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Department of Physiology and Biophysics, University of Illinois at Chicago, College of Medicine, Chicago, Illinois
Submitted 2 June 2005; accepted in final form 27 December 2005
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ABSTRACT |
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INTRODUCTION |
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) and the neurons in the VTA are classified into two major neuronal subgroups: dopaminergic (DA) and GABAergic neurons (Grace and Onn 1989; Johnson and North 1992). DA neurons constitute the majority of the neuronal population (70%) and 
-aminobutyric acid (GABA) neurons represent 30% of the neuronal population in the VTA (Johnson and North 1992) and DA VTA neurons are involved in the reinforcing effects of natural stimuli and drugs of abuse (Appel et al. 2004; Robinson and Berridge 2003; Wise 1987, 2002). The cell bodies of DA VTA neurons send terminal projections that synapse in the nucleus accumbens. Excitation of DA VTA neurons by drugs of abuse such as ethanol or nicotine (Brodie 1991; Brodie et al. 1999b) results in increased dopamine release in the nucleus accumbens (DiChiara and Imperato 1988; Weiss et al. 1993), which is important for the reinforcing effects of these drugs (Corrigall et al. 1994; Pfeffer and Samson 1988). DA VTA neurons have intrinsic pacemaker activity and several voltage-dependent ionic currents contribute to the excitability of these neurons (Brodie et al. 1999b; Grace and Onn 1989; Johnson and North 1992; Neuhoff et al. 2002).
M-current (IM) is a voltage-dependent, slow delayed rectifier K+ current that is activated at the subthreshold range of membrane potential and contributes to regulation of repetitive firing and excitability (Aiken et al. 1995; Brown and Adams 1980). IM has been shown to be modulated by drugs of abuse including cannabinoids, opiates, and ethanol (Koyama and Appel 2004; Moore et al. 1990, 1994; Schweitzer 2000). IM is mediated by current through KCNQ-type potassium channels (Wang et al. 1998). To date, five different KCNQ genes have been cloned and KCNQ2, 3, 4, and 5 channel subunits have been reported to mediate IM in various types of neurons (Lerche et al. 2000; Passmore et al. 2003; Selyanko et al. 2000; Shah et al. 2002; Shapiro et al. 2000; Søgaard et al. 2001; Wang et al. 1998). Immunohistochemical studies have shown that the KCNQ2 and KCNQ4 channel proteins are present in VTA neurons (Cooper et al. 2001; Kharkovets et al. 2000). Consistent with the presence of KCNQ channel proteins found in these anatomical studies, IM has been shown to be present in midbrain dopamine neurons with intracellular voltage-clamp recording in brain slices (Lacey et al. 1990), although it has not been studied in detail in these neurons.
The aims of the present study were 1) to characterize the electrophysiological parameters of IM in DA VTA neurons with voltage-clamp recording for comparison to IM in other types of neurons; 2) to determine the sensitivity of IM in DA VTA neurons to tetraethylammonium (TEA) and 10,10-bis(4-pyridinylmethyl)-9-(10H)-anthracenone (XE991), which has been shown to depend on the KCNQ channel subtypes that mediate the current in other types of neurons; and 3) to examine the physiological role of IM in the electrical behavior of DA VTA neurons in current-clamp experiments by blocking IM with XE991. The present study was performed in acutely dissociated rat DA VTA neurons, stripped of synaptic terminals (Koyama et al. 2005), and therefore the actions of the drugs tested reflect a direct postsynaptic action on these neurons. Nystatin-perforated patch-clamp recording was used to minimize intracellular dialysis and prevent the rundown of IM. Rundown occurs rapidly with whole cell patch-clamp recording because intracellular nucleotide phosphates are indispensable for maintenance of IM (Simmons and Schneider 1998). Some of the results of the present study were reported previously in abstract form (Koyama and Appel 2004).
