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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurochem. Author manuscript; available in PMC Jun 1, 2011.
Published in final edited form as:
PMCID: PMC2917206
NIHMSID: NIHMS220019

Serotonin (5-HT) 2C Receptor (5-HT2CR) Protein Expression is Enriched in Synaptosomal and Postsynaptic Compartments of Rat Cortex

Abstract

The action of serotonin (5-HT) at the 5-HT2C receptor (5-HT2CR) in cerebral cortex is emerging as a candidate modulator of neural processes that mediate core phenotypic facets of several psychiatric and neurological disorders. However, our understanding of the neurobiology of the cortical 5-HT2CR protein complex is currently limited. The goal of the present study was to explore the subcellular localization of the 5-HT2CR in synaptosomes and the postsynaptic density, an electron-dense thickening specialized for postsynaptic signaling and neuronal plasticity. Utilizing multiples tissues (brain, peripheral tissues), protein fractions (synaptosomal, postsynaptic density), and controls (peptide neutralization, 5-HT2CR stable-expressing cells), we established the selectivity of two commercially available 5-HT2CR antibodies and employed the antibodies in Western blot and immunoprecipitation studies of PFC and motor cortex, two regions implicated in cognitive, emotional and motor dysfunction. For the first time, we demonstrated the expression of the 5-HT2CR in postsynaptic density-enriched fractions from both PFC and motor cortex. Co-immunoprecipitation studies revealed the presence of PSD-95 within the 5-HT2CR protein complex expressed in PFC and motor cortex. Taken together, these data support the hypothesis that the 5-HT2CR is localized within the postsynaptic thickening of synapses and is therefore positioned to directly modulate synaptic plasticity in cortical neurons.

Keywords: Prefrontal cortex, motor cortex, 5-HT2C receptor, 5-HT2C receptor localization, postsynaptic density

INTRODUCTION

Compromised function of the serotonin (5-HT) system contributes to the symptomotology of many psychiatric disorders (e.g., addiction, depression, schizophrenia) and neurological diseases (e.g., Parkinson's disease; Giorgetti and Tecott 2004; Bubar and Cunningham 2008). The action of 5-HT at the 5-HT2C receptor (5-HT2CR) in cerebral cortex is emerging as a candidate modulator of neural processes that mediate core phenotypic facets of these disorders including cognitive, emotional and motor dysfunction (Fox and Brotchie 2000; Serretti et al. 2004). For example, the 5-HT2CR is localized to (Mengod et al. 1990; Liu et al. 2007) and controls the excitability of neurons in the prefrontal cortex (PFC) (Carr et al. 2002; Calcagno et al. 2009). Localized stimulation of the PFC 5-HT2CR has been observed to suppress the hypermotility, discriminative stimulus effects and reinstatement of self-administration of psychostimulants (Filip and Cunningham 2003; Pentkowski and Neisewander 2008; Leggio et al. 2009) while changes in expression profiles for the 5-HT2CR in PFC has been linked to addiction vulnerability (Dracheva et al. 2009) and schizophrenia (Dracheva et al. 2003). In contrast, a depression-like phenotype in rats is associated with decrements in 5-HT2CR protein in motor cortex (Leventopoulos et al. 2009), a cortical region essential for the translation of biologically-relevant stimuli into adaptive motor responses (Kosten et al. 2006). Although the motor cortex has also been shown to receive serotonergic innervation from the dorsal raphe nucleus (Blue et al. 1988), the functional role of 5-HT or the 5-HT2CR within motor cortex has not been as well investigated (Leventopoulos et al. 2009) as that within the PFC. Thus, while such studies provide some insight into the mechanisms through which the 5-HT2CR modulates cortical output circuits relevant to neuropsychiatric disorders, there is significant opportunity to gain a greater understanding of the role of the 5-HT2CR in cortical function and dysfunction.

Knowledge of the exact subcellular localization of the 5-HT2CR in cortical neurons has been largely inferred from research investigating the postsynaptic localization of its structurally-related homologue, the 5-HT2AR (Hamada et al. 1998; Rodriguez et al. 1999; Abbas et al. 2009). In addition, the congruence of 5-HT2CR mRNA with protein expression in cortical regions (e.g., frontal cortex, frontoparietal motor cortex, somatosensory cortex, anterior cingulate cortex) (Molineaux et al. 1989; Abramowski et al. 1995; Clemett et al. 2000; Lopez-Gimenez et al. 2002) as well as the persistence of 5-HT2CR protein expression in cortex after obliteration of serotonergic input (Sharma et al. 1997) also support a prominent postsynaptic localization for the 5-HT2CR. Consistent with these findings is the recent observation that postsynaptic density 95/Disc-large/Zonula occludens-1 (PDZ) domain containing proteins play a fundamental role in linking the C-terminus of the 5-HT2CR to its intracellular signaling cascades (Becamel et al. 2002; Becamel et al. 2004; Gavarini et al. 2004; Gavarini et al. 2006; Abbas et al. 2009). In fact, the PDZ domain binding protein postsynaptic density-95 (PSD-95) appears to be particularly important, not only for the regulation and expression of the 5-HT2CR, but also for the localization and organization of the 5-HT2CR synaptic protein network (Becamel et al. 2002; Becamel et al. 2004; Gavarini et al. 2006). A more complete understanding of such complex regulation of the 5-HT2CR postsynaptic complex in cortical regions requires precise identification of protein expression patterns in cellular compartments.

