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A-type K⁺ Current of Dopamine and GABA Neurons in the Ventral Tegmental Area

Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois

Submitted 14 December 2005; accepted in final form 10 April 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
A-type K⁺ current (I_A) is a rapidly inactivating voltage-dependent potassium current which can regulate the frequency of action potential (AP) generation. Increased firing frequency of ventral tegmental area (VTA) neurons is associated with the reinforcing effects of some drugs of abuse like nicotine and ethanol. In the present study, we classified dopamine (DA) and GABA VTA neurons, and investigated I_A properties and the physiological role of I_A in these neurons using conventional whole cell current- and voltage-clamp recording. DA VTA neurons had a mean firing frequency of 3.5 Hz with a long AP duration. GABA VTA neurons had a mean firing frequency of 16.7 Hz with a short AP duration. For I_A properties, the voltage-dependence of steady-state I_A activation and inactivation was similar in DA and GABA VTA neurons. I_A inactivation was significantly faster and became faster at positive voltages in GABA neurons than DA neurons. Recovery from inactivation was significantly faster in DA neurons than GABA neurons. I_A current density at full recovery was significantly larger in DA neurons than GABA neurons. In DA and GABA VTA neurons, latency to the first AP after the recovery from membrane hyperpolarization (repolarization latency) was measured. Longer repolarization latency was accompanied by larger I_A current density in DA VTA neurons, compared with GABA VTA neurons. We suggest that I_A contributes more to the regulation of AP generation in DA VTA neurons than in GABA VTA neurons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The ventral tegmental area (VTA) is thought to be critical for the mediation of reinforcing effects of drugs of abuse (Appel et al., 2004; 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 (I_A) 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 I_A 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 I_A 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 I_A is thought to be due to the heterogeneity of channels that underlie I_A. Kv1.4, Kv3.4, Kv4.1, Kv4.2 and Kv4.3 channels have been reported to mediate I_A (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 I_A 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 I_A of GABA VTA neurons and no comparison of I_A in the two major cell types of the VTA, DA, and GABA neurons. It is possible that different I_A 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 I_A 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 I_A properties and the physiological contribution of I_A in these neurons. Part of this work has appeared previously in abstract form (Koyama and Appel 2005).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Region specific cell dissociation

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 I_A 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 I_A. 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 I_A 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 (I_h). 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 I_A current density, I_A amplitude was divided by cell capacitance. I_A 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 I_A activation, the current (I) was converted to conductance by the following equation

G = I / (V - E_K)

Where G is the conductance at the test voltage of V and E_K is the reversal potential of K⁺ current, which was calculated to be –100 mV by the Nernst equation. I_A 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 I_A inactivation, the currents were fitted by the Boltzmann equation.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Classification of DA and GABA VTA neurons

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).

As illustrated in Fig. 1, we examined steady-state I_A activation in DA and GABA VTA neurons. Two types of prepulse protocols were used to obtain I_A by taking advantage of the sensitivity of I_A 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 (V_H) 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 V_H of –62 mV to –42 mV was given before various depolarizing test voltage steps from –92 mV to 18 mV in 10 mV increments. I_A was obtained by subtracting the currents in the second prepulse protocol from the currents in the first prepulse protocol.

View larger version (26K): [in this window] [in a new window]   FIG. 1. Steady-state activation of A-type K+ current (IA). A1, (top): outward currents of a DA VTA neuron in the first prepulse protocol. (bottom) Outward currents of the same DA VTA neuron in the second prepulse protocol. A2, IA obtained by digital subtraction of the outward currents in the second protocol from the outward currents in the first protocol. B1, (top): outward currents of a GABA VTA neuron in the first prepulse protocol (bottom). Outward currents of the same GABA VTA neuron in the second prepulse protocol. B2, IA obtained by digital subtraction of the outward currents in the second protocol from the outward currents in the first protocol. C: average current amplitude plotted as a function of test voltage for 6 DA VTA neurons (open circles) and 4 GABA VTA neurons (open triangles). D: average normalized IA conductance plotted as a function of test voltage for the DA VTA neurons (open circles) and GABA VTA neurons (open triangles) from C. Continuous lines represent curve fittings by a single Boltzmann function.

Activation and inactivation of I_A

As illustrated in Fig. 2, we examined I_A activation and inactivation. Because the short depolarizing prepulse effectively activates I_A but minimizes delayed rectifier K⁺ current activation (Bekkers 2000), we used two types of short prepulse protocols to obtain I_A. In the first protocol, a 100 ms-long prepulse from a V_H 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 V_H of –62 mV to –42 mV was given before various depolarizing test voltage steps from –52 mV to 8 mV in 10 mV increments. I_A was obtained by subtracting the currents in the second prepulse protocol from the currents in the first prepulse protocol.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Activation and inactivation of IA. A1: IA activation of a DA VTA neuron. A2, IA activation of a GABA VTA neuron. A3: average IA time-to-peak values plotted as a function of test voltage for 5 DA VTA neurons (open circles) and 7 GABA VTA neurons (open triangles). B1: IA inactivation of the same DA VTA neuron in A1. B2: IA inactivation of the same GABA VTA neuron in A2. B3: average IA inactivation time constants plotted as a function of test voltage for 5 DA VTA neurons (open circles) and 7 GABA VTA neurons (open triangles). Asterisks indicate significant difference on Student t-test (*P < 0.05, **P < 0.01).

Steady-state inactivation of I_A

As illustrated in Fig. 3, we examined the steady-state I_A 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 V_H 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 I_A. The depolarizing conditioning voltage steps decreased I_A amplitude in a DA VTA neuron (Fig. 3A₁) and a GABA VTA neuron (Fig. 3A₂). The peak amplitude of I_A was measured and normalized after subtracting a residual outward current. As shown in Fig. 3B, normalized I_A 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 (V_inact,0.5) was –84.5 ± 1.6 mV and average slope factor was 5.7 ± 0.6 (n = 7). In GABA VTA neurons, average V_inact,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 V_inact,0.5 (P > 0.3) or in slope factor (P > 0.7) between DA and GABA VTA neurons. We further examined steady-state I_A inactivation in a test solution contained normal Ca²⁺ concentration (2 mM Ca²⁺ and 1 mM Mg²⁺, see METHODS). Normalized I_A 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 V_inact,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 V_inact,0.5 with normal Ca²⁺ test solution was –75.3 ± 2.9 mV and average slope factor was 5.9 ± 0.2 (n = 4).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Steady-state inactivation of IA. A1: IA in a DA VTA neuron. (inset) Recording of IA from the same neuron on a faster time scale. A2, IA in a GABA VTA neuron. (inset) Recording of IA from the same neuron on a faster time scale. B: average normalized peak amplitudes of IA plotted as a function of conditioning voltage for 7 DA VTA neurons (open circles) and 9 GABA VTA neurons (open triangles). In a test solution containing normal Ca2+ concentration (2 mM), average normalized peak amplitudes of IA were also plotted as a function of conditioning voltage for 4 DA VTA neurons (filled circles) and 4 GABA VTA neurons (filled triangles). Continuous lines represent curve fittings by a single Boltzmann function. Bolzmann curves of steady-state IA activation for DA VTA neurons (continuous line) and GABA VTA neurons (dashed line) obtained in Fig. 1D are superimposed.

As illustrated in Fig. 4, we examined I_A recovery from inactivation in DA and GABA VTA neurons. Figure 4A₁ shows I_A recovery from inactivation in a DA neuron (top) and a GABA VTA neuron (bottom). Membrane potential was hyperpolarized from a V_H 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). I_A current amplitude of the DA VTA neuron was larger than that of the GABA VTA neuron. Figure 4A₂ shows the early part of I_A recovery from the same DA VTA neuron (top) and GABA VTA neuron (bottom). I_A recovery from inactivation was faster in the DA VTA neuron than the GABA neuron. A plot of I_A 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 I_A 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 I_A recovery time constants in DA and GABA VTA neurons. The average I_A 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 I_A density at full recovery in DA and GABA VTA neurons. The average I_A 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).

View larger version (32K): [in this window] [in a new window]   FIG. 4. IA recovery from inactivation and current density at full recovery. A1: (top) IA recovery from inactivation in a DA VTA neuron. (bottom) IA recovery from inactivation in a GABA VTA neuron. A2: early phase of IA recovery for the same DA VTA neuron (top) and GABA VTA neuron (bottom). B1: plots of IA recovery time from the DA VTA neuron in A1 (top). B2: plots of IA recovery time from the GABA VTA neuron in A1 (bottom). Continuous line represents a single exponential curve fitting. C: average recovery time constant in 9 DA VTA neurons and 8 GABA VTA neurons. D: average IA current density at full recovery in 9 DA VTA neurons and 8 GABA VTA neurons. IA current at full recovery was measured at 1000 ms in DA VTA neurons and 2000 ms in GABA VTA neurons. IA current density was obtained by dividing IA with cell capacitance. The asterisks represent significant differences (*P < 0.05, **P < 0.001).

