Received 4 October 2014; revised 22 November 2014; accepted 5 December 2014

ABSTRACT

Ventral tegmental area dopamine (DA VTA) neurons are important for the reinforcing effects of drugs of abuse such as ethanol and nicotine. We have previously shown that M-current (I_M) regu- lates the excitability of DA VTA neurons. Zinc (Zn²⁺) contributes to the regulation of neuronal ex- citation as a neuromodulator. In the present study, we investigated zinc effect on the properties of I_M and the spontaneous firing frequency of DA VTA neurons. The standard deactivation protocol was used to measure I_M during voltage-clamp recording with a hyperpolarizing voltage step to −40 mV from a holding potential (V_H) of −25 mV. Zn²⁺ (100 μM) inhibited I_M amplitude and I_M recovered completely from the inhibition after the washout of Zn²⁺. Zn²⁺ inhibited I_M in a concentration- dependent manner (IC₅₀: 5.8 μM). When hyperpolarizing voltage steps were given to −65 mV (in 10 mV increments) from a V_H of −25 mV, Zn²⁺ (100 μM) reduced I_M amplitude at each voltage and zinc inhibition of I_M was not voltage-dependent. Zn²⁺ increased the spontaneous firing frequency of DA VTA neurons in a concentration-dependent manner, suggesting that Zn²⁺ causes excitation of DA VTA neurons through an action on I_M. I_M of DA VTA neurons was inhibited by 100 μM divalent cations in increasing order of potency: Ba²⁺ (16%) < Co²⁺ (25%) < Ni²⁺ (40%) < Cd²⁺ (59%) < Zn²⁺ (67%). These results suggest that Zn²⁺ may exert physiologically significant regulation of neuronal excitability in DA VTA neurons.

Keywords:

Divalent Cation, Dopaminergic, Nystatin-Perforated Patch Recording, Zinc

1. Introduction

The ventral tegmental area dopamine (DA VTA) neurons send axons which synapse in the nucleus accumbens (NAcb) [1] and the excitation of DA VTA neurons results in increased dopamine release in the NAcb [2] - [4] , which is important for the reinforcing effects of drugs of abuse [5] [6] . M-current (I_M) is a voltage-dependent K⁺ current which is activated at the subthreshold range of membrane potential and contributes to the regulation of repetitive firing [7] [8] . I_M is mediated by current through KCNQ type potassium channels [9] . Among the five types of channel subunits (KCNQ1 to 5) [10] , immunohistochemical studies have shown that the KCNQ2 and KCNQ4 channel proteins are present in VTA neurons [11] [12] . DA VTA neurons have intrinsic pacemaker activity and a recent study has reported that the KCNQ4 channel subunit is the major component of I_M in DA VTA neurons and critical for the excitability of these neurons [13] . Thus, KCNQ channels (I_M) of DA VTA neurons may be a critical factor in the mediation of the reinforcing effect of drugs of abuse.

Zinc (Zn²⁺) is present in the midbrain in higher concentrations than in the blood and the total amount of Zn²⁺ is estimated to be 4225 ng in the substantia nigra (SN) in human brain [14] . The majority of Zn²⁺ is associated with a Zn²⁺-containing enzyme in cytoplasm [15] and the remainder of Zn²⁺ is present in presynaptic vesicles [16] . Zn²⁺ is thought to be co-released with neurotransmitter from presynaptic nerve terminals [17] and to act postsynaptically by the regulation of ligand-gated ion channels and voltage-dependent ion channels [18] . Extracellular Zn²⁺ inhibits I_M; the IC₅₀ values are 11 μM in rodent neuroblastoma x glioma hybrid cells [19] and 300 mM in bullfrog sympathetic neurons [20] . It has been reported that Zn²⁺ (10 - 100 μM) accelerates evoked action potential generation, suggesting that it increases excitability of midbrain DA neurons of the SN in a concentration-dependent manner [21] . We have previously shown that I_M underlies the fast and slow component of the action potential after hyperpolarization without affecting the middle component and prolongs the inter-spike interval to decrease the excitability of DA VTA neurons [22] . Therefore, it is hypothesized that relatively low concentrations of Zn²⁺ may inhibit I_M and increase the excitability of DA VTA neurons. This hypothesis was tested in the present study, investigating whether Zn²⁺ would modulate the properties of I_M and the excitability of DA VTA neurons through an action on I_M. In addition, we examined the potency of other divalent cations, barium (Ba²⁺), cadmium (Cd²⁺), cobalt (Co²⁺) and nickel (Ni²⁺) on I_M in DA VTA neurons.

