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Serotonin 5-Hydroxytryptamine_2C Receptor Signaling in Hypothalamic Proopiomelanocortin Neurons: Role in Energy Homeostasis in Females

Department of Physiology and Pharmacology (J.Q., C.X., M.A.B., O.K.R., M.J.K.), Department of Anesthesiology and Perioperative Medicine (O.K.R.), Center for the Study of Weight Regulation and Associated Disorders (J.G.M., W.F.), Division of Neuroscience, Oregon National Primate Research Center (M.A.B., O.K.R.), Oregon Health & Science University, Portland, Oregon

Received May 22, 2007; accepted July 10, 2007

	Abstract

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Hypothalamic proopiomelanocortin (POMC) neurons play a critical role in the regulation of energy balance, and there is a convergence of critical synaptic input including GABA and serotonin on POMC neurons to regulate their output. We found previously that 17β-estradiol (E₂) reduced the potency of the GABA_B receptor agonist baclofen to activate G protein-coupled inwardly rectifying potassium (GIRK) channels in hypothalamic POMC neurons through a membrane estrogen receptor (mER) via a G_q phospholipase C (PLC)-protein kinase C-protein kinase A pathway. We hypothesized that the mER and neurotransmitter receptor signaling pathways converge to control energy homeostasis. Because 5-HT_2C receptors mediate many of the effects of serotonin in POMC neurons, we elucidated the common signaling pathways of E₂ and 5-HT in guinea pigs using single-cell reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, and whole-cell patch recording. Both 5-hydroxytryptamine_2C (5-HT_2C) and 5-HT_2A receptors were coexpressed in POMC neurons. The 5-HT_2A/C agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) desensitized the GABA_B response in a dose-dependent manner, which was antagonized by the selective 5-HT_2C receptor antagonists 8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulphonamido) phenyl-5-oxopentyl]1,3,8-triazaspiro[4.5] decane-2,4-dione hydrochloride (RS102221) and 1,2,3, 4,10,14b-hexahydro-2-methyldibenzo [c,f]pyrazino[1,2-a]-azepine hydrochloride (ORG 3363). The 5-HT_2C receptor was G_q-coupled to PLC activation and hydrolysis of plasma membrane phosphatidylinositol bisphosphate to directly inhibit GIRK channel activity. Coapplication of the two agonists at their EC₅₀ concentrations (DOI, 20 µM, and E₂, 50 nM) produced additive effects. Although there was a significant gender difference in the effects of E₂ on baclofen responses, there was no gender difference in 5-HT_2C receptor-mediated effects. Finally, both DOI and estrogen (intracerebroventricular) inhibited feeding in ovariectomized female mice. Therefore, the G_q signaling pathways of the mER and 5-HT_2C receptors may converge to enhance synaptic efficacy in brain circuits that are critical for maintaining homeostatic functions.

It is interesting that serotonin neurons are targets of ovarian and testicular steroids (Bethea, 1993). These steroid effects have traditionally been attributed to activation of the nuclear receptors (McEwen, 2001). In nonhuman primates, estrogens up-regulate tryptophan hydroxylase in midbrain 5-HT neurons (Bethea et al., 1998), decrease serotonin transporter mRNA expression in midbrain raphe (Bethea et al., 1998), and decrease expression of the 5-HT_2C receptor in a number of hypothalamic nuclei (Gundlah et al., 1999). E₂ modulates many of the homeostatic functions through the transcription factors estrogen receptor- and -β (Couse and Korach, 1999). However, in contrast to the relatively slow genomic effects of E₂, we have identified a putative mER that is G_q-coupled to a PLC-PKC-PKA pathway (Qiu et al., 2003). E₂ reduces the potency of the GABA_B receptor agonist baclofen to activate G-protein-coupled inwardly rectifying K⁺ channels in hypothalamic neurons, and this membrane-delimited signaling pathway also plays a critical role in the control of energy homeostasis (Qiu et al., 2006).

It is noteworthy that serotoninergic drugs (i.e., selective serotonin reuptake inhibitors) and E₂ are effective in alleviating postmenopausal symptoms in women (Stearns et al., 2002). It may be that E₂ and serotonin, via mER and 5-HT_2C receptors, respectively, synergize to regulate energy metabolism in postpubertal female patients. Therefore, understanding the actions of E₂ and serotonin on POMC neurons may provide insight into fundamental differences between female and male patients in the hypothalamic control of feeding and energy homeostasis. In the present study, we sought to elucidate the cellular cascades activated by 5-HT compared with E₂ in hypothalamic POMC neurons using whole-cell recording, scRT-PCR, and real-time RT-PCR techniques and their functional consequences at the whole animal level. Our findings delineate the 5-HT_2C signaling pathway in male and female patients and determine its convergence with the mER signaling pathway in arcuate POMC neurons to control energy homeostasis.

	Materials and Methods

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Animals and Treatments. All animal procedures described in this study are in accordance with institutional guidelines based on the National Institutes of Health standards. Male and female Topeka guinea pigs (400–600 g), bred in our institutional breeding facility, and female multicolor guinea pigs (400–500 g; Elm Hill Breeding Labs, Chelmsford, MA) were used in these experiments. The guinea pigs were maintained under constant temperature (26°C) and light (on between 6:30 AM and 8:30 PM). Animals were housed individually with food and water provided ad libitum. They were gonadectomized under ketamine-xylazine anesthesia (33 and 6 mg/kg, respectively, s.c.) 5 to 7 days before experimentation and were given sesame oil vehicle (0.1 ml, s.c.) 24 h before experimentation. Serum estrogen concentrations were measured in the GDX female guinea pigs by radioimmunoassay (Oregon National Primate Research Center Radioimmunoassay Core, Beaverton, OR) from trunk blood collected on the day of experimentation and were <10 pg/ml. An additional group of female 11- to 12-week-old C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) used for feeding study were housed upon arrival on a 12-h light/dark cycle (lights off at 6:00 PM) with free access to standard pellet diet and water.

