How does dopamine signaling affect glutamate release?

Disturbances in effort-related decision-making caused by acute stress and the corticotropin-releasing factor



Acute stress activates numerous systems in a coordinated effort to promote homeostasis and can have different effects on mnemonic and cognitive functions depending on a variety of factors. Stress can alter various forms of cost-benefit decision-making, although the mechanisms that trigger these effects remain unclear. In the present study, we investigated how corticotropin releasing factor (CRF) can contribute to stress-related changes in cost / benefit decisions, using a task where well-trained rats switch between a lever with little effort and little reward ( LR) chose. two pellets) and a high power / high reward lever (HR; four pellets), with the power requirement increasing during a session (2, 5, 10 and 20 presses). One hour of restraint stress significantly reduced the preference for the HR option, but this effect was attenuated by infusions of the CRF antagonist Alpha-Helical CRF. Conversely, the central infusion of CRF mimicked the influence of stress on decision-making, increasing decision latencies and reducing reaction force. CKD infusions did not change the preference for larger or smaller rewards, but did reduce the response to foods given in a progressive ratio. CRF infusions in the ventral tegmental area recapitulated the effect of central CRF treatment and retention on selection behavior, suggesting that these effects may be mediated by disorders of dopamine transmission. These findings underscore the involvement of CKD in regulating effort-related decisions and suggest that increased CKD activity may contribute to motivational and decision-making disorders associated with stress-related psychiatric disorders such as depression.


Acute stress activates numerous systems in a coordinated reaction in order to promote the availability of energy and the adaptive behavior and to return the organism to homeostasis. At the same time, stress has been considered a key factor in a wide variety of psychiatric disorders, particularly depression. Among the many behaviors altered by acute stress, the effects on learning, memory, and cognition have been the subject of extensive research. Learning and memory can be influenced differently by acute stress, depending on a variety of factors such as the context, duration and time of the stressor (Shors et al., 1992; Cordero et al., 2003; Kim et al., 2007; de Quervain et al, 1998; Diamond and Rose, 1994). Similarly, stress can sometimes have adverse effects on certain executive functions mediated by the frontal lobes. It has been shown that working memory and various forms of cognitive flexibility are either impaired (Arnsten and Goldman-Rakic, 1998; Shansky et al., 2006; Arnsten, 2009; Butts et al., 2011, 2013) or increased (Yuen et al., 2006; Arnsten, 2009; Butts et al., 2011, 2013) al., 2009); Bryce and Howland, 2015) through acute stress. Now that it is recognized that stress-related psychiatric disorders are linked to cognitive impairment, understanding the mechanisms by which stress affects these functions can help clarify the relationship between psychopathology and cognitive dysfunction.

Decision-making processes that evaluate the relative costs and benefits of various measures can also be disrupted by acute stress. Studies in humans have shown that different forms of acute stress can lead to adverse or more automated decision-making patterns when choosing between options with different magnitudes and probabilities of receiving rewards (Starcke et al., 2008; Porcelli and Delgado 2009; Pabst et al, 2013). Similarly, studies by our group have shown that acute restraint stress in rats leads to a reliable and significant reduction in the preference for higher rewards, which is associated with higher effort costs and an increase in decision latencies (Shafiei et al., 2012). The observation is particularly interesting given that patients diagnosed with depression have less of a preference to work harder for greater rewards on a back-translated, effort-based decision task (Treadway et al., 2012). It is plausible, therefore, that elucidating the mechanisms by which acute stress can reduce the preference for more preferred yet more expensive rewards provides insight into the pathophysiology underlying motivational deficits and anergies associated with depression.

It is known that acute stress promotes corticosterone release and also improves dopamine transmission in the prefrontal cortex (Jedema and Moghaddam, 1994; Jackson and Moghaddam, 2004; Butts et al., 2011). However, the effects of stress on effort-related decision-making do not appear to be driven by these factors. The pharmacological antagonism of dopamine receptors did not change the effects of stress on exercise choice, and treatment with physiologically relevant doses of corticosterone did not recapitulate the effects of stress on this form of decision making (Shafiei et al., 2012). This indicates that other neurochemical mechanisms activated by stress can influence their effects on the cost-benefit decision. In this regard, corticotropin releasing factor (CRF) is a neurochemical signal that acts centrally to mediate many behavioral effects of acute stress, including stress-induced anxiety, aversion, and drug addiction (Müller et al., 2003; Cador et al. , 1992; Koob, 2010). CRF receptors are widespread in cortical and subcortical regions including the prefrontal cortex, nucleus accumbens and amygdala, and especially in mesencephalic regions including dopamine neurons in the ventral tegmental area (VTA; Van Pett et al., 2000; Bittencourt and Sawchenko, 2000). 2000). Acute stress can stimulate the release of CRF into the VTA (Wang et al., 2005) and the infusion of CRF into the VTA can stimulate both the reward-associated release of mesoaccumbene dopamine and motivation to work for food reward , weaken (Wanat et al., 2013).

The observation that CKD can attenuate the release of mesoaccumbene dopamine is interesting given the overwhelming evidence that intact dopamine function plays a key role in enabling animals to overcome the expense of the effort. The reduction in dopamine transmission, either systemically or within the nucleus accumbens, decreased the preference for the more elaborate option (Salamone et al., 1991, 1994; Floresco et al., 2008) in a manner similar to that of acute stress. With these considerations in mind, the present study was carried out to clarify a possible contribution of CRF transmission to the effects of acute stress on the cost / benefit decision. In particular, we investigated whether (i) blockade of CRF receptors can mitigate the effects of acute stress on decision-making, and (ii) intracerebral administration of CRF mimics the behavioral effects of stress.



