Unique Vibrational Characteristics and Structures of the Photoexcited Retinal Chromophore in Ion-Pumping Rhodopsins.

Photoisomerization of an all-trans-retinal chromophore triggers ion transport in microbial ion-pumping rhodopsins. Understanding chromophore structures in the electronically excited (S1) state provides insights into the structural evolution on the potential energy surface of the photoexcited state. In this study, we examined the structure of the S1-state chromophore in Natronomonas pharaonis halorhodopsin (NpHR), a chloride ion-pumping rhodopsin, using time-resolved resonance Raman spectroscopy. The spectral patterns of the S1-state chromophore were completely different from those of the ground-state chromophore, resulting from unique vibrational characteristics and the structure of the S1 state. Mode assignments were based on a combination of deuteration shifts of the Raman bands and hybrid quantum mechanics-molecular mechanics calculations. The present observations suggest a weakened bond alternation in the π conjugation system. A strong hydrogen-out-of-plane bending band was observed in the Raman spectra of the S1-state chromophore in NpHR, indicating a twisted polyene structure. Similar frequency shifts for the C═N/C═C and C-C stretching modes of the S1-state chromophore in NpHR were observed in the Raman spectra of sodium ion-pumping and proton-pumping rhodopsins, suggesting that these unique features are common to the S1 states of ion-pumping rhodopsins.

Optogenetic inhibition of the caudal substantia nigra inflates behavioral responding to uncertain threat and safety.

Defensive responding is adaptive when it approximates the current threat but maladaptive when it exceeds the current threat. Here we asked if the substantia nigra, a region consistently implicated in reward, is necessary to show appropriate levels of defensive responding in Pavlovian fear discrimination. Rats received bilateral transduction of the caudal substantia nigra with halorhodopsin or a control fluorophore and bilateral ferrule implants. Rats then behaviorally discriminated cues predicting unique foot shock probabilities (danger, p = 1; uncertainty, p = .25; and safety, p = 0). Green-light illumination (532 nm) during cue presentation inflated defensive responding of halorhodopsin rats-measured by suppression of reward seeking-to uncertainty and safety beyond control levels. Green-light illumination outside of cue presentation had no impact on halorhodopsin or control rat responding. The results reveal caudal substantia nigra cue activity is necessary to inhibit defensive responding to nonthreatening and uncertain threat cues. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.

The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.

Optogenetic ion pumps differ with respect to the secondary pattern of K+ redistribution.

We recently reported that strong activation of the optogenetic chloride pump, halorhodopsin leads to a secondary redistribution of K+ ions into the cell, through tonically open, "leak" K+ channels. Here we show that this effect is not unique to halorhodopsin but is also seen with activation of another electrogenic ion pump, archaerhodopsin. The two opsins differ however in the size of the rebound rise in extracellular potassium, [K+ ]o , after the end of activation, which is far larger with halorhodopsin than for archaerhodopsin activation. Multiple linear regression modeling indicates that the variance in the postillumination surge in [K+ ]o was explained both by the size of the preceding, illumination-induced drop in [K+ ]o and also by the type of opsin. These data provide additional support for the hypothesis that intense chloride-loading of cells, as occurs naturally following intense bursts of GABAergic synaptic bombardment, or artificially following halorhodopsin activation, is followed by extrusion of both Cl- and K+ coupled together. We discuss this with respect to the pattern of [K+ ]o rise that occurs at the onset of seizure-like events.

Substantia Nigra Pars Reticulata Projections to the Pedunculopontine Nucleus Modulate Dyskinesia.

