Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent.

Current tools for the inhibition of microRNA (miR) function are limited to modified antisense oligonucleotides, sponges and decoy RNA molecules and none have been used to understand miR function during development. CRISPR/Cas-mediated deletion of miR sequences within the genome requires multiple chromosomal deletions to remove all functional miR family members because of duplications. Here, we report a novel plasmid-based miR inhibitor system (PMIS) that expresses a new RNA molecule, which inhibits miR family members in cells and mice. The PMIS engineered RNA optimal secondary structure, flanking sequences and specific antisense miR oligonucleotide sequence bind the miR in a stable complex to inhibit miR activity. In cells, one PMIS can effectively inhibit miR family members that share the same seed sequence. The PMIS shows no off-target effects or toxicity and is highly specific for miRs sharing identical seed sequences. Transgenic mice expressing both PMIS-miR-17-18 and PMIS-miR-19-92 show similar phenotypes of miR-17-92-knockout mice. Interestingly, mice only expressing PMIS-miR-17-18 have developmental defects distinct from mice only expressing PMIS-miR-19-92 demonstrating usefulness of the PMIS system to dissect different functions of miRs within clusters. Different PMIS miR inhibitors can be linked together to knock down multiple miRs expressed from different chromosomes. Inhibition of the miR-17-92, miR-106a-363 and miR-106b-25 clusters reveals new mechanisms and developmental defects for these miRs. We report a new tool to dissect the role of miRs in development without genome editing, inhibit miR function in cells and as a potential new therapeutic reagent.

Welfare aspects of vertebrate pest control and culling: ranking control techniques for humaneness.

The management of vertebrate pests depends on the use of traps, pesticides, repellents and other methods, each of which can cause varying levels of pain and other negative experiences to animals. Vertebrate pest control is essential for managing the impacts of unwanted or over-abundant animals on human and animal health, ecological balance and economic interests. As the need for this management is unlikely to diminish over time, a framework has been developed for assessing the humaneness of each technique by considering their negative impacts on animal welfare so that these can be included in decision-making about the selection of techniques for a specific control operation. This information can also support evidence-based regulations directed at managing such animal welfare impacts. In this paper, the authors discuss this assessment framework, briefly review two assessments conducted using the framework and discuss ways in which Competent Authorities and others can use it and other means to improve animal welfare in vertebrate pest management.

The subcortical correlates of autistic traits in school-age children: a population-based neuroimaging study.

BACKGROUND: There is emerging evidence that the neuroanatomy of autism forms a spectrum which extends into the general population. However, whilst several studies have identified cortical morphology correlates of autistic traits, it is not established whether morphological differences are present in the subcortical structures of the brain. Additionally, it is not clear to what extent previously reported structural associations may be confounded by co-occurring psychopathology. To address these questions, we utilised neuroimaging data from the Adolescent Brain Cognitive Development Study to assess whether a measure of autistic traits was associated with differences in child subcortical morphology, and if any observed differences persisted after adjustment for child internalising and externalising symptoms. METHODS: Our analyses included data from 7005 children aged 9-10 years (female: 47.19%) participating in the Adolescent Brain Cognitive Development Study. Autistic traits were assessed using scores from the Social Responsiveness Scale (SRS). Volumes of subcortical regions of interest were derived from structural magnetic resonance imaging data. RESULTS: Overall, we did not find strong evidence for an association of autistic traits with differences in subcortical morphology in this sample of school-aged children. Whilst lower absolute volumes of the nucleus accumbens and putamen were associated with higher scores of autistic traits, these differences did not persist once a global measure of brain size was accounted for. LIMITATIONS: It is important to note that autistic traits were assessed using the SRS, of which higher scores are associated with general behavioural problems, and therefore may not be wholly indicative of autism-specific symptoms. In addition, individuals with a moderate or severe autism diagnosis were excluded from the ABCD study, and thus, the average level of autistic traits will be lower than in the general population which may bias findings towards the null. CONCLUSIONS: These findings from our well-powered study suggest that other metrics of brain morphology, such as cortical morphology or shape-based phenotypes, may be stronger candidates to prioritise when attempting to identify robust neuromarkers of autistic traits.

Biological mesh reconstruction of perineal wounds following enhanced abdominoperineal excision of rectum (APER).

