Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3(-/-) mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.

Deficiency in the monoamine degradation enzyme monoamine oxidase A (MAOA) or prenatal exposure to the monoamine uptake inhibitor cocaine alters behavior in humans and rodents, but the mechanisms are unclear. In MAOA knock-out mice, inhibiting serotonin synthesis during development can prevent abnormal segregation of axons in the retinogeniculate and somatosensory thalamocortical systems. To investigate this effect, we crossed MAOA knock-outs with mice lacking the serotonin transporter 5-HTT or the 5-HT1B receptor, two molecules present in developing sensory projections. Segregation was abnormal in 5-HTT knock-outs and MAOA/5-HTT double knock-outs but was normalized in MAOA/5-HT1B double knock-outs and MAOA/5-HTT/5-HT1B triple knock-outs. This demonstrates that the 5-HT1B receptor is a key factor in abnormal segregation of sensory projections and suggests that serotonergic drugs represent a risk for the development of these projections. We also found that the 5-HT1B receptor has an adverse developmental impact on beam-walking behavior in MAOA knock-outs. Finally, because the 5-HT1B receptor inhibits glutamate release, our results suggest that visual and somatosensory projections must release glutamate for proper segregation.

By taking up serotonin (5-hydroxytryptamine, 5-HT) released in the extracellular space, the 5-HT transporter (5-HTT) regulates central 5-HT neurotransmission. Possible adaptive changes in 5-HT neurotransmission in knock-out mice that do not express the 5-HT transporter were investigated with special focus on 5-HT1A and 5-HT1B receptors. Specific labelling with radioligands and antibodies, and competitive RT-PCR, showed that 5-HT1A receptor protein and mRNA levels were significantly decreased in the dorsal raphe nucleus (DRN), increased in the hippocampus and unchanged in other forebrain areas of 5-HTT-/- vs. 5-HTT+/+ mice. Such regional differences also concerned 5-HT1B receptors because a decrease in their density was found in the substantia nigra (-30%) but not the globus pallidus of mutant mice. Intermediate changes were noted in 5-HTT+/- mice compared with 5-HTT+/+ and 5-HTT-/- animals. Quantification of [35S]GTP-gamma-S binding evoked by potent 5-HT1 receptor agonists confirmed such changes as a decrease in this parameter was noted in the DRN (-66%) and the substantia nigra (-30%) but not other brain areas in 5-HTT-/- vs. 5-HTT+/+ mice. As expected from actions mediated by functional 5-HT1A and 5-HT1B autoreceptors, a decrease in brain 5-HT turnover rate after i.p. administration of ipsapirone (a 5-HT1A agonist), and an increased 5-HT outflow in the substantia nigra upon local application of GR 127935 (a 5-HT1B/1D antagonist) were observed in 5-HTT+/+ mice. Such effects were not detected in 5-HTT-/- mice, further confirming the occurrence of marked alterations of 5-HT1A and 5-HT1B autoreceptors in these animals.

Serotonin (5-HT) controls a wide range of biological functions. In the brain, its implication as a neurotransmitter and in the control of behavioral traits has been largely documented. At the periphery, its modulatory role in physiological processes, such as the cardiovascular function, is still poorly understood. The rate-limiting enzyme of 5-HT synthesis, tryptophan hydroxylase (TPH), is encoded by two genes, the well characterized tph1 gene and a recently identified tph2 gene. In this article, based on the study of a mutant mouse in which the tph1 gene has been inactivated by replacement with the ␤-galactosidase gene, we establish that the neuronal tph2 is expressed in neurons of the raphe nuclei and of the myenteric plexus, whereas the nonneuronal tph1, as detected by ␤-galactosidase expression, is in the pineal gland and the enterochromaffin cells. Anatomic examination of the mutant mice revealed larger heart sizes than in wild-type mice. Histological investigation indicates that the primary structure of the heart muscle is not affected. Hemodynamic analyses demonstrate abnormal cardiac activity, which ultimately leads to heart failure of the mutant animals. This report links loss of tph1 gene expression, and thus of peripheral 5-HT, to a cardiac dysfunction phenotype. The tph1 ؊/؊ mutant may be valuable for investigating cardiovascular dysfunction observed in heart failure in humans. S erotonin (5-hydroxytryptamine, 5-HT) was discovered in blood as a vasoconstrictor of large vessels (1). Subsequently, it has been found in the gastrointestinal tract as a contractile substance identical with enteramine (2), in the CNS as a neurotransmitter (3), and in the pineal gland as an intermediate in the synthesis of melatonin, the neurohormone implicated in the circadian rhythmicity of physiological functions (4). 5-HT is detected early during brain development, suggesting its involvement in neuronal proliferation, migration, and differentiation (5). 5-HT modulates a variety of behavioral functions, including regulation of sleep͞wakefulness, appetite, nociception, mood, stress, and maternal or sexual behavior (6). Altered regulation of 5-HT in human affects behavioral traits and personality disorders, such as impulsive aggression, manic depressive illness, anxiety and alcoholism, and neurological conditions, such as migraine (7-10).About 95% of the 5-HT in the periphery is in the gastrointestinal tract (11), where it initiates responses as diverse as nausea, intestinal secretion, and peristaltis and has been implicated in gastroenteric diseases, such as irritable bowel syndrome (12). The 5-HT originating from the gastrointestinal tract is stored in blood platelets and participates in blood coagulation and pressure and in homeostasis. In the heart, an increased 5-HT availability has been shown to produce arrhythmia, leading to heart block or to valvular fibroplasia (13). 5-HT has also been suggested to regulate cardiovascular development (14). Recently, disruption of 5HT-2B receptor revealed a role for 5-HT by means of...

