Glucocorticoid receptors in the prefrontal cortex regulate stress-evoked dopamine efflux and aspects of executive function

Abstract

Enhanced dopamine efflux in the prefrontal cortex is a well-documented response to acute stress. However, the underlying mechanism(s) for this response is unknown. Using in vivo microdialysis, we demonstrate that blocking glucocorticoid receptors locally within the rat prefrontal cortex results in a reduction in stress-evoked dopamine efflux. In contrast, blocking glucocorticoid receptors in the ventral tegmental area did not affect stress-evoked dopamine efflux in the prefrontal cortex. Additionally, local administration of corticosterone into the prefrontal cortex increased prefrontal dopamine efflux. The functional impact of enhanced dopamine efflux evoked by acute stress was demonstrated using a cognitive task dependent on the prefrontal cortex and sensitive to impairment in working memory. Notably, stress-induced impairments in cognition were attenuated by blockade of glucocorticoid receptors in the prefrontal cortex. Taken together, these data demonstrate that glucocorticoids act locally within the prefrontal cortex to modulate mesocortical dopamine efflux leading to the cognitive impairments observed during acute stress.

Exposure to acute stress increases dopamine (DA) efflux in the prefrontal cortex (PFC) and is associated with deficits in working memory performance (1–3). However, the mechanism by which stress increases DA efflux in the PFC remains to be determined. Glucocorticoid hormones, released following activation of the hypothalamic-pituitary-adrenal axis by acute stress, exert their effects on glucocorticoid receptors (GRs) and mineralcorticoid receptors (MRs). Glucocorticoids have a 10-fold greater affinity for MRs than for GRs, and therefore, MRs are substantially occupied at low basal glucocorticoid levels and primarily mediate basal or tonic actions of glucocorticoids (4, 5). GRs are only partially occupied at low basal glucocorticoid levels and become progressively saturated when glucocorticoid levels are elevated during the circadian peak or following exposure to stress (4, 5). The GR is expressed in both glial cells and neurons throughout the brain (5). Importantly, GRs are present on DA neurons in the ventral tegmental area (VTA) (6) and are also highly expressed in the PFC (4, 5, 7, 8).

We used in vivo microdialysis to investigate the role of GR in mediating the effects of acute stress on DA efflux in the PFC and to determine the anatomical site of action. The functional consequence of blocking the interaction between stress and DA efflux was also examined using a PFC-dependent cognitive task. We show that corticosterone activates GRs located in the medial PFC (mPFC), and not in the VTA, to enhance mesocortical DA efflux during stress. Furthermore, reducing the increase in mesocortical DA efflux by blocking prefrontal GRs prevented deficits in cognitive function observed following exposure to acute stress.

Results

GRs Modulate Stress-Evoked DA Efflux in the mPFC .

To investigate the role of the GR in stress-evoked DA efflux, in vivo microdialysis was used in combination with an acute mild tail-pinch stress and a GR antagonist, RU-38486. Tail-pinch stress was accompanied by a fivefold rise in plasma corticosterone (Fig. 1A) from 7.96 μg/dL (basal levels) to 41.54 μg/dL after 15 min of stress (n = 6 rats per condition). The increase in corticosterone release differed significantly from basal levels for the initial 45 min following tail-pinch stress [F(6, 35) = 10.35, P < 0.05, Tukey's test]. A separate study again confirmed a rapid and transient increase in DA efflux in the mPFC, reaching maximum values 15 min after the onset of tail-pinch stress and returning to basal values after 90 min (n = 6; Fig. 1B). Administration of RU-38486 (11.1 ng/μL) into the lateral ventricle (LV) using reverse microdialysis (n = 4) significantly attenuated stress-evoked DA efflux measured as mean (± SEM) area under the time–response curves (AUC) for vehicle- and RU-38486–treated rats. (Student t test, P < 0.05; Fig. 1C).

Fig. 1.

Activation of GRs mediates stress-evoked DA efflux in the mPFC. (A) Plasma corticosterone (CORT) concentration (μg/dL) in rats subjected to 15 min of tail-pinch stress (n = 6 for each time point). (B) Time course of the change of DA efflux from baseline measured from the mPFC in rats subjected to tail-pinch stress (15 min; gray-shaded box). RU-38486 or vehicle is perfused into the LV using reverse microdialysis (horizontal black line). All values are expressed as percentage change from baseline ± SEM (black circles; n = 6, vehicle, black triangles; n = 4, RU-38486). (C) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 90 min after the initiation of stress (*P < 0.05).

