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1.
Distribution of serotonin 5-HT1C receptor mRNA in adult rat brain   总被引:4,自引:0,他引:4  
B J Hoffman  E Mezey 《FEBS letters》1989,247(2):453-462
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Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.  相似文献   

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To date, there have been at least eight different receptors for the neurotransmitter serotonin (5-HT) identified in the central nervous system. These receptors fall into four pharmacological classes: 5-HT1, 5-HT2, 5-HT3 and 5-HT4. The 5-HT1 class has been shown to contain at least four pharmacologically distinct subtypes, 5-HT1A-D. Of these, cDNAs encoding the 5-HT1A and 5-HT1C receptors have been previously characterized. We now report the cloning and expression of a rat brain cDNA encoding another member of the 5-HT1 receptor family. Transient expression of this clone demonstrated high-affinity binding of [3H]5-HT with a pharmacological profile corresponding to that of the 5-HT1B subtype: 5-CT, 5-HT greater than propranolol greater than methysergide greater than rauwolscine greater than 8-OH-DPAT. In situ hybridization revealed expression of cognate mRNA within cells of the dorsal and median raphe nuclei, consistent with previous reports that the 5-HT1B receptor acts as an autoreceptor on 5-HT terminals in this species. mRNA expression was also detected in cells within the CA1 region of hippocampus, striatum, layer 4 of cortex and in the cerebellum, suggesting a previously unrecognized post-synaptic role for the 5-HT1B receptor.  相似文献   

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To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.  相似文献   

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Although acute food deprivation and chronic food restriction both result in body weight loss, they produce different metabolic states. To evaluate how these two treatments affect hypothalamic peptide systems involved in energy homeostasis, we compared patterns of hypothalamic neuropeptide Y (NPY), agouti-related protein (AgRP), proopiomelanocotin (POMC), and leptin receptor gene expression in acutely food-deprived and chronically food-restricted rats. Both acute food deprivation and chronic food restriction reduced body weight and circulating leptin levels and resulted in increased arcuate NPY and decreased arcuate POMC gene expression. Arcuate AgRP mRNA levels were only elevated in acutely deprived rats. NPY gene expression was increased in the compact subregion of the dorsomedial hypothalamus (DMH) in response to chronic food restriction, but not in response to acute food deprivation. Leptin receptor expression was not affected by either treatment. Double in situ hybridization histochemistry revealed that, in contrast to the situation in the arcuate nucleus, NPY and leptin receptor mRNA-expressing neurons were not colocalized in the DMH. Together, these data suggest that arcuate and DMH NPY gene expression are differentially regulated. DMH NPY-expressing neurons do not appear to be under the direct control of leptin signaling.  相似文献   

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5-hydroxytryptamine (5-HT) or serotonin 2A receptors play an important role in modulation of prefrontal cortex (PFC) activity and have been implicated in the physiopathology of psychiatric disorders. There is no quantitative information on the percentage of glutamatergic and GABAergic cells that express 5-HT(2A) receptors in human and monkey PFC. We have used double in situ hybridization to quantify the mRNA co-localization of 5-HT(2A) receptor with the glutamatergic transporter vesicular glutamate transporter 1, and with the GABAergic marker glutamic acid decarboxylase 65/67 and in parvalbumin and calbindin GABAergic cell populations. Our results show that nearly every glutamatergic cell (86-100%) in layers II-V expressed 5-HT(2A) receptor mRNA in both species. This percentage was lower in layer VI (13-31%). In contrast, not all the GABAergic interneurons (13-46%) expressed 5-HT(2A) receptor mRNA. This receptor was expressed in 45-69% of parvalbumin and in 61-87% of calbindin positive cells. These results indicate that, while the majority of glutamatergic neurons can be sensitive to 5-HT action via 5-HT(2A) receptors, this modulation occurs only in a limited population of GABAergic interneurons and provides new neuroanatomical information about the role played by serotonin through 5-HT(2A) receptors in the PFC and on the sites of action for drugs such as antipsychotics and antidepressants used in treatment of psychiatric disorders.  相似文献   

