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 cacl2 , 10 hepes , 5 bacl2 , 12 glucose , 0.001 tetrodotoxin , and glycine 0.02 , ph 7.37.4 , 300305 mosm / liter .
the internal solution contained ( in mm ) : 180 n - methyl - d - glucamine , 4 mgcl2 , 40 hepes , 0.5 1,2-bis(2-aminophenoxy)ethane - n , n , n,n-tetraacetic acid , 12 phosphocreatine , 3 na2atp , and 0.5 na2gtp , 0.1 leupeptin , ph 7.2 - 3 , 265270 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 24 m in bath solution . following seal rupture , series resistance ( 410 m )
attention was applied to monitor the series resistance , and recordings were stopped when a significant increase ( > 20% ) occurred .
the whole cell nmdar - mediated current was evoked by nmda ( 100 m ) application for 2 s every 30 s with neurons held at 60 mv .
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 .
, we performed the standard whole cell recording techniques in layer v pfc pyramidal neurons ( 21 , 23 ) .
patch pipettes ( 59 m ) were filled with the following internal solution ( in mm ) : 130 cesium methanesulfonate , 10 cscl , 4 nacl , 1 mgcl2 , 10 hepes , 5 egta , 2.2 qx-314 , 12 phosphocreatine , 5 mgatp , 0.5 na2gtp , 0.1 leupeptin , ph 7.27.3 , 265270 mosm / liter .
pfc slices ( 300 m ) were perfused at room temperature ( 2224 c ) , with artificial cerebrospinal fluid was bubbled with 95% o2/5% co2 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 gabaa receptors , respectively .
neurons were observed with a 40 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 membrane was tightly sealed with resistance ( 210 g ) . with additional suction ,
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 100m from the neuron upon recording . prior to stimulation , neurons ( clamped at 70 mv ) were depolarized to + 60 mv for 3 s to fully eliminate the voltage - dependent mg 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 .
the agents we used include serotonin , 8-hydroxy-2(di - n - propylamino)tetralin ( 8-oh - dpat ) , -me-5ht , ( )-2,5-dimethoxy-4-iodoamphetamine , ketanserin , benzothiazole , colchicine ( sigma ) , dynamin inhibitory peptide ( tocris ) , 4-amino-5-(4-chlorophenyl)-7-(t - butyl)pyrazolo[3,4-d]pyrimidine ( pp2 ) and 4-amino-7-phenylpyrazol[3,4-d]pyrimidine ( pp3 ) ( calbiochem ) .
they were made up as concentrated stocks in water or dmso and stored at 20 c .
animal treatment  young male rats ( 2528 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 1.01.4 mm lateral from midline , 2.2 mm anterior to bregma , and 3.3 mm dorsal to ventral .
small interfering rna  to knock down the endogenous -arrestin expression , we used the small interfering rna ( sirna ) that specifically targets -arrestin1 or -arrestin2 mrna ( 24 ) .
the sirna oligonucleotide sequences were : 5-ggcgagucuacgugacacutt-3 ( for -arrestin1 ) and 5-ggaccggaasguguuugugtt-3 ( for -arrestin2 ) .
the sirna ( purchased from ambion , austin , tx ) was co - transfected with enhanced gfp into cultured pfc neurons ( 11 days in vitro ) using the lipofectamine 2000 method .
biochemical , immunocytochemical , or electrophysiological experiments were performed in neurons after 23 days of transfection .
cultured pfc neurons ( 2  10 cells / cm , 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 mgso4 , 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 mgso4 , 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 protein were analyzed by western blotting using anti--tubulin ( sigma ) .
the intensity of tubulin bands was quantitatively analyzed with image ( national institutes of health ) .  
figure 1.activation of 5-ht2a / c receptors counteracts the 5-ht1a -induced reduction of nmdar - mediated ionic and synaptic currents in pfc pyramidal neurons .
a and c , plot of normalized peak nmdar currents ( a ) or nmdar - epsc ( c ) as a function of time and 8-oh - dpat ( 5-ht1a agonist , 20 m ) application in neurons treated with or without -me-5ht ( 5-ht2a / c agonist , 20 m ) . each point ( c ) represents the average peak ( mean  s.e . ) of three consecutive nmdar - epsc .
b and d , representative current traces taken from the records used to construct a or c ( at time points denoted by # ) .
scale bars : 100 pa , 1 s ( b ) ; 100 pa , 100 ms ( d ) .
e , plot of peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) and ketanserin ( 5-ht2a / c antagonist , 20 m ) .
