GLP-1-directed NMDA receptor antagonism for obesity treatment.

The N-methyl-D-aspartate (NMDA) receptor is a glutamate-activated cation channel that is critical to many processes in the brain. Genome-wide association studies suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity are important for body weight homeostasis1. Here we report the engineering and preclinical development of a bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycaemia and dyslipidaemia in rodent models of metabolic disease. GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects neuroplasticity in the hypothalamus and brainstem. Importantly, targeting of MK-801 to GLP-1 receptor-expressing brain regions circumvents adverse physiological and behavioural effects associated with MK-801 monotherapy. In summary, our approach demonstrates the feasibility of using peptide-mediated targeting to achieve cell-specific ionotropic receptor modulation and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for safe and effective obesity treatment.

An RNA-seq atlas of mouse brain areas during fasting and diet-induced obesity.

Mammalian energy homeostasis is primarilly regulated by the hypothalamus and hindbrain, with the hippocampus, midbrain nuclei, and other regions implicated by evidence from human genetics studies. To understand how these non-canonical brain regions respond to imbalances in energy homeostasis, we performed two experiments examining the effects of different diets in male C57BL6 mice. In our first study, groups of six pair-housed mice were given access to chow, high-fat diet or fasted for 16 hours. In our subsequent study, two groups of 10 mice were single-housed and given access to chow or fasted for 24 h. We recorded food intake for each cage, the change in body weight for each animal, and collected hypothalamus, hippocampus, superior colliculus, inferior colliculus, frontal cortex, and zona incerta-centric samples. We performed bulk RNA sequencing on 185 samples and validated them by a series of quality control assessments including alignment quality and gene expression profiling. We believe these studies capture the transcriptomic effects of acute fasting and high-fat diet in the rodent brain and provide a valuable reference.

Cross-species comparison of pregnancy-induced GDF15.

Growth differentiation factor 15 (GDF15) is a stress-induced cytokine. Although the exact physiological function of GDF15 is not yet fully comprehended, the significant elevation of circulating GDF15 levels during gestation suggests a potential role for this hormone in pregnancy. This is corroborated by genetic association studies in which GDF15 and the GDF15 receptor, GDNF family receptor alpha like (GFRAL) have been linked to morning sickness and hyperemesis gravidarum (HG) in humans. Here, we studied GDF15 biology during pregnancy in mice, rats, macaques, and humans. In contrast to macaques and humans, mice and rats exhibited an underwhelming induction in plasma GDF15 levels in response to pregnancy (∼75-fold increase in macaques vs. ∼2-fold increase in rodents). The changes in circulating GDF15 levels were corroborated by the magnitude of Gdf15 mRNA and GDF15 protein expression in placentae from mice, rats, and macaques. These species-specific findings may help guide future studies focusing on GDF15 in pregnancy and on the evaluation of pharmacological strategies to interfere with GDF15-GFRAL signaling to treat severe nausea and HG.NEW & NOTEWORTHY In the present study pregnancy-induced changes in circulating growth differentiation factor 15 (GDF15) in rodents, rhesus macaques, and humans are mapped. In sum, it is demonstrated that humans and macaques exhibit a tremendous increase in placental and circulating GDF15 during pregnancy. In contrast, GDF15 is negligibly increased in pregnant mice and rats, questioning a physiological role for GDF15 in pregnancy in rodents.

Linking Hypothermia and Altered Metabolism with TrkB Activation.