2. Materials and Methods

2.1. Preparation of Dissociated Neurons

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 US 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. Both male and female Fisher 344 rats (14 - 18 days old) were decapitated and the brain quickly removed. The brain was placed in an 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 and 5% CO₂. Transverse brain slices (350 - 400 μm thick) were made on a Vibratome (Series 1000 plus, St. Louis, MO, USA). The brain slices were incubated for 3 - 4 hr in an 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 and 5% CO₂ at 26˚C. VTA neurons were dissociated as previously described [22] . Specifically, brain slices were next incubated in a HEPES-buffered solution (see below) containing papain (15 - 18 U/ml) at 32˚C for 20 - 25 min. After papain treatment, the brain slices were further incubated in the ACSF for 20 - 40 min. The VTA neurons were dissociated by a vibrating stylus apparatus for dispersing cells from the brain slices. First the brain slice was transferred to a poly-_D-lysine- coated 35 mm culture dish (Becton Dickenson, Bedford, MA, USA) 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, the vibrating stylus was placed in the appropriate region with a micromanipulator. The stylus was made of glass capillary tubing (1.5 mm o.d.) pulled to a fine tip, fire-polished (200 - 400 μm in diameter) and mounted on the 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.

2.2. Nystatin-Perforated Patch Recording in Dissociated Neurons

Electrophysiological measurements were made with an Axopatch-1B patch-clamp amplifier (Axon Instruments, Union City, CA, USA). Microelectrodes were fabricated on a P-97 puller (Sutter Instrument Company, Novato, CA, USA), from LE16 glass capillaries (Dagan, Minneapolis, MN, USA) and heat-polished on a microforge (Narishige, Tokyo, Japan). The tip resistances of the electrodes were 3 - 7 MΩ when filled with a 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 290 mOsm). Nystatin-perforated patch recording was used to minimize the dialysis of intracellular contents and therefore prevent the rundown of I_M, as previously described [22] . Nystatin was dissolved in me- thanol at a concentration of 10 mg/ml. This nystatin stock solution was diluted with the 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 and capacitive transients were cancelled. When the access resistance had reached a steady level (15 - 30 MΩ), the recording was started. In case of the sudden change of the access resistance, the recording was stopped. Voltage-clamp recording was done in a HEPES-buffered 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) constantly bubbled with 100% O₂. The liquid junction potential between the pipette solution and the HEPES-buffered solution was estimated to be 5 mV [23] and the results have been corrected by this amount. 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, which is higher than the Nyquist’s critical sampling rate. Data acquisition was performed with a DigiData 1322A interface and pClamp software version 9.0 (Axon Instruments Inc., Union City, CA, USA). 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˚C - 25˚C).

2.3. Drug Application for Dissociated Neurons

Neurons were continuously bathed in the external solution and drugs were dissolved at final concentration in the same solution. Drug solutions were applied via a multiple channel manifold (MLF-4; ALA Scientific Instruments, Westbury, NY, USA). 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 (500 μm, i.d.), the tip of which was placed within 200 mm of the soma of the recorded neuron. Solutions flowed continuously through one manifold channel. Application of drug solutions was controlled by opening or closing valves connected to the reservoirs.

2.4. Preparation of Brain Slices

Following rapid removal of the brain, the tissue block containing the VTA was mounted in the Vibratome and submerged in the ice-cold cutting solution. Coronal sections (400 μm thick) were cut and the slice was placed on a mesh platform in the recording chamber. The slice was totally submerged in the ACSF maintained at a flow rate of 2 ml/min; the temperature in the recording chamber was kept at 35˚C. The ACSF was saturated with 95% O₂ and 5% CO₂ (pH = 7.4). Equilibration time of at least one hour was allowed after placement of the brain slice in the recording chamber before electrodes were placed in the tissue. Recording electrodes were placed in the VTA under visual control. Only those neurons which were anatomically located within the VTA and which conformed to the electrophysiological criteria for dopaminergic neurons [24] were studied. These criteria include broad action potentials and regular spontaneous firing frequency at 0.5 - 5 Hz.

2.5. Extracellular Recording in Brain Slices

Extracellular recording electrodes were fabricated from 1.5 mm diameter glass tubing and were filled with 0.9% NaCl. Tip resistance of the microelectrodes ranged from 3 to 8 MΩ. The Fintronics amplifier (Fintronics Inc., Orange, CT, USA) used in these recordings had a window discriminator, the output of which was fed to both a rectilinear pen recorder and a computer-based data acquisition system for on-line and off-line analysis of the data. The multiplexed output of the amplifier was displayed on an analog storage oscilloscope, for accurate adjustment of the window levels used to monitor single units. An IBM-PC-based data acquisition system was used to calculate, display and store the frequency of firing over 5 sec and 1 min intervals.

2.6. Drug Administration for Brain Slices

Drugs were added to the ACSF by means of a calibrated infusion pump from stock solutions 100 to 1000 times the desired final concentrations. The addition of drug solutions to the ACSF was performed in such a way as to permit the drug solution to mix completely with the ACSF before this mixture reached the recording chamber. The use of a calibrated, variable speed infusion pump permits the accurate addition of several concentrations of drugs from the same stock solution. Final concentrations were calculated from the ACSF flow rate, pump infusion speed and the concentration of drug stock solution. The small volume chamber (about 300 μl) used in this study permitted the rapid application and washout of drug solutions. Typically drugs reached equilibrium in the tissue after 2 to 3 minutes of application.

2.7. Drugs and Chemical Agents

BaCl₂, CdCl₂, CoCl₂, HEPES, NiCl₂, nystatin and ZnCl₂ were purchased from Sigma (Saint Louis, MO, USA). Papain was purchased from Worthington (Lakewood, NJ, USA).

2.8. Data Analysis and Curve Fitting

Action potentials were analyzed offline with pClamp 9.0 software (Axon Instruments Inc.). All average values are expressed as mean ± standard error of the mean (SE). Graphing and curve fitting of data was performed with Origin 7 software (OriginLab Corp., Northampton, MA, USA). Concentration-response curves for zinc were constructed by plotting percent inhibition of I_M 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 [25] :

where x is the concentration, y is the percent inhibition and y_max is the maximal value of y (at saturation); in the fitting procedure y_max was constrained not to exceed 100%. The term k is the IC₅₀ (the concentration giving half- maximal inhibition) and n (Hill slope) is the power term related to the slope of the curve. To assess the changes in spontaneous firing with drugs, drug effect was quantitated as the mean change in firing rate (normalized as the percentage of control) for 60 sec-long interval during the peak of the drug response as previously described [26] . The formula for this normalization is:

Data from dissociated cells with action potential amplitudes less than 50 mV were discarded. All average values are expressed as mean ± standard error (SE). Statistical comparison to assess significant differences was done by one-way ANOVA as appropriate followed by a Bonferroni correction. When needed, the Student-New- man-Keuls post hoc test was used to test multiple comparisons. Correlation was evaluated by linear regression with P < 0.05 being considered significant.

3. Results

3.1. Zinc (Zn²⁺) Inhibits M-Current (I_M) in Ventral Tegmental Area Dopamine (DA VTA) Neurons

After obtaining a stable perforated patch recording, DA VTA neurons were identified in current-clamp recording based on the electrophysiological characteristics by matching spontaneous firing frequency and action potential (AP) parameters, as described previously [22] . Then, I_M was measured in the same DA VTA neuron in voltage- clamp configuration.

