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Ulcerative colitis (UC) and Crohn’s disease (CD) are complex and heterogenous diseases characterized by chronic and progressive inflammation of the digestive tract. Single cell RNA sequencing combined with computational biology provides unprecedented insights into disease biology, informing novel therapeutic targets and precision medicine approaches. To identify potential mediators of persistent inflammation in inflammatory bowel disease (IBD), we performed single cell RNA sequencing on tissue samples collected from UC and CD patients before and after beginning adalimumab. In this study, 41 participants, including 21 UC patients, 17 CD patients and 3 healthy individuals, were enrolled at the University of Oxford’s Translational Gastroenterology Unit and Kennedy Institute of Rheumatology. Patients had moderate-to-severe disease based on symptoms and endoscopy, were biologic naïve, and planned to initiate adalimumab. Mucosal biopsies for single cell RNA sequencing were collected from multiple anatomic locations for each subject before beginning and after a minimum of 3 months of adalimumab, at which time treatment response was assessed. During the study, clinical response as defined by composite measures was observed in 8 UC and 9 CD patients. Differential cell composition and gene expression analysis of the single cell data revealed inflammatory monocyte subtypes as highly enriched in inflamed relative to uninflamed samples, and this cellular subset remained highly elevated in inflamed samples from adalimumab non-responders. These cells were characterized by expression of multiple cell-surface receptors with potential to drive pathology, including TREM1 (Triggering Receptor Expressed on Myeloid Cells 1), a transmembrane receptor that amplifies anti-bacterial neutrophil and monocyte mediated responses. TREM1+ inflammatory monocytes were highly enriched in UC and CD inflamed samples (Fig 1), both prior to adalimumab and in adalimumab non-responders. The enrichment of TREM1+ cells was specific to inflamed IBD tissue and was not observed in non-inflamed tissue from IBD patients or healthy subjects; enrichment of TREM1+ cells was also not observed in circulating peripheral blood mononuclear cells. TREM1+ neutrophils and monocytes in the gut mucosa amplify innate immune responses to bacteria in the context of epithelial barrier dysfunction and bacterial dysbiosis in IBD. When activated, TREM1 signaling induces multiple soluble mediators relevant for IBD pathology. Our single cell RNA sequencing data provide further evidence that TREM1 is an exciting therapeutic target for UC and CD, with inhibition of TREM1 signaling expected to address pathology while maintaining an anti-bacterial response. We therefore developed a highly potent and selective monoclonal antibody inhibitor of TREM-1, CEL383, with the Phase 1 clinical trial expected to start in 2023. Figure 1. tSNE plot of myeloid cells from inflamed IBD tissue samples demonstrating high TREM1 expression across inflammatory monocytes

Background Currently, 2 coprimary end points are used by health authorities to determine the effectiveness of therapeutic interventions in patients with Crohn’s disease (CD): symptomatic remission (patient-reported outcome assessment) and endoscopic remission (ileocolonoscopy). However, there is lack of accepted biomarkers to facilitate regulatory decision-making in the development of novel therapeutics for the treatment of CD. Methods With support from the Helmsley Charitable Trust, Critical Path Institute formed the Crohn’s Disease Biomarkers preconsortium (CDBpC) with members from the pharmaceutical industry, academia, and nonprofit organizations to evaluate the CD biomarker landscape. Biomarkers were evaluated based on biological relevance, availability of biomarker assays, and clinical validation data. Results The CDBpC identified the most critical need as pharmacodynamic/response biomarkers to monitor disease activity in response to therapeutic intervention. Fecal calprotectin (FC) and serum C-reactive protein (CRP) were identified as biomarkers ready for the regulatory qualification process. A number of exploratory biomarkers and potential panels of these biomarkers was also identified for additional development. Given the different factors involved in CD and disease progression, a combination of biomarkers, including inflammatory, tissue injury, genetic, and microbiome-associated biomarkers, will likely have the most utility. Conclusions The primary focus of the Inflammatory Bowel Disease Regulatory Science Consortium will be development of exploratory biomarkers and the qualification of FC and CRP for IBD. The Inflammatory Bowel Disease Regulatory Science Consortium, focused on tools to support IBD drug development, will operate in the precompetitive space to share data, biological samples for biomarker testing, and assay information for novel biomarkers.

