Interleukin-10 enhances activity of ventral tegmental area dopamine neurons resulting in increased dopamine release.

Dopamine transmission from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) regulates important aspects of motivation and is influenced by the neuroimmune system. The neuroimmune system is a complex network of leukocytes, microglia and astrocytes that detect and remove foreign threats like bacteria or viruses and communicate with each other to regulate non-immune (e.g neuronal) cell activity through cytokine signaling. Inflammation is a key regulator of motivational states, though the effects of specific cytokines on VTA circuitry and motivation are largely unknown. Therefore, electrophysiology, neurochemical, immunohistochemical and behavioral studies were performed to determine the effects of the anti-inflammatory cytokine interleukin-10 (IL-10) on mesolimbic activity, dopamine transmission and conditioned behavior. IL-10 enhanced VTA dopamine firing and NAc dopamine levels via decreased VTA GABA currents in dopamine neurons. The IL-10 receptor was localized on VTA dopamine and non-dopamine cells. The IL-10 effects on dopamine neurons required post-synaptic phosphoinositide 3-kinase activity, and IL-10 appeared to have little-to-no efficacy on presynaptic GABA terminals. Intracranial IL-10 enhanced NAc dopamine levels in vivo and produced conditioned place aversion. Together, these studies identify the IL-10R on VTA dopamine neurons as a potential regulator of motivational states.

Teaching Synaptic Transmission Using Primary Literature: A Skills-Focused Pedagogical Approach.

Neuroscience is a burgeoning and intensive undergraduate major at many institutions of higher education and several areas in neuroscience education need further development. One such needed development is an increased focus on the procurement of career-relevant skills in addition to the traditional acquisition of subject knowledge. Skill development is particularly challenging in neuroscience education as the subject's interdisciplinary nature provides an atypically broad range of potential careers for graduates. Skills common to many careers in neuroscience include the ability to understand and analyze quantitative data and to draw conclusions based on those analyses. Here is presented an active learning pedagogical approach involving the analysis of seminal articles in the primary scientific literature to provide practice in analyzing data and drawing conclusions from those data while at the same time learning the fundamental tenets of synaptic transmission. Articles were selected that highlight principles such as the role of Ca2+ in synaptic release, exocytosis, quantal release, and synaptic delay. Figures from these articles that can readily be used to teach these principles were selected, and questions that can help to guide students' analysis of the data are also suggested. Activities like this are needed in greater numbers to facilitate the process of helping students gain skills relevant to a productive career in neuroscience.

Seletalisib for Activated PI3Kδ Syndromes: Open-Label Phase 1b and Extension Studies.

Mutations in two genes can result in activated PI3Kδ syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3Kδ inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase 1b study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15-25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 n = 3; APDS2 n = 4) received seletalisib; five completed the phase 1b study. For the extension study, four patients entered, one withdrew consent (week 24), three completed ≥84 wk of treatment. In the phase 1b study, patients had improved peripheral lymphadenopathy (n = 2), lung function (n = 1), thrombocyte counts (n = 1), and chronic enteropathy (n = 1). Overall, effects were maintained in the extension. In the phase 1b study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase 1b study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase 1b study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk-benefit profile was maintained for ≤96 wk.

A phase 2 randomized, double-blind, placebo-controlled, proof-of-concept study of oral seletalisib in primary Sjögren's syndrome.

