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Articles in PresS. J Neurophysiol (August 15, 2007). doi:10.1152/jn.00824.2007
Withdrawal from intermittent ethanol exposure increases probability of burst firing in VTA neurons in vitro Running Head: Withdrawal from ethanol enhances VTA bursting F. Woodward Hopf1*, Miquel Martin2*, Billy T. Chen1, M. Scott Bowers1, Maysha M. Mohamedi1, and Antonello Bonci1 1
Ernest Gallo Clinic and Research Center, Dept. of Neurology, University of California, San Francisco, 2current address: Unit of Neuropharmacology, Pompeu Fabra University, Barcelona, Catalonia, Spain
Correspondence should be addressed to Antonello Bonci, Ernest Gallo Clinic and Research Center, University of California, San Francisco, Dept. of Neurology, 5858 Horton St., Suite 200, Emeryville, CA, 94608, phone: 510-985-3890, fax: 510-985-3101, e-mail:
[email protected]
Keywords: VTA, ethanol, Ih, channel, cross-sensitization, SK * indicates these authors contributed equally.
1 Copyright © 2007 by the American Physiological Society.
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Changing the activity of ventral tegmental area (VTA) dopamine neurons from pacemaker to burst firing is hypothesized to increase the salience of stimuli, such as an unexpected reward, and likely
contributes
to
withdrawal-associated
drug-seeking
behavior.
Accordingly,
pharmacological, behavioral and electrophysiological data suggest an important role of the VTA in mediating alcohol-dependent behaviors. However, the effects of repeated ethanol exposure on VTA dopamine neuron ion channel function are poorly understood. Here, we repeatedly exposed rats to ethanol (2 g/kg ethanol, i.p., twice per day for five days), then examined the firing patterns of VTA dopamine neurons in vitro after 7 days withdrawal. Compared to saline-treated animals, the function of the small calcium-dependent potassium channel (SK) was reduced in ethanoltreated animals. Consistent with a role for SK in regulation of burst firing, NMDA applied during firing facilitated the transition to bursting in ethanol-treated but not saline-treated animals; NMDA only consistently induced bursting in saline-treated animals when SK was inhibited. Also, enhanced bursting in ethanol-treated animals was not a result of differences in NMDAinduced depolarization. Further, Ih was also reduced in ethanol-treated animals, which delayed recovery from hyperpolarization, but did not account for the increased NMDA-induced bursting in ethanol-treated animals. Finally, repeated ethanol and withdrawal also enhanced the acute locomotor-activating effect of cocaine (15 mg/kg, i.p.). Thus, withdrawal after repeated ethanol exposure produced several alterations in the physiological properties of VTA dopamine neurons, which could ultimately increase the ability of VTA neurons to produce burst firing and thus might contribute to addiction-related behaviors.
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Introduction
In addition to other limbic brain structures, midbrain dopamine (DA) neurons from the ventral tegmental area (VTA) may play an important role in ethanol addiction. Acute exposure to ethanol can increase the firing rate of VTA DA neurons (Mereu et al. 1984; Brodie et al. 1990; Diana et al. 1992), and oral self-administration of ethanol increases DA release in a VTA target region, the nucleus accumbens (Gonzales and Weiss 1998). In contrast, withdrawal after chronic ethanol exposure decreases VTA cell firing and DA release in the nucleus accumbens for several days following removal from ethanol (Diana et al. 1993; Weiss et al. 1996; Bailey et al. 1998, 2001; Shen 2003), and re-exposure to ethanol during withdrawal restores nucleus accumbens DA release (Weiss et al. 1996). Behavioral studies have also shown that responses to ethanol are modified by pharmacological manipulations of DA transmission. For example, quinpirole microinjection into the VTA, which inhibits DA cell activity through activation of DA D2/D3 receptors, decreases the number of lever presses an animal will exert to obtain ethanol (Hodge et al. 1993). Several lines of evidence also suggest that the mesolimbic system is involved in ethanol seeking and relapse (Katner and Weiss 1999; McBride et al. 2002; Gonzales et al. 2004). Taken together, these results suggest that VTA DA cell activity contributes to the motivation to self-administer ethanol. From a physiological perspective, midbrain DA neurons present two patterns of activity, regular, pacemaker firing and burst firing (reviewed in Cooper 2002; Komendantov et al. 2004), which are considered important for control of different physiological responses. Tonic, pacemaker-like firing may be altered under conditions ranging from withdrawal from addictive drugs to schizophrenia (Weiss et al. 1992; Grace 2000). In addition, burst-like firing in VTA DA
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neurons, which may produce phasic increases in DA in the different target areas of the VTA (reviewed in Overton and Clark 1997), is thought to encode salience of important stimuli (Robinson and Berridge 2001) and may represent a teaching signal (Schultz 2002). A number of channels, including small conductance, calcium-activated potassium channels (SK) and inwardly rectifying hyperpolarization-activated cation channels (HCN, responsible for generating the Ih current) can regulate pacemaker and burst firing (Seutin et al. 1993; Johnson and Seutin 1997; Seutin et al. 2001; Neuhoff et al. 2002). However, little is known about the long-term effects of chronic ethanol treatment on the firing properties of VTA DA neurons. Furthermore, intermittent ethanol exposure can enhance the locomotor-activating effects of cocaine (Itzhak and Martin 1999), but the cellular mechanisms underlying hyperlocomotion following this pattern of ethanol exposure are poorly understood. In this study, we evaluated the function of different channels involved in control of firing in VTA DA neurons seven days after the end of a five-day repeated, intermittent ethanol treatment, and also the ability of repeated ethanol exposure and withdrawal to enhance locomotor activation by cocaine.
Materials and Methods
Ethanol treatment regimen. Male Sprague-Dawley rats were singly housed in a temperaturecontrolled (21±1oC) room on a 12-hour light/dark cycle (lights on at 07:00). Food and water were available ad libitum. Rats were acclimated to handling procedures starting three days before experiments began. All ethanol injections and behavioral testing were performed during the light cycle. Rats were injected with ethanol (2 g/kg, i.p., twice a day at 10:00 and 18:00, from a 20% v/v solution) or an equivalent volume of saline for five days, with injections starting at P21.
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Slice preparation. All electrophysiological recordings were performed in rats seven days after the end of repeated treatment with ethanol or saline (described above). To prepare VTA slices, animals were anesthetized with halothane and perfused transcardially with ~10 ml of nearly frozen (~0oC) perfusion solution at a rate of ~10 ml/min. This perfusion solution was saturated with 95% O2 and 5% CO2 and contained (in mM): 119 NaCl, 2.5 KCl, 1.0 NaH2PO4 , 6.1 MgCl2, 0.1 CaCl2, 26.2 NaHCO3, 1.25 glucose, 50 sucrose, 1 ascorbic acid, and 3 kynurenic acid. Horizontal slices of 230 µm containing the VTA were prepared with a VT1000S vibratome (Leica, Nussloch, Germany) in the same perfusion solution at 4oC. Slices were then placed in a holding chamber at 31-32oC containing artificial cerebro-spinal fluid (aCSF) saturated with 95% O2 and 5% CO2 and composed of (in mM): 119 NaCl, 2.5 KCl, 1.0 NaH2PO4, 1.3 MgCl2, 2.5 CaCl2, 26.2 NaHCO3 and 11 glucose. 1 mM ascorbic acid was added ~15 minutes before slices were placed in the recovery chamber. Slices were allowed to recover for at least 45 min before being placed in the recording chamber and superfused with oxygenated aCSF at 31-32oC with picrotoxin (100 µM) added to block GABAA receptor-mediated inhibitory post-synaptic currents.
