Activation of 5-HT2A/C Receptors Counteracts 5-HT1A Regulation of N-Methyl-D-aspartate Receptor Channels in Pyramidal Neurons of Prefrontal Cortex*

Abnormal serotonin-glutamate interaction in prefrontal cortex (PFC) is implicated in the pathophysiology of many mental disorders, including schizophrenia and depression. However, the mechanisms by which this interaction occurs remain unclear. Our previous study has shown that activation of 5-HT1A receptors inhibits N-methyl-d-aspartate (NMDA) receptor (NMDAR) currents in PFC pyramidal neurons by disrupting microtubule-based transport of NMDARs. Here we found that activation of 5-HT2A/C receptors significantly attenuated the effect of 5-HT1A on NMDAR currents and microtubule depolymerization. The counteractive effect of 5-HT2A/C on 5-HT1A regulation of synaptic NMDAR response was also observed in PFC pyramidal neurons from intact animals treated with various 5-HT-related drugs. Moreover, 5-HT2A/C stimulation triggered the activation of extracellular signal-regulated kinase (ERK) in dendritic processes. Inhibition of the β-arrestin/Src/dynamin signaling blocked 5-HT2A/C activation of ERK and the counteractive effect of 5-HT2A/C on 5-HT1A regulation of NMDAR currents. Immunocytochemical studies showed that 5-HT2A/C treatment blocked the inhibitory effect of 5-HT1A on surface NR2B clusters on dendrites, which was prevented by cellular knockdown of β-arrestins. Taken together, our study suggests that serotonin, via 5-HT1A and 5-HT2A/C receptor activation, regulates NMDAR functions in PFC neurons in a counteractive manner. 5-HT2A/C, by activating ERK via the β-arrestin-dependent pathway, opposes the 5-HT1A disruption of microtubule stability and NMDAR transport. These findings provide a framework for understanding the complex interactions between serotonin and NMDARs in PFC, which could be important for cognitive and emotional control in which both systems are highly involved.

Here we show that activation of 5-HT 1A and 5-HT 2A/C receptors in PFC pyramidal neurons regulates NMDAR channels in a counteractive manner by converging on the microtubule-based transport of NMDARs that is regulated by ERK. Given the importance of NMDAR and serotonin in mental processes under normal and pathological conditions, the complex regulation of NMDAR function by 5-HT 1A and 5-HT 2A/C receptors may provide a molecular and cellular mechanism underlying the role of serotonin in regulating cognitive and emotional behaviors.

EXPERIMENTAL PROCEDURES
Whole Cell Recordings-Whole cell current recordings of cultured PFC neurons employed standard voltage-clamp techniques as those we described previously (21,22). The external solution for recording NMDAR-mediated current contained (in mM): 127 NaCl, 20 CsCl, 1 CaCl 2 , 10 HEPES, 5 BaCl 2 , 12 glucose, 0.001 tetrodotoxin, and glycine 0.02, pH 7.3-7.4, 300 -305 mosM/liter. The internal solution contained (in mM): 180 N-methyl-D-glucamine, 4 MgCl 2 , 40 HEPES, 0.5 1,2-bis(2aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid, 12 phosphocreatine, 3 Na 2 ATP, and 0.5 Na 2 GTP, 0.1 leupeptin, pH 7.2-3, 265-270 mosM/liter. Recordings were obtained with an Axon Instruments (Union City, CA) 200B patch clamp amplifier that was monitored by an IBM PC running pClamp 8 with a DigiData 1320 series interface. Electrode resistances were normally 2-4 M⍀ in bath solution. Following seal rupture, series resistance (4 -10 M⍀) was compensated (70 -90%). Attention was applied to monitor the series resistance, and recordings were stopped when a significant increase (Ͼ20%) occurred. The whole cell NMDAR-mediated current was evoked by NMDA (100 M) application for 2 s every 30 s with neurons held at Ϫ60 mV. Drugs were applied with a "sewer pipe" system. The array of drug capillaries was positioned a few hundred micrometers from the cell under recording. Solution changes were controlled by the SF-77B fast-step solution stimulus delivery device (Warner Instruments, Hamden, CT). Data were analyzed with AxoGraph (Axon instruments) and KaleidaGraph (Albeck Software). ANOVA was performed to compare the differential degrees of current regulation between experimental groups subjected to different drug treatment. Data are expressed as the mean Ϯ S.E.
