Regulation of Energy Expenditure by Brainstem GABA Neurons.

Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.

Astrocytic Activation Generates De Novo Neuronal Potentiation and Memory Enhancement.

Astrocytes respond to neuronal activity and were shown to be necessary for plasticity and memory. To test whether astrocytic activity is also sufficient to generate synaptic potentiation and enhance memory, we expressed the Gq-coupled receptor hM3Dq in CA1 astrocytes, allowing their activation by a designer drug. We discovered that astrocytic activation is not only necessary for synaptic plasticity, but also sufficient to induce NMDA-dependent de novo long-term potentiation in the hippocampus that persisted after astrocytic activation ceased. In vivo, astrocytic activation enhanced memory allocation; i.e., it increased neuronal activity in a task-specific way only when coupled with learning, but not in home-caged mice. Furthermore, astrocytic activation using either a chemogenetic or an optogenetic tool during acquisition resulted in memory recall enhancement on the following day. Conversely, directly increasing neuronal activity resulted in dramatic memory impairment. Our findings that astrocytes induce plasticity and enhance memory may have important clinical implications for cognitive augmentation treatments.

Dentate gyrus is needed for memory retrieval.

The hippocampus is crucial for acquiring and retrieving episodic and contextual memories. In previous studies, the inactivation of dentate gyrus (DG) neurons by chemogenetic- and optogenetic-mediated hyperpolarization led to opposing conclusions about DG's role in memory retrieval. One study used Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-mediated clozapine N-oxide (CNO)-induced hyperpolarization and reported that the previously formed memory was erased, thus concluding that denate gyrus is needed for memory maintenance. The other study used optogenetic with halorhodopsin induced hyperpolarization and reported and dentate gyrus is needed for memory retrieval. We hypothesized that this apparent discrepancy could be due to the length of hyperpolarization in previous studies; minutes by optogenetics and several hours by DREADD/CNO. Since hyperpolarization interferes with anterograde and retrograde neuronal signaling, it is possible that the memory engram in the dentate gyrus and the entorhinal to hippocampus trisynaptic circuit was erased by long-term, but not with short-term hyperpolarization. We developed and applied an advanced chemogenetic technology to selectively silence synaptic output by blocking neurotransmitter release without hyperpolarizing DG neurons to explore this apparent discrepancy. We performed in vivo electrophysiology during trace eyeblink in a rabbit model of associative learning. Our work shows that the DG output is required for memory retrieval. Based on previous and recent findings, we propose that the actively functional anterograde and retrograde neuronal signaling is necessary to preserve synaptic memory engrams along the entorhinal cortex to the hippocampal trisynaptic circuit.

Indole induces anxiety-like behaviour in mice mediated by brainstem locus coeruleus activation.

The gut microbiota produces metabolites that enrich the host metabolome and play a part in host physiology, including brain functions. Yet the biological mediators of this gut-brain signal transduction remain largely unknown. In this study, the possible role of the gut microbiota metabolite indole, originating from tryptophan, was investigated. Oral administration of indole to simulate microbial overproduction of this compound in the gut consistently led to impaired locomotion and anxiety-like behaviour in both C3H/HeN and C57BL/6J mice. By employing c-Fos protein expression mapping in mice, we observed a noticeable increase in brain activation within the dorsal motor nucleus of the vagus nerve (DMX) and the locus coeruleus (LC) regions in a dose-dependent manner. Further immune co-labelling experiments elucidated that the primary cells activated within the LC were tyrosine hydroxylase positive. To delve deeper into the mechanistic aspects, we conducted chemogenetic activation experiments on LC norepinephrine neurons with two doses of clozapine N-oxide (CNO). Low dose of CNO at 0.5 mg/kg induced no change in locomotion but anxiety-like behaviour, while high dose of CNO at 2 mg/kg resulted in locomotion impairment and anxiety-like behaviour. These findings support the neuroactive roles of indole in mediating gut-brain communication. It also highlights the LC as a novel hub in the gut-brain axis, encouraging further investigations.