f , cumulative data ( mean  s.e . ) summarizing the percentage reduction of nmdar - epsc by 8-oh - dpat in the presence or absence of various 5-ht receptor agonists or antagonists . * ,
p < 0.001 , anova .   activation of 5-ht2a / c receptors counteracts the 5-ht1a -induced reduction of nmdar - mediated ionic and synaptic currents in pfc pyramidal neurons .
a and c , plot of normalized peak nmdar currents ( a ) or nmdar - epsc ( c ) as a function of time and 8-oh - dpat ( 5-ht1a agonist , 20 m ) application in neurons treated with or without -me-5ht ( 5-ht2a / c agonist , 20 m ) . each point ( c ) represents the average peak ( mean  s.e . ) of three consecutive nmdar - epsc .
b and d , representative current traces taken from the records used to construct a or c ( at time points denoted by # ) .
scale bars : 100 pa , 1 s ( b ) ; 100 pa , 100 ms ( d ) .
e , plot of peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) and ketanserin ( 5-ht2a / c antagonist , 20 m ) .
f , cumulative data ( mean  s.e . ) summarizing the percentage reduction of nmdar - epsc by 8-oh - dpat in the presence or absence of various 5-ht receptor agonists or antagonists . * ,
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 60 objective with a cooled charge - coupled device camera mounted on a nikon microscope .
a 50-m segment of dendrite was selected from the equal distance away from the soma of four to six individual neurons .
signal was counted as clusters when its intensity was 2- to 3-fold greater than the overall fluorescence on the dendritic shaft .
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 tris - buffered 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 napo4 , 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 ) .
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 - gabaar 2/3 ( chemicon ) .
activation of 5-ht1a and 5-ht2a / c receptors regulates nmdar - mediated ionic and synaptic currents in a counteractive manner  we have previously found that activation of 5-ht1a receptors reduces nmdar currents in pfc pyramidal neurons ( 21 ) . to examine the potential interactions between 5-ht1a and 5-ht2a / c receptors on nmdar functions , we tested the impact of 5-ht2 activation on 5-ht1a regulation of nmdar currents by preincubating pfc cultures with the 5-ht2a / c agonist -me-5ht ( 20 m , 1030 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-ht1a agonist 8-oh - dpat ( 20 m ) reversibly reduced nmdar currents ( 21.3  0.7% , n = 16 ) . however , in -me-5ht - treated neurons , the reduction of nmdar current by 8-oh - dpat was substantially attenuated ( 6.4  0.7% , n = 8) .
-me-5ht alone did not significantly affect nmdar currents ( 1 m : 4.0  0.8% , n = 5 ; 20 m : 5.7  1.4% , n = 7 ) .
another 5-ht2a / c agonist doi also had no effect on nmdar currents at low doses ( 0.05 m : 4.2  1.2% , n = 5 ; 0.1 m : 4.0  2.5% , n = 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-ht2a / c activation alone does not directly affect nmdar currents but opposes 5-ht1a regulation of nmdar currents in pfc pyramidal neurons .  
figure 2.inhibition of 5-ht2a / c receptors unmasks the 5-ht1a -mediated reduction of nmdar - epsc in response to 5-ht .
a , representative nmdar - epsc traces ( average of three trials ) showing the regulation of nmdar - epsc by 5-ht ( 10 , 40 , or 100 m ) in neurons treated with or without the 5-ht2a / c antagonist ketanserin ( ket , 20 m ) .
b , dose - response curves summarizing the percentage reduction of nmdar - epsc by different concentrations of 5-ht in neurons treated with or without ketanserin . * ,
0.001 , anova .   inhibition of 5-ht2a / c receptors unmasks the 5-ht1a -mediated reduction of nmdar - epsc in response to 5-ht .
a , representative nmdar - epsc traces ( average of three trials ) showing the regulation of nmdar - epsc by 5-ht ( 10 , 40 , or 100 m ) in neurons treated with or without the 5-ht2a / c antagonist ketanserin ( ket , 20 m ) .
b , dose - response curves summarizing the percentage reduction of nmdar - epsc by different concentrations of 5-ht in neurons treated with or without ketanserin . * ,
we further tested the influence of 5-ht2 activation on 5-ht1a regulation of nmdar - epsc mediated by synaptic nmda receptors in pfc slices .
as shown in fig . 1 ( c and d ) , application of 8-oh - dpat potently reduced the amplitude of nmdar - epsc ( 37.1  2.1% ; n = 10 ( fig .