Electrophysiology. Cells were visualized using infrared differential interference contrast video microscopy. Whole-cell current- and voltage-clamp recordings were made using a Multiclamp 700A or 700B amplifier and Clampex 9.2 (Axon Instruments, Foster City, CA). Electrodes (2.84.0 M ) generally contained: 130 mM KOH, 105 mM methanesulfonic acid, 17 mM hydrochloric acid, 20 mM HEPES, 0.2 mM EGTA, 2.8 mM NaCl, 2.5 mg/ml MgATP, and 0.25 mg/ml GTP, pH 7.2-7.4, 280-290 mOsm. A low level of the calcium buffer EGTA was included in the pipette solution in order to preserve calcium-dependent potassium currents during whole-
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cell current- and voltage-clamp recordings (Wolfart et al. 2001). For experiments examining brief (30 sec) bath application of NMDA under voltage clamp (Vholding = +40 mV), the internal solution was (in mM): 117 Cs-methanesulfonic acid, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 TEACl, 2.5 Mg2ATP, and 0.25 Mg2GTP, pH 7.2-7.4, 280-290 mOsm. The reversal potential of NMDA currents was not determined. Current-clamp data were acquired at 20 KHz and filtered at 2 KHz and voltage-clamp data were acquired at 10 KHz and filtered at 2 KHz. VTA DA neurons were identified by the presence of a large Ih current (Johnson and North 1992). Because Ih is present in both principal and tertiary VTA neurons (Margolis et al. 2006), we recognize that its presence does not unequivocally identify DA neurons in midbrain slices. However, in previous work (Ungless et al. 2001) and in the present study, this criterion was enough to obtain differences between control and experimental treatments. In addition, prior results from our laboratory (Borgland et al. 2006 and F. Sarti, unpublished observations) suggest a higher (~75%) correlation between the presence of Ih and tyrosine hydroxylase, indicative of DA neurons, in neurons just medial to the medial terminal nucleus of the accessory optic tract (MT), where nearly all the recordings in the present study were performed. Also, the decrease in Ih amplitude in ethanol animals was only ~40% (Fig. 7), and thus was not sufficient to prevent the use of Ih to identify putative DA neurons relative to GABA neurons, which do not have an Ih current (Johnson and North 1992; Margolis et al. 2006). The action potential waveform was analyzed using Clampfit 9.2 and custom software written in Python2.3 (www.python.org). The action potential threshold was defined as the voltage during the AP upstroke where the rate of rise exceeded 2 V/s. For pacemaker firing, neurons were considered able to fire in pacemaker mode if they were able to fire at least 10
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sequential APs with a coefficient of variation of the inter-spike interval of less than 20%. The average basal instantaneous firing frequency and inter-spike interval during periods of pacemaker firing were then determined across 30 successive action potentials just after breaking into a neuron, before any dialysis of the neuron with the intracellular pipette solution could occur. However, prolonged dialysis with the internal solution did not alter the firing pattern or action potential waveform of neurons (data not shown). In addition, due to variability among neurons in the basal pacemaker firing rate, neurons were brought to ~1 Hz by injecting DC current through the patch amplifier before experiments examining the effects of NMDA, ZD7288, or recovery from hyperpolarization. All voltage values were corrected for the liquid junction potential, estimated to be 10 mV using the Junction Null Calculator in Clampex 9.2, and also by direct measurement of the potential difference between internal and external solutions present after zeroing the pipette current. Bridge balance was used to compensate 60-80% of the series resistance. Also, because the average series resistance and instantaneous currents evoked by hyperpolarization (taken as a measure of input resistance) were the same between ethanol and saline animals (see below), any errors in compensation would be similar in both groups, and thus would not explain the differences in channel function observed here. Further, in order to estimate whether repeated ethanol exposure and withdrawal might have altered the size of VTA neurons, we estimated the whole-cell capacitance using a method adapted from Gentet et al. (2000). In Ih experiments, the voltage step from –60 mV to –70 mV was used for analysis of capacitance, where the series resistance was determined from the peak of the capacitive transient, the input resistance was determined 40 ms into plateau of the voltage response, the capacitive current transient just after application of the voltage step was best fit with two exponentials, and the fast exponential was
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used in the determination of cell capacitance. For SK experiments, the voltage step from –70 mV to –60 mV was used for capacitance analysis. Both methods gave equivalent measures of capacitance (data not shown). To investigate SK tail currents, neurons were held at –70 mV, and depolarizing current steps (400 ms, from –60 to –20 mV in 10 mV steps) were applied, with one sec between successive steps. Upon returning to –70 mV, a tail current was evident (see Fig. 3A). The peak magnitude of the SK tail current was determined for the steps to –20 mV, –30 mV, and –40 mV. In addition, the charge transfer for SK (in picocoulombs) was calculated by integrating (nanoamperes times milliseconds) the tail current evoked following the depolarizing pulse, beginning from the initiation of the tail current to 550 ms into the tail current, since the apaminsensitive component of the tail current should be < 5% by 550 ms into the tail current (Abel et al. 2004). The area under the curve was determined using GraphPad Prism (San Diego, CA). Finally, the rate of inactivation of the SK current was estimated in the tail current evoked after a –20 mV depolarization by fitting a single exponential from ~55 ms after the peak of the tail current to 550 ms after the peak of the tail current (Abel et al. 2004). To examine Ih, neurons were held at –60 mV and a series of voltage steps (500 ms, from –40 to –150 mV in 10 mV steps, with 4 sec between steps) was applied. In some Ih voltageclamp experiments, to reduce contamination from other currents and to better isolate Ih, we performed experiments under conditions where many currents other than Ih were inhibited using a modified internal solution (3 mM BAPTA added) and external solution (aCSF containing 2 mM TEA, 20 mM MgCl2, 0.5 µM TTX, and 2 mM 4-aminopyridine, with NaCl reduced in an equimolar fashion) (Liu et al. 2003).
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Ih was determined from the difference between the peak current response at the end of the pulse minus the instantaneous current response ~30 ms after the onset of the hyperpolarizing pulse (see Fig. 7A; Watts et al. 1996). In addition, we determined the Ih charge transfer using methods similar to those described above for SK, except that Ih charge transfer was determined by integrating the slowly activating inward current beginning from 30 ms after the initiation of the –140 mV hyperpolarizing voltage pulse (Neuhoff et al. 2002). We also determined the magnitude and charge transfer of the instantaneous current, which could represent inwardly rectifying potassium channels (IRK) and/or potassium leak channels (Watts et al. 1996; Wilson 2005). We did not perform experiments to determine the relative contribution of these currents to the instantaneous current. The voltage-dependence of activation of Ih was determined as described (Liu et al. 2003) using the equation G = I/(E-Erev), where –55 mV was used for the Erev for Ih, since it was not different between saline and ethanol animals (Fig. 8F). Data were fit with a Boltzmann equation (Liu et al. 2003) using GraphPad Prism in order to determine the half-activation voltage and slope factor. The Ih reversal potential was determined using described methods (Mayer and Westbrook 1983; Cathala and Paupardin-Tritsch 1997), where the reversal potential of Ih was estimated by comparing the instantaneous current evoked from a voltage of – 60 mV, where Ih was not active, and the instantaneous current evoked from a voltage of –80 mV, where Ih was activated (see Fig 8E). Since instantaneous currents evoked from –60 mV likely represent IRK/leak channels (Watts et al. 1996; Wilson 2005), while currents evoked from –80 mV represent Ih in addition to IRK/leak, one can estimate the reversal potential of Ih by determining the point where the two current-voltage curves intersect, which essentially represents the voltage where the Ih current is zero after the IRK/leak current is subtracted out.
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During experiments examining the effects of NMDA on bursting, it was necessary in many neurons to apply a small hyperpolarizing current to prevent depolarization block (see Seutin et al. 1993; Johnson and Seutin 1997). However, this applied current (values given for 20 µM NMDA) was not significantly different between ethanol (–31.1 ± 18.9 pA) and saline (–41.6 ± 14.4 pA) animals. In addition, the amount of holding current applied during NMDA exposure did not correlate with the development of bursting, measured as the coefficient of variance of the inter-spike interval (see below), in either saline or ethanol animals (r2 = 0.197 and 0.070 for ethanol and saline, respectively; n.s.).
Motor activity assay. Locomotor activity was measured in a 17 x 17-inch open field chamber lined with three 16-beam infrared arrays. Distance traveled (cm) was measured using Open Field Activity software (MED Associates, Inc., St. Albans, VT). Animals were habituated to the locomotor chamber during the fourth through sixth days after the last ethanol injection. Each day, the animals were allowed one hour free exploration, then were given an i.p. injection of saline (1 ml/kg) and then returned to the chamber for 30 min. This was followed by a second i.p. injection of saline and a final 30 min in the locomotor chamber. On the seventh day after ethanol injection, animals were allowed one hour free exploration, followed by an injection of saline and then 30 min in the locomotor chamber. The animals then received an i.p. injection of cocaine (15 mg/kg) and were returned to the chamber for a final 30 min. One concern with repeated injections is the possibility of stress-induced changes. However, as described below, saline and naïve neurons exhibited a similar afterhyperpolarization and voltage-clamp measures of SK and Ih, and saline animals exhibited a very similar locomotor response to cocaine as naïve animals. These results suggest that any stress related to multiple
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injections or handling procedures did not alter channel function or enhance the locomotor response to cocaine. As an additional indicator of possible stress due to injections, we examined the weight of the animals across the period of injection. Saline and ethanol animals showed a similar weight before injections (saline: 51.6 ± 1.4 g; ethanol: 52.2 ± 1.2 g, n = 25 both for saline and ethanol), but by the 5th day the weight of ethanol animals was slightly but significantly reduced (~10%) relative to saline animals (saline: 74.9 ± 1.6 g; ethanol: 68.4 ± 1.7 g, p < 0.05). The cause of this difference is unclear, but may relate to differences in activity of the animals, with two periods of intoxication and lethargy each day in ethanol but not saline animals. However, the weight of saline and ethanol animals was not significantly different after the 7 day withdrawal period (saline: 129.6 ± 3.4 g; ethanol: 121.0 ± 5.2 g), suggesting that repeated ethanol injection did not result in persisting weight loss due to stress associated with injection.
Reagents. All reagents were purchased from Sigma (St. Louis, MO), except for ZD7288, which was purchased from Tocris Cookson (Ellisville, MO). Reagents were prepared in a 1:1000 stock which was frozen in aliquots at –20oC, except for cocaine, which was made fresh on the day of experimentation. Apamin was prepared in distilled water, and ZD7288 in DMSO.
