Dopamine and Serotonin Interactively Modulate Prefrontal Cortex Neurons In Vitro

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    Mhigscrwidcat(1gliameffhathastaantio(11). Moreover, DA and 5-HT receptors coexist in the PFC (1214),an

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    00dod many of their intracellular signaling pathways overlap (15).Early work by Iyer and Bradberry (16) demonstrated a functionaleraction between 5-HT and DA systems in PFC as 5-HT applica-n (110mol/L) increased extracellular DA in a dose-dependentnner, an effect likely mediated by 5-HT1 receptors (1719). Indition, Westerink et al. (20) demonstrated that coadministration5-HT2Aantagonistwith aD2antagonist increasedDA release inmedial PFC to an extent that was significantly greater than that

    served after application of either antagonist alone (18). Thesedies suggest that it is not only the case that multiple mono-ines might be released simultaneously, but once released theyeract in a complex nontrivial manner.

    for 5 min, a 20-min washout, then bath application of the other mono-amine (either 5-HTorDA) for 5min, anda20-minwashout. For concomi-tant bath application of DA and 5-HT (n 13), baseline recordings weretaken for 10min before drug applications (5 min), followed by at least a20-min washout period. Drug controls were obtained by recording theeffectsofDAor5-HTaloneaftereithera10-min(DA[n10];5-HT[n12])or45-min(DA[n7];5-HT[n6])baselinerecordingperiod, followedbya 35-minwashout. Recording solution always contained 10Mbicucull-ine and 6-cyano-7-nitroquinoxaline-2,3-dione to block -aminobutyricacid (GABA)A and-amino-3-hydroxy-5-methylisoxazolepropionate cur-rents, respectively.


    Detailed descriptions of data analysis methods are provided inSupplement 1. We first quantified the effects of bath application ofeither 10 M 5-HT or 20 M DA on firing in response to a fixedcurrent pulse that would evoke approximately 46 spikes at base-line. As shown previously, DA (mean increase 30 6.3%, p .05)or 5-HT (mean increase 104.8 9.5%, p .001) application aloneincreased excitability (Figures 1A and 1B) (Figure S2 in Supplement

    m the Brain Research Center, Department of Psychiatry, University ofBritish Columbia, Vancouver, British Columbia, Canada.dress correspondence to Jeremy Seamans, Ph.D., University of BritishColumbia, Department of Psychiatry, Brain Research Center, KoernerPavilion, UBC Hospital, 2211WesbrookMall, room F-241, Vancouver, BCV6T 2R5, Canada; E-mail: Apr 19, 2010; revised Aug 4, 2010; Aug 9, 2010.

    BIOL PSYCHIATRY 2011;69:1204121106-3223/$36.00i:10.1016/j.biopsych.2010.08.007 2011 Society of Biological Psychiatryopamine and Serotonin Inrefrontal Cortex Neurons Inna C. Di Pietro and Jeremy K. Seamans

    ckground: Dopamine (DA) and serotonin (5-HT) are released in coth types of receptors, yet little is known about how they interact.

    thods: To characterize the nature of these interactions, the currenand/or 5-HT on pyramidal cells in layer V of the medial prefrontal c

    sults: Either DA or 5-HT applied in isolation increased the evokapplicationofDAand5-HTproducedeither a larger increase in excits never observed when either was given alone. Dopamine or 5-HTsequent application of the other monoamine.

    nclusions: These data reveal the unappreciated interactive natureDA and 5-HT can be different from their effects recorded in isolatioT might synergistically modulate cortical circuits during various ta

    y Words: Dopamine, electrophysiology, modulation, plasticity,frontal cortex, serotonin

    onoamines regulate prefrontal cortex (PFC) function, andtheir dysregulation plays an important role in a number ofpsychiatric and neurological disorders. Accordingly,

    hly selective serotonin (5-HT) reuptake inhibitors arewidely pre-ibed for depression, and catecholamine reuptake inhibitors areely prescribed for ADHD, whereas atypical antipsychotic medi-ions for schizophrenia target multiple monoamine receptors3). Despite the success of such drugs, basic research is still strug-ng to understand the rudimentary operating principles ofmono-inergic systems. In most cases, this involves investigating theect of a given monoamine in isolation. Although this approachs lead to a wealth of knowledge, it is important to bear in mindt a given monoamine is seldom released in isolation. For in-nce, a variety of events evoke both the release of dopamine (DA)d 5-HT, including stress (4,5), food consumption (6,7), condi-ned taste aversion (8), ethanol consumption (9,10), and exerciseractively Modulateitro

    nder similar circumstances, andmany psychiatric drugs bind to

    y used in vitro patch-clamp recordings tomeasure the effects ofx.