1f ) ) . however , this reduction was significantly attenuated in the presence of -me-5ht ( 10.4  0.7% ; n = 13 ( fig .
1f ) ) . to verify that 5-ht2a / c receptors were mediating this regulatory effect of -me-5ht ,
we pre - treated pfc slices with the specific 5-ht2a / c antagonist ketanserin ( 20 m ) . as shown in fig .
1e , -me-5ht failed to oppose the effect of 5-ht1a on nmdar - epsc in the presence of ketanserin ( 36.8  7.1% ; n = 5 ( fig .
1f ) ) . in contrast , the reduction of nmdar - epsc by 8-oh - dpat was intact in the presence of the 5-ht4 agonist benzothiazole ( 20 m , 33  2.9% ; n = 5 ( fig .
these results indicate that 5-ht2a / c receptor activation selectively counteracts 5-ht1a regulation of nmdar functions in pfc pyramidal neurons .
we next examined what would happen when both 5-ht1a and 5-ht2a / 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-ht2 antagonists .
as shown in fig . 2 ( a and b ) , different concentrations of serotonin reduced the amplitude of nmdar - epsc to different extents ( 10 m:21  2.3% , n = 4 ; 40 m : 28.5  4.9% , n = 4 ; 100 m : 29.4  4.8% , n = 5 ) . however , in the presence of the 5-ht2a / c antagonist ketanserin ( 20 m ) , the reduction of nmdar - epsc by serotonin was greatly augmented ( 10 m : 35.2  2.2% , n = 7 ; 40 m : 43.0  2.0% , n = 6 ; 100 m:47  1.9% , n = 7 ) .
it suggests that 5-ht2 receptor activation in response to serotonin masks part of the inhibitory action of 5-ht1a 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-ht1a and 5-ht2a / 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-ht1a or 5-ht2a / c agonists . as shown in fig .
3a , the amplitude of nmdar - epsc was significantly smaller in animals intraperitoneally injected with the 5-ht1a agonist 8-oh - dpat ( saline : 379.8  8.9 pa , n = 8 ; dpat : 166  11.5 pa , n = 11 ; p < 0.001 , anova ) , whereas it was largely unaltered by injecting the 5-ht2a / c agonist -me-5ht ( 349  12.2 pa , n = 10 ) .
however , the effect of 8-oh - dpat injection was blocked by injecting -me-5ht ( dpat + -me-5ht : 335  15.9 pa , n = 12 ) .
these results indicate that activation of 5-ht1a receptors in vivo down - regulates nmdar functions , and this effect of 5-ht1a is opposed by 5-ht2a / c receptor activation in vivo .
moreover , the amplitude of -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor - epsc was unchanged by 8-oh - dpat injection ( saline : 113  11.8 pa , n = 11 ; dpat : 112  9.9 pa , n = 10 ) , suggesting that 5-ht1a activation in pfc is specifically targeting postsynaptic nmdars rather than presynaptic glutamate release .  
figure 3.the synaptic nmda response is reduced in pfc pyramidal neurons from animals with systemic administration of 5-ht1a agonist or the serotonin re - uptake inhibitor fluoxetine , which is opposed by injecting 5-ht2a / c agonist .
a and b , dot plots showing the peak amplitude of nmdar - epsc recorded in pfc pyramidal neurons from animals with a single intraperitoneal injection of different drugs .
a , saline , 8-oh - dpat , -me-5ht , or 8-oh - dpat plus -me-5ht ; b , saline , fluoxetine , way-100635 , way-100635 plus fluoxetine , ketanserin , or ketanserin plus fluoxetine .
inset : representative nmdar - epsc traces recorded in pfc pyramidal neurons from animals with different drug injection .
c , plot of normalized peak nmdar - epsc showing the effect of bath application of 8-oh - dpat ( 40 m ) in pfc pyramidal neurons from animals intraperitoneally injected with fluoxetine ( 20 mg / kg ) or saline .  
the synaptic nmda response is reduced in pfc pyramidal neurons from animals with systemic administration of 5-ht1a agonist or the serotonin re - uptake inhibitor fluoxetine , which is opposed by injecting 5-ht2a / c agonist . a and b , dot plots showing the peak amplitude of nmdar - epsc recorded in pfc pyramidal neurons from animals with a single intraperitoneal injection of different drugs . a , saline , 8-oh - dpat , -me-5ht , or 8-oh - dpat plus -me-5ht ; b , saline , fluoxetine , way-100635 , way-100635 plus fluoxetine , ketanserin , or ketanserin plus fluoxetine .