Statistical analysis. All data is expressed as mean ± the standard error of the mean. Statistical analysis was performed with a two-tailed, unpaired t-test, except, where indicated, a 2-way, repeated measures ANOVA was utilized. Significance was determined using GraphPad Prism with confidence intervals of at least 95%. All exponentials were fit using Clampfit. During experiments aimed at examining changes in firing during NMDA application, the coefficient of variance of the inter-spike interval (CV-ISI) was determined from 100 successive action
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potentials. CV-ISI was calculated as the standard deviation divided by the mean, and expressed as a percentage (Zhang et al. 1994).
Results
In the present study, the effects of withdrawal from repeated ethanol treatment on the activity of VTA DA neurons were investigated. Animals were injected with ethanol (2 g/kg, i.p.) or an equivalent volume of saline, twice a day, for 5 days. Seven days after the final injection, brain slices were prepared, and several electrophysiological properties of VTA DA neurons were examined. Because a different number of neurons was recorded for each animal, we sought to avoid biasing our results by averaging, for each individual animal, all recordings of basal spike firing and voltage-clamp parameters, thus obtaining a single value for each particular animal. Therefore, unless otherwise indicated, data were from 29 saline animals and 25 ethanol animals.
Pacemaker activity was not altered after repeated ethanol treatment followed by seven days withdrawal We first investigated the basal pacemaker activity in VTA DA neurons. Here, neurons were considered able to fire in pacemaker mode if they were able to fire at least 10 sequential APs (see Methods). We recorded neurons meeting this criterion from 18 saline and 19 ethanol animals. The average basal instantaneous firing frequency and inter-spike interval (ISI) during periods of pacemaker firing were determined across 30 successive action potentials just after breaking into a neuron. When pacemaker firing occurred, there were no significant differences in the average instantaneous firing frequency (Fig. 1B; saline: 1.19 ± 0.07 Hz; ethanol: 1.27 ± 0.12 Hz; not
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significant, p > 0.05 (n.s.)) or the average ISI (Fig. 1C; saline: 958 ± 69 ms; ethanol: 967 ± 109 ms; n.s.) of neurons from saline and ethanol rats, with firing rates similar to those in several other studies using whole-cell recording (Marinelli et al. 2005; Margolis et al. 2006). In addition, although a resting membrane potential was difficult to determine because neurons were undergoing pacemaker firing, the average membrane potential during pacemaker firing was not different between saline and ethanol animals (saline: -59.1 ± 1.2 mV; ethanol: -59.3 ± 0.6 mV), in agreement with no change in many aspects of the action potential waveform (see below).
SK function was reduced in ethanol animals To further characterize the firing properties of VTA DA neurons, we examined the action potential waveform of neurons from saline and ethanol animals (n = 21 and 20, respectively). A fundamental feature of neuronal firing is the generation of action potentials, and analysis of specific components of the action potential waveform can often provide evidence that the function of a particular channel has been altered. For example, decreased Na+ channel function could be reflected by an increase in action potential threshold and a decrease in the action potential peak (Zhang et al. 1998). However, neither the threshold nor the peak of the action potential were different between saline and ethanol animals (Figs. 2A,B: threshold: saline: –43.5 ± 0.5 mV; ethanol: –44.1 ± 0.7 mV; peak: saline: 17.6 ± 1.4 mV; ethanol: 16.3 ± 1.5 mV; n.s.), suggesting no changes in Na+ channel function. Further, a reduction in the peak value of the afterhyperpolarization (AHP) is often observed during inhibition of fast-repolarizing potassium channels (such as BK and Kv3 delayed rectifier channels; Grillner and Mercuri 2002; Appel et al. 2003). However, the peak value of the AHP was not altered in ethanol animals (Figs. 2A,B; saline: –69.0 ± 1.7 mV; ethanol: –69.8 ± 0.8 mV; n.s.), suggesting no change in function of the
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fast-repolarizing channels. In addition, the action potential width, determined halfway between the action potential threshold and peak, was not different between saline and ethanol animals (saline: 1.27 ± 0.05 ms; ethanol: 1.29 ± 0.05 ms; n.s.). These data suggest that some of the basic electrophysiological properties of VTA DA neurons were not modified by withdrawal following repeated ethanol treatment. However, the time-to-peak of the AHP (TTP-AHP), determined relative to the action potential threshold, was significantly reduced in ethanol animals (Fig. 2C shows results from neurons treated with apamin: saline: 70.9 ± 8.7 ms; ethanol: 46.6 ± 7.0 ms; n = 5 for saline and 10 for ethanol, p < 0.05; significant differences were also apparent in an additional set of neurons from 17 saline and 14 ethanol animals in which apamin was not tested: saline: 56.0 ± 4.8 ms; ethanol: 37.5 ± 4.6 ms; p < 0.05). Although a number of potassium channels contribute to the AHP, the small conductance, calcium-dependent potassium current (SK) is of particular interest because it is hypothesized to regulate the transition from pacemaker to burst firing (Komendantov et al. 2004). Neurons from saline animals exhibited a very broad AHP with a greatly delayed TTPAHP, typical of naïve neurons with a strong SK (age-matched naïve: 61.2 ± 11.1 ms, n = 7; and see Shepard and Bunney 1988; Ping and Shepard 1996). Further, the SK-selective antagonist apamin (200 nM) significantly reduced the TTP-AHP in both saline and ethanol animals (Figs. 2D,F; p < 0.05; n = 5 for saline and 10 for ethanol), with a significantly greater change in the TTP-AHP in saline animals (Figs. 2E,F; saline: 60.6 ± 9.9 ms; ethanol 35.8 ± 6.3 ms; p < 0.05), and resulted in a similar TTP-AHP in both groups (Fig. 2D; saline: 10.4 ± 1.5 ms; ethanol 11.8 ± 1.1 ms; n.s.). Since SK was a dominant contributor to the TTP-AHP (see also Shepard and Bunney 1991; Ping and Shepard 1996), the significantly reduced TTP-AHP in ethanol animals suggests that withdrawal following repeated ethanol exposure reduced SK function.
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To more directly investigate the effects of ethanol withdrawal on SK function, we performed voltage-clamp experiments to better isolate the apamin-sensitive SK current. Neurons were held at –70 mV then depolarized for 400 ms with pulses ranging from –20 mV to –60 mV in 10 mV steps. Upon returning to –70 mV, a tail current due to slow de-activation of a channel was evident (see Fig. 3A, and see also Paul et al. 2003). Both the peak amplitudes of the tail currents evoked after depolarizing steps to –20, –30, and –40 mV and the tail current charge transfer, calculated by integrating the tail current evoked following the depolarizing pulse (see Materials and Methods), were significantly reduced in ethanol animals (Figs. 3A-D), with saline similar to age-matched naïve animals (naïve: 240 ± 40 pA for step to –20 mV, n = 7; compared to 239 ± 12 pA for saline and 178 ± 13 pA for ethanol). In addition, the tail current was inhibited by more than 90% by apamin in both saline and ethanol animals (analyzed for the depolarizing step to –20 mV; saline 92.9 ± 2.5% block; ethanol: 92.8 ± 2.5% block; n = 6 and 9 for saline and ethanol, respectively), indicating that SK is the primary channel active during the tail current following depolarization (see also Paul et al. 2003). In this regard, the apamin-sensitive component of the peak tail current was significantly reduced in ethanol relative to saline animals (Fig. 3E, saline: 60.6 ± 9.9 pA; ethanol: 34.8 ± 5.8 pA, p < 0.05), strongly suggesting that SK function was reduced in ethanol animals. Differences in SK function could result from changes in basic membrane properties. However, neither the series resistance (saline: 14.7 ± 0.9 M ; ethanol: 16.1 ± 0.8 M ; n.s.) nor the instantaneous currents evoked by hyperpolarization (see Figs. 7E,F, taken as a measure of input resistance) were altered in ethanol animals. In addition, we estimated whether cell capacitance might be different in neurons from saline and ethanol animals, perhaps indicating differences in cell size (see Neuhoff et al. 2002), following a method adapted from Gentet et al.
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(2000). Although there could be considerable error using this method due to space-clamp issues associated with low pass filtering and voltage-attenuation during patch-clamp of neurons in brain slice with intact dendrites (Major 1993; Spruston et al. 1993), as well as a lack of direct measurement of the cell surface area, we observed no differences in the fast exponential capacitive transient after a voltage step from –70 to –60 mV (saline: 0.99 ± 0.06 ms; ethanol: 1.08 ± 0.05 ms; n.s.) or the cell capacitance (saline: 76.7 ± 3.4 pF; ethanol: 77.6 ± 4.0 pF; n.s.). Further, differences between saline and ethanol animals in integrated currents of SK (Fig. 3C) and Ih (see below) persisted when normalized to the cell capacitance estimated in this way (data not shown). Taken together with a lack of differences in series resistance or the instantaneous currents evoked by hyperpolarization, these results suggest that apparent changes in SK function did not result from differences in basic membrane parameters. Reduced SK function in ethanol animals could be due to several factors, including altered kinetics or reversal potential. However, the time constant of inactivation (fit from 55 to 550 ms into the tail current; Abel et al. 2004) was not different between ethanol and saline animals (saline: 139 ± 6 ms; ethanol: 140 ± 7 ms; n.s.). Also, the reversal potential of SK (determined by depolarizing to –20 mV for 400 ms, then stepping the cell to voltages ranging from –70 mV to – 120 mV in 5-mV increments, in the presence of the Ih blocker ZD7288, 30 µM) was not different between groups (saline: –89.0 ± 3.2 mV; ethanol: –87.7 ± 1.9 mV; n = 4 for each group; n.s.). The deviation of the SK reversal potential relative to the EK+ derived from the Nerntz equation (~ –100 mV) could be due to small space clamp errors while patch-clamping neurons in a brain slice and/or localization of SK channels in the dendrites (Abel et al. 2004). Additionally, smaller SK amplitudes in ethanol animals might impair accurate determination of the reversal potential in those neurons. However, since the whole-cell capacitance, input resistance, and series
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resistance were similar between saline and ethanol animals, any such errors should be equivalent in both groups.