Despite major advances in the inflammatory bowel diseases field, biomarkers to enable personalized and effective management are inadequate. Disease course and treatment response are highly variable, with some patients experiencing mild disease progression, whereas other patients experience severe or complicated disease. Periodic endoscopy is performed to assess disease activity; as a result, it takes months to ascertain whether a treatment is having a positive impact on disease progression. Minimally invasive biomarkers for prognosis of disease course, prediction of treatment response, monitoring of disease activity, and accurate diagnosis based on improved disease phenotyping and classification could improve outcomes and accelerate the development of novel therapeutics. Rapidly developing technologies have great potential in this regard; however, the discovery, validation, and qualification of biomarkers will require partnerships including academia, industry, funders, and regulators. The Crohn’s & Colitis Foundation launched the IBD Biomarker Summit to bring together key stakeholders to identify and prioritize critical unmet needs; prioritize promising technologies and consortium approaches to address these needs; and propose harmonization approaches to improve comparability of data across studies. Here, we summarize the outcomes of the 2018 and 2019 meetings, including consensus-based unmet needs in the clinical and drug development context. We highlight ongoing consortium efforts and promising technologies with the potential to address these needs in the near term. Finally, we summarize actionable recommendations for harmonization, including data collection tools for improved consistency in disease phenotyping; standardization of informed consenting; and development of guidelines for sample management and assay validation. Taken together, these outcomes demonstrate that there is an exceptional alignment of priorities across stakeholders for a coordinated effort to address unmet needs of patients with inflammatory bowel diseases through biomarker science.

Environmental triggers is part of five focus areas of the Challenges in IBD research document, which also includes preclinical human IBD mechanisms, novel technologies, precision medicine and pragmatic clinical research. The Challenges in IBD research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the environmental triggers section is focused on the main research gaps in elucidating causality of environmental factors in IBD. Research gaps were identified in: 1) epidemiology of exposures; 2) identification of signatures of biological response to exposures; and 3) mechanisms of how environmental exposures drive IBD. To address these gaps, the implementation of longitudinal prospective studies to determine disease evolution and identify sub-clinical changes in response to exposures is proposed. This can help define critical windows of vulnerability and risk prediction. In addition, systems biology analysis and in silico modeling were proposed as approaches to integrate the IBD exposome for the identification of biological signatures of response to exposures, and to develop prediction models of the effects of environmental factors in driving disease activity and response to therapy. This research could lead to identification of biomarkers of exposures and new modalities for therapeutic intervention. Finally, hypothesis-driven mechanistic studies to understand gene-environment interactions and to validate causality of priority factors should be performed to determine how environment influences clinical outcomes.

Precision medicine is part of five focus areas of the Challenges in IBD research document, which also includes preclinical human IBD mechanisms, environmental triggers, novel technologies, and pragmatic clinical research. The Challenges in IBD Research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the precision medicine section is focused on highlighting the main gap areas that must be addressed to get closer to treatments tailored to the biological and clinical characteristics of each patient, which is the aim of precision medicine. The main gaps were identified in: 1) understanding and predicting the natural history of IBD: disease susceptibility, activity, and behavior; 2) predicting disease course and treatment response; and 3) optimizing current and developing new molecular technologies. Suggested approaches to bridge these gaps include prospective longitudinal cohort studies to identify and validate precision biomarkers for prognostication of disease course, and prediction and monitoring of treatment response. To achieve this, harmonization across studies is key as well as development of standardized methods and infrastructure. The implementation of state-of-the-art molecular technologies, systems biology and machine learning approaches for multi-omics and clinical data integration and analysis will be also fundamental. Finally, randomized biomarker-stratified trials will be critical to evaluate the clinical utility of validated signatures and biomarkers in improving patient outcomes and cost-effective care.