Immunohistochemical studies have provided important knowledge of 5-HT2CR protein expression (Abramowski et al. 1995; Clemett et al. 2000; Bubar and Cunningham 2007; Liu et al. 2007) as these techniques preserve cell morphology and tissue architecture. Additionally, Western blot and immunoprecipitation techniques are required to provide critical complementary information concerning the subcellular localization in cortical regions. The purpose of the present study was to investigate the subcellular localization of the 5-HT2CR in cortical synaptosomes and in the postsynaptic density ex vivo. To achieve this, we exploited protein biochemistry techniques to isolate protein fractions with increasing purification and enrichment for membrane to synaptosome to postsynaptic density protein. We employed semi-quantitative and qualitative protein identification methods to establish the postsynaptic localization of the 5-HT2CR in PFC and motor cortex ex vivo as well as to characterize the utility and validity of four commercially available 5-HT2CR antibodies (Supplementary Table 1) for use in subsequent Western blot and immunoprecipitation analyses. Since the 5-HT2CR is widely expressed in the CNS but not in the periphery (Julius et al. 1988a; Julius et al. 1988b), we compared membrane fractions derived from both brain and peripheral organs to assess whether the immunoreactive (IR) bands identified by these antibodies were unique to samples isolated from brain tissue as well as to quantify the relative distribution of the 5-HT2CR in the PFC and motor cortex.

Immunoprecipitation followed by immunoblot analyses of PFC and motor cortex synaptosomal fractions was conducted to further confirm the subcellular localization of the 5-HT2CR as well as to validate the chosen IR bands of interest for two antibodies that proved both consistent and selective for brain tissue compared to peripheral organs. To further demonstrate the postsynaptic localization of the 5-HT2CR in the PFC and motor cortex, we immunoprecipitated synaptosomal membrane proteins with the 5-HT2CR antibodies and then probed the resultant Western blots for PSD-95. Lastly, we compared IR band patterns produced by the 5-HT2CR antibodies in postsynaptic density samples taken from rat cortical tissues. These results demonstrate that the 5-HT2CR is enriched in the synaptosomal fraction of the PFC and motor cortex, particularly in the postsynaptic density, providing further evidence of the postsynaptic localization of cortical 5-HT2CR in the rat (Clemett et al. 2000; Lopez-Gimenez et al. 2002; Liu et al. 2007). The precise detection of the 5-HT2CR cellular and subcellular localization in motor cortex, PFC and other cortical regions will ultimately allow for a better understanding of the function and regulation of the cortical 5-HT2CR in the pathophysiology of complex neuropsychiatric disorders.

MATERIAL AND METHODS

Animals

Adult male Sprague-Dawley rats (virus antibody-free; Halan, Indianapolis, IN) weighing 225-280 g were used with food and water available ad libitum. The animal colony was maintained at a constant temperature (21-23°C) and humidity (45-50%) on a 12 hr light-dark cycle (lights on 0700-1900 hr). Rats were anesthetized [chloral hydrate solution (400 mg/kg)], decapitated, and brain (motor cortex, PFC and cerebellum), liver, kidney and lung were microdissected immediately on a cool tray (4°C) (Heffner et al. 1980). Samples were flash frozen in liquid nitrogen and stored at −80°C for subsequent protein extraction. All experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1986) and with the approval by the Institutional Animal Care and Use Committee (IACUC).

Cell lines and cell culture

Parental and 5-HT2CR-expressing (1C19) CHO cells were generous gifts of Drs. K. Berg and W. Clarke from the University of Texas Health Science Center at San Antonio. Reverse transcription of RNA followed by quantitative real time PCR confirmed that the 1C19 cell line expressed high levels of the 5-HT2CR transcript, but not the 5-HT2AR transcript and that the parental cell line did not express detectable amounts of either mRNA (data not shown; Berg et al. 2001). Protein expression in 1C19 cells was assessed at 200 fmol/mg protein which approximates physiological levels in brain (Berg et al. 2001). Cells were grown in GlutaMax-αMEM (Invitrogen, Carlsbad, CA) at 37°C, 5% CO2 and 85% relative humidity and were passaged when they reached 80% confluence.

Membrane fractionation: brain and peripheral tissues

A crude membrane fraction from brain and peripheral tissues was performed to assess the cellular and regional localization of the 5-HT2CR. All tissues (PFC, motor cortex, cerebellum, liver, kidney, and lung) were homogenized in 10 times w/v extraction buffer (pH 7.4) containing 10 mM HEPES, 1 mM EDTA, 2 mM EGTA, 500 μM DTT, protease inhibitor cocktail (10 μL/mL; Sigma-Aldrich, St. Louis, MO) and phosphatase inhibitor cocktails 1 and 2 (10 μL/mL each, Sigma-Aldrich). The homogenate was centrifuged at 1,000 g for 10 min at 4°C to pellet the nuclear fraction. The supernatant was removed and centrifuged at 20,000 g at 4°C for 30 min to pellet the membrane bound protein fraction. The membrane-enriched pellet was washed once, and then resuspended in 200 μL 1% SDS solution.

