted in larger photocurrentsterm at the somaterm and less neurotransmitterterm release than wild type GtACR2term. Thus, restricting the localization of light-gated chlorideterm channelsterm to the somatodendritic domain improves the inhibitory efficacy of these optogeneticterm·mth tools. Our results also suggest that light-gated chlorideterm channelsterm can be a useful tool for studying the physiological functions of chlorideterm electrochemical gradientsterm in specific brain regions, cell types, or subcellular domains that are otherwise difficult to access, such as small axonsterm and presynapticterm terminals.

To examine the efficacy of GtACR2term in mouse cortical excitatoryterm neuronsterm, we expressed a GtACR2-EYFP fusion protein (referred to as GtACR2term below) together with a red fluorescent protein, tdTomato, in layer 2/3 pyramidal neuronsterm of the mouse visual or somatosensory cortex by in utero electroporation of plasmids at embryonic day 14.5–15.5. We obtained acute coronal brain slices from 3 to 8 week-old mice and observed that GtACR2term was present in the somaterm, dendrites, and axonterm ( Figure 1A ). We performed whole-cell patch clamp recordings at the somaterm of neuronsterm expressing GtACR2term (GtACR2term + neuronsterm) with a K + -based pipette solution ( Figure 1B ). As previously reported ( Govorunova et al., 2015 ), activation of GtACR2term by wide-field blue light (455 nm) potently inhibited current-induced spiking in these neuronsterm ( Figure 1C ). However, when we voltage clamped the neuronsterm to record GtACR2-mediated photocurrentsterm, we unexpectedly found an inward current that was superimposed on the photocurrentterm. This inward current resembled an excitatoryterm postsynaptic current (EPSCterm; Figure 1D ). To further investigate this phenomenon, we recorded layer 2/3 pyramidal neuronsterm that did not express GtACR2term (GtACR2 – neuronsterm·def) with a Cs + -based pipette solution ( Figure 1E ). Activation of GtACR2term with a short light pulse (0.5–10 ms) generated inward currents in all recorded GtACR2 – neuronsterm·def that were voltage clamped at the reversal potentialterm for GABAergic inhibitionterm (−60 mV). The onsets of these inward currents followed the onset of the blue light by 3.19 ± 0.26 ms (mean ± s.e.m.term·stat, n = 25). These currents were abolished by the glutamatergic receptor antagonists, NBQX and CPP ( Figure 1F ), or the voltage-gated sodium channelterm blocker, tetrodotoxin (TTX; Figure 1G ), indicating that they were indeed monosynaptic EPSCsterm caused by the glutamateterm transmitter released from GtACR2term + neuronsterm. Activation of GtACR2term also produced inhibitory postsynaptic currents (IPSCsterm) in GtACR2 – neuronsterm·def that were voltage clamped at the reversal potentialterm for glutamatergic excitation (+10 mV). These IPSCsterm were disynaptic because they were abolished by NBQX and CPP ( Figure 1—figure supplement 1 ), indicating that activating GtACR2term in pyramidal neuronsterm can release sufficient glutamateterm to recruit inhibitory interneuronsterm.

Figure 1 with 3 supplements see all Download asset Open asset Light activation of GtACR2term causes excitatoryterm and inhibitory neuronsterm to release neurotransmittersterm.

( A ) A representative fluorescent image of the visual cortex showing GtACR2-EYFP and tdTomato expression in a subset of layer 2/3 pyramidal neuronsterm. Note the strong EYFP fluorescence in layer 5 that contains the axonsterm of layer 2/3 pyramidal neuronsterm. L, layer; WM, white matter. ( B ) Schematic of slice experiments in ( C,D ). GtACR2term in a subset of layer 2/3 pyramidal neuronsterm. ( C ) Membrane potentialsterm (upper panel) in response to somatic current injections (lower panel) from a GtACR2term + neuronterm. Blue light activation of GtACR2term suppressed the action potentials evoked by current injections ( n = 5 neuronsterm). ( D ) Blue light-induced membrane currents recorded at the membrane potentialsterm of −75, –77, −78, –79, and −80 mV from the same GtACR2term + neuronterm in ( C ). Note the EPSC-like inward currents (arrows) superimposed on the GtACR2-mediated photocurrentsterm ( n = 2 neuronsterm). ( E ) Schematic of slice experiments in ( F,G ). GtACR2term in a subset of layer 2/3 pyramidal neuronsterm. ( F ) Left, photoactivation of GtACR2term generated EPSCsterm in a GtACR2 – neuronterm·def, which were abolished by the glutamateterm receptor antagonists, NBQX and CPP. Right, summary graph of similar experiments ( n = 17, p<0.0001). ( G ) Left, photoactivation of GtACR2term generated EPSCsterm in a GtACR2 – neuronterm·def, which were abolished by the voltage-gated sodium channelterm blocker, TTX. Right, summary graph of similar experiments ( n = 8, p=0.02). ( H ) A representative fluorescent image of the visual cortex showing GtACR2-EYFP expression in Pv neuronsterm. ( I,J,K ) As in ( E,F,G ), but for GtACR2term in Pv neuronsterm. GtACR2term activation-induced IPSCsterm were abolished by the GABA A receptor antagonist, Gabazine ( J , n = 7, p=0.02) or TTX ( K , n = 10, p=0.002).

https://doi.org/10.7554/eLife.38506.003 To determine if the phenomenon of GtACR2-induced neurotransmitterterm release also occurs in GABAergic inhibitory neuronsterm, we expressed GtACR2term in parvalbumin-expressing (Pv) neuronsterm by injecting a Flpo recombinase-dependent adeno-associated virus (AAV) into the visual cortex of Pvalb-2A-Flpo mice ( Pvalb Flpo/+ ) ( Madisen et al., 2015 ) at postnatal day 1 ( Figure 1H ). Using acute brain slices from 3 to 6 week-old mice, we found that activation of GtACR2term in Pv neuronsterm generated IPSCsterm in all recorded GtACR2term – layer 2/3 pyramidal neuronsterm, and the IPSCsterm were abolished by Gabazine, a GABA A receptor antagonist, or TTX ( Figure 1I–K ). The onsets of the IPSCsterm followed the onset of the blue light by 2.47 ± 0.15 ms (mean ± s.e.m.term·stat, n = 17), indicating that they were monosynaptic IPSCsterm caused by the GABA transmitter released from GtACR2term + Pv neuronsterm.

We next determined how repetitive or prolonged activation of GtACR2term would affect neurotransmitterterm release. We activated GtACR2term in layer 2/3 pyramidal neuronsterm or Pv neuronsterm with a high-frequency train of light pulses and found that each light pulse produced reliable EPSCsterm or IPSCsterm, respectively ( Figure 1—figure supplement 2 ). Interestingly, continuous activation of GtACR2term in layer 2/3 pyramidal neuronsterm or Pv neuronsterm with a long pulse of light (e.g., 2 s) transiently generated large EPSCsterm or IPSCsterm, respectively, followed by various amounts of smaller synaptic responses ( Figure 1—figure supplement 3A,B,E,F,G ). Typically, more than 50% of the neurotransmitterterm release occurred within the first 100–200 ms of the light pulse ( Figure 1—figure supplement 3C,D,H,I ).

The finding of GtACR2-induced neurotransmitterterm release was unexpected, because the Nernst equilibrium potential of chlorideterm becomes lower than the action potential threshold in rodent cortical neuronsterm after the second postnatal week, and activation of chlorideterm channelsterm should inhibit, not promote, neurotransmitterterm release onto neighboring cells ( Ben-Ari, 2002 ; Owens et al., 1996 ). We thus sought to identify the cause of this paradoxical neurotransmitterterm release and considered three possibilities. First, GtACR2term may conduct cations to depolarize neuronsterm. Second, an increase in the intracellular chlorideterm or strong hyperpolarization induced by GtACR2term activation may lead to rebound spikes. Third, GtACR2-mediated chlorideterm currents may be excitatoryterm.

First, it was reported that GtACR2term did not conduct physiological cations ( Govorunova et al., 2015 ), but some other light-gated chlorideterm channelsterm retained certain cation conductance ( Berndt et al., 2014 ; Wietek et al., 2014 ). Thus, we sought to verify that in cortical neuronsterm, GtACR2term has a similar reversal potentialterm as a known chlorideterm channelterm. To accomplish this, we determined the reversal potentialterm of GtACR2-mediated photocurrentsterm in comparison with that of IPSCsterm mediated by the endogenous GABA A receptors. We used in utero electroporation to express GtACR2term in layer 2/3 pyramidal neuronsterm and a Cre recombinase-dependent AAV to express a red light-gated cation channelrhodopsin, ReaChR, in Pv neuronsterm of Pvalb-2A-Cre mice ( Pvalb Cre/+ ) ( Madisen et al., 2010 ). We performed whole-cell voltage clamp recordings at the somaterm of a GtACR2term + layer 2/3 pyramidal neuronterm and a nearby GtACR2term – pyramidal neuronterm simultaneously ( Figure 2—figure supplement 1A ). In the GtACR2term + neuronterm, we sequentially recorded the IPSCsterm induced by activating ReaChR in Pv neuronsterm via 617 nm light and the GtACR2-mediated photocurrentsterm activated by 455 nm light. Both IPSCsterm and photocurrentsterm were recorded at different membrane potentialsterm to determine their reversal potentialsterm in the same neuronterm ( Figure 2—figure supplement 1B,C ). 617 nm light does not activate GtACR2term ( Govorunova et al., 2015 ), whereas 455 nm light partially activates ReaChR ( Lin et al., 2013 ). Thus, to avoid Pv neuron-mediated IPSCsterm contaminating GtACR2-mediated photocurrentsterm, we monitored the IPSCsterm in the GtACR2 – neuronterm·def at the membrane potentialterm of +10 mV to ensure that the intensity of the 455 nm light was not sufficient to activate Pv neuronsterm and generate IPSCsterm ( Figure 2—figure supplement 1B ). We found that the reversal potentialsterm of GtACR2-mediated photocurrentsterm and GABAergic IPSCsterm were similar for each neuronterm and well below the action potential threshold ( Figure 2—figure supplement 1D ), indicating that GtACR2term does not conduct cations to cause neurotransmitterterm release.

