Abstract

Results

Extinction test performance

Rats received test sessions in which compounds of the training stimuli were presented under extinction. These compounds combined stimulus elements that dictated either the same (“congruent”) or different (“incongruent”) instrumental responses during initial training. The mean response rates (correct vs. incorrect) for each of the three trial types (single element, congruent, and incongruent) were analyzed.

Congruent performance

As shown in Figure 2A, rats made significantly more responses to the correct than incorrect lever during the congruent compound presentations (F_(1,23) = 66.4, P < 0.001). There were no significant differences between the groups, (F < 1), or a significant group × lever interaction (F_(1,23) = 2.55, P = 0.124).

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Figure 2.

Test levels of lever pressing during (A) congruent, (B) incongruent compound, and (C) single-element presentations in control and sucrose access rats. Data presented as log−1(x − 1) of mean transformed data. (D) Instrumental responding in the contexts associated with the nondevalued (black bars) and devalued (open bars) food rewards. Error bars represent SEM, Control N = 12, Sucrose N = 13. (*) P < 0.05, (**) P < 0.01.

Incongruent performance

Consistent with previous reports (Haddon and Killcross 2006; Reichelt et al. 2013a; Nelson et al. 2014), control rats used contextual cues to disambiguate the conflicting response information, responding according to the stimulus element that had previously been trained in that same test context (Fig. 2B). However, rats with a history of sucrose exposure did not make more responses to the correct than incorrect lever during the incongruent compound. Analysis revealed no significant effects of group (F < 1) or lever (F_(1,23) = 1.262, P = 0.273) but, importantly, the interaction between group and lever was significant (F_(1,23) = 4.909, P < 0.05). Inspection of the simple effects of the group × lever interaction revealed a significant effect of lever in control rats (F_(1,23) = 6.332, P < 0.05); however, there was no significant effect of lever in rats with a history of sucrose exposure (F < 1).

Single stimulus elements

Both groups showed accurate conditional discrimination performance when single training stimulus elements were presented (Fig. 2C), i.e., when tested on the stimulus elements acquired during training and when there was no conflict. Consequently, both groups produced more correct responses (F_(1,23) = 43.7, P < 0.001) with no effect of group or interaction (both F < 1).

Magazine activity

No significant differences were observed in overall magazine responding (F < 1) (mean entries per min [±SEM] Control: 8.1 [2.5]; Sucrose access: 8.9 [2.1]). When responding to stimuli was examined separately (mean entries per min [±SEM] Control: Congruent 6.3 [2.0], Incongruent 8.3 [2.4], Single element 8.5 [2.6]; Sucrose access: Congruent 7.6 [2.0], Incongruent 7.8 [1.8], Single element 9.6 [2.4]) a significant difference between stimulus types was observed (F_(2,46) = 3.4, P < 0.05), indicating that the single elements provoked more magazine entries than the congruent and incongruent compounds, but there was with no effect of group or interaction (both F < 1).

Context devaluation

To determine whether there were any differences between the groups in their ability to discriminate the two test contexts, we assessed the effect of outcome prefeeding on instrumental responding in the contexts. Rats from each group consumed comparable amounts of the reward pellet outcomes (mean [±SEM] Control: 10.7 g [0.7]; sucrose access 11.4 g [0.5]). Both groups (Fig. 2D) displayed intact context–outcome associations as they made fewer lever-press responses during extinction when prefed the outcome that had previously been earned in that test context compared with when prefed the outcome associated with the other context (F_(1,23) = 7.77, P = 0.01). There was no effect of group or any interaction with group (both F < 1). Magazine entries did not significantly differ (F < 1) between groups (mean [±SEM] Control: devalued 11.3 [2.4]; nondevalued 9.4 [1.4]; Sucrose access: devalued 11.5 [3.0]; nondevalued 9.5 [2.1]) and there was no overall effect of devaluation (F_(1,23) = 1.4, P = 0.251)

Parvalbumin immunohistochemistry

Rats exposed to intermittent sucrose throughout adolescence had significantly fewer parvalbumin immunopositive cells in the hippocampus, both overall (F_(1,29) = 27.6, P < 0.001) and in its subregions (CA1: F_(1,29) = 21.3, P < 0.001; CA3: F_(1,29) = 12.3, P = 0.001; dentate gyrus: F_(1,29) = 13.1, P = 0.001) as shown in Figure 4A,B. Rats exposed to intermittent sucrose throughout adolescence also had significantly fewer parvalbumin immunopositive cells in the mPFC overall (F_(1,29) = 10.4, P = 0.003) and in the prelimbic (PrL) PFC (F_(1,29) = 8.359, P < 0.01), but not in the infralimbic (IL) PFC (F_(1,29) = 3.97, P = 0.056); as shown in Figure 5A,B.