OBJECTIVES: This phase 2 proof-of-concept study (NCT02610543) assessed efficacy, safety and effects on salivary gland inflammation of seletalisib, a potent and selective PI3Kδ inhibitor, in patients with moderate-to-severe primary Sjögren's syndrome (PSS). METHODS: Adults with PSS were randomized 1:1 to seletalisib 45 mg/day or placebo, in addition to current PSS therapy. Primary end points were safety and tolerability and change from baseline in EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) score at week 12. Secondary end points included change from baseline at week 12 in EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) score and histological features in salivary gland biopsies. RESULTS: Twenty-seven patients were randomized (seletalisib n = 13, placebo n = 14); 20 completed the study. Enrolment challenges led to early study termination with loss of statistical power (36% vs 80% planned). Nonetheless, a trend for improvement in ESSDAI and ESSPRI [difference vs placebo: -2.59 (95% CI: -7.30, 2.11; P=0.266) and -1.55 (95% CI: -3.39, 0.28), respectively] was observed at week 12. No significant changes were seen in saliva and tear flow. Serious adverse events (AEs) were reported in 3/13 of patients receiving seletalisib vs 1/14 for placebo and 5/13 vs 1/14 discontinued due to AEs, respectively. Serum IgM and IgG concentrations decreased in the seletalisib group vs placebo. Seletalisib demonstrated efficacy in reducing size and organisation of salivary gland inflammatory foci and in target engagement, thus reducing PI3K-mTOR signalling compared with placebo. CONCLUSION: Despite enrolment challenges, seletalisib demonstrated a trend towards clinical improvement in patients with PSS. Histological analyses demonstrated encouraging effects of seletalisib on salivary gland inflammation and organisation. TRIAL REGISTRATION: https://clinicaltrials.gov, NCT02610543.

Selective targeting of PI3Kδ suppresses human IL-17-producing T cells and innate-like lymphocytes and may be therapeutic for IL-17-mediated diseases.

The delta isoform of phosphoinositide 3-kinase (PI3Kδ) regulates various lymphocyte functions. Considering the key pro-inflammatory role of IL-17A and IL-17F cytokines in psoriasis and spondyloarthritis (SpA), we investigated the potential of PI3Kδ blockade to suppress IL-17A, IL-17F and associated pro-inflammatory cytokines that could synergize with IL-17A and IL-17F. Using in vitro studies with primary human cells and ex vivo studies with inflamed target tissues, we assessed if seletalisib, a selective PI3Kδ inhibitor, suppresses cytokine production by T cells and innate-like lymphocytes, and if seletalisib modulates the inflammatory responses in stromal cell populations in psoriasis (human dermal fibroblasts (HDF)) and SpA (fibroblast-like synoviocytes (FLS)). In vitro, seletalisib inhibited the production of pro-inflammatory cytokines, including IL-17A and IL-17F, from peripheral blood mononuclear cells (PBMCs), T helper 17 (Th17) cells as well as γδ-T cells and mucosal-associated invariant T cells. This inhibition resulted in decreased inflammatory activation of HDF in co-culture systems. Seletalisib was also efficacious in inhibiting SpA PBMCs and synovial fluid mononuclear cells (SFMCs) from producing pro-inflammatory cytokines. Furthermore, supernatant derived from cultured seletalisib-treated Th17 cells showed reduced potency for activating inflammatory responses from cultured SpA FLS and decreased their osteogenic differentiation capacity. Finally, analysis of inflamed SpA synovial tissue biopsies revealed activation of the PI3K-Akt-mTOR pathway. We observed that ex vivo seletalisib treatment of inflamed synovial tissue reduced IL-17A and IL-17F expression. Collectively, inhibition of PI3Kδ reduces the production of pro-inflammatory cytokines from IL-17-producing adaptive and innate-like lymphocytes and thereby inhibits downstream inflammatory and tissue remodeling responses. PI3Kδ-targeting may therefore represent a novel therapeutic avenue for the treatment of IL-17-mediated chronic inflammatory diseases such as psoriasis and SpA.

On the Use of Differential Scanning Calorimetry for Thermal Hazard Assessment of New Chemistry: Avoiding Explosive Mistakes.

