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Multiple Effects of Dopamine on Layer V Pyramidal Cell Excitability in Rat Prefrontal Cortex ALLAN T. GULLEDGE AND DAVID B. JAFFE Division of Life Sciences, University of Texas at San Antonio, San Antonio, Texas 78249 Received 29 December 2000; accepted in final form 11 April 2001

Gulledge, Allan T. and David B. Jaffe. Multiple effects of dopamine on layer V pyramidal cell excitability in rat prefrontal cortex. J Neurophysiol 86: 586 –595, 2001. The mechanisms underlying the inhibitory effects of dopamine (DA) on layer V pyramidal neuron excitability in the prelimbic region of the rat medial prefrontal cortex were investigated. Under control conditions, DA depressed both action potential generation (driven by somatic current injection) and input resistance (RN). The presence of GABAA receptor antagonists blocked DA-induced depression of action potential generation and revealed a delayed increase in excitability that persisted for the duration of experimental recording, up to 20 min following the washout of DA. In contrast to spike generation, disinhibition did not affect the transient depression of RN produced by DA, suggesting independent actions of DA on spike generation and RN. Consistent with the hypothesis that DA acts to decrease pyramidal cell output via a GABAergic mechanism, DA increased the frequency of spontaneous inhibitory postsynaptic currents in both the absence and presence of TTX. Furthermore focal application of GABA to a perisomatic region mimicked the inhibitory effect of DA on spike production without affecting RN. Focal application of bicuculline to the same location reversed the inhibitory effect of bath-applied DA on spike generation, while again having no effect on RN. The depression of RN by DA was both occluded and mimicked by the Na⫹ channel blocker TTX, suggesting the involvement of a Na⫹ conductance in reducing pyramidal cell RN during the acute presence of DA. Together these data demonstrate that the acute presence of DA decreases pyramidal neuron excitability by two independent mechanisms. At the same time DA triggers a delayed and longer-lasting increase in excitability that is partially masked by synaptic inhibition.

The mammalian prefrontal cortex (PFC) is important for a form of short-term memory often described as “working memory” (Bauer and Fuster 1976; Goldman et al. 1971; Granon et al. 1994), and the ability of the PFC to facilitate working memory tasks is dependent on dopaminergic input from the ventral tegmental area (VTA) (Brozoski et al. 1979; Bubser and Schmidt 1990). In the rat, the cortical area most associated with both working memory behaviors and mesocortical dopamine (DA) input is the prelimbic region of the medial PFC (mPFC) (Fritts et al. 1998; Seamans et al. 1995, 1998). This area receives dense dopaminergic afferents (Berger et al. 1976; Descarries et al. 1987; Emson and Koob 1978; Van Eden et al. 1987), and pyramidal and nonpyramidal neurons in this region

can express members of both the D1 and D2 DA receptor families (Gaspar et al. 1995; Vincent et al. 1993, 1995). In primates and rats, local depletion of DA in the PFC produces deficits in working memory task completion (Brozoski et al. 1979; Bubser and Schmidt 1990). Moreover overstimulation of prefrontal DA receptors impairs working-memory performance (Arnsten et al. 1994, 1995; Druzin et al. 2000; Murphy et al. 1996; Romanides et al. 1999; Zahrt et al. 1997). Because these data suggest that proper working memory function depends on the maintenance of an optimal level of prefrontal DA, it is important to understand the effects of DA on the physiology of individual prefrontal neurons. At the cellular level there appear to be two seemingly conflicting effects of DA. Unit and intracellular recordings in vivo have shown that DA, whether released by VTA stimulation or exogenously applied, inhibits spontaneous and evoked firing of PFC neurons (Bernardi et al. 1982; Ferron et al. 1984; Godbout et al. 1991; Mantz et al. 1988; Pirot et al. 1992, 1996; Sesack and Bunney 1989). In agreement with these data, several in vitro studies have found DA inhibits the excitability of rat prefrontal pyramidal neurons (Geijo-Barrientos and Pastore 1995; Gulledge and Jaffe 1998; Zhou and Hablitz 1999). In contrast, a number of other in vitro studies have reported dramatic enhancement in the excitability of prefrontal pyramidal cells by DA (Ceci et al. 1999; Gorelova and Yang 2000; Henze et al. 2000; Penit-Soria et al. 1987; Yang and Seamans 1996). The purpose of the present study was to determine the mechanisms underlying the inhibitory effects of DA on action potential generation and input resistance (RN) of pyramidal neurons in the prelimbic region of the rat mPFC. Several factors suggest that DA may inhibit prefrontal pyramidal cell excitability indirectly by modulating GABAergic inhibition. First, anatomical studies have established direct DA innervation of GABAergic neurons in the PFC of both rats and monkeys (Benes et al. 1993, 1996; Sesack et al. 1995a,b, 1998; Smiley and Goldman-Rakic 1993; Verney et al. 1990). Second, DA can directly modulate the physiology of nonpyramidal, presumably GABAergic neurons in the rat PFC (Zhou and Hablitz 1999). Third, results from in vitro and in vivo studies demonstrate dopaminergic enhancement of extracellular GABA levels in the rat mPFC (Bourdelais and Deutch 1994; Del Arco and Mora 2000; Grobin and Deutch 1998; Retaux et

Address for reprint requests: D. B. Jaffe, Div. of Life Sciences, University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, TX 78249 (E-mail: [email protected]).