Many mechanisms have been proposed to explain acute antidepressant drug-induced activation of TrkB neurotrophin receptors, but several questions remain. In a series of pharmacological experiments, we observed that TrkB activation induced by antidepressants and several other drugs correlated with sedation, and most importantly, coinciding hypothermia. Untargeted metabolomics of pharmacologically dissimilar TrkB activating treatments revealed effects on shared bioenergetic targets involved in adenosine triphosphate (ATP) breakdown and synthesis, demonstrating a common perturbation in metabolic activity. Both activation of TrkB signaling and hypothermia were recapitulated by administration of inhibitors of glucose and lipid metabolism, supporting a close relationship between metabolic inhibition and neurotrophic signaling. Drug-induced TrkB phosphorylation was independent of electroencephalography slow-wave activity and remained unaltered in knock-in mice with the brain-derived neurotrophic factor (BDNF) Val66Met allele, which have impaired activity-dependent BDNF release, alluding to an activation mechanism independent from BDNF and neuronal activity. Instead, we demonstrated that the active maintenance of body temperature prevents activation of TrkB and other targets associated with antidepressants, including p70S6 kinase downstream of the mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3ß (GSK3ß). Increased TrkB, GSK3ß, and p70S6K phosphorylation was also observed during recovery sleep following sleep deprivation, when a physiological temperature drop is known to occur. Our results suggest that the changes in bioenergetics and thermoregulation are causally connected to TrkB activation and may act as physiological regulators of signaling processes involved in neuronal plasticity.

The GHB analogue HOCPCA improves deficits in cognition and sensorimotor function after MCAO via CaMKIIα.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA's effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA's selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.

The anorectic and thermogenic effects of pharmacological lactate in male mice are confounded by treatment osmolarity and co-administered counterions.

Lactate is a circulating metabolite and a signalling molecule with pleiotropic physiological effects. Studies suggest that lactate modulates energy balance by lowering food intake, inducing adipose browning and increasing whole-body thermogenesis. Yet, like many other metabolites, lactate is often commercially produced as a counterion-bound salt and typically administered in vivo through hypertonic aqueous solutions of sodium L-lactate. Most studies have not controlled for injection osmolarity and the co-injected sodium ions. Here, we show that the anorectic and thermogenic effects of exogenous sodium L-lactate in male mice are confounded by the hypertonicity of the injected solutions. Our data reveal that this is in contrast to the antiobesity effect of orally administered disodium succinate, which is uncoupled from these confounders. Further, our studies with other counterions indicate that counterions can have confounding effects beyond lactate pharmacology. Together, these findings underscore the importance of controlling for osmotic load and counterions in metabolite research.

Effects of acute exercise and exercise training on plasma GDF15 concentrations and associations with appetite and cardiometabolic health in individuals with overweight or obesity - A secondary analysis of a randomized controlled trial.

Growth Differentiation Factor 15 (GDF15) is seemingly involved in appetite control. Acute exercise increases GDF15 concentrations in lean humans, but acute and long-term effects of exercise on GDF15 in individuals with overweight/obesity are unknown. We investigated the effects of acute exercise and exercise training on GDF15 concentrations in individuals with overweight/obesity and associations with appetite and cardiometabolic markers. 90 physically inactive adults (20-45 years) with overweight/obesity were randomized to 6-months habitual lifestyle (CON, n=16), or isocaloric exercise of moderate (MOD, n=37) or vigorous intensity (VIG, n=37), 5 days/week. Testing was performed at baseline, 3, and 6 months. Plasma GDF15 concentrations, other metabolic markers, and subjective appetite were assessed fasted and in response to acute exercise before an ad libitum meal. Cardiorespiratory fitness, body composition, insulin sensitivity, and intraabdominal adipose tissue were measured. At baseline, GDF15 increased 18% (95%CI: 4; 34) immediately after acute exercise and 32% (16; 50) 60 min post-exercise. Fasting GDF15 increased 21% (0; 46) in VIG after 3 months (p=0.045), but this attenuated at 6 months (13% (-11; 43), p=0.316) and was unchanged in MOD (11% (-6; 32), p=0.224, across 3 and 6 months). Post-exercise GDF15 did not change in MOD or VIG. GDF15 was not associated with appetite or energy intake. Higher GDF15 was associated with lower cardiorespiratory fitness, central obesity, dyslipidemia, and poorer glycemic control. In conclusion, GDF15 increased in response to acute exercise but was unaffected by exercise training. Higher GDF15 concentrations were associated with a less favorable cardiometabolic profile but not with markers of appetite. This suggests that GDF15 increases in response to acute exercise independent of training state. Whether this has an impact on free-living energy intake and body weight management needs investigation.

