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Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois
Submitted 14 December 2005; accepted in final form 10 April 2006
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ABSTRACT |
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INTRODUCTION |
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; Wise 1987). The neurons in the VTA are classified into two major neuronal subgroups: dopamine (DA) and GABA neurons (Grace and Onn 1989; Johnson and North 1992; Korotkova et al. 2003). DA and GABA neurons have been reported to occupy 70% and 30% of the neuronal populations in the VTA, respectively (Johnson and North 1992). Morphologically, DA VTA neurons have large cell somata with multipolar or bipolar dendrite arborization, whereas GABA VTA neurons have small cell somata with multipolar dendrite arborization (Brodie et al. 1999; Grace and Onn 1989; Johnson and North 1992; Korotkova et al. 2003; Yang et al. 2001). Electrophysiologically, DA VTA neurons have slow firing frequencies (1–8 Hz) and long action potential (AP) durations (2–4 ms), whereas GABA VTA neurons have fast firing frequencies (>5 Hz) and short AP durations <2 ms (Grace and Onn 1989; Johnson and North 1992; Korotkova et al. 2003).
A rapidly inactivating A-type K+ current (IA) has been reported to regulate inter-AP intervals by damping the developing interspike depolarization and slowing the subsequent AP generation (Conner and Stevens 1971). In addition, the activation of IA has been reported to shorten AP duration (Zhang and McBain 1995b) and prolong the latency to the first AP after the recovery from membrane hyperpolarization (Shibata et al. 2000). Although IA has been found to be a transient outward K+ current, there are differences in conductance, gating kinetics and pharmacological properties (Bekkers 2000; Kanold and Manis 1999; Korngreen and Sakmann 2000; Luther et al. 2000; Saito and Isa 2000; Shibata et al. 2000; Song et al. 1998; Tkatch et al. 2000; Zhang and McBain 1995a). The diversity of IA is thought to be due to the heterogeneity of channels that underlie IA. Kv1.4, Kv3.4, Kv4.1, Kv4.2 and Kv4.3 channels have been reported to mediate IA (Baldwin et al. 1991; Covarrubias et al. 1991; Schroter et al. 1991; Serodio et al. 1996; Stuhmer et al. 1989).
There have been several studies which have identified IA in midbrain DA neurons (Hahn et al. 2003; Liss et al. 2001; Silva et al. 1990; Yang et al. 2001). However, there is little information on IA of GABA VTA neurons and no comparison of IA in the two major cell types of the VTA, DA, and GABA neurons. It is possible that different IA properties of DA and GABA VTA neurons may contribute to their different AP properties and firing frequencies. This information is also necessary for careful analysis of the effects of specific agents that alter IA in the function of the VTA. In addition, since some drugs of abuse (e.g., ethanol or nicotine) increase the rate of action potential generation in DA VTA neurons, it is important to understand factors which control firing frequency in these neurons. In the present study, we used both current- and voltage-clamp whole cell recording on acutely dissociated VTA neurons. We classified DA and GABA VTA neurons based on their firing frequencies and AP parameters and then investigated IA properties and the physiological contribution of IA in these neurons. Part of this work has appeared previously in abstract form (Koyama and Appel 2005).
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METHODS |
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Animals used in this study were treated in strict accordance with the U.S. National Institutes for Health Guide for the Care and Use of Laboratory Animals and all experimental methods were approved by the Animal Care Committee of the University of Illinois at Chicago. Fisher 344 rats (14–20 days old, both genders) were decapitated, and the brain was quickly removed. Since IA amplitude is detected at birth and increased with age, reaching 80% of maximal amplitude at 2 wk and a plateau at 3 wk in rat central neurons (Hattori et al. 2003), we used 14–20 day-old rats to examine IA. The brain was placed in the ice-cold cutting solution (in mM: 220 sucrose, 2.5 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.24 NaH2PO4, 26 NaHCO3 and 11 D-glucose), which was constantly bubbled with 95% O2-5% CO2. Transverse brain slices, at a thickness of 400 µm, were made using a Vibratome (Series 1000 plus, St. Louis, MO). The brain slices were incubated in an artificial cerebro-spinal fluid (ACSF) (in mM: 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.3 MgSO4 1.24 NaH2PO4, 26 NaHCO3 and 11 D-glucose; osmolarity 300 mOsm), which was constantly bubbled with 95% O2-5% CO2 at room temperature (23–25°C) for 30 min. The brain slices were then incubated in a HEPES-buffered bathing solution (see following text) containing papain (15–18 U/ml) at 32°C for 20–30 min. After papain treatment, the brain slices were further incubated in the ACSF for 20–40 min. The VTA neurons were dissociated using a vibrating stylus apparatus dispersing cells from the brain slices as previously described (Koyama et al. 2005). The cells were dispersed from the region located between the interfascicular nucleus and the medial lemniscus in the horizontal axis and between the paranigral nucleus and the red nucleus in the sagittal axis. 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.
