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A collaborative team of researchers in DPAG and Pharmacology led by Dr Lukas Krone have uncovered striking new data demonstrating that two widely used designer drugs used to turn populations of neurons on and off in the brain cause unexpected effects on sleep. These results demonstrate a critical need to improve chemogenetic approaches in behavioural studies.

Designer drugs used as 'inert' actuators of chemogenetic DREADD receptors to control the activity of selected neuronal populations have off-target effects on sleep in laboratory mice that may skew results of behavioural studies

The ability to remotely turn populations of neurons on and off in the brain is an important scientific method to assess their role in behaviour. During the sleep-wake cycle, selected neurons can be temporarily activated or inhibited in mice to systematically test which ones promote sleep or wakefulness. A commonly used technique is a ‘key-lock’ system called chemogenetics, in which researchers genetically modify specific neurons to contain a ‘designer receptor’ (also known as DREADD), which are then activated by specially created designer drugs.

Recent research has found that the most commonly used designer drug – clozapine-N-oxide (CNO) – can be partially converted into the antipsychotic drug clozapine that binds to many of the brain’s receptors. Despite the implications of this finding, it has been widely believed that CNO does not affect sleep-wake patterns. Therefore, many experiments have been conducted without control groups that receive the designer drug but do not express the designer receptors. This has led to a lack of concrete data to verify whether all changes to the sleep-wake cycle result from manipulating the targeted neurons or whether some alterations might result from direct effects of CNO or its metabolite clozapine. Despite widespread success of chemogenetics in both confirming known brain functions and revealing several new ones, some researchers have reported unexplained behavioural changes, such as reduced locomotor activity, when designer drugs were given to animals that do not have designer receptors.

A University of Oxford team, led by DPAG's Dr Lukas Krone, former DPhil student and presently an academic visitor, in Professor Vladyslav Vyazovskiy's group in collaboration with the Molnár group in DPAG and the Akerman group in the Department of Pharmacology, commenced a study to produce data to validate that CNO does not affect sleep-wake patterns in mice. Dr Krone said: “Chemogenetics is used by hundreds of researchers around the globe to probe the causal role of specific neuron populations in a wide range of animal behaviours, not only sleep, so our aim was to ensure that our own and other chemogenetic experiments are not confounded by effects of CNO without activation of the designer receptors.”

The research team applied three different doses of CNO – low, medium and high – to normal laboratory mice without designer receptors. They found that medium and high CNO doses systematically altered sleep patterns and brain activity during sleep. On these two doses, mice showed more consolidated sleep, characterised by longer sleep episodes, and fewer spontaneous awakenings from sleep. The mice also had less rapid eye movement (REM) sleep - the sleep stage considered to be important for processing of emotional memories. Furthermore, the electroencephalogram showed slower activity during non-rapid eye movement sleep (NREM) - the main sleep stage.

Researchers also tested a newer designer drug called compound-21 (C21) that cannot convert into the antipsychotic drug clozapine. They found that C21 also altered sleep in an almost identical pattern to CNO. This is understood to be the first study demonstrating the behavioural effects of a designer drug that cannot convert to clozapine. Dr Krone said: “This finding is particularly important because many research groups have changed their protocols to use C21 and other novel designer drugs instead of CNO under the assumption that the lack of conversion to clozapine would exclude the possibility of side effects induced by the designer drug.

Dr Krone continued: “Overall, our findings suggest that both designer drugs have direct sleep-modulating effects on the brain. This has important implications for the application of chemogenetics in neurosciences far beyond the field of sleep research.

“Our findings aid researchers across many fields to perform better experiments. Optimising the dosing of designer drugs, and using designer-receptor-free control groups that receive the same dose of any designer drug, are simple means to perform unbiased chemogenetic experiments.

“This approach clarifies which behavioural changes are indeed mediated by the activation or inhibition of the neuronal populations of interest and which are artifacts due to direct effects of the designer drugs. Through this refinement, experimental results become more reliable, which reduces the number of experiments and animals needed to draw a valid scientific conclusion.

“Our findings also help the development of improved chemogenetic tools. Because sleep regulation relies on the fine-tuning of many brain signals, sleep recordings in designer-receptor-free animals will provide an elegant method to test if novel designer drugs affect brain signalling.”

The team have made their ground breaking data and analysis code openly accessible for the scientific community to apply their analysis to their own findings. The project has also been selected for oral presentation at Euro Sleep 2022 and Dr Lukas Krone recognised with the Young Scientists Award by the European Sleep Research Society.

The full paper ‘Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice’ is available to read in eLife.