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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., Cellular activity level at the time of learning is thought to be a critical factor to determine which neurons are recruited to encode memory. Here, the authors show that competitive synaptic plasticity mechanisms influence which neurons will encode a fear memory.

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 (K-d,K-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 (K-d,K-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. SIGNIFICANCE Natronomonas pharaonis halorhodopsin (NpHR), a cytoplasmic membrane Cl- pump, was reconstituted into nanodisks (NDs) using endogenous and exogenous membranes. The photoreaction cycle was examined by absorption, circular dichroism spectra, and flash photolysis. Based on the thermodynamic and kinetic parameters of the photoreaction in detergent-solubilized NpHRs, NpHRs reconstituted into artificial lipid NDs, and NpHR reconstituted into native membrane NDs, we propose that the functional differences in these NpHRs are due to 1) the low content of bacterioruberin in the membrane, 2) the different charge on the membrane surface, 3) the suppression of the conformational changes associated with the Cl- release, 4) the conformational perturbation in the NpHR trimers, and 5) the lack of the intertrimer interactions of NpHR.

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

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.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.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.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.

Nature Communications 13(1), 6460 (2022). doi:10.1038/s41467-022-34019-9, 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$^−$, NO$_3$$^−$. 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., Published by Nature Publishing Group UK, [London]

Bacteriorhodopsin (bR) and halorhodopsin (hR) are both membrane proteins that transport ions across the cell membrane in halobacteria. Their ion transport function is triggered by photoactivated isomerization of the retinal protonated Schiff base (RPSB) chromophore. In spite of their similar structures, bR and hR exhibit widely differing RPSB isomerization rates and quantum yields (with bR being both faster and more efficient than hR). Previous simulations of photoisomerization in bR and hR using ab initio multiple spawning (AIMS) with QM/MM have successfully reproduced the experimentally observed ordering of quantum yields and isomerization rates, but the origin of these differences remains elusive. Here we investigate the role of electrostatic interactions in the protein pocket surrounding RPSB. We probe the influence of protein electrostatics by modifying the charge of the complex counterion in bR/hR to be more/less negative than the native state. We find that such modifications lead to bR-like behavior in hR and vice versa. This demonstrates the crucial role of electrostatic interactions in controlling the outcome of RPSB photoisomerization.

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.

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.

A detailed theoretical analysis of low-power, high-frequency and temporally precise optogenetic inhibition of neuronal spiking, with red-shifted opsins namely, NpHR, eNpHR3.0 and Jaws, has been presented. An accurate model for inhibition of spiking in these opsins expressed hippocampal neurons that includes the important rebound activity of chloride ions across the membrane has been formulated. The effect of various parameters including irradiance, pulse width, frequency, opsin-expression density and chloride concentration has been studied in detail. Theoretical simulations are in very good agreement with reported experimental results. The chloride concentration gradient directly affects the photocurrent and inhibition capacity in all three variants. eNpHR3.0 shows smallest inhibitory post-synaptic potential plateau at higher frequencies. The time delay between light stimulus and target spike is crucial to minimize irradiance and expression density thresholds for suppressing individual spike. Good practical values of photostimulation parameters have been obtained empirically for peak photocurrent, time delay and 100% spiking inhibition, at continuous and pulsed illumination. Under continuous illumination, complete inhibition of neural activity in Jaws-expressing neurons takes place at minimum irradiance of 0.2 mW mm-2 and expression density of 0.2 mS cm-2, whereas for pulsed stimulation, it is at minimum irradiance of 0.6 mW mm-2 and 5 ms pulse width, at 10 Hz. It is shown that Jaws and eNpHR3.0 are able to invoke single spike precise inhibition up to 160 and 200 Hz, respectively. The study is useful in designing new experiments, understanding temporal spike coding and bidirectional control, and curing neurological disorders.

Cl

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%.

Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.

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 A. 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.

Developments in optical imaging and optogenetics are transforming the functional investigation of neuronal networks throughout the brain. Recent studies in the neuroendocrine field have used genetic mouse models combined with a variety of light-activated optical tools as well as GCaMP calcium imaging to interrogate the neural circuitry controlling hormone secretion. The present review highlights the benefits and caveats of these approaches for undertaking both acute brain slice and functional studies in vivo. We focus on the use of channelrhodopsin and the inhibitory optogenetic tools, archaerhodopsin and halorhodopsin, in addition to GCaMP imaging of individual cells in vitro and neural populations in vivo using fiber photometry. We also address issues around the use of genetic vs viral delivery of encoded proteins to specific Cre-expressing cell populations, their quantification, and the use of conscious vs anesthetized animal models. To date, optogenetics and GCaMP imaging have proven useful in dissecting functional circuitry within the brain and are likely to become essential investigative tools for deciphering the different neural networks controlling hormone secretion.

