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IntroductionNeonatal intensive care units (NICUs) provide life-saving care for preterm and sick neonates, but many medical procedures are painful and stress-inducing. Even a routine NICU procedure, such as the “heel lancing” blood-draw procedure, is an acutely painful, repetitive manipulation that has lasting negative impacts on pain perception and anxiety responses. The intersection of nociception and negative affect occurs in a brain region called the central nucleus of the amygdala (CeA), and neurons expressing corticotropin-releasing factor (CRF) have been implicated in studies of both anxiety and pain.MethodsUsing a two-hit model of trauma-induced pain vulnerability—where repetitive needle prickings occur during the first week of life (“our NICU model”), followed by a second stressor (e.g., fear conditioning) during adolescence—our lab has observed a mechanical hypersensitivity in rats that endured our NICU model that manifests only after fear conditioning. We have also observed changes to expression and activation of CeA-CRF neurons after the NICU-like experience with an acute increase followed by a lasting reduction in the number of CRF cells in the right CeA of adolescent male rats. However, the relationship between these changes and the observed behavioral outcomes remains unclear, as does the function of the remaining CRF cell population. We hypothesize that the remaining population of CRF-expressing CeA neurons are functionally altered by early life pain and stress and primed to respond more readily, such that vulnerability to stress-induced hypersensitivity is increased.ResultsThrough chemogenetic inhibition of the amygdala, or specifically CeA-CRF neurons, we demonstrate that development of stress-induced mechanical hypersensitivity after our NICU model is completely reversed through silencing the amygdala. Inhibiting only CeA-CRF neurons during fear conditioning led to a partial reversal of the hypersensitivity, suggesting that other populations of cells also play critical roles. Nevertheless, we demonstrate that the NICU-like experience results in a lasting hyperexcitability of CeA-CRF neurons during adolescence, confirming that this population is affected by the early life manipulations.DiscussionIn all, this study suggests that CeA-CRF neurons may have pro-nociceptive properties that are exacerbated by early life pain and result in maladaptive responding to subsequent traumatic events.

Abstract The field of chemogenetics has rapidly expanded over the last decade, and engineered receptors are currently utilized in the lab to better understand molecular interactions in the nervous system. We propose that chemogenetic receptors can be used for far more than investigational purposes. The potential benefit of adding chemogenetic neuromodulation to the current neurosurgical toolkit is substantial. There are several conditions currently treated surgically, electrically, and pharmacologically in clinic, and this review highlights how chemogenetic neuromodulation could improve patient outcomes over current neurosurgical techniques. We aim to emphasize the need to take these techniques from bench to bedside.

Non-rapid eye movement (NREM) sleep, characterized by slow-wave electrophysiological activity, underlies several critical functions, including learning and memory. However, NREM sleep is heterogeneous, varying in duration, depth, and spatially across the cortex. While these NREM sleep features are thought to be largely independently regulated, there is also evidence that they are mechanistically coupled. To investigate how cortical NREM sleep features are controlled, we examined the astrocytic network, comprising a cortex-wide syncytium that influences population-level neuronal activity. We quantified endogenous astrocyte activity in mice over natural sleep and wake, then manipulated specific astrocytic G-protein-coupled receptor (GPCR) signaling pathways in vivo. We find that astrocytic Gi- and Gq-coupled GPCR signaling separately control NREM sleep depth and duration, respectively, and that astrocytic signaling causes differential changes in local and remote cortex. These data support a model in which the cortical astrocyte network serves as a hub for regulating distinct NREM sleep features. Sleep has many roles, from strengthening new memories to regulating mood and appetite. While we might instinctively think of sleep as a uniform state of reduced brain activity, the reality is more complex. First, over the course of the night, we cycle between a number of different sleep stages, which reflect different levels of sleep depth. Second, the amount of sleep depth is not necessarily even across the brain but can vary between regions. These sleep stages consist of either rapid eye movement (REM) sleep or non-REM (NREM) sleep. REM sleep is when most dreaming occurs, whereas NREM sleep is particularly important for learning and memory and can vary in duration and depth. During NREM sleep, large groups of neurons synchronize their firing to create rhythmic waves of activity known as slow waves. The more synchronous the activity, the deeper the sleep. Vaidyanathan et al. now show that brain cells called astrocytes help regulate NREM sleep. Astrocytes are not neurons but belong to a group of specialized cells called glia. They are the largest glia cell type in the brain and display an array of proteins on their surfaces called G-protein-coupled receptors (GPCRs). These enable them to sense sleep-wake signals from other parts of the brain and to generate their own signals. In fact, each astrocyte can communicate with thousands of neurons at once. They are therefore well-poised to coordinate brain activity during NREM sleep. Using innovative tools, Vaidyanathan et al. visualized astrocyte activity in mice as the animals woke up or fell asleep. The results showed that astrocytes change their activity just before each sleep–wake transition. They also revealed that astrocytes control both the depth and duration of NREM sleep via two different types of GPCR signals. Increasing one of these signals (Gi-GPCR) made the mice sleep more deeply but did not change sleep duration. Decreasing the other (Gq-GPCR) made the mice sleep for longer but did not affect sleep depth. Sleep problems affect many people at some point in their lives, and often co-exist with other conditions such as mental health disorders. Understanding how the brain regulates different features of sleep could help us develop better – and perhaps more specific – treatments for sleep disorders. The current study suggests that manipulating GPCRs on astrocytes might increase sleep depth, for example. But before work to test this idea can begin, we must first determine whether findings from sleeping mice also apply to people.

