Discussion
In this study, we sought to determine the contribution of dopamine D2 receptor-mediated signaling to the various stages of associative and reversal learning. D2R-/- mice demonstrated that they were capable of learning to locate and consume food pellets, indicating that their locomotor behavior was not detectably disrupted and their primary motivation to obtain a natural reinforcer (food pellet) was undisturbed. Rather, the impaired ability of D2R-/- mice to assign appropriate discriminative stimulus relationships in an operant discrimination task argues that D2R-mediated signaling contributes to the neuronal processes involved in attaching salience to environmental stimuli. The deficient capacity of D2R-/- mice to disengage inappropriate decision strategies strongly argues that mesolimbic dopamine signaling, mediated by dopamine D2Rs is essential for efficient reversal learning to occur.
Mice with or without intact dopamine D2R-mediated signaling displayed similar decreases in latencies to retrieve unhidden food pellets (Fig. 1) and learned to dig for food buried in unscented sand (Fig. 2). These findings are in complete agreement with earlier studies that utilized rats and fairly selective D2R antagonists [3-8], as well as even very extensive 6-OHDA lesions [1,10,11] in associative and operant learning paradigms. Our results add to a growing literature demonstrating a negligible role of dopamine, and now, specifically, D2Rs in the unconditioned or hedonic value of natural (food) reinforcers [1,2].
The comparatively poor skill of D2R-/- mice during discrimination trials suggests a role for D2Rs in acquisition of appropriate S+/S- relationships in operant associative learning. Several studies have demonstrated that both the acquisition [6,7] and expression [9] of associative learning are mediated by dopamine D1Rs. Most literature reviews identify dopamine D1Rs with dopamine-mediated learning and D2Rs with motor related behaviors [e.g. [24]]. Moreover, it has been reported that acute administration of the dopamine D2/3 antagonist, raclopride, actually improves acquisition of food-motivated associative learning [6]. However, only acute administrations of antagonists were given, and learning was not measured during complete D1R or D2R blockade [6]. Significantly, the reports cited above failed to address the technical limitations of the approach: i.e. that the antagonists used lack adequate subtype specificity and only partially blocked D2R-mediated signaling thus making it impossible to rigorously assess the role of D2R-mediated signaling in associative and reversal learning. Additionally, none of these studies addressed the observation that the effects of D2R antagonists on locomotion and learning depend on whether exposure is chronic or acute [e.g. [15,16]].
That D2R-mediated signaling orchestrates just motor components of learning is a conclusion that is potentially biased due to the practice of avoiding doses of drugs that induce catalepsy (and therefore measuring only partial blockade of dopamine D2Rs) and single administrations [e.g. [3]]. Quite possibly, the functional role of D2Rs in associative learning might be masked because of this concern about locomotor disruption [2,6,8]. Doses of raclopride as low as 0.5 mg/kg significantly disrupt motor behavior [25], although this peripheral dose is consistently used in learning paradigms [e.g. [6]]. One might then ask: "How then could the role of D2Rs in associative learning be dissociated from motor behavior?" Seminal experiments [26-28] have clearly demonstrated that repeated administration of catalepsy-inducing doses of D2R antagonists in rodents actually leads to a striking behavioral tolerance to catalepsy. These doses have been shown to occupy well over 80% of available D2Rs [29]. Future experiments measuring acquisition of associative learning in rodents that received chronic administration of D2R antagonists and demonstrated behavioral tolerance to their motor disrupting effects would be a logical test of this hypothesis. However, the realization that multiple dopamine receptor subtypes would be concurrently targeted with the presently commercially available antagonists, such as D2Rs, D3Rs, and D4Rs would have to be rectified. We would argue that the most parsimonious approach at this time is to utilize mice that have been genetically altered such that they are lacking one or both functional alleles of the specific receptor of interest. While they do have their limitations (e.g. developmental compensation and strain effects in mouse lines not backcrossed adequately to a parental strain for a minimum of 10 generations), use of our inbred (N20 generation) animals in the present study (where no differences in locomotor behavior were detected between D2R-/- and D2R+/+ mice; Figs. 1 &2) revealed a previously unappreciated role of D2R-mediated signaling in associative learning and attention that could not be measured with the currently available, acutely administered D2R antagonists.