Disrupting direct inputs from the dorsal subiculum to the granular retrosplenial cortex impairs flexible spatial memory in the rat.

In a changing environment, animals must process spatial signals in a flexible manner. The rat hippocampal formation projects directly upon the retrosplenial cortex, with most inputs arising from the dorsal subiculum and terminating in the granular retrosplenial cortex (area 29). The present study examined whether these same projections are required for spatial working memory and what happens when available spatial cues are altered. Consequently, injections of iDREADDs were made into the dorsal subiculum of rats. In a separate control group, GFP-expressing adeno-associated virus was injected into the dorsal subiculum. Both groups received intracerebral infusions within the retrosplenial cortex of clozapine, which in the iDREADDs rats should selectively disrupt the subiculum to retrosplenial projections. When tested on reinforced T-maze alternation, disruption of the subiculum to retrosplenial projections had no evident effect on the performance of those alternation trials when all spatial-cue types remained present and unchanged. However, the same iDREADDs manipulation impaired performance on all three alternation conditions when there was a conflict or selective removal of spatial cues. These findings reveal how the direct projections from the dorsal subiculum to the retrosplenial cortex support the flexible integration of different spatial cue types, helping the animal to adopt the spatial strategy that best meets current environmental demands.

Effect of Coffee and Chocolate Ingestion on Clozapine Dose and on Plasma Clozapine and Norclozapine Concentrations in Clinical Practice.

BACKGROUND: Some reports point to dietary caffeine intake as a cause of increased plasma clozapine concentrations in certain patients. METHODS: We compared clozapine dose and plasma clozapine and N-desmethylclozapine (norclozapine) concentrations in male and female smokers and nonsmokers in relation to reported (i) coffee (caffeine) and (ii) chocolate (caffeine and theobromine) intake in samples submitted for clozapine therapeutic drug monitoring, 1993-2017. RESULTS: There was information on coffee ingestion for 16,558 samples (8833 patients) from males and 5886 samples (3433 patients) from females and on chocolate ingestion for 12,616 samples (7568 patients) from males and 4677 samples (2939 patients) from females. When smoking was considered, there was no discernible effect of either coffee or chocolate ingestion either on the median dose of clozapine or on the median plasma clozapine and norclozapine concentrations in men and in women. However, cigarette smoking was associated with higher coffee and chocolate consumption. Although male nonsmokers who reported drinking 3 or more cups of coffee daily had significantly higher median plasma clozapine and norclozapine concentrations than those who drank less coffee, they were also prescribed a significantly higher clozapine dose. There was no clear effect of coffee ingestion on plasma clozapine and norclozapine in female nonsmokers. IMPLICATIONS: Inhibition of clozapine metabolism by caffeine at the doses of caffeine normally encountered in those treated with clozapine is unlikely even in male nonsmokers. Measurement of plasma caffeine in an appropriate sample should be considered in any future investigation into a presumed clozapine-caffeine interaction.

Plasma Clozapine and N -Desmethylclozapine (Norclozapine) Concentrations and the Clozapine/Norclozapine Ratio : Effect of Dose, Sex, and Cigarette Smoking.

BACKGROUND: Smoking enhances plasma clozapine clearance, but the magnitude of the effect across the dose and age ranges is unclear. METHODS: We audited clozapine dose and predose plasma clozapine and N -desmethylclozapine (norclozapine) concentrations by sex and smoking habit in samples submitted for clozapine TDM, 1996-2017. RESULTS: There were 105,316/60,792 and 34,288/31,309 samples from male/female smokers/nonsmokers, respectively. There were distinct dose-median plasma concentration trajectories for male/female smokers/nonsmokers across the range <50 to >850 mg d -1 . For both sexes, the percentage difference in median plasma clozapine in nonsmokers versus smokers averaged 50% but was greatest for men (76%) and women (59%) in the 151 to 250 mg d -1 dose band. In men, the percentage difference declined steadily to 34% at doses of ≥850 mg d -1 . In women, the difference after falling initially remained relatively constant at 40% to 54%. The pattern in median plasma clozapine/norclozapine ratio by plasma clozapine concentration and dose groups was independent of sex and smoking habit, but increased with plasma clozapine concentration (higher ratio at higher concentrations) and also changed with dose. Median plasma clozapine concentration and median clozapine dose by sex and smoking habit were similar up to age 60 years. Proportional weight gain was similar over time in smokers and nonsmokers of either sex. IMPLICATIONS: These data explain the variations in the effect size of starting or stopping smoking on plasma clozapine concentration at constant dose reported in different studies. Changes in smoking habit in patients prescribed clozapine require prompt dose adjustment.