Electrophysiological recording
Current- and voltage-clamp whole cell recording was performed using an Axopatch-1B patch-clamp amplifier (Axon Instruments, Union City, CA). Microelectrodes were fabricated using a P-87 puller (Sutter Instrument Company, Novato, CA) from LE16 glass capillaries (Dagan, Minneapolis, MN) and heat-polished on a microforge (Narishige, Tokyo). The tip resistances of the electrodes were 1.5–3 M
when filled with a pipette solution (in mM: 110 K-gluconate, 20 KCl, 4 MgATP, 0.3 Na2GTP, 0.2 ethylene glycol-bis-(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid [EGTA] and 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] [HEPES]; pH adjusted to 7.2 with KOH, final [K+]i = 139 mM, osmolality adjusted to 290 mOsm with sucrose). Cell capacitance was measured after the cancellation of the capacitative transients. Series resistance (3–7 M) was compensated for by 80% and periodically monitored. Membrane currents and voltage were filtered at 1 kHz by a –3 dB 4-pole filter and acquired at a sampling frequency of 10 kHz. Data acquisition was performed by a DigiData 1322A interface and pClamp software version 9.0 (Axon Instruments). The dissociated VTA neurons were visualized under phase-contrast on an inverted microscope (Diaphot 300, Nikon, Tokyo). All experiments were performed at room temperature (23–25°C).
Experimental solutions
Current-clamp recording was done with a HEPES-buffered bathing solution (in mM: 145 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES and 11 D-glucose; pH adjusted to 7.4 with NaOH, osmolarity 300 mOsm), which was constantly bubbled with 100% O2. Voltage-clamp recording to examine IA was done in a test solution (in mM: 145 NaCl, 2.5 KCl, 3 MgCl2, 10 HEPES and 11 D-glucose; pH adjusted to 7.4 with NaOH, osmolality 300 mOsm). For the test solution, Ca2+ was replaced by an equimolar Mg2+ to block voltage-dependent Ca2+ currents, 1 µM tetrodotoxin (TTX) was added to block voltage-dependent Na+ currents and 2 mM CsCl was included to block hyperpolarization-activated nonselective cation currents (Ih). In some experiments, we used a test solution containing normal Ca2+ concentration (in mM: 145 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 11 D-glucose, 1 µM TTX and 2 mM CsCl; pH adjusted to 7.4 with NaOH, osmolality 300 mOsm). The liquid junction potentials between the pipette solution and the extracellular solutions were estimated to be 12 mV (Neher 1992) and the results have been corrected by this amount. Osmolarity of the solutions was measured by a Vapro vapor pressure osmometer (Wescor, Logan, UT).
Drug application
Drug solutions were applied via a multiple channel manifold (Becton Dickinson). Each channel of the manifold was connected to a gravity-fed reservoir with tubing (860 µm, i.d.). The output of the manifold was connected to an outflow tube (580 µm, i.d.), 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 drugs and chemical agents used in this study 4-aminopyridine (4-AP), CdCl2, CsCl, EGTA, HEPES, MgATP, Na2GTP, papain and TTX were purchased from Sigma (Saint Louis, MO).