Second, we tested if GtACR2-induced neurotransmitterterm release could be due to rebound depolarization. An increase in the intracellular chlorideterm caused by GtACR2term activation may trigger rapid efflux of chlorideterm after the blue light illumination terminates. However, this possibility is unlikely because when GtACR2term was activated by a long pulse of blue light, neurotransmitterterm release occurred before the light illumination ended (see examples in Figure 1F,G and Figure 1—figure supplement 3 ). Another possibility is that the strong hyperpolarization induced by GtACR2term activates hyperpolarization-activated I h currents, which may depolarize the membrane potentialterm above the action potential threshold. However, pharmacological inhibitionterm of I h currents slightly increased the amplitudes of GtACR2-induced EPSCsterm ( Figure 2—figure supplement 2A,B ), most likely because inhibiting I h currents increases neuronal membrane resistances ( Robinson and Siegelbaum, 2003 ). Thus, GtACR2-induced neurotransmitterterm release is not caused by rebound depolarization.

Third, although GtACR2-mediated photocurrentsterm are inhibitory at the somaterm, it is possible that the chlorideterm concentrations are higher in some other cellular compartmentsterm, such that the electrochemical gradientterm causes chlorideterm to exit the cell upon GtACR2term channelterm opening, resulting in depolarization of the membrane potentialterm. To test this hypothesis, we pharmacologically inhibited the activity of Na + -K + -2Cl – cotransporter 1 (NKCC1) with bumetanide (50 or 100 µM) to decease the intracellular chloride concentrationsterm·var, as NKCC1 is responsible for transporting chlorideterm into neuronsterm ( Ben-Ari, 2017 ). When we activated GtACR2term in layer 2/3 pyramidal neuronsterm, the resulting EPSCsterm in GtACR2term – pyramidal neuronsterm were diminished by bath application of bumetanide ( Figure 2A,B ), indicating that GtACR2-induced neurotransmitterterm release requires high concentrations of intracellular chlorideterm. An alternative interpretation of this result would be that bumetanide blocks GtACR2term itself. To test this possibility, we simultaneously recorded the photocurrentsterm and EPSCsterm in GtACR2term + and GtACR2term – pyramidal neuronsterm, respectively. While bumetanide diminished the EPSCsterm in GtACR2 – neuronsterm·def, it had no effect on the photocurrentsterm in GtACR2term + neuronsterm ( Figure 2—figure supplement 3A,B ), thereby ruling out the possibility that bumetanide affects GtACR2term itself. Furthermore, when a cation channelterm, channelrhodopsin-2 (ChR2) ( Boyden et al., 2005 ; Li et al., 2005 ; Nagel et al., 2003 ), was expressed and activated in layer 2/3 pyramidal neuronsterm, the resulting EPSCsterm in ChR2 – neuronsterm were not affected by bumetanide ( Figure 2C,D ), indicating that reducing the intracellular chloride concentrationterm·var only affects chloride-mediated, and not cation-mediated, EPSCsterm. Finally, bumetanide also diminished the IPSCsterm resulting from GtACR2term activation in Pv neuronsterm ( Figure 2E,F ). Together, these results demonstrate that light activation of GtACR2term generates an excitatoryterm chlorideterm conductance in certain neuronal compartmentsterm to trigger neurotransmitterterm release.

Figure 2 with 3 supplements see all Download asset Open asset Reducing intracellular chloride concentrationsterm·var diminishes the neurotransmitterterm release induced by activation of GtACR2term or iC++.

( A ) Schematic of slice experiments in ( B ). GtACR2term in a subset of layer 2/3 pyramidal neuronsterm. ( B ) Left, photoactivation of GtACR2term generated EPSCsterm in a GtACR2 – neuronterm·def, which were diminished by decreasing intracellular chloride concentrationsterm·var with an NKCC1 blocker, bumetanide. Right, summary graph of similar experiments ( n = 10, p=0.002). ( C,D ) As in ( A,B ), but for ChR2. ChR2 activation-induced EPSCsterm were not affected by bumetanide ( n = 8, p=0.9). ( E,F ) As in ( A,B ), but for GtACR2term in Pv neuronsterm. GtACR2term activation-induced IPSCsterm were diminished by bumetanide ( n = 9, p=0.004). ( G,H ) As in ( A,B ), but for iC++. iC++-induced EPSCsterm were diminished by bumetanide ( n = 6, p=0.009). ( I,J ) As in ( E,F ), but for iC++. iC++-induced IPSCsterm were diminished by bumetanide ( n = 8, p=0.008).

https://doi.org/10.7554/eLife.38506.007 Since we expressed GtACR2term by in utero electroporation or neonatal AAV injection, we sought to determine if the excitatoryterm effect of GtACR2term was caused by the long-term expression of GtACR2term throughout development that somehow altered the chlorideterm homeostasis. To selectively express GtACR2term in adult neuronsterm, we in utero electroporated a Flpo-dependent plasmid into layer 2/3 pyramidal neuronsterm that will express GtACR2term only if Flpo is present ( Figure 2—figure supplement 2C,D ). A Flpo-expressing AAV was then injected into the electroporated mice at postnatal week 4 or 9 to turn on the GtACR2term expression. We obtained acute coronal brain slices 1–3 weeks after injecting Flpo-expressing AAV and found that light activation of GtACR2term, again, produced bumetanide-sensitive EPSCsterm in GtACR2 – neuronsterm·def ( Figure 2—figure supplement 2E ). Thus, it is unlikely that GtACR2term expression during neuronal development alters the chlorideterm homeostasis to render GtACR2term excitatoryterm, as acute expression of GtACR2term in mature neuronsterm had the same effect.

To determine if activation of other light-gated chlorideterm channelsterm can trigger neurotransmitterterm release, we examined iC++ and iChloC, two engineered blue light-gated chlorideterm channelsterm that were converted from cation channelrhodopsins ( Berndt et al., 2016 ; Wietek et al., 2015 ), and GtACR1, another natural anion channelrhodopsin from Guillardia theta ( Govorunova et al., 2015 ). We in utero electroporated plasmids to express iC++ in layer 2/3 pyramidal neuronsterm of the visual cortex and obtained acute coronal brain slices from 3 to 9 week-old mice. Similar to GtACR2term, light activation of iC++ generated EPSCsterm in iC++ – layer 2/3 pyramidal neuronsterm that were abolished by NBQX and CPP (amplitude reduced by 97.3 ± 0.9%, mean ± s.e.m.term·stat, n = 4). Bumetanide diminished the iC++-induced EPSCsterm ( Figure 2G,H ) without affecting the iC++-mediated photocurrentsterm ( Figure 2—figure supplement 3C,D ). We also expressed iC++ in Pv neuronsterm by injecting a Cre-dependent AAV into Pvalb Cre/+ mice and found that light activation of iC++ caused bumetanide-sensitive IPSCsterm in iC++ – layer 2/3 pyramidal neuronsterm ( Figure 2I,J ). Similarly, when we expressed iChloC in Pv neuronsterm, light activation of iChloC resulted in IPSCsterm in 10 out of 17 recorded iChloC – layer 2/3 pyramidal neuronsterm (183 ± 42 pA, mean ± s.e.m.term·stat, n = 10), presumably because iChloC generated smaller photocurrentsterm than iC++ (iChloC, 292 ± 62 pA, n = 7; iC++, 2182 ± 291 pA, n = 15; recorded at the membrane potentialterm of +10 mV; mean ± s.e.m.term·stat, p<0.0001, t test with Welch’s correctionterm·stat). Finally, activation of GtACR1 in layer 2/3 pyramidal neuronsterm produced EPSCsterm onto all recorded GtACR1 – pyramidal neuronsterm (149 ± 47 pA, mean ± s.e.m.term·stat, n = 11). Altogether, these results demonstrate that activation of different light-gated chlorideterm channelsterm in neuronsterm can trigger neurotransmitterterm release.

We hypothesized that the most likely neuronal compartmentsterm rendering GtACR2term excitatoryterm were the distal axonterm and presynapticterm terminals because of the following previous findings. First, activation of presynapticterm GABA A or glycine receptors enhanced neurotransmitterterm release at several synapses of the hippocampus, cerebellum, and brainstem ( Jang et al., 2006 ; Pugh and Jahr, 2011 ; Ruiz et al., 2010 ; Stell et al., 2007 ; Turecek and Trussell, 2001 ; Zorrilla de San Martin et al., 2017 ). Second, the chlorideterm concentrations were 4–5 times higher in the presynapticterm terminals of the Calyx of Held than the parent somaterm ( Price and Trussell, 2006 ). Third, there appeared to be an axo-somato-dendritic gradientterm in which the reversal potentialsterm of GABA from the axonterm to the somaterm and dendrites of cortical neuronsterm become progressively more negative ( Khirug et al., 2008 ). To test our hypothesis, we expressed GtACR2term in layer 2/3 pyramidal neuronsterm of the visual cortex in one hemisphere as described above and obtained acute coronal slices from the contralateral hemisphere ( Figure 3A ). GtACR2term was present in the long-range callosal projections in the contralateral hemisphere ( Figure 3—figure supplement 1A ), which enabled us to activate GtACR2term in the axonterm and presynapticterm terminals that were severed from their parent somasterm. Light activation of GtACR2term in the callosal projections generated EPSCsterm in layer 2/3 pyramidal neuronsterm of the contralateral cortex, which were diminished by TTX ( Figure 3—figure supplement 1B,C ) or bumetanide ( Figure 3B ). These results demonstrate that activation of GtACR2term in the axonterm and presynapticterm terminals is sufficient to trigger neurotransmitterterm release.

Figure 3 with 1 supplement see all Download asset Open asset GtACR2term activation directly depolarizes the distal axonterm and presynapticterm terminals and can result in antidromic spikes.

