Selectively bred lines provided us with a means for testing the hypothesis that heightened sensitivity to the locomotor stimulant effects of ethanol is genetically related to magnitude of dopamine response to ethanol and cocaine in the mesoaccumbens dopamine pathway. Pharmacological studies have suggested that this relationship exists (Jerlhag 2008
), and at least one microdialysis study comparing a pair of inbred strains that displays differential sensitivity to ethanol (2 g/kg) stimulation has found this association (Kapasova & Szumlinski 2008
), while another has not (Zapata et al. 2006
). However, those studies could not differentiate the genetic from non-genetic sources of the association. The FAST and SLOW lines are the only animals that have been specifically bred for behavioral stimulation to ethanol, and a substantial involvement of the basal ganglia in the segregation of the FAST and SLOW lines has been suggested (Demarest et al. 1999
). Here, the greater dopamine response to ethanol and to cocaine in FAST compared to SLOW mice that was found in both replicate sets of selected lines offers strong evidence for common genetic regulation of the behavioral and mesoaccumbens dopamine responses to these drugs.
Breeding for differential sensitivity to ethanol-induced locomotion also resulted in a difference in sensitivity to the sedative effects of ethanol, a difference that increased in magnitude across generations of selection (Phillips et al. 2002
). Thus, the FAST line represents a model of heightened sensitivity to ethanol-induced stimulation, reduced sensitivity to ethanol-induced sedation and increased dopamine response in at least one reward-related brain region. Inasmuch as the magnitude of increases in accumbal dopamine is associated with the intensity of reinforcement (Phillips et al. 1989
), FAST mice may be expected to exhibit increased sensitivity to the rewarding effects of ethanol. FAST mice exhibited increased voluntary ethanol consumption compared to SLOW mice, although the selected lines did not differ when tested for sensitivity to the conditioned rewarding effect of ethanol (0.8-2.0 g/kg) in a place conditioning paradigm (Risinger et al. 1994
). This suggests that the genes that influence these two putative measures of ethanol reward are at least partially genetically independent. There have been no studies of the rewarding effects of cocaine in FAST and SLOW mice, but we would expect greater reward sensitivity in FAST mice, given the current post-cocaine dopamine results and the positive association between ethanol and cocaine consumption in lines of mice selected for sensitivity to methamphetamine-induced locomotion (Kamens et al. 2006
). However, because measurements of drug reinforcement and reward can be influenced by taste factors, response to novelty, and other variables (Blednov et al. 2008
; Blizard & McClearn 2000
; Green & Grahame 2008
; Orsini et al. 2004
), alternative explanations would have to be carefully considered in comparisons of FAST and SLOW mice.
Mesolimbic dopamine as a genetic correlate of ethanol- and cocaine-induced locomotion
A fundamental difference in dopaminergic function between FAST and SLOW mice might explain their differential sensitivity, not just to psychostimulants like cocaine (Bergstrom et al. 2003
), but also to morphine (Bergstrom et al. 2003
; Holstein et al. 2005
), benzodiazepines and barbiturates (Palmer et al. 2002a
; Phillips et al. 1992
), and ketamine (Meyer & Phillips 2003
). While these studies were unable to determine whether there were differences in basal NAcc dopamine levels, data obtained from the potassium perfusion experiments indicated that the FAST and SLOW mice do not differ in the availability of releasable dopamine (Cosford et al. 1994
; Ripley et al. 1997
). However, the greater sensitivity of FAST compared to SLOW mice to the stimulant effects of cocaine and ethanol corresponded with larger increases in NAcc dopamine. This indicates that the mesolimbic dopamine system was altered during selective breeding for sensitivity to ethanol's locomotor effects. Consistent with this conclusion are data from our recent study of the electrical properties of dopamine neurons. In non-ethanol treated ventral midbrain slices, dopamine neuron pacemaker firing was faster, and ethanol produced a larger increase in spontaneous dopamine neuron firing, in FAST compared to SLOW mice (Beckstead & Phillips, accepted pending revision
That FAST mice exhibit profound locomotor stimulation to cocaine suggests that they are not engaging in significant stereotyped behaviors; however, greater sensitivity of SLOW mice to stereotypic effects of cocaine could be related to their blunted stimulant response. In addition, because degree of cocaine-induced stereotypy is associated with dopamine uptake inhibition in the NAcc (Budygin 2007
), one might expect to see greater dopamine levels after cocaine in the SLOW than FAST mice if the behavioral difference was due to heightened stereotypy in SLOW mice. We recently measured cocaine-induced stereotypy in FAST and SLOW mice (data not shown). In this study, we examined a battery of behaviors including chewing, exophthalmos (eye bulging), circling and line crossing after saline, 10, 20 or 40 mg/kg cocaine, and found little evidence for stereotypic behaviors induced by these doses. For line crossing, only the 40 mg/kg cocaine dose differentiated the FAST from SLOW mice, with FAST showing greater locomotor stimulation than SLOW, consistent with the data shown in .
