Download Ethanol Effects on Dopaminergic Ventral Tegmental Area Neurons

J Neurophysiol 100: 1202–1210, 2008.
First published July 9, 2008; doi:10.1152/jn.00994.2007.
Ethanol Effects on Dopaminergic Ventral Tegmental Area Neurons During
Block of Ih: Involvement of Barium-Sensitive Potassium Currents
John McDaid, Maureen A. McElvain, and Mark S. Brodie
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois
Submitted 5 September 2007; accepted in final form 5 July 2008
INTRODUCTION
activated cationic current (Ih) is a characteristic of mesencephalic dopamine neurons (Grace 1987; Neuhoff et al. 2002),
and we have reported that ethanol increases the amplitude of Ih
(Brodie and Appel 1998).
A recent report indicated that ethanol excitation in DA VTA
neurons of C57Bl/6J mice could be reduced by the compound
ZD7288, which is a selective blocker of Ih (Okamoto et al.
2006). This observation is different from that reported in rats,
in which no significant change in ethanol excitation was
observed in the presence of ZD7288 (Appel et al. 2003). In
light of this controversy, we felt it necessary to study this
phenomenon further. This study presents the results of studies
in two mouse strains and in F344 rats in an effort to determine
the role Ih may play in ethanol-induced excitation of DA VTA
neurons.
METHODS
Animals
Both rats and mice were used in these studies. Fischer 344 (F344;
90 –150 g) were obtained from Harlan Sprague-Dawley (Indianapolis,
IN); C57Bl/6J (C57; 3–5 wk old) and DBA/2J (DBA; 3–5 wk old)
mice were obtained from Jackson Laboratories (Bar Harbor, ME).
Animals used in this study were treated in strict accordance with the
National Institutes of 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.
Dopaminergic pathways are important in mediating the
rewarding and reinforcing properties of numerous drugs of
abuse (Koob et al. 1998; Wise 1996). Ethanol increases dopamine in target brain regions of the ventral tegmental area
(VTA) in rats and mice (Di Chiara and Imperato 1988; Zapata
et al. 2006). Ethanol directly stimulates dopaminergic (DA)
VTA neurons, increasing the spontaneous firing frequency in
dissociated neurons from rats (Brodie et al. 1999b) with an
excitatory potency that is similar to that observed in brain
slices of rats (Brodie et al. 1990) and mice (Brodie and Appel
2000). Excitation of DA VTA neurons may underlie the
rewarding/reinforcing effects of ethanol, so it is important to
understand how alcohol produces this effect. The mechanism
of this excitation is under study, but it is likely that an
ethanol-induced reduction of potassium current is involved
because the potassium channel blocker quinidine can block
ethanol excitation (Appel et al. 2003). In addition, ethanol
decreases M-current in DA VTA neurons (Koyama et al.
2007), and this action may be responsible in part for ethanolinduced excitation of DA VTA neurons. A hyperpolarization
Brain slices containing the VTA were prepared from the subject
animals as previously described (Brodie 2002; Brodie et al. 1999a).
Briefly, after rapid removal of the brain, the tissue was blocked
coronally to contain the VTA and substantia nigra; the cerebral
cortices and a portion of the dorsal mesencephalon were removed. The
tissue block was mounted in the vibratome and submerged in chilled
artificial cerebrospinal fluid (ACSF). Coronal sections (400 ␮m thick)
were cut, and the slice was placed onto a mesh platform in the
recording chamber. The slice was totally submerged in ACSF maintained at a flow rate of 2 ml/min; the temperature in the recording
chamber was kept at 35°C. The composition of the ACSF in these
experiments was (in mM) 126 NaCl, 2.5 KCl, 1.24 NaH2PO4, 2.4
CaCl2, 1.3 MgSO4, 26 NaHCO3, and 11 glucose. The ACSF was
saturated with 95% O2-5% CO2 (pH ⫽ 7.4). Equilibration time of at
least 1 h was allowed after placement of tissue in the recording
chamber before electrodes were placed in the tissue.
Address for reprint requests and other correspondence: M. S. Brodie, Dept.
of Physiology and Biophysics, Univ. of Illinois at Chicago, 835 South Wolcott,
Rm. E-202, M/C 901, Chicago, IL 60612-7342 (E-mail: [email protected]).
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.
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Preparation of brain slices
0022-3077/08 $8.00 Copyright © 2008 The American Physiological Society
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McDaid J, McElvain MA, Brodie MS. Ethanol effects on dopaminergic
ventral tegmental area neurons during block of Ih: involvement of
barium-sensitive potassium currents. J Neurophysiol 100: 1202–1210,
2008. First published July 9, 2008; doi:10.1152/jn.00994.2007. The
dopaminergic neurons of the ventral tegmental area (DA VTA neurons) are important for the rewarding and reinforcing properties of
drugs of abuse, including ethanol. Ethanol increases the firing frequency of DA VTA neurons from rats and mice. Because of a recent
report on block of ethanol excitation in mouse DA VTA neurons with
ZD7288, a selective blocker of the hyperpolarization-activated cationic current Ih, we examined the effect of ZD7288 on ethanol
excitation in DA VTA neurons from C57Bl/6J and DBA/2J mice and
Fisher 344 rats. Ethanol (80 mM) caused only increases in firing rate
in mouse DA VTA neurons in the absence of ZD7288, but in the
presence of ZD7288 (30 ␮M), ethanol produced a more transient
excitation followed by a decrease of firing. This same biphasic
phenomenon was observed in DA VTA neurons from rats in the
presence of ZD7288 only at very high ethanol concentrations (160 –
240 mM) but not at lower pharmacologically relevant concentrations.