( A ) Schematic of slice experiments in ( B ). GtACR2term in a subset of layer 2/3 pyramidal neuronsterm in the contralateral hemisphere. ( B ) Left, photoactivation of GtACR2term in the callosal axonsterm, severed from their somasterm, generated EPSCsterm in a GtACR2 – neuronterm·def, which were diminished by bumetanide. Right, summary graph of similar experiments ( n = 15, p<0.0001). ( C ) Schematic of slice experiments in ( D ). GtACR2term in a subset of layer 2/3 pyramidal neuronsterm. ( D ) Left, photoactivation of GtACR2term generated EPSCsterm in a GtACR2 – neuronterm·def, which were abolished by TTX, but partially recovered by the addition of voltage-gated potassium channelterm blockers, 4-AP (1.5 mM) and TEA (1.5 mM). Right, summary graph of similar experiments ( n = 10; TTX vs. control, p=0.02, average EPSCterm amplitude in TTX was 2% of control; TTX + 4-AP + TEA vs. TTX, p=0.0004, average EPSCterm amplitude in TTX + 4-AP + TEA was 35% of control). ( E,F ) As in ( C,D ), but for Pv neuronsterm. GtACR2term activation-induced IPSCsterm were abolished by TTX, but partially recovered by 4-AP and TEA ( n = 9; TTX vs. control, p=0.006, average IPSCterm amplitude in TTX was 0.9% of control; TTX + 4-AP + TEA vs. TTX, p=0.008, average IPSCterm amplitude in TTX + 4-AP + TEA was 23% of control). ( G ) Schematic of slice experiments in ( H ). GtACR2term in a subset of layer 2/3 pyramidal neuronsterm. ( H ) Photoactivation of GtACR2term generated antidromic spikes in GtACR2term + pyramidal neuronsterm, which were not affected by NBQX and CPP, but were abolished by TTX in both loose-patch (left panel) and whole-cell (right panel) recordings. In the whole-cell recordings, the resting membrane potentialsterm of those neuronsterm that generated antidromic spikes were −68.1 ± 1.7 mV (mean ± s.e.m.term·stat, n = 21). ( I,J ) As in ( G,H ), but for Pv neuronsterm. The antidromic spikes in GtACR2term + Pv neuronsterm were abolished by TTX. In the whole-cell recordings, the resting membrane potentialsterm of those neuronsterm that generated antidromic spikes were −60.0 ± 2.4 mV (mean ± s.e.m.term·stat, n = 7).

https://doi.org/10.7554/eLife.38506.011 If GtACR2-mediated chlorideterm currents are excitatoryterm in the presynapticterm terminals, then GtACR2term should be similar to ChR2, whose activation can directly depolarize the presynapticterm membrane in the absence of action potentials to trigger neurotransmitterterm release ( Petreanu et al., 2009 ). To test this prediction, we recorded EPSCsterm or IPSCsterm in GtACR2 – neuronsterm·def while activating GtACR2term in layer 2/3 pyramidal neuronsterm or Pv neuronsterm, respectively ( Figure 3C,E ). As described above, bath application of TTX abolished the EPSCsterm and IPSCsterm. However, when we further blocked voltage-gated potassium channelsterm by 4-aminopyridine (4-AP) and tetraethylammonium (TEA) to prolong membrane depolarization ( Petreanu et al., 2009 ), the EPSCsterm and IPSCsterm were partially recovered ( Figure 3D,F ). These results indicate that in the absence of action potentials, light activation of GtACR2term is sufficient to depolarize the presynapticterm membrane to open voltage-gated calcium channelsterm and trigger neurotransmitterterm release.

We further tested if GtACR2-induced axonal depolarization could evoke antidromic action potentials by performing extracellular loose-patch or whole-cell current clamp recordings at the somasterm of GtACR2term + pyramidal neuronsterm. We observed antidromic spikes in 9 out of 88 neuronsterm recorded in loose-patch configuration and 21 out of 56 neuronsterm recorded in whole-cell configuration in response to blue light stimulation ( Figure 3G,H ). In the whole-cell current clamp recordings, although the chlorideterm concentration in the patch pipette solution sets the Nernst equilibrium potential of chlorideterm around −85 mV (see Materials and methods), blue light induced a depolarization following the initial hyperpolarization. This observation is consistent with the notion that the depolarization antidromically propagated from the distal axonterm to the somaterm ( Figure 3H ). The antidromic spikes were not affected by NBQX and CPP, but were abolished by TTX ( Figure 3H ), indicating that the spikes were generated within the GtACR2term + neuronsterm, rather than by excitatoryterm inputs from other neuronsterm. Antidromic spikes were only observed in a subset of neuronsterm, likely because the GtACR2term expression levels are heterogeneous in different neuronsterm, and the hyperpolarization initiated at the somaterm can orthodromically propagate to counteract the antidromic spikes. Similarly, TTX-sensitive antidromic spikes were observed in a subset of GtACR2term + Pv neuronsterm (3 out of 11 neuronsterm in loose-patch configuration and 7 out of 17 neuronsterm in whole-cell configuration, Figure 3I,J ). These results show that GtACR2-induced axonal depolarization can be sufficient to elicit antidromic action potentials.

Activating GtACR2term and other light-gated chlorideterm channelsterm inhibits the somaterm but depolarizes the presynapticterm terminals to release neurotransmittersterm. This dichotomic effect can confound the utilization of these channelsterm as inhibitory optogeneticterm·mth tools for suppressing neuronal activity. We reasoned that reducing the trafficking of light-gated chlorideterm channelsterm into the axonterm and presynapticterm terminals should reduce or eliminate their depolarizing action. Thus, we sought to restrict GtACR2term within the somatodendritic domain of neuronsterm by fusing GtACR2term with a number of reported somatodendritic targeting motifsterm including a 26-amino acid Myosin Va-binding domain of Melanophilin (MBD) ( Lewis et al., 2009 ), a 32-amino acid cytoplasmic C-terminal motif of Neuroligin 1 (Nlgn1C) ( Rosales et al., 2005 ), a 16-amino acid dileucine-containing motif of potassium channelterm Kv4.2 (Kv4.2LL) ( Rivera et al., 2003 ), the N-terminal 150 residues of kainate receptor subunit 2 (KA2N) ( Shemesh et al., 2017 ), the C-terminal 17 residues of Telencephalin (TlcnC) ( Mitsui et al., 2005 ), and a 65-amino acid cytoplasmic C-terminal motif of potassium channelterm Kv2.1 (Kv2.1C) ( Lim et al., 2000 ; Wu et al., 2013 ). Each of these GtACR2 variantsterm·var ( Table 1 ), along with tdTomato, were expressed in layer 2/3 pyramidal neuronsterm of the visual cortex by in utero electroporation ( Figure 4A ). Since GtACR2term was tagged with EYFP or EGFP, we compared the EYFP or EGFP fluorescence in layer 5, which only contains the axonsterm of layer 2/3 pyramidal neuronsterm, with the EYFP or EGFP fluorescence in layer 2/3 to estimate the distribution of GtACR2term between the axonterm and somatodendritic domain. We normalized the EYFP or EGFP fluorescence ratio between layer 5 and layer 2/3 by the tdTomato fluorescence ratioterm·var between layer 5 and layer 2/3 to control for variations in the collateral axonsterm. Among tested motifs, TlcnC and Kv2.1C were most effective in targeting GtACR2term to the somaterm and dendrites ( Figure 4D ). As these two motifs may engage different trafficking mechanisms, we combined them to create two hybrid motifsterm·def, Kv2.1C-TlcnC and Kv2.1C-linker-TlcnC. Kv2.1C-linker-TlcnC turned out to be the best in restricting GtACR2term within the somatodendritic domain ( Figure 4A,D ). Interestingly, GtACR2-EYFP-Kv2.1C and GtACR2-EYFP-Kv2.1C-linker-TlcnC showed less intracellular aggregation than wild type GtACR2term ( Figure 4B ), suggesting that the somatodendritic targeting motifsterm also enhance the surface expression of GtACR2term. Finally, the EYFP fluorescence of the callosal projections in the contralateral hemisphere was also reduced for GtACR2-EYFP-Kv2.1C and GtACR2-EYFP-Kv2.1C-linker-TlcnC as compared to GtACR2-EYFP ( Figure 4C,E ), demonstrating that both somatodendritic targeting motifsterm decreased the trafficking of GtACR2term into the distal axonterm.

Figure 4 Download asset Open asset Targeting GtACR2term to neuronal somatodendritic domain.

( A ) Wild type (WT) GtACR2term and its variants tagged with EYFP or EGFP were expressed along with tdTomato in a subset of layer 2/3 pyramidal neuronsterm. Representative fluorescent images of the cortices showing the distribution of GtACR2term. Note the strong EYFP fluorescence in layer 5 for WT GtACR2term and weak EYFP fluorescence in layer 5 for some somatodendritically targeted GtACR2 variantsterm·var (e.g., Kv2.1C-linker-TlcnC). L, layer; WM, white matter. ( B ) Representative fluorescent images of electroporated neuronsterm showing that GtACR2-EYFP-Kv2.1C and GtACR2-EYFP-Kv2.1C-linker-TlcnC were more concentrated on the membranes at the somaterm and proximal dendrites (arrows) than GtACR2-EYFP. Note the intracellular aggregation of GtACR2-EYFP (arrow head). ( C ) Representative fluorescent images of the contralateral hemisphere showing less GtACR2-EYFP-Kv2.1C and GtACR2-EYFP-Kv2.1C-linker-TlcnC in the callosal projections than GtACR2-EYFP. The callosal projections were labeled by tdTomato. ( D ) Summary data of the experiments in ( A ). The EYFP or EGFP fluorescence ratio between layer 5 and layer 2/3 was normalized by the tdTomato fluorescence ratioterm·var between layer 5 and layer 2/3. A reduction in the normalized EYFP or EGFP fluorescence ratio indicates a shift of the EYFP or EGFP distribution from the axonterm to somatodendritic domain. ( E ) Summary data of the experiments in ( C ). The ratio of EYFP fluorescence to tdTomato fluorescence of the callosal projections in the contralateral hemisphere. A reduction in the ratio indicates less EYFP in the callosal axonsterm. The numbers of analyzed slices were indicated in the panels. The columns and error bars are mean ± s.e.m.term·stat **p<0.01, ****p<0.0001.

https://doi.org/10.7554/eLife.38506.013 Table 1 Amino acid sequences of somatodendritic targeting motifsterm.