BACKGROUND: Long-term use of levodopa for Parkinson's disease (PD) treatment is often hindered by development of motor complications, including levodopa-induced dyskinesia (LID). The substantia nigra pars reticulata (SNr) and globus pallidus internal segment (GPi) are the output nuclei of the basal ganglia. Dysregulation of SNr and GPi activity contributes to PD pathophysiology and LID. OBJECTIVE: The objective of this study was to determine whether direct modulation of SNr GABAergic neurons and SNr projections to the pedunculopontine nucleus (PPN) regulates PD symptoms and LID in a mouse model. METHODS: We expressed Cre-recombinase activated channelrhodopsin-2 (ChR2) or halorhodopsin adeno-associated virus-2 (AAV2) vectors selectively in SNr GABAergic neurons of Vgat-IRES-Cre mice in a 6-hydroxydopamine model of PD to investigate whether direct optogenetic modulation of SNr neurons or their projections to the PPN regulates PD symptoms and LID expression. The forepaw stepping task, mouse LID rating scale, and open-field locomotion were used to assess akinesia and LID to test the effect of SNr modulation. RESULTS: Akinesia was improved by suppressing SNr neuron activity with halorhodopsin. LID was significantly reduced by increasing SNr neuronal activity with ChR2, which did not interfere with the antiakinetic effect of levodopa. Optical stimulation of ChR2 in SNr projections to the PPN recapitulated direct SNr stimulation. CONCLUSIONS: Modulation of SNr GABAergic neurons alters akinesia and LID expression in a manner consistent with the rate model of basal ganglia circuitry. Moreover, the projections from SNr to PPN likely mediate the antidyskinetic effect of increasing SNr neuronal activity, identifying a potential novel role for the PPN in LID. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Circuit and Cell-Specific Contributions to Decision Making Involving Risk of Explicit Punishment in Male and Female Rats.

Decision making is a complex cognitive process that recruits a distributed network of brain regions, including the basolateral amygdala (BLA) and nucleus accumbens shell (NAcSh). Recent work suggests that communication between these structures, as well as activity of cells expressing dopamine (DA) D2 receptors (D2R) in the NAcSh, are necessary for some forms of decision making; however, the contributions of this circuit and cell population during decision making under risk of punishment are unknown. The current experiments addressed this question using circuit-specific and cell type-specific optogenetic approaches in rats during a decision making task involving risk of punishment. In experiment 1, Long-Evans rats received intra-BLA injections of halorhodopsin or mCherry (control) and in experiment 2, D2-Cre transgenic rats received intra-NAcSh injections of Cre-dependent halorhodopsin or mCherry. Optic fibers were implanted in the NAcSh in both experiments. Following training in the decision making task, BLA→NAcSh or D2R-expressing neurons were optogenetically inhibited during different phases of the decision process. Inhibition of the BLA→NAcSh during deliberation (the time between trial initiation and choice) increased preference for the large, risky reward (increased risk taking). Similarly, inhibition during delivery of the large, punished reward increased risk taking, but only in males. Inhibition of D2R-expressing neurons in the NAcSh during deliberation increased risk taking. In contrast, inhibition of these neurons during delivery of the small, safe reward decreased risk taking. These findings extend our knowledge of the neural dynamics of risk taking, revealing sex-dependent circuit recruitment and dissociable activity of selective cell populations during decision making.SIGNIFICANCE STATEMENT Until recently, the ability to dissect the neural substrates of decision making involving risk of punishment (risk taking) in a circuit-specific and cell-specific manner has been limited by the tools available for use in rats. Here, we leveraged the temporal precision of optogenetics, together with transgenic rats, to probe contributions of a specific circuit and cell population to different phases of risk-based decision making. Our findings reveal basolateral amygdala (BLA)→nucleus accumbens shell (NAcSh) is involved in evaluation of punished rewards in a sex-dependent manner. Further, NAcSh D2 receptor (D2R)-expressing neurons make unique contributions to risk taking that vary across the decision making process. These findings advance our understanding of the neural principles of decision making and provide insight into how risk taking may become compromised in neuropsychiatric diseases.

Indirect Effects of Halorhodopsin Activation: Potassium Redistribution, Nonspecific Inhibition, and Spreading Depolarization.