Drugs. All drugs were purchased from Calbiochem (La Jolla, CA) unless otherwise specified. tetrodotoxin (Alomone Labs, Jerusalem, Israel) was dissolved in Milli-Q H₂O and further diluted with 0.1% acetic acid (final concentration, 1 mM), pH 4 to 5. E₂ was purchased from Steraloids (Wilton, NH), recrystallized to ensure purity, and dissolved in 100% ethanol to a stock concentration of 1 mM. T and DHT (Steraloids) was also dissolved in 100% ethanol. The PKC inhibitors BIS (100 µM) and rottlerin (10 mM), the PLC inhibitor U73122 [GenBank] (20 mM), the less active analog U73343 [GenBank] (20 mM), the PI-4-kinase inhibitor wortmannin (10 mM), RS102221 hydrochloride (10 mM), and spiperone hydrochloride (20 mM; Tocris, Ellisville, MO) were dissolved in dimethyl sulfoxide. Phosphatidylinositol bisphosphate (PIP₂) was dissolved in the pipette solution at concentration of 5 µM. The solution was sonicated intermittently on ice for 30 min. Sonication was repeated each time before filling a new pipette. DOI (20 mM), m-CPP (10 mM), MK212 (10 mM), and ORG 3363 (20 mM; Organon NV, Oss, The Netherlands) were dissolved in H₂O. The G_q binding protein designed to mimic the C terminus of the G_q subunit and the G_s binding protein designed to mimic the C terminus of the G_s subunit were synthesized by PeptidoGenic Research (Livermore, CA). The peptide sequence for G_q peptide was Ac-LGLNLKEYNLV-OH, and the peptide sequence for G_s peptide was CRMHLRQYELL. The peptides were also dissolved in H₂O. Aliquots of the stock solutions were stored at -20°C until needed.

Electrophysiology. Adult Topeka guinea pigs were gonadectomized 6 to 10 days before each experiment. Each animal was quickly killed by decapitation, the brain rapidly removed from the skull, and a block containing the hypothalamus immediately dissected. The hypothalamic block was submerged in cold (4°C) oxygenated (95% O₂/5% CO₂) aCSF containing the following constituents: 124 mM NaCl, 5 mM KCl, 26 mM NaHCO₃, 2.6 mM NaH₂PO₄, 10 mM dextrose, 10 mM HEPES, 2 mM MgSO₄, and 2 mM CaCl₂ at 4°C. Coronal slices (300–350 µm) through the caudal-rostral extent of the arcuate nucleus were cut with the aid of a vibrating microtome. The slices were transferred to a multiwell auxiliary chamber containing oxygenated aCSF and kept there until electrophysiological recording after 2 h. During recording, slices were maintained in a chamber perfused via a peristaltic pump with warmed (35°C) oxygenated aCSF at a rate of 1.5 ml/min. Microelectrodes (resistances of 3–6 M) were fabricated from borosilicate glass pipettes (1.5 mm outer diameter) and filled with an internal solution, pH 7.30, containing the following constituents: 128 mM potassium gluconate, 10 mM NaCl, 2 mM MgCl₂,11 mM EGTA, 10 mM HEPES, 1 mM ATP, 0.25 mM GTP, and 0.25% biocytin. Standard whole-cell, voltage-clamp procedures were followed using an Axopatch 200A amplifier (Molecular Devices, Sunnyvale, CA). Signals were digitized with a Digidata 1200 and analyzed using pClamp 7.0 software (Molecular Devices). The liquid junctional potential of -10 mV was corrected in the data analysis. Current and voltage traces were also recorded on an analog chart recorder (Gould Instrument Systems, Cleveland, OH). After the formation of a >1 G seal, intracellular access was achieved by suction, and only those cells that showed less than 10% change in access resistance throughout the recording were included in this study. All of the responses to baclofen were measured in voltage clamp as outward currents (V_hold =-60 mV). For the electrophysiology analysis, only cells with gigaohm or better seals were included in this study.

The protocol for drug administration in the whole-cell patch voltage-clamp experiments (V_hold, -60 mV) was followed as described in a previous publication (Qiu et al., 2003). After seals were formed and the whole-cell configuration was obtained, slices were perfused with tetrodotoxin (1 µM) for 5 min. The first GABA_B receptor-mediated response was generated by perfusing baclofen (at EC₅₀ concentration of 5 µM) until a steady-state outward current was obtained (R₁). After drug washout, the current returned to its predrug resting level. The cells were then treated with serotonin receptor agonist drugs DOI and/or other drugs for 15 min, baclofen (5 µM) was perfused again, and R₂ was measured. The effects of serotonin receptor agonist drugs or other drugs on the baclofen response are expressed as a percentage of R₂ over R₁.

Composite dose-response curves were generated from the following logistic equation fitted by computer (Sigma Plot 8.0; SPSS Inc., Chicago, IL) to the data: I_max = 100 x ([agonist]^nH/([agonist]^nH + EC₅₀^nH)), where I_max is the maximum outward current for a given agonist, EC₅₀ represents the agonist potency, and n_H is the Hill slope.

Immunocytochemistry. After electrical recording, the slices were prepared for fluorescence immunocytochemistry as described previously (Qiu et al., 2003). In brief, the slices were fixed with 4% paraformaldehyde in Sorensen's phosphate buffer, pH 7.4, for 120 min, immersed overnight in 20% sucrose dissolved in Sorensen's buffer, and frozen in OCT embedding medium (Sakura Finetek, Torrance, CA) and prepared for immunocytochemistry as described previously (Kelly and Rønnekleiv, 1994). In brief, coronal sections (20 µm) were cut on a cryostat (model 1720 Digital Cryostat; Leitz, Wetzlar, Germany) and mounted on Fisher SuperFrost Plus slides (Fisher Scientific Co., Pittsburgh, PA). Sections were washed for 5 min with 0.1 M sodium phosphate buffer, pH 7.4, and then streptavidin-Cy2 (1:7500–1:10,000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was applied for 2 h. The reaction was terminated by washing with buffer. The slices were scanned for the injected neuron with a Nikon (Melville, NY) Eclipse 800 fluorescence microscope. After localization of the biocytin-filled neurons, the slides containing the appropriate sections were processed for the presence of β-endorphin using fluorescence immunohistochemistry as described previously (Kelly and Rønnekleiv, 1994). In brief, the sections with the biocytin-identified neurons were incubated overnight with a polyclonal β-endorphin antibody (kindly provided by Dr. Robert Eskay, National Institutes of Health, Bethesda, MD) at 1:5000 and washed in 0.1 M phosphate buffer followed by incubation with a donkey anti-rabbit IgG-Cy3 at 1:500 (Jackson ImmunoResearch Laboratories Inc.). The sections were washed with sodium phosphate buffer, and coverslips were applied using a glycerolglycine buffer (2:1), pH 8.6, containing 5% N-propylgallate (Sigma-Aldrich, St. Louis, MO) to reduce photobleaching. Immunostained cells were analyzed and photographed using a Nikon E800 microscope.

