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AbstractActivation of potassium (K+) currents plays a critical role in the control of programmed cell death. Because pituitary adenylate cyclase‐activating polypeptide (PACAP) has been shown to inhibit the apoptotic cascade in the cerebellar cortex during development, we have investigated the effect of PACAP on K+ currents in cultured cerebellar granule cells using the patch‐clamp technique in the whole‐cell configuration. Two types of outward K+ currents, a transient K+ current (IA) and a delayed rectifier K+ current (IK) were characterized using two different voltage protocols and specific inhibitors of K+ channels. Application of PACAP induced a reversible reduction of the IK amplitude, but did not affect IA, while the PACAP‐related peptide vasoactive intestinal polypeptide had no effect on either types of K+ currents. Repeated applications of PACAP induced gradual attenuation of the electrophysiological response. In the presence of guanosine 5′‐[γthio]triphosphate (GTPγS), PACAP provoked a marked and irreversible IK depression, whereas cell dialysis with guanosine 5′‐[βthio]diphosphate GDPβS totally abolished the effect of PACAP. Pre‐treatment of the cells with pertussis toxin did not modify the effect of PACAP on IK. In contrast, cholera toxin suppressed the PACAP‐induced inhibition of IK. Exposure of granule cells to dibutyryl cyclic adenosine monophosphate (dbcAMP) mimicked the inhibitory effect of PACAP on IK. Addition of the specific protein kinase A inhibitor H89 in the patch pipette solution prevented the reduction of IK induced by both PACAP and dbcAMP. PACAP provoked a sustained increase of the resting membrane potential in cerebellar granule cells cultured either in high or low KCl‐containing medium, and this long‐term depolarizing effect of PACAP was mimicked by the IK specific blocker tetraethylammonium chloride (TEA). In addition, pre‐incubation of granule cells with TEA suppressed the effect of PACAP on resting membrane potential. TEA mimicked the neuroprotective effect of PACAP against ethanol‐induced apoptotic cell death, and the increase of caspase‐3 activity observed after exposure of granule cells to ethanol was also significantly inhibited by TEA. Taken together, the present results demonstrate that, in rat cerebellar granule cells, PACAP reduces the delayed outward rectifier K+ current by activating a type 1 PACAP (PAC1) receptor coupled to the adenylyl cyclase/protein kinase A pathway through a cholera toxin‐sensitive Gs protein. Our data also show that PACAP and TEA induce long‐term depolarization of the resting membrane potential, promote cell survival and inhibit caspase‐3 activity, suggesting that PACAP‐evoked inhibition of IK contributes to the anti‐apoptotic effect of the peptide on cerebellar granule cells.

Alcoholism and alcohol abuse represent a significant medical and societal problem, and have been thoroughly investigated in humans as well as using animal models. A less well understood aspect of alcohol related disorders is the possible effect of this drug on offspring whose parents were exposed prior to conception. The zebrafish has been successfully employed in alcohol research, however, the effect of exposing the parents to alcohol before fertilization of the eggs on offspring has not been demonstrated in this species. In this proof of concept study, we attempt to address this hiatus. We exposed both adult male and female zebrafish to 0.0% (control) or 0.5% (vol/vol) alcohol chronically for 7 days, subsequently bred the fish within their respective treatment group, collected the fertilized eggs, allowed them to develop, and tested the behavior of free-swimming offspring at their age of 7-9 days post-fertilization. We conducted the analysis in two genetically distinct quasi-inbred strains of zebrafish, AB and TL. Although gross morphology and general activity of the fish appeared unaffected, we found significant behavioral alterations in offspring of alcohol exposed parents compared to offspring of control parents in both strains. These alterations included robustly increased duration and reduced frequency of immobility, increased turn angle, and increased intra-individual variance of turn angle in offspring of alcohol exposed parents in both strains. The mechanisms underlying these behavioral effects or whether the effects are due to exposure of the father, the mother, or both to alcohol are unknown. Nevertheless, our results now set the stage for future studies with zebrafish that will address these questions.

When rats are presented with a novel saccharin solution and immediately injected with either morphine or ethanol, they subsequently develop a conditioned taste aversion (CTA) to the saccharin solution which reflects the aversive component of the conditioning drug. The present study provides evidence which suggests that both morphine-induced and ethanol-induced CTAs can be blocked by the specific high-affinity binding opiate antagonist, naloxazone.