Ulcerative colitis is a chronic, debilitating disease characterized by damage to the mucous membrane (mucosa) lining the lower gastrointestinal tract. Endoscopic and histological evidence of healing of the damaged mucosa, referred to as mucosal healing, is increasingly accepted as an important endpoint in clinical practice and in clinical trials due to its association with improved clinical outcomes. In fact, in a draft guidance to industry, the Food and Drug Administration encouraged incorporating histologic evaluation in ulcerative colitis clinical trials. However, there is no community consensus regarding how to define mucosal healing and the field lacks standardized protocols for measuring this treatment target endpoint. Standardized assessment of histologic disease activity in clinical trials is challenging for many reasons, including variability in collection of mucosal biopsies (e.g., number and location), lack of fully validated histology scoring systems, and absence of standard definitions of histologic improvement and remission. The lack of standardized measurement of mucosal healing constitutes a major challenge for drug development and negatively impacts optimization of clinical management for patients. Therefore, the Biomarkers Consortium of the Foundation for the National Institutes of Health recently launched a consortium project, with governmental, nonprofit and industry partners, to address this gap by establishing a common methodology for a histologic measurement of mucosal healing in ulcerative colitis. The project will: 1) Establish the optimum number and locations of biopsies required to determine histological inflammation in ulcerative colitis; 2) Establish a standardized histopathologic measurement of mucosal healing that correlates with clinical outcomes; and 3) Develop an automated machine learning-based methodology for histopathological scoring of mucosal healing. The final results of this project will contribute to a consensus methodology with the goal of establishing a regulatory standard for future industry-sponsored clinical trials, with the potential to contribute to improved tools for clinical monitoring of disease activity in ulcerative colitis.

Novel technologies is part of five focus areas of the Challenges in IBD research document, which also includes preclinical human IBD mechanisms, environmental triggers, precision medicine and pragmatic clinical research. The Challenges in IBD research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the novel technologies section is focused on prioritizing unmet clinical needs in IBD that will benefit from novel technologies applied to: 1) non-invasive detection and monitoring of active inflammation and assessment of treatment response; 2) mucosal targeted drug delivery systems; and 3) prevention of post-operative septic complications and treatment of fistulizing complications. Proposed approaches include development of multiparametric imaging modalities and biosensors, to enable non invasive or minimally invasive detection of pro-inflammatory signals to monitor disease activity and treatment responses. Additionally, technologies for local drug delivery to control unremitting disease and increase treatment efficacy while decreasing systemic exposure are also proposed. Finally, research on biopolymers and other sealant technologies to promote post-surgical healing; and devices to control anastomotic leakage and prevent post-surgical complications and recurrences are also needed. ispartof: INFLAMMATORY BOWEL DISEASES vol:25 issue:Suppl 2 pages:S24-S30 ispartof: location:England status: published

Pragmatic clinical research is part of five focus areas of the Challenges in IBD research document, which also includes preclinical human IBD mechanisms, environmental triggers, novel technologies, and precision medicine. The Challenges in IBD research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the pragmatic clinical research section is focused on highlighting gaps that need to be addressed in order to optimize and standardize IBD care. Identified gaps include: 1) understanding the incidence and prevalence of IBD; 2) evaluating medication positioning to increase therapeutic effectiveness; 3) understanding the utility of therapeutic drug monitoring (TDM); 4) studying pain management; and 5) understanding healthcare economics and resources utilization. To address these gaps, there is a need to emphasize the use of emerging data sources and real-world evidence to better understand epidemiologic and therapeutic trends in IBD, expanding on existing data to better understand how and where we should improve care. Proposed approaches include epidemiological studies in ethnically and geographically diverse cohorts to estimate incidence and prevalence of IBD and impact of diversity on treatment patterns and outcomes. The implementation of new clinical trial design and methodologies will be essential to evaluate optimal medication positioning, appropriate use of TDM in adults and children, and multidisciplinary approaches to IBD pain management and its impact on healthcare resources.

