Skip to content
Other Publications (39)
- Alcoholism, Clinical and Experimental Research
- Pharmaceutical Research
- European Journal of Pharmacology
- Alcoholism, Clinical and Experimental Research
- Brain Research. Molecular Brain Research
- Journal of Neurochemistry
- Neuropharmacology
- Alcoholism, Clinical and Experimental Research
- Journal of Neurochemistry
- Journal of Drug Targeting
- Pharmaceutical Research
- The Journal of Biological Chemistry
- Neuropharmacology
- The Journal of Pharmacology and Experimental Therapeutics
- Journal of Neurochemistry
- Alcoholism, Clinical and Experimental Research
- Pharmaceutical Research
- The Journal of Biological Chemistry
- The Journal of Membrane Biology
- The Journal of Pharmacology and Experimental Therapeutics
- Pharmacology & Therapeutics
- Journal of Neurochemistry
- Alcoholism, Clinical and Experimental Research
- Alcoholism, Clinical and Experimental Research
- The Journal of Pharmacology and Experimental Therapeutics
- Neuropharmacology
- The Journal of Pharmacology and Experimental Therapeutics
- Neuropsychopharmacology : Official Publication of the American College of Neuropsychopharmacology
- The International Journal of Neuropsychopharmacology / Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP)
- Alcoholism, Clinical and Experimental Research
- Molecular Pharmacology
- Frontiers in Neuroscience
- Neuroreport
- Neurochemical Research
- The International Journal of Neuropsychopharmacology / Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP)
- Alcoholism, Clinical and Experimental Research
- Journal of Neuroimmunology
- Alcohol (Fayetteville, N.Y.)
- Alcoholism, Clinical and Experimental Research
Articles by Daryl L. Davies in JoVE
Other articles by Daryl L. Davies on PubMed
-
-
-
-
-
Effects of the Abused Solvent Toluene on Recombinant P2X Receptors Expressed in HEK293 Cells
Brain Research. Molecular Brain Research.
Jun, 2004 |
Pubmed ID: 15193425 ATP acts as a neurotransmitter in both the peripheral and central nervous systems by activating receptors in the P2Y and P2X families. P2Y receptors are coupled to intracellular signaling pathways, while P2X receptors contain an integral membrane-spanning pore and act as ion channels. Previous studies have established that certain abused drugs such as alcohol inhibit P2X receptors. In this study, we have examined the sensitivity of both homomeric and heteromeric P2X receptors to toluene, a commercial solvent widely used as a drug of abuse. P2X receptors were transiently expressed in HEK293 cells, and agonist-gated currents were measured using whole-cell patch clamp electrophysiology. Toluene potentiated currents in cells expressing homomeric P2X2 or P2X4 subunits when ATP concentrations were near or below the EC50 concentration. This potentiation was rapid in onset, voltage independent and was readily reversed upon washout of the toluene-containing solution. The toluene-induced potentiation of P2X2 currents was not altered by lowering the pH of the recording media to 5.5 or by including the heavy-metal chelator EDTA in the recording solution. At maximal ATP concentrations, toluene did not affect ATP-gated currents in cells expressing P2X2 or P2X4 receptors. ATP-gated currents were also markedly potentiated by toluene in cells transfected with both P2X4 and P2X6 subunits. In contrast, P2X3 receptor currents were inhibited by toluene at both low and high ATP concentrations. HEK293 cells transfected with both P2X2 and P2X3 subunits showed non-desensitizing currents when stimulated with alpha, beta-methylene ATP. In these cells, toluene potentiated currents only at sub-maximal concentrations of alpha, beta-methylene ATP. The results of this study suggest that the abused solvent toluene affects the function of P2X receptors in a subunit-dependent and agonist-dependent fashion.
-
-
-
-
-
-
-
-
-
Purinergic Type 2 Receptors at GABAergic Synapses on Ventral Tegmental Area Dopamine Neurons Are Targets for Ethanol Action
The Journal of Pharmacology and Experimental Therapeutics.
