5-HT3 receptors are membrane ion channels

The neurohormone 5-hydroxytryptamine (5HT or serotonin) exerts its effects by binding to several distinct receptors. One of these is the M-receptor of Gaddum and Picarelli, now called the 5-HT3 receptor, through which 5-HT acts to excite enteric neurons. Ligand-binding and functional studies have shown that the 5-HT3 receptor is widely distributed in peripheral and central nervous tissue and evidence suggests that the receptor might incorporate an ion channel permeable to cations. We now report the first recordings of currents through single ion channels activated by 5-HT3 receptors, in excised (outside-out) membrane patches from neurons of the guinea pig submucous plexus. Whereas application of acetylcholine activated predominantly a 40-pS channel, 5-HT caused unitary currents apparently through two channels of conductances of 15 and 9 pS, which were reversibly blocked by antagonists of the 5-HT3 receptor. Receptors for amine neurotransmitters, including 5-HT1 and 5-HT2, have previously been thought to transduce their effects through GTP-binding proteins: the direct demonstration that 5-HT3 receptors are ligand-gated ion channels implies a role for 5-HT, and perhaps other amines, as a 'fast' synaptic transmitter.     

A cytoplasmic region determines single-channel conductance in 5-HT3 receptors

5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.     

Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding

Functional serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into three subtypes: 5-HT1-like, 5-HT2 and 5-HT3 (ref. 1). Brain binding sites have been identified for both the 5-HT1 and 5-HT2 subtypes. Receptors of the 5-HT3 type have been characterized on isolated peripheral tissue models such as the rat vagus nerve, guinea-pig ileum and isolated rabbit heart. Using these models, selective 5-HT3 receptor antagonists such as MDL 72222 (ref. 5), ICS 205-930 (ref. 6), GR38032F (ref. 7) and BRL 43694 (ref. 8) have been developed. Recently, GR38032F, MDL 72222 and ICS 205-930 have been shown to have behavioural effects in rodents and primates that undoubtedly reflect an action in the central nervous system (refs 9-11 and unpublished observations), suggesting the existence of 5-HT3 receptors in the brain. Here we report direct evidence for the existence of 5-HT3 receptors in rat brain tissue and their distribution, based on high affinity binding of the potent 5-HT3 receptor antagonist 3H-GR65630 to homogenates of rat entorhinal cortex. Selective 5-HT3 receptor antagonists and agonists inhibited binding of 3H-GR65630 with high affinities which correlated well with their actions on the rat isolated vagus nerve. Binding was differentially distributed throughout the brain with high concentrations in cortical and limbic areas.     

Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.     

脑组织中5-HT及受体与运动的研究现状

运用文献资料法,系统地概括了脑内5-羟色胺（5-hydroxytryptamine,5-HT）及其受体的特性,并总结了近年来脑内5-HT及受体与运动的研究现状：1．5-HT作为一种抑制性神经递质可以降低从中枢向外周发放的冲动,导致中枢疲劳。2．5-HT的升高可能首先发生在某一脑区而非全脑。3．耐力运动中大脑5-HT含量升高且在疲劳时加剧。4．力竭运动可同时加快5-HT的合成和降解速度,脑内5-HT在运动即刻几乎没有明显变化。5．5-HT受体各亚型存在较大差异,有的起抑制性作用,有的则发挥兴奋性作用。     

Delta-promoted filopodia mediate long-range lateral inhibition in Drosophila

Drosophila thoracic mechanosensory bristles originate from cells that are singled out from 'proneural' groups of competent epithelial cells. Neural competence is restricted to individual sensory organ precursors (SOPs) by Delta/Notch-mediated 'lateral inhibition', whereas other cells in the proneural field adopt an epidermal fate. The precursors of the large macrochaetes differentiate separately from individual proneural clusters that comprise about 20-30 cells or as heterochronic pairs from groups of more than 100 cells, whereas the precursors of the small regularly spaced microchaetes emerge from even larger proneural fields. This indicates that lateral inhibition might act over several cell diameters; it was difficult to reconcile with the fact that the inhibitory ligand Delta is membrane-bound until the observation that SOPs frequently extend thin processes offered an attractive hypothesis. Here we show that the extension of these planar filopodia--a common attribute of wing imaginal disc cells--is promoted by Delta and that their experimental suppression reduces Notch signalling in distant cells and increases bristle density in large proneural groups, showing that these membrane specializations mediate long-range lateral inhibition.     

Modulation of visual cortical plasticity by acetylcholine and noradrenaline

During a critical period of postnatal development, the temporary closure of one eye in kittens will permanently shift the ocular dominance (OD) of neurones in the striate cortex to the eye that remains open. The OD plasticity can be substantially reduced if the cortex is infused continuously with the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) during the period of monocular deprivation, an effect that has been attributed to selective depletion of cortical noradrenaline. However, several other methods causing noradrenaline (NA) depletion leave the plasticity intact. Here we present a possible explanation for the conflicting results. Combined destruction of the cortical noradrenergic and cholinergic innervations reduces the physiological response to monocular deprivation although lesions of either system alone are ineffective. We also find that 6-OHDA can interfere directly with the action of acetylcholine (ACh) on cortical neurones. Taken together, our results suggest that intracortical 6-OHDA disrupts plasticity by interfering with both cholinergic and noradrenergic transmission and raise the possibility that ACh and NA facilitate synaptic modifications in the striate cortex by a common molecular mechanism.     