Separate cohorts of male Long Evans rats weighing 250-275 g at the start of training were used for all experiments. After one week of colonial acclimatization, the rats were housed individually in the operant chamber and restricted to 85% of their free feeding weight before training began. Water was provided ad libitum for the duration of the experiment. Body weight was monitored daily and rat chow was given every day immediately after the operant chamber training. All testing was conducted in accordance with the Canadian Council of Animal Care and the University of British Columbia Animal Care Committee.


Behavioral tests were carried out in operating chambers (30.5 × 24 × 21 cm; Med-Associates, St. Alban, VT, USA) which were enclosed in a soundproofing box. Each box was equipped with a fan to ensure ventilation and limit extraneous noise. The chamber was equipped with a central food container into which sugar pellets (45 mg; Bioserv, Frenchtown, NJ) were dispensed. There were two retractable levers on either side of the food container. The operant chamber was illuminated by a 100 mA house light located in the top center of the box opposite the food container. The experimental data were recorded by a personal computer which was connected to the working chambers via an interface.

Behavioral tasks

Expense discount

After the initial leverage training (see supplementary methods), various rat cohorts were trained 5–7 days per week in the effort-related decision-making task described above (Floresco et al., 2008; Ghods-Sharifi and Floresco, 2010). Each 32-minute daily training session consisted of 48 attempts, which were divided into four blocks. Experiments were started at intervals of 40 s by illuminating the house light and 2 s later by extending one or both levers. Each of the four blocks began with two forced voting attempts in which only one of the two levers was randomly extended. The remainder of the experiments were a free choice experiment in which both levers were presented and the rats chose between the two options. For all sessions and blocks, one lever was set as the Low Reward Lever (LR) and the other lever was set as the High Reward Lever (HR) (counterweight). After the levers were presented, the rats had to press one of the levers within 25 seconds of insertion to give an answer. Failure to respond to any of the levers was counted as a failure when the operant chamber returned to an intermediate state. Selection of the LR lever caused both levers to be withdrawn and the rat received two pellets. Conversely, if the rat selected the HR lever, only the LR lever was withdrawn and the HR lever remained extended until the rat had made the required number of presses to obtain four pellets or 25 seconds had passed since insertion. The number of presses required increased compared to the subsequent blocks, with the requirement initially set to 2 presses, and increased to 5, 10 and 20 presses for subsequent blocks. The house light remained lit for a further 4 s after the final pellet had been dispensed, after which the chamber was put into the test state. In the rare event that a rat did not complete the required number of presses within 25 seconds, the lever was withdrawn, no food was dispensed, and the chamber returned to the test condition. However, the choice of the rat continued to be included in the analysis. Other metrics included in the analysis were choice latency (the time between lever extension and choice) and the frequency of pressing the HR lever.

The rats were trained on the task until they, as a group, (i) selected the HR lever during the first test block in at least 75% of the freely selectable tests and (ii) showed stable basic discount levels on three consecutive days. Stability was analyzed using 3 × 4 repeated measures analysis of variance (ANOVA) with training day (3) and trial block (4) as subject internal factors. The animals were judged to achieve stable selection behavior when there was no major effect of the day or day × study block interaction (p> 0, 10).

Differentiation of reward sizes

A separate cohort of rats received initial lever press training, after which they were trained on easier, more rewarding size discrimination. In this task, the cost of the two levers was equated, so that a single press of the LR lever delivered two pellets and one press of the HR lever delivered four pellets. Each session consisted of 48 attempts (40 s interval between attempts) with 12 attempts per block (2 forced attempts followed by 10 free attempts). The rats were trained for 9 days and at the end of this period before the drug test showed a strong preference for the HR lever (~ 90%).

Progressive Ratio Tests

A separate cohort of animals was trained to operate a single lever on food dispensed on a progressive ratio plan. During the daily training sessions, the left lever in the operant chamber remained extended. During the initial training, responses were reinforced on an FR1 schedule for 2 days, followed by one day on an FR2 schedule and two days on an FR5 schedule. A closed relationship yielded a pellet. Next, the rats were trained on the progressive ratio scheme in which the number of presses required to obtain a pellet increased exponentially. The ratio was derived from that of Brown et al. (1998) and increased as follows: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 693, 737 and 901 presses. The rats were given a maximum of 20 minutes to complete each ratio and receive a reward. If a relationship was not completed within the specified time, the session ended. The main variables of interest were: (i) the total number of lever actuations during a session and (ii) the last ratio obtained before a session ended (breakpoint). The program also recorded the time intervals between the dispensing of each pellet and these values ​​were divided by the number of responses required to obtain that pellet to produce an average response rate for each ratio. Training was continued on this task for 10 days until the rats exhibited stable levels of lever pushing and hold-ups for three consecutive days as a group (ie, less than 15% variation within the group).


The rats were anesthetized using ketamine (100 mg / kg, IP) / xylazine (10 mg / kg, IP) and treated with an analgesic (Anafen, 10 mg / kg, SC) prior to surgery. The majority of the animals in this study were implanted with a unilateral cannula directed 1 mm dorsal to the right lateral ventricle (coordinates, flat skull: AP: -1.0 mm from the bregma; ML: -1.8 mm; DV: - 2.5 mm from the dura). Another group of rats were implanted with bilateral cannulas 1 mm dorsal to the VTA (coordinates: AP: -5.5 mm; ML: 0.7 mm; DV: -7.0 mm). Dental acrylate adhered to four stainless steel skull screws that held the cannula in place. Stainless steel obdurators that are flush with the end of the guide cannula were inserted after the operation. Postoperative procedures included daily weighing and subcutaneous analgesic administration for at least 2 days after surgery. The rats were given approximately 1 week to recover from the surgery before behavior training began (again). Rats trained for the effort reduction task were implanted after the initial training, while rats trained for tasks that require fewer sessions to achieve stable performance (i.e., reward size discrimination and progressive ratio test) were implanted before training were.

