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Official websites use. Share sensitive information only on official, secure websites. Many addictive drugs increase stress hormone levels. They also alter the propensity of organisms to prospectively select actions based on long-term consequences. We hypothesized that cocaine causes inflexible action by increasing circulating stress hormone levels, activating the glucocorticoid receptor GR. We trained mice to generate two nose pokes for food and then required them to update action-consequence associations when one response was no longer reinforced. Cocaine delivered in adolescence or adulthood impaired the capacity of mice to update action strategies, and inhibiting CORT synthesis rescued action flexibility. Next, we reduced Nr3c1 , encoding GR, in the orbitofrontal cortex OFC , a region of the brain responsible for interlacing new information into established routines. Nr3c1 silencing preserved action flexibility and dendritic spine abundance on excitatory neurons, despite cocaine. Spines are often considered substrates for learning and memory, leading to the discovery that cocaine degrades the representation of new action memories, obstructing action flexibility. Subject areas: Natural sciences, Biological sciences, Neuroscience, Behavioral neuroscience, Molecular neuroscience, Cellular neuroscience. Cocaine induces corticosteroid hormone release and decision-making biases. We find that hormone binding in the orbital cortex induces dendritic spine loss. One mechanism is by impairing the ability of mice to access reward memories. Natural sciences; Biological sciences; Neuroscience; Behavioral neuroscience; Molecular neuroscience; Cellular neuroscience. Stress is considered both a causal factor in, and consequence of, many neuropsychiatric illnesses including substance use disorders. In experimental animals, social defeat stress increases cocaine self-administration, 1 while cocaine elicits a stress response. For instance, repeated cocaine, 6 , 7 , 8 , 9 , 10 chronic stress, 11 , 12 and exogenous corticosterone CORT 13 , 14 degrade the capacity of organisms to select actions based on their consequences, causing a deferral to habit-like behavior. For example, Dias-Ferreira et al. CORT is a primary stress hormone cortisol in humans. It binds to high-affinity mineralocorticoid receptors MR at baseline and then additionally, low-affinity glucocorticoid receptors GR upon stress-induced adrenal CORT release. This brain region is important for integrating new learning into existing knowledge, enabling adaptive modification of behavioral action strategies when familiar expectancies change. Stressors, CORT, and psychostimulants all commonly cause dendritic spine attrition on excitatory OFC neurons and also obstruct action flexibility. Here, we tested the hypothesis that repeated cocaine, as would occur in individuals suffering from substance misuse, causes action inflexibilities by increasing circulating stress hormones and activating GRs. Further, these inflexibilities can be attributed to the inability of the OFC to use action memory traces to update action strategies, and not obviously impulsive- or anhedonic-like behavior. The over-arching hypothesis of this project was that cocaine-induced CORT contributes to cocaine-induced modifications in reward-seeking behavior. C Mice were trained to respond on 2 nose poke apertures for food. Responding at the other port remained reinforced. Behavioral flexibility was assessed the following day in a brief choice test. E Mice were trained to nose poke, with no differences between groups here or in other experiments. F Excess CORT blocked action flexibility, indicated by non-preferential responding at the choice test. Zero indicates no preference. J Effects of cocaine on sensitivity to reinforcer devaluation were also assessed. Control mice favored a valued over devalued pellet, but cocaine-exposed mice did not. We next used a task in which mice must update actions when familiar expectancies are violated. We trained mice to nose poke for food reinforcers and then reduced the likelihood that responding at 1 of 2 apertures would be reinforced, thus breaking the association between nose poking at that aperture and food delivery Figure 1 C. Successfully updating this association is evidenced by preferential responding during a subsequent choice test on the aperture at which the action-outcome association remained intact. In this experiment, mice were first delivered exogenous CORT, increasing blood serum CORT to a degree similar to that following cocaine, 26 followed by a washout period to assess long-term consequences Figure 1 D. Difference scores are reported in the rest of the main text. Acquisition curves i. Next, we assessed the effects of cocaine in the same task, anticipating that any behavioral inflexibilities would be attributable to cocaine-elicited CORT. Later testing revealed that cocaine obstructed action flexibility, as expected. Notably, we also observed the same effect in mice given cocaine in adolescence and then tested in adulthood Figure S2 , suggesting that cocaine-induced CORT release impacts action flexibility across multiple ages and drug administration procedures. This pattern of cocaine-induced response inflexibility has been framed as habit-like behavior. To substantiate this perspective, we assessed whether the same cocaine exposure procedure impacted the