Oct, 2008 |
Pubmed ID: 18583548 The current study investigated whether ethanol alters ATP activation of purinergic type 2 receptors (P2Rs) in the ventral tegmental area (VTA). The VTA is a key region of the brain that has been implicated in the development of alcohol addiction. We investigated the effects of ATP and ethanol on spontaneous inhibitory postsynaptic currents (sIPSCs) and the spontaneous firings in the VTA dopaminergic neurons, obtained using an enzyme-free procedure. These neurons preserved some functional GABA-releasing terminals after isolation. We found that ATP (1-200 microM) either increased or decreased the frequency of sIPSCs and the activity of VTA dopaminergic neurons. The effects of ATP on sIPSC frequency inversely correlated with its effects on dopaminergic neuron activity. The ATP-induced changes in sIPSC frequency were blocked by tetrodotoxin (a sodium channel blocker) and by suramin (a nonselective P2R antagonist). Furthermore, alpha,beta-methylene ATP, a selective P2X(1) and P2X(3) receptor agonist, increased sIPSC frequency, whereas adenosine 5'-[beta-thio]diphosphate, a preferential agonist of P2Y receptors, decreased sIPSC frequency. In experiments testing the effects of ethanol (10 and 40 mM) on sIPSCs, we found that ethanol significantly attenuated ATP-induced increase and enhanced ATP-induced decrease in sIPSC frequency. Taken together, the results demonstrate that multiple subtypes of P2Rs exist on GABA-releasing terminals that make synapses on VTA dopaminergic neurons. It seems that ATP increases sIPSC frequency involving P2X(1) and/or P2X(3) receptors, and ATP decreases sIPSC frequency involving P2YRs. These findings are also consistent with the notion that P2Rs at GABA-releasing terminals on VTA dopaminergic neurons are important targets for ethanol action.
-
-
-
-
Loop 2 Structure in Glycine and GABA(A) Receptors Plays a Key Role in Determining Ethanol Sensitivity
The Journal of Biological Chemistry.
Oct, 2009 |
Pubmed ID: 19656948 The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and gamma-aminobutyric acid type A receptors (GABA(A)Rs). To test this, we mutated Loop 2 in the alpha1 subunit of GlyRs and in the gamma subunit of alpha1beta2gamma2GABA(A)Rs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of alpha1GlyR subunits with Loop 2 from the deltaGABA(A)R (deltaL2), but not the gammaGABA(A)R subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the gamma subunit of GABA(A)Rs with deltaL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA(A) gamma-deltaL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA(A)Rs. The deltaL2 mutations did not affect GlyR or GABA(A)R sensitivity, respectively, to Zn(2+) or diazepam, which suggests that these deltaL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and deltaL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.
-
-
-
Molecular Targets and Mechanisms for Ethanol Action in Glycine Receptors
Pharmacology & Therapeutics.
Jul, 2010 |
Pubmed ID: 20399807 Glycine receptors (GlyRs) are recognized as the primary mediators of neuronal inhibition in the spinal cord, brain stem and higher brain regions known to be sensitive to ethanol. Building evidence supports the notion that ethanol acting on GlyRs causes at least a subset of its behavioral effects and may be involved in modulating ethanol intake. For over two decades, GlyRs have been studied at the molecular level as targets for ethanol action. Despite the advances in understanding the effects of ethanol in vivo and in vitro, the precise molecular sites and mechanisms of action for ethanol in ligand-gated ion channels in general, and in GlyRs specifically, are just now starting to become understood. The present review focuses on advances in our knowledge produced by using molecular biology, pressure antagonism, electrophysiology and molecular modeling strategies over the last two decades to probe, identify and model the initial molecular sites and mechanisms of ethanol action in GlyRs. The molecular targets on the GlyR are covered on a global perspective, which includes the intracellular, transmembrane and extracellular domains. The latter has received increasing attention in recent years. Recent molecular models of the sites of ethanol action in GlyRs and their implications to our understanding of possible mechanism of ethanol action and novel targets for drug development in GlyRs are discussed.