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al. 1991). DA-mediated increases in GABA release could be expected to both inhibit action potential generation and reduce RN in layer V pyramidal neurons. We tested the hypothesis that DA indirectly inhibits pyramidal cells by increasing local GABAergic tone within the rat prelimbic PFC. Evidence is presented suggesting that DA’s inhibitory actions on layer V pyramidal neurons are mediated through at least two distinct mechanisms and that this inhibition can mask a delayed increase in excitability. In addition, we show that dopaminergic reduction of RN is TTX sensitive, suggesting dopaminergic modulation of a Na⫹ conductance. A preliminary report has been presented in abstract form (Gulledge and Jaffe 1999). METHODS

Brain slices Animals were housed and handled in accord with the guidelines set by The American Physiological Society. Following decapitation of unanesthetized, 17- to 30-day-old Sprague-Dawley rats (Harlan, Indianapolis, IN), brains were removed and immersed in cold (0°C) artificial cerebral spinal fluid (ACSF, described in the following text) where NaCl was replaced with choline chloride. Coronal slices (300 ␮m) containing the prelimbic region of the mPFC were prepared with a vibratome. Brain slices were immediately (⬍5 min following decapitation) placed in a holding chamber of ACSF containing (in mM) 124 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 Na2HPO4, 2 MgCl2, 2 CaCl2, and 10 dextrose. Slices were allowed to incubate for ⱖ1 h at room temperature (⬃23°C) before being transferred, as needed, into a submerged recording chamber perfused with oxygenated ACSF (⬃2 ml/min). In some experiments, CaCl2 was removed and the amount of MgCl2 was increased to 6 mM. In other experiments, where spontaneous inhibitory postsynaptic currents (IPSCs) were monitored, the level of KCl was raised to 5.5 mM. All ACSF solutions were oxygenated continuously with a mixture of 95% O2-5% CO2. Experiments were performed at ⬃31°C.

Electrophysiology Whole cell recordings were made from visually identified layer V pyramidal cells from the prelimbic region of the rat mPFC using infrared video/differential interference contrast microscopy (Stuart et al. 1993) with a ⫻40 water-immersion objective. To ensure that pyramidal cells did not have cut apical dendrites, slices were chosen so that the apical dendrites were either in the same plane as the soma or descended into the slice. Patch pipettes (3–5 M⍀) were filled with (in mM) 120 K-gluconate, 10 HEPES, 0.1 EGTA, 20 KCl, 2 MgCl2, 3 Na2ATP, and 0.3 NaGTP (pH ⫽ 7.3). In some experiments, as noted, K-gluconate was replaced with KCl. Current- or voltage-clamp recordings were made using an Axoclamp 2B amplifier (Axon Instruments) and were digitized at 1, 3, 5, or 25 kHz using an ITC-16 interface (Instrutech) connected to a Power Macintosh computer running AxoData (Axon Instruments) acquisition software. Whole cell pipette series resistance was ⬍20 M⍀ and was bridge-compensated. No corrections were made for junction potentials, which should be ⬃10 mV with K-gluconate. Analyses of electrical responses were performed off-line using custom software written with Igor Pro (Wavemetrics). Only one neuron, experiencing a single drug treatment, was used from each brain slice. In all cases, only cells with a resting potential of at least ⫺60 mV, and stable baseline responses were given drug treatments.

Experimental paradigm Trains of action potentials were evoked by depolarizing current steps (1 s duration, 100 –300 pA) at 0.1 Hz. Current intensities were J Neurophysiol • VOL

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adjusted so that they evoked about six action potentials before any drug treatment (Gulledge and Jaffe 1998). RN was determined within the linear range of the steady-state voltage-current relationship, generated by a series of hyperpolarizing and depolarizing current injections (usually ⫺50 to ⫹50 pA). When a full range of steps could not be obtained, for example during brief pressure application of drugs (described below), RN was calculated from the response to single hyperpolarizing current steps (⫺50 or ⫺100 pA). DA-induced changes in resting potential were compensated with a bias current to maintain baseline levels during these measurements. After baseline measurements of action potential number and RN, DA (10 ␮M) was bath-applied for periods of ⬃5 min. Two gravity-fed perfusion lines were used to change solutions. New solutions reached the recording chamber in ⬃2 min and were removed in ⬃5 min (Gulledge and Jaffe 1998). In some experiments, as noted, drug solutions loaded into patch pipettes were pressure-ejected into the slice for periods of 10 –30 s (see Fig. 5). Both control and drug ACSF solutions were continuously oxygenated throughout experiments. In some control experiments, excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) were evoked with a monopolar stimulating electrode placed into layer V near the soma of the postsynaptic pyramidal cell. EPSPs were isolated using 10 ␮M bicuculline while IPSPs were recorded in the presence of 2 mM kynurenate. Evoked potentials were generated with a 50 ␮s pulse from a stimulus isolation unit controlled by a Master 8 pulse generator (AMPI, Israel). Stock solutions of DA and bicuculline methiodide were made fresh daily and mixed into oxygenated ACSF as needed. Stock solutions of TTX were kept as frozen aliquots until just prior to use. Ascorbate (10 ␮M) was added to all DA solutions to reduce oxidation. We previously found that this concentration of ascorbate has no effect on pyramidal neuron physiology (Gulledge and Jaffe 1998). All drugs were purchased from Sigma (St. Louis, MO).

