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The Journal of Neuroscience, March 22, 2006, 26(12):3229-3244; doi:10.1523/JNEUROSCI.4333-05.2006
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Cellular/Molecular
Dopaminergic Regulation of Neuronal Excitability through Modulation of Ih in Layer V Entorhinal Cortex
J. Amiel Rosenkranz andDaniel Johnston Center for Learning and Memory, University of Texas at Austin, Austin, Texas 78712
Abstract
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Abstract
Introduction
The entorhinal cortex (EC) is a significant component of the systems that underlie certain forms of memory formation and recall. Evidence has been emerging that the dopaminergic system in the EC facilitates these and other functions of the EC. The effects of dopamine (DA) on membrane properties and excitability of EC neurons, however, are not known. We used in vitro whole-cell patch-clamp recordings from layer V pyramidal neuronal somata and dendrites of the adult rat lateral EC to investigate the effects of DA on the excitability of these neurons. We found that brief application of DA caused a reduction in the excitability of layer V EC pyramidal neurons. This effect was attributable to voltage-dependent modification of membrane properties that can best be explained by an increase in a hyperpolarization-activated conductance. Furthermore, the effects of DA were blocked by pharmacological blockade of h-channels, but not by any of a number of other ion channels. These actions were produced by a D1 receptor-mediated increase of cAMP but were independent of protein kinase A. A portion of the actions of DA can be attributed to effects in the apical dendrites. The data suggest that DA can directly influence the membrane properties of layer V EC pyramidal neurons by modulation of h-channels. These actions may underlie some of the effects of DA on memory formation.
Key words: dopamine; entorhinal cortex; whole cell; patch clamp; rat; hyperpolarization-activated current; voltage sag
Introduction
The entorhinal cortex (EC) is part of a network that aids in the consolidation and recall of memories (Buzsaki, 1996; Murray et al., 1998
Similar to other cortical pyramidal neurons, deep layer EC neurons display a spectrum of ionic conductances, and electrophysiological features that are indicative of the presence of active conductances (Eder and Heinemann, 1996
Materials and Methods
Slice preparation. Male Sprague Dawley rats (4–6 weeks of age) were anesthetized with an intraperitoneal injection of a combination of ketamine (42 mg/ml), xylazine (8 mg/ml), and, in some experiments, acepromazine (1.4 mg/ml). No differences were observed in the results from animals in which acepromazine was included in the anesthetic mixture. Once anesthetized, rats were perfused intracardially with an ice-cold solution containing the following (in mM): 110 choline chloride, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 7 dextrose, 1.3 ascorbic acid, 3 pyruvic acid (all drugs were from Sigma, St. Louis, MO, unless otherwise specified). The solution was saturated with a 95% O2, 5% CO2 gas mixture. After perfusion, the rat was quickly decapitated, and the brain was removed and sliced in 350 µm sections in the same solution (Vibratome Series 1000; Vibratome, St. Louis, MO). Brain slices were then incubated at 37°C for 10 min in a solution containing the following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 2 MgCl2, 10 dextrose, 1.3 ascorbic acid, 3 pyruvic acid. After incubation, brain slices stabilized at room temperature for at least 50 min before transfer to the recording chamber.Whole-cell recordings. Brain slices were transferred to the recording chamber with a solution containing the following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 2 MgCl2, 10 dextrose, 0.075 sodium metabisulfite (an antioxidant that prevents degradation of DA). Layer V of the lateral EC was visually identified using infrared differential interference contrast microscopy. Pyramidal neurons were selected based on the protrusion of a long apical dendrite and a nonstellate appearance. Patch electrodes were constructed from borosilicate glass with an outer diameter of 2.0 mm and an inner diameter of 1.16 mm (Sutter Instruments, Novato, CA). Electrodes were pulled using a Flaming/Brown micropipette puller (model P-97; Sutter Instruments). Electrodes were filled with the following (in mM): 120 K+ methylsulfate, 20 KCl, 0.2 EGTA, 10 HEPES, 2 MgCl2, with a pH of 7.3. On the day of recordings, 4 mM Na2-ATP, 0.3 mM Tris-GTP, 7 mM phosphocreatine, and 0.1% Neurobiotin were added to the internal recording solution (yielding an osmolarity of
290 mOsm). The electrode junction potential was corrected in the bath, but the diffusion potential was not compensated after seal formation and membrane rupture. Based on relative ionic mobilities and charge, this potential was calculated to be 7–8 mV. Therefore, the actual membrane potentials could be up to 8 mV more hyperpolarized than the measured voltage. The liquid junction potential between the intracellular and extracellular solutions was also measured to be 3–4 mV using the method described by Neher (1992)
Synaptic stimulation. Synaptic inputs to the recorded neuron were activated by discrete electrical stimulation using theta glass pipettes with a tip size between 2–4 µm [filled with the following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 10 HEPES, 2 CaCl2, 2 MgCl2, 25 dextrose]. The electrodes were positioned within 20 µm of the apical dendrite at a distance of 150–200 µm from the soma. Stimulations were performed as single pulses (0.2 ms duration; 0.005–0.05 mA) or as trains (10 stimuli; 20 Hz).