GRs Within the mPFC Modulate Stress-Evoked DA Efflux.

To investigate the anatomical site of action for stress-evoked changes in DA efflux, RU-38486 was administered into the VTA or mPFC using reverse microdialysis, and DA efflux was measured in the mPFC. Application of RU-38486 (11.1 ng/μL) into the VTA did not alter stress-evoked DA efflux (n = 7 per condition; Fig. 2A). In contrast, stress-evoked increases in DA efflux were attenuated following reverse dialysis of RU-38486 into mPFC (n = 8 vehicle treatment group, n = 9 RU-38486 treatment group; Fig. 2B). Two-tailed Student t tests of mean AUC (± SEM) revealed no differences in DA levels when RU-38486 was perfused into the VTA (P > 0.05; Fig. 2D), whereas a significant attenuation was observed when this GR antagonist was administered into the mPFC (P < 0.05; Fig. 2E).

Fig. 2.

GRs in the mPFC mediate stress-evoked DA efflux. (A–C) Time course of the change of DA efflux from baseline measured from the mPFC while animals are subject to tail-pinch stress (15 min; gray-shaded box). Using reverse microdialysis, RU-38486 (horizontal black line) is perfused into the VTA (A) or mPFC (B) and lidocaine (Lido) is perfused into the VTA (C). All values are expressed as percentage change from baseline ± SEM (black circles; n = 5–7, vehicle, black triangles; n = 7–9, RU-38486 or lidocaine). (D–F) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 90 min after the initiation of stress (*P < 0.05).

To examine further the action of glucocorticoids in modulating intrinsic activity within the mesocortical DA pathway, DA cell bodies in the VTA were functionally inactivated by reverse dialysis of the sodium channel blocker lidocaine (20 mg/mL). Confirming previous experiments in our laboratory, basal DA levels in the mPFC were reduced significantly by lidocaine (9) (average decrease over the initial 45 min of perfusion: ∼27 ± 3% below baseline, P < 0.05; Fig. 2C). Furthermore, stress-evoked DA efflux in the mPFC was greatly reduced in animals treated with lidocaine compared with vehicle-treated controls (n = 5 vehicle treatment group, n = 8 lidocaine treatment group). Comparison of AUCs generated for the initial 90 min after stress exposure revealed a significant reduction of DA efflux after intra-VTA lidocaine administration (P < 0.05, two-tailed Student t test; Fig. 2F).

Corticosterone Enhances DA Efflux in the mPFC.

We next perfused corticosterone (50 μM) into the mPFC via reverse microdialysis. An immediate and sustained increase in mPFC DA efflux was observed in the corticosterone group (n = 8 per condition; Fig. 3A), as confirmed by a two-tailed Student t test on mean (± SEM) AUC for vehicle- and corticosterone-treated rats over 120 min (P < 0.05; Fig. 3B).

Fig. 3.

Corticosterone acts locally in the mPFC to enhance DA efflux. (A) Time course of the change of DA efflux from baseline measured from the mPFC while corticosterone or vehicle is administered into the mPFC via reverse dialysis. Application of the drug is shown with a horizontal black line. All values are expressed as percentage change from baseline ± SEM (black circles; n = 8, vehicle, black triangles; n = 8, corticosterone). (B) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 120 min after corticosterone perfusion (*P < 0.05).

GRs Within the mPFC Do Not Modulate DA Efflux Evoked by Feeding Behavior.

To determine if the attenuation of DA efflux by RU-38486 was selective to noxious stimuli, we used a food anticipation and consumption task shown previously to induce feeding-evoked DA efflux (10). Presentation and consumption of highly palatable food (Fruit Loops) in the present study caused a rapid and transient increase in DA efflux, with the highest levels observed 20 min after food consumption, returning to basal values after 60 min (n = 7 per condition; Fig. 4A). Administration of RU-38486 (11.1 ng/μL) into the mPFC did not attenuate DA efflux triggered by food anticipation or consumption. RU-38486 had no significant effect on DA efflux evoked by food presentation as measured by comparison of AUCs (P > 0.05, two-tailed Student t test; Fig. 4B).

Fig. 4.