Serotonin stimulates DNA synthesis in fibroblasts acting through 5-HT1B receptors coupled to a Gi-protein

Growth factors can be divided into two classes which act through distinct signal transduction pathways. One class including epidermal growth factor, platelet derived growth factor and fibroblast growth factor activates receptor tyrosine kinases, and the second class, including thrombin, bombesin, bradykinin and vasopressin activates a phosphoinositide-specific phospholipase C through GTP-binding proteins which can be inactivated by pertussis toxin. In Chinese hamster lung fibroblasts, thrombin-induced mitogenicity seems to correlate well with phospholipase C activation and both events are sensitive to pertussis toxin. Thrombin, like the other mitogens in this class, simultaneously inhibits adenylate cyclase. This involves an inhibitory G protein (Gi), a well established pertussis toxin substrate. The relative contributions of the two signalling pathways to mitogenicity has not been evaluated so far. We report here that the neurotransmitter serotonin (5-hydroxytryptamine), a contracting agent and mitogen for smooth muscle cells, activates phospholipase C, inhibits adenylate cyclase and stimulates DNA synthesis in fibroblasts. These events are sensitive to pertussis toxin. We show that the mitogenicity of 5-hydroxytryptamine can be uncoupled from phospholipase C activation that is mediated by 5-HT2 receptors, but correlates perfectly with inhibition of adenylate cyclase through 5-HT1B receptor. We propose that inhibition of adenylate cyclase or activation of an undefined effector system by Gi is important in 5-hydroxytryptamine induced DNA synthesis and contributes to the strong mitogenicity of the other members of this family of growth factors.     

Two chemosensory receptors together mediate carbon dioxide detection in Drosophila

Blood-feeding insects, including the malaria mosquito Anopheles gambiae, use highly specialized and sensitive olfactory systems to locate their hosts. This is accomplished by detecting and following plumes of volatile host emissions, which include carbon dioxide (CO2). CO2 is sensed by a population of olfactory sensory neurons in the maxillary palps of mosquitoes and in the antennae of the more genetically tractable fruitfly, Drosophila melanogaster. The molecular identity of the chemosensory CO2 receptor, however, remains unknown. Here we report that CO2-responsive neurons in Drosophila co-express a pair of chemosensory receptors, Gr21a and Gr63a, at both larval and adult life stages. We identify mosquito homologues of Gr21a and Gr63a, GPRGR22 and GPRGR24, and show that these are co-expressed in A. gambiae maxillary palps. We show that Gr21a and Gr63a together are sufficient for olfactory CO2-chemosensation in Drosophila. Ectopic expression of Gr21a and Gr63a together confers CO2 sensitivity on CO2-insensitive olfactory neurons, but neither gustatory receptor alone has this function. Mutant flies lacking Gr63a lose both electrophysiological and behavioural responses to CO2. Knowledge of the molecular identity of the insect olfactory CO2 receptors may spur the development of novel mosquito control strategies designed to take advantage of this unique and critical olfactory pathway. This in turn could bolster the worldwide fight against malaria and other insect-borne diseases.     

NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia

Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a potentially important therapeutic target in disorders in which demyelination is a prominent feature, such as multiple sclerosis, neurotrauma, infections (for example, HIV encephalomyelopathy) and aspects of ischaemic brain injury.     

Topographical rearrangement of acetylcholine receptors alters channel kinetics

Plasma membranes are dynamic structures of proteins and lipids. Protein-protein or protein-lipid interactions within the membrane are believed to have important roles in many membrane functions, including ion transport, enzyme activity and signal reception. The acetylcholine (ACh) receptor-channel complex in skeletal muscle membrane is one of the best known integral membrane proteins. Its ion transport function is accessible to direct measurement at the single-channel level by the use of the 'giga-seal' patch recording technique. Here we used an in situ electrophoresis technique to rearrange the topography of pre-existing ACh receptor-channels in the muscle membrane, and measured the single-channel kinetics of ACh-activated channels in two different molecular environments within the membrane: those in the diffusely distributed region and those in the ACh receptor clusters induced by the applied field. We found that the channel kinetics are significantly prolonged in the ACh receptor cluster compared with the non-clustered region of the same cell. This result strongly supports the notion that the function of a membrane ionic channel depends on the local molecular environment.     