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METHODS |
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Animals used in this study were treated in strict accordance with the American Physiological Society's Guiding Principles in the Care and Use of Animals and the U.S. National Institutes for Health Guide for the Care and Use of Laboratory Animals; the protocol for all experimental methods was approved by the Institutional Animal Care Committee of the University of Illinois at Chicago. Fisher 344 rats (14–20 days old, male and female) were decapitated and the brain quickly removed. As an inbred strain, Fisher 344 rats showed smaller animal-to-animal variation in ethanol sensitivity in our previous studies; we used Fisher 344 rats here for the comparison with future ethanol studies on IM. The brain was placed in ice-cold cutting solution (in mM: 220 sucrose, 2.5 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.24 NaH2PO4, 26 NaHCO3, 11 D-glucose), which was constantly bubbled with 95% O2-5% CO2. Transverse brain slices (400 µm thick) were made on a Vibratome (Series 1000 plus, St. Louis, MO). Brain slices were incubated for 30 min in artificial cerebrospinal fluid (aCSF) (in mM: 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.24 NaH2PO4, 26 NaHCO3, 11 D-glucose; osmolarity, 300 mOsm), which was constantly bubbled with 95% O2-5% CO2 at room temperature (23–25°C). The brain slices were then incubated in a HEPES-buffered solution (in mM: 145 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 11 D-glucose; pH 7.4 adjusted with NaOH; osmolarity, 300 mOsm) containing papain (15–18 U/ml) at 32°C for 20–25 min. After papain treatment, the brain slices were further incubated in aCSF for 20–40 min. VTA neurons were dissociated with a vibrating stylus apparatus for dispersing cells from the brain slices as previously described (Koyama et al. 2005). A brain slice was transferred to a poly-D-lysine–coated 35-mm culture dish (Becton Dickinson, Bedford, MA) containing the HEPES-buffered solution. A grid of nylon threads glued to a U-shaped metal frame was used to hold the brain slice down during cell dissociation. After the VTA was visually identified, a vibrating stylus was placed in the appropriate region with a micromanipulator. The stylus was made of glass capillary tubing (1.5 mm OD) pulled to a fine tip, fire-polished (200–400 µm in diameter), and mounted on a vibrating apparatus, which horizontally vibrated the stylus tip (excursions of 100–200 µm at 20–25 Hz). Once the cell dissociation procedure was completed (4–7 min), the brain slice was removed from the culture dish, and the dissociated neurons settled and adhered to the bottom of the dish within 20 min. The dissociation procedure with papain described above has been shown to yield acutely dissociated VTA neurons stripped of synaptic terminals (Koyama et al. 2005) and no spontaneous synaptic potentials or synaptic currents were recorded from these neurons in the present study.
Electrophysiological recording
Electrophysiological measurements were made with an Axopatch-1B patch-clamp amplifier (Axon Instruments, Union City, CA). Microelectrodes were fabricated on a P-87 puller (Sutter Instrument Company, Novato, CA), from LE16 glass capillaries (Dagan, Minneapolis, MN) and heat-polished on a microforge (Narishige, Tokyo, Japan). The tip resistances of the electrodes were 1–2.5 M
when filled with pipette solution (in mM: 60 K-acetate, 60 KCl, 1 CaCl2, 2 MgCl2, 40 HEPES; pH 7.2 adjusted with KOH, final [K+]i = 131 mM; osmolality was adjusted to 290 mOsm with sucrose). Nystatin-perforated patch-clamp recording (Akaike and Harata 1994) was used to minimize intracellular dialysis. Nystatin was dissolved in methanol at a concentration of 10 mg/ml. This nystatin stock solution was diluted with pipette solution to a final concentration of 100–200 µg/ml and the electrodes were backfilled with this solution. After the cell-attached configuration was attained, the access resistance was periodically monitored with hyperpolarizing voltage steps (amplitude 10 mV, duration 100 ms) and capacitive transients were cancelled. When the access resistance had reached a steady level (10.6 ± 0.4 M, n = 72), the recording was started. In case of the sudden change of the access resistance, the recording was stopped. Current- and voltage-clamp recordings were done in the HEPES-buffered solution constantly bubbled with 100% O2. The liquid junction potential between the pipette solution and the HEPES-buffered solution was estimated to be 5 mV (Neher 1992) and the results have been corrected by this amount. Membrane currents and voltage were filtered at 1 kHz by a –3-dB four-pole filter and acquired at a sampling frequency of 10 kHz. Data acquisition was performed with a DigiData 1322A interface and pClamp software version 9.0 (Axon Instruments). The dissociated VTA neurons were visualized under phase-contrast optics on an inverted microscope (Diaphot 300, Nikon, Tokyo, Japan). All experiments were performed at room temperature (23–25°C).
