addiction

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  • Created by: Sarah
  • Created on: 29-05-19 12:25
why is it so important to study addiction?
drug abuse produces enormous social economic and healthcare burdens
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how many alcohol related deaths were there in 2013 that weren't related to all the things like liver failure?
over 8000
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what are the 2 independent factors contributing to drug addiction?
1) sensitivity 2) development of tolerance
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what is sensitivity?
inability to tolerate the adverse effects of a drug from the start point- how well tolerated at first exposure
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what is the development of tolerance?
reduction in sensitivity when a drug is used repeatedly over time
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what does tolerance mean?
you need a higher amount of the drug to achieve the same level of response and adverse effects
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where do opiates work?
mu opioid receptors
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where does nicotine work?
acetylcholine recs
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where does cannabis work?
cannabinoid recs
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where does alcohol work?
lots of recs- the major being GABA +NMDA recs
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most if not all drugs of abuse act secondarily through what?
dopamine and the mesolimbic reward system
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what is the mesolimbic dopamine system involved in?
reinforcing natural rewards - feel good system
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what pathway is a key component of addiction?
cAMP pathway
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why is the cAMP pathway key in addiction?
neuropeptide binds to rec that activates Gs and get cAMP and PKA, PKA phosphorylates TFs including CBP that lead to LTP- LTP will reinforce memory
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what is CBP what does it do?
is a histone acetyltransferase which regulates neuronal plasticity AND a cofactor for CREB
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what is the main event happening in the cAMP pathway?
activation of PKA -> it can translocate to the nucleus to phosphorylate CREB which when phosphorylated can bind to CREB binding protein (CBP) a HAT to activate transcription of target genes
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how did they show that WT CREB protein can induce acetylation of histones?
a single point mutation in CBP completely abolishes its function as a HAT
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how did they look into the function of CBP in histone acetylation?
used a binary transgenic system where they generated transgenic mice having 2 different transgenes inserted into their genome
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what did the transgene in the forebrain encode encode?
one encoded a tetracycline transactivator protein tta and this was under the regulation of CamKII promoter which drives expression of TTA specifically in frontal neurons
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why did they use the CamKII promoter?
so it would only drive tetracycline transactivator in forebrain neurons
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what was the other transgene?
tet responsive promoter which drives the coding region and expression of CBP WT or mutated
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how did they turn on and off the transgene?
adding DOX (doxycycling) turns the transgene off- inhibits tTa so it can't bind to tet promoter and drive CBP expression
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what was happening in these mice normally?
these cells that express CamKII this tetracycline transactivator will be expressed and bind to the promoter and drive expression of CBP protein so CBPs expressed in frontal neurons
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what happens if DOX is added to turn off the transgene by inhibiting tetreacycline transactivator?
CamKII promoter will drive expression of tetracyline transactivator but it will be inhibited so it can't bind to tet promoter to drive CBP expression so CBP will be off
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why is it important to realise that many complex traits especially behaviour, memory and addiction are involve the action of multiple mechanisms?
not one mechanism responsible for addiction in this case it's possible to study the function of this particular gene as there's always a control group so can compare CBP when turned on/off = everything else in genome identical so must be CBP mutation
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using this transgene system what did they look at?
memory consolidation in CBP WT and mutant mice
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what did they find happened with CBP mutant mice?
CBP mutant mice had impaired consolidation of memory
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what does acute alcohol exposure do to dendritic spine density in the amgydala?
increases dendritic spine density
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what does acute alcohol exposure do to anxiety?
anxiolytic response produced (removes anxiety)
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when they were injected with alcohol how was their behaviour tested?
the open maze test for anxiety (open and closed branches time spent)
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what did acute alcohol exposure do the amount spent in open branches of the maze?
increased time spent in the open branch- reduced anxiety
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how long did it take for dendritic spine density to signicantly increase after acute alcohol exposure?
30 minutes
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what does acute exposure to alcohol activate?
CREB signalling pathway and increases BDNF expression
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what did they look at to find that CREB signalling pathway was causing the behaviour and dendritic spine changes in acute alchol exposure?
phospho-CREB shows the amount of activated CREB signalling, after acute exposure phospho-CREB increased and BDNF increased on mrna and protein level
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why would BDNF be increased?
because its downstream gene of the CREB signalling pathway
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where was this increase in BDNF nad increased phospho-CREB found?
medial and central amygdala- same regions were the dendritic spines were increased
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what else did acute ethanol exposure increase?
protein levels of CBP and acetylated histones H3 and H4
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what is CBP?
creb binding protein- cofactor for CREB and HAT
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where was CBP HAT increased?
in the same regions of the amydala (Central + medial). CBP acetylates Histone 2
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what does acute alcohol exposure inhibit?
