gain modulation by serotonin in pyramidal neurones of the rat prefrontal cortex

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J Physiol 566.2 (2005) pp 379–394 379 Gain modulation by serotonin in pyramidal neurones of the rat prefrontal cortex Zhong-wei Zhang and Dany Arsenault Centre de recherche Universit´ e Laval Robert-Giffard, D´ epartement de psychiatrie, Facult´ e de m´ edecine, Universit´ e Laval, Qu´ ebec, Canada Serotonin (5-HT) is widely implicated in brain functions and diseases. The vertebrate brain is extensively innervated by 5-HT fibres originating from the brain stem, and 5-HT axon terminals interact with other neurones in complex ways. The cellular mechanisms underlying 5-HT function in the brain are not well understood. The present study examined the effect of 5-HT on the responsiveness of neurones in the neocortex. Using patch-clamp recording in acute slices, we showed that 5-HT substantially increased the slope (gain) of the firing rate-current curve in layer 5 pyramidal neurones of the rat prefrontal cortex. The effect of 5-HT on gain is confined to the range of firing rate (0–10 Hz) that is known to be behaviourally relevant. 5-HT also changed current threshold for spike train generation, but this effect was inconsistent, and was independent of the effect on gain. The gain modulation by 5-HT was mediated by 5-HT 2 receptors, and involved postsynaptic mechanisms. 5-HT 2 -mediated gain increase could not be attributed to changes in the membrane potential, the input resistance or the properties of action potentials, but was associated with a reduction of the afterhyperpolarization and an induction of the slow afterdepolarization. Blocking Ca 2 + entry with Cd 2 + increased the gain by itself and blocked 5-HT 2 -mediated gain increase. Buffering [Ca 2 + ] i with 25 mM EGTA also substantially reduced 5-HT 2 -mediated gain increase. Noradrenaline, which blocked the after- hyperpolarization, also induced a moderate increase in gain. Together, our results suggest that 5-HT may regulate the dynamics of cortical circuits through multiplicative scaling. (Received 2 March 2005; accepted after revision 4 May 2005; first published online 5 May 2005) Corresponding author Z. W. Zhang: Centre de recherche U-Laval Robert-Giffard, 2601, de la canardi` ere, F-6500, Qu´ ebec, QC, Canada G1J 2G3. Email: [email protected] Serotonin (5-HT) is widely involved in brain functions and diseases. The central nervous system (CNS) in vertebrates is extensively innervated by 5-HT fibres arising from the raphe nuclei in the brain stem. 5-HT axon terminals interact with other neurones in complex ways, by binding of 5-HT to at least 14 distinct receptors (Aghajanian & Sanders-Bush, 2002; Cooper et al. 2003). 5-HT plays an important role in the regulation of behaviour. In cats, activity of 5-HT neurones in the brain stem is highest during periods of waking arousal, decreases progressively as the animal falls asleep, and is absent during rapid-eye-movement sleep (Jacobs & Fornal, 1999). Selective depletion of 5-HT in the prefrontal cortex (PFC) of monkeys induces cognitive inflexibility (Clarke et al. 2004); and 5-HT, via 5-HT 2A receptors, has been shown to contribute to working memory in the monkey PFC (Williams et al. 2002). In humans, dysfunction of the brain 5-HT system plays a critical role in depression; and many antidepressants are selective 5-HT uptake blockers, which enhance 5-HT transmission in the brain (Blier & de Montigny, 1999; Delgado, 2000; Bell et al. 2001). Together, such evidence suggests that at the system level, 5-HT facilitates motor and other executive functions of the CNS. The cellular mechanisms underlying brain 5-HT function are not well understood. Early in vivo studies showed that the predominant effect by 5-HT in the cerebral cortex is an inhibition of spontaneous firing (Krnjevic & Phillis, 1963; Reader et al. 1979). Later studies using intracellular recordings in brain slices revealed that 5-HT induces, often in the same cell, both inhibitory and excitatory responses (Segal, 1980; Andrade & Nicoll, 1987; Araneda & Andrade, 1991; Tanaka & North, 1993; Spain, 1994). The inhibitory effect, mediated by 5-HT 1A receptors, features a hyperpolarization associated with a reduction in cell input resistance. The excitatory effect of 5-HT involves 5-HT 2 receptors, and in most cases, consists of small, subthreshold depolarization associated with a slight increase in the input resistance. It is not clear how this apparently moderate excitation translates into significant enhancement in network activities. Excitatory effects of 5-HT have been extensively examined in pyramidal neurones in the neocortex C The Physiological Society 2005 DOI: 10.1113/jphysiol.2005.086066

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Page 1: Gain modulation by serotonin in pyramidal neurones of the rat prefrontal cortex

J Physiol 566.2 (2005) pp 379–394 379

Gain modulation by serotonin in pyramidal neuronesof the rat prefrontal cortex

Zhong-wei Zhang and Dany Arsenault

Centre de recherche Universite Laval Robert-Giffard, Departement de psychiatrie, Faculte de medecine, Universite Laval, Quebec, Canada

Serotonin (5-HT) is widely implicated in brain functions and diseases. The vertebrate brainis extensively innervated by 5-HT fibres originating from the brain stem, and 5-HT axonterminals interact with other neurones in complex ways. The cellular mechanisms underlying5-HT function in the brain are not well understood. The present study examined the effect of5-HT on the responsiveness of neurones in the neocortex. Using patch-clamp recording in acuteslices, we showed that 5-HT substantially increased the slope (gain) of the firing rate-currentcurve in layer 5 pyramidal neurones of the rat prefrontal cortex. The effect of 5-HT on gainis confined to the range of firing rate (0–10 Hz) that is known to be behaviourally relevant.5-HT also changed current threshold for spike train generation, but this effect was inconsistent,and was independent of the effect on gain. The gain modulation by 5-HT was mediated by5-HT2 receptors, and involved postsynaptic mechanisms. 5-HT2-mediated gain increase couldnot be attributed to changes in the membrane potential, the input resistance or the propertiesof action potentials, but was associated with a reduction of the afterhyperpolarization and aninduction of the slow afterdepolarization. Blocking Ca2+ entry with Cd2+ increased the gainby itself and blocked 5-HT2-mediated gain increase. Buffering [Ca2+]i with 25 mM EGTA alsosubstantially reduced 5-HT2-mediated gain increase. Noradrenaline, which blocked the after-hyperpolarization, also induced a moderate increase in gain. Together, our results suggest that5-HT may regulate the dynamics of cortical circuits through multiplicative scaling.

(Received 2 March 2005; accepted after revision 4 May 2005; first published online 5 May 2005)Corresponding author Z. W. Zhang: Centre de recherche U-Laval Robert-Giffard, 2601, de la canardiere, F-6500,Quebec, QC, Canada G1J 2G3. Email: [email protected]

Serotonin (5-HT) is widely involved in brain functions anddiseases. The central nervous system (CNS) in vertebratesis extensively innervated by 5-HT fibres arising from theraphe nuclei in the brain stem. 5-HT axon terminalsinteract with other neurones in complex ways, by bindingof 5-HT to at least 14 distinct receptors (Aghajanian &Sanders-Bush, 2002; Cooper et al. 2003).

