A new Opn4cre recombinase mouse line to target intrinsically photosensitive retinal ganglion cells (ipRGCs).

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in several physiological light responses. In this study we generate a new Opn4cre knock-in allele (Opn4cre(DSO)), in which cre is placed immediately downstream of the Opn4 start codon. This approach aims to faithfully reproduce endogenous Opn4 expression and improve compatibility with widely used reporters. We evaluated the efficacy and sensitivity of Opn4cre(DSO) for labeling in retina and brain, and provide an in-depth comparison with the extensively utilized Opn4cre(Saha) line. Through this characterization, Opn4cre(DSO) demonstrated higher specificity in labeling ipRGCs, with minimal recombination escape. Leveraging a combination of electrophysiological, molecular, and morphological analyses, we confirmed its sensitivity in detecting all ipRGC types (M1-M6). Using this new tool, we describe the topographical distributions of ipRGC types across the retinal landscape, uncovering distinct ventronasal biases for M5 and M6 types, setting them apart from their M1-M4 counterparts. In the brain, we find vastly different labeling patterns between lines, with Opn4cre(DSO) only labeling ipRGC axonal projections to their targets. The combination of off-target effects of Opn4cre(Saha) across the retina and brain, coupled with diminished efficiencies of both Cre lines when coupled to less sensitive reporters, underscores the need for careful consideration in experimental design and validation with any Opn4cre driver. Overall, the Opn4cre(DSO) mouse line represents an improved tool for studying ipRGC function and distribution, offering a means to selectively target these cells to study light-regulated behaviors and physiology.

Developmental adaptation of rod photoreceptor number via photoreception in melanopsin (OPN4) retinal ganglion cells.

Photoreception, a form of sensory experience, is essential for normal development of the mammalian visual system. Detecting photons during development is a prerequisite for visual system function - from vision's first synapse at the cone pedicle and maturation of retinal vascular networks, to transcriptional establishment and maturation of cell types within the visual cortex. Consistent with this theme, we find that the lighting environment regulates developmental rod photoreceptor apoptosis via OPN4-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Using a combination of genetics, sensory environment manipulations, and computational approaches, we establish a molecular pathway in which light-dependent glutamate release from ipRGCs is detected via a transiently expressed kainate receptor (GRIK3) in immature rods localized to the inner retina. Communication between ipRGCs and nascent inner retinal rods appears to be mediated by unusual hybrid neurites projecting from ipRGCs that sense light before eye-opening. These structures, previously referred to as outer retinal dendrites (ORDs), span the ipRGC-immature rod distance over the first postnatal week and contain the machinery for sensory detection (melanopsin, OPN4) and axonal/anterograde neurotransmitter release (Synaptophysin, and VGLUT2). Histological and computational assessment of human mid-gestation development reveal conservation of several hallmarks of an ipRGC-to-immature rod pathway, including displaced immature rods, transient GRIK3 expression in the rod lineage, and the presence of ipRGCs with putative neurites projecting deep into the developing retina. Thus, this analysis defines a retinal retrograde signaling pathway that links the sensory environment to immature rods via ipRGC photoreceptors, allowing the visual system to adapt to distinct lighting environments priory to eye-opening.

Dopamine signaling from ganglion cells directs layer-specific angiogenesis in the retina.

During central nervous system (CNS) development, a precisely patterned vasculature emerges to support CNS function. How neurons control angiogenesis is not well understood. Here, we show that the neuromodulator dopamine restricts vascular development in the retina via temporally limited production by an unexpected neuron subset. Our genetic and pharmacological experiments demonstrate that elevating dopamine levels inhibits tip-cell sprouting and vessel growth, whereas reducing dopamine production by all retina neurons increases growth. Dopamine production by canonical dopaminergic amacrine interneurons is dispensable for these events. Instead, we found that temporally restricted dopamine production by retinal ganglion cells (RGCs) modulates vascular development. RGCs produce dopamine precisely during angiogenic periods. Genetically limiting dopamine production by ganglion cells, but not amacrines, decreases angiogenesis. Conversely, elevating ganglion-cell-derived dopamine production inhibits early vessel growth. These vasculature outcomes occur downstream of vascular endothelial growth factor receptor (VEGFR) activation and Notch-Jagged1 signaling. Jagged1 is increased and subsequently inhibits Notch signaling when ganglion cell dopamine production is reduced. Our findings demonstrate that dopaminergic neural activity from a small neuron subset functions upstream of VEGFR to serve as developmental timing cue that regulates vessel growth.

