Number of rats undergoing PCA screening, CTTT acquisition and performance testing, and amperometric recordings (f, females).

Electrode in vitro calibration characteristics

Number of glutamate traces extracted for the analysis of glutamate peaks locked to turn cues, stop cues or reward delivery, per phenotype and sex.

a Distribution of PCA scores of rats eventually categorized as GTs or STs. A total of N=378 rats (215 females) underwent PCA screening. Final phenotypic classification was based on PCA score averages from the 4th and 5th test session (rats with intermediate PCA scores, INs, are not shown). The photos on the right depict lever contact triggered by the conditioned stimulus (CS) and indicative of sign-tracking behavior (top), versus and CS-triggered food port entry, signifying goal-tracking behavior (bottom). The distribution of PCA scores was unaffected by sex (b) but differed among rats obtained from the two vendors (c). Relatively more rats from Inotiv were classified as STs while rats from Taconic tended to emerge relatively more often as GTs (note that in c phenotype percentages are expressed based on the total number of rats per vendor). The distribution of vendor-specific PCA scores was not affected by sex (see Results; not shown).

Illustration of the CTTT, task rules, trial timeline, and of a cued turn. a shows a lateral view of 1.5-m long CTTT, with Faraday shielding comprised of copper wire mesh surrounding the motor and wooden frame of the Plexiglas-lined enclosure (both grounded), 2 mounted cue lights on both ends, 1 mounted SonAlert device centered (not visible here), and a copper reward port installed on either end of the treadmill. Web cameras were placed on each side of the treadmill for off-line analysis of the rat’s performance. b: Top-down view of the treadmill apparatus showing the position of the stimulus lights (green arrows), the SonAlert device to generate the tone (red arrow), the two copper reward ports (blue arrows), and a camera situated outside of the enclosure (yellow arrow). As illustrated in c, half of the rats were trained to turn in response to the light stimulus and to stop in response to the tone (upper two panels), and the other half acquired the reversed stimulus-response rules (lower 2 panels; note that in this example the treadmill moves from right to left – see arrows on the right – and, in case of turn cue would restart after the pause – see d – moving from left to right. d Trial events and timeline. The CTTT required rats to walk on a treadmill (walking speed: 9.6 cm/s). Presentation of a turn or stop cue for 2 s indicated that the treadmill stopped 1 s later, and restarted after a 5-s pause, in the reverse or same direction, respectively. Rats were trained using either the tone as the turn cue and the light as the stop cue, or vice versa. Successful turns and stops without turns, respectively, were rewarded by delivering a 45 mg banana pellet, on average 3.6 s following cue offset (see green square in d). Cued turns and cued stops were rewarded at the port located in front of the rat following completion of the turn or the stop, respectively. e shows photographs of a cued turn, initiated within the first second of cue onset (see lines connecting the individual photos to the task timeline in d; the upper four photographs were extracted from a top-down video and the lower four from a video taken using a camera situated at the backside of the apparatus (yellow arrow in b), facing the rat following completion of the turn). Successive presentation of cues was separated by a variable intertrial interval (ITI) of 60±45 s during which the rat continued walking the treadmill. Note that the rat shown in the photographs is a Long-Evans rat that was not part of the present experiments. This rat was used to generate these photos as the black-spotted fur provided better contrast against the white treadmill belt.

CTTT acquisition and asymptotic performance by GTs (n=29, 13 females) and STs (n=22, 12 females; see Table 1). a The number of training sessions to reach CTTT criterion performance, defined as 70% correct responses to either cue for two consecutive days, differed neither between the phenotypes nor the sexes. However, baseline performance, based on data from four test sessions conducted after rats had reached criterion performance, indicated that GTs scored more cued turns than STs (b). Moreover, a main effect of day reflected that all rats performed more cued turns on day 3 than on day 1 (post hoc Bonferroni test; c; note that c and d show response ratios but ANOVAs were conducted using arcsine transformed data because ratio data violated homoscedasticity). d The relative number of cued stops did not differ between the phenotypes. There were no main effects of sex and no significant interactions between the effects of phenotype, day and sex on cued turns and cued stops. e Relative to the time of cue onset, GTs initiated and completed cued turns later than STs. Reflecting the parallel effects of phenotype on initiation and completion times, the actual time needed to complete turns did not differ between the phenotypes (*,**,***: P<0.05, 0.01, 0.001).