Differential scanning calorimetry (DSC) is increasingly used as evidence to support a favourable safety profile of novel chemistry, or to highlight the need for caution. DSC enables preliminary assessment of the thermal hazards of a potentially energetic compound. However, unlike other standard characterisation methods, which have well defined formats for reporting data, the current reporting of DSC results for thermal hazard assessment has shown concerning trends. Around half of all results in 2019 did not include experimental details required to replicate the procedure. Furthermore, analysis for thermal hazard assessment is often only conducted in unsealed crucibles, which could lead to misleading results and dangerously incorrect conclusions. We highlight the specific issues with DSC analysis of hazardous compounds currently in the organic chemistry literature and provide simple "best practice" guidelines which will give chemists confidence in reported DSC results and the conclusions drawn from them.

Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren's syndrome.

BACKGROUND: The phosphatidylinositol 3-kinase delta isoform (PI3Kδ) belongs to an intracellular lipid kinase family that regulate lymphocyte metabolism, survival, proliferation, apoptosis and migration and has been successfully targeted in B-cell malignancies. Primary Sjögren's syndrome (pSS) is a chronic immune-mediated inflammatory disease characterised by exocrine gland lymphocytic infiltration and B-cell hyperactivation which results in systemic manifestations, autoantibody production and loss of glandular function. Given the central role of B cells in pSS pathogenesis, we investigated PI3Kδ pathway activation in pSS and the functional consequences of blocking PI3Kδ in a murine model of focal sialoadenitis that mimics some features of pSS. METHODS AND RESULTS: Target validation assays showed significant expression of phosphorylated ribosomal protein S6 (pS6), a downstream mediator of the phosphatidylinositol 3-kinase delta (PI3Kδ) pathway, within pSS salivary glands. pS6 distribution was found to co-localise with T/B cell markers within pSS aggregates and the CD138+ plasma cells infiltrating the glands. In vivo blockade of PI3Kδ activity with seletalisib, a PI3Kδ-selective inhibitor, in a murine model of focal sialoadenitis decreased accumulation of lymphocytes and plasma cells within the glands of treated mice in the prophylactic and therapeutic regimes. Additionally, production of lymphoid chemokines and cytokines associated with ectopic lymphoneogenesis and, remarkably, saliva flow and autoantibody production, were significantly affected by treatment with seletalisib. CONCLUSION: These data demonstrate activation of PI3Kδ pathway within the glands of patients with pSS and its contribution to disease pathogenesis in a model of disease, supporting the exploration of the therapeutic potential of PI3Kδ pathway inhibition in this condition.

Diazo-Transfer Reagent 2-Azido-4,6-dimethoxy-1,3,5-triazine Displays Highly Exothermic Decomposition Comparable to Tosyl Azide.

2-Azido-4,6-dimethoxy-1,3,5-triazine (ADT) was reported recently as a new "intrinsically safe" diazo-transfer reagent. This assessment was based on differential scanning calorimetry data indicating that ADT exhibits endothermic decomposition. We present DSC data on ADT that show exothermic decomposition with an initiation temperature ( Tinit) of 159 °C and an enthalpy of decomposition (Δ HD) of -1135 J g-1 (-207 kJ mol-1). We conclude that ADT is potentially explosive and must be treated with caution, being of comparable exothermic magnitude to tosyl azide (TsN3). A maximum recommended process temperature for ADT is 55 °C.

Seletalisib: Characterization of a Novel, Potent, and Selective Inhibitor of PI3Kδ.

Phosphoinositide 3-kinases (PI3K) are key signaling enzymes regulating cellular survival, development, and function. Expression of the PI3Kδ isoform is largely restricted to leukocytes and it plays a key role in immune cell development and function. Seletalisib is a novel small-molecule inhibitor of PI3Kδ that was evaluated in biochemical assays, cellular assays of adaptive and innate immunity, and an in vivo rat model of inflammation. Our findings show that seletalisib is a potent, ATP-competitive, and selective PI3Kδ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N-formyl peptide-stimulated but not phorbol myristate acetate-stimulated superoxide release from human neutrophils, consistent with a PI3Kδ-specific activity. No indications of cytotoxicity were observed in peripheral blood mononuclear cells (PBMCs) or other cell types treated with seletalisib. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. Seletalisib showed dose-dependent inhibition in an in vivo rat model of anti-CD3-antibody-induced interleukin 2 release. Collectively, these data characterize seletalisib as a selective PI3Kδ inhibitor and potential therapeutic candidate for the treatment of B-cell malignancies and autoimmune diseases driven by dysregulated proinflammatory cytokine secretion.