Analysis and statistics The effects of DA on excitability were determined by analyzing the number of spikes generated from the average of five traces obtained immediately before, at the end of, and five minutes after DA application. IPSCs were identified and selected as inward currents with fast rise times and an amplitude threshold of at least three standard deviations from the mean current noise. Fast rise times were selected from events ⬎1.5 to 3 times the standard deviation of a sweep’s second derivative. These parameters were adjusted on a cell-to-cell basis to maximize the accuracy of IPSC detection (to limit false hits) but were kept constant for all phases of an individual cell’s analysis. Statistical significance was determined using a two-tailed Student’s t-test for paired or unpaired samples, as appropriate. Numerical values are expressed as means ⫾ SE. RESULTS

As previously reported (Gulledge and Jaffe 1998), bath application of DA (10 ␮M) decreased both the number of action potentials generated by somatic current injection and the RN of layer V pyramidal cells in slices of rat mPFC (Fig. 1). The inhibitory effects of DA on spike generation and RN were evident within 3 min of application and reversed ⬃5 min following drug removal (Fig. 1C). In eight cells, DA significantly decreased spike generation by 81 ⫾ 8% (df ⫽ 7, t ⫽ 7.1, P ⬍ 0.05; Fig. 1, A, C, and E), while the latency to the first action potential (for 6 cells that still fired ⱖ1 spike in the presence of DA) increased from 52 ⫾ 6 to 74 ⫾ 11 ms (mean increase ⫽ 41 ⫾ 9%; df ⫽ 5, t ⫽ 3.3, P ⬍ 0.05; data not shown). DA reduced RN from a baseline mean of 63 ⫾ 9 M⍀ by 15 ⫾ 3% (df ⫽ 7 , t ⫽ 4.3, P ⬍ 0.05; Fig. 1, B, D, and F).

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tion depolarized layer V pyramidal cells by 2.0 ⫾ 0.5 mV (resting Vm ⫽ ⫺70 ⫾ 1 mV). This was not correlated with DA-mediated effects on RN (Fig. 1H) and did not significantly influence the inhibitory effects of DA on spike generation (Gulledge and Jaffe 1998). GABAA antagonists occlude the inhibitory effect of DA on spike generation To determine if DA inhibits pyramidal cell excitability by modulating GABAergic inhibition, DA was applied in the presence of GABAA blockers (Fig. 2). In 17 cells treated with 2 mM kynurenate (to block fast excitatory synaptic transmission and the subsequent activation of inhibitory neurons) and either 100 ␮M picrotoxin and 10 ␮M bicuculline or 20 ␮M bicuculline alone, DA failed to decrease the number of spikes generated by depolarizing current steps (mean change in DA ⫽ ⫹20 ⫾ 13% of baseline value; P ⬎ 0.05). The enhanced excitability became significant ⬃10 min after the washout of DA (mean change in “wash” ⫽ ⫹59 ⫾ 13%; df ⫽ 16, t ⫽ 4.3, P ⬍ 0.05; Fig. 2, B and C) and lasted for the duration of the recording (ⱕ20 min following DA removal). The inhibitory effect of DA was not affected by the presence of kynurenate alone (Gulledge and Jaffe 1998).

FIG. 1. Dopamine (DA) reversibly depresses pyramidal cell excitability. A: representative action potentials produced by a 250-pA current step before (left), during (middle), and after (right) bath application of 10 ␮M DA. Resting membrane potential for this cell was ⫺70 mV. B: subthreshold voltage responses to a series of depolarizing and hyperpolarizing current steps for the same cell as in A. DA reversibly decreased voltage responses in both the hyperpolarizing and depolarizing directions. C: time course of the effect of DA on spike generation for the same cell. D: voltage-current relationship for traces shown in B. - - -, linear fit to control voltage responses; —, linear fit to responses in the presence of DA. E and F: summary of the effect of DA on spike generation and RN for 8 cells. G: comparison of the effects of DA on voltage responses in depolarizing (■) and hyperpolarizing (u) directions. H: plot of DA-induced changes in membrane potential versus changes in RN. For this and subsequent figures, significant changes with respect to baseline (P ⬍ 0.05) are indicated by *.

Dopaminergic reduction of RN was symmetrical with respect to both depolarizing and hyperpolarizing directions. There was no significant difference, during DA application or following drug removal, between mean steady-state voltage responses pooled from depolarizing versus hyperpolarizing current steps (Fig. 1, D and G). As previously reported (Bernardi et al. 1982; Geijo-Barrientos and Pastore 1995; Gulledge and Jaffe 1998; Law-Tho et al. 1994; Penit-Soria et al. 1987; Shi et al. 1997) DA applicaJ Neurophysiol • VOL

FIG. 2. The inhibitory effect of DA on action potential generation is blocked by GABAA receptor antagonists. A: action potentials generated by a 250-pA current step before (left), during (middle), and after (right) the bath application of 10 ␮M DA to slices disinhibited by the presence of 20 ␮M bicuculline (with 2 mM kynurenic acid added to block recurrent excitation or inhibition). Baseline Vm for this cell was ⫺73 mV. B: time course of the effect of DA on pyramidal cell excitability for the same cell as in A. Note that DA induced a persistent increase in the number of spikes following the washout of DA. C and D: summary of the effect of DA on spike generation and RN for 17 cells exposed to GABAA antagonists. Although GABAA blockers occluded the inhibitory effect of DA on spike generation, they did not occlude DA’s effect on RN.