    xcitability of prefrontal cortex neurons, as shown previously.y thanwheneitherwasgiven aloneor a significant decrease thatprimed neurons to respond in an exaggerated manner to the

    euromodulation in cortex by showing that the combined effectsthe basis of these findings, we present a theory of how DA and

    Here we used patch-clamp recordings to investigate the effectsbath application of DA or 5-HT applied singly, concurrently, oruentially on the evokedfiring of deep layer PFCneurons in braines. We replicated previous studies by showing that DA or 5-HTld alone increase the excitability of these neurons (2126); how-r, when coapplied they either produced a larger increase initability than either alone or a decrease in firing that was neverserved when either was applied alone. Furthermore, the priorlication of either 5-HT or DA could prime PFC neurons to re-nd in an exaggeratedmanner to the other monoamine appliede 20 min later. Thus, 5-HT and DA can interact in unexpected

    ys to modulate the basic excitability of PFC neurons.

    thods andMaterials

    Detailed descriptions of brain slice preparation, whole-cellch-clamp recordings, and drugs are provided in Supplement 1.

    erimental ConditionsFor priming experiments, baseline recordings were taken for 10min,

  • N.C. Di Pietro and J.K. Seamans BIOL PSYCHIATRY 2011;69:12041211

  • 1) to an extent consistent with past studies. To assess the effectsacrfreDAincsloproincatres




    sum. Because the distribution of the effects was bimodal (Figure 2Bins


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    1206 BIOL PSYCHIATRY 2011;69:12041211 N.C. Di Pietro and J.K. Seamans

    wwoss a large activity range, we also analyzed the slope of thequency-current (F/I) curve or gain across multiple steps. Either(mean increase in slope .49 .10, p .001) or 5-HT (meanrease in slope 1.04 .17, p .001) application increased thepe of the F/I curve (Figure 1C), although the effects were mostminent on the initial slope (i.e., at lower current steps) (Figure S1Supplement 1). These effects of DA and 5-HT alone closely repli-e the findings of Thurley et al. (25) and Zhang andArsenault (26),pectively.Next we examined the effects of sequential application of 5-HTM) and DA (20M). Bath application of DAwhen preceded 20n earlier by 5-HT resulted in an increase in firing activity (meanrease383.9%) thatwasgreater inmagnitude thanwhenDAs applied at the same time point in the absence of 5-HT pretreat-nt (mean increase 22 3.0%,p .01) (Figure 1D). The slopeofF/I curve was not only increased by DAwhen it was followed byT pretreatment relative to its own baseline (mean increase 3 .15, p .01), but the relative increase in the slopes of primedls was significantly greater than the relative increase in thepes of nonprimed cells (Figure 1E) (p .05). This suggests thator 5-HT exposure made PFC neurons more sensitive to subse-ent DA application.When cells were first primed with DA, later application of 5-HTo led toanoverall increase infiring rates (mean increase77.4%) that was greater (p .03) than the increase observed in thesence of DA pretreatment, for a given current pulse (mean in-ase59.74.9%) (Figure1F). The slopeof theF/I curvewas alsoreasedby 5-HT (mean increase 0.53 .14,p .02)when itwaslowed by DA pretreatment, but in this case the relative increasethe slope of primed cells was not significantly greater than theative increase in the slopeof nonprimed cells (Figure 1G,p .05).wever, the initial slope of primed cells was significantly greatern baseline (Figure 1G, p .05), suggesting that although theming effect of 5-HT on DA occurred across the entire input/tput range, the priming effect of DA on 5-HT was limited to onlye or two current steps in the middle range of intensities.Finally we investigated the effects of simultaneous coapplica-n of DA (20M) and 5-HT (10M). Coapplication resulted in onetwo opposing effects; approximately one-half of the recordedls responded with a massive increase in firing rate (mean in-ase 211 43.7%, p .01; peaking at approximately 290%),ereas the remaining one-half of the cells were only inhibitedeandecrease69 22.7%,p .01) (Figure 2A and2B) (Figurein Supplement 1). For cells that were increased, the effect wasnificantly larger than that produced by either 5-HT (p .05) or(p .05) alone (Figure 2B) and was even larger than their linear

    ure 1. The effects of either dopamine (DA) or serotonin (5-HT) alone on evrons. (A) Left: baseline responses of twodifferent prefrontal cortex neurons(20 M) or (bottom) 5-HT (10 M), more spikes were evoked by the same cked spikes were increased significantly (asterisk and number sign) by eit). (C) Frequency-current (F/I) curves for eachcell and the slopesof these line

    pes of the lines fit to the F/I curves for separate groupsof cells duringbaselineincreased the initial slope (asterisks) but not second slope of the F/I curves, iotoninproducedan initial large increase in the responseof agroupofneuronpoint when DA was applied (gray line and diamonds). The DA-mediated