The movement of ions in and out of neurons can exert significant effects on neighboring cells. Here we report several experimentally important consequences of activation of the optogenetic chloride pump, halorhodopsin. We recorded extracellular K+ concentration ([K+]extra) in neocortical brain slices prepared from young adult mice (both sexes) which express halorhodopsin in pyramidal cells. Strong halorhodopsin activation induced a pronounced drop in [K+]extra that persisted for the duration of illumination. Pharmacological blockade of K+ channels reduced the amplitude of this drop, indicating that it represents K+ redistribution into cells during the period of hyperpolarization. Halorhodopsin thus drives the inward movement of both Cl- directly, and K+ secondarily. When the illumination period ended, a rebound surge in extracellular [K+] developed over tens of seconds, partly reflecting the previous inward redistribution of K+, but additionally driven by clearance of Cl- coupled to K+ by the potassium-chloride cotransporter, KCC2. The drop in [K+]extra during light activation leads to a small (2-3 mV) hyperpolarization also of other cells that do not express halorhodopsin. Its activation therefore has both direct and indirect inhibitory effects. Finally, we show that persistent strong activation of halorhodopsin causes cortical spreading depolarizations (CSDs), both in vitro and in vivo This novel means of triggering CSDs is unusual, in that the events can arise during the actual period of illumination, when neurons are being hyperpolarized and [K+]extra is low. We suggest that this fundamentally different experimental model of CSDs will open up new avenues of research to explain how they occur naturally.SIGNIFICANCE STATEMENT Halorhodopsin is a light-activated electrogenic chloride pump, which has been widely used to inhibit neurons optogenetically. Here, we demonstrate three previously unrecognized consequences of its use: (1) intense activation leads to secondary movement of K+ ions into the cells; (2) the resultant drop in extracellular [K+] reduces excitability also in other, nonexpressing cells; and (3) intense persistent halorhodopsin activation can trigger cortical spreading depolarization (CSD). Halorhodopsin-induced CSDs can occur when neurons are hyperpolarized and extracellular [K+] is low. This contrasts with the most widely used experimental models that trigger CSDs with high [K+]. Both models, however, are consistent with the hypothesis that CSDs arise following net inward ionic movement into the principal neuron population.

Structural insights into light-driven anion pumping in cyanobacteria.

Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl-, I-, NO3-. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics.

Mutations conferring SO42- pumping ability on the cyanobacterial anion pump rhodopsin and the resultant unique features of the mutant.

Membrane transport proteins can be divided into two types: those that bind substrates in a resting state and those that do not. In this study, we demonstrate that these types can be converted by mutations through a study of two cyanobacterial anion-pumping rhodopsins, Mastigocladopsis repens halorhodopsin (MrHR) and Synechocystis halorhodopsin (SyHR). Anion pump rhodopsins, including MrHR and SyHR, initially bind substrate anions to the protein center and transport them upon illumination. MrHR transports only smaller halide ions, Cl- and Br-, but SyHR also transports SO42-, despite the close sequence similarity to MrHR. We sought a determinant that could confer SO42- pumping ability on MrHR and found that the removal of a negative charge at the anion entrance is a prerequisite for SO42- transport by MrHR. Consistently, the reverse mutation in SyHR significantly weakened SO42- pump activity. Notably, the MrHR and SyHR mutants did not show SO42- induced absorption spectral shifts or changes in the photoreactions, suggesting no bindings of SO42- in their initial states or the bindings to the sites far from the protein centers. In other words, unlike wild-type SyHR, these mutants take up SO42- into their centers after illumination and release it before the ends of the photoreactions.

Glutamate inputs from the laterodorsal tegmental nucleus to the ventral tegmental area are essential for the induction of cocaine sensitization in male mice.

RATIONALE: Drug-induced potentiation of ventral tegmental area (VTA) glutamate signaling contributes critically to the induction of sensitization - an enhancement in responding to a drug following exposure which is thought to reflect neural changes underlying drug addiction. The laterodorsal tegmental nucleus (LDTg) provides one of several sources of glutamate input to the VTA. OBJECTIVE: We used optogenetic techniques to test either the role of LDTg glutamate cells or their VTA afferents in the development of cocaine sensitization in male VGluT2::Cre mice. These were inhibited using halorhodopsin during each of five daily cocaine exposure injections. The expression of locomotor sensitization was assessed following a cocaine challenge injection 1-week later. RESULTS: The locomotor sensitization seen in control mice was absent in male mice subjected to inhibition of LDTg-VTA glutamatergic circuitry during cocaine exposure. As sensitization of nucleus accumbens (NAcc) dopamine (DA) overflow is also induced by this drug exposure regimen, we used microdialysis to measure NAcc DA overflow on the test for sensitization. Consistent with the locomotor sensitization results, inhibition of LDTg glutamate afferents to the VTA during cocaine exposure prevented the sensitization of NAcc DA overflow observed in control mice. CONCLUSIONS: These data identify the LDTg as the source of VTA glutamate critical for the development of cocaine sensitization in male mice. Accordingly, the LDTg may give rise to the synapses in the VTA at which glutamatergic plasticity, known to contribute to the enhancement of addictive behaviors, occurs.