Black bold, amino acid sequences of somatodendritic targeting motifsterm; blue, GtACR2term; red, EYFP or EGFP; purple, linker.

https://doi.org/10.7554/eLife.38506.014 Motif Sequence MBD GtACR2term - EYFP - GSGSGTRGSGS - RDQPLNSKKKKRLLSFRDVDFEEDSD Nlgn1C GtACR2term - EYFP - VVLRTACPPDYTLAMRRSPDDIPLMTPNTITM Kv4.2LL GtACR2term - EYFP - FETQHHHLLHCLEKTT KA2N GtACR2term - GGSGGTGGSGGT - MPAELLLLLIVAFANPSCQVLSSLRMAAILDDQ TVCGRGERLALALAREQINGIIEVPAKARVEVDIFELQRDSQYETTDTMCQIL PKGVVSVLGPSSSPASASTVSHICGEKEIPHIKVGPEETPRLQYLRFASVSLYPS NEDVSLAVS - GASGGT - EGFP TlcnC GtACR2term - EYFP - AESPADGEVFAIQLTSS Kv2.1C GtACR2term - EYFP - QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVID MRSMSSIDSFISCATDFPEATRF Kv2.1C-TlcnC GtACR2term - EYFP - QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVID MRSMSSIDSFISCATDFPEATRF - AESPADGEVFAIQLTSS Kv2.1C-linker-TlcnC GtACR2term - EYFP - QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVID MRSMSSIDSFISCATDFPEATRF - GSGSGSGSGS - AESPADGEVFAIQLTSS To determine how targeting GtACR2term to the somatodendritic domain affects its photocurrentterm and ability to trigger neurotransmitterterm release, we compared the somatodendritically targeted GtACR2 variantsterm·var, GtACR2-EYFP-Kv2.1C and GtACR2-EYFP-Kv2.1C-linker-TlcnC, with wild type GtACR2term in the same litters of mice. We first recorded GtACR2term + layer 2/3 pyramidal neuronsterm and found that the blue light-activated photocurrentsterm of GtACR2-EYFP-Kv2.1C and GtACR2-EYFP-Kv2.1C-linker-TlcnC were 2.1–2.4 and 2.7–3.5 folds of GtACR2-EYFP photocurrentsterm, respectively ( Figure 5A–C and Figure 5—figure supplement 1A,C,E,G ). We then recorded the EPSCsterm in GtACR2term – layer 2/3 pyramidal neuronsterm in response to different strengths of blue light stimulation. The EPSCsterm evoked by activating GtACR2-EYFP-Kv2.1C or GtACR2-EYFP-Kv2.1C-linker-TlcnC were reduced by 52–60% or 65–77%, respectively, as compared to those evoked by activating GtACR2-EYFP ( Figure 5D–G and Figure 5—figure supplement 1B,D,F,H ). In a subset of experiments with GtACR2-EYFP-Kv2.1C or GtACR2-EYFP-Kv2.1C-linker-TlcnC, the lowest light stimulation strength could no longer evoke EPSCsterm in GtACR2 – neuronsterm·def, but still induced robust photocurrentsterm in GtACR2term + neuronsterm ( Figure 5—figure supplement 1E–H ). Furthermore, when photostimulating the callosal projections, the EPSCsterm in layer 2/3 pyramidal neuronsterm of the contralateral cortex were reduced by 54% and 73% for GtACR2-EYFP-Kv2.1C and GtACR2-EYFP-Kv2.1C-linker-TlcnC, respectively, as compared to GtACR2-EYFP ( Figure 5H,I ). These results show that the somatodendritic targeting motifsterm, especially Kv2.1C-linker-TlcnC, shift GtACR2term from the axonterm towards the somaterm and dendrites, thereby reducing the excitatoryterm action in the axonterm and presynapticterm terminals while enhancing the inhibitory currents at the somaterm and dendrites.

Figure 5 with 1 supplement see all Download asset Open asset Activation of somatodendritically targeted GtACR2 variantsterm·var generates larger photocurrentsterm but causes less neurotransmitterterm release than wild type GtACR2term.

( A ) Schematic of slice experiments in ( B,C ). GtACR2-EYFP, GtACR2-EYFP-Kv2.1C, or GtACR2-EYFP-Kv2.1C-linker-TlcnC in a subset of layer 2/3 pyramidal neuronsterm. ( B ) Somatic photocurrentsterm recorded at the membrane potentialterm of +10 mV from GtACR2-EYFP-, GtACR2-EYFP-Kv2.1C-, or GtACR2-EYFP-Kv2.1C-linker-TlcnC-expressing neuronsterm in response to a blue light pulse of 1 ms and 2.5 mW/mm 2 . ( C ) Summary graph showing the photocurrentterm peak amplitudes in response to a blue light pulse of 1 ms and 2.5 mW/mm 2 . ( D,E ) As in ( A,B ), but for recording EPSCsterm in GtACR2term – layer 2/3 pyramidal neuronsterm in response to a blue light pulse of 5 ms and 2.9 mW/mm 2 . ( F,G ) Summary graphs showing the EPSCterm amplitudes in response to a blue light pulse of 1 ms and 2.5 mW/mm 2 ( F ), 5 ms and 2.9 mW/mm 2 ( G , left panel), or 5 ms and 16.1 mW/mm 2 ( G , right panel). Note that at the stimulation strength of 1 ms and 2.5 mW/mm 2 ( F ), EPSCsterm were not observed in a subset of neuronsterm, particularly for GtACR2-EYFP-Kv2.1C-linker-TlcnC. ( H ) Schematic of slice experiments in ( I ). GtACR2-EYFP, GtACR2-EYFP-Kv2.1C, or GtACR2-EYFP-Kv2.1C-linker-TlcnC in a subset of layer 2/3 pyramidal neuronsterm in the contralateral hemisphere. ( I ) Summary graphs showing the EPSCterm amplitudes of GtACR2term – layer 2/3 pyramidal neuronsterm in response to stimulating the callosal axonsterm with a blue light pulse of 5 ms and 23.6 mW/mm 2 . The numbers of recorded neuronsterm were indicated in the panels. The columns and error bars are mean ± s.e.m.term·stat *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.38506.015 Optogeneticterm·mth suppression of neuronal activity and synaptic outputs is an essential approach for dissecting the roles of specific neuronsterm in brain functions. Light-gated chlorideterm channelsterm, particularly GtACR1 and GtACR2term, are increasingly used due to their large photocurrentsterm and high sensitivity to light ( Forli et al., 2018 ; Mardinly et al., 2018 ; Mauss et al., 2017 ; Mohamed et al., 2017 ; Mohammad et al., 2017 ). To use these tools to their full potentials, it is necessary that we understand their function and, importantly, their limitations. Our experiments reveal that wide-field activation of these channelsterm in cortical neuronsterm suppresses action potentials at the somaterm but also triggers neurotransmitterterm release at the presynapticterm terminals, thereby voiding inhibitionterm of neuronal activity. As demonstrated, the excitatoryterm action of chlorideterm channelsterm in the axonterm and presynapticterm terminals is due to the high intracellular chloride concentrationsterm·var that create a depolarizing chlorideterm electrochemical gradientterm. The depolarizing effect of presynapticterm chlorideterm channelsterm, GABA A or glycine receptors, has been documented at a few synapses in the hippocampus, cerebellum, and brainstem through application of agonists or antagonists ( Jang et al., 2006 ; Pugh and Jahr, 2011 ; Ruiz et al., 2010 ; Stell et al., 2007 ; Turecek and Trussell, 2001 ; Zorrilla de San Martin et al., 2017 ). Our results in cortical excitatoryterm and inhibitory neuronsterm expand previous findings and indicate that a presynapticterm depolarizing chlorideterm electrochemical gradientterm is likely a general property across brain regions and neuronal types. More importantly, our experiments directly demonstrate that activation of presynapticterm chlorideterm channelsterm is in fact excitatoryterm, as it can elicit action potentials with short latency, which was difficult to explicitly demonstrate by agonist application ( Pugh and Jahr, 2011 ). Thus, light-gated chlorideterm channelsterm can be targeted to different brain regions, cell types, and subcellular compartmentsterm to study chlorideterm electrochemical gradientsterm with unprecedented temporal precision.

Although the excitatoryterm effect of light-gated chlorideterm channelsterm is undesired for neuronal silencing, a potential utilization of their dual actions at the presynapticterm terminals and somaterm is to activate specific projections of neuronsterm while minimizing the effect of antidromic spikes. For example, long-range projection neuronsterm often target multiple brain areas, and sometimes it is desired to selectively excite the axonal terminals projecting to one particular area. If ChR2 is used, local activation of ChR2-expressing axonal terminals may generate antidromic spikes, which will affect other projections. However, if GtACR2term is used, one can simultaneously activate GtACR2term in the somaterm and axonal terminals. Axonal depolarization will result in neurotransmitterterm release, but the antidromic spikes will be reduced or suppressed by the hyperpolarization originating from the somatodendritic domain, which reduces the likelihood of activating other projections.

To create a better inhibitory optogeneticterm·mth tool, we tested a number of somatodendritic targeting motifsterm to confine GtACR2term in the somaterm and dendrites. We generated a hybrid motifterm·def, Kv2.1C-linker-TlcnC, that was more effective than the widely used Kv2.1C ( Baker et al., 2016 ; Mardinly et al., 2018 ; Wu et al., 2013 ). The Kv2.1C motif was recently used in a bioRxiv preprint ( Mahn et al., 2017 ) to target GtACR2term to the somatodendritic domain. This GtACR2 variantterm·var achieved a greater reduction of neurotransmitterterm release from the contralateral callosal projections of medial prefrontal cortical neuronsterm than what we observed with our GtACR2-EYFP-Kv2.1C and GtACR2-EYFP-Kv2.1C-linker-TlcnC in visual cortical neuronsterm. This quantitative difference is likely due to different expression levels, light stimulation strengths, or neuronal types (see discussion below), which reiterates the importance of precisely characterizing the effect of light-gated chlorideterm channelsterm, specific to each experimental design, when using them to manipulate neuronal activity.