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Relaxin 3 or insulin like peptide 7 has been identified as a new member of the insulin/relaxin superfamily. We recently reported that relaxin 3 was dominantly expressed in the brain, particularly in neurons of the nucleus incertus (NI) of the median dorsal tegmental pons and that it might act as a neurotransmitter. In the present study we investigated the developmental expression and serotonergic regulation of relaxin 3 gene in the rat brain. Relaxin 3 mRNA appeared at embryonic day 18 in the near region of the fourth ventricle, and was shown to have increased its density and the number of expressing neurons by in situ hybridization and RT-PCR examination. Relaxin 3 peptide was detected after birth by immunocytochemistry. Since the NI is located just caudal to the dorsal raphe nucleus where abundant serotonin (5-HT) neurons are present, we examined if 5-HT effects on the expression of relaxin 3. Relaxin 3 gene expression in the NI significantly increased after 5-HT depletion by p-chlorophenylalanine (PCPA) administration. We also observed the 5-HT1A receptor localization in relaxin 3 positive neurons of the NI. This result suggests that 5-HT negatively regulates the expression of relaxin 3 gene in the NI. The function of relaxin 3 neurons in the brain is influenced by the serotonergic activity.  相似文献   

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We describe a combined immunocytochemistry/in situ hybridization technique which allows for the simultaneous localization of protein and mRNA in a single cell. We have carried out these studies either on non-innervated skeletal myotubes or on myotubes which we have innervated with spinal cord explants or ciliary neurons. Our methods allowed us to detect acetylcholine receptor gene mRNA sequences which are expressed in low abundance within the cells and to determine the intracellular and intranuclear domains where these sequences are concentrated, as well as to identify neurons and their processes. The isotopic detection of RNA in combination with fluorescence microscopy produces high-resolution double-label images, with little background and good preservation of morphology, providing a powerful tool for detection of gene expression and protein content at the single-cell level.  相似文献   

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To study the effect of NPY deletion on the regulation of its receptors in the NPY knockout (NPY KO) mice, the expression and binding of NPY receptors were investigated by in situ hybridization and receptor autoradiography using (125)I-[Leu(31),Pro(34)]PYY and (125)I-PYY(3-36) as radioligands. A 6-fold increase in Y2 receptor mRNA was observed in the CA1 region of the hippocampus in NPY KO mice, but a significant change could not be detected for Y1, Y4, Y5 and y6 receptors. Receptor binding reveals a 60-400% increase of Y2 receptor binding in multiple brain areas. A similar increase in Y1 receptor binding was seen only in the hypothalamus. These results demonstrate the NPY receptor expression is altered in mice deficient for its natural ligand.  相似文献   

13.
Localization of aldolase C mRNA in brain cells   总被引:4,自引:0,他引:4  
The expression of aldolase C and aldolase A mRNA was assessed by Northern blot hybridization using RNAs purified from cultured rat and mouse brain neurons and astroglial cells. Neurons were found to contain about 4-fold more aldolase C mRNA and about twice as much aldolase A mRNA than astroglia. Analysis of the cellular localization of aldolase C mRNA by in situ hybridization to brain slices showed a predominantly neuronal labeling with an irregular distribution. A strong signal was observed in Purkinje cell somata and a weaker signal in subpopulations of neurons in cerebral cortex, striatum, hippocampus, hypothalamic nuclei and primary olfactory cortex.  相似文献   

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The actions of enteric 5-HT are terminated by 5-HT transporter (SERT)-mediated uptake, and gastrointestinal motility is abnormal in SERT -/- mice. We tested the hypothesis that adaptive changes in enteric 5-HT(3) receptors help SERT -/- mice survive despite inefficient 5-HT inactivation. Expression of mRNA encoding enteric 5-HT(3A) subunits was similar in SERT +/+ and -/- mice, but that of 5-HT(3B) subunits was fourfold less in SERT -/- mice. 5-HT(3B) mRNA was found, by in situ hybridization, in epithelial cells and enteric neurons. 5-HT evoked a fast inward current in myenteric neurons that was pharmacologically identified as 5-HT(3) mediated. The EC(50) of the 5-HT response was lower in SERT +/+ (18 microM) than in SERT -/- (36 microM) mice and desensitized rapidly in a greater proportion of SERT -/- neurons; however, peak amplitudes, steady-state current, and decay time constants were not different. Adaptive changes thus occur in the subunit composition of enteric 5-HT(3) receptors of SERT -/- mice that are reflected in 5-HT(3) receptor affinity and desensitization.  相似文献   

