Sensory detection of food rapidly modulates arcuate feeding circuits.

Hunger is controlled by specialized neural circuits that translate homeostatic needs into motivated behaviors. These circuits are under chronic control by circulating signals of nutritional state, but their rapid dynamics on the timescale of behavior remain unknown. Here, we report optical recording of the natural activity of two key cell types that control food intake, AgRP and POMC neurons, in awake behaving mice. We find unexpectedly that the sensory detection of food is sufficient to rapidly reverse the activation state of these neurons induced by energy deficit. This rapid regulation is cell-type specific, modulated by food palatability and nutritional state, and occurs before any food is consumed. These data reveal that AgRP and POMC neurons receive real-time information about the availability of food in the external world, suggesting a primary role for these neurons in controlling appetitive behaviors such as foraging that promote the discovery of food.

Natural neural projection dynamics underlying social behavior.

Social interaction is a complex behavior essential for many species and is impaired in major neuropsychiatric disorders. Pharmacological studies have implicated certain neurotransmitter systems in social behavior, but circuit-level understanding of endogenous neural activity during social interaction is lacking. We therefore developed and applied a new methodology, termed fiber photometry, to optically record natural neural activity in genetically and connectivity-defined projections to elucidate the real-time role of specified pathways in mammalian behavior. Fiber photometry revealed that activity dynamics of a ventral tegmental area (VTA)-to-nucleus accumbens (NAc) projection could encode and predict key features of social, but not novel object, interaction. Consistent with this observation, optogenetic control of cells specifically contributing to this projection was sufficient to modulate social behavior, which was mediated by type 1 dopamine receptor signaling downstream in the NAc. Direct observation of deep projection-specific activity in this way captures a fundamental and previously inaccessible dimension of mammalian circuit dynamics.

Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers.

The quantification of membrane-associated biomolecular interactions is crucial to our understanding of various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition of fluorescent labels, which complicates the quantification of the involved stoichiometries and dynamics because of low temporal resolution and the inherent limitations associated with labeling efficiency, photoblinking and photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method to the membrane remodeling GTPase, dynamin-1, reveals heterogeneous mixtures of dimer-based oligomers, oligomer-dependent mobilities, membrane affinities and (dis)association of individual complexes. These capabilities, together with assay-based advances for studying integral membrane proteins, will enable the elucidation of biomolecular mechanisms in and on lipid bilayers.

LASER FLARE PHOTOMETRY IN PRIMARY RHEGMATOGENOUS RETINAL DETACHMENT: An Evaluation of 2,487 Cases.

PURPOSE: Exploratory analysis associated with the prospective, multicenter, randomized PRIVENT trial. To characterize the associations between laser flare photometry and anatomical and epidemiological features of rhegmatogenous retinal detachment (RRD). METHODS: The authors measured laser flare values of all 3,048 prescreened patients excluding those with comorbidities. A mixed regression analysis evaluated the strength of the influencing factors like age, sex, lens status, and presence and extent of RRD on laser flare. RESULTS: Rhegmatogenous retinal detachment was more frequent in men (65.8%) than in women (34.2%, P < 0.001) and in right (52%) than in left eyes (48%, P = 0.045). Phakic RRD affected less quadrants and was less likely to be associated with macula-off status than pseudophakic RRD (48.4% vs. 58.0% macula off, 23% vs. 31% ≥3 quadrants, P < 0.001). Laser flare of affected eyes was significantly higher compared with fellow eyes (12.6 ± 15.2 vs. 8.3 ± 7.4 pc/ms, P < 0.001). The factors age, sex, lens status, presence of RRD, and the number of quadrants affected were independent influencing factors on laser flare. R 2 was 0.145 for phakic and 0.094 for pseudophakic eyes. CONCLUSION: The results indicate that there may be more factors affecting laser flare than previously assumed. This might limit flare as predictive value for PVR and retinal redetachment.

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

Wireless, battery-free subdermally implantable photometry systems for chronic recording of neural dynamics.