While GtACR2-EYFP-Kv2.1C-linker-TlcnC can still traffic to the axonterm to cause neurotransmitterterm release, it is thus far the most improved light-gated chlorideterm channelterm for optogeneticterm·mth inhibitionterm. Since it can generate much larger photocurrentsterm in the somatodendritic domain than what is necessary to suppress action potentials, one approach to use this tool is to reduce the overall GtACR2term expression level to further decrease its presence in the axonterm while still generating sufficient inhibitory photocurrentsterm at the somaterm. Supporting this idea, post hoc analysis of our experiments indicates that, for a given experiment, it is possible to identify a light stimulation strength that is low enough not to cause neurotransmitterterm release but still produce large inhibitory photocurrentsterm in GtACR2term + neuronsterm ( Figure 5—figure supplement 1 ). However, this stimulation strength must be empirically determined for every experiment. Furthermore, since during continuous activation of GtACR2term, large and synchronous neurotransmitterterm release occurs at the early phase of the light stimulation ( Figure 1—figure supplement 3 ), one may also take advantage of the late phase when neurotransmitterterm release is reduced but photocurrentterm is still robust for optogeneticterm·mth inhibitionterm experiments. Another approach is to selectively photostimulate GtACR2term at the somaterm by two-photon excitation ( Forli et al., 2018 ; Mardinly et al., 2018 ). However, it is difficult to apply this photoactivation approach to freely moving animals or deep brain areas. Therefore, it is imperative that we further engineer light-gated chlorideterm channelsterm to eliminate their excitatoryterm action in the axonal terminals. Future strategies include generating more effective somatodendritic targeting motifsterm, creating outwardly rectifying channelsterm, and the combination of both strategies. The GtACR2-EYFP-Kv2.1C-linker-TlcnC reported here enhances the available toolkit for optogeneticterm·mth inhibitionterm and will serve as the foundation for future improvement.

Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background ( M. musculus ) Pvalb-2A-Flpo Jackson Laboratory RRID: IMSR_JAX:022730 Strain, strain background ( M. musculus ) Pvalb-2A-Cre Jackson Laboratory RRID: IMSR_JAX:012358 Recombinant DNA reagent (plasmid) GtACR2term Govorunova et al., 2015 Addgene:67877 pLenti-UbiC- GtACR2-EYFP; obtained from Dr. John Spudich Recombinant DNA reagent (plasmid) GtACR1 Govorunova et al., 2015 Addgene:67795 pLenti-UbiC- GtACR1-EYFP; obtained from Dr. John Spudich Recombinant DNA reagent (plasmid) iC++ Berndt et al., 2016 pAAV-CaMKIIα-i C++-TS-EYFP or pAAV-EF1α-DIO-iC++ -TS-EYFP; obtained from Dr. Karl Deisseroth Recombinant DNA reagent (plasmid) Flpo XuepersName et al. (2014) Addgene:60662 pCAG-Flpo Recombinant DNA reagent (plasmid) ChR2 this paper Addgene:114367 pCAG-hChR2(H134R) -EYFP Recombinant DNA reagent (plasmid) Flpo-dependent GtACR2term this paper Addgene:114369 pAAV-EF1α-FRT-FLEX -GtACR2-EYFP Recombinant DNA reagent (plasmid) Flpo-dependent GtACR1 this paper Addgene:114370 pAAV-EF1α-FRT-FLEX -GtACR1-EYFP Recombinant DNA reagent (plasmid) GtACR2-EYFP-Kv2.1C this paper Addgene:114375 pAAV-EF1α-FRT- FLEX-GtACR2- EYFP-Kv2.1C Recombinant DNA reagent (plasmid) GtACR2-EYFP- Kv2.1C-linker-TlcnC this paper Addgene:114377 pAAV-EF1α-FRT- FLEX-GtACR2-EYFP- Kv2.1C-linker-TlcnC Transfected construct (adenoassociated virus) Flpo XuepersName et al. (2014) ; Penn Vector Core Addgene:60663 AAV9-hSyn-Flpo Transfected construct (adeno -associated virus) Flpo-dependent GtACR2term; GtACR2term This paper; Baylor College of Medicine Gene Vector Core Addgene:114369 AAV9-EF1α- FRT-FLEX -GtACR2-EYFP Transfected construct (adenoassociated virus) ReaChR This paper; Baylor College of Medicine Gene Vector Core Addgene:114368 AAV9-EF1α- DIO-ReaChR-P2A-dTomato Transfected construct (adenoassociated virus) iC++ This paper; Baylor College of Medicine Gene Vector Core AAV9-EF1α-DIOiC++-TS-EYFP; plasmid obtained from Dr. Karl Deisseroth Transfected construct (adeno-associated virus) iChloC This paper; Baylor College of Medicine Gene Vector Core AAV9-EF1α-DIOiChloC-T2A-mCherry; plasmid obtained from Drs. Matthew Caudill and Massimo Scanziani Chemical compound, drug Bumetanide (Ro 10–6338) Santa Cruz Biotechnology Santa Cruz:sc-200727 50 or 100 µM All procedures to maintain and use mice were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine. Mice were maintained on a 14 hr:10 hr light:dark cycle with regular mouse chow and water ad libitum . Experiments were performed during the light cycle. ICR (CD-1) female mice were purchased from Baylor College of Medicine Center for Comparative Medicine or Charles River Laboratories. C57BL6/J, Pvalb-2A-Cre , and Pvalb-2A-Flpo mice were obtained from Jackson Laboratory (stock numbers 000664, 012358, and 022730, respectively). Both male and female mice were used in the experiments. The mice were used at the age of 3–9 weeks for experiments, except for the conditional expression of GtACR2term in adults, where mice were used at the age of 10–12 weeks.

Plasmids pLenti-UbiC-GtACR2-EYFP (Addgene #67877) and pLenti-UbiC-GtACR1-EYFP (Addgene #67795) were obtained from Dr. John Spudich, pAAV-CaMKIIα-iC++-TS-EYFP and pAAV-EF1α-DIO-iC++-TS-EYFP from Dr. Karl Deisseroth, pAAV-EF1α-DIO-iChloC-T2A-mCherry from Drs. Matthew Caudill and Massimo Scanziani, pCAG-tdTomato from Anirvan Ghosh, and pCAG-Cre from Addgene (#13775). Plasmid pCAG-Flpo (Addgene #60662) was previously described ( XuepersName et al., 2014 ). All other plasmids were generated and deposited at Addgene as below. pCAG-hChR2(H134R)-EYFP (Addgene #114367) was created by replacing the EGFP in pCAG-EGFP (Addgene #11150) with the hChR2(H134R)-EYFP from pAAV-EF1α-DIO-hChR2(H134R)-EYFP (Addgene #20298). pAAV-EF1α-DIO-ReaChR-P2A-dTomato (Addgene #114368) was created by replacing the oChIEF(E163A T199C) in pAAV-EF1α-DIO-oChIEF(E163A T199C)-P2A-dTomato (Addgene #51094) with the ReaChR from pAAV-hSyn-FLEX-ReaChR-Citrine (Addgene #50955). pAAV-EF1α-FRT-FLEX-GtACR2-EYFP (Addgene #114369) and pAAV-EF1α-FRT-FLEX-GtACR1-EYFP (Addgene #114370) were created by replacing the mNaChBac-T2A-tdTomato in pAAV-EF1α-FRT-FLEX-mNaChBac-T2A-tdTomato (Addgene #60658) with the GtACR2-EYFP and GtACR1-EYFP from pLenti-UbiC-GtACR2-EYFP and pLenti-UbiC-GtACR1-EYFP, respectively. Motifs MBD and TlcnC were generated by PCR primers. Motifs Nlgn1C and Kv4.2LL were generated by PCR from pAAV-CAG-post-mGRASP-2A-dTomato (Addgene #34912) and a Kv4.2-expressing plasmid, respectively. Motif Kv2.1C was obtained from pAAV-EF1α-DIO-hChR2(H134R)-EYFP-Kv2.1C ( Wu et al., 2013 ). Motifs Kv2.1C-TlcnC and Kv2.1C-linker-TlcnC were generated by PCR from Kv2.1C and TlcnC. All motifs were then added to the C-terminus of the GtACR2-EYFP to create pAAV-EF1α-FRT-FLEX-GtACR2-EYFP-MBD (Addgene #114371), pAAV-EF1α-FRT-FLEX-GtACR2-EYFP-Nlgn1C (Addgene #114372), pAAV-EF1α-FRT-FLEX-GtACR2-EYFP-Kv4.2LL (Addgene #114373), pAAV-EF1α-FRT-FLEX-GtACR2-EYFP-TlcnC (Addgene #114374), pAAV-EF1α-FRT-FLEX-GtACR2-EYFP-Kv2.1C (Addgene #114375), pAAV-EF1α-FRT-FLEX-GtACR2-EYFP-Kv2.1C-TlcnC (Addgene #114376), and pAAV-EF1α-FRT-FLEX-GtACR2-EYFP-Kv2.1C-linker-TlcnC (Addgene #114377). pAAV-EF1α-FRT-FLEX-GtACR2-KA2N-EGFP (Addgene #114378) was created by replacing the EYFP in pAAV-EF1α-FRT-FLEX-GtACR2-EYFP with the KA2N-EGFP from pAAV-hSyn-soCoChR-EGFP (Addgene #107708).