Drug application. Chemicals were bath applied in the recording solution, and included DA (1–10 µM; all drugs were from Sigma unless otherwise specified), (±)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF81297) (10 µM), quinpirole (10 µM), R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) (5 µM), sulpiride (5 µM), TTX (1–2 µM), 4-AP (3 mM), CsCl (10 mM), CdCl2 (200 µM), BaCl2 (250 µM), tetraethylammonium (TEA) (1 mM), NiCl2 (50 µM), nimodipine (10 µM), 6,10-diaza-3(1,3)8,(1,4)-dibenzena-1,5(1,4)-diquinolinacy clodecaphane (UCL1684) (100 nM), forskolin (10–50 µM), 8-Br-cAMP (100–250 µM), and 4-ethylphenylamino-1,2-dimethyl-6-methylamino-pyrimidinium chloride (ZD7288) (20 µM). In a subset of experiments, as indicated, BAPTA tetrapotassium (20 mM), 9-(tetrahydro-2'-furyl)adenine (SQ22536) (1 mM; adenylyl cyclase blocker), (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg-3',2',1'-K1]pyrrolo[3,4–1][1,6]benzodiazocine-10-carboxylic acid (KT5720) (1.2 µM; protein kinase A blocker), or ZD7288 (20 µM) were included in the recording pipette. When mentioned, synaptic transmission was blocked by the inclusion of CNQX (10 µM), DL-AP-5 (50 µM), bicuculline (10 µM; in DMSO), and (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845) (2 µM; in DMSO) in the extracellular recording solution. The total concentration of DMSO in the bath remained <0.15%. On completion of the experiment, brain slices were transferred to 4% paraformaldehyde in 10% sucrose solution, in which they remained for at least 24 h. Slices were stained for Neurobiotin using the ABC Vectastain kit (Vector Laboratories, Burlingame, CA).
Data collection and analysis. Voltage signals were amplified and filtered at 2 kHz (SEC-05L; NPI, Tamm, Germany) and collected via an ITC18 interface board (Instrutech, Port Washington, NY), transmitting to an Apple computer (PowerPC G5: Apple, Cupertino, CA) running Igor Pro software (WaveMetrics, Lake Oswego, OR). Before formation of a seal, the electrode tip potential was negated, and the open-tip resistance was measured (typically 4–6 M
). After successful transition to whole-cell configuration, series resistance was monitored and compensated throughout the experiment using built-in bridge circuitry. Only recordings with a stable series resistance <15 M
Input resistance was determined from the linear fit of the neuronal voltage response to small (+40 to –40 pA; 500–700 ms) square-step direct-current injections from –70 mV. The initial membrane time constant of these small voltage responses was quantified by fitting with a least-squares fit to two exponentials. The voltage sag ratio in response to hyperpolarizing direct current steps of 700 ms was calculated as the peak voltage deflection divided by the amplitude of the steady-state voltage deflection. A hyperpolarizing direct current step that caused a peak 5–15 mV deflection was used for the purpose of this measurement. The normalized rebound voltage overshoot in response to hyperpolarizing direct current step was quantified as the peak rebound voltage deflection divided by the steady-state voltage deflection during the current step.