Drug and micro-infusion procedures

In a first experiment it was tested whether the effects of acute stress on decision-making could be attenuated by intracerebroventricular (ICV) administration of the unspecific CRF antagonist alpha-helical CRF (9–41; Tocris Bioscience). Previous studies have shown that ICV infusions of this compound negate the effect of acute stress on various behavioral measures at doses ranging from 5 to 50 µ. g (Krahn et al., 1986; Kalin et al., 1988; Berridge and Dunn, 1989; Cole et al. 1990; Nawata et al., 2012). In the present study, we infused 30 μg / 4 μl dissolved in distilled water.

Other experiments investigated whether central infusions of CRF could mimic the effects of acute restraint stress. In a first study, the effects of ICV infusions with three different CRF doses (rat / human; Tocris Bioscience; 0, 25, 1 and 3 μg) on ​​the effort reduction were examined. Previous experiments have seen changes in behavior following ICV infusions ranging from doses as low as 0.1 to 10 µ. g infused into the ventricular system (Dunn and Berridge, 1990; Cador et al., 1992; Adamec and McKay, 1993; Campbell et al., 2004; Van't Veer et al., 2012).Since the maximum solubility of CRF in artificial cerebrospinal fluid is 1 μg / 1 μl, the infusion volume for the 3 μg dose was set at 3 μl, while the volume for the other two doses was 1 μl. There were no significant differences in performance after 1 μl vs. 3 μl vehicle infusions (p> 0.05), so the data obtained after treatment with each of the three vehicle doses were averaged for the analyzes. Subsequent studies using CRF infusions with ICV only used the 3 µg dose. G. In another experiment, CRF was infused directly into the VTA (0.5 µg / 0.5 µl). This dose was determined with reference to the study by Wanat et al. (2013), which showed that it was effective in modifying sucrose response on a progressive ratio scheme.

Experimental microinfusion procedures

For the experiment with acute restraint stress and one-sided ICV infusion of alpha-helical CRF, the rats were trained on the task of reducing effort until they showed stable initial values ​​for voting behavior on three consecutive days. After recovery from surgery and retraining for stability, all cohorts of rats were given a sham infusion prior to the days of the microinfusion test. Here the injector was inserted into the guide cannula for the same duration as the infusion, but no infusion was administered. The rats were then exposed for the first of four days of testing: (i) vehicle / no stress; (ii) vehicle / restraint stress; (iii) Alpha Helix CKI / no stress, and; (iv) Alpha-Helix CNI / Retention Tension. On the test days, an infusion volume of 4 .mu.m was provided using a 30-gauge injector that extended 0.8 mm beyond the end of the guide cannula. l delivered over 3 min 20 s. The injector was attached to a hose attached to a microsyringe pump. The injector was left for an additional minute to ensure diffusion.

The days without stress and stress test took place as part of a two-day sequence, with the order of the infusion types in the rats being balanced. Therefore, on the first day of the sequence, the rats received vehicle or alpha-helical CRF infusions, returned to their home cages for 10 minutes, and were placed in a quiet room (no exposure condition) for 1 hour before being placed in the working chambers Testing. On the second day, the rats received the same infusion as the day before, returned to their home cage for 10 min, after which the rats were held in a plexiglass cylinder (83 × 133 × 197 mm; Harvard Apparatus, Massachusetts, USA) for 10 min for 1 h same space as the stress-free state. A desk fan was directed to the holders and was used to reduce the risk of hyperthermia. The length of the restraint system was adjusted to immobilize the rat without pain. After 1 hour of restraint, the rats were returned to their home cage, where they were undisturbed for 10 minutes, followed by placement in the operant chamber for testing. After this two day test sequence, the rats were trained back for stability (approximately 5 days) and then received the balanced infusion according to the same protocol. Therefore, rats that previously received alpha-helical CRF now received vehicle infusions and vice versa.

For the ICV central CRF experiments, all groups of animals were trained for stability and given a sham infusion (as described above) prior to testing. The central CRF infusions were either in a volume of 1 µg. l over 1 min 45 s or in a volume of 3 μl l administered over 3 min 34 s. The injector was left in place for an additional minute to allow diffusion. On the first day of a two-day test sequence, the rats were given a vehicle infusion and returned to their home cage for 10 minutes before behavioral testing. The following day the rats received a dose of CRF followed by 10 minutes in their home cage prior to the behavioral tests. In experiments with balanced infusions with several doses of CRF, the rats were retrained after the first test sequence until a stable initial behavior was achieved (2-5 days) before a further test sequence of the balanced infusions was received.

Intra-VTA infusion experiments were performed in a similar manner. Once stability was achieved, the rats were subjected to a sham infusion in which two injectors were inserted into the bilateral guide cannula for the same duration as the infusion. However, no infusion was given. On test days, infusions in a volume of 0.5 μl were administered over 1 min 15 s. After sham infusions, one group of rats received a bilateral vehicle infusion on the first day and another group a CRF infusion (0.5 μg). The rats were returned to their home cage for 10 minutes prior to the behavioral test. After 1-7 days of retraining, the rats received a balanced infusion of vehicle or CRF.


After testing, the rats were given CO 2 killed and the brain removed and fixed in a 4% formalin solution. The brains were at 50 µ m cut, mounted on gel-coated microscope slides and Nissl-stained with cresyl violet. Photomicrographs of representative examples of accurate and inaccurate placement in the lateral ventricular cannula are shown in Figures 1a and b. Figure 1c shows the exact placement of the cannulas in the VTA. Data from animals with placements either lateral or dorsal to the ventricle or VTA were removed from the analyzes.

Histology. (a) Accurate placement of the unilateral lateral ventricular cannula (ICV). (b) Missing placement of the unilateral lateral ventricular cannula (ICV). (c) Illustration of the exact placement of the VTA cannula.

