These studies are the first to show conditioned changes in FOS expression to a CS previously paired with ethanol in mice. In two independent studies, FOS expression during a drug-free test exposure to CS+ was significantly higher in experimental mice that had previously received ethanol coincident with the onset
of CS exposure (Before-S) than in unpaired (Delay-S) or naïve (Naïve-S) control mice in the VTAant (see -). Several other brain areas that showed conditioned increases in FOS were also identified in Experiments 1 (BST, SNC, DMH) and 2 (CeM, La, CA1, CA2, and DG). However, FOS expression was no different from control levels in experimental mice that had previously received ethanol coincident with termination
of CS exposure (After-S). Measurement of locomotor activity confirmed that our conditioning procedures were effective in producing behavioral differences between groups of mice ( and ). Before-S mice in both experiments showed locomotor sensitization across ethanol trials and conditioned increases in activity when tested under saline in the presence of CS+. Conversely, After-S mice showed suppressed locomotor activity across conditioning days and on test. Although place preference data were not obtained, these conditioned locomotor effects are consistent with previous studies in DBA/2J mice in which the Before procedure produced CPP and the After procedure produced CPA (Cunningham and Noble, 1992
; Cunningham et al., 1997
; Cunningham and Henderson, 2000
4.1. Conditioned changes in FOS
Differences between experimental (Before, After) and control (Delay, Naïve) groups in FOS expression elicited by CS+ presumably reflect the effects of prior conditioning. Moreover, because these differences were measured after a relatively large number of conditioning trials (six), it is reasonable to assume that they reflect neural processes involved in the expression of the ethanol-induced conditioned response rather than processes involved in the acquisition (i.e., learning) of that conditioned response. In Experiment 1, Before-S mice had higher levels of FOS expression in the BST and DMH compared to all other groups (). Moreover, Before-S mice had significantly higher levels in the SNC and VTAant compared to Delay-S and Naïve-S controls. (The difference between Before-S and After-S mice for the VTAant region approached significance, p < .06). Experiment 2 replicated the finding of higher FOS activation in VTAant in Before-S-mice compared to Delay-S-mice, but did not replicate the finding of group differences in SNC or DMH (). Although the group difference in BST fell short of the criterion for significance in Experiment 2 (p = .09, two-tailed), the direction of the effect was identical to that seen in Experiment 1. In addition, Experiment 2 yielded several other brain areas in which experimental mice had higher levels of FOS expression than control mice, including the CeM and La portions of the amygdala and the CA1, CA3, and DG regions of the hippocampus. In two cases (CA1, CA3), the group difference occurred only when mice were tested with saline (i.e., Before-S vs. Delay-S), which was also true for the effects seen in BST and VTAant. In the other three cases (DG, CeM, La), however, the conditioning effect was apparent in both saline- and ethanol-tested mice, suggesting that the ability of CS+ to increase FOS expression did not depend on drug state.
In aggregate, these experiments implicate portions of the mesolimbic dopamine pathway (VTAant), extended amygdala (CeM, La, BST), hypothalamus (DMH), and hippocampus (CA1, CA2, DG) as potential mediators of conditioned responses to ethanol-paired cues. Acute ethanol exposure has previously been shown in mice to increase FOS expression in two of these brain areas, both of which are part of the extended amygdala. More specifically, injection of ethanol (1.5-4 g/kg) has been reported to induce FOS in the central nucleus of the amygdala (CeA: Demarest et al., 1999
; Hitzemann and Hitzemann, 1997
; Ryabinin and Wang, 1998
) and BST (Demarest et al., 1999
; Ryabinin and Wang, 1998
) in mice. Thus, the present studies show that an ethanol-paired CS+ is able to induce FOS in brain areas that are directly activated by ethanol exposure. However, ethanol-induced induction of FOS in a particular brain area does not necessarily endow CS+ with the ability to produce conditioned increases in FOS in that same area. For example, although acute injection of ethanol at 2 g/kg has been reported to increase FOS expression in AcbC of DBA/2J mice (Hitzemann and Hitzemann, 1997
), we found no evidence of a conditioned FOS response in this brain area.
The present studies also found that an ethanol-paired CS+ acquired the ability to induce FOS in several brain areas that have previously been reported to be unaffected by acute or chronic ethanol exposure in DBA/2J mice (VTA: Hitzemann and Hitzemann, 1997
; La, CA1, CA2, DG: Ryabinin and Wang, 1998
). The conditioned FOS response in VTAant, which was significant in both experiments, is of particular interest given the previously reported sensitivity of VTA dopamine neurons to direct activation by ethanol as measured by extracellular single unit recordings in a mouse brain slice preparation (Brodie, 2002
; Brodie and Appel, 2000
). As part of the mesolimbic dopamine system, the VTA has been widely implicated in the neurocircuitry involved in mediating the reinforcing effects of ethanol and other abused drugs (Koob et al., 1998
; McBride et al., 1999). The finding that rats will self-administer ethanol directly into VTA provides compelling evidence that this brain site is importantly involved in mediating ethanol’s reinforcing effects (Gatto et al., 1994
; Rodd et al., 2004
). Thus, despite the lack of a direct ethanol effect on FOS expression in the VTA of DBA/2J mice (Hitzemann and Hitzemann, 1997
), our finding of conditioned FOS increases in this brain area appears consistent with many other findings implicating this area in the mediation of ethanol’s primary reinforcing effects. Involvement of the VTA in ethanol-induced conditioning is also consistent with findings from several studies suggesting that this brain area plays an important role in place preference conditioning induced by other abused drugs (Gholami et al., 2003
; Harris and Aston-Jones, 2003
; Neumaier et al., 2002
; Popik and Kolasiewicz, 1999
). In all of these studies, effects on CPP were believed to be due to modulation of VTA dopamine cells. It is not known, however, whether the VTA neurons activated in the present studies are dopaminergic.
Conditioned increases in FOS were also observed in two structures within the extended amygdala: BST (Exp. 1) and CeM (Exp. 2). Both of these areas receive projections from the VTA (Swanson, 1982
) and both shows increases in FOS expression after acute ethanol exposure (Demarest et al., 1999
; Hitzemann and Hitzemann, 1997
; Ryabinin and Wang, 1998
). Moreover, ethanol (like morphine, nicotine and cocaine) produces dose-dependent increases in extracellular dopamine in the BST (Carboni et al., 2000
). The extended amygdala has been implicated in the primary reinforcing effects of ethanol (Koob, 2003
; McBride, 2002
). For example, infusion of a GABAA
antagonist into either the CeA or BST has been reported to decrease ethanol self-administration in rats (Hyytiä and Koob, 1995
). Moreover, infusion of a dopamine D1
antagonist into the BST has been found to reduce ethanol self-administration (Eiler et al., 2003
) and to interfere with the acquisition and expression of morphine-induced CPP (Zarrindast et al., 2003
). The extended amygdala has also been more broadly implicated in associative learning processes (Balleine and Killcross, 2006
; Fanselow and Poulos, 2005
). Thus, development of an ethanol-induced conditioned FOS response within the extended amygdala appears to be consistent with the larger role this brain area plays in learning and drug reinforcement.
Because several previous studies have shown that acute ethanol exposure increases FOS in BST (Demarest et al., 1999
; Ryabinin and Wang, 1998
), the finding that test session exposure to ethanol reduced the FOS response in this brain area was somewhat unexpected (Before-S vs. Before-E, ). However, because there was no difference in the FOS response in BST between the Delay-S and Delay-E groups (), it appears that repeated exposure to ethanol (during the conditioning phase) reduced the FOS response in BST to acute ethanol, much the same as repeated ethanol exposure has been reported to reduce the FOS response in CeA to acute ethanol (Ryabinin and Wang, 1998
). Nevertheless, despite losing its ability to directly induce FOS in BST, test exposure to ethanol was able to suppress the FOS increase in BST. One potential interpretation of this finding is that the FOS increase in BST in the Before-S group may have been triggered by the omission of an expected ethanol injection (see Section 4.4 for further discussion of this possibility).
The finding of conditioned FOS increases in hippocampus (CA1, CA3, DG) in Experiment 2 was somewhat surprising given that acute ethanol exposure has previously been shown either to have no effect (Ryabinin and Wang, 1998
) or to reduce FOS expression (Ryabinin, 1998
) in hippocampus. Nevertheless, the finding of ethanol-induced conditioned increases in the hippocampus appears to be consistent with previous reports of increased FOS in hippocampal areas of rats exposed to an environment in which they had previously self-administered ethanol (CA3: Topple et al., 1998
) or cocaine (CA1, DG: Neisewander et al., 2000
). Increased FOS in hippocampal areas has also been reported for rats (CA1, CA3, DG: Wisłowska-Stanek et al., 2005
) and mice (Milanovic et al., 1998
) exposed to a context previously paired with electric shock in a fear conditioning procedure. Thus, it appears that activation of FOS in the hippocampus may develop as a conditioned response to cues previously paired with a variety of biologically important events.
Our finding of a conditioned FOS increase in the amygdala but no conditioned change in the nucleus accumbens is consistent with the results of one early study that examined cocaine-induced conditioning in rats (Brown et al., 1992
). However, these outcomes are opposite to those reported in several subsequent studies of conditioning induced by cocaine (Franklin and Druhan, 2000
; Hotsenpiller et al., 2002
; Miller and Marshall, 2005
) or morphine (Schroeder and Kelley, 2002
) in rats. In all of these other studies, a conditioned increase in FOS was observed in the nucleus accumbens (core), but not in the amygdala. In a recent study of methamphetamine-induced conditioning in outbred mice, conditioned increases in FOS were found in the nucleus accumbens shell and in both the basolateral and basomedial amygdala (Rhodes et al., 2005
). In that study, which appears to be the only previous study to examine BST, conditioned FOS increases were also found in that brain area, consistent with the outcome of our studies. Unfortunately, none of these other studies examined FOS changes in the brain area showing the most robust conditioned effect in both of our studies (VTAant). It is not clear whether the discrepancies between our studies and these other studies are due to differences in species (i.e., rats vs. mice), drug (cocaine vs. morphine vs. methamphetamine vs. ethanol) or methodology.