Drug application
Neurons were continuously bathed in control HEPES-buffered solution and drugs were dissolved at final concentration in the same solution. Drug solutions were applied by a multiple channel manifold (Becton Dickinson, Bedford, MA). Each channel of the manifold was connected to a gravity-fed reservoir with tubing (860 µm, ID). The output of the manifold was connected to an outflow tube (580 µm, ID), the tip of which was placed within 200 µm of the soma of the recorded neuron. Solution flowed continuously through one manifold channel. Application of drug solutions was controlled by opening or closing valves connected to the reservoirs.
Source of drugs and chemical agents
The following drugs and chemical agents were used in this study. Nystatin and TEA were purchased from Sigma (St. Louis, MO). Papain was purchased from Worthington (Lakewood, NJ), and XE991 dihydrochloride was purchased from Tocris-Cookson (Ellisville, MO).
Data analysis and curve fitting
Action potentials were analyzed off-line with pClamp 9.0 software (Axon Instruments). All average values are expressed as mean ± SE. Statistical comparison was done by Student's t-test to assess significant differences. Graphing and curve fitting of data were performed with Origin 7 software (OriginLab, Northampton, MA). The inward relaxation current, which was attributed to deactivation of IM, was fitted by a single exponential function of the form
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is the decay time constant.
Concentration–response curves for XE991 and TEA were constructed by plotting percentage inhibition of IM as a function of drug concentration plotted on a log scale. Smooth curves were fit to these data with the Hill equation of the form
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RESULTS |
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In the present study, we recorded 72 spontaneously active dopamine neurons dissociated from the VTA with nystatin-perforated patch current-clamp recording. To identify VTA neurons as dopaminergic, we analyzed cell capacitance, spontaneous firing frequency, and action potential shape parameters in all of these neurons as shown in Table 1 and Fig. 1A. In addition, in some of the same experiments, we measured input resistance and estimated resting membrane potential in 20 typical neurons out of the 72 DA VTA neurons (Table 1).
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). Hyperpolarizing voltage steps (1-s duration) were given from a holding potential of –25 to –65 mV (in 10-mV increments) as shown in Fig. 1B (the currents recorded in response to this protocol are shown below in Fig. 1B, left). IM was measured as the inward relaxation current caused by deactivation of IM during the voltage step as shown in Fig. 1B (right). Figure 1C shows the current–voltage relationship of IM for 13 DA VTA neurons. The mean IM amplitude was voltage dependent and the maximal IM amplitude (64.9 ± 7.4 pA) was measured at –45 mV. The deactivation time constant of IM was determined by fitting the current measured at each voltage with a single exponential function. Figure 1D shows the mean deactivation time constant of IM as a function of voltage (n = 13). The mean IM deactivation time constant was 105.6 ± 10.4 ms (at –65 mV), 138.4 ± 13.2 ms (at –55 mV), 193.5 ± 24.5 ms (at –45 mV), and 241.8 ± 30.1 ms (at –35 mV), indicating that it was voltage dependent. Note that the deactivation time constant was a linear function of voltage (correlation coefficient r = 0.99) and was shorter at more negative membrane potentials (faster deactivation). DA VTA neurons have a prominent A-type transient K+ current that was inactivated at the holding potential of –25 mV and therefore did not obscure the measurement of IM with the voltage protocol shown in Fig. 1. A-current activation can be seen after the offset of the larger hyperpolarizing voltage steps in Fig. 1B (left).
XE991 inhibition of IM
XE991 has been shown to be a potent and selective inhibitor of IM in native neurons and currents from expressed KCNQ channels (Wang et al. 1998). Figure 2A1 (top) shows that a maximal concentration of XE991 (30 µM) greatly reduced IM in a typical DA VTA neuron. This effect was accompanied by a reduction of the baseline outward current. Figure 2A1 (bottom) shows the XE991-sensitive current obtained by digital subtraction of the XE991-resistant component (top right) from the control current (top left). The deactivation time constant of XE991-sensitive current was 190.0 ms and that of the control IM was 186.1 ms. In these experiments, IM was monitored with a 1-s-long hyperpolarizing voltage step from a holding potential of –25 to –40 mV (Fig. 2B1, inset). To determine the time course of the XE991 inhibition, IM was monitored with this voltage step every 20 s. Figure 2A2 shows the inhibition by 30 µM XE991 of the mean IM measured in seven DA VTA neurons versus time. The mean maximal reduction of IM amplitude with 30 µM XE991 was 90.2 ± 2.3% and this reduction was partially reversible after the washout of XE991. The concentration dependency of XE991 inhibition is shown in Fig. 2, B1 and B2. Note that XE991 reduced both IM and the baseline outward current in a concentration-dependent manner in this typical DA VTA neuron (Fig. 2B1). Figure 2B2 shows the pooled concentration–response curve that plots mean percentage inhibition of IM amplitude versus log concentration of XE991 from seven DA VTA neurons. The mean inhibition of IM by XE991 was 27.2 ± 3.8% (at 0.3 µM), 51.4 ± 2.9% (at 1 µM), 81.4 ± 1.7% (at 3 µM), and nearly complete block of IM was seen with the two highest concentrations tested: 86.6 ± 2.5% (at 10 µM) and 90.2 ± 2.3% (at 30 µM). A smooth curve was fit to the mean data in Fig. 2B2 with the Hill equation (see METHODS). The IC50 for XE991 was 0.71 µM and the power term n (Hill slope), which is related to the steepness of curve, was 1.14. The goodness of fit R2 was 0.988.