5-HT plays an important role in the regulation ofbehaviour. In cats, activity of 5-HT neurones in the brainstem is highest during periods of waking arousal, decreasesprogressively as the animal falls asleep, and is absentduring rapid-eye-movement sleep (Jacobs & Fornal, 1999).Selective depletion of 5-HT in the prefrontal cortex (PFC)of monkeys induces cognitive inflexibility (Clarke et al.2004); and 5-HT, via 5-HT2A receptors, has been shownto contribute to working memory in the monkey PFC(Williams et al. 2002). In humans, dysfunction of thebrain 5-HT system plays a critical role in depression; andmany antidepressants are selective 5-HT uptake blockers,which enhance 5-HT transmission in the brain (Blier & deMontigny, 1999; Delgado, 2000; Bell et al. 2001). Together,

such evidence suggests that at the system level, 5-HTfacilitates motor and other executive functions of the CNS.

The cellular mechanisms underlying brain 5-HTfunction are not well understood. Early in vivo studiesshowed that the predominant effect by 5-HT in thecerebral cortex is an inhibition of spontaneous firing(Krnjevic & Phillis, 1963; Reader et al. 1979). Later studiesusing intracellular recordings in brain slices revealed that5-HT induces, often in the same cell, both inhibitoryand excitatory responses (Segal, 1980; Andrade & Nicoll,1987; Araneda & Andrade, 1991; Tanaka & North, 1993;Spain, 1994). The inhibitory effect, mediated by 5-HT1A

receptors, features a hyperpolarization associated with areduction in cell input resistance. The excitatory effect of5-HT involves 5-HT2 receptors, and in most cases, consistsof small, subthreshold depolarization associated with aslight increase in the input resistance. It is not clear how thisapparently moderate excitation translates into significantenhancement in network activities.

Excitatory effects of 5-HT have been extensivelyexamined in pyramidal neurones in the neocortex

C© The Physiological Society 2005 DOI: 10.1113/jphysiol.2005.086066

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380 Z. w. Zhang and D. Arsenault J Physiol 566.2

(Araneda & Andrade, 1991; Spain, 1994). In additionalto its effect on membrane potential, 5-HT was found toincrease the steady-state firing rate, presumably throughan inhibition of the afterhyperpolarization, and anenhancement of the afterdepolarization. Moreover, 5-HTalso increased the slope (gain) of the firing rate-currentcurve in some cortical pyramidal neurones (Araneda &Andrade, 1991; Spain, 1994). This effect of 5-HT on gainmodulation, however, has not been examined in any detail.Quantitative data on 5-HT-mediated gain modulation arestill not available, and little is known about the underlyingmechanisms. In the present study, we examined the effectsof 5-HT in layer 5 pyramidal neurones of the rat PFC.We found that 5-HT, via 5-HT2 receptors, consistentlyincreased the gain of neurones. This effect was limited tolow spike frequency, and was independent of the effects onmembrane potentials and input resistance, but required arise in [Ca2+]i.

Methods

Slice preparation

Brain slices were prepared from Sprague-Dawley rats ofeither sex aged P21 to P35 (with the day of birth asP0) as previously described (Zhang, 2004). Briefly, ratswere deeply anaesthetized with ketamine and xylazine,and decapitated. The brain was removed quickly (<60 s),and placed in ice-cold solution containing (mm): 210sucrose, 3.0 KCl, 1.0 CaCl2, 3.0 MgSO4, 1.0 NaH2PO4,26 NaHCO3, 10 glucose, saturated with 95% O2 and 5%CO2. Coronal slices including the prelimbic area werecut at 300 µm on a vibrating tissue slicer (VT 1000s,Leica, Germany), and kept in artificial cerebral spinal fluid(ACSF) containing (mm): 124 NaCl, 3.0 KCl, 1.5 CaCl2, 1.3MgCl2, 1.0 NaH2PO4, 26 NaHCO3, 20 glucose, saturatedwith 95% O2 and 5% CO2 at room temperature. Slices wereallowed to recover for at least 1 h before any recording. Allprocedures were performed according to the guidelines ofthe Canadian Council on Animal Care, and were approvedby the Animal Care Committee at Laval University.

For recording, a slice was transferred to a submerge-typechamber where it was continuously exposed to ACSF,saturated with 95% O2 and 5% CO2 and flowing at rate of2.0 ± 0.2 ml min−1. The slice was viewed first with a 4×objective and the prelimbic area of the PFC was localizedas the area between the forceps minor corpus callosumand the midline (Paxinos & Watson, 1998). Layers 1, 2/3,5 and 6 of the prelimbic area were then viewed undernear infrared illumination with a 40× water-immersionobjective (Fluor, 40×/0.80 W, Nikon, Mississauga, ON)and a CCD camera (IR-1000, MTI, Michigan City, IN,USA). Layer 5 pyramidal neurones were identified by theirlarge size and apical dendrite.

Patch-clamp recording

Experiments were conducted at 30–32◦C unless indicatedotherwise. Electrodes were pulled from thick wallborosilicate glass (1.5/0.84 mm, WPI, Sarasota, FL, USA)on a horizontal puller (P-97, Sutter Instruments, Novato,CA, USA). The standard pipette solution contained (mm):100 K-gluconate, 15 KCl, 4 ATP-Mg, 0.3 GTP-Na2,10 creatine phosphate, 0.5 EGTA, 20 Hepes (pH 7.4with KOH, 280 ± 3 mOsmol with sucrose). In someexperiments, K-gluconate was replaced with equimolarK-methanesulphonate. Electrodes had resistances between4 and 7 M�. Liquid junction potential, estimated to be12 mV, was not corrected. The seal resistance was greaterthan 5 G�. Whole-cell recordings were made at the somawith a Multiclamp 700A amplifier (Axon Instruments,Union City, CA, USA). For current-clamp recordings, theseries resistance (Rs), usually between 15 and 45 M�, wascompensated using the bridge balance. To establish thefiring rate curve, current steps of 3 s long were applied onceevery 15 s, with increments of 10 or 20 pA. Experimentswere conducted using the Axograph 4.9 program (AxonInstruments). Data were filtered at 1 or 2 kHz, and digitizedat 4 or 8 kHz.

Drugs and drug delivery

All agents were applied by changing the bath perfusatefrom standard ACSF to modified ACSF to which variousdrugs were simply added. Unless indicated otherwise,all solutions were continuously bubbled with 95% O2

and 5% CO2. In the experiments with Cd2+, recordingswere carried out at 24 ± 1◦C with a solution containing(mm): 145 NaCl, 3.0 KCl, 1.5 CaCl2, 1.3 MgCl2, 20glucose and 10 Hepes (pH. 7.2), gassed with O2. Tominimize degradation, 5-HT or noradrenaline was addedto ACSF containing 20–40 µm sodium metabisulphate,and both overhead and microscope lights were turned offduring the recording. Sodium metabisulphate by itself hadno effect on the excitability of neurones. All chemicalswere purchased from Sigma-Aldrich Canada (Oakville,ON). K-methanesulphonate was obtained by titratingmethanesulphonic acid with KOH.