Preclinical human IBD mechanisms is part of five focus areas of the Challenges in IBD research document, which also include environmental triggers, novel technologies, precision medicine and pragmatic clinical research. The Challenges in IBD research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the preclinical human IBD mechanisms manuscript is focused on highlighting the main research gaps in the pathophysiological understanding of human IBD. These research gap areas include: 1) triggers of immune responses; 2) intestinal epithelial homeostasis and wound repair; 3) age-specific pathophysiology; 4) disease complications; 5) heterogeneous response to treatments; and 6) determination of disease location. As an approach to address these research gaps, the prioritization of reverse translation studies is proposed in which clinical observations are the foundation for experimental IBD research in the lab, and for the identification of new therapeutic targets and biomarkers. The use of human samples in validating basic research findings and development of precision medicine solutions is also proposed. This prioritization aims to put emphasis on relevant biochemical pathways and humanized in vitro and in vivo models that extrapolate meaningfully to human IBD, to eventually yield first-in-class and effective therapies.

OBJECTIVE Despite their origins in different germ layers, pancreatic islet cells share many common developmental features with neurons, especially serotonin-producing neurons in the hindbrain. Therefore, we tested whether these developmental parallels have functional consequences. RESEARCH DESIGN AND METHODS We used transcriptional profiling, immunohistochemistry, DNA-binding analyses, and mouse genetic models to assess the expression and function of key serotonergic genes in the pancreas. RESULTS We found that islet cells expressed the genes encoding all of the products necessary for synthesizing, packaging, and secreting serotonin, including both isoforms of the serotonin synthetic enzyme tryptophan hydroxylase and the archetypal serotonergic transcription factor Pet1. As in serotonergic neurons, Pet1 expression in islets required homeodomain transcription factor Nkx2.2 but not Nkx6.1. In β-cells, Pet1 bound to the serotonergic genes but also to a conserved insulin gene regulatory element. Mice lacking Pet1 displayed reduced insulin production and secretion and impaired glucose tolerance. CONCLUSIONS These studies demonstrate that a common transcriptional cascade drives the differentiation of β-cells and serotonergic neurons and imparts the shared ability to produce serotonin. The interrelated biology of these two cell types has important implications for the pathology and treatment of diabetes.

During pregnancy, the energy requirements of the fetus impose changes in maternal metabolism. Increasing insulin resistance in the mother maintains nutrient flow to the growing fetus, whereas prolactin and placental lactogen counterbalance this resistance and prevent maternal hyperglycemia by driving expansion of the maternal population of insulin-producing beta cells. However, the exact mechanisms by which the lactogenic hormones drive beta cell expansion remain uncertain. Here we show that serotonin acts downstream of lactogen signaling to stimulate beta cell proliferation. Expression of serotonin synthetic enzyme tryptophan hydroxylase-1 (Tph1) and serotonin production rose sharply in beta cells during pregnancy or after treatment with lactogens in vitro. Inhibition of serotonin synthesis by dietary tryptophan restriction or Tph inhibition blocked beta cell expansion and induced glucose intolerance in pregnant mice without affecting insulin sensitivity. Expression of the G alpha(q)-linked serotonin receptor 5-hydroxytryptamine receptor-2b (Htr2b) in maternal islets increased during pregnancy and normalized just before parturition, whereas expression of the G alpha(i)-linked receptor Htr1d increased at the end of pregnancy and postpartum. Blocking Htr2b signaling in pregnant mice also blocked beta cell expansion and caused glucose intolerance. These studies reveal an integrated signaling pathway linking beta cell mass to anticipated insulin need during pregnancy. Modulators of this pathway, including medications and diet, may affect the risk of gestational diabetes.