A circuit from hippocampal CA2 to lateral septum disinhibits social aggression.

Although the hippocampus is known to be important for declarative memory, it is less clear how hippocampal output regulates motivated behaviours, such as social aggression. Here we report that pyramidal neurons in the CA2 region of the hippocampus, which are important for social memory, promote social aggression in mice. This action depends on output from CA2 to the lateral septum, which is selectively enhanced immediately before an attack. Activation of the lateral septum by CA2 recruits a circuit that disinhibits a subnucleus of the ventromedial hypothalamus that is known to trigger attack. The social hormone arginine vasopressin enhances social aggression by acting on arginine vasopressin 1b receptors on CA2 presynaptic terminals in the lateral septum to facilitate excitatory synaptic transmission. In this manner, release of arginine vasopressin in the lateral septum, driven by an animal's internal state, may serve as a modulatory control that determines whether CA2 activity leads to declarative memory of a social encounter and/or promotes motivated social aggression.

The coding of valence and identity in the mammalian taste system.

The ability of the taste system to identify a tastant (what it tastes like) enables animals to recognize and discriminate between the different basic taste qualities1,2. The valence of a tastant (whether it is appetitive or aversive) specifies its hedonic value and elicits the execution of selective behaviours. Here we examine how sweet and bitter are afforded valence versus identity in mice. We show that neurons in the sweet-responsive and bitter-responsive cortex project to topographically distinct areas of the amygdala, with strong segregation of neural projections conveying appetitive versus aversive taste signals. By manipulating selective taste inputs to the amygdala, we show that it is possible to impose positive or negative valence on a neutral water stimulus, and even to reverse the hedonic value of a sweet or bitter tastant. Remarkably, mice with silenced neurons in the amygdala no longer exhibit behaviour that reflects the valence associated with direct stimulation of the taste cortex, or with delivery of sweet and bitter chemicals. Nonetheless, these mice can still identify and discriminate between tastants, just as wild-type controls do. These results help to explain how the taste system generates stereotypic and predetermined attractive and aversive taste behaviours, and support the existence of distinct neural substrates for the discrimination of taste identity and the assignment of valence.

Localized chemogenetic silencing of inhibitory neurons: a novel mouse model of focal cortical epileptic activity.

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. GABAergic silencing using Clozapine-N-Oxide (CNO) demonstrated reliable induction of local epileptiform events in the electroencephalogram signal of awake freely moving mice. Anesthetized mice experiments showed consistent induction of focal epileptiform-events in both the barrel cortex (BC) and the medial prefrontal cortex (mPFC), accompanied by high-frequency oscillations, a known characteristic of human seizures. Epileptiform-events showed propagation indication with favored propagation pathways: from the BC on 1 hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, sensory whisker-pad stimulation evoked BC epileptiform events post-CNO, highlighting the potential use of this model in studying sensory-evoked seizures. Combined, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile, and reliable model of focal cortical epileptic activity suitable for systematically studying cortical ictogenesis in different cortical areas.

Chemogenetic modulation of the medial prefrontal cortex regulates resistance to acute stress-induced cognitive impairments.

