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

Jessica Messier, Hongmei Chen, Zhao-Lin Cai, Mingshan Xue

Preprint posted on 25 May 2018

Article now published in eLife at


High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins

Mathias Mahn, Lihi Gibor, Katayun Cohen-Kashi Malina, Pritish Patil, Yoav Printz, Shir Oring, Rivka Levy, Ilan Lampl, Ofer Yizhar

Preprint posted on 8 December 2017

Article now published in Nature Communications at

Advanced soma-targeting of light-gated chloride channels delivers the most potent inhibitory optogenetic control to date.

Selected by Mahesh Karnani


Recent advances have enhanced optogenetic inhibition by targeting a powerful light-gated chloride channel mostly to the somatodendritic compartment of neurons.



Optogenetics, i.e., the use of light controlled membrane proteins to manipulate electrical activity of neurons, is a decisive technique in circuit neuroscience and beyond. Particularly the millisecond precision and cell specificity of this perturbation method enable profound advances in circuit studies. Neuronal activation is achieved routinely with channelrhodopsin variants to suit the illumination wavelength and speed required. However, neuronal inhibition with optogenetics is less trivial1. This is fundamentally due to the different transmembrane driving forces of ions available for inhibition compared to those for depolarisation. Reversal potentials of inhibiting anion channel currents are more varied and closer to a typical resting membrane potential than the reversal potentials of depolarising cation channel currents. This causes problems for inhibition. One problem is that chloride channels are commonly excitatory at axon terminals while being inhibitory in somatodendritic domains of mature neurons. In contrast, cation channels like channelrhodopsin will depolarise and elicit action potentials regardless of subcellular compartment. Besides channels, ionic pumps may be used to manipulate the membrane potential. This has been a useful strategy for optogenetic inhibition, and experimenters have greatly benefited from the light activated inward chloride pump halorhodopsin, and the outward proton pump archeorhodopsin. These come with some disadvantages though – the hyperpolarising current achieved with pumps is less effective at inhibition than inhibitory ion channels, and archeorhodopsin has an unforeseen side-effect of exciting some axons due to calcium influx triggered by increased intracellular pH2. For these reasons, the high conductance anion channelrhodopsin GtACR2 is a promising advance in optogenetic inhibition, if its expression can be excluded from axons where it would be excitatory.


Key findings

These preprints show that GtACR2 is excitatory in axons, in complementary sets of experiments mostly in cultured neurons and in vivo (Mahn et al.), and in brain slices with pharmacological manipulations (Messier et al.). Both preprints demonstrate light activated postsynaptic currents in neurons that are innervated by GtACR2 bearing fibers. Messier and colleagues went on to show these currents were sensitive to synaptic blockers and were abolished by action potential blockade with tetrodotoxin. The currents reappeared when 4-aminopyridine and tetraethylammonium were added on top of tetrodotoxin to prolong membrane depolarisation – a tell-tale sign of direct depolarisation of presynaptic terminals (see Figure). Both studies also observed antidromic (initiated in the axon) spikes during light induced hyperpolarisation in the somata of GtACR2 bearing cells, in line with axonal depolarisation.

Besides GtACR2 and its variant GtACR1, two other light-gated chloride channels, iC++ and iChloC, performed similarly, though GtACR2 was more powerful. This is why both studies selected GtACR2 for a further modification that targets it to the somato-dendritic compartment. Mahn and colleagues fused GtACR2 to the Kv2.1 C-terminal segment (Kv2.1C), a strategy that has been used in two recent publications3,4. Messier and colleagues screened eight different soma-targeting motifs, including Kv2.1C, and found greatest somatodendritic specificity with a hybrid targeting motif Kv2.1C-linker-TlcnC. Both studies found several-fold increased somatic inhibitory current and decreased axonal activation with their soma-targeting constructs.

Excitatory postsynaptic currents elicited by a chloride conducting opsin reported by two laboratories. Two panels from left from Messier et al., 2018, Figure 3 (under CC-BY 4.0), and two panels from right from Mahn et al., 2017, Figure 5 (with permission from the author).


Why I chose these preprints 

I am a believer in the transformative power of optogenetics, but sometimes I note more seasoned researchers ‘stroking their beards’ tentatively when these beliefs are laid bare. I think this is because optogenetics is a relatively young technique and as such we are still unclear of all the caveats. The ambiguity of effects of inhibitory opsins has prompted a few studies before these preprints5,2. On the other hand, we might question how physiologically relevant it is to use excitatory opsins to activate a population of neurons at millisecond synchrony out of the blue from the circuit’s perspective. Might it be more normative for a circuit to gradually ramp up its activity and find its own resonant frequency than to receive a constant frequency pulse train? Is it relevant to average across repeated trials given that there are likely plasticity effects from such a powerful activation?