In Vivo Neocortical [K]o Modulation by Targeted Stimulation of Astrocytes.

A normally functioning nervous system requires normal extracellular potassium ion concentration ([K]o). Throughout the nervous system, several processes, including those of an astrocytic nature, are involved in [K]o regulation. In this study we investigated the effect of astrocytic photostimulation on [K]o. We hypothesized that in vivo photostimulation of eNpHR-expressing astrocytes leads to a decreased [K]o. Using optogenetic and electrophysiological techniques we showed that stimulation of eNpHR-expressing astrocytes resulted in a significantly decreased resting [K]o and evoked K responses. The amplitude of the concomitant spreading depolarization-like events also decreased. Our results imply that astrocytic membrane potential modification could be a potential tool for adjusting the [K]o.

Synaptic plasticity-dependent competition rule influences memory formation.

Memory is supported by a specific collection of neurons distributed in broad brain areas, an engram. Despite recent advances in identifying an engram, how the engram is created during memory formation remains elusive. To explore the relation between a specific pattern of input activity and memory allocation, here we target a sparse subset of neurons in the auditory cortex and thalamus. The synaptic inputs from these neurons to the lateral amygdala (LA) are not potentiated by fear conditioning. Using an optogenetic priming stimulus, we manipulate these synapses to be potentiated by the learning. In this condition, fear memory is preferentially encoded in the manipulated cell ensembles. This change, however, is abolished with optical long-term depression (LTD) delivered shortly after training. Conversely, delivering optical long-term potentiation (LTP) alone shortly after fear conditioning is sufficient to induce the preferential memory encoding. These results suggest a synaptic plasticity-dependent competition rule underlying memory formation.

Lipid Dynamics in Diisobutylene-Maleic Acid (DIBMA) Lipid Particles in Presence of Sensory Rhodopsin II.

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein-protein interaction and oligomerization in a constraining environment.

Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin.

We report a simple and direct fluorimetric vesicle-based method for measuring the transport rate of the light-driven ions pumps as specifically applied to the chloride pump, halorhodopsin, from Natronomonas pharaonis (pHR). Previous measurements were cell-based and methods to determine average single channel permeability challenging. We used a water-in-oil emulsion method for directional pHR reconstitution into two different types of vesicles: lipid vesicles and asymmetric lipid-block copolymer vesicles. We then used stopped-flow experiments combined with fluorescence correlation spectroscopy to determine per protein Cl- transport rates. We obtained a Cl- transport rate of 442 (±17.7) Cl-/protein/s in egg phosphatidyl choline (PC) lipid vesicles and 413 (±26) Cl-/protein/s in hybrid block copolymer/lipid (BCP/PC) vesicles with polybutadine-polyethylene oxide (PB12PEO8) on the outer leaflet and PC in the inner leaflet at a photon flux of 1450 photons/protein/s. Normalizing to a per photon basis, this corresponds to 0.30 (±0.07) Cl-/photon and 0.28 (±0.04) Cl-/photon for pure PC and BCP/PC hybrid vesicles respectively, both of which are in agreement with recently reported turnover of ~500 Cl-/protein/s from flash photolysis experiments and with voltage-clamp measurements of 0.35 (±0.16) Cl-/photon in pHR-expressing oocytes as well as with a pHR quantum efficiency of ~30%.

Functional Mechanism of Cl--Pump Rhodopsin and Its Conversion into H+ Pump.

Cl--pump rhodopsin is the second discovered microbial rhodopsin. Although its physiological role has not been fully clarified, its functional mechanism has been studied as a model for anion transporters. After the success of neural activation by channel rhodopsin, the first Cl--pump halorhodopsin (HR) had become widely used as a neural silencer. The emergence of artificial and natural anion channel rhodopsins lowered the importance of HRs. However, the longer absorption maxima of approximately 585-600 nm for HRs are still advantageous for applications in mammalian brains and collaborations with neural activators possessing shorter absorption maxima. In this chapter, the variation and functional mechanisms of Cl- pumps are summarized. After the discovery of HR, Cl--pump rhodopsins were confined to only extremely halophilic haloarchaea. However, after 2014, two Cl--pump groups were newly discovered in marine and terrestrial bacteria. These Cl- pumps are phylogenetically distinct from HRs and have unique characteristics. In particular, the most recently identified Cl- pump has close similarity with the H+ pump bacteriorhodopsin and was converted into the H+ pump by a single amino acid replacement.