The medial prefrontal cortex (mPFC) has been implicated in regulating resistance to the effects of acute uncontrollable stress. We previously showed that mPFC-lesioned animals exhibit impaired object recognition memory after acute exposure to a brief stress that had no effect in normal animals. Here, we used designer receptors exclusively activated by designer drugs to determine how modulating mPFC activity affects recognition-memory performance under stressful conditions. Specifically, animals with chemogenetic excitation or inhibition of the mPFC underwent either a brief ineffective stress (20-min restraint + 20 tail shocks) or a prolonged effective stress (60-min restraint + 60 tail shocks). Subsequent recognition memory tests showed that animals with chemogenetic mPFC inhibition exposed to brief stress showed impairment in an object recognition memory task, whereas those with chemogenetic mPFC excitation exposed to prolonged stress did not. Thus, the present findings the decreased mPFC activity exacerbates acute stress effects on memory function whereas increased mPFC activity counters these stress effects provide evidence that the mPFC bidirectionally modulates stress resistance.

A study of the pharmacokinetics of clozapine and its metabolites by the dynamics of its distribution in the oral fluid of healthy volunteers.

Clozapine (CLZ) -related accidents or crimes are common in the world. Oral fluid drug detection is a convenient measure of dealing with things like that. There has not been any literature reported detailedly the representation rule of clozapine and its metabolites in oral fluid so far. The study aimed to describe the pharmacokinetics of CLZ and its metabolites N-desmethylclozapine and clozapine-N-oxide in human oral fluid after a single 12.5 mg oral dose of CLZ. Twenty-nine volunteers, including 20 males and 9 females, were recruited, and 2 mL oral fluid was collected from each participant at post-consumption time-points of prior (zero), 0.5, 1.5, 3, 5, 8, 12, 24, 36, 51, 82, and 130 h, respectively. Analytes of interest were extracted with solid-phase extraction and analyzed with liquid chromatography tandem mass spectrometry method. Pharmacokinetic parameters were calculated using the pharmacokinetic software DAS according to the non-compartment model. The maximum concentration, the time of maximum concentration, oral clearance, and the elimination half-life of clozapine were 16.57 ± 9.63 ng/mL, 4.53 ± 3.61 h, 57.65 ± 23.77 L/h and 53.58 ± 52.28 h, respectively. The maximum concentration, the time of maximum concentration, and the elimination half-life of the metabolite N-desmethylclozapine were 3.08 ± 1.19 ng/mL, 9.38 ± 9.33 h and 62.67 ± 82.57 h, respectively; of clozapine-N-oxide were 1.15 ± 0.36 ng/mL, 4.53 ± 2.19 h and 19.15 ± 23.11 h, respectively. It was the first study on the pharmacokinetics of CLZ and its metabolites in the oral fluid of Chinese healthy volunteers, and it provided a basis for the therapeutic drug monitoring and toxicological interpretation in clozapine-related cases.

Mechanisms and plasticity of chemogenically induced interneuronal suppression of principal cells.

How do firing patterns in a cortical circuit change when inhibitory neurons are excited? We virally expressed an excitatory designer receptor exclusively activated by a designer drug (Gq-DREADD) in all inhibitory interneuron types of the CA1 region of the hippocampus in the rat. While clozapine N-oxide (CNO) activation of interneurons suppressed firing of pyramidal cells, unexpectedly the majority of interneurons also decreased their activity. CNO-induced inhibition decreased over repeated sessions, which we attribute to long-term synaptic plasticity between interneurons and pyramidal cells. Individual interneurons did not display sustained firing but instead transiently enhanced their activity, interleaved with suppression of others. The power of the local fields in the theta band was unaffected, while power at higher frequencies was attenuated, likely reflecting reduced pyramidal neuron spiking. The incidence of sharp wave ripples decreased but the surviving ripples were associated with stronger population firing compared with the control condition. These findings demonstrate that DREADD activation of interneurons brings about both short-term and long-term circuit reorganization, which should be taken into account in the interpretation of chemogenic effects on behavior.

The Dopaminergic Cells in the Median Raphe Region Regulate Social Behavior in Male Mice.

