Rescue of impaired blood-brain barrier in tuberous sclerosis complex patient derived neurovascular unit.

BACKGROUND: Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene and dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). METHODS: We generated TSC disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. RESULTS: Using microphysiological systems, we demonstrate that a BBB generated from TSC2 heterozygous mutant cells shows increased permeability. This can be rescued by wild type astrocytes or by treatment with rapamycin, an mTOR kinase inhibitor. CONCLUSION: Our results demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of cell lineages contributing to TSC pathogenesis and informs future therapeutics.

Comparison of Wild-Type and High-risk PNPLA3 variants in a Human Biomimetic Liver Microphysiology System for Metabolic Dysfunction-associated Steatotic Liver Disease Precision Therapy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a worldwide health epidemic with a global occurrence of approximately 30%. The pathogenesis of MASLD is a complex, multisystem disorder driven by multiple factors including genetics, lifestyle, and the environment. Patient heterogeneity presents challenges for developing MASLD therapeutics, creation of patient cohorts for clinical trials and optimization of therapeutic strategies for specific patient cohorts. Implementing pre-clinical experimental models for drug development creates a significant challenge as simple in vitro systems and animal models do not fully recapitulate critical steps in the pathogenesis and the complexity of MASLD progression. To address this, we implemented a precision medicine strategy that couples the use of our liver acinus microphysiology system (LAMPS) constructed with patient-derived primary cells. We investigated the MASLD-associated genetic variant PNPLA3 rs738409 (I148M variant) in primary hepatocytes, as it is associated with MASLD progression. We constructed LAMPS with genotyped wild type and variant PNPLA3 hepatocytes together with key non-parenchymal cells and quantified the reproducibility of the model. We altered media components to mimic blood chemistries, including insulin, glucose, free fatty acids, and immune activating molecules to reflect normal fasting (NF), early metabolic syndrome (EMS) and late metabolic syndrome (LMS) conditions. Finally, we investigated the response to treatment with resmetirom, an approved drug for metabolic syndrome-associated steatohepatitis (MASH), the progressive form of MASLD. This study using primary cells serves as a benchmark for studies using patient biomimetic twins constructed with patient iPSC-derived liver cells using a panel of reproducible metrics. We observed increased steatosis, immune activation, stellate cell activation and secretion of pro-fibrotic markers in the PNPLA3 GG variant compared to wild type CC LAMPS, consistent with the clinical characterization of this variant. We also observed greater resmetirom efficacy in PNPLA3 wild type CC LAMPS compared to the GG variant in multiple MASLD metrics including steatosis, stellate cell activation and the secretion of pro-fibrotic markers. In conclusion, our study demonstrates the capability of the LAMPS platform for the development of MASLD precision therapeutics, enrichment of patient cohorts for clinical trials, and optimization of therapeutic strategies for patient subgroups with different clinical traits and disease stages.

Gravity-perfused airway-on-a-chip optimized for quantitative BSL-3 studies of SARS-CoV-2 infection: barrier permeability, cytokine production, immunohistochemistry, and viral load assays.

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Rescue of Impaired Blood-Brain Barrier in Tuberous Sclerosis Complex Patient Derived Neurovascular Unit.

Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene. Dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). We generated disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Using these microphysiological systems, we demonstrate that the BBB generated from TSC2 heterozygous mutant cells shows increased permeability which can be rescued by wild type astrocytes and with treatment with rapamycin, an mTOR kinase inhibitor. Our results further demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of the cell lineages contributing to TSC pathogenesis.

Application of a Human Blood Brain Barrier Organ-on-a-Chip Model to Evaluate Small Molecule Effectiveness against Venezuelan Equine Encephalitis Virus.

The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.

Predicting susceptibility to SARS-CoV-2 infection based on structural differences in ACE2 across species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the global pandemic of coronavirus disease-2019 (COVID-19). SARS-CoV-2 is a zoonotic disease, but little is known about variations in species susceptibility that could identify potential reservoir species, animal models, and the risk to pets, wildlife, and livestock. Certain species, such as domestic cats and tigers, are susceptible to SARS-CoV-2 infection, while other species such as mice and chickens are not. Most animal species, including those in close contact with humans, have unknown susceptibility. Hence, methods to predict the infection risk of animal species are urgently needed. SARS-CoV-2 spike protein binding to angiotensin-converting enzyme 2 (ACE2) is critical for viral cell entry and infection. Here we integrate species differences in susceptibility with multiple in-depth structural analyses to identify key ACE2 amino acid positions including 30, 83, 90, 322, and 354 that distinguish susceptible from resistant species. Using differences in these residues across species, we developed a susceptibility score that predicts an elevated risk of SARS-CoV-2 infection for multiple species including horses and camels. We also demonstrate that SARS-CoV-2 is nearly optimal for binding ACE2 of humans compared to other animals, which may underlie the highly contagious transmissibility of this virus among humans. Taken together, our findings define potential ACE2 and SARS-CoV-2 residues for therapeutic targeting and identification of animal species on which to focus research and protection measures for environmental and public health.