    nificantly greater (as denotedby the asterisk) than that observed in cells receares). (E) Slopes of the F/I curves for the same cells shown inD. The asteriskgroup that had been pretreated with 5-HT than the group that had not. (F)fixed current pulse that slightly decayedduring the following 20minuntil trease in excitability in these cells was greater (as denoted by the asterisk) thaexperiment (black line and squares). (G) Slopesof the F/I curves for the sameor two current steps in the middle range of intensities; hence the slope, a grouping criterion of 25% in firing rate was used toegorize cells as either excited or inhibited by DA and 5-HT coap-ation.Examination of the effects across current intensities for excitedls indicated that only the initial slope was significantly increasedcoapplication (2.86 .9 vs. .61 .23, p .03) and not theond slope (1.52 .36 vs. 1.6 .15) (Figures 2C and 2D). Theibitory effects of concomitant 5-HTandDAapplicationwere alsost evident on the initial slope (1.17 .1 vs. .29 .1) and not theond slope of the F/I curves (1.33 .14 vs. 1.33 .28) (Figures 2C2D). Therefore, although DA and 5-HT were always excitatory

    en given alone, together they produced a larger excitation or apletely novel mode of suppression, approximately one-half oftime.


    The present study investigated the integrative effects of DA andT on the electrophysiological properties of PFC neurons. Onee of comodulation took the form of priming, in that prior appli-ion of DAor 5-HT potentiated the subsequent effect of the othernoamine. A second type of comodulation was bidirectional inure, because in some cells coapplication caused an increase initability that was greater than the increase produced by eitherurotransmitter alone, whereas in the other cells a profound de-ssion in spikingoccurred. This formof comodulationwasuniquehat adecrease infiringwasnever observedwhen5-HTorDAwasen alone.

    tential Cellular MechanismsThe present data are consistent with past studies where DA orT was applied in isolation to PFC neurons in that DA, at thesecentrations, typically increased evoked excitability by approxi-tely 30%, whereas 5-HT increased it by approximately 100%% (21,23,2528). However, thepresent study is the first to exam-the combined effects of these twomonoamines on the evokedng properties of PFC neurons in brain slices. Under these condi-ns, the most striking effect was the bidirectionality of cell re-nses.It is currently unclear as to why some cells exhibited an increasexcitability and others exhibited a decrease upon coapplicationDA and 5-HT. One reason might be related to the age of themals used, with the assumption that monoamines affect PFCuronsdifferently at different stages of development (29). Accord-ly, Zhang (30) showed a developmental decline of 5-HTin-ced (10M)excitatory effects in layer Vpyramidal neurons of the

    excitability and the priming effects of 5-HT and DA on prefrontal cortexxed amplitude intracellular current pulse. Right: after application of (top)t pulse in the two neurons. (B) In separate groups of cells, the number ofA (black line and diamonds) or 5-HT (gray line and boxes) (mean calculated. Thehistogramshows theaverage (andSEM)first and secondds andduring the 5-minperiodof 5-HTorDAapplication. Both 5-HT andting that the effects weremainly in response to smaller current steps. (D)fixedcurrentpulse that slowlydecayedduring the following20minuntilase in excitability in the cells that received prior 5-HT application wasonlyDAat a similar timepoint 35min into the experiment (black line andates that the relative change in the initial F/I slope by DA was greater inmine produced an initial increase in the response of a group of neuronsintwhen5-HTwas applied (gray line anddiamonds). The 5-HTmediatedt observed in cells receiving only 5-HT at a similar time point 35min intoshown inF. Note that theprimingeffect ofDAon5-HTwas limited toonlyseems less pronounced than for nonprimed cells.catplic


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    N.C. Di Pietro and J.K. Seamans BIOL PSYCHIATRY 2011;69:12041211 1207ure 2. Coapplication of dopamine (DA) or serotonin (5-HT) produce qualitatively and quantitatively different effects from those produced by either alone. (A)resentative examples of the twoclasses of effects producedbyDAand5-HT coapplication. Left: baseline responses of twodifferent prefrontal cortex neurons toedamplitude intracellularcurrentpulse.Right:aftercoapplicationofDAplus5-HT,manyfewerspikeswereevokedbythesamecurrentpulse inoneneuron(top),ereas in theother,manymore spikeswereevoked. (B) In response toafixedcurrent step, coapplication increased thenumberof spikes inapproximatelyone-halfhe cells (gray line and triangles) and significantly decreased the number of spikes in the other one-half (black line and open circles). Dopamine (gray line andmonds) or 5-HT (gray dashed line and solid squares) alone only increased e...


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