Recording cell-specific neuronal activity while monitoring behaviors of freely moving subjects can provide some of the most significant insights into brain function. Current means for monitoring calcium dynamics in genetically targeted populations of neurons rely on delivery of light and recording of fluorescent signals through optical fibers that can reduce subject mobility, induce motion artifacts, and limit experimental paradigms to isolated subjects in open, two-dimensional (2D) spaces. Wireless alternatives eliminate constraints associated with optical fibers, but their use of head stages with batteries adds bulk and weight that can affect behaviors, with limited operational lifetimes. The systems introduced here avoid drawbacks of both types of technologies, by combining highly miniaturized electronics and energy harvesters with injectable photometric modules in a class of fully wireless, battery-free photometer that is fully implantable subdermally to allow for the interrogation of neural dynamics in freely behaving subjects, without limitations set by fiber optic tethers or operational lifetimes constrained by traditional power supplies. The unique capabilities of these systems, their compatibility with magnetic resonant imaging and computed tomography and the ability to manufacture them with techniques in widespread use for consumer electronics, suggest a potential for broad adoption in neuroscience research.

Relevance of Laser Flare Photometry in Retinal Pathologies. / Relevanz der Laser-Flare-Photometrie bei retinalen Pathologien.

Laser flare photometry provides a non-invasive and objective measurement of the Tyndall effect in the anterior chamber. The laser flare value (measured in photon number per millisecond [pc/ms]) thus quantifies the extent of disruption to the blood-aqueous barrier and can be used in clinical applications to monitor uveitis therapy or to measure the postoperative degree of inflammation. Standardised performance must be observed during measurement. Publications of the last 35 years on laser flare photometry deal not only with the measurement procedure but also with its use in clinical practice for different ophthalmological pathologies. Likewise, various influencing factors have already been investigated and described that must be considered when measuring and evaluating laser flare values. The focus of this article is the relevance of laser flare photometry in retinal pathologies. In recently published studies, the level of objective tyndallometry in primary rhegmatogenous retinal detachment is shown to depend on lens status, symptom duration, and extent of retinal detachment. The greater is the area of the retina affected, the greater the disruption of the blood-aqueous barrier appears to be. Elevated laser flare values have also been considered as a predictor for the development of proliferative vitreoretinopathy (PVR). However, based on current knowledge, this assumption must be put into perspective. According to current data, objective tyndallometry can be used to monitor the progression of intraocular inflammation and to quantify the blood-aqueous barrier, and the values correlate with the extent and anatomical features, as well as the symptom duration in retinal detachment. Many influencing factors have already been identified. But further evaluation is desirable and needed. It is still unclear whether laser flare values can be used in the future as a predictor for sequelae such as PVR development.

The Value of Laser Flare Photometry as a Monitoring Tool for Uveitis. / Der Stellenwert von Laser-Flare-Messungen zur Verlaufsbeurteilung von Uveitiden.

Laser flare (LF) photometry (P) is used to quantify the protein concentration in the aqueous humor, and therefore assess the blood-aqueous humor barrier. LFP is more reliable than the clinical assessment of the Tyndall effect, and is thus especially useful in the follow-up of uveitis patients. In active uveitis, LFP correlates well with the anterior chamber cell grading. Various studies have shown that high LF values are associated with an increased risk of uveitic complications, such as macular edema, glaucoma, and posterior synechiae. LFP can also be used to assess the response to anti-inflammatory treatments as well as the optimal timing and selection of the surgical technique for intraocular surgeries.

Dorsal Raphe Dopamine Neurons Signal Motivational Salience Dependent on Internal State, Expectation, and Behavioral Context.

