Binge drinking is associated with increased risk for cerebrovascular spasm and stroke. Acute exposure to ethanol at concentrations obtained during binge drinking constricts cerebral arteries in several species, including humans, but the mechanisms underlying this action are largely unknown. In a rodent model, we used fluorescence microscopy, patch-clamp electrophysiology, and pharmacological studies in intact cerebral arteries to pinpoint the molecular effectors of ethanol cerebrovascular constriction. Clinically relevant concentrations of ethanol elevated wall intracellular Ca 2؉ concentration and caused a reversible constriction of cerebral arteries (EC50 ‫؍‬ 27 mM; Emax ‫؍‬ 100 mM) that depended on voltage-gated Ca 2؉ entry into myocytes. However, ethanol did not directly increase voltage-dependent Ca 2؉ currents in isolated myocytes. Constriction occurred because of an ethanol reduction in the frequency (؊53%) and amplitude (؊32%) of transient Ca 2؉ -activated K ؉ (BK) currents. Ethanol inhibition of BK transients was caused by a reduction in Ca 2؉ spark frequency (؊49%), a subsarcolemmal Ca 2؉ signal that evokes the BK transients, and a direct inhibition of BK channel steady-state activity (؊44%). In contrast, ethanol failed to modify Ca 2؉ waves, a major vasoconstrictor mechanism. Selective block of BK channels largely prevented ethanol constriction in pressurized arteries. This study pinpoints the Ca 2؉ spark͞BK channel negative-feedback mechanism as the primary effector of ethanol vasoconstriction.M oderate-heavy episodic alcohol intake, such as in binge drinking, remains a major public health problem (1, 2). Moderate-heavy drinking is associated, independently of any other factor, with an increased risk for stroke and deaths from ischemic stroke (3, 4). Binge drinkers are significantly predisposed to brain hemorrhage, cerebrovascular spasm, and stroke (3, 5).Cerebrovascular disease associated with moderate-heavy alcohol intake is independent of beverage type and alcohol metabolism but linked to ethanol (EtOH) itself (6, 7). Strong evidence for a dose-response relationship between EtOH intake and risk for stroke suggests causality (8). EtOH cerebral artery constriction is considered responsible for cerebral vasospasm, ischemia, and stroke in moderate-heavy drinkers (6, 9). Acute EtOH at legally intoxicating (Ն20 mM) blood levels in naive subjects constricts cerebral arteries in several species, including humans (7, 9).Rats are excellent models to study EtOH cerebral artery constriction and stroke (7,10,11). Evidence from this and other species indicates that EtOH constricts cerebral arteries by acting primarily on the smooth muscle (7,11,12). However, the molecular mechanisms mediating EtOH cerebral artery constriction remain largely unidentified.In cerebrovascular smooth muscle, an elevation in global intracellular Ca 2ϩ ([Ca 2ϩ ] ic ) leads to contraction (13). Ca 2ϩ mobilization in response to EtOH may result from direct potentiation of mechanisms leading to Ca 2ϩ influx͞release from internal organel...

Advances in tip-enhanced Raman spectroscopy (TERS) have demonstrated ultrahigh spatial resolution so that the vibrational modes of individual molecules can be visualized. The spatial resolution of TERS is determined by the confinement of the plasmon-induced field in the junction; however, the conditions necessary for achieving the high spatial confinement required for imaging individual molecules are not fully understood. Here, we present a systematic theoretical study of TERS imaging of single molecules, using a hybrid atomistic electrodynamics-quantum mechanical method. This approach provides a consistent treatment of the molecule and the plasmonic near field under conditions where they cannot be treated separately. In our simulations, we demonstrate that TERS is capable of resolving intricate molecule vibrations with atomic resolution, although we find that TERS images are extremely sensitive to the near field in the junction. Achieving the atomic resolution requires the near field to be confined within a few ångstroms in diameter and the near-field focal plane to be in the molecule plane. Furthermore, we demonstrate that the traditional surface selection rule of Raman spectroscopy is altered due to the significant field confinement that leads to significant field-gradient effects in the Raman scattering. This work provides insights into single-molecule imaging based on TERS and Raman scattering of molecules in nanojunctions with atomic dimensions.

High-conductance, Ca 2+ -activated and voltage-gated (BK) channels set neuronal firing. They are almost universally activated by alcohol, leading to reduced neuronal excitability and neuropeptide release and to motor intoxication. However, several BK channels are inhibited by alcohol, and most other voltage-gated K + channels are refractory to drug action. BK channels are homotetramers (encoded by Slo1) that possess a unique transmembrane segment (S0), leading to a cytosolic S0-S1 loop. We identified Thr107 of bovine slo (bslo) in this loop as a critical residue that determines BK channel responses to alcohol. In addition, the activity of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) in the cell controlled channel activity and alcohol modulation. Incremental CaMKIImediated phosphorylation of Thr107 in the BK tetramer progressively increased channel activity and gradually switched the channel alcohol responses from robust activation to inhibition. Thus, CaMKII phosphorylation of slo Thr107 works as a 'molecular dimmer switch' that could mediate tolerance to alcohol, a form of neuronal plasticity.High-conductance Ca 2+ -activated K + (BK) channels are key to various physiological processes 1 . In most tissues, BK channels consist of pore-forming (α, encoded by Slo1 or KCNMA1) and accessory (β 1-4 ) subunits 2 . The role of BK channels in a wide variety of processes is primarily ensured by slo isoforms and their post-translational modifications, which adjust the channels' biophysical characteristics to specific cell processes 3 .Neuronal BK channels participate in repolarization and fast after-hyperpolarization of action potentials, setting neuron firing properties 4 . It has been speculated that a drug-induced increase in Ca 2+ -activated K + currents might contribute to the depression of central neurons by ethanol and by sedatives and/or hypnotics 5 . Indeed, acute exposure to intoxicating concentrations of ethanol potentiates BK channels in neurons and neuroendocrine cells 6 , decreasing excitability 7 and neuropeptide release and producing eventual diuresis 8 . In addition, ethanol potentiation of neuronal BK channels underlies alcohol-induced motor incoordination in Caenorhabditis elegans 9 .Ethanol potentiation of BK channels, however, is not universal. Ethanol concentrations that activate neuronal BK channels inhibit aortic BK channels 10,11 . Ethanol also inhibits BK currents in cerebral artery myocytes and thus causes cerebral artery constriction 12 , a major mechanism underlying ischemic stroke linked to binge drinking 13 . Although several of the pathophysiological and behavioral consequences of ethanol modulation of BK channels have

Tip-enhanced Raman spectroscopy (TERS) exhibits new selection rule and sub-nanometer spatial resolution, which is attributed to the plasmonic near-field confinement. Despite recent advances in simulations of TERS spectra under highly confined fields, a simply physical mechanism has remained elusive. In this work we show that single-molecule TERS images can be explained by local sub-molecular density changes induced by the confined near-field during the Raman process. The local sub-molecular density changes determine the spatial resolution in TERS and the gradient-based selection rule. Using this approach we find that the four-fold symmetry of meso -tetrakis(3,5-di- tert -butylphenyl)porphyrin (H 2 TBPP) TERS images observed in experiments arises from the combination of degenerate normal modes localized in the functional side groups rather than the porphyrin ring as previously considered. As an illustration of the potential of the method, we demonstrate how this new theory can be applied to microscopic structure characterization.

Atomically terminated and nanoscopically smooth silver tips effectively focus light on the angstrom scale, allowing tip-enhanced Raman spectromicroscopy (TER-sm) with single molecule sensitivity and submolecular spatial resolution. Through measurements carried out on cobalt-tetraphenylporphyrin (CoTPP) adsorbed on Au(111), we highlight peculiarities of vibrational spectromicroscopy with light confined on the angstrom scale. Field-gradient-driven spectra, orientational fingerprinting, and sculpting of local fields by atomic morphology of the junction are elucidated through measurements that range from 2D arrays at room temperature to single molecule manipulations at 5 K. Notably, vibrational Stark tuning within molecules, reflecting intramolecular charge distributions, becomes accessible when light is more localized than the interrogated normal modes. The Stark images of CoTPP reveal that it is saddled, and the distortion is accompanied by charge transfer to gold through the two anchoring pyrroles.