W8 - Neurotransmitters Systems II: GABA & Glycine

Question 1

What is GABA?

Answer 1

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central
nervous system (CNS).
* GABA was first identified in the mammalian nervous system in 1950
* In 1957, GABA was shown to inhibit action potential firing in crayfish neurons
* It is now known that GABA is the major inhibitory neurotransmitter in the human brain
* Approximately one third of synapses utilise GABA as their neurotransmitter
* GABA most commonly found as an inhibitory
neurotransmitter in local circuit interneurons

Question 2

What is GABA synthesis and storage?

Answer 2

Glucose is synthesised to glutamate

Glutamate
Glutamate decarboxylase (GAD) and Pyridoxal phosphate(derived from vitamin B6) (cofactor) catalyses the reaction from glutamate to GABA.
GABA

Synthesised in the nerve terminals
Transported into vesicles by vesicular inhibitory amino acid transporters (VIAAT).
Glutamate is stored in Round vesicles.
GABA is stored on Oval vesicles.

Lack of B6 can prevent this conversion leading to seizures due to a lack of neuroinhibitory drive.

Question 3

How does GABA re-uptake and degradation happen?

Answer 3

Once GABA is synthesised and stored, it is released by exocytosis in order to bind to post synaptic GABA receptors. This activity eventually needs to be terminated. This occurs via the reuptake and degradation.

Re-uptake:
Neurons and glial contain high-affinity Na+
dependent GABA re-uptake transporters (GATs)
 Neurons = GAT-1
 Glial cells = GAT-3

Degradation:
GABA
GABA transaminase (GABA-T) converts GABA to
Succinic semialdehyde
Succinic semialdehyde, dehydrogenase (SSADH) further converts it into
Succinic acid

Question 4

What are Neurotransmitter receptors?

Answer 4

There are two broad families of neurotransmitter receptor – ligand-gated ion channels (ionotropic) and G-protein coupled receptors (metabotropic).

Both are located on the plasma membrane of post synaptic cells, with an extracellular domain that detects the presence of neurotransmitters in the Synaptic cleft.

Ionotrophic - has a membrane spanning domain that forms the ion channel. Neurotransmitter binding to the receptor, opens this channel and allows for ions to pass through the membrane where it can increase in excitatory neurotranmitters or decrease in inhibitory neurotransmitters. Works in milliseconds.

Metabotrophic - 7 transmembrane domain shape with an extracellular domain for neurotransmitter binding. It activates molecular switches called G proteins with a, B and Y subunits. Then dissociates from the receptor and interact with ion channels or bind to other affected proteins. Slower pace.

Question 5

What are the GABA receptors?

Answer 5

GABA binds at both ionotropic and metabotropic GABA receptors.
IONOTROPIC <–> METABOTROPIC
GABAA RECEPTOR GABAB RECEPTOR
There is also a GABAC receptor, an ionotrophic one similar in structure and function, but is insensitive to a number of drugs that act on it.

Question 6

What are Ligand-gated Cl- channel, Pentameric structure, Multiple binding sites?

Answer 6

Ligand-gated Cl- channel:

Pentameric structure:
Six a subtypes (a 1–6)
Three B subtypes (B 1-3)
Three y subtypes (y1-3)
Also δ ε π θ subunits
2α 2β γ most common configuration

Multiple binding sites:
- Agonists/antagonists e.g. GABA
- Benzodiazepine binding site
- Channel blockers e.g. picrotoxin - acts as a CNS stimulant
- Channel modulators e.g. GA
- Allosteric modulators e.g. barbiturates

GABA A is post synaptic and a key drug target due to the number of different binding sites present on the receptor.

Question 7

What are the G-Protein coupled GABAB receptors

Answer 7

Dimers
Heteromers GABAB1 & GABAB2

GABAB receptors assemble as hetromeres comprised of GABAB1 and GABAB2 subunits
Neurotransmitter binding activated the G protein Gi/o, which dissociates from the GABAB receptor. G protein activates a secondary messenger pathway inhibiting an enzyme called adenylatcyclase, which reduces levels of the secondary messenger cyclic ANP leading to the activation of potassium channels. This leads to an E.flux of potassium ions out of the cell. They also block the voltage gated calcium channels, which is crucial for the action potential. Together this serves to cause hyperpolarisation.

Question 8

What are inhibitory neurotransmitters and hyperpolarisation?

Answer 8

Inhibitory neurotransmitters (e.g. GABA) can cause neuronal membrane hyperpolarisation –
displacement of a membrane potential towards a more negative value.

Hyperpolarisation involves the displacement of a membrane potential towards a more negative value. This inhibits action potential firing by increasing the stimulus required to fire that action potential. This occurs via an influx of negatively charged chloride ions. Also occurs via the influx of positively charged potassium ions.

Question 9

What is the cerebellum?

Answer 9

The cerebellum (or “little brain”) is a prominent hindbrain structure – it accounts for approximately 10% of the human brains volume.

Function:
* The cerebellum does not initiate movement but detects differences in “motor error” between an intended movement and the actual movement
* Aids the motor cortex to produces precise and co-ordinated movement

Is the function of the cerebellum conserved?
* It has been shown that, for example,
the cerebellum is important in
synchronisation of movement with
musical rhythm
* This may be widespread across the
animal kingdom…

Question 10

What is GABA projections in the cerebellum?

Answer 10

Purkinje cells are are a class of GABAergic neurons that comprise the principle projection
neurons of the cerebellar cortex.