NMDA-induced burst firing was enhanced in ethanol animals In addition to shaping the AHP, SK can regulate the firing pattern of VTA DA neurons. In particular, studies have demonstrated that SK inhibition facilitates the transition to bursting after application of NMDA (Seutin et al. 1993; Johnson and Seutin 1997). Entry of Na+ and Ca2+ following activation of NMDA receptors produces a depolarization and an increase in intracellular Ca2+ which, in addition to the inactivation of SK (which reduces the AHP), may facilitate the transition from pacemaker firing to burst firing (Seutin et al. 1993; Johnson and Seutin 1997; Komendantov et al. 2004). Since our action potential waveform analysis and voltage-clamp data suggest that SK function was reduced in VTA DA neurons from ethanol animals, we studied whether reduced SK function in ethanol animals would facilitate the ability of NMDA to induce bursting activity. To more precisely quantify bursting, we determined the coefficient of variation of the inter-spike interval (CV-ISI), a measure of spike train irregularity, before and after NMDA application. CV-ISI was defined as the standard deviation divided by the mean of the ISI distribution (Zhang et al. 1994) and was calculated across 100 action potentials. The change from pacemaker to bursting activity increases the CV-ISI, where the CV-ISI is small for very regular spike trains (like pacemaker) and larger in irregular or bursting spike trains. Bath application of NMDA (20 µM) for 10 min increased the firing frequency in neurons from saline animals, but generally did not induce bursting activity, indicated by no difference in the CV-ISI before and after application of 20 µM NMDA (Figs. 4A,D; pre-NMDA: 23.8 ± 4.8 %; post-NMDA: 31.1 ± 9.6 %; n = 10; n.s.). In contrast, the same dose of NMDA (20 µM; n =
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11) induced bursting activity in ethanol animals, indicated by a significant increase in CV-ISI (Figs. 4A,D; pre-NMDA: 27.5 ± 5.5 %; post-NMDA: 79.4 ± 16.1 %; p < 0.05), with a significant difference in the CV-ISI between saline and ethanol after 20 µM NMDA (p < 0.05). Example histograms of the ISI distribution before and after 20 µM NMDA are shown in Figs. 4B and C, and demonstrate that NMDA enhanced firing rate but maintained a regular firing pattern in saline animals, while NMDA produced an ISI distribution with both short ISIs (during a burst) and longer ISIs (between bursts) in ethanol animals. A lower dose of NMDA (10 µM) increased firing rate but did not produce bursting or alter the CV-ISI in either group (Fig. 4D; saline: 20.2 ± 7.5 % post-NMDA; ethanol: 33.0 ± 10.7 % post-NMDA; n = 7 and 6 for saline and ethanol, respectively). A lack of bursting in saline animals was not due to insufficient NMDA receptor activation, since 50 µM NMDA also did not significantly increase the CV-ISI in saline animals (Fig. 4D; 34.1 ± 14.1 % post-NMDA; n = 5). However, neurons from saline animals exhibited bursting and a significantly elevated CV-ISI when 20 µM NMDA was combined with SK inhibition with apamin (Figs. 4D, 5B; 78.8 ± 6.2 % post-NMDA; n = 5; p < 0.05), in agreement with studies showing bursting after NMDA and apamin in combination (Seutin et al. 1993; Johnson and Seutin 1997). Although NMDA alone generally did not elicit bursting in saline neurons, bursting was occasionally observed (an example is shown in Fig. 5B). Thus, to further define whether bursting was induced within a particular neuron, a criterion was defined whereby individual neurons would be considered bursting if they had a CV-ISI greater than the mean plus 2 standard deviations of the CV-ISI values taken from neurons from saline animals before NMDA addition, yielding a threshold of a 68.5% CV-ISI (Fig. 5A). With this criterion, bursting was observed in saline animals in 2 of 10 neurons exposed to 20 µM NMDA, 1 of 5 neurons exposed to 50 µM
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NMDA, but 4 of 5 neurons exposed to 20 µM NMDA combined with apamin (Fig. 5A). Further, bursting was observed in 8 of 11 neurons from ethanol animals exposed to 20 µM NMDA (Fig. 5A). Thus, NMDA alone can induce bursting in some neurons from saline animals, but the incidence of bursting in 20 µM NMDA was significantly greater in ethanol animals (p < 0.05, chi-squared test). Further, inhibition of SK at the same time as depolarization with NMDA strongly facilitated the transition to bursting in saline animals, and generated bursting that was very similar to that observed in ethanol animals exposed only to NMDA (Figs. 4D, 5B). Since there was variability among neurons regarding the pre-NMDA baseline (Fig. 5A), we also calculated a CV-ISI ratio, defined as the ratio of the CV-ISI after NMDA to the CV-ISI before NMDA. The CV-ISI ratio was significantly greater in ethanol relative to saline neurons (Fig. 5C; saline: 1.3 ± 0.3; ethanol: 3.0 ± 0.6; p < 0.05), in agreement with a greater increase in variability in ISIs that typifies burst firing (see Figs. 4A-D). In addition, we observed that the CV ratio was significantly negatively correlated with the SK tail current (Fig. 5D; r2 = 0.387; p < 0.05). Since neurons from ethanol animals that exhibited smaller SK function showed a significantly larger increase in the CV ratio, this suggests that reduced SK function might be responsible for the enhanced NMDA-induced transition to bursting in ethanol animals. Finally, differences in NMDA-induced bursting could result from different NMDAdependent receptor activation. To examine this possibility, we determined the current response to a 30-sec exposure to NMDA (Vholding = +40 mV) under voltage-clamp with a Cs+-based internal solution (Borgland et al. 2006). Bath application of 10 or 20 µM NMDA produced a dosedependent increase in evoked current that was similar in neurons from saline and ethanol animals (Fig. 6; n = 6 for all groups and doses), suggesting that NMDA receptor activation was not different between the two groups. Thus, bursting induced by NMDA was significantly facilitated
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in ethanol animals, with no change in NMDA receptor activity, while neurons from saline animals could exhibit bursting with 20 µM NMDA combined with apamin, but generally did not exhibit bursting after NMDA alone. Taken together, these results strongly suggest that the transition to burst firing was facilitated in ethanol animals, and that the reduced SK function in ethanol animals might play a key role in this enhanced transition to bursting.