Electrochemical measurement scheme, electrode calibration and placement. a Schematic illustration of a ceramic backbone with four platinum-iridium (Pt/Ir) recording sites, organized in two pairs, with each recording site measuring 333 µm long and 15 µm wide. The upper pair was fabricated to measure currents reflecting glutamate concentration (red, also b), while the lower pair served as sentinels for background current recording and subtraction (yellow, also c). Glutamate oxidase (GO) was immobilized onto the upper (b), but not lower (c), pair of recording sites. After GO coating, a non-conducting polymer, m-(1,3)-phenylenediamine was electroplated onto both sites to prevent the transfer of small electroactive organic molecules to the Pt sites. Application of a 0.7 V constant potential versus an Ag/AgCl reference electrode (not shown) served to oxidize hydrogen peroxide at individual recording sites (e.g., Burmeister et al., 2000; Parikh et al., 2010b), yielding current proportional to the concentration of glutamate at the recording site. d In vitro calibration (representative example) indicated the sensitivity, linearity of the response to increasing concentrations of glutamate, and selectivity of GO-coated recording sites (red; see Table 1 for calibration data). The current recorded via the sentinel site did not respond to glutamate and the meta-(1,3)-phenylenediamine (mPD) barrier completely prevented ascorbic acid (AA) from generating current, Addition of dopamine (DA) increased the current by about 0.5 pA (note that the ordinate used to graph the current obtained via the GO-immobilized site (200 pA) obscures the visualization of such a small increase). For the 3/15 electrodes that exhibited a dopamine response (>0.1 pA; see Table 2), net currents were normalized by the dopamine response (Burmeister and Gerhardt, 2001). e Electrodes were implanted into the DMS; (see Methods for stereotaxic coordinates of accepted placements; the circles in e indicate the location of microelectrode tips placed within this area (red circles: placements in rats which were recorded during CTTT performance; purple circles: placements in rats in which fronto-striatal projections were silenced; note that placements along the anterior-posterior axis were flattened onto the section shown in e).

Glutamate concentrations locked to turn and stop cues and reward delivery. a and b show representative recording traces from GTs and STs (5 each), respectively, recorded during cued turns. Filled circles indicate peaks (defined in Methods), open black symbols were associated with the execution of the turn, symbols surrounded by a black circle with treadmill stops and the onset of a 5-s pause (see also Fig. 2d), and symbols surrounded by a green circle mark the delivery of reward following a cued turn. Currents recorded during the cue-on period (reddish background) were used to determine cue-locked peak characteristics, and those recorded during a 2-s period following reward delivery were used to compute reward-locked glutamate peaks. These current traces illustrate the predominance of single turn cue-locked peaks in GTs (a), contrasting with the more frequent presence of 2 or 3 turn cue-locked peaks in STs (b). Furthermore, reward delivery more reliably evoked glutamate peaks in STs (b). LMM-based analysis of glutamate traces (see Table 3 for the number of traces per phenotype and sex included in this analyses) indicated significantly higher turn cue-locked maximum peaks in GTs when followed by actual turns (c). Furthermore, single glutamate peaks were observed more frequently in GTs than in STs, while 2 or 3 peaks during the cue period tended to be more frequent in STs (X2 test; d). In contrast to glutamate currents recorded during the cue period, maximum glutamate concentrations were higher in STs during all other response categories (missed turns, e; cued stops f; note that errors following stop cues, that is, false turns, were extremely rare and thus peaks obtained from these trials were not analyzed), and following reward delivery during cued turn trials (g; c,e,f,g depict estimated marginal means (EMMs), and 95% CI; main effects of phenotype: **,***: P<0.01, 0.001).

Relative probabilities for cued turns in GTs given the presence of individual and combined properties of turn cue-locked glutamate peaks (based on a total of 548 traces, 364 recorded during cued turns and 184 during misses, 318 from 8 GTs and 230 from 7 STs). a: The ordinate depicts relative turn probabilities derived from contingency table analyses. A value of 1 indicates that GTs were as likely as STs to turn (inserted horizontal line), while a value of 2 that GTs were twice as likely as STs to turn. For each relative turn probability value, the associated significance level, derived from contingency table analyses and reflecting the degree of dissimilarity of the proportion of turns and misses in GTs versus STs, is indicated by the symbol color (no fill, not significant, n.s.; blue, P<0.05; green, P<0.01; magenta, P<0.001; red: P<0.0001). The three curves in a depict the probability of a cued turn in GTs, relative to STs, based on: 1) turn cue-locked maximum peak glutamate concentrations (abscissa; middle curve, circles); 2) such concentrations derived from the presence of a single turn cue-locked peak (top curve, rhombi); and 3) such concentrations in conjunction with the presence of multiple (2 or 3) turn cue-locked peaks (bottom curve, triangles). In a, the data points labeled b,c,d and e mark data points for which representative traces are shown in b-e (all traces are from cued turns in GTs, as the plot in a indicates the probability of GTs to turn relatively to STs). For example, the data point next to the label b in a indicates that in the presence of a single glutamate peak of about 2.0 µM, GTs were 1.5 times as likely as STs to turn (significantly different from as likely to turn as STs at P<0.0001). A trace exemplifying this data point is shown in b. Concerning the significance of the three probability curves in a: 1) middle curve, circles: regardless of other glutamate peak number, increasing maximum peak concentrations, beginning with 2.8 µM glutamate, yielded significantly different proportions of turns and misses in GTs and STs and rising relative probabilities for GTs to execute a turn (“GLU max peaks ≥x”; the slope of the linear regression of all three curves was significantly different from zero; see Results). For example, for maximum peak concentrations ≥4 µM, GTs were 1.24 times as likely as STs to turn. 2) top curve, rhombi: the presence of a single turn cue-locked peak strongly increased the relative probability for GTs to turn (see Results). In conjunction with increasing amplitudes of these peaks (“1 peak only ∪ ≥x”), these probabilities did further increase, reaching, for example, 1.73 for glutamate concentrations ≥4 µM. 3) bottom curve, triangles: the presence of 2 or 3 turn cue-evoked peaks (no more than 3 turn cue-locked peaks were observed) significantly lowered the relative probability for a turn in GTs below 1 or, conversely, indicated that STs were more likely to turn as GTs. However, in conjunction with rising max amplitude threshold levels (“2 and 3 peaks ∪ max peaks ≥x”), the relative probabilities of GTs to turn increased, so that at maximum peak concentrations of ≥6 µM glutamate, the proportions of turns and misses no longer differed significantly between the phenotypes. The vertical brackets on the right symbolize significant differences between the relative probabilities of the three trace characteristics (H(3)=33.41, P<0.0001; Kruskal–Wallis test; multiple comparisons: *,****, P<0.05, 0.0001).