Dispersed Single-Cell RT-PCR. Arcuate single-cell harvest from guinea pig hypothalamic slices was performed as described previously (Qiu et al., 2003). In brief, coronal hypothalamic slices (350 µm) were cut on a vibrating microtome and placed in an auxiliary chamber containing oxygenated aCSF. The slices were allowed to recover for 1 to 2 h in the chamber before dispersion. The arcuate nucleus of the hypothalamus was microdissected and incubated in 2 to 3 ml of aCSF containing 1 mg/ml protease XIV (Sigma-Aldrich) for 15 min at 37°C. The tissue was then washed three times in 1 volume of low-calcium aCSF and two times in normal aCSF. The cells were isolated by trituration with flame-polished Pasteur pipettes, dispersed on a dish, and perfused continuously with aCSF at a rate of 1.5 ml/min. Cells were visualized using a Nikon inverted microscope, and individual neurons were patched and harvested into the patch pipette by applying negative pressure. The content of the pipette was expelled into a siliconized microcentrifuge tube containing 5 µl of the following solution: 0.5 µl of 10x buffer (100 mM Tris-HCl, 500 mM KCl, and 1% Triton X-100; Promega, Madison, WI), 15 U of RNasin (Promega), 0.5 µl of 100 mM DTT, and DEPC-treated water (Ambion, Austin, TX). In addition, hypothalamic tissue was homogenized, and total RNA was extracted using the RNeasy kit (QIAGEN, Valencia, CA) according to the protocol of the manufacturer. The harvested cell solution and 25 ng of hypothalamic total RNA in 1 µl were denatured for 5 min at 65°C and cooled on ice for 5 min, and then single-stranded cDNA was synthesized from cellular RNA by adding 50 U of MLVRT (Applied Biosystems, Foster City, CA), 1.5 µl of 10x buffer, 2 mM MgCl₂, 0.2 µl of dNTPs, 15 U of RNasin, 10 mM DTT, 100 ng of random hexamers (Promega), and DEPC-treated water to a final volume of 20 µl. Cells and tissue RNA used as negative controls were processed as described above but without MLVRT. The reaction mixtures were incubated at 42°C for 60 min, denatured at 99°C for 5 min, and cooled on ice for 5 min.

Primers listed in Table 1 were designed using the Clone Manager Software (Scientific and Educational Software, Cary, NC) and synthesized by Invitrogen (Carlsbad, CA). PCR was performed using 3 µl of cDNA template (2 µl for GAPDH) from each RT reaction in a 30 µl of PCR reaction volume containing the following: 3 µl of 10x buffer, 2.4 µl of 25 mM MgCl₂ (2 mM final concentration), 0.2 mM dNTPs, 0.2 µM forward and reverse primers, 2 U of TaqDNA polymerase (Promega), and 0.22 µg of TaqStart antibody (Clontech, Mountain View, CA). TaqDNA polymerase and TaqStart antibody were combined and incubated at room temperature for 5 min, and the remainder of the reaction contents was added to the tube and incubated at 94°C for 2 min. PCR reactions for 5-HT_1A, 5-HT_2A, and 5-HT_2C went through 42 to 47 cycles of amplification according to the following protocols: 20-s denaturation (94°C), 30-s annealing (59–62°C), 30-s elongation (72°C), with a final 72°C extension for 5 min. POMC and GAPDH PCR went through 45 and 37 cycles of amplification, respectively, in two steps: 30-s denaturation (94°C), 45-s annealing (65–67°C), with a final 72°C extension for 5 min. Ten microliters of the PCR products were visualized with ethidium bromide on a 1.5% agarose gel.

Real-Time RT-PCR. Total RNA was extracted from the microdissected arcuate nucleus of GDX female guinea pigs (n = 6) using the RNAqueous-Micro kit (Ambion). The RNA was treated with DNase I using the DNA-free kit (Ambion) according to manufacturer's instructions and quantified using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). Reverse transcription was carried out with 200 ng of total RNA using 50 U RT (Applied Biosystems), 1.5 µl of 10x buffer, 2 mM MgCl₂, 0.2 mM dNTPs, 15 U of RNasin, 10 mM DTT, 100 ng of random hexamers, and DEPC-treated water to a final volume of 20 µl. As a negative control, RNA was processed as described above but without MLVRT. The reaction mixtures were incubated at 42°C for 60 min, denatured at 99°C for 5 min, and cooled on ice for 5 min.

qPCR was performed using an equivalent of 1.5 ng of total RNA (3 µl of a 1:20 dilution of cDNA template), 0.5 µM forward and reverse primers, and the Power SybrGreen PCR Master Mix (Applied Biosystems) in a 20-µl reaction volume. The qPCR reaction for 5-HT_2C, 5-HT_2A, 5-HT_1A, and GAPDH contained 10 µl of 2x Master Mix, 0.5 µM forward and reverse primers, 3 µl of cDNA, and nuclease-free water to a 20 µl final volume. qPCR was performed on samples in the ABI Prism 7500 Fast machine in triplicate under the following conditions: 95°C, 10 min; 40 cycles of amplification at 95°C, 15 s, and 60°C, 1 min followed by a dissociation step for melting point analysis with 35 cycles of 95°C for 15 s, 60°C to 95°C in increments of 1°C for 1 min, and 95°C for 15 s. Standard curves using diluted cDNA from guinea pig hypothalamus (1:5, 1:10, 1:50, 1:100, 1:500) (Fig. 6e) were prepared to determine the efficiency of the primers. The slopes of the standard curves for 5-HT_2C, 5-HT_2A, 5-HT_1A, and GAPDH were -3.1, -3.2, -3.2, and -3.3, respectively (Fig. 6e). The efficiency was calculated for each primer pair using the following formula: E = 10^(-1/m) - 1, where m = slope (Livak and Schmittgen, 2001; Pfaffl, 2001). The efficiencies were 100% for all transcripts. The similar efficiencies between the primer pairs allowed us to make quantitative estimates between 5-HT_2C, 5-HT_2A, and 5-HT_1A mRNA expression. The amplification data were analyzed by the ABI 7500 System version 1.3.0 software and calculated using the C_T method (Livak and Schmittgen, 2001).