inset : representative nmdar - epsc traces recorded in pfc pyramidal neurons from animals with different drug injection .
c , plot of normalized peak nmdar - epsc showing the effect of bath application of 8-oh - dpat ( 40 m ) in pfc pyramidal neurons from animals intraperitoneally injected with fluoxetine ( 20 mg / kg ) or saline .  
figure 4.activation of 5-ht2a / c receptors attenuates the 5-ht1a -induced microtubule depolymerization and the effect of microtubule depolymerizer on nmdar - epsc .
a , western blot analysis of free tubulin in lysates of cultured pfc neurons treated with or without 8-oh - dpat ( 40m , 30 min ) in the absence or presence of -me-5ht ( 20 m , added 10 min before 8-oh - dpat treatment ) .
free tubulin level was normalized to control (  ) , based on the intensity of the tubulin band from western blot analyses .
c , plot of normalized peak nmdar - epsc showing the effect of microtubule depolymerizer colchicine ( 30 m ) in the absence or presence of -me-5ht ( 20 m ) .
d , cumulative data ( mean  s.e . ) illustrating the percent reduction of nmdar - epsc by colchicine in neurons with or without the exposure to -me-5ht . * , p < 0.05 , anova .   activation of 5-ht2a / c receptors attenuates the 5-ht1a -induced microtubule depolymerization and the effect of microtubule depolymerizer on nmdar - epsc .
a , western blot analysis of free tubulin in lysates of cultured pfc neurons treated with or without 8-oh - dpat ( 40m , 30 min ) in the absence or presence of -me-5ht ( 20 m , added 10 min before 8-oh - dpat treatment ) .
free tubulin level was normalized to control (  ) , based on the intensity of the tubulin band from western blot analyses .
c , plot of normalized peak nmdar - epsc showing the effect of microtubule depolymerizer colchicine ( 30 m ) in the absence or presence of -me-5ht ( 20 m ) .
d , cumulative data ( mean  s.e . ) illustrating the percent reduction of nmdar - epsc by colchicine in neurons with or without the exposure to -me-5ht . * , p < 0.05 , anova .
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 : 403.5  15.8 pa , n = 11 ; fluox : 195.1  12.4 pa , n = 14 ; p < 0.001 , anova ) .
this effect of fluoxetine was blocked by injecting the 5-ht1a antagonist way-100635 ( way : 367  12.9 pa , n = 12 ; way + fluox : 390  15.8 pa , n = 12 ) , suggesting the mediation by 5-ht1a receptors . moreover
, the reducing effect of fluoxetine on nmdar - epsc was potentiated by injecting the 5-ht2a / c antagonist ketanserin ( ket : 392  12.3 pa , n = 11 ; ket + fluox : 59.6  5.2 pa , n = 12 ; p < 0.001 , anova ) , revealing the counteracting effect of 5-ht2a / c on 5-ht1a regulation of synaptic nmda responses .
moreover , the effect of bath application of 8-oh - dpat ( 40 m ) on nmdar - epsc was occluded in animals injected with fluoxetine , as compared with saline controls ( fig .
3c , saline : 40.2  1.9% , n = 5 , fluox : 10.5  2.5% , n = 6 ) , confirming the common mechanism underlying the in vivo fluoxetine effect and in vitro 8-oh - dpat effect on nmdars .
in contrast to the strong effect in pfc pyramidal neurons , fluoxetine injection failed to affect nmdar - epsc in striatal medium spiny neurons ( saline : 375  15.9 pa , n = 8 ; fluox : 351  13.9 pa , n = 10 ) , suggesting the specificity of serotonergic regulation of nmdars in pfc .  
figure 5.activation of 5-ht2a / c receptors induces the phosphorylation and activation of erk in neuronal dendrites in cultured pfc neurons . a and b , immunocytochemical images of cultured pfc neurons stained with phospho - erk ( a , green ) or erk ( b , green ) plus the nucleus marker topro3 ( red ) in the absence ( vehicle ) or presence of -me-5ht ( 20 m , 3 min ) or glutamate ( 100 m , 3 min ) .  
activation of 5-ht2a / c receptors induces the phosphorylation and activation of erk in neuronal dendrites in cultured pfc neurons . a and b , immunocytochemical images of cultured pfc neurons stained with phospho - erk ( a , green ) or erk ( b , green ) plus the nucleus marker topro3 ( red ) in the absence ( vehicle ) or presence of -me-5ht ( 20 m , 3 min ) or glutamate ( 100 m , 3 min ) .  
