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Photoreceptor degeneration sufficient to produce severe visual loss often spares the inner retina. This raises the hope that treatments using optogenetics or electrical stimulation, which generate a replacement light input signal in surviving neurons, may restore vision. The success of these approaches is dependent on the capacity of surviving circuits in the early stages of the visual system to generate and propagate an appropriate visual code in the face of neuroanatomical remodelling. To determine the capacity of surviving circuits in advanced retinal degeneration to present an appropriate visual code, we generated a transgenic mouse expressing the optogenetic actuator ReaChR in ON bipolar cells (second order neurons in the visual projection). After crossing this with the rd1 model of photoreceptor degeneration, we compared ReaChR derived responses with photoreceptor-driven responses in wildtype (WT) mice in retinal ganglion cells and visual thalamus. The ReaChR-driven responses in rd1 animals showed low photosensitivity, but in other respects generated a visual code that was very similar to WT. Furthermore, ReaChR rd1 units in the retina had high response reproducibility and showed sensitivity normalisation to code contrast stably across different background intensities. At the single unit level, ReaChR-derived responses exhibited broadly similar variation in light response polarity, contrast sensitivity and temporal frequency tuning as WT. Units from WT and ReaChR rd1 mice clustered together when subjected to unsupervised community detection based on stimulus-response properties. Our data reveal an impressive ability for surviving circuitry to recreate a rich visual code following advanced retinal degeneration and are promising for regenerative medicine in the central nervous system.

Degenerative retinal disorders are a diverse family of diseases commonly leading to irreversible photoreceptor death, while leaving the inner retina relatively intact. Over recent years, innovative gene replacement therapies aiming to halt the progression of certain inherited retinal disorders have made their way into clinics. By rendering surviving retinal neurons light sensitive optogenetic gene therapy now offers a feasible treatment option that can restore lost vision, even in late disease stages and widely independent of the underlying cause of degeneration. Since proof-of-concept almost fifteen years ago, this field has rapidly evolved and a detailed first report on a treated patient has recently been published. In this article, we provide a review of optogenetic approaches for vision restoration. We discuss the currently available optogenetic tools and their relative advantages and disadvantages. Possible cellular targets will be discussed and we will address the question how retinal remodelling may affect the choice of the target and to what extent it may limit the outcomes of optogenetic vision restoration. Finally, we will analyse the evidence for and against optogenetic tool mediated toxicity and will discuss the challenges associated with clinical translation of this promising therapeutic concept.

During inherited retinal degenerations (IRDs), vision is lost due to photoreceptor cell death; however, a range of optogenetic tools have been shown to restore light responses in animal models. Restored response characteristics vary between tools and the neuronal cell population to which they are delivered: the interplay between these is complex, but targeting upstream neurons (such as retinal bipolar cells) may provide functional benefit by retaining intraretinal signal processing. In this study, our aim was to compare two optogenetic tools: mammalian melanopsin (hOPN4) and microbial red-shifted channelrhodopsin (ReaChR) expressed within two subpopulations of surviving cells in a degenerate retina. Intravitreal adeno-associated viral vectors and mouse models utilising the Cre/lox system restricted expression to populations dominated by bipolar cells or retinal ganglion cells and was compared with non-targeted delivery using the chicken beta actin (CBA) promoter. In summary, we found bipolar-targeted optogenetic tools produced faster kinetics and flatter intensity-response relationships compared with non-targeted or retinal-ganglion-cell-targeted hOPN4. Hence, optogenetic tools of both mammalian and microbial origins show advantages when targeted to bipolar cells. This demonstrates the advantage of bipolar-cell-targeted optogenetics for vision restoration in IRDs. We therefore developed a bipolar-cell-specific gene delivery system employing a compressed promoter with the potential for clinical translation.

This book brings us back to the post-Vietnam era, when the US Air Force launched two new, state-of-the-art fighter aircraft: the F-15 Eagle and the F-16 Fighting Falcon. It was an era when debates about aircraft superiority went public — and these were not uncontested discussions. This book delves deep into the fighter pilot culture that gave rise to both designs, showing how a small but vocal group of pilots, engineers, and analysts in the Department of Defense weaponized their own culture to affect technological development and larger political change. The design and advancement of the F-15 and F-16 reflected this group's nostalgic desire to recapture the best of World War I air combat. Known as the “Fighter Mafia,” and later growing into the media-savvy political powerhouse “Reform Movement,” it believed that American weapons systems were too complicated and expensive, and thus vulnerable. The group's leader was Colonel John Boyd, a contentious former fighter pilot heralded as a messianic figure by many in its ranks. He and his group advocated for a shift in focus from the multi-role interceptors the Air Force had designed in the early Cold War towards specialized air-to-air combat dogfighters. Their influence stretched beyond design and into larger politicized debates about US national security, debates that still resonate today. A biography of fighter pilot culture and the nostalgia that drove decision-making, the book deftly engages both popular culture and archives to animate the movement that shook the foundations of the Pentagon and Congress.

This chapter establishes a working definition for fighter pilot culture and how it operates. It describes how and why the fighter pilot emerged as a distinct cultural category during World War I and explains the key mechanisms by which that culture was able to persevere and evolve for over sixty years, through the Vietnam War and beyond. The chapter looks at militaries as large organizations, which generate cultures in specific ways. It zooms in on the formation of subcultures in the Air Force, centered broadly on the two main “families” of aircraft: large, crewed bombers, and single-seat fighters. The chapter underscores that the division between bomber and fighter culture was a hallmark of the Air Force for most of its existence, at least through the late 1960s. Fighter pilots in those years tended to be marginalized. As the fighter subculture gained more of a voice and more influence within the Air Force, the service began to produce technologies that were more in line with that culture's values. The culture of fighter pilots was passed down through word of mouth, behavioral expectations, peer pressure, training, popular media and fiction, shared rituals, and institutional structures.

