DNA targeting of rhinal cortex D2 receptor protein reversibly blocks learning of cues that predict reward

Abstract

When schedules of several operant trials must be successfully completed to obtain a reward, monkeys quickly learn to adjust their behavioral performance by using visual cues that signal how many trials have been completed and how many remain in the current schedule. Bilateral rhinal (perirhinal and entorhinal) cortex ablations irreversibly prevent this learning. Here, we apply a recombinant DNA technique to investigate the role of dopamine D2 receptor in rhinal cortex for this type of learning. Rhinal cortex was injected with a DNA construct that significantly decreased D2 receptor ligand binding and temporarily produced the same profound learning deficit seen after ablation. However, unlike after ablation, the D2 receptor-targeted, DNA-treated monkeys recovered cue-related learning after 11-19 weeks. Injecting a DNA construct that decreased N-methyl-d-aspartate but not D2 receptor ligand binding did not interfere with learning associations between the cues and the schedules. A second D2 receptor-targeted DNA treatment administered after either recovery from a first D2 receptor-targeted DNA treatment (one monkey), after N-methyl-d-aspartate receptor-targeted DNA treatment (two monkeys), or after a vector control treatment (one monkey) also induced a learning deficit of similar duration. These results suggest that the D2 receptor in primate rhinal cortex is essential for learning to relate the visual cues to the schedules. The specificity of the receptor manipulation reported here suggests that this approach could be generalized in this or other brain pathways to relate molecular mechanisms to cognitive functions.

Fig. 1.

The visually cued reward schedules task. (a) Schematic diagram of a three-trial schedule with brightness cues. On each trial, the monkey was required to respond when a visual target changed from red to green (see Materials and Methods for details). The monkey initiated each trial by touching a lever. A trial was scored as correct if the monkey released the lever 200–1,000 ms after the green target (visual target) appeared. If the trial was correctly performed a blue spot (correct trial indicator) replaced the green target. A drop of water (reward) was delivered only after the third trial in the schedule had been correctly completed. A visual cue was assigned to each trial; the cue indicated how many trials had been performed and how many trials remained to be completed before a reward was delivered (relative workload). In the example shown here, the light gray rectangle is the cue for schedule state 1/3, the dark gray rectangle is the cue for state 2/3, and the black rectangle is the cue for state 3/3, where the schedule fraction has the trial number in the numerator and schedule length in the denominator. (b) The five visual cue sets used in this study. The schedule states corresponding to the cues are shown in the top row.

Fig. 2.

Error rates of monkeys performing the visually cued reward schedules in the different conditions. Each bar represents the mean error rate for that schedule state; the error bars are SEMs. * marks the conditions in which the error rates were distinguishable (single-factor ANOVA, P < 0.05) across the schedule states, indicating that the monkeys were using the cues to adjust their behavior (see text for details). (a) Error rates of monkeys using brightness cues in the fourth week of testing after the cue's introduction before any treatment. (b) Error rates of different groups of monkeys using length cues in the eighth week after delivery of DNA constructs into the rhinal cortex, i.e., the fourth week after introduction of the length cue. Groups of monkeys were injected with the following: (i) a mixture of D2- and NMDA receptor-targeting constructs (Length Cues, D2+NMDA); (ii) D2 receptor-targeting construct (Length Cues, D2), (iii) NMDA receptor-targeting construct (Length Cues, NMDA), and (iv) vector (Length Cues, Control). Monkeys that received either the D2+NMDA receptor-targeting constructs or the D2 receptor-targeting construct were only impaired in learning associations between visual cues and the workload expected before reward. (c) Error rates of the four monkeys that received the D2 receptor-targeting construct after behavioral recovery. Data were obtained during the first week after performance had recovered from the effect of either the D2- and NMDA-targeting construct mixture or D2 receptor-targeting construct alone (≈12–20 weeks after injection) (Length Cues, Recovered), and during the third week after new cues have been introduced to the same monkeys (Pattern Cues). (d) Error rates of the monkeys that received the second treatment of D2 receptor-targeting construct (D2, n = 3; and mixture of D2 and NMDA, n = 1). Data (Pattern Cues 2, D2) were obtained during the eighth week after treatment and show that the monkeys did not use the cues to adjust their behavior. Data (Pattern Cues 2, Recovered) collected from the same monkeys during the 12th week after injection, which is the first week after performance had recovered from the effect of the treatments, and during the third week after new cues have been introduced (Pattern Cues 3).

Fig. 3.

Receptor binding autoradiography. (a) Autoradiograph of a single brain section from the monkey treated with DNA construct targeting the D2 receptor showing D2 receptor ligand binding with [¹²⁵I]iodosulpiride. The rhinal cortex in the left hemisphere (between the two arrows) was treated with DNA targeting the D2 receptor. There is a significant decrease in D2 receptor ligand binding density in the rhinal cortex of the treated relative to the untreated hemisphere. rs indicates rhinal sulcus, amts indicates anterior middle temporal sulcus, and A marks the amygdala. (b) D2 receptor ligand optical density in the rhinal cortex of the monkey treated with the D2-targeting DNA construct. Shown are the means and SEMs for the average density of D2 receptor ligand binding in the D2 receptor construct-treated rhinal cortex (treated) and in the untreated rhinal cortex (control). The treated side has a significantly lower density of D2 receptor ligand binding than the untreated side. (c) NMDA receptor ligand optical density in the rhinal cortex of the monkey treated with the NMDA-targeting DNA construct. The NMDA receptor-targeting DNA treatment depresses MK801 binding significantly. (d) D2 receptor ligand optical density in the rhinal cortex of the monkey treated with the NMDA receptor-targeting DNA construct. NMDA receptor-targeting DNA treatment did not affect the D2 receptor ligand binding (paired t test, P = 0.6). This finding indicates that treatment with the DNA construct targeting the NMDA receptor has no nonspecific effect on D2 receptor ligand binding. * indicates a significant difference between two hemispheres (paired t test, P < 0.05).