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In contrast, GABAA antagonists did not interfere with DA’s effects on RN or Vm. Under conditions where fast synaptic transmission was blocked, pyramidal cell RN was significantly higher than in control conditions (with GABAA and glutamate receptors blocked, mean RN ⫽ 105 ⫾ 11 M⍀; df ⫽ 23, t ⫽ 2.3, P ⬍ 0.05). DA application reversibly decreased RN of these neurons by 10 ⫾ 3% (df ⫽ 16, t ⫽ 3.1, P ⬍ 0.05; Fig. 2D). This effect was not statistically different from that of DA in control conditions. Likewise disinhibition did not affect the depolarization produced by exposure to DA (mean baseline Vm ⫽ ⫺71 ⫾ 1 mV; mean change ⫽ ⫹1.6 ⫾ 0.4 mV; df ⫽ 15, t ⫽ 3.3, P ⬍ 0.05). These data suggest that DA’s effect on action potential generation is via a mechanism independent of its effects on RN and Vm. Dopamine and spontaneous IPSCs We next tested the hypothesis that DA affects the frequency or amplitude of GABAergic IPSCs. In 17 neurons, spontaneous IPSCs (sIPSCs) were recorded in the presence of 2 mM kynurenate (Fig. 3, A–C). Because the reversal potential for Cl⫺ in cells filled with K-gluconate-based pipette solutions is close to the resting potential, intracellular Cl⫺ concentration was increased with a KCl-based pipette solution (see METHODS). Additionally, in some experiments, extracellular KCl was increased to 5.5 mM. Mean sIPSC frequency was 7 ⫾ 1 Hz. Bath-applied DA (10 ␮M) reversibly increased the frequency of sIPSCs by 22 ⫾ 10% (df ⫽ 16, t ⫽ 2.3, P ⬍ 0.05; Fig. 3C) but did not significantly change sIPSC amplitude (mean change ⫽ ⫹13 ⫾ 8%). Previous in vitro studies indicate that DA modulates either spike-mediated (Retaux et al. 1991; Zhou and Hablitz 1999) or spike-independent (Penit-Soria et al. 1987) GABA release onto prefrontal neurons. To confirm if DA enhances spike-independent GABA release, we recorded miniature IPSCs (mIPSCs) in 10 cells exposed to 1 ␮M TTX and 2 mM kynurenate (Fig. 3, D–F). During these experiments, extracellular KCl was maintained at 2.5 mM. Baseline mIPSC frequencies in TTX (5 ⫾ 1 Hz) were not significantly different from sIPSC frequencies, described in the preceding text. Bath application of 10 ␮M DA significantly enhanced mIPSC frequency by 29 ⫾ 11% (df ⫽ 9, t ⫽ 2.4, P ⬍ 0.05; Fig. 3F) while not significantly affecting mIPSC amplitude (mean change ⫽ ⫹19 ⫾ 12%). In contrast to sIPSCs, DA-mediated increases in mIPSC frequency persisted following washout of DA [mean change in “wash” ⫽ ⫹29 ⫾ 9% (df ⫽ 9, t ⫽ 5.7, P ⬍ 0.05)]. Calcium independence If DA can enhance TTX-insensitive release of GABA, DA might still depress spike generation under conditions in which spike-mediated transmitter release is reduced. We tested this possibility by conducting experiments where evoked synaptic transmission was blocked by removing calcium from the ACSF while at the same time increasing external magnesium to 6 mM. To test whether transmission was indeed blocked, several experiments were performed to demonstrate that Ca2⫹ removal blocked evoked IPSPs and EPSPs. Synaptic responses were produced by 50 ␮s stimulation of a monopolar stimulating electrode placed in layer V near the soma of the recorded pyramidal cell in the presence of either 2 mM kynurenate J Neurophysiol • VOL

FIG. 3. DA increases the frequency of spontaneous and miniature IPSCs in layer V pyramidal cells. A: sequential traces of voltage-clamp recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) in the presence of 2 mM kynurenic acid before (left), during (middle), and after (right) the application of 10 ␮M DA. B: representative time course of DA’s effect on sIPSCs. C: summary of results from 17 cells treated with DA. D: sequential traces of miniature IPSCs (mIPSCs) recorded in the presence of kynurenate and 1 ␮M TTX before (left), during (middle), and after (right) DA application (10 ␮M). E: representative time course of the effect of DA on mIPSCs. F: summary of results from 10 cells treated with DA in the presence of TTX.