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Data analysis

The main dependent variable obtained from the cost discounting task was the ratio of HR lever choices for each block, taking experimental omissions into account. For this measure, the ratio of the number of HR decisions divided by the total number of attempts in which a decision was made was calculated. Additional measures included the frequency of pushing the lever on the HR lever, the dial delay, and omission of attempts. The selection data for the CRF antagonist experiment were analyzed using an ANOVA of 4 (treatment) x 4 (block) replicate measurements. Selection data for intracranial CRF infusion experiments were analyzed using ANOVAs with two-way replicate measurements, with dose and block as factors within the subject. One- or two-way replicate ANOVA measurements were used to analyze the other performance measurements. If necessary, several comparisons were made with Dunnett or Tukey tests (//

For the Progressive Ratio experiment, the main dependent variables were the total number of lever presses and the hold-up point (the last ratio obtained); These were compared with one-way ANOVAs. Lever pressure rate was analyzed using a two-way indoor subject ANOVA with treatment and ratio (the first four ratios all animals achieved after both treatments and the rate during the last ratios achieved for each animal) as the inner ratio Factors.


Alpha-helical CRF improves the impact of stress on tedious decision-making

In our first experiment we tried to replicate the effects of 1 hour of acute restraint stress on the choice of effort and to investigate whether the central CRF activity mediates this diversion of the preference behavior. Nineteen rats were tested on the effort reduction task with data removed from two animals due to missed placements (final n = 17). Analysis of the selection data revealed a significant main treatment effect (F (3, 48) = 2.81, p = 0.05), but no significant treatment × block interaction (F (9, 144) = 1, 23, ns). As shown in Figure 2a and b (left), acute stress resulted in a significant decrease in the choice of HR compared to the no-stress conditions (Tukey's, p <0.05). In addition, the performance after vehicle or alpha-helix CRF treatments without stress conditions was comparable (p> 0.90; Figure 2a, circles vs. triangles). Of particular interest, however, is that the preference for the HR option after treatment with alpha-helical CRF / stress does not differ significantly from the conditions for vehicle / no stress (p> 0.60) or alpha-helical CRF / no stress ( p> 0.90) difference. Figure 2a and b, right), although in these post-hoc analyzes the difference between the two stress conditions (vehicle vs. alpha-helical CRF) was also not statistically significant (p <0.40). Based on the observation that acute stress alone significantly reduced the preference for the HR option compared to the corresponding stress-free condition, while stress + alpha-helical CRF did not, we interpret these data to mean the effects of acute stress to be implied on effort -Discounting were weakened by CRF antagonism.

The CRF receptor antagonism blocks the effects of acute restraint stress on effort reduction. (a) The ordinate shows the percentage choice of the HR lever over the four test blocks and the abscissa shows the four test blocks with increasing effort. One hour of reluctance after infusing the ICV vehicle decreased the choice of HR option across all experimental blocks in relation to the vehicle's conditions / no stress (stars mean p <0.05, Tukey tests). However, the infusion of alpha-helical CKI attenuated the stress-induced reduction, with the HR option being preferred. (b) The same data as in (a) were recorded for clarity to highlight the stress-related decrease in HR option choice after vehicle infusions (left) and the relative lack of choice after treatment with alpha-helical CKD (right) . (c) Elective latency across the four treatment conditions and four experimental blocks. (d) The rates of pressure on the HR lever did not differ between the different treatment conditions.

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After vehicle infusions, the restraint voltage increases the dial latency compared to the no-stress conditions (t (16) = 2.46, p <0.05). However, the overall analysis of these data did not reveal any significant differences between the various conditions for this measure (main treatment effect: F (3 48) = 2.33, p = 0.086; treatment × block interaction F (9 144) = 0.68, ns Figure 2c). Likewise, there were no differences between the treatments with regard to the lever pressure on the HR lever (F (3, 48) = 0, 67, ns; Figure 2d) or experimental omissions (F (3, 48) = 1, 34, ns ; Range of group means = 0, 4–5, 3). Taken together, these results show that acute stress lowers the preference for the "high effort" / "high reward" options and redirects the choice to the "low effort" / "low reward" options. However, this effect was attenuated by prior administration of a CRF antagonist, suggesting that the ability of acute stress, the tendency to work harder for greater reward, is due, at least in part, to an increase in central CRF transmission .

The central CRF infusion mimics the acute stress in terms of choice, which is related to the effort

After uncovering a key role for CKD in mediating the effects of stress on cost / benefit decisions, we next examined whether central CKD administration could mimic the effects of acute stress on effort reduction. We tested the effect of three doses of CRF (0, 25, 1 and 3 μg) dissolved in either 1 μl or 3 μl of artificial cerebrospinal fluid in 17 rats. Data from four of these rats were removed from the analyzes due to missing cannula placements (final n = 13). The performance after 1 vs 3 μl vehicle infusions did not differ (F (2, 24) = 1, 19, ns), therefore the data from all three vehicle infusion days were combined for the analysis. Analysis of the selection data revealed a significant main treatment effect (F (3, 36) = 8, 36, p <0.001; treatment × block interaction (F (9, 108) = 1, 37, ns; Figure 3a). Multiple Comparisons with Dunnett's tests confirmed that the doses of 0, 25 μg or 1 μg did not change the choice in relation to the vehicle, but the higher CRF dose (3 μg) significantly reduced the choice of the HR option (p <0, 01) of the CNI increased electoral latency across all trial blocks (main effect of treatment: F (3, 36) = 4, 79, p <0, 01 and Dunnett's, p <0, 01; treatment × block interaction: F (9, 108) = 1, 43, ns; Figure 3b) and decreased lever depression rates (F (3, 36) = 30, 95, p <0.001 and Dunnett's p <0.05, Figure 3c) the 3-μg dose also increased the test omissions (9, 7 ± 2, 8) compared to vehicle (1, 0 +/- 0, 6), whereas the other two doses were not (0.25 µg = 0.1 + / 0.1 ; 1, 0 µg = 0, 4 +/- 0, 2; F (3, 36) = 10, 15, p <0.001 and Dunnett's, p <0.05). Taken together, these data suggest that the administration of exogenous CKD decreases the preference for animals to work harder for greater rewards, increases latency to make choices, and also decreases responsiveness.

