A novel visuospatial priming task for rats with relevance to Tourette syndrome and modulation of dopamine levels

Abstract

Individuals with Tourette syndrome (TS) exhibit deficits in inhibitory information processing which may reflect impaired neural mechanisms underlying symptoms and which can be detected using a negative priming (NP) task. NP is the normal reduction of performance when identifying target stimuli that appear where non-target stimuli appeared previously. TS subjects exhibit diminished NP and their NP levels predict their response to behavioral therapy. Here we review relevant literature on this issue and also report a novel rat NP task. In the latter, rats respond to target stimuli (continuous light) while ignoring non-target stimuli (blinking light). Each trial was preceded by a prime in which target and non-target stimuli were briefly presented. Performance was challenged by shortening prime duration and by administering amphetamine. During the short prime challenge, rats exhibited lower accuracy in NP vs. baseline trials, indicative of inhibitory information processing. Modulation by amphetamine administration indicates that this drug had rate-dependent effects. Evidence is provided of individual differences in NP and response to the drug, with priming being reduced in high NP rats, while it was increased in low NP subjects. The rat NP task represents a novel and suitable tool for investigating the neural bases of inhibitory information processing and its dysfunction in TS.

Figure 1

Task schematic for the negative priming task exemplifying the different trial types. Training and testing are conducted in 9-hole operant testing chambers enclosed in ventilated sound-attenuating chambers (Med Associates Inc., St. Albans, VT and Lafayette Instrument Company, Lafayette, IN). Each testing chamber contains a curved rear wall with nine contiguous apertures. Metal inserts cover six of the apertures, leaving open apertures 3, 5, and 7, so that the rodent faces a curved wall with three equidistant apertures. Each aperture contains an infrared beam at the entrance to detect nosepoke responses and a LED stimulus light at the rear to present stimuli. Liquid reinforcement in the form of strawberry milkshake (Nesquik® plus non-fat milk, 40 μl) can be delivered into a magazine located in the opposite wall via peristaltic pump; an infrared beam detects head entries into the magazine. A house light is located in the middle of the chamber ceiling. The control of stimuli and recording of responses is managed by a SmartCtrl Package 8-In/16-Out with additional interfacing by MED-PC for Windows (Med Associates Inc., St. Albans, VT) using custom programming. During the prime trial, the target stimulus (continuous light) and the non-target stimulus (5 Hz flashing light) are briefly presented simultaneously in pseudorandom locations. After a short period of time (interstimulus interval), a probe trial follows. In the case of a baseline trial, the location of the target stimulus in the probe trial is unrelated to the location of either stimulus in the prime trial. In the case of a negative priming trial, the target stimulus in the probe trial is located in the same aperture that contained the non-target stimulus during the preceding prime trial.

Figure 2

Flowchart of negative priming (NP) task performance. Every trial begins with the illumination of the magazine (reward delivery area) light. The magazine light is extinguished once the rat performs a head entry into the magazine. Exiting the magazine initiates a 4 s intertrial interval. Nosepoking in any aperture during this interval (i.e., before presentation of the stimuli) results in a premature response being recorded and the error sequence (see below) being initiated. Otherwise, at the end of the intertrial interval, the prime trial begins, during which the target stimulus (continuous light) and the non-target stimulus (flashing light) are simultaneously presented for 0.5 s. No responses are required during the prime trial; any nosepokes performed by the rat are recorded but have no consequence. The stimuli are then extinguished for a 0.3 s interstimulus interval. The probe trial then begins, in which the target and non-target stimuli are again simultaneously presented in different locations. If the target stimulus is presented in the aperture that held the non-target stimulus during the preceding prime trial, the current trial constitutes an NP trial; otherwise, it is a baseline trial (see Fig. 1). Any following computer inputs are tallied separately for baseline and NP trials. If the rat nosepokes into the aperture containing the target stimulus, a correct response is recorded and a liquid reward (strawberry milkshake) is delivered into a now illuminated magazine. Head entry into the magazine to consume the reward again extinguishes the magazine light, and exiting initiates a new trial and intertrial interval. Alternatively, if the rat nosepokes into the aperture containing the non-target stimulus, a false alarm is recorded; if it nosepokes into an unlit aperture, an incorrect response is recorded; if it fails to perform a nosepoke during the duration of stimulus presentation, an omission is recorded. All of these errors initiate the error sequence: all stimulus lights are extinguished and the house light is illuminated for a 6 s timeout, and no food reward is delivered. After completion of the timeout interval, the house light is extinguished and the magazine light illuminated so that the next trial can begin.