The anion pumping cycle of halorhodopsin from Natronomonas pharaonis (pHR) is initiated when the all-trans/15-anti isomer of retinal is photoisomerized into the 13-cis/15-anti configuration. A recent crystallographic study suggested that a reaction state with 13-cis/15-syn retinal occurred during the anion release process, i.e., after the N state with the 13-cis/15-anti retinal and before the O state with all-trans/15-anti retinal. In this study, we investigated the retinal isomeric composition in a long-living reaction state at various bromide ion concentrations. It was found that the 13-cis isomer (csHR′), in which the absorption spectrum was blue-shifted by ∼8 nm compared with that of the trans isomer (taHR), accumulated significantly when a cold suspension of pHR-rich claret membranes in 4 M NaBr was illuminated with continuous light. Analysis of flash-induced absorption changes suggested that the branching of the trans photocycle into the 13-cis isomer (csHR′) occurs during the decay of an O-like sta...

Early research on the four microbial rhodopsins discovered in the archaeal Halobacterium salinarum revealed a structural template that served as a scaffold for two different functions: light-driven ion transport and phototaxis. Bacteriorhodopsin and halorhodopsin are proton and chloride pumps, respectively, while sensory rhodopsin I and II are responsible for phototactic behavior of the archaea. Halorhodopsins have been identified in various other species. Besides this group of archaeal halorhodopsins distinct chloride transporting rhodopsins groups have recently been identified in other organism like Flavobacteria or Cyanobacteria. Halorhodopsin from Natronomonas pharaonis is the best-studied homologue because of its facile expression and purification and its advantageous properties, which was the reason to introduce this protein as neural silencer into the new field of optogenetics. Two other major families of genetically encoded silencing proteins, proton pumps and anion channels, extended the repertoire of optogenetic tools. Here, we describe the functional and structural characteristics of halorhodopsins. We will discuss the data in light of common principles underlying the mechanism of ion pumps and sensors and will review biophysical and biochemical aspects of neuronal silencers.

Aspartic acid 103 (D103) of sensory rhodopsin II from Halobacterium salinarum (HsSRII, or also called phoborhodopsin) corresponds to D115 of bacteriorhodopsin (BR). This amino acid residue is functionally important in BR. This work reveals that a substitution of D103 with asparagine (D103N) or glutamic acid (D103E) can cause large changes in HsSRII photocycle. These changes include (1) shortened lifetime of the M intermediate in the following order: the wild-type > D103N > D103E; (2) altered decay pathway of a 13-cis O-like species. The 13-cis O-like species, tentatively named Px, was detected in HsSRII photocycle. Px appeared to undergo branched reactions at 0°C, leading to a recovery of the unphotolyzed state and formation of a metastable intermediate, named P370, that slowly decayed to the unphotolyzed state at room temperature. In wild-type HsSRII at 0°C, Px mainly decayed to the unphotolyzed state, and the decay reaction toward P370 was negligible. In mutant D103E at 0°C, Px decayed to P370, while the recovery of the unphotolyzed state became unobservable. In mutant D103N, the two reactions proceeded at comparable rates. Thus, D103 of HsSRII may play an important role in regulation of the photocycle of HsSRII.

Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl−-pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl− pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl−-transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl−-pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR.

We revealed the chloride ion pumping mechanism in halorhodopsin from Natronobacterium pharaonis ( pHR) by exploring sequential structural changes in the retinal chromophore during its photocycle using time-resolved resonance Raman (RR) spectroscopy on the nanosecond to millisecond time scales. A series of RR spectra of the retinal chromophore in the unphotolyzed state and of the three intermediates of pHR were obtained. Using singular value decomposition analysis of the C═C and C-C stretch bands in the time-resolved RR spectra, we identified the spectra of the K, L, and N intermediates. We focused on structural markers of the RR bands to explore the structure of the retinal chromophore. In the unphotolyzed state, the retinal chromophore is in the planar all- trans, 15- anti geometry. The bound ion affects the polyene chain but does not interact with the protonated Schiff base. In the observed intermediates, the chromophore is in the 13- cis configuration. The chromophore in the K intermediate is distorted due to the photoisomerization of retinal. The hydrogen bond is weak in the unphotolyzed state and in the K intermediate, resulting in exchange of the hydrogen-bond acceptor to a water molecule in the K-to-L transition, relaxation of the polyene chain distortion, and generation of an alternative distortion near the Schiff base. The bound halide ion interacts with the protonated Schiff base through the water molecule bound to the protonated Schiff base. In the L-to-N transition, the hydrogen acceptor of the protonated Schiff base switches from the water molecule to another species, although the strong hydrogen bond of the protonated Schiff base remains. This paper reports the first observation of sequential changes in the RR spectra in the pHR photocycle, provides information on the structural evolution of the retinal chromophore, and proposes a model for chloride ion translocation in pHR.