First-in-human studies of seletalisib, an orally bioavailable small-molecule PI3Kδ inhibitor for the treatment of immune and inflammatory diseases.

PURPOSE: PI3Ks are potential therapeutic targets in immune-inflammatory diseases. These studies aimed to investigate the safety, tolerability and PK profile of seletalisib, a selective inhibitor of PI3Kδ in humans. METHODS: These phase I, randomised, double-blind, placebo-controlled, single-centre studies (NCT02303509, NCT02207595) evaluated single and multiple oral doses of seletalisib (5-90 mg QD and 30 mg BID) in healthy adults and subjects with mild-to-moderate psoriasis (Study-1). Pharmacodynamic effects on markers of inflammation were assessed via changes in ex vivo basophil degranulation and histological assessment of psoriatic skin biopsies. RESULTS: Seletalisib was well tolerated at doses ≤15 mg (Study-1) and ≤45 mg QD (Study-2) for 14 days. No safety concerns or dose-limiting toxicities were identified (Study-1). Incidence of gastrointestinal-related AEs was not dose related but higher incidences of rash AEs were associated with higher-dose seletalisib (Study-2 rash AEs: 18 in 12 seletalisib-treated subjects versus 1 in 1 placebo-treated subject). Mean seletalisib plasma concentration-time profiles increased with increasing doses after single and multiple dosing, with no major deviations from dose-proportionality. There was no unexpected accumulation or loss of exposure after multiple dosing (time-independent pharmacokinetic profile). Apparent t 1/2 values were supportive of once-daily dosing (geometric mean t1/2: Study-1, 17.7-21.1 h; Study-2, 18.1-22.4 h). No clinically significant food effect was observed (Study-1). Pharmacodynamic findings demonstrated ex vivo inhibition of basophil degranulation, improvements in histological assessment of skin biopsies and other markers of psoriatic biology and preliminary evidence of target engagement in psoriatic skin tissue. CONCLUSIONS: Seletalisib safety, tolerability and pharmacokinetic/pharmacodynamic profiles support its continued clinical development in immune-inflammatory diseases.

Caspase-3-Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging.

Mouse models of neurodegenerative diseases such as Alzheimer's disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and with time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates with increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly correlated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain.

High-accuracy X-ray absorption spectra from mM solutions of nickel (II) complexes with multiple solutions using transmission XAS.

A new approach is introduced for determining X-ray absorption spectroscopy (XAS) spectra on absolute and relative scales using multiple solutions with different concentrations by the characterization and correction of experimental systematics. This hybrid technique is a development of standard X-ray absorption fine structure (XAFS) along the lines of the high-accuracy X-ray extended range technique (XERT) but with applicability to solutions, dilute systems and cold cell environments. This methodology has been applied to determining absolute XAS of bis(N-n-propyl-salicylaldiminato) nickel(II) and bis(N-i-propyl-salicylaldiminato) nickel(II) complexes with square planar and tetrahedral structures in 15 mM and 1.5 mM dilute solutions. It is demonstrated that transmission XAS from dilute systems can provide excellent X-ray absorption near-edge structure (XANES) and XAFS spectra, and that transmission measurements can provide accurate measurement of subtle differences including coordination geometries. For the first time, (transmission) XAS of the isomers have been determined from low-concentration solutions on an absolute scale with a 1-5% accuracy, and with relative precision of 0.1% to 0.2% in the active XANES and XAFS regions after inclusion of systematic corrections.

Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes.

Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute critically to extracellular matrix synthesis during development and throughout life. In glaucoma, and in related disorders affecting the optic nerve and the optic nerve head, pathological changes include altered astrocyte gene and protein expression resulting in their activation and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA in order to slow glaucoma and related pathologies are of high clinical relevance. Herein, we describe the development of standardized methods that will allow for the systematic advancement of such glioprotective strategies. These include isolation, purification and culture of primary adult rat ONHAs, optimized immunocytochemical protocols for cell type validation, as well as plate reader-based assays determining cellular viability, proliferation and the intracellular redox state. We validated and standardized our protocols by performing a glioprotection study using primary ONHAs. Specifically, we measured protection against exogenously-applied oxidative stress using tert-butylhydroperoxide (tBHP) as a model of disease-mediated oxidative stress in the retina and optic nerve head by the prototypic antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Levels of oxidative stress were increased in the response to exogenously applied tBHP and were assessed by 6-carboxy-2', 7' dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Normalized DCFDA fluorescence showed a maximal 5.1-fold increase; the half-maximal effect (EC50) for tBHP was 212 ± 25 µM. This was paralleled very effectively in the assays measuring cell death and cell viability with half-maximal effects of 241 ± 20 µM and 194 ± 5 µM for tBHP in the lactate dehydrogenase (LDH) release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays, respectively. Pre-treatment with 100 µM Trolox decreased the sensitivity of ONHAs to tBHP. Half-maximal effects increased to 396 ± 12 µM tBHP in the LDH release assay and to 383 ± 3 µM tBHP in the MTT assay. Vehicle treatment (0.1% v/v ethanol) did not significantly affect cellular responses to tBHP. Antioxidant treatment increases ONHA viability and reduces the deleterious effects of oxidative stress. Our experiments provide important feasibility data for utilizing primary rat ONHAs in plate reader-based assays assessing novel therapeutics for glioprotection of the optic nerve and the optic nerve head in glaucoma and related disorders. Furthermore, our novel, standardized protocols have the potential to be readily adapted to high-throughput and high-content testing strategies.

Identification and optimisation of a series of tetrahydrobenzotriazoles as metabotropic glutamate receptor 5-selective positive allosteric modulators that improve performance in a preclinical model of cognition.

Herein we describe a series of tetrahydrobenzotriazoles as novel, potent metabotropic glutamate receptor subtype 5 (mGlu5) positive allosteric modulators (PAMs). Exploration of the SAR surrounding the tetrahydrobenzotriazole core ultimately led to the identification of 29 as a potent mGlu5 PAM with a low maximal glutamate potency fold shift, acceptable in vitro DMPK parameters and in vivo PK profile and efficacy in the rat novel object recognition (NOR) assay. As a result 29 was identified as a suitable compound for progression to in vivo safety evaluation.

Antioxidant drug therapy approaches for neuroprotection in chronic diseases of the retina.

The molecular pathways contributing to visual signal transduction in the retina generate a high energy demand that has functional and structural consequences such as vascularization and high metabolic rates contributing to oxidative stress. Multiple signaling cascades are involved to actively regulate the redox state of the retina. Age-related processes increase the oxidative load, resulting in chronically elevated levels of oxidative stress and reactive oxygen species, which in the retina ultimately result in pathologies such as glaucoma or age-related macular degeneration, as well as the neuropathic complications of diabetes in the eye. Specifically, oxidative stress results in deleterious changes to the retina through dysregulation of its intracellular physiology, ultimately leading to neurodegenerative and potentially also vascular dysfunction. Herein we will review the evidence for oxidative stress-induced contributions to each of the three major ocular pathologies, glaucoma, age-related macular degeneration, and diabetic retinopathy. The premise for neuroprotective strategies for these ocular disorders will be discussed in the context of recent clinical and preclinical research pursuing novel therapy development approaches.