Membrane fractionation: CHO cells

A crude membrane fraction from parental CHO and 1C19 cells was performed to assess the specificity of 5-HT2CR antibodies. Cells were rinsed and scraped in ice-cold Krebs (125 mM NaCl, 1.2 mM KCl, 1.2 mM MgSO4, 1.2 mM CaCl2, 22 mM Na2CO3, 1 mM NaH2PO4, 10 mM glucose) and then centrifuged at 1000 rpm for 2 min to pellet the cells. The pelleted cells were resuspended and homogenized in 200 μL extraction buffer and subjected to differential centrifugation as described above. The membrane-enriched pellet was washed once, and then resuspended in 200 μL resuspension buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease and phosphatase inhibitors, and 1% dodecyl maltoside).

Synaptosomal Preparation: PFC and motor cortex

To validate the IR bands identified with D-12 and N-19 in a subcellular fraction that expresses high levels of 5-HT2CR, synaptosomal fractions were prepared from PFC or motor cortex brain sections homogenized (10 times w/v) in ice cold Krebs buffer containing 0.32 M sucrose plus protease inhibitor cocktail and phosphatase inhibitor 1 and 2 cocktails (10 μL/mL Sigma-Aldrich). The homogenate was then centrifuged at 1,000 g at 4°C for 10 min. The supernatant was collected and centrifuged at 16,000 g at 4°C for 20 minutes to pellet the crude synaptosomes; the resultant pellet was re-suspended in Krebs buffer plus 1% dodecyl maltoside.

Postsynaptic density-enriched fractionation

To assess the subcellular localization of the 5-HT2CR in the motor cortex, postsynaptic density-enriched fractionation was performed as previously described (Phillips et al. 2001; Liu et al. 2007; Moron et al. 2007). We chose to perform this fractionation initially in motor cortex rather than PFC as this brain region is larger in size when harvested, such that pooling of samples is not required to recover the maximal amount of protein from each subcellular fraction. Motor cortex was homogenized in 0.32 M sucrose solution containing 0.1 mM CaCl2 and protease and phosphatase inhibitor cocktails 1 and 2 (10 μL/mL each). An aliquot (total homogenate) was set aside and the remaining homogenate was diluted with 6 mL 2 M sucrose and 2.5 mL 0.1 mM CaCl2. The resulting solution was mixed by inversion, transferred to a 25 mL ultracentrifuge tube, overlaid with 1 M sucrose solution, then ultracentrifuged in a fixed angle rotor at 100,000 g for 3 hrs at 4°C. Synaptosomes were located at the interface of the 1.25 M and the 1 M sucrose interface and were removed by careful pipetting. An aliquot (synaptosomal fraction) was taken and the remaining solution (approximately 2.5 mL) was diluted with 15.6 mL hypotonic solution [13.5 mL 0.1 mM CaCl2, 0.3 mL 1 M Tris pH 6 buffer, 1.5 mL 10% Triton X-100, 150 μL each of protease and phosphatase inhibitor cocktails]. This solution was mixed by inversion and incubated on a shaker for 20 min at 4°C. The synaptic junctions were collected by centrifugation at 48,000 g for 20 min at 4°C. The supernatant was discarded; the pellet was resuspended in 15.6 mL hypotonic solution and incubated on a shaker for 20 min at 4°C. The supernatant was discarded and the pellet was resuspended in 10 mL of pH 8 buffer [20 mM Tris, 1% Triton X-100] to disrupt the pre-to-post synaptic connection. The solution was incubated on a shaker for 20 min at 4°C. The postsynaptic density-enriched fraction was collected by centrifugation at 48,000 g for 20 min at 4°C, washed once in 10 mL pH 8 buffer with incubation on a shaker for 20 min at 4°C and recentrifuged. The supernatant was discarded; the remaining pellet (postsynaptic density fraction) was resuspended in 1% SDS.

Western blotting

Equal amounts of protein [(Total protein concentration determined with the BCA assay (Pierce, Rockford, IL), reduced with Laemmli sample buffer and heated for 20 min at 70°C] were separated by SDS-PAGE using 10% or 4-12% Bis-Tris gels (Invitrogen, San Diego, CA) for 2-3 hrs at 120V. Following gel electrophoresis, proteins were transferred to a PVDF membrane (BioRad, Hercules, CA) via a wet-transfer electroblotting apparatus (BioRad) for 3 hr at 100V. Western blot assays were conducted as follows: membranes were blocked with Odyssey blocking buffer (LI-COR® Biosciences; 1:1 in Tris Buffered Saline (TBS), pH 7.4) for 1-2 hr followed by incubation with primary antibody for 1 hr to overnight (see below). Membranes were rinsed 3 × 10 min in TBS + 0.1% Tween-20 (TBS-T), incubated with secondary antibody for 45 min, then rinsed 3 × 10 min in TBS-T prior to incubation with TBS. Fluorescent dyes were detected with the Odyssey® Infrared Imaging System (LI-COR® Biosciences).