The longer latency ethanol-induced inhibition was not observed in DA
VTA neurons from mice or rats in the presence of barium (100 ␮M),
which blocks G protein–linked potassium channels (GIRKs) and other
inwardly rectifying potassium channels. Ethanol may have a direct
effect to increase an inhibitory potassium conductance, but this effect
of ethanol can only decrease the firing rate if Ih is blocked.
ETHANOL EXCITATION, INHIBITION, AND H-CURRENTIN THE VTA
Cell identification
Drug administration
Drugs were added to the ACSF by means of a calibrated infusion
pump from stock solutions 100 –1,000 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
ACSF before this mixture reached the recording chamber. Final
concentrations were calculated from ACSF flow rate, pump infusion
rate, and concentration of drug stock solution. The small volume
chamber (⬃300 ␮l) used in these studies permitted the rapid application and washout of drug solutions. Typically, drugs reach equilibrium in the tissue after 2–3 min of application.
A stock solution of 95% ethanol (vol/vol USP) was used in the
pump, and infusion of ethanol never exceeded 1% of the flow rate of
the ACSF. Ethanol was administered for 6 min to ensure that measurements were made after the full ethanol concentration was reached
in the tissue, and the peak drug effect was attained.
The behaviorally active range for blood ethanol concentrations in
the rat extends from 40 (sedation) to 90 mM (loss of righting reflex)
(Majchrowicz and Hunt 1976); the lethal blood ethanol concentration
in rats is ⬃200 mM (LD50 ⫽ 202 mM) (Haggard et al. 1940). Rats
will self-administer 44 –55 mM ethanol directly into the VTA, indicating that this concentration is reinforcing in the whole animal
(Rodd-Henricks et al. 2000), whereas mice with continuous access to
ethanol can achieve plasma ethanol concentrations as high as 120 mM
(Jelic et al. 1998). This study primarily examined an ethanol concentration of 80 mM, a pharmacologically relevant, sublethal concentration in both the rat and mouse. Note that lower ethanol concentrations
have been shown to have effects on behavioral performance. We used
80 mM ethanol and higher concentrations in these studies because
these concentrations of ethanol produce reliable and robust excitation.
Lower concentrations of ethanol (20 – 40 mM) have been shown to
produce qualitatively similar excitation.
ZD7288 was purchased from Tocris (Ellisville, MO). Most of the
salts used to prepare the extracellular media were purchased from
Sigma (St. Louis, MO). Sulpiride and bicuculline were also purchased
from Sigma. Barium chloride was purchased from Fisher Scientific
(Fair Lawn, NJ).
Extracellular recording
Extracellular recording electrodes were made from 1.5-mm-diam
glass tubing with filament and were filled with 0.9% NaCl. Tip
resistance of the microelectrodes ranged from 4 to 8 M⍀. The
Fintronics amplifier used in these recordings includes a window
discriminator, the output of which was fed to both a rectilinear pen
recorder, and a computer-based data acquisition system that was used
for on-line and off-line analysis of the data. The multiplexed output of
J Neurophysiol • VOL
the Fintronics 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-s and 1-min
intervals. Firing rate was determined before and during drug application. Firing rate was calculated over a 1-min interval immediately
before drug administration and a 1-min interval during the peak drug
effect; drug-induced changes in firing rate were expressed as the
percentage change from the control firing rate according to the formula
[(FRD ⫺ FRC)/FRC] ⫻ 100, where FRD is the firing rate during the
peak drug effect and FRC is the control firing rate. The change in firing
rate thus is expressed as a percentage of the initial firing rate, which
controls for small changes in firing rate that may occur over time. This
formula was used to calculate both excitatory and inhibitory drug
effects. Excitation was defined as the peak increase in firing rate
produced by the drug (e.g., ethanol) greater than the predrug baseline.
Inhibition was defined as the lowest firing rate below the predrug
baseline; in cases in which no inhibition of firing rate was observed,
the value of 0% inhibition was used.
For comparison of the time course of effects on firing rate, the data
were normalized and averaged. Firing rates over 1-min intervals were
calculated and normalized to the 1-min interval immediately before
ethanol administration. These normalized data were averaged by
synchronizing the data to the ethanol administration period, and
graphs of the averaged data were made.
Statistical analysis
Averaged numerical values were expressed as the mean ⫾ SE. The
significance of firing rate changes before and after a single drug
concentration was assessed with a paired t-test. For effects of multiple
drug concentrations or more than one drug, an appropriate one- or
two-way ANOVA was used, followed by Student-Newman-Keuls
post hoc comparisons when needed (Kenakin 1987). Statistical analyses were performed with SigmaStat software (Systat, San Jose, CA).