Advances in blood-brain barrier modeling in microphysiological systems highlight critical differences in opioid transport due to cortisol exposure.

BACKGROUND: The United States faces a national crisis involving opioid medications, where currently more than 130 people die every day. To combat this epidemic, a better understanding is needed of how opioids penetrate into the central nervous system (CNS) to facilitate pain relief and, potentially, result in addiction and/or misuse. Animal models, however, are a poor predictor of blood-brain barrier (BBB) transport and CNS drug penetration in humans, and many traditional 2D cell culture models of the BBB and neurovascular unit have inadequate barrier function and weak or inappropriate efflux transporter expression. Here, we sought to better understand opioid transport mechanisms using a simplified microfluidic neurovascular unit (NVU) model consisting of human brain microvascular endothelial cells (BMECs) co-cultured with astrocytes. METHODS: Human primary and induced pluripotent stem cell (iPSC)-derived BMECs were incorporated into a microfluidic NVU model with several technical improvements over our previous design. Passive barrier function was assessed by permeability of fluorescent dextrans with varying sizes, and P-glycoprotein function was assessed by rhodamine permeability in the presence or absence of inhibitors; quantification was performed with a fluorescent plate reader. Loperamide, morphine, and oxycodone permeability was assessed in the presence or absence of P-glycoprotein inhibitors and cortisol; quantification was performed with mass spectrometry. RESULTS: We first report technical and methodological optimizations to our previously described microfluidic model using primary human BMECs, which results in accelerated barrier formation, decreased variability, and reduced passive permeability relative to Transwell models. We then demonstrate proper transport and efflux of loperamide, morphine, and oxycodone in the microfluidic NVU containing BMECs derived from human iPSCs. We further demonstrate that cortisol can alter permeability of loperamide and morphine in a divergent manner. CONCLUSIONS: We reveal a novel role for the stress hormone cortisol in modulating the transport of opioids across the BBB, which could contribute to their abuse or overdose. Our updated BBB model represents a powerful tool available to researchers, clinicians, and drug manufacturers for understanding the mechanisms by which opioids access the CNS.

The Microbiome and the Gut-Liver-Brain Axis for Central Nervous System Clinical Pharmacology: Challenges in Specifying and Integrating In Vitro and In Silico Models.

The complexity of integrating microbiota into clinical pharmacology, environmental toxicology, and opioid studies arises from bidirectional and multiscale interactions between humans and their many microbiota, notably those of the gut. Hosts and each microbiota are governed by distinct central dogmas, with genetics influencing transcriptomics, proteomics, and metabolomics. Each microbiota's metabolome differentially modulates its own and the host's multi-omics. Exogenous compounds (e.g., drugs and toxins), often affect host multi-omics differently than microbiota multi-omics, shifting the balance between drug efficacy and toxicity. The complexity of the host-microbiota connection has been informed by current methods of in vitro bacterial cultures and in vivo mouse models, but they fail to elucidate mechanistic details. Together, in vitro organ-on-chip microphysiological models, multi-omics, and in silico computational models have the potential to supplement the established methods to help clinical pharmacologists and environmental toxicologists unravel the myriad of connections between the gut microbiota and host health and disease.

Functional Coupling of Human Microphysiology Systems: Intestine, Liver, Kidney Proximal Tubule, Blood-Brain Barrier and Skeletal Muscle.

Organ interactions resulting from drug, metabolite or xenobiotic transport between organs are key components of human metabolism that impact therapeutic action and toxic side effects. Preclinical animal testing often fails to predict adverse outcomes arising from sequential, multi-organ metabolism of drugs and xenobiotics. Human microphysiological systems (MPS) can model these interactions and are predicted to dramatically improve the efficiency of the drug development process. In this study, five human MPS models were evaluated for functional coupling, defined as the determination of organ interactions via an in vivo-like sequential, organ-to-organ transfer of media. MPS models representing the major absorption, metabolism and clearance organs (the jejunum, liver and kidney) were evaluated, along with skeletal muscle and neurovascular models. Three compounds were evaluated for organ-specific processing: terfenadine for pharmacokinetics (PK) and toxicity; trimethylamine (TMA) as a potentially toxic microbiome metabolite; and vitamin D3. We show that the organ-specific processing of these compounds was consistent with clinical data, and discovered that trimethylamine-N-oxide (TMAO) crosses the blood-brain barrier. These studies demonstrate the potential of human MPS for multi-organ toxicity and absorption, distribution, metabolism and excretion (ADME), provide guidance for physically coupling MPS, and offer an approach to coupling MPS with distinct media and perfusion requirements.