figure 6.the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function is dependent on the -arrestin / erk pathway
. a , top : western blots of -arrestin 1/2 in pfc cultures transfected without or with sirna against -arrestin 1/2 or a scrambled sirna .
b , top : western blots of phospho - erk in the absence or presence of -me-5ht ( 20 m , 3 min ) in pfc cultures transfected with or without -arrestin 1/2 sirna . bottom : quantification of phospho - erk under different conditions .
c , plot of peak nmdar currents as a function of time and 8-oh - dpat ( 20m ) application in the presence of-me-5ht ( 20 m ) in gfp - positive neurons transfected with or without sirna against -arrestin 1/2 .
d , cumulative data ( mean  s.e . ) summarizing the percentage reduction of nmdar currents by 8-oh - dpat in the absence or presence of-me-5ht under different conditions . * ,
p < 0.001 , anova .   the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function is dependent on the -arrestin / erk pathway
. a , top : western blots of -arrestin 1/2 in pfc cultures transfected without or with sirna against -arrestin 1/2 or a scrambled sirna .
b , top : western blots of phospho - erk in the absence or presence of -me-5ht ( 20 m , 3 min ) in pfc cultures transfected with or without -arrestin 1/2 sirna . bottom : quantification of phospho - erk under different conditions .
c , plot of peak nmdar currents as a function of time and 8-oh - dpat ( 20m ) application in the presence of-me-5ht ( 20 m ) in gfp - positive neurons transfected with or without sirna against -arrestin 1/2 .
d , cumulative data ( mean  s.e . ) summarizing the percentage reduction of nmdar currents by 8-oh - dpat in the absence or presence of-me-5ht under different conditions . * ,
. 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  10.1 pa , n = 11 ; fluox : 162  13.2 pa , n = 11 , p < 0.001 , anova ) .
the opposing regulation of nmda receptors by 5-ht1a and 5-ht2 receptors depends on microtubule stability 
next , we investigated the potential mechanism by which 5-ht2 receptors counteract the effect of 5-ht1a on nmdar currents in pfc neurons .
previously , we have found that 5-ht1a disrupts nmda receptor trafficking by destabilizing microtubule integrity ( 21 ) .
thus , we examined whether microtubule dynamics is the convergent target of 5-ht1a- and 5-ht2a - 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 -me5ht .
4 ( a and b ) , application of 8-oh - dpat ( 40 m , 20 min ) caused a potent increase in free tubulin ( 1.7  0.3-fold increase , n = 3 , p < 0.001 , anova ) , however , this effect was significantly blocked by -me-5ht ( 20 m , 0.6  0.2-fold increase , n = 3 ) , indicating that 5-ht2 activation could increase microtubule stability and prevent 5-ht1a - induced microtubule depolymerization .
we then tested whether the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents is due to the opposing regulation of microtubule dynamics by 5-ht1a and 5-ht2 receptors .
4 ( c and d ) , bath application of the microtubule depolymerizer , colchicine ( 30 m ) , gradually reduced nmdar - epsc in pfc slices ( 38.0  2.9% , n = 5 , also see ref .
22 ) , mimicking and occluding the 5-ht1a effect ( 21 ) . however , this inhibitory effect of colchicine was largely attenuated in the presence of -me-5ht ( 20 m , 11.2  13.4% , n = 6 ) .
these results suggest that activation of 5-ht1a and 5-ht2 receptors could regulate nmdar currents in a counteractive manner by converging on microtubule dynamics .
activation of 5-ht2 receptors induces erk activation in neuronal processes  how could 5-ht2 activation increase microtubule stability ?
evidence has shown that activation of erk stabilizes microtubule integrity ( 27 , 28 ) . moreover , our previous findings have suggested that 5-ht1a reduces erk activity , which in turn reduces map2 phosphorylation and microtubule stability ( 21 ) .
thus , we speculate that 5-ht2 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-ht2a / 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 ) .
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-ht2-activated erk is mainly targeted to the cytoplasm rather than nucleus . among all experimental groups ,
these data suggest that 5-ht2 receptors induce erk activation , which may oppose the down - regulation of erk by 5-ht1a receptors .  
figure 7.the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function involves clathrin / dynamin - mediated endocytosis . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the cell - permeable ( myristoylated ) or non - permeable dynamin inhibitory peptide ( both 50 m ) , or a cell - permeable scrambled control peptide ( 50 m ) .
this scrambled peptide was fused with the protein transduction domain of the human immunodeficiency virus tat protein ( ygrkkrrqrrr , 53 ) , which rendered it cell - permeable .