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Figure 4.

Results of parvalbumin immunohistochemistry in dorsal hippocampus. (A) Mean number of parvalbumin immunopositive cells in control and sucrose exposed rats in the hippocampus overall (A) and in the hippocampal subfields CA1, CA3, and dentate gyrus (B). (C) Photomicrographs show representative hippocampal staining of control and sucrose exposed rats, 4× magnification, marker represents 250 µm. (D) Regions of the hippocampus studied. (E) Photomicrograph showing parvalbumin stained neurons in the CA1 of a control rat at 20× magnification, marker represents 25 µm.

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Figure 5.

Results of parvalbumin immunohistochemistry in PFC. (A) Mean number of parvalbumin immunopositive cells in control and sucrose exposed rats in the PFC overall (A) and in the PFC subfields prelimbic PFC (PrL) and infralimbic PFC (IL) (B) Photomicrographs show representative PFC staining of control and sucrose exposed rats. (C) Regions of the mPFC studied. (D) Photomicrographs show representative parvalbumin stained neurons in the PrL PFC of a control rat and sucrose exposed rat 20× magnification, marker represents 25 µm.

Discussion

This experiment exposed young rats to a 10% sucrose solution for a limited period of time each day across their adolescence and examined their subsequent performance on tasks that required the use of (1) contextual information to disambiguate response conflict and (2) of spatial relations between objects to identify what has changed in the environment. Conflict control was examined by simultaneously presenting pairs of stimuli that had each controlled opposing (“incongruent”) responses due to their initial conditional discrimination training in different contexts. Consistent with previous reports (Haddon and Killcross 2006; Marquis et al. 2007; Reichelt et al. 2013a), control rats were able to use contextual information to resolve this conflict task. Rats that had been exposed to intermittent sucrose across adolescence learned the biconditional discriminations just as well as control rats, but, in contrast to the control rats, failed to use the contextual information to discriminate between the context-appropriate and context-inappropriate levers during incongruent trials. No differences were observed in latency to make responses during the discriminations, suggesting that the performance deficit to incongruent compounds in the sucrose exposed rats was not due to increased impulsivity. Furthermore, the observation that daily limited exposure to sucrose across adolescence exerts long-term effects upon PFC function in the form of impairments in response-conflict resolution further extends the known effects of high sucrose diet consumption beyond that of the hippocampus. This conclusion derives from the finding that rat hippocampal lesions can enhance, rather than disrupt, incongruent-trial performance on the present Stroop-based task (Haddon and Killcross 2007) potentially due to the hippocampus not being required to utilize contextual cues as a task-setting cue to guide responding. However, PFC-dependent control appears to be best characterized as an explicit goal-directed process in which contextual or task-setting cues are used as the basis of higher-order rules to enable the appropriate control of choice responses (Haddon and Killcross 2007). Thus, it is possible that high sucrose diet in adolescence may exert long-term adverse effects on a variety of neural structures involved in high-order cognitive control of behavior, including the PFC.