Figure 3

Decreasing the duration of the prime stimulus disrupted negative priming (NP). On the day of the increased prime duration challenge, accuracy priming values became less positive (A), reflecting less NP, although this effect did not reach statistical significance [F(2, 24) = 1.06, p > 0.1]; no significant difference between accuracy in baseline and NP trials [t(12) = 0.94, p > 0.1] was detected on the day of the challenge (B). Likewise, there was a trend for correct latency priming values to become less negative on the day of the challenge [F(2, 24) = 2.78, p = 0.08], also reflecting less NP (C); correct response latencies during NP trials were actually shorter than during baseline trials (the opposite pattern from that predicted by NP) during the challenge [t(12) = 3.34, p < 0.01] (D). Data were analyzed using repeated-measures analyses of variance (ANOVA) to compare the priming values during challenge to the sessions before and after the challenge. When statistically significant effects were found in the ANOVAs, post hoc comparisons among means were conducted using Newman-Keuls tests. Paired two-tailed t-tests were used to compare performance during baseline and NP trials on the duration challenge days. The level of significance was set at 0.05 throughout. Data were analyzed using GraphPad Prism® (GraphPad, San Diego, CA) and Sigmaplot® (Systat Software Inc., San Jose, CA, USA). Values are expressed as mean ± SEM. Asterisks (**p < 0.01) denote significant differences compared with baseline trials.

Figure 4

Decreasing the duration of the prime stimulus enhanced negative priming (NP). On the day of the decreased prime duration challenge, accuracy priming values became more positive, reflecting more NP (A). Comparison of accuracy priming values before, during, and after the challenge showed a significant main effect of day [F(2, 24) = 5.35, p < 0.05]. Post hoc testing confirmed that accuracy priming values on the challenge day were significantly larger compared to both the three days preceding the challenge (p < 0.05) and the day after the challenge (p < 0.05). Rats exhibited significantly higher accuracy in baseline trials compared with NP trials [t(12) = 4.29, p < 0.01] on the day of the challenge (B). Similarly, correct latency priming values became more negative on the day of the challenge, also reflecting more NP (C), although this effect did not reach statistical significance [F<1, ns] and there was no significant difference between correct response latencies in baseline and NP trials [t(12) = 0.91, p > 0.1] on the day of the challenge (D). Data were analyzed using repeated-measures analyses of variance (ANOVA) to compare the priming values during challenge to the sessions before and after the challenge. When statistically significant effects were found in the ANOVAs, post hoc comparisons among means were conducted using Newman-Keuls tests. Paired two-tailed t-tests were used to compare performance during baseline and NP trials on the duration challenge days. Values are expressed as mean ± SEM. Asterisks (**p < 0.01) denote significant differences compared with days before and after the challenge (A) or compared with baseline trials (B).

Figure 5

The effect of the decreased prime stimulus challenge to enhance negative priming (NP) was sustained upon repeated challenge. On the day of the second decreased prime duration challenge, accuracy priming values again became more positive, reflecting more NP (A), with a very strong trend towards differences between the accuracy priming values obtained before, during, and after the challenge [F(2, 24) = 3.40, p = 0.05]. Rats again exhibited significantly higher accuracy in baseline trials compared with NP trials [t(12) = 3.66, p < 0.01] on the day of the challenge (B). Although there was a tendency for correct latency priming values to become more negative on the day of the challenge, also reflecting more NP (C), this tendency persisted on the day after the challenge, and did not result in statistically significant differences in response latency priming values on the different days [F(2, 24) = 2.19, p > 0.1]. There was no significant difference between correct response latencies in baseline and NP trials [t(12) = 0.38, p > 0.1] on the day of the challenge (D). Data were analyzed using repeated-measures analyses of variance (ANOVA) to compare the priming values during challenge to the sessions before and after the challenge. When statistically significant effects were found in the ANOVAs, post hoc comparisons among means were conducted using Newman-Keuls tests. Paired two-tailed t-tests were used to compare performance during baseline and NP trials on the duration challenge days. Values are expressed as mean ± SEM. Dagger symbol (†) denotes trend toward difference between with days before, during, and after the challenge; asterisks (**p < 0.01) denote significant differences compared with baseline trials.