Electrophysiological Recordings in Slices-To record NMDAR-mediated synaptic transmission, we performed the standard whole cell recording techniques in layer V PFC pyramidal neurons (21,23). Patch pipettes (5-9 M⍀) were filled with the following internal solution (in mM): 130 cesium methanesulfonate, 10 CsCl, 4 NaCl, 1 MgCl 2 , 10 HEPES, 5 EGTA, 2.2 QX-314, 12 phosphocreatine, 5 MgATP, 0.5 Na 2 GTP, 0.1 leupeptin, pH 7.2-7.3, 265-270 mosM/liter. PFC slices (300 m) were perfused at room temperature (22-24°C), with artificial cerebrospinal fluid was bubbled with 95% O 2 /5% CO 2 containing 6-cyano-7-nitroquinoxaline-2,3-dione (20 M) and bicuculline (10 M) to block ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid/kainite receptors and GABA A receptors, respectively. Neurons were observed with a 40ϫ water-immersion lens and illuminated with near infrared IR light, and the image was captured with an IR-sensitive charge-coupled device camera. All recordings were performed using a Multiclamp 700A amplifier. Upon application of negative pressure, the mem-brane was tightly sealed with resistance (2-10 G⍀). With additional suction, the membrane was disrupted into the whole cell configuration. The access resistances ranged from 13 to 18 M⍀ with 50 -70% compensation. Evoked currents were generated with a pulse from a stimulation isolation unit controlled by an S48 pulse generator (Astro-Med). A bipolar stimulating electrode (Fredrick Haer Company) was positioned ϳ100 m from the neuron upon recording. Prior to stimulation, neurons (clamped at Ϫ70 mV) were depolarized to ϩ60 mV for 3 s to fully eliminate the voltage-dependent Mg 2ϩ block of NMDAR. Age-matched saline controls were done side-by-side with drug-injected animals on each day of experiments. To minimize variations between slices, the stimulus with the same intensity was delivered by the stimulating electrode placed at the same location. Data analyses were performed with the Clampfit software (Axon Instruments).
Animal Treatment-Young male rats (25-28 days old) were administered intraperitoneally with the drugs as described in the text. For stereotaxic injection, rats were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal) and then mounted into a stereotaxic apparatus (David Kopf Instruments). Fluoxetine (fluox) (2 l, 0.34 mg/ml, dissolved in saline) were injected unilaterally into the PFC region using a Hamilton syringe (22-gauge needle) at a rate of 0.5 l/min. The coordinates of the lateral PFC used are 1.0 -1.4 mm lateral from midline, 2.2 mm anterior to Bregma, and 3.3 mm dorsal to ventral.
Measurement of Free Tubulin-Free tubulin from PFC cultures was extracted as described previously (21). Cultured PFC neurons (2 ϫ 10 5 cells/cm 2 , 14 days in vitro) in 3.5-cm dishes were washed twice at 37°C with 1 ml of microtubule stabilizing buffer containing (0.1 M MES (pH 6.75), 1 mM MgSO 4 , 2 mM EGTA, 0.1 mM EDTA, and 4 M glycerol). Cultures were then incubated at 37°C for 5 min in 600 l of soluble tubulin extraction buffer (0.1 M MES (pH 6.75), 1 mM MgSO 4 , 2 mM EGTA, 0.1 mM EDTA, 4 M glycerol, and 0.1% Triton X-100) with the addition of protease inhibitor mixture tablets (Roche Applied Science). The soluble extract was centrifuged at 37°C for 2 min, and the supernatant was collected. Equal amounts of total pro-tein were analyzed by Western blotting using anti-␣-tubulin (Sigma). The intensity of tubulin bands was quantitatively analyzed with Image (National Institutes of Health).

