The mGluR2 positive allosteric modulator AZD-8529 dose-dependently reduced checking behaviour

Both functional Observing Lever Presses (OLPs) and dysfunctional Extra Observing Lever Presses (eOLPs) varied markedly across rats, so the data were square root transformed, and responding under drug was compared to responding during the two sessions prior to dosing (‘Baseline, BL’) and the two sessions following dosing (‘Rebaseline, RBL’).

As expected, when rats were dosed with saline vehicle, there were no differences in responding under drug for either OLPs [Figure 2a, b; Session: F(1.8,31.1) = 2.40, p = .10] or eOLPs [Figure 2c, d; Session: F(2,132) = 1.68, p = .19]. As we have observed previously, rats classified as sign-trackers showed higher levels of checking, both for OLPs [Phenotype: F(1,66) = 6.37, p = .014, η2 = 0.09; Session x Phenotype: F(1.81,119.5) = 3.83, p = .028, η2 = 0.06] and eOLPs [Phenotype: F(1,66) = 11.8, p = .001, η2 = 0.15]. A similar lack of effect on checking was found for the 0.3 mg/kg dose [OLPs, Session: F < 1; eOLPs, Session: F < 1] and the 1 mg/kg dose [OLPs, Session: F < 1; eOLPs, Session: F < 1].

Fig. 2

The mGluR2 positive allosteric modulator AZD-8529 dose-dependently reduced functional and dysfunctional checking. Rats were classified as sign-trackers and goal-trackers and trained on the Observing Response Task prior to testing with acute doses of the mGluR2 positive allosteric modulator AZD-8529 in a Latin square design. All rats received treatment with vehicle (0 mg/kg), and a subset of the different AZD-8529 doses. The number of rats receiving each dose is represented by numbers at the base of the bar for the drug dosing day; the same rats are represented in the ‘baseline’ and ‘rebaseline’ bars for each dose. In rats classified as sign-trackers, the 3 mg/kg and 10 mg/kg doses of AZD-8529 reduced (a) functional Observing Lever Presses (OLPs) and (c) dysfunctional extra Observing Lever Presses (eOLPs). A similar effect was observed in goal-trackers, for both (b) functional OLPs and (d) dysfunctional eOLPs. BL, baseline sessions; AZD: treatment with AZD-8529 treatment; RBL, rebaseline sessions. Data are shown as means ± s.e.m.

The 3 mg/kg dose of AZD-8529 reduced checking behaviour compared to baseline, for both OLPs [Session: F(1.76,86.0) = 8.96, p < .001, η2 = 0.15] and eOLPs [Session: F(1.87,91.5) = 5.46, p = .007, η2 = 0.10]. Although sign-trackers checked more overall, the 3 mg/kg dose of AZD-8529 was effective at reducing dysfunctional checking in both sign-trackers and goal-trackers [eOLPs, Phenotype: F(1,49) = 5.68, p = .021, η2 = 0.10; Session x Phenotype: F < 1]. As we observed previously, sign-trackers and goal-trackers showed similar levels of functional checking [OLPs, Phenotype: F(1,49) = 1.56, p = .22] and AZD-8529 reduced functional checking similarly in goal-trackers and sign-trackers [OLPs, Session x Phenotype: F < 1].

The 10 mg/kg dose of AZD-8529 also reduced checking behaviour compared to baseline, for OLPs [Session: F(1.83,54.9) = 37.0, p < .001, η2 = 0.55] and eOLPs [Session: F(1.85,55.5) = 28.1, p < .001, η2 = 0.48]. At this dose, dysfunctional checking was affected more in sign-trackers than goal-trackers [eOLPs, Session x Phenotype: F(1.85,55.5) = 3.62, p = .037, η2 = 0.11], with Sidak-corrected pairwise comparisons showing that while sign-trackers showed higher levels of dysfunctional checking during the baseline sessions [p = .029], their dysfunctional checking was normalised to the same level as goal-trackers while receiving the 10 mg/kg dose [p = .69] and during the post-drug sessions [p = .20].