IntroductionExposure to chronic hypoxia leads to the development of pulmonary hypertension (PH) owing to persistent vasoconstriction and structural remodeling of pulmonary vessels. Proliferation of vascular smooth muscle cells (SMCs) is an important component of pulmonary vessel remodeling that results in increased thickness of the medial muscular coat in normally muscular arteries and in extension of muscle into smaller and more peripheral arteries (1). The mechanism by which hypoxia induces pulmonary SMCs' proliferation, however, is not well understood. One current hypothesis is that hypoxia may directly affect the expression of specific genes involved in pulmonary vascular SMC growth (2).The serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) in pulmonary vascular SMCs has many attributes suggesting that it may be a key determinant of pulmonary vessel remodeling. In addition to contributing to the uptake and subsequent inactivation of 5-HT passing through the lung, 5-HTT mediates the proliferation of pulmonary vascular SMCs through its ability to internalize indoleamine (3, 4). The requirement of 5-HTT as a mediator of 5-HT mitogenic activity appears specific for pulmonary vascular SMCs, since it has not been reported in other cell types (5). Moreover, exposure of pulmonary vascular SMCs to hypoxia increases 5-HTT expression and activity (3, 6), an effect associated with potentiation of the mitogenic action of 5-HT (6). Increased 5-HTT gene expression also occurs in remodeled pulmonary vessels of rats during PH development associated with chronic exposure to hypoxia (6). Because 5-HTT is a target for drugs that recently have been shown to increase the risk of PH development in humans (7), it may be of clinical relevance; therefore elucidation of its role in pulmonary vascular SMC proliferation is of interest. However, direct evidence for a role of 5-HTT in vessel remodeling during PH is lacking.The purpose of this study was to investigate whether 5-HTT deficiency affects the development of pulmonary vascular remodeling and PH during chronic hypoxia. We used mice with targeted disruption of the 5-HTT gene (5-HTT -/-) (8) and investigated their hemo- Hypoxia is a well-recognized stimulus for pulmonary blood vessel remodeling and pulmonary hypertension development. One mechanism that may account for these effects is the direct action of hypoxia on the expression of specific genes involved in vascular smooth muscle cell (SMC) proliferation. Previous studies demonstrated that the serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) mediates the mitogenic activity of 5-HT in pulmonary vascular SMCs and is overexpressed during hypoxia. Thus, 5-HT-related mitogenic activity is increased during hypoxia. Here, we report that mice deficient for 5-HTT (5-HTT -/-) developed less hypoxic pulmonary hypertension and vascular remodeling than paired 5-HTT +/+ controls. When maintained under normoxia, 5-HTT -/--mutant mice had normal hemodynamic parameters, low blood 5-HT levels, deficient platelet 5-HT uptake, and u...

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Background-The mechanism of pulmonary artery smooth muscle cell (PA-SMC) hyperplasia in idiopathic pulmonary artery hypertension (iPH) may involve both an inherent characteristic of PA-SMCs and abnormal control by external stimuli. We investigated the role of pulmonary microvascular endothelial cells (P-ECs) in controlling PA-SMC growth. Methods and Results-Serum-free medium of quiescent P-ECs elicited marked PA-SMC proliferation, and this effect was greater with P-ECs from patients with iPH than from control subjects and greater with PA-SMCs from these patients than from control subjects. Fluoxetine, which inhibits serotonin-induced mitogenesis by blocking the serotonin transporter, and p-chlorophenylalanine, which inhibits serotonin synthesis by blocking tryptophan hydroxylase (TPH), caused a similar 60% reduction in the growth-promoting effect of P-EC media, whereas endothelin receptor blockers had no effect. Assays of TPH activity in P-EC medium based on p-chlorophenylalanine-sensitive 5-hydroxytryptophan accumulation or serotonin determination indicated serotonin synthesis by P-ECs and an increase in this TPH-dependent process in iPH. Expression of the tph1 gene encoding the peripheral form of the TPH enzyme was increased in lungs and P-ECs from patients with iPH. Lung TPH1 immunostaining was confined to the pulmonary vessel intima. Conclusions-P-ECs produce paracrine factors governing PA-SMC growth. Serotonin, the main P-EC-derived growth factor, is overproduced in iPH and contributes to PA-SMC hyperplasia.

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