(IPSPs, n ⫽ 3) or 10 ␮M bicuculline (EPSPs, n ⫽ 3). The removal of Ca2⫹ effectively, and reversibly, eliminated evoked responses in all six cells (data not shown). Under Ca2⫹-free conditions, DA had the same effect on pyramidal cell excitability as under control conditions (Fig. 4). In the absence of Ca2⫹, DA significantly depressed action potential generation by 68 ⫾ 12% (Fig. 4, A and B; df ⫽ 5, t ⫽ 5.6, P ⬍ 0.05) and reduced RN by 11 ⫾ 3% (Fig. 4C; baseline RN ⫽ 72 ⫾ 14 M⍀; df ⫽ 5, t ⫽ 4.7, P ⬍ 0.05). Additionally, DA depolarized these cells 1.5 ⫾ 0.6 mV (baseline Vm ⫽ ⫺68 ⫾ 1 mV; df ⫽ 5, t ⫽ 2.5, P ⬍ 0.05). This change in Vm, like the effects on spiking and RN, was not significantly different from changes seen in control cells treated with DA in normal ACSF. These results support the hypothesis that DA inhibits spike generation in pyramidal cells by enhancing TTXinsensitive GABAergic synaptic inhibition while at the same

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tion. We first examined the effect of focal bicuculline application (20 ␮M) into the same basal region as described in the preceding text, under control conditions (Fig. 6A). In four cells, focally applied bicuculline produced modest facilitation of spike generation (mean increase was only 1.1 ⫾ 0.8 spikes). Next, as observed previously, for seven pyramidal neurons exposed to 10 ␮M DA, there was a 69 ⫾ 8% decrease in spikes, a 15 ⫾ 4% decrease in RN (baseline RN ⫽ 71 ⫾ 13 M⍀), and a 2.3 ⫾ 0.3 mV depolarization of Vm (baseline Vm ⫽ ⫺72 ⫾ 1 mV). With DA in the bath, focally applied bicuculline (10 –30 s) partially reversed the effects of DA on spike generation, but not RN (Fig. 6, B–D). The number of spikes generated with bicuculline present was significantly increased by 3.9 ⫾ 0.7 spikes (Fig. 6C; df ⫽ 6, t ⫽ 5.5, P ⬍ 0.05), returning the number of action potentials generated to within 10 ⫾ 21% of baseline values. Bicuculline’s effect on spike FIG. 4. DA depresses action potential generation and RN in pyramidal neurons in the absence of extracellular Ca2⫹ (replaced with 6 mM external Mg2⫹). A: action potentials generated in the absence of Ca2⫹ by a 300 pA current step before (top), during (middle), and after (bottom) bath application of DA. Baseline Vm was ⫺65 mV. B and C: summary of the effect of DA on action potential generation and RN for 6 neurons in which DA was applied in the absence of external Ca2⫹.

time ruling out dopaminergic modulation of voltage-gated Ca2⫹ channels. Exogenous GABA mimics the effect of dopamine on spike inhibition The data presented in the preceding text point to a GABAergic mechanism for DA-dependent depression of spike generation that is, at least in part, independent of its modulation of RN. Anatomical studies in primate PFC (Sesack et al. 1998) suggest DA could preferentially increase GABA release near spikegenerating sites in the axon. If this is the case, exogenous GABA introduced near the axon should mimic the inhibitory effects of DA on action potential generation while having little or no effect on somatic RN. To test this hypothesis, we performed experiments in which GABA (2 ␮M) was pressureejected (10 to 30 s duration) to a location where we would expect to affect the pyramidal neuron’s axon initial segment (Fig. 5A) (Williams and Stuart 2000). In five cells, focal application of GABA reversibly and repeatedly reduced the number of spikes produced by depolarizing current injection. The mean change in spike number was ⫺59 ⫾ 5% (df ⫽ 4, t ⫽ 5.0, P ⬍ 0.05), an amount not significantly different from the inhibition produced by bath-applied DA. More importantly, the decrease in spike generation produced by focal GABA application was not accompanied by a decrease in pyramidal cell RN (Fig. 5, B–E), suggesting that the effect of GABA was indeed localized and distal from the soma or dendrites. The mean change in RN measured from the voltage responses to single hyperpolarizing current steps, was only ⫺5 ⫾ 2% (mean baseline RN ⫽ 98 ⫾ 8 M⍀; df ⫽ 4, t ⫽ 2.1, P ⬎ 0.05). Additionally, GABA application did not significantly impact pyramidal cell membrane potentials (mean baseline Vm ⫽ ⫺71 ⫾ 1 mV; mean change ⫽ ⫹0.7 ⫾ 0.4 mV). If DA acts to reduce action potential generation by enhancing GABA release at perisomatic locations, localized disinhibition should prevent dopaminergic effects on spike generaJ Neurophysiol • VOL

FIG. 5. Focal GABA application mimics the inhibitory effect of DA on spike generation but not RN. A: photomicrograph of pressure ejection pipette filled with 2 ␮M GABA (arrow) relative to a patched pyramidal neuron soma (asterisk) whose primary apical dendrite is extending downward. Scale bar ⫽ 25 ␮m. B: action potentials generated by a 250 pA current step before (top) and during (bottom) brief (20 s) application of GABA. Baseline Vm for this neuron was ⫺69 mV. C: expanded traces from B, superimposed. While 2 ␮M GABA reduced the number of spikes generated by the current step (thick trace), it did not change RN (note the lack of change in the hyperpolarizing test pulse before depolarization). D: summary of the inhibitory effects of bath-applied DA and focally applied GABA on spike generation in layer V pyramidal cells. The inhibitory effect of GABA on spike generation was not significantly different from that produced by DA. E: summary of the effects of bath-applied DA and focally applied GABA on RN. GABA application produced significantly less reduction of RN than bath-applied DA.