Background Clostridium difficile infection (CDI), a complication of antibiotic-induced injury to the gut microbiome, is a prevalent and dangerous cause of infectious diarrhea. Antimicrobial therapy for CDI is typically effective for acute symptoms, but up to one third of patients later experience recurrent CDI. Fecal-derived microbiota transplantation (FMT) can ameliorate the underlying dysbiosis and is highly effective for recurrent CDI. Traditional methods of FMT are limited by patient discomfort, risk and inefficient procedures. Many individuals with recurrent CDI have extensive comorbidities and advanced age. Widespread use of FMT requires strategies that are non-invasive, scalable and applicable across healthcare settings. Methods A method to facilitate microbiota transfer was developed. Fecal samples were collected and screened for potential pathogens. Bacteria were purified, concentrated, cryopreserved and formulated into multi-layered capsules. Capsules were administered to patients with recurrent CDI, who were then monitored for 90 days. Results Thirteen women and six men with recurrent CDI were provided with microbiota transfer with orally administered capsules. The procedure was well tolerated. Thirteen individuals responded to a single course. Four patients were cured after a second course. There were 2 failures. The cumulative clinical cure rate of 89% is similar to the rates achieved with reported fecal-derived transplantation procedures. Conclusions Recurrent CDI represents a profound dysbiosis and a debilitating chronic disease. Stable cure can be achieved by restoring the gut microbiome with an effective, well-tolerated oral capsule treatment. This strategy of microbiota transfer can be widely applied and is particularly appropriate for frail patients. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-0930-z) contains supplementary material, which is available to authorized users.

Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes.

Systemic infection can initiate or exacerbate central nervous system (CNS) pathology, even in the absence of overt invasion of bacteria into the CNS. Recent epidemiological studies have demonstrated that human survivors of sepsis have an increased risk of long-term neurocognitive decline. There is thus a need for improved understanding of the physiological mechanisms whereby acute sepsis affects the CNS. In particular, MyD88-dependent activation of brain microvascular endothelial cells and a resulting loss of blood-brain barrier integrity have been proposed to play an important role in the effects of systemic inflammation on the CNS. Signaling through the vagus nerve has also been considered to be an important component of CNS responses to systemic infection. Here, we demonstrate that blood-brain barrier permeabilization and hippocampal transcriptional responses during polymicrobial sepsis occur even in the absence of MyD88-dependent signaling in cerebrovascular endothelial cells. We further demonstrate that these transcriptional responses can occur without vagus nerve input. These results suggest that redundant signals mediate CNS responses in sepsis. Either endothelial or vagus nerve activation may be individually sufficient to transmit systemic inflammation to the central nervous system. Transcriptional activation in the forebrain in sepsis may be mediated by MyD88-independent endothelial mechanisms or by non-vagal neuronal pathways.

Background Multicellular organisms are characterized by a remarkable diversity of morphologically distinct and functionally specialized cell types. Transgenic techniques for the manipulation of gene expression in specific cellular populations are highly useful for elucidating the development and function of these cellular populations. Given notable similarities in developmental gene expression between pancreatic β-cells and serotonergic neurons, we examined the pattern of Cre-mediated recombination in the nervous system of a widely used mouse line, Pdx1-cre (formal designation, Tg(Ipf1-cre)89.1Dam), in which the expression of Cre recombinase is driven by regulatory elements upstream of the pdx1 (pancreatic-duodenal homeobox 1) gene. Methods Single (hemizygous) transgenic mice of the pdx1-creCre/0 genotype were bred to single (hemizygous) transgenic reporter mice (Z/EG and rosa26R lines). Recombination pattern was examined in offspring using whole-mount and sectioned histological preparations at e9.5, e10.5, e11.5, e16.5 and adult developmental stages. Results In addition to the previously reported pancreatic recombination, recombination in the developing nervous system and inner ear formation was observed. In the central nervous system, we observed a highly specific pattern of recombination in neuronal progenitors in the ventral brainstem and diencephalon. In the rostral brainstem (r1-r2), recombination occurred in newborn serotonergic neurons. In the caudal brainstem, recombination occurred in non-serotonergic cells. In the adult, this resulted in reporter expression in the vast majority of forebrain-projecting serotonergic neurons (located in the dorsal and median raphe nuclei) but in none of the spinal cord-projecting serotonergic neurons of the caudal raphe nuclei. In the adult caudal brainstem, reporter expression was widespread in the inferior olive nucleus. In the adult hypothalamus, recombination was observed in the arcuate nucleus and dorsomedial hypothalamus. Recombination was not observed in any other region of the central nervous system. Neuronal expression of endogenous pdx1 was not observed. Conclusions The Pdx1-cre mouse line, and the regulatory elements contained in the corresponding transgene, could be a valuable tool for targeted genetic manipulation of developing forebrain-projecting serotonergic neurons and several other unique neuronal sub-populations. These results suggest that investigators employing this mouse line for studies of pancreatic function should consider the possible contributions of central nervous system effects towards resulting phenotypes.

