A Presynaptic Role for Nitric Oxide at a GABAergic Synapse

Amacrine cells are a class of retinal interneurons that process the visual signal in the inner retina. Several subtypes of amacrine cells express nitric oxide synthase and produce nitric oxide (NO), making NO a possible regulator of amacrine cell function. My dissertation research tests the hypothesis that NO alters amacrine cell GABAergic synaptic output. To investigate this, I made whole-cell voltage clamp recordings of cultured chick amacrine cells receiving synaptic input from other amacrine cells and Ca2+ imaging of amacrine cell dendrites, which can be presynaptic. I find that NO-dependent increases in GABAergic spontaneous postsynaptic current (sPSC) frequency are independent of soluble guanylate cyclase and action potentials. Removal of extracellular Ca2+ and buffering of cytosolic Ca2+ both inhibit the response to NO. In Ca2+ imaging experiments, I confirm that NO increases dendritic Ca2+ by activating a Ca2+ influx pathway. Neither NO-dependent dendritic Ca2+ elevation nor increase in sPSC frequency are dependent upon Ca2+ release from stores. NO also enhances evoked GABAergic responses, and because voltage-gated Ca2+ channel function is not altered by NO, the enhanced evoked release is likely due to the combination of voltage-dependent Ca2+ influx and the voltage-independent, NO-dependent Ca2+ influx. Insight into the identity of the Ca2+ channel involved in the NO response was provided by characteristics unique to the transient receptor potential canonical (TRPC) channel subunits 4 and 5: the NO-dependent increase in sPSC frequency was dependent on downstream activity of PLC, blocked by 2 mM La3+ and enhanced by 10 µM La3+. The TRPC inhibitor ML204, which preferentially blocks TRPC4, had no effect on the NO response at 10 µM, but 20 µM ML204 blocked the NO response. The TRPC inhibitor clemizole, which preferentially blocks TRPC5, blocked NO-dependent dendritic Ca2+ elevations and the increase in sPSC frequency. Genetic knockdown of TRPC5 in cultured amacrine cells using the CRISPR/Cas9 system confirms that TRPC5 mediates NO-dependent dendritic Ca2+ elevations and the increase in sPSC frequency. These results suggest that NO-dependent activation of TRPC5 at amacrine cell presynaptic sites will enhance vesicular GABA release and increase inhibition onto postsynaptic cells

Nitric oxide promotes GABA release by activating a voltage-independent Ca\u3csup\u3e2+\u3c/sup\u3e influx pathway in retinal amacrine cells

© 2017 the American Physiological Society. Retinal amacrine cells express nitric oxide (NO) synthase and produce NO, making NO available to regulate the function of amacrine cells. Here we test the hypothesis that NO can alter the GABAergic synaptic output of amacrine cells. We investigate this using whole cell voltage clamp recordings and Ca2 imaging of cultured chick retinal amacrine cells. When recording from amacrine cells receiving synaptic input from other amacrine cells, we find that NO increases GABAergic spontaneous postsynaptic current (sPSC) frequency. This increase in sPSC frequency does not require the canonical NO receptor, soluble guan-ylate cyclase, or presynaptic action potentials. However, removal of extracellular Ca2+ and buffering of cytosolic Ca2+ both inhibit the response to NO. In Ca2+ imaging experiments, we confirm that NO increases cytosolic Ca2+ in amacrine cell processes by activating a Ca2+ influx pathway. Neither the increase in sPSC frequency nor the cytosolic Ca2+ elevations are dependent upon Ca2+ release from stores. NO also enhances evoked GABAergic responses. Because voltage-gated Ca2+ channel function is not altered by NO, the increased evoked response is likely due to the combined effect of voltage-dependent Ca2+ influx adding to the NO-dependent, voltage-independent, Ca2+ influx. Insight into the identity of the Ca2+ influx pathway is provided by the transient receptor potential canonical (TRPC) channel inhibitor clemizole, which prevents the NO-dependent increase in sPSC frequency and cytosolic Ca2+ elevations. These data suggest that NO production in the inner retina will enhance Ca2+-dependent GABA release from amacrine cells by activating TRPC channel(s). NEW & NOTEWORTHY Our research provides evidence that nitric oxide (NO) promotes GABAergic output from retinal amacrine cells by activating a likely transient receptor potential canonical-mediated Ca2+ influx pathway. This NO-dependent mechanism promoting GABA release can be voltage independent, suggesting that, in the retina, local NO production can bypass the formal retinal circuitry and increase local inhibition