The GDF15-GFRAL pathway is dispensable for the effects of metformin on energy balance.

Metformin is a blood-glucose-lowering medication with physiological effects that extend beyond its anti-diabetic indication. Recently, it was reported that metformin lowers body weight via induction of growth differentiation factor 15 (GDF15), which suppresses food intake by binding to the GDNF family receptor α-like (GFRAL) in the hindbrain. Here, we corroborate that metformin increases circulating GDF15 in mice and humans, but we fail to confirm previous reports that the GDF15-GFRAL pathway is necessary for the weight-lowering effects of metformin. Instead, our studies in wild-type, GDF15 knockout, and GFRAL knockout mice suggest that the GDF15-GFRAL pathway is dispensable for the effects of metformin on energy balance. The data presented here question whether metformin is a sufficiently strong stimulator of GDF15 to drive anorexia and weight loss and emphasize that additional work is needed to untangle the relationship among metformin, GDF15, and energy balance.

Acute and long-term effects of psilocybin on energy balance and feeding behavior in mice.

Psilocybin and other serotonergic psychedelics have re-emerged as therapeutics for neuropsychiatric disorders, including addiction. Psilocybin induces long-lasting effects on behavior, likely due to its profound ability to alter consciousness and augment neural connectivity and plasticity. Impaired synaptic plasticity in obesity contributes to 'addictive-like' behaviors, including heightened motivation for palatable food, and excessive food seeking and consumption. Here, we evaluate the effects of psilocybin on feeding behavior, energy metabolism, and as a weight-lowering agent in mice. We demonstrate that a single dose of psilocybin substantially alters the prefrontal cortex transcriptome but has no acute or long-lasting effects on food intake or body weight in diet-induced obese mice or in genetic mouse models of obesity. Similarly, sub-chronic microdosing of psilocybin has no metabolic effects in obese mice and psilocybin does not augment glucagon-like peptide-1 (GLP-1) induced weight loss or enhance diet-induced weight loss. A single high dose of psilocybin reduces sucrose preference but fails to counter binge-like eating behavior. Although these preclinical data discourage clinical investigation, there may be nuances in the mode of action of psychedelic drugs that are difficult to capture in rodent models, and thus require human evaluation to uncover.

Beta-Hydroxybutyrate Suppresses Hepatic Production of the Ghrelin Receptor Antagonist LEAP2.

INTRODUCTION: Liver-expressed antimicrobial peptide-2 (LEAP2) is an endogenous ghrelin receptor antagonist, which is upregulated in the fed state and downregulated during fasting. We hypothesized that the ketone body beta-hydroxybutyrate (BHB) is involved in the downregulation of LEAP2 during conditions with high circulating levels of BHB. METHODS: Hepatic and intestinal Leap2 expression were determined in 3 groups of mice with increasing circulating levels of BHB: prolonged fasting, prolonged ketogenic diet, and oral BHB treatment. LEAP2 levels were measured in lean and obese individuals, in human individuals following endurance exercise, and in mice after BHB treatment. Lastly, we investigated Leap2 expression in isolated murine hepatocytes challenged with BHB. RESULTS: We confirmed increased circulating LEAP2 levels in individuals with obesity compared to lean individuals. The recovery period after endurance exercise was associated with increased plasma levels of BHB levels and decreased LEAP2 levels in humans. Leap2 expression was selectively decreased in the liver after fasting and after exposure to a ketogenic diet for 3 weeks. Importantly, we found that oral administration of BHB increased circulating levels of BHB in mice and decreased Leap2 expression levels and circulating LEAP2 plasma levels, as did Leap2 expression after direct exposure to BHB in isolated murine hepatocytes. CONCLUSION: From our data, we suggest that LEAP2 is downregulated during different states of energy deprivation in both humans and rodents. Furthermore, we here provide evidence that the ketone body, BHB, which is highly upregulated during fasting metabolism, directly downregulates LEAP2 levels. This may be relevant in ghrelin receptor-induced hunger signaling during energy deprivation.

GDF15 in Appetite and Exercise: Essential Player or Coincidental Bystander?