This chapter looks at the many visions involved in the F-X program. The Aeronautical Systems Division, the Office of the Secretary of Defense, and some members of Tactical Air Command and the Air Staff wanted the F-X to be a multi-role fighter emphasizing ground attack, while fighter advocates coming from the knights of the air tradition fought to make it a lightweight, agile air-to-air machine. The program became a battleground for competing interests. The chapter tackles the contributions of John Boyd, Pierre Sprey, and Robert Titus, among others, in defining the F-X in a way that satisfied their desire for a true fighter. The chapter also discusses how General Felix Michael Rogers and General Glenn Kent contributed to the elimination of many of the more extreme requirements for the F-X. By the spring of 1967, the Air Force had worked up a design of a lighter aircraft to weigh forty thousand pounds. Furthermore, the chapter explores how the Navy's designs for a new fighter, code-named the VFAX, pressured the Air Force and continued to influence the design of the F-X throughout its development. Another external source of pressure that shaped the F-X toward an air-to-air focus was the Domodedovo air show in Moscow in July 1967, an event that revealed a new arsenal of Soviet fighter aircraft for the first time. In February 1968, Chuck Myers drafted a review of the F-X program and pushed to move away from “the multi-purpose monster” toward what he called “the right fighter.”

This chapter provides an overview of how the F-15 Eagle and F-16 Fighting Falcon, launched by the United States Air Force, had changed aerial warfare. It briefly discusses how the fighter pilot culture developed during World War I when romantic and heroic idealization of the fighter pilot meant a great deal to the American public and the pilots themselves. The chapter also describes the five core elements of the mythic construction of an idealized fighter: aggressiveness, independence, heroic imagery, technology, and community. Concepts of masculinity are intertwined in all five elements. Moreover, the chapter looks at the F-15 Eagle and F-16 Fighting Falcon, originally designed to focus on air-to-air combat, as the strongest examples of the influence of culture on technology within the United States Air Force. It emphasizes the link between culture and technology in military hardware, highlighting that all technologies are the results of humans making specific choices in a particular historical moment and context.

This chapter looks back at the time of national anxiety and self-doubt in the US, in the wake of the fall of Saigon in 1975 and the subsequent economic turmoil. During that time, the Military Reform movement was popularized by James Fallows, the Washington editor for The Atlantic magazine. Fallows met extensively with John Boyd and the other Reformers and published “The Muscle-Bound Superpower” in 1979. This was the first full public presentation of the Reformers' views — that military technology had gotten too complex, and the system was too full of corrupt insiders. The chapter explores how the Reformers' efforts soon drew allies from within the military and in Congress.

This chapter tackles how the Fighter Mafia's frustration with the lightweight fighter (LWF) drove the group to look beyond the Air Force and attempt to effect change in the entire military. The Fighter Mafia cultivated the allegory of themselves and the Air Force as competing religious orders. Referring to their efforts as “the Lord's work,” they took on the role of religious zealots, fighting against what they saw as corrupt orthodoxy. In the 1980s, the Fighter Mafia evolved into a larger group, known as the Military Reform movement. The movement published conference papers, briefings, newspapers, articles in professional and popular magazines, and books. Its concerns went beyond aircraft, encompassing budgets, procurement policy, research and development processes, and doctrine. Yet, they still spoke of the same elements: agility, maneuverability, the ability to surprise, and the conviction that machines needed to be simple. This reform movement incorporated many of the original fighter pilot stereotypes — strong individualism; distrust or resistance to authority; a focus on specific technologies that addressed the movement's values; protection of its community; use of heroic and religious imagery; and above all, aggressiveness.

This chapter looks at how a small community of fighter advocates influenced the development of the F-15 Eagle fighter, which itself was the result of a long and tumultuous development process. These pilots advocated for a dedicated air-to-air fighter that could re-create the glory days of fighter combat — an idealized vision of World War I, which they sometimes referred to as “the white scarf stuff.” The chapter looks at the principles held by Navy test pilot Lieutenant Charles “Chuck” E. Myers Jr., who said a true fighter should excel in air-to-air dogfighting at close range. He advocated for a redefinition of the fighter role to emphasize dogfighting, rejecting the Air Force doctrine that air superiority would not be achieved through air-to-air attrition but by destroying enemy planes and bases on the ground. During that time, the Air Force and Navy had just committed to a shift away from gun-based air combat. Both services were buying large numbers of F-4s, a multi-role interceptor that could excel at ground attack yet carried no gun. The chapter also looks at John Boyd, one of the more controversial figures in the history of the US Air Force. It tackles his advocacy for fighter aircraft and his concept of “energy maneuverability” as a quantifiable way to aid in the evaluation and design of fighters. Finally, the chapter explores the so-called rebirth of the air fighting mission.

This chapter discusses how Lieutenant Colonel John Boyd, Pierre Sprey, and their associates viewed the F-15 Eagle as too big, too sophisticated, and too expensive. The group, which saw themselves as guerrilla fighters against the US government, advocated for an entirely new aircraft that culminated in the F-16 Fighting Falcon. On July 18, 1968, Sprey had written a memo to General James Ferguson, commander of AFSC, outlining the problems with the F-X, proposing large-scale changes to bring down the weight and size, remove most or all the avionics, and increase its maneuverability. But Ferguson took no action, prompting Sprey to carry the debate into public. The chapter also explores the activities of the Fighter Mafia, an underground group, initially consisting of Boyd, Sprey, Chuck Myers, and Thomas Christie. Other key members were Harry Hillaker, the chief of General Dynamics' preliminary design division in Fort Worth, Texas, and test pilot Colonel Everest Riccioni who had flown P-38 and P-51 fighters in World War II. It was Riccioni who floated the idea of the group calling themselves the “Fighter Mafia,” with himself as their “Godfather.” This “mafia” tried to hide their activities, fearing that they might be overheard. Any reference to work on the F-XX was called “the Lord's work,” a coded term. Along the way, the Fighter Mafia's extremism also began to alienate their former allies.