Metabolic consequences of inflammatory disruption of the blood-brain barrier in an organ-on-chip model of the human neurovascular unit.

BACKGROUND: Understanding blood-brain barrier responses to inflammatory stimulation (such as lipopolysaccharide mimicking a systemic infection or a cytokine cocktail that could be the result of local or systemic inflammation) is essential to understanding the effect of inflammatory stimulation on the brain. It is through the filter of the blood-brain barrier that the brain responds to outside influences, and the blood-brain barrier is a critical point of failure in neuroinflammation. It is important to note that this interaction is not a static response, but one that evolves over time. While current models have provided invaluable information regarding the interaction between cytokine stimulation, the blood-brain barrier, and the brain, these approaches-whether in vivo or in vitro-have often been only snapshots of this complex web of interactions. METHODS: We utilize new advances in microfluidics, organs-on-chips, and metabolomics to examine the complex relationship of inflammation and its effects on blood-brain barrier function ex vivo and the metabolic consequences of these responses and repair mechanisms. In this study, we pair a novel dual-chamber, organ-on-chip microfluidic device, the NeuroVascular Unit, with small-volume cytokine detection and mass spectrometry analysis to investigate how the blood-brain barrier responds to two different but overlapping drivers of neuroinflammation, lipopolysaccharide and a cytokine cocktail of IL-1ß, TNF-α, and MCP1,2. RESULTS: In this study, we show that (1) during initial exposure to lipopolysaccharide, the blood-brain barrier is compromised as expected, with increased diffusion and reduced presence of tight junctions, but that over time, the barrier is capable of at least partial recovery; (2) a cytokine cocktail also contributes to a loss of barrier function; (3) from this time-dependent cytokine activation, metabolic signature profiles can be obtained for both the brain and vascular sides of the blood-brain barrier model; and (4) collectively, we can use metabolite analysis to identify critical pathways in inflammatory response. CONCLUSIONS: Taken together, these findings present new data that allow us to study the initial effects of inflammatory stimulation on blood-brain barrier disruption, cytokine activation, and metabolic pathway changes that drive the response and recovery of the barrier during continued inflammatory exposure.

Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor.

The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier.

Coordinated messenger RNA/microRNA changes in fibroblasts of patients with major depression.

BACKGROUND: Peripheral biomarkers for major psychiatric disorders have been an elusive target for the last half a century. Dermal fibroblasts are a simple, relevant, and much underutilized model for studying molecular processes of patients with affective disorders, as they share considerable similarity of signal transduction with neuronal tissue. METHODS: Cultured dermal fibroblast samples from patients with major depressive disorder (MDD) and matched control subjects (n = 16 pairs, 32 samples) were assayed for genome-wide messenger RNA (mRNA) expression using microarrays. In addition, a simultaneous quantitative polymerase chain reaction-based assessment of >1000 microRNA (miRNA) species was performed. Finally, to test the relationship between the mRNA-miRNA expression changes, the two datasets were correlated with each other. RESULTS: Our data revealed that MDD fibroblasts, when compared with matched control subjects, showed a strong mRNA gene expression pattern change in multiple molecular pathways, including cell-to-cell communication, innate/adaptive immunity, and cell proliferation. Furthermore, the same patient fibroblasts showed altered expression of a distinct panel of 38 miRNAs, which putatively targeted many of the differentially expressed mRNAs. The miRNA-mRNA expression changes appeared to be functionally connected, as the majority of the miRNA and mRNA changes were in the opposite direction. CONCLUSIONS: Our data suggest that combined miRNA-mRNA assessments are informative about the disease process and that analyses of dermal fibroblasts might lead to the discovery of promising peripheral biomarkers of MDD that could be potentially used to aid the diagnosis and allow mechanistic testing of disturbed molecular pathways.

Metabolic consequences of interleukin-6 challenge in developing neurons and astroglia.

