Pressure-dependent modulation of inward-rectifying K+ channels: implications for cation homeostasis and K+ dynamics in glaucoma.

Glaucoma is the leading cause of blindness worldwide, resulting from degeneration of retinal ganglion cells (RGCs), which form the optic nerve. Prior to structural degeneration, RGCs exhibit physiological deficits. Müller glia provide homeostatic regulation of ions that supports RGC physiology through a process called K+ siphoning. Recent studies suggest that several retinal conditions, including glaucoma, involve changes in the expression of K+ channels in Müller glia. To clarify whether glaucoma-related stressors directly alter expression and function of K+ channels in Müller glia, we examined changes in the expression of inwardly rectifying K+ (Kir) channels and two-pore domain (K2P) channels in response to elevated intraocular pressure (IOP) in vivo and in vitro in primary cultures of Müller glia exposed to elevated hydrostatic pressure. We then measured outcomes of cell health, cation homeostasis, and cation flux in Müller glia cultures. Transcriptome analysis in a murine model of microbead-induced glaucoma revealed pressure-dependent downregulation of Kir and K2P channels in vivo. Changes in the expression and localization of Kir and K2P channels in response to elevated pressure were also found in Müller glia in vitro. Finally, we found that elevated pressure compromises the plasma membrane of Müller glia and induces cation dyshomeostasis that involves changes in ion flux through cation channels. Pressure-induced changes in cation flux precede both cation dyshomeostasis and membrane compromise. Our findings have implications for Müller glia responses to pressure-related conditions, i.e., glaucoma, and identify cation dyshomeostasis as a potential contributor to electrophysiological impairment observed in RGCs of glaucomatous retina.

Phenotypes of primary retinal macroglia: Implications for purification and culture conditions.

Many neurodegenerations, including those of the visual system, have complex etiologies that include roles for both neurons and glia. In the retina there is evidence that retinal astrocytes play an important role in neurodegeneration. There are several approaches for isolating and growing primary retinal astrocytes, however, they often lead to different results. In this study, we examined the influence of culture conditions on phenotypic maturation of primary, purified retinal glia. We compared retinal astrocytes and Müller glia purified by immunomagnetic separation, as differentiation between these astrocyte subtypes is critical and immuno-based methods are the standard practice of purification. We found that while time in culture impacts the health and phenotype of both astrocytes and Müller glia, the phenotypic maturation of retinal astrocytes was most impacted by serum factors. These factors appeared to actively regulate intermediate filament phenotypes in a manner consistent with the induction of astrocyte-mesenchymal transition (AMT). This propensity for retinal astrocytes to shift along an AMT continuum should be considered when interpreting resulting data. Our goal is that this study will help standardize the field so that studies are replicable, comparable, and as accurate as possible for subsequent interpretation of findings.

Reversal of morphine-induced cell-type-specific synaptic plasticity in the nucleus accumbens shell blocks reinstatement.

Drug-evoked plasticity at excitatory synapses on medium spiny neurons (MSNs) of the nucleus accumbens (NAc) drives behavioral adaptations in addiction. MSNs expressing dopamine D1 (D1R-MSN) vs. D2 receptors (D2R-MSN) can exert antagonistic effects in drug-related behaviors, and display distinct alterations in glutamate signaling following repeated exposure to psychostimulants; however, little is known of cell-type-specific plasticity induced by opiates. Here, we find that repeated morphine potentiates excitatory transmission and increases GluA2-lacking AMPA receptor expression in D1R-MSNs, while reducing signaling in D2-MSNs following 10-14 d of forced abstinence. In vivo reversal of this pathophysiology with optogenetic stimulation of infralimbic cortex-accumbens shell (ILC-NAc shell) inputs or treatment with the antibiotic, ceftriaxone, blocked reinstatement of morphine-evoked conditioned place preference. These findings confirm the presence of overlapping and distinct plasticity produced by classes of abused drugs within subpopulations of MSNs that may provide targetable molecular mechanisms for future pharmacotherapies.

Mechanisms underlying the activation of G-protein-gated inwardly rectifying K+ (GIRK) channels by the novel anxiolytic drug, ML297.

ML297 was recently identified as a potent and selective small molecule agonist of G-protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels. Here, we show ML297 selectively activates recombinant neuronal GIRK channels containing the GIRK1 subunit in a manner that requires phosphatidylinositol-4,5-bisphosphate (PIP2), but is otherwise distinct from receptor-induced, G-protein-dependent channel activation. Two amino acids unique to the pore helix (F137) and second membrane-spanning (D173) domain of GIRK1 were identified as necessary and sufficient for the selective activation of GIRK channels by ML297. Further investigation into the behavioral effects of ML297 revealed that in addition to its known antiseizure efficacy, ML297 decreases anxiety-related behavior without sedative or addictive liabilities. Importantly, the anxiolytic effect of ML297 was lost in mice lacking GIRK1. Thus, activation of GIRK1-containing channels by ML297 or derivatives may represent a new approach to the treatment of seizure and/or anxiety disorders.

US physician perspective on the use of biomarker and ctDNA testing in patients with melanoma.