Immunocytochemical Staining-
The detection of surface GFP-NR2B (25) was performed as how we described before (21,22). Briefly, cultured PFC neurons were treated with various agents after transfection, and then fixed in 4% paraformaldehyde for 30 min at room temperature without permeabilization. After incubating in 5% bovine serum albumin to block nonspecific staining, cells were incubated with the anti-GFP antibody (1:100, Chemicon, Temecula, CA) for 1 h at room temperature. After three washes in phosphate-buffered saline, cells were incubated with a rhodamine-conjugated secondary antibody (1: 200, Sigma) for 1 h at room temperature. After washing in phosphate-buffered saline, coverslips were mounted on slides with Vectashield mounting media (Vector Laboratories, Burlingame, CA). Fluorescence images were detected using a 60ϫ objective with a cooled charge-coupled device camera mounted on a Nikon microscope.
The surface GFP-NR2B clusters were analyzed with ImageJ software. All specimens were imaged under identical conditions and analyzed with identical parameters. A 50-m segment of dendrite was selected from the equal distance away from the soma of four to six individual neurons. To define dendritic clusters, a single threshold was selected manually. Signal was counted as clusters when its intensity was 2-to 3-fold greater than the overall fluorescence on the dendritic shaft. Three to four independent experiments were performed. Quantitative analyses were performed blindly without knowledge of experimental conditions.
Biochemical Measurement of Surface Receptors-After treatment, PFC slices were incubated with artificial cerebrospinal fluid containing 1 mg/ml Sulfo-NHS-LC-Biotin (Pierce) for 20 min on ice. The slices were then rinsed three times in Trisbuffered saline to quench the biotin reaction, followed by homogenization in 300 l of modified radioimmune precipitation assay buffer (1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid, 50 mM NaPO 4 , 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml leupeptin). The homogenates were centrifuged at 14,000 ϫ g for 15 min at 4°C. 15 g of homogenates was removed to measure total proteins. For surface protein detection, 150 g of homogenates was incubated with 100 l of 50% NeutrAvidin-Agarose (Pierce) for 2 h at 4°C, and bound proteins were resuspended in 25 l of SDS sample buffer and boiled. Quantitative Western blots were performed on both total and biotinylated (surface) proteins using anti-NR1, anti-NR2B (Upstate Biotechnology, Lake Placid, NY), and anti-GABA A R ␤2/3 (Chemicon).

Activation of 5-HT 1A and 5-HT 2A/C Receptors Regulates NMDAR-mediated Ionic and Synaptic Currents in a Counter-
active Manner-We have previously found that activation of 5-HT 1A receptors reduces NMDAR currents in PFC pyramidal neurons (21). To examine the potential interactions between 5-HT 1A and 5-HT 2A/C receptors on NMDAR functions, we tested the impact of 5-HT 2 activation on 5-HT 1A regulation of NMDAR currents by preincubating PFC cultures with the 5-HT 2A/C agonist ␣-Me-5HT (20 M, 10 -30 min). Application of NMDA (100 M) elicited an inward current that was partially desensitized and was completely eliminated by the NMDA receptor antagonist D-aminophosphonovalerate (50 M). As shown in Fig. 1 (A and B), application of the 5-HT 1A agonist 8-OH-DPAT (20 M) reversibly reduced NMDAR currents (21.3 Ϯ 0.7%, n ϭ 16). However, in ␣-Me-5HT-treated neurons, the reduction of NMDAR current by 8-OH-DPAT was substantially attenuated (6.4 Ϯ 0.7%, n ϭ 8). ␣-Me-5HT alone did not significantly affect NMDAR currents (1 M: 4.0 Ϯ 0.8%, n ϭ 5; 20 M: 5.7 Ϯ 1.4%, n ϭ 7). Another 5-HT 2A/C agonist DOI also had no effect on NMDAR currents at low doses (0.05 M: 4.2 Ϯ 1.2%, n ϭ 5; 0.1 M: 4.0 Ϯ 2.5%, n ϭ 5), which are different from the inhibitory effect of DOI shown before (26). The discrepancy may be due to different experimental procedures in recording NMDA-induced currents. In a previous study (26), microdrops of NMDA (1 mM, every 15 min) were applied to PFC slices, which did not allow the accurate detection of peak NMDA currents because of the slow diffusion of the ligand. Moreover, it was not a pure postsynaptic preparation like dissociated neurons, which could have indirect effects due to changes in the circuit. Our results suggest that 5-HT 2A/C activation alone does not directly affect NMDAR currents but opposes 5-HT 1A regulation of NMDAR currents in PFC pyramidal neurons.