According to previous studies, the median raphe region (MRR) is known to contribute significantly to social behavior. Besides serotonin, there have also been reports of a small population of dopaminergic neurons in this region. Dopamine is linked to reward and locomotion, but very little is known about its role in the MRR. To address that, we first confirmed the presence of dopaminergic cells in the MRR of mice (immunohistochemistry, RT-PCR), and then also in humans (RT-PCR) using healthy donor samples to prove translational relevance. Next, we used chemogenetic technology in mice containing the Cre enzyme under the promoter of the dopamine transporter. With the help of an adeno-associated virus, designer receptors exclusively activated by designer drugs (DREADDs) were expressed in the dopaminergic cells of the MRR to manipulate their activity. Four weeks later, we performed an extensive behavioral characterization 30 min after the injection of the artificial ligand (Clozapine-N-Oxide). Stimulation of the dopaminergic cells in the MRR decreased social interest without influencing aggression and with an increase in social discrimination. Additionally, inhibition of the same cells increased the friendly social behavior during social interaction test. No behavioral changes were detected in anxiety, memory or locomotion. All in all, dopaminergic cells were present in both the mouse and human samples from the MRR, and the manipulation of the dopaminergic neurons in the MRR elicited a specific social response.

Plasma Clozapine and Norclozapine After Use of Either Crushed Tablets or Suspension Compared With Tablets.

BACKGROUND: With clozapine, either crushed tablets suspended in an aqueous medium or proprietary suspension is sometimes prescribed as an alternative to tablets, but bioequivalence data are scant. METHODS: We compared clozapine dose, and plasma clozapine and N -desmethylclozapine (norclozapine) concentrations after use of either tablets or crushed tablets/suspension in samples submitted for clozapine therapeutic drug monitoring, 1993 to 2017. RESULTS: There were 846 patients (1646 samples) given crushed tablets/suspension and 6065 patients (10,779 samples) given tablets. The median dose (mg d -1 ) was significantly higher in men (500 vs 450) and women (500 vs 400) given crushed tablets/suspension, but the median plasma clozapine and norclozapine concentrations (mg L -1 ) were significantly lower (men: 0.29 and 0.22 vs 0.39 and 0.28; women: 0.35 and 0.26 vs 0.50 and 0.32, respectively). A subgroup of 480 patients was prescribed either crushed tablets/suspension (1016 samples) or tablets (1259 samples) at different times. The median dose was again significantly higher in men (500 vs 500) and women (500 vs 450), but the median plasma clozapine and norclozapine concentrations were significantly lower (men: 0.29 and 0.22 vs 0.32 and 0.24; women: 0.30 and 0.24 vs 0.38 and 0.29, respectively). IMPLICATIONS: Poor adherence, sedimentation of suspension before use, and incomplete dosage are potential contributors to the lower median plasma clozapine and norclozapine concentrations observed after use of either crushed clozapine tablets or suspension as compared with tablets. Those administering crushed tablets/suspension should be aware of these factors.

Metabolite Profiling of Clozapine in Patients Switching Versus Maintaining Treatment: A Retrospective Pilot Study.

PURPOSE/BACKGROUND: Pharmacokinetics may be of relevance for the risk of clozapine discontinuation. We compared metabolite profiles, accounting for smoking habits, in patients switching versus maintaining clozapine treatment at therapeutic concentrations. METHODS/PROCEDURES: Adult patients with clozapine serum levels above 1070 nmol/L (350 ng/mL) were retrospectively included from a Norwegian therapeutic drug monitoring service during 2018-2020. Inclusion criteria were (1) known smoking habits, (2) blood sample drawn within 10 to 30 hours after last clozapine intake, and (3) detectable levels of N -desmethylclozapine, clozapine -N -oxide, clozapine-5 N -glucuronide, or clozapine- N + - glucuronide. Patients comedicated with cytochrome P450 enzyme inducers, inhibitors, or valproic acid were excluded. The high-resolution mass spectrometry assay enabled detection of 21 clozapine metabolites. Metabolite profiles were compared between patients switching treatment (switchers), measured as clozapine being replaced by another antipsychotic drug in blood samples, versus maintaining clozapine treatment (nonswitchers) during the study period. FINDINGS/RESULTS: Of the 84 patients fulfilling the study criteria, 7 patients (8.3%) were identified as clozapine switchers. After correcting for smoking habits, the clozapine-5 N -glucuronide/clozapine ratio was 69% lower ( P < 0.001), while the clozapine- N + -glucuronide/clozapine-5 N -glucuronide ratio was 143% higher ( P = 0.026), respectively, in switchers versus nonswitchers. The other metabolite ratios did not significantly differ between switchers and nonswitchers. IMPLICATIONS/CONCLUSIONS: The present study found a significantly reduced 5 N -glucuronidation phenotype in patients switching from clozapine at therapeutic serum concentrations (>1070 nmol/L) to other antipsychotic drugs. This may indicate that glucuronidation, as a potential detoxification mechanism, is related to clozapine tolerability. However, the causality of this observation needs to be investigated in future studies with larger patient populations.