Previously, it has been suggested that acute ethanol (alcohol) administration can result in concentration-dependent vasoconstriction and decreased cerebral blood flow. Here, we present in vivo results using rapid (240 nm/min) optical backscatter measurements, with an intact cranial preparation in the rat, indicating that acute infusion of ethanol directly into the rat brain rapidly produces dose-dependent vasoconstriction of the cerebral microcirculation associated with a pronounced reduction in tissue blood content, pronounced rises in deoxyhemoglobin, significantly increased levels of reduced cytochrome oxidase and microvascular damage as the dose increases. Furthermore, we present in vivo experiments demonstrating the capability of magnesium ions (Mg(2+)) to attenuate and prevent these deleterious responses. Optical backscatter spectra (500-800 nm) were obtained by directing a single sending and receiving fiber to a portion of the left parietal cranium (in anesthetized rats), shaved to a translucent appearance to facilitate optical penetration. In the absence of added Mg(2+), infusion of a 10% solution of ethanol at 0.34 ml/min ( approximately 26.8 mg/min) produced prompt vasoconstriction as evidenced by a greater than 90% loss of oxyhemoglobin from the field-of-view and increases in levels of reduced cytochrome oxidase to between 50% and >90%. These effects were partially, to nearly completely, attenuated by the addition of MgCl(2) to the infusate containing added ethanol. Of special interest was the observation that attenuation of the vasoconstrictive effect of ethanol by Mg(2+) persisted despite a subsequent ethanol challenge without added Mg(2+). The results obtained demonstrate that, depending on dose, ethanol can produce prompt and severe vasoconstriction of the intact cerebral microcirculation and that infusion of moderate doses of Mg(2+) can largely attenuate and prevent this response. We conclude that appreciable, graded changes in cerebral cytochrome oxidase aa(3), blood volume and the state of hemoglobin occur at minimal tissue levels of ethanol which can be modulated by Mg(2+).

Summary: The potential role of genetic factors in the etiology of posttraumatic and alcohol‐associated seizures was studied in 289 male patients with recurrent seizures and in 174 individuals who had never experienced a seizure. The incidence of seizures in first‐degree relatives of probands was compared with that in relatives of unaffected individuals. Relatives of patients with alcoholassociated seizures had a rate ratio of 2.45 [95% confidence interval (CI) 1.41–4.251, whereas no excess incidence was noted among relatives of posttraumatic epilepsy patients (rate ratio 1.20, 0.64–2.25 CI). Relatives of probands with both antecedents showed an intermediate rate ratio of 1.72 (0.92–3.20 CI). Among probands with alcohol‐associated seizures, the rate ratio of 2.05 for patients with alcohol‐related seizures (i.e., spontaneously occurring seizures in association with chronic alcohol abuse) was slightly higher than that of 1.85 for probands with alcohol withdrawal seizures. Trauma severity had a slight impact on the incidence of affected relatives; patients with severe head injuries had a rate ratio of 0.73 and probands with milder trauma had a rate ratio of 0.99. The results indicate a limited, if any, role of genetic predisposition in development of posttraumatic seizures. Alcoholrelated seizures, however, showed familial aggregation of unprovoked seizures, suggesting an involvement of genetic factors in the origin of such seizures.

Using two inbred strains of mice which have similar rates of alcohol metabolism, we asked whether prenatal alcohol exposure would cause greater incidence and severity of defects in the development of two forebrain fiber tracts, the corpus callosum and the anterior commissure, in mice prone to these defects (BALB/c) than in mice not prone to these defects (C57BL/6). Pregnant animals were fed 0.6 kcal/g body weight of a Sustacal-based liquid diet containing 0, 15, 17.5, 20, or 25% ethanol-derived calories from day 7 to fetal assessment on day 18 of gestation. Most of alcohol's greatest effects and the greatest strain differences in alcohol's effects on fetal variables were produced by the 17.5% diet. This dose had inhibitory effects on fetal body, brain, and midsagittal corpus callosum and anterior commissure growth. All these effects, except that on brain weight, were significantly greater in C57s than in BALBs. When the results were compared with prenatal growth curves for normal untreated mice, the effect of alcohol on corpus callosum but not anterior commissure growth was largely explained by its effects on overall development. The 17.5% diet had a greater specific effect on size of the anterior commissure in C57s than BALBs but increased the incidence and severity of its permanent dysmorphology in BALBs more than in C57s. Anterior commissure size and morphology may be sensitive indicators of alcohol's effects on prenatal brain development. Hereditary differences in rate of maternal alcohol metabolism no doubt have important consequences for risks arising from prenatal alcohol exposure. However, this study clearly indicates that inherited factors, other than those that influence rate of alcohol metabolism, are important influences on the overall fetal response and the specific responses of the anterior commissure to prenatal alcohol exposure.