c , plot of normalized peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) in cells dialyzed with the dynamin inhibitory peptide ( 50 m ) or a scrambled control peptide ( 50 m ) .
inset , representative traces of nmdar - epsc taken at time points denoted by # .
d , cumulative data ( mean  s.e . ) summarizing the percentage reduction of nmdar - epsc by 8-oh - dpat in the absence or presence of -me-5ht in neurons injected with or without the dynamin inhibitory peptide . * ,
the counteractive effect of 5-ht2a / c on 5-ht1a regulation of nmdar function involves clathrin / dynamin - mediated endocytosis . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the cell - permeable ( myristoylated ) or non - permeable dynamin inhibitory peptide ( both 50 m ) , or a cell - permeable scrambled control peptide ( 50 m ) .
this scrambled peptide was fused with the protein transduction domain of the human immunodeficiency virus tat protein ( ygrkkrrqrrr , 53 ) , which rendered it cell - permeable .
c , plot of normalized peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) in cells dialyzed with the dynamin inhibitory peptide ( 50 m ) or a scrambled control peptide ( 50 m ) .
inset , representative traces of nmdar - epsc taken at time points denoted by # .
d , cumulative data ( mean  s.e . ) summarizing the percentage reduction of nmdar - epsc by 8-oh - dpat in the absence or presence of -me-5ht in neurons injected with or without the dynamin inhibitory peptide . * ,
the -arrestin - mediated pathway is involved in the counteractive effects of 5-ht2 receptors on 5-ht1a regulation of nmdar currents  next , we sought to identify the signaling mechanism underlying 5-ht2 activation of erk .
recently , it has been shown that some g proteins form a signaling complex with the multifunctional adaptor and transducer molecule , -arrestins 1/2 , which recruits and activates components of the mitogen - activated protein kinase cascades ( 31 , 32 ) . to examine the possibility that 5-ht2 receptors activate erk through the -arrestin pathway , we transfected pfc cultures with sirna against -arrestin1 or -2 to knock down their expression .
6a , the protein level of -arrestin1 or -2 was selectively reduced by -arrestin1 sirna or -arrestin2 sirna , respectively , but not by a scrambled sirna .
the -me-5ht - induced activation of erk1/2 was markedly blocked by knockdown of -arrestin1 or -2 ( fig .
-arrestin is highly expressed in postsynaptic synapses of glutamatergic neurons ( 33 ) , where both 5-ht receptors and nmda receptors are abundant . to test whether 5-ht2 activation of erk is required for the counteracting effect of 5-ht2 on 5-ht1a modulation of nmdar channels
, we examined the effect of 8-oh - dpat on nmdar currents in the presence of -me-5ht in pfc cultures transfected with sirna against -arrestin1 or -2 .
no significant differences in nmdar current densities were found in neurons with different transfections ( gfp : 20.1  1.3 pa / picofarad , n = 11 ; -arrestin1 sirna : 20  1.2 pa / picofarad , n = 7 ; -arrestin2 sirna : 19.5  0.8 pa / picofarad , n = 11 ) .
in addition , the effect of 8-oh - dpat on nmdar currents was the same in all experimental groups .
however , -me-5ht failed to counteract the 8-oh - dpat reduction of nmdar currents in gfp - positive neurons transfected with -arrestin1 or -arrestin2 sirna ( fig .
6d : -arrestin2 sirna : 20.0  1.1% , n = 11 ) , as compared with neurons transfected with gfp alone ( 7.4  1.2% ;
taken together , these data suggest that -arrestins are involved in the counteractive effect of 5-ht2 on 5-ht1a regulation of nmdar currents in pfc neurons .
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-ht2 on 5-ht1a 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 inhibitory 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-ht1a reduction of nmdar - epsc ( 35.8  6.3% , n = 5 ( fig . 7 , c and d ) ) .
together , these data suggest that dynamin - based 5-ht2 receptor endocytosis is involved in 5-ht2 activation of erk and 5-ht2 opposing of 5-ht1a regulation of nmdar currents .  
figure 8.inhibition of src prevents 5-ht2a / c from opposing 5-ht1a regulation of nmdar currents . a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the src kinase inhibitor pp2 or its inactive homolog pp3 ( both 10 m , added 30 min before -me-5ht treatment ) .
c , plot of normalized peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) in cells injected with pp2 ( 20 m ) or pp3 ( 20 m ) .
inset , representative traces of nmdar - epsc ( average of three trials ) taken at time points denoted by # .