Encephalopsin (OPN3) is required for normal refractive development and the GO/GROW response to induced myopia.

Purpose: Myopia, or nearsightedness, is the most common form of refractive error and is increasing in prevalence. While significant efforts have been made to identify genetic variants that predispose individuals to myopia, these variants are believed to account for only a small portion of the myopia prevalence, leading to a feedback theory of emmetropization, which depends on the active perception of environmental visual cues. Consequently, there has been renewed interest in studying myopia in the context of light perception, beginning with the opsin family of G-protein coupled receptors (GPCRs). Refractive phenotypes have been characterized in every opsin signaling pathway studied, leaving only Opsin 3 (OPN3), the most widely expressed and blue-light sensing noncanonical opsin, to be investigated for function in the eye and refraction. Methods: Opn3 expression was assessed in various ocular tissues using an Opn3eGFP reporter. Weekly refractive development in Opn3 retinal and germline mutants from 3 to 9 weeks of age was measured using an infrared photorefractor and spectral domain optical coherence tomography (SD-OCT). Susceptibility to lens-induced myopia was then assessed using skull-mounted goggles with a -30 diopter experimental and a 0 diopter control lens. Mouse eye biometry was similarly tracked from 3 to 6 weeks. A myopia gene expression signature was assessed 24 h after lens induction for germline mutants to further assess myopia-induced changes. Results: Opn3 was found to be expressed in a subset of retinal ganglion cells and a limited number of choroidal cells. Based on an assessment of Opn3 mutants, the OPN3 germline, but not retina conditional Opn3 knockout, exhibits a refractive myopia phenotype, which manifests in decreased lens thickness, shallower aqueous compartment depth, and shorter axial length, atypical of traditional axial myopias. Despite the short axial length, Opn3 null eyes demonstrate normal axial elongation in response to myopia induction and mild changes in choroidal thinning and myopic shift, suggesting that susceptibility to lens-induced myopia is largely unchanged. Additionally, the Opn3 null retinal gene expression signature in response to induced myopia after 24 h is distinct, with opposing Ctgf, Cx43, and Egr1 polarity compared to controls. Conclusions: The data suggest that an OPN3 expression domain outside the retina can control lens shape and thus the refractive performance of the eye. Prior to this study, the role of Opn3 in the eye had not been investigated. This work adds OPN3 to the list of opsin family GPCRs that are implicated in emmetropization and myopia. Further, the work to exclude retinal OPN3 as the contributing domain in this refractive phenotype is unique and suggests a distinct mechanism when compared to other opsins.

Diurnal regulation of metabolism by Gs-alpha in hypothalamic QPLOT neurons.