Resveratrol and calcium signaling: molecular mechanisms and clinical relevance.

Resveratrol is a naturally occurring compound contributing to cellular defense mechanisms in plants. Its use as a nutritional component and/or supplement in a number of diseases, disorders, and syndromes such as chronic diseases of the central nervous system, cancer, inflammatory diseases, diabetes, and cardiovascular diseases has prompted great interest in the underlying molecular mechanisms of action. The present review focuses on resveratrol, specifically its isomer trans-resveratrol, and its effects on intracellular calcium signaling mechanisms. As resveratrol's mechanisms of action are likely pleiotropic, its effects and interactions with key signaling proteins controlling cellular calcium homeostasis are reviewed and discussed. The clinical relevance of resveratrol's actions on excitable cells, transformed or cancer cells, immune cells and retinal pigment epithelial cells are contrasted with a review of the molecular mechanisms affecting calcium signaling proteins on the plasma membrane, cytoplasm, endoplasmic reticulum, and mitochondria. The present review emphasizes the correlation between molecular mechanisms of action that have recently been identified for resveratrol and their clinical implications.

A case for neuroprotection in ophthalmology: developments in translational research.

Cellular aging occurs by the lifelong accumulation of oxidative damage leading to neuronal apoptosis, termed 'neurodegeneration', and the functional deficits of aging. Loss of visual function is one of the most important quality of life measures for older adults. We discuss recent clinical and laboratory advances in the neuroprotective treatment of the aging eye with particular emphasis on the three major ocular neurodegenerative conditions: glaucoma, age-related macular degeneration (AMD), and diabetic retinopathy (DR).

Purification of retinal ganglion cells using low-pressure flow cytometry.

Purified Retinal Ganglion Cells (RGCs) for in vitro study have been a valuable tool in the study of neural regeneration and in the development of therapies to treat glaucoma. Traditionally, RGCs have been isolated from early postnatal rats and mice, and more recently from human in vitro derived retinal organoids using a two-step immunopanning technique based upon the expression of Thy-1. This technique, however, limits the time periods from which RGCs can be isolated, missing the earliest born RGCs at which time the greatest stage of axon growth occurs, as well as being limited in its use with models of retinal degeneration as Thy-1 is downregulated following injury. While fluorescence associated cell sorting (FACS) in combination with new optogenetically labeled RGCs would be able to overcome this limitation, the use of traditional FACS sorters has been limited to genomic and proteomic studies, as RGCs have little to no survival post-sorting. Here we describe a new method for RGC isolation utilizing a combined immunopanning-fluorescence associated cell sorting (IP-FACS) protocol that initially depletes macrophages and photoreceptors, using immunopanning to enrich for RGCs before using low-pressure FACS to isolate these cells. We demonstrate that RGCs isolated via IP-FACS when compared to RGCs isolated via immunopanning at the same age have similar purity as measured by antibody staining and qRT-PCR; survival as measured by live dead staining; neurite outgrowth; and electrophysiological properties as measured by calcium release response to glutamate. Finally, we demonstrate the ability to isolate RGCs from early embryonic mice prior to the expression of Thy-1 using Brn3b-eGFP optogenetically labeled cells. This method provides a new approach for the isolation of RGCs for the study of early developed RGCs, the study of RGC subtypes and the isolation of RGCs for cell transplantation studies.

Direct electronic linearization for camera-based spectral domain optical coherence tomography.

An electronic method of k-space linearization for an analog camera for use in optical coherence tomography is demonstrated. The method applies a chirp to the data transfer clock signal of the camera in order to temporally compensate for diffraction that is nonlinear in wavenumber. The optimum parameters are obtained experimentally and theoretically and are shown to be in good accordance. Close to maximum measurable axial range, by applying this method, the FWHM of the point spread function is reduced by a factor of 5.6 and sensitivity is increased by 9.8 dB.