GABA-A Receptors and Addiction Flashcards
Describe the GABAA receptor
Major inhibitory receptor in the mammalian brain
Both GABA-AR and glycine receptors are transmitter-gated anion (Cl-) conducting ion channels
What mediates fast inhibition within the spinal cord?
GABAARs and stychnine sensitive glycine receptors
What is the resultant action of inhibition of GABAAR?
Proconvulsant
Anxiogenic
Examples include bicuculline and picrotoxin
What is the resultant action of enhancement of GABAR?
Positive allosteric modulators (PAMs) such as diazepam Anticonvulsant Anxiolytic Sedative Analgesic
What subunits make up the GABAA receptors?
2 alpha
2 beta
1 gamma
What IV anaesthetics will modulate GABA activity?
Etomidate
Propofol
Barbiturates
What properties will drugs that enhance GABAAR function show?
Anxiolytic Anticonvulsant Analgesic Amnesic Sedative/ hypnotic: GA
Where do BZDs bind on GABAR?
Interface between the alpha and gamma subunit
What is the action of flumeazenil?
Antagonist at GABAR
Blocks effect of diazepam and beta-carboline
What is a “NAM”?
Negative allosteric modulator
Describe a “NAM”
Beta-carbolines e.g. DMCD
Do PAMs or NAMs have an effect in the absence of GABA?
No; GABA must be present
These drugs will either enhance GABA or inhibit GABA; have no action on the actual GABA receptor themselves
Do oocytes express GABAR?
No
In which direction does Cl- move through a GABAR?
Inward; hyperpolarization of the cell
What was the outcome of the first cloning of GABAr subunits alpha 1 and beta 1 in frog oocytes?
Alpha 1 and beta 1 GABAR subunits are activated by GABA. blocked by picrotoxin and bicuculline, and enhanced by barbiturates
No effect by BZD
This lead to the search for additional subunits e.g. the gamma 2 subunit
What is the basic composition of GABAR?
5 transmembrane crossing subunits
Majority contain 2 beta, 2 alpha and 1 gamma
How many subtypes are there for GABAR?
19; alpha 1-6 beta 1-3 gamma 1-3 delta pi
Are GABAR uniformly distributed in the brain and spinal cord
No;
For example;
alpha 1 is heavily expressed in the cortex, cerebellum and hippocampus
alpha 5 is heavily expressed in the hippocampus
delta subunit; granule cells of cerebellum and thalamus
What is the significance of GABAr receptor isoforms?
Distinct physiological and pharmacological patterns
Exhibit a heterogenous expression pattern in the CNS
Which GABAR isoform in particular is sensitive to BZD?
Alpha 1, beta 1, gamma 2
In terms of specific subunits of the GABAR; what confers BZD sensitivity?
Alpha 1, 2, 3 and 5 are sensitive
4 and 6 are not
Alpha 1,2,3 and 5 have a H (histidine) residue at position 101 of extracellular amino acid chain whereas 4 and 6 have an R (arginine; +ve charge) residue
Mutation of alpha 6 R101 to H produces a diazepam sensitive receptor
Mutation of alpha1H101 to R blocks diazepam sensitivity
What experiment was set up to determine if certain alpha receptors play a role in the multitude of side effects of BZD?
Alpha H-R “knock in” BZD insensitive mice
Alpha1 H101r
Alpha 2 H101R
Alpha 5 H101R
What was the results of the alpha 1H101R mutant mouse?
Autoradiography revealed that the alpha 1 mutation caused a loss of 3H-flumazenil binding in areas known to express the alpha 1 subunit
In the alpha1H101R mouse, diazepam was no longer sedative but retained its anxiolytic properties
What do the results of the alpha H101R suggest?
The sedative effects of BZD are mediated by alpha 1 subunits
How is anxiety tested in mouse?
Light/ dark box
What were the results of the alpha 2H101R mutant mouse?
Diazepam was no longer anxiolytic but sedative
What do the results of alpha 2H101R suggest?
The anxiolytic properties of BZD are mediated by alpha 2 subunits
What are BZD used to treat?
Sleep disorders
Anxiety disorders
Epilepsy
Muscle spasms
What are common side effects of BZDs?
Sedation
Amnesia
Dependence
Addiction
Through the use of the “knock in” mice; describe the role of GABAr subtypes in BZD properties
Sedation; alpha 1 Anxiolysis; alpha 2 Muscle relaxation; alpha2,3,5 Anticonvulsive; alpha 1 Amnesia; alpha 1, 5 Addiction; alpha 1 and 2
Can you develop tolerance to BZD?
Yes; this manifests as a withdrawal syndrome: sleep disturbances, irritability, anxiety, panic attacks
What is the action of flumazenil?
BZD antagonist
Helpful in overdose of diazepam
What can increase the risk of overdose of BZD?
Taken in conjunction with alcohol and opioids
What nT do the majority of neurons in the nucleus accumbens project?