The central CRF infusion mimics the effect of acute restraint stress on the effort reduction. (a) ICV infusions with a CRF dose of 3 μg reduced the preference for the HR option compared to the vehicle infusion, whereas the two lower CRF doses had no influence on selection behavior (0, 25 and 1 μg). (b) The 3 μg CRF dose increased the elective latency compared to the vehicle infusion in all four experimental blocks. (c) Following the 3 μg dose of CRF, the rates of lever depressions on the HR lever were also decreased. Stars indicate p <0.05 compared to vehicle.

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The central CRF infusion does not interfere with the distinction between the reward sizes

To further clarify the way in which increased CRF activity affects decision making, we performed a control experiment using reward size discrimination to determine whether these treatments produced a more fundamental deficit in preferring larger or smaller rewards. A separate group of nine rats was trained in a simpler decision-making task where the cost of the two levers were equated so that a single press of the HR or LR lever provided four or two reward pellets, respectively. As shown in Figure 4a, the ICV infusion of 3 µg has g CRF did not significantly change decision-making in this simpler task (F (1, 8) = 0.86, ns). Interestingly, these treatments increased dial latencies significantly (main effect of treatment: F (1, 8) = 8, 76, p <0.05; treatment × block interaction: F (3, 24) = 2. 50, p = 0.08; Figure 4b) a manner similar to the effect of reluctance to perform this task (Shafiei et al., 2012). However, experimental omissions were not affected (vehicle = 0; CRF = 1, 4 +/- 0.8; t (8) = 1, 77, ns). Increasing central CRF activity does not affect the preference for larger versus smaller rewards at the same cost, nor does it interfere with spatial discrimination skills. However, as observed in the effort discounting experiment, these treatments increased the latencies to take actions that would bring a reward.

The central infusion of CRF does not affect reward size discrimination, but it does reduce the motivation to advocate for the reward in a progressive ratio reinforcement plan. (a) CRF did not change the preference for the larger versus the smaller rewards during a reward size differentiation if there were no additional costs involved. (b) Response latencies averaged over the four test blocks. The CRF infusion increased the latency to make a choice. (c) The ICV-CRF infusion (3 μg) reduced the total number of lever pushes performed during the progressive ratio abandonment compared to the vehicle infusion. (d) The infusion of CRF (3 µg) decreased the break point / total number of reward pellets received during the session. (e) The number of presses / s (rate) for the first four gear ratios and the last gear ratio obtained. The CRF infusion did not affect the rate during the lower ratios or the last ratio obtained, but CRF decreased the squeeze rate during the higher ratios. Stars denote p <0.05.

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Central CRF infusion and instrumental response on a progressive ratio plan

An increase in central CRF activity did not change the subjective value of objectively higher rewards. However, the possibility remained that increased CRF activity may have impaired motivation to exert greater effort in order to receive a reward. To remedy this, we tested the effects of infusing CRF on instrument response to food delivered using a progressive ratio reinforcement regimen, a common motivation assay for rodents. In well-trained rats (n = 8), ICV administration of CRF (3 μg) reduced the total number of lever presses (F (1, 7) = 17, 86, p <0.01; Figure 4c) and the break point (or the last ratio reached); F (1,7) = 19,60, p <0,01; Fig. 4d) relative to the performance after the vehicle.

To analyze lever compression rates, we took into account the fact that most rats had a lower knee point after CKD infusions compared to control treatments. As such, we analyzed the rate according to the ratio completed for all rats under both conditions (vehicle and CRF infusion), which in this case was the fourth ratio (six presses required), as well as obtained the press rate for the last ratio . CRF infusions reduced response rates (main treatment effect: F (1, 7) = 13, 38, p <0.01; treatment interaction × ratio: F (4, 28) = 12, 68, p <0.001) . A simple analysis of the main effects showed that rats exhibited comparable lever pressure rates in the first two session ratios, but reacted more slowly in the later ratios after CRF treatments compared to the vehicle (p <0.05; 4e). Note that while trying to reduce effort, the same dose of CRF resulted in both a decrease in lever pressure and an increase in latency to elicit a response. Therefore, it is possible that the effects of CKD on the average response rates in the Progressive Ratio experiment were in part also caused by an increase in the latency period for the re-initialization of the reaction after consuming a food pellet. Taken together, these data suggest that infusions of CRF reduce the motivation to react in high ratios in order to receive a reward for food.

Intra-VTA-CRF infusion and expense discount

Our finding that ICV infusions of CRF postpone the selection of high-cost, high-reward options prompted us to investigate the potential brain nuclei in which CRF may mediate these effects. For this purpose, we focused on the dopamine cell body region in the VTA, since (i) the VTA expresses CRF receptors (Van Pett et al., 2000), (ii) CRF is released into the VTA during episodes of acute stress (Wang et al (2005) and (iii) dopamine plays a crucial role in promoting the selection of larger, more expensive rewards (Salamone et al., 1991; Sokolowski and Salamone, 1998; Aberman and Salamone, 1999; Salamone et al., 1994; Cousins And Salamone) 1994; Floresco et al. 2008). Eleven rats were trained and tested in the task of reducing effort. two were removed due to cannula placements outside the VTA (final n = 9). Intra-VTA administration of CRF (0.5 μg) significantly reduced the choice of the HR option compared to vehicle infusion (F (1, 8) = 7, 56, p <0.05; Figure 5a). Interestingly, these treatments had no effect on dial-up latency (main effect of treatment: F (1, 8) = 0.32, ns; treatment × block interaction: F (3, 24) = 0.51, ns; Figure 5b) or omissions ( Vehicle) = 0.1 +/- 0.1; CRF = 1.1 +/- 0.2; F (1, 8) = 2, 17, ns). Intra-VTA CRF treatments caused a slight decrease in lever compression rates as seen after ICV administration of this compound, but analysis of these data only revealed an effect that approached statistical significance (F (1, 8) = 4, 57, p = 0, 07; 5c).

