Altered Membrane Properties Research Articles

Identification of an amphipathic helix within IFITM3 that promotes inhibition of influenza virus

Abstract Interferon-induced transmembrane protein 3 (IFITM3) inhibits influenza virus fusion with endosomes by altering cellular membrane properties through mechanisms that are largely uncharacterized. In order to identify regions of secondary structure within IFITM3 that may contribute to its mechanism of action, we performed structural predictions using the I-TASSER and PSSpred algorithms. Insterestingly, we identified a 10-amino acid region of likely helicity that possesses an amphipathic distribution of amino acids. Consistent with the known role of amphipathic helices in altering membrane properties, we observed that cells expressing a helix-deleted mutant of IFITM3 were infected with influenza virus at a 4-fold higher rate than cells expressing WT IFITM3. Confirming that the amphipathicity of this helix contributes to antiviral activity, single, double and triple alanine mutations of the three residues making up the hydrophilic face of the helix progressively decreased antiviral activity of IFITM3, as did single polar mutations in the hydrophobic face of the helix, or single amino acid insertions that disrupted amphipathicity. Amino acid sequence alignments suggest that amphipathicity in this region is highly conserved in IFITMs throughout evolution. Overall, we utilized bioinformatics methods coupled with functional infectivity assays to provide the first evidence that IFITM3 utilizes an amphipathicity-based mechanism for inhibition of viruses.

Spatio-Temporal Regulation of PKC Isoforms Imparts Signaling Specificity.

Nishizuka and colleagues discovered protein kinase C (PKC) as a calcium-dependent, lipid cofactor-sensitive protein kinase (1). Initially known as the only physiological effector for tumor-promoting phorbol esters, this ubiquitous enzyme eventually took center stage in cellular signaling. Part of the AGC kinase branch of the kinome, PKC is a family of serine/threonine kinases comprising 11 isoforms encoded by 9 genes and grouped into 4 classes – classical (cPKCs-α, βI, βII, γ), novel (nPKCs-δ, ϵ, η, θ), atypical (aPKCs-ζ, ι/λ), and PKCμ (a form between novel and atypical isoforms) (Box 1). Their considerable structural homology, overlapping substrate specificities, and biochemical properties indicated at least partial enzymatic redundancy and rendered the task of identifying isoform-specific functions challenging. The initial phase of PKC research correlated the unique structural features of the isoforms with their functions (Box 1) (2). But, with their structural overlaps and hugely varying functions in different models, specificity of the isoforms became a confounding puzzle demanding stringent isoform-specific regulation to avoid functional redundancy. To define this stringency, subsequent PKC research focused on the upstream and downstream regulatory mechanisms (3, 4), highlighting the importance of subcellular distribution as a function of time (5). As simultaneous activation of all PKC isoforms would be energetically and spatially conflicting for decoding the message received by the cell surface receptor, cell type, and stimulus-specific selective and sequential activation deemed justified (6). The recent phase of PKC research propounds a PKC-signaling module (7, 8) wherein an inter-isoform network regulates the PKC isoforms’ activity (Box 2; Figure ​Figure1B).1B). Here, we propose activator- and cofactor-specific sequential activation of PKC isoforms in a spatio-temporal model wherein selective subcellular compartmentalization quantitatively determines the isoform-specific effector functions. Box 1 Structural features of PKC isoforms defining functional specificity. PKC serve as a paradigm for the reversible regulation of membrane localization by the concerted action of two membrane-targeting modules. C1: a cysteine-rich region of approximately 50 residues present in all PKC isozymes. In cPKCs and nPKCs, it is present as a tandem repeat C1A and C1B. aPKCs contain a single copy of the domain termed atypical because it does not bind phorbol esters. The domain contains two pulled apart β-sheet forming the ligand-binding pocket. Two zinc atoms are coordinated by two histidines and six cysteines at opposite ends of the primary sequence stabilizing the domain. In aPKCs, one face of the ligand-binding pocket is compromised, so that the module cannot bind phorbol esters or DAG. Ligand binding dramatically alters the surface properties of the module (C1B). The ligand caps the hydrophilic ligand-binding pocket, so that the top-third of the C1 domain presents a continuous hydrophobic surface thus achieving membrane targeting by simply altering membrane properties. C2: an independent membrane-targeting module that binds calcium in cPKCs, but not in nPKCs. C2 domain is a β-strand rich globular domain with loops formed by sequences at opposite ends of the primary structure. Two topological variants exist – Type I for domains that follow the C1 domain (cPKCs) and Type II for domains that precede C1 (nPKCs). In the calcium-responsive C2 domains of cPKCs, the pocket is lined by multiple aspartic acid residues that coordinate two to three calcium ions that act as a bridge between the C2 domain and the phospholipid head groups of the membrane. CR, cysteine rich; PS, pseudosubstrate; DAG, diacylglycerol; PMA, phorbol-12-myristate 13-acetate; PB1, Phox–Bem 1; C, constant regions; V, variable regions.