Female ICR mice were crossed with male C57BL6/J, Pvalb-2A-Cre , or Pvalb-2A-Flpo mice to obtain timed pregnancies. In utero electroporation was performed as previously described ( XuepersName et al., 2014 ) with a square-wave pulse generator (Gemini X2, BTX Harvard Bioscience). To express GtACR2term, GtACR1, iC++, or ChR2 in layer 2/3 pyramidal neuronsterm, pLenti-UbiC-GtACR2-EYFP, pLenti-UbiC-GtACR1-EYFP, pAAV-CaMKIIα-iC++-TS-EYFP, or pCAG-hChR2(H134R)-EYFP (all 2 µg/µl) was used, respectively. In a few experiments, pAAV-EF1α-FRT-FLEX-GtACR2-EYFP (2 µg/µl) with pCAG-Flpo (0.2 µg/µl), pAAV-EF1α-FRT-FLEX-GtACR1-EYFP (2 µg/µl) with pCAG-Flpo (0.2 µg/µl), or pAAV-EF1α-DIO-iC++-TS-EYFP (2 µg/µl) with pCAG-Cre (0.2 µg/µl) was used to express GtACR2term, GtACR1, or iC++, respectively. To express somatodendritically targeted GtACR2 variantsterm·var in layer 2/3 pyramidal neuronsterm and compare them with wild type GtACR2term, the pAAV-EF1α-FRT-FLEX constructs described above were used (all 2 µg/µl) with pCAG-Flpo (0.2 µg/µl). pCAG-tdTomato (0.1 µg/µl) was included in all experiments. The plasmid concentrations stated above were final concentrations in the plasmid mix. Transfected pups were identified by the transcranial fluorescence of tdTomato with a MZ10F stereomicroscope (Leica) 1–2 days after birth.

All recombinant AAV serotype 9 vectors were produced by the Gene Vector Core at Baylor College of Medicine except AAV9-hSyn-Flpo (Addgene #60663), which was produced by the Penn Vector Core ( XuepersName et al., 2014 ). To express GtACR2term, ReaChR, iC++, or iChloC in Pv neuronsterm, 200–250 nl of the following recombinant AAV serotype 9 vectors at their respective titer were injected into the visual cortex of Pvalb Flpo/+ (for GtACR2term) or Pvalb Cre/+ (for ReaChR, iC++, or iChloC) mice at postnatal day 1 as previously described ( XuepersName et al., 2014 ): AAV9-EF1α-FRT-FLEX-GtACR2-EYFP (3.8 × 10 13 genome copies/ml), AAV9-EF1α-DIO-ReaChR-P2A-dTomato (7.0 × 10 13 genome copies/ml), AAV9-EF1α-DIO-iC++-TS-EYFP (3.71 × 10 13 genome copies/ml), and AAV9-EF1α-DIO-iChloC-2A-mCherry (3.7 × 10 14 genome copies/ml). To conditionally express GtACR2term in juvenile and adult neuronsterm, mice previously electroporated with plasmid pAAV-EF1α-FRT-FLEX-GtACR2-EYFP into layer 2/3 pyramidal neuronsterm were injected with 200 nl of AAV9-hSyn-Flpo (1.2 × 10 12 genome copies/ml) at postnatal day 23, 60, or 64. Injection was performed as previously described ( XuepersName et al., 2014 ) with an UltraMicroPump III and a Micro4 controller (World Precision Instruments).

Mice were anesthetized by an intraperitoneal injection of a ketamine and xylazine mix (80 mg/kg and 16 mg/kg, respectively) and transcardially perfused with cold (0–4°C) slice cutting solution containing 80 mM NaCl, 2.5 mM KCl, 1.3 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 4 mM MgCl 2 , 0.5 mM CaCl 2 , 20 mM D-glucose, 75 mM sucrose and 0.5 mM sodium ascorbate (315 mosmol, pH 7.4, saturated with 95% O 2 /5% CO 2 ). Brains were removed and sectioned in the cutting solution with a VT1200S vibratome (Leica) to obtain 300 μm coronal slices. Slices were incubated in a custom-made interface holding chamber containing slice cutting solution saturated with 95% O 2 /5% CO 2 at 34°C for 30 min and then at room temperature for 20 min to 10 hr until they were transferred to the recording chamber.

Recordings were performed on submerged slices in artificial cerebrospinal fluid (ACSF) containing 119 mM NaCl, 2.5 mM KCl, 1.3 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 1.3 mM MgCl 2 , 2.5 mM CaCl 2 , 20 mM D-glucose and 0.5 mM sodium ascorbate (305 mosmol, pH 7.4, saturated with 95% O 2 /5% CO 2 , perfused at 3 ml/min) at 30–32°C. For whole-cell recordings, a K + -based pipette solution containing 142 mM K + -gluconate, 10 mM HEPES, 1 mM EGTA, 2.5 mM MgCl2, 4 mM ATP-Mg, 0.3 mM GTP-Na, 10 mM Na2-phosphocreatine (295 mosmol, pH 7.35) or a Cs + -based pipette solution containing 121 mM Cs + -methanesulfonate, 1.5 mM MgCl 2 , 10 mM HEPES, 10 mM EGTA, 4 mM Mg-ATP, 0.3 mM Na-GTP, 10 mM Na 2 -Phosphocreatine, and 2 mM QX314-Cl (295 mosmol, pH 7.35) was used. Membrane potentialsterm were not corrected for liquid junction potential (experimentally measured as 12.5 mV for the K + -based pipette solution and 9.5 mV for the Cs + -based pipette solution).

Neuronsterm were visualized with video-assisted infrared differential interference contrast imaging, and fluorescent neuronsterm were identified by epifluorescence imaging under a water immersion objective (40×, 0.8 numerical aperture) on an upright SliceScope Pro 1000 microscope (Scientifica) with an infrared IR-1000 CCD camera (DAGE-MTI). Data were low-pass filtered at 4 kHz and acquired at 10 kHz with an Axonterm Multiclamp 700B amplifier and an Axonterm Digidata 1550 Data Acquisition System under the control of Clampex 10.7 (Molecular Devices). Data were analyzed offline using AxoGraph X (AxoGraph Scientific). For the photostimulation of GtACR2-, iC++-, iChloC-, or ChR2-expressing neuronsterm, blue light was emitted from a collimated light-emitting diode (LED) of 455 nm, whereas for the photostimulation of GtACR1- or ReaChR-expressing neuronsterm, red light was emitted from a LED of 617 nm. The LEDs were driven by a LED driver (Mightex) under the control of an Axonterm Digidata 1550 Data Acquisition System and Clampex 10.7. Light was delivered through the reflected light fluorescence illuminator port and the 40× objective.

Synaptic currents and photocurrentsterm were recorded in the whole-cell voltage clamp mode with the Cs + -based patch pipette solution. Only recordings with series resistance below 20 MΩ were included. EPSCsterm and IPSCsterm were recorded at the reversal potentialterm for IPSCsterm (−60 mV) and EPSCsterm (+10 mV), respectively, unless stated otherwise. Photocurrentsterm were recorded at +10 mV unless stated otherwise. For short light pulse stimulationterm·var, pulse duration (0.5–10 ms) and intensity (2.3–23.6 mW/mm 2 ) were adjusted for each recording to evoke small (to minimize voltage-clamp errors) but reliable monosynaptic EPSCsterm or IPSCsterm. Disynaptic IPSCsterm were evoked using the same light pulses that were used for evoking the corresponding monosynaptic EPSCsterm. Light pulses were delivered at 30 s interstimulus intervals. For long light pulse stimulation, blue light was delivered for 2 s at 60 s interstimulus intervals. To obtain the cumulative charge transfer curves, the EPSCterm traces were high-pass filtered at 0.3 Hz to minimize the effect of slow changes in the baselines, except for two neuronsterm that were filtered at 0.5 Hz or 1.3 Hz. It was not necessary to high-pass filter the IPSCterm traces. The traces were baselined before the light onset and then integrated to calculate the cumulative charge transfers. Antidromic spikes in GtACR2term + neuronsterm were recorded with the K+-based patch pipette solution in whole-cell current clamp mode or with ACSF as the patch pipette solution in the loose-patch current clamp mode.

For pharmacology experiments, the baseline synaptic currents were recorded for at least 3 min in the absence of any drug. The drugs were then added to the ACSF at the following concentrations: TTX (1 μM), NBQX (10 μM), ( RS )-CPP (10 μM), SR95531 (Gabazine, 10 μM), ZD7288 (20 μM), bumetanide (50 or 100 μM), TEA (1.5 mM), and 4-AP (1.5 mM). The synaptic currents were recorded for at least 3 min in the presence of drugs. For ZD7288, which did not inhibit GtACR2-induced neurotransmitterterm release, the efficacy of the drug was monitored by examining the I h current of cortical layer 5 pyramidal neuronsterm.

Fluorescent images were taken from live brain slices, except for the conditional expression of GtACR2term in adults, where images were taken from fixed brain slices. Live brain slices were prepared as described for slice electrophysiologyterm·mth. For the fixed brain slices, mice were anesthetized by an intraperitoneal injection of a ketamine and xylazine mix (80 mg/kg and 16 mg/kg, respectively) and transcardially perfused with phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS (pH 7.4). Brains were further fixed overnight in 4% paraformaldehyde, cryoprotected with 30% sucrose in PBS, and frozen in optimum cutting-temperature medium until sectioning. A HM 450 Sliding Microtome (Thermo Scientific) was used to section the brains to obtain 30–50 mm coronal slices. Images were acquired on an Axio Zoom.V16 Fluorescence Stereo Zoom Microscope (Zeiss) and processed using National Institutes of Health ImageJ.

To determine the EYFP (or EGFP) fluorescence ratio between layer 5 and layer 2/3, one or two 350 µm-wide rectangular regions that were perpendicular to the pia and spanned all 6 cortical layers were selected in the most transfected regions of each slice. The mean EYFP (or EGFP) fluorescence was measured for layer 5 and layer 2/3 within the selected area. The mean tdTomato fluorescence was measured similarly for layer 5 and layer 2/3. The mean background fluorescence was measured from a nearby rectangular region (140.5 µm by 90.8 µm) where no cellular EYFP (or EGFP) and tdTomato fluorescence was present. The normalized EYFP (or EGFP) fluorescence ratio between layer 5 and layer 2/3 was calculated by L a y e r 5 E Y F P - B a c k g r o u n d E Y F P L a y e r 2 / 3 E Y F P - B a c k g r o u n d E Y F P / L a y e r 5 t d T o m a t o - B a c k g r o u n d t d T o m a t o L a y e r 2 / 3 t d T o m a t o - B a c k g r o u n d t d T o m a t o .

To determine the ratio of EYFP fluorescence to tdTomato fluorescence in the callosal projections, one or two rectangular regions that contained the tdTomato-labeled axonsterm were selected in each slice to measure the mean EYFP and tdTomato fluorescence. The mean background fluorescence was measured in a nearby cortical area spanning the same layers. The ratio of EYFP fluorescence to tdTomato fluorescence was calculated by F l u o r e s c e n c e E Y F P - B a c k g r o u n d E Y F P F l u o r e s c e n c e t d T o m a t o - B a c k g r o u n d t d T o m a t o .