INTRODUCTION: An abdominoperineal excision of rectum (APER) may be required for rectal tumours less than 6 cm from the anal verge. Recently, the cylindrical APER has been used to prevent the "surgical waist" and so decrease margin involvement. However, removal of the levators leaves a large defect. Myocutaneous flaps [e.g. vertical rectus abdominis (VRAM)] are often used to fill the cylindrical resection defect, but have disadvantages associated with operative time, expertise and morbidity. We report our early experience of pelvic floor reconstruction with a biological mesh following cylindrical APER. METHODS: Data on consecutive patients having cylindrical APER between January 2008 and November 2010 were collected. Outcomes were compared between a VRAM reconstruction group and a mesh group. RESULTS: In 15 consecutive patients with low rectal cancer, five patients had VRAM pelvic floor reconstruction prior to ten patients having biosynthetic mesh repairs. The median operative time for the VRAM cohort was 405 min, compared with 259 min for the mesh (p = 0.0013). The median length of postoperative stay was 20 days for VRAM and 10 days for the mesh group (p = 0.067). There were four early complications for the VRAM group compared with seven for the mesh cohort (p = 0.37). The median cost per patient for the VRAM cohort was £11,075 compared to a median cost of £6,513 for the Mesh (p = 0.0097). CONCLUSION: The use of a biological mesh for pelvic floor reconstruction following cylindrical APER is feasible with morbidity comparable to VRAM reconstruction. There is significant cost-saving using a biosynthetic mesh, mainly due to reduced length of stay.

Prone position in covid-19: Can we tackle rising dead space?

Little is known about the impact of proning on oxygenation and ventilatory efficiency on patients with severe Covid-19. In this retrospective observational study we calculated Pa/FiO2 ratio (P/F) as a marker of oxygenation and dead space fraction (Vd/Vt) to assess ventilation. 12 patients who were proned twice or more were included. There was a significant improvement in P/F ratio when prone (110.18 ± 28.11) compared to supine (88.95 ± 19.34) (p < 0.01). There was no improvement in Vd/Vt on proning (p > 0.05). Vd/Vt as a function of time displayed a positive linear correlation in those who did not survive (n = 9) (Rs = 0.48, p < 0.01) but no observed correlation in those who survived (n = 3) (Rs = 0.002, p = 0.97). Our findings indicate that prone position in patients with Covid-19 has little effect on dead space fraction but does improve oxygenation. Rise in dead space with time appears to be a prognostic factor for death in patients with severe Covid- 19.

Effect of class II antigen matching on renal allograft survival in miniature swine.

The benefit of class II major histocompatibility complex (MHC) antigen matching to renal allograft survival, in the absence of immunosuppression, has been studied in partially inbred miniature swine. Permanent (greater than 6 mo) renal allograft survival was found in 30% of recipients of either class II only or fully matched grafts. Analysis of the survival of the class II-only matched grafts by specific recipient/donor haplotype combinations indicated that survival was regulated by at least three genetic factors, including antigen gene dose, a class I MHC allele-dependent effect, and non-MHC-linked immune response phenomenon. Animals accepting class II-matched kidneys developed spontaneous tolerance to the graft, despite mounting an initial immune response marked by renal damage and the development of serum cytotoxic antibodies directed at the donor MHC antigens. The antibodies were only of the IgM class, suggesting that conversion of the humoral response to IgG was blocked. After acceptance of the kidney, three out of five animals showed specific prolongation of donor skin grafts. At the time of rejection of these skin grafts, no decrease in renal function nor reappearance of anti-donor antibodies was observed.

Evidence that increased 5-HT release evokes region-specific effects on blood-oxygenation level-dependent functional magnetic resonance imaging responses in the rat brain.

This study aimed to determine the potential of in vivo functional magnetic resonance imaging (fMRI) methods as a non-invasive means of detecting effects of increased 5-HT release in brain. Changes in blood-oxygenation level-dependent (BOLD) contrast induced by administration of the 5-HT-releasing agent, fenfluramine, were measured in selected brain regions of halothane-anesthetized rats. Initial immunohistochemical measurements of the marker of neural activation, Fos, confirmed that in halothane-anesthetized rats fenfluramine (10 mg/kg i.v.) evoked cellular responses in cortical regions which were attenuated by pre-treatment with the 5-HT synthesis inhibitor p-chlorophenylalanine (300 mg/kg i.p. once daily for 2 days). Fenfluramine-induced Fos was demonstrated in numerous glutamatergic pyramidal neurons (Fos/excitatory amino acid carrier 1 (EAAC1) co-labeled), but also a small number of GABA interneurons (Fos/glutamic acid decarboxylase (GAD)(67) colabeled). Fenfluramine (10 mg/kg i.v.) evoked changes in BOLD signal intensity in a number of cortical and sub-cortical regions with the greatest effects being observed in the nucleus accumbens (-13.0%+/-2.7%), prefrontal cortex (-10.1%+/-3.2%) and motor cortex (+2.3%+/-1.0%). Pre-treatment with p-chlorophenylalanine, significantly attenuated the response to fenfluramine (10 mg/kg i.v.) in all regions with the exception of the motor cortex which showed a trend. These experiments demonstrate that increased 5-HT release evokes region-specific changes in the BOLD signal in rats, and that this effect is attenuated in almost all regions by 5-HT depletion. These findings support the use of fMRI imaging methods as a non-invasive tool to study 5-HT function in animal models, with the potential for extension to clinical studies.