View larger version (27K): [in this window] [in a new window]   Fig. 6. 5-HT2C receptor mRNA is highly expressed in POMC neurons. a, representative gel illustrating the mRNA expression of 5-HT2C, 5-HT2A, and 5-HT1A receptors in arcuate neurons. All neurons expressed GAPDH (data not shown). One cell contained no MLVRT as a negative control (-RT). Other controls included aCSF from the vicinity of the harvested cells and a water blank (data not shown). b, diagram showing the distribution and coexpression of 5-HT2C, 5-HT2A, and 5-HT1A receptor transcripts in arcuate POMC neurons from GDX female guinea pigs. 5-HT2C and 5-HT2A were detected in the majority of POMC neurons, 84 and 76% respectively, whereas 5-HT1A receptor mRNA was detected in only 42% of POMC neurons. c, qPCR analysis of 5-HT2C, 5-HT2A, and 5-HT1A receptor mRNA expression in the microdissected arcuate nucleus using the SYBR green method. Cycle number is plotted against the normalized fluorescence intensity (Mean Delta Rn) to visualize the PCR amplification. The cycle threshold (Ct) value (broken line) is the point in the amplification at which the sample values were calculated. d, the superimposed melting curves for GAPDH, 5-HT2A, 5-HT2C, and 5-HT1A depict a single product. e, the standard curve regression line produced slope values of -3.1, -3.2, and -3.2, which translate into similar efficiencies of 100%. f, quantitative analysis of 5-HT2C, 5-HT2A, and 5-HT1A receptor mRNA expression in the arcuate nucleus from oil-treated GDX female guinea pigs (mean ± S.E.M.; n = 6). 5-HT2A and 5-HT1A are compared with 5-HT2C (one-way ANOVA: a, p < 0.05; b, p < 0.001; c, p < 0.001).

-CT

Feeding Study. Eleven- to twelve-week-old female mice anesthetized by intraperitoneal injection of 0.15 ml of mouse cocktail (ketamine-xylazine-saline, 1:1:8) were ovariectomized and then kept anesthetized with isoflurane during the icv cannulation procedure. The mice were placed into a stereotaxic instrument (Cartesian Instruments, Bend, OR), the cranial surface cleaned, and a cannula placed into the third ventricle as described previously (Cepoi et al., 2004). In brief, a small hole was drilled, and a sterile stainless steel guide cannula (25 gauge, 1.1 cm long, with an obturator stylet placed within) was implanted at midline, 0.825 mm posterior to bregma and 4.8 mm below bregma based on Franklin and Paxinos (1997). Mice were housed individually and allowed to recover for 10 days. Thereafter, the animals were adapted repeatedly for at least 3 weeks to the experimental procedure, which included a brief restraint in a procedure bag during which time the icv injection was performed. To test the effects of the compounds on feeding after an overnight fast, mice were placed in clean cages with bedding material and free access to water but without food for 16 h (5:00 PM to 9:00 AM). At the end of the fast, each mouse was lightly restrained, the obturator stylet was removed from the guide cannulae, and saline (0.9% NaCl), E₂ (0.012 nmol) or DOI (110 nmol) in 2-µl total volume was infused over a 1-min period. Another 1-min period was allowed for diffusion of the drugs before removing the injection needle. The mice were put back into their cages with a preweighed food pellet (Purina Mouse Chow, 5144; Purina, St. Louis, MO). Body weight and pellet weights were determined at 1, 2, 6, and 24 h after injection. The correct cannulae placement was confirmed by injecting methylene blue dye at the end of the study and visualizing the location of the dye in brain slices.

Statistical Analysis. Comparisons between groups were performed using a one-way or two-way ANOVA and Bonferroni post test for the tissue analysis and whole-animal experiments and a one-way ANOVA for the electrophysiological experiments with post hoc Newman-Keuls paired analysis. Differences were considered statistically significant if the probability of error was <5%.

