A bistable inhibitory optoGPCR for multiplexed optogenetic control of neural circuits.

Information is transmitted between brain regions through the release of neurotransmitters from long-range projecting axons. Understanding how the activity of such long-range connections contributes to behavior requires efficient methods for reversibly manipulating their function. Chemogenetic and optogenetic tools, acting through endogenous G-protein-coupled receptor pathways, can be used to modulate synaptic transmission, but existing tools are limited in sensitivity, spatiotemporal precision or spectral multiplexing capabilities. Here we systematically evaluated multiple bistable opsins for optogenetic applications and found that the Platynereis dumerilii ciliary opsin (PdCO) is an efficient, versatile, light-activated bistable G-protein-coupled receptor that can suppress synaptic transmission in mammalian neurons with high temporal precision in vivo. PdCO has useful biophysical properties that enable spectral multiplexing with other optogenetic actuators and reporters. We demonstrate that PdCO can be used to conduct reversible loss-of-function experiments in long-range projections of behaving animals, thereby enabling detailed synapse-specific functional circuit mapping.

Optimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors.

Neuromodulatory signaling via G protein-coupled receptors (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. The recent development of optogenetic tools to induce G protein-mediated signaling provides the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signaling in vivo remains challenging. Here we optimize the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). These optoDopRs exhibit high signaling specificity and light sensitivity in vitro. In vivo, we show receptor and cell type-specific effects of dopaminergic signaling in various behaviors, including the ability of optoDopRs to rescue the loss of the endogenous receptors. This work demonstrates that optoXRs can enable optical control of neuromodulatory receptor-specific signaling in functional and behavioral studies.

Aion is a bistable anion-conducting channelrhodopsin that provides temporally extended and reversible neuronal silencing.

Optogenetic silencing allows to reveal the necessity of selected neuronal populations for various neurophysiological functions. These range from synaptic transmission and coordinated neuronal network activity to control of specific behaviors. An ideal single-component optogenetic silencing tool should be switchable between active and inactive states with precise timing while preserving its activity in the absence of light until switched to an inactive state. Although bistable anion-conducting channelrhodopsins (ACRs) were previously engineered to reach this goal, their conducting state lifetime was limited to only a few minutes and some ACRs were not fully switchable. Here we report Aion, a bistable ACR displaying a long-lasting open state with a spontaneous closing time constant close to 15 min. Moreover, Aion can be switched between the open and closed state with millisecond precision using blue and orange light, respectively. The long conducting state enables overnight silencing of neurons with minimal light exposure. We further generated trafficking-optimized versions of Aion, which show enhanced membrane localization and allow precisely timed, long-lasting all-optical control of nociceptive responses in larvae of Drosophila melanogaster. Thus, Aion is an optogenetic silencing tool for inhibition of neuronal activity over many hours which can be switched between an active and inactive state with millisecond precision.

A neuropeptidergic circuit gates selective escape behavior of Drosophila larvae.

Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.

BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons.

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.

Efficient optogenetic silencing of neurotransmitter release with a mosquito rhodopsin.

Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity. However, existing inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homolog of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents induces a reversible ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at presynaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

Siboglinidae Tubes as an Additional Niche for Microbial Communities in the Gulf of Cádiz-A Microscopical Appraisal.

Siboglinids were sampled from four mud volcanoes in the Gulf of Cádiz (El Cid MV, Bonjardim MV, Al Gacel MV, and Anastasya MV). These invertebrates are characteristic to cold seeps and are known to host chemosynthetic endosymbionts in a dedicated trophosome organ. However, little is known about their tube as a potential niche for other microorganisms. Analyses by scanning and transmission electron microscopy showed dense biofilms on the tube in Al Gacel MV and Anastasya MV specimens by prokaryotic cells. Methanotrophic bacteria were the most abundant forming these biofilms as further supported by 16S rRNA sequence analysis. Furthermore, elemental analyses with electron microscopy and energy-dispersive X-ray spectroscopy point to the mineralization and silicification of the tube, most likely induced by the microbial metabolisms. Bacterial and archaeal 16S rRNA sequence libraries revealed abundant microorganisms related to these siboglinid specimens and certain variations in microbial communities among samples. Thus, the tube remarkably increases the microbial biomass related to the worms and provides an additional microbial niche in deep-sea ecosystems.

Cyanobacterial Mats in Calcite-Precipitating Serpentinite-Hosted Alkaline Springs of the Voltri Massif, Italy.