RESULTS
Neuronal characteristics
Spontaneously active neurons that conformed to the criteria
set by this study and the literature for DA VTA neurons (see
METHODS) were recorded in a series of extracellular recording
experiments. Data from one slice per animal and one neuron
per slice are used in these results. Baseline firing rates of DA
VTA neurons from F344 rats and C57 and DBA mice were
similar to those previously reported (Brodie and Appel 2000;
Brodie et al. 1995). Baseline firing rates for F344 rats were in
the range of 0.83–2.21 Hz, with a mean of 1.39 ⫾ 0.15 Hz, n ⫽
12. For C57 mice, baseline firing rates ranged from 0.66 to 4.49
Hz, with a mean firing rate of 1.89 ⫾ 0.16 Hz, n ⫽ 31, whereas
for DBA mice, the baseline firing rate was from 0.66 to 3.06
Hz, with a mean of 1.76 ⫾ 0.13 Hz, n ⫽ 21. Ethanol was added
to the superfusate at concentrations of 80 –240 mM. Most
experiments conducted in this study involved the use of ethanol
at a concentration of 80 mM. In F344 rats, 80 mM ethanol
reversibly increased the spontaneous firing rate of DA VTA
neurons by 19.76 ⫾ 1.68%. For C57 mice, 80 mM ethanol
increased firing by 16.55 ⫾ 5.63%, and for DBA mice, the
increase was 24.68 ⫾ 2.89%; the excitatory effect of ethanol
was significantly greater in DBA mice than C57 mice [1-way
ANOVA; F(2,59) ⫽ 5.24, P ⬍ 0.01]. The larger effect of
ethanol on DA VTA neurons from DBA mice compared with
those from C57 mice is consistent with data from a previous
study from this laboratory (Brodie and Appel 2000).
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The VTA was clearly visible in the fresh tissue as a gray area
medial to the darker substantia nigra and separated from the nigra by
white matter. Recording electrodes were placed in the VTA under
visual control. DA neurons have been shown to have electrophysiological characteristics very different from non-DA neurons in the
mesencephalon (Grace and Bunney 1984; Lacey et al. 1989). Only
those neurons that were anatomically located within the VTA and that
conformed to the criteria for DA neurons established in the literature
and in this laboratory (Lacey et al. 1989; Mueller and Brodie 1989)
were studied. These criteria include broad action potentials, slow
spontaneous firing rate (0.5–5 Hz), and a regular interspike interval.
Cells were not tested with opiate agonists as has been done by other
groups to further characterize and categorize VTA neurons (Margolis
et al. 2006a). It should be noted that some neurons with the characteristics we used to identify DA VTA neurons may not, in fact, be
dopamine containing (Margolis et al. 2006b).
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J. MCDAID, M. A. MCELVAIN, AND M. S. BRODIE
J Neurophysiol • VOL
A
Control
3.0
ZD7288
Ethanol 80 mM
Firing rate (Hz)
2.5
2.0
Ethanol 80 mM
1.5
1.0
0.5
0.0
15
20
25
30
35
95 100 105 110 115
Time (min)
Control
B
20
ZD7288
e
e
15
10
5
i
i
0
-5
-10
-15
-20
-25
-30
Control
C
30
ZD7288
e
e
20
10
i
i
0
-10
-20
-30
-40
-50
1. Effects of the selective Ih blocker ZD7288 (30 ␮M) on the firing
rate response to 80 mM ethanol in dopaminergic neurons of the ventral
tegmental area (DA VTA neurons) from C57 or DBA mice. A: a typical
ratemeter graph of an extracellular single unit recording of firing rate of a
single C57 DA VTA neuron. Firing rate is plotted as a function of time
(vertical bars), and the horizontal bars indicate the duration of application of
ethanol. Before application of ZD7288, 80 mM ethanol produced an increase
in the firing rate with no subsequent inhibition. Note that 30 ␮M ZD7288
(ZD7288) decreased the baseline firing rate by 42.72%. In the presence of
ZD7288, there was a biphasic effect of ethanol; brief ethanol-induced excitation was followed by inhibition of firing below baseline levels. This inhibition
recovered on washout of ethanol. B and C: pooled data from experiments such
as that shown in Fig. 1A. For each ethanol response, both excitatory (e)
responses and inhibitory (i) responses were measured. Bars labeled “e”
represent mean peak excitatory responses, and bars labeled “i” represent mean
peak inhibitory responses to ethanol. B: ethanol (80 mM) effect on firing
frequency of DA VTA neurons from C57 mice in the absence (Control) and
presence (ZD7288) of 30 ␮M ZD7288. C: ethanol (80 mM) effect on firing
frequency of DA VTA neurons from DBA mice in the absence (Control) and
presence (ZD7288) of 30 ␮M ZD7288.
FIG.
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ZD7288 (30 ␮M) has been shown to completely block the
hyperpolarization activated cationic current (Ih) in DA VTA
neurons of both mice and rats (Neuhoff et al. 2002; Okamoto
et al. 2006); based on this evidence from the literature, we used
ZD7288 at 30 ␮M to assess the effect of block of Ih on ethanol
excitation in our experiments. Superfusion of ZD7288 (30 ␮M)
completely blocks Ih within 10 min of bath application (Okamoto et al. 2006); because ZD7288 significantly decreased the
firing rate in both C57 and DBA DA VTA neurons, we tested
ethanol 15–20 min after application of ZD7288 to allow the
firing rate to stabilize at a new, lower baseline. In C57 mice,
ZD7288 decreased spontaneous firing by 33.28 ⫾ 5.33%
(paired t-test, t ⫽ 5.01, df ⫽ 7, P ⬍ 0.005, n ⫽ 8); in DBA
mice, there was a decrease of 28.94 ⫾ 5.44% (paired t-test, t ⫽
3.99, df ⫽ 14, P ⬍ 0.005, n ⫽ 15). Ethanol was not tested until
the lower baseline firing rate induced by ZD7288 was established and stable.