Ih function was reduced and recovery from hyperpolarization impaired in ethanol animals, but reduced Ih did not account for enhanced transition to bursting The Ih current can also regulate the activity of VTA DA neurons (Seutin et al. 2001; Neuhoff et al. 2002; Okamoto et al. 2006). To measure Ih, we performed voltage-clamp experiments where a series of hyperpolarizing voltage steps (500 ms, ranging from –40 to –150 mV in 10 mV steps) was applied to VTA neurons from a holding potential of –60 mV, and the magnitude of Ih determined from the slowly developing current sag (Cathala and Paupardin-Tritsch 1997; Neuhoff et al. 2002; Liu et al. 2003). We also determined the Ih charge transfer by integrating the area under the slowly activating inward current in the –140 mV hyperpolarizing voltage pulse (see Materials and Methods). Finally, to better isolate Ih, we performed some voltage-clamp experiments under conditions where many currents other than Ih were inhibited using modified internal and external solutions (see Materials and Methods and Liu et al. 2003). Figures 7A and B show examples of the current response to a step from –60 mV to –140 mV in both experimental groups. The Ih current was significantly decreased in ethanol compared with saline animals, measured both in terms of peak current (Figs. 7A-D; p < 0.01) and Ih charge transfer (Fig. 7G, measured for step to –140 mV; p < 0.05), and using either standard intracellular and extracellular solutions (Figs. 7A,C,E; saline: n = 11, ethanol: n = 9) or solutions
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that provide better isolation of Ih (Figs. 7B,D,F; saline: n = 6, ethanol: n = 8). Also, the Ih current in standard solutions in saline neurons was similar to that in neurons from age-matched naïve rats (measured for step to –140 mV, naïve: 625 ± 74 pA, n = 7; compared with 670 ± 59 pA from saline and 476 ± 61 pA from ethanol). In addition, Ih was determined in neurons from the additional 18 saline and 16 ethanol animals using a current step from –80 mV to –140 mV. Although the Ih measured in this manner was significantly reduced in ethanol relative to saline animals (saline: 382 ± 20 pA; ethanol: 321 ± 18 pA; p < 0.05), Ih is partially active at –80 mV (Cathala and Paupardin-Tritsch 1997; Neuhoff et al. 2002; Liu et al. 2003), and thus these experiments were not used for kinetic analyses. Finally, instantaneous currents were not altered in ethanol relative to saline animals, measured either as peak currents or charge transfer during a hyperpolarizing step from –60 mV (Figs. 7E,F,H), perhaps suggesting no changes in IRK and/or potassium leak channels (Watts et al. 1996; Wilson 2005). Although total Ih currents were reduced in ethanol animals, several kinetic parameters of Ih were not altered. First, the voltage-dependence of activation of Ih, fit with a Boltzmann equation (see Materials and Methods), was not different between saline and ethanol animals (Figs. 8A,B), with no differences in the half-activation voltage (Fig. 8C) or slope factor (Fig. 8D). Thus, ethanol reduced the total Ih current without altering the voltage-dependence of activation of Ih (see also Okamoto et al. 2006). Repeated ethanol exposure also did not alter the reversal potential of Ih (Figs. 8E-F; n = 6 for saline and 9 for ethanol), which was estimated, as described (Mayer and Westbrook 1983, Cathala and Paupardin-Tritsch 1997), by comparing the instantaneous currents evoked from a holding voltage of –60 mV, where Ih is not active, and the instantaneous currents evoked from a holding voltage of –80 mV, where Ih is activated (Cathala and Paupardin-Tritsch 1997; Neuhoff et al. 2002; Liu et al. 2003). In particular, one can estimate
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the reversal potential of Ih from the point where the two current-voltage curves intersect; since currents from a –60 mV holding potential contain IRK/leak but little Ih, while currents from a – 80 mV holding potential contain both IRK/leak and Ih due to activation of Ih at –80 mV, the intersection of the current-voltage curves from –60 and –80 mV essentially represents the voltage where the Ih current is zero after the IRK/leak current is subtracted out. Ih has been reported to alter burst firing in other types of neurons (Wilson 2005). Thus, we examined the effects of the Ih antagonist ZD7288 (30 µM) on firing or NMDA modulation of bursting after 10 min exposure to ZD7288, after which time >90% of Ih should be inhibited (Fig. 9C; Gasparini and DiFrancesco 1997; Okamoto et al. 2006; Gu et al. 2007). In saline animals, ZD7288 did not facilitate a transition to bursting when NMDA was applied subsequent to ZD7288, indicated by no significant increase in the CV-ISI (Fig. 9A; baseline 31.1 ± 6.6 %; after ZD7288 addition: 25.1 ± 5.5 %; after ZD7288 and NMDA: 32.3 ± 10.8 %; n.s.; n = 5). Thus, the reduced Ih in ethanol animals cannot account for the facilitated transition to bursting by NMDA in ethanol animals. In addition, ZD7288 did not alter pacemaker firing, measured by determining the instantaneous firing frequency, in either saline animals (Fig. 9B; before ZD7288: 0.75 ± 0.06 Hz; after ZD7288: 0.72 ± 0.14 Hz; n = 8; n.s.) or ethanol animals (before ZD7288: 0.86 ± 0.05 Hz; after ZD7288: 0.98 ± 0.10 Hz; n = 8; n.s.), in agreement with no differences in pacemaker firing (Fig. 1). One function of Ih is to facilitate recovery from hyperpolarizing events (Satoh and Yamada 2000; Neuhoff et al. 2002; Wilson 2005; Puopolo et al. 2007). Therefore, to further investigate the effects of ethanol withdrawal on Ih, a set of hyperpolarizing steps (300 ms, ranging from –50 to –300 pA) was applied to the neurons during pacemaker firing, and the time to recover the firing activity was measured. Recovery from hyperpolarization was significantly
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delayed in ethanol animals relative to saline animals (examples shown in Fig. 9D; n = 10 for saline and 9 for ethanol). Using a two-way, repeated measures ANOVA, we found a significant effect of treatment (ethanol vs. saline; F(1,17) = 8.344; p < 0.05), the hyperpolarizing step (F(5,85) = 28.690; p < 0.001), and the interaction between the two factors (treatment by hyperpolarizing steps; F(5,85) = 4.365; p < 0.01). These results support the suggestion that the reduced Ih function, apparent in voltage-clamp experiments (Fig. 7), also impaired the ability of VTA DA neurons from ethanol animals to recover from hyperpolarizing events. In addition, as shown in Figures 9E and F, ZD7288 significantly delayed the recovery from hyperpolarization in saline animals (F(1,14) = 7.073; p < 0.05; two-way, repeated measures ANOVA; n = 8). In strong contrast, ZD7288 had no effect on the recovery from hyperpolarization in ethanol animals (Figs. 9E,G; F(1,14) = 0.227; n.s.; two-way, repeated measures ANOVA; n = 8). These results suggest that the reduction in Ih may impair recovery from hyperpolarization in ethanol animals, and are in agreement with studies suggesting a critical role for Ih in DA neurons in recovery from hyperpolarization (Neuhoff et al. 2002; Puopolo et al. 2007).
Withdrawal from repeated ethanol treatment enhanced the locomotor response to cocaine Finally, we wanted to establish whether the repeated ethanol treatment used here could be sufficient to produce physiological changes in ethanol animals. In this regard, drug-induced locomotor sensitization has been associated with some aspects of drug reward, dependence and relapse (Robinson and Berridge 2001), and repeated ethanol exposure has been shown to produce a cross-sensitization to the locomotor-activating effects of cocaine (Itzhak and Martin 1999). Thus, we examined whether the ethanol exposure protocol utilized here enhanced the motor response to cocaine, examined after 7 days of withdrawal.
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Animals withdrawn following repeated ethanol or saline were habituated to the openfield locomotor chamber for three days (see Materials and Methods) before administration of cocaine on the seventh day of withdrawal. Ethanol animals exhibited a significantly greater locomotor response to an acute injection of cocaine (15 mg/kg, i.p.) relative to saline animals or naïve animals (Fig. 10; n = 16, 13, and 11 for saline, ethanol, and naïve animals, respectively; p < 0.05). No differences were observed between groups in the locomotor activity during a 60-min period of habituation, or in response to an acute injection of saline (Fig. 10). Further, saline animals exhibited a very similar locomotor response to cocaine as naïve animals, suggesting that any stress related to multiple injections or handling procedures did not enhance the locomotor response to cocaine. Taken together, these results indicate that the repeated ethanol exposure and withdrawal paradigm utilized here altered channel function and also produced a crosssensitization to the acute locomotor-activating effects of cocaine.
Discussion
Here, we show that pacemaker firing of VTA DA neurons was not altered after withdrawal from repeated ethanol exposure, but that the probability of burst firing after NMDA-induced depolarization was significantly increased in ethanol animals. Enhanced NMDA-induced bursting may have resulted from decreased SK function in ethanol animals. Ih currents were also decreased in ethanol animals, and may underlie the reduced recovery following hyperpolarization, but likely cannot account for the enhanced bursting in ethanol animals. Finally, repeated treatment with ethanol significantly enhanced the locomotor-activating effects of cocaine, administered after seven days of withdrawal, providing evidence that the ethanol
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exposure procedure used here was sufficient to produce physiological changes in ethanol animals. Thus, decreased SK function after repeated ethanol exposure may enhance the ability of VTA dopamine neurons to produce burst firing, and thus may contribute to addiction-related behaviors through increasing the salience of drug-related stimuli and by supporting crosssensitization with other drugs of abuse.
Pacemaker activity was not altered in VTA DA neurons from ethanol animals Pacemaker firing is under strong control of a number of currents including Ca2+ channels and Atype potassium channels (see Marinelli et al. 2006; Puopolo et al. 2007) and the Na+/K+-ATPase (Johnson et al. 1992). However, studies of SK and Ih regulation of pacemaker activity in midbrain DA neurons have yielded mixed results. When pacemaker frequencies are more similar to those observed here, SK inhibition with apamin did not alter pacemaker firing (Seutin et al. 1993; Brodie et al. 1999; Johnson and Wu 2004; Waroux et al. 2005), although, with more rapid firing, apamin can increase firing rates (Shepard and Bunney 1988; Ping and Shepard 1996; Wolfart et al. 2001; Waroux et al. 2005). Further, Ih-dependent depolarization enhanced firing in some studies (Seutin et al. 2001; Okamoto et al. 2006), but other results suggest a less consistent relationship between Ih and pacemaker activity (Neuhoff et al. 2002; Liu et al. 2003), and Ih can reduce firing by decreasing the input resistance (Fan et al. 2005). Thus, the currents responsible for the strong oscillatory drive in VTA neurons could override the contribution of Ih and SK during pacemaker firing, perhaps also explaining the lack of difference in pacemaker activity between saline and ethanol animals despite functional changes in several currents. Previous in vivo and in vitro observations during withdrawal following chronic ethanol found reduced firing rates in adult midbrain DA neurons after 1-3 days (Diana et al. 1993, 1996;
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Bailey et al. 1998; Shen 2003; but see Brodie 1999) or 6 days of withdrawal (Bailey et al. 2001), with no changes in firing rates of active DA neurons 3 or more weeks after repeated ethanol exposure (Bailey et al. 2001; Shen et al. 2007), although the number of neurons firing was reduced (Shen et al. 2007). Here, we observed no changes in pacemaker activity 7 days after repeated ethanol treatment. Taken together, these observations suggest that withdrawal from chronic ethanol treatment decreases pacemaker activity for several days (but see Brodie 1999), but that this effect is absent after 7 or more days of withdrawal. However, it should be noted that our studies utilized juvenile animals, and thus our results might not fully reflect those collected from adult animals, although differences in species (mice vs. rat) and route of ethanol administration (liquid diet vs. intermittent i.p. injections) could also explain apparent discrepancies between our studies and those of Bailey and colleagues (2001).