Neurons in the hypothalamic preoptic area (POA) regulate multiple homeostatic processes, including thermoregulation and sleep, by sensing afferent input and modulating sympathetic nervous system output. The POA has an autonomous circadian clock and may also receive circadian signals indirectly from the suprachiasmatic nucleus. We have previously defined a subset of neurons in the POA termed QPLOT neurons that are identified by the expression of molecular markers (Qrfp, Ptger3, LepR, Opn5, Tacr3) that suggest receptivity to multiple stimuli. Because Ptger3, Opn5, and Tacr3 encode G-protein coupled receptors (GPCRs), we hypothesized that elucidating the G-protein signaling in these neurons is essential to understanding the interplay of inputs in the regulation of metabolism. Here, we describe how the stimulatory Gs-alpha subunit (Gnas) in QPLOT neurons regulates metabolism in mice. We analyzed Opn5cre; Gnasfl/fl mice using indirect calorimetry at ambient temperatures of 22°C (a historical standard), 10°C (a cold challenge), and 28°C (thermoneutrality) to assess the ability of QPLOT neurons to regulate metabolism. We observed a marked decrease in nocturnal locomotion of Opn5cre; Gnasfl/fl mice at both 28°C and 22°C, but no overall differences in energy expenditure, respiratory exchange, or food and water consumption. To analyze daily rhythmic patterns of metabolism, we assessed circadian parameters including amplitude, phase, and MESOR. Loss-of-function GNAS in QPLOT neurons resulted in several subtle rhythmic changes in multiple metabolic parameters. We observed that Opn5cre; Gnasfl/fl mice show a higher rhythm-adjusted mean energy expenditure at 22°C and 10°C, and an exaggerated respiratory exchange shift with temperature. At 28°C, Opn5cre; Gnasfl/fl mice have a significant delay in the phase of energy expenditure and respiratory exchange. Rhythmic analysis also showed limited increases in rhythm-adjusted means of food and water intake at 22°C and 28°C. Together, these data advance our understanding of Gαs-signaling in preoptic QPLOT neurons in regulating daily patterns of metabolism.

Ventral striatum dopamine release encodes unique properties of visual stimuli in mice.

The mesolimbic dopamine system is an evolutionarily conserved set of brain circuits that play a role in attention, appetitive behavior, and reward processing. In this circuitry, ascending dopaminergic projections from the ventral midbrain innervate targets throughout the limbic forebrain, such as the ventral striatum/nucleus accumbens (NAc). Dopaminergic signaling in the NAc has been widely studied for its role in behavioral reinforcement, reward prediction error encoding, and motivational salience. Less well characterized is the role of dopaminergic neurotransmission in the response to surprising or alerting sensory events. To address this, we used the genetically encoded dopamine sensor dLight1 and fiber photometry to explore the ability of striatal dopamine release to encode the properties of salient sensory stimuli in mice, such as threatening looming discs. Here, we report that lateral NAc (LNAc) dopamine release encodes the rate and magnitude of environmental luminance changes rather than the visual stimulus threat level. This encoding is highly sensitive, as LNAc dopamine could be evoked by light intensities that were imperceptible to human experimenters. We also found that light-evoked dopamine responses are wavelength-dependent at low irradiances, independent of the circadian cycle, robust to previous exposure history, and involve multiple phototransduction pathways. Thus, we have further elaborated the mesolimbic dopamine system's ability to encode visual information in mice, which is likely relevant to a wide body of scientists employing light sources or optical methods in behavioral research involving rodents.

Comprehensive Behavioral Analysis of Opsin 3 (Encephalopsin)-Deficient Mice Identifies Role in Modulation of Acoustic Startle Reflex.

Opsin-3 (Opn3, encephalopsin) was the first nonvisual opsin gene discovered in mammals. Since then, several Opn3 functions have been described, and in two cases (adipose tissue, smooth muscle) light sensing activity is implicated. In addition to peripheral tissues, Opn3 is robustly expressed within the central nervous system, for which it derives its name. Despite this expression, no studies have investigated developmental or adult CNS consequences of Opn3 loss-of-function. Here, the behavioral consequences of mice deficient in Opn3 were investigated. Opn3-deficient mice perform comparably to wild-type mice in measures of motor coordination, socialization, anxiety-like behavior, and various aspects of learning and memory. However, Opn3-deficient mice have an attenuated acoustic startle reflex (ASR) relative to littermates. This deficit is not because of changes in hearing sensitivity, although Opn3 was shown to be expressed in auditory and vestibular structures, including cochlear outer hair cells. Interestingly, the ASR was not acutely light-dependent and did not vary between daytime and nighttime trials, despite known functions of Opn3 in photoreception and circadian gene amplitude. Together, these results demonstrate the first role of Opn3 on behavior, although the role of this opsin in the CNS remains largely elusive.