Because sensitivity to 4-AP is characteristic of each I_A 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 I_A sensitivity to 4-AP. Figure 5 A shows 4-AP effect on I_A in a DA VTA neuron (top) and a GABA VTA neuron (bottom). Although 4-AP reduced I_A amplitude in a concentration-dependent manner in both neurons, I_A of the GABA neuron was more sensitive to 4-AP than that of the DA VTA neuron. Figure 5B shows average % inhibition of I_A by 4-AP in DA VTA neurons (open circles) and GABA VTA neurons (open triangles). In DA VTA neurons, 4-AP inhibited I_A 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 I_A 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). I_A sensitivity to each concentration of 4-AP was significantly different between DA and GABA VTA neurons (P < 0.05).

View larger version (17K): [in this window] [in a new window]   FIG. 5. 4-AP effect on IA in VTA neurons. A: (top) IA of a DA VTA neuron before and after treatment with 0.3, 1 and 3 mM 4-AP. (bottom) IA of a GABA VTA neuron before and after treatment with 0.3, 1 and 3 mM 4-AP. IA was activated by 1 s-long hyperpolarizing voltage step from a VH of –42 mV to –92 mV and the membrane potential was returned to –42 mV. Dashed lines indicate initial baselines. IA amplitude was measured from the initial baseline to IA peak. B: average IA inhibition rate by 4-AP for 6 DA VTA neurons (open circles) and 4 GABA VTA neurons (open triangles). Percent inhibition in IA was calculated as [(IA,control –IA,4-AP) / IA,control] x 100. When 4-AP was applied at concentration more than 1 mM, the osmolarity was adjusted by reducing the concentration of extracellular NaCl.

Another pharmacological characteristic of I_A 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 I_A inactivation. Cd²⁺ reduced I_A amplitude in a DA VTA neuron (Fig. 6A₁, top). In this neuron, 200 µM Cd²⁺ shifted V_{inact, 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 I_A inactivation in 5 DA VTA neurons. Cd²⁺ significantly shifted V_{inact, 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 I_A amplitude in a GABA VTA neuron (Fig. 6B₁, top). In this neuron, 200 µM Cd²⁺ shifted V_{inact, 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 I_A inactivation in 4 GABA VTA neurons. Cd²⁺ significantly shifted V_inact,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).

View larger version (33K): [in this window] [in a new window]   FIG. 6. Cd2+ effect on the steady-state IA inactivation. A1, (top) IA of a DA VTA neuron before (left) and after (right) treatment with Cd2+. (bottom) Normalized IA amplitude as a function of conditioning voltage before (open circles) and after (filled circles) treatment with Cd2+ in the same DA VTA neuron. Continuous lines represent curve fittings by a single Boltzmann function. A2, Cd2+ effect on average Vinac,0.5 (left) and average slope factor (right) in 5 DA VTA neurons. B1, (top) IA of a GABA VTA neuron before (left) and after (right) treatment with Cd2+. (bottom) Normalized IA amplitude plotted as a function of conditioning voltage before (open triangles) and after (filled triangles) treatment with Cd2+ in the same GABA VTA neuron. Continuous lines represent curve fittings by a single Boltzmann function. B2, Cd2+ effect on average Vinac, 0.5 (left) and average slope factor (right) in 4 GABA VTA neurons. IA was obtained by the same voltage protocol shown in Fig. 3A. The asterisks indicate significant difference on paired Student's t-test (P < 0.01).

We examined whether I_A 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).

View larger version (36K): [in this window] [in a new window]   FIG. 7. Repolarization latency and IA in VTA neurons. A1: membrane response to hyperpolarizing current injection in a DA VTA neuron. A series of four 1 s long hyperpolarizing current pulses (-30, –50, –70 and –90 pA) were injected. Continuous current was not injected. A2: membrane response to hyperpolarizing current injection in a GABA VTA neuron. A3: average repolarization latency in DA VTA neurons (open circles, n = 13) and GABA VTA neurons (open triangles, n = 6). B1, top: large IA in a DA VTA neuron at –42 mV. Bottom: recording of IA from the same neuron on a faster time scale. B2, top: small IA in a GABA VTA neuron at –42 mV. Bottom: recording of IA from the same neuron on a faster time scale. B3: relationship between IA density evoked at –42 mV and repolarization latency induced by the hyperpolarizing current pulse of –90 pA in DA neurons (open circles, n = 9) and GABA neurons (open triangles, n = 5).

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).

