eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications - PubMed (original) (raw)
eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications
Viviana Gradinaru et al. Brain Cell Biol. 2008 Aug.
Abstract
Temporally precise inhibition of distinct cell types in the intact nervous system has been enabled by the microbial halorhodopsin NpHR, a fast light-activated electrogenic Cl(-) pump. While neurons can be optically hyperpolarized and inhibited from firing action potentials at moderate NpHR expression levels, we have encountered challenges with pushing expression to extremely high levels, including apparent intracellular accumulations. We therefore sought to molecularly engineer NpHR to achieve strong expression without these cellular side effects. We found that high expression correlated with endoplasmic reticulum (ER) accumulation, and that under these conditions NpHR colocalized with ER proteins containing the KDEL ER retention sequence. We screened a number of different putative modulators of membrane trafficking and identified a combination of two motifs, an N-terminal signal peptide and a C-terminal ER export sequence, that markedly promoted membrane localization and ER export defined by confocal microscopy and whole-cell patch clamp. The modified NpHR displayed increased peak photocurrent in the absence of aggregations or toxicity, and potent optical inhibition was observed not only in vitro but also in vivo with thalamic single-unit recording. The new enhanced NpHR (eNpHR) allows safe, high-level expression in mammalian neurons, without toxicity and with augmented inhibitory function, in vitro and in vivo.
Figures
Fig. 1
Protein aggregation and photocurrents altered by changes in intracellular targeting of NpHR. (A) Table of screened intracellular targeting strategies using N- and C-terminal peptides fused to NpHR. (B) The fraction of neurons containing one or more NpHR aggregates was determined in cultured hippocampal neurons transduced with quantitatively titer-matched viral CaMKII_α_::NpHR-EYFP constructs, and allowed to express for 10 days. Compared to the unmodified NpHR (orange), some constructs (gray) had little effect on aggregates while others (blue) partially reduced aggregate incidence. Notably, for one construct (*) aggregates and toxicity were virtually abolished (only a single possible aggregate observed in >400 neurons). Data shown are relative to wild-type NpHR, and the number of neurons sampled for each construct is shown in parenthesis after the construct label. (C) Functionality was assessed by whole-cell patch clamp (for details see “Methods” and Fig. 3B). All the constructs were functional as indicated by photocurrents that were comparable to the original NpHR, but one construct, the optimal construct from (B) (*), gave rise to significantly higher photocurrent per cell than all other variants (eNpHR). Data shown are relative to wild-type NpHR, and the number of neurons sampled for each construct is shown in parenthesis after the construct label.
Fig. 2
Intracellular targeting of eNpHR. (A) Primary structure of the selected construct (eNpHR) showing addition of the N-terminal signal peptide derived from nAChR and the C-terminal ER export signal derived from Kir2.1. Expression here was driven by the CaMKII_α_ promoter and visualized by fusion to EYFP. (B) Top row: Untargeted NpHR (green) colocalized with somatic ER (KDEL ER protein staining in red; overlap indicated in yellow and by arrows) and also notably aggregated in ER-rich regions of the dendrites (overlap indicated in yellow and by arrowheads). Bottom row: Little colocalization of eNpHR with somatic ER staining could be found, and indeed pronounced accumulations of ER staining in eNpHR dendrites were not observed. Right column: representative images of neuronal populations expressing NpHR and eNpHR. The neurons were infected with quantitatively titer-matched CaMKII_α_-NpHR-EYFP or CaMKII_α_-eNpHR-EYFP.
Fig. 3
Summary of eNpHR functional properties. Summary of electrophysiological properties of NpHR and eNpHR in cultured hippocampal neurons. Top: Representative confocal images in cultured hippocampal neurons revealed that like a typical membrane protein, eNpHR did not appear to fill cytoplasm like NpHR (left)(right). NpHR and eNpHR were expressed for 10 days in cultured hippocampal neurons. Insets: magnified views of selected regions. Bottom: 593 nm light (yellow bar) induced outward photocurrents (top right: sample traces in voltage clamp), with eNpHR evoking significantly stronger photocurrents per cell than NpHR (left bar graph; NpHR: 38.9 ± 6.8 pA; eNpHR: 68.1 ± 7.2 pA; unpaired _t_-test P = 0.008). Viral titers were quantitatively matched across groups (see “Methods”). Membrane input resistance was similar for all neurons patched (right bar graph; NpHR: 113.5 ± 13.9 mΩ; eNpHR: 116.8 ± 13.9 mΩ; unpaired _t_-test P = 0.87). Values plotted are mean ± SEM; n = 12 for NpHR, n = 10 for eNpHR. Bottom right: Illumination with yellow light as expected sufficed to inhibit spiking induced by current injection in eNpHR+ neurons.
Fig. 4
in vivo function of eNpHR at high expression level. (A) Confocal images showing NpHR and eNpHR expression in rodent hippocampal CA1. NpHR aggregations were clearly visualized after 10 days of strong expression of high titer virus (left) while eNpHR showed no signs of aggregates or toxicity (right) with quantitatively matched viral titers (see “Methods”). Lower panels: magnified views of the dendritic layer. Compared to NpHR, eNpHR revealed not only absence of aggregates but also more membrane localization in distal processes in vivo. (B) Simultaneous optical stimulation and electrophysiology in living mice demonstrates eNpHR potency in vivo: single unit recordings in deep brain structures. Yellow illumination delivered by the optrode method (Gradinaru et al., 2007) in vivo inhibited electrical activity in thalamus previously transduced with eNpHR by lentiviral stereotactic injection (middle trace). Top trace: same thalamic region, recording without illumination. Bottom trace: control recording 1 mm ventral and anterior from the eNpHR injection site. As expected, in non-transduced tissue, light did not inhibit recorded spikes. Confocal image: eNpHR expression in the thalamus (same animal). Inset: expanded view of a spike from unit (*) represented in the top trace.