Body weight gain in clozapine-treated patients: Is norclozapine the culprit?

The antipsychotic drug clozapine is associated with weight gain. The proposed mechanisms include blocking of serotonin (5-HT2a/2c ), dopamine (D2 ) and histamine (H1 ) receptors. Clozapine is metabolized by cytochrome P450 1A2 (CYP1A2) to norclozapine, a metabolite with more 5-HT2c -receptor and less H1 blocking capacity. We hypothesized that norclozapine serum levels correlate with body mass index (BMI), waist circumference and other parameters of the metabolic syndrome. We performed a retrospective cross-sectional study in 39 patients (female n = 8 (20.5%), smokers n = 18 (46.2%), average age 45.8 ± 9.9 years) of a clozapine outpatient clinic in the Netherlands between 1 January 2017 and 1 July 2020. Norclozapine concentrations correlated with waist circumference (r = 0.354, P = .03) and hemoglobin A1c (HbA1c) (r = 0.34, P = .03). In smokers (smoking induces CYP1A2), norclozapine concentrations correlated with waist circumference (r = 0.723, P = .001), HbA1c (r = 0.49, P = .04) and BMI (r = 0.63, P = .004). Elucidating the relationship between norclozapine and adverse effects of clozapine use offers perspectives for interventions and treatment options.

Characterization of Clozapine-Responsive Human T Cells.

Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vß 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.

The rostromedial tegmental nucleus is essential for non-rapid eye movement sleep.

The rostromedial tegmental nucleus (RMTg), also called the GABAergic tail of the ventral tegmental area, projects to the midbrain dopaminergic system, dorsal raphe nucleus, locus coeruleus, and other regions. Whether the RMTg is involved in sleep-wake regulation is unknown. In the present study, pharmacogenetic activation of rat RMTg neurons promoted non-rapid eye movement (NREM) sleep with increased slow-wave activity (SWA). Conversely, rats after neurotoxic lesions of 8 or 16 days showed decreased NREM sleep with reduced SWA at lights on. The reduced SWA persisted at least 25 days after lesions. Similarly, pharmacological and pharmacogenetic inactivation of rat RMTg neurons decreased NREM sleep. Electrophysiological experiments combined with optogenetics showed a direct inhibitory connection between the terminals of RMTg neurons and midbrain dopaminergic neurons. The bidirectional effects of the RMTg on the sleep-wake cycle were mimicked by the modulation of ventral tegmental area (VTA)/substantia nigra compacta (SNc) dopaminergic neuronal activity using a pharmacogenetic approach. Furthermore, during the 2-hour recovery period following 6-hour sleep deprivation, the amount of NREM sleep in both the lesion and control rats was significantly increased compared with baseline levels; however, only the control rats showed a significant increase in SWA compared with baseline levels. Collectively, our findings reveal an essential role of the RMTg in the promotion of NREM sleep and homeostatic regulation.

A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors.

Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma, and mutations in Gq-linked G protein-coupled receptors have been identified recently in numerous human malignancies. How Gα(q) and its coupled receptors transduce mitogenic signals is still unclear because of the complexity of signaling events perturbed upon Gq activation. Using a synthetic-biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, and thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-ß. Indeed, whereas many biological responses elicited by Gq depend on the transient activation of second-messenger systems, Gq utilizes a hard-wired protein-protein-interaction-based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth.