Optogenetic Inhibition of the Orbitofrontal Cortex Disrupts Inhibitory Control during Stop-Change Performance in Male Rats.

Historically, the orbitofrontal cortex (OFC) has been implicated in a variety of behaviors ranging from reversal learning and inhibitory control to more complex representations of reward value and task space. While modern interpretations of the OFC's function have focused on a role in outcome evaluation, these cognitive processes often require an organism to inhibit a maladaptive response or strategy. Single-unit recordings from the OFC in rats performing a stop-change task show that the OFC responds strongly to STOP trials. To investigate the role that the OFC plays in stop-change performance, we expressed halorhodopsin (eNpHR3.0) in excitatory neurons in the OFC and tested rats on the stop-change task. Previous work suggests that the OFC differentiates between STOP trials based on trial sequence (i.e., gS trials: STOP trials preceded by a GO vs sS trials: STOP trials preceded by a STOP). We found that yellow light activation of the eNpHR3.0-expressing neurons significantly decreased accuracy only on STOP trials that followed GO trials (gS trials). Further, optogenetic inhibition of the OFC speeded reaction times on error trials. This suggests that the OFC plays a role in inhibitory control processes and that this role needs to be accounted for in modern interpretations of OFC function.

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.

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.

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.

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.

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.

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.

Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn.

Nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate spinal interneurons that express the calcium-binding protein calretinin (CR). We show that these interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relay signals to lamina I projection neurons. Photoactivation of CR interneurons in vivo resulted in a significant nocifensive behavior that was morphine sensitive, caused a conditioned place aversion, and was enhanced by spared nerve injury. Furthermore, halorhodopsin-mediated inhibition of these interneurons elevated sensory thresholds. Our results suggest that dorsal horn circuits that involve excitatory CR neurons are important for the generation and amplification of pain and identify these interneurons as a future analgesic target.

The Blue-Green Sensory Rhodopsin SRM from Haloarcula marismortui Attenuates Both Phototactic Responses Mediated by Sensory Rhodopsin I and II in Halobacterium salinarum.

Haloarchaea utilize various microbial rhodopsins to harvest light energy or to mediate phototaxis in search of optimal environmental niches. To date, only the red light-sensing sensory rhodopsin I (SRI) and the blue light-sensing sensory rhodopsin II (SRII) have been shown to mediate positive and negative phototaxis, respectively. In this work, we demonstrated that a blue-green light-sensing (504 nm) sensory rhodopsin from Haloarcula marismortui, SRM, attenuated both positive and negative phototaxis through its sensing region. The H. marismortui genome encodes three sensory rhodopsins: SRI, SRII and SRM. Using spectroscopic assays, we first demonstrated the interaction between SRM and its cognate transducer, HtrM. We then transformed an SRM-HtrM fusion protein into Halobacterium salinarum, which contains only SRI and SRII, and observed that SRM-HtrM fusion protein decreased both positive and negative phototaxis of H. salinarum. Together, our results suggested a novel phototaxis signalling system in H. marismortui comprised of three sensory rhodopsins in which the phototactic response of SRI and SRII were attenuated by SRM.

Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin.

Mastigocladopsis repens halorhodopsin (MrHR) is a Cl--pumping rhodopsin that belongs to a distinct cluster far from other Cl- pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I- similar to Cl- but cannot transport it. I--bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl--pumping photocycle, Cl- moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I--bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl--binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl--binding affinity nor the absorption spectrum, but completely eliminated the Cl--pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H+ release was detected for photolyzed MrHR by using an indium­tin oxide electrode, which acts as a good time-resolved pH sensor. This H+ release disappeared in the I--bound form of the wild-type and Cl--bound form of the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl- movement to the CP channel.

Light-Driven Chloride Transport Kinetics of Halorhodopsin.

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl- transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl-/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.

Hyperpolarization by activation of halorhodopsin results in enhanced synaptic transmission: Neuromuscular junction and CNS circuit.