Comparison of clotrimazole 1% cream with 50% tea tree oil extract in a cream base for the treatment of tinea corporis/cruris: A randomized controlled trial

Background: Tinea cruris and corporis are common diseases and can be widespread. Estimated lifetime probability of contracting the disease is as high as 10-20%. Dermatophytic infections are one of the three most frequently observed skin diseases among poor communities in tropical countries. Tea tree oil exerts its antifungal activity by altering membrane properties and compromising membrane associated function resulting in clinical improvement and mycological cure of superficial dermatophytes. Objectives: The aim of this study is to determine the efficacy and safety of tea tree oil 50% in a cream base compared with clotrimazole 1% cream for the treatment of tinea corporis or cruris. Material and Methods: Sixty patients were enrolled in the study and randomized into tea tree oil and clotrimazole groups with 30 patients in each group. Both groups were instructed to apply cream two times a day after cleaning with soap for 4 weeks. The patients were asked to follow-up every 2 weeks for photography, microscopic and clinical assessment. Before enrollment, patient signed a written consent form. Results: Using intention to treat analysis, the assessment of mycological cure, clinical cure, global physician assessments and overall patient perception were done. There were no statistical difference of all the parameters between tea tree oil group and clotrimazole group. Conclusion: Tea tree oil 50% cream is safe and as effective as clotrimazole 1% cream for the treatment of tinea corporis or cruris.NJDVL Vol. 13, No. 1, 2015 Page: 24-30

Migraine Triggers and Oxidative Stress: A Narrative Review and Synthesis.

Blau theorized that migraine triggers are exposures that in higher amounts would damage the brain. The recent discovery that the TRPA1 ion channel transduces oxidative stress and triggers neurogenic inflammation suggests that oxidative stress may be the common denominator underlying migraine triggers. The aim of this review is to present and discuss the available literature on the capacity of common migraine triggers to generate oxidative stress in the brain. A Medline search was conducted crossing the terms "oxidative stress" and "brain" with "alcohol," "dehydration," "water deprivation," "monosodium glutamate," "aspartame," "tyramine," "phenylethylamine," "dietary nitrates," "nitrosamines," "noise," "weather," "air pollutants," "hypoglycemia," "hypoxia," "infection," "estrogen," "circadian," "sleep deprivation," "information processing," "psychosocial stress," or "nitroglycerin and tolerance." "Flavonoids" was crossed with "prooxidant." The reference lists of the resulting articles were examined for further relevant studies. The focus was on empirical studies, in vitro and of animals, of individual triggers, indicating whether and/or by what mechanism they can generate oxidative stress. In all cases except pericranial pain, common migraine triggers are capable of generating oxidative stress. Depending on the trigger, mechanisms include a high rate of energy production by the mitochondria, toxicity or altered membrane properties of the mitochondria, calcium overload and excitotoxicity, neuroinflammation and activation of microglia, and activation of neuronal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. For some triggers, oxidants also arise as a byproduct of monoamine oxidase or cytochrome P450 processing, or from uncoupling of nitric oxide synthase. Oxidative stress is a plausible unifying principle behind the types of migraine triggers encountered in clinical practice. The possible implications for prevention and for understanding the nature of the migraine attack are discussed.

Bilayer Effects of Antimalarial Compounds.

Because of the perpetual development of resistance to current therapies for malaria, the Medicines for Malaria Venture developed the Malaria Box to facilitate the drug development process. We tested the 80 most potent compounds from the box for bilayer-mediated effects on membrane protein conformational changes (a measure of likely toxicity) in a gramicidin-based stopped flow fluorescence assay. Among the Malaria Box compounds tested, four compounds altered membrane properties (p< 0.05); MMV007384 stood out as a potent bilayer-perturbing compound that is toxic in many cell-based assays, suggesting that testing for membrane perturbation could help identify toxic compounds. In any case, MMV007384 should be approached with caution, if at all.

Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance.

In many organisms "Universal Stress Proteins" (USPs) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8Å and 2.4Å resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing.

Bilayer Perturbation is a Predictive Parameter for Antimalarial Development

There is a constant need for new malaria drugs because of the perpetual development of resistance to current therapies. To facilitate the costly drug development process and to avail pharmaceutical company-vetted drug candidates to academicians who wish to explore novel strategies for preventing and treating malaria, the Medicines for Malaria Venture developed the Malaria Box. To assist in this effort, and to reduce the expenses required for drug development, we tested the 80 most potent compounds from the Malaria Box for their bilayer-perturbing effects, as sensed by a membrane-spanning channel, as a proxy for a generic membrane protein. Specifically, we used a gramicidin-based stopped-flow fluorescence assay to probe whether the compounds altered lipid bilayer properties at the concentrations where they are used to inhibit/kill the malaria parasites. Membrane-active compounds are expected to be ubiquitous modifiers of membrane protein conformational changes, meaning that they are predicted to be toxic (above some threshold concentration). Among the compounds tested, four altered membrane properties (p<0.05), and one of them MMV007384 was a potent bilayer-perturbing compound that should be used with caution, if at all. This compound was indeed known to be toxic in cell-based screens, suggesting that one can use the gramicidin-based fluorescence to test for cell toxicity.

Impact of in situ physical and chemical cleaning on PVDF membrane properties and performances

Appropriate selection of cleaning agent is an important factor to achieve a better cleaning efficiency and this topic has become an ongoing discussion. This work assesses the impacts of sodium hydroxide (NaOH) and sodium hypochlorite (NaOCl) aqueous solution towards polyvinylidene fluoride (PVDF) stability at the typical concentrations used in membrane cleaning. The cleaned membranes were characterised using field emission scanning electron microscopy (FESEM), Fourier transform infrared (FTIR), pure water flux measurement, contact angle, protein retention and tensile testing. Membrane cleaned at elevated temperature and higher concentration presented a higher water flux than the virgin membrane which can be a worrying sign of alteration in membrane properties. The FTIR spectra indicated that the alteration in chemical composition of the membrane causes a reduction in the degree of hydrophilicity. The mechanical properties of the membrane were compromised based on the declination of tensile strength. The findings from this work suggest that the usage of NaOCl as compared to NaOH causes a more detrimental effect towards the stability of the PVDF membrane.

Saturated Fatty Acids Alter the Late Secretory Pathway by Modulating Membrane Properties

Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic β-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.

Protection of a Ceramide Synthase 2 Null Mouse from Drug-induced Liver Injury: ROLE OF GAP JUNCTION DYSFUNCTION AND CONNEXIN 32 MISLOCALIZATION

Very long chain (C22-C24) ceramides are synthesized by ceramide synthase 2 (CerS2). A CerS2 null mouse displays hepatopathy because of depletion of C22-C24 ceramides, elevation of C16-ceramide, and/or elevation of sphinganine. Unexpectedly, CerS2 null mice were resistant to acetaminophen-induced hepatotoxicity. Although there were a number of biochemical changes in the liver, such as increased levels of glutathione and multiple drug-resistant protein 4, these effects are unlikely to account for the lack of acetaminophen toxicity. A number of other hepatotoxic agents, such as d-galactosamine, CCl4, and thioacetamide, were also ineffective in inducing liver damage. All of these drugs and chemicals require connexin (Cx) 32, a key gap junction protein, to induce hepatotoxicity. Cx32 was mislocalized to an intracellular location in hepatocytes from CerS2 null mice, which resulted in accelerated rates of its lysosomal degradation. This mislocalization resulted from the altered membrane properties of the CerS2 null mice, which was exemplified by the disruption of detergent-resistant membranes. The lack of acetaminophen toxicity and Cx32 mislocalization were reversed upon infection with recombinant adeno-associated virus expressing CerS2. We establish that Gap junction function is compromised upon altering the sphingolipid acyl chain length composition, which is of relevance for understanding the regulation of drug-induced liver injury.