We next examined what would happen when both 5-HT 1A and 5-HT 2A/C receptors are co-activated by serotonin. If both signals oppose each other, then blocking one receptor activation would unmask the effect of the other. Thus, we tested the effect of serotonin on NMDAR-EPSC in PFC slices treated with or without 5-HT 2 antagonists. As shown in Fig . It suggests that 5-HT 2 receptor activation in response to serotonin masks part of the inhibitory action of 5-HT 1A receptors on NMDAR channels.
To assess whether the serotonergic regulation of NMDARs, which we found in vitro, is also occurring in vivo, we tested whether endogenous serotonin, via the activation of 5-HT 1A and 5-HT 2A/C receptors, could regulate NMDAR functions in a similar manner in intact animals. First, we examined NMDAR-EPSC in PFC slices from animals intraperitoneally injected with 5-HT 1A or 5-HT 2A/C agonists. As shown in Fig. 3A, the amplitude of NMDAR-EPSC was significantly smaller in animals intraperitoneally injected with the 5-HT 1A agonist 8-OH- Moreover, the amplitude of ␣-amino-3-hydroxy-5-methyl-4isoxazoleproprionic acid receptor-EPSC was unchanged by 8-OH-DPAT injection (saline: 113 Ϯ 11.8 pA, n ϭ 11; DPAT: 112 Ϯ 9.9 pA, n ϭ 10), suggesting that 5-HT 1A activation in PFC is specifically targeting postsynaptic NMDARs rather than presynaptic glutamate release.
Next, we examined NMDAR-EPSC in PFC slices from animals intraperitoneally injected with the serotonin re-uptake inhibitor fluoxetine (20 mg/kg) to elevate endogenous 5-HT levels at synapses. As shown in Fig. 3B, the amplitude of NMDAR-EPSC in fluoxetine-injected animals was significantly smaller, compared with saline controls (saline: 403.   neurons, fluoxetine injection failed to affect NMDAR-EPSC in striatal medium spiny neurons (saline: 375 Ϯ 15.9 pA, n ϭ 8; fluox: 351 Ϯ 13.9 pA, n ϭ 10), suggesting the specificity of serotonergic regulation of NMDARs in PFC.
To further test the effect of 5-HT on NMDARs in PFC networks in vivo without affecting other circuits, we also performed stereotaxic injection of fluoxetine to PFC to elevate the local 5-HT levels. We found that the amplitude of NMDAR-EPSC in PFC pyramidal neurons localized at the proximity of injection sites was significantly smaller, compared with saline controls (saline: 401 Ϯ 10.1 pA, n ϭ 11; fluox: 162 Ϯ 13.2 pA, n ϭ 11, p Ͻ 0.001, ANOVA).
The Opposing Regulation of NMDA Receptors by 5-HT 1A and 5-HT 2 Receptors Depends on Microtubule Stability-Next, we investigated the potential mechanism by which 5-HT 2 receptors counteract the effect of 5-HT 1A on NMDAR currents in PFC neurons. Previously, we have found that 5-HT 1A disrupts NMDA receptor trafficking by destabilizing microtubule integrity (21). Thus, we examined whether microtubule dynamics is the convergent target of 5-HT 1A -and 5-HT 2A -mediated signaling. To test this, we compared the level of free (depolymerized) tubulin in PFC cultures subjected to 8-OH-DPAT treatment in the absence or presence of ␣-Me-5HT. As shown in Fig. 4 (A and B), application of 8-OH-DPAT (40 M, 20 min) caused a potent increase in free tubulin (1.7 Ϯ 0.3-fold increase, n ϭ 3, p Ͻ 0.001, ANOVA), however, this effect was significantly blocked by ␣-Me-5HT (20 M, 0.6 Ϯ 0.2-fold increase, n ϭ 3), indicating that 5-HT 2 activation could increase microtubule stability and prevent 5-HT 1A -induced microtubule depolymerization.