As shown in Figs. 1 and 2A, Ih blockade with ZD7288 did
not significantly reduce the peak excitatory effect of 80 mM
ethanol in DA VTA neurons of C57 mice, but a significant
inhibitory effect of ethanol was observed [2-way repeatedmeasures ANOVA; F(7,31) ⫽ 6.33, P ⬍ 0.05, n ⫽ 8; post hoc
Student-Newman-Keuls, P ⬎ 0.05 for ethanol excitation before and after ZD7288, P ⬍ 0.05 for inhibition before and after
ZD7288]. This observation that, in ZD7288, the excitatory
effect of ethanol was followed by an inhibitory effect is
important because this phenomenon was not observed before Ih
block. The inhibitory effect of ethanol in ZD7288 was quantified using the same method used to calculate the excitatory
effect of ethanol. As seen in Figs. 1A and 2A, the excitatory
effect of 80 mM ethanol in the C57 DA VTA neuron consists
typically of a sustained plateau of excitation during the period
of drug application; however, in the presence of ZD7288,
similar peak excitatory effect is reached, but this is not sustained and gradually becomes an inhibitory effect before the
end of the 6-min period of application. A similar time course of
ethanol effect on firing rate in ZD7288 was observed in DA
VTA neurons of DBA mice (Fig. 2B); for DA VTA neurons
from DBA mice, the peak excitatory effect of ethanol was
reduced significantly [2-way repeated-measures ANOVA,
F(14,59) ⫽ 26.76, P ⬍ 0.001, n ⫽ 15; post hoc StudentNewman-Keuls, P ⬍ 0.05]. Of the eight C57 DA VTA neurons
tested, only one neuron did not show ethanol-induced inhibition in ZD7288; mean data presented include the results in all
eight cells tested, whether they were inhibited or not. Of 15
DBA neurons similarly tested, 2 neurons did not show ethanolinduced inhibition, and 2 neurons stopped firing completely in
the presence of ethanol and ZD7288; mean data presented
include the results in all 15 cells tested.
To compare the results of these studies more directly to the
earlier study of ZD7288 and ethanol in DA VTA neurons from
C57 mice (Okamoto et al. 2006), we averaged the normalized
responses over 1-min intervals to facilitate comparison of
responses in the absence and presence of ZD7288. These
normalized data were averaged and are displayed in the graphs
in Fig. 2. For DA VTA neurons from C57 mice under control
conditions, the typical pure excitation induced by 80 mM
ethanol is observed; in the presence of 30 ␮M ZD7288, a
transient excitation is followed by inhibition, which reversed at
7–9 min after washout (Fig. 2A). In DA VTA neurons from
DBA mice (Fig. 2B), clear excitation was observed under
Ethanol excitation (e)
or inhibition (i) (%)
Effect of ZD7288 on ethanol excitation of C57 and DBA
mouse DA VTA neurons
Ethanol excitation (e)
or inhibition (i) (%)
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ETHANOL EXCITATION, INHIBITION, AND H-CURRENTIN THE VTA
A
Barium
Ethanol 80
A
Ethanol 80 mM
1.2
ZD7288
6
Firing rate (Hz)
Normalized firing rate
1.4
1.0
0.8
Control
ZD7288
0.6
-2
0
2
4
6
8
10
12
Ethanol 80
4
2
0
14
10
Time (min)
1.0
0.8
0.6
Control
ZD7288
-2
0
2
4
6
8
40
50
60
10
12
Ethanol 80 mM
1.4
Normalized firing rate
Normalized firing rate
B
1.2
0.4
30
Time (min)
Ethanol 80 mM
1.4
20
1.2
1.0
0.8
Barium
Barium + ZD7288
0.6
-2
14
0
2
Time (min)
4
6
8
10
12
14
16
Time (min)
control conditions, but in the presence of 30 ␮M ZD7288, a
brief excitation was also followed by a profound and prolonged
inhibition. Note that, in DA VTA neurons from both strains of
mice, the normalized firing rate was ⬍1.0 in the presence of
ethanol, and this inhibition of firing persisted after the ethanol
was removed from the perfusate but did show reversal with
washout. It is unclear why the inhibition outlasts the duration
of ethanol application in DA VTA neurons from both strains of
mice.
Effect of barium on ethanol excitation/inhibition of C57 DA
VTA neurons in the absence and presence of ZD7288
Figure 3 shows the effect of the prior application of barium
(100 ␮M) on the response to 80 mM ethanol in the absence and
presence of ZD7288 (30 ␮M) in DA VTA neurons of C57
mice. Barium has been shown to block dopamine and baclofenmediated GIRK currents in xenopus oocytes (Werner et al.
1996) and in DA neurons from substantia nigra and VTA (Cruz
et al. 2004; Lacey et al. 1988) while having no effect on Ih (van
et al. 2005). Barium also blocks other inwardly rectifying
J Neurophysiol • VOL
40
C
Ethanol excitation (%)
FIG. 2. Pooled time-course of the effects of 80 mM ethanol on firing
frequency for C57 and DBA VTA DA neurons before (F) and during (䡺) the
application of ZD7288 (30 ␮M). Firing rate over 1-min intervals for each cell
was calculated and normalized to the firing rate immediately before the
administration of ethanol (time ⫽ 0). The data for the firing rates over these
1-min intervals was averaged across the DA VTA neurons tested in the
absence and presence of ZD7288. A: in C57 VTA DA neurons, the initial peak
excitatory effect of 80 mM ethanol was not altered significantly by ZD7288
(30 ␮M), but during ethanol application, there was a decrease in the sustained
excitatory effect followed by a decrease of the firing rate below baseline; this
effect gradually recovered during the washout of ethanol. B: in DA VTA
neurons from DBA mice, there was a decrease in the peak and sustained
excitatory effect of 80 mM ethanol in the presence of ZD7288 (30 ␮M), and,
as in C57 mice, there was a statistically significant decrease of the firing rate
below baseline, which did not fully recover.
35
30
25
20
15
10
5
0
Control
Barium
Barium +
ZD7288
FIG. 3. Effects of barium (100 ␮M) on the response to ethanol of DA VTA
neurons from C57 mice in the absence and presence of 30 ␮M ZD7288. A: a
typical ratemeter graph of an extracellular single unit recording of firing rate of
a single C57 DA VTA neuron. Firing rate is plotted as a function of time
(vertical bars), and the horizontal bars indicate the duration of application of
the agents as labeled. Barium (100 ␮M) was present during the entire time of
the recording shown. Ethanol (80 mM) increased the firing rate before the
addition of ZD7288 and also in the presence of ZD7288 without subsequent
ethanol-induced inhibition. Note that the baseline firing rate was reduced by
31.01% in the presence of 30 ␮M ZD7288 (ZD7288), but the inhibitory effect
of 80 mM ethanol was not observed. B: pooled time-course data from
experiments such as that shown in A, illustrating the effects of 80 mM ethanol
on firing frequency for C57 VTA DA neurons in the presence of barium (100
␮M) before (F) and during (䡺) the application of ZD7288 (30 ␮M). Firing
rates over 1-min intervals for each cell was calculated and normalized to the
firing rate immediately before the administration of ethanol (time ⫽ 0). The
data for the firing rates over these 1-min intervals was averaged across the DA
VTA neurons tested in the absence and presence of ZD7288. C: pooled data
comparing peak excitatory effects of 80 mM ethanol in control conditions
(Control), in the presence of 100 ␮M barium (Barium), and in the presence of
100 ␮M barium and 30 ␮M ZD7288 (Barium ⫹ ZD7288). There was no
difference statistical difference observed in the ethanol-induced excitation
observed under these 3 conditions (1-way repeated-measures ANOVA,
P ⬎ 0.05).
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B
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J. MCDAID, M. A. MCELVAIN, AND M. S. BRODIE
potassium channels (Morishige et al. 1993, 1994). Barium (100
␮M) increased the baseline firing rate of C57 DA VTA neurons
by 45.93 ⫾ 7.43% (paired t-test, t ⫽ ⫺3.89, df ⫽ 4, P ⬍ 0.05,
n ⫽ 5) but did not significantly increase the excitatory response to 80 mM ethanol [1-way repeated-measures ANOVA,
F(2,14) ⫽ 2.72, P ⬎ 0.05, n ⫽ 5]. In the presence of barium,
there was no inhibitory effect of ethanol in ZD7288 in any of
the cells tested (5/5), and ethanol produced a pattern of sustained excitation as is usually seen in the absence of ZD7288.
Effect of ZD7288 on ethanol excitation of F344 rat DA
VTA neurons
A
3.0
ZD7288
Control
Ethanol 80 mM
Firing rate (Hz)
2.5
2.0
Ethanol 80 mM
Effect of barium on ethanol excitation/inhibition of DA VTA
neurons from F344 rats in the absence and presence
of ZD7288
1.5
1.0
As barium blocked ethanol-induced inhibition in the presence of ZD7288 in mouse DA VTA neurons, we examined the
effect of barium on the F344 DA VTA ethanol excitation/inhibition response in ZD7288. Because the magnitude of the inhibitory
response to 200 mM ethanol was the closest to that produced by
80 mM ethanol in C57 mice (cf. Fig. 5B with Fig. 1B), 200 mM
ethanol was chosen as the test concentration in these DA VTA
neurons from F344 rats. Barium (100 ␮M) alone increased the
baseline firing frequency by 65.67 ⫾ 13.78% (paired t-test, t ⫽
⫺6.22, df ⫽ 2, P ⬍ 0.05, n ⫽ 3). The excitatory response to 200
mM ethanol was not significantly greater in the presence of
barium [1-way repeated-measures ANOVA, F(2,8) ⫽ 1.14, P ⬎
0.05, n ⫽ 3], and, similar to C57 mice (cf. Fig. 6A with Fig. 3A),
no inhibitory response was observed, and the normal pattern of
sustained ethanol excitation was restored.
0.5
0.0
30
35
40
45 135 140 145 150
Time (min)
Ethanol excitation (%)
B
28
24
20
16
12
8
4
DISCUSSION
0
Control
ZD7288
FIG. 4. Effects of the selective Ih blocker ZD7288 (30 ␮M) on the firing
rate response to 80 mM ethanol in DA VTA neurons from F344 rats. A: a
typical ratemeter graph of an extracellular single unit recording of firing rate of
a single F344 rat DA VTA neuron. Firing rate is plotted as a function of time
(vertical bars), and the horizontal bars indicate the duration of application of
ethanol. Note that 80 mM ethanol (EtOH 80) produced excitation without
subsequent inhibition in the absence and presence of 30 ␮M ZD7288
(ZD7288), in contrast to the biphasic response to ethanol seen in DA VTA
neurons from C57 and DBA mice in the presence of ZD7288 (cf., Fig. 1).