ability of mice to update action strategies when outcome values changed Figure 1 I. In this task, mice must integrate the sensory properties of rewards into goal representations to engage in flexible goal seeking. A failure to do so is classically considered habitual behavior. The OFC is an expansive and functionally heterogeneous structure. Mice were trained to respond for food, then had to update the association between nose poking and pellet delivery when one response was no longer reinforced, as above. Cocaine-exposed mice exhibited non-preferential responding, as expected. Meanwhile, GR reduction rescued preferential responding in cocaine-exposed mice. E Impulsive-like behavior was next assessed in the same mice using a delayed reinforcement task. Mice could preferentially respond for a large over small reinforcer. F When a delay was introduced between responding for the large reinforcer and reinforcer delivery, mice switched preference to the small reinforcer. G Groups did not differ in sucrose consumption. Response inflexibilities following cocaine could conceivably be attributable to multiple stress-related sequelae, including impulsive- or anhedonic-like behavior. To disentangle these possibilities, mice were next trained to nose poke at 1 aperture, resulting in the delivery of 5 pellets large reinforcer or another aperture, resulting in 1 pellet small reinforcer. Following this training, a delay between nose poking for the large reinforcer and pellet delivery was introduced, increasing across sessions. Another explanation for cocaine-induced response inflexibilities, aside from impulsive- or anhedonic-like behavior, is deficiencies in the learning and memory processes required for mice to update action strategies. A neuronal ensemble in the VLO that encodes action memories was recently identified. These cells are active when mice experience unexpected non-reinforcement, and then must be re-activated later in order for mice to flexibly deviate from familiar response strategies. We rationalized that if cocaine deteriorates new memory or weakens memory retrieval, stimulating this memory trace population may restore action flexibility. If cocaine instead ablates action memory, which is another possibility, then stimulation would have no effects. With viral vectors thus validated, we proceeded to behavioral testing. Cocaine alone caused non-preferential responding even when one familiar behavior was no longer reinforced, as expected. This was despite reactivation of memory trace neurons at the choice test, as measured by c-Fos in viral transduced cells Figure S4. A Timeline. The rationale was that if cocaine weakened action memory representations, then stimulating these neurons may reinstate action flexibility. B Representative viral vector infusion. D Control mice favored a reinforced behavior, and cocaine obstructed response preference, as expected. F Response rates dropped considerably from the beginning to end of the session in control mice, reflected by large difference scores. H Line fits with individual data points. The slope of the cocaine-alone group did not differ from 0, indicating no change across time. Simple linear regression analyses were also applied bottom row. To summarize, cocaine destabilizes action memories — likely memory to inhibit a familiar response. In this case, reinforcers are delivered only when mice inhibit responding, so the adaptive response is to depress responding. We next calculated the difference between response rates during the last 5 min relative to the first 5 min. To assess response rates in another way, we used simple linear regression analyses to fit lines to each group. We then assessed whether each line was significantly different than zero, indicating a change in responding. Taken together, our findings indicate that cocaine impedes the ability of OFC neurons to stabilize novel action strategies, as opposed to ablating action memory entirely. In addition to their overlapping effects on behavioral responses, both cocaine and CORT cause attrition of dendritic spines on excitatory neurons in the VLO. Given our findings that cocaine imperils memory, and dendritic spines are often considered substrates of learning and memory, we lastly hypothesized that cocaine-induced spine loss was due to activation of GRs. Here, YFP identifies excitatory layer V neurons and enables high-resolution single cell imaging Figure 4 A , and mCherry signifies viral vector transduction. Dendritic spine densities were lower in cocaine-exposed mice, which was rescued by viral-mediated GR reduction. Interestingly, GR reduction alone in the absence of cocaine also reduced dendritic spine density, bringing to mind poor learning in these mice cf. In other words, both groups that struggled to update action strategies also suffered spine attrition on excitatory OFC neurons. GR reduction alone also reduced overall dendritic spine densities in the VLO. C Cocaine caused attrition of all spine subtypes, with prevention of thin-type spine loss by GR reduction. D Cocaine and GR reduction alone also increased dendritic spine lengths. Meanwhile, cocaine-induced lengthening was prevented by GR reduction. Bars represent means. Dark gray dots represent individual mice, and light gray triangles represent individual dendrites in B and C. Dots represent individual dendrites in D. Next, we compared dendritic spine morphological subtypes across groups — stubby-type, mushroom-type, versus thin-type. We then assessed the distribution of spine lengths, agnostic to the spine types. Other morphometric measures did not differ between groups not shown. Here, we examined mechanisms by which cocaine biases organisms toward inflexible, familiar action strategies. Cocaine-induced response biases were not obviously attributable to impulsive- or anhedonic-like behavior, sequelae linked to stress systems, but rather, the destabilization of memory traces in the VLO that store action memories for later retrieval and action flexibility. Here, we investigated the learning and memory processes by which behaviors are flexibly updated in dynamic environments. Mice were first trained to respond in two nose-poke apertures for food pellets delivered into a separate magazine. Then, responding was no longer reinforced at one aperture, and instead, pellets were delivered non-contingently. Thus, for one nose-poking behavior, familiar reinforcement conditions were maintained, while for the other, those expectations were violated. Nose-poking is instrumental in nature, as opposed to a Pavlovian response based on the stimulus properties of the nose-poke aperture. Cocaine causes failures in action updating in this and similar tasks in rodents 6 , 7 , 33 and humans 9 , 10 alike. Prior stressor exposure appears to be a major contributor in humans, 9 and is itself sufficient to induce the same behavioral inflexibilities. Indeed, blocking CORT synthesis prior to cocaine administration fully rescued flexible behavior in cocaine-exposed mice. Notably, CORT synthesis inhibition can also blunt cocaine-induced locomotor sensitization, as well as self-administration and reinstatement of drug seeking. One important aspect of this report is that we replicated evidence that cocaine increases circulating CORT, as previously reported with repeated 2 , 3 , 4 and acute 34 exposure. We measured CORT levels halfway through our repeated cocaine exposure procedure to emphasize that GRs are likely being repeatedly activated with repeated cocaine exposure. Cocaine-induced CORT release may be due to cocaine increasing monoamines in the synapse. For instance, norepinephrine binding to receptors on neurons that release corticotropin-releasing hormone in the paraventricular nucleus of the hypothalamus leads to adrenocorticotropin hormone binding in the adrenal glands, ultimately causing CORT release. What might account for cocaine-induced action inflexibility? A number of possibilities are plausible, particularly given this discovery of stress hormone involvement. It is conceivable that cocaine caused impulsive-like behavior, 41 , 42 which caused mice to be unable to preferentially respond at the previously reinforced aperture. We tested this possibility with a delayed reinforcement task and found that cocaine was without effect at the time of testing, which was notably well after cocaine exposure. Another consideration is that mice here could have been primed to detect changes in task parameters, given their experiences with other behavioral tests. It is also conceivable that cocaine caused response biases by inducing anhedonic-like behavior. Yet, we found no effects in a sucrose consumption test well-suited to detect anhedonic-like behavior, 26 if it exists. Action inflexibility in the task used here has often been interpreted as being habit-like — meaning, driven by stimulus-response associations that are insensitive to outcomes. To substantiate this perspective, we turned to a classical reinforcer devaluation task, in which case reinforcer value is reduced and habitual behavior is inferred if mice do not modify their behaviors accordingly. We included female mice throughout this report because prior reports overwhelmingly utilized males. To our knowledge, ours is amongst the first time this phenomenon has been comprehensively demonstrated in adult, female mice, as opposed to adult males or adolescent mice of both sexes. Mammals may perform habitual behaviors by virtue of extensive familiarity with a task, or because they are unable to learn, maintain, or recall the action-outcome links that support competing goal-directed actions. Medial prefrontal cortical regions like the prelimbic cortex form action-outcome links, and the OFC, particularly ventrolateral compartment, is necessary for updating and maintaining associations when they change and retrieving new memories, thus enabling action flexibility. VLO neurons form stable representations of new action strategies when familiar expectations are violated. These memory traces are then retrieved when mice seek rewards in the future. To disentangle these possibilities, we selectively induced chemogenetic constructs in cells active when familiar expectations were violated, which allowed us to stimulate those memory trace cells later, after cocaine exposure. Stimulating memory trace cells in cocaine-exposed mice improved action flexibility. Thus, cocaine did not ablate action memory, since it could be made accessible by virtue of chemogenetic cell stimulation. Cocaine also did not grossly obstruct the reactivation of memory trace neurons, since these neurons expressed c-Fos upon memory retrieval. Thus, we imagine that the representation of action memories is corrupted by cocaine, such that greater stimulation than typical is required for memory retrieval. One consideration, though, is that c-Fos offers limited resolution by which to