GABAErgic
Describe the connection between the VTA and the nucleus accumbens
VTA sends a dopaminergic projection (pleasure) to the N. Acc
This activity is under the local control of GABAergic interneurons
BZD influence the activity of the VTA interneurons to impact upon dopamine release in N. Acc
Describe the difference between GABAa and GABAb
GABAA - transmitter gated anion channel; fast inhibition
GABAB; GPCR. 2 subunits; beta 1 (GABA binding site) and GABA2 (G protein linked) = slow transmission
How is GABA synthesized?
Alpha-ketoglutarate
Converted to glutamate via GABA-T
Converted to GABA by GAD (glutamic acid decarboxylase)
What drug blocks GABA transporter?
Tiagabine; an anticonvulsant
Which transporter permits entry of GABA into vesicles at the prestynaptic termina?
VGAT; vesicular GABA amino acid transporter
What is the role of GABAB receptors on the postsynaptic membrane?
Allows efflux of potassium (slow hyperpolarization)
High GABA release; they can act back on presynaptic GABAB receptors which through their G protein linked to voltage gated calcium channels, reduce the GABA release from a 2nd action potential
Form of autoregulation of GABA release
What is the purpose of the patch clamp technique?
Holds the membrane potential at a fixed value to allow recording of activity of neurons under voltage clamp condition
How many receptors are present in a synapse?
30-40
How do “PAM’s” enhance GABAergic transmission?
They make it more favourable for chloride channels at post synaptic membrane to remain in the open state
This enhances neuronal inhibition
What mediates tonic inhibition?
Extrasynaptic GABAA receptors
Less prone to desensitization
Activated by very low concentrations of ambient GABA overspill
Slow, tonic current
Major impact on the excitability of neurons
What subunit predominates in the GABAergic neurons in the VTA?
Alpha 1
What subunit predominates in the dopaminergic neurons in the VTA?
Alpha 3
In a normal state, describe the action of GABA on dopamine in the VTA
GABA fires to exert an inhibitory effect on dopamine
This reduces dopamine transmission to the nucleus accumbens
Under BZD control, describe the action of GABA on dopamine in the VTA
Diazepam acts on alpha 1 on GABA neuron
Decreases firing rate of GABAergic interneurons by enhancing inhibition
Consequently, less GABA is released to dopamine neurons
The firing of dopamine neurons will therefore increase
DISINHIBITION
Increased DA to N. Acc
The BZs enhance phasic inhibition on the GABAergic interneuron, causing a decreased GABA release on DA neuron
This disinhibition results in increased DA too N. Acc
What are the effects of midazolam and flumazenil on in vivo firing rate of DA and GABA neurons in WT neurons?
Midazolam decreases the firing rate of GABA on WT neurons (reversed by flumazenil)
This consequently increases DA firing rate (reversed by flumazenil)
What are the effects of midazolam and flumazenil on in vivo firing rate of DA and GABA neurons in a1(H101R) mice?
Midazolam has no effect on firing rate of GABAergic interneurons and consequently little to no effect on DA
In terms of addiction, what effect does the a1(H101R) have on mice?
Due to the fact that in a1(H101R) mice; midazolam has no effect on DA (and therefore no effect on N. Acc) - there is no pleasure from midazolam
In a study, wild type but NOT a1(H101R) mice preferentially self administer midazolam
Both alpha 1 and alpha 2 are required for midazolam self-administration
Does the alpha 2 receptors of GABA have any receptors in the VTA?
Very little; the locus are focussed at the N. Acc
Alpha 1 = VTA (GABAergic interneurons)
Alpha 2 = N. Acc - selective knockdown of alpha2 unit expression in N. Acc abolished midazolam self-administration
How do opioids result in disinhibition to increase accumbal DA?
Decrease GABA release on dopamine neurons via mu opioid GPCR receptors opening K+ channels resulting in hyperpolarization +/- decrease opening of calcium channels
How do cannabinoids result in disinhibition to increase accumbal DA?
Decrease release of GABA on to DA via cannabinoid GPCR CB1 receptors to decrease opening probability of Ca2+ channels
How do BZDs result in disinhibition to increase accumbal DA?
Act of alpha 1 GABAARs to decrease release onto dopamine neurons; therefore increasing their excitability
How does nicotine result in increased accumbal dopamine?
Activates nAChRs (alpha4beta2) on DA neurons resulting in depolarization and increased dopamine release
How does cocaine and amphetamine influence the accumbal dopamine?
Influence dopamine transporters/ release and thereby increasing the extracellular conc of dopamine
What conditions have been shown to be affected by early life adversity (ELA)?
Depression
Anxiety
Cognitive impairment
Drug abuse
Is there a genetic component for drug abuse?
Studies of twins reveals 50% heritability for alcoholism and 60-70& for cocaine and opiate abuse
What genes have been implicated in addictive behaviour?
GABAR2 gene
Haplotypes with cocaine, heroin and alcohol addiction and particularly evident for individuals with prior childhood trauma
What is the NAc associated with?
Reward
Depression
Stress
Where are synaptic alpha-2 GABAARs expressed?
NAc medium spiny neuron (MSN)
What evidence is there to show that alpha 2 plays a role in addictive behaviour?
Cocaine behaviour is significantly altered in the alpha 2 knockout mouse
Midazolam self-administration is reduced in mice with a decreased expression of the alpha 2 subunit in the NAc
How is early life adversity created in mouse models?