Catalysis of the olivine to spinel transformation by high clinoenstatite.

Although enstatite is a major constituent of the Earth's upper mantle and subducting lithosphere, most kinetic studies of olivine phase transformations have typically involved single-phase polycrystalline aggregates. Transmission electron microscopy investigations of olivine to spinel and modified spinel (beta phase) reactions in the (Mg, Fe)(2)SiO(4)-(Mg,Fe)SiO(3) system show that transformation of olivine in the stability field of spinel plus phase begins with coherent nucleation of spinel on high-clinoenstatite grains. These observations demonstrate that high clinoenstatite can catalyze the transformation by enhancing nucleation kinetics and therefore imply that secondary phases can influence reaction kinetics during high-pressure mineral transformations.

A post-stishovite SiO2 polymorph in the meteorite Shergotty: implications for impact events.

Transmission electron microscopy and electron diffraction show that the martian meteorite Shergotty, a shocked achondrite, contains a dense orthorhombic SiO2 phase similar to post-stishovite SiO2 with the alpha-PbO2 structure. If an SiO2 mineral exists in Earth's lower mantle, it would probably occur in a post-stishovite SiO2 structure. The presence of such a high-density polymorph in a shocked sample indicates that post-stishovite SiO2 structures may be used as indicators of extreme shock pressures.

Evidence that genetic variation in 5-HT transporter expression is linked to changes in 5-HT2A receptor function.

Variability in expression of the 5-HT transporter (5-HTT) gene in the human population has been associated with a range of behavioural phenotypes. The underlying mechanisms are unclear but may involve changes in 5-HT receptor levels and/or signalling. The present study used a novel 5-HTT overexpressing transgenic mouse to test the hypothesis that variability in 5-HTT expression may alter 5-HT(2A) receptor function. In wildtype mice, the 5-HT(2) receptor agonist DOI increased regional brain mRNA expression of two immediate early genes (c-fos and Arc), and induced head twitches, and both effects were abolished by pre-treatment with the 5-HT(2A) receptor antagonist MDL 100907. In 5-HTT overexpressing mice, DOI induced a greater increase in both c-fos and Arc mRNA expression in cortical brain regions, and more head twitches, compared to wildtype mice. Autoradiographic and in situ hybridisation experiments showed that 5-HT(2A) receptor binding sites and 5-HT(2A) receptor mRNA did not differ between transgenic and wildtype mice. Finally, the transgenic mice had lower regional brain 5-HT levels compared to wildtype mice. This depletion of 5-HT may underpin the increase in 5-HT(2A) receptor function because in wildtype mice 5-HT depletion using the 5-HT synthesis inhibitor, p-chlorophenylalanine, enhanced the head twitch response to DOI. These data demonstrate that elevated 5-HTT expression is accompanied by increased 5-HT(2A) receptor function, an effect possibly mediated by decreased availability of synaptic 5-HT. Variation in levels of 5-HTT expression may therefore be a source of variability in 5-HT(2A) receptor function, which may be an important modifier of 5-HTT-linked phenotypes.

Mice overexpressing the 5-hydroxytryptamine transporter show no alterations in feeding behaviour and increased non-feeding responses to fenfluramine.

RATIONALE: The 5-HT transporter (5-HTT) is implicated in the regulation of appetite. Expression of the 5-HTT varies in the human population, and this variation may determine both individual differences in feeding and abnormal feeding behaviours such as eating disorders. OBJECTIVES: The effects of 5-HTT expression on feeding and satiety were examined in a transgenic mouse model of 5-HTT overexpression. MATERIALS AND METHODS: We measured free-feeding food intake and observed the behavioural satiety sequence (BSS) after food deprivation in mice at baseline and after administration of the anorectic drug fenfluramine. RESULTS: 5-HTT overexpressing mice were both lighter and shorter than their wildtype littermates. Despite this size difference, food intake by transgenic and wildtype mice did not differ. There was no effect of genotype on the BSS or on food intake during the test at baseline. Increasing doses of fenfluramine reduced food intake in a similar manner in both transgenic and wildtype mice. After 0.3 and 1 mg/kg fenfluramine, the temporal pattern of the BSS was the same for both groups, whereas 3 and 10 mg/kg fenfluramine disrupted the BSS. In transgenic mice, this disruption was evident at the 3 mg/kg dose, while in wildtypes, it emerged only at the 10-mg/kg dose. CONCLUSION: These data suggest that overexpression of the 5-HTT does not lead to alterations in feeding or satiety in food-deprived mice but does increase the occurrence of other non-feeding behaviours in response to the 5-HT releasing agent fenfluramine.

