DREADDs

Question 1

controlling neural activity

Answer 1

Neural activity is the foundation of the nervous system
* Manipulating neural activity allows us to breakdown the function of the nervous system, the role of specific cells and circuits, and behaviour
* Historically, lesion studies or direct electrical stimulation were the main ways of manipulating neural activity
* Global vs local control

Question 2

chemogenetics

Answer 2

• A process where macromolecules can be engineered to interact with a previously unrecognised small molecules
• A method where proteins are engineered to interact with small molecular chemical actuators that
  they did not previously recognise
• Wide range of engineered proteins:
  -Kinases
  -Non-kinase enzymes
  -GPCRs
  -Ligand gated ion channels

Question 3

chemogenetic receptor criteria

Answer 3

1) Not be receptive to endogenous ligands
2) Have minimal/no endogenous activity in the absence of ligand binding
3) Have high affinity for the ligand

Question 4

DREADDS

Answer 4

• A subclass of engineered GPCRs
• Activation of DREADDs alters neuronal activity
• Clozapine-N-Oxide (CNO), a pharmacologically inert* metabolite of the antipsychotic drug clozapine is commonly used to activate DREADDs
• DREADDs were first derived by mutating the human M3 muscarinic receptor (hM3)
• hM3Dq refers to human M3 muscarinic DREADD receptor couple to Gq (one of the excitatory G proteins)

Question 5

hM3Dq

Answer 5

• Activation of hM3Dq enhances neuronal firing by activating Gq signalling in neuronal and non-neuronal cells
• CNO activation of hM3Dq depolarises neurons which can be used to drive behaviours (e.g. feeding)
• Activation of hM3Dq also induces intracellular calcium release > can be used to alter astrocyte function/activity

Question 6

hM4Di

Answer 6

• hM4Di refers to human muscarinic DREADD receptor
• Used to inihibit neural activity by activating Gi-mediated signalling
• Activates potassium channels and hyperpolarises cells
• Can “silence” synapses by inhibiting neurotransmitter release

Question 7

silencing synapses with hM4Di

Answer 7

• Applying CNO to presynaptic neurons transfected with hM4Di increases the rate of synaptic failure (the rate at which presynaptic action potential firing fails to produce a current in the postsynaptic cell)

Question 8

axon selective hM4Di

Answer 8

• hM4Di causes hyperpolarisation (K+ efflux) and synaptic release inhibition
• If hM4Di is selectively located on axons/axonal terminals, its primary effect will be to inhibit synaptic release

Question 9

K opioid derived DREADD (KORD)

Answer 9

• Newer subtype of DREADD
• Activated by salvorin B which is pharmacologically inert and has no known other molecular target
• Activation of KORD induces hyperpolarisation and inhibits neurotransmitter release
• Strong inhibitor of neurotransmitter release
• Can be combined with a CNOactivated hM3Dq DREADD for bidirectional control of neural activity

Question 10

CNO

Answer 10

• Activates DREADDs with low nanomolar concentrations of CNO
  – ~0.5-10mg/kg
• Long lasting- 1 injection lasts ~60 minutes in vivo
  – EC50~8.1nM
• Low doses of CNO used when you want short lasting DREADD activation
• CNO can back metabolise to clozapine and could have clozapine side effects, but the extent of back metabolism is low at the concentrations of CNO administered
• Studies have shown that CNO is not as exclusive as initially reported
• Reports of off-target effects throughout the CNS
• Some targets include serotonin receptors, muscarinic receptors, histamine receptors, dopamine receptors
• higher doesnt always result in changes in behaviour

Question 11

CNO alternatives

Answer 11

compound 21
deschloroclozapine

Question 12

compound 21

Answer 12

• A more potent agonist of the hM3Dq DREADD than CNO
  – EC50 ~ 2.95
• Can use lower concentrations for a similar response
  <0.5mg/kg
• Doesn’t seem to metabolise into CNO or CLZ
• Minimal off-target behavioural effects

Question 13

deschloroclozapine

Answer 13

• Can be used a very low doses
  – ~0.001-0.1mg/kg
• Minimal off target effects

Question 14

salvorin B

Answer 14

• Salvorin A is a psychotropic agent
• Salvorin B is an inert ligand
• Short lasting effect (~5 minutes)

Question 15

DREADDs to interrogate behaviour

Answer 15

• Express DREADDs in specific cell types and in brain regions/locations
• The time of DREADD activation can be controlled (i.e. when the drug is administered). Can observe behaviours before/during/after DREADD activation
• Salvorin B activated DREADDs are more useful when you want to observe the effects of transiently changing neural activity
• CNO activated DREADDs are more useful when you want to observe the effect of tonically changing neural activity (e.g. serotonergic neurons)

Question 16

DREADDs in drug development

Answer 16

• DREADDs can identify which GPCR pathways are useful therapeutic targets
• “Biased” drug design
• Targeting specific cell types, within a certain circuit narrows down the effect and consequences of targeting a specific G protein pathway

Question 17

synthetic ion channel: GluCl

Answer 17

• Aimed at modifying the membrane potential
• Modified glutamate gated chloride channel
• Ivermectin (anti-parasitic drug) activates GluCl
• Ivermectin activation occurs at low concentrations (5mg/kg)
• High concentrations of ivermectin activates GABAA
  receptors>tremors and paralysis
• Long lasting behavioural effects (days) after a single administration

Question 18

glycinergic channel

Answer 18

• Glycine gated chloride channels regulate inhibitory neurotransmission in the spinal cord and brainstem
• Glycine gated chloride channels also expressed non-synaptically in the brain
• Very low concentrations (10nM) increases the amount of chloride ion flow/post-synaptic inhibition
• So far, only appears to be effective in regulating spinal cord neurons