(1) Background: Microbial communities in terrestrial, calcifying high-alkaline springs are not well understood. In this study, we investigate the structure and composition of microbial mats in ultrabasic (pH 10-12) serpentinite springs of the Voltri Massif (Italy). (2) Methods: Along with analysis of chemical and mineralogical parameters, environmental DNA was extracted and subjected to analysis of microbial communities based upon next-generation sequencing. (3) Results: Mineral precipitation and microbialite formation occurred, along with mat formation. Analysis of the serpentinite spring microbial community, based on Illumina sequencing of 16S rRNA amplicons, point to the relevance of alkaliphilic cyanobacteria, colonizing carbonate buildups. Cyanobacterial groups accounted for up to 45% of all retrieved sequences; 3-4 taxa were dominant, belonging to the filamentous groups of Leptolyngbyaceae, Oscillatoriales, and Pseudanabaenaceae. The cyanobacterial community found at these sites is clearly distinct from creek water sediment, highlighting their specific adaptation to these environments.

Ret and Substrate-Derived TGF-ß Maverick Regulate Space-Filling Dendrite Growth in Drosophila Sensory Neurons.

Dendrite morphogenesis is a highly regulated process that gives rise to stereotyped receptive fields, which are required for proper neuronal connectivity and function. Specific classes of neurons, including Drosophila class IV dendritic arborization (C4da) neurons, also feature complete space-filling growth of dendrites. In this system, we have identified the substrate-derived TGF-ß ligand maverick (mav) as a developmental signal promoting space-filling growth through the neuronal Ret receptor. Both are necessary for radial spreading of C4da neuron dendrites, and Ret is required for neuronal uptake of Mav. Moreover, local changes in Mav levels result in directed dendritic growth toward regions with higher ligand availability. Our results suggest that Mav acts as a substrate-derived secreted signal promoting dendrite growth within not-yet-covered areas of the receptive field to ensure space-filling dendritic growth.

Transcriptomic and metabolic analyses reveal salvage pathways in creatine-deficient AGAT(-/-) mice.

Skeletal muscles require energy either at constant low (e.g., standing and posture) or immediate high rates (e.g., exercise). To fulfill these requirements, myocytes utilize the phosphocreatine (PCr)/creatine (Cr) system as a fast energy buffer and shuttle. We have generated mice lacking L-arginine:glycine amidino transferase (AGAT), the first enzyme of creatine biosynthesis. These AGAT(-/-) (d/d) mice are devoid of the PCr/Cr system and reveal severely altered oxidative phosphorylation. In addition, they exhibit complete resistance to diet-induced obesity, which is associated with a chronic activation of AMP-activated protein kinase in muscle and white adipose tissue. The underlying metabolic rearrangements have not yet been further analyzed. Here, we performed gene expression analysis in skeletal muscle and a serum amino acid profile of d/d mice revealing transcriptomic and metabolic alterations in pyruvate and glucose pathways. Differential pyruvate tolerance tests demonstrated preferential conversion of pyruvate to alanine, which was supported by increased protein levels of enzymes involved in pyruvate and alanine metabolism. Pyruvate tolerance tests suggested severely impaired hepatic gluconeogenesis despite increased availability of pyruvate and alanine. Furthermore, enzymes of serine production and one-carbon metabolism were significantly up-regulated in d/d mice, indicating increased de novo formation of one-carbon units from carbohydrate metabolism linked to NAD(P)H production. Besides the well-established function of the PCr/Cr system in energy metabolism, our transcriptomic and metabolic analyses suggest that it plays a pivotal role in systemic one-carbon metabolism, oxidation/reduction, and biosynthetic processes. Therefore, the PCr/Cr system is not only an energy buffer and shuttle, but also a crucial component involved in numerous systemic metabolic processes.

Differential regulation of AMPK activation in leptin- and creatine-deficient mice.

AMP-activated protein kinase (AMPK) is a key sensor and regulator of energy homeostasis. Previously, we demonstrated that intracellular energy depletion by L-arginine:glycine amidinotransferase (AGAT) deficiency resulted in AMPK activation and protected from metabolic syndrome. In the present study, we show tissue-specific leptin dependence of AMPK activation by energy depletion. We investigated leptin-dependent AMPK regulation in AGAT- and leptin-deficient (d/d ob/ob) mice. Like ob/ob mice, but unlike d/d mice, d/d ob/ob mice were obese and glucose intolerant. Therefore, leptin is a prerequisite for resistance to metabolic syndrome in AGAT-deficient mice. Quantitative Western blots revealed a 4-fold increase in AMPK activation in skeletal muscle of d/d ob/ob mice (P<0.001). However, AMPK activation was absent in white adipose tissue (WAT) and liver. Compared with blood glucose levels in ob/ob mice, fasting levels were still reduced and therefore did not show leptin dependence (wild-type, 79.4±3.9 mg/dl; d/d, 68.4±3.2 mg/dl; P<0.05). In ob/ob mice and wild-type mice, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), in combination with leptin, augmented glucose tolerance compared with AICAR alone, whereas no improvement was found under conditions of high-fat-diet feeding. These findings reveal a previously unknown synergistic AMPK activation by leptin and intracellular energy depletion, suggesting that AMPK activation can be therapeutically effective in metabolic syndrome only if leptin sensitivity is preserved.