Data analysis

The AxoGraph 4.9 and Origin 7 (OriginLab, Nothampton,MA, USA) were used for analysis.

Action potentials were detected using the eventdetection package of the AxoGraph. Events with peakamplitude of 60 mV or higher, and a rise time of about0.5 ms were detected automatically, and the results wereanalysed with Origin 7. The steady-state spike rate wasestimated by counting the number of spikes during the last2.5 s of the 3 s step, and the result was plotted versus the

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J Physiol 566.2 Serotonin increases the gain of neurones 381

intensity of the injected current (F–I curve). The slope ofthe F–I curve (referred to as gain) was estimated by linearfit. Input resistance was estimated by applying small hyper-polarizing current pulses. The action potential thresholdwas measured using a cursor, by inspecting a 5 ms segmentaround the rising phase of action potential.

Throughout, means are given ± s.e.m. Means werecompared using paired or unpaired two-tailed Student’st test.

Results

Stable whole-cell recordings were obtained from 160layer 5 pyramidal neurones from 51 rats aged between P21and P35 (with P0 as the day of birth). Only neurones withresting potential more negative than −55 mV, and inputresistance greater than 30 M� were examined. Eighty-fourper cent of the neurones were regular spiking cells; theremaining (16%) were bursting cells which showed threeor more grouped spikes at the beginning of a supra-threshold current step. Only regular spiking cells wereincluded in this study.

Gain modulation by 5-HT in layer 5 pyramidalneurones

The input–output relationship was examined undercurrent clamp by applying 3 s steps once every 15 swith increments of 10 or 20 pA. Suprathreshold currentsteps evoked trains of action potentials with littleaccommodation after the first few spikes. An exampleis illustrated in Fig. 1A. The spike frequency increasedwith current intensity. Bath application of 5-HT (20 µm)depolarized the cell by 3 mV, and induced significantfluctuations in the resting potential (Fig. 1B), presumablydue to presynaptic effects of 5-HT (Aghajanian & Marek,1997; Zhou & Hablitz, 1999; Lambe et al. 2000). 5-HT alsoreduced the current threshold for spike train generation(referred to as sensitivity hereafter): the minimal currentintensity required for generating two or more spikesdecreased from 100 pA for the control, to 70 pA in thepresence of 5-HT (Fig. 1A and B). The effect of 5-HT wasreversible after 15–20 min wash (Fig. 1C). The steady-statespike rate was estimated by counting the number of spikesduring the last 2.5 s of the 3 s step, and the result was plottedversus the intensity of the injected current (F–I curve).Since previous studies in free-moving rats showed thatneurones in the PFC usually fire at less than 15 Hz (Gillet al. 2000; Baeg et al. 2003), we focused at the rangebetween 0 and 20 Hz. As illustrated in Fig. 2A, the F–Icurve before 5-HT application (control, �) was linearbetween 0 and 20 Hz, with a slope of 120 Hz nA−1.Application of 5-HT increased significantly the slope ofthe F–I curve (Fig. 2A, �). The effect of 5-HT on the slope

was mostly confined to the range of spike rate between0 and 10 Hz: the slope was 222 Hz nA−1 between 0 and12 Hz – an increase of 85% over the control. This effectwas reversible after 15–20 min wash (Fig. 2A, �).

Similar effects of 5-HT on the initial slope wereobserved in 22 other cells examined. Collectively, theinitial slope (0–10 Hz) before 5-HT application (control)was 118 ± 6 Hz nA−1; during 5-HT application, it was294 ± 19 Hz nA−1 (n = 23; P < 0.0001 versus control,paired t test), which corresponds to an increase of156 ± 15% (n = 23) over the control. The recovery after15 min wash was 105 ± 4% of the control (n = 23; P > 0.1versus control, paired t test). The firing pattern of neuroneswas not affected by 5-HT.

In contrast to its effect on the initial slope, 5-HThad inconsistent effects on the sensitivity of neurones.Based on the minimum current required for spike traingeneration, the sensitivity was increased in 10 out of the23 cells, decreased in seven cells, and unchanged in theremaining six cells. An example where 5-HT inducedboth a reduction in the sensitivity and an increase in theinitial slope is illustrated in Fig. 1D–F and in Fig. 2B. Thedecrease in sensitivity was associated with a reduction inthe input resistance (RN), rather than a hyperpolarizationof membrane potential. Out of the seven cells wherea decrease in sensitivity was observed, only one cellshowed a significant hyperpolarization (−2.5 mV) during5-HT application, while the others were either slightlydepolarized (<3 mV) or unchanged. On the other hand, allseven cells showed substantial reductions in RN (70 ± 4%of the control, n = 7). In comparison, 5-HT had littleeffect on the input resistance in cells that showed anincrease in sensitivity (105 ± 3% of the control, n = 10).The difference in 5-HT-induced RN changes between thetwo groups (sensitivity increased versus decreased) wasstatistically significant (P < 0.01, unpaired t test).

Since 5-HT induced a small but significantdepolarization (<5 mV) in the majority of cellstested, we tested the effect of membrane depolarizationon the slope of the F–I curve. Depolarization of 5–7 mVby injections of DC currents shifted the curve to the left,but had no effect on the slope of the curve (101 ± 2%of the control, n = 6 cells; P > 0.5, paired t test). Toexamine further the effect of depolarization, we heldthe membrane potential constant at −60 mV before andduring 5-HT application. The effect of 5-HT on the slopeof the F–I curve persisted under such condition (Fig. 2C).Similar results were obtained in all four cells tested, withthe initial slope increased by 131 ± 33% over the control(n = 4).

To examine whether internal dialysis may causetime-dependent change in gain, we examined F–I curvesat 10–15 min and 40–50 min after break-in. There was nosignificant change in the slope of the curve (97 ± 3%, n = 5cells; P > 0.4, paired t test).

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Figure 1. 5 HT increases the response of layer 5 pyramidal neurones to depolarizing current stepsA, trains of action potentials in response to 3 s current steps of +100, +120, and +140 pA (top to bottom). Thesteady-state frequencies were 1.2, 3.6 and 6.4 Hz, respectively. Resting potentials at −61 mV. B, bath applicationof 5-HT (20 µM) depolarized the cell by 3 mV, and increased the responses to current injections. The steady-statefrequencies were 2.0, 7.2 and 10.8 Hz for steps of +70, +90 and +110 pA, respectively (top to bottom). Restingpotentials at −58 mV. C, recovery from the effect of 5-HT after 15 min wash. The steady-state frequencies were2.0, 4.4 and 7.2 Hz for steps of +100, +120 and +140 pA, respectively. Resting potentials at −61 mV. A–C wereobtained from the same cell. D–F were obtained from another cell where 5-HT (20 µM) increased the currentthreshold for spike train (from 120 to 160 pA) with little effect on the resting potential (−59 mV throughout therecording). The effect was reversible after 15 min wash (F).