We used devaluation of one outcome to assess whether the sugar intake across adolescence had impaired subsequent ability to discriminate between contexts, thereby impairing the use of contextual information to solve response conflict in the Stroop-like task (Bar and Aminoff 2003). However, we found that rats that had received sucrose across their adolescence showed greater instrumental responses in the context associated with the nondevalued outcome than in the context associated with the now devalued outcome. This indicates that the history of sucrose had not impaired the subsequent ability to use contextual information to guide responding in a goal-directed manner. This is a novel finding, as previous studies have provided evidence that goal-directed instrumental actions are disrupted in rats provided with 2 h daily access to a high fat/high sugar condensed milk solution (Furlong et al. 2014) or to a 10% sucrose solution (Kendig et al. 2013), suggesting that 2 h daily access to these beverages accelerates the development of habitual instrumental behaviors compared with rats with constant sucrose access. Acceleration of habitual responses has also been observed following sensitization of the dopaminergic system by d-amphetamine (Nelson and Killcross 2006). Previous studies have indicated that frequent intake of high-fat and high-sugar foods is associated with decreased dopamine D2-receptor sensitivity and expression in the striatum of rodents and obese humans, paralleling the effects seen after chronic exposure to psychostimulants (Bello et al. 2002, 2003). D2 receptors are implicated in controlling behavioral flexibility (Yawata et al. 2012) and their down-regulation therefore could contribute to loss of goal-directed control.

Thus, in light of our observations that sucrose consumption impairs behavioral flexibility and previous studies indicating goal-directed instrumental behavioral deficits following sucrose consumption, it is possible that the expression of frontostriatal D2 receptors are chronically down-regulated by daily restricted access to sucrose consumption. This suggestion warrants further exploration by analysis of dopamine receptor mRNA and potentially through administration of D2 receptor agonists to ameliorate cognitive deficits.

Rats exposed to sucrose for 2 h a day during adolescence also exhibited a subsequent deficit in an object-in place memory task. In this task, rats are first exposed to four objects, A, B, C, and D occupying distinct locations in a familiar environment. After a brief retention interval, they are reexposed to the objects, two of which, B and C, now occupy each other's previous locations, while the other two, A and D, continue to occupy their original locations. Control rats spent more time exploring the objects whose locations were changed, B and C, than the objects that remained in the same locations. In contrast, rats exposed to sucrose across adolescence spent just as much time exploring both pairs of objects. Previous studies have provided evidence that this object-in-place recognition memory involves several brain regions. These include the hippocampus, which codes spatial relations between the objects (Barker and Warburton 2011), the perirhinal cortex, which constructs a representation of the identities of the objects (Barker et al. 2007), the mPFC, which may integrate the spatial and object information (Kesner and Ragozzino 2003; Barker et al. 2007), as well as interactions between these regions, especially, those between hippocampus and the mPFC (Barker and Warburton 2011). The object-in-place memory deficit observed in sucrose exposed rats could not be attributed to hyperactivity, as the sucrose fed rats did not differ in their levels of locomotor behavior from control rats, nor to a lack of motivation to explore as the sucrose fed rats did not differ in the total time spent exploring from control rats.

Immunohistochemical analysis performed 8 wk after termination of the intermittent sucrose access revealed a significant deficit in parvalbumin-immunoreactive cell density in the hippocampus and PFC. Consistent with these findings, other studies have reported correlations between reductions in parvalbumin expression and impairments in fear conditioning (Courtin et al. 2014) and attentional set formation (Bissonette et al. 2014). Studies in postmortem schizophrenia patients and rodent models have also demonstrated a reduction in parvalbumin expression in the mPFC (Lewis et al. 2005) and the hippocampus (Lodge et al. 2009; Konradi et al. 2011). A recent study by Boley et al (2014) indicated that a lentiviral delivered short hairpin RNA-induced knockdown of parvalbumin expression in the ventral hippocampus in rats led to downstream increases in spontaneous dopamine neuron population activity in the VTA and an enhanced locomotor response to amphetamine administration. Sugar-induced alteration of the dopamine system has been previously reported in rats maintained on a daily 20-min sugar-feeding schedule, this alteration included increased dopamine turnover in the nucleus accumbens (Hajnal and Norgren 2002) and up-regulation of DA transporter mRNA in the ventral tegmental area and nucleus accumbens (Bello et al. 2002, 2003). Dopamine is critical for modulating cortical GABA mediated inhibition and glutamate mediated excitation in the PFC and other cortical regions (O'Donnell 2010). This dopamine directed control of the excitation–inhibition balance within the cortex and hippocampus is refined during adolescence (Tseng and O'Donnell 2007; O'Donnell 2010). Thus, alterations in dopamine turnover and activity may contribute to the observed loss of parvalbumin-expressing GABA-interneurons, leading to alterations in the maturation of circuits during adolescence that are responsible for excitation–inhibition balance in the PFC, leading to cortical disinhibition and behavioral impairments (O'Donnell 2011).