The molecular clockwork of mammalian cells.

Most organisms contain self-sustained circadian clocks. These clocks can be synchronized by environmental stimuli, but can also oscillate indefinitely in isolation. In mammals this is true at the molecular level for the majority of cell types that have been examined. A core set of "clock genes" form a transcriptional/translational feedback loop (TTFL) which repeats with a period of approximately 24 h. The exact mechanism of the TTFL differs slightly in various cell types, but all involve similar family members of the core cohort of clock genes. The clock has many outputs which are unique for different tissues. Cells in diverse tissues will convert the timing signals provided by the TTFL into uniquely orchestrated transcriptional oscillations of many clock-controlled genes and cellular processes.

Retinal patterns and the cellular repertoire of neuropsin (Opn5) retinal ganglion cells.

Obtaining a parts list of the sensory components of the retina is vital to understand the effects of light in behavior, health, and disease. Rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) are the best described photoreceptors in the mammalian retina, but recent functional roles have been proposed for retinal neuropsin (Opn5)-an atypical opsin. However, little is known about the pattern of Opn5 expression in the retina. Using cre (Opn5cre ) and cre-dependent reporters, we uncover patterns of Opn5 expression and find that Opn5 is restricted to retinal ganglion cells (RGCs). Opn5-RGCs are nonhomogenously distributed through the retina, with greater densities of cells located in the dorsotemporal quadrant. In addition to the local topology of these cells, using cre-dependent AAV viral tracing, we surveyed their central targets and found that they are biased towards image-forming and image-stabilizing regions. Finally, molecular and electrophysiological profiling reveal that Opn5-RGCs comprise previously defined RGC types that respond optimally to edges and object-motion (F-mini-ONs, HD2, HD1, LEDs, ooDSRGCs, etc.). Together, these data describe the second collection of RGCs that express atypical opsins in the mouse, and expand the roles of image-forming cells in retinal physiology and function.

Lessons of the month 2: Retinal vasculitis: a first presentation of Takayasu's arteritis.

Takayasu's arteritis is a chronic, systemic, large-vessel vasculitis affecting the aorta and its primary branches. However, coronary, renal and pulmonary arteries and small vessel involvement has been documented. We describe a rare case of Takayasu's arteritis with extensive supra-aortic arch disease, manifesting with bilateral occlusive retinal vasculitis as a first presentation. This is elicited by fundus findings of vascular sheathing and fundus fluoresceine angiography evidence of retinal vessel occlusion and peripheral capillary non-perfusion.

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice.

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus-induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

QPLOT Neurons-Converging on a Thermoregulatory Preoptic Neuronal Population.

The preoptic area of the hypothalamus is a homeostatic control center. The heterogeneous neurons in this nucleus function to regulate the sleep/wake cycle, reproduction, thirst and hydration, as well as thermogenesis and other metabolic responses. Several recent studies have analyzed preoptic neuronal populations and demonstrated neuronal subtype-specific roles in suppression of thermogenesis. These studies showed similar thermogenesis responses to chemogenetic modulation, and similar synaptic tracing patterns for neurons that were responsive to cold, to inflammatory stimuli, and to violet light. A reanalysis of single-cell/nucleus RNA-sequencing datasets of the preoptic nucleus indicate that these studies have converged on a common neuronal population that when activated, are sufficient to suppress thermogenesis. Expanding on a previous name for these neurons (Q neurons, which reflect their ability to promote quiescence and expression of Qrfp), we propose a new name: QPLOT neurons, to reflect numerous molecular markers of this population and to capture its broader roles in metabolic regulation. Here, we summarize previous findings on this population and present a unified description of QPLOT neurons, the excitatory preoptic neuronal population that integrate a variety of thermal, metabolic, hormonal and environmental stimuli in order to regulate metabolism and thermogenesis.