We then tested whether the counteractive effect of 5-HT 2 on 5-HT 1A regulation of NMDAR currents is due to the opposing regulation of microtubule dynamics by 5-HT 1A and 5-HT 2 receptors. As shown in Fig. 4 (C and D), bath application of the microtubule depolymerizer, colchicine (30 M), gradually reduced NMDAR-EPSC in PFC slices (38.0 Ϯ 2.9%, n ϭ 5, also see Ref. 22), mimicking and occluding the 5-HT 1A effect (21). However, this inhibitory effect of colchicine was largely attenuated in the presence of ␣-Me-5HT (20 M, 11.2 Ϯ 13.4%, n ϭ 6). These results suggest that activation of 5-HT 1A and 5-HT 2 receptors could regulate NMDAR currents in a counteractive manner by converging on microtubule dynamics.
Activation of 5-HT 2 Receptors Induces ERK Activation in Neuronal Processes-How could 5-HT 2 activation increase microtubule stability? Evidence has shown that activation of ERK stabilizes microtubule integrity (27,28). Moreover, our previous findings have suggested that 5-HT 1A reduces ERK activity, which in turn reduces MAP2 phosphorylation and  microtubule stability (21). Thus, we speculate that 5-HT 2 receptors might activate ERK, leading to increased phosphorylation of MAP2 and its association with microtubules. To test this, we measured the activation of ERK1/2 in response to 5-HT 2A/C agonists with an antibody that recognizes activated EKR1/2, which are doubly phosphorylated at Thr-202/Tyr-204 in the activation loop of the kinases (29). As a positive control, we also treated neurons with glutamate, which was reported to activate ERK (30). As shown in Fig. 5A, application of ␣-Me-5HT (20 M, 3 min) or glutamate (100 M, 3 min) increased ERK phosphorylation, as compared with vehicle-treated neurons. Unlike glutamate treatment, the ␣-Me-5HT-activated ERK did not co-localize with TOPRO3 (a nucleus marker) staining, suggesting that 5-HT 2 -activated ERK is mainly targeted to the cytoplasm rather than nucleus. Among all experimental groups, total ERK levels remained unchanged (Fig. 5B). These data suggest that 5-HT 2 receptors induce ERK activation, which may oppose the down-regulation of ERK by 5-HT 1A receptors.
The ␤-Arrestin-mediated Pathway Is Involved in the Counteractive Effects of 5-HT 2 Receptors on 5-HT 1A Regulation of NMDAR Currents-Next, we sought to identify the signaling mechanism underlying 5-HT 2 activation of ERK. Recently, it has been shown that some G proteins form a signaling complex with the multifunctional adaptor and transducer molecule, ␤-ar-restins 1/2, which recruits and activates components of the mitogenactivated protein kinase cascades (31,32). To examine the possibility that 5-HT 2 receptors activate ERK through the ␤-arrestin pathway, we transfected PFC cultures with siRNA against ␤-arrestin1 or -2 to knock down their expression. As shown in Fig. 6A, the protein level of ␤-arres-tin1 or -2 was selectively reduced by ␤-arrestin1 siRNA or ␤-arrestin2 siRNA, respectively, but not by a scrambled siRNA. The ␣-Me-5HTinduced activation of ERK1/2 was markedly blocked by knockdown of ␤-arrestin1 or -2 (Fig. 6B). The total ERK level was not affected among all experimental conditions. It suggests that 5-HT 2 receptors activate ERK through a mechanism depending on ␤-arrestins.