Abstract: Ethanol can alter the affinity of mouse striatal opiate receptors for their ligands, and the present studies were aimed at determining the importance of the receptor microenvironment for this effect of ethanol. Changing the temperature of the binding assay, and thus altering the properties of neuronal membrane lipids, resulted in changes in the observed affinity of striatal binding sites for dihydromorphine (DHM), but not for d‐Ala2, d‐Leu5‐enkephalin (ENK). The changes in temperature also differentially altered the response of the two binding sites to ethanol. Two other factors that regulate opiate receptor affinity, Na+ and GTP, also affected the response to ethanol. High concentrations of ethanol were more effective at decreasing receptor affinity for both DHM and ENK when the binding assays were performed in the presence of GTP or Na+. In addition, at 37°C and in the presence of GTP or Na+, DHM binding, but not ENK binding, was significantly inhibited by a low, physiologically attainable concentration of ethanol. Our results suggest that the response of opiate receptors to ethanol is influenced by the microenvironment of the receptors, including the physical state of the membrane lipids and/or by the nature of the interactions of receptors with “coupling proteins” within the membrane. The differential responses of μ and δ receptors to temperature and to ethanol suggest that these receptors reside in specific membrane environments. Under physiological conditions, several different factors may contribute to a selective action of ethanol on particular subtypes of opiate receptors.

Atherosclerosis (AS), a lipid-induced inflammatory condition of the arteries, is a primary contributor to atherosclerotic cardiovascular diseases including stroke. Arctium lappa L. leaf (ALL), an edible and medicinal herb in China, has been documented and commonly used for treating stroke since the ancient times. However, the elucidations on its anti-AS effects and molecular mechanism remain insufficient.To investigate the AS-ameliorating effects and the underlying mechanism of action of an ethanolic extract of leaves of Arctium lappa L. (ALLE).ALLE was reflux extracted using with 70% ethanol. An HPLC method was established to monitor the quality of ALLE. High fat diet (HFD) and vitamin D3-induced experimental AS in rats were used to determine the in vivo effects; and oxidized low-density lipoprotein-induced RAW264.7 macrophage foam cells were used for in vitro assays. Simvatatin was used as positive control. Biochemical assays were implemented to ascertain the secretions of lipids and pro-inflammatory mediators. Haematoxylin-eosin (H&E) and Oil red O stains were employed to assess histopathological alterations and lipid accumulation conditions, respectively. CCK-8 assays were used to measure cytotoxicity. Immunoblotting assay was conducted to measure protein levels.ALLE treatment significantly ameliorated lipid deposition and histological abnormalities of aortas and livers in AS rats; improved the imbalances of serum lipids including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C); notably attenuated serum concentrations of inflammation-associated cytokines/molecules including TNF-α, IL-6, IL-1β, VCAM-1, ICAM-1and MMP-9. Mechanistic studies demonstrated that ALLE suppressed the phosphorylation/activation of PI3K, Akt and NF-κB in AS rat aortas and in cultured foam cells. Additionally, the PI3K agonist 740Y-P notably reversed the in vitro inhibitory effects of ALLE on lipid deposition, productions of TC, TNF-α and IL-6, and protein levels of molecules of PI3K/Akt and NF-κB singnaling pathways.ALLE ameliorates HFD- and vitamin D3-induced experimental AS by modulating lipid metabolism and inflammatory responses, and underlying mechanisms involves inhibition of the PI3K/Akt and NF-κB singnaling pathways. The findings of this study provide scientific justifications for the traditional application of ALL in managing atherosclerotic diseases.

Brain-Machine Interfaces (BMIs) have shown great potential for generating prosthetic control signals. Translating BMIs into the clinic requires fully implantable, wireless systems; however, current solutions have high power requirements which limit their usability. Lowering this power consumption typically limits the system to a single neural modality, or signal type, and thus to a relatively small clinical market. Here, we address both of these issues by investigating the use of signal power in a single narrow frequency band as a decoding feature for extracting information from electrocorticographic (ECoG), electromyographic (EMG), and intracortical neural data. We have designed and tested the Multi-modal Implantable Neural Interface (MINI), a wireless recording system which extracts and transmits signal power in a single, configurable frequency band. In prerecorded datasets, we used the MINI to explore low frequency signal features and any resulting tradeoff between power savings and decoding performance losses. When processing intracortical data, the MINI achieved a power consumption 89.7% less than a more typical system designed to extract action potential waveforms. When processing ECoG and EMG data, the MINI achieved similar power reductions of 62.7% and 78.8%. At the same time, using the single signal feature extracted by the MINI, we were able to decode all three modalities with less than a 9% drop in accuracy relative to using high-bandwidth, modality-specific signal features. We believe this system architecture can be used to produce a viable, cost-effective, clinical BMI.