The hypothalamic link between arousal and sleep homeostasis in mice.

Sleep and wakefulness are not simple, homogenous all-or-none states but represent a spectrum of substates, distinguished by behavior, levels of arousal, and brain activity at the local and global levels. Until now, the role of the hypothalamic circuitry in sleep-wake control was studied primarily with respect to its contribution to rapid state transitions. In contrast, whether the hypothalamus modulates within-state dynamics (state "quality") and the functional significance thereof remains unexplored. Here, we show that photoactivation of inhibitory neurons in the lateral preoptic area (LPO) of the hypothalamus of adult male and female laboratory mice does not merely trigger awakening from sleep, but the resulting awake state is also characterized by an activated electroencephalogram (EEG) pattern, suggesting increased levels of arousal. This was associated with a faster build-up of sleep pressure, as reflected in higher EEG slow-wave activity (SWA) during subsequent sleep. In contrast, photoinhibition of inhibitory LPO neurons did not result in changes in vigilance states but was associated with persistently increased EEG SWA during spontaneous sleep. These findings suggest a role of the LPO in regulating arousal levels, which we propose as a key variable shaping the daily architecture of sleep-wake states.

Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice.

Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools for remote control of targeted cell populations using chemical actuators that bind to modified receptors. Despite the popularity of DREADDs in neuroscience and sleep research, potential effects of the DREADD actuator clozapine-N-oxide (CNO) on sleep have never been systematically tested. Here, we show that intraperitoneal injections of commonly used CNO doses (1, 5, and 10 mg/kg) alter sleep in wild-type male laboratory mice. Using electroencephalography (EEG) and electromyography (EMG) to analyse sleep, we found a dose-dependent suppression of rapid eye movement (REM) sleep, changes in EEG spectral power during non-REM (NREM) sleep, and altered sleep architecture in a pattern previously reported for clozapine. Effects of CNO on sleep could arise from back-metabolism to clozapine or binding to endogenous neurotransmitter receptors. Interestingly, we found that the novel DREADD actuator, compound 21 (C21, 3 mg/kg), similarly modulates sleep despite a lack of back-metabolism to clozapine. Our results demonstrate that both CNO and C21 can modulate sleep of mice not expressing DREADD receptors. This implies that back-metabolism to clozapine is not the sole mechanism underlying side effects of chemogenetic actuators. Therefore, any chemogenetic experiment should include a DREADD-free control group injected with the same CNO, C21, or newly developed actuator. We suggest that electrophysiological sleep assessment could serve as a sensitive tool to test the biological inertness of novel chemogenetic actuators.

Scientists have developed ways to remotely turn on and off populations of neurons in the brain to test the role they play in behaviour. One technique that is frequently used is chemogenetics. In this approach, specific neurons are genetically modified to contain a special 'designer receptor' which switches cells on or off when its corresponding 'designer drug' is present. Recent studies have shown that the drug most commonly used in these experiments, clozapine-N-oxide (CNO), is broken down into small amounts of clozapine, an antipsychotic drug that binds to many natural receptors in the brain and modulates sleep. Nevertheless, CNO is still widely believed to not affect animals' sleep-wake patterns which in turn could influence a range of other brain activities and behaviours. However, there have been reports of animals lacking designer receptors still displaying unusual behaviours when administered CNO. This suggests that the breakdown of CNO to clozapine may cause off-target effects which could be skewing the results of chemogenetic studies. To investigate this possibility, Traut, Mengual et al. treated laboratory mice that do not have a designer receptor with three doses of CNO, and one dose of a new designer drug called compound-21 (C21) that is not broken down to clozapine. They found that high and medium doses of CNO, but also C21 altered the sleep-wake patterns of the mice and their brain activity during sleep. These findings show that CNO and C21 both have sleep-modulating effects on the brain and suggest that these effects are not only due to the production of clozapine, but the drugs binding to off-target natural receptors. To counteract this, Traut, Mengual et al. recommend optimizing the dose of drugs given to mice, and repeating the experiment on a control group which do not have the designer receptor. This will allow researchers to determine which behavioural changes are the result of turning on or off the neuron population of interest, and which are artefacts caused by the drug itself. They also suggest testing how newly developed designer drugs impact sleep before using them in behavioural experiments. Refining chemogenetic studies in these ways may yield more reliable insights about the role specific groups of cells have in the brain.

The role of snare proteins in cortical development.

Neural communication in the adult nervous system is mediated primarily through chemical synapses, where action potentials elicit Ca2+ signals, which trigger vesicular fusion and neurotransmitter release in the presynaptic compartment. At early stages of development, the brain is shaped by communication via trophic factors and other extracellular signaling, and by contact-mediated cell-cell interactions including chemical synapses. The patterns of early neuronal impulses and spontaneous and regulated neurotransmitter release guide the precise topography of axonal projections and contribute to determining cell survival. The study of the role of specific proteins of the synaptic vesicle release machinery in the establishment, plasticity, and maintenance of neuronal connections during development has only recently become possible, with the advent of mouse models where various members of the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex have been genetically manipulated. We provide an overview of these models, focusing on the role of regulated vesicular release and/or cellular excitability in synaptic assembly, development and maintenance of cortical circuits, cell survival, circuit level excitation-inhibition balance, myelination, refinement, and plasticity of key axonal projections from the cerebral cortex. These models are important for understanding various developmental and psychiatric conditions, and neurodegenerative diseases.

Gene Transfer with AAV9-PHP.B Rescues Hearing in a Mouse Model of Usher Syndrome 3A and Transduces Hair Cells in a Non-human Primate.

Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP.B, produces efficient transgene expression of a GFP reporter in both inner and outer hair cells of neonatal mice. We show also that AAV9-PHP.B mediates almost complete transduction of inner and outer HCs in a non-human primate. In a mouse model of Usher syndrome type 3A deafness (gene CLRN1), we use AAV9-PHP.B encoding Clrn1 to partially rescue hearing. Thus, we have identified a vector with promise for clinical treatment of hereditary hearing disorders, and we demonstrate, for the first time, viral transduction of the inner ear of a primate with an AAV vector.