Because dynamin-dependent endocytosis of G protein-coupled receptor is required for G protein-coupled receptor/␤arrestin-induced ERK signaling (34), we further examined the role of dynamin in the counteractive effect of 5-HT 2 on 5-HT 1A regulation of NMDAR currents. PFC cultures were treated with a dynamin inhibitory peptide, which competes for binding to amphiphysin and hence inhibits the clathrin/dynamin-dependent endocytosis (35). As shown in Fig. 7 (A and B), treatment of PFC cultures with the membrane-permeable dynamin inhibi- tory peptide (50 M) markedly blocked the ␣-Me-5HT-induced ERK activation, whereas the membrane-impermeable dynamin inhibitory peptide or a cell-permeable scrambled control peptide was ineffective. Furthermore, in neurons injected with the dynamin inhibitory peptide (50 M), ␣-Me-5HT failed to counteract the 5-HT 1A reduction of NMDAR-EPSC (35.8 Ϯ 6.3%, n ϭ 5 (Fig. 7, C and D)). Together, these data suggest that dynamin-based 5-HT 2 receptor endocytosis is involved in 5-HT 2 activation of ERK and 5-HT 2 opposing of 5-HT 1A regulation of NMDAR currents.
Previous studies have suggested that Src-mediated tyrosine phosphorylation of dynamin is involved in G protein-coupled receptor-induced ERK signaling (31,36). To test whether Src kinase activity is required for 5-HT 2 activation of ERK and the counteractive effect of 5-HT 2 on 5-HT 1A regulation of NMDAR currents, we measured the effect of ␣-Me-5HT in PFC neurons treated with the Src kinase inhibitor, PP2. As shown in Fig. 8 (A  and B), application of PP2 (20 M), but not the inactive analog PP3 (20 M), significantly blocked the ␣-Me-5HT-induced ERK phosphorylation. The total ERK level was not affected in cells subjected to different treatments. Moreover, in the presence of PP2, but not PP3, ␣-Me-5HT lost the ability to oppose 8-OH-DPAT reduction of NMDAR-EPSC (Fig. 8C: PP2: 32.7 Ϯ 3.7%, n ϭ 7; Fig. 8D: PP3: 8.8 Ϯ 3.3%, n ϭ 4). Taken together, these results suggest that Src kinase activation is involved in 5-HT 2 signaling.
Activation of 5-HT 2 Receptors Opposes 5-HT 1A Reduction of Surface NR2B Subunits in a ␤-Arrestin-dependent Manner-If 5-HT 1A and 5-HT 2A/C regulate microtubule dynamics in a counteractive manner, it is possible that they may alter NMDAR trafficking on microtubules in an opposite way. To test this, we performed immunocytochemical experiments in cultured PFC neurons transfected with GFPtagged NR2B subunits (the GFP tag is positioned at the extracellular N terminus of NR2B). Surface NR2B receptors were detected with the anti-GFP primary antibody, followed by rhodamine-conjugated secondary antibody in non-permeabilized conditions. Consistent with our previous findings (21), application of 8-OH-DPAT (40 M, 5 min), caused a marked reduction in the number and size of surface NR2B clusters (Fig. 9, A and B), as compared with control neurons (cluster density: 43.6 Ϯ 1.7 clusters/50 m in controls versus 22.7 Ϯ 0.8 clusters/50 m in DPAT-treated cells; cluster size: 0.33 Ϯ 0.02 m 2 in controls versus 0.18 Ϯ 0.02 m 2 in DPAT-treated cells (Fig. 9G). Treatment with ␣-Me-5HT (40 M, 30 min) significantly prevented the 8-OH-DPAT-induced reduction of surface NR2B subunits (cluster density: 40.0 Ϯ 2.7 clusters/50 m; cluster size: 0.3 Ϯ 0.03 m 2 (Fig.  9, C and G)). The ␣-Me-5HT treatment itself did not affect the distribution of surface NR2B clusters (data not shown). The fluorescence intensity of surface NR2B clusters (average gray value per pixel) was not significantly changed in neurons subject to various treatments (Fig. 9G). The total amount of recombinant NR2B receptor (GFP channel) was unaltered.