• Purkinje cells have elaborate dendritic trees that receive convergent input from cells in the molecular layer
• Purkinje cells send GABAergic projections to deep cerebellar neurons
• Purkinje cell output to the deep cerebellar neurons generates an error connection signal that can modify movements
• This provides the basis for real-time control of
  precise and synchronous movement

Question 11

What is the balance of GABA and glutamate?

Answer 11

• GABA and glutamate are the major neurotransmitters in the brain – both work together to control the brain’s overall level of excitation.
• Remarkably, in one step, the major excitatory neurotransmitter in the brain is converted into the major inhibitory neurotransmitter in the brain!

GABA = inhibitory
Glutamate = Excitatory

Glutamate
Glutamate
decarboxylase (GAD)
Pyridoxal phosphate
(derived from vitamin B6) (co-factor)
GABA

Question 12

What is Epilepsy and how is the disruption of the balance between Glutamate and GABA related?

Answer 12

Epilepsy is a brain disorder characterised by periodic and unpredictable seizures mediated
by the rhythmic firing of large groups of neurons.

If there is too much excitation, the GABA system can be targeted to increase inhibition to restore balance.

• GABAA receptor enhancers - increases GABA neurotransmission by acting as positive allosteric modulators at GABAA receptors.

-GAT blockers - blocks the reuptake of GABA into the presynaptic neurone increasing the availability of GABA in the synaptic cleft.

-GABA transaminase inhibitor - Inhibits GABA transaminase. Catalyses the breakdown of GABA to increase its availability.

• GAD modulators - increases the activity of the enzyme GAD to increase the conversion of glutamate to GABA.
• Prodrug - inactive precursors like Progabide can be metabolised by the body into GABA to increase GABA availibility.

Other anti-epileptics directly decrease excitation.

Question 13

How is anxiety and GABA neurotransmission related?

Answer 13

Anxiety can be defined as a feeling of unease (e.g. worry or fear), which can range from mild
to severe.

Anxiety disorders (e.g. generalised anxiety disorder, panic disorder). Anxiolytics (e.g. benzodiazepines – GABAA receptor).
These drugs act as positive allosteric modulators at the GABAA receptor to increase GABA neurotransmission.

Question 14

What is glycine?

Answer 14

• Glycine was first identified in the spinal cord and brainstem in 1965
• In the 1967, glycine was shown to inhibit action potential firing in spinal neurons - much of this was performed using spinal cord neurones from cats.
• Glycine most commonly found as an inhibitory
  neurotransmitter in the ventral horn, the location for spinal interneuron terminals
• However, our understanding of the glycine receptor is lagging behind the GABA receptors – in part due to limited allosteric modulators of the receptor

Question 15

What is glycine synthesis?

Answer 15

3-phosphoglycerate(glycolysis)
Serine
Serine hydroxymethyl-transferase
Glycine

Synthesised in the nerve terminals.
Transported into vesicles by vesicular inhibitory
amino acid transporters (VIAAT). This is the same as GABA.

Glutamate is stored in Round vesicles
GABA and Glycine are stored in Oval vesicles.

Question 16

How does Glycine re-uptake and degradation work?

Answer 16

Re-uptake:
Neurons and glial contain high-affinity Na+
dependent glycine re-uptake transporters (GlyTs)
 Glial cells = GlyT-1
 Neurons = GlyT-2
Both of these function to transport glycine from synaptic cleft back into the liver cells or neurone for subsequent degredation.

Mutations in genes encoding for some of these transporters can result in hypoglycemia - neonatal disease characterised by lethargy and seizures.

Degradation:
Various enzymes responsible for the breakdown
of glycine – including the reversal of glycine
biosynthesis:
Glycine
Serine hydroxymethyl-transferase
Serine

Question 17

How do the glycine receptor function?

Answer 17

Ligand-gated Cl- channel:
Upon glycine binding to the ion channel allows the influx of negatively charged Cl- ions. This can lead to hyper polarisation when the membrane potential becomes more negative than the resting potential. This inhibits action potential firing by increasing the stimulus required.

Pentameric structure:
Four α subtypes (α1 – α4)
One β subtype
3α12β or 4α1β most common configuration
Agonist/antagonist binding sites unclear –
although plant alkaloid strychnine potently
blocks glycine receptors

Glycine activity terminated upon reuptake by glycine reuptake transporter (GlyT) before it is subsequently degraded.
The Glycine receptor is both presynaptic and post synaptic.

Question 18

What are the Glycine and NMDA receptors?

Answer 18

Glycine can act as a co agonist at the glutamate NMDA receptor. The ligand for this receptor is glutamate (GluN2 receptor) in addition to either Glycine or D-serine (GluN1) for ion channel opening to occur.

Question 19

What is Hyperkplexia and how does it relate to neurotransmission?

Answer 19

Hyperekplexia is a rare disorder characterised by hypertonia (increased muscle tone) and an
exaggerated startle response.
* Symptoms can manifest in relation to unexpected stimuli (e.g. loud noises)
Role of Glycine:
* Gene mutations (e.g. glycine receptors, glycine transporters) can disrupt normal glycinergic neurotransmission - so it is diminished.
* Can lead to neuronal hyperexcitability (by impairing glycinergic inhibition)
* Leads to hypertonia and exaggerated startle response

Question 20

What is special about Startle (myotonic, fainting) goats?

Answer 20

• In startle goats, there is a decreased muscle chloride conductance – can be caused by glycine
  receptor mutations
• As the goats mature, GABAA receptors are upregulated to compensate for this muscle chloride conductance - which can relieve the startle response in these animals.