-
-
Alcohol-binding Sites in Distinct Brain Proteins: the Quest for Atomic Level Resolution
Alcoholism, Clinical and Experimental Research.
Sep, 2011 |
Pubmed ID: 21676006 Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmitter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a "molecular lubricant" for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol.
-
Implication of the Purinergic System in Alcohol Use Disorders
Alcoholism, Clinical and Experimental Research.
Apr, 2011 |
Pubmed ID: 21223299 In the central nervous system, adenosine and adenosine 5'-triphosphate (ATP) play an important role in regulating neuronal activity as well as controlling other neurotransmitter systems, such as, GABA, glutamate, and dopamine. Ethanol increases extracellular adenosine levels that regulate the ataxic and hypnotic/sedative effects of ethanol. Interestingly, ethanol is known to increase adenosine levels by inhibiting an ethanol-sensitive adenosine transporter, equilibrative nucleoside transporter type 1 (ENT1). Ethanol is also known to inhibit ATP-specific P2X receptors, which might result in such similar effects as those caused by an increase in adenosine. Adenosine and ATP exert their functions through P1 (metabotropic) and P2 (P2X-ionotropic and P2Y-metabotropic) receptors, respectively. Purinergic signaling in cortex-striatum-ventral tegmental area (VTA) has been implicated in regulating cortical glutamate signaling as well as VTA dopaminergic signaling, which regulates the motivational effect of ethanol. Moreover, several nucleoside transporters and receptors have been identified in astrocytes, which regulate not only adenosine-ATP neurotransmission, but also homeostasis of major inhibitory-excitatory neurotransmission (i.e., GABA or glutamate) through neuron-glial interactions. This review will present novel findings on the implications of adenosine and ATP neurotransmission in alcohol use disorders.
-
-
-
-
Sociocommunicative and Sensorimotor Impairments in Male P2X4-deficient Mice
Neuropsychopharmacology : Official Publication of the American College of Neuropsychopharmacology.
Sep, 2013 |
Pubmed ID: 23604007 Purinergic P2X receptors are a family of ligand-gated ion channels gated by extracellular adenosine 5'-triphosphate (ATP). Of the seven P2X subtypes, P2X4 receptors (P2X4Rs) are richly expressed in the brain, yet their role in behavioral organization remains poorly understood. In this study, we examined the behavioral responses of P2X4R heterozygous (HZ) and knockout (KO) mice in a variety of testing paradigms designed to assess complementary aspects of sensory functions, emotional reactivity, and cognitive organization. P2X4R deficiency did not induce significant alterations of locomotor activity and anxiety-related indices in the novel open field and elevated plus-maze tests. Conversely, P2X4R KO mice displayed marked deficits in acoustic startle reflex amplitude, as well as significant sensorimotor gating impairments, as assessed by the prepulse inhibition of the startle. In addition, P2X4R KO mice displayed enhanced tactile sensitivity, as signified by a lower latency in the sticky-tape removal test. Moreover, both P2X4R HZ and KO mice showed significant reductions in social interaction and maternal separation-induced ultrasonic vocalizations in pups. Notably, brain regions of P2X4R KO mice exhibited significant brain-regional alterations in the subunit composition of glutamate ionotropic receptors. These results collectively document that P2X4-deficient mice exhibit a spectrum of phenotypic abnormalities partially akin to those observed in other murine models of autism-spectrum disorder. In conclusion, our findings highlight a putative role of P2X4Rs in the regulation of perceptual and sociocommunicative functions and point to these receptors as putative targets for disturbances associated with neurodevelopmental disorders.
-
Pharmacological Insights into the Role of P2X4 Receptors in Behavioural Regulation: Lessons from Ivermectin
The International Journal of Neuropsychopharmacology / Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP).