GRs in the mPFC do not mediate DA efflux to an incentive stimulus. (A) Time course of the change of DA efflux from baseline measured from the mPFC during a food anticipation and consumption task (gray-shaded box). RU-38486 or vehicle is perfused into the mPFC using reverse microdialysis (horizontal black line). All values are expressed as percentage change from baseline ± SEM (black circles; n = 7, vehicle, black triangles; n = 7, RU-38486). (B) Histogram depicting mean AUC (± SEM) for the change in DA efflux relative to baseline for 70 min after presentation of food (*P < 0.05).

Blockade of Prefrontal GRs Reverses Stress-Evoked Cognitive Deficits.

Given that optimal DA levels in the PFC are required for the integration of information held in working memory into a prospective plan to retrieve food in a complex environment (11, 12), we hypothesized that an increase in DA efflux arising from acute tail-pinch stress would impair performance on a delayed win-shift (DSWSh) task, and that treatment with RU-38486 would reverse this effect. Furthermore, we hypothesized that performance on a nondelayed variant of the DSWSh task, which is not dependent on the mPFC (13), would not be impaired by exposure to acute stress. To test the first hypothesis, rats were given bilateral infusions into the mPFC of either RU-38486 (100 ng/μL) or vehicle before acute tail-pinch stress during the delay period (Fig. 5A). As shown in Fig. 5B, RU-38486 or vehicle treatment alone had no effect on memory for the correct location of food during the retrieval phase of the task (n = 8 per condition). In contrast, retrieval-phase errors were increased after exposure to tail-pinch stress during the delay period, and this was reversed by infusion of RU-38486 into the mPFC. A two-way repeated-measures ANOVA for the factors of treatment and day revealed a significant main effect of day [F(3, 28) = 14.38, P < 0.05]. Tukey's post hoc analysis confirmed that rats made significantly more retrieval-phase errors after stress exposure compared with the day prior (P < 0.05). Planned comparisons revealed that vehicle-treated animals made significantly more errors after exposure to stress compared with RU-38486–treated or control animals (P < 0.05). Furthermore, animals subjected to tail-pinch stress made significantly more across-phase errors (P < 0.05, two-tailed Student t tests; Fig. 5C). RU-38486 did not alter motor or motivational processes, as all food pellets were consumed, and performance timing (a measure of total time to complete the acquisition phase) did not differ between treatment groups (P > 0.05, two-tailed Student t tests; Fig. 5D). A nondelayed random foraging task was used subsequently to investigate whether acute stress impairs navigation in a spatial environment without knowledge of the location of food (Fig. 5E). Performance on this task was unaffected by exposure to acute tail-pinch stress before the test phase (n = 6), as confirmed by a two-tailed Student t test comparing mean (± SEM; P > 0.05, Fig. 5F).

Fig. 5.

Blockade of GRs in the mPFC reverses stress-induced working memory impairments. (A) Illustration of the DSWSh task. (B) Errors made on DSWSh task after exposure to tail-pinch stress (15 min) compared with errors made the day prior and administration RU-38486 or vehicle into the mPFC (n = 8 for all treatment conditions). (C) Type of errors divided into across phase and within phase. (D) Latencies to complete the acquisition phase. (E) Illustration of the random foraging task. (F) Errors made on the random foraging task after exposure to tail-pinch stress (15 min; n = 6, *P < 0.05).

Discussion

Prefrontal GRs Mediate DA Efflux During Stress.

Our data demonstrate a unique mechanism by which DA efflux is enhanced in the mPFC during acute stress. Administration of the GR antagonist RU-38486 into the LV or mPFC significantly attenuated DA efflux in the mPFC induced by exposure to acute tail-pinch stress. Previous studies suggest that glucocorticoids may alter DA efflux through activation of GRs on DA neurons in the VTA, leading to changes in DA synthesis and neuronal firing (14–17). However, we find no effect on prefrontal DA efflux following acute stress when RU-38486 is administered locally into the VTA. Local application of corticosterone directly into the mPFC did evoke a significant increase in DA efflux, supporting a role for local activation of GRs in the mPFC in regulating DA efflux.