	Results

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Gender Differences in the Estrogen-Mediated Desensitization of the GABA_B Response in Hypothalamic Neurons. Whole-cell recordings were made in arcuate neurons (n = 153) from GDX male and female guinea pigs. A subgroup of these neurons (n = 56) was identified using dual-labeling immunocytochemistry (Fig. 1), and 40% of the neurons were β-endorphin-positive (i.e., POMC neurons). For the electrophysiology analysis, only cells with gigaohm or better seals were included in this study. There was no difference in the mean resting membrane potential (male guinea pigs: -55.5 ± 1.3 mV (n = 21) versus female guinea pigs: -55.0 ± 0.5 mV (n = 102), I_hold = 0 pA) or the mean input resistance (male guinea pigs: 0.96 ± 0.3 G versus female guinea pigs: 0.92 ± 0.1 G) between the two groups. Similar to our previous findings in mice (Qiu et al., 2006), there were no gender differences in the mean outward current induced by 5 µM baclofen (male mice: 37.7 ± 3.8 pA, n = 43; female mice: 34.7 ± 2.4 pA, n = 88). However, there was a significant gender difference in the effects of E₂ on the baclofen response in GDX guinea pigs. POMC neurons from GDX male guinea pigs were less sensitive to the effects of estrogen by approximately 15% (Fig. 1). T at a higher concentration (1 µM) could attenuate the baclofen response (Fig. 1, a and b) in GDX male guinea pigs but still was not as efficacious as E₂. However, DHT (1 µM), which cannot be aromatized to E₂, had no effect on the baclofen response (Fig. 1, a and b), suggesting an estrogenic response.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Gender differences in the effects of E2 on baclofen responses in GDX guinea pigs. a, representative traces of the baclofen response in the presence of T (1 µM) or DHT (1 µM) in male arcuate (POMC) neurons. b, bar graphs summarizing the effects of the E2 (100 nM), T (1 µM), and DHT (1 µM) in arcuate (POMC) neurons. E2 was more effective in female than in male arcuate (POMC) neurons. T at a higher concentration (1 µM) could mimic, but DHT could not mimic the effects of E2. Bars represent the mean ± S.E.M. of 3 to 11 cells tested per group. *, p < 0.05, E2 in female versus male neurons, and DHT versus testosterone in male neurons; #, p < 0.05, T versus control. Because there was no difference in female (96.6 ± 6%, n = 8) and male (96.0 ± 5%, n = 3) control cells, the data were combined for the control bar. c, a representative POMC neuron that responded to DOI. Arcuate neurons were filled with biocytin during the whole-cell recording. Left, biocytin-streptavidin-Cy2 labeling of a small fusiform arcuate neuron. Scale bar, 20 µm. Middle, immunocytochemical staining of β-endorphin in the same neuron (arrow). Right, overlay of stains shown left and middle.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Activation of 5-HT2C receptors desensitizes the GABAB response in POMC neurons. a, representative traces of the effects of DOI and ORG 3363 on the baclofen responses. b, bar graphs summarizing the effects of 5-HT2C receptor agonists MK212 (10 µM) and m-CPP (10 µM), 5-HT2A/C receptor agonist DOI (20 µM), 5-HT2A receptor antagonist spiperone (20 µM), and 5-HT2C receptor antagonists ORG 3363 (10 µM) and RS102221 (20 µM). Bars represent the mean ± S.E.M. of 4 to 14 cells. *, p < 0.05, and **, p < 0.01, versus vehicle control.

5-HT_2C Receptor-Mediated Attenuation of the GABA_B Response Was Dependent on Activation of G_q. Early studies showed that 5-HT₂ receptor subtypes couple to G_q protein and activate PLC (de Chaffoy de Courcelles et al., 1985; Conn et al., 1986). Therefore, we next examined the involvement of specific signaling proteins in the DOI-mediated modulation. Because 5-HT_2A/C receptors are G_q protein-coupled and PKC is the downstream pathway, we first examined whether activation of PKC is critical for DOI modulation of the GABA_B response using a PKC inhibitor. If activation of the PKC pathway is involved, then the effect of DOI on GABA_B responses should be blocked by inhibiting PKC. To test this, we applied a PKC inhibitor, BIS, which is a selective inhibitor of PKC that does not distinguish between the conventional, novel, and atypical isoforms of PKC, and a selective PKC inhibitor, rottlerin. We have established that BIS (100 nM) and rottlerin (5 µM) block the inhibition of baclofen responses by E₂ (Qiu et al., 2003). But as shown in Fig. 3, after 15 min of dialysis with BIS (100 nM), the DOI-induced reduction of the GABA_B response was not attenuated. Furthermore, both BIS (100 nM) and rottlerin (5 µM) did not block the inhibition of baclofen response by m-CPP, a selective 5-HT_2C agonist (p > 0.05, m-CPP, 71.17 ± 2.24, n = 3, versus m-CPP + BIS, 73.16 ± 3.06, n = 7; m-CPP versus m-CPP + rottlerin 68.92 ± 4.01, n = 3). These results indicate that the suppression of the GABA_B response by DOI requires PLC activation but not the activation of PKC. To examine whether the DOI-mediated inhibition of the GABA_B response depended on the activation of G_q, arcuate neurons were dialyzed with a peptide (11 amino acids) that mimics the C-terminal binding site of G_q. A similar strategy has been used by Carr et al. (2002) to abrogate 5-HT₂ receptor signaling in prefrontal cortical neurons. Indeed, in cells dialyzed with this peptide (200 µM), the DOI-mediated reduction of the GABA_B response was blocked significantly (Fig. 3, a and b) compared with cells dialyzed with a control peptide (11 amino acids) that mimics the C-terminal domain of G_s (Fig. 3, a and b).

View larger version (31K): [in this window] [in a new window]   Fig. 3. 5-HT2C receptor-mediated attenuation of the GABAB response depends on activation of Gq but not PKC. Cells were dialyzed for 15 min before baclofen application with BIS (100 nM), Gq (200 µM), or Gs peptide (200 µM), which were included in the patch pipette solution. a, representative traces of the baclofen responses in the presence of PKC inhibitor BIS or Gq peptide. b, bar graphs summarizing the effects of the broad-spectrum PKC inhibitor BIS and G inhibitors. Bars represent the mean ± S.E.M. of 4 to 14 cells. *, p < 0.05, Gq peptide plus DOI versus Gs peptide plus DOI; and **, p < 0.01 versus vehicle control. c and d, 5-HT2C receptor-mediated attenuation of the GABAB response depends on activation of PLC. c, representative traces of the baclofen responses in the presence of PIP2 or the PLC inhibitor U73122. [GenBank] Cells were dialyzed for 15 min before baclofen application with PIP2 (5 µM), which was included in the patch pipette solution. d, bar graphs summarizing the effects of PIP2, PLC inhibitor U73122 [GenBank] (10 µM) and the inactive analog of U73122 [GenBank] , U73343 [GenBank] (10 µM), and PI-4-kinase inhibitor wortmannin (wort, 10 µM). Bars represent the mean ± S.E.M. of 3 to 14 cells. *, p < 0.05, U73122 [GenBank] plus DOI versus U73343 [GenBank] plus DOI; **, p < 0.01 versus vehicle control; ***, p < 0.005 versus vehicle control; and ###, p < 0.005 Wort versus DOI plus Wort.

