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Ethanol-induced changes in G-proteins gene expression UEL Research & Knowledge Exchange Conference 2013 Stefano Casalotti Oghenetega Umukoro School of.

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Presentation on theme: "Ethanol-induced changes in G-proteins gene expression UEL Research & Knowledge Exchange Conference 2013 Stefano Casalotti Oghenetega Umukoro School of."— Presentation transcript:

1 Ethanol-induced changes in G-proteins gene expression UEL Research & Knowledge Exchange Conference 2013 Stefano Casalotti Oghenetega Umukoro School of Health, Sport and Bioscience

2 More informative title: Investigations of cellular and molecular effect of alcohol in a Drosophila (fruit fly) model.

3 Journalistic style Alcohol addiction: Fruit flies are used to investigate the effect of alcohol on the brain

4 Brain scientists enjoy getting young flies drunk

5 Tax payers’ money ‘wasted’ on drunken flies

6 Ethanol-induced changes in G-proteins gene expression Alcohol addiction Changes in gene expression G-proteins

7 Alcohol

8 Addiction

9 What is addiction ? Addiction Tolerance Craving Withdrawal Relapse

10 Tolerance Following multiple stimulation: –Reduced response to the same stimulus Or: –The need for a higher stimulus to obtain the same response

11 Tolerance to alcohol in flies Sedation Time Exposure

12 What is the mechanism of tolerance ? Stimulus Response Stimulus Response

13 What has changed at…. ? ? …brain level? …network level? …cellular level?

14 Neurochemical Transmission DA Pre-synapse Post-synapse Neurotransmitter e.g Dopamine Dopamine Receptors

15 Neurochemical Transmission DA Pre-synapse Post-synapse Dopamine Receptors

16 Neurochemical Transmission DA Pre-synapse Post-synapse Dopamine Receptors

17 Neurochemical Transmission DA Pre-synapse Post-synapse Drugs of Addiction

18 Acute and chronic drug effect and gene expression

19 Acute/ chronic effect StimulusGene expression (mRNA) Example AcuteIncreaseMorphine  cFos Chronic + AcuteReduced increase Morphine  cFos Which gene should be studied?

20 G proteins       GTP

21 Molecular effect of alcohol Alcohol Receptors (e.g. GABA-R) Receptors (e.g. GABA-R) Cell membrane fluidity Cell membrane fluidity Dopamine release ?? Dopamine Receptors/ G-proteins

22 Quantification of G-Proteins gene expression = measurement of mRNA for G-proteins Method: Real Time,Reverse Transcriptase Polymerase Chain Reaction q-RT-PCR

23 Primer design 6 Gα : Gs, Go, Gq, Gi, Gf and cta 3 Gβ : Gβ13F, Gβ5 and Gβ76C 2 Gγ: Gγ1 and Gγ30A

24 Primer specificity

25 RNA dose dependancy

26 Changes in G protein expression in ethanol treated drosophila (acute effect) Relative Gene Expression Gq G  5 G  76C Control Treated

27 Type of changes Acute effect (naïve flies) Acute effect (‘cronic’ flies) Cronic effect (‘cronic’ flies)

28 System complexity G protein coupled Receptors Dopamine R Opiate R Serotonin R GABA Glutamate Etc,etc G protein coupled Receptors Dopamine R Opiate R Serotonin R GABA Glutamate Etc,etc G proteins Alpha  s  i  o ….. Beta  1  2  3 ….. Gamma  1  2  3 ……. G proteins Alpha  s  i  o ….. Beta  1  2  3 ….. Gamma  1  2  3 ……. Effectors Adenylate cyclase PLC K+ channels Effectors Adenylate cyclase PLC K+ channels

29 Rationale for studying G proteins gene expression       GTP       Normal Tolerance Effect A Effect B Protein X Protein Y

30 System variability (are all flies equal?) Relative Gene Expression Gq G  5 G  76C Control Treated

31 Onset of sedation induced by alcohol Time (sec)

32 Onset of sedation induced by alcohol Time (sec) Early Female Flies Late Female Flies

33 Onset of sedation induced by alcohol Time (sec) Early Males Flies Late Males Flies

34 ‘Behavioural’ sub-groups of flies

35 Genetic differences (Genetic crosses) Early male Early female Late male Late female F1 Early F1 Late

36 Changes in gene expression on a variable genetic background Ethanol Gene Expression

37 Other ‘addiction’ research at UEL Substance use and misuse ‘ network’ SUM-UEL

38 SUM Substance Use and Misuse Substance Use and Misuse Poverty Physical Addiction Peer pressure Experimentation Crime Education (lack of) Psychological wellbeing Genetics

39 Cognitive Studies Clinical Interventions Ethnographic Studies Toxicology Pharmacology Training Education Social Policies UEL SUM Network UEL SUM Network Child Development

40 Get in touch SUM ‘network members’ Dr Daniel Briggs LSS Dr. Stefano Casalotti. HSB Dr Olivia Corcoran HSB Dr. Lynne DawkinsPsychology Prof. Angela Harden. HSB Stephanie Lynch Psychology John McCarthy. Student Services Prof. Mark McDermott Psychology Dr. Olive McKeown. HSB Prof. Derek Moore Psychology Dr Winston Morgan. HSB Dr Barbara Pendry. HSB Dr Alice Sampson LSS Dr. Kirstie Soar.Psychology Dr. John Turner Psychology Oghenetega Umukoro HSB Contact: John Turner Stefano Casalotti

41 Thank you Oghenetega Umukoro (PhD Student) UEL – Start up-grant


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