Growth differentiation factor 15 (GDF15) has recently moved to the forefront of metabolism research. When administered pharmacologically, GDF15 reduces food intake and lowers body weight via the hindbrain-situated receptor GFRAL (glial cell-derived neurotrophic factor family receptor alpha-like). Endogenous GDF15 is a ubiquitous cellular stress signal that can be produced and secreted by a variety of cell types. Circulating levels are elevated in a series of disease states, but also in response to exogenous agents such as metformin, colchicine, AICAR, and cisplatin. Recently, exercise has emerged as a relevant intervention to interrogate GDF15 physiology. Prolonged endurance exercise increases circulating GDF15 to levels otherwise associated with certain pathological states and in response to metformin treatment. The jury is still out on whether GDF15 is a functional "exerkine" mediating organ-to-brain crosstalk or whether it is a coincidental bystander. In this review, we discuss the putative physiological implication of exercise-induced GDF15, focusing on the potential impact on appetite and metabolism.

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain.

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

α-Synuclein Responses in the Laterodorsal Tegmentum, the Pedunculopontine Tegmentum, and the Substantia Nigra: Implications for Early Appearance of Sleep Disorders in Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder associated with insoluble pathological aggregates of the protein α-synuclein. While PD is diagnosed by motor symptoms putatively due to aggregated α-synuclein-mediated damage to substantia nigra (SN) neurons, up to a decade before motor symptom appearance, patients exhibit sleep disorders (SDs). Therefore, we hypothesized that α-synuclein, which can be present in monomeric, fibril, and other forms, has deleterious cellular actions on sleep-control nuclei. OBJECTIVE: We investigated whether native monomer and fibril forms of α-synuclein have effects on neuronal function, calcium dynamics, and cell-death-induction in two sleep-controlling nuclei: the laterodorsal tegmentum (LDT), and the pedunculopontine tegmentum (PPT), as well as the motor-controlling SN. METHODS: Size exclusion chromatography, Thioflavin T fluorescence assays, and circular dichroism spectroscopy were used to isolate structurally defined forms of recombinant, human α-synuclein. Neuronal and viability effects of characterized monomeric and fibril forms of α-synuclein were determined on LDT, PPT, and SN neurons using electrophysiology, calcium imaging, and neurotoxicity assays. RESULTS: In LDT and PPT neurons, both forms of α-synuclein induced excitation and increased calcium, and the monomeric form heightened putatively excitotoxic neuronal death, whereas, in the SN, we saw inhibition, decreased intracellular calcium, and monomeric α-synuclein was not associated with heightened cell death. CONCLUSION: Nucleus-specific differential effects suggest mechanistic underpinnings of SDs' prodromal appearance in PD. While speculative, we hypothesize that the monomeric form of α-synuclein compromises functionality of sleep-control neurons, leading to the presence of SDs decades prior to motor dysfunction.

Pharmacological but not physiological GDF15 suppresses feeding and the motivation to exercise.

Growing evidence supports that pharmacological application of growth differentiation factor 15 (GDF15) suppresses appetite but also promotes sickness-like behaviors in rodents via GDNF family receptor α-like (GFRAL)-dependent mechanisms. Conversely, the endogenous regulation of GDF15 and its physiological effects on energy homeostasis and behavior remain elusive. Here we show, in four independent human studies that prolonged endurance exercise increases circulating GDF15 to levels otherwise only observed in pathophysiological conditions. This exercise-induced increase can be recapitulated in mice and is accompanied by increased Gdf15 expression in the liver, skeletal muscle, and heart muscle. However, whereas pharmacological GDF15 inhibits appetite and suppresses voluntary running activity via GFRAL, the physiological induction of GDF15 by exercise does not. In summary, exercise-induced circulating GDF15 correlates with the duration of endurance exercise. Yet, higher GDF15 levels after exercise are not sufficient to evoke canonical pharmacological GDF15 effects on appetite or responsible for diminishing exercise motivation.

Thyroid hormone receptor α in skeletal muscle is essential for T3-mediated increase in energy expenditure.