The action potential rise time was quantified as the peak of the first derivative (dV/dT) of the action potential waveform. The action potential threshold was defined as the peak of the third derivative of the action potential waveform. The action potential amplitude was defined as the difference between the action potential peak voltage and the initial membrane potential. The rheobase current was defined as the amplitude of the current injection to evoke a single action potential. Excitability was quantified as the number of action potentials in response to a fixed amplitude square step current injection. A slow afterhyperpolarizing potential (sAHP) was evoked by 5–10 suprathreshold direct current steps of 2–5 ms duration at 50 Hz, at a membrane potential of approximately –55 mV. The peak amplitude of the sAHP was measured relative to the baseline membrane potential. The fast afterhyperpolarizing potential (fAHP) evoked by a single action potential was measured as the peak hyperpolarizing deflection from action potential threshold. PSP amplitude was measured as the peak voltage deflection in response to synaptic stimulation. PSP summation was quantified as the ratio of the last PSP to the first PSP. Paired-pulse facilitation was quantified as the amplitude of the second PSP divided by the amplitude of the first PSP. In some experiments, a current shaped similar to a synaptic current was injected into the neuron (
PSP, current shape described by A(t/
All planned comparisons were made with paired t tests, with an
Results
A total of 241 neurons was recorded from layer V of lateral EC that displayed electrophysiological characteristics consistent with previously identified EC layer V pyramidal neurons (Hamam et al., 2002
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Figure 1. Dopamine decreases the excitability of layer V EC pyramidal neurons. A1–A3, Example of a Neurobiotin-filled pyramidal neuron from layer V of lateral EC. Also shown is the electrophysiological response of that filled neuron to a set of subthreshold step current injections from –100 to +100 pA in 25 pA steps, and a step current injection of 200 pA that evokes action potentials. The filled neuron displays morphological and electrophysiological similarities to previously described layer V EC pyramidal neurons. B1–B3, Application of DA (10 µM) decreased the excitability of layer V EC pyramidal neurons, measured as the number of action potentials evoked by a set current step (B1) from the same membrane potential (–70 mV; n = 11). This effect of DA persisted in the presence or absence of blockers of synaptic transmission (B2) (10 µM CNQX, 10 µM bicuculline, 2 µM CGP55845, 50 µM APV; n = 27) and increased the rheobase current (B3) (n = 27). C1, C2, DA decreased the excitability of layer V EC pyramidal neurons to a much lesser extent when the membrane was permitted to depolarize as a result of DA application (n = 6). D, After a 2–3 min application of DA (applied during black bar in plot), the excitability was reduced, and partially recovered after a prolonged washout. This plot was constructed from the data from 12 neurons whose excitability was continuously monitored. The last six data points are from eight of those neurons, whose excitability was monitored for a prolonged period of time. *Significant p value of at least 0.05. Error bars indicate SE.
DA modulation of excitability
Excitability was measured as the number of action potentials evoked by a fixed amplitude of current injection (700 ms duration) from a membrane potential of –70 mV. A current amplitude was chosen that evoked three to five action potentials. After short (2–3 min) bath application of 10 µM DA, there was a significant decrease in the number of evoked action potentials (Fig. 1B) (baseline, 3.86 ± 0.23 action potentials; post-DA, 1.13 ± 0.35 action potentials; n = 11; t = 8.59; p < 0.001; df = 11; paired t test). This was accompanied by an increase in the rheobase current (baseline, 117.3 ± 12.4 pA; post-DA, 129.6 ± 12.4 pA; n = 11; t = –4.50; p = 0.0011; df = 10; paired t test). In most instances, a small,In a separate set of experiments, excitability was examined in the absence of a holding current. This allowed the membrane potential to depolarize after DA application (n = 6; baseline, –68.8 ± 0.9 mV; post-DA, –65.8 ± 0.9 mV; t = –5.81; p = 0.0021; df = 5; paired t test). Nevertheless, there was a significant, albeit smaller, decrease in excitability (Fig. 1C) (baseline, 3.9 ± 0.16 action potentials; post-DA, 3.5 ± 0.15 action potentials; t = 2.88; p = 0.034; df = 5; paired t test).
In some preliminary observations, it was noted that application of DA was accompanied by changes in the occurrence of postsynaptic potentials. Because alterations of synaptic conductances can conceivably induce changes of excitability, these and all subsequent experiments were performed in the presence of blockers for glutamatergic and GABAergic inputs, unless otherwise noted (see Materials and Methods). Application of 10 µM DA still resulted in a decrease of excitability (baseline, 3.65 ± 0.23 action potentials; post-DA, 1.22 ± 0.21 action potentials; n = 27; t = 7.39; p < 0.001; df = 26; paired t test), an increase in rheobase current (baseline, 91.9 ± 4.9 pA; post-DA, 107.0 ± 6.1 pA; n = 27; t = –6.27; p < 0.0001; df = 26; paired t test) (Fig. 1B), and a 2–3 mV depolarization. The persistence of these effects of DA when synaptic inputs were blocked indicated that they were likely attributable to direct postsynaptic actions. The onset of the effect on excitability could be observed within 3–4 min after the start of the DA application and persisted after the 2–3 min DA application (Fig. 1D). The effects of DA on excitability reversed to near-baseline values after washout periods close to 15 min.