Similar articles
- Improved expression of halorhodopsin for light-induced silencing of neuronal activity.
Zhao S, Cunha C, Zhang F, Liu Q, Gloss B, Deisseroth K, Augustine GJ, Feng G. Zhao S, et al. Brain Cell Biol. 2008 Aug;36(1-4):141-54. doi: 10.1007/s11068-008-9034-7. Epub 2008 Oct 17. Brain Cell Biol. 2008. PMID: 18931914 Free PMC article. - Light-induced silencing of neural activity in Rosa26 knock-in mice conditionally expressing the microbial halorhodopsin eNpHR2.0.
Imayoshi I, Tabuchi S, Hirano K, Sakamoto M, Kitano S, Miyachi H, Yamanaka A, Kageyama R. Imayoshi I, et al. Neurosci Res. 2013 Jan;75(1):53-8. doi: 10.1016/j.neures.2012.03.008. Epub 2012 Mar 23. Neurosci Res. 2013. PMID: 22465523 - Multimodal fast optical interrogation of neural circuitry.
Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, Deisseroth K. Zhang F, et al. Nature. 2007 Apr 5;446(7136):633-9. doi: 10.1038/nature05744. Nature. 2007. PMID: 17410168 - Optogenetic investigation of neuropsychiatric diseases.
Huang F, Tang B, Jiang H. Huang F, et al. Int J Neurosci. 2013 Jan;123(1):7-16. doi: 10.3109/00207454.2012.728651. Epub 2012 Oct 16. Int J Neurosci. 2013. PMID: 23002710 Review. - Light-activated channels in acute seizures.
Kokaia M. Kokaia M. Epilepsia. 2011 Oct;52 Suppl 8:16-8. doi: 10.1111/j.1528-1167.2011.03226.x. Epilepsia. 2011. PMID: 21967352 Review.
Cited by
- A Systematic Review to Compare Electrical, Magnetic, and Optogenetic Stimulation for Peripheral Nerve Repair.
Kaluskar P, Bharadwaj D, Iyer KS, Dy C, Zheng M, Brogan DM. Kaluskar P, et al. J Hand Surg Glob Online. 2024 Jun 29;6(5):722-739. doi: 10.1016/j.jhsg.2024.03.005. eCollection 2024 Sep. J Hand Surg Glob Online. 2024. PMID: 39381397 Free PMC article. Review. - Optogenetics and Targeted Gene Therapy for Retinal Diseases: Unravelling the Fundamentals, Applications, and Future Perspectives.
Kulbay M, Tuli N, Akdag A, Kahn Ali S, Qian CX. Kulbay M, et al. J Clin Med. 2024 Jul 19;13(14):4224. doi: 10.3390/jcm13144224. J Clin Med. 2024. PMID: 39064263 Free PMC article. Review. - Optogenetic manipulation of lysosomal physiology and autophagy-dependent clearance of amyloid beta.
Zeng W, Li C, Wu R, Yang X, Wang Q, Lin B, Wei Y, Li H, Shan G, Qu L, Cang C. Zeng W, et al. PLoS Biol. 2024 Apr 23;22(4):e3002591. doi: 10.1371/journal.pbio.3002591. eCollection 2024 Apr. PLoS Biol. 2024. PMID: 38652732 Free PMC article. - Optogenetic Inhibition of Rat Anterior Cingulate Cortex Impairs the Ability to Initiate and Stay on Task.
Vázquez D, Maulhardt SR, Stalnaker TA, Solway A, Charpentier CJ, Roesch MR. Vázquez D, et al. J Neurosci. 2024 May 15;44(20):e1850232024. doi: 10.1523/JNEUROSCI.1850-23.2024. J Neurosci. 2024. PMID: 38569923 Free PMC article. - Engineering luminopsins with improved coupling efficiencies.
Slaviero AN, Gorantla N, Simkins J, Crespo EL, Ikefuama EC, Tree MO, Prakash M, Björefeldt A, Barnett LM, Lambert GG, Lipscombe D, Moore CI, Shaner NC, Hochgeschwender U. Slaviero AN, et al. Neurophotonics. 2024 Apr;11(2):024208. doi: 10.1117/1.NPh.11.2.024208. Epub 2024 Mar 29. Neurophotonics. 2024. PMID: 38559366 Free PMC article.
References
- Aravanis AM, Wang LP, Zhang F, Meltzer LA, Mogri MZ, Schneider MB, Deisseroth K. An optical neural interface: In vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural. Eng. 2007;4:S143–S156. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- DP1 OD000616/OD/NIH HHS/United States
- DP1 OD000616-01/OD/NIH HHS/United States
- DP1 OD000616-02/OD/NIH HHS/United States
- DP1 OD000616-03/OD/NIH HHS/United States
- DP1 OD000616-04/OD/NIH HHS/United States
LinkOut - more resources
Full Text Sources
Other Literature Sources