BACKGROUND: Maternal immune activation and subsequent interleukin-6 (IL-6) induction disrupt normal brain development and predispose the offspring to developing autism and schizophrenia. While several proteins have been identified as having some link to these developmental disorders, their prevalence is still small and their causative role, if any, is not well understood. However, understanding the metabolic consequences of environmental predisposing factors could shed light on disorders such as autism and schizophrenia. METHODS: To gain a better understanding of the metabolic consequences of IL-6 exposure on developing central nervous system (CNS) cells, we separately exposed developing neuron and astroglia cultures to IL-6 for 2 hours while collecting effluent from our gravity-fed microfluidic chambers. By coupling microfluidic technologies to ultra-performance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS), we were able to characterize the metabolic response of these CNS cells to a narrow window of IL-6 exposure. RESULTS: Our results revealed that 1) the use of this technology, due to its superb media volume:cell volume ratio, is ideally suited for analysis of cell-type-specific exometabolome signatures; 2) developing neurons have low secretory activity at baseline, while astroglia show strong metabolic activity; 3) both neurons and astroglia respond to IL-6 exposure in a cell type-specific fashion; 4) the astroglial response to IL-6 stimulation is predominantly characterized by increased levels of metabolites, while neurons mostly depress their metabolic activity; and 5) disturbances in glycerophospholipid metabolism and tryptophan/kynurenine metabolite secretion are two putative mechanisms by which IL-6 affects the developing nervous system. CONCLUSIONS: Our findings are potentially critical for understanding the mechanism by which IL-6 disrupts brain function, and they provide information about the molecular cascade that links maternal immune activation to developmental brain disorders.

Sex Is a major determinant of neuronal dysfunction in neurofibromatosis type 1.

OBJECTIVE: Children with neurofibromatosis-1 (NF1) are at risk for developing numerous nervous system abnormalities, including cognitive problems and brain tumors (optic pathway glioma). Currently, there are few prognostic factors that predict clinical manifestations or outcomes in patients, even in families with an identical NF1 gene mutation. In this study, we leveraged Nf1 genetically engineered mice (GEM) to define the potential role of sex as a clinically relevant modifier of NF1-associated neuronal dysfunction. METHODS: Deidentified clinical data were analyzed to determine the impact of sex on optic glioma-associated visual decline in children with NF1. In addition, Nf1 GEM were employed as experimental platforms to investigate sexually dimorphic differences in learning/memory, visual acuity, retinal ganglion cell (RGC) death, and Nf1 protein (neurofibromin)-regulated signaling pathway function (Ras activity, cyclic adenosine monophosphate [cAMP], and dopamine levels). RESULTS: Female patients with NF1-associated optic glioma were twice as likely to undergo brain magnetic resonance imaging for visual symptoms and 3× more likely to require treatment for visual decline than their male counterparts. As such, only female Nf1 GEM exhibited a decrement in optic glioma-associated visual acuity, shorter RGC axons, and attenuated cAMP levels. In contrast, only male Nf1 GEM showed spatial learning/memory deficits, increased Ras activity, and reduced dopamine levels. INTERPRETATION: Collectively, these observations establish sex as a major prognostic factor underlying neuronal dysfunction in NF1, and suggest that sex should be considered when interpreting future preclinical and clinical study results.

The role of cannabinoid 1 receptor expressing interneurons in behavior.

Schizophrenia is a devastating neurodevelopmental disorder that affects approximately 1% of the population. Reduced expression of the 67-kDa protein isoform of glutamic acid decarboxylase (GAD67) is a hallmark of the disease and is encoded by the GAD1 gene. In schizophrenia, GAD67 downregulation occurs in multiple interneuronal subpopulations, including the cannabinoid receptor type 1 positive (CNR1+) cells, but the functional consequences of these disturbances are not well understood. To investigate the role of the CNR1-positive GABA-ergic interneurons in behavioral and molecular processes, we employed a novel, miRNA-mediated transgenic mouse approach. We silenced the Gad1 transcript using a miRNA engineered to specifically target Gad1 mRNA under the control of Cnr1 bacterial artificial chromosome. Behavioral characterization of our transgenic mice showed elevated and persistent conditioned fear associated with an auditory cue and a significantly altered response to an amphetamine challenge. These deficits could not be attributed to sensory deficits or changes in baseline learning and memory. Furthermore, HPLC analyses revealed that Cnr1/Gad1 mice have enhanced serotonin levels, but not dopamine levels in response to amphetamine. Our findings demonstrate that dysfunction of a small subset of interneurons can have a profound effect on behavior and that the GABA-ergic, monoamine, and cannabinoid systems are functionally interconnected. The results also suggest that understanding the function of various interneuronal subclasses might be essential to develop knowledge-based treatment strategies for various mental disorders including schizophrenia and substance abuse.