Fine Tuning of Calcium Constitutive Entry by Optogenetically-Controlled Membrane Polarization: Impact on Cell Migration.

Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on channels, such as transient receptor potential (TRP) channels, to be constitutively opened and strongly depend on membrane potential and a calcium driving force. We developed an optogenetic approach based on the expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using C2C12 cells, we found that halorhodopsin can be used to achieve a finely tuned control of membrane polarization. Escalating the membrane polarization by incremental changes in light led to a concomitant increase in CCE through transient receptor potential vanilloid 2 (TRPV2) channels. Moreover, light-induced calcium entry through TRPV2 channels promoted cell migration. Our study shows for the first time that by modulating CCE and related physiological responses, such as cell motility, halorhodopsin serves as a potentially powerful tool that could open new avenues for the study of CCE and associated cellular behaviors.

Structure-Based Functional Modification Study of a Cyanobacterial Chloride Pump for Transporting Multiple Anions.

Understanding the structure and functional mechanisms of cyanobacterial halorhodopsin has become increasingly important, given the report that Synechocystis halorhodopsin (SyHR), a homolog of the cyanobacterial halorhodopsin from Mastigocladopsis repens (MrHR), can take up divalent ions, such as SO42-, as well as chloride ions. Here, the crystal structure of MrHR, containing a unique "TSD" chloride ion conduction motif, was determined as a homotrimer at a resolution of 1.9 Å. The detailed structure of MrHR revealed a unique trimeric topology of the light-driven chloride pump, with peculiar coordination of two water molecules and hydrogen-mediated bonds near the TSD motif, as well as a short B-C loop. Structural and functional analyses of MrHR revealed key residues responsible for the anion selectivity of cyanobacterial halorhodopsin and the involvement of two chloride ion-binding sites in the ion conduction pathway. Alanine mutant of Asn63, Pro118, and Glu182 locating in the anion inlet induce multifunctional uptake of chloride, nitrate, and sulfate ions. Moreover, the structure of N63A/P118A provides information on how SyHR promotes divalent ion transport. Our findings significantly advance the structural understanding of microbial rhodopsins with different motifs. They also provide insight into the general structural framework underlying the molecular mechanisms of the cyanobacterial chloride pump containing SyHR, the only molecule known to transport both sulfate and chloride ions.

Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids.

We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl- binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl--releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl- uptake was faster than that of MF NpHR. These differences in the Cl--releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl- release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl--binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR' state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl- pumping.

Light-induced silencing of neural activity in Rosa26 knock-in and BAC transgenic mice conditionally expressing the microbial halorhodopsin eNpHR3.

An engineered light-inducible chloride pump, Natronomonas pharaonis halorhodopsin 3 (eNpHR3) enables temporally and spatially precise inhibition of genetically defined cell populations in the intact nervous tissues. In this report, we show the generation of new mouse strains that express eNpHR3-EYFP fusion proteins after Cre- and/or Flp-mediated recombination to optically inhibit neuronal activity. In these mouse strains, Cre/Flp recombination induced high levels of opsin expression. We confirmed their light-induced activities by brain slice whole-cell patch clamp experiments. eNpHR3-expressing neurons were optically hyperpolarized and silenced from firing action potentials. In prolonged silencing of action potentials, eNpHR3 was superior to eNpHR2, a former version of the engineered pump. Thus, these eNpHR3 mouse strains offer reliable genetic tools for light-induced inhibiting of neuronal activity in defined sets of neurons.

Optogenetic Modulation for the Treatment of Traumatic Brain Injury.

Although research involving traumatic brain injury (TBI) has traditionally focused on the acute clinical manifestations, new studies provide evidence for chronic and progressive neurological sequelae associated with TBI, highlighting the risk of persistent, and sometimes life-long, consequences for affected patients. Several treatment modalities to date have demonstrated efficacy in experimental models. However, there is currently no effective treatment to improve neural structure repair and functional recovery of TBI patients. Optogenetics represents a potential molecular tool for neuromodulation and monitoring cellular activity with unprecedented spatial resolution and millisecond temporal precision. In this review, we discuss the conceptual background and preclinical evidence of optogenetics for neuromodulation, and translational applications for TBI treatment are considered.