Taken together, these findings indicate that perturbations in parvalbumin expression are associated with cognitive deficits and alterations in dopamine signaling. These findings also provide additional support for the suggestion that sucrose access during adolescence can produce enduring neuropathological changes in the rat and that these changes underpin our observed deficits in response conflict resolution and object-in-place recognition memory. However, further studies are needed to establish the extent of changes, whether these extend to other regions involved in reward learning and behavioral flexibility, and, indeed, whether such changes are causally related to the cognitive deficits observed here.

High-sugar diets have been implicated in weight gain and obesity, by increasing energy consumption to an extent that exceeds energy output, thereby distorting mechanisms controlling energy balance (DiMeglio and Mattes 2000). Behavior was tested 6 wk after sucrose exposure ceased, thus it is likely that any effects on blood sugar had dissipated at this time. Blood glucose levels were not measured in this study, however it has recently been shown that rats exposed to 10% sucrose continuously for 30 d had a decreased ability to tolerate an oral glucose load (Sheludiakova et al. 2012). Furthermore, a recent study indicated that plasma insulin levels remained elevated after sucrose access ceased in adolescent, but not adult sucrose exposed rats (Hsu et al. 2015).

Importantly, the impairments in cognitive control observed in our current study indicate that sucrose access for a limited period each day can induce subsequent deficits in decision-making and behavioral flexibility. The present results may have implications for mental health and neurodevelopmental disorders although it must be acknowledged that a range of variables contribute to mental health in people and that disorders of mental health have a complex atiology. Nevertheless, the finding that daily consumption of sucrose in rats led to various cognitive deficits provides support for the proposal that sugar consumption in children and adolescents may likewise have adverse effects on cognitive development. The World Health Organisation's guideline for sugar intake is ∼25 g per day (10% of total energy intake), but a single can of standard soft drinks may contain as much as 40–50 g of sugar. There is growing evidence that the pathophysiology of many mental disorders might emerge from maturational aberrations of normal neurodevelopmental changes in the adolescent brain, particularly in the PFC (Paus et al. 2008). Thus, high levels of sugar-sweetened beverages consumed during adolescence may pose a risk factor for the development of mental health disorders, perhaps by impairing neurocognitive functions affecting decision-making and memory.

Materials and Methods

Behavioral procedure

Pretraining

Rats received two 48-min sessions of magazine training. Food pellet rewards (grain or sucrose) were delivered on a random time 120-sec schedule. Magazine training occurred in a morning session in one context (e.g., rod floors with grain pellets) and, 3 h later, an afternoon session in the alternative context (e.g., mesh floors and sucrose pellets). This allocation of contexts and their associated outcome remained consistent throughout training and was counterbalanced across animals (see below). After magazine training, the rats were trained to respond during alternating presentations of the left and right levers. Each session lasted 48 min and consisted of twenty-four 60 sec trials (12 each of the left and right lever presentation). Rats received two training sessions per day, one in the morning and the other, 3 h later, in the afternoon. On the first day lever pressing was reinforced on a continuous schedule (CRF) and on the second day was reinforced on a random interval 15-sec schedule (RI15). Rats then progressed to biconditional discrimination training.

Biconditional discrimination training

Rats were trained on two simultaneous biconditional discriminations (see Table 1). All elements were counterbalanced across rats. For half the rats, the floor of context 1 (C1) consisted of stainless steel rods, while the floor of context 2 (C2) consisted of stainless steel mesh; for the other half of the rats, the mesh floor was C1 and the floor with rods was C2. Previous studies (Reichelt et al. 2013b) have shown that these contexts with their distinct floors are highly discriminable for rodents. Half of the rats in each of these allocations received the auditory discrimination in C1 and the visual discrimination in C2, and this allocation was reversed for the other half of each subset of rats. During the auditory discrimination rats received presentations of auditory (A) cues (tone or clicker [A1 or A2]) during which the correct lever-press responding (left or right [LP1 or LP2], counterbalanced) led to an outcome (O1; food pellets). During the visual discrimination lever-press responding (LP1 and LP2, counterbalanced) led to reward (O2; sucrose pellets) in the presence of visual cues (V) (illumination of the house light, or two stimulus lights plus the magazine light [V1 and V2]). Rats received one morning session in C1 and one afternoon session in C2.