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ined spike threshold, the second derivative of the spike rising phase, action potential amplitude, and spike half-width, no significant effects of DA (or DA washout) were observed in any of the preceding experimental conditions (data not shown). However, it is possible that DA might modulate subthreshold states of Na⫹ channels and/or a persistent Na⫹ conductance, while not affecting fast Na⫹ currents underlying spike kinetics (Ahern et al. 2000; Cepeda et al. 1995; Hammarstro¨m and Gage 1999). In cortical pyramidal cells, voltage responses can be amplified in both the depolarizing and hyperpolarizing directions, respectively, by the activation and deactivation of inward Na⫹ currents active near rest (Cepeda et al. 1995; French et al. 1990; Schwindt and Crill 1995; Stuart 1999). Depression of this current by DA would decrease steady-state voltage responses, resulting in reduction of the measured RN. To test this hypothesis, 10 ␮M DA was bath-applied to 15 cells in the presence of 1 ␮M TTX (Fig. 7). Under these conditions, DA failed to reduce pyramidal cell RN (baseline RN ⫽ 83 ⫾ 9 M⍀; mean change ⫽ ⫹1 ⫾ 3%). This lack of effect in the presence of TTX was significantly different from changes in RN seen in control cells treated with DA alone (Fig. 7D; df ⫽ 21, t ⫽ 4.6, P ⬍ 0.05). TTX did not occlude the DA-induced depolarization of Vm (baseline Vm ⫽ ⫺71 ⫾ 1 mV; mean change ⫽ ⫹1.1 ⫾ 0.5 mV; df ⫽ 14, t ⫽ 2.3, P ⬍ 0.05). Changes in Vm with DA application in the presence of TTX were not signifFIG. 6. Focally applied bicuculline reverses DA-induced inhibition of spike generation but not RN. A: spikes generated by a 150 pA current injection before (left) and during (right) focal application of 20 ␮M bicuculline in the absence of DA. Bicuculline induced a modest increase in spikes (from 1 to 3). B: spikes generated in the same cell as shown in A by a 200 pA current injection before (top) and during (middle) the bath application of 10 ␮M DA. With DA still present in the bath, focal application of bicuculline (bottom) partially reversed DA-induced inhibition of spike generation. Baseline Vm for this neuron was ⫺74 mV. C and D: summary of effects on spike generation and RN for 7 experiments in which bicuculline was focally applied following the bath application of DA.

number in the presence of DA was significantly greater than under control conditions (df ⫽ 9, t ⫽ 2.6, P ⬍ 0.05). In contrast, in the presence of DA, focal application of bicuculline did not significantly affect RN. During bicuculline application, RN remained depressed by 10 ⫾ 4% from control values, an amount not significantly different from found under control or disinhibited conditions (see Figs. 1F and 2D). Additionally, focal bicuculline application did not significantly affect pyramidal cell Vm (mean change in Vm during bicuculline puff ⫽ ⫺0.2 ⫾ 0.2 mV). These data are consistent with the hypothesis that DA inhibits pyramidal neuron excitability via modulation of GABAergic synaptic transmission, that this modulation is localized to the spike generating zone, and that DA’s inhibitory effect on action potential generation is, in part, independent of its effects on RN and Vm. TTX blocks the inhibitory effect of dopamine on RN Our observation that DA still depressed RN under disinhibited conditions suggests that its effect on membrane conductance is independent of a GABAergic mechanism. A likely candidate mechanism for DA’s inhibitory actions is through modulation of voltage-gated Na⫹ channels. When we examJ Neurophysiol • VOL

FIG. 7. Dopaminergic reduction of pyramidal cell RN is TTX sensitive. A: membrane potential changes to a variety of positive and negative current steps before (top) and after (bottom) the application of 10 ␮M DA for a cell exposed to 1 ␮M TTX. Baseline Vm for this neuron was ⫺75 mV. B: plot of the voltage-current relationship before (E), during (●), and after (䊐) bath application of 10 ␮M DA. C: summary of the lack of effect of DA on RN for 15 cells treated with 1 ␮M TTX. D: comparison of control cells treated with DA with those exposed to DA after treatment with 1 ␮M TTX. TTX significantly occluded the effect of DA on RN.