Disinfection of a polyamide nanofiltration membrane using ethanol

It is imperative that nanofiltration membranes are disinfected before they are used for laboratory-scale bacterial adhesion or biofouling experiments, yet currently no suitable disinfection protocol exists. This study aimed to determine if an ethanol treatment at a minimum inhibitory concentration (MIC) could be used to effectively disinfect nanofiltration membranes without altering membrane properties which could affect research. Two strains of bacteria, Pseudomonas fluorescens and Staphylococcus sp., were exposed to a range of ethanol concentrations to determine the MIC required for a 4log10 reduction in bacteria. In parallel, ethanol's effects on the filtration, surface and mechanical properties of a Dow Filmtec NF90 membrane were analysed. A 1.5h treatment with 40% ethanol was shown to effectively disinfect the membrane without significantly affecting any of the membrane's properties tested. This treatment protocol can now be safely used to disinfect the studied membrane prior to bacterial adhesion or biofouling experiments. This study also acts as a guideline for researchers using other membranes to determine a suitable disinfection protocol for their needs.

Remodeling of liver phospholipidomic profile in streptozotocin-induced diabetic rats

Lipid homeostasis in liver is known to be altered with diabetes mellitus, ultimately leading to liver damage and related complications. The present work aimed to evaluate changes in the liver phospholipid profile after 4months of uncontrolled hyperglycemia. Twenty Wistar rats were divided into two groups: control and streptozotocin-treated (T1DM). After 4months, animals were sacrificed and morphological characterization of liver was performed and related with serum markers of hepatic damage. Lipid extracts were obtained from liver and phospholipid (PL) classes were quantified. Lipid molecular species were determined by LC–MS and LC–MS/MS, and fatty acids by GC–MS. Concomitantly with signs of hepatic damage we found variations in the relative amount of phospholipid classes in T1DM, characterized by a decrease in PLs with choline head group, and by an increase in the relative content of other PL classes. A remodeling in PL fatty acyl chains was observed in T1DM liver, with a similar pattern to all the PL classes, and consisting in the reduction of 16:0 and an increase of 18:0 and 18:2 acyl chains. The observed changes in T1DM lipid profile may contribute to the altered membrane properties underlying hepatic damage, worsening the metabolic alterations that characterize T1DM.

Virtual neurophysiology laboratories for life science education: action potentials and voltage-/patch-clamp recordings

Understanding neurophysiology is for many students a particularly difficult task, especially the interpretation of different measures their physiological meaning and their interrelations. It could be of great help if the students could do the recordings themselves, according to the didactically advantageous strategy learning by doing. When such experiments are not possible in regular students' courses, for whatever reasons (too difficult, no resources etc.), virtual laboratories can become valuable alternatives. During recent years, we have developed a series of virtual laboratories (Virtual Physiology), among them three neurophysiology tools. There is SimNerv for clinically still most relevant extracellular recordings of compound actions potentials. A second program, SimPatch, is for whole-cell current recordings from different types of neurons, also allowing the application of channel blockers or changing ion concentrations. These programs are providing realistically appearing laboratories on the computer screen that allow the students performing physiological and pharmacological experiments almost as in real life. All stimulus and recording parameters of the virtual devices are freely adjustable and a mathematical algorithm guarantees for the appropriate reaction of the virtual preparations. A third series of applications, SimNeuron, has been designed in a more simple, easy to overlook form to demonstrate the relations between current-clamp, whole-cell voltage-clamp and single-channel patch-clamp recordings. This program also allows changing the model neuron's parameters to examine how altered membrane properties are reflected in the different recordings. All these programs are designed with the major goal to train the students' understanding and know-how, although factual knowledge is a prerequisite for systematic experimentation in the virtual labs. With this respect, detailed tutorial and protocol form are provided, supplemented by additional simulations and animation of specific features. Figure 1 SimPatch, the Virtual Lab Figure 2 SimNeuron menu and recording examples from the current-clamp, whole-cell voltage-clamp lab, single channel patch-clamp lab (closed and open states with gating) with I-V curves for K+- (blue), Na+- (red) and unspecific cation (black) ion channels

Intracardiac origin of heart rate variability, pacemaker funny current and their possible association with critical illness.