TRPC5 is required for the NO-dependent increase in dendritic Ca \u3csup\u3e2+\u3c/sup\u3e and GABA release from chick retinal amacrine cells

© 2018 American Physiological Society. All rights reserved. GABAergic signaling from amacrine cells (ACs) is a fundamental aspect of visual signal processing in the inner retina. We have previously shown that nitric oxide (NO) can elicit release of GABA independently from activation of voltage-gated Ca 2+ channels in cultured retinal ACs. This voltage-independent quantal GABA release relies on a Ca 2+ influx mechanism with pharmacological characteristics consistent with the involvement of the transient receptor potential canonical (TRPC) channels TRPC4 and/or TRPC5. To determine the identity of these channels, we evaluated the ability of NO to elevate dendritic Ca 2+ and to stimulate GABA release from cultured ACs under conditions known to alter the function of TRPC4 and 5. We found that these effects of NO are phospholipase C dependent, have a biphasic dependence on La 3+ , and are unaffected by moderate concentrations of the TRPC4-selective antagonist ML204. Together, these results suggest that NO promotes GABA release by activating TRPC5 channels in AC dendrites. To confirm a role for TRPC5, we knocked down the expression of TRPC5 using CRISPR/Cas9-mediated gene knockdown and found that both the NO-dependent Ca 2+ elevations and increase in GABA release are dependent on the expression of TRPC5. These results demonstrate a novel NO-dependent mechanism for regulating neurotransmitter output from retinal ACs. NEW & NOTEWORTHY Elucidating the mechanisms regulating GABAergic synaptic transmission in the inner retina is key to understanding the flexibility of retinal ganglion cell output. Here, we demonstrate that nitric oxide (NO) can activate a transient receptor potential canonical 5 (TRPC5)-mediated Ca 2+ influx, which is sufficient to drive vesicular GABA release from retinal amacrine cells. This NO-dependent mechanism can bypass the need for depolarization and may have an important role in processing the visual signal by enhancing retinal amacrine cell GABAergic inhibitory output

Bayesian Optimization with Conformal Prediction Sets

Bayesian optimization is a coherent, ubiquitous approach to decision-making under uncertainty, with applications including multi-arm bandits, active learning, and black-box optimization. Bayesian optimization selects decisions (i.e. objective function queries) with maximal expected utility with respect to the posterior distribution of a Bayesian model, which quantifies reducible, epistemic uncertainty about query outcomes. In practice, subjectively implausible outcomes can occur regularly for two reasons: 1) model misspecification and 2) covariate shift. Conformal prediction is an uncertainty quantification method with coverage guarantees even for misspecified models and a simple mechanism to correct for covariate shift. We propose conformal Bayesian optimization, which directs queries towards regions of search space where the model predictions have guaranteed validity, and investigate its behavior on a suite of black-box optimization tasks and tabular ranking tasks. In many cases we find that query coverage can be significantly improved without harming sample-efficiency.Comment: For code, see https://www.github.com/samuelstanton/conformal-bayesopt.gi

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl \u3csup\u3e–\u3c/sup\u3e from acidic organelles in amacrine cells

© 2017 the American Physiological Society. γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligandgated ion channels support the influx of Cl – . However, the electrochemical gradient for Cl – across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl – levels support inhibition, whereas higher levels of cytosolic Cl – can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl – from acidic organelles and transiently elevates cytosolic Cl – , making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl – . We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage- clamp recordings of currents through GABA-gated Cl – channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl – . To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl – from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl – from acidic organelles. NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl – from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl – levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission