Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway.

Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma-infected mice 5days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin (5-HT) and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and 5-HT metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.

Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

BACKGROUND AND PURPOSE: The current lack of disease-modifying therapeutics to manage neurological and neurodegenerative conditions justifies the development of more efficacious agents. One distinct pathway leading to neuronal death in these conditions and which represents a very promising and attractive therapeutic target is parthanatos, involving overactivation of PARP-1. We therefore sought to identify small molecules that could be neuroprotective by targeting the pathway. EXPERIMENTAL APPROACH: Using HeLa cells, we developed and optimized an assay for high-throughput screening of about 5120 small molecules. Structure-activity relationship (SAR) study was carried out in HeLa and SH-SY5Y cells for molecules related to the initial active compound. The neuroprotective ability of each active compound was tested in cortical neuronal cultures. KEY RESULTS: 4'-Methoxyflavone (4MF) showed activity by preventing the decrease in cell viability of HeLa and SH-SY5Y cells caused by the DNA-alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which induces parthanatos. A similar compound from the SAR study, 3',4'-dimethoxyflavone (DMF), also showed significant activity. Both compounds reduced the synthesis and accumulation of poly (ADP-ribose) polymer and protected cortical neurones against cell death induced by NMDA. CONCLUSIONS AND IMPLICATIONS: Our data reveal additional neuroprotective members of the flavone class of flavonoids and show that methoxylation of the parent flavone structure at position 4' confers parthanatos-inhibiting activity while additional methoxylation at position 3', reported by others to improve metabolic stability, does not destroy the activity. These molecules may therefore serve as leads for the development of novel neurotherapeutics for the management of neurological and neurodegenerative conditions.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

Spatial and functional relationship between poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in the brain.

Poly(ADP-ribose) polymerases (PARPs) are members of a family of enzymes that utilize nicotinamide adenine dinucleotide (NAD(+)) as substrate to form large ADP-ribose polymers (PAR) in the nucleus. PAR has a very short half-life due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). PARP-1 mediates acute neuronal cell death induced by a variety of insults including cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism, and CNS trauma. While PARP-1 is localized to the nucleus, PARG resides in both the nucleus and cytoplasm. Surprisingly, there appears to be only one gene encoding PARG activity, which has been characterized in vitro to generate different splice variants, in contrast to the growing family of PARPs. Little is known regarding the spatial and functional relationships of PARG and PARP-1. Here we evaluate PARG expression in the brain and its cellular and subcellular distribution in relation to PARP-1. Anti-PARG (alpha-PARG) antibodies raised in rabbits using a purified 30 kDa C-terminal fragment of murine PARG recognize a single band at 111 kDa in the brain. Western blot analysis also shows that PARG and PARP-1 are evenly distributed throughout the brain. Immunohistochemical studies using alpha-PARG antibodies reveal punctate cytosolic staining, whereas anti-PARP-1 (alpha-PARP-1) antibodies demonstrate nuclear staining. PARG is enriched in the mitochondrial fraction together with manganese superoxide dismutase (MnSOD) and cytochrome C (Cyt C) following whole brain subcellular fractionation and Western blot analysis. Confocal microscopy confirms the co-localization of PARG and Cyt C. Finally, PARG translocation to the nucleus is triggered by NMDA-induced PARP-1 activation. Therefore, the subcellular segregation of PARG in the mitochondria and PARP-1 in the nucleus suggests that PARG translocation is necessary for their functional interaction. This translocation is PARP-1 dependent, further demonstrating a functional interaction of PARP-1 and PARG in the brain.

Influence of duration of focal cerebral ischemia and neuronal nitric oxide synthase on translocation of apoptosis-inducing factor to the nucleus.

Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 h of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 h of permanent ischemia. However, as little as 20 min of reperfusion after 1 h of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 h of reperfusion. In contrast, 20 min of reperfusion after 2 h of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 h of reperfusion. With brief occlusion of 30 min duration, nuclear AIF remained undetectable at both 20 min and 6 h and became evident only after 24 h of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 h), but is markedly delayed after brief ischemia (30 min). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.

Mining for survival genes.

Many stressful, but not lethal, stimuli activate endogenous protective mechanisms that significantly decrease the degree of injury to subsequent injurious stimuli. This protective mechanism is termed preconditioning and tolerance. It occurs across organ systems including the brain and nervous system. Preconditioning has been investigated in cell and animal models and recently been shown to potentially occur in human brain. Learning more about these powerful endogenous neuroprotective mechanisms could help identify new approaches to treat patients with stroke and other central nervous system disorders or injury. Cell and animal models are helping us to better understand the network response of gene and protein expression that activates the neuroprotective response.

EndoG is dispensable in embryogenesis and apoptosis.

The mitochondrial protein, endonuclease G (EndoG), is one of the endonucleases implicated in DNA fragmentation during apoptosis. It has been shown to translocate from the mitochondria to the nucleus upon cell death stimuli. These observations suggest that EndoG is a mitochondrial cell death effector, and that it possibly acts as a cell death nuclease, similar to DNA fragmentation factor. To better understand the role of EndoG in development and apoptosis, we generated EndoG null mice by homologous gene targeting without disruption of D2Wsu81e. EndoG null mice are viable and develop to adulthood with no obvious abnormalities. Fibroblasts generated from the EndoG null mice show no difference in susceptibility when induced to die by a variety of intrinsic and extrinsic apoptotic stimuli. Additionally, EndoG null mice are equally sensitive to excitotoxic stress. These data suggest that EndoG is not essential for early embryogenesis and apoptosis.

Nitric oxide, S-nitrosylation and neurodegeneration.

Nitric oxide is a critically important signaling molecule, controlling a wide range of pathways and biological processes. Highly reactive nitric oxide mediates its function through reaction with different molecules directly or indirectly. One of these modifications is the S-nitrosylation of cysteine residues in proteins. S-nitrosylation is emerging as an important redox signaling mechanism and has been found to regulate a broad range of biologic, physiologic and cellular functions. One of the major findings in this area recently is the linkage of nitrosative stress to various neurodegenerative disorders. Oxidative stress has long been regarded as a prime mediator in the development of neurodegeneration as various indices of oxidative stress are readily observed in postmortem studies. A causative role for nitrosative stress in neurodegeneration is just now being appreciated. The direct connection of S-nitrosylation to the pathogenesis of Parkinson's disease in recent studies further provide insights into how imbalance in nitric oxide metabolism can contribute to the development of selective injury and disease.

Poly(ADP-ribosyl)ation regulation of life and death in the nervous system.

Poly(ADP-ribosyl)ation is required by multicellular eukaryotes to ensure genomic integrity under conditions of mild to moderate genotoxic stress. However, severe stress following acute neuronal injury causes overactivation of poly(ADP-ribose) polymerase-1, which results in unregulated poly(ADP-ribose) (PAR) synthesis and widespread neuronal cell death. Once thought to be a necrotic cell death resulting from energy failure, PARP-1 activation is now known to induce the nuclear translocation of apoptosis-inducing factor, which results in caspase-independent cell death. Conversely, poly(ADP-ribose) glycohydrolase, once thought to contribute to neuronal injury, now appears to have a protective role as demonstrated by recent studies utilizing gene disruption technology. Thus, the emerging mechanism dictating the fate of neurons appears to involve the regulation of PAR levels in neurons. Therefore, therapies targeting poly(ADP-ribosyl)ation in the treatment of neurodegenerative conditions such as stroke and Parkinson's disease are required to inhibit PAR synthesis and/or facilitate its degradation.

Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease.

Parkinson disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. Here we show that parkin interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. We further show that familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1.

Identification by mRNA differential display of two up-regulated genes as candidate mediators of AIDS dementia.