Antibodies

Supplementary table 1 lists the suppliers and key features of each of the 5-HT2CR antibodies raised against either the N-terminus or the C-terminus of the receptor purchased from various commercial sources and employed in the present study. Fig. 1 is a schematic showing the 5-HT2CR topology and the epitope targets for the four commercially tested antibodies (adapted from (Julius et al. 1988a;Julius et al. 1988b). PVDF membranes were incubated overnight with primary antibody as follows: mouse monoclonal D-12 [sc-17797, Santa Cruz; 1:100]; goat polyclonal N-19 [sc-15081, Santa Cruz; 1:250]; goat polyclonal C-20 [sc-1464, Santa Cruz; 1:200]; rabbit polyclonal CH [AB5655, Chemicon International, Temecula, CA; 1:500]. Other primary antibodies (all from Chemicon International) were incubated at room temperature for 1 hr including monoclonal mouse anti-PSD-95 [MAB1598, 1:2,000]; monoclonal mouse anti-syntaxin [MAB336, 1:5,000]; monoclonal mouse anti-SNAP-25 [AB5871, 1:1,000]; monoclonal mouse anti-β-actin [MAB1501, 1:5000]. Secondary antibodies (used at 1:10,000 or 1:20,000 dilution for 1 hr at RT) included infrared-labeled goat anti-mouse (IRDye™680; 926-32220, LI-COR® Biosciences, Lincoln, NE); goat anti-rabbit (IRDye™800; 827-08365, LI-COR® Biosciences); donkey anti-goat (IRDye™800CW; 605-731-125, Rockland Immunochemicals, Inc., Gilbertsville, PA) and sheep anti-mouse (IRDye™680, Rockland Immunochemicals, Inc.).

Figure 1
Schematic representation of the 5-HT2CR topology and the target regions for the commercially available anti-5-HT2CR antibodies. Adapted from Julius et al. 1988a.

Immunoprecipitation assays

D-12 and N-19 antibodies (45 μg per reaction) were covalently crosslinked onto protein A/G resin according to the manufacturer's instructions (Pierce© Crosslink Immunoprecipitation Kit, Pierce Biotechnology, Rockford, IL). Prior to the immunoprecipitation, 400-500 μg of synaptosomal protein was incubated for 1 hr at RT with Control Agarose Resin© to reduce non-specific protein binding. The pre-cleared synaptosomal lysate was incubated with the antibody-crosslinked resin overnight at 4°C with constant shaking. The suspension was centrifuged at 1,000 g for 30 sec and the flow-through was collected (unbound antigen). The bound antigen-antibody-crosslinked resin was washed twice with IP Lysis/Wash buffer, then once with 1X Conditioning Buffer©. The complex was incubated for 5 min at room temperature in Elution Buffer©. Neutralization buffer (1 M Tris pH 9.5; 1/3 of the Elution buffer volume) was added to the collection tube to adjust the eluate to physiological pH. Samples were centrifuged and the flow-through was collected. The antibody-coupled resin was regenerated with 1X Coupling Buffer© and stored at 4°C for future use. The eluted antigen was subjected to cold acetone precipitation (4 vol acetone for 3 hrs at −20°C). The sample was centrifuged at 15,000 g for 10 min at 4°C, the acetone was decanted and the precipitated protein was resuspended in 2X loading buffer and subjected to SDS-PAGE. Immunoblotting for D-12, N-19 and PSD-95 was performed as described above.

Data Analysis

Membranes were imaged using the Odyssey® Infrared Imaging System (LI-COR® Biosciences) at 700 and/or 800 nm at 169 μm resolution. The integrated intensity of each band was analyzed with the Odyssey® Infrared Imaging System Application version 2.1 Software. The ratio of 5-HT2CR band intensity to β-actin band intensity was determined for each sample for normalization (see Results for details of band selection). A one-way Analysis of Variance [ANOVA, GraphPad InStat® software for Windows V.3.01 (GraphPad Software Inc., La Jolla, CA)] was used to assess differences in the density of 5-HT2CR protein expression in motor cortex, PFC, cerebellum, liver, kidney, and lung. When an overall significant F ratio was observed, preplanned comparisons across tissue samples were conducted using Dunnett's procedure, with the motor cortex set as the standard (control) for comparisons and an experiment wise error rate α = 0.05.

RESULTS

Tissue distribution of the 5-HT2CR

Based upon expression of the 5-HT2CR protein in cortical and other brain regions identified in radioligand binding studies (Molineaux et al. 1989; Abramowski et al. 1995; Lopez-Gimenez et al. 2002) and immunohistochemical analyses, we expected to see a rank order of PFC > motor cortex > cerebellum for 5-HT2CR expression as an internal validation measure for the antibody. To further explore the regional localization of the 5-HT2CR and to identify IR bands of interest, we also assessed the band pattern from membrane fractions in three peripheral organs (liver, kidney, and lung) that should not express the 5-HT2CR (Julius et al. 1988a; Julius et al. 1988b). In light of the aforementioned studies, a selective 5-HT2CR antibody should label protein(s) or produce more intense IR staining in cortical regions relative to the cerebellum and all peripheral organs (Fig. 2).