Application of DA also resulted in a small decrease of the measured input resistance (Fig. 2A) (RN baseline, 105.3 ± 6.7 M
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Figure 2. Dopamine modulates membrane properties of layer V EC pyramidal neurons. A, Application of DA (10 µM) reduced the input resistance, measured from –70 mV as the response to small current steps (–40 to +40 pA). B, Application of DA also sped the membrane time constant (Tau), measured by least-squares fitting of two exponentials to the beginning of the voltage response to a small current step (traces displayed are an average of 10 consecutive sweeps). The time course of the change of membrane time constant is plotted below the trace. The bottom plots demonstrate the fitted exponential (thin black line) for the averaged voltage response displayed in B and the residuals. C, Group data for the effects of DA on RN (n = 32) and Tau (n = 32). *Significant p value of at least 0.05. Error bars indicate SE.
In a separate group of neurons, the effects of DA were examined with the membrane potential held at –70 mV with continuous direct current, and also at a more depolarized potential that was still subthreshold to spontaneous action potential firing (close to –50 mV, smaller direct current steps were used to evoke action potentials). The effect of DA on excitability was eliminated at the depolarized membrane potential (Fig. 3A,B) (n = 5; –70 mV baseline, 3.7 ± 0.2; post-DA, 0.63 ± 0.3; t = 8.34; p = 0.001; df = 4; paired t test; close to threshold baseline, 3.8 ± 0.2; post-DA 4.3 ± 0.3; t = 2.6; p = 0.06; df = 7; paired t test). To examine this more systematically, the membrane potential was shifted through a range of voltages (–85 to –50 mV). A current intensity was used that evoked approximately four action potentials from a baseline membrane potential of –70 mV. To exclude the possibility that the effects of DA were specific to a small range of current intensities, two different amplitudes of current steps were also used, while shifting the membrane potential through this range of voltages. The two other current intensities evoked one action potential from a baseline membrane potential of –70 mV or approximately eight action potentials from –70 mV. As above, application of DA reduced excitability measured from –70 mV at these three frequencies. However, the reduction of excitability observed after application of DA was reduced at depolarized potentials, often reversing at more depolarized potentials (Fig. 3C) (n = 7). This may be consistent with two possibilities: (1) DA may modulate an ionic conductance that is deactivated by prolonged depolarization, or (2) the effect is simply dependent on the amplitude of the direct current step used to evoke action potentials, and the effects will not be observed with a wider range of steps. To determine whether this alone, independent of the membrane potential, could explain the results, a series of direct current step amplitudes was examined, from a potential close to –70 mV. The effects of DA remained throughout a range of stimulus amplitudes (Fig. 3B2) (n = 12). However, the effects were larger with greater amplitude direct current steps. One possibility is that DA modulated a current that was both dependent on the absolute "resting" potential as well as the relative change of potential during a direct current step. These features are consistent with a current that is voltage dependent, in that the effects of DA were greatest when greater changes of voltage were used, and the current was deactivated by prolonged depolarization, as the effects were minimized at continuous depolarized potentials.
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Figure 3. The effects of dopamine depend on the membrane voltage. A1, A2, Application of DA reduced the excitability of layer V EC neurons, when action potentials were evoked from –70 mV, but not from a more depolarized membrane potential close to action potential threshold (–55 to –50 mV in this plot; n = 8). B1, B2, The effects of DA on excitability persisted over a range of stimulation intensities (n = 12; the x-axis represents multiplicatives of the threshold stimulation intensity). The action potentials were evoked by current injection at a baseline membrane potential of –70 mV. C, The voltage dependence of the effects of DA on excitability were maintained over a series of firing frequencies when the neuron was shifted through a range of membrane potentials (n = 7) (for more details, see Results). In this panel, three different amplitudes of step current injection were alternatingly used while the membrane potential was shifted through a range of voltages: stimulation intensity 1, a current injection that evokes one AP from a baseline membrane potential of –70 mV; stimulation intensity 2, a current injection that evokes approximately four APs from a baseline membrane potential of –70 mV; and stimulation intensity 3, a current injection that evokes approximately eight APs from a baseline membrane potential of –70 mV. *Significant p value of at least 0.05. Error bars indicate SE.