Figure 6

Positive correlations of performance between first and second short prime duration challenges. Individual accuracy priming values for each animal from the two short prime duration challenges were compared using Spearman correlations. Values from the first and second decreased prime duration challenges were significantly positively correlated (r = 0.66, p < 0.05), indicating that rats exhibiting high rates of negative priming (NP) in the first challenge also did so during the second challenge, and likewise for rats exhibiting low rates of NP.

Figure 7

Amphetamine (AMPH) exhibited rate-dependent effects on negative priming (NP). Two-way ANOVA detected a significant effect of Trial Type on accuracy [F(1, 72) = 5.22, p < 0.05]; however, post hoc testing did not reveal any significant differences in accuracy between baseline and NP trials overall at any amphetamine dose (A). No main effects of Amphetamine Dose or Trial Type x Amphetamine Dose interaction were observed overall. However, when animals were examined separately based on their initial NP rate in the drug-free state, significant patterns emerged. A three-way ANOVA of Median Split Group, Trial Type, and Amphetamine Dose revealed a very strong trend towards a significant interaction [F(3, 33) = 2.90, p = 0.05] between these factors. When accuracy priming values were calculated and compared for the two groups, a significant Median Split Group x Amphetamine Dose interaction [F(3, 33) = 2.98, p < 0.05] was observed. Post hoc testing indicated that groups differed in priming values only after saline administration (p < 0.05), while this difference was lost after AMPH administration at any dose. In high NP rats, two-way ANOVA revealed a significant effect of Trial Type on accuracy [F(1, 42) = 13.47, p < 0.01]. Post hoc testing revealed that animals exhibited significantly lower accuracy in NP trials compared to baseline trials after saline treatment (p < 0.01). This NP effect was absent after amphetamine administration at any dose (B). Examination of accuracy priming values shows that high NP rats exhibited a robustly positive accuracy priming value after saline treatment that was attenuated by amphetamine treatment at every dose (C), although this effect did not reach statistical significance [F<1.7, ns]. In low NP rats, no significant effect of Trial Type was detected [F<1, ns]. However, a tendency toward lower accuracy in baseline trials compared to NP trials after saline administration was observed. This pattern was reversed after the 0.25 mg/kg dose of amphetamine; here, rats exhibited lower accuracy in NP trials compared to baseline trials, suggesting a strengthened NP effect. Accuracy differences between baseline and NP trials were absent after the higher amphetamine doses (D). While no significant differences in accuracy priming values for low NP were found [F<1.3, ns], inspection of the data suggests that rats did not exhibit an NP effect after saline administration, whereas after administration of the 0.25 mg/kg amphetamine dose, the accuracy difference became positive, indicating a stronger NP effect. At higher amphetamine doses, no NP effect was observed (E). No main effect of Amphetamine Dose and no interaction were found in either high NP or low NP rats. No significant effect of Trial Type or Amphetamine Dose and no Trial Type x Amphetamine Dose interaction on correct response latency were observed (data not shown). Data were analyzed using two-way ANOVA, with the two factors Trial Type (baseline or NP) and Amphetamine Dose. Post hoc comparisons of significant effects were conducted using Bonferroni tests. Values are expressed as mean ± SEM. Asterisks (**p < 0.01) denote significant differences compared with baseline trials. BL, baseline; NP, negative priming.

Figure 8

Schematic of the Yerkes-Dodson law in relation to the effects of amphetamine observed in this study. Variables of the original Yerkes-Dodson law are marked along the solid axes; variables relating to this study are marked along the dotted axes. As described in the original Yerkes-Dodson law, low arousal levels are associated with low performance. At higher arousal levels, performance improves up to a point; however, at very high arousal levels, performance deteriorates. Analogously, in the hypothetical pattern underlying the findings in our study, low levels of dopamine transmission are associated with low levels of negative priming (NP). As dopamine levels increase, NP at first increases also, e.g. in low NP (O) rats administered amphetamine (☆). Increasing dopamine levels past a presumed optimal point, e.g. in high NP rats (★) administered amphetamine (●), results in NP deteriorating.