The role of nitric oxide ( • NO) as a regulatory diffusible molecule in the brain requires the evaluation of its concentration dynamics. In this work, we have developed microelectrodes suitable for real time electrochemical measurements of • NO in vitro. Nafion and o-phenylenediamine were used to modify the surface of carbon fiber microelectrodes (8 m diameter; ≈100 m tip length). Coating with Nafion was done at 170 • C and the o-phenylenediamine solution was electropolimerized on the carbon surface. • NO peak potential (+0.78 ± 0.03 V versus Ag/AgCl) was determined by square wave voltammetry with • NO solutions prepared from the-generating compound diethylenetriamine/nitric oxide (DETA/NO). Microelectrodes were calibrated by amperometry at a potential of +0.90 V versus Ag/AgCl. They showed good sensitivity (954 ± 217 pA/M; n = 6) and linearity to • NO in the concentration range of 100-1000 nM. They were also characterized in terms of detection limit (6 ± 2 nM, n = 4), response time at 50% (1 s), and selectivity against interferents, such as nitrite (780 ± 84:1, n = 6), ascorbic acid (750 ± 187:1, n = 6) or dopamine (18 ± 2:1, n = 6). Injections of 1 mM l-glutamate, 1 mM l-arginine, and 0.1 mM N-methyl-d-aspartate did not produce changes in background current. Finally, the microelectrodes were used to measure • NO concentration dynamics in rat hippocampal brain slices stimulated with l-glutamate and N-methyl-d-aspartate. Taken together, the data indicate that the microelectrodes exhibit the proper sensitivity and selectivity for studies of • NO dynamics in brain slices (in vitro) and possibly in whole brain (in vivo) recordings.

Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.

Nitric oxide (NO Å ) is a diffusible regulatory molecule involved in a wide range of physiological and pathological events. At the tissue level, a local and temporary increase in NO Å concentration is translated into a cellular signal. From our current knowledge of biological synthesis and decay, the kinetics and mechanisms that determine NO Å concentration dynamics in tissues are poorly understood. Generally, NO Å mediates its effects by stimulating (e.g., guanylate cyclase) or inhibiting (e.g., cytochrome oxidase) transition metal-containing proteins and by post-translational modification of proteins (e.g., formation of nitrosothiol adducts). The borderline between the physiological and pathological activities of NO Å is a matter of controversy, but tissue redox environment, supramolecular organization and compartmentalisation of NO Å targets are important features in determining NO Å actions. In brain, NO Å synthesis in the dependency of glutamate NMDA receptor is a paradigmatic example; the NMDA-subtype glutamate receptor triggers intracellular signalling pathways that govern neuronal plasticity, development, senescence and disease, suggesting a role for NO Å in these processes. Measurements of NO Å in the different subregions of hippocampus, in a glutamate NMDA receptor-dependent fashion, by means of electrochemical selective microsensors illustrate the concentration dynamics of NO Å in the sub-regions of this brain area. The analysis of NO Å concentration-time profiles in the hippocampus requires consideration of at least two interrelated issues, also addressed in this review. NO Å diffusion in a biological medium and regulation of NO Å activity. Ó 2004 Elsevier Ltd. All rights reserved.Abbreviations: CAPON, Carboxy-terminal PDZ ligand of nNOS; DOPAC, dihydroxyphenylacetic acid; EPR, electron paramagnetic resonance; LTP, long-term potentiation; NMDA, N-methyl-D D -aspartate; NOS, nitric oxide synthase; PDZ, PSD-95 discs large/ZO-1 homology domain; PSD-95, post-synaptic density protein 95

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