Comparisons of choroidal thickness and volume in eyes with central serous chorioretinopathy to that of control eyes determined by ultra-widefield optical coherence tomography.

PURPOSE: To compare choroidal thickness and volume in eyes with central serous chorioretinopathy (CSC) and healthy control eyes over a wide area of the fundus using ultra-widefield optical coherence tomography (UWF-OCT). METHODS: Thirty-three eyes of 29 patients with CSC and 36 eyes of 21 healthy controls were examined retrospectively. Choroidal images were obtained with a prototype UWF-OCT device with a field of view of 105° or approximately 31.5-mm wide by 10.9-mm deep. Choroidal thickness and volume were measured in the images of 12 radial scans (every 15°) from the horizontal scan. The "new index" of the extent of focal choroidal protrusion was defined as the maximum steepness of choroidal thickness (MSCT). RESULTS: Choroidal volume in CSC eyes was significantly larger than in control eyes within the central 50° (P < 0.001). However, there was no significant difference in choroidal volume in the peripheral 50 to 105° (P = 0.071). The MSCTs were significantly steeper in CSC eyes than in control eyes at scan lines 1, 6, 7, 8, and 10 (P < 0.01, P < 0.001, P < 0.05, P < 0.01, P < 0.05). CONCLUSIONS: The choroid in CSC eyes was thickened only at the posterior pole, and its protrusion was significant mainly in the vertical direction. Focal choroidal thickening at the posterior pole, which we speculate includes congenital scleral changes, may affect the pathophysiology of CSC.

Forward genetic screen of Caenorhabditis elegans mutants with impaired sleep reveals a crucial role of neuronal diacylglycerol kinase DGK-1 in regulating sleep.

The sleep state is widely observed in animals. The molecular mechanisms underlying sleep regulation, however, remain largely unclear. In the nematode Caenorhabditis elegans, developmentally timed sleep (DTS) and stress-induced sleep (SIS) are 2 types of quiescent behaviors that fulfill the definition of sleep and share conserved sleep-regulating molecules with mammals. To identify novel sleep-regulating molecules, we conducted an unbiased forward genetic screen based on DTS phenotypes. We isolated 2 mutants, rem8 and rem10, that exhibited significantly disrupted DTS and SIS. The causal gene of the abnormal sleep phenotypes in both mutants was mapped to dgk-1, which encodes diacylglycerol kinase. Perhaps due to the diminished SIS, dgk-1 mutant worms exhibited decreased survival following exposure to a noxious stimulus. Pan-neuronal and/or cholinergic expression of dgk-1 partly rescued the dgk-1 mutant defects in DTS, SIS, and post-stress survival. Moreover, we revealed that pkc-1/nPKC participates in sleep regulation and counteracts the effect of dgk-1; the reduced DTS, SIS, and post-stress survival rate were partly suppressed in the pkc-1; dgk-1 double mutant compared with the dgk-1 single mutant. Excessive sleep observed in the pkc-1 mutant was also suppressed in the pkc-1; dgk-1 double mutant, implying that dgk-1 has a complicated mode of action. Our findings indicate that neuronal DGK-1 is essential for normal sleep and that the counterbalance between DGK-1 and PKC-1 is crucial for regulating sleep and mitigating post-stress damage.

ER proteostasis regulators cell-non-autonomously control sleep.

Sleep is regulated by peripheral tissues under fatigue. The molecular pathways in peripheral cells that trigger systemic sleep-related signals, however, are unclear. Here, a forward genetic screen in C. elegans identifies 3 genes that strongly affect sleep amount: sel-1, sel-11, and mars-1. sel-1 and sel-11 encode endoplasmic reticulum (ER)-associated degradation components, whereas mars-1 encodes methionyl-tRNA synthetase. We find that these machineries function in non-neuronal tissues and that the ER unfolded protein response components inositol-requiring enzyme 1 (IRE1)/XBP1 and protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor-2α (eIF2α)/activating transcription factor-4 (ATF4) participate in non-neuronal sleep regulation, partly by reducing global translation. Neuronal epidermal growth factor receptor (EGFR) signaling is also required. Mouse studies suggest that this mechanism is conserved in mammals. Considering that prolonged wakefulness increases ER proteostasis stress in peripheral tissues, our results suggest that peripheral ER proteostasis factors control sleep homeostasis. Moreover, based on our results, peripheral tissues likely cope with ER stress not only by the well-established cell-autonomous mechanisms but also by promoting the individual's sleep.

Intracellular Ca2+ dynamics in the ALA neuron reflect sleep pressure and regulate sleep in Caenorhabditis elegans.

The mechanisms underlying sleep homeostasis are poorly understood. The nematode Caenorhabditis elegans exhibits 2 types of sleep: lethargus, or developmentally timed, and stress-induced sleep. Lethargus is characterized by alternating cycles of sleep and motion bouts. Sleep bouts are homeostatically regulated, i.e., prolonged active bouts lead to prolonged sleep bouts. Here we reveal that the interneuron ALA is crucial for homeostatic regulation during lethargus. Intracellular Ca2+ in ALA gradually increased during active bouts and rapidly decayed upon transitions to sleep bouts. Longer active bouts were accompanied by higher intracellular Ca2+ peaks. Optogenetic activation of ALA during active bouts caused transitions to sleep bouts. Dysfunction of CEH-17, which is an LIM homeodomain transcription factor selectively expressed in ALA, impaired the characteristic patterns of ALA intracellular Ca2+ and abolished the homeostatic regulation of sleep bouts. These findings indicate that ALA encodes sleep pressure and contributes to sleep homeostasis.

PERIPHERAL CHOROIDAL THICKNESS DETERMINED BY WIDE-FIELD OPTICAL COHERENCE TOMOGRAPHY IN EYES WITH CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: To determine the central and peripheral choroidal thickness in eyes with central serous chorioretinopathy (CSC) and to compare these thicknesses values with those of control normal eyes. METHODS: Wide-field optical coherence tomographic images of 24 eyes of 19 patients with CSC and 14 normal eyes of 7 individuals were recorded. A 20-mm vertical scan through the fovea was obtained with the Xephilio optical coherence tomographic S1 (Canon, Japan), a wide-field optical coherence tomographic device. The subfoveal choroidal thickness and the thickness at 5 mm superior (S5) and inferior (I5), 7 mm superior (S7) and inferior (I7), 8.5 mm superior (S8) and inferior (I8), and 10 mm superior (S10) and inferior (I10) from the fovea in the CSC eyes and normal eyes were compared. RESULTS: There was no significant difference in the age ( P = 0.8) or the refractive error ( P = 0.7) between the CSC and normal eyes. The choroidal thickness was significantly thicker in the eyes with CSC than that in the normal eyes at subfoveal choroidal thickness ( P < 0.01), S5 ( P = 0.01), and S7 ( P = 0.02). However, there was no significant difference in the choroidal thickness at the more peripheral points (all P > 0.1). CONCLUSION: The thickened choroid in CSC was observed at the fovea and the area just superior to the fovea. The pathogenesis of CSC may be associated with the choroidal thickening confined to the fovea and superior foveal area.

Morphological differences of choroid in central serous chorioretinopathy determined by ultra-widefield optical coherence tomography.

PURPOSE: The purpose of this study was to compare the morphology of the central and peripheral choroid of eyes with central serous chorioretinopathy (CSC) to that of normal eyes using ultra-widefield optical coherence tomography (UWF-OCT). METHODS: We reviewed the medical records of 29 eyes of 25 patients (23 men, 2 women; average age 44.4 years) with CSC and 34 eyes of 22 healthy subjects (19 men, 3 women; average age, 49.5 years) with normal eyes. The images obtained by a prototype swept source UWF-OCT (Topcon, Tokyo, Japan) of about 31.5-mm wide and a depth of 10.9 mm were analyzed. The choroidal thickness was measured for each sector of the eye using the conventional automated layer analysis method. The local morphological differences were quantified by the maximum steepness (µm/deg) which was obtained by differentiating the changes in the choroidal thickness from the periphery to the fovea. Only the vertical scans were evaluated to avoid the influence of the optic disc. RESULTS: The choroid was thicker in the macular area than the peripheral area in both normal and CSC eyes. The choroid at the subfovea was significantly thicker in the CSC eyes than that of the normal eyes (P < 0.0001); however, the difference at the periphery was not significant. The mean of the maximum steepness of the choroidal thickness was 20.8 ± 3.8 µm/deg in the CSC eyes which was significantly steeper than the 16.0 ± 4.6 µm/deg in healthy eyes (P < 0.0001). CONCLUSION: The choroid in CSC eyes has a steeper slope around the posterior pole. UWF-OCT can be used to evaluate the abnormalities of the choroidal structures from the posterior pole to the periphery in eyes with CSC.