Data analysis and curve fitting
Action potentials were analyzed off-line by pClamp software version 9.0 (Axon Instruments). Data from cells containing AP amplitude <50 mV were discarded. All average values are expressed as mean ± SE. Statistical comparison to assess significant differences was done by paired or unpaired Student's t-test. 4-AP effect on DA and GABA VTA neurons were evaluated with a two-way ANOVA. To calculate IA current density, IA amplitude was divided by cell capacitance. IA inactivation was fitted by a single exponential function
y = Aexp(-x/t)
where A is amplitude obtained from the beginning of the fit and t is decay time constant. To analyze the steady-state IA activation, the current (I) was converted to conductance by the following equation
G = I / (V - EK)
Where G is the conductance at the test voltage of V and EK is the reversal potential of K+ current, which was calculated to be –100 mV by the Nernst equation. IA conductance was normalized to the maximal conductance and fitted by the Boltzmann equation, using Origin software version 7 (Origin Lab., Northampton, MA)
y = A2 + (A1 –A2) / {1 + exp(x –x0) / dx}
where y is the conductance at the test voltage of x, A1 is the minimal conductance and set to be 0, A2 is the maximal conductance and set to be 1, x0 is the membrane potential for half-activation and dx is the slope factor. To analyze the steady-state IA inactivation, the currents were fitted by the Boltzmann equation.
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RESULTS |
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In the present study, we recorded 84 VTA neurons in whole cell current-clamp recording; 56 DA neurons and 28 GABA neurons; all neurons which we examined generated action potentials (APs) spontaneously. As shown in Table 1, DA VTA neurons had lower firing frequencies than GABA VTA neurons. DA VTA neurons had smaller AP amplitudes, wider AP half-width, more positive AP threshold potentials and more positive peak afterhyperpolarization (AHP) than GABA VTA neurons, whereas there was no significant difference in AHP amplitude in these neurons (Table 1).
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As illustrated in Fig. 1, we examined steady-state IA activation in DA and GABA VTA neurons. Two types of prepulse protocols were used to obtain IA by taking advantage of the sensitivity of IA to different voltages (Bekkers 2000; Han et al. 2003; Luther et al. 2000; Saito and Isa 2000; Song et al. 1998; Zhang and McBain 1995a; Yang et al. 2001). In the first protocol, a 500 ms-long prepulse from a holding potential (VH) of –62 mV to –102 mV was given before various depolarizing test voltage steps from –92 mV to 18 mV in 10 mV increments. In the second protocol, a 500 ms-long prepulse from a VH of –62 mV to –42 mV was given before various depolarizing test voltage steps from –92 mV to 18 mV in 10 mV increments. IA was obtained by subtracting the currents in the second prepulse protocol from the currents in the first prepulse protocol.
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Activation and inactivation of IA
As illustrated in Fig. 2, we examined IA activation and inactivation. Because the short depolarizing prepulse effectively activates IA but minimizes delayed rectifier K+ current activation (Bekkers 2000), we used two types of short prepulse protocols to obtain IA. In the first protocol, a 100 ms-long prepulse from a VH of –62 mV to –102 mV was given before various depolarizing test voltage steps from –52 mV to 8 mV in 10 mV increments. In the second protocol, a 100 ms-long prepulse from a VH of –62 mV to –42 mV was given before various depolarizing test voltage steps from –52 mV to 8 mV in 10 mV increments. IA was obtained by subtracting the currents in the second prepulse protocol from the currents in the first prepulse protocol.