Phosphodiesterase 4 inhibition affects both the direct and indirect pathway: an electrophysiological study examining the tri-phasic response in the substantia nigra pars reticulata.

Fronto-striatal circuits constitute the neurobiological basis of many neuropsychiatric disorders. Part of the intracellular signaling within these circuits, including its dopaminergic modulation, is regulated by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling cascade. Based on the overall expression in human fronto-striatal circuitry, we tested the effects of a cAMP selective phosphodiesterase 4 (PDE4) inhibitor on the tri-phasic response in the dorsomedial substantia nigra pars reticulata (SNr) upon stimulation of the infralimbic cortex in rats. Our results show for the first time that stimulation of the cognitive infralimbic cortex leads to a tri-phasic response in SNr neurons. In addition and in line with previous biochemical and behavioral studies, PDE4 inhibition by roflumilast affects the direct pathway as well as the indirect pathway of which the latter appears more sensitive than the former.

Correction to: Cardiopulmonary exercise testing (CPET) in the United Kingdom-a national survey of the structure, conduct, interpretation and funding.

[This corrects the article DOI: 10.1186/s13741-017-0082-3.].

Ebselen has lithium-like effects on central 5-HT2A receptor function.

BACKGROUND AND PURPOSE: Lithium's antidepressant action may be mediated by inhibition of inositol monophosphatase (IMPase), a key enzyme in Gq -protein coupled receptor signalling. Recently, the antioxidant agent ebselen was identified as an IMPase inhibitor. Here, we investigated both ebselen and lithium in models of the 5-HT2A receptor, a Gq -protein coupled receptor involved in lithium's actions. EXPERIMENTAL APPROACH: 5-HT2A receptor function was assessed in mice by measuring the behavioural (head-twitches, ear scratches) and molecular (cortical immediate early gene [IEG] mRNA; Arc, c-fos, Egr2) responses to 5-HT2A receptor agonists. Ebselen and lithium were administered either acutely or repeatedly prior to assessment of 5-HT2A receptor function. Because lithium and 5-HT2A receptor antagonists augment the action of selective serotonin reuptake inhibitors (SSRIs), ebselen was tested for this activity by co-administration with the SSRI citalopram in microdialysis (extracellular 5-HT) experiments. KEY RESULTS: Acute and repeated administration of ebselen inhibited behavioural and IEG responses to the 5-HT2A receptor agonist DOI. Repeated lithium also inhibited DOI-evoked behavioural and IEG responses. In comparison, a selective IMPase inhibitor (L-690330) attenuated the behavioural response to DOI whereas glycogen synthase kinase inhibitor (AR-A014418) did not. Finally, ebselen enhanced the increase in extracellular 5-HT induced by citalopram, and also increased regional brain 5-HT synthesis. CONCLUSIONS AND IMPLICATIONS: Our data demonstrated lithium-mimetic effects of ebselen in different experimental models of 5-HT2A receptor function, probably mediated by IMPase inhibition. This evidence of lithium-like neuropharmacological effects of ebselen adds further support for the clinical testing of ebselen in mood disorders, including as an antidepressant augmenting agent.

Cardiopulmonary exercise testing (CPET) in the United Kingdom-a national survey of the structure, conduct, interpretation and funding.

