Nucleus accumbens shell and core dopamine responsiveness to sucrose in rats: role of response contingency and discriminative/conditioned cues

Animals

Male Sprague–Dawley rats (Harlan Italy, Udine, Italy) weighing 250–275 g were housed in groups of six per cage with standard food (MIL topi e ratti, GLP diets; Stefano Morini, S. Polo D'Enza, RE, Italy) and water ad libitum for at least 1 week in the central animal room, under constant temperature (23 °C), humidity (60%) and a 12-h light/dark cycle (lights on from 08:00 to 20:00 h).

All animal experiments were conducted in accordance with the statement that was revised and approved by the Society for Neuroscience in January 1995, with the guidelines for the care and use of experimental animals of the European Economic Commission (EEC Council 86/609; DL 27.01.1992, no. 116), and in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council 2003).

Surgery

Rats were anaesthetized by 400 mg/kg, i.p. of chloral hydrate (Carlo Erba, Milano, Italy). A guide cannula (Plastics One, Roanoke, VA, USA) was stereotaxically implanted at the following coordinates: NAc shell (A: 2.0; L: 1 from bregma; V: −3.6 from dura) and NAc core (A: 1.6; L: 1.9 from bregma; V: −3.4 from dura) according to the atlas of Paxinos & Watson (1998) (Fig. 1). Guide cannulas were plugged with a dummy cannula.

After surgery, rats were housed in individual cages (45 × 21 × 24 cm) under the same conditions as above. Rats were left to recover for 10 days. During the first 5 days they were administered with gentamicin sulphate (40 mg/kg, s.c.). Rats were manipulated once a day for 5 min during the whole training period.

After recovery, rats were fed 15 g of standard food (MIL topi e ratti, GLP diets; Stefano Morini) in order to keep their weight at around 90% of their ad-libitum weight. Water was available ad libitum for the whole duration of the experiments.

Microdialysis

Sucrose

Sucrose pellets of 45 mg each were utilized as food (Test Diet, Richmond, IN, USA).

Operant and yoked training

At 10 days after guide cannula implant, rats (n = 30) were randomly assigned to operant and yoked groups. Each yoked rat was paired to one operant rat. Rats were trained every day for 2 weeks, apart from weekends. Sessions lasted 1 h and took place between 09:00 and 14:00 h in acoustically isolated and ventilated operant cages (Coulbourn Instruments, Allentown, NJ, USA). Two nose-poke holes were placed on one wall at 2 cm from the cage floor. The active nose-poke was illuminated by a green/yellow light and the inactive nose-poke by a red light. The food dispenser was placed between the nose-poke holes. On the same wall was located a loudspeaker emitting a tone of 4500 Hz.

Operant rats were trained to respond to sucrose under an FR1 schedule. Yoked rats received sucrose contingent upon responding by the operant rat to which they were paired. The number of nose-pokes made and rewards earned were recorded by Graphic State 2 software (Coulbourn Instruments).

For operant rats each 1-h session was composed of a cyclic alternation of three phases.

Microdialysis after operant and yoked training

After the completion of training, operant and yoked rats were tested on three microdialysis sessions performed each day on three consecutive days. Operant and yoked sessions were started as soon as the DA basal dialysate levels had become stable (i.e. after approximately 1 h).

Day 1 of testing was an operant FR1 session and, as such, sucrose was presented in a response-contingent manner, as in all of the preceding training sessions. Because of this we did not find it necessary to introduce a further group tested under non-contingent sucrose pellet presentation on day 1.

Reverse order group

In order to control for the influence of session order on the response pattern of dialysate DA, an additional group of nine rats was trained on FR1 responding for sucrose and then tested on a reverse order, i.e. on day 2 under non-contingent sucrose pellet presentation and on day 3 under extinction (see Supporting Information).

Histology

At the end of all experimental procedures, rats were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and transcardially perfused with 50 mL of saline, followed by 100 mL of 4% paraformaldehyde solution. The brains were then removed and post-fixed for 2 days. The brains were cut in alternate 40-μm-thick and 100-μm-thick serial coronal slices on a Vibratome (Technical Products International, St Louis, MO, USA). To establish the location of the dialysis probes, 100-μm-thick sections were examined under a stereomicroscope, whereas the 40-μm-thick sections were processed for tyrosine hydroxylase immunohistochemistry, as previously described (Lecca et al., 2006a).The location of the probes was reconstructed and referred to the atlas of Paxinos & Watson (1998) (Fig. 1).