The ability to recognize motivationally salient events and adaptively respond to them is critical for survival. Here, we tested whether dopamine (DA) neurons in the dorsal raphe nucleus (DRN) contribute to this process in both male and female mice. Population recordings of DRNDA neurons during associative learning tasks showed that their activity dynamically tracks the motivational salience, developing excitation to both reward-paired and shock-paired cues. The DRNDA response to reward-predicting cues was diminished after satiety, suggesting modulation by internal states. DRNDA activity was also greater for unexpected outcomes than for expected outcomes. Two-photon imaging of DRNDA neurons demonstrated that the majority of individual neurons developed activation to reward-predicting cues and reward but not to shock-predicting cues, which was surprising and qualitatively distinct from the population results. Performing the same fear learning procedures in freely-moving and head-fixed groups revealed that head-fixation itself abolished the neural response to aversive cues, indicating its modulation by behavioral context. Overall, these results suggest that DRNDA neurons encode motivational salience, dependent on internal and external factors.SIGNIFICANCE STATEMENT Dopamine (DA) contributes to motivational control, composed of at least two functional cell types, one signaling for motivational value and another for motivational salience. Here, we demonstrate that DA neurons in the dorsal raphe nucleus (DRN) encode the motivational salience in associative learning tasks. Neural responses were dynamic and modulated by the animal's internal state. The majority of single-cells developed responses to reward or paired cues, but not to shock-predicting cues. Additional experiments with freely-moving and head-fixed mice showed that head-fixation abolished the development of cue responses during fear learning. This work provides further characterization on the functional roles of overlooked DRNDA populations and an example that neural responses can be altered by head-fixation, which is commonly used in neuroscience.

Depth-resolved fiber photometry with a single tapered optical fiber implant.

Fiber photometry is increasingly utilized to monitor fluorescent sensors of neural activity in the brain. However, most implementations are based on flat-cleaved optical fibers that can only interface with shallow tissue volumes adjacent to the fiber. We exploit modal properties of tapered optical fibers (TFs) to enable light collection over an extent of up to 2 mm of tissue and multisite photometry along the taper. Using a single TF, we simultaneously observed distinct dopamine transients in dorsal and ventral striatum in freely moving mice performing a simple, operant conditioning task. Collection volumes from TFs can also be engineered in both shape and size by microstructuring the nonplanar surface of the taper, to optically target multiple sites not only in the deep brain but, in general, in any biological system or organ in which light collection is beneficial but challenging because of light scattering and absorption.

High-density multi-fiber photometry for studying large-scale brain circuit dynamics.

Animal behavior originates from neuronal activity distributed across brain-wide networks. However, techniques available to assess large-scale neural dynamics in behaving animals remain limited. Here we present compact, chronically implantable, high-density arrays of optical fibers that enable multi-fiber photometry and optogenetic perturbations across many regions in the mammalian brain. In mice engaged in a texture discrimination task, we achieved simultaneous photometric calcium recordings from networks of 12-48 brain regions, including striatal, thalamic, hippocampal and cortical areas. Furthermore, we optically perturbed subsets of regions in VGAT-ChR2 mice by targeting specific fiber channels with a spatial light modulator. Perturbation of ventral thalamic nuclei caused distributed network modulation and behavioral deficits. Finally, we demonstrate multi-fiber photometry in freely moving animals, including simultaneous recordings from two mice during social interaction. High-density multi-fiber arrays are versatile tools for the investigation of large-scale brain dynamics during behavior.

MACULAR PIGMENT OPTICAL DENSITY ASSESSED BY HETEROCHROMATIC FLICKER PHOTOMETRY IN EYES AFFECTED BY PRIMARY EPIRETINAL MEMBRANE.

PURPOSE: To compare macular pigment optical density (MPOD) in healthy eyes versus eyes affected by primary epiretinal membrane (ERM) in different stages and to assess the relation between MPOD and optical coherence tomography findings. METHODS: Prospective cross-sectional study of 62 eyes of 62 patients affected by unilateral primary ERM. Contralateral healthy eyes from the same patients were used as a control group. Main outcome measures were MPOD, ERM stage, central foveal thickness (CFT), outer nuclear layer thickness (ONLT), integrity of outer retinal bands, and presence of central bouquet abnormalities. RESULTS: In the study group, mean CFT was 444 ± 75 µm and mean ONLT was 245 ± 40 µm, whereas in the control group, mean CFT was 230 ± 21 µm and mean ONLT was 102 ± 14 µm (P < 0.001). Mean MPOD was 0.86 ± 0.07 in eyes with ERM and 0.48 ± 0.09 in contralateral healthy eyes (P < 0.001). Macular pigment optical density was associated with CFT (P = 0.006) and ONLT (P < 0.001), whereas no significant associations were observed between MPOD and outer retinal bands integrity (P = 0.14) and central bouquet abnormalities (P = 0.08). CONCLUSION: Macular pigment optical density increased in eyes affected by primary ERM proportionally to CFT and, especially, ONLT. Probably, centripetal forces exerted by ERM contraction on the retinal surface lead to a progressive foveal packing of foveal Müller cells.