Jun, 2013 |
Pubmed ID: 23174033 Purinergic ionotropic P2X receptors are a family of cation-permeable channels that bind extracellular adenosine 5'-triphosphate. In particular, convergent lines of evidence have recently highlighted P2X(4) receptors as a potentially critical target in the regulation of multiple nervous and behavioural functions, including pain, neuroendocrine regulation and hippocampal plasticity. Nevertheless, the role of the P2X(4) receptor in behavioural organization remains poorly investigated. To study the effects of P2X(4) activation, we tested the acute effects of its potent positive allosteric modulator ivermectin (IVM, 2.5-10 mg/kg i.p.) on a broad set of paradigms capturing complementary aspects of perceptual, emotional and cognitive regulation in mice. In a novel open field, IVM did not induce significant changes in locomotor activity, but increased the time spent in the peripheral zone. In contrast, IVM produced anxiolytic-like effects in the elevated plus maze and marble burying tasks, as well as depression-like behaviours in the tail-suspension and forced swim tests. The agent induced no significant behavioural changes in the conditioned place preference test and in the novel object recognition task. Finally, the drug induced a dose-dependent decrease in sensorimotor gating, as assessed by pre-pulse inhibition (PPI) of the acoustic startle reflex. In P2X(4) knockout mice, the effects of IVM in the open field and elevated plus maze were similar to those observed in wild type mice; conversely, the drug significantly increased startle amplitude and failed to reduce PPI. Taken together, these results suggest that P2X(4) receptors may play a role in the regulation of sensorimotor gating.
-
-
-
P2X4 Receptors (P2X4Rs) Represent a Novel Target for the Development of Drugs to Prevent And/or Treat Alcohol Use Disorders
Frontiers in Neuroscience.
2014 |
Pubmed ID: 25009459 Alcohol use disorders (AUDs) have a staggering socioeconomic impact. Few therapeutic options are available, and they are largely inadequate. These shortcomings highlight the urgent need to develop effective medications to prevent and/or treat AUDs. A critical barrier is the lack of information regarding the molecular target(s) by which ethanol (EtOH) exerts its pharmacological activity. This review highlights findings implicating P2X4 receptors (P2X4Rs) as a target for the development of therapeutics to treat AUDs and discusses the use of ivermectin (IVM) as a potential clinical tool for treatment of AUDs. P2XRs are a family of ligand-gated ion channels (LGICs) activated by extracellular ATP. Of the P2XR subtypes, P2X4Rs are expressed the most abundantly in the CNS. Converging evidence suggests that P2X4Rs are involved in the development and progression of AUDs. First, in vitro studies report that pharmacologically relevant EtOH concentrations can negatively modulate ATP-activated currents. Second, P2X4Rs in the mesocorticolimbic dopamine system are thought to play a role in synaptic plasticity and are located ideally to modulate brain reward systems. Third, alcohol-preferring (P) rats have lower functional expression of the p2rx4 gene than alcohol-non-preferring (NP) rats suggesting an inverse relationship between alcohol intake and P2X4R expression. Similarly, whole brain p2rx4 expression has been shown to relate inversely to innate 24 h alcohol preference across 28 strains of rats. Fourth, mice lacking the p2rx4 gene drink more EtOH than wildtype controls. Fifth, IVM, a positive modulator of P2X4Rs, antagonizes EtOH-mediated inhibition of P2X4Rs in vitro and reduces EtOH intake and preference in vivo. These findings suggest that P2X4Rs contribute to EtOH intake. The present review summarizes recent findings focusing on the P2X4R as a molecular target of EtOH action, its role in EtOH drinking behavior and modulation of its activity by IVM as a potential therapy for AUDs.
-
Multiday Administration of Ivermectin is Effective in Reducing Alcohol Intake in Mice at Doses Shown to Be Safe in Humans
Neuroreport.