A cursory analysis of our data might suggest to some that the genetic manipulation of the drd2 locus conferred a gross olfactory impairment to the D2R-/- mice. Rather, we argue that this is not likely because the D2R-/- mice did learn to retrieve the food pellets from the dishes and eventually learned to accurately discriminate odors. Recent electrophysiological data demonstrate that dopamine D2R's are located on glutamatergic terminal axons of olfactory nerves and depress excitatory input of the olfactory nerve to the mitral cells of the olfactory bulb [30,31]. Consequently, our data, and data from other studies, suggest that the complete lack of D2Rs in the olfactory bulb does not prevent transduction of olfactory stimuli; rather it affects the ability to habituate, or tune, olfactory nerve activity associated with repeatedly encountered concentrations of chemical stimuli [32].

The performance of D2R-/- mice during reversal learning is deficient revealing a role for dopamine D2R-mediated signaling in tasks requiring behavioral flexibility
Perseverative behavioral patterns characterized the D2R-/- mice relative to D2R+/+ mice during reversal learning sessions (Figure 4), manifested during early reversal trials (Figure 6) and persisted across several sessions (Figure 7). These findings are significant because D2R-/- mice respond to food reinforcers and ultimately form and maintain odor-driven S+/S- relationships (a putative D1R-mediated behavior [6]) just as D2R+/+ mice (both groups achieved ≥ 80% discrimination accuracy, the dopamine D1Rs in our mice were not targeted). A future extension of this study would be to test performance over a fixed number of trials and determine patterns of error rates during acquisition of the discrimination and reversal learning tasks. This manipulation would control the number of errors based on trials performed to determine if the apparent difference in absolute number of errors demonstrated by the mutant mice (the putative performance deficit) is simply a reflection of extra trials. Nonetheless, the inability of D2R-/- mice to disengage from previously established S+/S- contingencies (responding to a discriminative stimulus; Fig. 4) strongly argues that mesolimbic dopamine, and in particular dopamine D2R-mediated signaling, modulates the process of alerting the subject that familiar contingencies are now associated unexpected consequences.
Schultz and colleagues have demonstrated that dopamine cells display consistent tonic firing patterns during maintenance of associative learning tasks [33]. However, phasic burst activity of dopaminergic cells occurs when discrepancies between predicted and actual reinforcement contingencies transpire [14]. This robust increase of dopaminergic cell activation in response to unpredicted outcomes has been referred to as an "error" signal [14]. To date, the molecular basis of this error signal has not been identified. The inability of the D2R-/- mice to desist responding to a previously reinforced stimulus suggests that the dopamine D2R might in fact be the focal point of this error-signaling cascade.
Electrophysiological data further support the hypothesis that signaling mediated by dopamine D2Rs tunes D1R-mediated mesocorticolimbic output. Calabresi et al. [34] demonstrated that tetanic stimulation of dorsal striatum slices prepared from D2R-/- mice is associated with enhanced EPSP and as a result increased striatal synaptic efficacy. In contrast, stimulation of dorsal striatum slices from wild-type mice resulted in IPSP activity, long-term depression, and decreased neuronal activity of striatal efferents [34]. Carlsson and colleagues [35] have speculated that dopamine D2R stimulation in the striatum serves to "brake" or diminish excitatory corticostriatal signaling and plasticity. Indeed, perseverative behavior is associated with over activity of the dorsal striatum in rodents [36] and over activity of the caudate in patients with ADHD [37] and a strong inverse correlation of D2R binding with compulsive behavior has been reported [22]. Importantly, our data indicate that the poor performances displayed by the D2R-/- mice are manifestations of reversal learning deficits and not gross motivational or sensory impairments. We therefore argue that D2Rs participate in signaling or alerting the organism of learning contingency changes during reversal learning and sculpt ongoing goal-directed behavior.