It is important to consider whether increased FOS seen in several brain areas in the Before-S mice was due to their conditioned locomotor behavior. Previous studies have shown inconsistent results on this issue. Thus, on one hand, several studies have demonstrated dissociation of conditioned locomotion and conditioned FOS induction (Hotsenpiller et al., 2002
; Mead et al., 1999
; Schroeder et al., 2000
). On the other hand, Rhodes et al (2005)
found a positive correlation between locomotion and FOS expression in several areas. Importantly, however, the latter investigators found no correlation between locomotor activity and expression of FOS in BST. Moreover, locomotor activity was not significantly correlated with FOS-positive cell counts in either BST or VTAant in our study (data not shown). Thus, it is unlikely that the ethanol-induced conditioned increases in FOS in the present studies were simply a byproduct of the conditioned increase in activity.
4.3. Comparison of Before and After Groups
The results of Experiment 1 showing different patterns of neural activation for Before-S and After-S mice are consistent with the hypothesis that ethanol-induced CPP and CPA are mediated by different underlying processes (Cunningham et al., 2002
). In general, the FOS profiles for these two groups differ in that Before-S mice had more activation in the BST and DMH compared to After-S mice (as well as both control groups), and FOS differences in the VTAant approached significance at p < .06. However, no After-S mice were tested in Experiment 2, so these differences could not be reexamined. Activation of reward circuitry (VTAant and BST) in Before-S mice but not in After-S mice fits with the notion that CPP reflects rewarding effects of ethanol whereas CPA does not.
Somewhat surprisingly, there were no significant differences in FOS expression between the After-S group and either control group (Delayed-S, Naïve-S). In other words, FOS expression in the brain areas we examined was not sensitive to the neural changes that presumably underlie the conditioned activity suppressing and aversive effects of a CS that immediately precedes injection of ethanol (Cunningham et al., 1997
). This unexpected outcome may be due to the exclusion of brain stem regions from the FOS analysis. Ethanol-induced taste aversion conditioning has previously been shown by others to increase FOS expression in the nucleus of the solitary tract, the parabrachial nucleus, and the area postrema in subjects subsequently presented with an ethanol-paired tastant (Thiele et al., 1996
). In light of data suggesting a genetic correlation between ethanol-induced conditioned taste aversion and ethanol-induced CPA (Cunningham and Ignatoff, 2000
), it is possible that brain regions activated by the CS+ in the After-S group may have been missed by not examining FOS changes in the brain stem.
4.5. Final Caveats
Given the discrepancies between studies in the brain areas showing conditioned FOS responses, primary emphasis should be placed on VTAant, which showed clear and consistent differences between the Before and Delay groups in both experiments. Based on the significant group difference in Experiment 1 and the strong trend in Experiment 2, BST should also receive strong consideration as a candidate brain area involved in ethanol-induced Pavlovian conditioning. The reasons behind the discrepancies in other brain areas are not clear, but they may be the result of batch effects in FOS immunohistochemistry (e.g., Rhodes et al., 2005
). Although the degree of concordance between experiments was lower than expected, it should be noted that the present study is among the first in which drug-induced conditioned changes in FOS expression are reported from two independent experiments involving the same comparison groups. In most previously published studies (Brown et al., 1992
; Franklin and Druhan, 2000
; Hotsenpiller et al., 2002
; Miller and Marshall, 2005
; Neisewander et al., 2000
; Rhodes et al., 2005
; Schroeder et al., 2001
; Schroeder and Kelley, 2002
; Topple et al., 1998
), effects of the drug conditioning procedure on FOS expression were examined in only one experiment, providing no opportunity to assess the reliability of observed group differences in specific brain areas.
Because the conditioning procedure used for Before mice has previously been shown to produce CPP, there is a strong likelihood that the brain regions showing conditioned changes in FOS in these studies are normally involved in the expression of CPP. However, the correlational nature of this mapping technique encourages caution when interpreting these results. For example, although these studies consistently showed increased FOS induction in the VTAant in response to an ethanol-paired CS+, they do not provide direct evidence that this region mediates or modulates expression of CPP. Experimental manipulations that target this candidate structure (e.g., lesions or microinjections) are required to more completely determine their influence on approach and contact with ethanol-paired stimuli. Recently, the importance of the VTA in modulating expression of ethanol-induced CPP was confirmed in a study (Bechtholt and Cunningham, 2005
) that showed a reduction in expression of CPP following intra-VTA infusionof an opioid antagonist (methylnaloxonium) or GABAB
Finally, the present studies do not address whether regions involved in the acquisition
(initial learning) of an ethanol conditioned response differ from those mediating the expression
of that response. It is likely that patterns of neural activity elicited by a drug-paired stimulus shift across learning trials (Phillips et al., 2003
). Because the present studies examined tissue collected after a relatively large number of conditioning trials (six), they are probably most relevant to understanding brain systems involved in the expression
of ethanol-induced conditioning. Future studies must address their possible role in the acquisition of ethanol-induced conditioned responses.