Thus, the mGluR2 positive allosteric modulator AZD-8529 reduced both functional and dysfunctional checking behaviour at the 3 mg/kg and 10 mg/kg doses.

The 3 mg/kg dose of AZD-8529, which reduced checking, left generalised measures of task performance intactDiscrimination between the active and inactive levers, and overall rates of lever pressing

To assess the impact of AZD-8529 on task performance more generally, several generalised measures of task performance were compared for each of the doses of AZD-8529 administered. As would be expected for the vehicle-treated condition, rats showed higher rates of lever pressing on the active over the inactive lever across the baseline, drug testing and rebaseline sessions [Figure 3a-d; Lever: F(1,66) = 68.4, p <.001, η2 = 0.51]. Rats tended to respond less during the rebaseline session, but this was the case for both levers [Session: F(2,132) = 17.4, p <.001, η2 = 0.21; Lever x Session: F < 1]. Sign-trackers tended to respond more than goal-trackers [Phenotype: F(1,66) = 5.66, p =.02, η2 = 0.08]. Similar effects were observed at the 0.3 mg/kg dose, with rats discriminating between the active and inactive levers, and responding less during the rebaseline sessions [Lever: F(1,15) = 10.8, p =.005, η2 = 0.42; Session: F(1.62,24.3) = 6.77, p =.007, η2 = 0.31; Lever x Session: F < 1]. Rats also discriminated between the active and inactive levers at the 1 mg/kg dose of AZD-8529 [Lever: F(1,34) = 16.2, p <.001, η2 = 0.32]. Thus, for the doses of AZD-8529 that did not reduce checking behaviour, lever discrimination remained unimpaired.

Fig. 3

The reductions in checking produced by AZD-8529 were selective at the 3 mg/kg dose but not the 10 mg/kg dose. The effects of AZD-8529 on generalised measures of task performance were assessed. For both sign-trackers (a, c, e, g, i) and goal-trackers (b, d, f, h, j) the 10 mg/kg, but not the 3 mg/kg, dose of AZD-8529 reduced rates of (a, b) active lever pressing and (b, d) inactive lever pressing, and (e, f) discrimination between the two levers with the cue light on. For both sign-trackers and goal-trackers, (g, h) discrimination with the cue light off remained at chance. (i, j) The 10 mg/kg dose of AZD-8529 also produced a small reduction in the number of rewards earned. Group sizes are shown by the numbers at the base of each bar for the drug dosing day; the same rats are represented in the ‘baseline’ and ‘rebaseline’ bars for each dose. The dotted lines in (e), (f), (g) and (h) represent responding at chance (50%). Data are shown as means ± s.e.m.

Of the two doses of AZD-8529 that reduced checking behaviour, only the 3 mg/kg dose left overall rates of lever pressing intact. Rats continued to discriminate between the active and inactive levers on the 3 mg/kg dose [Lever: F(1,49) = 39.1, p <.001, η2 = 0.44], with no overall change in lever pressing across the baseline, drug treatment and rebaselining sessions [Session: F < 1]. The rate of active lever pressing reduced slightly under drug treatment [Lever x Session: F(2,98) = 3.95, p =.022, η2 = 0.08]. Sidak-corrected pairwise comparisons revealed that rats continued to discriminate between the active and inactive levers across the baseline, drug dosing and rebaseline sessions [active vs. inactive lever, all p’s < 0.001] but that active lever pressing reduced slightly during the 3 mg/kg dosing session compared to the baseline session [p =.023], though not the rebaseline session [p =.41]. By contrast, treatment with the 10 mg/kg dose of AZD-8529 reduced rates of lever pressing compared to the baseline and rebaseline sessions. While rats continued to discriminate between the active and inactive levers [Lever: F(1,30) = 30.5, p <.001, η2 = 0.50], responding was reduced during the drug treatment session [Session: F(2,60) = 30.5, p <.001, η2 = 0.50; Sidak-corrected pairwise comparisons showed reduced responding during the drug treatment session compared to the baseline and rebaselining sessions, all p’s < 0.001].