SK function was reduced and burst firing enhanced in ethanol animals Several lines of evidence suggest that repeated ethanol exposure decreased SK function in VTA neurons. The time-to-peak of the AHP (TTP-AHP) was significantly reduced in ethanol animals, with no apparent changes in several other parameters of the action potential waveform. The TTPAHP was also greatly reduced by the SK antagonist apamin (Shepard and Bunney 1988; Ping and Shepard 1996), suggesting a primary contribution of SK to the TTP-AHP. Further, SKmediated tail currents (Paul et al. 2003) were significantly reduced in ethanol animals, with a significant correlation between the TTP-AHP and the tail current magnitude in ethanol animals. Taken together, these results strongly suggest that SK function was reduced in ethanol animals. Further experiments will be required to determine whether differences in SK function in ethanol
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animals reflect changes in number or regulation of SK or of the calcium channels required for SK activation (Bond et al. 2005). In addition to regulation of the AHP, SK inhibition facilitates NMDA-induced burst firing (Seutin et al. 1993; Johnson and Seutin 1997; Waroux et al. 2005). Here, NMDA (20 µM) increased pacemaker frequency in saline animals, but induced burst firing in ethanol animals, which could be explained by decreased SK activity facilitating NMDA induction of bursting in ethanol animals. Differential NMDA induction of burst firing was not due to differences in NMDA receptor activation, since the current response to NMDA, measured under voltage clamp, was similar in saline and ethanol animals. In addition, control neurons were capable of bursting, since SK inhibition in combination with NMDA elicited bursting in saline animals, as is widely observed in control neurons (Seutin et al. 1993; Johnson and Seutin 1997). Thus, neurons from saline animals could exhibit bursting, especially when SK was inhibited during NMDA application, but NMDA induction of bursting was greatly facilitated in ethanol animals, perhaps due to decreased SK function. In addition, NMDA induction of bursting in ethanol neurons was significantly negatively correlated with the SK tail current magnitude, strongly supporting the possibility that reduced SK function might be responsible for the enhanced NMDA-induced transition to bursting in ethanol animals. An increased ability of the glutamate system in the VTA to induce bursting activity after repeated drug exposure may contribute to drug seeking and enhance the vulnerability to relapse during ethanol withdrawal. Glutamatergic afferents into the VTA can regulate the shift from pacemaker to burst firing (Chergui et al. 1994; Floresco et al. 2001; Grillner and Mercuri 2002), which increases DA release in midbrain target regions and may contribute to some aspects of drug-induced reinstatement (Grace 2000; Tupala and Tiihonen 2004; Schmidt and Pierce 2006).
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Ih function was reduced and recovery from hyperpolarization impaired in ethanol animals Repeated ethanol exposure and 7 days withdrawal also significantly reduced hyperpolarizationevoked Ih currents, which could significantly influence firing properties. For example, Ih drives certain aspects of oscillatory activity in striatal cholinergic interneurons (Wilson 2005), and thus decreased Ih might retard bursting by reducing the depolarizing drive (Wilson 2005). However, NMDA-induced bursting was enhanced in ethanol animals, and Ih inhibition in saline animals did not enhance NMDA-induced bursting, suggesting that differences in Ih did not account for the increased NMDA-induced transition to bursting in ethanol animals. In addition, Ih can facilitate recovery from hyperpolarization (Satoh and Yamada 2000; Neuhoff et al. 2002; Wilson 2005). Firing is delayed after strong hyperpolarization (see Fig. 9D) due to an A-type potassium current (Koyama and Appel 2006), as Ih activation at hyperpolarized potentials can provide depolarization to drive neurons to voltages permissive for firing. Here, neurons from ethanol animals showed impaired recovery of firing after hyperpolarization, in agreement with voltageclamp observations suggesting decreased Ih function. Also, the Ih antagonist ZD7288 significantly delayed recovery of firing following hyperpolarization in saline animals, but had no effect in ethanol animals, suggesting that reduced Ih in ethanol animals was responsible for the delayed recovery from hyperpolarization. Reduced Ih was not due to altered voltage-dependence of activation, in agreement with a previous study showing a dissociation between changes in total Ih current and voltage-dependence of activation of Ih (Okamoto et al. 2006). Several papers have raised the possibility that ZD7288 exhibits non-specific effects through targets other than Ih (Chevaleyre and Castillo 2002; Wilson 2005). The concentration of ZD7288 employed here is widely used, and has been reported to not effect a number of
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physiological parameters including instantaneous currents during hyperpolarization (Harris and Constanti, 1995; Liu et al. 2003; Okamoto et al. 2006; but see Wilson 2005), AP potential waveform except for the AHP (Harris and Constanti, 1995; Gasparini and DiFrancesco 1997; and data not shown), and several forms of synaptic plasticity (Gasparini and DiFrancesco 1997; Chevaleyre and Castillo 2002; but see Mellor et al. 2002). In addition, in several studies, nonspecific effects of ZD7288 were generally evident at higher ZD7288 concentrations and/or after much longer exposure times (Chevaleyre and Castillo 2002; Mellor et al. 2002; Wilson 2005) than those employed here. We found that ZD7288 did not alter pacemaker firing or NMDA modulation of firing, but did alter recovery from hyperpolarization in saline but not ethanol animals, suggesting a more selective effect of ZD7288 on VTA DA neuron firing. We should note that it is unclear whether the Ih modulation of recovery from hyperpolarization is directly relevant in vivo, since the VTA in vivo generally does not exhibit such stereotyped pauses in firing, compared, for example, to striatal cholinergic interneurons (see Wilson 2005). The purpose of measuring the recovery from hyperpolarization was not to claim that a pause such as that observed by the strong hyperpolarizing current step actually occurs in vivo in the VTA. Instead, our results suggest that, under conditions of strong hyperpolarization (e.g., with strong activation of IRK), a reduction in Ih will facilitate the potency of hyperpolarization for reducing firing, as has been demonstrated by Liu and colleagues (2005). However, several groups have found a reduction in VTA firing during omission of an expected reward (see Schultz 2002); the underlying cellular mechanism is unclear, and could result from circuit interactions, but neuro-modulator activation of IRK might also contribute to this pause in firing (but see Ji and Shepard 2007).
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At present, the mechanisms through which repeated ethanol exposure might produce lasting changes in SK and Ih function remain unclear. Expression of both SK and HCN subunits could exhibit persistent plasticity (Seroussi et al. 2002; Chaplain et al. 2003; Kye et al. 2007), and SK subunit expression can be regulated by transcriptional modulators (Kye et al. 2007; see also Sun et al. 2001). Since ethanol can activate a number of transcriptional pathways (Mulligan et al. 2006), we speculate that protracted changes in channel function after repeated ethanol could reflect altered channel expression. In addition, it is possible that decreased Ih function after repeated ethanol exposure and withdrawal might represent a compensatory mechanism opposing the actions of reduced SK channel function on firing; for example, substantia nigra neurons exhibit greater function of both Ih and SK relative to VTA (Wolfart et al. 2001; Neuhoff et al. 2002), suggesting a possible counterbalancing role of Ih and SK in midbrain DA neuron firing.
Repeated ethanol treatment enhanced locomotor activation by cocaine Here, repeated ethanol exposure enhanced the locomotor activation produced when cocaine was acutely administered after 7 days of withdrawal, in agreement with a previously described crosssensitization to the locomotor-activating effects of cocaine after repeated ethanol (Itzhak and Martin 1999). We should note that not all studies examining repeated ethanol injection have observed subsequent enhanced locomotor activation by cocaine (Wise et al. 1996, Lessov and Phillips 2003). However, as noted by Lessov and Phillips (2003), the drug treatment schedules and time of testing for sensitization vary widely across these studies, making comparisons difficult. In addition, since all these studies were performed in mice, ours is the first to demonstrate in rats that repeated ethanol and 7 days withdrawal can significantly enhance the locomotor-activating effects of cocaine, perhaps representing a form of cross-sensitization.