I_M was measured in the standard deactivation protocol [8] with 1 sec-long hyperpolarizing voltage step from a holding potential (V_H) of −25 mV to −40 mV. I_M was measured as the inward relaxation current caused by deactivation of I_M during the voltage step (Figure 1(a)). Figure 1(a1) shows I_M before, during and after treatment with 100 mM Zn²⁺ in a typical DA VTA neuron. Zn²⁺ reduced I_M amplitude and also reduced the sustained outward current present at −25 mV as indicated by the inward shift in the baseline current. Just after the termination

of the hyperpolarizing voltage step from −25 mV to −40 mV, a transient outward current was recorded in the presence of Zn²⁺ (Figure 1(a1), middle panel). This current is likely to be transient A-type K⁺ current (I_A), because DA VTA neurons exhibit prominent I_A [27] and Zn²⁺ shifts the voltage-dependency of steady-state I_A inactivation to the depolarizing direction [28] . In the presence of zinc, I_A can be activated at a membrane potential of −40 mV. Figure 1(a2) shows the measurement of the inward relaxation current caused by deactivation of I_M during the voltage step; the difference between the instantaneous current at the beginning (I_in) and the steady- state current at the end of the voltage step (I_ss). Figure 1(b) shows the average time course of normalized I_M amplitude before, during and after application of 100 μM Zn²⁺ in DA VTA neurons. I_M recovered completely after the washout of Zn²⁺ in DA VTA neurons.

3.2. Concentration-Dependent Inhibition of I_M by Zn²⁺

Figure 2(a) shows that Zn²⁺ caused a concentration-dependent reduction of I_M amplitude in a typical DA VTA neuron. Zn²⁺ also caused a concentration-dependent reduction of the baseline sustained outward current. The 300 μM concentration appeared to produce a inhibitory effect near to or at the maximum, since it did not inhibit I_M substantially more than the 100 mM concentration. Zn²⁺ at 300 μM did not produce complete inhibition of I_M. Figure 2(b) shows the pooled concentration-response curve which plots normalized I_M amplitude versus log concentration of Zn²⁺ from DA VTA neurons. The Hill equation was used to fit a smooth curve to the mean data in Figure 2(b). The IC₅₀ for Zn²⁺ was 5.8 mM and the Hill slope was 0.8.

3.3. Zinc Inhibition of I_M Is Not Voltage-Dependent

We then examined whether zinc inhibition of I_M was voltage-dependent. Figure 3(a) shows I_M induced by a series of hyperpolarizing voltage steps before, during, and after treatment with 100 mM Zn²⁺. Zn²⁺ inhibited I_M amplitude measured with all four hyperpolarizing voltage steps in 6 DA VTA neurons. I_A activation can be seen after the offset of the larger voltage steps in control and washout, while I_A is not prominent after the offset of the

larger voltage steps in the presence of Zn²⁺ (Figure 3(a)). This is likely due to a Zn²⁺-induced shift in the voltage-dependency of steady-state I_A activation in the depolarizing direction [29] . Figure 3(b) shows the relationship between membrane voltage and I_M amplitude before, during, and after treatment with 100 mM Zn²⁺ for DA VTA neurons. Zn²⁺ inhibited I_M amplitude at any voltage examined. There was no correlation between membrane voltage and the % I_M inhibition by Zn²⁺ (Figure 3(c)).

3.4. Zn²⁺ Increases the Spontaneous Firing Frequency of DA VTA Neurons

The effect of Zn²⁺ on the spontaneous firing frequency of DA VTA neurons was measured with extracellular single-unit recording of these neurons in brain slices. The percentage increase in firing frequency produced by Zn²⁺ was calculated. Figure 4 shows the concentration-response relationship between Zn²⁺ and the firing rate change by Zn²⁺ from DA VTA neurons. Average Zn²⁺-induced increase in firing frequency was 17.3% ± 10.6% with 10 mM Zn²⁺ and 49.5% ± 7.6% with 50 mM Zn²⁺ (n = 5).