Desensitization of GABA_B Response Was Dependent on 5-HT_2C Activation of PLC and Hydrolysis of Plasma Membrane PIP₂. In light of the above results for a primary role for G_q in DOI-mediated inhibition, we tested whether the activation of PLC might also play a role. Because the specific PKC inhibitor BIS did not block the effects of DOI, this indicated that the PLC, which is downstream from the activation of G_q protein and a well known G_q effector, may be responsible for the DOI effect. Therefore, we focused on pathways downstream of PLC to further elucidate the DOI-mediated signaling pathway. To determine whether the activation of PLC is required for the DOI-induced inhibition of the GABA_B response, neurons were treated with the broad-spectrum PLC inhibitor U73122 [GenBank] (10 µM), which was perfused in the extracellular bathing media. Under these conditions, there was no difference between the first baclofen response and second one in the presence of U73122 [GenBank] (U73122 [GenBank] -treated versus control group, 95.57 ± 2.44, n = 3 and 96.48 ± 5.9, n = 5), but the DOI-mediated reduction of GABA_B response was blocked (Fig. 3, c and d), whereas the less active PLC inhibitor U73343 [GenBank] at the same concentration had no effect (Fig. 3, c and d). Furthermore, whole-cell dialysis with PIP₂ (5 µM) also attenuated the DOI-mediated inhibition, and there were no significant differences between the first baclofen responses with and without PIP₂ dialysis. Moreover, the addition of PI-4-kinase inhibitor wortmannin at 10 µM potentiated the inhibition of DOI on the baclofen response (Fig. 3d). Therefore, the 5-HT_2C receptor signals through PLC and PIP₂, which is different from the mER signaling pathway (Qiu et al., 2003). Because DOI and E₂ act through divergent G_q signaling pathways, we investigated the convergence of the 5-HT_2A/C receptor agonist DOI and E₂ on the GABA_B response. Concentration-response curves (Fig. 4b) showed that DOI rapidly attenuated the GABA_B response in a concentration-dependent manner with 50% inhibition at 16.5 µM for DOI. Therefore, based on the previously published EC₅₀ value (46.0 nM) for the E₂-mediated desensitization of the GABA_B response (Qiu et al., 2006), we coapplied both agonists at their EC₅₀ concentrations and found that the effects were additive (Fig. 4, a and c).

View larger version (15K): [in this window] [in a new window]   Fig. 4. Effects of DOI and E2 on GABAB response were additive. a, representative traces of the baclofen response in the absence or presence of DOI (20 µM) alone or DOI plus E2 (50 nM) in arcuate (POMC) neurons. b, cells were perfused with different concentrations of DOI (0, 10, 50, 100, and 300 µM). Data are presented as mean ± S.E.M. (n = 4–14 cells/data point). Based on a logistics equation fit to the data points (see Materials and Methods), the EC50 value of the inhibition of baclofen response by DOI was 16.5 µM. The Hill slope for DOI was 1.8. c, bar graphs summarizing the effects of DOI (20 µM) and E2 (50 nM). E2 or DOI at their EC50 concentrations could significantly inhibit the baclofen response, and the effects were additive. Bars represent the mean ± S.E.M. of 5 to 14 cells. **, p < 0.01, and ***, p < 0.005, versus vehicle control; #, p < 0.05, E2 or DOI versus DOI plus E2.

View larger version (25K): [in this window] [in a new window]   Fig. 5. 5-HT2A/C receptor agonist and estrogen inhibit feeding in GDX female mice. a, 5-HT2A/C receptor agonist DOI reduces food intake in GDX female mice. DOI (110 nmol) given icv to overnight food-deprived, ovariectomized mice produced a potent inhibition of cumulative food intake. Data are shown as means ± S.E.M., n = 6 for each group. *, p < 0.05, **, p < 0.01, and ***, p < 0.005, compared with saline vehicle-treated mice at all time points measured (two-way ANOVA and Bonferroni post test). b, 5-HT2A/C receptor agonist DOI reduced body weight gain in GDX female mice. DOI (110 nmol) given icv to overnight food-deprived GDX female mice produced a potent inhibition of body weight gain. Data are shown as mean ± S.E.M., n = 6 for each group. *, p < 0.05, ***, p < 0.005, compared with saline vehicle-treated mice at all time points measured (two-way ANOVA and Bonferroni post test). c and d, E2, similar to DOI, reduced feeding at the 6-h time point. Bar graphs summarizing the effects of DOI and E2 (0.012 nmol) on food intake (c) and body weight gain (d). Bars represent the mean ± S.E.M., n = 5 to 7 mice per group; ***, p < 0.005, **, p < 0.01, DOI or E2 group versus their respective control groups.

Expression of 5-HT₂ Receptor mRNA Transcripts in Arcuate (POMC) Neurons from GDX Guinea Pigs. Based on our electrophysiology results, we examined which serotonin receptors are expressed in arcuate (POMC) neurons in the guinea pig. Previous findings have identified 5-HT_2C receptors in male mouse POMC neurons (Heisler et al., 2002). Using scRT-PCR, we measured 5-HT_2C, 5-HT_2A, and 5-HT_1A receptor transcripts in 54 male and 57 female arcuate neurons, including POMC neurons from 4 animals each (Fig. 6a). Overall, the distribution of these receptors in arcuate neurons was similar in male (69 ± 13, 47 ± 6, and 51 ± 8%; n = 4) and female neurons (81 ± 12, 62 ± 8, and 37 ± 6; n = 4) for 5-HT_2C, 5-HT_2A, and 5-HT_1A receptors, respectively. Based on the frequency of distribution of 5-HT_2C, 5-HT_2A, and 5-HT_1A receptor mRNAs specifically in POMC-positive neurons in GDX female guinea pigs (n = 38 cells), 84% of these neurons expressed 5-HT_2C receptor mRNA, 76% expressed 5-HT_2A receptor mRNA, and 42% expressed 5-HT_1A receptor mRNA (Fig. 6b). The degree of coexpression of these receptors in POMC neurons was also determined as illustrated in Fig. 6b and was found to be 71% for 5-HT_2A and 5-HT_2C and 26% for all three receptors (Fig. 6b). Using RT and real-time PCR to quantify the level of expression of serotonin receptor mRNAs in the microdissected arcuate nucleus, we found that 5-HT_2A receptors were most highly expressed followed by 5-HT_2C receptors (Fig. 6, c–f; p < 0.05). The expression of 5-HT_1A was significantly lower than that of 5-HT_2A and 5-HT_2C receptor transcripts (p < 0.001; Fig. 6, c–f). Therefore, the scRT-PCR and qPCR data support the electrophysiological data that 5-HT_2C and 5-HT_2A receptors are highly expressed in the arcuate and coexpressed in the majority of POMC neurons.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we have characterized the signaling pathway of the serotonin 5-HT_2C receptor in hypothalamic (POMC) arcuate neurons. Activation of this G_q-coupled receptor desensitized the GABA_B response in neurons. Short-term activation of the mER with E₂ also desensitized the GABA_B receptor response, but unlike the mER signaling pathway, the 5-HT_2C receptor pathway led to direct hydrolysis of PIP₂ to affect GIRK channel function. Although there was a clear gender difference in the efficacy of E₂ to activate mER, there was no difference in the 5-HT_2C-mediated response between male and female guinea pigs. Finally, E₂ and the 5-HT_2C agonist DOI, albeit at a higher dose, were effective in reducing food intake and body weight gain in fasted, GDX female guinea pigs, which highlights the physiological importance of the mER and 5-HT_2C signaling pathways in mediating POMC neuronal excitability.