Figure 2
Specificity of anti-5-HT2CR antibodies for brain

The IR bands selected for quantification corresponded to bands of interest identified in preliminary analyses conducted in the PSD-enriched fraction of the motor cortex (data not shown), and, as such, the motor cortex was chosen as the control standard for preplanned comparisons using the Dunnett's procedure. A one-way ANOVA revealed a main effect of tissue type on intensity of the following quantified IR bands: D-12 [46 kDa; F(5,19)=17.84, p<0.0001, Fig. 2A]; N-19 [52 kDa; F(5,21)=21.60, p<0.0001, Fig. 2B]; N-19 [60 kDa; F(5,22)= 9.96, p<0.0001, Fig. 2B]; C-20 [41 kDa, F(5,21)=16.47, p<0.0001; Fig. 2C]; CH [41 kDa; F(5,19)= 17.84, p<0.0001, Fig. 2D]. No main effect of tissue type on intensity of the 60 kDa band detected using the CH antibody was observed [F(5,21)=0.15, p=0.98; Fig. 2D].

An intense IR band was detected with the D-12 5-HT2CR antibody (Fig. 2A) at an apparent MW of 46 kDa in the motor cortex and PFC and to a lesser extent in the cerebellum. The intensity of the 46 kDa band was significantly higher in the PFC than in the motor cortex while IR detected at 46 kDa in the liver, kidney, and lung membranes with D-12 was negligible and significantly less than that observed in motor cortex (Fig 2A; p<0.05). The N-19 5-HT2CR antibody revealed a faint IR band at 52 kDa in all three brain regions as well as an intense IR band with an apparent MW of 60 kDa in the motor cortex, PFC, and to a lesser extent in the cerebellum. Expression of these two N-19-labeled bands was all but non-existent in the liver, kidney, or lung samples (Fig. 2B; p<0.05 vs. motor cortex). An N-19-labeled 41 kDa IR band was also detected in brain, liver, kidney and lung samples; however, this band was too faint to be accurately quantified (data not shown). The C-20 5-HT2CR antibody produced an IR band at 41 kDa in all three brain regions, but also intensely cross-reacted with proteins from the peripheral organ membrane fractions. In fact, expression of the C-20-labeled 41 kDa IR band in liver was significantly higher than that detected in the motor cortex (Fig. 2C; p<0.05). The CH 5-HT2CR antibody detected an intensely IR band at apparent MW of 41 kDa in motor cortex, PFC and to a lesser extent in the cerebellum (Fig. 2D). The intensity of the 41 kDa band in the PFC was significantly higher than in the motor cortex (p<0.05). Little to no immunoreactivity was detected in liver, kidney, or lung membrane fractions at 41 kDa with the CH antibody (Fig. 2D; p<0.05 vs. motor cortex). The intensity of the 60 kDa band detected using the CH antibody was virtually identical across brain and peripheral tissue samples (Fig. 2D; p>0.05). A CH-labeled 52 kDa band was also present in both peripheral and brain tissues, but was too faint to be accurately quantified (data not shown).

The high levels of C-20-labeled expression at 41 kDa detected in membrane fractions of peripheral tissue, reduced confidence that the C-20 antibody selectively recognizes the 5-HT2CR protein in cortex. Control studies indicated that the CH primary antibody exhibited a high cross-reactivity with antibodies raised in goat, i.e., the N-19 antibody (data not shown). Due to its high cross-reactivity and requirement for two reliable 5-HT2CR antibodies for immunoprecipitation studies, we chose to employ the D-12 and N-19 antibodies. The CH antibody was excluded from the remainder of our studies. The results presented in Fig. 2 served as a guide for selecting the IR bands of interest to analyze in subsequent studies. Based on our results, we examined the 46 kDa IR band labeled by the D-12 antibody and the 52 and 60 kDa IR bands labeled by the N-19 antibody.

Regional distribution of 5-HT2CR protein expression in cortical synaptosomes

The relative subcellular distribution of the 5-HT2CR in PFC and motor cortex was further investigated using a synaptosomal preparation. The synaptosome contains the presynaptic molecular machinery for the uptake, storage and release of neurotransmitters as well as the postsynaptic milieu that is composed of receptors and downstream effectors of neuronal transmission without contamination from membranous organelles and soma found in crude membrane protein fractions (Breukel et al. 1997). Detection of the N-19-labeled 5-HT2CR IR bands at 52 and 60 kDa in the PFC as well as several higher IR bands at MWs >100 kDa were dependent upon protein concentration indicating primary antibody specificity (Fig. 3A). We also performed Western blot analysis with the N-19 antibody in the presence and absence of its blocking peptide (Fig. 3B) to determine if the N-19 IR band pattern can be neutralized. The blocking peptide for the D-12 is not currently available from the manufacturer, so similar studies were not possible with D-12.

Figure 3
Western blot analysis determining the detection limits of 5-HT2CR with the N-19 antibody in the synaptosomal fraction of PFC and motor cortex

In agreement with our previous immunohistochemical study (Liu et al. 2007), pre-incubation with the N-19 blocking peptide neutralized N-19 immunoreactivity as evidenced by a decrease in the intensity of the 52 and 60 kDa bands from synaptosomal PFC samples (Fig. 3B). In the motor cortex, the N-19 antibody revealed a protein concentration-dependent detection of the 5-HT2CR IR bands at 52 and 60 kDa in the motor cortex as well as several higher IR bands at MWs >100 kDa (Fig. 3C), which were decreased in intensity following antibody neutralization (Fig. 3D).