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J Physiol 566.2 Serotonin increases the gain of neurones 383

Previous studies have shown that intracellular gluconateinhibits K+ conductance (Zhang et al. 1994; Velumianet al. 1997), which would alter the input–outputrelationship of the neurones. To test if the effect of 5-HTon the F–I curve is affected by the presence of intracellulargluconate, we replaced gluconate with methanesulphonatein the recording pipette. 5-HT had a similar effecton the F–I curve (Fig. 2D). The initial slope was94 ± 7 Hz nA−1 before (control) and 256 ± 34 Hz nA−1

during 5-HT application (n = 6), which represents anincrease of 170 ± 28% over the control. These valuesare comparable with the results obtained with gluconate(P > 0.1, unpaired t test). This finding suggests thatthe effect of 5-HT on the F–I curve was not affectedsignificantly by the presence of intracellular gluconate.

Figure 2. 5-HT increases the gain at low spike ratesA, F–I curves of a neurone before (control, �), during 5-HT application ( �), and after 20 min wash (�). The insertshows data points between 0 and 12 Hz before (�) and during 5-HT application ( �). Data were fitted to linearfunction which yielded slopes of 120 and 222 Hz nA−1 for the control and during 5-HT, respectively (R2 > 0.98in both cases). After 20 min wash, the initial slope retuned to the control value at 110 Hz nA−1. B, F–I curves ofanother neurone before (control, �), and during 5-HT application ( �). The initial slopes (from 0 to 10 Hz) were118 and 344 Hz nA−1 for the control and during 5-HT, respectively. 5-HT decreased the sensitivity of the neurone,but increased the gain. C, F–I curves of a neurone before (control, �) and during 5-HT application ( �). The restingmembrane potential was maintained at −60 mV throughout the recording with DC injection. The initial slopes was114 and 248 Hz nA−1 for the control and during 5-HT, respectively. D, F–I curves of a neurone before (control, �)and during 5-HT application ( �). Intracellular gluconate was replaced with equimolar methanesulphonate in thisexperiment. The initial slopes were 98 and 348 Hz nA−1 for the control and during 5-HT, respectively.

Role of 5-HT2 receptors in the gainmodulation by 5-HT

Previous studies have shown that 5-HT2 receptors mediateexcitatory effects of 5-HT2 in layer 5 pyramidal neurones(Araneda & Andrade, 1991; Tanaka & North, 1993). Todetermine the role of 5-HT2 receptors in gain modulation,we first used the selective 5-HT2 receptor antagonistketanserin. Ketanserin (1 µm) by itself had no effect onthe F–I curve (initial slope 103 ± 5% of the control, n = 6cells), but blocked the effects of 5-HT (20 µm) on the initialslope (Fig. 3A). Similar results were observed in all six cellstested. Collectively, the initial slope increased by 14 ± 3%in the presence of both 5-HT and ketanserin (n = 6,Fig. 3D). This corresponds to 9% of the increase induced

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by 5-HT alone (P < 0.001, unpaired t test). Ketanserin alsoblocked 5-HT-induced depolarization in these cells.

The effect of 5-HT on the initial slope was mimickedby the selective 5-HT2 receptor agonist α-methyl-5-HT(α-me-5HT). Bath application of α-me-5HT (20 µm)substantially increased the initial slope of the F–I curve,with little effect on the late portion of the curve(Fig. 3B). Similar results were observed in seven othercells examined. Collectively, α-me-5HT (20 µm) increasedthe initial slope by 155 ± 22% over the control (n = 8,Fig. 3D). Like 5-HT, α-me-5HT also induced moderatedepolarization (<5 mV) and substantial fluctuation inthe resting membrane potential. However, α-me-5HThad little effect on the input resistance (106 ± 5% ofthe control, n = 8; P > 0.1, paired t test). The sensitivityof neurones was increased in four out of eight cells,

Figure 3. 5-HT2 receptors are responsible for the gain modulation by 5-HTA, ketanserin blocks the gain modulation by 5-HT. The initial slopes of F–I curves were 118 Hz nA−1 for ketanserinalone, and 126 Hz nA−1 for 5-HT (20 µM) and ketanserin. In the presence of ketanserin, 5-HT decreased slightlythe sensitivity of the neurone, presumably due to the remaining 5-HT1A effects. B, effects of α-me-5HT (20 µM)on the F–I curve in another neurone. The initial slopes were 99 Hz nA−1 for the control and 220 Hz nA−1 duringα-me-5HT application. There was also a slight increase in the sensitivity during α-me-5HT application. C, effectsof 5-HT (20 µM) on the F–I curves in the presence of WAY100135 (100 nM). The initial slopes were 115 Hz nA−1

for WAY100135 alone, and 316 Hz nA−1 for 5-HT and WAY100135. D, summary of the results obtained with5-HT (20 µM) alone, 5-HT (20 µM) plus ketaserin (1 µM), α-me-5HT (20 µM), and 5-HT plus WAY100135 (100 nM).The initial slopes were normalized to those of controls. The broken line indicates the level of control (100%). Thenumber of cells in each group is given in parentheses.

unchanged in three cells, and decreased in one cell inresponse to α-me-5HT.

Previous studies have shown that the majority oflayer 5 pyramidal neurones in the PFC express both5-HT1A and 5-HT2 receptors (Araneda & Andrade, 1991;Amargos-Bosch et al. 2004). Accordingly, we examinedthe effect of 5-HT in the presence of the selective 5-HT1A

antagonist WAY100135. WAY100135 (100 nm) by itselfhad no effect on the F–I curve (initial slope 97 ± 3% ofthe control, n = 6 cells). In the presence of WAY100135(100 nm), 5-HT (20 µm) substantially increased the initialslope of the F–I curve (Fig. 3C). Collectively, the initialslope was increased by 167 ± 13% over the control(n = 6 cells; Fig. 3D), which is comparable with those ofα-me-5HT and of 5-HT alone (P > 0.1, unpaired t test).This result suggests that 5-HT1A receptors are not involved

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J Physiol 566.2 Serotonin increases the gain of neurones 385

in the gain modulation by 5-HT. In the presence ofWAY100135, the sensitivity of neurones was increased infour out of six cells, and unchanged in the other two cellsin response to 5-HT.

Together, these results suggest that 5-HT2 receptors areresponsible for the gain modulation by 5-HT. Therefore,we used the selective 5-HT2 agonist α-me-5HT in allsubsequent experiments.