New treatments have increased survival of patients with melanoma, and methods to monitor patients throughout the disease process are needed. Circulating tumor DNA (ctDNA) is a predictive and prognostic biomarker that may allow routine, real-time monitoring of disease status. We surveyed 44 US physicians to understand their preferences and practice patterns for biomarker and ctDNA testing in their patients with melanoma. Tumor biomarker testing was often ordered in stage IIIA-IV patients. Barriers to biomarker testing include insufficient tissue (60%) and lack of insurance coverage (54%). ctDNA testing was ordered by 16-18% of physicians for stages II-IV. Reasons for not using ctDNA testing included lack of prospective data (41%), ctDNA testing used for research only (18%), and others. Physicians (≥74%) believed that ctDNA assays could help with monitoring and treatment selection throughout the disease process. Physicians consider ctDNA testing potentially valuable for clinical decision-making but cited concerns that should be addressed.

Impairment of Membrane Repolarization Accompanies Axon Transport Deficits in Glaucoma.

Glaucoma is a leading cause of blindness worldwide, resulting from degeneration of retinal ganglion cells (RGCs), which form the optic nerve. In glaucoma, axon transport deficits appear to precede structural degeneration of RGC axons. The period of time between the onset of axon transport deficits and the structural degeneration of RGC axons may represent a therapeutic window for the prevention of irreversible vision loss. However, it is unclear how deficits in axon transport relate to the electrophysiological capacity of RGCs to produce and maintain firing frequencies that encode visual stimuli. Here, we examined the electrophysiological signature of individual RGCs in glaucomatous retina with respect to axon transport facility. Utilizing the Microbead Occlusion Model of murine ocular hypertension, we performed electrophysiological recordings of RGCs with and without deficits in anterograde axon transport. We found that RGCs with deficits in axon transport have a reduced ability to maintain spiking frequency that arises from elongation of the repolarization phase of the action potential. This repolarization phenotype arises from reduced cation flux and K+ dyshomeostasis that accompanies pressure-induced decreases in Na/K-ATPase expression and activity. In vitro studies with purified RGCs indicate that elevated pressure induces early internalization of Na/K-ATPase that, when reversed, stabilizes cation flux and prevents K+ dyshomeostasis. Furthermore, pharmacological inhibition of the Na/K-ATPase is sufficient to replicate pressure-induced cation influx and repolarization phase phenotypes in healthy RGCs. These studies suggest that deficits in axon transport also likely reflect impaired electrophysiological function of RGCs. Our findings further identify a failure to maintain electrochemical gradients and cation dyshomeostasis as an early phenotype of glaucomatous pathology in RGCs that may have significant bearing on efforts to restore RGC health in diseased retina.

Impact of Graphene on the Efficacy of Neuron Culture Substrates.

How graphene influences the behavior of living cells or tissues remains a critical issue for its application in biomedical studies, despite the general acceptance that graphene is biocompatible. While direct contact between cells and graphene is not a requirement for all biomedical applications, it is often mandatory for biosensing. Therefore, it is important to clarify whether graphene impedes the ability of cells to interact with biological elements in their environment. Here, a systematic study is reported to determine whether applying graphene on top of matrix substrates masks interactions between these substrates and retinal ganglion cells (RGCs). Six different platforms are tested for primary RGC cultures with three platforms comprised of matrix substrates compatible with these neurons, and another three having a layer of graphene placed on top of the matrix substrates. The results demonstrate that graphene does not impede interactions between RGCs and underlying substrate matrix, such that their positive or negative effects on neuron viability and vitality are retained. However, direct contact between RGCs and graphene reduces the number, but increases basal activity, of functional cation channels. The data indicate that, when proper baselines are established, graphene is a promising biosensing material for in vitro applications in neuroscience.

Cocaine and Amphetamine Induce Overlapping but Distinct Patterns of AMPAR Plasticity in Nucleus Accumbens Medium Spiny Neurons.

Repeated exposure to psychostimulant drugs such as cocaine or amphetamine can promote drug-seeking and -taking behavior. In rodent addiction models, persistent changes in excitatory glutamatergic neurotransmission in the nucleus accumbens (NAc) appear to drive this drug-induced behavioral plasticity. To study whether changes in glutamatergic signaling are shared between or exclusive to specific psychostimulant drugs, we examined synaptic transmission from mice following repeated amphetamine or cocaine administration. Synaptic transmission mediated by AMPA-type glutamate receptors was potentiated in the NAc shell 10-14 days following repeated amphetamine or cocaine treatment. This synaptic enhancement was depotentiated by re-exposure to amphetamine or cocaine. By contrast, in the NAc core only repeated cocaine exposure enhanced synaptic transmission, which was subsequently depotentiated by an additional cocaine but not amphetamine injection during drug abstinence. To better understand the drug-induced depotentiation, we replicated these in vivo findings using an ex vivo model termed 'challenge in the bath,' and showed that drug-induced decreases in synaptic strength occur rapidly (within 30 min) and require activation of metabotropic glutamate receptor 5 (mGluR5) and protein synthesis in the NAc shell, but not NAc core. Overall, these data demonstrate the specificity of neuronal circuit changes induced by amphetamine, introduce a novel method for studying drug challenge-induced plasticity, and define NAc shell medium spiny neurons as a primary site of persistent AMPA-type glutamate receptor plasticity by two widely used psychostimulant drugs.