Thus, while the 10 mg/kg dose of AZD-8529 reduced checking, it also reduced overall rates of lever pressing. By contrast, the 3 mg/kg dose of AZD-8529 reduced checking without impairing ongoing responding on the active and inactive levers.

Discrimination with the cue light on and off

The doses of AZD-8529 that did not affect checking behaviour also did not impair the ability of rats to respond on the correct lever in the presence of the cue light. Under vehicle treatment, rats more readily discriminated between the active and inactive levers when the light cue was illuminated [Figure 3e-h; Cue: F(1,66) = 60.2, p <.001, η2 = 0.48], with greater accuracy in the presence of the light cue being observed in baseline, drug treatment and rebaseline sessions [Session: F < 1; Cue x Session: F < 1]. This was also the case for the 0.3 mg/kg dose [Cue: F(1,15) = 45.5, p <.001, η2 = 0.75; Session: F < 1; Cue x Session: F < 1] and the 1 mg/kg dose of AZD-8529 [Cue: F(1,34) = 64.3, p <.001; η2 = 0.65; Session: F < 1; Cue x Session: F(1.69,57.6) = 1.02, p =.36].

The 3 mg/kg dose of AZD-8529, which reduced checking behaviour but left the rate of lever pressing unimpaired, left overall cue-guided responding intact [Cue: F(1,49) = 21.2, p <.001, η2 = 0.30]. Responding was slightly reduced in the drug treatment session relative to the baseline session, but not the rebaseline session [Session: F(1.43,70.0) = 4.79, p =.02, η2 = 0.09; pairwise comparisons showed a reduction in discrimination between the baseline and drug treatment sessions (p =.024) but not the drug treatment and rebaseline sessions (p =.23)]. Importantly, rats treated with the 3 mg/kg dose of AZD-8529 continued to show better discrimination in the presence of the cue [Cue x Session: F(1.33,65.1) = 2.24, p =.13]. This was not the case for the 10 mg/kg dose of AZD-8529, which markedly impaired discrimination between the levers when the cue was on [Cue: F(1,30) = 3.08, p =.09; Session: F(1.25,37.6) = 9.00, p =.003, η2 = 0.23; Cue x Session: F(1.23,36.8) = 9.42, p =.002, η2 = 0.24].

Thus, consistent with the effects on lever pressing, the 10 mg/kg dose of AZD-8529 produced generalised deficits on task performance in addition to reducing checking behaviour, while the 3 mg/kg dose did not.

Rewards earned

The number of rewards (Fig. 3i, j) earned on task was generally consistent across individuals, although there were some reductions in the number of rewards earned across the course of a week of testing, i.e. between baselining and rebaselining sessions. However, this did not appear attributable to drug treatment, as this effect was also seen following injections of vehicle [Session: F(1.71,113) = 8.33, p <.001, η2 = 0.11; pairwise comparisons showed that responding was lower in the rebaselining sessions compared to both the baseline and vehicle treatment sessions (p’s < 0.007) which, importantly, did not differ from each other (p =.87)]. A similar effect was observed for the 1 mg/kg dose of AZD-8529 [Session: F(1.94,65.9) = 9.66, p <.001, η2 = 0.22], though for this dose, pairwise comparisons revealed that the rewards earned during the rebaselining sessions were lower than the baseline (p =.001), but that these did not differ from the number of rewards earned under drug treatment (p =.088), which in turn did not differ from the rewards earned during the baseline sessions (p =.059). There were no differences in the number of rewards earned under the 0.3 mg/kg dose of AZD-8529 [Session: F < 1].

The 3 mg/kg dose of AZD-8529 did not reduce the number of rewards earned compared to the baseline and rebaseline sessions [Session: F < 1]. By contrast, the 10 mg/kg dose reduced the number of rewards earned during the drug treatment session [Session: F(2,60) = 4.65, p =.013, η2 = 0.13] as compared to the rebaselining sessions (p =.012), though not the baselining sessions (p =.059).