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It has been proposed that sensitization represents an increase in the positive value attributed to a drug or drug-associated stimulus, and sensitization has thus been considered a model of altered drug seeking (Robinson and Berridge 2001). In addition, sensitization may result from persisting neuroadaptations in mesolimbic brain regions (Vanderschuren and Kalivas 2000; Nestler 2001; Vezina 2004), including the VTA (e.g., Ungless et al. 2001; Liu et al. 2005). Although the VTA is critical for the development of sensitization for many abused drugs, the direct role of the VTA in expression of sensitization may depend on the particular drug of abuse, where a clearer role for opiates but a less clear role for psychostimulants has been described (Vanderschuren and Kalivas 2000; Leite-Morris et al. 2004). In addition, a recent study showed that ethanol-related sensitization in mice can be apparent without increased dopamine release, perhaps dissociating the dopaminergic system from enhanced locomotor activation by ethanol after repeated ethanol in mice (Zapata et al. 2006). Although little is known about the cellular mechanisms underlying enhanced locomotor activation after repeated ethanol, reduced SK and facilitation of NMDA-induced bursting might contribute to the enhanced locomotor activation by cocaine after repeated ethanol exposure that we and others (Itzhak and Martin 1999) have observed.
Conclusion We have shown here that repeated ethanol exposure and 7 days withdrawal led to several alterations in regulation of firing of VTA DA neurons. In particular, several lines of evidence indicate that SK function was reduced in ethanol relative to saline animals. Interestingly, neurons from ethanol animals showed an increased propensity for bursting during exposure to NMDA, with no change in the amount of NMDA-dependent depolarization measured under voltage-
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clamp conditions, suggesting that the reduced SK function could be responsible for the enhanced bursting in ethanol animals. Repeated ethanol exposure also led to reduced Ih function, which impaired the ability of VTA neurons to recover from hyperpolarization, but likely did not account for the enhanced bursting in ethanol animals. Also, repeated ethanol exposure increased the locomotor activation by acute cocaine, confirming that the ethanol exposure procedure used here was able to produce functional physiological changes. Thus, repeated ethanol exposure and withdrawal produced enhanced bursting of VTA DA neurons during withdrawal from repeated ethanol exposure, which may contribute to other addiction-related behaviors through increasing the salience of drug-related stimuli.
Acknowledgements
We thank D. Harada and G. Brush for custom-designed analysis software and L. Daitch for proofreading. This work was supported by funds provided by the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco (AB) and by the Department of the Army, Grant # W81XWH-05-1-0213 (AB). The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014, is the awarding and administering acquisition office. The content of the information represented does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred.
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Figure Legends
Figure 1. Seven days withdrawal after repeated ethanol treatment did not alter pacemaker firing in VTA DA neurons. A, Representative examples of recordings of spontaneous neuronal activity from saline and ethanol rats. B and C, No changes in instantaneous firing frequency or the interspike interval (ISI) were observed.
Figure 2. Seven days withdrawal after repeated ethanol treatment did not alter several parameters of the action potential waveform, but did decrease the time-to-peak of the afterhyperpolarization (TTP-AHP). A, Example action potentials from a saline and an ethanol rat. B, No changes were observed in the action potential threshold or peak or in the AHP peak. C and D, Examples (C) and grouped data (D) showing a decreased TTP-AHP in ethanol relative to saline animals. Arrowheads indicate AHP peak. D-F, The small conductance, calcium-dependent potassium channel (SK)-selective inhibitor apamin (200 nM) significantly reduced the TTP-AHP (D), suggesting a significant contribution of SK to the TTP-AHP, with a significantly greater reduction in the TTP-AHP in ethanol relative to saline animals (E). * p < 0.01 vs. saline. # p < 0.01 vs. before apamin. “AP”, “bas”, and “apa” indicate action potential, basal, and apamin, respectively. F, Examples showing greater reduction of the TTP-AHP by apamin in saline relative to ethanol animals.
Figure 3. Seven days withdrawal after repeated ethanol treatment decreased the activity of SK. A, Depolarizing current steps resulted in a tail current upon return to the –70 mV holding potential. The left trace shows an example from a saline animal of the full current response to the
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–20 mV depolarization. The right traces show examples of the post-depolarization tail current in saline and ethanol animals, before and after apamin. B, The peak of the tail current, determined for the voltage steps to –20, –30, and –40 mV, was significantly smaller in ethanol animals. C, The tail current charge transfer was also significantly reduced in ethanol animals. D, Apamin significantly reduced the tail current in both saline and ethanol animals. “bas” and “apa” indicate basal and apamin, respectively. E, The apamin-sensitive portion of the tail current was significantly greater in saline versus ethanol animals. * p < 0.05 saline vs. ethanol. # p < 0.01 vs. before apamin.
Figure 4. NMDA-induced burst firing in VTA DA neurons from ethanol animals. A, Examples of spontaneous firing and the effects of NMDA (20 µM) bath application on the activity of VTA DA neurons. NMDA increased the firing rate in neurons from saline animals, but produced a burst-firing pattern of activity in animals treated with ethanol. B and C, Example histograms showing the distribution of inter-spike intervals (ISI) in saline (B) and ethanol (C) animals before and after application of NMDA. D, The coefficient of variation of the ISI (CV-ISI) was used to evaluate the effects of NMDA on the pattern of neuronal activity. A significant increase in the CV-ISI was observed in ethanol animals with 20 µM NMDA, suggesting a change from regular firing to bursting activity. 10, 20, and 50 µM NMDA did not alter the CV-ISI in saline animals, although combined 20 µM NMDA and apamin (200 nM) did significantly increase the CV-ISI in saline animals. * p < 0.05 compared to basal firing. # p < 0.05 compared to saline with 20 µM NMDA.
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Figure 5. NMDA-induced burst firing in VTA DA neurons was variable across cells. A, Raw data of the CV-ISI values showing that NMDA alone did elicit bursting in some saline neurons, although the prevalence was significantly greater in ethanol neurons, and was significantly enhanced in saline animals by inhibition of SK with apamin concurrent with NMDA application. The dotted line indicates threshold of a 68.5% CV-ISI, derived from the mean plus two times the standard deviation of the CV-ISI distribution in saline animals before NMDA. B, Examples of bursting in neurons from saline animals after 20 µM NMDA (left) or 20 µM NMDA plus SK inhibition with apamin (right). C, The CV-ISI ratio, defined as the ratio of the CV-ISI after NMDA to the CV-ISI before NMDA, was significantly greater in ethanol relative to saline neurons. D, The CV-ISI ratio was significantly negatively correlated with the peak SK tail current (regression line shown), suggesting that reduced SK function might be responsible for the enhanced NMDA-induced transition to bursting in ethanol animals. * p < 0.05 compared to basal firing.
Figure 6. Examples (A) and grouped data (B) showing that the current response to a 30 sec exposure to 10 or 20 µM NMDA was dose-dependent, but not different between neurons from saline and ethanol animals. Recordings were made under voltage-clamp conditions with a Cs+based internal solution, with a holding potential of +40 mV.
Figure 7. Withdrawal following repeated ethanol exposure decreased Ih currents in VTA neurons. A and B, Examples showing a 500-ms hyperpolarizing step from –60 to –140 mV, with a significant decrease in ethanol animals in the Ih-dependent slowly developing current sag but not the initial, instantaneous current, with (A) standard external and internal solutions (“Stand.
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solns.”), or (B) solutions modified to facilitate isolation of Ih (“Ih isol. solns.”; see Materials and Methods). C and D, Reduced Ih currents in ethanol animals were apparent when using Stand. solns. (C) or Ih isol. solns (D). E and F, Instantaneous currents were not altered in ethanol relative to saline animals. G and H, Analysis of currents by determining the charge transfer (for the voltage step to –140 mV) also showed significantly reduced Ih currents (G) but no differences in instantaneous currents (H) in ethanol animals. * p < 0.05.
Figure 8. Decreased Ih currents following repeated ethanol and withdrawal occurred without changes in several kinetic parameters of Ih. A and B, No differences in voltage-dependence of activation of Ih were apparent between saline and ethanol animals, in either standard external and internal solutions (“Stand. solns.”), or solutions modified to facilitate isolation of Ih (“Ih isol. solns.”; see Materials and Methods). The V1/2 (C ) and slope factor (D) for voltage dependence of activation of Ih were not altered in ethanol animals. E, Example demonstrating estimation of the Ih reversal potential by comparing the instantaneous currents evoked from a holding voltage of –60 mV, where Ih is not active, and the instantaneous currents evoked from a holding voltage of –80 mV, where Ih is activated (see Materials and Methods). F, The reversal potential of Ih was not altered in ethanol animals.
Figure 9. Reduced Ih function during withdrawal following repeated ethanol exposure did not facilitate burst firing or alter pacemaker firing, but did reduce the capability of VTA DA neurons to recover pacemaker activity after a hyperpolarizing step. A, Inhibition of Ih with ZD7288 (ZD, 30 µM) did not facilitate NMDA-induced bursting in saline animals. B, ZD7288 did not alter pacemaker frequency in saline or ethanol animals. C, Example and time-course showing
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significant reduction of Ih by ZD7288 within 10 min of exposure in a saline neuron. D-G, A series of hyperpolarizing steps was applied, and the time to recover pacemaker activity was determined (indicated by the dark bars over the traces). D, Examples of response to a 50 pA hyperpolarizing step, showing a slight hyperpolarization, and a 300 pA hyperpolarizing step, showing that the time to recover pacemaker activity after the hyperpolarizing step was significantly increased in VTA DA neurons from ethanol animals. E-G, Example traces (E) and grouped data (F,G) showing that ZD7288 significantly delayed recovery from hyperpolarization in saline animals (F), but had no effect in ethanol animals (G).
