Chemical Genetics and DREADD

How do scientists manipulate neurons| chemical genetics

The brain is working every second, no one wants it to stop spinning. But if scientists want to study the function of a certain brain region, they must make that brain region 'stop spinning'. The functioning of the brain relies on the discharge of billions of neurons, and stopping or inactivating a brain area at a microscopic level is equivalent to inactivating neurons within that area.

Brain function depends on neurons

The methods of inactivating neurons can generally be classified into two categories: reversible and irreversible. If a method only temporarily inactivates neurons and restores normal function after a period of time, then this method is reversible. On the contrary, it is irreversible.

In reversible technology, there is a technique called chemical genetics.

Chemical Genetics and DREADD

Chemical genetics is a product of the combination of chemical drugs and genetic technology, which refers to the use of genetic technology to modify biological proteins and explore the interaction between mutated proteins and chemical drug molecules. DREADD is the most commonly used chemical genetic technique by neuroscientists to manipulate neuronal responses.

The full name DREADD is long and difficult to pronounce, but it clearly expresses its meaning: artificially designed receptors are only activated by artificially designed drugs, Designer Receptor Exclusively Activated by Designer Drugs， The English abbreviation is DREADD.

Specifically, a protein receptor is artificially designed and expressed in neurons through viral vectors. This receptor specifically binds to a specific artificially designed drug and can activate or inhibit neuronal firing upon binding. After drug metabolism, the function of neurons will be restored.

DREADD schematic diagram. Left: endogenous receptors and ligands, right: artificially mutated receptors and drugs

Artificially designed drugs can be ingested through simple methods such as intramuscular, intravenous, intraperitoneal, or subcutaneous injection, or even oral administration. Drugs enter the bloodstream, cross the blood-brain barrier and enter brain tissue, ultimately binding to artificially designed protein receptors to exert their effects.

Chemical Genetics Workflow

There is a type of DREADD system that frequently appears in neuroscience research, which is the CNO-hM4Di/hM3Dq system.

CNO-hM4Di/hM3Dq system

The full name of CNO is clozapine N-oxide, which is an artificially designed drug in the DREADD system. And hM4Di and hM3Dq are artificially designed protein receptors, which are mutated human derived muscarinic receptors.

Muscarinic receptors are endogenous receptors for the neurotransmitter acetylcholine and are widely present in animal bodies. The muscarinic receptor is a G protein coupled receptor. When acetylcholine binds to the muscarinic receptor, the receptor is activated, conformational changes occur, and the coupled G protein is further activated. The G protein then triggers downstream reactions, ultimately activating or inhibiting neurons.

Muscarinic receptors are G protein coupled receptors. M1, M3, and M5 receptors activate neurons, while M2 and M4 inhibit neurons

Whether acetylcholine activates or inhibits neurons depends on the specific subtype of muscarinic receptors. If it is an M4 muscarinic receptor, coupled with Gi protein, the subsequent reaction of Gi protein will ultimately inhibit neurons. If it is M3 type, it attracts Gq protein, and downstream reactions of Gq protein will ultimately activate neurons.

In order to accurately activate or inhibit neurons, people mutate muscarinic receptors at specific sites. After mutation, muscarinic receptors no longer bind to endogenous acetylcholine and are specifically activated by the artificial drug CNO. The downstream reactions after activation remain unchanged.

CNO

The mutated human M4 muscarinic receptor is called hM4Di, which binds to CNO and inhibits neurons. The mutated M3 muscarinic receptor is called hM3Dq, which activates neurons when it binds to CNO.

CNO and hM4Di bind to suppress neuronal hyperpolarization, while hM3Dq binds to trigger neuronal firing

Internal mechanism

For a long time, scientists believed that CNOs entering the body cross the blood-brain barrier and act on neurons, but a 2017 article in Science changed people's view.

CNO cannot cross the blood-brain barrier.

Scientists injected CNO containing radioactive element C11 into mice and then used positron emission tomography (PET) technology to detect the location of C11 in the body. It was found that there was no trace of C11 starlight in the mouse brain, which means that CNOs with C11 did not enter the brain.