Diagnosis of central serous chorioretinopathy by deep learning analysis of en face images of choroidal vasculature: A pilot study.

PURPOSE: To diagnose central serous chorioretinopathy (CSC) by deep learning (DL) analyses of en face images of the choroidal vasculature obtained by optical coherence tomography (OCT) and to analyze the regions of interest for the DL from heatmaps. METHODS: One-hundred eyes were studied; 53 eyes with CSC and 47 normal eyes. Volume scans of 12×12 mm square were obtained at the same time as the OCT angiographic (OCTA) scans (Plex Elite 9000 Swept-Source OCT®, Zeiss). High-quality en face images of the choroidal vasculature of the segmentation slab of one-half of the subfoveal choroidal thickness were created for the analyses. The 100 en face images were divided into 80 for training and 20 for validation. Thus, we divided it into five groups of 20 eyes each, trained the remaining 80 eyes in each group, and then calculated the correct answer rate for each group by validation with 20 eyes. The Neural Network Console (NNC) developed by Sony and the Keras-Tensorflow backend developed by Google were used as the software for the classification with 16 layers of convolutional neural networks. The active region of the heatmap based on the feature quantity extracted by DL was also evaluated as the percentages with gradient-weighted class activation mapping implemented in Keras. RESULTS: The mean accuracy rate of the validation was 95% for NNC and 88% for Keras. This difference was not significant (P >0.1). The mean active region in the heatmap image was 12.5% in CSC eyes which was significantly lower than the 79.8% in normal eyes (P<0.01). CONCLUSIONS: CSC can be automatically diagnosed by DL with high accuracy from en face images of the choroidal vasculature with different programs, convolutional layer structures, and small data sets. Heatmap analyses showed that the DL focused on the area occupied by the choroidal vessels and their uniformity. We conclude that DL can help in the diagnosis of CSC.

Corollary discharge promotes a sustained motor state in a neural circuit for navigation.

Animals exhibit behavioral and neural responses that persist on longer timescales than transient or fluctuating stimulus inputs. Here, we report that Caenorhabditis elegans uses feedback from the motor circuit to a sensory processing interneuron to sustain its motor state during thermotactic navigation. By imaging circuit activity in behaving animals, we show that a principal postsynaptic partner of the AFD thermosensory neuron, the AIY interneuron, encodes both temperature and motor state information. By optogenetic and genetic manipulation of this circuit, we demonstrate that the motor state representation in AIY is a corollary discharge signal. RIM, an interneuron that is connected with premotor interneurons, is required for this corollary discharge. Ablation of RIM eliminates the motor representation in AIY, allows thermosensory representations to reach downstream premotor interneurons, and reduces the animal's ability to sustain forward movements during thermotaxis. We propose that feedback from the motor circuit to the sensory processing circuit underlies a positive feedback mechanism to generate persistent neural activity and sustained behavioral patterns in a sensorimotor transformation.

Sleep Architecture in Mice Is Shaped by the Transcription Factor AP-2ß.