Table 1.

Experimental design indicating biconditional training and test stimuli

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Each session lasted 48 min in total and consisted of 24 trials (six of each trial type; A1 and A2 or V1 and V2) with a variable intertrial interval (range 40–60 sec; mean, 50 sec). Stimuli were presented for 60 sec, during which both levers were present. Reinforcement was unavailable for the first 10 sec (CS1) and was then available during the final 50-sec period (CS2) on the RI15 schedule. The levers were retracted at the end of the 60 sec stimulus presentation and remained retracted during the intertrial interval. Rats received 16 sessions of each stimulus condition, two sessions per day, for 16 d.

Test sessions and probe stimuli

Test stimuli were congruent and incongruent stimulus compounds or single training stimulus elements previously associated with each training context as described previously (Reichelt et al. 2013a). For example, if C1 was previously associated with auditory stimuli A1 and A2, these single stimulus elements were presented during the test; in the alternative context (C2) visual stimuli V1 and V2 were presented as single elements. The compound test stimuli were categorized as either congruent or incongruent depending on the responses previously required during training. Congruent stimulus compounds were composed of stimulus elements which had each signaled the same lever-press response–outcome relation during training on the biconditional discrimination task. Thus, the elements previously presented in A1V1 and A2V2 compounds had each signaled the same lever-press responses in training (LP1 and LP2, respectively). Incongruent stimulus compounds, A1V2 and A2V1 were composed of stimulus elements which had signaled different, and thus conflicting, lever-press responses during biconditional discrimination training. “Correct” responding to incongruent compounds is defined by responding according to the stimulus element previously trained in the appropriate context. Thus, if during the test session the context had been previously associated with the visual discrimination, the visual stimulus element (V1 or V2) of the incongruent compound (A1V2 or A2V1) was the correct cue. For example, if presentations of A1V2 occurred in the context where A1 had been presented during the biconditional training, selection of the lever appropriate to A1, rather than V2, was deemed to be correct. Therefore, for the rats to respond correctly to the incongruent stimulus element compounds they must utilize contextual information provided by the test context to disambiguate responding.

Following acquisition of the biconditional discrimination, rats received probe test trials within training sessions. Rats received two test sessions per day, one in each context. Test sessions consisted of four extinction probe trials consisting of presentations of congruent and incongruent audio-visual compounds of training stimuli (A1V1, A2V2, A1V2, A2V1) and eight reinforced training trials of single training elements associated with the context (A1, A2 or V1, V2), presented in a manner identical to training trials. Test session duration was 24 min and trial order was randomized; each extinction probe trial was presented once during the test session. Testing took place over 4 d.

Analysis

To assess whether the history of sucrose access selectively disrupted incongruent, but not congruent compound performance, within-subjects ANOVAs were performed on congruent and incongruent compound responses separately. As the variance of test data was found to increase with the mean, data were subject to a logarithmic transformation (Log₁₀ (x + 1)) to correct for the heterogeneity of variance before performing any further analyses (Howell 2013).

Selective reward devaluation on contextual conditioning

Rats prefed one food reward (i.e., grain or sucrose pellets) to satiety show a selective reduction in instrumental responding for the sated reward associated with that particular context (Haddon and Killcross 2006; Reichelt et al. 2013b). To assess whether the sucrose history influenced this contextually specific devaluation effect, we subjected rats to two 10-min extinction sessions during which both levers were available. Prior to each extinction session, the rats received 40-min free access to one of the two rewards (20 g of sucrose or grain pellets) earned during training. This access occurred in individual cages located in a separate room. Each rat was tested in both contexts (Context 1 and Context 2), with testing order (devalued versus nondevalued) counterbalanced. We expected that control rats would produce more lever-press responses in the context associated with the nondevalued outcome. The question of interest was whether those with a history of sucrose access would also behave in this outcome value-specific manner.

Acknowledgments

Footnotes

References