histone deacetylases
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why would HDACs be inhibited?
so CBP can acetylate more histones and open up chromatin to increase neuropeptide Y expression for anxiolytic effects (acetylation + chromatin remodeling -> gene transc -> behaviour)
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what's the model for increasing neuropeptide expression to have anxiolytic effects?
acute alcohol exposures -> activates cAMP pathway -> PKA phos CREB -> CBP can bind to CREB -> CBP active +HDACs inactive -> open up chromatin -> increase BDNF+ neuropeptide Y -> anzxiolytic
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why is it important we understand the chronic phase of addiction?
because after chronic exposure it is nearly impossible to stop seeking more drugs
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what does chronic alcohol exposure do to dendritic spine density?
restores dendritic spine density = same as the control
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what happens to dendritic spine density in withdrawal?
drops to significantly below the control
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what does chronic alcohol exposure due to all the acetylation of chromatin?
acetylation of histone 3,4 and the amount of CBP protein that acetylates histone 2 are the same as the control
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what happened to acetylation of histone 3, 4 and CBP with withdrawal?
they are significantly reduced below control
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what did chronic alcohol exposure do to BDNF?
restores its expression back to the control through neuroadaptation
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what does withdrawal do to BDNF expression?
significant reduction in both mRNA and protein levels of BDNF
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what did this lead to the model of?
acute alcohol exposure opens up chromatin through CBP action but also through inhibition of HDACs, this changes expression of multiple proteins involved in behaviour and memory formation including BDNF+NPY. Neuroadaptation with chronic changes reverg
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what causes the problem? why?
withdrawal as the changes are reversed chromatin becomes more compacted, acetylation of histones reduced expression of mols reduced so witthdrawal beh as seek more drug to reverse changes and open up chromatin again
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is it that simple?
not that simple changes aren't limited to amygdala more complex and mutliple other epigenetic changes
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what changes did they find in the liver with alcohol exposure?
cultured rat hepatocytes- ethanol decreased levels of H3K9 methylation and increased H3K4 methylation = both open chromatin which coresponds to increased expression of alcohol dehydrogenase which metabolises alcohol
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What phosphorylation was changed in the lvier?
increased phosphorylation of histone H3 at serine 10 and serine 28
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what did chronic alcohol do to the rat hepatocytes cell culture?
decreased site specific demethylation in cortical neurons and so probably changed gene expression
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these changes in methylation (decreased in cortical neurons) persisted after withdrawal may have an effect of why drugs addicts relapse many years after withdrawal, why?
because they persist the history of alcohol abuse is kept in the bod even after the withdrawal which may be why you get relapse
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what happened if there was chronic intermittent alcohol exposure to the cell culture system?
chronic application of ethanol altered regulation of the methylation pathway by inhibiting methionine adenosyltransferases- enzyme that converts to methionine to SAM to add methyl groups
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so effectively chronic intermittent alcohol exposure would do what?
wouldn't have SAM to donate methyl groups- links to the hypomethylation of DNA seen in chronic alcohol abuse
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what is SAM required for?
main donor of methyl groups for DNA methylation- leads to a global DNA hypomethylation links chronic alcohol abuse with hypomethylation of the DNA
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what might this explain?
why chronic alcohol abuse patients are more prone to developing liver cancers as hypmethylation of the genome especially LINE1 elements is associated with genome instability
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what did acute cocaine treatment do histone acetylation?
increased histone acetylation of specific promoters at H4
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what were they looking at to see acetylation changes?
looking for acetylation only on specific gene promoters not whole genome scale changes
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what are cFos and BDNF downstream of?
the cAMP pathway
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what was seen 30 mins after injection of cocaine?
massive changes in histone H4 acetylation at the promoter of BDNF + forsB and Cfos- maintained and increased after 90 minutes
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when do they start disappearing?
after 3 hours
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what was there no change in?
histone 3 acetylation
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what did this tell us?
acute cocaine treatment induces an increase in specifically acetylation of histone 4 but not histone 3.
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chronic cocaine treatment induces an increase in histone acetylation of specific promoters at where?
H3
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how did they find this?
compared the acetylation of histone 3 or 4 to the control (injected with saline) or the chronic injection of cocaine- mice were allowed to self-administer cocaine lever to inject them as self administration+drug seeking is addiction behavioural trait
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when is the self administration method usually used?
to look for therapeutic approaches to heal addiction
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what did they find with chronic or self administered cocaine exposure over a long time?
there was a large increase in acetylation of histone 3 at BDNF and fosB but no changes in the promoter of cFos
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what was there no change in with chronic cocaine?
acetylation of histone H3 at cFos + there were no changes anymore in acetylation of histone 4 in any of those genes
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what did crhonic but not acute cocaine treatment decrease the function of?
HDAC5
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how is HDAC5 inhibited with chronic cocaine exposure?
it's hyperphosphorylated
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what happened to phosphorylation of HDAC5 in acute cocaine exposure? how did they show this?
no changes in HDAC5 phosphorylation shown by WB of antibodies that recognise phosphorylated HDAC5