Optogenetics play a significant role in neuroscientific research by providing a tool for understanding neural circuits and brain functions. Natronomonas pharaonis halorhodopsin (NpHR) actively pumps chloride ions into the cells and hyperpolarizes neuronal membranes in response to yellow light. In this study, we generated transgenic Drosophila expressing NpHR under the control of the Gal4/UAS system and virus-infected mice expressing NpHR to explore the effect of dendritic cell factor 1 (Dcf1) on the behavior mediated by the mushroom body in Drosophila and the dentate gyrus (DG) in mice. Study of optogenetic behavior showed that NpHR suppressed the behavior in Drosophila larvae and mice, whereas Dcf1 rescued this suppression. These results suggest that Dcf1 plays an important role in behavior induced by the mushroom body and the hippocampus and provides novel insights into their functions. J. Cell. Biochem. 118: 4210-4215, 2017. © 2017 Wiley Periodicals, Inc.

Background Optogenetic silencing techniques have expanded the causal understanding of the functions of diverse neuronal cell types in both the healthy and diseased brain. A widely used inhibitory optogenetic actuator is eNpHR3.0, an improved version of the light-driven chloride pump halorhodopsin derived from Natronomonas pharaonis. A major drawback of eNpHR3.0 is related to its pronounced inactivation on a time-scale of seconds, which renders it unsuited for applications that require long-lasting silencing. Results Using transgenic mice and Xenopus laevis oocytes expressing an eNpHR3.0-EYFP fusion protein, we here report optimized photo-stimulation techniques that profoundly increase the stability of eNpHR3.0-mediated currents during long-term photo-stimulation. We demonstrate that optimized photo-stimulation enables prolonged hyperpolarization and suppression of action potential discharge on a time-scale of minutes. Conclusions Collectively, our findings extend the utility of eNpHR3.0 to the long-lasting inhibition of excitable cells, thus facilitating the optogenetic dissection of neural circuits.

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.

From the low-temperature absorption and Raman measurements of halorhodopsin from

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., eLife digest Despite being unpleasant, pain is critical to survival because it acts as a warning for damage or impending harm. Day-to-day pain like a stubbed toe or a pricked finger is called acute pain. It alerts the body to harm but only lasts a short time and usually goes away on its own. Pain that persists more than three months after the damaged tissue has healed is known as chronic pain, and it is a serious problem that is often difficult to treat. Learning more about the causes of chronic pain is necessary to help develop more effective therapies. Nerve pathways in the spinal cord help process pain and other sensory information from the skin and relay it to the brain. These pathways include sensory fibers that carry pain information from the body to the spinal cord, as well as cells that relay this information to the brain. But not much is known about how the nerves and cells in this region prioritize or refine sensory information before sending it to the brain. Now, Smith et al. have used mice to show that nerve cells in the spinal cord that produce the protein calretinin can act as a pain amplifier, causing it to persist. A technique called optogenetics was used to turn on calretinin nerve cells by exposing them to light. This caused the mice to behave like they were in pain even though they had not been harmed, and the behaviour stopped when they were treated with morphine, a powerful painkiller. Further experiments showed that calretinin nerve cells form a highly interconnected network in the spinal cord. These results show that calretinin nerve cells can ‘jump-start’ the pain pathway within the spinal cord, even when there is no painful stimulation of the skin. Turning on these cells even briefly causes behaviours associated with prolonged pain. By revealing that networks of calretinin nerve cells in the spinal cord act like an in-built pain amplifier, the experiments identify these cells as a potential target for new treatments for chronic pain.

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.

In this study, we used the peristaltic crawling ofDrosophilalarvae as a model to study how motor patterns are regulated by central circuits. We built an experimental system that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). We then investigated the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 in a middle segment (A4, A5, or A6), but not other segments, dramatically decreased the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 in a posterior segment (A6 or A7) increased the frequency of the motor waves. Since peripheral nerves mediating sensory feedback were severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. Our results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation were lost inshaking-B2(shakB2) orogre2, gap-junction mutations inDrosophila, or upon acute application of the gap junction blocker carbenoxolone, implicating electrical synapses in the signaling from MNs. Cell-type-specific RNAi suggestedshakBandogrefunction in MNs and interneurons, respectively, during the signaling. Our results not only reveal an unexpected role for MNs in motor pattern regulation, but also introduce a powerful experimental system that enables examination of the input–output relationship among the component neurons in this system.SIGNIFICANCE STATEMENTMotor neurons are generally considered passive players in motor pattern generation, simply relaying information from upstream interneuronal circuits to the target muscles. This study shows instead that MNs play active roles in the control of motor generation by conveying information via gap junctions to the central pattern-generating circuits in larvalDrosophila, providing novel insights into motor circuit control. The experimental system introduced in this study also presents a new approach for studying intersegmentally coordinated locomotion. Unlike traditional electrophysiology methods, this system enables the simultaneous recording and manipulation of populations of neurons that are genetically specified and span multiple segments.