Optogenetics offers a unique method to regulate the activity of select neural circuits. However, the electrophysiological consequences of targeted optogenetic manipulation upon the entire circuit remain poorly understood. Analysis of the sensory-CNS-motor circuit in Drosophila larvae expressing eHpHR and ChR2-XXL revealed unexpected patterns of excitability. Optical stimulation of motor neurons targeted to express eNpHR resulted in inhibition followed by excitation of body wall contraction with repetitive stimulation in intact larvae. In situ preparations with direct electrophysiological measures showed an increased responsiveness to excitatory synaptic activity induced by sensory stimulation within a functional neural circuit. To ensure proper function of eNpHR and ChR2-XXL they were expressed in body wall muscle and direct electrophysiological measurements were obtained. Under eNpHR induced hyperpolarization the muscle remained excitable with increased amplitude of excitatory postsynaptic synaptic potentials. Theoretical models to explain the observations are presented. This study aids in increasing the understanding of the varied possible influences with light activated proteins within intact neural circuits.

Natronomonas pharaonis halorhodopsin Ser81 plays a role in maintaining chloride ions near the Schiff base.

Optogenetic technologies have often been used as tools for neuronal activation or silencing by light. Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven chloride ion pump. Upon light absorption, a chloride ion passes through the cell membrane, which is accompanied by the temporary binding of a chloride ion with Thr126 at binding site-1 (BS1) near the protonated Schiff base in NpHR. However, the mechanism of stabilization of the binding state between a chloride ion and BS1 has not been investigated. Therefore, to identify a key component of the chloride ion transport pathway as well as to acquire dynamic information about the chloride ion-BS1 binding state, we performed a rough analysis of the chloride ion pathway shape followed by molecular dynamics (MD) simulations for both wild-type and mutant NpHR structures. The MD simulations showed that the hydrogen bond between Thr126 and the chloride ion was retained in the wild-type protein, while the chloride ion could not be retained at and tended to leave BS1 in the S81A mutant. We found that the direction of the Thr126 side chain was fixed by a hydroxyl group of Ser81 through a hydrogen bond and that Thr126 bound to a chloride ion in the wild-type protein, while this interaction was lost in the S81A mutant, resulting in rotation of the Thr126 side chain and reduction in the interaction between Thr126 and a chloride ion. To confirm the role of S81, patch clamp recordings were performed using cells expressing NpHR S81A mutant protein. Considered together with the results that the NpHR S81A-expressing cells did not undergo hyperpolarization under light stimulation, our results indicate that Ser81 plays a key role in chloride migration. Our findings might be relevant to ongoing clinical trials using optogenetic gene therapy in blind patients.

Retinal Configuration of ppR Intermediates Revealed by Photoirradiation Solid-State NMR and DFT.

Pharanois phoborhodopsin (ppR) from Natronomonas pharaonis is a transmembrane photoreceptor protein involved in negative phototaxis. Structural changes in ppR triggered by photoisomerization of the retinal chromophore are transmitted to its cognate transducer protein (pHtrII) through a cyclic photoreaction pathway involving several photointermediates. This pathway is called the photocycle. It is important to understand the detailed configurational changes of retinal during the photocycle. We previously observed one of the photointermediates (M-intermediates) by in situ photoirradiation solid-state NMR experiments. In this study, we further observed the 13C cross-polarization magic-angle-spinning NMR signals of late photointermediates such as O- and N'-intermediates by illumination with green light (520 nm). Under blue-light (365 nm) irradiation of the M-intermediates, 13C cross-polarization magic-angle-spinning NMR signals of 14- and 20-13C-labeled retinal in the O-intermediate appeared at 115.4 and 16.4 ppm and were assigned to the 13-trans, 15-syn configuration. The signals caused by the N'-intermediate appeared at 115.4 and 23.9 ppm and were assigned to the 13-cis configuration, and they were in an equilibrium state with the O-intermediate during thermal decay of the M-intermediates at -60°C. Thus, photoirradiation NMR studies revealed the photoreaction pathways from the M- to O-intermediates and the equilibrium state between the N'- and O-intermediate. Further, we evaluated the detailed retinal configurations in the O- and N'-intermediates by performing a density functional theory chemical shift calculation. The results showed that the N'-intermediate has a 63° twisted retinal state due to the 13-cis configuration. The retinal configurations of the O- and N'-intermediates were determined to be 13-trans, 15-syn, and 13-cis, respectively, based on the chemical shift values of [20-13C] and [14-13C] retinal obtained by photoirradiation solid-state NMR and density functional theory calculation.