Potentiation of cortical inhibition by visual deprivation

The fine-tuning of circuits in sensory cortex requires sensory experience during an early critical period. Visual deprivation during the critical period has catastrophic effects on visual function, including loss of visual responsiveness to the deprived eye, reduced visual acuity, and loss of tuning to many stimulus characteristics. These changes occur faster than the remodelling of thalamocortical axons, but the intracortical plasticity mechanisms that underlie them are incompletely understood. Long-term depression of excitatory intracortical synapses has been proposed as a general candidate mechanism for the loss of cortical responsiveness after visual deprivation. Alternatively (or in addition), the decreased ability of the deprived eye to activate cortical neurons could be due to enhanced intracortical inhibition. Here we show that visual deprivation leaves excitatory connections in layer 4 (the primary input layer to cortex) unaffected, but markedly potentiates inhibitory feedback between fast-spiking basket cells (FS cells) and star pyramidal neurons (star pyramids). Further, a previously undescribed form of long-term potentiation of inhibition (LTPi) could be induced at synapses from FS cells to star pyramids, and was occluded by previous visual deprivation. These data suggest that potentiation of inhibition is a major cellular mechanism underlying the deprivation-induced degradation of visual function, and that this form of LTPi is important in fine-tuning cortical circuitry in response to visual experience.     

GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels

The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca2+-dependent cellular functions including exocytosis and contractility. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline- and gamma-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins); or (2) intracellular administration of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins. Our findings provide the first direct demonstration of the G-protein-mediated inhibition of voltage-dependent calcium channels by neurotransmitters. This mode of transmitter action may explain the ability of noradrenaline and GABA to presynaptically inhibit Ca2+-dependent neurosecretion from DRG sensory neurones.     

Cloning of adiponectin receptors that mediate antidiabetic metabolic effects

Adiponectin (also known as 30-kDa adipocyte complement-related protein; Acrp30) is a hormone secreted by adipocytes that acts as an antidiabetic and anti-atherogenic adipokine. Levels of adiponectin in the blood are decreased under conditions of obesity, insulin resistance and type 2 diabetes. Administration of adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice. Conversely, adiponectin-deficient mice exhibit insulin resistance and diabetes. This insulin-sensitizing effect of adiponectin seems to be mediated by an increase in fatty-acid oxidation through activation of AMP kinase and PPAR-alpha. Here we report the cloning of complementary DNAs encoding adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) by expression cloning. AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver. These two adiponectin receptors are predicted to contain seven transmembrane domains, but to be structurally and functionally distinct from G-protein-coupled receptors. Expression of AdipoR1/R2 or suppression of AdipoR1/R2 expression by small-interfering RNA supports our conclusion that they serve as receptors for globular and full-length adiponectin, and that they mediate increased AMP kinase and PPAR-alpha ligand activities, as well as fatty-acid oxidation and glucose uptake by adiponectin.     

水体中5-HT对仔蟹生长以及内源5-HT水平的影响

已有研究表明外源给予一定量的5-羟色胺（5-HT）可以调控甲壳动物的发育.本实验将同期变态的Ⅰ期仔蟹（仔Ⅰ）单只浸泡于浓度分别为1,3和5μg/mL的5-HT中,浸泡时间分别为1,2和4d,每组20个平行,每个平行1只,待变态成Ⅱ期仔蟹（仔Ⅱ）后观察各组的存活率、变态时间、规格（壳长、壳宽和湿重）,并测体内5-HT含量...     

NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury

Injury to oligodendrocyte processes, the structures responsible for myelination, is implicated in many forms of brain disorder. Here we show NMDA (N-methyl-D-aspartate) receptor subunit expression on oligodendrocyte processes, and the presence of NMDA receptor subunit messenger RNA in isolated white matter. NR1, NR2A, NR2B, NR2C, NR2D and NR3A subunits showed clustered expression in cell processes, but NR3B was absent. During modelled ischaemia, NMDA receptor activation resulted in rapid Ca2+-dependent detachment and disintegration of oligodendroglial processes in the white matter of mice expressing green fluorescent protein (GFP) specifically in oligodendrocytes (CNP-GFP mice). This effect occurred at mouse ages corresponding to both the initiation and the conclusion of myelination. NR1 subunits were found mainly in oligodendrocyte processes, whereas AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor subunits were mainly found in the somata. Consistent with this observation, injury to the somata was prevented by blocking AMPA/kainate receptors, and preventing injury to oligodendroglial processes required the blocking of NMDA receptors. The presence of NMDA receptors in oligodendrocyte processes explains why previous studies that have focused on the somata have not detected a role for NMDA receptors in oligodendrocyte injury. These NMDA receptors bestow a high sensitivity to acute injury and represent an important new target for drug development in a variety of brain disorders.     

Embryonic acetylcholine receptors guarantee spontaneous contractions in rat developing muscle

Many proteins are expressed in distinct embryonic and adult forms. However, in most cases we do not know why the embryonic form of proteins is required. This question can be readily addressed for the acetylcholine receptor (AChR) because developmentally specified modifications of this ligand-gated ion channel can be directly related to changes in membrane currents. In developing rat soleus muscle, spontaneous transmitter release causes miniature end-plate currents (m.e.p.cs) to flow into the muscle cell. We show here that these m.e.p.cs in neonatal soleus trigger spontaneous contractions. By injecting m.e.p.cs into young fibres, we showed that only embryonic m.e.p.cs can trigger such contractions; adult m.e.p.cs do not last long enough. Developing muscle fibres must be active for synapse and muscle differentiation. Our experiments indicate that the embryonic form of the AChR is essential for spontaneous contractile activity and may therefore be required for normal neuromuscular development.