Another reason these studies are crucial is that targeting opsins exclusively to somata is the way forward for true single-cell resolution optical control3,4,6,7. It is clear from many studies that off-target activation of the neuropil structures (i.e., dendrites and axons) adjacent to somata is a caveat for single cell manipulation even with state-of-the-art optical systems. Sparse, soma-specific expression offers a solution.


What next?

These studies honestly and constructively address the challenges inherent in optogenetic inhibition. The authors have made considerable progress toward reliable optogenetic inhibition by targetting the opsin to somatodendritic domain. The achieved soma-specific expression is not total in either case however. Higher soma-specificity would be highly welcome, but how could this be achieved? Messier and colleagues mention engineering inward rectification to the chloride channel, seeking better soma-targeting motifs or simply reducing overall expression level of the current mostly soma-targeted protein such that the axonal activation would be minimal. One might imagine that for pyramidal neurons targeting opsin expression to the axon-initial-segment could yield further improvement. Another possibility may be some form of two-component strategy involving a soma-specific structure (nuclear membrane protein interacting with plasma-membrane?). On the other hand, just for reliable inhibition in general, a light-gated potassium channel might be optimal1, as the potassium equilibrium potential tends to remain hyperpolarised in axons.



  1. Wiegert, J. S., Mahn, M., Prigge, M., Printz, Y. & Yizhar, O. Silencing Neurons: Tools, Applications, and Experimental Constraints. Neuron 95, 504–529 (2017).
  2. Mahn, M., Prigge, M., Ron, S., Levy, R. & Yizhar, O. Biophysical constraints of optogenetic inhibition at presynaptic terminals. Nat. Neurosci. 19, 554–556 (2016).
  3. Forli, A. et al. Two-Photon Bidirectional Control and Imaging of Neuronal Excitability with High Spatial Resolution In Vivo. Cell Rep. 22, 3087–3098 (2018).
  4. Mardinly, A. R. et al. Precise multimodal optical control of neural ensemble activity. Nat. Neurosci. 21, 881–893 (2018).
  5. El-Gaby, M. et al. Archaerhodopsin Selectively and Reversibly Silences Synaptic Transmission through Altered pH. Cell Rep. 16, 2259–2268 (2016).
  6. Shemesh, O. A. et al. Temporally precise single-cell-resolution optogenetics. Nat. Neurosci. 20, 1796–1806 (2017).
  7. Baker, C. A., Elyada, Y. M., Parra, A. & Bolton, M. M. Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin. Elife 5, (2016).


Posted on: 22 July 2018

(1 votes)

Author's response

Jessica Messier shared about Targeting light-gated chloride channels to neuronal somatodendritic domain reduces their excitatory effect in the axon

Thank you for allowing our pre-print manuscript to receive feedback via preLights. We believe our work, along with the work of Mahn and colleagues, provide complementary and convincing data that shows that, at least in some neuronal subtypes, activation of light-gated chloride channels can result in neurotransmitter release due to an excitatory axonal chloride gradient. Both our group and Mahn et al. attempted to decrease the unwanted neurotransmitter release by targeting GtACR2 to the somatodendritic compartment. While the work from Mahn et al. suggests that their somatodendritic-targeted version essentially eliminated antidromic spikes and neurotransmitter release, we found a less dramatic effect, with both the same motif Mahn et al. tested (Kv2.1C) and our “best” motif (Kv2.1C-linker-TlcnC). This could be due to the fact that Mahn et al. used a construct that also had an additional trafficking signal (“ts”) between GtACR2 and the fluorescent protein or because they were activating GtACR2 in different neurons than us. Regardless, these results support the fact that experimenters should test their “tools” in their own experimental paradigms to make sure they are working as expected. 


