Optogenetics 2 Flashcards
What is chemogenetics?
Use unique (inert) ligands to activate engineered receptors
What is chemogenetics used for?
Can use this approach to reversibly alter function of neurons in circuits and alter behaviour
What is a common chemogenetic approach?
DREADDs
What does DREADDS stand for?
Designer Receptors Exclusively Activated by Designer Drugs
What are DREADDS?
Receptors are engineered to interact with a pharmacologically inert ligand (designer drug)
Administration of the designer drug follows non-invasive control of function of cells
Cell-type specific expression of the exogenous receptor with the use of promotors
What is the major difference between optogenetics and chemogenetics?
Optogenetics provides precise temporal control of neuronal firing with light pulses, whereas chemogenetics provides the ability to modulate neuronal firing for several hours with the single administration of a designer drug
What are DREADDS based on?
Based on human muscarinic acetylcholine G-protein-coupled receptors
What variants of DREADDs have been made?
hM3Dq
hM4Di
== Can excite or inhibit neural activity depending on the choice of DREADD
How does hM3Dq work?
hM3Dq is a modified form of the human M3 muscarinic (hM3) receptor. It can be activated by the inert clozapine metabolite clozapine-N-oxide (CNO), engaging the Gq signaling pathway
Phospholipase C broken down into DAG and IP3
DAG goes on to actiavte protein kinase C whilst IP3 induces the release of calcium ions
Protein kinase C and Ca2+ release causes an increase in protein phosphorylation and activates Ca2+ binding proteins leading to depolarisation
How does hM4Di work?
hM4Di is a modified form of the human M4 muscarinic (hM4) receptor. It can be activated by the inert clozapine metabolite clozapine-N-oxide (CNO), engaging the Gi signaling pathway
inhibiting adenylyl cyclase activity, decreasing the production of cAMP from ATP, which, in turn, results in decreased activity of cAMP-dependent protein kinase. Therefore, the ultimate effect of Gi is the inhibition of the cAMP-dependent protein kinase, increasing protein phosphorylation can causing hyperpolarisation
What ligand specifically activates DREADDs?
CNO = clozapine-N-oxide
How do you express DREADDs?
- Injection of AAV vectors encoding DREADD into brain - use of promotor for cell-type specificity of expression – enables region specific expression
- Transgenic mice – use of cre-recombinase system to ensure cell-type specificity
- Cre-dependent AAV encoding DREADD
How is CNO administered?
Can be administered by injection directly into the brain or given orally - can activate the DREADDs non-invasively
What did Gomez et al. (2017) find?
Demonstrated that in vivo CNO does not cross the BBB instead, it is metabolised to clozapine which then crosses BBB. Clozapine is a typical antipsychotic and therefore works on dopamine receptors and other non-DREADD targets
Clozapine can act on non-DREADD targets so need to be careful when interpreting results - need adequate controls
What did Schulz et al. (2012) find regarding combining methods?
Demonstrated that it was possible to simultaneously measure fMRI BOLD and cellular calcium responses to electrical paw stimulation in their set up
Combined fmri with fiber-optic recordings of fluorescent calcium indicator signals to investigate this relationship in rat somatosensory cortex
Used a 7m long optic fibre connecting the mouse to a photomultiplier collecting the calcium responses