This chapter focuses on how the new doctrines and technologies developed after Vietnam were put to the test in Iraq in 1991. Operation Desert Storm was based on extensive use of the technologies and strategies that the Fighter Mafia and the Reformers had opposed. John Boyd and his fellow Reformers had predicted a future war in which large fleets of aircraft battled for air superiority in ferocious dogfights. They argued that this hypothetical scenario demanded smaller, simpler aircraft, relying on the flying skill of the individual pilot. Furthermore, they assumed that the large all-weather radar of the F-15 Eagle was unnecessary and made it vulnerable. The Reformers also assumed that missiles would be useless. However, the skies over Iraq in 1991 debunked these assumptions as radar played a key role in gaining and maintaining air superiority; air-to-air combat did not involve the types of aircraft that the Reformers thought it should; and according to Western intelligence sources, Iraqi fighter pilots were poorly trained and inexperienced. For many observers, the Gulf War proved the Reformers wrong. On the other hand, the Reformers themselves took it as a validation. Most of the Reformers' ideas went untested in the Gulf War. What is clear is that the Reformers' vision was not applicable to all wars.

This chapter dives deeper into the five elements of fighter pilot culture, the various ways in which they expressed themselves, and how the culture evolved through various contexts — from World War I through the Vietnam era. It looks at aggressiveness as the element that sets fighter pilots apart, citing key figures who illustrated this tenet such as Kiffin Rockwell, the first American pilot to shoot down a German aircraft; Victor Chapman, the first US pilot to be killed in aerial combat; and Colonel James Jabara, the first US pilot to achieve ace status in Korea. The chapter also tackles individualism, pointing out that preference for solo flying and self-reliance reflected a key component of fighter pilot culture. A fighter pilot could easily feel isolated, encased in the cockpit, and alone at the controls of a powerful machine of war. Next, the chapter discusses the third core element of this culture: technology. It highlights how the fighter pilot culture is tied specifically to technologies that enhance the role of air-to-air combat — giving more importance to agility and maneuverability over other characteristics such as range or payload. Focusing on the element of heroic imagery, the chapter describes how fighter pilots tended to talk of themselves in heroic terms. Furthermore, it looks at the tight-knit community of fighter pilots that often sees itself as under threat from outsiders and is carefully guarded. Last, the chapter tackles how masculinity is intertwined with all these defining elements.

This chapter discusses how the Military Reform debate declined and has been confined to ever-smaller sectors after 1991. Key caucus members lost enthusiasm and by the mid-1990s, the Military Reform Caucus disbanded. Chuck Spinney, along with some members of the movement, tried to keep it going and some of them remained active in government and increasingly associated with the radical wing of conservative US politics. The ideals of the Military Reform movement and the Fighter Mafia did not disappear. They still influenced debates on defense technology four decades after the height of their influence. The chapter also weaves the impact of the Military Reform movement to present-day decisions of those in power and reviews its important contributions. Moreover, the chapter underscores that the Reformers became trapped by nostalgia — constantly looking backward and seeking to recreate earlier forms of combat, especially the gun-battle aerial dogfights of the world wars.

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3Sci/+ retina show an increased sensitivity of ipRGC light responses.

Human opsin-based photopigments have great potential as light-sensitisers, but their requirement for phototransduction cascade-specific second messenger proteins may restrict their functionality in non-native cell types. In this study, eight chimeric human opsins were generated consisting of a backbone of either a rhodopsin (RHO) or long-wavelength-sensitive (LWS) opsin and intracellular domains from Gq/11-coupled human melanopsin. Rhodopsin/melanopsin chimeric opsins coupled to both Gi and Gq/11 pathways. Greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in Gi activation. Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeras were dependent upon exogenous chromophore to function. By contrast, wild-type LWS opsin and LWS opsin/melanopsin chimeras showed only weak Gi activation in response to light, whilst Gq/11 pathway activation was not detected. Immunocytochemistry (ICC) demonstrated that chimeric opsins with more intracellular domains of melanopsin were less likely to be trafficked to the plasma membrane. This study demonstrates the importance of Gα coupling efficiency to the speed of cellular responses and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies., Summary: Combining different domains of human visual opsins and melanopsin creates functionally unique chimeric opsins with potential optogenetic applications.

Purpose Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. Methods Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. Results A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. Conclusion Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies., Highlights • Bipolar cells are attractive targets for therapeutic optogenetics in IRDs. • This is the first cell specific transcriptomic analysis of bipolar cells in an IRD model. • Bipolar cells maintain expression of genes essential to act as targets for optogenetics. • Protein staining relating to four candidate genes (Anxa7, Cntn1, Srm2, Sulf2) is confirmed using immunohistochemistry.

Purpose: To characterize the retinal expression and localization of Kcne2, an ancillary (β) ion-channel subunit with an important role in fine-tuning cellular excitability. Methods: We analyzed available single-cell transcriptome data from tens of thousands of murine retinal cells for cell-type-specific expression of Kcne2 using state-of-the-art bioinformatics techniques. This evidence at the transcriptome level was complemented with a comprehensive immunohistochemical characterization of mouse retina (C57BL/6, ages 8–12 weeks) employing co-labeling techniques and cell-type-specific antibody markers. We furthermore examined how conserved the Kcne2 localization pattern in the retina was across species by performing immunostaining on zebrafish, cowbird, sheep, mice, and macaque. Results: Kcne2 is distinctly expressed in cone photoreceptors and rod bipolar cells. At a subcellular level, the bulk of Kcne2 immunoreactivity can be observed in the outer plexiform layer. Here, it localizes into cone pedicles and likely the postsynaptic membrane of the rod bipolar cells. Thus, the vast majority of Kcne2 immunoreactivity is observed in a thin band in the outer plexiform layer. In addition to this, faint Kcne2 immunoreactivity can also be observed in cone inner segments and the somata of a small subset of cone ON bipolar cells. Strikingly, the localization of Kcne2 in the outer plexiform layer was preserved among all of the species studied, spanning at least 300 million years of evolution of the vertebrate kingdom. Conclusions: The data we present here suggest an important and specific role for Kcne2 in the highly specialized photoreceptor-bipolar cell synapse.