B: the pooled responses of DA VTA neurons from F344 rats to ethanol (80
mM) alone (Control) or in the presence of 30 ␮M ZD7288 (ZD7288).
J Neurophysiol • VOL
Ethanol excitation of DA VTA neurons is likely to be
important in the rewarding and reinforcing properties of ethanol (Koob et al. 1998; Wise 1996). We have previously shown
that the firing frequency of DA VTA neurons is increased by
ethanol in vitro, both in brain slices (Brodie et al. 1990) and in
dissociated neurons (Brodie et al. 1999b); ethanol increases the
firing rate of DA VTA neurons in vivo as well (Gessa et al.
1985). This study indicated that blockade of Ih using the
selective Ih blocker ZD7288 shows an inhibitory action of
ethanol that follows the excitatory action of ethanol. This
inhibition was produced only in the presence of ZD7288, and,
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In DA VTA neurons from F344 rats, the peak excitatory
effect of 80 mM ethanol was not altered by Ih blockade with 30
␮M ZD7288 (paired t-test, P ⬎ 0.05), nor was there any
inhibitory effect of 80 mM ethanol on the firing of F344 rats
neurons (Fig. 4), in contrast to the ethanol-induced inhibition
seen in the presence of ZD7288 in DA VTA neurons of C57
and DBA mice (cf., Fig. 1). These results are consistent with
data from a previous study from this laboratory (Appel et al.
2003). A small but significant increase in firing rate was
produced by ZD7288 alone (1.19 ⫾ 0.14 Hz before ZD7288;
1.38 ⫾ 0.16 Hz in 30 ␮M ZD7288; paired t-test, P ⬍ 0.05, n ⫽
6). Some effects of ethanol in rat brain slices were only
observed at ethanol concentrations far in excess of 200 mM, a
concentration associated with respiratory failure in rats (Haggard et al. 1940). As we have recently shown depolarization
blockade of firing produced by high ethanol concentrations
(200 – 400 mM) and concentrations of longer chain alcohols
(Appel et al. 2006), we examined the effect of ZD7288 on
excitation produced by high ethanol concentrations (160 –240
mM) (Fig. 5). As shown in Fig. 5A, the excitatory effect of a
high concentration of ethanol (200 mM) on a F344 DA VTA
neuron in the absence of ZD7288 typically consisted of a
sustained plateau of excitation during the period of drug
application; however, in the presence of 30 ␮M ZD7288, a
similar peak excitatory effect was reached, but this was not
sustained, and gradually the firing rate was reduced below
baseline before the end of the 6-min period of ethanol application. This effect is similar to that observed with 80 mM
ethanol in DA VTA neurons from C57 and DBA mice. For the
population of cells tested, an increase of firing of 45.56 ⫾ 4.08
(n ⫽ 3) was produced by 160 mM ethanol; 200 (n ⫽ 3) and 240
mM (n ⫽ 3) elicited similar large and reversible increases in
the firing frequency (Fig. 5B). In the presence of ZD7288, the
excitatory effect of ethanol at 200 and 240 mM ethanol was
reduced, and each ethanol excitation was followed by significant inhibition [2-way repeated-measures ANOVA, F(1,35) ⫽
19.97, P ⬍ 0.05; post hoc Student-Newman-Keuls, P ⬍ 0.05].
ETHANOL EXCITATION, INHIBITION, AND H-CURRENTIN THE VTA
3.0
Ethanol 200 mM
2.5
Ethanol 200 mM
2.0
1.5
1.0
0.5
Effects of ZD7288 on DA VTA firing rate
0.0
5
10
15
40
45
50
Time (min)
B
80
Ethanol excitation (e)
or inhibition (i) (%)
60
ZD7288
Control
e e
e
40
e
e
20
e
i
i
i
A
0
160 200 240
-20
160
160
200
10
Barium
240
ZD7288
200
8
-40
-60
240
FIG. 5. Effects of the selective Ih blocker ZD7288 (30 ␮M) on the firing
rate response to high concentrations of ethanol (160, 200, and 240 mM) in DA
VTA neurons from F344 rats. A: a typical ratemeter graph of an extracellular
single unit recording of the firing rate of a single F344 rat DA VTA neuron.
Firing rate is plotted as a function of time (vertical bars), and the horizontal
bars indicate the duration of application of the agents as labeled. Before
application of ZD7288, 200 mM ethanol produced a firing rate increase
without subsequent inhibition. The addition of 30 ␮M ZD7288 produced an
increase in the baseline firing rate of 27.7% immediately before 200 mM
ethanol was reapplied. In the presence of 30 ␮M ZD7288, ethanol excitation
was followed by an ethanol-induced inhibition. B: the pooled responses of DA
VTA neurons from F344 rats to ethanol (160, 200, and 240 mM) alone
(Control) or to ethanol in the presence of 30 ␮M ZD7288 (ZD7288). Bars
labeled “e” represent mean peak excitatory responses, and bars labeled “i”
represent mean peak inhibitory responses to ethanol. ZD7288 decreased the
peak excitatory (e) response to 200 and 240 mM ethanol (2-way repeatedmeasures ANOVA, P ⬍ 0.05) in DA VTA neurons from F344 rats. There was
no inhibitory effect (i) of ethanol at any of these concentrations before the
application of ZD7288.
although it was observed in mouse DA VTA neurons at close
to pharmacological concentrations, it was only observed in rat
DA VTA neurons at high concentrations that would be lethal
in vivo. This inhibition of firing produced by ethanol could be
blocked by barium in both mouse and rat DA VTA neurons,
suggesting an involvement of inwardly rectifying potassium
(IRK) channels in this phenomenon, possibly GIRK channels.