The molecular mechanism regulating sleep largely remains to be elucidated. In humans, families that carry mutations in TFAP2B, which encodes the transcription factor AP-2ß, self-reported sleep abnormalities such as short-sleep and parasomnia. Notably, AP-2 transcription factors play essential roles in sleep regulation in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster Thus, AP-2 transcription factors might have a conserved role in sleep regulation across the animal phyla. However, direct evidence supporting the involvement of TFAP2B in mammalian sleep was lacking. In this study, by using the CRISPR/Cas9 technology, we generated two Tfap2b mutant mouse strains, Tfap2bK144 and Tfap2bK145 , each harboring a single-nucleotide mutation within the introns of Tfap2b mimicking the mutations in two human kindreds that self-reported sleep abnormalities. The effects of these mutations were compared with those of a Tfap2b knockout allele (Tfap2b-). The protein expression level of TFAP2B in the embryonic brain was reduced to about half in Tfap2b+/- mice and was further reduced in Tfap2b-/- mice. By contrast, the protein expression level was normal in Tfap2bK145/+ mice but was reduced in Tfap2bK145/K145 mice to a similar extent as Tfap2b-/- mice. Tfap2bK144/+ and Tfap2bK144/K144 showed normal protein expression levels. Tfap2b+/- female mice showed increased wakefulness time and decreased nonrapid eye movement sleep (NREMS) time. By contrast, Tfap2bK145/+ female mice showed an apparently normal amount of sleep but instead exhibited fragmented NREMS, whereas Tfap2bK144/+ male mice showed reduced NREMS time specifically in the dark phase. Finally, in the adult brain, Tfap2b-LacZ expression was detected in the superior colliculus, locus coeruleus, cerebellum, and the nucleus of solitary tract. These findings provide direct evidence that TFAP2B influences NREMS amounts in mice and also show that different mutations in Tfap2b can lead to diverse effects on sleep architecture.

Progressive Changes in Sleep and Its Relations to Amyloid-ß Distribution and Learning in Single App Knock-In Mice.

Alzheimer's disease (AD) patients often suffer from sleep disturbances. Alterations in sleep, especially rapid eye movement sleep (REMS), can precede the onset of dementia. To accurately characterize the sleep impairments accompanying AD and their underlying mechanisms using animal models, it is crucial to use models in which brain areas are affected in a manner similar to that observed in the actual patients. Here, we focused on AppNL-G-F mice, in which expression levels and patterns of mutated amyloid precursor protein (APP) follow the endogenous patterns. We characterized the sleep architecture of male AppNL-G-F homozygous and heterozygous mice at two ages (six and 12 months). At six months, homozygous mice exhibited reduced REMS, which was further reduced at 12 months together with a slight reduction in non-REMS (NREMS). By contrast, heterozygous mice exhibited an overall normal sleep architecture. Homozygous mice also exhibited decreased electroencephalogram γ to δ power ratio during REMS from six months, resembling the electroencephalogram slowing phenomenon observed in preclinical or early stages of AD. In addition, homozygous mice showed learning and memory impairments in the trace fear conditioning (FC) at both ages, and task performance strongly correlated with REMS amount at 12 months. Finally, histologic analyses revealed that amyloid-ß accumulation in the pontine tegmental area and ventral medulla followed a course similar to that of the REMS reduction. These findings support the notion that changes in REMS are an early marker of AD and provide a starting point to address the mechanism of sleep deficits in AD and the effects on cognition.

Widely Distributed Neurotensinergic Neurons in the Brainstem Regulate NREM Sleep in Mice.

Classical transection studies suggest that, in addition to the hypothalamus, the brainstem is essential for non-rapid eye movement (NREM) sleep. The circuits underlying this function, however, have remained largely unknown. We identified a circuit distributed in the midbrain, pons, and medulla that promotes NREM sleep in mice. We focused on the sublaterodorsal tegmentum, an area implicated in dual regulation of REM and NREM sleep. Transcriptomic and genetic analyses revealed that neurons positive for the neuropeptide neurotensin promote NREM sleep. Further analyses identified downstream NREM sleep-promoting neurons in the dorsal deep mesencephalic nucleus, the lateral part of the periaqueductal gray, and the medial vestibular nucleus that were also neurotensinergic. Infusion of neurotensin into the fourth ventricle induced NREM sleep-like cortical activity, whereas mice deficient for neurotensin exhibited increased REM sleep, implicating the involvement of the neuropeptide itself. These findings identify a widely distributed NREM sleep-regulating circuit in the brainstem with a common molecular property.