Purpose: To report on a severe case of presumed giant cell arteritis (GCA) presenting with partial and complete ophthalmic artery occlusion along with bilateral central retinal vein occlusions (CRVO). Observations: A 73-year-old female presented with bilateral complete vision loss of sudden onset. The patient also experienced a mild frontal headache prior to onset of vision loss. Fundus examination revealed bilateral central retinal artery occlusion (CRAO) and CRVO. Subsequent fluorescein angiography indicated partial right ophthalmic artery occlusion and complete left ophthalmic artery occlusion. Acute phase reactants were elevated. The patient was clinically diagnosed with GCA and intravenous (IV) steroids were initiated. Four days later, a temporal artery biopsy (TAB) was performed and resulted as negative for granulomatous inflammation. The patient did not regain vision and remained with no light perception (NLP) in both eyes. Conclusions: and Importance: This case highlights the discrepancy between clinical diagnosis and pathologic tissue diagnosis in a patient that presented with such extensive ocular vasculitic disease. Such extensive bilateral disease has not been reported. In addition, there are few studies regarding the effect of pulse-dosed IV steroids on TAB results. This case report suggests that the gradual histologic changes that occur over one or two weeks while on oral steroids may occur over three to four days while on high dose IV steroids, necessitating early biopsy. Keywords: Giant cell arteritis, Temporal arteritis, Intravenous, Bilateral, Ophthalmic artery occlusion, Central retinal artery occlusion, Central retinal vein occlusion, Biopsy negative

We determined whether deficiency of neuronal SOCS3 (suppressor of cytokine signaling 3)—a potential negative regulator of leptin signaling—amplifies the chronic effects of leptin on food intake, energy expenditure, glucose, and blood pressure (BP) and protects against adverse cardiometabolic effects of obesity. BP and heart rate were recorded by telemetry, and oxygen consumption (V O 2 ) was monitored in 22-week-old mice with nervous system SOCS3 deficiency (SOCS3-Nestin-Cre) and control mice (SOCS3 flox/flox ) fed normal or high-fat–high-fructose diet from 6 to 22 weeks of age. Compared with controls, SOCS3-Nestin-Cre mice had lower plasma glucose (124±7 versus 146±10 mg/dL), consumed less food (3.0±0.4 versus 3.6±0.2 g/d), and had similar V O 2 (77±6 versus 73±3 mL/kg per minute) and BP (103±3 versus 107±3 mm Hg) but higher heart rate (666±15 versus 602±17 bpm). In mice fed the normal diet, leptin infusion for 7 days caused similar reductions in food intake (2.3±0.1 versus 2.4±0.2 g) but greater increases in BP (15±3 versus 7±2 mm Hg) in SOCS3-Nestin-Cre compared with controls. Leptin reduced blood glucose concentrations in both groups. Male or female SOCS3-Nestin-Cre fed high-fat–high-fructose diet exhibited less weight gain, body fat, and liver steatosis and greater energy expenditure and heart rate compared with controls. Female SOCS3-Nestin-Cre mice fed high-fat–high-fructose diet had higher BP compared with controls. Thus, neuronal SOCS3 seems to play an important role in cardiometabolic regulation because neuronal SOCS3 deficiency reduced body weight and food intake while amplifying leptin’s effects on appetite and BP and attenuating the adverse metabolic effects of high-fat–high-fructose diet.

Melanopsin is a blue light-sensitive opsin photopigment involved in a range of non-image forming behaviours, including circadian photoentrainment and the pupil light response. Many naturally occurring genetic variants exist within the human melanopsin gene (OPN4), yet it remains unclear how these variants affect melanopsin protein function and downstream physiological responses to light. Here, we have used bioinformatic analysis and in vitro expression systems to determine the functional phenotypes of missense human OPN4 variants. From 1242 human OPN4 variants collated in the NCBI Short Genetic Variation database (dbSNP), we identified 96 that lead to non-synonymous amino acid substitutions. These 96 missense mutations were screened using sequence alignment and comparative approaches to select 16 potentially deleterious variants for functional characterisation using calcium imaging of melanopsin-driven light responses in HEK293T cells. We identify several previously uncharacterised OPN4 mutations with altered functional properties, including attenuated or abolished light responses, as well as variants demonstrating abnormal response kinetics. These data provide valuable insight into the structure–function relationships of human melanopsin, including several key functional residues of the melanopsin protein. The identification of melanopsin variants with significantly altered function may serve to detect individuals with disrupted melanopsin-based light perception, and potentially highlight those at increased risk of sleep disturbance, circadian dysfunction, and visual abnormalities. Electronic supplementary material The online version of this article (10.1007/s00018-018-2813-0) contains supplementary material, which is available to authorized users.