Selectivity of primary antibodies

The disappearance of all bands following pre-adsorption of the antibody with the N-19 blocking peptide (Fig. 3) indicates that all bands on the immunoblot, including the target 52 and 60 kDa bands that represent 5-HT2CR, are a result of binding of the antibody to the peptide epitope. Thus, to further validate the selectivity of the N-19 as well as the D-12 antibody for the 5-HT2CR, we performed Western blots with D-12 and N-19 on membrane protein extracted from cells that either stably express (1C19 CHO cells) or do not express the human 5-HT2CR (parental CHO cells; Berg et al. 2001). We chose this approach as opposed to assessment of antibody validity in cortical samples taken from the only available conditional 5-HT2CR knockout mouse (Tecott et al. 1995) because 5-HT2CR protein is detectable in PFC at the same level in these knockout mice relative to their wildtype controls (unpublished observations; see Lopez-Gimenez et al. 2002).

The D-12 antibody detected the 46 kDa IR band selectively in the 1C19 cell line (Fig. 4A). The N-19 antibody detected a 52 kDa band only in the 1C19 cell line, while a 41 kDa band was detected in both the parental and 1C19 cell lines (Fig. 4B). The presence of the 41 kDa band in the parental CHO cells (which do not possess 5-HT2CR mRNA) raises sufficient doubt about the identity of the 41 kDa band detected by N-19. On the other hand, the 60 kDa band detected in rat brain preparations by N-19 (Fig. 2B) was not detected in the parental or 1C19 cell lines (Fig. 4B), indicating that the N-19-labeled 60 kDa IR band represents a 5-HT2CR isoform unique to rat relative to the 1C19 cell line, which is stably transfected with the human 5-HT2CR.

Figure 4
Western blot analysis showing the specificity of 5-HT2CR antibodies for the 5-HT2CR in CHO cells

Postsynaptic 5-HT2CR in PFC

Immunoprecipitation followed by immunoblot analyses of synaptosomal fractions from PFC were conducted to further confirm the subcellular localization of the 5-HT2CR as well as to validate the chosen IR bands of interest for the D-12 and N-19 antibody, respectively. The PFC was chosen for these experiments because the band intensity was found to be the greatest in the PFC (Fig. 2). We expected that synaptosomal fractions enriched by pulldown with a given 5-HT2CR antibody followed by immunoblotting with an alternative 5-HT2CR antibody would result in the detection of the predicted IR bands labeled with the 5-HT2CR antibody employed for immunoblotting. In PFC synaptosomes enriched for the 5-HT2CR by immunoprecipitation with the D-12 antibody, immunoblot detection with the D-12 antibody produced an intense IR band at the appropriate MW of 46 kDa and a faint band at 52 kDa (Fig. 5A). As expected, immunoblotting with the N-19 antibody produced antibody-appropriate IR bands of 52 and 60 kDa in samples immunoprecipitated with the D-12 antibody as well as the intense non-specific IR band at 41 kDa seen previously in CHO cells without the 5-HT2CR (Fig. 5B). For PFC synaptosomes immunoprecipitated with the N-19 antibody, immunoblotting with the D-12 antibody produced the predicted IR band at 46 kDa (Fig 5C). PFC synaptosomal samples immunoprecipitated with the N-19 antibody and immunoblotted with the N-19 antibody produced IR bands at the MWs of 52 and 60 kDa as well as the non-specific IR band at 41 kDa (Fig 5D). Preliminary studies performed with synaptosomes from motor cortex show similar results albeit the intensity of the bands was reduced compared to PFC samples (data not shown), as predicted by the less abundant expression of 5-HT2CR (Fig. 2). Non-specific binding of the D-12 and the N-19 5-HT2CR antibodies to the proteins associated to the control resin was not observed (data not shown).

Figure 5
Immunoprecipitation studies with anti-5-HT2CR N-19 or D-12 antibody in PFC synaptosomes

PSD-95 is a member of the membrane-associated guanylate kinase (MAGUK) scaffolding family of proteins which has been positively identified to be associated with receptors and cytoskeletal elements solely in the postsynaptic density of neurons (Banker et al. 1974; Hunt et al. 1996) and is known to complex with the C terminus of the 5-HT2CR in vitro (Becamel et al. 2002; Becamel et al. 2004; Gavarini et al. 2004; Gavarini et al. 2006) and in mouse striatum and hippocampus ex vivo (Abbas et al. 2009). Therefore, identification of PSD-95 in brain samples immunoprecipitated with a 5-HT2CR antibody would further validate that the protein recognized by the 5-HT2CR antibody is 5-HT2CR and would confirm a postsynaptic localization for the 5-HT2CR ex vivo. The PSD-95 antibody produced the expected IR band (95 kDa) in PFC synaptosomal samples immunoprecipitated with the D-12 (Fig. 6A) as well as the N-19 5-HT2CR antibody (Fig. 6B). The PSD-95 antibody also detected a faint band at 100 kDa in synaptosomes immunoprecipitated with the D-12 antibody (Fig. 6A).