BACKGROUND: Cardiopulmonary exercise testing (CPET) is an exercise stress test with concomitant expired gas analysis that provides an objective, non-invasive measure of functional capacity under stress. CPET-derived variables predict postoperative morbidity and mortality after major abdominal and thoracic surgery. Two previous surveys have reported increasing utilisation of CPET preoperatively in England. We aimed to evaluate current CPET practice in the UK, to identify who performs CPET, how it is performed, how the data generated are used and the funding models. METHODS: All anaesthetic departments in trusts with adult elective surgery in the UK were contacted by telephone to obtain contacts for their pre-assessment and CPET service leads. An online survey was sent to all leads between November 2016 and March 2017. RESULTS: The response rate to the online survey was 73.1% (144/197) with 68.1% (98/144) reporting an established clinical service and 3.5% (5/144) setting up a service. Approximately 30,000 tests are performed a year with 93.0% (80/86) using cycle ergometry. Colorectal surgical patients are the most frequently tested (89.5%, 77/86). The majority of tests are performed and interpreted by anaesthetists. There is variability in the methods of interpretation and reporting of CPET and limited external validation of results. CONCLUSIONS: This survey has identified the continued expansion of perioperative CPET services in the UK which have doubled since 2011. The vast majority of CPET tests are performed and reported by anaesthetists. It has highlighted variation in practice and a lack of standardised reporting implying a need for practice guidelines and standardised training to ensure high-quality data to inform perioperative decision making.

Pharmacological validation of a semi-automated in vitro hippocampal brain slice assay for assessment of seizure liability.

INTRODUCTION: Drug-induced seizures are a serious, life-threatening adverse drug reaction (ADR) that can result in the failure of drugs to be licensed for clinical use or withdrawn from the market. Seizure liability of potential drugs is traditionally assessed using animal models run during the later phases of the drug discovery process. Given the low throughput, high animal usage and high compound requirement associated with these assays, it would be advantageous to identify higher throughput, in vitro models that could be used to give an earlier assessment of seizure liability. The hippocampal brain slice is one possibility but conventionally allows recording from only one slice at a time. The aim of this study was to validate a semi-automated system (Slicemaster, Scientifica UK Ltd) which allows concurrent electrophysiological recording from multiple brain slices. METHODS: Conventional electrophysiological recording techniques were used to record electrically evoked synaptic activity from rat hippocampal brain slices. Population spikes (PS) were evoked at 30 s intervals by electrical stimulation of the Schaffer collateral pathway and were recorded using extracellular electrodes positioned in the CA1 cell body layer. Responses were quantified as PS areas (the area above and below the 0 mV line). The effects of eight validation compounds known to cause seizures in vivo and/or in the clinic were assessed. RESULTS: Seven out of eight compounds evoked a concentration-dependent increase in population spike (PS) area that was statistically significant at higher concentrations (P<0.05; ANOVA). At the highest test concentration the percentage effects (mean+/-s.e.m.), relative to vehicle, were: picrotoxin 212.9+/-28.8, pentylenetrazole (PTZ) 181.4+/-24.7, 4-AP 328.9+/-48.6, aminophylline 124.5+/-5.9, chlorpromazine 122.1+/-9.8, SNC-80 132.1+/-12.6 and penicillin 174.7+/-14.1. Physostigmine had no significant effect on PS area although a concentration-dependent change in the morphology of the response was evident. DISCUSSION: All validation compounds evoked a statistically significant effect on synaptic activity in the rat hippocampal slice. Although similar effects have been described previously, this is the first time that the effects of a pharmacologically diverse set of compounds have been assessed using a standardised brain slice assay. Given the low compound usage and relatively high throughput associated with this assay, the hippocampal brain slice assay may facilitate earlier testing of convulsant liability than is currently possible using in vivo models.

Oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells resistant to PI3K inhibition.

Compounds targeting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling are being investigated in multiple clinical settings, but drug resistance may reduce their benefit. Compound rechallenge after drug holidays can overcome such resistance, yet little is known about the impact of drug holidays on cell biochemistry. We found that PI3K inhibitor (PI3Ki)-resistant cells cultured in the absence of PI3Ki developed a proliferative defect, increased oxygen consumption and accumulated reactive oxygen species (ROS), leading to lactate production through hypoxia-inducible factor-1α. This metabolic imbalance was reversed by mammalian target of rapamycin complex 1 (mTORC1) inhibitors. Interestingly, neither AKT nor c-MYC was involved in mediating the metabolic phenotype, despite the latter contributing to resistant cells' proliferation. These data suggest that an AKT-independent PI3K/mTORC1 axis operates in these cells. The excessive ROS hampered cell division, and the metabolic phenotype made resistant cells more sensitive to hydrogen peroxide and nutrient starvation. Thus, the proliferative defect of PI3Ki-resistant cells during drug holidays is caused by defective metabolic adaptation to chronic PI3K/mTOR pathway inhibition. This metabolic imbalance may open the therapeutic window for challenge with metabolic drugs during drug holidays.

In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism.

BACKGROUND AND PURPOSE: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503. EXPERIMENTAL APPROACH: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907. KEY RESULTS: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity. CONCLUSIONS AND IMPLICATIONS: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.