The present series of experiments demonstrate that antagonism of nAChRs prevents the ability of sucrose-associated cues to act as a conditioned reinforcer in the stringent acquisition of a new response paradigm. These data are consistent with our previous demonstration of an important role for nAChRs in the conditioned reinforcing properties of ethanol cues (Löf et al. 2007
) and the ability of nicotine administration to enhance conditioned reinforcement (Olausson et al. 2004b
). Together, these findings suggest that nAChRs act as a common substrate that can modulate conditioned reinforcement processes.
Experiment 1 demonstrated that the nonselective nAChR antagonist mecamylamine blocked the conditioned reinforcing properties of a sucrose-associated stimulus that acted as a reinforcer for new learning in control rats in a dose-dependent manner. Indeed, the same dose of systemic mecamylamine (1.0 mg/kg i.p.) blocks the conditioned reinforcing effects also of alcohol-associated stimuli in the same experimental setup (Löf et al. 2007
). While in vitro studies suggest that mecamylamine at higher doses also inhibits N
-aspartate receptor complex-mediated currents (O’Dell and Christensen 1988
) and blocks N
-aspartate-induced noradrenaline release from hippocampal rat slices (Snell and Johnson 1989
), pharmacological studies demonstrate that mecamylamine, at doses equivalent to that used in the present experiment, does not interact with these receptors in vivo (Ericson et al. 2003
). Thus, the present observations suggest that mecamylamine (1.0 mg/kg i.p.) reduced or blocked the conditioned reinforcing effects of the sucrose-associated cues by antagonizing nAChRs. Importantly, this assumption was supported by data from the second experiment that confirmed a central role of these receptors in the neurobiological processes subserving responding with conditioned reinforcement.
Experiment 2 examined the effects of the selective competitive nAChR antagonist MLA at two different doses on responding with conditioned reinforcement for sucrose-associated conditioned stimuli. Here, a systemic MLA injection (6.0 or 3.0 mg/kg i.p., 10 min prior to testing) reduced the conditioned reinforcing properties of the sucrose-associated CS such that responses on the CR lever and NCR lever were not different following drug administration. These results suggest that the nAChR antagonist MLA blocks responding with conditioned reinforcement, consistent with the observations following mecamylamine administration in Experiment 1. MLA has generally been regarded as a selective α7 nAChR antagonist but was recently demonstrated to block also α3/α6β2β3* nAChRs at a similar concentration range (Klink et al. 2001
; Mogg et al. 2002
; Salminen et al. 2004
). The α3/α6β2β3* receptors, but not the α7 subtype, are further antagonized by α-CtxMII, a compound that was recently demonstrated to block the conditioned reinforcing effects of ethanol-associated cues (Löf et al. 2007
). This may indicate that the α3/α6β2β3* nAChRs are primarily responsible for the effects reported here. These converging effects of α3/α6β2β3* nAChR antagonists support the notion of a common basis for the impact of reward-related cues on behavior (Kelley and Berridge 2002
; Nie and Janak 2003
) and suggest a role for the α3/α6β2β3* nAChR subtypes.
Although the mecamylamine (1.0 mg/kg) injection in Experiment 1 abolished the conditioned reinforcing effect observed following PBS treatment, an unpaired t
test analysis of the number of responses on the active lever following mecamylamine (1.0 mg/kg) demonstrated a nonsignificant difference compared to PBS treatment. When the same statistical comparison was made in Experiment 2, there was a significantly lower response on the active lever following administration of the high MLA dose (6.0 mg/kg i.p., p
≤0.05), but not the low dose (3.0 mg/kg), as compared to PBS. This outcome is in line with the differences in the total number of lever presses between MLA dose (6.0 mg/kg i.p.) and PBS and may indicate an unspecific effect, such as suppression of locomotor activity, of the higher MLA dose. While both antagonists tested here should be specific for nAChRs at the present doses, MLA has been reported to be less selective for the specific nAChRs at higher concentrations. However, since it is difficult to precisely determine the achieved brain concentrations of these drugs, we cannot completely exclude the possibility that MLA also interferes with the function at additional nAChR receptor subtypes and configurations. Nevertheless, a pharmacokinetic study reported that an MLA dose of 5.4 mg/kg i.p. results in brain drug levels of approximately 50–100 nM (Turek et al. 1995
), levels sufficient to displace α-CtxMII in binding studies (Mogg et al. 2002
) and to inhibit α7-nAChR-mediated responses in vitro (Alkondon and Albuquerque 1993
; Yu and Role 1998
While the present study investigated the effects of systemic administration of nAChR antagonists, the involvement of α3/α6β2β3* nAChRs in mediating the reinforcing effects of alcohol-associated cues was found to be localized to the VTA (Löf et al. 2007
). We have therefore hypothesized that the presentation of an alcohol-associated cue results in the release of acetylcholine into the VTA and a subsequent release of dopamine in the nucleus accumbens (nAc; Löf et al. 2007
). Subpopulations of nAc neurons that respond to sucrose cues require the dopaminergic projections from the VTA to promote reward-seeking behavior (Yun et al. 2004
). Intermediate levels of MLA binding sites have been found in several of the brain areas involved in reward and motivation, such as the VTA (Mugnaini et al. 2002
). We thus speculate that the effects of nAChR antagonism on conditioned reinforcement to sucrose observed in the present study could involve MLA-sensitive nAChRs on dopaminergic cell bodies in the VTA.
In conclusion, the present set of experiments demonstrates that MLA-sensitive nAChRs are required for the conditioned reinforcing properties of cues associated with a palatable and rewarding food source. These receptors may be identical to the α6* and/or α3β2* nAChRs that were recently demonstrated to be involved in the impact of alcohol cues on reward-related behaviors (Löf et al. 2007
). Together, these observations suggest neuronal nAChRs as common mediators of the impact of cues on incentive processes and suggest that antagonists of these nAChR subtypes should be explored as future medications for relapse prevention and overeating.