CNO cannot cross the blood-brain barrier (the blue hollow area represents the mouse brain)

Furthermore, scientists sacrificed mice to extract their blood, peripheral organs, and brain tissue, and then detected traces of radioactive isotope C11. Like PET testing, scientists only found C11 in blood and peripheral organs, and found nothing in the brain.

That's strange. After all, a pile of published articles have shown that the combination of CNO and hM4Di/hM3Dq can effectively inhibit or excite neurons in the brain.

When data cannot be fully explained, scientists begin to propose new hypotheses.

CNO will be converted into Clozapine in the body. Has Clozapine fulfilled its mission for CNO?

Clozapine (left) and CNO (right)

Firstly, scientists also bound the reflective isotope C11 to clozapine and injected it into mice. When the PET scan image was presented, scientists found that the Milky Way lit up in the mouse brain. That is to say, clozapine can cross the blood-brain barrier and enter the brain.

Clozapine can cross the blood-brain barrier and enter the brain (the yellow part is the mouse brain)

If clozapine wants to complete tasks for CNO, it must also be able to bind with hM4Di and hM3Dq. Scientists express hM4Di at a specific location in the mouse brain. If hM4Di has a strong affinity for chlorpromazine, then the location of hM4Di expression will also be the site of chlorpromazine aggregation. After 50 minutes of injection of chlorpromazine, PET technology and immunohistochemistry staining both illuminated the aggregated chlorpromazine at the expression site of hM4Di, indicating that chlorpromazine can indeed bind with high affinity to hM4Di.

Clozapine accumulates at the position of hM4Di

The final step is to verify whether clozapine combined with hM4Di can alter animal behavior. Firstly, scientists expressed hM4Di in the nucleus accumbens of mice, which is an important component of the brain's reward pathway that receives input from the dopamine central VTA. Previous experiments have shown that if the nucleus accumbens is inhibited, mice will become wilted and inactive. On the contrary, by activating the nucleus accumbens, mice become hyperactive.

If clozapine can bind to hM4Di and inhibit neurons in the nucleus accumbens, the amount of exercise in mice will be significantly reduced, and the experimental results are also consistent.

A small amount of clozapine (CLZ) can inhibit the movement of mice

Compared to CNO, achieving the same behavioral effects in mice requires only one percent of the dosage of chlorpromazine. Moreover, CNO only takes effect after being ingested into the body for a period of time, and this delay is exactly the time required for CNO to transform into clozapine in the body.

The above series of experiments indicate that CNO is converted into clozapine in vivo, and then binds to hM4Di to inhibit neuronal responses.

Side effects of CNO and Clozapine

High concentrations of CNO and clozapine not only bind to hM4Di and hM3Dq, but also compete with endogenous ligands to bind to endogenous receptors, resulting in side effects. These endogenous receptors include dopamine D1, D2 receptors, acetylcholine muscarinic M1, M3, M4 receptors, serotonin 2A receptors, etc. And the side effects are usually manifested in behavior as animal lethargy and reduced activity.

Since CNO and clozapine have side effects, are there better drugs available?

Chlorpromazine (DCZ)

In 2019, Japanese scientist Takafumi Minamimoto discovered another drug: clozapine (DCZ). As the name suggests, removing chlorine from the molecules of chlorpromazine.

Left: Clozapine, right: Clozapine

Chlorpromazine and chlorpromazine have extremely similar structures and have higher affinity with hM4Di and hM3Dq. Meanwhile, it has weak affinity with endogenous dopamine receptors, acetylcholine receptors, and serotonin receptors. That is to say, clozapine has higher selectivity and fewer side effects, making it a rising star drug of the new generation.

In monkey brains, the binding of chlorpromazine (DCZ) to hM4Di is more specific

Summarize

The methods of chemical genetics cause minimal damage to animals. After the virus is expressed in the brain, each experiment only requires muscle or intravenous injection of drugs to inhibit or excite one brain area, which is very suitable for human medical translation.

But chemical genetics also has drawbacks, such as poor time accuracy. The medication will work continuously for several hours, during which the neurons remain in an abnormal state. The cognition of the brain usually changes rapidly, with several cognitive processes occurring within seconds or even within a second. To separate these transient and sequential cognitive processes, there is an urgent need for faster and more flexible reversible means.

So, optogenetic technology stood out.