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Previous studies reported that different KCNQ channel proteins have different sensitivities to TEA (Hadley et al. 2000; Shapiro et al. 2000; Wang et al. 1998), and therefore it was of interest to examine the TEA sensitivity of IM in DA VTA neurons. Figure 3A shows that TEA caused a concentration-dependent reduction in IM in this typical DA VTA neuron. TEA also caused a concentration-dependent reduction in the baseline outward current. The voltage protocol used to measure IM is shown in the inset. Figure 3B shows the pooled concentration–response curve that plots mean percentage inhibition of IM amplitude versus log concentration of TEA from five DA VTA neurons. The mean inhibition of IM by TEA was 13.5 ± 3.2% (at 0.3 mM), 32.3 ± 3.9% (at 1 mM), 74.0 ± 5.3% (at 3 mM), and nearly complete block of IM (97.6 ± 0.5%) was seen with 10 mM TEA. A smooth curve was fit to the mean data in Fig. 3B with the Hill equation (see METHODS). The IC50 for TEA was 1.51 mM and the power term n (Hill slope) was 1.49. The goodness of fit R2 was 0.990. The sensitivity to TEA has been used as an indication of KCNQ subunit composition, and thus for comparison, we also used the Hill equation to fit the log concentration–response curves and to determine the IC50 and Hill slope for each of the five DA VTA neurons individually. The mean of the IC50 values determined for each individual experiment was 1.56 ± 0.27 mM and the mean Hill slope was 1.58 ± 0.08. These were very similar to the IC50 and Hill slope determined from the pooled log concentration–response curve shown in Fig. 3B described above.
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The specific antagonist XE991 was used to block IM to assess the contribution of IM to the shape of the evoked action potential in DA VTA neurons. These experiments were performed with nystatin-perforated patch recording in current-clamp mode. Continuous DC hyperpolarizing current was injected to prevent spontaneous action potential firing and to maintain the baseline membrane potential constant throughout the experiment. A small depolarizing current pulse (+10 pA, inset) was used to evoke one or two action potentials before and after XE991 treatment. Figure 4A1 shows action potentials evoked in a typical DA VTA neuron before (left) and in the presence of 10 µM XE991 (right). A large hyperpolarizing current pulse (–70 pA) activated a prominent time-dependent inward rectification (voltage sag) arising from the presence of Ih, indicating that this neuron was a dopamine neuron (Figs. 4A1 and 5A1). Blockade of IM with 10 µM XE991 shortened the latency to the first evoked action potential (arrows) and reduced the interspike interval (ISI) (Fig. 4A1). Figure 4 A2 and A3 show the first evoked action potentials before and after treatment with 10 µM XE991 from the same neuron, on a short (20-ms) and long (340-ms) timescale, respectively. XE991 reduced the fast and slow components of the afterhyperpolarization (AHP) without affecting the middle component of AHP. Figure 4B illustrates measurement of the evoked action potential shape parameters shown in Fig. 4A3 and Table 2. AHP-1, AHP-2, and AHP-3 correspond to the fast, middle, and slow components of AHP mentioned above. Table 2 shows the effects of 1, 3, and 10 µM XE991 on evoked action potential shape parameters in DA VTA neurons. XE991 significantly reduced the amplitude of the fast (AHP-1) and slow (AHP-3) components, without affecting the amplitude of the middle component (AHP-2). The size of these effects appeared to be concentration dependent. XE991 (1–10 µM) did not affect evoked action potential amplitude, half-width (duration at half-amplitude), or threshold. In addition, 10 µM XE991 did not significantly change Ih, as measured by the amplitude of the voltage sag induced by a hyperpolarizing current step of –70 pA (39.7 ± 5.4 mV in control, 37.8 ± 5.0 mV with XE991; paired Student's t-test, P > 0.4, n = 7). In five DA VTA neurons, 30 µM XE991 caused a small prolongation of action potential half-width (1.4 ± 0.2 ms in control, 1.6 ± 0.3 ms in XE991; paired Student's t-test significant, P < 0.05); this effect was not seen at lower concentrations. As seen with 1–10 µM XE991, 30 µM XE991 also caused a significant reduction in AHP-1 (26.9 ± 2.7 mV in control, 15.8 ± 1.8 mV in XE991; paired Student's t-test, P < 0.01) and AHP-3 (12.8 ± 1.8 mV in control, 8.5 ± 2.0 mV in XE991; paired Student's t-test, P < 0.05) with no effect on AHP-2.
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To examine the contribution of IM to the ISI in DA VTA neurons, a larger depolarizing current pulse than that in Fig. 4 was used to evoke a train of several action potentials in control and in the presence of XE991. Figure 5A shows action potentials evoked by a +30 pA depolarizing current pulse before (left) and in the presence of 10 µM XE991 (right) in the same DA VTA neuron as in Fig. 4A. Measurements were made of the first ISI evoked in each case (arrows). Figure 5A2 shows evoked action potentials recorded from the same DA VTA neuron before (black line) and after 10 µM XE991 treatment (gray line), superimposed for comparison of the first ISIs. Note that XE991 reduces the slow AHP, which leads to earlier occurrence of the second action potential, i.e., a shortening of the ISI. Figure 5B summarizes the mean effect of XE991 on the first ISI in seven DA VTA neurons. XE991 (10 µM) significantly reduced the first ISI (278.0 ± 44.2 ms in control, 221.6 ± 44.6 ms in XE991; paired Student's t-test, P < 0.01). As can be seen in Fig. 5A1, XE991 also increased the number of action potentials evoked by the +30 pA depolarizing current pulse. In seven DA VTA neurons, the mean number of action potentials evoked by this current pulse was 4.14 ± 0.55 in control and 5.57 ± 0.81 in 10 µM XE991, which was a significant increase (paired Student's t-test, P < 0.01).
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DISCUSSION |
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; Grace and Onn 1989; Johnson and North 1992; Neuhoff et al. 2002). Furthermore, with nystatin-perforated patch voltage-clamp recording, it was possible to study IM with the standard IM deactivation protocol, without the rundown of this current seen in whole cell recordings. The presence of IM in DA VTA neurons found in our study agrees with the earlier observation of IM in mesencephalic DA neurons recorded with sharp intracellular electrodes from brain slices, with a similar voltage protocol (Lacey et al. 1990).
XE991 has been shown to be a potent and selective inhibitor of IM in native neurons and expressed KCNQ channels (Wang et al. 1998). In the present study, XE991 (10–30 µM) caused about 90% block of the current measured with the standard IM deactivation voltage protocol, confirming the identity of this current as IM in DA VTA neurons. The IC50 for XE991 in DA VTA neurons was 0.71 µM, which is closest to the IC50 value for XE991 inhibition of expressed KCNQ2 channels (0.71 µM), but is also similar to expressed KCNQ2/3 heteromeric channels (0.6 µM) and native IM (0.98 µM) in sympathetic ganglion neurons (Wang et al. 1998). IC50 values reported for XE991 inhibition of expressed KCNQ4 and KCNQ5 channels are higher: 5.5 µM (Søgaard et al. 2001) and 65 µM (Schroeder et al. 2000), respectively.