The intra-VTA CRF infusion mimics the effect of acute restraint stress and central CRF infusion on the effort reduction. (a) Intra-VTA CRF infusion (0.5 μg) decreased the preference for the HR option compared to the vehicle infusion. (b) Election latencies across the four test blocks. There was no effect of intra-VTA-CRF on election latency. (c) The response rates to the HR lever were averaged over all four blocks. Intra-VTA-CRF caused a minor but insignificant effect on the reaction force rate. Stars denote p <0.05.

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This set of experiments provides strong evidence that CRF mediates stress-related changes in effort-related decision making. An hour of stress reduces the preference for the higher, more expensive reward, but this effect was attenuated by previous administration of the CRF antagonist Alpha-Helical CRF. In addition, centralized management of CRF itself reduced the preference for larger, more costly rewards, but did not affect the preference for larger and smaller rewards at the same cost. On the other hand, the CRF administration reduced motivation to work for a reward, which is reflected in a reduced response to a reward under a progressive ratio plan. Finally, changes in the assessment of the subjective cost costs seem to involve CRF, which act in the VTA as a CRF infusion into this cell nucleus, and to recapitulate the selection behavior profile that is caused by both acute restraint stress and central CRF infusion.

Stress, cost / benefit decision making and CRF

Earlier work from our laboratory showed that 1 hour of restraint stress significantly reduced the preference for larger rewards, which are associated with higher effort costs (Shafiei et al., 2012). It is well known that this form of acute stress increases circulating corticosterone levels and also increases dopamine outflow in regions such as the prefrontal cortex and amygdala (Imperato et al., 1991; Jackson and Moghaddam, 2004; Del Arco et al., 2015 ). In an attempt to elucidate the neurochemical mechanisms underlying this effect, however, we found that systemic treatment with corticosterone at doses that produced plasma levels comparable to those produced by stress failed to recapitulate this effect . Similarly, stress-induced voting behavior disorders do not appear to be due to increased dopamine transmission, since pretreatment with the dopamine antagonist flupenthixol could not block the effect of acute stress on this measure (Shafiei et al., 2012). We now show that the CRF transmission plays a fundamental role in conveying the stress-related changes in the preference for the choice of effort, since the pretreatment of rats with the CRF antagonist alpha-helical CRF significantly weakened the effect of restraint stress, while this drug had no effect on behavior on its own.

The above-mentioned findings were supplemented by a subsequent experiment in which it was investigated whether the administration of exogenous CRF is sufficient to reorganize effort-related decision-making, similar to stress through restraint. High (3 μg) but not lower (0, 25 μg or 1 μg) doses of CRF in the lateral ventricle significantly reduced the preference for the HR option during the effort reduction. This dose-response effect differs somewhat from previous studies in which it was reported that lower doses of CRF can alter behavior in a number of anxiety assays (Campbell et al., 2004; Dunn and Berridge, 1990). The fact that only the 3 mcg dose was effective suggests that more complex behaviors may only be prone to greater increases in circulating CKD compared to relatively simpler or spontaneous behaviors associated with anxiety, desperation, or social inquiry. Taken together, these results indicate that CKD in the context of acute stress influences the subjective assessment during effort-related decision-making and modifies subjective selection bias in the direction of lower costs and yet lower rewards.

The increase in central CRF transmission activates the HPA axis and promotes the release of corticosterone. An increased corticosterone level can in turn contribute centrally to the modulation of various forms of learning and knowledge. With this in mind, we believe it is unlikely that the effects of CRF treatments on decision-making were mediated by increased corticosterone release. As discussed above, administration of exogenous corticosterone did not affect the discounting of effort (Shafiei et al., 2012). In addition, previous studies have shown that a broad dose range of centrally infused CRF (0.1–10 μg) activates the HPA axis and increases plasma corticosterone levels to a similar extent (Campbell et al., 2004; Cador et al., 1992). In comparison, only the 3 μg dose of CRF changed the selection behavior in the present study, whereas the treatment with the lower dose, at which an increase in the plasma corticosterone level was also to be expected, had no effect. Therefore, it is more likely that the mechanisms underlying these effects of increased CRF transmission are independent of their effects on activation of the HPA axis and increases in plasma corticosterone levels.

Cognitive / motivational changes caused by increased CRF activity

Numerous cognitive and / or motivational processes can be impaired by increased CRF activity, which could deviate the choice from the larger, more costly reward. For example, these treatments may have altered processes related to the objective evaluation of various rewards and result in a more general impairment of the preference for larger versus smaller rewards, as observed after inactivation of the nucleus accumbens shell or reduction in GABA transmission in the prefrontal cortex (Stopper and Floresco, 2011; Piantadosi et al., 2016). To remedy this, we used a reward size differentiation that equates the cost of the two leverage options. Here, the central infusion of CRF had no effect on the selection of objectively larger rewards, a zero result similar to that observed after acute restraint stress (Shafiei et al., 2012). This lack of efficacy also suggests that it is unlikely that changes in behavior induced by CKD infusions are due to changes in satiety (Stopper and Floresco, 2014) or other non-specific impairments. More fundamental reward processes that influence the choice between larger and smaller rewards are therefore left relatively unaffected by increased CRF activity.