Figure 6
Postsynaptic localization of the 5-HT2CR in PFC

Localization of the 5-HT2CR to the postsynaptic density in motor cortex

Aliquots from the various stages of a purified subcellular fractionation from motor cortex were analyzed to confirm expected enrichment by Western blot for appropriate marker proteins (Fig. 7). The motor cortex was chosen for these experiments as the motor cortex dissection is larger in area than the PFC, and a larger tissue sample (with minimal pooling of samples) is required to obtain a workable level of postsynaptic density proteins for accurate and precise Western blot analysis. The highest levels of PSD-95 expression was observed in the synaptic junction and the postsynaptic density-enriched fraction (Fig. 7), as expected (Phillips et al. 2001; Liu et al. 2007; Moron et al. 2007). In contrast, syntaxin, which selectively labels the presynaptic active zone (Bennett and Scheller 1994), was only detected in the total homogenate and synaptosomal fractions (Fig. 7). Expression of SNAP-25, another presynaptic active zone marker, was observed at dramatically higher levels in the total homogenate and in the synaptosomal fraction, than in the synaptic junction and postsynaptic density-enriched fractions (Fig. 7). Thus, the synaptosomal- and postsynaptic density-enriched fractionation protocol described herein yielded an enrichment of proteins known to be localized to the postsynaptic density with a reduction of presynaptic active zone proteins in agreement with previously published studies (Phillips et al. 2001; Liu et al. 2007; Moron et al. 2007).

Figure 7
Validation of synaptosomal enrichment from motor cortex

The D-12 antibody produced the expected intense IR band of 46 kDa, a very faint band around 52 kDa, and an additional faint IR band above 100 kDa in the postsynaptic density enriched protein fraction from motor cortex (Fig. 8, lane 1). The N-19 antibody produced the expected 52 and 60 kDa bands of intermediate intensity. The N-19 antibody also yielded a very faint band at 46 kDa and a very strong IR band above 100 kDa, which appears to be the same as that faintly detected by the D-12 antibody (Fig. 8, lane 2). These data with the N-19 antibody complement and extend our previous work reporting an enrichment of 5-HT2CR in postsynaptic density samples from the PFC (Liu et al. 2007).

Figure 8
Band patterns of 5-HT2CR in the postsynaptic density from motor cortex

DISCUSSION

We clearly demonstrate the presence of the 5-HT2CR in the postsynaptic density of PFC and motor cortex in the present study, as illustrated by the intensity of the specific IR bands in enriched samples. The postsynaptic density is a main region within the synapse that receives and transduces synaptic information, including receptors and their intracellular signaling components, as well as proteins involved in the modulation and maintenance of synaptic structure (Sheng and Hoogenraad 2007). The co-immunoprecipitation of the 5-HT2CR and PSD-95, a known protein partner of the 5-HT2CR (Becamel et al. 2002; Becamel et al. 2004; Gavarini et al. 2004; Gavarini et al. 2006; Abbas et al. 2009), further supports the hypothesis that the 5-HT-2CR is localized to the postsynaptic thickening of axo-dendritic synapses (Becamel et al. 2004). The low abundance of 5-HT2CR detected in tissues lacking a prominent population of the 5-HT2CR (cerebellum, peripheral organs) further lends credence to the conclusion that the analyzed IR bands accurately reflect variations in the level of 5-HT2CR protein expression across tissues (Hoffman and Mezey 1989; Molineaux et al. 1989; Clemett et al. 2000). The 5-HT2CR affords a dynamic influence over a variety of cortically-mediated autonomic, motor and limbic function and our present results suggest that the actions of 5-HT at the 5-HT2CR in these cortical regions occurs in the postsynaptic specialization of neuronal synapses.

The 5-HT2CR, which contains 460 amino acids, presents a theoretical MW of 51,916 Daltons (Julius et al. 1988a). Previous results with laboratory-produced 5-HT2CR antibodies have reported IR bands ranging in MW from 41 kDa to 97 kDa in membrane fractions of rat choroid plexus, frontal cortex, and PFC (Abramowski and Staufenbiel 1995; Sharma et al. 1997; Liu et al. 2007) as well as in the 3T3/2C cell line (Backstrom et al. 1995). In the present studies, the C-terminus targeted antibody (D-12) yielded only a single, highly intense IR band within the range of the predicted MW of the 5-HT2CR (46 kDa); conversely, the N-terminus targeted antibody (N-19) produced several IR bands within the predicted 5-HT2CR MW range (52 and 60 kDa). This multiplicity of IR bands has been attributed to the fact that the expression and function of the 5-HT2CR, like many G-protein coupled receptors, is dynamically regulated by a multitude of conditions, including but not limited to RNA editing (Niswender et al. 1998; Sanders-Bush et al. 2003), post-translational regulation [e.g., glycosylation, phosphorylation, (Abramowski and Staufenbiel 1995; Sharma et al. 1997)] and desensitization/resensitization processes (Berg et al. 1998; Berg et al. 2001; Gavarini et al. 2004; Gavarini et al. 2006). Deglycosylation experiments conducted in studies employing laboratory-produced 5-HT2CR antibodies demonstrated that the 52 kDa and 60 kDa bands most likely represent glycosylated monomers of the 5-HT2CR, while the 46 kDa band represents the native 5-HT2CR monomer (Backstrom et al. 1995; Sharma et al. 1997; Mancia et al. 2008). Interestingly, in PFC synaptosomes enriched for the 5-HT2CR by immunoprecipitation with either the D-12 or N-19 antibody, we detected D-12 labeled IR bands at both 46 kDa and 52 kDa, suggesting that enriching the samples may enhance the detection of the glycosylated form (i.e., 52 kDa) of the receptor by the D-12 antibody. These results imply that glycosylation of the receptor may alter recognition of the protein by the commercially available 5-HT2CR antibodies employed in this study. We also detected various IR bands >100 kDa; these high molecular weight bands may represent ex vivo evidence of the formation of dimers of the 5-HT2CR protein (Herrick-Davis et al. 2004; Mancia et al. 2008). More in-depth studies are required to explore the differential post-translational modifications of and subsequent alterations in the function of the 5-HT2CR regionally within the cortex.