L-arginine:glycine amidinotransferase deficiency protects from metabolic syndrome.

Phosphorylated creatine (Cr) serves as an energy buffer for ATP replenishment in organs with highly fluctuating energy demand. The central role of Cr in the brain and muscle is emphasized by severe neurometabolic disorders caused by Cr deficiency. Common symptoms of inborn errors of creatine synthesis or distribution include mental retardation and muscular weakness. Human mutations in l-arginine:glycine amidinotransferase (AGAT), the first enzyme of Cr synthesis, lead to severely reduced Cr and guanidinoacetate (GuA) levels. Here, we report the generation and metabolic characterization of AGAT-deficient mice that are devoid of Cr and its precursor GuA. AGAT-deficient mice exhibited decreased fat deposition, attenuated gluconeogenesis, reduced cholesterol levels and enhanced glucose tolerance. Furthermore, Cr deficiency completely protected from the development of metabolic syndrome caused by diet-induced obesity. Biochemical analyses revealed the chronic Cr-dependent activation of AMP-activated protein kinase (AMPK), which stimulates catabolic pathways in metabolically relevant tissues such as the brain, skeletal muscle, adipose tissue and liver, suggesting a mechanism underlying the metabolic phenotype. In summary, our results show marked metabolic effects of Cr deficiency via the chronic activation of AMPK in a first animal model of AGAT deficiency. In addition to insights into metabolic changes in Cr deficiency syndromes, our genetic model reveals a novel mechanism as a potential treatment option for obesity and type 2 diabetes mellitus.

A homozygous SCN5A mutation in a severe, recessive type of cardiac conduction disease.

Cardiac sodium channels are key players in the generation and propagation of action potentials in the human heart. Heterozygous mutations in the SCN5A gene have been found to be associated with long QT syndrome, Brugada syndrome, and sinus node dysfunction (SND). Recently, overlapping arrhythmia phenotypes have been reported as well. Here we describe a novel recessive SCN5A mutation in a family originating from the German minority in White Russia. Four affected children with a history of early cardiac arrhythmia encompassing SND, conduction disease, and severe ventricular arrhythmias, are homozygous carriers of a novel SCN5A missense mutation (p.I230T) in the channel protein. Interestingly, the heterozygous mutation carriers had neither significant ECG abnormalities nor a history of cardiac events. Heterologous expression of SCN5A(I230T) channels revealed normal protein transport but altered biophysical sodium channel properties. Voltage range of both activation and inactivation were shifted in a way that resulted in decreased sodium current and loss of channel function. In conclusion, we describe a rare clinical condition with a novel SCN5A mutation causing a new type of complex cardiac arrhythmia. Unlike most previously reported sodium channelopathies, this overlap syndrome displays recessive inheritance characteristics and does not seem to follow simple Mendelian rules.

Gamma-aminobutyric acid type B receptors are constitutively internalized via the clathrin-dependent pathway and targeted to lysosomes for degradation.

Receptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of gamma-aminobutyric acid, type B (GABAB) receptors. Therefore, we analyzed internalization of GABAB receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40-50% of cell surface receptors was detected. Stimulation of GABAB receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonist-induced internalization. The data suggest that GABAB receptors were endocytosed via the classical dynamin- and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABAB receptors via clathrin-coated pits, which resulted in lysosomal degradation of the receptors.

C-terminal HERG (LQT2) mutations disrupt IKr channel regulation through 14-3-3epsilon.