Previous studies investigating the effects of systemic administration of corticosterone on DA efflux in the mPFC have produced conflicting results. Administration of corticosterone via drinking water or s.c. injections increased, decreased, or had no effect on DA efflux (14, 18–21). The present study used reverse microdialysis to administer drugs locally into different brain regions. This approach limited any glucocorticoid secretion that may be triggered by s.c. or i.p. injections (22). It is important to note that rat serum lacks the specific binding protein, α-1-acid glycoprotein, for RU-38486, thereby limiting its diffusion across the blood–brain barrier (23). Accordingly, the concentration of RU-38486 reaching the rat brain after systemic administration is only 28% of the serum levels (23), which may explain why Imperato et al. (24) failed to observe any effect of RU-38486 when examining the role of GRs in stress-evoked DA efflux in the mPFC.

Emerging evidence from the drug addiction field supports the suggestion that glucocorticoids act on GRs on DA terminal regions rather than on DA cell bodies in the VTA. Ambroggi et al. (25) used two mouse models in which GR was specifically knocked out in either DA neurons or postsynaptic neurons expressing the DA D1 receptor to determine which location of GR mediated the effects of glucocorticoids on cocaine self-administration. Deletion of GR in postsynaptic neurons innervated by DA terminals was associated with reduced spontaneous neural activity, firing rate, and frequency of burst events in DA neurons (25). The authors attributed this effect to the disruption of feedback control by postsynaptic neurons in the PFC or nucleus accumbens on VTA DA neurons. Our findings support this mechanism, because functional inactivation of DA cell bodies in the VTA with lidocaine led to a significant reduction in stress-evoked DA efflux. Importantly, it has recently been demonstrated that activation of the DA system during stress is inhibited after infusion of TTX into the ventral hippocampus (26). Brief periods of high-frequency stimulation to the ventral hippocampus lead to a robust and long-lasting increase in DA efflux in the mPFC that is partially blocked by reverse microdialysis of AMPA/kainite receptor antagonists into the mPFC (9). These findings suggest that increased mesocortical DA efflux observed during acute stress may be driven by ventral hippocampus stimulation of mPFC, resulting in increased excitatory drive to the VTA. Glucocorticoids released after acute stress may enhance this glutamatergic transmission in PFC pyramidal neurons through a GR-dependent mechanism (27). Furthermore, the importance of this pathway is further underscored by the recent finding that stress disrupts long-term potentiation in the hippocampal–PFC pathway (28, 29).

Our data demonstrate a localized role of GRs in the mPFC; and although we favor a direct effect on dopaminergic terminals or alternatively on glutamatergic afferents from the hippocampus or amygdala within the PFC, we cannot exclude the involvement of GRs in other brain regions in the modulation of prefrontal DA efflux by glucocorticoids. Lesions to the central and basolateral nuclei of the amygdala inhibit DA efflux in the PFC during stress (30, 31) and also reduce working memory impairments induced by systemic administration or direct injection into the mPFC of a glucocorticoid agonist (32). Glucocorticoids are also implicated in modulating DA metabolism (20, 21). However, neither the enzymatic activity nor gene expression levels of catechol-O-methyltransferase or monoamine oxidase in the mPFC change after treatment with corticosterone (33). More recent evidence suggests that corticosterone can inhibit a high-capacity monoamine uptake transport system, which plays a key role in monoamine clearance (34). Future studies to examine the intracellular mechanisms governing the release of DA by GRs in the mPFC are warranted.

Blockade of Prefrontal GRs Restores Deficits in Executive Function After Acute Stress.

The DSWSh task was used to examine the effect of acute stress in modulating prefrontal-dependent executive function processes. Previous studies from our laboratory using in vivo microdialysis in conjunction with this task have demonstrated that DA efflux in the PFC occurs during both the acquisition (acquiring the information before the delay) and retrieval (using the information following the delay) phases of the task (11). Optimal performance was associated with increased DA efflux during the retrieval phase, whereas inaccurate recall was negatively correlated with the magnitude of DA efflux. This result led us to investigate whether enhanced DA efflux after tail-pinch stress would impair performance during the retrieval phase of this task and whether RU-38486 could restore performance given its ability to reduce stress-evoked DA efflux in the mPFC. Control studies have confirmed our microdialysis probes have a permeability efficiency in the range of 15% to 20%. Accordingly, the continuous perfusion of RU-38486 at a concentration of 11.1 ng/μL, infused through the probe at a rate of 1 μL/min, over 45-min period, would deliver ∼100 ng/μL of drug into the mPFC. To ensure use of a comparable dose in the DSWSh working memory study, injections of a 100 ng/μL dose of RU-38486 were given 30–40 min before the acquisition phase. We found that exposure to acute tail-pinch stress during the delay period impaired accurate recall and importantly, microinfusion of RU-38486 into the mPFC significantly reduced the numbers of stress-induced errors. The effect of RU-38486 was not due to alterations in motor or motivational processes, because response latencies were unaffected. Furthermore, these effects appear to be related specifically to acute stress, because microdialysis data confirmed that elevated DA efflux during anticipation or consumption of food was not affected by administration of RU-38486 into the mPFC.