Finally, we tested whether the change on NMDAR-EPSC amplitudes in PFC neurons by acute fluoxetine treatment of intact animals can be accounted for by the altered number of NMDA receptors on the cell membrane. Surface biotinylation experiments (23) were performed to measure levels of surface NR1 and NR2B in PFC slices from animals intraperitoneally injected with fluoxetine (20 mg/kg). Surface proteins were labeled with Sulfo-NHS-LC-biotin, and then biotinylated surface proteins were separated from non-labeled intracellular proteins by reaction with NeutrAvidin beads. Surface and total proteins were subjected to electrophoresis and probed with an antibody against the NR1 or NR2B subunit. As shown in Fig. 10  (A and B), the surface levels of NR1 and NR2B in PFC slices were significantly lower in animals exposed to fluoxetine (NR1: 70 Ϯ 3% of control; NR2B: 60 Ϯ 6% of control; n ϭ 4). Consistent with electrophysiological results, the fluoxetine-induced reduction of surface NMDARs was more prominent in animals co-injected with the 5-HT 2A/C antagonist ketanserin (NR1: 29 Ϯ 3% of control; NR2B: 30 Ϯ 1% of control; n ϭ 4) and was largely blocked in animals co-injected with the 5-HT 1A antagonist WAY-100635 (NR1: 82 Ϯ 5% of control; NR2B: 84 Ϯ 1% of control; n ϭ 4). The surface GABA A R ␤ 2 subunit level, as well as the total NR1 or NR2B level, in PFC slices was unchanged by any of these drug administrations. Taken together, these results suggest that endogenous serotonin regulates PFC NMDAR surface expression in vivo through 5-HT 1A and 5-HT 2A/C receptors in a counteractive manner.
NR1 and NR2 subunits have to be co-assembled before leaving the endoplasmic reticulum and being transported along microtubules in dendrites to synapses. Our previous study (21) shows that 5-HT 1A receptors primarily target NR2B subunit-containing NMDA receptors, consistent with NR2B being the cargo of the microtubule motor KIF17. With the application of fluoxetine plus ketanserin, the surface levels of NR1 and NR2B were reduced to a similar degree (surface NR2A was also reduced, data not shown). It suggests that this reduction likely results from endogenous NR1/ NR2B heteromers and NR1/ NR2A/NR2B triheteromers.

DISCUSSION
It is well known that many antidepressants and antipsychotics exert their actions by inhibiting serotonin reuptake and thus enhance serotonergic transmission (37,38). Because serotonin has multiple receptor subtypes, systemic elevation of serotonin can induce diverse physiological effects in neurons (2). The molecular mechanisms for serotonin to regulate cellular targets through different subtypes of receptors remain to be identified. We have previously found that 5-HT 2 and 5-HT 4 receptors are linked to regulate GABA A receptors (9, 39), whereas 5-HT 1A receptors are linked to regulate NMDA receptors in PFC neurons (21). The specific coupling of different 5-HT receptors to distinct ion channels allows serotonin to regulate multiple targets in a precise but flexible manner. Here, we provide evidence showing that the 5-HT 1A regulation of NMDARs can be modified by 5-HT 2A/C receptor activation, which provides a mechanism to fine-tune the effect of serotonin on NMDAR-mediated synaptic transmission and plasticity.
Under normal physiological conditions, which of the one or more 5-HT receptors in PFC pyramidal neurons that are activated by serotonin is determined by the serotonergic projection from dorsal raphe to cellular compartments rich in different subtypes of receptors (40). Agonist binding studies indicate that 5-HT 1 and 5-HT 2 receptors have different affinities (nanomo- lar versus low micromolar) to 5-HT, suggesting that 5-HT 1 receptor activation may play a dominant role in response to serotonin. Microdialysis studies have shown that synaptic concentration of serotonin can reach up to 6 mM (41), suggesting that sometimes all 5-HT receptors could be fully activated at synapses. Both 5-HT 1A and 5-HT 2A receptors are expressed at dendritic shafts and spines of PFC pyramidal neurons (42,43), where NMDA receptors are abundant, prompting us to speculate that both receptors may interact with NMDA receptors in synergistic or opposite ways. Our studies in PFC pyramidal neurons from both acute slices and intact animals treated with various 5-HT-related drugs indicate that blocking 5-HT 2A/C receptors unmasks the ability of 5-HT 1A receptors to reduce NMDAR currents, suggesting that 5-HT 1A and 5-HT 2A/C receptors converge on NMDAR channels in a counteractive manner. Similar to serotonin, it has been shown that, in response to dopamine, D 1 and D 2 receptors that are linked to distinct signaling cascades regulate cortical GABAergic inhibition in an opposing manner (44).