Excitability: ion channels
Layer V EC neurons are likely to express an array of voltage-dependent and -independent conductances. The results above suggest that a voltage-dependent conductance may be involved in the effects of DA. To further understand what ion channels may be modulated by DA, and thereby alter excitability, the contributions of voltage-dependent and -independent ion channels were pharmacologically probed. Application of 4-AP (3 mM), a blocker of the voltage-dependent delayed K+ current (ID) (Hermann and Gorman, 1981
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Table 1. Effects of ion channel blockers on excitability
Although these data demonstrate that numerous ion channels may be involved in regulating the excitability of layer V EC neurons, the effects of DA on excitability were only blocked by preapplication of ZD7288 (20 µM) or cesium (10 mM) (Figs. 4
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Figure 4. Blockade of select K-channels does not block the actions of dopamine on the excitability of layer V EC pyramidal neurons. Preapplication of TEA (1 mM; n = 6), 4-AP (3 mM; n = 8), or barium (250 µM; n = 7) did not block the DA-induced reduction of excitability of layer V EC pyramidal neurons. These pharmacological agents were applied to the bath at least 15 min before DA was applied. *Significant p value of at least 0.05. Error bars indicate SE.
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Table 2. Suppression of the DAergic effects on excitability by pharmacological blockade of h-channels
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Figure 5. Manipulation of calcium-related events does not block the actions of DA on the excitability of layer V EC pyramidal neurons. Blockade of a subset of voltage-dependent calcium channels by cadmium (200 µM; n = 5) (A) or nickel/nimodipine (50/10 µM) (B) did not attenuate the DA-mediated reduction of neuronal excitability in layer V EC pyramidal neurons. Neither did inclusion of a high concentration of BAPTA (20 µM; n = 5) in the recording pipette (C) or blockade of BK channels by UCL1684 (100 nM; n = 6) (D). *Significant p value of at least 0.05. Error bars indicate SE.
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Figure 6. Blockade of h-channels by nonspecific and specific antagonists reduces the effects of dopamine on excitability of layer V EC pyramidal neurons. Preapplication of cesium (10 mM; n = 9) (A) or ZD7288 (20 µM; n = 11) (B) attenuated the effects of DA (10 µM) on neuronal excitability (C1). This effect was maintained over a range of current injection intensities (C2) and a range of membrane potentials (D), indicating that blockade of Ih abolished the voltage-dependent attenuation of excitability caused by DA. In these experiments, there was even a tendency toward an enhancement of excitability after DA application when h-channels were blocked. Cesium (E) and ZD7288 (F) also blocked the effects of DA on input resistance. G, Group data demonstrate that preapplication of 10 mM cesium (n = 9) or 20 µM ZD7288 (n = 11) attenuated the DA-mediated reduction of input resistance (F). Error bars indicate SE.
The change in input resistance can be a significant contributor to the alterations of excitability. Therefore, we examined which ion channels normally contribute to the apparent input resistance of these neurons close to resting potentials (Table 3). Application of barium (250 µM), TEA (1 mM), Cs+ (10 mM), or ZD7288 (20 µM) all increased the input resistance, measured close to –70 mV. Application of 4-AP (3 mM) produced only a small change of input resistance measured at –70 mV. Application of Ni2+ (50 µM), nimodipine (10 µM), and UCL1684 (100 nM), blockers of Ca2+ currents and Ca2+-activated K+ currents, had no significant effects on the measured input resistance. Thus, of all the compounds that exert a significant effect on excitability, only those that block h-currents, sustained K+ currents, or IRK channels also appeared to alter the input resistance near the resting membrane potential.
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Table 3. Blockade of DAergic effects on input resistance by blockers of h-channels
The effects of DA on input resistance measured at –70 mV were blocked by ZD7288 (20 µM) or by cesium (10 mM), but not by any of the other compounds listed above (Fig. 3, Table 3), indicating that the effects of DA on the apparent input resistance were likely attributable to modulation of h-channels. Furthermore, these results provide additional evidence for an association between the effects of DA on RN and on excitability. In addition, the effects of DA on the membrane time constant were blocked by ZD7288 (20 µM; baseline with ZD7288, 70.8 ± 11.4 ms; post-DA with ZD7288, 73.9 ± 13.9 ms; n = 11; t = 0.907; p = 0.399; paired t test).To further investigate the target of DAergic modulation observed in this study, other electrophysiological parameters were compared before and after DA. The transient Na+ current (INa) was monitored by measuring the peak amplitude and rise time of the action potential peak before and after DA. There was no significant difference in the action potential amplitude, although many neurons displayed a tendency for an increase (baseline, 104.8 ± 1.1 mV; post-DA, 105.7 ± 1.0 mV; n = 27; t = 0.27; p = 0.788; df = 26; paired t test). The action potential rise time did not significantly change (baseline, 96.8 ± 5.53; post-DA, 99.8 ± 5.77; t = –0.75; df = 17; p = 0.46). There was no significant change in the action potential threshold after application of DA (baseline, –38.3 ± 0.8 mV; post-DA, –39.0 ± 0.8 mV; n = 18; t = 1.89; p = 0.075; df = 17; paired t test).