BACKGROUND: In the dementia associated with acquired immunodeficiency syndrome (AIDS), indirect pathomechanisms are important mediators of progressive neuronal injury and variable candidate molecules of potential pathogenetic importance have been identified. MATERIALS AND METHODS: In an attempt to characterize additional mediators of human immunodeficiency virus type 1 (HIV-1)-induced neurotoxicity in vivo we have adapted the mRNA differential display technique to monitor the gene expression pattern in postmortem cortical tissue from AIDS patients with (n = 7) and without (n = 8) cognitive impairment as well as from HIV-1 seronegative controls (n = 4). RESULTS: Out of 29 differentially expressed cDNAs, two cDNA clones had confirmed variation of transcriptional regulation as assessed by reverse Northern analysis and gene-specific reverse transcription polymerase chain reaction (RT-PCR) and were up-regulated in the cortex of patients with AIDS dementia. Nucleotide sequence analysis of the two cDNAs identified known genes not previously associated with the pathogenesis of AIDS dementia, including the neurotrophin receptor tyrosine kinase receptor B (TrkB) and the potassium channel human open rectifyer K+ channel (ORK) homologous open reading frame (HOHO1). CONCLUSIONS: The altered expression of these transcripts may contribute to AIDS dementia through the enhancement of microglial activation and immunologic nitric oxide synthase (iNOS) activity by abnormal neurotrophic regulation and interference with membrane excitability through disturbance of local ion homeostasis.

Neuroprotective effect of sigma(1)-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) is linked to reduced neuronal nitric oxide production.

BACKGROUND AND PURPOSE: The potent final sigma(1)-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) provides neuroprotection in experimental stroke. We tested the hypothesis that PPBP attenuates striatal tissue damage after middle cerebral artery occlusion (MCAO) by a mechanism involving reduction of ischemia-evoked nitric oxide (NO) production. Furthermore, we determined whether the agent fails to protect ischemic brain when neuronal nitric oxide synthase (nNOS) is genetically deleted or pharmacologically inhibited (selective nNOS inhibitor, 7-nitroindazole [7-NI]). METHODS: Halothane-anesthetized adult male Wistar rats were subjected to 2 hours of MCAO by the intraluminal filament occlusion technique. All physiological variables were controlled during the ischemic insult. In vivo striatal NO production was estimated via microdialysis by quantification of local, labeled citrulline recovery after labeled arginine infusion. In a second series of experiments, nNOS null mutants (nNOSKOs) and the genetically matched wild-type (WT) strain were treated with 90 minutes of MCAO. Brains were harvested at 22 hours of reperfusion for measurement of infarction volume by triphenyltetrazolium chloride histology. RESULTS: PPBP attenuated infarction volume at 22 hours of reperfusion in cerebral cortex and striatum and markedly attenuated NO production in ischemic and nonischemic striatum during occlusion and early reperfusion. Treatment with 7-NI mimicked the effects of PPBP. In WT mice, infarction volume was robustly decreased by both PPBP and 7-NI, but the efficacy of PPBP was not altered by pharmacological nNOS inhibition in combined therapy. In contrast, PPBP did not decrease infarction volume in nNOSKO mice. CONCLUSIONS: These data suggest that the mechanism of neuroprotection of PPBP in vivo is through attenuation of nNOS activity and ischemia-evoked NO production. Neuroprotective effects of PPBP are lost when nNOS is not present or is inhibited; therefore, PPBP likely acts upstream from NO generation and its subsequent neurotoxicity.

Neuroimmunophilins: novel neuroprotective and neuroregenerative targets.

Cyclosporin A (CsA) and FK506 (tacrolimus) are immunosuppresants that are widely used in organ transplantation. CsA is an 11-member cyclic peptide, whereas FK506 is a macrolide antibiotic. Recently, these powerful and useful compounds have become of great interest to neuroscientists for their unique neuroprotective and neuroregenerative effects. These drugs and nonimmunosuppressive analogs protect neurons from the effects of glutamate excitotoxicity, focal ischemia, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic cell death. They also stimulate functional recovery of neurons in a variety of neurologic injury paradigms. These drugs exert their effects via immunophilins, the protein receptors for these agents. The immunophilin ligands show particular promise as a novel class of neuroprotective and neuroregenerative agents that have the potential to treat a variety of neurologic disorders.

Neuroimmunophilin ligands exert neuroregeneration and neuroprotection in midbrain dopaminergic neurons.