Serotonin affects arcuate (POMC) neuronal activity through multiple receptor-mediated mechanisms. Both 5-HT_2A and 5-HT_2C receptors are localized to the medial basal hypothalamus, and activation of these G_q-coupled receptors excites these neurons (Wright et al., 1995; Heisler et al., 2002). In particular, POMC neurons are excited in response to d-fenfluramine, 5-HT, or 5-HT_2C receptor agonists like m-CPP and MK212 (Heisler et al., 2002). However, the underlying mechanism(s) for the excitation of hypothalamic neurons has not been elucidated. Our data show that the G_q/PLC-coupled pathway of 5-HT_2A/C receptors causes an inhibition of the baclofen response, and we provide the first evidence that depletion of PIP₂, rather than the phosphorylation of the channel, is a key step in this pathway based on the following: First, PKC inhibitors did not block the inhibition of the baclofen response by the 5-HT_2A/C receptor agonist DOI or 5-HT_2C receptor agonist m-CPP. Second, the DOI-mediated reduction of the GABA_B response was significantly attenuated by the PLC inhibitor U73122 [GenBank] compared with cells perfused with the less active inhibitor U73343. [GenBank] Third, whole-cell dialysis with PIP₂ attenuated the DOI inhibition of the GABA_B response, and the addition of wortmannin greatly potentiated the inhibition of DOI on baclofen responses. Finally, intracellular dialysis with a peptide fragment of G_q abrogated the 5-HT_2C receptor interaction with G-protein, which indicates that 5-HT_2A/C receptors are specifically coupled to G_q protein. Thus, we conclude that 5-HT_2A/C receptor-mediated inhibition of GIRK channels involves PLC activation by G subunits of the G_q family and that the receptor-mediated hydrolysis of plasma membrane PIP₂ is the critical mediator. It is worth noting that although both 5-HT_2A and 5-HT_2C receptors are G_q-coupled, the inhibition of the baclofen response in POMC neurons is mainly through the 5-HT_2C receptor because the 5-HT_2C-selective agonists m-CPP and MK212 attenuated baclofen responses in POMC neurons. Moreover, the selective 5-HT_2C antagonists ORG 3363 and RS102221 but not the 5-HT_2A antagonist spiperone potently blocked the actions of DOI in guinea pig arcuate (POMC) neurons, indicating that the inhibition of the GABA_B response is through the 5-HT_2C receptor. This is compatible with previous findings that the 5-HT_2C receptor agonists show greater efficacy for activating the PLC pathway, whereas 5-HT_2A receptor agonists have relatively greater efficacy for activating the phospholipase A₂ pathway (Berg et al., 1998; Kurrasch-Orbaugh et al., 2003). The PLC hydrolysis of PIP₂ and inhibition of GIRK channels is not unique to 5-HT_2C receptors because other G_q-coupled receptors have different propensities for activating this pathway depending on the subcellular localizations of the G-protein-coupled receptor relative to the proximity of PLC and GIRK channels (Cho et al., 2005).

It is interesting that GABA_B, µ-opioid, and 5-HT_1A receptors are all expressed in POMC neurons (Kelly et al., 1992; Lagrange et al., 1994; Qiu et al., 2003; present findings). All of these receptors are G_i/o-coupled to activation of GIRK channels, which uniformly inhibit POMC neuronal activity. Similar to our findings with the 5-HT_2C receptor-mediated desensitization of the GABA_B response in POMC neurons, activation of 5-HT_2A receptors can desensitize 5-HT_1A receptors and increase the excitability of CRH neurons, as measured by ACTH release (Zhang et al., 2001). The heterologous desensitization in these paraventricular nucleus neurons has not been characterized but may be via PKC-mediated phosphorylation of GIRK channels (Brown et al., 2005) or by PLC-mediated PIP₂ depletion (Brown et al., 2005; Cho et al., 2005). Therefore, the PLC-mediated PIP₂ depletion maybe a common signaling pathway for 5-HT_2A/C receptors in hypothalamic neurons.

View larger version (36K): [in this window] [in a new window]   Fig. 7. A cellular model of the rapid signaling of E2 and 5-HT in hypothalamic neurons. Schematic overview showing the mER and 5-HT2A/C receptor-mediated modulation of neurotransmitter regulated, G protein-coupled receptors in arcuate (POMC) neurons. Black is used to summarize the results of the current experiments. The cellular cascade activated by E2 (Qiu et al., 2003) is shown in gray. E2 activates a mER that is Gq/11-coupled to activation of PLC that catalyzes the hydrolysis of membrane-bound PIP2 to IP3 and DAG. Calcium is released from intracellular stores (endoplasmic reticulum) by IP3, and DAG activates PKC. Through phosphorylation, adenylyl cyclase VII (ACVII) activity is up-regulated by PKC. The generation of cAMP activates PKA, which can rapidly uncouple GABAB and µ-opioid (µ) receptors from their effector system through phosphorylation of a downstream effector molecule (e.g., the inwardly rectifying K+ channel, or GIRK). 5-HT2C receptor-mediated inhibition of GIRK channels involves PLC activation by G subunits of the Gq/11 family, and receptor-mediated hydrolysis of plasma membrane PIP2 (PIP2 depletion) is the critical mediator. Therefore, 5-HT2C receptor and mER intracellular signaling pathways converge to disinhibit arcuate (POMC) neurons.