Choroidal neovascularization imaging using multiple en face optical coherence tomography angiography image averaging.

PURPOSE: To determine the effects of averaging five en face optical coherence tomography angiographic (OCTA) images on the quality of the images in eyes with a choroidal neovascularization (CNV). METHODS: Twenty-seven eyes of 25 patients (18 men, 7 women; average age 71.0 years) with a CNV were examined by OCTA (OCT HS-100, Canon. Japan). A 3 × 3-mm image including the CNV was recorded and automatically segmented between the retinal outer layers. Analyses were performed on a single image (S-image) and the average of five single images of the same area (A-images). The region of the CNV was selected by ImageJ, and the peak signal-to-noise ratio (PSNR), the vascular density (VD), fractal dimension (FD), and the noise component using band pass filter (BPF) processing of the S- and A-images of each case were compared. RESULTS: The average PSNR for the A-images was 14.0 which was significantly higher than the 12.2 for the S-images (P < 0.01). However, the average VD was 33.6% for the S-images and 34.8% for the A-images (P > 0.1). The average FD was 1.67 for the S-images and 1.54 for the A-images (P < 0.01). The mean luminance difference obtained by subtracting the luminance of the A-image from the S-image after BPF processing was 10.41 ± 14.66 db which was positive for all eyes. CONCLUSIONS: The better quality of the A-images of a CNV and absence of a significant difference in the vascular density indicates that the improvement was due to the removal of the same signal levels of the noise component and blood vessels.

Visualizing large choroidal blood flow by subtraction of the choriocapillaris projection artifacts in swept source optical coherence tomography angiography in normal eyes.

Optical coherence tomography angiography (OCTA) seems not to image the choroidal blood flow pattern in the normal individual because of the OCT light attenuation. Our purpose in the current study was to visualize the large choroidal blood flow pattern after subtraction of the choriocapillaris projection artifact in normal eyes non-invasively by swept source (SS) OCTA. Sixty-one eyes of 45 individuals (19 men, 26 women) without ocular disease were examined by SS-OCTA (AngioPlex Elite 9000, Zeiss, Germany). A 12 × 12 mm macular area was scanned. Subfoveal choroidal thickness (SCT) was measured, and the choroidal blood flow pattern in a slab of 30 µm width at one-half of SCT was analyzed. In examining the choroidal blood flow pattern, a slab that was between 30 to 60 µm posterior to the retinal pigment epithelium, in which the choriocapillaris blood flow was most clearly imaged, was used for the subtraction of the projection artifacts from the choriocapillaris on the stromal area of choroid. The ratio (%) of the choroidal blood flow area in the whole choroidal region was calculated after binarization. Thirty-four eyes of 27 individuals (12 men, 15 women) were also examined by spectral domain OCTA (SD-OCTA). After the subtraction, the middle and large choroidal blood flow were clearly visible in SS-OCTA in all eyes. The mean SCT was 297 ± 61 µm, and the mean ratio of the choroidal blood flow area was 27.3 ± 8.2%, which was significantly correlated with SCT (R = 0.738, P < 0.01). SD-OCTA did not show the choroidal blood flow pattern. In conclusion, removal of the projection artifacts of choriocapillaris can make the choroidal blood flow visible in SS-OCTA of normal eyes. Because the ratio of choroidal blood flow area was correlated with SCT, the choroidal blood flow might be an important factor related to the choroidal thickness.

Descending pathway facilitates undulatory wave propagation in Caenorhabditis elegans through gap junctions.

Descending signals from the brain play critical roles in controlling and modulating locomotion kinematics. In the Caenorhabditis elegans nervous system, descending AVB premotor interneurons exclusively form gap junctions with the B-type motor neurons that execute forward locomotion. We combined genetic analysis, optogenetic manipulation, calcium imaging, and computational modeling to elucidate the function of AVB-B gap junctions during forward locomotion. First, we found that some B-type motor neurons generate rhythmic activity, constituting distributed oscillators. Second, AVB premotor interneurons use their electric inputs to drive bifurcation of B-type motor neuron dynamics, triggering their transition from stationary to oscillatory activity. Third, proprioceptive couplings between neighboring B-type motor neurons entrain the frequency of body oscillators, forcing coherent bending wave propagation. Despite substantial anatomical differences between the motor circuits of C. elegans and higher model organisms, converging principles govern coordinated locomotion.