Mother and pups transferred to a different cage 2 days after birth
New cage has reduced bedding and restricted access to saw dust
In ELS mice compared to WT; the mother leaves the nest much more times resulting in reduced bonding between mother and pups
After 9 days (P9); they get moved back into a normal cage and are left to grow
Stress HPA axis is greatly changed in these mice; CRF release in hypothalamus is much higher
How does cocaine affect mice?
Increases locomotor activity
Distance travelled in wheel is measured as a marker for cocaine
What is the significance of an increased distance travelled from day 1 to day 10 in mice given cocaine?
Behavioural sensitization; each day cocaine is administered; the distance travelled increases and the effects of cocaine is more pronounced
Thought to be due to a form of synaptic plasticity that occurs in drug abuse
What was seen in ELA mince when exposed to cocaine?
There was a high distance travelled on the 1st day and a similarly high distance travelled on the 2nd day
No behavioural sensitisation
The acute response of cocaine is amplified but sensitisation is blunted
What is seen in alpha 2 -/- mice when exposed to cocaine?
Acute response amplified
Sensitization is blunted
What are the similarities between the ELA mouse and a2 -/- mouse?
Both ELA and a2-/- exhibit an enhanced acute locomotor effect to cocaine and reduced sensitization (reduced synaptic plasticity in regards to drug abuse behaviour)
Which GABAA receptor subunits are expressed in N.Acc MSN?
Alpha 1/2 beta gamma 2 = synaptic, BZD sensitive
Alpha 4 beta delta (extrasynaptic) = BZD insensitive
Is there a link between ELA and alpha 2 subunit expression?
Yes; ELA selectively decreases GABAAR A2-subunit in the NAc
In terms of mice models, what is the link between ELA and GABAAR a2 subunit
Prior ELA selectivity decreases GABAAR a2 subunit immunoreactivity in the adult (P60) mouse NAc
Decreased mean density (synapses with no alpha 2) along side a decreased area (reduced alpha 2 receptors expressed in synapse)
Was there a change in alpha1 subunit expression in ELA mice models?
No; highly selective against alpha 2 subunits
Summarize the results of ELA and alpha 2 subunit expression
Reduced expression of alpha 2 subunit will:
Enhance acute locomotor effects of cocaine
Supress sensitisation to cocaine
ELA is highly sensitive for a2by2
What is the GABAA2 a2 subunit associated with?
Addiction
Stress
Anxiety
What is the evidence that extrasynaptic delta-GABAARs are a direct target for ethanol?
High dose of ethanol (100mM) enhanced GABA response
30mM ethanol had no effect on dentate gyrus cell tonic current
What is the behaviourally relevant concentrations of ethanol?
10-30 mM
Are the effects of acute ethanol on anxiety and sedation altered in delta knock out mice as expected?
No; no effect
However these mice drink less alcohol suggesting a role for delta-GABAARs/ tonic currents in the rewarding/reinforcing effects of alcohol
What is the evidence that alcohol will influence the production of GABA indirectly?
Ethanol has been proven to influence and enhance neurosteroid production extrasynaptically
Neurosteroids in brain and spinal cord are potent allosteric modulators of GABAAR.
What are neurosteroids synthesized from?
Cholesterol
What is the role of neurosteroids (5alpha-3alpha THPROG)
Potent positive allosteric modulators of GABAAR
Bind to site and enhance their function
For phasic inhibition; prolong the MIPSC
Can act on extrasynaptic receptors to enhance tonic currents
Can be synthesized by neurons, glial cells or by peripheral derived progesterone which crosses BBB
Auto-control via endogenous neurosteroids
What is the functional evidence of alcohol increasing neurosteroid levels?
Ethanol immediately has no effect
Slowly, the IPSC delays - increases GABAAR inhibition
Further evidence; 5-alpha reductase inhibitor blocks the effect of delay of IPSC
Conclude; the effect of ethanol on the IPSC decay is produced by increased neurosteroid levels
GABAAR alpha 2 point mutation mice prevents neurosteroid modulation
The modified mouse compared to wild type exhibits reduced ethanol preference
What is the role of accumbal extrasynaptic GABAARs in mediating the effects of alcohol
Link between GABAA receptors and ethanol administration in N. Acc
Micro perfusion of bicuculline into accumbens will reduce ethanol self-administration
Accumbal injection of an interference RNA (decrease expression of delta or alpha 4 subunit) will decrease ethanol intake in rats
What is the role of the beta 1 subunit and ethanol consuption?
A mouse carried a single point mutation of GABAR B1 subunit
This mouse showed a preference for ethanol and increased ethanol consumption
Why does the mutation in beta 1 subunit of GABAAR result in increased ethanol consumption?
Mutations result in the spontaneous activity of GABAAR in the absence of GABA nT
Mutant mice exhibit large tonic currents in the accumbens due to spontaneous opening of beta-GABAARs
What has been shown to decrease ethanol consumption in mice?
Decreased expression of accumbal delta or alpha 4 subunits (extrasynaptic)
Local micro perfusion of bicuculline into accumbens