d , cumulative data ( mean  s.e . ) showing the percent reduction of nmdar - epsc by 8-oh - dpat in the absence or presence of -me-5ht under different conditions . * ,
0.001 , anova .   inhibition of src prevents 5-ht2a / c from opposing 5-ht1a regulation of nmdar currents .
a , western blots of phospho - erk and total erk in pfc cultures treated with or without -me-5ht ( 20 m , 3 min ) in the absence or presence of the src kinase inhibitor pp2 or its inactive homolog pp3 ( both 10 m , added 30 min before -me-5ht treatment ) .
c , plot of normalized peak nmdar - epsc showing the effect of 8-oh - dpat ( 20 m ) in the presence of -me-5ht ( 20 m ) in cells injected with pp2 ( 20 m ) or pp3 ( 20 m ) .
inset , representative traces of nmdar - epsc ( average of three trials ) taken at time points denoted by # .
d , cumulative data ( mean  s.e . ) showing the percent reduction of nmdar - epsc by 8-oh - dpat in the absence or presence of -me-5ht under different conditions . * ,
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-ht2 activation of erk and the counteractive effect of 5-ht2 on 5-ht1a 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  3.7% , n = 7 ; fig . 8d : pp3 : 8.8  3.3% , n = 4 ) . taken together , these results suggest that src kinase activation is involved in 5-ht2 signaling .
activation of 5-ht2 receptors opposes 5-ht1a reduction of surface nr2b subunits in a -arrestin - dependent manner  if 5-ht1a and 5-ht2a / 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 gfp - tagged 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  1.7 clusters/50 m in controls versus 22.7  0.8 clusters/50 m in dpat - treated cells ; cluster size : 0.33  0.02 m in controls versus 0.18  0.02 m in dpat - treated cells ( fig .
treatment with -me-5ht ( 40 m , 30 min ) significantly prevented the 8-oh - dpat - induced reduction of surface nr2b subunits ( cluster density : 40.0  2.7 clusters/50 m ; cluster size : 0.3  0.03 m ( fig .
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 .
next , we examined the signaling molecules involved in 5-ht2 blockade of 5-ht1a reduction of surface nr2b clusters . to test this ,
we co - transfected pfc cultures with -arrestin2 sirna and gfp - tagged nr2b subunits . as shown in fig .
9 ( d and e ) , transfection of -arrestin2 alone did not affect the distribution of surface nr2b clusters ( cluster density : 38.1  3.1 clusters/50 m ; cluster size : 0.29  0.01 m ( fig .
9 g ) ) or the reducing effect of 8-oh - dpat on nr2b clusters ( cluster density : 22.8  0.8 clusters/50 m ; cluster size : 0.16  0.03 m ( fig .
9 g ) ) . however , knockdown of -arrestin2 prevented -me-5ht from blocking the 8-oh - dpat - induced reduction of surface nr2b clusters on dendrites ( cluster density : 23.4  1.6 clusters/50 m ; cluster size : 0.17  0.02 m ( fig .
these results suggest that activation of 5-ht2 receptors opposes 5-ht1a reduction of the surface nr2b level through a -arrestin - dependent mechanism . 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  3% of control ; nr2b : 60  6% of control ; n = 4 ) .
consistent with electrophysiological results , the fluoxetine - induced reduction of surface nmdars was more prominent in animals co - injected with the 5-ht2a / c antagonist ketanserin ( nr1 : 29  3% of control ; nr2b : 30  1% of control ; n = 4 ) and was largely blocked in animals co - injected with the 5-ht1a antagonist way-100635 ( nr1 : 82  5% of control ; nr2b : 84  1% of control ; n = 4 ) .
the surface gabaar 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-ht1a and 5-ht2a / c receptors in a counteractive manner .  
figure 9.activation of 5-ht2a / c receptors opposes the 5-ht1a -induced decrease of surface nr2b clusters on neuronal dendrites .
a  f , immunocytochemical images of surface gfp - nr2b clusters in pfc cultures transfected with or without-arrestin2 sirna .
cultures were untreated ( control ) or treated with 8-oh - dpat ( 40 m , 5 min ) in the absence or presence of -me-5ht ( 20 m , added 10 min before 8-oh - dpat treatment ) .
enlarged versions of the boxed regions of dendrites are shown beneath each of the images .
g , quantitative analysis of surface gfp - nr2b clusters ( cluster density , size , and intensity ) along dendrites under different treatments . * , p < 0.01 , anova .   activation of 5-ht2a / c receptors opposes the 5-ht1a -induced decrease of surface nr2b clusters on neuronal dendrites .
a  f , immunocytochemical images of surface gfp - nr2b clusters in pfc cultures transfected with or without-arrestin2 sirna .
cultures were untreated ( control ) or treated with 8-oh - dpat ( 40 m , 5 min ) in the absence or presence of -me-5ht ( 20 m , added 10 min before 8-oh - dpat treatment ) .
enlarged versions of the boxed regions of dendrites are shown beneath each of the images .
g , quantitative analysis of surface gfp - nr2b clusters ( cluster density , size , and intensity ) along dendrites under different treatments . * , p < 0.01 , anova . 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-ht1a 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 .