Heart rate variability (HRV) is an indirect estimator of autonomic modulation of heart rate and is considered a risk marker in critical illness, particularly in heart failure and severe sepsis. A reduced HRV has been found in critically ill patients and has been associated with neuro-autonomic uncoupling or decreased baroreflex sensitivity. However, results from human and animal experimental studies indicate that intracardiac mechanisms might also be responsible for interbeat fluctuations. These studies have demonstrated that different membrane channel proteins and especially the so-called ‘funny’ current (If), an hyperpolarization-activated, inward current that drives diastolic depolarization resulting in spontaneous activity in cardiac pacemaker cells, are altered during critical illness. Furthermore, membrane channels kinetics seem to have significant impact upon HRV, whose early decrease might reflect a cellular metabolic stress. In this review article we present research findings regarding intracardiac origin of HRV, at the cellular level and in both isolated sinoatrial node and whole ex vivo heart preparations. In addition, we will review results from various experimental studies that support the interrelation between If and HRV during endotoxemia. We suggest that reduced HRV during sepsis could also be associated with altered pacemaker cell membrane properties, due to ionic current remodeling.

Intracardiac Origin of Heart Rate Variability, Pacemaker Funny Current and their Possible Association with Critical Illness

Heart rate variability (HRV) is an indirect estimator of autonomic modulation of heart rate and is considered a risk marker in critical illness, particularly in heart failure and severe sepsis. A reduced HRV has been found in critically ill patients and has been associated with neuro-autonomic uncoupling or decreased baroreflex sensitivity. However, results from human and animal experimental studies indicate that intracardiac mechanisms might also be responsible for interbeat fluctuations. These studies have demonstrated that different membrane channel proteins and especially the so-called ‘funny’ current (If), an hyperpolarization-activated, inward current that drives diastolic depolarization resulting in spontaneous activity in cardiac pacemaker cells, are altered during critical illness. Furthermore, membrane channels kinetics seem to have significant impact upon HRV, whose early decrease might reflect a cellular metabolic stress. In this review article we present research findings regarding intracardiac origin of HRV, at the cellular level and in both isolated sinoatrial node and whole ex vivo heart preparations. In addition, we will review results from various experimental studies that support the interrelation between If and HRV during endotoxemia. We suggest that reduced HRV during sepsis could also be associated with altered pacemaker cell membrane properties, due to ionic current remodeling. Keywords: Endotoxin, funny current, heart rate, heart rate variability, ivabradine, sepsis, sinoatrial node

Alcohol-induced oxidative/nitrosative stress alters brain mitochondrial membrane properties

Chronic alcohol consumption causes numerous biochemical and biophysical changes in the central nervous system, in which mitochondria is the primary organelle affected. In the present study, we hypothesized that alcohol alters the mitochondrial membrane properties and leads to mitochondrial dysfunction via mitochondrial reactive oxygen species (mROS) and reactive nitrogen species (RNS). Alcohol-induced hypoxia further enhances these effects. Administration of alcohol to rats significantly increased the mitochondrial lipid peroxidation and protein oxidation with decreased SOD2 mRNA and protein expression was decreased, while nitric oxide (NO) levels and expression of iNOS and nNOS in brain cortex were increased. In addition, alcohol augmented HIF-1α mRNA and protein expression in the brain cortex. Results from this study showed that alcohol administration to rats decreased mitochondrial complex I, III, IV activities, Na(+)/K(+)-ATPase activity and cardiolipin content with increased anisotropic value. Cardiolipin regulates numerous enzyme activities, especially those related to oxidative phosphorylation and coupled respiration. In the present study, decreased cardiolipin could be ascribed to ROS/RNS-induced damage. In conclusion, alcohol-induced ROS/RNS is responsible for the altered mitochondrial membrane properties, and alcohol-induced hypoxia further enhance these alterations, which ultimately leads to mitochondrial dysfunction.

Cholesterol through the Looking Glass: ABILITY OF ITS ENANTIOMER ALSO TO ELICIT HOMEOSTATIC RESPONSES

How cholesterol is sensed to maintain homeostasis has been explained by direct binding to a specific protein, Scap, or through altering the physical properties of the membrane. The enantiomer of cholesterol (ent-cholesterol) is a valuable tool in distinguishing between these two models because it shares nonspecific membrane effects with native cholesterol (nat-cholesterol), but not specific binding interactions. This is the first study to compare ent- and nat-cholesterol directly on major molecular parameters of cholesterol homeostasis. We found that ent-cholesterol suppressed activation of the master transcriptional regulator of cholesterol metabolism, SREBP-2, almost as effectively as nat-cholesterol. Importantly, ent-cholesterol induced a conformational change in the cholesterol-sensing protein Scap in isolated membranes in vitro, even when steps were taken to eliminate potential confounding effects from endogenous cholesterol. Ent-cholesterol also accelerated proteasomal degradation of the key cholesterol biosynthetic enzyme, squalene monooxygenase. Together, these findings provide compelling evidence that cholesterol maintains its own homeostasis not only via direct protein interactions, but also by altering membrane properties.