Thus, of the doses of AZD-8529 tested, only the 3 mg/kg dose selectively reduced checking behaviour while leaving other measures of task performance intact.

The mGluR2/3 agonist LY404039 dose-dependently reduced checking behaviour

A subset of the rats that had been tested with AZD-8529 were subsequently tested with different doses of the mGluR2/3 agonist LY404039 after a washout period. As before, checking behaviour was highly variable across individuals, so the data were square root transformed prior to analysis.

As expected, when administered with the double distilled water vehicle, levels of functional checking remained consistent across the baseline, testing and rebaselining sessions [Figure 4a, b; Session: F(1.71,59.9) = 2.81, p =.076]. Functional checking was similarly unaffected by the 0.3 mg/kg dose of LY404039 [Session: F(2,68) = 2.31, p =.11]. However, functional checking was reduced by the 1 mg/kg dose [Session: F(1.66,58.0) = 18.9, p <.001, η2 = 0.35; pairwise comparisons showed that checking in the drug testing session was lower than the baseline and rebaseline sessions (all p’s < 0.001), which did not differ from each other (p =.99)].

Fig. 4

The mGluR2/3 agonist LY404039 dose-dependently reduced functional and dysfunctional checking. A subset of the rats tested with AZD-8529 were subsequently tested with LY404039 in a Latin square design. All rats received treatment with vehicle (0 mg/kg), and a subset of the different LY404039 doses. The number of rats receiving each dose is represented by numbers at the base of the bar for the drug dosing day; the same rats are represented in the ‘baseline’ and ‘rebaseline’ bars for each dose. In rats classified as sign-trackers, the 1 mg/kg dose of LY404039 reduced (a) functional Observing Lever Presses (OLPs) and (c) dysfunctional extra Observing Lever Presses (eOLPs). A similar effect was observed in goal-trackers, for both (b) functional OLPs and (d) dysfunctional eOLPs. BL, baseline sessions; LY: treatment with LY404039 treatment; RBL, rebaseline sessions. Data are shown as means ± s.e.m.

A similar pattern was observed with dysfunctional checking (Fig. 4c, d). As expected, the vehicle injection produced no change in dysfunctional checking [Session: F < 1], and the 0.3 mg/kg dose also left dysfunctional checking intact [Session: F < 1]. However, as for functional checking, the 1 mg/kg dose reduced dysfunctional checking relative to the baseline and rebaseline sessions [Session: F(2,70) = 14.1, p <.001, η2 = 0.29; pairwise comparisons showed reduced dysfunctional checking in the drug testing session relative to the baseline and rebaseline sessions (p’s < 0.001), which did not differ from each other (p =.95)].

Thus, the mGluR2/3 agonist LY404039 reduced both functional and dysfunctional checking behaviour at the 1 mg/kg dose.

The 1 mg/kg dose of LY404039, which reduced checking, left generalised measures of task performance intactDiscrimination between the active and inactive levers, and overall rates of lever pressing

As would be expected, the vehicle treatment had no impact on the preference of rats for the active lever, or overall rates of lever pressing on the Observing Response Task (Fig. 5a-d). Rats pressed the active lever more [Lever: F(1,35) = 66.9, p <.001, η2 = 0.66] and there were no differences in their rates of lever pressing across the baseline, test and rebaseline sessions [Session: F < 1; Lever x Session: F < 1]. This was also the case for the 0.3 mg/kg dose [Lever: F(1,34) = 100.6, p <.001, η2 = 0.75; Session: F(1.59,54.1) = 2.50, p =.10; Lever x Session: F < 1], which had also had no impact on checking behaviour.