The author team shared about High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins

Thank you for featuring our pre-print on preLights. In our view, our study and that of Messier and colleagues highlight three important points. The first and perhaps most important point, is that the chloride reversal potential in the axonal compartment needs to be further investigated. The current pervasive opinion mostly regards evidence of an excitatory chloride reversal potential in the axonal compartment (Haam, 2012) as exceptions rather than the rule. An excitatory chloride reversal potential in the axonal compartment would have important implications on the functional role of presynaptic GABAA receptors (Ruiz, 2003 & 2010; Woodruff, 2006; Alle, 2007; Pugh, 2011). To emphasize this point, we used short, high light-power illumination pulses and thereby maximized light onset-trigged action potential initiation. From the perspective of optogenetic tool applications, such transient excitation at the beginning of a longer light pulse might be tolerable, much akin to (and perhaps less problematic than) the rebound excitation observed following optogenetic silencing with a wide range of inhibitory tools (Raimondo 2012, Chuong 2014, Mahn 2016). Furthermore, we show that efficient soma silencing is possible at lower light powers than needed to reliably evoke antidromic spiking. Second, as Messier et al. state in their comment, these studies, along with other previous studies (Raimondo, 2012; Herman, 2014; Mahn, 2016), are an important reminder that optogenetic manipulations should be performed in combination with electrophysiological methods to allow for the careful characterization of the effect of a given manipulation. Finally, soma-targeting using the Kv2.1 targeting signal, along with a trafficking signal, greatly enhanced membrane targeting while at the same time reducing distal axonal excitation. In contrast, expression of a GtACR1-Kv2.1 fusion at high levels was shown to cause cell death (Mardinly, 2018: Fig. 2d) and expression of GtACR2-Kv2.1 potentially led to less efficient axonal exclusion (Messier, 2018). While the tools described in the current study do not completely abolish axonal excitation, we believe that they provide several advantages over the current state-of-the-art optogenetic inhibition by ion pumps, especially when large brain volumes need to be addressed, or when silencing needs to be performed over longer time periods.


Haam J, Popescu IR, Morton LA, Halmos KC, Teruyama R, Ueta Y, Tasker JG, GABA is excitatory in adult vasopressinergic neuroendocrine cells. J. Neurosci. (2012), doi: 10.1523/JNEUROSCI.3826-11.2012

Ruiz A, Ruth FF, Scott R, Walker MC, Rusakov DA, Kullmann DM, GABAA Receptors at Hippocampal Mossy Fibers. Neuron (2003), doi: 10.1016/s0896-6273(03)00559-2

Ruiz A, Campanac E, Scott RS, Rusakov DA, Kullmann DM, Presynaptic GABAA receptors enhance transmission and LTP induction at hippocampal mossy fiber synapses. Nat. Neurosci. (2010), doi: 10.1038/nn.2512

Woodruff AR, Monyer H, Sah P, GABAergic Excitation in the Basolateral Amygdala. J. Neurosci. (2006), doi: 10.1523/jneurosci.3389-06.2006

Alle H, Geiger JRP, GABAergic spill-over transmission onto hippocampal mossy fiber boutons. J. Neurosci. (2007), doi: 10.1523/JNEUROSCI.4996-06.2007

Pugh JR, Jahr CE, Axonal GABAA receptors increase cerebellar granule cell excitability and synaptic activity. J. Neurosci. (2011), doi: 10.1523/JNEUROSCI.4506-10.2011

Raimondo JV, Kay L, Ellender TJ, Akerman CJ, Optogenetic silencing strategies differ in their effects on inhibitory synaptic transmission. Nat. Neurosci. (2012), doi: 10.1038/nn.3143

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. Noninvasive optical inhibition with a red-shifted microbial rhodopsin, Nat. Neurosci. (2014), doi: 10.1038/nn.3752

Mahn M, Prigge M, Ron S, Levy, R, Yizhar O, Biophysical constraints of optogenetic inhibition at presynaptic terminals. Nat. Neurosci. (2016), doi: 10.1038/nn.4266

Herman AM, Huang L, Murphey DK, Garcia I, Arenkiel BR, Cell type-specific and time-dependent light exposure contribute to silencing in neurons expressing Channelrhodopsin-2. eLIFE (2014), doi: 10.7554/eLife.01481

Mardinly AR, Oldenburg IA, Pégard NC, Sridharan S, Lyall EH, Chesnov K, Brohawn SG, Waller L, Adesnik H, Precise multimodal optical control of neural ensemble activity. Nat. Neurosci. (2018), doi: 10.1038/s41593-018-0139-8

Messier J, Chen H, Cai ZL, Xue M, Targeting light-gated chloride channels to neuronal somatodendritic domain reduces their excitatory effect in the axon. BioRxiv (2018), doi: 10.1101/331165


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