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modulating a TTX-sensitive conductance independent of GABA receptor activation, and DA triggers a delayed increase in excitability that is, in part, masked by synaptic inhibition. Taken together, these data indicate that the overall effect of DA on pyramidal cell excitability may depend on phasic changes in DA levels and the tone of GABAergic inhibition. Dopamine and action potential generation

FIG. 8. Bath application of TTX mimics the effect of DA on RN. A: membrane potential responses to a range of positive and negative current steps before (top) and after (bottom) the bath application of 1 ␮M TTX. The baseline Vm for this neuron was ⫺72 mV. B: plot of the voltage-current relationship before (E) and after (●) TTX application. Solid lines are the results from linear regression. C: summary of the effect of TTX on normalized RN (n ⫽ 8). D: comparison of control cells treated with DA alone and cells exposed to TTX alone.

icantly different from the changes in Vm observed with DA alone. If DA decreases RN by inhibiting a resting Na⫹ conductance, it follows that the application of TTX should mimic the effect of DA on RN and that TTX application itself should hyperpolarize pyramidal cells. To test these predictions, in eight cells RN was measured before and after bath application of 1 ␮M TTX under conditions in which fast synaptic transmission was blocked with bicuculline (20 ␮M) and kynurenate (2 mM; Fig. 8). As was found for cells treated with DA alone, TTX application produced a decrease in the voltage-current relationship in both the depolarizing and hyperpolarizing directions (Fig. 8B), resulting in a 17 ⫾ 3% decrease in RN (mean baseline RN ⫽ 125 ⫾ 26 M⍀; df ⫽ 7, t ⫽ 3.8, P ⬍ 0.05). The decrease in RN seen with TTX application was similar in magnitude to the effect seen in control cells treated with DA alone (Fig. 8D). Bath application of TTX had no effect on pyramidal cell Vm (baseline Vm ⫽ ⫺73 ⫾ 1 mV; mean change ⫽ ⫹0.1 ⫾ 0.6 mV). Therefore the effect of TTX on RN, but not Vm (see ⫹ DISCUSSION), is consistent with the reduction of a Na conductance. DISCUSSION

The major conclusions of the present study are that the acute presence of DA depresses action potential generation primarily via an increase in GABAergic inhibition, DA reduces RN by J Neurophysiol • VOL

Three lines of evidence presented here indicate that the inhibitory effect of DA on spike generation in layer V pyramidal neurons is mediated, in part, by an increase in GABAergic synaptic transmission. First, blockade of GABAA receptors occludes the inhibitory effect of DA on spike generation. Second, the inhibitory effect of DA on spike generation is mimicked by focal GABA application to sites near the spike-generating zone. Third, DA increases the frequencies of spontaneous and miniature IPSCs in layer V pyramidal cells. The GABAA-independent effect of DA on RN suggests a second mechanism for modulating membrane excitability (discussed in the following text). In this study, we employed bicuculline methiodide to block GABAA receptors. Because bicuculline blocks channels mediating the slow afterhyperpolarization (sAHP) (Khawaled et al. 1999), it is important to note that our data are not consistent with an alternative hypothesis where DA enhances the sAHP to decrease excitability. To our knowledge, subcortical modulators such as DA typically depress the sAHP (Malenka and Nicoll 1986), leading to enhanced excitability. Moreover involvement of a sAHP was ruled out by the fact that DA increased the latency to the first spike produced by depolarizing current injection. Additionally, we found that DA, in many cases, depressed firing down to one or no spikes (see Fig. 1). A sAHP strong enough to depress subsequent spikes is not likely to be generated by a single action potential, and no indications of a sAHP were evident following the limited number of spikes generated under bicuculline-free conditions. Our finding that bicuculline blocks DA-mediated spike inhibition in vitro supports earlier in vivo results demonstrating bicuculline sensitivity of DA-induced inhibition of spontaneous firing of prefrontal neurons (Pirot et al. 1992). Additionally, the dopaminergic enhancement of spontaneous IPSCs presented here is similar to previous findings that DA can increase extracellular GABA levels in PFC (Bourdelais and Deutch 1994; Del Arco and Mora 2000; Grobin and Deutch 1998; Retaux et al. 1991) and enhance IPSC frequency onto both pyramidal and nonpyramidal cells (Zhou and Hablitz 1999). We should note that in our experiments DA only had a modest effect on IPSC frequency (⬃20% increase), while Zhou and Hablitz (1999) found DA to produce large increases in both the frequency and amplitude of spontaneous IPSCs (⬃250 and ⬃155%, respectively). Disparity between the results of these studies may reflect differences in prefrontal region (anterior cingulate versus prelimbic PFC) and cell type (layer I and III pyramidal and nonpyramidal neurons versus layer V pyramidal cells). Finally, DA has been shown to directly enhance the excitability of prefrontal nonpyramidal neurons (Zhou and Hablitz 1999), and dopaminergic modulation of inhibitory neuron excitability is fully consistent with a GABAergic mechanism for spike inhibition. However, the observations that DA increases mIPSC frequency in the pres-

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ence of TTX and still depresses spike generation in Ca2⫹-free ACSF suggest that increases in nonpyramidal cell firing rates may not be required. If DA modulates spike-independent GABA release, wouldn’t a change in RN be expected in the presence of TTX? Two sets of experiments suggest that DA-induced modulation of GABA release may have minimal impact on somatic RN. First, mIPSC frequencies were only modestly enhanced by DA (⬍30% increase) and IPSCs are only one of many conductances contributing to the total membrane conductance. Second, even direct application of exogenous GABA, at concentrations sufficient to reduce spike generation, did not significantly depress RN (see Fig. 5). This experiment demonstrates that modulation of excitability at a site distant from the soma, such as the initial node (Williams and Stuart 2000), can have little, if any, effect on total input conductance. This explanation is consistent with anatomical data demonstrating that some GABAergic neurons form axoaxonic synapses onto pyramidal cells (Anderson et al. 1995; Kawaguchi and Kubota 1998; Williams et al. 1992) and that GABAergic neurons are innervated by dopaminergic fibers (Benes et al. 1993; Sesack et al. 1995a,b, 1998; Smiley and Goldman-Rakic 1993; Verney et al. 1990).