Statistics

Statistical analysis was carried out by Statistica for Windows. Basal dialysate DA was calculated as the mean of the last three consecutive samples, differing by no more than 10%, collected during the 60-min period preceding experimental sessions. Basal dialysate DA values (expressed as fmol/5 μL dialysate) were compared between groups by one-way anova. Changes in dialysate DA were expressed as a percentage of the respective baseline values and were analysed by two-way anova for repeated measures. To assess the normality assumption we considered separate normal Q–Q plots of our response variable for each combination of the groups (levels) of the two independent variables, as well as Q–Q plots of the residuals. None of the plots indicated a significant violation of the normality assumption. Results from treatments showing significant overall changes were subjected to post-hoc Tukey's test with P < 0.05 as statistically significant.

Habituation of nucleus accumbens shell dopamine responsiveness to sucrose feeding

The habituation of DA responsiveness to feeding of palatable foods has been shown by us for a salty food (Fonzies^®) as well as for a sweet food (Kinder^®) (Bassareo & Di Chiara, 1997, 1999b). In order to establish whether habituation also applies to sucrose, dialysate DA was monitored every day for 3 days during feeding of experimenter-administered sucrose pellets in rats that had never been previously exposed to sucrose. Figure 2 shows the time-course of dialysate DA in the NAc shell on three successive daily sucrose-feeding trials. The basal value of NAc shell DA (mean ± SEM) was 27 ± 3 fmol/sample (n = 16). This value did not change over days in the same subject (two-way anova, F_2,13 = 0.028; P = 0.97).

As shown in Fig. 2, NAc shell DA started to increase during sucrose consumption (between 5 and 10 min), peaking at 5 min after the end of pellet delivery. Rats continued to explore the food magazine for a few minutes after the last pellet delivery.

Two-way anova showed an effect of day (F_2,13 = 17.28; P < 0.01) and time (F_12,156 = 12.64; P < 0.01), and a day × time interaction (F_24,156 = 5.44; P ≤ 0.01). Tukey's test showed that sucrose feeding increased dialysate shell DA on the first day but not on the following days.

Acquisition of FR1 responding for sucrose

Figure 3 shows the number of cumulative active and inactive nose-pokes during training of FR1 responding for sucrose. Nose-poking increased selectively on the active hole, reaching asymptote on the seventh session. No difference was observed in the temporal profile of acquisition or in asymptotic responding in the group implanted in the shell as compared with the group implanted in the core. Three-way anova showed a main effect of nose-poke (F_1,32 = 130.54; P < 0.01) and session (F_9,288 = 24.59; P < 0.01), and a nose-poke × session interaction (F_9,288 = 18.24; P < 0.01).

Results superimposable to those above were obtained in the reverse order group (Fig. S1).

Monitoring dialysate dopamine after training

Monitoring dialysate dopamine in FR1-trained (master) rats

The basal values of DA (means ± SEM) were 25 ± 3 fmol/sample (n = 20) in the NAc shell and 26 ± 4 fmol/sample (n = 26) in the core. As shown by two-way anova, basal dialysate DA did not change within subjects over the 3 days of testing, in both the shell (F_2,17 = 0.58; P = 0.56) and core (F_2,17 = 0.0001; P = 1).

Responding for sucrose

Figure 4A shows the time-course of dialysate DA in the NAc shell and core and the number of active nose-pokes during FR1 responding for sucrose. Responding for sucrose increased dialysate DA only in the shell. Core DA did not change for the whole session. Shell DA peaked on the first sample and returned to the basal level on the sixth sample. Responding remained high for one more sample beyond the increase of DA in the shell. Two-way anova showed an effect of area (F_1,18 = 15.83; P < 0.01) and time (F_6,108 = 7.77; P < 0.01), and an area × time interaction (F_6,108 = 4.79; P ≤ 0.01). Post-hoc test showed an increase of dialysate DA in the NAc shell but not in the core.

Figure S2A shows that similar results were obtained under responding for sucrose in the reverse order group.

Responding under extinction

Figure 4B shows the time-course of dialysate DA in the NAc shell and core, and that of active nose-pokes under extinction in the presence of cues signalling sucrose availability and associated with sucrose delivery.

Responding under extinction was short-lasting and was associated with a selective increase of DA in the shell. NAc core DA did not change for the whole session. Shell DA peaked on the first sample and remained high for one more sample. As in the case of responding for sucrose, the high rate of responding exceeded by one sample the increase of dialysate DA in the shell. Two-way anova showed an effect of area (F_1,14 = 8.46; P = 0.011) and time (F_6,84 = 11.29; P < 0.01), and an area × time interaction (F_6,84 = 9.75; P < 0.01). Post-hoc test showed that DA increased in the NAc shell but not in the core.

Figure S2C shows that when the extinction session was performed on the third day, i.e. on the day after that of non-contingent sucrose presentation (reverse order group), results similar to those above (Fig. 4B) were obtained.