Thyroid hormones are important for homeostatic control of energy metabolism and body temperature. Although skeletal muscle is considered a key site for thyroid action, the contribution of thyroid hormone receptor signaling in muscle to whole-body energy metabolism and body temperature has not been resolved. Here, we show that T3-induced increase in energy expenditure requires thyroid hormone receptor alpha 1 (TRα1 ) in skeletal muscle, but that T3-mediated elevation in body temperature is achieved in the absence of muscle-TRα1 . In slow-twitch soleus muscle, loss-of-function of TRα1 (TRαHSACre ) alters the fiber-type composition toward a more oxidative phenotype. The change in fiber-type composition, however, does not influence the running capacity or motivation to run. RNA-sequencing of soleus muscle from WT mice and TRαHSACre mice revealed differentiated transcriptional regulation of genes associated with muscle thermogenesis, such as sarcolipin and UCP3, providing molecular clues pertaining to the mechanistic underpinnings of TRα1 -linked control of whole-body metabolic rate. Together, this work establishes a fundamental role for skeletal muscle in T3-stimulated increase in whole-body energy expenditure.

Pharmacological targeting of α3ß4 nicotinic receptors improves peripheral insulin sensitivity in mice with diet-induced obesity.

AIMS/HYPOTHESIS: Treatment with the α3ß4 nicotinic acetylcholine receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), improves glucose tolerance in diet-induced obese (DIO) mice, but the physiological and molecular mechanisms are unknown. METHODS: DMPP (10 mg/kg body weight, s.c.) was administered either in a single injection (acute) or daily for up to 14 days (chronic) in DIO wild-type (WT) and Chrnb4 knockout (KO) mice and glucose tolerance, tissue-specific tracer-based glucose metabolism, and insulin signalling were assessed. RESULTS: In WT mice, but not in Chrnb4 KO mice, single acute treatment with DMPP induced transient hyperglycaemia, which was accompanied by high plasma adrenaline (epinephrine) levels, upregulated hepatic gluconeogenic genes, and decreased hepatic glycogen content. In contrast to these acute effects, chronic DMPP treatment in WT mice elicited improvements in glucose tolerance already evident after three consecutive days of DMPP treatment. After seven days of DMPP treatment, glucose tolerance was markedly improved, also in comparison with mice that were pair-fed to DMPP-treated mice. The glycaemic benefit of chronic DMPP was absent in Chrnb4 KO mice. Chronic DMPP increased insulin-stimulated glucose clearance into brown adipose tissue (+69%), heart (+93%), gastrocnemius muscle (+74%) and quadriceps muscle (+59%), with no effect in white adipose tissues. After chronic DMPP treatment, plasma adrenaline levels did not increase following an injection with DMPP. In glucose-stimulated skeletal muscle, we detected a decreased phosphorylation of the inhibitory Ser640 phosphorylation site on glycogen synthase and a congruent increase in glycogen accumulation following chronic DMPP treatment. CONCLUSIONS/INTERPRETATION: Our data suggest that DMPP acutely induces adrenaline release and hepatic glycogenolysis, while chronic DMPP-mediated activation of ß4-containing nAChRs improves peripheral insulin sensitivity independently of changes in body weight via mechanisms that could involve increased non-oxidative glucose disposal into skeletal muscle.

Glucometabolic consequences of acute and prolonged inhibition of fatty acid oxidation.

Excessive circulating FAs have been proposed to promote insulin resistance (IR) of glucose metabolism by increasing the oxidation of FAs over glucose. Therefore, inhibition of FA oxidation (FAOX) has been suggested to ameliorate IR. However, prolonged inhibition of FAOX would presumably cause lipid accumulation and thereby promote lipotoxicity. To understand the glycemic consequences of acute and prolonged FAOX inhibition, we treated mice with the carnitine palmitoyltransferase 1 (CPT-1) inhibitor, etomoxir (eto), in combination with short-term 45% high fat diet feeding to increase FA availability. Eto acutely increased glucose oxidation and peripheral glucose disposal, and lowered circulating glucose, but this was associated with increased circulating FAs and triacylglycerol accumulation in the liver and heart within hours. Several days of FAOX inhibition by daily eto administration induced hepatic steatosis and glucose intolerance, specific to CPT-1 inhibition by eto. Lower whole-body insulin sensitivity was accompanied by reduction in brown adipose tissue (BAT) uncoupling protein 1 (UCP1) protein content, diminished BAT glucose clearance, and increased hepatic glucose production. Collectively, these data suggest that pharmacological inhibition of FAOX is not a viable strategy to treat IR, and that sufficient rates of FAOX are required for maintaining liver and BAT metabolic function.