Non-alcoholic fatty liver disease is the most rapidly growing form of liver disease and if left untreated can result in non-alcoholic steatohepatitis, ultimately resulting in liver cirrhosis and failure. Biliverdin reductase A (BVRA) is a multifunctioning protein primarily responsible for the reduction of biliverdin to bilirubin. Also, BVRA functions as a kinase and transcription factor, regulating several cellular functions. We report here that liver BVRA protects against hepatic steatosis by inhibiting glycogen synthase kinase 3β (GSK3β) by enhancing serine 9 phosphorylation, which inhibits its activity. We show that GSK3β phosphorylates serine 73 (Ser(P)73) of the peroxisome proliferator-activated receptor α (PPARα), which in turn increased ubiquitination and protein turnover, as well as decreased activity. Interestingly, liver-specific BVRA KO mice had increased GSK3β activity and Ser(P)73 of PPARα, which resulted in decreased PPARα protein and activity. Furthermore, the liver-specific BVRA KO mice exhibited increased plasma glucose and insulin levels and decreased glycogen storage, which may be due to the manifestation of hepatic steatosis observed in the mice. These findings reveal a novel BVRA-GSKβ-PPARα axis that regulates hepatic lipid metabolism and may provide unique targets for the treatment of non-alcoholic fatty liver disease.

The discovery of melanopsin-expressing photosensitive retinal ganglion cells (pRGCs) has led to a fundamental change in our understanding of retinal light detection. pRGCs perform a broad range of non-visual functions — most notably mediating circadian entrainment to the environmental light/dark cycle. However, over the last two decades it has become clear that the melanopsin system is far more complex than first realised, influencing a wide range of physiology and behaviour, including pupillary constriction, light aversion, sleep, learning and memory and even mood. Here we provide an overview of the key areas of physiology known to be mediated by melanopsin pRGCs, as well as emerging translational opportunities.

Deposition fat in adipocytes has become key to understanding how obesity and insulin resistance impact morbidity and mortality. Several studies have shown a decreased amount of body fat in patients...

Over the past two decades there have been significant advances in our understanding of both the anatomy and function of the melanopsin system. It has become clear that rather than acting as a simple irradiance detector the melanopsin system is in fact far more complicated. The range of behavioural systems known to be influenced by melanopsin activity is increasing with time, and it is now clear that melanopsin contributes not only to multiple non-image forming systems but also has a role in visual pathways. How melanopsin is capable of driving so many different behaviours is unclear, but recent evidence suggests that the answer may lie in the diversity of melanopsin light responses and the functional specialisation of photosensitive retinal ganglion cell (pRGC) subtypes. In this review, we shall overview the current understanding of the melanopsin system, and evaluate the evidence for the hypothesis that individual pRGC subtypes not only perform specific roles, but are functionally specialised to do so. We conclude that while, currently, the available data somewhat support this hypothesis, we currently lack the necessary detail to fully understand how the functional diversity of pRGC subtypes correlates with different behavioural responses, and ultimately why such complexity is required within the melanopsin system. What we are lacking is a cohesive understanding of how light responses differ between the pRGC subtypes (based not only on anatomical classification but also based on their site of innervation); how these diverse light responses are generated, and most importantly how these responses relate to the physiological functions they underpin.

Chronic, low level treatment with a carbon monoxide releasing molecule (CO-RM), CORM-A1, has been shown to prevent the development of obesity in response to a high fat diet. The objective of this study was to test the hypothesis that chronic, low level treatment with this CO-RM can reverse established obesity via a mechanism independent of food intake. Dietary induced obese mice were treated with CORM-A1, the inactive compound iCORM-A1, or saline every 48 hours for 30 weeks while maintained on a high fat (60%) diet. Chronic treatment with CORM-A1 resulted in a 33% decrease from initial body weight over the 30 week treatment period while treatment with iCORM and saline were associated with 18 and 25% gain in initial body weight over the same time frame. Chronic treatment with CORM-A1 did not affect food intake or activity but resulted in a significant increase in metabolism. CORM-A1 treatment also resulted in lower fasting blood glucose, improvement in insulin sensitivity and decreased heptatic steatosis. Chronic treatment with CO releasing molecules can reverse dietary induced obesity and normalize insulin resistance independent of changes in food intake or activity. These findings are likely though a mechanism which increases metabolism.

Optogenetic strategies to restore vision in patients who are blind from end-stage retinal degenerations aim to render remaining retinal cells light sensitive once photoreceptors are lost. Here, we assessed long-term functional outcomes following subretinal delivery of the human melanopsin gene (OPN4) in the rd1 mouse model of retinal degeneration using an adenoassociated viral vector. Ectopic expression of OPN4 using a ubiquitous promoter resulted in cellular depolarisation and ganglion cell action potential firing. Restoration of the pupil light reflex, behavioural light avoidance and the ability to perform a task requiring basic image recognition were restored up to 13 months following injection. These data suggest that melanopsin gene therapy via a subretinal route may be a viable and stable therapeutic option for the treatment of end-stage retinal degeneration in humans.

Induction of heme oxygenase-1 (HO-1) has been demonstrated to decrease body weight and improve insulin sensitivity in several models of obesity in rodents. To further study the role of HO-1 in adipose tissue, we created an adipose-specific HO-1 knockout mouse model. Male and female mice were fed either a control or a high-fat diet for 30 weeks. Body weights were measured weekly and body composition, fasting blood glucose and insulin levels were determined every six weeks. Adipocyte-specific knockout of HO-1 had no significant effect on body weight in mice fed a high-fat diet but increased body weight in female mice fed a normal-fat diet. Although body weights were not different in females fed a high fat diet, loss of HO-1 in adipocytes resulted in significant alterations in body composition. Adipose-specific HO-1 knockout resulted in increased fasting hyperglycemia and insulinemia in female but not male mice on both diets. Adipose-specific knockout of HO-1 resulted in a significant loss of HO activity and a decrease in the protein levels of adiponectin in adipose tissue. These results demonstrate that loss of HO-1 in adipocytes has greater effects on body fat and fasting hyperglycemia in a sex-dependent fashion and that expression of HO-1 in adipose tissue may have a greater protective role in females as compared to males.