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when did hyperphosphorylation of HDAC5 return to normal after chronic cocaine treatment?
after a day
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how did they know where HDAC5 is normally localised?
HDAC5 normally localised in the nucleus (tagged HDAC5 and colocalised with DAPII)
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where did HDAC5 go after chronic cocaine exposure?
all over the cytoplasm
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what did they suggest?
phosphorylation of HDAC5 lead to its extrusion from the nucleus
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what does this mean for cell funciton?
if HDAC5 is excluded from the nucleus it can't function there so it can't deacetylate histone 3 so it becomes hyperacetylated after chronic cocaine exposure
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when else was the same changes to HDAC5 hapening?
after exposure to chronic stress
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what did chronic but not acute cocaine treatment do to phospho- CREB?
increases phosphorylated CREB
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what were the 3 conditions used when looking at the amount of phospho-CREB with cocaine?
control = saline, restricted = saline injection, extended = chronic application
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how did they quantify phosphorylated CREB?
WB
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what was the increase in phospho-CREB associated with?
expression of micro RNAs 212 and 132
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how does miRNA 212+132 interact with the CREB pathway?
positive feedback loop, CREB upregulates these microRNAs and they increase phosphorylation of CREB
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how are actul changes with cocaine much more complex?
they are not limited to the cAMP pathway and histone acetylation they're much broader both histone and DNA methylation has also been implicated in regulating cocaine induced changes
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similarities between alcohol and cocaine exposure?
1) both at least partly act through cAMP pathway and CREB protein- has roles in memory formation 2) both not limited to histone acetylation but most studies but includes multiple other epigenetic changes eg DNA+histone methylation
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differences between alcohol and cocaine exposure?
1) ethanol chronic exposure leads to neuroadaptation and restores the epigenetic context of histone acetylation- with withdrawal is prob. 2) chronic cocaine exposure particular changes induced in histone acetylation in chronic but not acute
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what experiment did they do with TSA (HDAC inhibitor)
mice were injected with ethanol or saline in the control and were injected with TSA
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what did they find?
withdrawal reduces histone acetylation (compared to control) and ethanol inhibits HDACs so asked if we inhibit HDAC in withdrawal can we restore withdrawal symptoms so looked at acetylation of H3, H3 and CBP levels
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what were they measuring?
looked at acetylation of histone 3, 4 and amount of CBP protein
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what happened to the changes in acetylation?
in the chronic alcohol injected group there was no change but there was a massive decrease in acetylation with withdrawal
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what did TSA do to the withdrawal group?
With TSA inhibitor restored acetylation in the withdrawal group
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what happened with TSA in withdrawal in relation to CBP?
histone acetylation of 3 + 4 levels returned to normal (same as control) but CBP protein levels didn't
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in withdrawal where do the mice like to be in the open maze test?
in the closed branches so they're anxious and hiding
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what happened to the anxiety when they were going through withdrawal but injected with a TSA inhibitor?
restored it they, performed more active where they chose between light and dark components
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what does this mean for TSA?
has some potential for treating at least some of the withdrawal syndromes and in particular anxiety that is occurring with withdrawal in chronic drug abuse
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what was shown to be altered in both cases of chronic drug abuse?
histone acetylation
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what did they look at a therapeutic treatment for?
histone acetylation
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when they crossed 2 inbred stains of mice for many generations what did they find about ethanol?
one of the mice lines prefers ethanol and the other is non-preferring so they compared alcohol preferring and non-preferring rats
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how did they compare these rats?
they were allowed to self apply ethanol as they had 2 bottles in their cage and one is ethanol and they chose what to drink from- 3 days had 7% ethanol then increased to 9% and choose between them for 4 days
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then what were they injected with?
TSA- HDAC inhibitor or control
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when were the preferring rats preferring the bottle with ethanol?
1-3 days and 4-7 days
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what happened if they were injected with TSA?
their preference for ethanol drops- they seek less ethanol which suggests a potential for HDAC inhibition for treatment of withdrawal
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how did they see if an increase in histone acetylation that is the case of chronic cocaine might have therapeutic potential?
used garcinol to inhibit histone acetyltransferases, rats allowed to self-administer cocaine for 10 days and at the same time shown 2 conditional stimuli so they associate it
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what happened after rats had associated 2 stimuli with cocaine application?
stopped application so they had lever to self administer cocaine but it didn't have cocaine in and they were not shown any stimuli allowed withdrawal for 8 days
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after 8 days of not having any stimuli or cocaine what were they doing?
not pressing the level
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when they showed the stimuli they started pressing the lever a lot again, what happened when they were given HAT inhibitor?
the seeking behaviour was significantly reduced
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Card 2

Front

how many alcohol related deaths were there in 2013 that weren't related to all the things like liver failure?

Back

over 8000

Card 3

Front

what are the 2 independent factors contributing to drug addiction?

Back

Preview of the front of card 3

Card 4

Front

what is sensitivity?

Back

Preview of the front of card 4

Card 5

Front

what is the development of tolerance?

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