GABA

Question 1

Which isoforms does diazepam have an enhancing effect on?

Answer 1

α1- , α2-, α3- and α5βγ2 GABA A receptors

Question 2

What are the therapeutic effects and the side effects of α1βγ2 GABA A receptors?

Answer 2

Therapeutic effects:
- Sedation
- Anti-convulsant

Side effects:
- Amnesia
- Addiction

Question 3

What are the therapeutic effects and the side effects of α2βγ2 GABA A receptors?

Answer 3

Therapeutic effects:
Anxiolysis and muscle-relaxant

Side effects:
Addiction

Question 4

What are the therapeutic effects and the side effects of α5-, α3βγ2 GABA A receptors?

Answer 4

Therapeutic effects:
Muscle relaxant

Side effects:
Amnesia (only α5 isoform)

Question 5

What is benzodiazepine used to treat?

Answer 5

Sleep disorders, Anxiety disorders, Epilepsy and Muscle spasms

α1- , α2-, α3- and α5βγ2 GABA A receptors therapeutic effects combined

Question 6

What are the side effects of benzodiazepines?

Answer 6

Sedation, Amnesia, Dependence, Addiction

α1- , α2-, α3- and α5βγ2 GABA A receptors side effects combined

Question 7

example of GABAAR Inhibitors and their effects

Answer 7

Exampels of inhibitors: bicuculine and picrotoxin
Effects: proconvulsant and anxiogenic

binds to GABA binding site or channel pore

Question 8

Which type of GABAAR binding molecules target benzodiazepine site?

Answer 8

• Positive Allosteric Modulator (PAM), e.g. diazepam
• Negative Allosteric Modulator (NAM), e.g. β-carboline (DMCM)
• Antagonists, e.g. flumazenil

PAM = enhances effect of GABA
NAM = suppresses the effect of GABA
Antagonist = blocks effect of PAM and NAM but no effect on its own

Question 9

How did they find out diazepam enhances the function of α1-, α2-, α3-, & α5βγ2-GABAARs, but not α4- or α6-GABAARs?

Answer 9

α1/α6 chimaera
* α1-, α2-, α3-, & α5βγ2-GABAARs have a H residue at position 101, whereas α4 & α6 have an R residue at this position
* Mutation of α6 R101 to H produces diazepam sensitive receptor

αH-R “knock in” benzodiazepine-insensitive mice = α1/2/3H to R

Question 10

Which GABAAR subtypes mediate the anxiolytic & sedative effects of
diazepam & how was that shown?

Answer 10

knock in mice
* α2H101R => no longer anxiolytic but is sedative
* α1H101R => no longer sedative but is anxiolytic

Anxiety was tested using light/dark box.

Question 11

Which benzodiazepine is widely abused?

Answer 11

Xanax (alprazolam)

often taken in conjuction with other substances - increases risk of overdose

Question 12

What is tonic inhibition?

Answer 12

• Mediated by extrasynaptic GABA receptors
• low concentrations of GABA is sufficient for activation (background inhibitors)
• bicuculine forces extrasynaptic receptors to close

Question 13

What is phasic inhibition?

Fast Phasic Inhibition

Answer 13

• Activity of GABA receptors in the synapse region
• Only occurs when there is a release of GABA from presynaptic terminal

Question 14

How was GABAAR subtypes expressed in VTA GABA interneurons and dopamine identified?

Answer 14

• Use of patch clamp technique on reporter mice
• mIPSCs were recorded from GABA and dopamine neurons
• In the GABA neurons, midazolam prolongs the decay time in wild type mice and no effect on α1H101R mice
• In the dopamine neurons, midazolam polongs the decay time in both wildtype and α1H101R mice - further studies show that dopamine neurons have α3 subtype of GABAAR.

Markers: GAD67: GABA neurons,Tyrosine Hydrolase: dopamine neurons

Question 15

What are the effects of benzodiazepam on the VTA?

Answer 15

• GABA neurons: BZ enhances phasic inhibition on GABAergic interneurons = decreased GABA release on dopamine neurons
• Dopamine neurons: decreased GABA release => increase in excitation of DA neuron => increase dopamine release in the Nucleus Accumbens (NA)

Question 16

Explain the self administration of midazolam test and why it was done?

Answer 16

• Mice is give 2 bottle choice of 1) sucrose or 2) sucrose + midazolam
• Wildtype mice has preference to bottle (2)
• α1 H101R mice do not show preference to midazolam

Engin et al 2014

Question 17

What is the role of α2-GABAAR in midazolam self-administration?

Answer 17

Require both α1- & α2-GABAARs for midazolam self-administration.
However, there is limited expression of α2-GABAARs in the VTA,
but plenty of expression in the nucleus accumbens.

Selective knockdown of α2-subunit expression in the nucleus accumbens
abolished midazolam self-administration.

Engin et al 2014

Question 18

How does drug of abuse act on the VTA-NA pathway to increase accumbal dopamine?

Answer 18

• Opioids, Cannabinoids and Benzodiazepines act through disinhibition.
• Nicotine and Cocaine act through dopamine.

Question 19

Reference for ELA

Answer 19

Gunn et al 2013

Question 20

What is the effect of cocaine on α2 0/0 mice and ELA mice in locomotion test?

Answer 20

Both ELA & α20/0 mice exhibit an enhanced acute locomotor effect to cocaine (Day 1) & reduced sensitization (Day 10)

the effect of cocaine is enhanced in day1 and remains enhanced in day10

Gunn et al 2013

Question 21

What is the subunit composition of GABAA receptor in the medium spiny neuron (MSN) in nucleus accumbens?

Answer 21

• Synaptic receptors: α1βγ2 and α2βγ2
• Extrasynaptic receptors: α4βδ

Dixon et al 2010
Maguire et al 2014
Anstee et al 2013

Question 22

How was the GABAAR subunit composition in the MSN of NA identified?

Answer 22

Using selective inhibitors:
* Zolpidem : α1
* Gaboxadol: α4βδ
* DS2: δ
* Ethomidate: β2/β3

Question 23

In mice using a combination of immunohistochemistry & electrophysiology, how would you investigate the impact of prior early life adversity upon GABAAR subtype expression/function?

Answer 23

• Prior ELA selectively decreases GABAAR α2-subunit immunoreactivity in the adult (P60) mouse NAc
  [markers for α2, MSN(DARPP2) and GABA(VGAT) were used to determine this]
• ELA, in common with the α2-/- decreases mIPSC amplitude & frequency of P60 accumbal core MSNs.

Mitchell et al 2018