Presynaptic effects of 5-HT on gain modulation

Previous studies have shown that 5-HT, through 5-HT2

receptors, induces large increase of spontaneous activitiesat both glutamatergic and GABA-ergic synapses inpyramidal neurones in the PFC (Aghajanian & Marek,1997; Zhou & Hablitz, 1999; Lambe et al. 2000; Lambe &

Figure 4. Presynaptic effects are not required for 5-HT-induced gain modulationA, effects of α-me-5HT (20 µM) on the firing rate curve in the presence of kynurenic acid (KN, 1 mM) and picrotoxin(PTX, 0.1 mM). The initial slopes were 122 Hz nA−1 before and 396 Hz nA−1 during α-me-5HT application.B, effects of α-me-5HT (20 µM) on the F–I curve in the presence of D-APV (50 µM), CNQX (10 µM), and PTX(0.1 mM). The initial slopes were 112 Hz nA−1 before and 272 Hz nA−1 during α-me-5HT application. C, effectsof α-me-5HT on the firing rate curve in the presence of KN (1 mM). The initial slopes were 110 Hz nA−1 beforeand 464 Hz nA−1 during application. D, summary of results obtained with α-me-5HT (20 µM), α-me-5HT (20 µM)in the presence of KN (1 mM) and PTX (0.1 mM), α-me-5HT (20 µM) in the presence of D-APV, CNQX and PTX,and α-me-5HT (20 µM) in the presence of KN (1 mM). The broken line indicates the level of control (100%). Thenumber of cells is given in parentheses.

Aghajanian, 2001). As suggested in recent studies (Chanceet al. 2002; Fellous et al. 2003; Shu et al. 2003), suchincreases in background synaptic activities may modulatethe gain of neurones. To determine the role of presynapticeffects on gain modulation by 5-HT, we examined theeffects of α-me-5HT in the presence of kynurenic acid(KN) and picrotoxin (PTX), antagonists for ionotropicglutamate and GABAA receptors, respectively. KN (1 mm)and PTX (0.1 mm) blocked the fluctuation of membranepotential induced by α-me-5HT, but had little effect on theincrease in the initial slope caused by α-me-5HT (Fig. 4A).Similar results were obtained from five cells examined.In the presence of KN (1 mm) and PTX (0.1 mm), themean initial slopes were 122 ± 10 Hz nA−1 before, and358 ± 41 Hz nA−1 during α-me-5HT application (n = 5).This represents an increase of 195 ± 31% over the control,

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which is comparable with that obtained in the absence ofKN and PTX (Fig. 4D; P > 0.05, unpaired t test). In thepresence of KN and PTX, the sensitivity was increased inthree of five cells, and unchanged in two cells.

To exclude possible non-specific effects of KN, we usedin some experiments selective antagonists of glutamatereceptors 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)and d-(−)amino-5-phosphonopentanoic acid (d-APV).In the presence of CNQX (10 µm), d-APV (50 µm) andPTX (0.1 mm), α-me-5HT (20 µm) increased the initialslope of the F–I curve by 192 ± 19% over the control

Figure 5. Lack of effect of α-me-5HT on the current-voltagerelationship in the presence of TTXA, changes in membrane potential in response to current steps of 0.10and 0.22 nA before (upper traces) and during the application ofα-me-5HT (lower traces). TTX (0.4 µM) was present throughout therecording. B, current-voltage relationships before and duringα-me-5HT. The slopes were 70 M� before and 68 M� duringα-me-5HT. A and B were from the same cell.

(Fig. 4B; n = 5), comparable with that obtained with KNand PTX (Fig. 4D; P > 0.1, unpaired t test).

We also examined the effect of α-me-5HT in thepresence of KN alone. KN (1 mm) substantially reduced thefluctuation in membrane potential induced by α-me-5HT,but had little effect on the change in the initial slope causedbyα-me-5HT (Fig. 4B). The mean increase in the slope was218 ± 32% (n = 7), comparable with that obtained in thepresence of KN and PTX (P > 0.05, Fig. 4C). However,unlike with both KN and PTX, none of cells showed anincrease in sensitivity in the presence of KN. The sensitivitywas either unchanged (three of seven cells), or decreased(four of seven cells) in response to α-me-5HT in thepresence of KN.

Together, these results suggest that the presynapticeffects mediated by 5-HT2 receptors are not required forthe gain modulation by 5-HT.

Postsynaptic mechanisms involved in the gainmodulation by 5-HT

5-HT may change the slope of F–I curve by affecting RN

at membrane potentials near the spike threshold. Thispossibility was examined using the sodium channel blockertetrodotoxin (TTX). In the presence of TTX (0.4 µm),2 s current steps of +20 to +300 pA were applied beforeand during α-me-5HT application (Fig. 5A). The changein membrane potential was measured for the last 1.5 sportion of the step, and was plotted versus the intensityof injected current. α-Me-5HT (20 µm) had little effecton the current–voltage relationship (Fig. 5B). The slopewas 65 ± 7 M� before, and 61 ± 5 M� during α-me-5HTapplication (n = 4 cells; P > 0.3, paired t test). Theseresults suggest that changes in RN are not involved in thegain modulation by α-me-5HT.

We then examined the possibility that 5-HT-inducedgain modulation may be caused by changes inthe properties of action potentials. We comparedaction potentials evoked by current steps beforeand during α-me-5HT application. Application ofα-me-5HT (20 µm) had no effect on spike threshold:the mean thresholds were −40.2 ± 1.0 mV beforeand −39.3 ± 1.1 mV during α-me-5HT (n = 11 cells;P > 0.05, paired t test). The rise time, height, andhalf-width of action potentials also did not changeduring α-me-5HT application (Fig. 6A). The meanvalues were 0.38 ± 0.02 ms (control) and 0.38 ± 0.02 ms(α-me-5HT) for the rise time, 76.3 ± 1.8 mV (control)and 75.8 ± 2.1 mV (α-me-5HT) for spike height, and0.70 ± 0.02 ms (control) and 0.70 ± 0.02 ms (α-me-5HT)for half-width (n = 11; P > 0.1 for all three groups, pairedt test).

Application of α-me-5HT did reduce the after-hyperpolarization (AHP, Fig. 6B). To examine

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quantitatively the effect of α-me-5HT on AHP, currentpulses of 50 ms were applied to induce trains of spikes.The resting membrane potential was maintained at−60 mV throughout the recording by injections ofDC currents, and the amplitude of current pulse wasadjusted so that three spikes were induced at all times,with the last spike located just before the end of thecurrent pulse. Under such conditions, each train ofspikes induced an AHP that lasted for about 1 s (Fig. 7).Applications of α-me-5HT (20 µm) reduced the peakamplitude of AHP by 1.0 ± 0.2 mV (n = 7 cells). Moreimportantly, α-me-5HT substantially induced a slowafterdepolarization (sADP; Fig. 7). The peak amplitude ofsADP was 1.8 ± 0.2 mV (n = 7 cells). Both the reductionof AHP and the induction of sADP may contribute to thegain modulation by 5-HT.

