Noninvasive optical inhibition with a red-shifted microbial rhodopsin

Noninvasive optical inhibition with a red-shifted microbial rhodopsin Chuong Miri Busskamp Matthews Acker Sørensen Young Klapoetke

Tag layer of the DHSS Meta-Tagging Project. The article text is reproduced from the staged copy named in sourceDesc and is not altered; the tags are held separately in standOff so that overlapping and nested spans are preserved.

Noninvasive optical inhibition with a red-shifted microbial rhodopsin 2014 Nature neuroscience 10.1038/nn.3752 Chuong2014-noninvasive-optical-inhibition-with-a-red-sh Chuong2014-noninvasive-optical-inhibition-with-a-red-sh.txt, the project's staged copy 6731987776548e95548e45f2cb83b1b13d4584df not-redistributable neuroscience

the paper's own words

the tag layer; a sentence written by the tagger about the paper, always anchored to a quotation but not itself a quotation

0 spans located by searching the stored text for the quote's exact words, allowing any whitespace between them how to silence neurons over large volumes and at depth without putting anything into the brain Quotation withheld because this record is not cleared for redistribution. whether a red-shifted chloride pump can be engineered to give enough photocurrent for inhibition through intact skull Quotation withheld because this record is not cleared for redistribution. Jaws, a cruxhalorhodopsin with the most red-shifted spectrum of any inhibitor, giving roughly threefold photocurrents at red wavelengths and enabling transcranial silencing 1-3 mm deep in awake mice Quotation withheld because this record is not cleared for redistribution. method Quotation withheld because this record is not cleared for redistribution. optogenetic neural inhibition Quotation withheld because this record is not cleared for redistribution. system-or-tool-building system-or-tool-building Quotation withheld because this record is not cleared for redistribution. mechanisms Haemoglobin absorbs red light less than shorter wavelengths, so red-shifting the opsin's action spectrum lets usable light reach deeper brain tissue, which is what makes large-volume and transcranial inhibition possible. Quotation withheld because this record is not cleared for redistribution. mechanisms A minority of recorded neurons increase firing under illumination because inhibition of upstream neurons removes their drive (network disinhibition), not because the opsin excites them. Quotation withheld because this record is not cleared for redistribution. red light reaches deeper because hemoglobin, myoglobin and lipid absorb it less than blue or yellow Quotation withheld because this record is not cleared for redistribution. the gain came from two point mutations plus trafficking sequences borrowed from a potassium channel Quotation withheld because this record is not cleared for redistribution. rebound after illumination is attributed variously to hyperpolarisation-activated currents or to chloride accumulation shifting the reversal potential Quotation withheld because this record is not cleared for redistribution. ramping the light down rather than switching it off halves the rebound rate and delays it fourfold Quotation withheld because this record is not cleared for redistribution. the few neurons that increased firing are attributed to inhibition of upstream neurons rather than a direct effect Quotation withheld because this record is not cleared for redistribution. The noninvasive results cannot be shown to be direct rather than mediated by network effects or illumination of superficial apical dendrites. what this limits: the noninvasive results specifically: WE CANNOT EXPLICITLY RULE OUT THE POSSIBILITY THAT THE OBSERVED NONINVASIVE RESULTS WERE THE RESULT OF INDIRECT NETWORK ACTIVITY OR APICAL DENDRITIC ILLUMINATION Quotation withheld because this record is not cleared for redistribution. Transcranial illumination cannot be confined to deep tissue with this setup. what this limits: any claim of depth-specific inhibition: IT IS NOT POSSIBLE TO NONINVASIVELY ILLUMINATE ONLY DEEP VOLUMES WITH OUR EXPERIMENTAL SETUP WITHOUT ALSO TARGETING SURFACE AND INTERMEDIATE LAYERS Quotation withheld because this record is not cleared for redistribution. Jaws still produces post-illumination rebound spiking, which constrains experimental design. what this limits: experimental designs built on this inhibitor: JAWS DOES YIELD POST-ILLUMINATION REBOUND, WHICH LIKELY HAS IMPLICATIONS FOR CAREFUL EXPERIMENT DESIGN Quotation withheld because this record is not cleared for redistribution. Experiments were essentially unblinded and unrandomised. what this limits: every comparison in the paper: NO BLINDING WAS CARRIED OUT, WITH THE EXCEPTION OF COTRANSFECTED OPSIN-GFP AND TDTOMATO DATA SETS Quotation withheld because this record is not cleared for redistribution. the method cannot illuminate deep tissue without also illuminating everything above it, so direct deep inhibition cannot be separated from surface or dendritic effects what this limits: the same scope as items 0 and 1 - surface and intermediate layers are illuminated too, so deep effects are not isolated Quotation withheld because this record is not cleared for redistribution. Jaws produces MORE post-illumination rebound than the inhibitors it replaces what this limits: the choice between inhibitors: Quotation withheld - more post-illumination rebound than the tools it replaces Quotation withheld because this record is not cleared for redistribution. myelination still affects light