Optogenetic induction of chronic glucocorticoid exposure in early-life leads to blunted stress-response in larval zebrafish.

Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood.

Pesticide exposure and the microbiota-gut-brain axis.

The gut microbiota exist within a dynamic ecosystem shaped by various factors that includes exposure to xenobiotics such as pesticides. It is widely regarded that the gut microbiota plays an essential role in maintaining host health, including a major influence on the brain and behaviour. Given the widespread use of pesticides in modern agriculture practices, it is important to assess the long-term collateral effects these xenobiotic exposures have on gut microbiota composition and function. Indeed, exposure studies using animal models have shown that pesticides can induce negative impacts on the host gut microbiota, physiology and health. In tandem, there is a growing body of literature showing that the effects of pesticide exposure can be extended to the manifestation of behavioural impairments in the host. With the increasing appreciation of the microbiota-gut-brain axis, in this review we assess whether pesticide-induced changes in gut microbiota composition profiles and functions could be driving these behavioural alterations. Currently, the diversity of pesticide type, exposure dose and variation in experimental designs hinders direct comparisons of studies presented. Although many insights presented, the mechanistic connection between the gut microbiota and behavioural changes remains insufficiently explored. Future experiments should therefore focus on causal mechanisms to examine the gut microbiota as the mediator of the behavioural impairments observed in the host following pesticide exposure.

Microbial-derived metabolites induce actin cytoskeletal rearrangement and protect blood-brain barrier function.

The gut microbiota influences host brain function, but the underlying gut-brain axis connections and molecular processes remain unclear. One pathway along this bidirectional communication system involves circulating microbially derived metabolites, such as short-chain fatty acids (SCFAs), which include butyrate and propionate. Brain endothelium is the main interface of communication between circulating signals and the brain, and it constitutes the main component of the blood-brain barrier (BBB). Here, we used a well-established in vitro BBB model treated with physiologically relevant concentrations of butyrate and propionate with and without lipopolysaccharide (LPS) to examine the effects of SCFAs on the actin cytoskeleton and tight junction protein structure. Both SCFAs induced distinct alterations to filamentous actin directionality. SCFAs also increased tight junction protein spikes and protected from LPS-induced tight-junction mis-localization, improved BBB integrity, and modulated mitochondrial network dynamics. These findings identify the actin cytoskeletal dynamics as another target further illuminating how SCFAs can influence BBB physiology.

Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Activity in Behavior.

Over the past 15 years, optogenetic methods have revolutionized neuroscientific and cell biological research, also in the nematode Caenorhabditis elegans. In this chapter, we give an update about current optogenetic tools and methods to address neuronal activity and inhibition, as well as second messenger signaling, based on microbial rhodopsins. We address channelrhodopsins and variants thereof, which conduct cations or anions, for depolarization and hyperpolarization of the membrane potential. Also, we cover ion pumping rhodopsins, like halorhodopsin, Mac, and Arch. A recent addition to rhodopsin-based optogenetics is voltage imaging tools that allow fluorescent readout of membrane voltage (directly, via fluorescence of the rhodopsin chromophore retinal, or indirectly, via electrochromic FRET). Last, we report on a new addition to the optogenetic toolbox, which is rhodopsin guanylyl cyclases, as well as mutated variants with specificity for cyclic AMP. These can be used to regulate intracellular levels of cGMP and cAMP, which are important second messengers in sensory and other neurons. We further show how they can be combined with cyclic nucleotide-gated channels in two-component optogenetics, for depolarization or hyperpolarization of membrane potential. For all tools, we present protocols for straightforward experimentation to address neuronal activation and inhibition, particularly at the neuromuscular junction, and for combined optogenetic actuation and Ca2+ imaging. We also provide protocols for usage of rhodopsin guanylyl and adenylyl cyclases. Finally, we list a number of points to consider when designing and conducting rhodopsin-based optogenetic experiments.

Bistable Photoswitch Allows in Vivo Control of Hematopoiesis.

Optical control has enabled functional modulation in cell culture with unparalleled spatiotemporal resolution. However, current tools for in vivo manipulation are scarce. Here, we design and implement a genuine on-off optochemical probe capable of achieving hematopoietic control in zebrafish. Our photopharmacological approach first developed conformationally strained visible light photoswitches (CS-VIPs) as inhibitors of the histone methyltransferase MLL1 (KMT2A). In blood homeostasis MLL1 plays a crucial yet controversial role. CS-VIP 8 optimally fulfils the requirements of a true bistable functional system in vivo under visible-light irradiation, and with unprecedented stability. These properties are exemplified via hematopoiesis photoinhibition with a single isomer in zebrafish. The present interdisciplinary study uncovers the mechanism of action of CS-VIPs. Upon WDR5 binding, CS-VIP 8 causes MLL1 release with concomitant allosteric rearrangements in the WDR5/RbBP5 interface. Since our tool provides on-demand reversible control without genetic intervention or continuous irradiation, it will foster hematopathology and epigenetic investigations. Furthermore, our workflow will enable exquisite photocontrol over other targets inhibited by macrocycles.