Gilbert’s syndrome in humans is derived from a polymorphism (TA repeat) in the hepatic UGT1A1 gene that results in decreased conjugation and increased levels of unconjugated bilirubin. Recently, we have shown that bilirubin binds directly to the fat-burning nuclear peroxisome proliferator-activated receptor-α (PPARα). Additionally, we have shown that serine 73 phosphorylation [Ser(P)73] of PPARα decreases activity by reducing its protein levels and transcriptional activity. The aim of this study was to determine whether humanized mice with the Gilbert’s polymorphism (HuUGT*28) have increased PPARα activation and reduced hepatic fat accumulation. To determine whether humanized mice with Gilbert’s mutation (HuUGT*28) have reduced hepatic lipids, we placed them and C57BL/6J control mice on a high-fat (60%) diet for 36 wk. Body weights, fat and lean mass, and fasting blood glucose and insulin levels were measured every 6 wk throughout the investigation. At the end of the study, hepatic lipid content was measured and PPARα regulated genes as well as immunostaining of Ser(P)73 PPARα from liver sections. The HuUGT*28 mice had increased serum bilirubin, lean body mass, decreased fat mass, and hepatic lipid content as well as lower serum glucose and insulin levels. Also, the HuUGT*28 mice had reduced Ser(P)73 PPARα immunostaining in livers and increased PPARα transcriptional activity compared with controls. A chronic but mild endogenous increase in unconjugated hyperbiliubinemia protects against hepatic steatosis through a reduction in Ser(P)73 PPARα, causing an increase in PPARα transcriptional activity.

Acute light exposure exerts various effects on physiology and behaviour. Although the effects of light on brain network activity in humans are well demonstrated, the effects of light on cognitive performance are inconclusive, with the size, as well as direction, of the effect depending on the nature of the task. Similarly, in nocturnal rodents, bright light can either facilitate or disrupt performance depending on the type of task employed. Crucially, it is unclear whether the effects of light on behavioural performance are mediated via the classical image-forming rods and cones or the melanopsin-expressing photosensitive retinal ganglion cells. Here, we investigate the modulatory effects of light on memory performance in mice using the spontaneous object recognition task. Importantly, we examine which photoreceptors are required to mediate the effects of light on memory performance. By using a cross-over design, we show that object recognition memory is disrupted when the test phase is conducted under a bright light (350 lux), regardless of the light level in the sample phase (10 or 350 lux), demonstrating that exposure to a bright light at the time of test, rather than at the time of encoding, impairs performance. Strikingly, the modulatory effect of light on memory performance is completely abolished in both melanopsin-deficient and rodless–coneless mice. Our findings provide direct evidence that melanopsin-driven and rod/cone-driven photoresponses are integrated in order to mediate the effect of light on memory performance.

Circadian rhythms optimize physiology and behavior to the varying demands of the 24 h day. The master circadian clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and it regulates circadian oscillators in tissues throughout the body to prevent internal desynchrony. Here, we demonstrate for the first time that, under standard 12 h:12 h light/dark (LD) cycles, object, visuospatial, and olfactory recognition performance in C57BL/6J mice is consistently better at midday relative to midnight. However, under repeated exposure to constant light (rLL), recognition performance becomes desynchronized, with object and visuospatial performance better at subjective midday and olfactory performance better at subjective midnight. This desynchrony in behavioral performance is mirrored by changes in expression of the canonical clock genes Period1 and Period2 (Per1 and Per2), as well as the immediate-early gene Fos in the SCN, dorsal hippocampus, and olfactory bulb. Under rLL, rhythmic Per1 and Fos expression is attenuated in the SCN. In contrast, hippocampal gene expression remains rhythmic, mirroring object and visuospatial performance. Strikingly, Per1 and Fos expression in the olfactory bulb is reversed, mirroring the inverted olfactory performance. Temporal desynchrony among these regions does not result in arrhythmicity because core body temperature and exploratory activity rhythms persist under rLL. Our data provide the first demonstration that abnormal lighting conditions can give rise to temporal desynchrony between autonomous circadian oscillators in different regions, with different consequences for performance across different sensory domains. Such a dispersed network of dissociable circadian oscillators may provide greater flexibility when faced with conflicting environmental signals. SIGNIFICANCE STATEMENT A master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus regulates physiology and behavior across the 24 h day by synchronizing peripheral clocks throughout the brain and body. Without the SCN, these peripheral clocks rapidly become desynchronized. Here, we provide a unique demonstration that, under lighting conditions in which the central clock in the SCN is dampened, peripheral oscillators in the hippocampus and olfactory bulb become desynchronized, along with the behavioral processes mediated by these clocks. Multiple clocks that adopt different phase relationships may enable processes occurring in different brain regions to be optimized to specific phases of the 24 h day. Moreover, such a dispersed network of dissociable circadian clocks may provide greater flexibility when faced with conflicting environmental signals (e.g., seasonal changes in photoperiod).

Two-pore domain (K2P) potassium channels perform essential roles in neuronal function. These channels produce background leak type potassium currents that act to regulate resting membrane potential and levels of cellular excitability. 15 different K2P channels have been identified in mammals and these channels perform important roles in a wide number of physiological systems. However, to date there is only limited data available concerning the expression and role of K2P channels in the retina. In this study we conduct the first comprehensive study of K2P channel expression in the retina. Our data show that K2P channels are widely expressed in the mouse retina, with variations in expression detected at different times of day and throughout postnatal development. The highest levels of K2P channel expression are observed for Müller cells (TWIK-1, TASK-3, TRAAK, and TREK-2) and retinal ganglion cells (TASK-1, TREK-1, TWIK-1, TWIK-2 and TWIK-3). These data offer new insight into the channels that regulate the resting membrane potential and electrical activity of retinal cells, and suggests that K2P channels are well placed to act as central regulators of visual signalling pathways. The prominent role of K2P channels in neuroprotection offers novel avenues of research into the treatment of common retinal diseases.