propagation, so red penetrance is not uniform across preparations what this limits: the transfer of the penetrance advantage between preparations: MYELINATION MAY STILL AFFECT OPTICAL PROPAGATION IN NEURAL TISSUE Quotation withheld because this record is not cleared for redistribution. the two beneficial mutations do not transfer to the halorhodopsin they were modelled on, so the engineering is scaffold-specific what this limits: the generality of the engineering strategy: THE TWO HOMOLOGOUS POINT MUTATIONS DID NOT ENHANCE THE N. PHARAONIS HALORHODOPSIN - the mutations do not transfer Quotation withheld because this record is not cleared for redistribution. even improved, the restored retinal spiking reaches about 190 Hz against a wild-type range of 15-450 Hz what this limits: the vision-restoration claim: THE RESULTANT SPIKING RATES ARE LIMITED TO LESS THAN 200 HZ, SUBSTANTIALLY LESS THAN THE FULL WILD-TYPE RANGE OF 15-450 HZ Quotation withheld because this record is not cleared for redistribution. Monte Carlo simulation of light propagation with Henyey-Greenstein and Gegenbauer-kernel anisotropic scattering phase functions, imported to predict how deep red light penetrates brain tissue and skull Quotation withheld because this record is not cleared for redistribution. a rhodopsin from a halophilic archaeon, screened as a scaffold rather than studied for itself Quotation withheld because this record is not cleared for redistribution. trafficking sequences taken from the Kir2.1 potassium channel to improve membrane delivery Quotation withheld because this record is not cleared for redistribution. Monte Carlo modelling of light transport, used to justify the wavelength choice before any protein was made Quotation withheld because this record is not cleared for redistribution. yellow light at 593 nm, close to the spectral peak of both opsins, used to identify which neurons express the opsin, with paired recordings of the same neurons across wavelengths eliminates: mistaking a non-expressing neuron for an expressing one when assessing performance at the red-shifted wavelength · outcome (stated by the tagger, no quotation): held Quotation withheld because this record is not cleared for redistribution. paired recordings with an acutely inserted 200-um fiber as a positive control against transcranial illumination through the intact skull - marked PARTIAL because the paper reports in its own words that this did not settle the question eliminates: hyperpolarisation of superficial apical dendrites, producing somatic or downstream network inhibition, as the explanation of the noninvasive result - the paper states it CANNOT EXPLICITLY RULE OUT that possibility · outcome (stated by the tagger, no quotation): partial Quotation withheld because this record is not cleared for redistribution. Jaws Jaws Quotation withheld because this record is not cleared for redistribution. photocurrents photocurrents Quotation withheld because this record is not cleared for redistribution. rebound rebound Quotation withheld because this record is not cleared for redistribution. point mutations point mutations Quotation withheld because this record is not cleared for redistribution. optogenetic inhibition here means a light-driven ION PUMP moving chloride inward, not a channel and not a synaptic silencer - the class matters because rebound and reversal behave differently Quotation withheld because this record is not cleared for redistribution. noninvasive means no implanted optical device; the skull is intact but the virus was still injected surgically, and the paper's no-surgery scenario is explicitly future work with a transgenic line Quotation withheld because this record is not cleared for redistribution. red-shifted is relative and small in absolute terms - a 14 nm shift from the standard halorhodopsin - and the practical gain comes from where that sits on the tissue absorption curve Quotation withheld because this record is not cleared for redistribution. percent inhibition here is computed over neurons SHOWING suppression, with unchanged and increased-firing units reported separately rather than folded in Quotation withheld because this record is not cleared for redistribution. Jaws and Jaws-ER2 are two molecules differing in trafficking sequence and are reported separately throughout Quotation withheld because this record is not cleared for redistribution. claim_type: method a second reading reaching the same value. Recorded: method — note: a second reading reaching the same value from a different sentence. Not applied - a person decides. Quotation withheld because this record is not cleared for redistribution. method: screening of a rhodopsin class, two point mutations and trafficking sequences, then comparison against existing inhibitors in culture, retina, slice and awake cortex a second reading DIFFERING from the recorded value. Recorded: system-or-tool-building — note: a second reading, verbatim-verified, disagreeing with the value on the record. Not applied - a person decides. Quotation withheld because this record is not cleared for redistribution. phenomenon: light-driven hyperpolarisation of genetically targeted neurons a second reading DIFFERING from the recorded value. Recorded: optogenetic neural inhibition — note: a second reading, verbatim-verified, disagreeing with the value on the record. Not applied - a person decides. Quotation withheld because this record is not cleared for redistribution. builds-on → the authors' earlier identification of red-shifted cruxhalorhodopsins from