On the other hand, increased CRF activity may have altered subjective assessments of expense costs and may have impaired motivation to work for rewards or the willingness to respond in high ratios. To counter this possibility, we tested how CRF administration affects responding to rewards provided on a progressive odds schedule. The central infusion of CRF significantly reduced the motivation to work for rewards here. A comparison of these results with the data from efforts to reduce effort suggests that increased CRF activity disrupts motivational processes that promote sustainable behavioral patterns that are necessary to overcome the effort costs that separate an organism from the desired rewards. Consistent with this idea, CRF infusions also reduced the rate of pushing the lever across tasks, especially when response demands were high, presumably due to an increased tendency to detach from the lever for long periods of time and / or react again. Rather than reducing the incentive value of larger rewards, increased CRF transmission appears to increase the perceived overhead cost that may be required to obtain it. Alternatively, it could be argued that CKD or acute stress adversely affect numerous motivational aspects in a non-discriminatory manner, which in turn changes effort-related decision making and the response to the progressive ratio. However, it is unlikely that this would explain the entire behavioral profile described in this study. If so, these manipulations would be expected to change behavior on any task that requires goal-directed behavior, including reward size discrimination. Unlike other manipulations that result in a more general decrease in motivation (e.g. prefeeding before a test session (Stopper and Floresco, 2014)), treatment for CKD or stress did not change the preference for larger or smaller rewards at the same cost (Shafiei et al., 2000). 2012). In this respect, we think it is more likely that these manipulations change the motivational processes, which contribute to stimulating behavior and enabling an organism to make greater efforts to achieve a goal, a little more selectively.

It should be noted that while infusions of CRF and 1 hour restraint stress in our hands resulted in comparable changes in decision making, similar restraint stresses did not affect foods dispensed on the progressive ratio scheme (Shafiei et al., 2012) . In this regard, it has been reported that different types of stressors have different effects on progressive relationship response. Wanat et al. (2013) reported that 20 minutes of restraint stress combined with intra-VTA vehicle infusions reduced holdover points from baseline in a two-lever task where the active lever between sessions and the reinforcement plan was more liberal than that used here. In contrast, treatment with the pharmacological stressor yohimbine increased the breakpoints and this effect was caused by a CRF 1- Antagonists weakened (Liu, 2015). It is interesting to note that chronic corticosterone exposure significantly decreases the hold-up point and number of lever presses in a progressive ratio task in a manner similar to the central CRF infusion described here (Olausson et al., 2013). Therefore, the dose of CRF that was effective in the present study may be more similar to changes in CRF activity induced by more intense or chronic stress manipulation compared to 1 hour of restraint.

CRF infusions not only influenced voting behavior and response rates, but also led to reliable increases in decision latencies, which paralleled the previously reported effects of acute restraint stress (Shafiei et al., 2012). Increased deliberation times after CKD infusions or acute stress were not only evident in more complex decisions in which the expense costs were assessed, but also in a relatively simpler discrimination of the reward size. In this latter case, these manipulations did not affect voting behavior, suggesting that the mechanisms by which increased CRF transmission alters election latencies are inseparable from those involved in biasing voting direction. It has been reported that central CRF infusions in particular increase election latency during attention tests (Van't Veer et al., 2012; Beard et al., 2015). These results suggest that the ability of acute stress to induce "indecision" and increase processing times for action selection is also mediated, in part, by increased central CRF transmission.

CRF actions in VTA and effort-related decision-making

In a final experiment, we tried to identify neural loci where the CRF might alter motivation in relation to exercise-related decisions. To this end, we targeted the midbrain dopamine neurons in the VTA, as dopamine has a known role in promoting the preference for larger, more expensive rewards (Salamone et al., 1991; Sokolowski and Salamone, 1998; Salamone et al ., 1994); Cousins ​​and Salamone, 1994; Denk et al., 2005; Floresco et al., 2008). CRF is released in the VTA during episodes of acute stress (Wang et al., 2005) and previous results suggest that stress-induced motivational disorders are mediated through activation of CRF receptors in the VTA (Wanat et al., 2013). Intra-VTA-CRF lowers the breakpoint in a progressive ratio task in a similar way to ICV-CRF infusions in the present study, and the blockade of VTA-CRF receptors improves the motivation deficits caused by acute stress (Wanat et al., 2013).Consistent with the above results, we found that infusing CRF into the VTA significantly reduced the preference for the higher, more costly reward in the effort reduction task. The fact that acute restraint stress, ICV or intra-VTA CRF infusion all produced a similar effect on the choice of effort provides strong evidence that increased CRF release within the VTA due to stress-related events increases the cost / benefit - Can influence decision.

The question remains, how can CRF modulate the activity of VTA dopamine neurons to alter decision-making? CRF obtained from external sources, including the bed core of the stria terminalis (Rodaros et al., 2007; Vranjkovic et al., 2014) or within the VTA itself (Grieder et al., 2014), act in complex, often contradicting ways and may increase or decrease the excitatory and / or inhibitory transmission in the VTA by numerous mechanisms. In vitro studies have shown that CRF 1 Receptors act presynaptically on glutamatergic terminals to increase the glutamatergic drive on dopamine neurons (Williams et al., 2014) and post-synaptic actions to increase the EPSCs and trigger rates of dopamine neurons (Wanat et al., 2008)). CRF 2 Receptors, in conjunction with CRF-binding protein, can also facilitate NMDAR-mediated synaptic transmission to dopamine neurons (Ungless et al., 2003). These results complement reports suggesting that increased CRF activity may promote terminal dopamine release (Lavicky and Dunn, 1993; Holly et al., 2015). In contrast, there is evidence that the activation of VTA CRF 2 -Receptors can increase GABA release, which acts on both postsynaptic GABA-A receptors and presynaptic GABA-B heteroreceptors, which are located on glutamate terminals to decrease glutamate release (Williams et al, 2014) . This latter effect is expected to decrease the burning of dopamine neurons. Taken together, these studies demonstrate the complexity with which various CRF receptors act to shape the neuronal excitability of VTA.