Sep, 2014 |
Pubmed ID: 25004078 Ivermectin (IVM), an FDA approved anthelmintic agent, can significantly reduce ethanol intake in mice following acute administration. The current study evaluates the sustainability and safety of multiday IVM administration in reducing 10% v/v ethyl alcohol (10E) intake in mice at a dose shown to be safe in humans. We tested the effect of 10-day administration of IVM (3.0 mg/kg/day; intraperitoneally) on reducing 10E intake in C57BL/6J mice using a 24-h, two-bottle choice paradigm. On the 10th day of IVM administration, mice were sacrificed at 0, 0.5, 2, 8, 32, 48, and 72 h after injection. Brain tissue and plasma samples were collected and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Analysis of variance (ANOVA) was used to assess the effect of 10-day IVM administration on 10E intake, 10E preference, water intake, and total fluid intake with Dunnett's multiple comparison post-hoc test. Individual Student's t-tests were also used to further quantify changes in these dependent variables. IVM significantly decreased 10E intake over a 9-day period (P
-
-
-
Structural Models of Ligand-gated Ion Channels: Sites of Action for Anesthetics and Ethanol
Alcoholism, Clinical and Experimental Research.
Mar, 2014 |
Pubmed ID: 24164436 The molecular mechanism(s) of action of anesthetic, and especially, intoxicating doses of alcohol (ethanol [EtOH]) have been of interest even before the advent of the Research Society on Alcoholism. Recent physiological, genetic, and biochemical studies have pin-pointed molecular targets for anesthetics and EtOH in the brain as ligand-gated ion channel (LGIC) membrane proteins, especially the pentameric (5 subunit) Cys-loop superfamily of neurotransmitter receptors including nicotinic acetylcholine (nAChRs), GABAA (GABAA Rs), and glycine receptors (GlyRs). The ability to demonstrate molecular and structural elements of these proteins critical for the behavioral effects of these drugs on animals and humans provides convincing evidence for their role in the drugs' actions. Amino acid residues necessary for pharmacologically relevant allosteric modulation of LGIC function by anesthetics and EtOH have been identified in these channel proteins. Site-directed mutagenesis revealed potential allosteric modulatory sites in both the trans-membrane domain (TMD) and extracellular domain (ECD). Potential sites of action and binding have been deduced from homology modeling of other LGICs with structures known from crystallography and cryo-electron microscopy studies. Direct information about ligand binding in the TMD has been obtained by photoaffinity labeling, especially in GABAA Rs. Recent structural information from crystallized procaryotic (ELIC and GLIC) and eukaryotic (GluCl) LGICs allows refinement of the structural models including evaluation of possible sites of EtOH action.
-
-
Oral Delivery of Ivermectin Using a Fast Dissolving Oral Film: Implications for Repurposing Ivermectin As a Pharmacotherapy for Alcohol Use Disorder
Alcohol (Fayetteville, N.Y.).
Sep, 2015 |
Pubmed ID: 26095588 Individuals suffering from an alcohol-use disorder (AUD) constitute a major health concern. Preclinical studies in our laboratory show that acute and chronic intraperitoneal (i.p.) administration of ivermectin (IVM) reduces alcohol intake and preference in mice. To enable clinical investigation to use IVM for the treatment of an AUD, development of an oral formulation that can be used in animals as well as long-term preclinical toxicology studies are required. The present work explores the use of a promising alternative dosage form of IVM, fast-dissolving oral films (Cure Pharmaceutical®), to test the efficacy and safety of oral IVM in conjunction with alcohol exposure. We tested the effect of IVM (0.21 mg) using a fast-dissolving oral film delivery method on reducing 10% v/v alcohol (10E) intake in female C57BL/6 mice using a 24-h access two-bottle choice paradigm for 6 weeks (5 days per week). Differences in ethanol intake, preference for ethanol, water intake, fluid intake, food intake, changes in mouse and organ weights, as well as histological changes to kidney, liver, and brain were analyzed. The IVM group drank significantly less ethanol over the 30-day period compared to the placebo (blank strip) and the no-treatment groups. Organ weights did not differ between the groups. Histological evaluation showed no differences in the brain and kidney between groups. In the liver, there was a slight increase in the incidence of microvesicular fatty and degenerative changes of the animals receiving the thin strips. No overt hepatocellular necrosis or perivascular inflammation was noted. Overall, these data support the use of this novel method of oral drug delivery for longer-term studies and should facilitate FDA required preclinical testing that is necessary to repurpose IVM for treatment of an AUD.
-
Get cutting-edge science videos from JoVE sent straight to your inbox every month.