Figure 10. Withdrawal from repeated ethanol injection significantly enhanced the acute locomotor response to cocaine (15 mg/kg, i.p.) relative to animals repeatedly injected with saline and to naïve animals. Locomotor activity during the first hour of habituation and in response to acute saline injection was not changed, suggesting a selective cross-sensitization of ethanol withdrawal to the locomotor-activating effects of acute cocaine. Also, a similar cocaine-induced locomotor response in saline and naïve animals suggests that repeated injection and handling did not alter the locomotor-activating effects of acute cocaine.
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Figure 1. Seven days withdrawal after repeated ethanol treatment did not alter pacemaker firing in VTA DA neurons. A, Representative examples of recordings of spontaneous neuronal activity from saline and ethanol rats. B and C, No changes in instantaneous firing frequency or the inter-spike interval (ISI) were observed. 88x72mm (600 x 600 DPI)
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Figure 2. Seven days withdrawal after repeated ethanol treatment did not alter several parameters of the action potential waveform, but did decrease the time-to-peak of the afterhyperpolarization (TTP-AHP). A, Example action potentials from a saline and an ethanol rat. B, No changes were observed in the action potential threshold or peak or in the AHP peak. C and D, Examples (C) and grouped data (D) showing a decreased TTP-AHP in ethanol relative to saline animals. Arrowheads indicate AHP peak. D-F, The small conductance, calcium-dependent potassium channel (SK)-selective inhibitor apamin (200 nM) significantly reduced the TTP-AHP (D), suggesting a significant contribution of SK to the TTP-AHP, with a significantly greater reduction in the TTP-AHP in ethanol relative to saline animals (E). * p < 0.01 vs. saline. # p < 0.01 vs. before apamin. AP, bas, and apa indicate action potential, basal, and apamin, respectively. F, Examples showing greater reduction of the TTP-AHP by apamin in saline relative to ethanol animals.
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88x127mm (600 x 600 DPI)
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Figure 3. Seven days withdrawal after repeated ethanol treatment decreased the activity of SK. A, Depolarizing current steps resulted in a tail current upon return to the -70 mV holding potential. The left trace shows an example from a saline animal of the full current response to the -20 mV depolarization. The right traces show examples of the postdepolarization tail current in saline and ethanol animals, before and after apamin. B, The peak of the tail current, determined for the voltage steps to -20, -30, and -40 mV, was significantly smaller in ethanol animals. C, The tail current charge transfer was also significantly reduced in ethanol animals. D, Apamin significantly reduced the tail current in both saline and ethanol animals. bas and apa indicate basal and apamin, respectively. E, The apamin-sensitive portion of the tail current was significantly greater in saline versus ethanol animals. * p < 0.05 saline vs. ethanol. # p < 0.01 vs. before apamin. 87x124mm (600 x 600 DPI)
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Figure 4. NMDA-induced burst firing in VTA DA neurons from ethanol animals. A, Examples of spontaneous firing and the effects of NMDA (20 M) bath application on the activity of VTA DA neurons. NMDA increased the firing rate in neurons from saline animals, but produced a burst-firing pattern of activity in animals treated with ethanol. B and C, Example histograms showing the distribution of inter-spike intervals (ISI) in saline (B) and ethanol (C) animals before and after application of NMDA. D, The coefficient of variation of the ISI (CV-ISI) was used to evaluate the effects of NMDA on the pattern of neuronal activity. A significant increase in the CV-ISI was observed in ethanol animals with 20 M NMDA, suggesting a change from regular firing to bursting activity. 10, 20, and 50 M NMDA did not alter the CV-ISI in saline animals, although combined 20 M NMDA and apamin (200 nM) did significantly increase the CV-ISI in
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saline animals. * p < 0.05 compared to basal firing. # p < 0.05 compared to saline with 20 M NMDA. 88x141mm (600 x 600 DPI)
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Figure 5. NMDA-induced burst firing in VTA DA neurons was variable across cells. A, Raw data of the CV-ISI values showing that NMDA alone did elicit bursting in some saline neurons, although the prevalence was significantly greater in ethanol neurons, and was significantly enhanced in saline animals by inhibition of SK with apamin concurrent with NMDA application. The dotted line indicates threshold of a 68.5% CV-ISI, derived from the mean plus two times the standard deviation of the CV-ISI distribution in saline animals before NMDA. B, Examples of bursting in neurons from saline animals after 20 M NMDA (left) or 20 M NMDA plus SK inhibition with apamin (right). C, The CV-ISI ratio, defined as the ratio of the CV-ISI after NMDA to the CV-ISI before NMDA, was significantly greater in ethanol relative to saline neurons. D, The CV-ISI ratio was significantly negatively correlated with the peak SK tail current (regression line shown), suggesting that reduced SK function might be responsible for the enhanced NMDA-
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induced transition to bursting in ethanol animals. * p < 0.05 compared to basal firing. 87x120mm (600 x 600 DPI)
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Figure 6. Examples (A) and grouped data (B) showing that the current response to a 30 sec exposure to 10 or 20 M NMDA was dose-dependent, but not different between neurons from saline and ethanol animals. Recordings were made under voltage-clamp conditions with a Cs+-based internal solution, with a holding potential of +40 mV. 87x89mm (600 x 600 DPI)
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Figure 7. Withdrawal following repeated ethanol exposure decreased Ih currents in VTA neurons. A and B, Examples showing a 500-ms hyperpolarizing step from -60 to -140 mV, with a significant decrease in ethanol animals in the Ih-dependent slowly developing current sag but not the initial, instantaneous current, with (A) standard external and internal solutions (Stand. solns.), or (B) solutions modified to facilitate isolation of Ih (Ih isol. solns.; see Materials and Methods). C and D, Reduced Ih currents in ethanol animals were apparent when using Stand. solns. (C) or Ih isol. solns (D). E and F, Instantaneous currents were not altered in ethanol relative to saline animals. G and H, Analysis of currents by determining the charge transfer (for the voltage step to -140 mV) also showed significantly reduced Ih currents (G) but no differences in instantaneous currents (H) in ethanol animals. * p < 0.05. 88x200mm (600 x 600 DPI)
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Figure 8. Decreased Ih currents following repeated ethanol and withdrawal occurred without changes in several kinetic parameters of Ih. A and B, No differences in voltagedependence of activation of Ih were apparent between saline and ethanol animals, in either standard external and internal solutions (Stand. solns.), or solutions modified to facilitate isolation of Ih (Ih isol. solns.; see Materials and Methods). The V1/2 (C ) and slope factor (D) for voltage dependence of activation of Ih were not altered in ethanol animals. E, Example demonstrating estimation of the Ih reversal potential by comparing the instantaneous currents evoked from a holding voltage of -60 mV, where Ih is not active, and the instantaneous currents evoked from a holding voltage of -80 mV, where Ih is activated (see Materials and Methods). F, The reversal potential of Ih was not altered in ethanol animals. 88x135mm (600 x 600 DPI)
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Figure 9. Reduced Ih function during withdrawal following repeated ethanol exposure did not facilitate burst firing or alter pacemaker firing, but did reduce the capability of VTA DA neurons to recover pacemaker activity after a hyperpolarizing step. A, Inhibition of Ih with ZD7288 (ZD, 30 M) did not facilitate NMDA-induced bursting in saline animals. B, ZD7288 did not alter pacemaker frequency in saline or ethanol animals. C, Example and time-course showing significant reduction of Ih by ZD7288 within 10 min of exposure in a saline neuron. D-G, A series of hyperpolarizing steps was applied, and the time to recover pacemaker activity was determined (indicated by the dark bars over the traces). D, Examples of response to a 50 pA hyperpolarizing step, showing a slight hyperpolarization, and a 300 pA hyperpolarizing step, showing that the time to recover pacemaker activity after the hyperpolarizing step was significantly increased in VTA DA neurons from ethanol animals. E-G, Example traces (E) and grouped data (F,G) showing that ZD7288
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significantly delayed recovery from hyperpolarization in saline animals (F), but had no effect in ethanol animals (G). 88x231mm (600 x 600 DPI)
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Figure 10. Withdrawal from repeated ethanol injection significantly enhanced the acute locomotor response to cocaine (15 mg/kg, i.p.) relative to animals repeatedly injected with saline and to naïve animals. Locomotor activity during the first hour of habituation and in response to acute saline injection was not changed, suggesting a selective crosssensitization of ethanol withdrawal to the locomotor-activating effects of acute cocaine. Also, a similar cocaine-induced locomotor response in saline and naïve animals suggests that repeated injection and handling did not alter the locomotor-activating effects of acute cocaine. 86x53mm (600 x 600 DPI)