Beta-adrenergic receptor-mediated cAMP or protein kinase A (PKA)-dependent modulation of cardiac potassium currents controls ventricular action potential duration (APD) at faster heart rates. HERG (KCNH2) gene mutations are associated with congenital long-QT syndrome (LQT2) and affect IKr activity, a key determinant in ventricular repolarization. Physical activity or emotional stress often triggers lethal arrhythmias in LQT2 patients. Beta-adrenergic stimulation of HERG channel activity is amplified and prolonged in vitro by the adaptor protein 14-3-3epsilon. In LQT2 families, we identified three novel heterozygous HERG mutations (G965X, R1014PfsX39, V1038AfsX21) in the C-terminus that led to protein truncation and loss of a PKA phosphorylation site required for binding of 14-3-3epsilon. When expressed in CHO cells, the mutants produced functional HERG channels with normal kinetic properties. We now provide evidence that HERG channel regulation by 14-3-3epsilon is of physiological significance in humans. Upon co-expression with 14-3-3epsilon, mutant channels still bound 14-3-3epsilon but did not respond with a hyperpolarizing shift in voltage dependence as seen in wild-type channels. Co-expression experiments of wild-type and mutant channels revealed dominant-negative behavior of all three HERG mutations. Simulations of the effects of sympathetic stimulation of HERG channel activity on the whole-cell action potential suggested a role in rate-dependent control of APD and an impaired ability of mutant cardiac myocytes to respond to a triggered event or an ectopic beat. In summary, the attenuated functional effects of 14-3-3epsilon on C-terminally truncated HERG channels demonstrate the physiological importance of coupling beta-adrenergic stimulation and HERG channel activity.

Subtype-selective interaction with the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) regulates cell surface expression of GABA(B) receptors.

The metabotropic gamma-aminobutyric acid, type B (GABA(B)) receptors mediate the slow component of GABAergic transmission in the brain. Functional GABA(B) receptors are heterodimers of the two subunits GABA(B1) and GABA(B2), of which GABA(B1) exists in two main isoforms, GABA(B1a) and GABA(B1b). The significance of the structural heterogeneity of GABA(B) receptors, the mechanism leading to their differential targeting in neurons as well as the regulation of cell surface numbers of GABA(B) receptors, is poorly understood. To gain insights into these processes, we searched for proteins interacting with the C-terminal domain of GABA(B2). Here, we showed that the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) directly interacts with GABA(B) receptors in a subtype-selective manner to regulate cell surface expression of GABA(B1a)/GABA(B2) receptors upon co-expression in HEK 293 cells. The interaction of CHOP with GABA(B1a)/GABA(B2) receptors resulted in their intracellular accumulation and in a reduced number of cell surface receptors. This regulation required the interaction of CHOP via two distinct domains with the heterodimeric receptor; its C-terminal leucine zipper associates with the leucine zipper present in the C-terminal domain of GABA(B2), and its N-terminal domain associates with an as yet unidentified site on GABA(B1a). In conclusion, the data indicated a subtype-selective regulation of cell surface receptors by interaction with the transcription factor CHOP.

Contribution of N- and C-terminal Kv4.2 channel domains to KChIP interaction [corrected].

Association of Shal gene-related voltage-gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C-terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine-scanning mutagenesis within the proximal 40 amino acid portion and a structure-based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C-terminus was truncated at various positions. Wild-type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2-induced modulation of Kv4.2 currents was studied with whole-cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N-terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C-terminal deletions also had marked effects on KChIP2-dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C-terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose 'anchoring' at T1- and C-terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.

N-type inactivation features of Kv4.2 channel gating.

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Delta40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K+. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Delta40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Delta40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.

Identification and functional characterization of a novel KCNE2 (MiRP1) mutation that alters HERG channel kinetics.

Long-QT syndrome (LQTS) may cause syncope and sudden death due to cardiac tachyarrhythmia. Chromosome 7-linked LQTS (LQT2) has been correlated with mutations in the human ether-a-go-go-related gene (HERG). HERG forms voltage-gated K channels that may be associated with Mink-related peptide 1 (MiRP1), an auxiliary beta-subunit. The channels mediate currents that resemble native I(Kr). Mutations in the KCNE2 gene encoding MiRP1 may also cause LQTS. In this study, the frequency of mutations in KCNE2 of 150 unrelated LQTS patients without known genotype and of 100 controls was analyzed using single-strand conformation polymorphism analysis and direct sequencing. We identified a novel missense mutation, V65 M, in the KCNE2 gene of a 17-year-old female with syncope and LQTS. Expression studies in Chinese hamster ovary cells revealed that mutant and wild-type MiRP1 co-localized with HERG subunits and formed functional channels. However, mutant HERG/MiRP1(V65M) channels mediated currents with an accelerated inactivation time course compared with wild-type channels. The accelerated inactivation time course of HERG/MiRP1(V65M) channels may decrease I(Kr) current density of myocardial cells, thereby impairing the ability of myocytes to repolarize in response to sudden membrane depolarizations such as extrasystoles.