All reported sample numbers ( n ) represent biological replicatesterm·def that are the numbers of recorded neuronsterm for electrophysiologyterm·mth or the numbers of analyzed regions of interest (ROI) for fluorescent images. Statistical analyses were performed with Prism 7 (GraphPad Software). We first determined whether the data were normally distributed by performing the D’Agostino and Pearson test, Shapiro-Wilk test, and KS testterm·stat. If all data within one experiment passed all three normality teststerm·stat, we then performed the statistical test that assumes a Gaussian distribution. Otherwise, we performed the statistical test that assumes a non-Gaussian distribution. All statistical tests were two-tailed with an alpha of 0.05.

Wilcoxan matched-pairs signed rank testterm·stat was used for Figure 1F,K ; Figure 2B,D,F,J ; Figure 2—figure supplement 2B,E ; Figure 2—figure supplement 3B (EPSCsterm); Figure 3B ; and Figure 3—figure supplement 1C . Paired t testterm·stat was used for Figure 1G,J ; Figure 2H ; Figure 2—figure supplement 1D ; and Figure 2—figure supplement 3B (photocurrentsterm); Figure 2—figure supplement 3D . t test with Welch’s correctionterm·stat was used for Figure 5C (WT vs. Kv2.1C). Mann-Whitney testterm·stat was used for Figure 5C (WT vs. Kv2.1C-linker-TlcnC); Figure 5F,G,I ; Figure 5—figure supplement 1C,D . Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey multiple comparisonsterm·stat with multiplicity adjusted P values was used for Figure 3D,F . Kruskal-Wallis test with Dunn’s multiple comparisonsterm·stat with multiplicity adjusted P values was used for Figure 4D . Ordinary one-way ANOVA with Dunnet’s multiple comparison test with multiplicity adjusted P values was used for Figure 4E . The details of all statistical tests, numbers of replicates and mice, and P values were reported in Supplementary file 1 .

Add a comment + Open annotations. The current annotation count on this page is being calculated .

All data generated or analyzed during this study are included in the manuscript and supporting files.

Allen BD Singer AC Boyden ES (2015) Principles of designing interpretable optogeneticterm·mth behavior experiments Learning & Memory 22 :232–238.

https://doi.org/10.1101/lm.038026.114 PubMed Google Scholar Baker CA Elyada YM Parra A Bolton MM (2016) Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin eLife 5 :11981.

https://doi.org/10.7554/eLife.14193 PubMed Google Scholar Ben-Ari Y (2002) Excitatoryterm actions of gaba during development: the nature of the nurture Nature Reviews Neuroscience 3 :728–739.

https://doi.org/10.1038/nrn920 PubMed Google Scholar Ben-Ari Y (2017) NKCC1 chlorideterm importer antagonists attenuate many neurological and psychiatric disorders Trends in Neurosciences 40 :536–554.

https://doi.org/10.1016/j.tins.2017.07.001 PubMed Google Scholar Berndt A Lee SY Ramakrishnan C Deisseroth K (2014) Structure-guided transformation of channelrhodopsin into a light-activated chlorideterm channelterm Science 344 :420–424.

https://doi.org/10.1126/science.1252367 PubMed Google Scholar Berndt A Lee SY Wietek J Ramakrishnan C Steinberg EE Rashid AJ Kim H Park S Santoro A Frankland PW Iyer SM Pak S Ährlund-Richter S Delp SL Malenka RC Josselyn SA Carlén M Hegemann P Deisseroth K (2016) Structural foundations of optogeneticsterm·mth: determinants of channelrhodopsin ion selectivity PNAS 113 :822–829.

https://doi.org/10.1073/pnas.1523341113 PubMed Google Scholar Boyden ES Zhang F Bamberg E Nagel G Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity Nature Neuroscience 8 :1263–1268.

https://doi.org/10.1038/nn1525 PubMed Google Scholar Chow BY Han X Dobry AS Qian X Chuong AS Li M Henninger MA Belfort GM Lin Y Monahan PE Boyden ES (2010) High-performance genetically targetable optical neural silencing by light-driven proton pumpsterm Nature 463 :98–102.

https://doi.org/10.1038/nature08652 PubMed Google Scholar Chuong AS Miri ML Busskamp V Matthews GA Acker LC Sørensen AT Young A Klapoetke NC Henninger MA Kodandaramaiah SB Ogawa M Ramanlal SB Bandler RC Allen BD Forest CR Chow BY Han X Lin Y Tye KM Roska B Cardin JA Boyden ES (2014) Noninvasive optical inhibitionterm with a red-shifted microbial rhodopsin Nature Neuroscience 17 :1123–1129.

https://doi.org/10.1038/nn.3752 PubMed Google Scholar Forli A Vecchia D Binini N Succol F Bovetti S Moretti C Nespoli F Mahn M Baker CA Bolton MM Yizhar O Fellin T (2018) Two-Photon bidirectional control and imaging of neuronal excitability with high spatial resolution in Vivo Cell Reports 22 :3087–3098.

https://doi.org/10.1016/j.celrep.2018.02.063 PubMed Google Scholar Govorunova EG Sineshchekov OA Janz R Liu X Spudich JL (2015) NEUROSCIENCE. natural light-gated anion channelsterm: a family of microbial rhodopsins for advanced optogeneticsterm·mth Science 349 :647–650.

https://doi.org/10.1126/science.aaa7484 PubMed Google Scholar Govorunova EG Sineshchekov OA Li H Spudich JL (2017a) Microbial rhodopsins: diversity, mechanisms, and optogeneticterm·mth applications Annual Review of Biochemistry 86 :845–872.

https://doi.org/10.1146/annurev-biochem-101910-144233 PubMed Google Scholar Govorunova EG Sineshchekov OA Rodarte EM Janz R Morelle O Melkonian M Wong GK Spudich JL (2017b) The expanding family of natural anion channelrhodopsins reveals large variations in kinetics, conductance, and spectral sensitivity Scientific Reports 7 :43358.

https://doi.org/10.1038/srep43358 PubMed Google Scholar Han X Boyden ES (2007) Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution PLoS ONE 2 :e299.

https://doi.org/10.1371/journal.pone.0000299 PubMed Google Scholar Han X Chow BY Zhou H Klapoetke NC Chuong A Rajimehr R Yang A Baratta MV Winkle J Desimone R Boyden ES (2011) A high-light sensitivity optical neural silencer: development and application to optogeneticterm·mth control of non-human primate cortex Frontiers in Systems Neuroscience 5 :18.

https://doi.org/10.3389/fnsys.2011.00018 PubMed Google Scholar Jang IS Nakamura M Ito Y Akaike N (2006) Presynapticterm GABAA receptors facilitate spontaneous glutamateterm release from presynapticterm terminals on mechanically dissociated rat CA3 pyramidal neuronsterm Neuroscience 138 :25–35.

https://doi.org/10.1016/j.neuroscience.2005.11.001 PubMed Google Scholar Khirug S Yamada J Afzalov R Voipio J Khiroug L Kaila K (2008) GABAergic depolarization of the axonterm initial segment in cortical principal neuronsterm is caused by the Na-K-2Cl cotransporter NKCC1 Journal of Neuroscience 28 :4635–4639.

https://doi.org/10.1523/JNEUROSCI.0908-08.2008 PubMed Google Scholar Lewis TL Mao T Svoboda K Arnold DB (2009) Myosin-dependent targeting of transmembrane proteins to neuronal dendrites Nature Neuroscience 12 :568–576.

https://doi.org/10.1038/nn.2318 PubMed Google Scholar Li X Gutierrez DV Hanson MG Han J Mark MD Chiel H Hegemann P Landmesser LT Herlitze S (2005) Fast noninvasive activation and inhibitionterm of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin PNAS 102 :17816–17821.

https://doi.org/10.1073/pnas.0509030102 PubMed Google Scholar Lim ST Antonucci DE Scannevin RH Trimmer JS (2000) A novel targeting signal for proximal clustering of the Kv2.1 K+ channelterm in hippocampal neuronsterm Neuronterm 25 :385–397.

https://doi.org/10.1016/S0896-6273(00)80902-2 PubMed Google Scholar Lin JY Knutsen PM Muller A Kleinfeld D Tsien RY (2013) ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogeneticterm·mth excitation Nature Neuroscience 16 :1499–1508.

https://doi.org/10.1038/nn.3502 PubMed Google Scholar Madisen L Garner AR Shimaoka D Chuong AS Klapoetke NC Li L van der Bourg A Niino Y Egolf L Monetti C Gu H Mills M Cheng A Tasic B Nguyen TN Sunkin SM Benucci A Nagy A Miyawaki A Helmchen F Empson RM Knöpfel T Boyden ES Reid RC Carandini M Zeng H (2015) Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance Neuronterm 85 :942–958.

https://doi.org/10.1016/j.neuronterm.2015.02.022 PubMed Google Scholar Madisen L Zwingman TA Sunkin SM Oh SW Zariwala HA Gu H Ng LL Palmiter RD Hawrylycz MJ Jones AR Lein ES Zeng H (2010) A robust and high-throughput cre reporting and characterization system for the whole mouse brain Nature Neuroscience 13 :133–140.

https://doi.org/10.1038/nn.2467 PubMed Google Scholar Mahn M Gibor L Malina KC-K Patil P Printz Y Oring S Levy R Lampl I Yizhar O (2017) High-efficiency optogeneticterm·mth silencing with soma-targeted anion-conducting channelrhodopsins bioRxiv .

https://doi.org/10.1101/225847 Google Scholar Mahn M Prigge M Ron S Levy R Yizhar O (2016) Biophysical constraints of optogeneticterm·mth inhibitionterm at Presynapticterm terminals Nature Neuroscience 19 :554–556.