The transmembrane pump halorhodopsin in halophilic archaea translocates chloride ions from the extracellular to the cytoplasmic side upon illumination. In the ground state a tightly bound chloride ion occupies the primary chloride-binding site (CBS I) close to the protonated Schiff base that links the retinal chromophore to the protein. The light-triggeredtrans–cisisomerization of retinal causes structural changes in the protein associated with movement of the chloride ion. In reverse, chemical depletion of CBS I inNatronomonas pharaonishalorhodopsin (NpHR) through deprotonation of the Schiff base results in conformational changes of the protein: a state thought to mimic late stages of the photocycle. Here, crystals ofHalobacterium salinarumhalorhodopsin (HsHR) were soaked at high pH to provoke deprotonation of the Schiff base and loss of chloride. The crystals changed colour from purple to yellow and the occupancy of CBS I was reduced from 1 to about 0.5. In contrast toNpHR, this chloride depletion did not cause substantial conformational changes in the protein. Nevertheless, two observations indicate that chloride depletion could eventually result in structural changes similar to those found inNpHR. Firstly, the partially chloride-depleted form ofHsHR has increased normalizedBfactors in the region of helix C that is close to CBS I and changes its conformation inNpHR. Secondly, prolonged soaking ofHsHR crystals at high pH resulted in loss of diffraction. In conclusion, the conformation of the chloride-free protein may not be compatible with this crystal form ofHsHR despite a packing arrangement that hardly restrains helices E and F that presumably move during ion transport.

Like other microbial rhodopsins, the light driven chloride pump halorhodopsin from Natronomonas pharaonis (pHR) contains a mixture of all-trans/15-anti and 13-cis/15-syn isomers in the dark adapted state. A recent crystallographic study of the reaction states of pHR has shown that reaction states with 13-cis/15-syn retinal occur in the anion pumping cycle that is initiated by excitation of the all-trans isomer. In this study, we investigated interconversions among different isomeric states of pHR in the absence of chloride ions. The illumination of chloride free pHR with red light caused a large blue shift in the absorption maximum of the retinal visible band. During this "red adaptation", the content of the 11-cis isomer increased significantly, while the molar ratio of the 13-cis isomer to the all-trans isomer remained unchanged. The results suggest that the thermally activated interconversion between the 13-cis and the all-trans isomers is very rapid. Diffraction data from red adapted crystals showed that accommodation of the retinal chromophore with the 11-cis/15-syn configuration was achieved without a large change in the retinal binding pocket. The measurement of absorption kinetics under illumination showed that the 11-cis isomer, with a λmax at 565 nm, was generated upon excitation of a red-shifted species (λmax = 625 nm) that was present as a minor component in the dark adapted state. It is possible that this red-shifted species mimics an O-like reaction state with 13-cis/15-syn retinal, which was hypothesized to occur at a late stage of the anion pumping cycle.

Maladaptive aggressive behavior is associated with a number of neuropsychiatric disorders1 and is thought to partly result from inappropriate activation of brain reward systems in response to aggressive or violent social stimuli2. Nuclei within the ventromedial hypothalamus3–5, extended amygdala6 and limbic7 circuits are known to encode initiation of aggression; however, little is known about the neural mechanisms that directly modulate the motivational component of aggressive behavior8. To address this, we established a mouse model to measure the valence of aggressive inter-male social interaction with a smaller subordinate intruder as reinforcement for the development of conditioned place preference (CPP). Aggressors (AGG) develop a CPP, while non-aggressors (NON) develop a conditioned place aversion (CPA), to the intruder-paired context. Further, we identify a functional GABAergic projection from the basal forebrain (BF) to the lateral habenula (lHb) that bi-directionally controls the valence of aggressive interactions. Circuit-specific silencing of GABAergic BF-lHb terminals of AGG with halorhodopsin (NpHR3.0) increases lHb neuronal firing and abolishes CPP to the intruder-paired context. Activation of GABAergic BF-lHb terminals of NON with channelrhodopsin (ChR2) decreases lHb neuronal firing and promotes CPP to the intruder-paired context. Lastly, we show that altering inhibitory transmission at BF-lHb terminals does not control the initiation of aggressive behavior. These results demonstrate that the BF-lHb circuit plays a critical role in regulating the valence of inter-male aggressive behavior and provide novel mechanistic insight into the neural circuits modulating aggression reward processing.

International audience; 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.

A novel method is presented to screen for suitable crystallization conditions and produce large amounts of microcrystals of membrane proteins in lipidic cubic phase for serial crystallography experiments., Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

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 indium‑tin 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.

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.

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.