Enhanced cholesterol removal ability of lactobacilli via alteration of membrane permeability upon ultraviolet radiation

This study aimed to evaluate the effects of ultraviolet radiation (UVA, UVB, and UVC) on the cholesterol removal ability of lactobacilli and altered membrane properties. Viability of lactobacilli decreased (P < 0.05) immediately upon UV treatment due to oxidative damage of the cellular membrane, as induced by lipid peroxidation. UVB and UVC showed higher (P < 0.05) effects in inducing lipid peroxidation compared to UVA. However, the viability of lactobacilli increased significantly (P < 0.05) upon fermentation at 37°C for 20 h, which was attributed to the formation of transient pores and membrane permeabilization. These alterations also led to increased (P < 0.05) assimilation of cholesterol and incorporation of cholesterol into the cellular membrane upon fermentation. The ratio of membrane cholesterol:phospholipids (C:P) also increased upon UV treatment, accompanied by increased saturation of cholesterol in the polar head, apolar tail and the interface regions of the membrane lipid bilayer.

Long-Lasting Alterations in Membrane Properties, K+ Currents, and Glutamatergic Synaptic Currents of Nucleus Accumbens Medium Spiny Neurons in a Rat Model of Alcohol Dependence

Chronic alcohol exposure causes marked changes in reinforcement mechanisms and motivational state that are thought to contribute to the development of cravings and relapse during protracted withdrawal. The nucleus accumbens (NAcc) is a key structure of the mesolimbic dopaminergic reward system. Although the NAcc plays an important role in mediating alcohol-seeking behaviors, little is known about the molecular mechanisms underlying alcohol-induced neuroadaptive changes in NAcc function. The aim of this study was to investigate the effects of chronic intermittent ethanol (CIE) treatment, a rat model of alcohol withdrawal and dependence, on intrinsic electrical membrane properties and glutamatergic synaptic transmission of medium spiny neurons (MSNs) in the NAcc core during protracted withdrawal. We show that CIE treatment followed by prolonged withdrawal increased the inward rectification of MSNs observed at hyperpolarized potentials. In addition, MSNs from CIE-treated animals displayed a lower input resistance, faster action potentials (APs), and larger fast afterhyperpolarizations (fAHPs) than MSNs from vehicle-treated animals, all suggestive of increases in K+-channel conductances. Significant increases in the Cs+-sensitive inwardly rectifying K+-current accounted for the increased input resistance, while increases in the A-type K+-current accounted for the faster APs and increased fAHPs in MSNs from CIE rats. We also show that the amplitude and the conductance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated mEPSCs were enhanced in CIE-treated animals due to an increase in a small fraction of functional postsynaptic GluA2-lacking AMPARs. These long-lasting modifications of excitability and excitatory synaptic receptor function of MSNs in the NAcc core could play a critical role in the neuroadaptive changes underlying alcohol withdrawal and dependence.

Mechanisms contributing to the dopamine induction of crawl-like bursting in leech motoneurons

Dopamine (DA) activates fictive crawling behavior in the medicinal leech. To identify the cellular mechanisms underlying this activation at the level of crawl-specific motoneuronal bursting, we targeted potential cAMP-dependent events that are often activated through DA(1)-like receptor signaling pathways. We found that isolated ganglia produced crawl-like motoneuron bursting after bath application of phosphodiesterase inhibitors (PDIs) that upregulated cAMP. This bursting persisted in salines in which calcium ions were replaced with equimolar cobalt or nickel, but was blocked by riluzole, an inhibitor of a persistent sodium current. PDI-induced bursting contained a number of patterned elements that were statistically similar to those observed during DA-induced fictive crawling, except that one motoneuron (CV) exhibited bursting during the contraction rather than the elongation phase of crawling. Although DA and the PDIs produced similar bursting profiles, intracellular recordings from motoneurons revealed differences in altered membrane properties. For example, DA lowered motoneuron excitability whereas the PDIs increased resting discharge rates. We suggest that PDIs (and DA) activate a sodium-influx-dependent timing mechanism capable of setting the crawl rhythm and that multiple DA receptor subtypes are involved in shaping and modulating the phase relationships and membrane properties of cell-specific members of the crawl network to generate crawling.