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Steady-state inactivation of IA
As illustrated in Fig. 3, we examined the steady-state IA inactivation in DA and GABA VTA neurons. One second long conditioning voltage steps from –122 mV to –32 mV (in 10 mV increments) were given from a VH of –62 mV before a test step to –27 mV. There was no significant difference in time-to-peak value at –22 mV between subtracted and unsubtracted raw currents. For DA VTA neurons, the time-to-peak was 3.1 ± 0.2 ms using subtracted currents and 3.2 ± 0.2 ms using raw currents (P > 0.3, n = 5). For GABA VTA neurons, time-to-peak was 2.3 ± 0.2 ms using subtracted currents and 2.5 ± 0.2 ms using raw currents (P > 0.05, n = 7). We used raw currents for analyzing the steady-state inactivation of IA. The depolarizing conditioning voltage steps decreased IA amplitude in a DA VTA neuron (Fig. 3A1) and a GABA VTA neuron (Fig. 3A2). The peak amplitude of IA was measured and normalized after subtracting a residual outward current. As shown in Fig. 3B, normalized IA amplitude was plotted as a function of conditioning voltage for DA VTA neurons (open circles) and GABA VTA neurons (open triangles). These plots were well fitted by a single Boltzmann function. In DA VTA neurons, average half-inactivating voltage (Vinact,0.5) was –84.5 ± 1.6 mV and average slope factor was 5.7 ± 0.6 (n = 7). In GABA VTA neurons, average Vinact,0.5 was –85.2 ± 1.2 mV and average slope factor was 6.4 ± 0.2 (n = 9). There was no significant difference in Vinact,0.5 (P > 0.3) or in slope factor (P > 0.7) between DA and GABA VTA neurons. We further examined steady-state IA inactivation in a test solution contained normal Ca2+ concentration (2 mM Ca2+ and 1 mM Mg2+, see METHODS). Normalized IA amplitude was plotted as a function of conditioning voltage for DA VTA neurons (filled circles) and GABA VTA neurons (filled triangles) (Fig. 3B). The plots were well fitted by a single Boltzmann function. In DA VTA neurons, average Vinact,0.5 with normal Ca2+ test solution was –74.5 ± 2.7 mV and average slope factor was 6.8 ± 0.3 (n = 4). In GABA VTA neurons, average Vinact,0.5 with normal Ca2+ test solution was –75.3 ± 2.9 mV and average slope factor was 5.9 ± 0.2 (n = 4).
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As illustrated in Fig. 4, we examined IA recovery from inactivation in DA and GABA VTA neurons. Figure 4A1 shows IA recovery from inactivation in a DA neuron (top) and a GABA VTA neuron (bottom). Membrane potential was hyperpolarized from a VH of –42 mV to –92 mV in various durations (from 5 ms to 3000 ms) before the membrane potential was returned to –42 mV (Fig. 4A1). IA current amplitude of the DA VTA neuron was larger than that of the GABA VTA neuron. Figure 4A2 shows the early part of IA recovery from the same DA VTA neuron (top) and GABA VTA neuron (bottom). IA recovery from inactivation was faster in the DA VTA neuron than the GABA neuron. A plot of IA peak current amplitude as a function of time was made (Fig. 4B1) from the DA VTA neuron shown in Fig. 4A1 (top); recovery time constant was 41.7 ms being fitted by a single exponential function. Similarly, we plotted IA peak current amplitude as a function of time (Fig. 4B2) from the GABA VTA neuron shown in Fig. 4A1 (bottom); recovery time constant was 220.9 ms being fitted by a single exponential function. Figure 4C shows average IA recovery time constants in DA and GABA VTA neurons. The average IA recovery time constant of DA VTA neurons was 49.5 ± 4.3 ms (n = 9) and that of GABA VTA neurons was 306.0 ± 29.8 ms (n = 8); these values were significantly different (P < 0.001). Figure 4D shows average IA density at full recovery in DA and GABA VTA neurons. The average IA current density of DA VTA neurons was 67.8 ± 18.0 pA/pF (n = 9) and that of GABA VTA neurons was 14.4 ± 2.5 pA/pF (n = 8); these values were significantly different (P < 0.05).
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Because sensitivity to 4-AP is characteristic of each IA subtype (Bekkers 2000; Hahn et al. 2003; Kanold and Manis 1999; Korngreen and Sakmann 2000; Liss et al. 2001; Luther et al. 2000; Saito and Isa 2000; Shibata et al. 2000; Song et al., 1998; Tkatch et al., 2000; Zhang and McBain 1995-a), we examined IA sensitivity to 4-AP. Figure 5 A shows 4-AP effect on IA in a DA VTA neuron (top) and a GABA VTA neuron (bottom). Although 4-AP reduced IA amplitude in a concentration-dependent manner in both neurons, IA of the GABA neuron was more sensitive to 4-AP than that of the DA VTA neuron. Figure 5B shows average % inhibition of IA by 4-AP in DA VTA neurons (open circles) and GABA VTA neurons (open triangles). In DA VTA neurons, 4-AP inhibited IA by 14.2 ± 3.8% at 0.3 mM, 23.7 ± 2.4% at 1 mM and 37.8 ± 3.4% at 3 mM (n = 6). In GABA VTA neurons, 4-AP inhibited IA by 34.5 ± 5.6% at 0.3 mM, 70.3 ± 7.5% at 1 mM and 78.5 ± 7.7% at 3 mM (n = 4). IA sensitivity to each concentration of 4-AP was significantly different between DA and GABA VTA neurons (P < 0.05).
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Another pharmacological characteristic of IA has been reported to be Cd2+ modulation of voltage-dependency in steady-state activation and inactivation (Song et al. 1998; Kuo and Chen 1999; Tkatch et al. 2000). As illustrated in Fig. 6, we examined 200 µM Cd2+ effect on steady-state IA inactivation. Cd2+ reduced IA amplitude in a DA VTA neuron (Fig. 6A1, top). In this neuron, 200 µM Cd2+ shifted Vinact, 0.5 in a positive direction by 20.2 mV without affecting slope factor (4.9 in control, 4.6 with Cd2+) (Fig. 6A1, bottom). Figure 6A2 summarizes Cd2+ effect on steady-state IA inactivation in 5 DA VTA neurons. Cd2+ significantly shifted Vinact, 0.5 in a positive direction (-85.7 ± 1.2 mV in control, –63.2 ± 1.8 mV with Cd2+; P < 0.01) (Fig. 6A2, left) without significant effect on slope factor (5.0 ± 0.2 in control, 5.8 ± 0.5 with Cd2+; P > 0.1) (Fig. 6A2, right). Similarly, Cd2+ reduced IA amplitude in a GABA VTA neuron (Fig. 6B1, top). In this neuron, 200 µM Cd2+ shifted Vinact, 0.5 in a positive direction by 23.6 mVwithout affecting slope factor (5.8 in control, 5.1 with Cd2+) (Fig. 6B1, bottom). Figure 6B2 summarizes Cd2+ effect on steady-state IA inactivation in 4 GABA VTA neurons. Cd2+ significantly shifted Vinact,0.5 in a positive direction (-83.0 ± 2.2 mV in control, –61.0 ± 2.3 mV with Cd2+; P < 0.01) (Fig. 6B2, left) without significant effect on slope factor (6.0 ± 0.3 in control, 6.2 ± 0.8 with Cd2+; P > 0.7) (Fig. 6B2, right).
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We examined whether IA contributed to repolarization latency, which is designated as the duration from the end of hyperpolarizing current step to the first AP. In whole cell current-clamp recording, 1 s long hyperpolarizing current pulses, amplitude of which was –30, –50, –70, and –90 pA, were injected. Figure 7 A1 shows a DA VTA neuron with a prominent time-dependent inward rectification (voltage-sag) during strong hyperpolarizing current injection followed by long repolarization latency. Figure 7A2 shows a GABA VTA neuron with a smaller voltage-sag during strong hyperpolarizing current injection followed by very short reporalization latency. Figure 7A3 shows the average repolarization latency of each hyperpolarizing current in DA and GABA VTA neurons. In DA VTA neurons, average repolarization latency was 692.2 ± 131.5 ms at -90 pA, 712.7 ± 133.5 ms at –70 pA, 714.1 ± 147.8 ms at –50 pA and 636.5 ± 121.6 ms at –30 pA (Fig. 7A3, open circles, n = 13). In GABA VTA neurons, average repolarization latency was 14.0 ± 3.6 ms at –90 pA, 13.5 ± 3.0 ms at –70 pA, 15.2 ± 3.5 ms at –50 pA and 13.5 ± 2.8 ms at –30 pA (Fig. 7A3, open triangles, n = 6). Repolarization latency at each hyperpolarizing current was significantly different between DA and GABA VTA neurons (P < 0.05).
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Time- and voltage-dependent spike generation delay in DA VTA neurons
As shown in Fig. 8, depolarizing current injection induced a train of spikes with the delay of spike generation in DA VTA neurons. Figure 8A shows membrane response to depolarizing current (70 pA) injection at various times after spontaneous AP generation in a DA VTA neuron. When depolarizing current pulse was initiated at relatively positive membrane potential after recovery from AHP, the delay to the first spike was short (Fig. 8A, top). When depolarizing current pulse was initiated just after spontaneous AP near the peak of AHP, the delay to the first spike remained short (Fig. 8A, middle). By contrast, when depolarizing current pulse was initiated during the AHP of spontaneous AP with enough time for membrane hyperpolarization, the delay to the first spike was long (Fig. 8A, bottom). We recorded 15 trials of depolarizing current pulse injection in DA VTA neurons and obtained the total of 75 data points from 5 neurons. The graph in Fig. 8B shows the relationship between the membrane potential at which depolarizing current was injected and the delay to the first spike. The delay to the first spike became longer at membrane potentials negative to –55 mV (Fig. 8B). The graph in Fig. 8C is the relationship between the duration from AP to the initiation of depolarizing current injection and the delay to the first spike. The longest delay is detected between 30 to 120 ms (Fig. 8C).