15.
The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT(7) receptor family. Expression of the Am5-HT(7) receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT(7) is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC(50) = 1.1-1.8 nm). The Am5-HT(7) receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.  相似文献   

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The hippocampus receives major noradrenergic and serotoninergic (5-HT) innervations which interact with corticosteroid-sensitive cells. However, the subregional localization of these actions and the corticosteroid receptor types involved have not been defined and current ligand binding techniques for estimating corticosteroid receptors are hampered by several methodological limitations. We have developed in situ hybridization histochemical techniques to allow specific and sensitive estimation of glucocorticoid (GR) and mineralocorticoid receptor (MR) mRNA expression in rat hippocampus. Investigation of the effects of 5,7-dihydroxytryptamine lesions of 5-HT neurons showed significantly reduced GR and MR mRNA expression in some hippocampal subregions. Both abnormal 5-HT neurotransmission and excessive corticosteroid secretion are associated with major affective disorders, particularly depression. The crucial interaction between these two systems may occur, at least in part, at the level of regulation of hippocampal corticosteroid receptor expression.  相似文献   

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Serotonin 1A (5-HT(1A)) receptors are found in high densities in prefrontal cortex. However, their distribution within cortical cell populations is unknown in both humans and primates. We used double in situ hybridization histochemistry to quantify the percentage of glutamatergic and GABAergic neurons expressing 5-HT(1A) receptors in human and monkey prefrontal cortex. Moreover, in the case of the monkey, we also quantified the parvalbumin and calbindin GABAergic subpopulations expressing this receptor. 5-HT(1A) receptor mRNAs were expressed in about 80% of glutamatergic neurons in external layers II and upper III, and in around 50% in layer VI; they were also present in approximately 20% of GABAergic neurons in both species. Although they were found in up to 43% of the calbindin cell subpopulation they were rarely present in parvalbumin cells in monkey prefrontal cortex. The knowledge of the phenotype of the prefrontal cortex (PFC) cells expressing 5-HT(1A) will help understanding serotonin actions in PFC.  相似文献   

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Treatment of rodents with exogenous leptin increases SOCS-3 mRNA levels in the arcuate nucleus (ARC) and dorsomedial nucleus (DMN) of the hypothalamus. To determine if SOCS-3 gene activity in the hypothalamus could be influenced by changes in physiological levels of circulating leptin, we performed in situ hybridization (ISH) and immunostaining for SOCS-3 expression in fed vs. fasted (48 h) rats. The ARC and DMN were the only regions of the diencephalon that showed SOCS-3 ISH and the autoradiographic ISH signal for SOCS-3 mRNA was visibly less in the ARC and DMN of fasted rats. The ISH signal for SOCS-3 mRNA was decreased 70% in the ARC and 90% in the DMN (to background levels) when animals were fasted (P<0.01), consistent with decreased immunostaining for SOCS-3 protein observed in the fasted rats. Double fluorescence ISH (FISH) analyses showed colocalization of SOCS-3 mRNA with mRNAs for NPY and POMC in the ARC. These findings are consistent with increased leptin signaling to the NPY and POMC neurons in the ARC by physiological levels of circulating leptin during normal feeding. Therefore, changes in SOCS-3 mRNA levels in the ARC and DMN can be viewed as an indicator of relative physiological leptin signaling to the hypothalamus and also identify cells responding directly to leptin signaling through its cognate receptor.  相似文献   

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用PRV和NPY免疫荧光双标记法研究了大鼠孤束核中NPY样神经元对咽肌运动神经元的调控。PRV注射大鼠咽肌后,在孤束核的中介亚核和中间亚核中可见许多PRV和NPY双标记细胞。首次证明了大鼠孤束核中的NPY样神经元和咽肌运动神经元的联系。推测NPY可能对咽肌运动的精确调控有关。  相似文献   

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