Background: Serotonin (5-HT) is a neurotransmitter with important roles in the regulation of neurobehavioral processes, particularly those regulating affect in humans. Drugs that potentiate serotonergic neurotransmission by selectively inhibiting the reuptake of serotonin (SSRIs) are widely used for the treatment of psychiatric disorders. Although the regulation of serotonin synthesis may be an factor in SSRI efficacy, the effect of chronic SSRI administration on 5-HT synthesis is not well understood. Here, we describe effects of chronic administration of the SSRI citalopram (CIT) on 5-HT synthesis and content in the mouse forebrain. Methodology/Principal Findings: Citalopram was administered continuously to adult male C57BL/6J mice via osmotic minipump for 2 days, 14 days or 28 days. Plasma citalopram levels were found to be within the clinical range. 5-HT synthesis was assessed using the decarboxylase inhibition method. Citalopram administration caused a suppression of 5-HT synthesis at all time points. CIT treatment also caused a reduction in forebrain 5-HIAA content. Following chronic CIT treatment, forebrain 5-HT stores were more sensitive to the depleting effects of acute decarboxylase inhibition. Conclusions/Significance: Taken together, these results demonstrate that chronic citalopram administration causes a sustained suppression of serotonin synthesis in the mouse forebrain. Furthermore, our results indicate that chronic 5-HT reuptake inhibition renders 5-HT brain stores more sensitive to alterations in serotonin synthesis. These results suggest that the regulation of 5-HT synthesis warrants consideration in efforts to develop novel antidepressant strategies.

Serotonin (5-HT) is a neuromodulator with important roles in the regulation of physiological processes, particularly those regulating affect in humans. Drugs which selectively potentiate serotonergic neurotransmission by inhibiting the reuptake of serotonin (SSRIs) are widely used for the treatment of psychiatric disorders. It is thought that the therapeutic efficacy of SSRIs is limited by homeostatic mechanisms which counteract the ability of the drug to potentiate serotonergic neurotransmission. Here, I describe my work on two such mechanisms. First, I describe a role for 5-HT 2C receptors in counteracting the physiological and behavioral effect of SSRIs. In serotonin 2C receptor null mutant mice, extracellular 5-HT was unaltered at baseline. Upon administration of the SSRI fluoxetine, extracellular 5-HT rapidly increased in both wild-type and null mutant mice; this effect was potentiated in mutant mice. Furthermore, mutant mice had a greater response to fluoxetine in a behavioral assay for antidepressant effect. Pharmacological blockade of 5-HT 2C receptors also potentiated the effects of fluoxetine and another SSRI, citalopram, in wild-type mice and rats. In a second line of investigation, we investigated the effects of chronic citalopram treatment on 5-HT synthesis, which is an important factor in the clinical effects of SSRIs. Administration of citalopram for 2 days, 14 days or 28 days suppressed 5-HT synthesis. 5-HT content was not significantly reduced by citalopram at any time point. However, chronic citalopram did cause a significant reduction in forebrain 5-HT content when monoamine synthesis was completely inhibited by acute administration of an amino acid decarboxylase inhibitor. 5-HIAA content was reduced in citalopram-treated brains at all time points. These results demonstrate a sustained suppression of serotonin synthesis by chronic citalopram administration and suggest that serotonergic neurons are particularly dependent on de novo synthesis of 5-HT when the reuptake of 5-HT is inhibited. In technical appendices I describe novel tools for the study of serotonin biology. Taken together, these studies improve our understanding of the physiological actions of SSRIs and suggest strategies for improving their efficacy.