H. marismortui and H. vallismortis (the paper's refs 1, 18) Quotation withheld because this record is not cleared for redistribution. extends → previous halorhodopsin silencers (eNpHR, eNpHR3.0) Quotation withheld because this record is not cleared for redistribution. replicates → previous reports of a minority of neurons increasing firing during optogenetic inhibition (the paper's refs 3, 40, 41) Quotation withheld because this record is not cleared for redistribution. extends → earlier hyperpolarising pumps, which addressed about a cubic millimetre Quotation withheld because this record is not cleared for redistribution. contradicts → the standing position that noninvasive optical inhibition was not achievable Quotation withheld because this record is not cleared for redistribution. critiques → existing inhibitors' red-light performance, at 10-30% of peak capacity Quotation withheld because this record is not cleared for redistribution. extends → eNpHR-based cone reactivation in retinitis pigmentosa models, by widening the restored spike frequency range Quotation withheld because this record is not cleared for redistribution. confirms → prior reports that a minority of neurons increase firing under optogenetic inhibition Quotation withheld because this record is not cleared for redistribution. EVIDENCE CORRECTED (RE-GROUNDED) · Jaws —is-a→ cruxhalorhodopsin Jaws cruxhalorhodopsin EVIDENCE CORRECTED: RE-GROUNDED. The old span described a screen of the class without naming Jaws. This sentence names both IN APPOSITION - 'a red-shifted cruxhalorhodopsin, Jaws' - which is kind-membership stated as directly as a paper ever states it. — strength of the relation: 1 Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but 'Jaws' is not named in this span, which identifies Halo57 as the cruxhalorhodopsin Jaws was engineered from; the relation holds only through that chain. Quotation withheld because this record is not cleared for redistribution. point mutations —causes→ photocurrents point mutations photocurrents direction on the measure: increases · strength of the relation: 1 · note: SIGN: increases, without moving the action spectrum - the two properties were separable Quotation withheld because this record is not cleared for redistribution. Jaws —causes→ rebound Jaws rebound holds when: relative to eNpHR3.0 and ArchT - the comparison is between opsins, not against no opsin · direction on the measure: increases · strength of the relation: 0.9 · note: SIGN: worse than the tools it replaces. The paper reports the axis on which its own tool loses Quotation withheld because this record is not cleared for redistribution. ANOVA ANOVA Quotation withheld because this record is not cleared for redistribution. ANOVA with Dunnett's post hoc test ANOVA with Dunnett's post hoc test Quotation withheld because this record is not cleared for redistribution. paired t-test paired t-test Quotation withheld because this record is not cleared for redistribution. Shapiro-Wilk normality test Shapiro-Wilk normality test Quotation withheld because this record is not cleared for redistribution. Kolmogorov-Smirnov test Kolmogorov-Smirnov test Quotation withheld because this record is not cleared for redistribution. principal component analysis principal component analysis Quotation withheld because this record is not cleared for redistribution. ANOVA with Newman-Keuls post hoc test ANOVA with Newman-Keuls post hoc test Quotation withheld because this record is not cleared for redistribution. Kolmogorov-Smirnov test Kolmogorov-Smirnov test Quotation withheld because this record is not cleared for redistribution. paired t -test paired t -test Quotation withheld because this record is not cleared for redistribution. Dunnett Dunnett Quotation withheld because this record is not cleared for redistribution. Jaws produced significantly larger red-light photocurrents than the existing silencers eNpHR3.0 and ArchT at every light power tested in cultured neurons. Quotation withheld because this record is not cleared for redistribution. Jaws strongly suppressed visually evoked cortical activity in vivo and restored light-driven retinal ganglion spiking in retinitis pigmentosa model mice. Quotation withheld because this record is not cleared for redistribution. Jaws can mediate noninvasive transcranial optical inhibition of neurons deep in the brain of awake mice. Quotation withheld because this record is not cleared for redistribution. Transcranial red-light illumination cut firing by roughly 92% in suppressed units across motor, somatosensory, insular and piriform cortex. Quotation withheld because this record is not cleared for redistribution. Gradually ramping the light down cut mean rebound firing from about 52 to 23 Hz, suggesting that shaping light pulses may reduce rebound. Quotation withheld because this record is not cleared for redistribution. Jaws gave significantly larger red-light photocurrents than eNpHR3.0 or ArchT at every power tested Quotation withheld because this record is not cleared for redistribution. visually evoked activity fell by about 86% in Jaws-expressing cortex Quotation withheld because this record is not cleared for redistribution. under red light Jaws inhibited about 89-97% while eNpHR3.0 fell to 27.5%, against 76% at its own yellow peak Quotation withheld because this record is not cleared for redistribution. transcranial illumination through intact skull suppressed firing by about 92% in neurons showing suppression, 1-3 mm deep Quotation withheld because this record is not cleared