Currents mediated by the expressed KCNQ1–5 channel subunits differ considerably in their sensitivity to inhibition by TEA (Hadley et al. 2000; Lerche et al. 2000) and therefore TEA sensitivity has been used as an indication of possible subunit composition of native IM currents (Hadley et al. 2003; Wang et al. 1998). Homomeric KCNQ2 channels have an IC50 value for TEA inhibition in the range of 0.16–0.3 mM (Hadley et al. 2000; Shapiro et al. 2000; Wang et al. 1998). The IC50 value of TEA for homomeric KCNQ4 channels is 3.0 mM (Hadley et al. 2000). Interestingly, the IC50 value for TEA inhibition of IM in DA VTA neurons found in the present study was 1.56 mM, which lies between the IC50 values for TEA of KCNQ2 channels and KCNQ4 channels. Similarly, the Hill slope calculated for the TEA inhibition curves for DA VTA neurons in the present study was 1.58, which is also intermediate between the Hill coefficients reported for KCNQ2 (0.9) and KCNQ4 (2.0) channels (Hadley et al. 2000). Both KCNQ2 and KCNQ4 subunits have been shown to be present in the VTA in immunohistochemical studies (Cooper et al. 2001; Kharkovets et al. 2000). A study in ooctyes concluded that coexpression of KCNQ2 and KCNQ4 subunits did not lead to formation of functional heteromeric channels (Kubisch et al. 1999). The IC50 for TEA for homomeric KCNQ3 is >30 mM (Hadley et al. 2000). KCNQ1 channels have been detected in heart, kidney, and cochlea, but not in brain (Robbins 2001; Wang et al. 1996; Wangemann 2002). Taken together, these data raise the possibility that both homomeric KCNQ2 and homomeric KCNQ4 channels contribute to IM in DA VTA neurons.
Although KCNQ3 subunits have not been demonstrated in the VTA, the IC50 for TEA inhibition of heteromeric KCNQ2/3 channels is 3.5–3.8 mM (Hadley et al. 2000; Wang et al. 1998), which is close to the IC50 for homomeric KCNQ4 channels (3.0 mM). KCNQ3 has also been shown to form heteromeric channels with KCNQ4 when these subunits are coexpressed (Kubisch et al. 1999). Therefore the contribution of KCNQ2/3 and KCNQ 3/4 heteromeric channels to IM in DA VTA neurons cannot be conclusively excluded at this time. The present study shows that 10 mM TEA causes essentially complete inhibition (97.6%) of IM in DA VTA neurons. By contrast, homomeric KCNQ3, homomeric KCNQ5, and heteromeric KCNQ3/5 channels are very insensitive to TEA with estimated IC50 values of >30, 71, and 200 mM, respectively (Hadley et al. 2000; Lerche et al. 2000; Schroeder et al. 2000; Shapiro et al. 2000), making it very unlikely that these channels contribute to IM in DA VTA neurons.
Hill slopes >1 are generally believed to indicate a cooperativity of binding. The difference between the interaction of TEA with the KCNQ channel compared with XE991 may explain why TEA reduced IM by 98% and XE991 reduced IM by 90%. The 8% difference may indicate an additional action of TEA on a KCNQ channel that is related to the cooperativity of binding of TEA (Hill slope 1.58) compared with the lack of cooperativity induced by the lower Hill slope (1.14) seen with XE991. Additional studies will be necessary to investigate this difference in detail.
IM amplitude in DA VTA neurons was found to be voltage dependent in the present study, similar to native IM measured in sympathetic ganglion and hippocampal neurons (Brown and Adams 1980; Schweitzer 2000; Shah et al. 2002 Wang et al. 1998). The maximal IM amplitude in the DA VTA neurons was obtained at –45 mV with the deactivation protocol. The deactivation time constant was voltage dependent in DA VTA neurons, becoming shorter at more hyperpolarized membrane potentials, as has been observed for native IM currents in other neuronal cell types (Brown and Adams 1980; Passmore et al. 2003; Wang et al. 1998). The time course of IM deactivation in DA VTA neurons was well fitted with a single exponential and the value of the deactivation time constant was 138 ms at –55 mV. This is in contrast to deactivation of native IM currents in other types of neurons, which have a biphasic (double-exponential) time course (Passmore et al. 2003; Shah et al. 2002; Wang et al. 1998). The deactivation time constant in DA VTA neurons seems to be closest to the fast component of the deactivation time constant in sympathetic neurons, which was reported to be 145 ms at –50 mV (Wang et al. 1998). The slow deactivation time constant observed in native IM in other types of neurons is typically >500 ms. The absence of this slow component of deactivation in our experiments could be attributable to a difference in the types of KCNQ channels underlying IM in DA VTA neurons, as compared with IM in these other types of neurons. It is possible, however, that we did not detect a slow component in our studies as a result of methodological differences; we determined the deactivation time constant from the exponential fit to IM (inward relaxation) measured with the deactivation protocol (Fig. 1B), whereas some of the other studies had larger IM and determined the deactivation time constant from tail currents following an activation protocol.