Non-contingent sucrose presentation

Figure 4C shows the time-course of DA in the NAc shell and core following non-contingent sucrose presentation. In contrast to responding for sucrose and to extinction, non-contingent sucrose presentation was associated with an increase of dialysate DA in both the NAc shell and core. The increase was maximal in both areas from the first sample and the DA time-course was almost superimposable in the two areas. In the NAc core the increase over the basal level was maintained for two more samples (seventh and eighth) compared with shell DA. Bars show the number of pellets presented every 5 min. Two-way anova showed an effect of time (F_8,48 = 10.61; P < 0.01). Post-hoc test showed an increase of DA in both the shell and core.

Figure S2B shows that when the non-contingent sucrose session was performed on the day before that of the extinction session (reverse order group), similar results to those above (Fig. 4C) were obtained.

Monitoring dialysate dopamine in yoked rats

The basal values of DA (means ± SEM) in 5-min samples were 24 ± 2 fmol/sample (n = 14) in the NAc shell and 25 ± 2 fmol/sample (n = 12) in the core. As shown by two-way anova, basal DA did not change within subjects over the 3 days of testing, in both the shell (F_2,8 = 0.76; P = 0.50) and core (F_2,9 = 0.0001; P = 1).

Yoked sucrose pellet presentation

Figure 5A shows the time-course of dialysate DA in the NAc shell and core, and the number of nose-pokes of yoked rats during presentation of sucrose pellets contingent upon responding of rats to which they were paired. Yoked presentation of sucrose pellets increased DA in the shell from the first sample and with a delay of one sample in the core but the maximal increase and duration (up to the seventh sample) was similar in the two areas. Two-way anova showed a main effect of time (F_12,72 = 29.61; P < 0.01) and an area × time interaction (F_12,72 = 9.56; P ≤ 0.01). Post-hoc test showed that dialysate DA increased in the shell and core, with a higher response in the shell on the first sample.

Yoked extinction

Figure 5B shows the time-course of dialysate DA in the NAc shell and core, and of nose-pokes in rats yoked to operant rats under extinction. Yoked extinction resulted in a selective increase of shell DA lasting up to the fifth sample. Two-way anova showed an effect of area (F_1,6 = 35.90; P < 0.01) and time (F_12,72 = 13.76; P < 0.01), and an area × time interaction (F_12,72 = 11.22; P < 0.01). Post-hoc test showed an increase of DA in the NAc shell but not in the core.

Yoked non-contingent sucrose pellet presentation

Figure 5C shows the time-course of DA in the shell and core during sucrose presentation non-contingent with responding of the operant paired rat but at the same mean rate of the operant sessions. Bars show the number of pellets presented every 5 min. Two-way anova showed an effect of time (F_12,72 = 44.31; P < 0.01) and an area × time interaction (F_12,72 = 2.76; P < 0.01). Post-hoc test showed that, although dialysate DA increased to a similar level in the shell and core, the increase took place earlier in the shell and lasted longer than in the core.

Responding for sucrose

Our observation that FR1 responding for sucrose was associated with a rapid increase of DA in the shell and no change in the core is at variance with Sokolowski et al. (1998) who reported that, during responding for food, dialysate DA increased to a similar extent in the shell and core, with a higher DA response in the shell only during the post-instrumental phase. In the present study, changes in shell DA increased over the basal level from the first sample and only during high nose-poking. Experimental differences (food instead of sucrose as reward and bar-pressing instead of nose-poking) might account for these differences. However, a recent study from the same laboratory by Segovia et al. (2011), in rats trained on FR1 to respond for food pellets (Bioserv), failed to observe changes in dialysate DA in both the shell and core. However, in rats bar-pressing for food on a random-ratio of 10 paradigm, Ostlund et al. (2011) observed an increase of DA in the shell and core with no differences between the two areas.

Cheng & Feenstra (2006) reported that, in rats learning to respond for sucrose/cellulose pellets during two sessions (2 h apart), dialysate DA increased to a similar extent in the shell and core, but, on the first session, rats that learned to criterion showed a higher shell DA response compared with rats that did not learn. These results are difficult to compare with ours due to the short duration of the training period.

With regard to voltammetry, there are basic differences from microdialysis that make it difficult to compare the results. Thus, voltammetry estimates changes in DA signals in a very short time interval (usually 100 ms) over or below background noise rather than basal DA levels, whereas microdialysis estimates absolute basal and stimulated levels of DA over a 100× longer interval (5–20 min; Robinson et al., 2008). As a result of this, voltammetry would not detect slow changes, taking place over tens of minutes, such as changes in basal NAc DA in rats responding for cocaine, whereas microdialysis may not detect changes resulting from discrete and isolated cues signalling the start of a behavioural sequence.

In spite of these basic differences, Cacciapaglia et al. (2012) reported that presentation of visual/auditory cues signalling sucrose availability elicits a larger phasic increase of DA in the shell than in the core. This response is followed by a slower response, selectively in the shell, that coincides with sucrose reward. If one considers the differences in time-scale between microdialysis (5 min) and voltammetric measurements (100 ms), the observations of Cacciapaglia et al. (2012) are not in contrast to ours. Thus, changes in NAc core DA might be too small in absolute terms to modify basal dialysate DA, whereas this may not be the case for NAc shell DA, which is released both in response to conditioned stimuli (CS) preceding sucrose delivery as well to sucrose itself.