Timeline of experimental procedures and effects of CNO on CTTT performance. a: Following initial handling and screening to identify the rat’s phenotype (17 GTs, 8 females; 12 STs, 6 females; Table 1), rats were infused with either a Cre-dependent inhibitory DREADD or the empty control vector (both expressing mCherry; b) into the lower layers of the prelimbic cortex, and a retrogradely transported, Cre-expressing plasmid into the DMS (expressing eGFP). These rats continued to undergo CTTT performance training, followed either by (c) a test of the effects of vehicle or CNO on CTTT performance (the blue syringes symbolizing CNO administration) or by (d) implantation of an microelectrode array for the measurement of glutamate concentrations in the DMS and following vehicle or CNO administration. Following recovery from that surgery, the effects of CNO and vehicle on performance and performance-associated glutamate concentrations were assessed. In GTs, administration of CNO significantly reduced the relative number of cued turns (e), but not cued stops (f; e and f show individual data, mean and 95% CI; post hoc multiple comparisons: ***,****: P<0.001, 0.0001, Tukey’s Honest Significant Difference test). CNO had no effects on cued turns or stops in rats expressing an empty control vector (see Results). In GTs, CNO administration attenuated cue-locked maximum peak glutamate concentrations during (residual) cued turns (g) and decreased the number of traces with just 1 glutamate peak during the cue period while increasing the frequency of 2 peaks during this period (h). Following the administration of CNO, residual turns took significantly more time to be initiated in GTs when compared with vehicle-treated GTs and CNO-treated STs (i). When followed by failures to turn, or misses, turn cue-locked maximum peak concentrations were not affected in GTs but reduced in STs (significant interaction between the effects of phenotype and CNO; j). Maximum peak glutamate concentrations locked to reward delivery again were higher in STs than in GTs, and CNO reduced these concentrations in both phenotypes (main effect of CNO, no interaction (k; (multiple comparisons: *,**,***: P<0.05, 0.01, 0.001).

Expression of eGFP (green channel), indicating the expression of the retrograde Cre-expressing vector in the DMS and, following retrograde transport, in the medial frontal cortex. The presence of the amplified mCherry fluorescent reporter signal (red channel) in cortex indicated the expression of the inhibitory hM4Di DREADD vector. a-d: Illustration of expression efficacy ratings for eGFP in the striatum (a,b) and of mCherry in the prelimbic cortex (c,d), with a top score of 5 indicating complete or near-complete expression exclusively in the DMS projection field of prelimbic cortex efferent neurons (Mailly et al., 2013) and prelimbic cortex, respectively. The example of eGFP expression in the DMS in e (cc, corpus callosum) received a score of 2 (illustrated in a) because the expression field was located in part lateral to the prelimbic cortex projection field. f depicts an example of mCherry expression in the prelimbic cortex that was assigned a scores of 4 as it was restricted to the deeper layers in the prelimbic cortex and extended dorsally into cingulate cortex. In GTs, but not STs, mCherry expression scores in cortex were significantly correlated with the efficacy of CNO to reduce cued turns (g; note that the ordinate in g depicts the difference between the proportion of turns in vehicle-minus CNO-treated rats, so that higher scores indicate greater CNO effects, and therefore higher expression scores were correlated with greater CNO effects). h depicts the retrogradely transported eGFP and the expression of mCherry, including the co-expressing of both fluorochromes, primarily in layer 5 of the prelimbic cortex. The brightened rectangular region in h is enlarged in i and shows neurons co-expressing nucleolar eGFP and cytoplasmic mCherry (white arrows in i). In GTs, but not STs, the proportion of double-labeled neurons in prelimbic cortex was significantly correlated with the efficacy of CNO to attenuate cued turns (j; note that higher scores on the ordinate depicts greater CNO effects).