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DISCUSSION |
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Biophysical properties of IA in VTA neurons
There was no significant difference in half-activating and half-inactivating voltages and slope factors for IA between DA and GABA VTA neurons. In our study on steady-state IA inactivation, half-inactivating voltage was –85 mV in Ca2+-free test solution and –75 mV in normal Ca2+-containing test solution. Extracellular 2 mM Ca2+ shifted steady-state IA inactivation to a positive direction by 10 mV. This observation in our study is consistent with previous studies on midbrain DA neurons showing that half-inactivating voltage is –79 to –64 mV in the extracellular solutions containing normal Ca2+ concentration (Hahn et al. 2003; Liss et al. 2001). Previous studies on IA of midbrain DA neurons have reported that half-activating voltage is –27 to –25 mV (Hahn et al., 2003; Liss et al., 2001). In our present study on DA VTA neurons, half-activating voltage was –38 mV. Although there is 10 mV difference in half-activating voltage between previous and our studies, this difference is likely due to the different concentration of extracellular Ca2+; the extracellular solutions in previous studies contained normal Ca2+ concentration (Hahn et al. 2003; Liss et al. 2001) and we used a Ca2+-free test solution to block voltage-dependent Ca2+ currents (see METHODS).
IA activation and inactivation were significantly faster in GABA VTA neurons than DA VTA neurons. In addition, IA inactivation was more voltage-dependent in GABA VTA neurons than DA VTA neurons. Previous studies on midbrain DA neurons have reported that IA inactivation time constant is 20 to 53 ms and IA inactivation does not exhibit prominent voltage-dependency (Hahn et al. 2003; Liss et al. 2001; Silva et al. 1990). In our present study on DA VTA neurons, IA inactivation time constant was 24 to 29 ms and exhibited weak voltage-dependency, and this is consistent with previous studies (Hahn et al. 2003; Liss et al. 2001). In GABA VTA neurons, IA inactivation was faster at membrane potentials positive to the AP threshold of these neurons. IA has been reported to contribute to AP duration (Zhang and McBain 1995b), but shortening of the AP duration due to IA may be relatively small in GABA VTA neurons. One factor to be considered is the difference in cell capacitance, which could contribute to the apparent difference in time-to-peak between DA and GABA VTA neurons.
The time constant of IA recovery was significantly faster in DA VTA neurons than GABA VTA neurons. Previous studies on midbrain DA neurons have reported that the IA recovery time constant is 20 to 30 ms (Hahn et al. 2003; Liss et al. 2001; Silva et al. 1990). In our present study, the IA recovery time constant was 50 ms in DA VTA neurons. The difference in IA recovery time constant may be due to different intracellular Ca2+ buffering ability that is determined by the composition of Ca2+ and EGTA in pipette solutions used. Intracellular Ca2+/calmodulin-dependent protein kinase can regulate the rate of IA recovery from inactivation (Sergeant et al. 2005), so the intracellular Ca2+ concentration is an important factor to be considered with respect of to IA inactivation. IA current density at full recovery of DA VTA neurons was 4.7-fold larger than that of GABA VTA neurons. A previous study has reported that large IA is critical for the regulation of spontaneous firing frequency in substantia nigra DA neurons (Liss et al. 2001). In GABA VTA neurons, because IA recovery time constant was longer than inter-AP interval and IA current density at full recovery was small, IA may be less important for the regulation of spontaneous firing in these neurons than midbrain DA neurons.