•Combined brain perturbation and neuroimaging can reveal causal brain mechanisms.•Overview of perturbation methods used with non-human primate neuroimaging.•Methodological considerations of the different techniques are discussed.•Translational potential and future directions are laid out and critically assessed. Brain perturbation studies allow detailed causal inferences of behavioral and neural processes. Because the combination of brain perturbation methods and neural measurement techniques is inherently challenging, research in humans has predominantly focused on non-invasive, indirect brain perturbations, or neurological lesion studies. Non-human primates have been indispensable as a neurobiological system that is highly similar to humans while simultaneously being more experimentally tractable, allowing visualization of the functional and structural impact of systematic brain perturbation. This review considers the state of the art in non-human primate brain perturbation with a focus on approaches that can be combined with neuroimaging. We consider both non-reversible (lesions) and reversible or temporary perturbations such as electrical, pharmacological, optical, optogenetic, chemogenetic, pathway-selective, and ultrasound based interference methods. Method-specific considerations from the research and development community are offered to facilitate research in this field and support further innovations. We conclude by identifying novel avenues for further research and innovation and by highlighting the clinical translational potential of the methods.

The locus coeruleus (LC) projects throughout the brain and spinal cord and is the major source of central noradrenaline. It remains unclear whether the LC acts functionally as a single global effector or as discrete modules. Specifically, while spinal-projections from LC neurons can exert analgesic actions, it is not known whether they can act independently of ascending LC projections. Using viral vectors taken up at axon terminals, we expressed chemogenetic actuators selectively in LC neurons with spinal (LC:SC) or prefrontal cortex (LC:PFC) projections. Activation of the LC:SC module produced robust, lateralised anti-nociception while activation of LC:PFC produced aversion. In a neuropathic pain model, LC:SC activation reduced hind-limb sensitisation and induced conditioned place preference. By contrast, activation of LC:PFC exacerbated spontaneous pain, produced aversion and increased anxiety-like behaviour. This independent, contrasting modulation of pain-related behaviours mediated by distinct noradrenergic neuronal populations provides evidence for a modular functional organisation of the LC.

INTRODUCTION Alzheimer's disease (AD) primarily affects episodic memory, which relies on the medial temporal lobe, including the hippocampus and lateral entorhinal cortex (LEC). However, it remains unclear whether memory deficits in AD reflect disrupted encoding of new experiences or impaired retrieval of previously stored information. METHODS APPJ20 transgenic mice were used to investigate memory deficits. Neuronal populations activated during the learning phase of associative and non‐associative tasks were tagged to express the excitatory chemogenetic receptor hM3Dq. Chemogenetic activation of these tagged neurons was performed during the recall phase of the tasks. RESULTS Chemogenetic reactivation of LEC or dentate gyrus (DG) learning‐tagged neurons rescued memory performance in associative and non‐associative tasks, respectively. Neuronal activation, assessed using c‐Fos as a marker, revealed a specific deficit in the reactivation of neurons recruited during learning. DISCUSSION Chemogenetic reactivation of neuronal ensembles in the LEC and DG restored memory performance, suggesting that memory deficits in APPJ20 mice are associated with a failure in the endogenous reactivation of learning‐relevant neurons. Highlights APPJ20 mice exhibited early entorhinal synaptic dysfunction and impaired episodic‐like memory retrieval. At a later stage, hippocampal synaptic function became impaired, leading to altered non‐associative memory performance. The analysis of neuronal activation using c‐Fos revealed a specific impairment of the subpopulation recruited during memory encoding. Chemogenetic reactivation of LEC learning‐tagged neurons rescued associative memory performance in 2‐month‐old APPJ20 mice, while promoting dendritic spine maturation and stabilization in LEC neurons. Chemogenetic reactivation of DG learning‐tagged neurons in 6‐month‐old APPJ20 mice restored non‐associative memory retrieval. This study supports the hypothesis that during AD progression, memory is encoded but not accessible through natural cues alone.

A growing body of evidence suggests that dopamine plays a role in sleep–wake regulation, but the dopamine-producing brain areas that control sleep–wake states are unclear. In this study, we chemogenetically activated dopamine neurons in the ventral midbrain of mice to examine the role of these neurons in sleep–wake regulation. We found that activation of dopamine neurons in the ventral tegmental area (VTA), but not in the substantia nigra, strongly induced wakefulness, although both cell populations expressed the neuronal activity marker c-Fos after chemogenetic stimulation. Analysis of the pattern of behavioral states revealed that VTA activation increased the duration of wakefulness and decreased the number of wakefulness episodes, indicating that wakefulness was consolidated by VTA activation. The increased wakefulness evoked by VTA activation was completely abolished by pretreatment with the dopamine D 2 /D 3 receptor antagonist raclopride, but not by the D 1 receptor antagonist SCH23390. These findings indicate that the activation of VTA dopamine neurons promotes wakefulness via D 2 /D 3 receptors.

Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene (Agtr2) expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. Agtr2-expressing macrophages (MΦs) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral MΦs as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain.

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.

A study that monitored the expression and function of designer receptors called DREADDs in macaque monkeys for a period of three years demonstrates that they are effective in long-term studies of nonhuman primates.