A recent report indicated that ethanol excitation in DA VTA
neurons from C57 mice could be antagonized by ZD7288
(Okamoto et al. 2006). Our study demonstrated no significant
effect of ZD7288 on the peak excitatory response of DA VTA
neurons in C57 mice. However, we did observe inhibition in
firing after ethanol excitation in both C57 and DBA mice.
Because ethanol produces both excitation and inhibition in C57
mice in the presence of ZD7288, these effects may cancel out
overall, resulting in an apparent blockade of ethanol-induced
excitation of firing rate. This may be the case with the results
J Neurophysiol • VOL
Ethanol 200 mM
Ethanol 200 mM
6
4
2
0
10
20
30
40
50
Time (min)
B
120
100
80
60
40
20
0
Control
Barium
Barium +
ZD7288
FIG. 6. Effects of barium (100 ␮M) on the response to 200 mM ethanol of
DA VTA neurons from F344 rats in the absence or presence of 30 ␮M
ZD7288. A: a typical ratemeter graph of an extracellular single-unit recording
of firing rate of a single F344 rat DA VTA neuron. Firing rate is plotted as a
function of time (vertical bars), and the horizontal bars indicate the duration of
application of the agents as labeled. Barium (100 ␮M) was present throughout
this recording. Before ZD7288 administration, 200 mM ethanol (EtOH 200)
produced an increase in firing rate. In the presence of ZD7288, 200 mM
ethanol produced a greater increase in firing rate, but no subsequent inhibition.
B: the pooled responses of DA VTA neurons from F344 rats to 200 mM
ethanol alone (Control), in the presence of 100 ␮M barium (Barium), or in the
presence of 30 ␮M ZD7288 and barium (Barium ⫹ ZD7288). There was no
statistically significant difference in the ethanol-induced excitation under these
3 conditions (1-way repeated-measures ANOVA, P ⬎ 0.05).
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In both C57 and DBA DA VTA neurons, ZD7288 decreased
the baseline firing rate by similar amounts; this effect may have
been caused by the blockade of Ih because the firing rate effect
of ZD7288 showed the same dose dependency as the reduction
in Ih (Okamoto et al. 2006). A link between spontaneous firing
rate and Ih is reinforced by the time required for the firing to
reach a new baseline in ZD7288, an effect that closely correlates with the time taken for ZD7288 to fully block Ih (Harris
55
Firing rate (Hz)
Firing rate (Hz)
of Okamoto et al. 2006. Other technical reasons for the discrepancy between that previous study and this study may
include the plane of the brain slice section (coronal vs. horizontal) and recording method used (extracellular vs. whole cell
patch), but it is clear from this study and our earlier work
(Appel et al. 2003) that ZD7288 does not block ethanol
excitation but may reveal an inhibitory phenomenon that can
counteract the ongoing excitation.
ZD7288
Ethanol excitation (%)
A
1207
1208
J. MCDAID, M. A. MCELVAIN, AND M. S. BRODIE
Role of Ih in DA VTA neurons
In many neurons, Ih is a pacemaker current (Pape 1996) and
is necessary for regular firing of some central neurons. This
does not seem to be true in DA VTA neurons, because 30 ␮M
ZD7288 reduces but does not completely inhibit the spontaneous activity of these neurons. This inhibition produced by
ZD7288 is noticeably larger in DA VTA neurons from mice
but more modest and variable in DA VTA neurons from rats.
Higher concentrations of ZD7288 have been shown to block
spontaneous activity, but the conclusion of that study suggested a non–Ih-related mechanism for that cessation of firing
(Seutin et al. 2001). Although the effect of opening of Ih
channels is to produce a depolarizing influence, a more important role of these channels in the VTA may be their characteristic opening in response to hyperpolarizing membrane potentials and the resultant decrease in membrane resistance. In
response to a hyperpolarizing potential in a portion of the
membrane (near a GIRK channel, for example), Ih channels
open and decrease the membrane resistance in that membrane
region. An increase in membrane conductance (decreased
membrane resistance) caused by activation of that Ih may
decrease the efficiency of passive transmission of that hyperpolarization over the membrane. The lowest threshold for
generation of an action potential is at the axon hillock, so it is
the ultimate site at which firing frequency is controlled. Decreased efficiency of passive transmission of hyperpolarizing
current toward the axon hillock would decrease the influence of
J Neurophysiol • VOL
that hyperpolarizing current on firing rate. Simply stated,
opening of h channels would shunt out or short circuit that
hyperpolarization. A similar role for Ih in the subiculum has
been proposed (van et al. 2006). Blockade of Ih by ZD7288
would eliminate this shunting, and as a result of this reduction
of Ih, hyperpolarizing influences (like the opening of potassium
channels) would have a greater effect on firing frequency.