Retinal ganglion cell interactions shape the developing mammalian visual system.

Retinal ganglion cells (RGCs) serve as a crucial communication channel from the retina to the brain. In the adult, these cells receive input from defined sets of presynaptic partners and communicate with postsynaptic brain regions to convey features of the visual scene. However, in the developing visual system, RGC interactions extend beyond their synaptic partners such that they guide development before the onset of vision. In this Review, we summarize our current understanding of how interactions between RGCs and their environment influence cellular targeting, migration and circuit maturation during visual system development. We describe the roles of RGC subclasses in shaping unique developmental responses within the retina and at central targets. Finally, we highlight the utility of RNA sequencing and genetic tools in uncovering RGC type-specific roles during the development of the visual system.

Violet-light suppression of thermogenesis by opsin 5 hypothalamic neurons.

The opsin family of G-protein-coupled receptors are used as light detectors in animals. Opsin 5 (also known as neuropsin or OPN5) is a highly conserved opsin that is sensitive to visible violet light1,2. In mice, OPN5 is a known photoreceptor in the retina3 and skin4 but is also expressed in the hypothalamic preoptic area (POA)5. Here we describe a light-sensing pathway in which POA neurons that express Opn5 regulate thermogenesis in brown adipose tissue (BAT). We show that Opn5 is expressed in glutamatergic warm-sensing POA neurons that receive synaptic input from several thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5-null mice show overactive BAT, increased body temperature, and exaggerated thermogenesis when cold-challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type mice but not in Opn5-null mice. Direct measurements of intracellular cAMP ex vivo show that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light-sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis.

Adaptive Thermogenesis in Mice Is Enhanced by Opsin 3-Dependent Adipocyte Light Sensing.

Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.

Procalcitonin and Other Common Biomarkers Do Not Reliably Identify Patients at Risk for Bacterial Infection After Congenital Heart Surgery.

OBJECTIVES: Following surgery, it is difficult to distinguish a postoperative inflammatory reaction from infection. This study examined the predictive value of the biomarkers; procalcitonin, C-reactive protein, lactate, neutrophils, lymphocytes, platelets, and the biphasic activated partial thromboplastin time waveform in diagnosing bacterial infection following cardiac surgery. DESIGN: Prospective, observational study. SETTING: A regional, PICU in the United Kingdom. PATIENTS: Three-hundred sixty-eight children under the age of 16 admitted to the PICU for elective cardiac surgery were enrolled in the study. INTERVENTIONS: All biomarker measurements were determined daily until postoperative day 7. Children were assessed for postoperative infection until day 28 and divided into four groups: bacterial infection, culture-negative sepsis, viral infection, and no infection. We used the Kruskal-Wallis test, chi-square test, analysis of variance, and area under the curve in our analysis. MEASUREMENTS AND MAIN RESULTS: In total, 71 of 368 children (19%) developed bacterial infection postoperatively, the majority being surgical site infections. In those with bacterial infection, procalcitonin was elevated on postoperative days 1-3 and the last measurement prior to event compared with those without bacterial infection. The most significant difference was the last measurement prior to event; 0.72 ng/mL in the bacterial infection group versus 0.13 ng/mL in the no infection group (for all groups; p < 0.001). Longitudinal profiles of all biomarkers were indistinct in the bacterial infection and nonbacterial infection groups except in those with culture-negative infections who had distinct procalcitonin kinetics on postoperative days 1-4. Children with culture-negative sepsis required longer ventilatory support and PICU stay and were more likely to develop complications than the other groups. CONCLUSIONS: None of the biomarkers studied within 3 days of infection distinguished between infection and postoperative inflammatory reaction. However, procalcitonin kinetics peaked on postoperative day 2 and fell more sharply than C-reactive protein kinetics, which peaked at postoperative day 3. The monitoring of procalcitonin kinetics following cardiac surgery may help guide rational antimicrobial use.