Fig. 5

The reductions in checking produced by LY404039 were selective at the 1 mg/kg dose. The effects of LY404039 on generalised measures of task performance were assessed. For both sign-trackers (a, c, e, g, i) and goal-trackers (b, d, f, h, j) the 1 mg/kg dose of LY404039, which reduced checking, did not affect rates of (a, b) active lever pressing, or (b, d) inactive lever pressing, or (e, f) discrimination between the two levers with the cue light on. For both sign-trackers and goal-trackers, (g, h) discrimination with the cue light off remained at chance. (i, j) The number of rewards earned on task was not affected by the LY404039 doses tested. Group sizes are shown by the numbers at the base of each bar for the drug dosing day; the same rats are represented in the ‘baseline’ and ‘rebaseline’ bars for each dose. The dotted lines in (e), (f), (g) and (h) represent responding at chance (50%). Data are shown as means ± s.e.m

The 1 mg/kg dose of LY404039, which reduced checking behaviour, did not affect the preference of rats for the active lever, or rates of lever pressing in the drug testing session. Rats continued to prefer the active over the inactive lever [Lever: F(1,35) = 76.0, p <.001; η2 = 0.69]. Although the rates of lever pressing differed across the baseline, drug testing and rebaselining sessions [Session: F(1.56,54.5) = 4.57, p =.022, η2 = 0.12; Lever x Session: F(2,70) = 3.63, p =.032, η2 = 0.09; Lever x Session x Phenotype: F(2,70) = 3.18, p =.048, η2 = 0.08], pairwise comparisons revealed no difference in the rate of lever pressing between the baseline and drug testing sessions [p =.18], but rather that rates of lever pressing were reduced in the rebaselining sessions, as compared to the pre-drug baseline [p <.001]. This reduction in the rate of lever pressing during the rebaseline sessions was specific to the active lever, on which responding was lower in the rebaseline sessions compared to both the baseline [p =.043] and the drug testing [p <.001] sessions, while rates of inactive lever pressing were unaffected [all p’s > 0.20]. These differences were driven by changes in the rate of lever pressing by the sign-tracking rats, which showed lower responding on the active lever in the rebaseline session compared to the baseline and drug testing sessions [p’s < 0.01] while the goal-tracking rats showed no change [all p’s > 0.11].

Discrimination with the cue light on and off

A similar pattern of results was observed for the capacity of rats to use the cue light to guide their responding towards the currently active lever (Fig. 5e-h). Under vehicle treatment, this remained unaffected [Cue: F(1,35) = 43.5, p <.001, η2 = 0.55; Session: F < 1; Cue x Session: F < 1], as was also the case with the 0.3 mg/kg dose of LY404039 [Cue: F(1,34) = 29.8, p <.001, η2 = 0.47; Session: F(2,68) = 2.33, p =.11; Cue x Session: F(2,68) = 2.18, p =.12]. Discrimination also remained intact under the 1 mg/kg dose of LY404039 [Cue: F(1,35) = 46.0, p <.001, η2 = 0.57; Session: F(1.35,47.3) = 1.05, p =.33; Cue x Session: F < 1].

Rewards earned

The number of rewards earned on task was also affected dose-dependently by LY404039 (Fig. 5i, j). As expected, the number of rewards earned was similar under vehicle as compared to the baseline and rebaseline sessions [Session: F(1.38,48.2) = 1.23, p =.29], with goal-trackers earning more rewards overall [Phenotype: F(1,35) = 4.44, p =.042, η2 = 0.11; Session x Phenotype: F < 1]. The 0.3 mg/kg dose of LY404039 led to a slight increase in the number of rewards earned relative to the baseline sessions [Session: F(2,68) = 3.88, p =.025, η2 = 0.10; pairwise comparisons revealed that the number of rewards earned was higher in the drug test session than the baseline (p =.039) though not the rebaseline (p =.22) sessions]. The 1 mg/kg dose, which reduced checking, did not affect the number of rewards earned [Session: F < 1].

Thus, the 1 mg/kg dose of LY404039 reduced both functional and dysfunctional checking without impairing generalised task performance.