In contrast to the DSWSh task, performance on a nondelayed random foraging task was unaffected by acute stress, thus providing further insight into the neural circuitry mediating cognitive processes that are affected by stress. Projections from the ventral hippocampus to the prelimbic region of the mPFC are important for performance on the DSWSh task in which foraging is guided by knowledge of the probable location of food (35). Conversely, afferent input from the ventral hippocampus to the nucleus accumbens mediates efficient foraging behavior in the absence of information about the location of food (35). In this context it is noteworthy that administration of hydrocorticosterone to healthy human subjects led to deficits in cognitive processes mediated by the PFC (working memory) but not the hippocampus (declarative memory) (36). Furthermore, blocking D1 receptors in mPFC impairs performance on the DSWSh but not the random foraging task (12). D1 receptors also play an important role in modulating hippocampal-evoked activity of PFC neurons (37), suggesting that stress-induced increase in DA efflux in the PFC may disrupt D1 receptor modulation of hippocampal inputs to the PFC, thereby impairing performance on spatial working memory tasks.

Our data extend previous findings on the impairment of working memory performance by acute stress (1–3). The DSWSh task we used differs in important ways from delayed alternation tasks used in other studies. First, the rat must remember trial-unique spatial information required to form an effective foraging strategy in an eight-arm radial maze (compared with simple alternation between two arms on a T-maze). Second, our task allows a longer delay (15 min) than the 15–30 s in the delayed alternation task. Finally, inactivation of the PFC before the retrieval phase (but not before the acquisition phase) severely impairs performance, confirming a role for the PFC in accessing previously acquired information from short-term memory and its integration into a prospective foraging strategy (13). Therefore, acute stress may impair both the ability to actively retain information over a short delay (as assessed using the delayed alternation task) and/or executive functioning related to memory-based prospective planning as observed on the DSWSh task. Rats subjected to an acute stressor committed mainly across-phase errors consistent with a strong preference to revisit previously baited arms. Therefore, stress may disrupt the ability of the mPFC to suppress responses to familiar stimuli previously associated with reward (i.e., the four-baited arms from the acquisition phase), an effect consistent with disruption of behavioral flexibility (38).

It is important to acknowledge that the impact of glucocorticoids on cognitive processes is complex. Indeed, exposure to a more intense acute stressor can facilitate working memory performance. Yuen et al. (27) report that 20 min of forced swim stress enhanced working memory on a delayed alternation task when tested 4 h and 1 d poststress—an effect attributed to GR-mediated potentiation of NMDAR- and AMPAR-mediated synaptic currents in PFC pyramidal neurons (27). It is worth noting that this study used adolescent, rather than adult, rats. As the impact of acute stress may depend on the developmental stage of the animal (39), further studies examining the effect of acute stress on age-dependent changes in working memory function are warranted. Enhanced glutamatergic transmission allowing for recurrent excitation within PFC pyramidal neuronal networks is essential for working memory (40). Excessive DA levels suppress firing of PFC neurons engaged in a working memory task (41), and therefore, the differential effects of acute stress exposure on working memory may be related to the timing of the stressor relative to the memory task (42, 43). The present study focused on the immediate consequences of acute stress on cognitive function, as this may be a time when individuals are particularly vulnerable to the effects of recent stress. Further support for prefrontal GRs in the disruptive effects of acute stress on working memory performance is provided by a report of working memory deficits on a delayed alternation task after prior microinfusion of corticosterone into the mPFC (44). This effect is also reversed by coadministration of a GR, but not an MR, antagonist. This impairment of working memory may reflect an increased DA efflux similar to that reported in the present study after reverse dialysis of corticosterone into the mPFC.

