Ethanolinduced changes in Gproteins gene expression UEL Research

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

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

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

Brain scientists enjoy getting young flies drunk

Tax payers’ money ‘wasted’ on drunken flies

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

Alcohol

Addiction

What is addiction ? Addiction Tolerance Craving Withdrawal Relapse

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

Sedation Time Tolerance to alcohol in flies Exposure

What is the mechanism of tolerance ? Stimulus Response Stimulus Response

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

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

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

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

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

Acute and chronic drug effect and gene expression

Acute/ chronic effect Stimulus Gene expression (m. RNA) Example Acute Increase Morphine c. Fos Chronic + Acute Reduced increase Morphine c. Fos Which gene should be studied?

G proteins a GTP b g

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

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

Primer design 6 Gα : Gs, Go, Gq, Gi, Gf and cta 3 Gβ : Gβ 13 F, Gβ 5 and Gβ 76 C 2 Gγ: Gγ 1 and Gγ 30 A

Primer specificity

RNA dose dependancy

Relative Gene Expression Changes in G protein expression in ethanol treated drosophila (acute effect) 2. 0 1. 5 Control Treated 1 0. 5 0. Gq Gb 5 Gb 76 C

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

System complexity G protein coupled Receptors Dopamine R Opiate R Serotonin R GABA Glutamate Etc , etc G proteins Alpha as ai ao …. . Beta b 1 b 2 b 3 …. . Gamma g 1 g 2 g 3 ……. Effectors Adenylate cyclase PLC K+ channels

Rationale for studying G proteins gene expression a b g GTP Effect A a b g Effect B GTP Protein X Protein Y Normal Tolerance

Relative Gene Expression System variability (are all flies equal? ) 2. 0 1. 5 Control Treated 1 0. 5 0. Gq Gb 5 Gb 76 C

Onset of sedation induced by alcohol Time (sec)

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

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

‘Behavioural’ sub-groups of flies

Genetic differences (Genetic crosses) F 1 Early male Early female F 1 Late male Late female

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

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

SUM Psychological wellbeing Po s ic ve et rty en G Education (lack of) al ic n ys tio Ph dic pe Ex Peer pressure rim en ta t Ad io n Crime Substance Use and Misuse

C hild De vel opm ent Tra Ed inin uc g ati on gn ud iti ie ve s St Co gy icolo Tox cology rma Pha UEL SUM Network hic rap nog s die Eth Stu Clinical Interventions l a i c o s S e i ic l o P

Get in touch SUM ‘network members’ • • • • Dr Daniel Briggs Dr. Stefano Casalotti. Dr Olivia Corcoran Dr. Lynne Dawkins Prof. Angela Harden. Stephanie Lynch John Mc. Carthy. Prof. Mark Mc. Dermott Dr. Olive Mc. Keown. Prof. Derek Moore Dr Winston Morgan. Dr Barbara Pendry. Dr Alice Sampson Dr. Kirstie Soar. Dr. John Turner Oghenetega Umukoro LSS HSB Psychology Student Services Psychology HSB HSB LSS Psychology HSB Contact: John Turner 2@uel. ac. uk Stefano Casalotti stefano 1@uel. ac. uk

Thank you • Oghenetega Umukoro (Ph. D Student) • UEL – Start up-grant