Requirement for Ca2+ influx and risein intracellular [Ca2+]

Previous studies have shown that the AHP and sADPin cortical neurones are dominated by Ca2+-activatedK+ currents (IK(Ca)) and Ca2+-dependent non-selective

Figure 6. Effects of α-me-5HT on action potentialsA, action potentials before (control, in black) and during α-me-5HT(20 µM, in grey). Each trace is the average of 5–7 consecutive spikeslocated at the second half of the 3 s step (with the mean frequency ofabout 7.5 Hz for both control and during α-me-5HT). Actionpotentials were aligned at the onset. B, the same data as in A with theAHP. The peaks of action potentials were truncated. The slow AHP wasreduced by α-me-5HT (in grey).

cation currents (ICAN), respectively (Schwindt et al. 1988b;Caeser et al. 1993; Haj-Dahmane & Andrade, 1998; Sah& Faber, 2002). Both types of current can be inhibitedby blocking Ca2+ influx during action potentials. Wetherefore examined the effects of Cd2+ (200 µm) on theinput–output relationship. Because Cd2+ slowly causedprecipitations in phosphate-free ACSF, presumably dueto the formation of CdCO3, a Hepes-based solution (seeMethods) was used. Recordings were done at 23 ± 1◦C(room temperatures) to attenuate the deterioration ofslices observed at 32◦C in the Hepes solution. Under suchconditions, α-me-5HT (20 µm) increased the slope ofthe F–I curve (Fig. 8A; 157 ± 4% of the control, n = 4cells; P < 0.005, paired t test). Cd2+ (200 µm) by itselfincreased the slope of the F–I curve (Fig. 8B; 158 ± 6%,n = 4 cells; P < 0.001, paired t test). In the presence ofCd2+, α-me-5HT had no effect on the gain (Fig. 8C;102 ± 3% of that with Cd2+ alone, n = 4 cells; P > 0.5,paired t test). Consistent with the effects on gain, Cd2+

(200 µm) substantially reduced the AHP (Fig. 8D), andα-me-5HT (20 µm) did not affect the afterpotentials inthe presence of Cd2+ (Fig. 8D; n = 5 cells). These findingssuggest that an influx of Ca2+ is required for 5-HT-inducedgain modulation, thus consistent with the hypothesis thatICAN is involved. The fact that Cd2+ by itself increased thegain suggests that an inhibition of IK(Ca) can lead to anincrease in gain.

To determine whether a rise in [Ca2+]i is requiredfor 5-HT2-mediated gain increase, cells were loaded with

Figure 7. Effects of α-me-5HT on AHP and sADPMembrane potential was maintained at −60 mV with DC currentinjection, and a train of three spikes was evoked by a 50 ms currentpulse applied at 0.1 Hz. Application of α-me-5HT (20 µM, in grey)reduced the AHP and induced a sADP. The lower traces show AHPsand sADPs on a faster scale. Each trace was the average of 7–8consecutive responses. The presynaptic effect of α-me-5HT wasattenuated by the presence of CNQX (10 µM) during the recording.

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25 mm EGTA in the recording pipette (replacing equalmolar of K-gluconate). These experiments were done innormal ACSF at 30–32◦C. Loading with 25 mm EGTAwas effective within 10 min of seal break in, as the latephase of action potential repolarization became slower. F–Icurves were examined 15 min after seal break in. Figure 9Aillustrates an example. The mean slope for the controlwas 174 ± 12 Hz nA−1 (n = 8 cells), which was 60% higherthan that obtained with the standard intracellular solution(P < 0.001, unpaired t test). Application of α-me-5HT(20 µm) increased the initial slope by only 38 ± 10%(n = 8 cells), which was 75% less than the amount ofincrease observed with the standard intracellular solution(P < 0.001, unpaired t test). Loading with 25 mm EGTAalso reduced or abolished 5-HT2-induced sADP (Fig. 9B;0.13 ± 0.05 mV, n = 7 cells). These findings are consistent

Figure 8. Cd2+ occluded the effect of α-me-5HT on gainAll experiments were conducted in a Hepes-buffered solution at 24 ± 1◦C. A, α-me-5HT (20 µM) increased theslope of the firing rate curve from 92 to 144 Hz nA−1 (156%). B, Cd2+ (200 µM) increased the slope of the firingrate curve from 79 to 131 Hz nA−1 (166%). C, in the presence of Cd2+ (200 µM), α-me-5HT (20 µM) had littleeffect on the slope of the firing rate curve (111%). B and C were from the same cell. D, effects of Cd2+ (200 µM)and α-me-5HT (20 µM) on the afterpotentials in a neurone. Membrane potential was maintained at −60 mV withDC current injection, and a train of three spikes was evoked by a 50 ms current pulse applied at 0.1 Hz. Cd2+(200 µM, red) reduced most of the AHP; in the presence of Cd2+, α-me-5HT (20 µM, green) had no effect on theafterpotentials.

with the hypothesis that 5-HT2-mediated gain increaseinvolves a reduction of IK(Ca) and an induction of ICAN.

Role of ICAN in 5-HT-induced gain modulation

We tested flufenamic acid (FFA), a widely used blockerof Ca2+-dependent non-selective cation channels (CAN).In the presence of 50 µm FFA (with 0.1% DMSO),α-me-5HT (20 µm) increased the sADP by 1.1 ± 0.2 mV(n = 5 cells), which was less than that observed withoutFFA (1.8 ± 0.2 mV, n = 7; P < 0.02, unpaired t test). FFA(50 µm) by itself had no effect on the slope of the F–Icurve (103 ± 3% of the control, n = 7 cells; P > 0.1,paired t test), and slightly reduced the effect of α-me-5HT(20 µm) on the initial slope (112 ± 23% over the control,n = 5). At 200 µm (also with 0.1% DMSO) however,

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FFA by itself strongly inhibited the generation of spiketrains, and the effect was reversible (n = 4 cells). In thepresence of 200 µm FFA, the spike threshold rose byabout 10 mV over the control (11.4 ± 1.7 mV, n = 4 cells;P < 0.01, paired t test), suggesting an inhibitory effect ofFFA on voltage-gated Na+ channels. Therefore, we didnot examine the effect of high concentrations of FFA on5-HT-induced gain increase.

Role of IK(Ca) in 5-HT-induced gain modulation

Several K+ channels including BK and SK are involvedin the AHP (Sah & Faber, 2002). We first examine therole of BK channels using the selective blocker iberiotoxin(IBTX). IBTX (100 nm) attenuated the late phase of actionpotential repolarization without affecting the AHP (n = 4cells, Fig. 10A). IBTX by itself had little effect on theslope of the F–I curve (104 ± 6%, n = 4 cells; P > 0.5,paired t test). Furthermore, the effect of α-me-5HT on

Figure 9. Loading with 25 mM EGTA markedly reduced5-HT2-mediated gain increase, and abolished the enhancementof sADP by α-me-5HTA, firing rate curves before (control, �) and during the application ofα-me-5HT (20 µM; �). The initial slopes (0–10 Hz) were 150 Hz nA−1

for control and 173 Hz nA−1 for α-me-5HT. B, membrane potentialsfollowing brief spike trains (3 spikes during 50 ms) before (control,black) and during α-me-5HT (20 µM; grey). Resting membranepotentials were maintained at −60 mV with DC current injection. Thepresynaptic effect of α-me-5HT was attenuated by the presence ofCNQX (10 µM) during the recording.

the initial slope was not blocked by IBTX (196 ± 33% overthe control, n = 4 cells).

We then examined the role of SK channels using theselective blocker apamin. Apamin (200 nm) attenuated anearly phase of the AHP without affecting the slow phaseof AHP (n = 4 cells, Fig. 10B). Apamin by itself had littleeffect on the slope of the F–I curve (89 ± 3%, n = 4 cells;P > 0.5, paired t test), and did not block the effect ofα-me-5HT (210 ± 44% over the control, n = 4 cells).

Together, these results suggest that neither BK nor SKchannels are involved in the gain modulation by 5-HT.

Figure 10. Effects of iberiotoxin (100 nM) and apamin (200 nM)on the AHPA, upper traces are superimposed action potentials before (black) andduring 100 nM iberiotoxin (grey, indicated by the arrow); lower tracesare AHPs induced by current steps of 100 ms in duration that evokedseven spikes. Iberiotoxin attenuated the repolarization of the actionpotential without any effect on the AHP. B, apamin (grey) reduced theinitial phase of the AHP with little effect on the late phase. AHPs wereinduced by 100 ms current steps that evoked eight spikes.

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Figure 11. NA did not occlude the effect of α-me-5HT on gainA, AHPs following trains of action potentials before (control, black)and during NA application (grey). The duration of spike train and thenumber of spikes were the same in both cases. The broken lineindicates the baseline level (−60 mV). NA completely blocked sAHPwith little effect on ADP. B, F–I curves before (control, �) and duringNA ( �). The initial slopes were 112 Hz nA−1 for the control, and256 Hz nA−1 for NA. A and B were obtained from the same cell. C, F–Icurves before (control, �), during 10 µM NA ( �), and during 10 µM NAand 20 µM α-me-5HT (�). The initial slopes were 89 Hz nA−1 for thecontrol, 144 Hz nA−1 for NA alone, and 332 Hz nA−1 for α-me-5HTplus NA.

Noradrenaline (NA) has been shown to inhibit thesAHP with little effect on the sADP (McCormick &Prince, 1988; Sah, 1996). Therefore, we tested the effectsof NA on the gain of neurones. NA (10 µm) completelyblocked the sAHP (Fig. 11A), and induced a moderateincrease in gain (Fig. 11B). Similar results were obtainedin 12 cells tested, and collectively, NA increased the gainby 97 ± 17% (n = 12). In five cells, α-me-5HT (20 µm)was applied together with NA (10 µm) following anapplication of NA. Application of α-me-5HT inducedan additional increase in gain (Fig. 11C; 94 ± 28% overthat of NA, n = 5; P < 0.001, paired t test). This latterobservation is consistent with the idea that sAHP is notthe only conductance involved in 5-HT2-mediated gainmodulation.

Discussion

In this study, we showed that 5-HT substantially increasedthe gain of layer 5 pyramidal neurones in the ratPFC. 5-HT-induced gain increase was limited to firingfrequencies less than 15 Hz, and was independent of theeffects on membrane potentials and on input resistance.The effect of 5-HT on gain was mediated through5-HT2 receptors, and involved postsynaptic mechanismsincluding a reduction of the sAHP and an induction of thesADP.

Effects of 5-HT on the gain of neurones

In contrast to the strong excitatory effect observed inimmature neurones, 5-HT induces either moderatemembrane depolarization or hyperpolarization inpyramidal neurones of the adult cortex (Tanaka & North,1993; Zhang, 2003; Beique et al. 2004). The effects of 5-HTon repetitive firing have been examined in detail in layer 5pyramidal neurones of the rat PFC (Araneda & Andrade,1991) and the cat motor cortex (Spain, 1994). In bothstudies, 5-HT was found to significantly increase the firingrate of neurones in response to depolarizing current steps.However, it was not entirely clear whether this increase inexcitability was due to changes in sensitivity or gain. In therat PFC, the increase in firing rate was more pronouncedat higher spike frequency (Araneda & Andrade, 1991).Although this result would imply an effect of 5-HT ongain, no quantitative analysis was performed, and itwas not clear whether the effect was observed in themajority of the neurones. In the cat motor cortex, 5-HTinduced a small increase in gain in only a population ofpyramidal neurones (Spain, 1994). Our results confirmedand extended these previous findings. We showed that5-HT consistently and substantially increased the slope ofthe firing-rate curve in layer 5 pyramidal neurones in therat PFC. This increase by 5-HT was limited to the range

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from 0 to about 10 Hz in spike frequency, and higher than15 Hz, the slope was either unchanged or slightly reducedduring 5-HT application (Fig. 2A). This latter finding mayhave important functional implications. Recent studiesusing chronic recording in free-moving rats have shownthat neurones in the PFC usually fire at 1–3 Hz, and thefiring rate increases to about 10 Hz while performingsustained visual attention or working memory tasks (Gillet al. 2000; Baeg et al. 2003). Thus, our results suggestthat 5-HT increases the gain of PFC neurones in a rangeof frequency that is behaviourally relevant.

5-HT receptors involved in the effects of 5-HT

The pharmacology of 5-HT receptors involved in gainmodulation has not been examined in previous studies.Our results suggest that 5-HT increased the gain of PFCneurones through activation of 5-HT2 receptors. Thisconclusion is drawn from two observations. First, the effectof 5-HT on gain was blocked by the 5-HT2 antagonistketanserin. Second, the selective 5-HT2 agonist α-me-5HTmimicked the effect of 5-HT on gain. Our results did notdistinguish between 5-HT2A and 5-HT2C receptors; bothhave been shown to be expressed by layer 5 pyramidalneurones (Cornea-Hebert et al. 1999; Carr et al. 2002).

Previous studies in slices showed that 5-HT, through5-HT2 receptors, induces large increases in spontaneoustransmission at both excitatory and inhibitory synapses(Aghajanian & Marek, 1997; Zhou & Hablitz, 1999; Lambeet al. 2000; Lambe & Aghajanian, 2001). These presynapticeffects of 5-HT were not required in 5-HT2-mediated gainincrease, because blocking both excitatory and inhibitorytransmission had little effect on 5-HT-induced increasein gain. Postsynaptic mechanisms were therefore requiredfor the gain modulation by 5-HT. However, our resultsdid not exclude a role of spontaneous synaptic activity ongain modulation in vivo. The presynaptic effects inducedby 5-HT or α-me-5HT were probably much lower in slicesdue to the loss of synapses, thus not sufficient to producea significant change in gain.

Unlike the effect on gain, 5-HT induced an increase, adecrease, or no change in current threshold (sensitivity)for spike train generation. Both 5-HT1A and 5-HT2

receptors were involved. Activation of 5-HT1A receptorshyperpolarizes the cell and reduces the input resistanceby opening K+ conductance (Davies et al. 1987; Spain,1994), which leads to a reduction in sensitivity. The effectmediated by 5-HT2 receptors may be more complex. Onthe one hand, activation of 5-HT2 receptors increasesthe sensitivity through depolarization, and a reductionof K+ conductance (VanderMaelen & Aghajanian, 1980).On the other hand, the large increase in spontaneousexcitatory and inhibitory transmission following 5-HT2

receptor activation would result in a reduction in the

input resistance, thus a shunting inhibition. Indeed, halfof the neurones examined here showed either no change(3/8 cells) or a slight decrease (1/8 cell) in sensitivityin response to the selective 5-HT2 agonist α-me-5HT,although it induced modest depolarization in all cellstested. Our results suggest that although both excitatoryand inhibitory transmissions contribute to the shuntinginhibition, GABAA-mediated synaptic response may bepredominant, because in the presence of kynurenic acid,the majority of neurones (4/7) showed a decrease insensitivity in response to α-me-5HT.

Role of Ca2+-activated K+ channels in the gainmodulation by 5-HT

The AHP in neurones is dominated by Ca2+-dependentK+ channels activated following Ca2+ influx during actionpotentials. There are three components: the fast AHP(fAHP) that is mediated by BK channels and blocked byiberiotoxin, the medium AHP (mAHP) that is mediatedby SK channels and blocked by apamin, and the slowAHP (sAHP) for which molecular identities of channelsremain elusive (Schwindt et al. 1988b; Sah & Faber, 2002).The sAHP is particularly important for neuromodulation,since it is the target of several neurotransmitters includingacetylcholine (ACh), noradrenaline (NA), and 5-HT(Andrade & Nicoll, 1987; Schwindt et al. 1988a; Foehringet al. 1989; Knopfel et al. 1990; Sah & Isaacson, 1995).Indeed, both ACh and NA have been shown to increasespike frequency and attenuate spike accommodation(Benardo & Prince, 1982; Madison & Nicoll, 1982;McCormick & Prince, 1987, 1988; Foehring et al. 1989).

A reduction of sAHP was involved in the gainmodulation by 5-HT. This conclusion is drawn fromthree observations. First, α-me-5HT reduced the sAHP.Second, Cd2+ or loading with 25 mm EGTA increased thegain, suggesting that an inhibition of IK(Ca) is sufficientto enhance the gain. Finally, NA (10 µm), which blockedthe sAHP, also produced an increase in gain, but didnot occlude the effect on gain by α-me-5HT. This latterobservation suggests that although a reduction of sAHPmay be required for the effect of α-me-5-HT on gain, it isnot the only conductance involved (see below).

Previous studies have shown that 5-HT reducesthe sAHP through different mechanisms. CyclicAMP-dependent mechanisms, following activation of5-HT4 or 5-HT7 receptors, are involved in the reductionof sAHP in the hippocampus and thalamus (Torres et al.1996; Bacon & Beck, 2000; Goaillard & Vincent, 2002).5-HT also reduces sAHP in pyramidal neurones in theneocortex (Araneda & Andrade, 1991; Spain, 1994),and the effect has been attributed to 5-HT2 receptors(Araneda & Andrade, 1991). Consistent with previousfindings in the neocortex, our results suggested a role for

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5-HT2 receptors in the reduction of sAHPs in pyramidalneurones in the PFC. A recent study showed that activationof 5-HT2 receptors reduced L-type voltage-gated Ca2+

conductance in the same population of neurones in the ratPFC (Day et al. 2002). Whether this reduction of L-typeCa2+ conductance can account for all the reduction in thesAHP needs to be further examined.

Role of ICAN in 5-HT-induced gain modulation

Consistent with previous findings in cortical neuroneswith 5-HT (Araneda & Andrade, 1991; Spain, 1994),our results showed an induction of sADP by α-me-5HT.Using a short spike train as trigger, we found that sADP,absent under control conditions, was induced in thepresence of α-me-5HT. Compared with the sAHP, thesADP had a much slower decay time (7–8 s versus 1 sor less), suggesting that the reduction of the sAHP byα-me-5HT can contribute to the initial part of the sADP,but it is not the cause of sADP being observed in thepresence of α-me-5HT. In many parts of the brainincluding the neocortex, the sADP is mediated by CANchannels (Egorov et al. 2002; Ghamari-Langroudi &Bourque, 2002; Schiller, 2004). Our results obtained with25 mm EGTA and Cd2+ suggest that a rise in [Ca2+]i, pre-sumably through voltage-gated Ca2+ channels (VGCC),is required for the induction of the sADP by α-me-5HT.How does this requirement for VGCC reconcile with the5-HT2-mediated inhibitory effect on VGCC? A simpleexplanation is that activation of 5-HT2 receptors may leadto a large increase in calcium sensitivity of CAN channelsso that a much smaller increase in [Ca2+]i is sufficient forits activation.

5-HT has been shown to enhance the hyper-polarization-activated cation conduction (Ih) and toinhibit a voltage- and time-dependent K+ current(m-current) (Colino & Halliwell, 1987; McCormick& Williamson, 1989; McCormick & Pape, 1990). Ourresults suggest that neither Ih nor m-current is involvedin 5-HT-induced gain modulation. 5-HT-inducedenhancement of Ih is mediated by 5-HT7 or 5-HT4

receptors (Cardenas et al. 1999; Chapin & Andrade, 2001;Bickmeyer et al. 2002), whereas 5-HT2 receptors areresponsible for the gain modulation. The lack of effectof α-me-5HT on the I–V relationship (Fig. 5) suggeststhat m-current is not modulated by 5-HT2 receptoractivation.

Functional Implications of 5-HT-inducedgain modulation

Previous studies have shown a strong correlation betweenthe behavioural state and the level of 5-HT transmissionin the brain. In cats, activity of 5-HT neurones in thebrain stem is highest during periods of waking arousal,

reduced as the animal falls asleep, and absent duringrapid-eye-movement sleep (Jacobs & Fornal, 1999). Inhumans, antidepressants such as fluoxetine are selective5-HT uptake blockers that enhance 5-HT transmission(Delgado, 2000; Bell et al. 2001). These findings suggestthat a key role of 5-HT transmission is to enhancemotor and other executive functions of the brain. How5-HT accomplishes this role is not clear. According tothe available evidence, there is as much inhibition asexcitation by 5-HT throughout the brain. Our resultsshowed that 5-HT consistently increased the gain of layer 5pyramidal neurones, and the effect was independentof those on membrane potential and input resistance.Interestingly, the effect of 5-HT on gain was confined toa range of firing rate that is associated with behaviour.These findings suggest a mechanism by which 5-HTcan selectively amplify behaviourally relevant excitatoryinputs, thus enhancing the response of cortical neuroneswhile having little effect on the basal level of activity.

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Acknowledgements

We thank Dr Kresimir Krnjevic for comments on an early versionof the manuscript. This work was supported by a grant from theCanadian Institutes of Health Research (CIHR). Z.W.Z. is a CIHRNew Investigator.

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