Serotonin 5-HT_2C receptors have also been strongly implicated in inhibiting feeding. For example, the selective 5-HT_2C antagonist RS102221 increases food intake and body weight when injected intraperitoneally (Bonhaus et al., 2006), and 5-HT_2C receptor-deficient mice are hyperphagic, obese, and refractory to threshold anorexic doses of d-fenfluramine (Tecott et al., 1995; Vickers et al., 1999), a drug that blocks the reuptake of 5-HT and stimulates its release (Heal et al., 1998). In contrast, 5-HT_2A receptor knockout mice do not exhibit an obesity phenotype, so one may assume that 5-HT_2A receptors are not critically involved in energy homeostasis (Zhou et al., 2005). We found that 5-HT_2C mRNA was highly expressed and colocalized with 5-HT_2A receptor in POMC neurons. This would disagree with in situ hybridization results in rhesus (female) monkeys showing that 5-HT_2C receptor mRNA is highly expressed in the arcuate region, whereas the 5-HT_2A receptor mRNA is more localized to the PVN (Gundlah et al., 1999). In addition, Heisler et al. (2002) found that up to 80% of POMC neurons express 5-HT_2C receptor mRNA. However, the role of the 5-HT_2C and 5-HT_2A receptors in different physiological processes may lie in their coupling to distinctive signaling pathways.

Based on our cellular electrophysiological data, we have proposed a model for the convergence of the mER and 5-HT_2C signaling pathways in arcuate (POMC) neurons (Fig. 7). It is noteworthy that the downstream signaling pathway of baclofen inhibition by 5-HT_2C receptor agonists in arcuate (POMC) neurons is different from mER in which E₂ desensitizes the GABA_B response via a G_q-PLC-PKC-PKA pathway (Qiu et al., 2003). These differences may be due to the different compartmentalization of the receptors. It has been reported that the signaling components for G-protein activation in neurons are compartmentalized or preassembled (Lober et al., 2006). G-protein-coupled receptors, G-proteins, and other effector molecules can preassemble into stable signaling complexes (Rebois and Hebert, 2003). Therefore, the 5-HT_2C receptor and mER may be preassembled into complexes with different downstream effectors that converge on GIRK channels. For example, Brown and colleagues (2005) have shown that although protein kinases (i.e., PKC) mediate the inhibition of GIRK_1/2 channels by the muscarinic M₃ receptor, resynthesis of PIP₂ is required for complete recovery from inhibition. Therefore, PIP₂ turnover is critical for GIRK channel function. Our data support this idea because 5-HT_2C receptors can inhibit the GIRK channels by an independent pathway from mER, but the two pathways converge on the same population of GIRK channels as shown by the additive effects of the two agonists given together. As proof of principle, we have found that both E₂ and DOI are effective to inhibit feeding in GDX female mice, and as predicted from the cellular findings, E₂ was more potent than DOI to inhibit food intake and weight gain. Therefore, the G_q signaling pathways of mER and 5-HT_2C receptors may converge to enhance synaptic efficacy in brain circuits that are critical for maintaining homeostatic functions.

	Acknowledgements

 
We recognize the skilled technical assistance of Rebecka Amodei and Scott Kuhn. We recognize Dr. Jan Kelder (Organon NV) for his generous gift of ORG 3363.

	Footnotes

 
This study was supported by United States Public Health Service grants NS43330, NS38809, DK68098, and DK62179.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.107.038083.

ABBREVIATIONS: POMC, proopiomelanocortin; E₂,17β-estradiol; GABA_B, -amino butyric acid B receptor; GIRK, G protein-coupled inwardly rectifying potassium; mER, membrane estrogen receptor; PLC, phospholipase C; PKC, protein kinase C ; PCR, polymerase chain reaction; PKA, protein kinase A; 5-HT, 5-hydroxytryptamine, serotonin; RT-PCR, reverse transcription-polymerase chain reaction; scRT-PCR, single-cell reverse transcription-polymerase chain reaction; qPCR, quantitative polymerase chain reaction; DOI, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; RS102221, 8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulphonamido) phenyl-5-oxopentyl] 1,3,8-triazaspiro[4.5] decane-2,4-dione hydrochloride; ORG 3363, 1,2,3,4,10,14b-hexahydro-2-methyldibenzo [c,f]pyrazino[1,2-a]azepine hydrochloride, R-enantiomer; PIP₂, phosphatidylinositol 4,5 bisphosphate; icv, intracerebroventricular; PVN, paraventricular nucleus; GDX, gonadectomized; BIS, bisindolymaleimide I hydrochloride; DHT, dihydrotestosterone; wortmannin, (1S,6br,9aS,11R,11bR) 11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo [4,3,2-de]indeno[4,5,-h]-2-h-2-benzopyran-3,6,9-trione; spiperone, 8-[4-(4-fluorophenyl)-4-oxobutyl]-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one hydrochloride; m-CPP, 1-(3-chlorophenyl)piperazine hydrochloride; MK212, 6-chloro-2-(1-piperazinyl) pyrazine hydrochloride; aCSF, artificial cerebral spinal fluid; DTT, dithiothreitol; DEPC, diethylpyrocarbonate; MLVRT, murine leukemia virus reverse transcriptase; C_T, cycle threshold; PI, phosphatidylinositol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ANOVA, analysis of variance; T, testosterone; IP₃, inositol 1,4,5 triphosphate; DAG, diacylglycerol; U73122 [GenBank] , 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; U73343 [GenBank] , 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione.

Address correspondence to: Dr. Jian Qiu, Department of Physiology and Pharmacology, L334, Oregon Health and Science University, Portland, OR 97239-3098. E-mail: qiuj{at}ohsu.edu

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