Light plays a critical role in the regulation of numerous aspects of physiology and behaviour, including the entrainment of circadian rhythms and the regulation of sleep. These responses involve melanopsin (OPN4)-expressing photosensitive retinal ganglion cells (pRGCs) in addition to rods and cones. Nocturnal light exposure in rodents has been shown to result in rapid sleep induction, in which melanopsin plays a key role. However, studies have also shown that light exposure can result in elevated corticosterone, a response that is not compatible with sleep. To investigate these contradictory findings and to dissect the relative contribution of pRGCs and rods/cones, we assessed the effects of light of different wavelengths on behaviourally defined sleep. Here, we show that blue light (470 nm) causes behavioural arousal, elevating corticosterone and delaying sleep onset. By contrast, green light (530 nm) produces rapid sleep induction. Compared to wildtype mice, these responses are altered in melanopsin-deficient mice (Opn4-/-), resulting in enhanced sleep in response to blue light but delayed sleep induction in response to green or white light. We go on to show that blue light evokes higher Fos induction in the SCN compared to the sleep-promoting ventrolateral preoptic area (VLPO), whereas green light produced greater responses in the VLPO. Collectively, our data demonstrates that nocturnal light exposure can have either an arousal- or sleep-promoting effect, and that these responses are melanopsin-mediated via different neural pathways with different spectral sensitivities. These findings raise important questions relating to how artificial light may alter behaviour in both the work and domestic setting., Light can produce either sleep or arousal in mice. This study reveals that these opposing effects depend upon the wavelength of light and appear to involve separate pathways, both modulated by the photopigment melanopsin., Author Summary Light exerts profound effects on our physiology and behaviour, setting our biological clocks to the correct time and regulating when we are asleep and we are awake. The photoreceptors mediating these responses include the rods and cones involved in vision, as well as a subset of photosensitive retinal ganglion cells (pRGCs) expressing the blue light-sensitive photopigment melanopsin. Previous studies have shown that mice lacking melanopsin show impaired sleep in response to light. However, other studies have shown that light increases glucocorticoid release—a response typically associated with stress. To address these contradictory findings, we studied the responses of mice to light of different colours. We found that blue light was aversive, delaying sleep onset and increasing glucocorticoid levels. By contrast, green light led to rapid sleep onset. These different behavioural effects appear to be driven by different neural pathways. Surprisingly, both responses were impaired in mice lacking melanopsin. These data show that light can promote either sleep or arousal. Moreover, they provide the first evidence that melanopsin directly mediates the effects of light on glucocorticoids. This work shows the extent to which light affects our physiology and has important implications for the design and use of artificial light sources.

The mammalian retina contains three classes of photoreceptor. In addition to the rods and cones, a subset of retinal ganglion cells that express the putative sensory photopigment melanopsin are intrinsically photosensitive. Functional and anatomical studies suggest that these inner retinal photoreceptors provide light information for a number of non-image-forming light responses including photoentrainment of the circadian clock and the pupil light reflex. Here, we employ a newly developed mouse model bearing lesions of both rod and cone phototransduction cascades (Rho−/−Cnga3−/−) to further examine the function of these non-rod non-cone photoreceptors. Calcium imaging confirms the presence of inner retinal photoreceptors inRho−/−Cnga3−/−mice. Moreover, these animals retain a pupil light reflex, photoentrainment, and light induction of the immediate early genec-fosin the suprachiasmatic nuclei, consistent with previous findings that pupillary and circadian responses can employ inner retinal photoreceptors.Rho−/−Cnga3−/−mice also show a light-dependent increase in the number of FOS-positive cells in both the ganglion cell and (particularly) inner nuclear layers of the retina. The average number of cells affected is several times greater than the number of melanopsin-positive cells in the mouse retina, suggesting functional intercellular connections from these inner retinal photoreceptors within the retina. Finally, however, while we show that wild types exhibit an increase in heart rate upon light exposure, this response is absent inRho−/−Cnga3−/−mice. Thus, it seems that non-rod non-cone photoreceptors can drive many, but not all, non-image-forming light responses.

Non-visual photoreception in mammals is primarily mediated by two splice variants that derive from a single melanopsin (OPN4M) gene, whose expression is restricted to a subset of retinal ganglion cells. Physiologically, this sensory system regulates the photoentrainment of many biological rhythms, such as sleep via the melatonin endocrine system and pupil constriction. By contrast, melanopsin exists as two distinct lineages in non-mammals, opn4m and opn4x, and is broadly expressed in a wide range of tissue types, including the eye, brain, pineal gland and skin. Despite these findings, the evolution and function of melanopsin in early vertebrates are largely unknown. We, therefore, investigated the complement of opn4 classes present in the genome of a model deep-sea cartilaginous species, the elephant shark (Callorhinchus milii), as a representative vertebrate that resides at the base of the gnathostome (jawed vertebrate) lineage. We reveal that three melanopsin genes, opn4m1, opn4m2 and opn4x, are expressed in multiple tissues of the elephant shark. The two opn4m genes are likely to have arisen as a result of a lineage-specific duplication, whereas “long” and “short” splice variants are generated from a single opn4x gene. By using a heterologous expression system, we suggest that these genes encode functional photopigments that exhibit both “invertebrate-like” bistable and classical “vertebrate-like” monostable biochemical characteristics. We discuss the evolution and function of these melanopsin pigments within the context of the diverse photic and ecological environments inhabited by this chimaerid holocephalan, as well as the origin of non-visual sensory systems in early vertebrates.