Optogenetic tools for manipulation of cyclic nucleotides functionally coupled to cyclic nucleotide-gated channels.

BACKGROUND AND PURPOSE: The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarizers based on K+ currents. EXPERIMENTAL APPROACH: For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide-gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels. KEY RESULTS: We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established photoactivatable membrane-bound adenylyl cyclases, based on mutated versions ("A-2x") of Blastocladiella and Catenaria ("Be," "Ca") CyclOp, as N-terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two-component optogenetics, we introduced the cAMP-gated K+ -channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the B. emersonii cGMP-gated K+ -channel BeCNG1 together with BeCyclOp. CONCLUSION AND IMPLICATIONS: We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Microbiota-brain interactions: Moving toward mechanisms in model organisms.

Changes in the microbiota are associated with alterations in nervous system structure-function and behavior and have been implicated in the etiology of neuropsychiatric and neurodegenerative disorders. Most of these studies have centered on mammalian models due to their phylogenetic proximity to humans. Indeed, the germ-free mouse has been a particularly useful model organism for investigating microbiota-brain interactions. However, microbiota-brain axis research on simpler genetic model organisms with a vast and diverse scientific toolkit (zebrafish, Drosophila melanogaster, and Caenorhabditis elegans) is now also coming of age. In this review, we summarize the current state of microbiota-brain axis research in rodents and humans, and then we elaborate and discuss recent research on the neurobiological and behavioral effects of the microbiota in the model systems of fish, flies, and worms. We propose that a cross-species, holistic and mechanistic approach to unravel the microbiota-brain communication is an essential step toward rational microbiota-based therapeutics to combat brain disorders.

Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.

Anatomy, development, and plasticity of the neurosecretory hypothalamus in zebrafish.

The paraventricular nucleus (PVN) of the hypothalamus harbors diverse neurosecretory cells with critical physiological roles for the homeostasis. Decades of research in rodents have provided a large amount of information on the anatomy, development, and function of this important hypothalamic nucleus. However, since the hypothalamus lies deep within the brain in mammals and is difficult to access, many questions regarding development and plasticity of this nucleus still remain. In particular, how different environmental conditions, including stress exposure, shape the development of this important nucleus has been difficult to address in animals that develop in utero. To address these open questions, the transparent larval zebrafish with its rapid external development and excellent genetic toolbox offers exciting opportunities. In this review, we summarize recent information on the anatomy and development of the neurosecretory preoptic area (NPO), which represents a similar structure to the mammalian PVN in zebrafish. We will then review recent studies on the development of different cell types in the neurosecretory hypothalamus both in mouse and in fish. Lastly, we discuss stress-induced plasticity of the PVN mainly discussing the data obtained in rodents, but pointing out tools and approaches available in zebrafish for future studies. This review serves as a primer for the currently available information relevant for studying the development and plasticity of this important brain region using zebrafish.

Food Sensation Modulates Locomotion by Dopamine and Neuropeptide Signaling in a Distributed Neuronal Network.

Finding food and remaining at a food source are crucial survival strategies. We show how neural circuits and signaling molecules regulate these food-related behaviors in Caenorhabditis elegans. In the absence of food, AVK interneurons release FLP-1 neuropeptides that inhibit motorneurons to regulate body posture and velocity, thereby promoting dispersal. Conversely, AVK photoinhibition promoted dwelling behavior. We identified FLP-1 receptors required for these effects in distinct motoneurons. The DVA interneuron antagonizes signaling from AVK by releasing cholecystokinin-like neuropeptides that potentiate cholinergic neurons, in response to dopaminergic neurons that sense food. Dopamine also acts directly on AVK via an inhibitory dopamine receptor. Both AVK and DVA couple to head motoneurons by electrical and chemical synapses to orchestrate either dispersal or dwelling behavior, thus integrating environmental and proprioceptive signals. Dopaminergic regulation of food-related behavior, via similar neuropeptides, may be conserved in mammals.

Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.

Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Activity in Behavior.