3.5. I_M Inhibition by Ba²⁺, Co²⁺, Ni²⁺ and Cd²⁺

Finally, we examined whether divalent cations other than Zn²⁺ modulated I_M (Figure 5). Ba²⁺, Co²⁺, Ni²⁺ and Cd²⁺ at a concentration of 100 mM all reduced I_M amplitude and induced an inward shift of the baseline outward current in DA VTA neurons, however their potencies for I_M inhibition were different (Figures 5(a)-(d)). Just after the termination of the hyperpolarizing voltage step from −25 mV to −40 mV, I_A was recorded in the pres-

ence of Cd²⁺ (Figure 5(d), middle panel), because Cd²⁺, like Zn²⁺, shifts the voltage-dependency of steady-state I_A inactivation in the depolarizing direction [28] . Figure 6(a) shows the average time course of normalized I_M amplitude before, during and after application of 100 mM divalent cations in DA VTA neurons. I_M of DA VTA neurons were inhibited by 100 mM extracellular divalent cations in increasing order of potency: Ba²⁺ < Co²⁺ < Ni²⁺ < Cd²⁺ < Zn²⁺. Average maximal inhibition of I_M was 15.6% ± 3.0 % with Ba²⁺ (n = 7), 24.9% ± 2.2 % with Co²⁺ (n = 6), 39.5% ± 4.2% with Ni²⁺ (n = 7), 59.0% ± 3.5% with Cd²⁺ (n = 6), and 66.8% ± 2.8% with Zn²⁺ (n = 6).

4. Discussion

Extracellular Zn²⁺ inhibited I_M in a concentration-dependent manner with IC₅₀ value of 5.8 mM in DA VTA neurons; the IC₅₀ value is smaller than that reported for rodent neuroblastoma x glioma hybrid cells (11 mM) [19] or bullfrog sympathetic neurons (300 mM) [20] . We estimate the maximal inhibition of I_M by zinc to be about 62% with 38% of this current remaining unblocked by Zn²⁺ in DA VTA neurons. Since our previous study has confirmed the inward relaxation current obtained by the same voltage protocol in the present study to be I_M in DA VTA neurons [22] , I_M of these neurons can be classified into Zn²⁺-sensitive and Zn²⁺-insensitive components. Previous immunohistochemical studies have shown that KCNQ2 and KCNQ4 channel proteins are present in VTA neurons [11] [12] . Hansen et al. (2006) have reported that the KCNQ4 channel subunit is the main component of I_M in DA VTA neurons, and found weak immunoreactivity of the KCNQ2 channel subunit and lack of KCNQ3 channel immunoreactivity in VTA neurons [13] . Since the KCNQ2 and the KCNQ4 channel subunits cannot be coassembled as a functional heteromeric channel in vitro [30] , the KCNQ2 and the KCNQ4 channels may contribute to I_M independently, as homomers, in a DA VTA neuron. KCNQ4 channels may under- lie the Zn²⁺-sensitive component of I_M and KCNQ2 channels may underlie the remaining Zn²⁺-insensitive com- ponent of I_M. It is unlikely that the KCNQ5 channel component is a significant contributor to I_M in DA VTA neurons, since KCNQ5 channels are potentiated by Zn²⁺ in a concentration-dependent manner [31] .

In DA VTA neurons, zinc inhibition of I_M was not voltage-dependent and the Hill slope was near 1 (0.8) for the concentration-dependent zinc inhibition of I_M. These observations suggest a single Zn²⁺ site of action and one that is different from the voltage-sensitive region of the M-channels. Furthermore, it seems likely that the site of action of zinc in DA VAT neurons is not the M-channel pore, since the mechanism for voltage-dependent I_M inhibition with divalent cation (Ba²⁺) has been reported to be KCNQ channel pore blocking [32] . The voltage-dependency for the action of divalent cations on KCNQ/M-current differ among experimental preparations. In neuroblastoma x glioma hybrid cells [19] and the rod photoreceptor of tiger salamander [33] , I_M inhibition by Ba²⁺ or Cd²⁺ shows voltage-dependency. Expressed KCNQ2/KCNQ3 heteromeric channels are inhibited by Ba²⁺ in a voltage-independent manner [34] .