While more study is needed to clarify the exact mechanisms by which CRF modulates the activity of VTA dopamine neurons, the fact remains that ICV or intra-VTA infusions of CRF make effort decision-making in a similar fashion to that Dopamine antagonism either systemically or in a similar way alter the nucleus accumbens (Salamone et al., 1991, 1994; Floresco et al., 2008; Nowend et al., 2001; Farrar et al., 2010). This similarity suggests that, despite the complex CRF effects in the VTA, the net effect of improved CRF transmission in these situations may be to decrease the activity of mesoaccumbens dopamine. In support of this idea, dopamine release in the nucleus accumbens was attenuated by intra-VTA CRF infusion elicited by rewards according to a progressive ratio scheme, while the phasic dopamine responses to predictive cues were unaffected, highlighting how activation of VTA-CRF receptors in restrained animals can change the release of mesoaccumbene dopamine in a stimulus-specific manner (Wanat et al., 2013). Conversely, a recent report by Twining et al. (2015) that the administration of a CRF antagonist to the VTA reversed the aversive flavor-induced reduction in dopamine transmission in the nucleus accumbens. Similarly, ICV administration of a CRF increases 1- Antagonists the activity of dopamine neurons and the release of mesoaccumbens dopamine, suggesting a tonic inhibitory role of CRF on the activity of VTA dopamine neurons (Lodge and Grace, 2005). Taken together, these earlier findings suggest that CRF in the VTA may, at least in some cases, act stimulus specifically to decrease dopamine release, possibly by reducing glutamate and / or increasing GABA release on dopamine neurons in the VTA. For the purposes of the present study, CKD infusions may have dampened dopamine release triggered by receiving rewards. In particular, the suppression of the reward-associated dopamine signal by stimulating the lateral habenula also reduced the choice of larger, uncertain rewards (Stopper et al., 2014). A similar mechanism could explain the results of the present study, in which increased CRF activity in the VTA could attenuate reward-associated dopamine signaling and decrease the perceived value of rewards relative to the cost of effort required to obtain them. This, in turn, can reduce the tendency to choose the more expensive options in subsequent decisions.

Although intra-VTA CRF infusions changed voting behavior, these manipulations did not increase election latencies, in contrast to the effects produced by either ICV-CRF or acute stress. The timing and effective doses of CRF in these experiments can explain these discrepancies. For example, central infusions of exogenous CRF were given as a bolus 10 minutes prior to the behavioral test. In comparison, it is to be expected that acute restraint stress leads to a slower accumulation of CRF within the VTA and other target nuclei. On the other hand, it should be noted that stress-related increases in voting times are evidently mediated by increased dopamine transmission, as this effect was blocked by the administration of a dopamine antagonist prior to exposure (Shafiei et al., 2012). . Although the terminal regions where elevated dopamine levels can mediate this effect are unclear, stimulation of D increases 2 - Receptors in the prefrontal cortex or in the basolateral amygdala reduce the latency of choice in other types of cost / benefit decisions (St. Onge et al., 2011); Larkin et al., 2015). It is interesting to note that acute stress potentiates DOPAC metabolism and extracellular dopamine levels in the nucleus accumbens and in the prefrontal cortex (Abercrombie et al., 1989; Dunn and Berridge, 1990; Holly et al., 2015; Imperato et al., 1991; Matsuzaki et al, 1989) in a manner similar to the ICV-CRF infusion (0.2-20 µg; Dunn, 1988; Dunn and Berridge, 1987). However, intra-VTA CRF infusions decrease dopamine metabolism in the PFC and increase DA metabolism in the NAc 60 minutes after the infusion (Kalivas et al., 1987). In addition, the intra-accumbens CRF infusion (0, 1 and 1 μg) acts locally to increase dopamine release (Lemos et al., 2012). This suggests that CKD can have conflicting effects on dopamine transmission, depending on the neural loci involved. In addition, depending on the region it may be acting in, CRF infusion may have a two-phase effect on DA transmission. It seems reasonable, therefore, to suggest that stress-related increases in CRF transmission can delay decision-making processes by improving terminal dopamine release through mechanisms that are independent of their effects within the VTA. The spectrum of behavioral changes caused by increased CRF activity (e.g., changing choice behavior towards latencies to make this choice) appears to be anatomically dissociable, further highlighting how CRF can act in a circuit-specific manner, to modulate different aspects of motivation behavior.

Summary and Clinical Implications

The results of the present study provide new insights into the neural mechanisms by which acute stress can alter cost-benefit decision-making and motivational processes, and identify a key role for increased CRF transmission in mediating its effects on decision bias, choice delays, and responses Force. In addition, these results may also have important implications for understanding the pathophysiology that drives certain symptoms of stress-related psychiatric illnesses, particularly major depressive disorder. Negative affect and depressed mood are the most recognized phenotypes of depression. However, individuals with this disorder also suffer from a number of energy-related deficiencies, including a substantial lack of motivation, or anergia, with depressed patients reluctant to expend effort in exchange for pleasurable experiences (Tylee et al, 1999; Stahl, 2002) . Indeed, it is this lack of motivation, and not an inability to experience pleasure per se, that is the most debilitating symptom of depression, rendering sufferers unable to participate in everyday activities (Salamone et al, 2015). With this in mind, depressed patients tested on a back-translated assay of effort-based decision-making show a marked reduction in preference for larger rewards associated with greater effort costs (Treadway et al, 2012) and increased decision latencies on various tests of executive functioning (Rubinsztein et al, 2000; Murphy et al, 2001). These deficits are remarkably similar to the effects of acute restraint stress or CRF infusions reported here. Given that abnormal increases in central CRF activity have been implicated in the pathophysiology of depression (Nemeroff et al, 1984; Banki et al, 1987; Hauger et al, 2009; Binder and Nemeroff, 2009), the present findings provide support for the notion that aberrant hyperactive CRF transmission may be a key driving force underlying the decline in motivation seen in human depression, potentially via its effects on dopamine transmission. As such, development of compounds that can attenuate CRF activity and in particular, its effects on the dopamine system, may hold promise for treating motivational deficits associated with depression and other stress-related psychiatric disorders.

Funding and Disclosure

The authors do not declare any conflict of interest.

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