Stress is a major factor contributing to the development, recurrence, and treatment outcome in affective disorders, including bipolar disorder (45, 46). Pronounced neurocognitive dysfunction is commonly reported in patients with bipolar disorder (47, 48) and may be attributed to alterations in PFC functioning that occurs as a result of interactions between stress and DA function (49). Our preclinical data indicate that prefrontal activation of GRs during acute stress enhances mesocortical DA efflux, resulting in cognitive deficits. Attenuation of this elevation in mesocortical DA by blockade of prefrontal GRs reduced stress-induced cognitive impairment and therefore may represent a unique therapeutic target for treatment of psychiatric disorders. Before chronic treatment with GR antagonists can be contemplated, many factors remain to be clarified given the complex nature of glucocorticoid effects on cognition. Particular attention should be paid to an apparent inverted U-shape function between levels of glucocorticoids and hippocampal-dependent cognitive performance (50, 51). Limited duration of treatment with GR antagonists may be warranted, given our observation of improved neurocognitive function following 7 d of adjunctive treatment with RU-38486 in patients with bipolar disorder (48). Similarly, short-term intervention with RU-38486 has also proven to be beneficial in the treatment for psychotic depression (52). Thus, acute blockade of GRs may represent a novel strategy for treatment of psychiatric disorders for which stress appears to be a significant mitigating factor.

Materials and Methods

Animals.

Male Sprague–Dawley rats (Charles River) were pair-housed except for those subjected to a food restriction protocol. The colony was maintained at 21 °C with a 12-h light/dark cycle (lights on at 7:00 PM). Rats had free access to rat chow and water unless otherwise stated. All experimental protocols were approved by the Committee on Animal Care, University of British Columbia, and conducted in compliance with guidelines provided by the Canadian Council of Animal Care.

Drugs.

Stock solutions were made and dissolved in artificial cerebrospinal fluid (aCSF): RU-38486 (Tocris Biosciences) dissolved in ethanol (EtOH). Corticosterone (Tocris Biosciences) dissolved in DMSO. Neither EtOH nor DMSO exceeded 0.1% when dissolved in aCSF. Lidocaine hydrochloride monohydrate (Sigma-Aldrich) was dissolved in aCSF.

In Vivo Microdialysis.

Rats (∼280–310 g) were implanted with stainless steel guide cannulae (19 gauge × 15 mm) directly above the mPFC (3.0 mm AP and ±0.6 mm ML from bregma, 4.6 mm DV from dura) and/or LV (−0.8 mm AP, ±1.4 mm ML, 4.5 mm DV) and VTA (−5.8 mm AP, ±0.6 mm ML, 8.0 mm DV) (53). One week after surgery, microdialysis probes were implanted 14–16 h before experiments. Probes were concentric in design with silica inlet/outlet lines and a dialysis surface consisting of a semipermeable membrane 1 or 2 mm in length (340 μM outer diameter; 65,000 molecular weight cutoff; Filtral 12; Hospal). Typical in vitro probe recovery of an external DA standard is 18 ± 1% at room temperature. Microdialysis probes were connected to an Instech dual-channel liquid swivel and perfused continuously at 1.0 μL/min with aCSF [10 mM sodium phosphate, 1.2 mM CaCl₂, 3.0 mM KCl, 1.0 mM MgCl₂, 147.0 mM NaCl (pH 7.4)] using a 2.5-mL gas-tight syringe (Hamilton) and a syringe pump (model 22; Harvard Apparatus). Samples were collected at 15-min intervals. Following establishment of a stable baseline of DA (four samples, <10% variability), the perfusion into the LV, VTA, or mPFC was switched to one containing RU-38486, corticosterone, or lidocaine, and DA was continuously sampled from the mPFC. At 45 min after drug administration, animals were subjected to tail-pinch stress (clothespin with Velcro covering on base of tail) for 15 min, and samples were continually collected for another 120 min.

Food Anticipation and Consumption Task.

Rats were food restricted to 90% of their free-feeding body weight. Training took place in a chamber divided by a removable perforated Plexiglas screen as previously described (10). Dialysis samples were collected at 10-min intervals from the mPFC. Once a stable baseline was established, the animals were perfused with RU-38486 directly into the mPFC for 40 min. Three grams of Froot Loops (Kellogg Canada Inc.) was then placed behind the screen for a 10-min appetitive period. The screen was removed, and animals had access to the food for 10 min, after which the remaining food, if any, was replaced by another 3 g of the same food for a total of up to 6 g over 20 min.

HPLC.