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity γ-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

Profiling of GABAA and GABAB receptor expression in the myometrium of the human uterus.

AIMS: γ-aminobutyric acid (GABA) mediates its physiological effects through the GABAA and GABAB receptors. In this study the putative expression of GABAAR and GABABR subunits in human myometrium tissue was investigated. MAIN METHODS: The expression levels of the 19 GABAAR subunits (α1-α6, ß1-ß3, γ1-γ3, δ, ε, π, θ, ρ1-ρ3) and the three GABABR subunits (GABAB1a, GABAB1b, GABAB2) were characterized by RT-qPCR analysis on two commercial samples and six samples derived from surgically removed myometrial tissues from different women. We probed for functional GABAAR expression in primary human myometrial smooth muscle cells (HMSMCs) by whole-cell patch-clamp electrophysiology. KEY FINDINGS: The absolute mRNA levels of the 22 GABAAR and GABABR genes varied considerably across the eight samples, but a pronounced overlap existed between the specific subunits detected in the samples, with α2, ß2, ß3, ε, π, θ, GABAB1a and GABAB1b mRNAs being detected in most samples. The expression profile of GABAAR and GABABR subunit mRNAs in HMSMCs correlated with that observed in the eight tissue samples, albeit the subunit transcripts were detected at lower relative levels. Neither muscimol nor GABA evoked significant currents in these cells in the patch-clamp recordings. SIGNIFICANCE: While the expression of the GABAB1 subunits on their own is unlikely to give rise to functional GABABR expression, the GABAAR subunits identified at mRNA level would be able to form functional receptors in the human myometrial tissue. Although GABAAR-mediated currents could not be recorded from HMSMCs in this study, this suggests a role for GABAergic transmission in the human myometrium.

Reduced cortical serotonin 5-HT2A receptor binding and glutamate activity in high compulsive drinker rats.

Serotonin2A receptors and glutamate signaling have been implicated in the pathophysiology and treatment of compulsive spectrum disorders. Schedule-Induced Polydipsia (SIP), characterized by excessive drinking under intermittent food reinforcement schedules, is a valid model for studying the compulsive phenotype in rats. We explored the expression, function, and neurochemistry of 5-HT2A receptors in the frontal cortex (FC) of rats with individual differences to compulsivity. Rats were selected for high (HD) versus low (LD) drinking on SIP. First, we measured 5-HT2A, 5-HT1A, and mGlu2/3 receptors and serotonin transporter binding in different brain regions. Second, we assessed the effect of microinfusion into the medial prefrontal cortex (mPFC) of the 5-HT2A/C receptor agonist DOI, the mGlu2/3 agonist LY379268, and the combination of DOI with the 5-HT2A receptor antagonist M100907 and the 5-HT2C receptor antagonist SB242084. Finally, we measured the serotonin and glutamate efflux in mPFC in basal condition and after DOI local application. The compulsive HD rats showed a specific reduction of 5-HT2A receptor binding in FC compared to LD rats. The highest dose of DOI reduced compulsive drinking in HD rats on SIP, whereas LY379268 did not induce any significant effect. The 5-HT2A receptor antagonist M100907 reversed the DOI induced reduction on compulsive drinking in HD rats while blocking the 5-HT2C receptor did not affect SIP. Compulsive HD rats showed increased serotonin and decreased glutamate efflux in basal conditions that were modified by the DOI application. These findings indicate that reduced 5-HT2A receptor binding and glutamate neurochemical mechanisms may underlie compulsive behavior vulnerability.