Our previous study has shown that activation of 5-HT 1A receptors suppresses NMDAR currents by reducing microtubule stability and the ensuing NMDAR trafficking along dendritic microtubules (21). In this study, we found that activation of 5-HT 2A/C receptor opposes the ability of 5-HT 1A to induce microtubule depolymerization, suggesting that microtubule dynamics and the microtubule-based NMDAR transport are regulated by 5-HT 1A and 5-HT 2A/C receptors in a counteractive fashion.
The stability of microtubules is regulated by different microtubule-associated proteins (MAPs) in distinct neuronal compartments. The phosphorylation state of MAP2, a dendrite-specific MAP, determines the ability of MAP2 to associate and stabilize dendritic microtubules (45). One key signaling molecule that regulates MAP2 phosphorylation is ERK (28). Our previous study suggests that 5-HT 1A activation suppresses ERK activity, which leads to the decreased MAP2 phosphorylation, MAP2-microtubule interaction, and microtubule stability (21). It is possible that 5-HT 1A and 5-HT 2A/C oppositely regulates ERK activity, thereby controlling microtubule stability in a counteractive manner. Consistently, our biochemical and immunocytochemical data show that 5-HT 2A/C activation potently enhances ERK activity in the dendrites of PFC cultures.
How does the 5-HT 2A/C receptor activate ERK? Phospholipase C and inositol 1,4,5-trisphosphate, two downstream molecules of classic 5-HT 2 signaling, are not involved in this regulation (data not shown). Recent studies have suggested that the scaffolding protein ␤-arrestin, which binds to the third intracellular loop of certain G protein-coupled receptors, induces ERK activation (31,32). 5-HT 2A receptors are found to bind purified ␤-arrestin (46). However, whether 5-HT 2A activates mitogen-activated protein kinase via ␤-arrestin pathway is essentially unknown. Our data show that knockdown of ␤-ar-restin1/2 not only blocks the 5-HT 2A/C -induced ERK activation but also eliminates the counteractive effect of 5-HT 2A/C on 5-HT 1A reduction of NMDAR currents. In addition, we found that the 5-HT 2A/C action is dependent on Src activation and clarthin/dynamin-mediated endocytosis of the receptor. Taken together, 5-HT 2A , via the ␤-arrestin/Src/dynamin cascade, induces ERK activation to oppose the effect of 5-HT 1A on NMDAR functions.
Several mechanisms have been proposed for the regulation of NMDAR functions, including altering the phosphorylation state and biophysical properties of the channel (47,48) and changing NMDAR trafficking and channel numbers at the membrane (49,50). Our previous finding suggests that 5-HT 1A decreases surface NR2B clusters in a microtubule-dependent manner (21), consistent with the role of cytoskeleton-based transport on NMDAR insertion to the plasma membrane (51,52). Our immunocytochemical data show that the 5-HT 1A reduction of surface NR2B clusters is attenuated by pretreatment with a 5-HT 2A/C agonist, confirming that 5-HT 1A and 5-HT 2A/C oppositely regulate NMDAR trafficking. Moreover, 5-HT 2A/C opposes the 5-HT 1A reduction of surface NR2B clusters in a ␤-arrestin-dependent manner, consistent with the ␤-arrestin dependence of the 5-HT 2A/C action on 5-HT 1A regulation of NMDAR currents. Surface biotinylation experiments using PFC from intact animals subject to acute fluoxetine treatment have also confirmed the results found in cultured neurons.
Based on the experimental data, we speculate that, in response to serotonin, both 5-HT 1A and 5-HT 2A/C receptors localized in PFC pyramidal neurons are activated, which converge to regulate NMDAR trafficking and function in an opposite manner, by coupling to distinct signaling cascades and differentially affecting microtubule stability. This study may provide significant insights into the complex regulation of NMDAR-mediated synaptic transmission and plasticity by different serotonin receptors in the PFC network.