To examine currents that underlie the afterhyperpolarization potential (AHP), both the fAHP evoked by a single action potential and the sAHP evoked by a train of action potentials were measured before and after DA. The amplitude of the fAHP decreased after application of DA (Fig. 7A) (baseline, 10.5 ± 0.6 mV; post-DA, 9.6 ± 0.5 mV; n = 27; t = 2.76; p = 0.011; df = 26; paired t test). A reduction of fAHP would not be expected to cause the decrease of excitability observed after application of DA. In conjunction with the pharmacological evidence that demonstrates that the effects of DA were not reduced when BK channels were blocked, this suggests that BK channels are unlikely to be the target of DA in its actions that reduce excitability. The amplitude of the sAHP, measured from a membrane potential of near –55 mV, was also decreased after application of DA (Fig. 7B) (baseline, 8.4 ± 0.9 mV; post-DA, 5.9 ± 0.8 mV; n = 10; t = –2.99; p = 0.015; df = 9; paired t test), as seen in other studies (Malenka and Nicoll, 1986
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Figure 7. Application of dopamine enhances voltage sag in response to a hyperpolarizing current step. Application of 10 µM DA reduced the amplitude of the fAHP evoked by a single action potential (A) (n = 27) and reduced the amplitude of the sAHP evoked by a train of action potentials (B) (n = 10). Therefore, it is unlikely that changes in fAHP or sAHP could account for the DA-induced reduction of excitability. The voltage sag that can be observed after hyperpolarization of the membrane with current steps was enhanced after DA application (C) (n = 32), measured as the ratio between the voltage deflection at steady-state and peak voltage changes. Additionally, the voltage overshoot observed after cessation of the current step was also enhanced after application of DA (D) (n = 23). The black traces are baseline conditions, and the red traces are after DA application. *Significant p value of at least 0.05. Error bars indicate SE.
The voltage sag during prolonged hyperpolarizing direct current steps and the rebound overshoot after cessation of the current step are hallmarks of the presence of Ih. To obtain independent, nonpharmacological evidence for a role of h-channels, the voltage sag ratio in response to a prolonged (700 ms) hyperpolarizing direct current step and the normalized voltage rebound were examined. The voltage sag persisted in the presence of barium (250 µM), so it is unlikely to be attributable to M-currents or KIRs in these neurons (Armstrong et al., 1982A subset of key experiments was replicated at more physiological temperatures (
Modulation of dendritic excitability
Several studies from other brain regions indicate that most of pyramidal neuronal h-channels are preferentially located in the apical dendrites, HCN channels are found on the apical dendrites of pyramidal neurons (Lorincz et al., 2002
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Figure 8. Dopamine modulates dendritic excitability and voltage sag. A, The amplitude of the voltage sag in response to a prolonged hyperpolarizing step current, a reflection of h-channel activation, increases at more distal apical dendritic recording sites. This plot depicts somatic recordings (at 0 µm; mean ± 2 SDs) and the sag ratio recorded from the apical dendrite at variable distances from the soma. B, Application of DA (10 µM) increases the amplitude of the voltage sag (n = 10; 150–225 µm from the soma). The amplitude of this effect is greater in the dendritic recordings than in the somatic recordings. C, In parallel with the enhanced voltage sag, the excitability of the dendrites is also reduced after application of DA (n = 12; 150–225 µm from the soma). The black traces are baseline conditions, and the red traces are after DA application. *Significant p value of at least 0.05. Error bars indicate SE.
Excitability: DA receptor subtype and second messenger system
To determine which DA receptors are involved in the reduction of excitability and input resistance, two approaches were used: specific agonists for DA D1 or D2 receptors were used, and specific antagonists for DA D1 or D2 receptors were applied at least 10 min before application of DA. Application of the DA D1 agonist SKF81297 (10 µM) mimicked the effects of DA, decreasing the excitability of layer V EC pyramidal neurons (Fig. 9A) (baseline, 3.27 ± 0.48 action potentials; post-SKF81297, 1.47 ± 0.39 action potentials; n = 6; t = 4.58; p = 0.0059; df = 5; paired t test). In addition, preapplication of the DA D1 antagonist SCH23390 (5 µM) prevented the DA-induced decrease in excitability usually observed with application of 10 µM DA (Fig. 9B) (baseline SCH23390, 3.70 ± 0.37 action potentials; post-DA, 3.93 ± 0.43; n = 5; t = –2.18; p = 0.117; df = 4; paired t test). The effects of DA were not mimicked by the DA D2 agonist quinpirole (10 µM), which tended to increase the excitability of layer V EC pyramidal neurons (Fig. 9C) (baseline, 3.51 ± 0.18 action potentials; post-quinpirole, 4.14 ± 0.18 action potentials; n = 6; t = –2.22; p = 0.077; df = 5; paired t test). Preapplication of the DA D2 antagonist sulpiride (5–10 µM) did not significantly reduce the effects of DA (10 µM) on excitability (Fig. 9D) (baseline sulpiride, 3.76 ± 0.31 action potentials; post-DA, 3.00 ± 0.51; n = 5; t = 2.26; p = 0.87; df = 4; paired t test). This is consistent with a DA D1 receptor-mediated effect of DA in reducing layer V EC neuronal excitability.