Summary Photoreceptive, melanopsin-expressing retinal ganglion cells (mRGCs) encode ambient light (irradiance) for the circadian clock, the pupillomotor system, and other influential behavioral/physiological responses. mRGCs are activated both by their intrinsic phototransduction cascade and by the rods and cones. However, the individual contribution of each photoreceptor class to irradiance responses remains unclear. We address this deficit using mice expressing human red cone opsin, in which rod-, cone-, and melanopsin-dependent responses can be identified by their distinct spectral sensitivity. Our data reveal an unexpectedly important role for rods. These photoreceptors define circadian responses at very dim “scotopic” light levels but also at irradiances at which pattern vision relies heavily on cones. By contrast, cone input to irradiance responses dissipates following light adaptation to the extent that these receptors make a very limited contribution to circadian and pupillary light responses under these conditions. Our data provide new insight into retinal circuitry upstream of mRGCs and optimal stimuli for eliciting irradiance responses., Highlights ► Red cone knockin (Opn1mwR) mice reveal rod, cone, and melanopsin phases to NIF vision ► Rods drive circadian responses to very low irradiances (scotopic threshold) ► Circadian responses to light in the photopic range can be rod driven ► Light adaptation limits the influence of cones on NIF vision

In the mammalian retina, besides the conventional rod–cone system, a melanopsin-associated photoreceptive system exists that conveys photic information for accessory visual functions such as pupillary light reflex and circadian photo-entrainment1–7. On ablation of the melanopsin gene, retinal ganglion cells that normally express melanopsin are no longer intrinsically photosensitive8. Furthermore, pupil reflex8, light-induced phase delays of the circadian clock9,10 and period lengthening of the circadian rhythm in constant light9,10 are all partially impaired. Here, we investigated whether additional photoreceptive systems participate in these responses. Using mice lacking rods and cones, we measured the action spectrum for phase-shifting the circadian rhythm of locomotor behaviour. This spectrum matches that for the pupillary light reflex in mice of the same genotype11, and that for the intrinsic photosensitivity of the melanopsin-expressing retinal ganglion cells7. We have also generated mice lacking melanopsin coupled with disabled rod and cone photo-transduction mechanisms. These animals have an intact retina but fail to show any significant pupil reflex, to entrain to light/dark cycles, and to show any masking response to light. Thus, the rod–cone and melanopsin systems together seem to provide all of the photic input for these accessory visual functions.

The mammalian retina contains directly photosensitive retinal ganglion cells (RGCs), which use the photopigment melanopsin. The generation of mice lacking melanopsin has been invaluable in elucidating the function of these cells. These animals display deficiencies in circadian photoentrainment, the pupil light reflex, and the circadian regulation of the cone pathway. Interpreting the results from such gene knock-out models is always complicated by neuronal plasticity and the potential for restructuring of neuronal networks. Until now, the study of photosensitive RGCs has lacked an acute inhibitor. 2-Aminoethoxydiphenylborane (2-APB) is an antagonist at IP3receptors and an inhibitor of canonical transient receptor potential ion channels (TRPCs). Here, we show that 2-APB is an extremely potentin vitroinhibitor of the photosensitive RGCs and that its effect is independent of store-dependent Ca2+release. The identification of canonical TRPC6 and TRPC7 ion channels in melanopsin-expressing ganglion cells suggests that 2-APB may act directly on a TRPC ion channel. Importantly, using the pupil light reflex as a functional assay, we show that 2-APB inhibits photosensitive RGC activityin vivo. Collectively, our data further elucidate the phototransduction pathway in the photosensitive RGCs and demonstrate that 2-APB can be used to silence activity in these cells bothin vitroandin vivo.

Melanopsin expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of ocular photoreceptors and mediate a range of non-image forming responses to light. Melanopsin is a G protein coupled receptor (GPCR) and existing data suggest that it employs a membrane bound signalling cascade involving Gnaq/11 type G proteins. However, to date the precise identity of the Gα subunits involved in melanopsin phototransduction remains poorly defined. Here we show that Gnaq, Gna11 and Gna14 are highly co-expressed in pRGCs of the mouse retina. Furthermore, using RNAi based gene silencing we show that melanopsin can signal via Gnaq, Gna11 or Gna14 in vitro, and demonstrate that multiple members of the Gnaq/11 subfamily, including Gna14 and at least Gnaq or Gna11, can participate in melanopsin phototransduction in vivo and contribute to the pupillary light responses of mice lacking rod and cone photoreceptors. This diversity of G protein interactions suggests additional complexity in the melanopsin phototransduction cascade and may provide a basis for generating the diversity of light responses observed from pRGC subtypes. Electronic supplementary material The online version of this article (doi:10.1007/s00018-014-1664-6) contains supplementary material, which is available to authorized users.

Research on circadian biology over the past decade has paid increasing attention to the photoreceptor mechanisms that align the molecular clock to the 24-h light/dark cycle, and some of the results to emerge are surprising. For example, the rods and cones within the mammalian eye are not required for entrainment. A population of directly light-sensitive ganglion cells exists within the retina and acts as brightness detectors. This article provides a brief history of the discovery of these novel ocular photoreceptors and then describes the methods that have been used to study the photopigments mediating these responses to light. Photopigment characterization has traditionally been based on a number of complementary approaches, but one of the most useful techniques has been action spectroscopy. A photopigment has a discrete absorbance spectrum, which describes the probability of photons being absorbed as a function of wavelength, and the magnitude of any light-dependent response depends on the number of photons absorbed by the photopigment. Thus, a description of the spectral sensitivity profile (action spectrum) of any light-dependent response must, by necessity, match absorbance spectra of the photopigment mediating the response. We provide a step-by-step approach to conducting action spectra, including the construction of irradiance response curves, the calculation of relative spectral sensitivities, and photopigment template fitting, and discuss the underlying assumptions behind this approach. We then illustrate action spectrum methodologies by an in-depth analysis of action spectra obtained from rodless/coneless (rd/rd cl) mice and discuss, for the first time, the full implications of these findings.