for redistribution. transdural illumination suppressed firing by about 86% in medial prefrontal cortex Quotation withheld because this record is not cleared for redistribution. efficacy was similar across several millimetres of depth and across baseline firing rates Quotation withheld because this record is not cleared for redistribution. in the retinitis pigmentosa model Jaws drove mean ganglion spiking of 189.5 Hz against 67.8 Hz for eNpHR, roughly tripling the bandwidth Quotation withheld because this record is not cleared for redistribution. REPORTED AGAINST ITSELF: Jaws produced greater post-illumination rebound than eNpHR3.0 or ArchT Quotation withheld because this record is not cleared for redistribution. ramping the light down cut mean rebound firing from 52 Hz to 23 Hz Quotation withheld because this record is not cleared for redistribution. the two homologous point mutations did not enhance N. pharaonis halorhodopsin Quotation withheld because this record is not cleared for redistribution. CONTROL NULL: expressing Jaws did not alter basal cell properties in culture or in slice, and its kinetics matched other inhibitors Quotation withheld because this record is not cleared for redistribution. CONTROL BY DESIGN: cells were selected by an independent tdTomato marker so the photocurrent comparison could not be driven by opsin expression level Quotation withheld because this record is not cleared for redistribution. DECLARED UNRESOLVABLE: surface and intermediate layers are necessarily illuminated too, so direct deep inhibition cannot be separated from dendritic or network effects Quotation withheld because this record is not cleared for redistribution. the conclusion is downgraded accordingly to highly suggestive of direct inhibition rather than demonstrated Quotation withheld because this record is not cleared for redistribution. optogenetic inhibition optogenetic inhibition Quotation withheld because this record is not cleared for redistribution. Jaws Jaws Quotation withheld because this record is not cleared for redistribution. Halo57 Halo57 Quotation withheld because this record is not cleared for redistribution. cruxhalorhodopsin cruxhalorhodopsin Quotation withheld because this record is not cleared for redistribution. rebound rebound Quotation withheld because this record is not cleared for redistribution. retinitis pigmentosa retinitis pigmentosa Quotation withheld because this record is not cleared for redistribution. Jaws Jaws Quotation withheld because this record is not cleared for redistribution. light power light power Quotation withheld because this record is not cleared for redistribution. degree of suppression degree of suppression Quotation withheld because this record is not cleared for redistribution. uninfected dentate granule cells uninfected dentate granule cells Quotation withheld because this record is not cleared for redistribution. photocurrents photocurrents Quotation withheld because this record is not cleared for redistribution. rebound rebound Quotation withheld because this record is not cleared for redistribution. transcranial transcranial Quotation withheld because this record is not cleared for redistribution. 86 ± 3% — reduction of visually evoked cortical activity in Jaws-expressing visual cortex under yellow light Quotation withheld because this record is not cleared for redistribution. 67.8 ± 7.0 Hz versus 189.5 ± 15.9 Hz — mean retinal ganglion cell spiking frequency driven through eNpHR versus through Jaws in blind mouse retina (a threefold bandwidth increase) Quotation withheld because this record is not cleared for redistribution. 14-nm — red shift of the Jaws action spectrum relative to the N. pharaonis halorhodopsin family Quotation withheld because this record is not cleared for redistribution. 10–30% — fraction of peak capacity at which pre-existing optogenetic inhibitors operate under red light Quotation withheld because this record is not cleared for redistribution. 1–3 mm — depth below the brain surface at which neurons were inhibited transcranially in awake mice Quotation withheld because this record is not cleared for redistribution. 14 nm — spectral shift of Jaws relative to the standard halorhodopsin - small in absolute terms, decisive on the absorption curve Quotation withheld because this record is not cleared for redistribution. 10-30% — fraction of peak capacity at which existing inhibitors work under red light Quotation withheld because this record is not cleared for redistribution. 27.5 ± 30.3% — eNpHR3.0 red-light inhibition - note the spread exceeds the mean Quotation withheld because this record is not cleared for redistribution. 189.5 vs 67.8 Hz — mean restored ganglion spiking with Jaws against eNpHR, against a wild-type range of 15-450 Hz Quotation withheld because this record is not cleared for redistribution. 52 to 23 Hz — rebound firing before and after ramping the light down Quotation withheld because this record is not cleared for redistribution. 1-3 mm — depth range at which transcranial inhibition was effective Quotation withheld because this record is not cleared for redistribution. 13 suppressed, 9 unchanged, 3 increased — unit outcomes under transcranial illumination, reported separately Quotation withheld because this record is not cleared for redistribution.

The full text of this article is not redistributable, so it is not included here. The evidence sentences are withheld with it; the tag layer and its grounding status remain. The article itself is at https://doi.org/10.1038/nn.3752.