The IM/KCNQ antagonist XE991 (10 µM) blocked IM in DA VTA neurons by about 90%. XE991 was then used in nystatin-perforated patch current-clamp experiments, to study the contribution of IM to the action potential shape and interspike trajectory in DA VTA neurons. XE991 (1–10 µM) did not affect the threshold, amplitude, or duration (at half-amplitude) of the evoked action potential. XE991 shortened the latency to the first evoked action potential and shortened the ISI. Furthermore, XE991 reduced the amplitude of the fast AHP and slow AHP without affecting the amplitude of the middle component of the AHP, which followed the evoked action potential. XE991 reduction of the slow AHP causes a steeper voltage trajectory, leading to earlier occurrence of the next action potential and a shortening of the ISI.
Although a study has reported that a relatively high concentration of linopirdine, an IM inhibitor structurally related to XE991, also blocks some types of K+ currents in addition to IM (Schnee and Brown 1998), it seems unlikely that the relatively low concentrations of XE991 (1–10 µM) used in the present study modulated relevant currents other than IM and affected spike frequency with the change of AP shape and AHP amplitude. In the present study, Ih (as measured by voltage-sag amplitude) was not significantly affected by 10 µM XE991 (Figs. 4 and 5), indicating that XE991 did not affect Ih at this concentration. Consistent with our result, it has been reported that 30 µM linopirdine does not change Ih in hippocampal neurons (Schnee and Brown 1998). It has been reported that the IC50 values of XE991 for types of K+ channels (eag1, erg1, erg3, Kv1.2) other than KCNQ channels are in the range of 49 to >100 µM (Wang et al. 1998). Kv4.3 channels mediate IA in midbrain dopamine neurons (Liss et al. 2001) but the IC50 of XE991 for Kv4.3 channels is 43 µM (Wang et al. 1998). Therefore it is unlikely that 1–10 µM XE991 affects types of K+ channels in DA VTA neurons other than KCNQ channels and changes the shape of AP in DA VTA neurons. Our previous study has shown that 200 nM apamin selectively blocks only the middle component of AHP (100 ms after AP peak) without affecting the fast and slow components of AHP in DA VTA neurons (Brodie et al. 1999a). Thus IM and SK currents contribute to the AHP differently in DA VTA neurons and thus it is unlikely that SK current is the target of 1–10 µM XE991. This conclusion is supported by the finding that 10 µM XE991 does not affect IAHP, which is also mediated by the SK current, in sympathetic neurons (Wang et al. 1998).
Based on our voltage-clamp experiments on IM in DA VTA neurons and the XE991-induced changes in the spike shape and ISI, the role of IM in DA VTA neurons can be understood as follows. From the voltage dependency of IM deactivation, the deactivation time constant is predicted to be smaller than 100 ms at –70 mV, which corresponds to the membrane potential at the peak of the fast AHP of the evoked action potential in DA VTA neurons. At this negative membrane potential, IM would contribute only to the fast phase near the peak of the AHP. In the subsequent phase, IM is likely to be fully deactivated and therefore would not contribute to the middle AHP. As the membrane potential depolarizes, IM would be reactivated during the slow AHP, decreasing membrane resistance and delaying the generation of the subsequent action potential. Because the activation time constants of KCNQ2, KCNQ3, and KCNQ4 channels have been reported to be more than 40 ms at +40 mV (Lerche et al. 2000; Selyanko et al. 2000; Søgaard et al. 2001), IM would not be further activated during the very short time from threshold through the depolarizing phase of the action potential. This could explain why inhibition of IM with 1–10 µM XE991 did not affect action potential amplitude or duration in DA VTA neurons. The duration of the evoked action potential was prolonged by 30 µM XE991, which suggests a nonspecific effect of XE991 at this high concentration.
In conclusion, the present study indicates that IM is active near the resting membrane potential to decrease excitability and during the slow AHP to decrease the ISI in DA VTA neurons. In addition to the physiological role of IM, KCNQ channels are a potential target for modulation by drugs. Ethanol has been shown to reduce IM in hippocampal neurons (Moore et al. 1990) and, in our preliminary studies, in DA VTA neurons (Koyama and Appel 2004). It is possible that this action could contribute to the excitatory effect of ethanol on DA VTA neurons and therefore to its reinforcing effects in the mesolimbic pathway.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. B. Appel, Department of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612-7342 (E-mail: sappel{at}uic.edu)
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