This article is a part of Brainwaves, a Neuroscience Opinion column written by Leah Fogarty. Explore the rest of the series here.
The last 15 years have seen huge advances in neuroscience tools, with chemogenetics being no exception. Chemogenetics is a technique involving the engineering of molecules to allow the remote, reversible control of brain cells through the injection of an activating agent. It allows the temporary activation or inhibition of neurons, showing how specific cells affect specific behaviours. Chemogenetics was first used in 2007 and has been credited with dramatically improving our understanding of the neural control of behaviour. This understanding is critical for establishing the relationship between brain function and behaviour. This can help lead to effective treatment for disorders such as schizophrenia, addiction, and Parkinson’s disease.
Designer Receptors Exclusively Activated by Designer Drug (DREADD) technology is the main chemogenetic technique in use. DREADDs are modified G-protein coupled receptors, a type of receptor naturally found in humans that are usually responsible for slower, but longer-lasting cellular signalling. DREADDs work because they prefer to be activated by synthetic compounds instead of naturally circulating molecules in the body. Usually, DREADDS are selectively activated by a molecule called clozapine-N-Oxide (CNO).
Chemogenetics have allowed scientists to successfully model human diseases, such as Parkinson’s, in animals. This advance means that the effects of specific neurons in disease can be studied. Modulating the neurons using chemogenetics also allows researchers to determine possible therapeutic targets for treatment of the disease. Other research teams have used DREADDs to study drug seeking and sensitisation behaviour. Studies in non-human primates have used DREADDs to show that changing the connection between the orbitofrontal and rhinal cortex disrupts relative reward value. This finding offers a potential route for the treatment of addiction disorders.
Furthermore, chemogenetics have successfully been used to model drug-resistant epilepsy in preclinical trials. In this model, seizures are caused from a discrete brain area and are currently treated predominantly by resective surgery, which involves the removal of the brain area causing the seizures. Resective surgery is not ideal, because the brain areas causing epilepsy often lie close to areas associated with motor function and memory, meaning these major areas can be damaged during surgery. The use of chemogenetics offers an alternative approach with (hopefully) fewer side effects. It has been shown that DREADDs and CNO decrease neuronal excitability in specific brain areas associated with seizure activity, thereby reducing the chance of epileptic attack. While chemogenetics are an undeniably useful tool, they come with caveats that should not be forgotten.
For example, chemogenetics are not effective for treating diseases with complicated underlying causes. Chemogenetic tools are specific and have high spatial resolution, but they are not ideal for investigating neuropsychiatric disorders, which often have a complex origin. While chemogenetics has been able to reverse certain deficits associated with neuropsychiatric conditions, such as attention deficit hyperactivity disorder (ADHD), chemogenetics has not been able to uncover the main cause or attempt to fully cure these diseases. This means that chemogenetics can only help to find a cure for simpler conditions with a clear cause.
Chemogenetics can only help to find a cure for simpler conditions with a clear cause.
DREADDs target specific neurons using cell-specific promoters, which initiates gene transcription in specific neurons. Delivery using a virus as a vector allows the DREADD to reach the cell. The delivery of this vector can be optimised through recombinase strategies, which increase the specificity of DREADD expression by restricting their expression to only neurons with specific genetic markers. Nonetheless, this comes with caveats. Firstly, recombinase technology can lead to ‘leaky’ expression, where DREADDs continue to be expressed in areas where they have not been guided to. Furthermore, Cre, one type of recombinase system, has been shown to demonstrate tumour-causing, off-target effects in vivo, which is particularly worrying. Moreover, a drug with the capacity to cause cancer is unlikely to ever get approved. These potentially harmful effects raises questions about whether this is a worthwhile pursuit for drug development.
Potential clinical applications of chemogenetics will naturally rely on chronic or repeated drug administration to determine the efficacy, dosage, and safety of potential novel drugs. Research evaluating the effects of longer-term administration only began 10 years after the introduction of chemogenetics. In one study, Roth, the creator of DREADDs, found that chronic activation of serotonin has an antidepressant and anxiolytic effect, while short-term activation of the same neurons increases feelings of anxiety. This is not completely surprising, since short-term use of antidepressants increasing serotonin neurotransmission can cause feelings of anxiety, but longer-term use combats anxiety disorders. Nonetheless, this finding does suggest that long-term application of chemogenetic technology may induce neural adaptations which cannot occur when administration is only short term. While this warrants further investigation, the possibility that chemogenetics may lead to neural plasticity is exciting.
The possibility that chemogenetics may lead to neural plasticity is exciting.
On the other hand, other studies report the same, null, or even negative results on depressive behaviour after chronic administration. This lack of consistency shows that we cannot extrapolate findings from shorter-term studies to longer-term applications, and the chronic administration of CNO must be fully investigated. To me, it seems clear that the priority should be uncovering the molecular basis of chronic neuromodulation via DREADDs. This could answer questions regarding the diversity of effects after chronic activation of DREADDs. Researchers have suggested neural plasticity, varying feedback mechanisms, and receptor desensitisation as possible contributors.
CNO vs Clozapine
Evidence is unclear as to whether CNO can cross the blood-brain barrier in all species: it is possible in humans
CNO has also been found to not be entirely pharmacologically inert in itself. A 2016 study by MacLaren et al. showed that large doses of CNO reduced amphetamine-induced hyperlocomotion, which occurs when an animal continues to move without stopping. Even small doses of CNO can disrupt the response to loud acoustic stimuli, since animals given CNO had a reduced startle response. This means that an effective dosage of CNO, where off-target effects are minimised, must be established. MacLaren et al. also suggest that the inclusion of behavioural control groups, where an animal is given CNO but does not express DREADDs, may limit the confounding effects.
An alternative to CNO-activated DREADDs is kappa-opioid (KORD) DREADDS activated by a molecule known as salvinorin B. Yet, CNO still remains the favoured DREADD activator among scientists. This is likely because it is widely commercially available and is a better activator. It is evident that further research is needed to minimise the confounding factors associated with the use of CNO, should scientists prefer to use CNO-activated DREADDs.
Chemogenetics is a promising tool that can help to establish a better understanding of the relationship between neural circuitry and behaviour, with potential clinical applications. Nonetheless, the caveats of chemogenetics have clearly been overlooked.
Chemogenetics is a promising tool that can help to establish a better understanding of the relationship between neural circuitry and behaviour, with potential clinical applications. Nonetheless, the caveats of chemogenetics have clearly been overlooked. For example, we still do not know if we can trust results about CNO given all the confounding effects. Moreover, it still remains to be seen if including extra control groups will be enough to counter these confounders.
Perhaps chemogenetics are not the innovative miracle they seem.
The use of chemogenetics is also limited and cannot be used to determine causes or treatments for more complex neuropsychiatric disorders. Perhaps chemogenetics are not the innovative miracle they seem. Further research needs to be done to improve the specificity of DREADD technology, as well as to uncover the mechanisms underlying the effects seen upon chronic activation of DREADDs. To me, the most concerning possibility is the potential of tumours as an off-target effect. While chemogenetics are a useful tool to study behaviour, there can never be an effective clinical translation if the technology is known to cause tumours. The priority of researchers is to try and find out how these tumours are forming, or to try to minimise the risk. These caveats do not make chemogenetics pointless, but they do need to be addressed to make the most of this emerging technology.