ethanol-induced changes in g-proteins gene expression
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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:. - PowerPoint PPT PresentationTRANSCRIPT
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
Tolerance to alcohol in flies
1st 2nd0
5
10
15
20
25*
Expsoure
ST
50 (
min
)
Sed
atio
n T
ime
Exposure
What is the mechanism of tolerance ?
StimulusResponse
Stimulus
Stimulus
Stimulus
StimulusResponse
What has changed at….
?
…brain level?
…network level?
…cellular level?
Neurochemical Transmission
DADA
DA
Pre-synapse
Post-synapse
Neurotransmitter e.g Dopamine
DopamineReceptors
Neurochemical Transmission
DADA
DA
Pre-synapse
Post-synapse
Dopamine
DopamineReceptors
Neurochemical Transmission
DADA
DA
Pre-synapse
Post-synapse
Dopamine
DopamineReceptors
Neurochemical Transmission
DADA
DA
Pre-synapse
Post-synapse
Drugs of Addiction
Acute and chronic drug effectand gene expression
Acute/ chronic effect
Stimulus Gene expression (mRNA)
Example
Acute Increase Morphine cFos
Chronic + Acute Reduced increase
Morphine cFos
Which gene should be studied?
G proteins
a b gGTP
Molecular effect of alcohol
Alcohol
Receptors(e.g. GABA-R)
Cell membranefluidity
Dopamine release
??
Dopamine Receptors/ G-proteins
Quantification of G-Proteins gene expression=
measurement of mRNA 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 : Gβ13F, Gβ5 and Gβ76C
2 Gγ: Gγ1 and Gγ30A
Primer specificity
RNA dose dependancy
Changes in G protein expression in ethanol treated drosophila
(acute effect) R
elat
ive
Gen
e E
xpre
ssio
n
Gq Gb5 Gb76C
ControlTreated
2.0
1.5
1
0.5
0.
Type of changes
Acute effect (naïve flies)
Acute effect (‘cronic’ flies)
Cronic effect (‘cronic’ flies)
System complexity
G protein coupled
Receptors
Dopamine ROpiate RSerotonin RGABAGlutamateEtc ,etc
G proteinsAlphaasaiao…..Betab1b2b3…..
Gammag 1 g 2 g 3
…….
Effectors
Adenylate cyclase
PLC
K+ channels
Rationale for studying G proteins gene expression
a b gGTP
a b gGTP
Normal Tolerance
Effect A Effect B
Protein XProtein Y
System variability(are all flies equal?)
Rel
ativ
e G
ene
Exp
ress
ion
Gq Gb5 Gb76C
ControlTreated
2.0
1.5
1
0.5
0.
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)
Early male Early female Late male Late female
F1 Early F1 Late
Changes in gene expression on a variable genetic background
Ethanol
Gene Expression
Other ‘addiction’ research at UEL
Substance use and misuse ‘ network’
SUM-UEL
SUM
Substance Use
and Misuse
Pover
ty
Physical
Addiction
Peer pressure Ex
perim
enta
tion
Cri
me
Ed
uca
tion
(lack
of)
Psychological wellbeing G
enetics
Cogni
tive
Studi
es
Clinical Interventions E
thnographic
StudiesToxicology
Pharm
acology Trai
ning
E
duca
tion
Social
Policies
UEL SUM
Network
Child Development
Get in touchSUM ‘network members’ • Dr Daniel Briggs LSS• Dr. Stefano Casalotti. HSB• Dr Olivia Corcoran HSB• Dr. Lynne Dawkins Psychology• 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 [email protected]
Stefano [email protected]
Thank you• Oghenetega Umukoro (PhD Student)• UEL – Start up-grant