Optogenetics was introduced as a new technology in the neurosciences about a decade ago (Zemelman et al., Neuron 33:15-22, 2002; Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005; Zemelman et al., Proc Natl Acad Sci USA 100:1352-1357, 2003). It combines optics, genetics, and bioengineering to render neurons sensitive to light, in order to achieve a precise, exogenous, and noninvasive control of membrane potential, intracellular signaling, network activity, or behavior (Rein and Deussing, Mol Genet Genomics 287:95-109, 2012; Yizhar et al., Neuron 71:9-34, 2011). As C. elegans is transparent, genetically amenable, has a small nervous system mapped with synapse resolution, and exhibits a rich behavioral repertoire, it is especially open to optogenetic methods (White et al., Philos Trans R Soc Lond B Biol Sci 314:1-340, 1986; De Bono et al., Optogenetic actuation, inhibition, modulation and readout for neuronal networks generating behavior in the nematode Caenorhabditis elegans, In: Hegemann P, Sigrist SJ (eds) Optogenetics, De Gruyter, Berlin, 2013; Husson et al., Biol Cell 105:235-250, 2013; Xu and Kim, Nat Rev Genet 12:793-801, 2011). Optogenetics, by now an "exploding" field, comprises a repertoire of different tools ranging from transgenically expressed photo-sensor proteins (Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005) or cascades (Zemelman et al., Neuron 33:15-22, 2002) to chemical biology approaches, using photochromic ligands of endogenous channels (Szobota et al., Neuron 54:535-545, 2007). Here, we will focus only on optogenetics utilizing microbial rhodopsins, as these are most easily and most widely applied in C. elegans. For other optogenetic tools, for example the photoactivated adenylyl cyclases (PACs, that drive neuronal activity by increasing synaptic vesicle priming, thus exaggerating rather than overriding the intrinsic activity of a neuron, as occurs with rhodopsins), we refer to other literature (Weissenberger et al., J Neurochem 116:616-625, 2011; Steuer Costa et al., Photoactivated adenylyl cyclases as optogenetic modulators of neuronal activity, In: Cambridge S (ed) Photswitching proteins, Springer, New York, 2014). In this chapter, we will give an overview of rhodopsin-based optogenetic tools, their properties and function, as well as their combination with genetically encoded indicators of neuronal activity. As there is not "the" single optogenetic experiment we could describe here, we will focus more on general concepts and "dos and don'ts" when designing an optogenetic experiment. We will also give some guidelines on which hardware to use, and then describe a typical example of an optogenetic experiment to analyze the function of the neuromuscular junction, and another application, which is Ca(2+) imaging in body wall muscle, with upstream neuronal excitation using optogenetic stimulation. To obtain a more general overview of optogenetics and optogenetic tools, we refer the reader to an extensive collection of review articles, and in particular to volume 1148 of this book series, "Photoswitching Proteins."

Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

AzoCholine Enables Optical Control of Alpha 7 Nicotinic Acetylcholine Receptors in Neural Networks.

Nicotinic acetylcholine receptors (nAChRs) are essential for cellular communication in higher organisms. Even though a vast pharmacological toolset to study cholinergic systems has been developed, control of endogenous neuronal nAChRs with high spatiotemporal precision has been lacking. To address this issue, we have generated photoswitchable nAChR agonists and re-evaluated the known photochromic ligand, BisQ. Using electrophysiology, we found that one of our new compounds, AzoCholine, is an excellent photoswitchable agonist for neuronal α7 nAChRs, whereas BisQ was confirmed to be an agonist for the muscle-type nAChR. AzoCholine could be used to modulate cholinergic activity in a brain slice and in dorsal root ganglion neurons. In addition, we demonstrate light-dependent perturbation of behavior in the nematode, Caenorhabditis elegans.

Cysteine dioxygenase 1 is a tumor suppressor gene silenced by promoter methylation in multiple human cancers.

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma.

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.

Association of early phase of colorectal carcinogenesis with STAT3 activation and its relevance in apoptosis regulation.

Expression of STAT3/pSTAT3 in colorectal cancer (CRC) patients of Indian origin was studied to assess its significance in early detection and apoptosis regulation. Colorectal tissues with malignant lesions were STAT3/pSTAT3 positive in 66% of the cases and among these positive cases, well differentiated, moderately differentiated and poorly differentiated cancers were 86%, 60% and 0% respectively. All CRC specimens studied were immunoreactive with anti-carcinoembryonic antigen antibody. Cells purified from CRC tissues exhibited greater STAT3/pSTAT3 reactivity than peripheral blood mononuclear cells (PBMC) from healthy individuals, which served as control. apoptotic index (AI) was comparatively low in tissue specimens with STAT3/pSTAT3 expression. CRC cells with a comparatively less number of apoptotic cells, expressed a minimum number of Caspase-3 positive cells (4.73%), in comparison to healthy-PBMC (12.63%). CRC cells with high STAT3/pSTAT3 staining had cells with greater percentage of Bcl2 reactivity (23.05%), but less positivity with Caspase3 antibody (2.05%). Overall data suggests that CRC population was STAT3/pSTAT3 immunoreactive in a stage specific manner and STAT3 protects cancerous colorectal epithelial cells from apoptosis. Bcl-2, Cyclin D1 and Caspase-3 control the activity of apoptosis regulator, STAT3.

ATM kinase is a master switch for the Delta Np63 alpha phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage.

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of Delta Np63 alpha associated with its increased phosphorylated status. Since Delta Np63 alpha usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of Delta Np63 alpha in HNSCC cells upon cisplatin exposure. We found that Delta Np63 alpha is phosphorylated in the time-dependent fashion at the following positions: S385, T397 and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of Delta Np63 alpha in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of Delta Np63 alpha residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of Delta Np63 alpha degradation. Finally, we demonstrated that the Delta Np63 alpha protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of Delta Np63 alpha following DNA damage.