It should be pointed out, however, that, as in the case of microdialysis, large discrepancies are found between voltammetry studies. Thus, in contrast to Brown et al. (2011), Cacciapaglia et al. (2012) observed phasic activation of DA in the core but no change in the shell after presentation of discriminative cues signalling food availability. In turn, whereas Brown et al. (2011) report an increase of phasic DA in the core but not in the shell in response to food, Roitman et al. (2008) and Wheeler et al. (2011) found just the opposite.

From this analysis it appears that large discrepancies do exist not only between our study and those of other laboratories but even between studies from the same laboratory. Nonetheless, the differences between the shell and core in the responsiveness of DA transmission observed in the present study are consistent with those reported in the voltammetric study of Cacciapaglia et al. (2012).

Extinction in master and yoked rats

During extinction sessions, all cues, either preceding or following responding, were still present, except that the pellet was prevented from falling into the dispenser. Under these conditions, rat responses were still emitted on the active nose-poke and DA increased selectively in the shell, just as in rats earning sucrose contingent upon their response. The same pattern of selective shell DA activation was observed in rats trained while yoked to the operant rats and therefore exposed to the same cues except that, in this case, any conditioning of sucrose-related cues would have been acquired by pavlovian rather than instrumental learning.

As far as concerns the literature, to our knowledge, no microdialysis studies are available comparing shell and core DA responsiveness to food-related cues presented under extinction by rats trained to respond for food. As for voltammetry, the study of Cacciapaglia et al. (2012) has been discussed previously.

Non-contingent sucrose feeding (including yoked feeding)

A main purpose of the present study was to compare the effect of response-contingent and response non-contingent sucrose feeding on in vivo DA transmission in the NAc shell and core. In naive rats repeatedly fed with sucrose pellets presented non-contingently, dialysate DA increased on the first trial and this response habituated on a second and third trial (24 and 48 h apart). These observations extend to sucrose the observations of previous studies from our laboratory and those of others made with various palatable foods (Bassareo & Di Chiara, 1997, 1999a,b; Gambarana et al., 2003; Rada et al., 2005; Danielli et al., 2010).

In rats previously trained to respond for sucrose on FR1, response non-contingent sucrose feeding elicited a robust and sustained increase in dialysate DA in the shell and core. Thus, in spite of the fact that these rats had been eating sucrose daily during training, no habituation of NAc shell DA responsiveness to sucrose was observed. However, this lack of habituation of shell DA transmission might only be apparent as, as observed during extinction sessions, NAc shell DA can be activated in trained rats, either yoked or operant, by conditioned/discriminative stimuli. These stimuli are likely to replace unconditioned stimuli as determinants of NAc shell DA activation.

As to the DA response in the NAc core, the fact that it is consistently and robustly observed under non-contingent sucrose feeding but is absent under extinction and in yoked rats exposed to sucrose-related cues suggests that it is either unconditioned in nature or is due to conditioned stimuli arising from stimuli intrinsic to sucrose (e.g. taste) and therefore evoked only by sucrose feeding. This conclusion makes even more striking the fact that, under responding for sucrose, when rats eat sucrose as a result of their instrumental behaviour, activation of DA transmission is observed in the shell but not in the core. We suggest therefore that the responsiveness of NAc core DA transmission to sucrose is actively suppressed during instrumental responding.

These observations are consistent with the hypothesis that DA responsiveness in the NAc shell and core is driven by different stimuli and is differentially affected by response contingency. This in turn suggests that DA transmission in these two areas plays different roles in instrumental behaviour. The robust and selective activation of DA transmission in the shell during responding for sucrose and under extinction is consistent with a role of conditioned stimuli and with the property of encoding a prediction of the intrinsic rewarding value of the action outcome (sucrose). Because of this, the activation of shell DA transmission might be intrinsically rewarding, whereas DA release in other terminal areas (NAc core, dorsal striatum and prefrontal cortex) might play more cognitive functions, such as signalling reward for learning purposes (Schultz et al., 1997). This hypothesis is consistent with the rewarding and reinforcing properties of amphetamine-like and cocaine-like psychostimulants, which preferentially increase DA in the NAc shell (Di Chiara & Bassareo, 2007; Lecca et al., 2007a) and depend for their reinforcing properties on DA receptors in this area (Pisanu et al., 2014). The suppression of DA activation in the core during responding for sucrose might be related to the need to prevent species-specific, automatic actions that would interfere with goal-directed action under conditions, such as those of FR1 responding, that do not require a decision among different reward options (Saddoris et al., 2013).