Immunosuppressant drugs, like FK506, and nonimmunosuppressant compounds like, GPI1046 and L685818, are immunophilin ligands that specifically bind to immunophilins, like FK506 binding protein 12 (FKBP12). Several lines of evidence show that these ligands exert neurotrophic properties in neural injury models and in PC12 cells. However, the mechanism of the neurotrophic function of the immunophilin ligands is poorly known. In the present study, we use MPP+ and 6-OHDA toxicity models to examine both neuroprotective and neuroregenerative effects of immunophilin ligands on primary cultures of midbrain dopaminergic neurons. We find that FK506, GPI1046 and L685818 at concentrations from 0.01 to 1 microM partially, but significantly, protect dopaminergic neurons against both MPP+ and 6-OHDA toxicity. By Western blot analysis, we also find that all three compounds prevent tyrosine hydroxylase (TH) loss induced by MPP+ and 6-OHDA treatments. Morphologic analysis of dopaminergic neurons, by immunocytochemistry, shows that MPP+ and 6-OHDA cause the retraction and loss of neuronal processes, while FK506, GPI1046 and L685818 promote regeneration of these processes as indicated by increases in process number and length. To examine if FKBP12 is required for neurotrophic effects of immunophilin ligands, we cultured dopaminergic neurons from FKBP12 knockout mice and find that FK506 still protects dopaminergic neurons against MPP+ toxicity. These results suggest that FKBP12 is not essential for the neurotrophic properties of immunophilin ligands, and immunophilin ligands are a new class of neuroprotective and neuroregenerative agents that may have therapeutic potential in a variety of neurological disorders.

Critical role for nitric oxide signaling in cardiac and neuronal ischemic preconditioning and tolerance.

Preconditioning to ischemic tolerance is a phenomenon in which brief episodes of a subtoxic insult induce a robust protection against the deleterious effects of subsequent, prolonged, lethal ischemia. The subtoxic stimuli that constitute the preconditioning event are quite diverse, ranging from brief ischemic episodes, spreading depression or potassium depolarization, chemical inhibition of oxidative phosphorylation, exposure to excitotoxins and cytokines. The beneficial effects of preconditioning were first demonstrated in the heart; it is now clear that preconditioning can induce ischemic tolerance in a variety of organ systems including brain, heart, liver, small intestine, skeletal muscle, kidney, and lung. There are two temporally and mechanistically distinct types of protection afforded by preconditioning stimuli, acute and delayed preconditioning. The signaling cascades that initiate the acute and delayed preconditioning responses may have similar biochemical components. However, the protective effects of acute preconditioning are protein synthesis-independent, mediated by post-translational protein modifications, and are short-lived. The effects of delayed preconditioning require new protein synthesis and are sustained for days to weeks. Elucidation of the molecular mechanisms that are involved in preconditioning and ischemic tolerance and identification of drugs that mimic this protective response have the potential to improve the prognosis of patients at risk for ischemic injury. This article focuses on recent findings on the effects of ischemic preconditioning in the cardiac and nervous systems and discusses potential targets for a successful therapeutic approach to limit ischemia-reperfusion injury.

Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity.

Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional coactivator CREB binding protein, CBP. We found that CBP was depleted from its normal nuclear location and was present in polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescued polyglutamine-induced neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic gain of function, underlying the pathogenesis of polyglutamine disorders.

FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle.

FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor beta (TGF-beta) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12(-/-)) mice manifest cell cycle arrest in G(1) phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-beta1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-beta receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-beta receptor construct. TGF-beta receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12(-/-) cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12(-/-) cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-beta receptor signaling.

Brain serotonin dysfunction accounts for aggression in male mice lacking neuronal nitric oxide synthase.

Genetically engineered mice with targeted disruption of the neuronal nitric oxide synthase (nNOS) gene established the inhibitory role of nitric oxide (NO) in male impulsive aggressive behavior. This was later confirmed by using selective nNOS inhibitors in male wild-type mice. The molecular mechanisms accounting for the aggressive behavior caused by the lack of neuronally derived NO is not known. Recent studies suggest that central serotonergic neuronal circuits and particularly 5-HT(1A) and 5-HT(1B) receptors play a prominent role in the regulation of aggression. Accordingly, we investigated whether the aggressiveness caused by the lack of nNOS might be because of alterations in serotonergic function. We now demonstrate that the excessive aggressiveness and impulsiveness of nNOS knockout mice is caused by selective decrements in serotonin (5-HT) turnover and deficient 5-HT(1A) and 5-HT(1B) receptor function in brain regions regulating emotion. These results indicate an important role for NO in normal brain 5-HT function and may have significant implications for the treatment of psychiatric disorders characterized by aggressiveness and impulsivity.