Our extracellular recording study revealed that a relatively low concentration of Zn²⁺ (50 mM) significantly increased the spontaneous firing frequency of DA VTA neurons. Taken together with the present voltage-clamp analysis, zinc inhibited I_M and subsequently increased the excitability of DA VTA neurons, leading to increase in firing frequency of these neurons (Figure 7). Consistent with our present study, it has been reported that Zn²⁺ (10 - 100 mM) increases the firing frequency during evoked spike trains in midbrain DA neurons of the SN in a

concentration-dependent manner [21] . Several types of voltage-dependent ion currents contribute to the spontaneous activity of midbrain DA neurons. A high voltage-activated (HVA) Ca²⁺ current (I_Ca) underlies the spontaneous oscillatory potential [35] . A low voltage-activated (LVA) transient I_Ca is tightly coupled to a small conductance Ca²⁺-activated K⁺ (SK) current which underlies the middle component of the after hyperpolarization [36] . A transient A-type K⁺ current (I_A) is critical for the regulation of inter-action potential trajectory by generating a time- and voltage-dependent repolarization delay [26] [37] . I_M underlies the fast and slow components without affecting the middle component of AHP and prolongs inter-action potential intervals [22] . Many studies have reported that Zn²⁺ modulates all the types of ion currents described above. Zn²⁺ inhibits HVA I_Ca with the IC₅₀ value of 21 μM and slows current activation [38] . Zn²⁺ inhibits LVA I_Ca with the IC₅₀ value from 11 to 55 μM [39] [40] . Zn²⁺ (10 - 1000 μM) shifts both steady-state activation and inactivation of I_A to a depolarizing direction and slows current activation [27] [28] [41] . Thus, the Zn²⁺-induced excitation of midbrain DA neurons is likely to be the sum of zinc effects on HVA I_Ca, LVA I_Ca, I_A and I_M. Since I_M is more sensitive to Zn²⁺ (IC₅₀ = 5.8 μM) than HVA I_Ca, LVA I_Ca or I_A and Zn²⁺ inhibition of I_M was not voltage-dependent, Zn²⁺ can inhibit I_M potently at any membrane potential during the spontaneous activity of DA VTA neurons and increase the excitability of these neurons as shown in the present study.

I_M of DA VTA neurons were inhibited by 100 μM extracellular divalent cations with the following of order of potency: Zn²⁺ > Cd²⁺ > Ni²⁺ > Co²⁺ > Ba²⁺. Similarly to the results with Zn²⁺, the onset of I_M inhibition by Ba²⁺, Cd²⁺, Co²⁺ or Ni²⁺ was fast and I_M recovered completely after the washout of these divalent cations. In neuro- blastoma x glioma hybrid cells, Cd²⁺, Ni²⁺ and Zn²⁺ exhibit a similar order of potency for the inhibition of I_M but Ba²⁺ is twice as potent as Co²⁺ [19] . In the rod photoreceptor of tiger salamander, I_M is inhibited by Ba²⁺ with the IC₅₀ value of 7.6 mM without apparent sensitivity to 5 mM Co²⁺ and Zn²⁺ [32] . Again, the specific KCNQ subunits, and their combination in functional channels, in each tissue may dictate the sensitivity to inhibition by these divalent cations.

Considering the fact that the concentration of Zn²⁺ is relatively high in the brain, we suggest that Zn²⁺ exerts physiologically significant regulation of neuronal excitability through an action on I_M. It has been shown that Zn²⁺ can be co-released with glutamate from presynaptic terminals, and the concentration of Zn²⁺ in the synapse has been estimated to be between 10 and 100 µM [42] [43] . Our results indicate that the concentration of Zn²⁺ that is achieved during synaptic transmission could be expected to have effects on I_M in addition to directly affecting glutamate neurotransmission. Therefore, inhibition of I_M may be a critical aspect of the neuromodulatory action of Zn²⁺.

Acknowledgements

This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA05846 (to S.B.A.). This study was partly supported by Grants-in-Aid for Scientific Research (C) (Nos. 22500685 and 25350166 to S.K.) from the Japan Society for the Promotion of Science (JSPS) and the Mishima Kaiun Memorial Foundation, Japan (to S.K.). This study was also supported by funds (No. 106006 to S.K.) from the General Research Institute of Fukuoka University.

Competing Interests

The authors have declared that no competing interests exist.

References

NOTES

^*Corresponding author.