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-ht2 and 5-ht4 receptors are linked to regulate gabaa receptors ( 9 , 39 ) , whereas 5-ht1a 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-ht1a regulation of nmdars can be modified by 5-ht2a / 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-ht1 and 5-ht2 receptors have different affinities ( nanomolar versus low micromolar ) to 5-ht , suggesting that 5-ht1 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-ht1a and 5-ht2a 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-ht2a / c receptors unmasks the ability of 5-ht1a receptors to reduce nmdar currents , suggesting that 5-ht1a and 5-ht2a / c receptors converge on nmdar channels in a counteractive manner .
similar to serotonin , it has been shown that , in response to dopamine , d1 and d2 receptors that are linked to distinct signaling cascades regulate cortical gabaergic inhibition in an opposing manner ( 44 ) .  
figure 10.the surface nmdar level is reduced in pfc from fluoxetine - injected animals . a , western blot analysis showing the surface and total nr1 , nr2b , and gabaar 2 subunits in lysates of pfc slices taken from animals receiving a single intraperitoneal injection of saline , fluoxetine , ketanserin , ketanserin plus fluoxetine , way-100635 , or way-100635 plus fluoxetine ( all drugs are 20 mg / kg ) .
b , quantification of surface nmdar subunits in pfc slices from animals treated with different drugs . * , p < 0.01 ; * * , p < 0.001 ; anova .   the surface nmdar level is reduced in pfc from fluoxetine - injected animals . a , western blot analysis showing the surface and total nr1 , nr2b , and gabaar 2 subunits in lysates of pfc slices taken from animals receiving a single intraperitoneal injection of saline , fluoxetine , ketanserin , ketanserin plus fluoxetine , way-100635 , or way-100635 plus fluoxetine ( all drugs are 20 mg / kg ) .
b , quantification of surface nmdar subunits in pfc slices from animals treated with different drugs . * , p < 0.01 ; * * , p < 0.001 ; anova . our previous study has shown that activation of 5-ht1a 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-ht2a / c receptor opposes the ability of 5-ht1a to induce microtubule depolymerization , suggesting that microtubule dynamics and the microtubule - based nmdar transport are regulated by 5-ht1a and 5-ht2a / 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 ) .
our previous study suggests that 5-ht1a activation suppresses erk activity , which leads to the decreased map2 phosphorylation , map2-microtubule interaction , and microtubule stability ( 21 ) .
it is possible that 5-ht1a and 5-ht2a / c oppositely regulates erk activity , thereby controlling microtubule stability in a counteractive manner .
consistently , our biochemical and immunocytochemical data show that 5-ht2a / c activation potently enhances erk activity in the dendrites of pfc cultures .
phospholipase c and inositol 1,4,5-trisphosphate , two downstream molecules of classic 5-ht2 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-ht2a receptors are found to bind purified -arrestin ( 46 ) . however , whether 5-ht2a activates mitogen - activated protein kinase via -arrestin pathway is essentially unknown .
our data show that knockdown of -arrestin1/2 not only blocks the 5-ht2a / c - induced erk activation but also eliminates the counteractive effect of 5-ht2a / c on 5-ht1a reduction of nmdar currents . in addition
, we found that the 5-ht2a / c action is dependent on src activation and clarthin / dynamin - mediated endocytosis of the receptor .
taken together , 5-ht2a , via the -arrestin / src / dynamin cascade , induces erk activation to oppose the effect of 5-ht1a 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-ht1a 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-ht1a reduction of surface nr2b clusters is attenuated by pretreatment with a 5-ht2a / c agonist , confirming that 5-ht1a and 5-ht2a / c oppositely regulate nmdar trafficking .
moreover , 5-ht2a / c opposes the 5-ht1a reduction of surface nr2b clusters in a -arrestin - dependent manner , consistent with the -arrestin dependence of the 5-ht2a / c action on 5-ht1a 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-ht1a and 5-ht2a / 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 .
 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 -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 -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 -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 . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION