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. 2005 May 5;566(Pt 2):379–394. doi: 10.1113/jphysiol.2005.086066

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

Zhong-wei Zhang 1, Dany Arsenault 1
PMCID: PMC1464765  PMID: 15878946

Abstract

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-HT2 receptors, and involved postsynaptic mechanisms. 5-HT2-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 Ca2+ entry with Cd2+ increased the gain by itself and blocked 5-HT2-mediated gain increase. Buffering [Ca2+]i with 25 mm EGTA also substantially reduced 5-HT2-mediated gain increase. Noradrenaline, which blocked the afterhyperpolarization, also induced a moderate increase in gain. Together, our results suggest that 5-HT may regulate the dynamics of cortical circuits through multiplicative scaling.

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-HT2A 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-HT1A receptors, features a hyperpolarization associated with a reduction in cell input resistance. The excitatory effect of 5-HT involves 5-HT2 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 (Araneda & Andrade, 1991; Spain, 1994). In additional to its effect on membrane potential, 5-HT was found to increase the steady-state firing rate, presumably through an inhibition of the afterhyperpolarization, and an enhancement of the afterdepolarization. Moreover, 5-HT also increased the slope (gain) of the firing rate-current curve in some cortical pyramidal neurones (Araneda & Andrade, 1991; Spain, 1994). This effect of 5-HT on gain modulation, however, has not been examined in any detail. Quantitative data on 5-HT-mediated gain modulation are still not available, and little is known about the underlying mechanisms. In the present study, we examined the effects of 5-HT in layer 5 pyramidal neurones of the rat PFC. We found that 5-HT, via 5-HT2 receptors, consistently increased the gain of neurones. This effect was limited to low spike frequency, and was independent of the effects on membrane potentials and input resistance, but required a rise in [Ca2+]i.

Methods

Slice preparation

Brain slices were prepared from Sprague-Dawley rats of either sex aged P21 to P35 (with the day of birth as P0) as previously described (Zhang, 2004). Briefly, rats were deeply anaesthetized with ketamine and xylazine, and decapitated. The brain was removed quickly (<60 s), and placed in ice-cold solution containing (mm): 210 sucrose, 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 were cut 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, 1.3 MgCl2, 1.0 NaH2PO4, 26 NaHCO3, 20 glucose, saturated with 95% O2 and 5% CO2 at room temperature. Slices were allowed to recover for at least 1 h before any recording. All procedures were performed according to the guidelines of the Canadian Council on Animal Care, and were approved by the Animal Care Committee at Laval University.

For recording, a slice was transferred to a submerge-type chamber where it was continuously exposed to ACSF, saturated with 95% O2 and 5% CO2 and flowing at rate of 2.0 ± 0.2 ml min−1. The slice was viewed first with a 4× objective and the prelimbic area of the PFC was localized as the area between the forceps minor corpus callosum and the midline (Paxinos & Watson, 1998). Layers 1, 2/3, 5 and 6 of the prelimbic area were then viewed under near infrared illumination with a 40× water-immersion objective (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 their large size and apical dendrite.

Patch-clamp recording

Experiments were conducted at 30–32°C unless indicated otherwise. Electrodes were pulled from thick wall borosilicate 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 7.4 with KOH, 280 ± 3 mOsmol with sucrose). In some experiments, K-gluconate was replaced with equimolar K-methanesulphonate. Electrodes had resistances between 4 and 7 MΩ. Liquid junction potential, estimated to be 12 mV, was not corrected. The seal resistance was greater than 5 GΩ. Whole-cell recordings were made at the soma with a Multiclamp 700A amplifier (Axon Instruments, Union City, CA, USA). For current-clamp recordings, the series resistance (Rs), usually between 15 and 45 MΩ, was compensated using the bridge balance. To establish the firing rate curve, current steps of 3 s long were applied once every 15 s, with increments of 10 or 20 pA. Experiments were conducted using the Axograph 4.9 program (Axon Instruments). Data were filtered at 1 or 2 kHz, and digitized at 4 or 8 kHz.

Drugs and drug delivery

All agents were applied by changing the bath perfusate from standard ACSF to modified ACSF to which various drugs were simply added. Unless indicated otherwise, all solutions were continuously bubbled with 95% O2 and 5% CO2. In the experiments with Cd2+, recordings were carried out at 24 ± 1°C with a solution containing (mm): 145 NaCl, 3.0 KCl, 1.5 CaCl2, 1.3 MgCl2, 20 glucose and 10 Hepes (pH. 7.2), gassed with O2. To minimize degradation, 5-HT or noradrenaline was added to ACSF containing 20–40 μm sodium metabisulphate, and both overhead and microscope lights were turned off during the recording. Sodium metabisulphate by itself had no effect on the excitability of neurones. All chemicals were purchased from Sigma-Aldrich Canada (Oakville, ON). K-methanesulphonate was obtained by titrating methanesulphonic 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 event detection package of the AxoGraph. Events with peak amplitude of 60 mV or higher, and a rise time of about 0.5 ms were detected automatically, and the results were analysed with Origin 7. The steady-state spike rate was estimated by counting the number of spikes during the last 2.5 s of the 3 s step, and the result was plotted versus the intensity of the injected current (F–I curve). The slope of the F–I curve (referred to as gain) was estimated by linear fit. Input resistance was estimated by applying small hyperpolarizing current pulses. The action potential threshold was measured using a cursor, by inspecting a 5 ms segment around the rising phase of action potential.

Throughout, means are given ± s.e.m. Means were compared using paired or unpaired two-tailed Student's t test.

Results

Stable whole-cell recordings were obtained from 160 layer 5 pyramidal neurones from 51 rats aged between P21 and P35 (with P0 as the day of birth). Only neurones with resting potential more negative than −55 mV, and input resistance greater than 30 MΩ were examined. Eighty-four per cent of the neurones were regular spiking cells; the remaining (16%) were bursting cells which showed three or more grouped spikes at the beginning of a suprathreshold current step. Only regular spiking cells were included in this study.

Gain modulation by 5-HT in layer 5 pyramidal neurones

The input–output relationship was examined under current clamp by applying 3 s steps once every 15 s with increments of 10 or 20 pA. Suprathreshold current steps evoked trains of action potentials with little accommodation after the first few spikes. An example is illustrated in Fig. 1A. The spike frequency increased with current intensity. Bath application of 5-HT (20 μm) depolarized the cell by 3 mV, and induced significant fluctuations in the resting potential (Fig. 1B), presumably due to presynaptic effects of 5-HT (Aghajanian & Marek, 1997; Zhou & Hablitz, 1999; Lambe et al. 2000). 5-HT also reduced the current threshold for spike train generation (referred to as sensitivity hereafter): the minimal current intensity required for generating two or more spikes decreased from 100 pA for the control, to 70 pA in the presence of 5-HT (Fig. 1A and B). The effect of 5-HT was reversible after 15–20 min wash (Fig. 1C). The steady-state spike rate was estimated by counting the number of spikes during the last 2.5 s of the 3 s step, and the result was plotted versus the intensity of the injected current (F–I curve). Since previous studies in free-moving rats showed that neurones in the PFC usually fire at less than 15 Hz (Gill et al. 2000; Baeg et al. 2003), we focused at the range between 0 and 20 Hz. As illustrated in Fig. 2A, the F–I curve before 5-HT application (control, ▪) was linear between 0 and 20 Hz, with a slope of 120 Hz nA−1. Application of 5-HT increased significantly the slope of the F–I curve (Fig. 2A, ^). The effect of 5-HT on the slope was mostly confined to the range of spike rate between 0 and 10 Hz: the slope was 222 Hz nA−1 between 0 and 12 Hz – an increase of 85% over the control. This effect was reversible after 15–20 min wash (Fig. 2A, ▵).

Figure 1. 5 HT increases the response of layer 5 pyramidal neurones to depolarizing current steps.

Figure 1

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

Figure 2. 5-HT increases the gain at low spike rates.

Figure 2

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A, F–I curves of a neurone before (control, ▪), during 5-HT application (^), and after 20 min wash (▵). The insert shows data points between 0 and 12 Hz before (▪) and during 5-HT application (^). Data were fitted to linear function which yielded slopes of 120 and 222 Hz nA−1 for the control and during 5-HT, respectively (R2 > 0.98 in both cases). After 20 min wash, the initial slope retuned to the control value at 110 Hz nA−1. B, F–I curves of another neurone before (control, ▪), and during 5-HT application (^). The initial slopes (from 0 to 10 Hz) were 118 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 resting membrane potential was maintained at −60 mV throughout the recording with DC injection. The initial slopes was 114 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 this experiment. The initial slopes were 98 and 348 Hz nA−1 for the control and during 5-HT, respectively.

Similar effects of 5-HT on the initial slope were observed in 22 other cells examined. Collectively, the initial slope (0–10 Hz) before 5-HT application (control) was 118 ± 6 Hz nA−1; during 5-HT application, it was 294 ± 19 Hz nA−1 (n = 23; P < 0.0001 versus control, paired t test), which corresponds to an increase of 156 ± 15% (n = 23) over the control. The recovery after 15 min wash was 105 ± 4% of the control (n = 23; P > 0.1 versus control, paired t test). The firing pattern of neurones was not affected by 5-HT.

In contrast to its effect on the initial slope, 5-HT had inconsistent effects on the sensitivity of neurones. Based on the minimum current required for spike train generation, the sensitivity was increased in 10 out of the 23 cells, decreased in seven cells, and unchanged in the remaining six cells. An example where 5-HT induced both a reduction in the sensitivity and an increase in the initial slope is illustrated in Fig. 1D–F and in Fig. 2B. The decrease in sensitivity was associated with a reduction in the input resistance (RN), rather than a hyperpolarization of membrane potential. Out of the seven cells where a decrease in sensitivity was observed, only one cell showed a significant hyperpolarization (−2.5 mV) during 5-HT application, while the others were either slightly depolarized (<3 mV) or unchanged. On the other hand, all seven cells showed substantial reductions in RN (70 ± 4% of the control, n = 7). In comparison, 5-HT had little effect on the input resistance in cells that showed an increase in sensitivity (105 ± 3% of the control, n = 10). The difference in 5-HT-induced RN changes between the two groups (sensitivity increased versus decreased) was statistically significant (P < 0.01, unpaired t test).

Since 5-HT induced a small but significant depolarization (<5 mV) in the majority of cells tested, we tested the effect of membrane depolarization on the slope of the F–I curve. Depolarization of 5–7 mV by 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). To examine further the effect of depolarization, we held the membrane potential constant at −60 mV before and during 5-HT application. The effect of 5-HT on the slope of the F–I curve persisted under such condition (Fig. 2C). Similar results were obtained in all four cells tested, with the initial slope increased by 131 ± 33% over the control (n = 4).

To examine whether internal dialysis may cause time-dependent change in gain, we examined F–I curves at 10–15 min and 40–50 min after break-in. There was no significant change in the slope of the curve (97 ± 3%, n = 5 cells; P > 0.4, paired t test).

Previous studies have shown that intracellular gluconate inhibits K+ conductance (Zhang et al. 1994; Velumian et al. 1997), which would alter the input–output relationship of the neurones. To test if the effect of 5-HT on the F–I curve is affected by the presence of intracellular gluconate, we replaced gluconate with methanesulphonate in the recording pipette. 5-HT had a similar effect on the F–I curve (Fig. 2D). The initial slope was 94 ± 7 Hz nA−1 before (control) and 256 ± 34 Hz nA−1 during 5-HT application (n = 6), which represents an increase of 170 ± 28% over the control. These values are comparable with the results obtained with gluconate (P > 0.1, unpaired t test). This finding suggests that the effect of 5-HT on the F–I curve was not affected significantly by the presence of intracellular gluconate.

Role of 5-HT2 receptors in the gain modulation by 5-HT

Previous studies have shown that 5-HT2 receptors mediate excitatory effects of 5-HT2 in layer 5 pyramidal neurones (Araneda & Andrade, 1991; Tanaka & North, 1993). To determine the role of 5-HT2 receptors in gain modulation, we first used the selective 5-HT2 receptor antagonist ketanserin. Ketanserin (1 μm) by itself had no effect on the F–I curve (initial slope 103 ± 5% of the control, n = 6 cells), but blocked the effects of 5-HT (20 μm) on the initial slope (Fig. 3A). Similar results were observed in all six cells tested. 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 by 5-HT alone (P < 0.001, unpaired t test). Ketanserin also blocked 5-HT-induced depolarization in these cells.

Figure 3. 5-HT2 receptors are responsible for the gain modulation by 5-HT.

Figure 3

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A, ketanserin blocks the gain modulation by 5-HT. The initial slopes of F–I curves were 118 Hz nA−1 for ketanserin alone, and 126 Hz nA−1 for 5-HT (20 μm) and ketanserin. In the presence of ketanserin, 5-HT decreased slightly the 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 during α-me-5HT application. There was also a slight increase in the sensitivity during α-me-5HT application. C, effects of 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 with 5-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%). The number of cells in each group is given in parentheses.

The effect of 5-HT on the initial slope was mimicked by 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 other cells examined. Collectively, α-me-5HT (20 μm) increased the initial slope by 155 ± 22% over the control (n = 8, Fig. 3D). Like 5-HT, α-me-5HT also induced moderate depolarization (<5 mV) and substantial fluctuation in the resting membrane potential. However, α-me-5HT had little effect on the input resistance (106 ± 5% of the control, n = 8; P > 0.1, paired t test). The sensitivity of neurones was increased in four out of eight cells, unchanged in three cells, and decreased in one cell in response to α-me-5HT.

Previous studies have shown that the majority of layer 5 pyramidal neurones in the PFC express both 5-HT1A and 5-HT2 receptors (Araneda & Andrade, 1991; Amargos-Bosch et al. 2004). Accordingly, we examined the effect of 5-HT in the presence of the selective 5-HT1A antagonist WAY100135. WAY100135 (100 nm) by itself had no effect on the F–I curve (initial slope 97 ± 3% of the control, n = 6 cells). In the presence of WAY100135 (100 nm), 5-HT (20 μm) substantially increased the initial slope of the F–I curve (Fig. 3C). Collectively, the initial slope was increased by 167 ± 13% over the control (n = 6 cells; Fig. 3D), which is comparable with those of α-me-5HT and of 5-HT alone (P > 0.1, unpaired t test). This result suggests that 5-HT1A receptors are not involved in the gain modulation by 5-HT. In the presence of WAY100135, the sensitivity of neurones was increased in four out of six cells, and unchanged in the other two cells in response to 5-HT.

Together, these results suggest that 5-HT2 receptors are responsible for the gain modulation by 5-HT. Therefore, we used the selective 5-HT2 agonist α-me-5HT in all subsequent 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 activities at both glutamatergic and GABA-ergic synapses in pyramidal neurones in the PFC (Aghajanian & Marek, 1997; Zhou & Hablitz, 1999; Lambe et al. 2000; Lambe & Aghajanian, 2001). As suggested in recent studies (Chance et al. 2002; Fellous et al. 2003; Shu et al. 2003), such increases in background synaptic activities may modulate the gain of neurones. To determine the role of presynaptic effects on gain modulation by 5-HT, we examined the effects of α-me-5HT in the presence of kynurenic acid (KN) and picrotoxin (PTX), antagonists for ionotropic glutamate and GABAA receptors, respectively. KN (1 mm) and PTX (0.1 mm) blocked the fluctuation of membrane potential induced by α-me-5HT, but had little effect on the increase 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), the mean initial slopes were 122 ± 10 Hz nA−1 before, and 358 ± 41 Hz nA−1 during α-me-5HT application (n = 5). This represents an increase of 195 ± 31% over the control, which is comparable with that obtained in the absence of KN and PTX (Fig. 4D; P > 0.05, unpaired t test). In the presence of KN and PTX, the sensitivity was increased in three of five cells, and unchanged in two cells.

Figure 4. Presynaptic effects are not required for 5-HT-induced gain modulation.

Figure 4

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A, 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, effects of α-me-5HT on the firing rate curve in the presence of KN (1 mm). The initial slopes were 110 Hz nA−1 before and 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%). The number of cells is given in parentheses.

To exclude possible non-specific effects of KN, we used in some experiments selective antagonists of glutamate receptors 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) and PTX (0.1 mm), α-me-5HT (20 μm) increased the initial slope of the F–I curve by 192 ± 19% over the control (Fig. 4B; n = 5), comparable with that obtained with KN and PTX (Fig. 4D; P > 0.1, unpaired t test).

We also examined the effect of α-me-5HT in the presence of KN alone. KN (1 mm) substantially reduced the fluctuation in membrane potential induced by α-me-5HT, but had little effect on the change in the initial slope caused by α-me-5HT (Fig. 4B). The mean increase in the slope was 218 ± 32% (n = 7), comparable with that obtained in the presence of KN and PTX (P > 0.05, Fig. 4C). However, unlike with both KN and PTX, none of cells showed an increase in sensitivity in the presence of KN. The sensitivity was either unchanged (three of seven cells), or decreased (four of seven cells) in response to α-me-5HT in the presence of KN.

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

Postsynaptic mechanisms involved in the gain modulation by 5-HT

5-HT may change the slope of F–I curve by affecting RN at membrane potentials near the spike threshold. This possibility was examined using the sodium channel blocker tetrodotoxin (TTX). In the presence of TTX (0.4 μm), 2 s current steps of +20 to +300 pA were applied before and during α-me-5HT application (Fig. 5A). The change in membrane potential was measured for the last 1.5 s portion of the step, and was plotted versus the intensity of injected current. α-Me-5HT (20 μm) had little effect on the current–voltage relationship (Fig. 5B). The slope was 65 ± 7 MΩ before, and 61 ± 5 MΩ during α-me-5HT application (n = 4 cells; P > 0.3, paired t test). These results suggest that changes in RN are not involved in the gain modulation by α-me-5HT.

Figure 5. Lack of effect of α-me-5HT on the current-voltage relationship in the presence of TTX.

Figure 5

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A, changes in membrane potential in response to current steps of 0.10 and 0.22 nA before (upper traces) and during the application of α-me-5HT (lower traces). TTX (0.4 μm) was present throughout the recording. B, current-voltage relationships before and during α-me-5HT. The slopes were 70 MΩ before and 68 MΩ during α-me-5HT. A and B were from the same cell.

We then examined the possibility that 5-HT-induced gain modulation may be caused by changes in the properties of action potentials. We compared action potentials evoked by current steps before and during α-me-5HT application. Application of α-me-5HT (20 μm) had no effect on spike threshold: the mean thresholds were −40.2 ± 1.0 mV before and −39.3 ± 1.1 mV during α-me-5HT (n = 11 cells; P > 0.05, paired t test). The rise time, height, and half-width of action potentials also did not change during α-me-5HT application (Fig. 6A). The mean values 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, and 0.70 ± 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, paired t test).

Figure 6. Effects of α-me-5HT on action potentials.

Figure 6

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A, action potentials before (control, in black) and during α-me-5HT (20 μm, in grey). Each trace is the average of 5–7 consecutive spikes located at the second half of the 3 s step (with the mean frequency of about 7.5 Hz for both control and during α-me-5HT). Action potentials were aligned at the onset. B, the same data as in A with the AHP. The peaks of action potentials were truncated. The slow AHP was reduced by α-me-5HT (in grey).

Application of α-me-5HT did reduce the afterhyperpolarization (AHP, Fig. 6B). To examine quantitatively the effect of α-me-5HT on AHP, current pulses of 50 ms were applied to induce trains of spikes. The resting membrane potential was maintained at −60 mV throughout the recording by injections of DC currents, and the amplitude of current pulse was adjusted so that three spikes were induced at all times, with the last spike located just before the end of the current pulse. Under such conditions, each train of spikes induced an AHP that lasted for about 1 s (Fig. 7). Applications of α-me-5HT (20 μm) reduced the peak amplitude of AHP by 1.0 ± 0.2 mV (n = 7 cells). More importantly, α-me-5HT substantially induced a slow afterdepolarization (sADP; Fig. 7). The peak amplitude of sADP was 1.8 ± 0.2 mV (n = 7 cells). Both the reduction of AHP and the induction of sADP may contribute to the gain modulation by 5-HT.

Figure 7. Effects of α-me-5HT on AHP and sADP.

Figure 7

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Membrane potential was maintained at −60 mV with DC current injection, and a train of three spikes was evoked by a 50 ms current pulse 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 AHPs and sADPs on a faster scale. Each trace was the average of 7–8 consecutive responses. The presynaptic effect of α-me-5HT was attenuated by the presence of CNQX (10 μm) during the recording.

Requirement for Ca2+ influx and rise in intracellular [Ca2+]

Previous studies have shown that the AHP and sADP in cortical neurones are dominated by Ca2+-activated K+ currents (IK(Ca)) and Ca2+-dependent non-selective 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 inhibited by blocking Ca2+ influx during action potentials. We therefore examined the effects of Cd2+ (200 μm) on the input–output relationship. Because Cd2+ slowly caused precipitations in phosphate-free ACSF, presumably due to the formation of CdCO3, a Hepes-based solution (see Methods) was used. Recordings were done at 23 ± 1°C (room temperatures) to attenuate the deterioration of slices observed at 32°C in the Hepes solution. Under such conditions, α-me-5HT (20 μm) increased the slope of the F–I curve (Fig. 8A; 157 ± 4% of the control, n = 4 cells; P < 0.005, paired t test). Cd2+ (200 μm) by itself increased the slope of the F–I curve (Fig. 8B; 158 ± 6%, n = 4 cells; P < 0.001, paired t test). In the presence of Cd2+, 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), and α-me-5HT (20 μm) did not affect the afterpotentials in the presence of Cd2+ (Fig. 8D; n = 5 cells). These findings suggest that an influx of Ca2+ is required for 5-HT-induced gain modulation, thus consistent with the hypothesis that ICAN is involved. The fact that Cd2+ by itself increased the gain suggests that an inhibition of IK(Ca) can lead to an increase in gain.

Figure 8. Cd2+ occluded the effect of α-me-5HT on gain.

Figure 8

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All experiments were conducted in a Hepes-buffered solution at 24 ± 1°C. A, α-me-5HT (20 μm) increased the slope of the firing rate curve from 92 to 144 Hz nA−1 (156%). B, Cd2+ (200 μm) increased the slope of the firing rate curve from 79 to 131 Hz nA−1 (166%). C, in the presence of Cd2+ (200 μm), α-me-5HT (20 μm) had little effect 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 with DC 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 the afterpotentials.

To determine whether a rise in [Ca2+]i is required for 5-HT2-mediated gain increase, cells were loaded with 25 mm EGTA in the recording pipette (replacing equal molar of K-gluconate). These experiments were done in normal ACSF at 30–32°C. Loading with 25 mm EGTA was effective within 10 min of seal break in, as the late phase of action potential repolarization became slower. F–I curves were examined 15 min after seal break in. Figure 9A illustrates an example. The mean slope for the control was 174 ± 12 Hz nA−1 (n = 8 cells), which was 60% higher than 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 of increase observed with the standard intracellular solution (P < 0.001, unpaired t test). Loading with 25 mm EGTA also reduced or abolished 5-HT2-induced sADP (Fig. 9B; 0.13 ± 0.05 mV, n = 7 cells). These findings are consistent with the hypothesis that 5-HT2-mediated gain increase involves a reduction of IK(Ca) and an induction of ICAN.

Figure 9. Loading with 25 mm EGTA markedly reduced 5-HT2-mediated gain increase, and abolished the enhancement of sADP by α-me-5HT.

Figure 9

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A, firing rate curves before (control, ▪) and during the application of α-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 potentials following brief spike trains (3 spikes during 50 ms) before (control, black) and during α-me-5HT (20 μm; grey). Resting membrane potentials were maintained at −60 mV with DC current injection. The presynaptic effect of α-me-5HT was attenuated by the presence of CNQX (10 μm) during the recording.

Role of ICAN in 5-HT-induced gain modulation

We tested flufenamic acid (FFA), a widely used blocker of 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 without FFA (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–I curve (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, FFA by itself strongly inhibited the generation of spike trains, and the effect was reversible (n = 4 cells). In the presence of 200 μm FFA, the spike threshold rose by about 10 mV over the control (11.4 ± 1.7 mV, n = 4 cells; P < 0.01, paired t test), suggesting an inhibitory effect of FFA on voltage-gated Na+ channels. Therefore, we did not examine the effect of high concentrations of FFA on 5-HT-induced gain increase.

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

Several K+ channels including BK and SK are involved in the AHP (Sah & Faber, 2002). We first examine the role of BK channels using the selective blocker iberiotoxin (IBTX). IBTX (100 nm) attenuated the late phase of action potential repolarization without affecting the AHP (n = 4 cells, Fig. 10A). IBTX by itself had little effect on the slope of the F–I curve (104 ± 6%, n = 4 cells; P > 0.5, paired t test). Furthermore, the effect of α-me-5HT on the initial slope was not blocked by IBTX (196 ± 33% over the control, n = 4 cells).

Figure 10. Effects of iberiotoxin (100 nm) and apamin (200 nm) on the AHP.

Figure 10

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A, upper traces are superimposed action potentials before (black) and during 100 nm iberiotoxin (grey, indicated by the arrow); lower traces are AHPs induced by current steps of 100 ms in duration that evoked seven spikes. Iberiotoxin attenuated the repolarization of the action potential without any effect on the AHP. B, apamin (grey) reduced the initial phase of the AHP with little effect on the late phase. AHPs were induced by 100 ms current steps that evoked eight spikes.

We then examined the role of SK channels using the selective blocker apamin. Apamin (200 nm) attenuated an early phase of the AHP without affecting the slow phase of AHP (n = 4 cells, Fig. 10B). Apamin by itself had little effect 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 of α-me-5HT (210 ± 44% over the control, n = 4 cells).

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

Noradrenaline (NA) has been shown to inhibit the sAHP with little effect on the sADP (McCormick & Prince, 1988; Sah, 1996). Therefore, we tested the effects of NA on the gain of neurones. NA (10 μm) completely blocked the sAHP (Fig. 11A), and induced a moderate increase in gain (Fig. 11B). Similar results were obtained in 12 cells tested, and collectively, NA increased the gain by 97 ± 17% (n = 12). In five cells, α-me-5HT (20 μm) was applied together with NA (10 μm) following an application of NA. Application of α-me-5HT induced an additional increase in gain (Fig. 11C; 94 ± 28% over that of NA, n = 5; P < 0.001, paired t test). This latter observation is consistent with the idea that sAHP is not the only conductance involved in 5-HT2-mediated gain modulation.

Figure 11. NA did not occlude the effect of α-me-5HT on gain.

Figure 11

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A, AHPs following trains of action potentials before (control, black) and during NA application (grey). The duration of spike train and the number of spikes were the same in both cases. The broken line indicates the baseline level (−60 mV). NA completely blocked sAHP with little effect on ADP. B, F–I curves before (control, ▪) and during NA (^). The initial slopes were 112 Hz nA−1 for the control, and 256 Hz nA−1 for NA. A and B were obtained from the same cell. C, F–I curves before (control, ▪), during 10 μm NA (^), and during 10 μm NA and 20 μmα-me-5HT (▴). The initial slopes were 89 Hz nA−1 for the control, 144 Hz nA−1 for NA alone, and 332 Hz nA−1 for α-me-5HT plus NA.

Discussion

In this study, we showed that 5-HT substantially increased the gain of layer 5 pyramidal neurones in the rat PFC. 5-HT-induced gain increase was limited to firing frequencies less than 15 Hz, and was independent of the effects on membrane potentials and on input resistance. The effect of 5-HT on gain was mediated through 5-HT2 receptors, and involved postsynaptic mechanisms including a reduction of the sAHP and an induction of the sADP.

Effects of 5-HT on the gain of neurones

In contrast to the strong excitatory effect observed in immature neurones, 5-HT induces either moderate membrane depolarization or hyperpolarization in pyramidal neurones of the adult cortex (Tanaka & North, 1993; Zhang, 2003; Beique et al. 2004). The effects of 5-HT on repetitive firing have been examined in detail in layer 5 pyramidal neurones of the rat PFC (Araneda & Andrade, 1991) and the cat motor cortex (Spain, 1994). In both studies, 5-HT was found to significantly increase the firing rate of neurones in response to depolarizing current steps. However, it was not entirely clear whether this increase in excitability was due to changes in sensitivity or gain. In the rat PFC, the increase in firing rate was more pronounced at higher spike frequency (Araneda & Andrade, 1991). Although this result would imply an effect of 5-HT on gain, no quantitative analysis was performed, and it was not clear whether the effect was observed in the majority of the neurones. In the cat motor cortex, 5-HT induced a small increase in gain in only a population of pyramidal neurones (Spain, 1994). Our results confirmed and extended these previous findings. We showed that 5-HT consistently and substantially increased the slope of the firing-rate curve in layer 5 pyramidal neurones in the rat PFC. This increase by 5-HT was limited to the range from 0 to about 10 Hz in spike frequency, and higher than 15 Hz, the slope was either unchanged or slightly reduced during 5-HT application (Fig. 2A). This latter finding may have important functional implications. Recent studies using chronic recording in free-moving rats have shown that neurones in the PFC usually fire at 1–3 Hz, and the firing rate increases to about 10 Hz while performing sustained visual attention or working memory tasks (Gill et al. 2000; Baeg et al. 2003). Thus, our results suggest that 5-HT increases the gain of PFC neurones in a range of frequency that is behaviourally relevant.

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

The pharmacology of 5-HT receptors involved in gain modulation has not been examined in previous studies. Our results suggest that 5-HT increased the gain of PFC neurones through activation of 5-HT2 receptors. This conclusion is drawn from two observations. First, the effect of 5-HT on gain was blocked by the 5-HT2 antagonist ketanserin. Second, the selective 5-HT2 agonist α-me-5HT mimicked the effect of 5-HT on gain. Our results did not distinguish between 5-HT2A and 5-HT2C receptors; both have been shown to be expressed by layer 5 pyramidal neurones (Cornea-Hebert et al. 1999; Carr et al. 2002).

Previous studies in slices showed that 5-HT, through 5-HT2 receptors, induces large increases in spontaneous transmission at both excitatory and inhibitory synapses (Aghajanian & Marek, 1997; Zhou & Hablitz, 1999; Lambe et al. 2000; Lambe & Aghajanian, 2001). These presynaptic effects of 5-HT were not required in 5-HT2-mediated gain increase, because blocking both excitatory and inhibitory transmission had little effect on 5-HT-induced increase in gain. Postsynaptic mechanisms were therefore required for the gain modulation by 5-HT. However, our results did not exclude a role of spontaneous synaptic activity on gain modulation in vivo. The presynaptic effects induced by 5-HT or α-me-5HT were probably much lower in slices due to the loss of synapses, thus not sufficient to produce a significant change in gain.

Unlike the effect on gain, 5-HT induced an increase, a decrease, or no change in current threshold (sensitivity) for spike train generation. Both 5-HT1A and 5-HT2 receptors were involved. Activation of 5-HT1A receptors hyperpolarizes the cell and reduces the input resistance by opening K+ conductance (Davies et al. 1987; Spain, 1994), which leads to a reduction in sensitivity. The effect mediated by 5-HT2 receptors may be more complex. On the one hand, activation of 5-HT2 receptors increases the sensitivity through depolarization, and a reduction of K+ conductance (VanderMaelen & Aghajanian, 1980). On the other hand, the large increase in spontaneous excitatory and inhibitory transmission following 5-HT2 receptor activation would result in a reduction in the input resistance, thus a shunting inhibition. Indeed, half of the neurones examined here showed either no change (3/8 cells) or a slight decrease (1/8 cell) in sensitivity in response to the selective 5-HT2 agonist α-me-5HT, although it induced modest depolarization in all cells tested. Our results suggest that although both excitatory and inhibitory transmissions contribute to the shunting inhibition, GABAA-mediated synaptic response may be predominant, because in the presence of kynurenic acid, the majority of neurones (4/7) showed a decrease in sensitivity in response to α-me-5HT.

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

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

A reduction of sAHP was involved in the gain modulation by 5-HT. This conclusion is drawn from three observations. First, α-me-5HT reduced the sAHP. Second, Cd2+ or loading with 25 mm EGTA increased the gain, suggesting that an inhibition of IK(Ca) is sufficient to enhance the gain. Finally, NA (10 μm), which blocked the sAHP, also produced an increase in gain, but did not occlude the effect on gain by α-me-5HT. This latter observation suggests that although a reduction of sAHP may be required for the effect of α-me-5-HT on gain, it is not the only conductance involved (see below).

Previous studies have shown that 5-HT reduces the sAHP through different mechanisms. Cyclic AMP-dependent mechanisms, following activation of 5-HT4 or 5-HT7 receptors, are involved in the reduction of 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 the neocortex (Araneda & Andrade, 1991; Spain, 1994), and the effect has been attributed to 5-HT2 receptors (Araneda & Andrade, 1991). Consistent with previous findings in the neocortex, our results suggested a role for 5-HT2 receptors in the reduction of sAHPs in pyramidal neurones in the PFC. A recent study showed that activation of 5-HT2 receptors reduced L-type voltage-gated Ca2+ conductance in the same population of neurones in the rat PFC (Day et al. 2002). Whether this reduction of L-type Ca2+ conductance can account for all the reduction in the sAHP needs to be further examined.

Role of ICAN in 5-HT-induced gain modulation

Consistent with previous findings in cortical neurones with 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 the presence of α-me-5HT. Compared with the sAHP, the sADP had a much slower decay time (7–8 s versus 1 s or less), suggesting that the reduction of the sAHP by α-me-5HT can contribute to the initial part of the sADP, but it is not the cause of sADP being observed in the presence of α-me-5HT. In many parts of the brain including the neocortex, the sADP is mediated by CAN channels (Egorov et al. 2002; Ghamari-Langroudi & Bourque, 2002; Schiller, 2004). Our results obtained with 25 mm EGTA and Cd2+ suggest that a rise in [Ca2+]i, presumably 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 the 5-HT2-mediated inhibitory effect on VGCC? A simple explanation is that activation of 5-HT2 receptors may lead to a large increase in calcium sensitivity of CAN channels so that a much smaller increase in [Ca2+]i is sufficient for its activation.

5-HT has been shown to enhance the hyperpolarization-activated cation conduction (Ih) and to inhibit a voltage- and time-dependent K+ current (m-current) (Colino & Halliwell, 1987; McCormick & Williamson, 1989; McCormick & Pape, 1990). Our results suggest that neither Ih nor m-current is involved in 5-HT-induced gain modulation. 5-HT-induced enhancement 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 are responsible for the gain modulation. The lack of effect of α-me-5HT on the I–V relationship (Fig. 5) suggests that m-current is not modulated by 5-HT2 receptor activation.

Functional Implications of 5-HT-induced gain modulation

Previous studies have shown a strong correlation between the behavioural state and the level of 5-HT transmission in the brain. In cats, activity of 5-HT neurones in the brain stem is highest during periods of waking arousal, reduced as the animal falls asleep, and absent during rapid-eye-movement sleep (Jacobs & Fornal, 1999). In humans, antidepressants such as fluoxetine are selective 5-HT uptake blockers that enhance 5-HT transmission (Delgado, 2000; Bell et al. 2001). These findings suggest that a key role of 5-HT transmission is to enhance motor and other executive functions of the brain. How 5-HT accomplishes this role is not clear. According to the available evidence, there is as much inhibition as excitation by 5-HT throughout the brain. Our results showed that 5-HT consistently increased the gain of layer 5 pyramidal neurones, and the effect was independent of those on membrane potential and input resistance. Interestingly, the effect of 5-HT on gain was confined to a range of firing rate that is associated with behaviour. These findings suggest a mechanism by which 5-HT can selectively amplify behaviourally relevant excitatory inputs, thus enhancing the response of cortical neurones while having little effect on the basal level of activity.

Acknowledgments

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

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