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Figure 9. The actions of dopamine on the excitability of layer V EC pyramidal neurons is likely mediated by activation of D1 receptors. Application of the DA D1 agonist SKF81297 (10 µM; n = 6) mimicked the effects of dopamine on excitability (A), and the effects of DA were attenuated by preapplication of the DA antagonist SCH23390 (5 µM; n = 5) (B). The effects of DA were not mimicked by the DA D2 agonist quinpirole (10 µM; n = 6) (C), and the effects of DA were not blocked by preapplication of the DA D2 antagonist sulpiride (5–10 µM; n = 5) (D). *Significant p value of at least 0.05. Error bars indicate SE.
Traditionally, DA receptors are thought to affect cellular events by modulation of cAMP levels via G-protein-mediated alteration of adenylyl cyclase activity. To determine whether the effects of DA are mediated via the cAMP pathway, activators and inhibitors of the cAMP pathway were used. The effects of DA (10 µM) were greatly attenuated when the blocker of adenylyl cyclase, SQ22536, (1 mM) was included in the recording pipette, suggesting that the effects of DA were likely mediated by DA-induced activation of adenylyl cyclase (Fig. 10) (baseline, 3.65 ± 0.20 action potentials; post-DA, 3.19 ± 0.30 action potentials; n = 6; t = 1.48; p = 0.20; df = 5; paired t test). It is known that cAMP can exert actions directly on ion channels or can activate protein kinase A, which phosphorylates many ion channels (Gershon et al., 1992
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Figure 10. The actions of dopamine on the excitability of layer V EC pyramidal neurons appear to be mediated by a cAMP-dependent, but PKA-independent pathway. A, To examine the second messenger system that may be involved, 1 mM SQ22536 was included in the pipette to block the activation of adenylyl cyclase by G-proteins, 100–250 µM 8-Br-cAMP or 50 µM forskolin was used to mimic DA-mediated enhancement of adenylyl cyclase, or 1.2 µM KT5720 was included in the pipette to block activation of PKA by cAMP. B, The effects of DA on excitability were blocked when SQ22536 was included in the pipette (n = 6), indicating that a G-protein-mediated cascade is activated by DA. Neither 8-Br-cAMP (n = 5) nor forskolin (n = 10) alone mimic the effects of DA, yet they did mimic the effects of DA when the PKA blocker KT5720 was included in the pipette (8-Br-cAMP, n = 6; forskolin, n = 6). This may indicate that there are pathways in these neurons by which cAMP can act independently of PKA, and furthermore, this pathway exerts a similar action as DA. Finally, the effects of DA on excitability were not occluded by inclusion of KT5720 in the pipette (n = 7), indicating, along with the previous results, that the actions of DA on excitability are likely mediated by direct cAMP-mediated modulation of h-channels, or other protein. *Significant p value of at least 0.05. Error bars indicate SE.
Modulation of synaptic summation
To investigate the functional effects of DA-mediated modulation of h-channels on EPSP integration, we stimulated trains of EPSPs (3–9 mV EPSPs; 10 stimuli at 50 ms intervals) before and after applying DA. In these experiments, the concentration of CNQX was decreased to 0.1–1 µM (a minimal amount of CNQX was necessary to avoid epileptiform activity evoked by synaptic stimulation when GABAA receptors were blocked; blockade of GABAA receptors may artificially prolong the duration of the EPSP, and therefore the contribution of h-channels to synaptic integration). EPSPs measured at –70 mV displayed only a small degree of temporal summation (Fig. 11A). Application of 10 µM DA further reduced EPSP summation (Fig. 10A) (baseline, 1.4 ± 0.2; post-DA, 1.2 ± 0.3; n = 6; t = 2.49; p = 0.05; df = 5; paired t test) without a significant effect on EPSP amplitude or paired pulse facilitation (baseline amplitude, 5.6 ± 1.5 mV; post-DA, 4.9 ± 1.0 mV; n = 6; t = 0.99; p = 0.37; df = 5; paired t test; baseline paired-pulse facilitation, 1.56 ± 0.12; post-DA, 1.52 ± 0.11; n = 6; t = 0.90; p = 0.41; df = 5; paired t test).