The goal to identify the subcellular localization of the 5-HT2CR in PFC and motor cortex was coupled, by necessity, to the need to validate commercially-available antibodies suitable for the ex vivo analysis of the 5-HT2CR in cortical brain regions. Utilizing a variety of tissues (e.g., brain and peripheral tissues), sample preparations (e.g., membrane, synaptosomal and postsynaptic density-enriched fractions), and control experiments (e.g., peptide neutralization and 5-HT2CR stably-expressing cell line), our straightforward and critical analyses identified the utility of two, commercially available 5-HT2CR antibodies based upon the criteria outlined for validation of antibodies specific for G-protein coupled receptors (Pradidarcheep et al. 2008; Michel et al. 2009). Thus, to date, the D-12 and N-19 5-HT2CR antibodies are the first to prove suitable for application in semi-quantitative analyses of 5-HT2CR protein expression in cortical tissue ex vivo.

Protein-protein interactions between membrane-localized receptors and intracellular signaling molecules exert control over neuronal function (Aarts et al., 2002; Cui et al., 2007) and theoretically provide a rich source of vastly over-looked targets that may play a role in neuropsychiatric disorders. The physical association of PSD-95 with the 5-HT2CR has been shown to promote internalization and trafficking of the 5-HT2CR in vitro (Becamel et al. 2002; Becamel et al. 2004; Gavarini et al. 2004; Gavarini et al. 2006) and is essential for 5-HT2CR signaling in vivo (Abbas et al. 2009). The presence of the 5-HT2CR:PSD-95 complex has now been identified specifically in PFC and motor cortex in our studies. Given the contribution of PSD-95 to synaptic plasticity and maturation of excitatory synapses (El-Husseini et al. 2000; Ehrlich et al. 2007), the 5-HT2CR:PSD-95 interaction is poised to influence neuronal excitability and transmission, and, therefore, cortical function and output (Abbas et al. 2009). Future investigations of the involvement of the 5-HT2CR:PSD-95 interaction in various neuropsychiatric disease states are needed to elucidate targeting this protein:protein interaction for development of highly efficacious therapeutics.

In conclusion, we demonstrate the expression of the 5-HT2CR in postsynaptic density-enriched fractions from both PFC and motor cortex (Sharma et al. 1997; Clemett et al. 2000; Liu et al. 2007; Abbas et al. 2009). While the presence of 5-HT2CR in the postsynaptic density does not rule out the possibility that the 5-HT2CR may also be localized to perisynaptic or extrasynaptic regions of cortical neurons, this finding does support the hypothesis that the 5-HT2CR is positioned to directly modulate synaptic plasticity in cortical neurons (Sheng and Hoogenraad 2007). Our careful and detailed approach paves the way for in-depth and semi-quantitative analyses of the precise detection of the 5-HT2CR subcellular localization in cortical regions as well as trafficking and regulation of the 5-HT2CR at the cortical synapse following genetic and pharmacological manipulations, as a means to further our understanding of cortical 5-HT2CR function and regulation in the pathophysiology of complex neuropsychiatric disorders.

Supplementary Material

Supp Fig S1-2 & Table S1

ACKNOWLEDGEMENTS

We thank Drs. Kelly Berg and William Clarke from the University of Texas Health Science Center at San Antonio for providing the CHO cells as well as Drs. Ronald Emeson and Randi Ulbricht from the Center for Molecular Neuroscience at Vanderbilt University School of Medicine for providing the immunoprecipitation protocol. This research was supported by the National Institute on Drug Abuse grants DA006511, DA000260, DA020087, the Peter F. McManus Charitable Trust (KAC) and the Jeane B. Kempner Postdoctoral Scholar Award (NCA). A portion of these data were presented by Maria Fe Lanfranco in partial fulfillment of the requirements for the Ph.D. degree.

Abbreviations

5-HT
serotonin
5-HT2CR
serotonin 2C receptor
PSD-95
postsynaptic density-95
PFC
prefrontal cortex
IR
immunoreactivity
MW
molecular weight

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