DA in microdialysates was separated using HPLC with electrochemical detection. Four systems were used, each consisting of an ESA 582 pump (Bedford), a pulse damper (Scientific Systems Inc.), a Rheodyne Inert Manual Injector (model 7725i, 50-mL injection loop), a Tosoh Bioscience Super ODS TSK column (2-μm particle, 2 mm × 10 mm) an Antec Leyden Intro Electrochemical Detector and VT-03 flow cell with a Ag/AgCl reference electrode (V_applied = +650 mV). The mobile phase [100 mM sodium acetate buffer, 40 mg/L EDTA and 4 mg/L of SDS (variable between 3.5 and 4.5 mg/L), pH 4.1, 10% methanol] flowed through the system at 0.17 mL/min. EZChrome Elite software (Scientific Software) was used to acquire and analyze chromatographic data. The average concentration of basal DA in dialysates from the mPFC uncorrected for probe recovery was 0.16 ± 0.01 nM.

Corticosterone Radioimmunoassay.

Blood samples were taken once from animals used in the microdialysis experiments via tail nick 15, 30, 45, 60, and 75 min after initiation of stress. Basal corticosterone levels were measured immediately after removal of animals from colony room. Samples were taken from the dark phase between 09:00 AM and 1:00 PM. Blood samples were centrifuged at 1,336 × g for 10 min at 4 °C, and plasma was stored at −20 °C until assayed. Total corticosterone (bound + free) levels were measured by radioimmunoassay using a commercially available assay kit (MP Biomedicals) according to the manufacturer's instructions. The antiserum cross-reacts 100% for corticosterone. The minimum detectable corticosterone concentration was 7.7 ng/mL, and the intra- and interassay coefficients of variation were 7.1% and 7.2%, respectively.

DSWSh and Nondelayed Random Foraging Task.

Rats were food restricted to 90% of their free-feeding body weight. Training on both tasks were conducted on an eight-arm radial arm maze as previously described (12, 13, 35) (Fig. 5 A and E).

DSWSh task.

Rats (∼280–310 g) were implanted with bilateral microinjection cannulae (Plastics One Inc.) directly above the mPFC (3.0 mm AP, ±0.6 mm ML, −3.6 mm DV). One week after surgery, animals received one training trial per day consisting of an acquisition phase, a delay period, and a retrieval phase. During the acquisition phase, a novel set of four arms were opened, and upon retrieval of all four food pellets (45 mg; Bioserv) from these arms, animals were retained in the last arm visited and lights were turned off, signaling the delay phase. During the retrieval phase, all eight arms were open, and food pellets were located in the arms that were blocked before the delay. Efficient error-free performance is achieved by visiting each of the four previously blocked arms. Errors were scored as reentries into arms that were previously entered either during the acquisition or retrieval phases. Rats were trained with a 15-min delay, until criterion was reached (one error or less for two consecutive training days). On the test day, animals were randomly preassigned into different treatment groups to receive bilateral infusions (3 μL/min for 20 s; Harvard Apparatus pump) of RU-38486 or vehicle (1% EtOH, pH 5.0) into the mPFC 30–40 min (33.50 ± 0.76) before the start of the DSWSh task. During the delay phase, animals were subjected to acute tail-pinch stress for the entire 15 min or given no stress. The latencies to complete the acquisition phase were recorded to ensure RU-38486 had no effect on motor or motivational responses. Retrieval phase error types were categorized as either across-phase errors (defined as any initial entry into an arm that had been visited during the acquisition phase) or within-phase errors (defined as any reentry into an arm that had been entered in the retrieval phase).

Nondelayed random foraging task.

During each daily session, animals were trained to locate food pellets placed at random in a novel set of four of the eight arms. Animals were trained to a criterion of one reentry error per daily trial for four consecutive days. The following day, animals were exposed to tail-pinch stress in their home cage for 15 min. Immediately after the stress exposure, animals were placed on the maze for the test trial. Errors were scored as reentries into arms entered previously within a trial.

Histology.

Following all experiments, rats were killed after being deeply anesthetized with isoflurane. Brains were promptly removed and stored in 20% wt/vol sucrose and 4% vol/vol paraformaldehyde solution for a minimum of 1 wk. Brains were then sliced into 50 μM coronal sections, stained with cresyl violet, and examined for verification of probe or microinjection cannulae placement (Fig. S1).