Pharmacology of IA in VTA neurons
IA of DA and GABA VTA neurons was sensitive to block by 4-AP. Previous studies on IA of midbrain DA neurons have reported that 4 to 10 mM 4-AP blocks IA (Hahn et al. 2003; Liss et al. 2001; Silva et al. 1990; Yang et al. 2001). IA was more sensitive to 4-AP in GABA VTA neurons than DA VTA neurons. In the CNS, it has been reported that there are neurons which exhibit IA with higher 4-AP sensitivity (IC50, 1 to 1.8 mM) (Saito and Isa 2000; Song et al. 1998; Zhang and McBain 1995a) and neurons which exhibit IA with lower 4-AP sensitivity (IC50, 2.4 to 4.2 mM) (Korngreen and Sakmann 2000; Saito and Isa 2000).
Extracellular Cd2+ shifted steady-state IA inactivation to a positive direction in DA and GABA VTA neurons. This effect of Cd2+ could cause the activation of IA at more positive membrane potentials and could decrease the excitability of DA and GABA VTA neurons. Among divalent cations, Cd2+, Co2+ and Zn2+ have been reported to shift the voltage-dependency of steady-state IA inactivation in a positive direction (Kuo and Chen 1999; Silva et al. 1990; Song et al. 1998). In substantia nigra DA neurons, both 200 µM Cd2+ and 5 mM Co2+ have been reported to shift the voltage dependency of steady-state IA inactivation in a positive direction by 30 mV (Silva et al. 1990).
Possible involvement of Kv4.3 channels and auxiliary modulation
Specific types of Kv channels are likely to be involved in DA and GABA VTA neurons, and the identification of the specific type of Kv channel is based on their IA properties, such as recovery time constant <1 s, half-activating voltage of –38 to –35 mV and Cd2+ modulation of the voltage-dependency of steady-state inactivation. These characteristics of IA in DA and GABA VTA neurons are consistent with Kv4 channels (An et al. 2000; Decher et al. 2004; Patel et al. 2004; Wickenden et al. 1999). It seems unlikely that KV1.4 channels are involved in DA and GABA VTA neurons. Kv1.4 channels have been reported to have a long recovery time constant more than 5 s and are resistant to the effect of Cd2+ on the voltage-dependency of steady-state inactivation (Wickenden et al. 1999). Of the three members of the Kv4 channel family (Kv4.1, Kv4.2, and Kv4.3) that could be responsible for IA, it seems likely that Kv4.3 channels are involved in DA and GABA VTA neurons. An anatomical study has reported that the midbrain expresses Kv4.3 gene transcripts selectively (Serodio and Rudy 1998). In addition, both midbrain DA and GABA neurons have been reported to express Kv4.3 a subunit mRNA (Hahn et al. 2003; Liss et al. 2001). By contrast, midbrain did not express Kv4.1 and Kv4.2 gene transcripts (Serodio and Rudy 1998) and neither Kv4.1 nor Kv4.2 a subunit mRNA was detected in midbrain DA neurons (Liss et al. 2001). Kv3.4 channels have been reported to be very sensitive to 4-AP with an IC50 of 20–500 µM and have a half-activating voltage of +10 mV (Rudy and McBain 2001). It is unlikely that Kv3.4 channels are involved in DA VTA neurons. Although IA of GABA VTA neurons was sensitive to 4-AP as similar extent as Kv3.4 channels, Vact, 0.5 of IA in these neurons is more negative than that of Kv3.4 channels by 45 mV. Therefore it is also unlikely that Kv3.4 channels contribute to IA much in GABA VTA neurons.
Although IA of DA and GABA VTA neurons had similar voltage-dependency for steady-state activation and inactivation, there were differences in kinetics, recovery time constant and current density at full recovery. It is possible that these different IA properties in DA and GABA VTA neurons may be due to the auxiliary modulation of IA, rather than a difference in the channel types. Potassium channel interacting proteins (KChIPs), which are Ca2+-binding proteins, have been reported to regulate Kv4.3 channel properties by increasing inactivation time constant, accelerating recovery from inactivation and augmenting current density (An et al. 2000; Decher et al. 2004; Nadal et al. 2001; Patel et al. 2004). Furthermore, it has been reported that several types of KChIP isoforms contribute to different Kv4.3 channel properties (Decher et al. 2004; Nadal et al. 2001; Patel et al. 2004). Thus different types of KChIP isoforms may modulate Kv4.3 channels involved in DA and GABA VTA neurons and contribute to the heterogeneity of IA in these neurons. Since intracellular Ca2+ buffering affect IA kinetics through KChIPs (Patel et al. 2002), future studies could determine whether different Ca2+ buffering conditions alter the channel kinetics in a predictable manner in one or both types of DA and GABA VTA neurons.