compensate for small changes in Vm produced by depressing the inward current. Although the relationship between DA, TTX, and pyramidal cell Vm is as yet unresolved, there is substantial evidence linking DA with Na⫹ channel regulation. Generally, DA has an inhibitory influence on Na⫹ currents. In both striatal (Calabresi et al. 1987; Cepeda et al. 1995; Schiffmann et al. 1998), and hippocampal (Cantrell et al. 1997, 1999) neurons, DA depresses Na⫹ currents. While DA has purely inhibitory effects on Na⫹ conductances in other cell types, there is not yet consensus on the effect of DA on Na⫹ currents in prefrontal cells. Our results are most consistent with the observations of Geijo-Barrientos and Pastore (1995) and Geijo-Barrientos (1999), who report DA-induced decreases of subthreshold Na⫹ conductances (see also Maurice et al. 2000). In contrast, the work of Yang and colleagues indicates that DA activates a delayed increase in a persistent Na⫹ current (Gorlova and Yang 2000; Yang and Seamans 1996). The most unifying hypothesis is that DA has a biphasic effect on subthreshold Na⫹ conductances. Differential experimental conditions, including whether excitability is assessed in the acute presence of DA or after exposure to DA, or variations in the level of synaptic inhibition, may dictate whether the excitatory or inhibitory effects of DA are observed.

Effects of dopamine on TTX-sensitive conductance

Dopamine and working memory

Dopaminergic reduction of RN was not affected by GABAA antagonists but was occluded after blockade of Na⫹ channels with TTX. There are two possible explanations for the TTX sensitivity of dopaminergic modulation of RN: DA depresses the constitutive, TTX-sensitive release of another neuromodulator that normally increases pyramidal cell RN, or the acute presence of DA, inhibits a resting Na⫹ current in layer V pyramidal cells. How might the closing of a Na⫹ channel lead to an increase in conductance? Cortical pyramidal cells have resting voltage-gated Na⫹ currents that amplify both depolarizing and hyperpolarizing membrane responses (French et al. 1990; Schwindt and Crill 1995; Stuart 1999). A reduction in this current by DA would appear as a decrease in the steadystate voltage response and be interpreted as a decrease in RN. If DA does reduce a resting Na⫹ conductance, why doesn’t DA, or the application of TTX itself, hyperpolarize layer V pyramidal cells? Fig. 1H suggests that DA modulates RN and Vm independently. Additionally, DA-induced depolarization of pyramidal neurons may mask any hyperpolarizing effect of Na⫹ channel closure. This depolarization, produced by DA, may not involve activation of DA receptors but may result from nonspecific effects of DA on other receptor types within the PFC (Shi et al. 1997). DA-induced depolarization, however, does not explain why the application of TTX alone does not hyperpolarize prefrontal neurons. It is possible that TTX application modulates the relative endogenous concentrations of DA and other neuromodulators that influence pyramidal cell resting potentials, as well as RN. Additionally, one must consider that reducing a resting Na⫹ conductance would likely decrease resting intracellular Na⫹ concentrations. In turn, a number of factors, including depolarization of the equilibrium potential for Na⫹, decreasing the electrogenic contribution the Na⫹/K⫹ ATPase, or reducing outward current generated by Na⫹-activated K⫹ currents (Schwindt et al. 1989), could all

The data presented here point to at least three distinct mechanisms for dopaminergic modulation of prefrontal pyramidal cells. First, DA inhibits action potential generation by enhancing spontaneous inhibitory synaptic input. Second, DA decreases RN via reduction of a TTX-sensitive conductance. Both of these inhibitory effects are expressed during acute exposure to DA. Third, DA triggers a delayed and prolonged enhancement of excitability, as has been previously reported (Gorelova and Yang 2000; Henze et al. 2000; Yang and Seamans 1996). This increased excitability may be related to the “rebound” excitation we previously found when inhibition was intact (Gulledge and Jaffe 1998). It is possible that prolonged increases in excitability, observed only under disinhibited conditions, are normally masked by the persistent increase in TTX-insensitive IPSCs reported here. One hypothesis for working memory involves the formation of reverberatory circuits that actively maintain information for a short period of time (Amit 1995). Indeed, a well-established feature of prefrontal neurons is enhanced output during specific phases of working memory tasks (Funahashi et al. 1989; Fuster and Alexander 1971; Kubota and Niki 1971). While computational models suggest that transient changes in either membrane excitability or synaptic connectivity (Camperi and Wang 1998; Durstewitz et al. 1999; Lisman et al. 1998; Wang 1999) may maintain sustained activity, a level of synaptic inhibition is required to constrain firing to only those neurons providing behaviorally significant output (Camperi and Wang 1999). The acute presence of DA, through its inhibitory actions on TTXsensitive conductances and enhancement of GABAergic transmission, may serve to gate appropriate firing by allowing output only from those neurons receiving the strongest excitatory drive. Delayed increases in excitability may regulate tonic levels of excitability. In this way, DA release during workingmemory tasks may not only set the stage for reverberatory

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