Excitatory motor neurons are local oscillators for backward locomotion.

Cell- or network-driven oscillators underlie motor rhythmicity. The identity of C. elegans oscillators remains unknown. Through cell ablation, electrophysiology, and calcium imaging, we show: (1) forward and backward locomotion is driven by different oscillators; (2) the cholinergic and excitatory A-class motor neurons exhibit intrinsic and oscillatory activity that is sufficient to drive backward locomotion in the absence of premotor interneurons; (3) the UNC-2 P/Q/N high-voltage-activated calcium current underlies A motor neuron's oscillation; (4) descending premotor interneurons AVA, via an evolutionarily conserved, mixed gap junction and chemical synapse configuration, exert state-dependent inhibition and potentiation of A motor neuron's intrinsic activity to regulate backward locomotion. Thus, motor neurons themselves derive rhythms, which are dually regulated by the descending interneurons to control the reversal motor state. These and previous findings exemplify compression: essential circuit properties are conserved but executed by fewer numbers and layers of neurons in a small locomotor network.

CLINICAL FINDINGS OF EYES WITH MACULAR EDEMA ASSOCIATED WITH BRANCH RETINAL VEIN OCCLUSION REFRACTORY TO RANIBIZUMAB.

PURPOSE: To determine the relationship between the clinical findings and the response to ranibizumab therapy in eyes with macular edema associated with branch retinal vein occlusion. METHODS: We reviewed the medical records of 68 patients with macular edema associated with a branch retinal vein occlusion. The patients were placed in the refractory group if the central foveal thickness remained more than 250 µm throughout the 6-month study period despite the ranibizumab therapy; otherwise, they were placed in the responsive group. RESULTS: Sixty (88.2%) of 68 eyes were placed in the responsive group and the other 8 eyes (11.8%) were placed in the refractory group. At the pretreatment examination, fluorescein angiography showed extensive leakage from occluded vessels in 52 (86.7%) of the 60 eyes in the responsive group and focal leakages from microaneurysms or dilated capillaries in the other 8 eyes (13.3%). In the refractory group, 7 (87.5%) of 8 eyes had only focal leakage and 1 eye (12.5%) had extensive leakage (P < 0.0001). The mean initial subfoveal choroidal thickness in the eyes with branch retinal vein occlusion in the responsive group was significantly thicker than that in the fellow eyes (278.0 ± 90.5 µm, 249.9 ± 94.4 µm; P < 0.0001). On the other hand, the mean initial subfoveal choroidal thickness in the refractory group was not significantly different from that of the fellow eyes (P = 0.4002). CONCLUSION: The dye leakage pattern in the fluorescein angiography images and choroidal thickness may be associated with response to ranibizumab therapy.

An extrasynaptic GABAergic signal modulates a pattern of forward movement in Caenorhabditis elegans.

As a common neurotransmitter in the nervous system, γ-aminobutyric acid (GABA) modulates locomotory patterns in both vertebrates and invertebrates. However, the signaling mechanisms underlying the behavioral effects of GABAergic modulation are not completely understood. Here, we demonstrate that a GABAergic signal in C. elegans modulates the amplitude of undulatory head bending through extrasynaptic neurotransmission and conserved metabotropic receptors. We show that the GABAergic RME head motor neurons generate undulatory activity patterns that correlate with head bending and the activity of RME causally links with head bending amplitude. The undulatory activity of RME is regulated by a pair of cholinergic head motor neurons SMD, which facilitate head bending, and inhibits SMD to limit head bending. The extrasynaptic neurotransmission between SMD and RME provides a gain control system to set head bending amplitude to a value correlated with optimal efficiency of forward movement.