View larger version (15K): [in this window] [in a new window]   FIG. 8. Delay of spike generation in DA VTA neurons. A: a depolarizing current pulse (+70 pA) was injected at various times after a spontaneous AP; a train of spikes were induced with the delay to the first spike in the train. (top) Depolarizing current was injected from relatively positive membrane potential after recovery from AHP. (middle) Depolarizing current was injected just after a spontaneous AP at time near AHP peak. (bottom) Depolarizing current was injected during the AHP of a spontaneous AP. Arrows indicate the delay to the first spike. B: plots of the delay to the first spike as a function of membrane potential at which depolarizing current pulse was injected. C: plots of the delay to the first spike as a function of the duration from a spontaneous AP to the initiation of depolarizing current pulse on the logarithmic scale. All data points were obtained from 5 DA VTA neurons. For each neuron, 15 trials of depolarizing current injection at various times after spontaneous AP generation were performed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

Biophysical properties of I_A in VTA neurons

There was no significant difference in half-activating and half-inactivating voltages and slope factors for I_A between DA and GABA VTA neurons. In our study on steady-state I_A 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 I_A 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 I_A 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).

I_A activation and inactivation were significantly faster in GABA VTA neurons than DA VTA neurons. In addition, I_A inactivation was more voltage-dependent in GABA VTA neurons than DA VTA neurons. Previous studies on midbrain DA neurons have reported that I_A inactivation time constant is 20 to 53 ms and I_A 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, I_A 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, I_A inactivation was faster at membrane potentials positive to the AP threshold of these neurons. I_A has been reported to contribute to AP duration (Zhang and McBain 1995b), but shortening of the AP duration due to I_A 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 I_A recovery was significantly faster in DA VTA neurons than GABA VTA neurons. Previous studies on midbrain DA neurons have reported that the I_A 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 I_A recovery time constant was 50 ms in DA VTA neurons. The difference in I_A 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 I_A recovery from inactivation (Sergeant et al. 2005), so the intracellular Ca²⁺ concentration is an important factor to be considered with respect of to I_A inactivation. I_A 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 I_A is critical for the regulation of spontaneous firing frequency in substantia nigra DA neurons (Liss et al. 2001). In GABA VTA neurons, because I_A recovery time constant was longer than inter-AP interval and I_A current density at full recovery was small, I_A may be less important for the regulation of spontaneous firing in these neurons than midbrain DA neurons.

Pharmacology of I_A in VTA neurons

I_A of DA and GABA VTA neurons was sensitive to block by 4-AP. Previous studies on I_A of midbrain DA neurons have reported that 4 to 10 mM 4-AP blocks I_A (Hahn et al. 2003; Liss et al. 2001; Silva et al. 1990; Yang et al. 2001). I_A 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 I_A 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 I_A 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 I_A inactivation to a positive direction in DA and GABA VTA neurons. This effect of Cd²⁺ could cause the activation of I_A 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 I_A 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 I_A 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 I_A 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 I_A 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 K_V1.4 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 I_A, 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 I_A of GABA VTA neurons was sensitive to 4-AP as similar extent as Kv3.4 channels, V_{act, 0.5} of I_A 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 I_A much in GABA VTA neurons.

Although I_A 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 I_A properties in DA and GABA VTA neurons may be due to the auxiliary modulation of I_A, 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 I_A in these neurons. Since intracellular Ca²⁺ buffering affect I_A 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 I_A and spontaneous firing properties in DA VTA neurons, we suggest that I_A 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 I_A 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 I_A (25% of maximal I_A) is available for activation at AHP peak (-68 mV) in these neurons. Since average I_A 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 I_A to recover from inactivation during the inter-spike interval in these neurons. In addition, I_A current density at full recovery was large in DA VTA neurons, suggesting that the maximal magnitude of I_A can be activated during the depolarizing phase following AHP. In substantia nigra DA neurons, it has been reported that I_A 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 I_A is thought to be important for the regulation of spontaneous firing frequency in midbrain DA neurons. On the other hand, we suggest that I_A probably does not affect the spontaneous firing of GABA neurons. Since the average I_A 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 I_A 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 I_A current density in voltage-clamp configuration. In DA VTA neurons, since I_A had rapid activation (5 ms) and large magnitude with the inactivation time constant of about 30 ms at subthreshold membrane potentials, I_A 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 I_h 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 I_h is 843–1286 ms (Neuhoff et al. 2002), which are larger than the activation time constant of I_A presented in the present study. Therefore I_A is thought to have a more critical influence on repolarization in DA VTA neurons. Similarly, the effect of I_A 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 I_A current density in voltage-clamp configuration. In GABA VTA neurons, since I_A had small magnitude with the inactivation time constant of about 15 ms at subthreshold membrane potentials, I_A 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, I_A 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 I_A to recover from inactivation was 50 ms, I_A 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 I_A.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA05846 to S. B. Appel.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors thank Dr. Mark S. Brodie for helpful discussion.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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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