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Figure 11. Dopaminergic modulation of EPSP summation and APs evoked by EPSPs depends on h-channels. A, EPSPs displayed a small degree of temporal summation at rest and at hyperpolarized potentials (black traces). Application of DA caused an additional reduction in summation of EPSPs (red traces; n = 6). B, Inclusion of 20 µM ZD7288 in the recording pipette caused a gradual decrease in the voltage sag measured during a small hyperpolarizing current step and a co-occurring enhancement of EPSP summation. This was quantified as ratio of the amplitude of the last EPSP to the first EPSP, as exemplified in the traces and plot of data from this neuron. C, When 20 µM ZD7288 was included in the recording pipette, summation was greatly enhanced compared with control neurons (n = 7; black traces). Furthermore, application of DA resulted in an increase of EPSP summation (red traces; n = 7). D, When the membrane potential is depolarized with constant DC, EPSPs can summate enough to evoke action potentials. In control conditions, application of DA resulted in a reduction in the probability of an action potential evoked by a train of EPSPs (n = 5; left traces). When ZD7288 was included in the recording pipette, application of DA resulted in an increase of the probability of action potentials evoked by EPSPs (right traces; n = 7) The group data are quantified in E (probability measured from EPSPs evoked at –65 mV in this plot). The black traces are baseline conditions, and the red traces are after DA application. *Significant p value of at least 0.05. Error bars indicate SE.
To examine the role of h-channels in the effects of DA on EPSP summation and EPSP–action potential coupling, 20 µM ZD7288 was included in the recording pipette. Inclusion of ZD7288 in the pipette resulted in a gradual decrease in the measured voltage sag accompanied by an increase in the summation of EPSPs after initiation of whole-cell recordings (Fig. 11B). Control neurons displayed a stable sag ratio over a similar time course (1.13 ± 0.03 atIn addition, when summation of a train of EPSPs was great enough to result in action potential firing, DA reduced the probability of action potential firing (Fig. 11D) (baseline action potential probability, 1.16 ± 0.13 AP/train; post-DA, 0.45 ± 0.16 AP/train; n = 5; t = 10.96; p < 0.001; df = 4; paired t test; EPSPs evoked from –65 mV were used for this comparison). In contrast, when ZD7288 was included in the recording pipette, the probability of action potential firing evoked by EPSP trains was increased (Fig. 11D) (baseline action potential probability, 1.1 ± 0.1 AP/train; post-DA, 1.7 ± 0.3 AP/train; n = 5; t = –3.76; p = 0.02; df = 4; paired t test). Thus, it appears that DA can reduce the summation of a train of large EPSPs, and reduce EPSP–action potential coupling, in a manner that depends on h-channels.
To exclude a potential presynaptic effect of DA, as well as to examine modulation of inputs to the dendrites,
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Figure 12. Dopamine modulates
Discussion
The DA system is known to have a potent impact on functions that depend on the EC, such as recall and consolidation of memory (Ardenghi et al., 1997Application of DA caused a decrease of the excitability of layer V EC pyramidal neurons. This action of DA displayed a unique voltage dependence indicative of the participation of voltage-gated ion channels. Our data indicate that several voltage-dependent ion channels can regulate the excitability of layer V EC pyramidal neurons, including TEA- and 4-AP-sensitive K+ channels, and h-channels. However, our data are consistent with DAergic modulation of h-channels, and not these other channels, in the regulation of excitability. After application of DA, several indicators of h-channel activity were enhanced, including voltage sag and rebound voltage overshoot. The accompanying decrease of RN and faster membrane time constant indicate enhancement of a conductance that is active near Vrest. Furthermore, the suppressive effects of DA were reduced or reversed at depolarized potentials, and were pharmacologically blocked by ZD7288 and cesium. Thus, although h-currents were not directly measured, and voltage sag is only an indirect measure of h-channel activity, it is likely that h-channels underlie the effects of DA on excitability. Other ion channels are unlikely to account for the observed effects, because none of the K+ or Ca2+ channel blockers used attenuated the actions of DA.
Although our evidence indicates that modulation of h-channels may be the main process by which DA alters excitability in these neurons, it is unlikely to be the only channel modulated by DA. Specifically, when h-channels were inactivated by depolarization or pharmacologically blocked, an increase, not a decrease, of excitability was observed after application of DA. Potential explanations include an increase of INa or the observed decreases in the fast