Portrait imaging relighting system based on a simplified photometric stereo method.

This study proposes a portrait image relighting system based on a simplified photometric stereo method. The system, comprising a controllable digital single lens reflex camera and five polarized flashlights, can obtain a color shade-less image and synthesize a normal map from shaded images. When calibrating the photometric stereo, the normal map is taken as a linear combination of shaded images and clamped with respect to specific normal directions on a white-coated sphere. The relit images were generated through inverse rendering in a predefined virtual environment. To evaluate personal preference, 24 adult subjects were recruited to conduct subjective assessments comparing the deep portrait relighting method results. From experiments regarding different scenarios, we concluded that the proposed system based on a simplified photometric stereo performs acceptably for relighting portrait images.

Single molecule mass photometry of nucleic acids.

Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.

Bovine Respiratory Syncytial Virus (BRSV) Infection Detected in Exhaled Breath Condensate of Dairy Calves by Near-Infrared Aquaphotomics.

Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. This study evaluated NIR spectra (n = 240) of exhaled breath condensate (EBC) from dairy calves (n = 5) undergoing a controlled infection with BRSV. Changes in the organization of the aqueous phase of EBC during the baseline (pre-infection) and infected (post-infection and clinically abnormal) stages were found in the WAMACS (water matrix coordinates) C1, C5, C9, and C11, likely associated with volatile and non-volatile compounds in EBC. The discrimination of these chemical profiles by PCA-LDA models differentiated samples collected during the baseline and infected stages with an accuracy, sensitivity, and specificity >93% in both the calibration and validation. Thus, biochemical changes occurring during BRSV infection can be detected and evaluated with NIR-aquaphotomics in EBC. These findings form the foundation for developing an innovative, non-invasive, and in-field diagnostic tool to identify BRSV infection in cattle.

A classification approach to estimating human circadian phase under circadian alignment from actigraphy and photometry data.

The time of dim light melatonin onset (DLMO) is the gold standard for circadian phase assessment in humans, but collection of samples for DLMO is time and resource-intensive. Numerous studies have attempted to estimate circadian phase from actigraphy data, but most of these studies have involved individuals on controlled and stable sleep-wake schedules, with mean errors reported between 0.5 and 1 hour. We found that such algorithms are less successful in estimating DLMO in a population of college students with more irregular schedules: Mean errors in estimating the time of DLMO are approximately 1.5-1.6 hours. We reframed the problem as a classification problem and estimated whether an individual's current phase was before or after DLMO. Using a neural network, we found high classification accuracy of about 90%, which decreased the mean error in DLMO estimation-identifying the time at which the switch in classification occurs-to approximately 1.3 hours. To test whether this classification approach was valid when activity and circadian rhythms are decoupled, we applied the same neural network to data from inpatient forced desynchrony studies in which participants are scheduled to sleep and wake at all circadian phases (rather than their habitual schedules). In participants on forced desynchrony protocols, overall classification accuracy dropped to 55%-65% with a range of 20%-80% for a given day; this accuracy was highly dependent upon the phase angle (ie, time) between DLMO and sleep onset, with the highest accuracy at phase angles associated with nighttime sleep. Circadian patterns in activity, therefore, should be included when developing and testing actigraphy-based approaches to circadian phase estimation. Our novel algorithm may be a promising approach for estimating the onset of melatonin in some conditions and could be generalized to other hormones.

Photometric Determination of Iron in Pharmaceutical Formulations Using Double-Beam Direct Injection Flow Detector.