Role of barium-sensitive currents in excitatory and inhibitory
effects of ethanol
Many inwardly rectifying potassium channels (IRKs) are
sensitive to blockade by barium, and one candidate for the
barium-sensitive current in DA VTA neurons is a GIRK
current. The effect of ethanol on other IRKs has not been
studied as extensively as ethanol effects on GIRKs. A comparison of the direct effects of ethanol on GIRKs and IRK
channels expressed in xenopus oocytes showed an effect on the
GIRK but not IRK channels (Kobayashi et al. 1999). An
enhancement of GIRK function by ethanol has been shown at
concentrations as low as 10 mM in xenopus oocytes and 75
mM in rat cerebellar granule cells in culture, and this enhancement is thought to be strongest in the GIRKs expressing the
GIRK2 subunit (Lewohl et al. 1999), which is the predominant
GIRK subunit expressed in the VTA (Karschin et al. 1996;
Liao et al. 1996). Stimulation of dopamine D2 receptors leads
to inhibition of DA VTA firing because of activation of GIRK
channels (Lacey et al. 1988); this raises the possibility that
ethanol-induced excitation of dopamine neurons could lead to
the increased release of dopamine onto postsynaptic D2 autoreceptors, which may inhibit firing. We applied the D2 receptor
antagonist sulpiride (10 ␮M) at a concentration that blocks the
inhibitory effects of bath applied dopamine, but a significant
inhibitory effect of ethanol persisted in the presence of 10 ␮M
sulpiride and 30 ␮M ZD7288 [data not shown, 2-way repeatedmeasures ANOVA, F(2,47) ⫽ 8.81, P ⬍ 0.005; post hoc
Student-Newman-Keuls, P ⬍ 0.05, n ⫽ 8]. Because numerous
neurotransmitters activate GIRKs in the VTA (e.g., adenosine,
GABA via GABAB receptors, etc.), it is not possible to rule out
the specific action of ethanol on all possible receptors that
might produce inhibition by GIRK activation. One important
non–GIRK-mediated inhibitory influence on DA VTA neurons
is GABA acting at GABAA receptors. Because the sensitivity
of GABAA receptors may be altered by ethanol (Weight et al.
1992), we tested the GABAA antagonist bicuculline against the
inhibitory effect of ethanol, but this did not block the inhibitory
effect of ethanol in the presence of ZD7288 [data not shown,
2-way repeated-measures ANOVA, F(2,23) ⫽ 2.43, P ⬎ 0.05,
n ⫽ 4]. Barium was the only agent we found that completely
blocked the inhibitory effect of ethanol in ZD7288; this may
indicate that ethanol can directly affect a barium-sensitive
current, thus bypassing any second messenger system (Kobayashi et al. 1999). Previously, we showed that Ih blockade with
30 ␮M ZD7288 increased the inhibitory effect of dopamine on
DA VTA cell firing (Liu et al. 2003); this action of ZD7288
may also be a consequence of the reduced ability of the cell
membrane to shunt out the hyperpolarizing effects of D2
receptor–mediated GIRK currents after a reduction of Ih (Liu
et al. 2003).
A similar enhancement of the inhibitory effect of dopamine
has been observed in the presence of a reduced Ih after repeated
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and Constanti 1995; Okamoto et al. 2006). Note that excessively high (ⱖ100 ␮M) concentrations of ZD7288 have been
shown to shut off spontaneous firing of DA neurons completely
(Neuhoff et al. 2002; Seutin et al. 2001), an effect that may be
related to the ability of ZD7288 to decrease synaptic transmission (Chen 2004; Chevaleyre and Castillo 2002; Inaba et al.
2006) and/or exocytosis (Gonzalez-Iglesias et al. 2006). Careful studies of the kinetics of ZD7288 and the effects on firing
rate may be needed to determine the relationship between
blockade of Ih and firing rate changes. In contrast to the effects
in DA VTA neurons from mice, in DA VTA neurons from
Fischer344 rats, ZD7288 produced a small but significant
increase in the baseline firing rate; ZD7288 produced decreases
in firing in other similar studies (Appel et al. 2003; Seutin et al.
2001).
The difference in the magnitude of the inhibitory responses
of rat and mouse VTA neurons may be caused by a greater role
of Ih in mouse DA VTA neurons. Relative age differences
between the rats and mice are unlikely to be the reason for
these differences. Although careful comparison of age-related
and strain/species-related differences is beyond the scope of
this study, qualitatively similar results were observed in DA
VTA neurons from C57 mice 8 –12 wk old (data not shown).
Clearly, the large decrease in firing rate produced by ZD7288
alone in this study suggests that firing frequency is significantly
regulated by Ih in mice. In contrast, in the rat DA VTA
neurons, ZD7288 had a small excitatory (this study) to a small
inhibitory (Appel et al. 2003) effect on spontaneous firing rate.
The greater role of Ih in mouse DA VTA neurons may also
account for the observation of the apparent ethanol activation
of barium-sensitive potassium channels at lower ethanol concentrations in mouse neurons than in rat neurons.
ETHANOL EXCITATION, INHIBITION, AND H-CURRENTIN THE VTA
ACKNOWLEDGMENTS
We thank L. L. Roesch.
Present address of J. McDaid: Department of Anesthesia and Critical Care,
University of Chicago, 5841 South Maryland Avenue, Chicago, IL 606371447.
GRANTS
This work was supported by National Institute of Alcohol Abuse and
Alcoholism Grants AA-09125 to M. S. Brodie and AA-05846 to Sarah B.
Appel.
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