The quantitative relationship between drug-induced locomotor stimulation and elevated accumbal extracellular dopamine levels does not appear straightforward. In FAST mice there was a substantial difference in the extent to which dopamine was increased by ethanol (~30%) compared to cocaine (~350%), whereas the behavioral activation induced by these drugs was similar. SLOW mice displayed behavioral differences to ethanol versus cocaine that were correlated more closely with changes in dopamine. It is possible that degree of behavioral stimulation is limited by a ceiling effect, in which a mouse cannot increase locomotor activity above a certain level. Thus, in the presence of larger increases in dopamine, a larger amount of behavior may not be expressed.
It is well-known that non-dopaminergic processes influence the ethanol response (Heinz et al. 2003
; Phillips & Shen 1996
; Vengeliene et al. 2008
), whereas NAcc dopamine is a primary modulator of cocaine-induced stimulation (Amalric & Koob 1993
; Zhang et al. 2001
). For example, the stimulant response to ethanol has been associated with its N-methyl-D-aspartate receptor antagonist (Meyer & Phillips 2003
) and GABAergic properties (Shen et al. 1998
) in FAST and SLOW mice. These properties of ethanol, or another dopamine pathway (e.g., ventral tegmental area to amygdala; Demarest et al. 1999
), could be responsible for a portion of the locomotor response. There were increases in dopamine in SLOW mice treated with ethanol, even though these mice did not show stimulation. This suggests dissociation between ethanol-induced activity and ethanol-induced increases in dopamine levels. However, it may be that the neural substrates of ethanol-induced depression mask the behavioral effects of ethanol-induced dopamine in SLOW mice.
There was no evidence for an acute effect of ethanol or cocaine on NAcc glutamate levels in the current studies. A glutamatergic input into the accumbens from other brain areas such as the prefrontal cortex has been demonstrated (LaLumiere & Kalivas 2008
; Pennartz et al. 1994
), and was confirmed in these studies by the ability of 100 mM potassium-containing aCSF to stimulate increases in glutamate in these mice. Some studies have reported an acute effect of ethanol on NAcc glutamate levels (Dahchour et al. 2000
; Kapasova & Szumlinski 2008
; Selim & Bradberry 1996
) in mice or rats, while at least one has reported no effect (Dahchour et al. 1994
) in rats. Changes in glutamate in our study may have been difficult to detect because of rapid uptake or the sampling time of our experiments. However, results from a recent paper are consistent with ours for the same time period following an acute injection of 2 g/kg ethanol (Kapasova & Szumlinski 2008
), although increases in glutamate at later time points or after repeated injections of ethanol were seen. At least one study has reported a difference in ethanol-induced changes in glutamate levels in the NAcc in rats selectively bred for high and low sensitivity to the sedative effects of 2 g/kg ethanol (Dahchour et al. 2000
), which suggests that the glutamatergic response to ethanol is genetically correlated with this measure of behavioral sensitivity. However, our data do not corroborate this and suggest that glutamate levels in the NAcc are not related to the enhanced sensitivity to ethanol-induced locomotor depression in SLOW mice. These disparities may be due to differences in the effects of ethanol in rats and mice.
Conclusions and Future Directions
The sensitivity of the mesolimbic dopamine system to ethanol and cocaine was altered by selectively breeding for sensitivity to the locomotor stimulant effects of ethanol. These data are unique in that they demonstrate a genetically-determined relationship, but they are in agreement with the growing body of literature that suggests that the ventral tegmental area-NAcc pathway is a common substrate for the locomotor stimulant and reinforcing properties of ethanol and other abused drugs (Amalric & Koob 1993
; Di Chiara & Imperato 1986
; Rodd-Henricks et al. 2000
; Tzschentke & Schmidt 2000
). However, additional studies are needed to characterize the nature of the differences between FAST and SLOW mice, such as studies using the no-net-flux method to measure basal neurotransmitter levels and quantitative methods to determine whether transient changes in extracellular dopamine are due to changes in vesicular release or in dopamine uptake and clearance (Chefer et al. 2003
). Studies such as these would provide greater insight into the molecular substrates that influence the genetic relationship between ethanol's dopamine-enhancing properties and its ability to induce behavioral stimulation.
While no specific genes have been definitively shown to underlie differences in sensitivity to the locomotor stimulant effects of ethanol, quantitative trait locus analyses have suggested specific chromosomal regions and candidate genes in those regions (Downing et al. 2006
; Kamens et al. 2008
; Palmer et al. 2002b
; Xu et al. 2002
). Some of this work recently led us to examine the expression of several nicotinic acetylcholine receptor subunit genes in the FAST and SLOW mice, and to identify a difference in the expression of the α6 and β4 subunit genes (Kamens & Phillips 2008
). Whether differences in the expression or function of these genes could influence the dopamine response of FAST and SLOW mice is a question for future exploration.