In this work, an innovative, flow-through, double-beam, photometric detector with direct injection of the reagents (double-DID) was used for the first time for the determination of iron in pharmaceuticals. For stable measurement of the absorbance, double paired emission-detection LED diodes and a log ratio precision amplifier have been applied. The detector was integrated with the system of solenoid micro-pumps. The micro-pumps helped to reduce the number of reagents used and are responsible for precise solution dispensing and propelling. The flow system is characterized by a high level of automation. The total iron was determined as a Fe(II) with photometric detection using 1,10-phenanthroline as a complexing agent. The optimum conditions of the propose analytical procedure were established and the method was validated. The calibration graph was linear in the range of 1 to 30 mg L-1. The limit of detection (LOD) was 0.5 mg L-1. The throughput of the method was 90 samples/hour. The repeatability of the method expressed as the relative standard deviation (R.S.D.) was 2% (n = 10). The method was characterized by very low consumption of reagents and samples (20 µL each) and a small amount of waste produced (about 540 µL per analysis). The proposed flow method was successfully applied for determination of iron in pharmaceutical products. The results were in good agreement with those obtained using the manual UV-Vis spectrophotometry and with values claimed by the manufacturers. The flow system worked very stably and was insensitive to bubbles appearing in the system.

Photometry unlocks 3D information from 2D localization microscopy data.

We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry.

Measurements of biomolecular interactions are crucial to understand the mechanisms of the biological processes they facilitate. Bulk-based methods such as ITC and SPR provide important information on binding affinities, stoichiometry, and kinetics of interactions. However, the ensemble averaging approaches are not able to probe the intrinsic heterogeneity often displayed by biological systems. Interactions that involve cooperativity or result in the formation of multicomponent complexes pose additional experimental challenges. Single-molecule techniques have previously been applied to solve these problems. However, single-molecule experiments are often technically demanding and require labeling or immobilization of the molecules under study. A recently developed single-molecule method, mass photometry (MP), overcomes these limitations. Here we applied MP to measure the affinities of biomolecular interactions. We have demonstrated how MP allows the user to study multivalent complexes and quantify the affinities of different binding sites in a single measurement. Results obtained from this single-molecule technique have been validated by ITC and BLI. The quality and information content of the MP data, combined with simple and fast measurements and low sample consumption makes MP a new preferred method for measuring strong protein-protein interactions.

Dexmedetomidine Activation of Dopamine Neurons in the Ventral Tegmental Area Attenuates the Depth of Sedation in Mice.

BACKGROUND: Dexmedetomidine induces a sedative response that is associated with rapid arousal. To elucidate the underlying mechanisms, the authors hypothesized that dexmedetomidine increases the activity of dopaminergic neurons in the ventral tegmental area, and that this action contributes to the unique sedative properties of dexmedetomidine. METHODS: Only male mice were used. The activity of ventral tegmental area dopamine neurons was measured by a genetically encoded Ca indicator and patch-clamp recording. Dopamine neurotransmitter dynamics in the medial prefrontal cortex and nucleus accumbens were measured by a genetically encoded dopamine sensor. Ventral tegmental area dopamine neurons were inhibited or activated by a chemogenetic approach, and the depth of sedation was estimated by electroencephalography. RESULTS: Ca signals in dopamine neurons in the ventral tegmental area increased after intraperitoneal injection of dexmedetomidine (40 µg/kg; dexmedetomidine, 16.917 [14.882; 21.748], median [25%; 75%], vs. saline, -0.745 [-1.547; 0.359], normalized data, P = 0.001; n = 6 mice). Dopamine transmission increased in the medial prefrontal cortex after intraperitoneal injection of dexmedetomidine (40 µg/kg; dexmedetomidine, 10.812 [9.713; 15.104], median [25%; 75%], vs. saline, -0.498 [-0.664; -0.355], normalized data, P = 0.001; n = 6 mice) and in the nucleus accumbens (dexmedetomidine, 8.543 [7.135; 11.828], median [25%; 75%], vs. saline, -0.329 [-1.220; -0.047], normalized data, P = 0.001; n = 6 mice). Chemogenetic inhibition or activation of ventral tegmental area dopamine neurons increased or decreased slow waves, respectively, after intraperitoneal injection of dexmedetomidine (40 µg/kg; delta wave: two-way repeated measures ANOVA, F[2, 33] = 8.016, P = 0.002; n = 12 mice; theta wave: two-way repeated measures ANOVA, F[2, 33] = 22.800, P < 0.0001; n = 12 mice). CONCLUSIONS: Dexmedetomidine activates dopamine neurons in the ventral tegmental area and increases dopamine concentrations in the related forebrain projection areas. This mechanism may explain rapid arousability upon dexmedetomidine sedation.