https://doi.org/10.1038/nn.4266 PubMed Google Scholar Malyshev AY Roshchin MV Smirnova GR Dolgikh DA Balaban PM Ostrovsky MA (2017) Chlorideterm conducting light activated channelterm GtACR2term can produce both cessation of firing and generation of action potentials in cortical neuronsterm in response to light Neuroscience Letters 640 :76–80.

https://doi.org/10.1016/j.neulet.2017.01.026 PubMed Google Scholar Mardinly AR Oldenburg IA Pégard NC Sridharan S Lyall EH Chesnov K Brohawn SG Waller L Adesnik H (2018) Precise multimodal optical control of neural ensemble activity Nature Neuroscience 21 :881–893.

https://doi.org/10.1038/s41593-018-0139-8 PubMed Google Scholar Marty A Llano I (2005) Excitatoryterm effects of GABA in established brain networks Trends in Neurosciences 28 :284–289.

https://doi.org/10.1016/j.tins.2005.04.003 PubMed Google Scholar Mauss AS Busch C Borst A (2017) Optogeneticterm·mth neuronal silencing in Drosophila during visual processing Scientific Reports 7 :13823.

https://doi.org/10.1038/s41598-017-14076-7 PubMed Google Scholar Mitsui S Saito M Hayashi K Mori K Yoshihara Y (2005) A novel phenylalanine-based targeting signal directs telencephalin to neuronal dendrites Journal of Neuroscience 25 :1122–1131.

https://doi.org/10.1523/JNEUROSCI.3853-04.2005 PubMed Google Scholar Mohamed GA Cheng RK Ho J Krishnan S Mohammad F Claridge-Chang A Jesuthasan S (2017) Optical inhibitionterm of larval zebrafish behaviour with anion channelrhodopsins BMC Biology 15 :103.

https://doi.org/10.1186/s12915-017-0430-2 PubMed Google Scholar Mohammad F Stewart JC Ott S Chlebikova K Chua JY Koh TW Ho J Claridge-Chang A (2017) Optogeneticterm·mth inhibitionterm of behavior with anion channelrhodopsins Nature Methods 14 :271–274.

https://doi.org/10.1038/nmeth.4148 PubMed Google Scholar Nagel G Szellas T Huhn W Kateriya S Adeishvili N Berthold P Ollig D Hegemann P Bamberg E (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channelterm PNAS 100 :13940–13945.

https://doi.org/10.1073/pnas.1936192100 PubMed Google Scholar Owens DF Boyce LH Davis MB Kriegstein AR (1996) Excitatoryterm GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging The Journal of Neuroscience 16 :6414–6423.

https://doi.org/10.1523/JNEUROSCI.16-20-06414.1996 PubMed Google Scholar Petreanu L Mao T Sternson SM Svoboda K (2009) The subcellular organization of neocortical excitatoryterm connections Nature 457 :1142–1145.

https://doi.org/10.1038/nature07709 PubMed Google Scholar Price GD Trussell LO (2006) Estimate of the chlorideterm concentration in a central glutamatergic terminal: a gramicidin perforated-patch study on the calyx of Held Journal of Neuroscience 26 :11432–11436.

https://doi.org/10.1523/JNEUROSCI.1660-06.2006 PubMed Google Scholar Pugh JR Jahr CE (2011) Axonal GABAA receptors increase cerebellar granule cell excitability and synaptic activity Journal of Neuroscience 31 :565–574.

https://doi.org/10.1523/JNEUROSCI.4506-10.2011 PubMed Google Scholar Raimondo JV Kay L Ellender TJ Akerman CJ (2012) Optogeneticterm·mth silencing strategies differ in their effects on inhibitory synaptic transmission Nature Neuroscience 15 :1102–1104.

https://doi.org/10.1038/nn.3143 PubMed Google Scholar Rivera JF Ahmad S Quick MW Liman ER Arnold DB (2003) An evolutionarily conserved dileucine motif in Shal K+ channelsterm mediates dendritic targeting Nature Neuroscience 6 :243–250.

https://doi.org/10.1038/nn1020 PubMed Google Scholar Robinson RB Siegelbaum SA (2003) Hyperpolarization-activated cation currents: from molecules to physiological function Annual Review of Physiology 65 :453–480.

https://doi.org/10.1146/annurev.physiol.65.092101.142734 PubMed Google Scholar Rosales CR Osborne KD Zuccarino GV Scheiffele P Silverman MA (2005) A cytoplasmic motif targets neuroligin-1 exclusively to dendrites of cultured hippocampal neuronsterm European Journal of Neuroscience 22 :2381–2386.

https://doi.org/10.1111/j.1460-9568.2005.04400.x PubMed Google Scholar Ruiz A Campanac E Scott RS Rusakov DA Kullmann DM (2010) Presynapticterm GABAA receptors enhance transmission and LTP induction at hippocampal mossy fiber synapses Nature Neuroscience 13 :431–438.

https://doi.org/10.1038/nn.2512 PubMed Google Scholar Shemesh OA Tanese D Zampini V Linghu C Piatkevich K Ronzitti E Papagiakoumou E Boyden ES Emiliani V (2017) Temporally precise single-cell-resolution optogeneticsterm·mth Nature Neuroscience 20 :1796–1806.

https://doi.org/10.1038/s41593-017-0018-8 PubMed Google Scholar Stell BM Rostaing P Triller A Marty A (2007) Activation of presynapticterm GABA(A) receptors induces glutamateterm release from parallel fiber synapses Journal of Neuroscience 27 :9022–9031.

https://doi.org/10.1523/JNEUROSCI.1954-07.2007 PubMed Google Scholar Trigo FF Marty A Stell BM (2008) Axonal GABAA receptors The European Journal of Neuroscience 28 :841–848.

https://doi.org/10.1111/j.1460-9568.2008.06404.x PubMed Google Scholar Turecek R Trussell LO (2001) Presynapticterm glycine receptors enhance transmitter release at a mammalian central synapse Nature 411 :587–590.

https://doi.org/10.1038/35079084 PubMed Google Scholar Wietek J Beltramo R Scanziani M Hegemann P Oertner TG Wiegert JS Simon Wiegert J (2015) An improved chloride-conducting channelrhodopsin for light-induced inhibitionterm of neuronal activity in vivo Scientific Reports 5 :14807.

https://doi.org/10.1038/srep14807 PubMed Google Scholar Wietek J Wiegert JS Adeishvili N Schneider F Watanabe H Tsunoda SP Vogt A Elstner M Oertner TG Hegemann P (2014) Conversion of channelrhodopsin into a light-gated chlorideterm channelterm Science 344 :409–412.

https://doi.org/10.1126/science.1249375 PubMed Google Scholar Wu C Ivanova E Zhang Y Pan ZH (2013) rAAV-mediated subcellular targeting of optogeneticterm·mth tools in retinal ganglion cells in vivo PLoS One 8 :e66332.

https://doi.org/10.1371/journal.pone.0066332 PubMed Google Scholar XuepersName M Atallah BV Scanziani M (2014) Equalizing excitation-inhibition ratios across visual cortical neuronsterm Nature 511 :596–600.

https://doi.org/10.1038/nature13321 PubMed Google Scholar Zhang F Wang LP Brauner M Liewald JF Kay K Watzke N Wood PG Bamberg E Nagel G Gottschalk A Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry Nature 446 :633–639.

https://doi.org/10.1038/nature05744 PubMed Google Scholar Zorrilla de San Martin J Trigo FF Kawaguchi SY (2017) Axonal GAB AA receptors depolarize presynapticterm terminals and facilitate transmitter release in cerebellar Purkinje cells The Journal of Physiology 595 :7477–7493.

https://doi.org/10.1113/JP275369 PubMed Google Scholar Author details Jessica E MessierpersName Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States The Cain Foundation Laboratories, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, Texas, United States Contribution Conceptualization, Resources, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing Competing interests No competing interests declared "This ORCID iD identifies the author of this article:" 0000-0002-5865-7043 Hongmei ChenpersName Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States The Cain Foundation Laboratories, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, Texas, United States Contribution Resources, Investigation Competing interests No competing interests declared Zhao-Lin CaipersName Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States The Cain Foundation Laboratories, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, Texas, United States Contribution Validation, Investigation, Writing—review and editing Competing interests No competing interests declared "This ORCID iD identifies the author of this article:" 0000-0003-4034-2884 Mingshan XuepersName Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States The Cain Foundation Laboratories, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, Texas, United States Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States Contribution Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing For correspondence Mingshan.XuepersName@bcm.edu Competing interests No competing interests declared "This ORCID iD identifies the author of this article:" 0000-0003-1463-8884 Mingshan XuepersName Jessica E MessierpersName Mingshan XuepersName The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank Elena Govorunova and John Spudich for discussions and the pLenti-UbiC-GtACR2-EYFP and pLenti-UbiC-GtACR1-EYFP plasmids, Anirvan Ghosh for the pCAG-tdTomato plasmid, Karl Deisseroth for the pAAV-CaMKIIα-iC++-TS-EYFP and pAAV-EF1α-DIO-iC++-TS-EYFP plasmids, Matthew Caudill and Massimo Scanziani for the pAAV-EF1α-DIO-iChloC-T2A-mCherry plasmid, Zhuo-Hua Pan for the pAAV-EF1α-DIO-hChR2(H134R)-EYFP-Kv2.1C plasmid, Edward Boyden for the pAAV-hSyn-soCoChR-EGFP plasmid, Paul Pfaffinger for the Kv4.2-expressing plasmid, Shuyun Deng and Kazuhiro Oka at the Baylor College of Medicine Gene Vector Core for recombinant AAV vector production, and Hsiao-Tuan Chao, Matthew Caudill, and the members of the XuepersName Laboratory for comments on earlier versions of the manuscript. This work was supported in part by the Whitehall Foundation (grant #2015-05-54 to MX) and the National Institutes of Health (grant R01NS100893 to MX). JEM is part of the Baylor College of Medicine Medical Scientist Training Program and a McNair MD/PhD Student Scholar supported by the McNair Medical Institute at the Robert and Janice McNair Foundation. MX is a Caroline DeLuca Scholar.

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures to maintain and use mice were approved in the Animal Research Protocol AN-6544 by the Institutional Animal Care and Use Committee at Baylor College of Medicine.

© 2018, MessierpersName et al.

This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.