Physiological implication
Considering the biophysical characteristics of IA and spontaneous firing properties in DA VTA neurons, we suggest that IA can contribute to the spontaneous firing of these neurons. Under the low calcium conditions used in these experiments, analysis of steady-state inactivation in DA VTA neurons indicates that about 5–10% of IA is available for activation at the AHP peak (-68 mV). By the analysis of steady-state inactivation in DA VTA neurons under more physiological conditions in which the extracellular solution contains 2 mM Ca2+, 608 pA IA (25% of maximal IA) is available for activation at AHP peak (-68 mV) in these neurons. Since average IA time constant for recovery from inactivation was 50 ms and average spontaneous firing frequency was 3.3 Hz (duration between APs, 303 ms) in DA VTA neurons, the membrane remains hyperpolarized for sufficient time for IA to recover from inactivation during the inter-spike interval in these neurons. In addition, IA current density at full recovery was large in DA VTA neurons, suggesting that the maximal magnitude of IA can be activated during the depolarizing phase following AHP. In substantia nigra DA neurons, it has been reported that IA charge density is negatively correlated with spontaneous firing frequency and a Kv4 channel blocker, heptapodatoxin, increases spontaneous firing frequency in a concentration-dependent manner (Liss et al. 2001). Thus IA is thought to be important for the regulation of spontaneous firing frequency in midbrain DA neurons. On the other hand, we suggest that IA probably does not affect the spontaneous firing of GABA neurons. Since the average IA recovery time constant from inactivation was 306 ms and the average spontaneous firing frequency was 16 Hz (duration between APs, 63 ms) in GABA VTA neurons, the membrane is not hyperpolarized for sufficient time for recovery of IA from inactivation between APs in these neurons.
When hyperpolarizing currents were injected, DA VTA neurons exhibited delay to the initiation of the first spike in current-clamp recording. In addition, the same DA VTA neurons exhibited large IA current density in voltage-clamp configuration. In DA VTA neurons, since IA had rapid activation (5 ms) and large magnitude with the inactivation time constant of about 30 ms at subthreshold membrane potentials, IA could contribute as a hyperpolarizing driving force to delay to the initiation of the first spike. Conversely, low-threshold Ca2+ current (T-type Ca2+ current) (Wolfart and Roeper 2002) and Ih tail current can contribute as a depolarizing driving force at recovery of membrane hyperpolarization in midbrain DA neurons (Neuhoff et al. 2002). However, the activation of T-type Ca2+ current is 23 ms (Wolfart and Roeper 2002) and the activation of Ih is 843–1286 ms (Neuhoff et al. 2002), which are larger than the activation time constant of IA presented in the present study. Therefore IA is thought to have a more critical influence on repolarization in DA VTA neurons. Similarly, the effect of IA to delay repolarization has been reported in cerebellar granule neurons (Shibata et al. 2000). On the other hand, GABA VTA neurons exhibited short repolarization latency in current-clamp recording and small IA current density in voltage-clamp configuration. In GABA VTA neurons, since IA had small magnitude with the inactivation time constant of about 15 ms at subthreshold membrane potentials, IA may not contribute significantly to the delay of repolarization as a hyperpolarizing driving force.
When depolarizing current was injected at various times after spontaneous AP generation, a train of spikes were evoked and the delay of first spike was detected in DA VTA neurons. This delay of spike generation became longer when depolarizing current was initiated at membrane potentials negative to –55 mV. In the similar voltage rage, IA of DA VTA neurons could be available for activation under physiological conditions. The long delay to the first spike was induced by the duration of 30–120 ms measured from spontaneous AP to the initiation of depolarizing current injection. Since the time constant of IA to recover from inactivation was 50 ms, IA could be activated during the time window between 30 to 120 ms. Therefore the component for the time- and voltage-dependent delay of spike generation induced in DA VTA neurons is probably IA.
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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. Koyama, Dept. of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-7342. Tel.: 312-996-0650, Fax.: 312-996-1414, Email: koyama{at}uic.edu
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