discern differences in degrees of activity in neurons, 47 , 48 so it is possible that cocaine impacts memory trace neuron reactivation in a subtle fashion. Cocaine and CORT both cause a loss of dendritic spines on excitatory neurons in the OFC, 49 , 50 including projection-defined neurons necessary for memory retrieval. Perturbations in either or both systems could result in dendritic spine loss. Importantly, BDNF in the VLO is necessary for the encoding and retrieval of action memories 25 and is therefore, in conjunction with its role in dendritic spine plasticity, likely to be involved in action memory stability and its recall. GRs play an important role in learning and memory processes, including long-term memory storage and retrieval. Relatively little is known about GR or mineralocorticoid receptor levels following cocaine exposure. Future investigations could help disentangle these possibilities. Further information and requests for resources should be directed to the lead contact, Shannon Gourley shannon. Any additional information required to reanalyze the data reported in this paper may be made available from the lead contact upon request. Initial pharmacological studies Figure 1 utilized female mice, replicating prior, foundational experiments conducted in males see discussion for references. Subsequent experiments used both sexes. Sex differences were not detected. Experiments took place between and The head was cleaned, skin cut, and skull leveled. Mice were sutured and allowed to recover for 3 weeks, allowing for viral vector expression. These doses and timing were derived from prior investigations revealing that cocaine impacts response strategies, biasing rodents towards habit-based behaviors. Importantly, this dose does not by itself impact responding in this task. Control mice consumed tap water. Water bottles were weighed daily, and mice weighed every other day. Water bottles were refilled with fresh water or newly prepared CORT solution every 3 days. Mice were exposed to CORT for 14 days, followed by a day washout period. Blood serum CORT was collected and measured 1 day following the final cocaine injection, as described. Albans, VT equipped with 2 nose poke apertures and a food magazine. Mice required 10—14 sessions to acquire, and the last 10 sessions are shown. For experiments in Figures 1 and S2 , mice received 2 additional days of training according to a random interval RI s schedule of reinforcement to increase response rates. Mice then proceeded to satiety-specific devaluation or the test of response flexibility. Satiety-specific devaluation was used to assess whether mice could modify response strategies due to changes in the value of expected outcomes. Following ad libitum feeding 90 min , mice were placed in the operant conditioning chambers for a 10 min choice test conducted in extinction. Mice that successfully update their response strategies will decrease responding for the devalued pellet. Pellet vs. This test was conducted after instrumental response training, first using a single reinforcer purified grain-based pellet; for Figures 1 C—1G , then with 2 separate, equally preferred reinforcers purified grain and chocolate for the rest of the manuscript, given that goal-seeking behavior is often thought to involve the integration of specific reward features into goal representations. Responses at the available aperture resulted in no programmed consequence. The following day, both apertures were available in a 10 min choice test conducted in extinction. Preferential responding at the nose poke that was previously reinforced indicates a flexible response strategy, deviating from equivalent responding during training. Meanwhile, comparable responding at both apertures indicates a failure to update action strategies. Mice in the final behavioral experiment reported here underwent the 2 25 min sessions described above, and 4OHT was administered following the non-reinforced session to induce chemogenetic constructs in neurons active during this period. These neurons form memory traces for new action strategies, and chemogenetic receptors allow for their later manipulation. Mice were then re-trained daily for 5 days using an FR1 schedule of reinforcement to reignite responding. The test of response flexibility protocol was repeated as described above, with the reinforced and non-reinforced apertures held constant. A choice test followed the next day, with CNO delivered prior to the test. Finally, mice were again re-trained using an FR1 schedule of reinforcement for 2 days to reignite responding before an omission test, described below. A delayed reinforcement task was used to assess impulsive-like behavior. First, mice had access to 2 active nose poke apertures for 30 min. Nose poking at 1 aperture resulted in the delivery of 5 pellets large reinforcer while nose poking at the other aperture resulted in delivery of a single pellet small reinforcer. Following nose poke responses, there was a 25s time-out. Mice were considered to have acquired once they preferentially responded for the large reinforcer. Mice then began the delay phase of the task. Across 7 days, they experienced an increasing delay 10s, 20s, 30s, 45s, 60s, 80s, s between nose poking for the large reinforcer and delivery of the pellets, and responding was monitored. Conditions were unchanged for the small reinforcer. Instrumental omission was used to further assess the ability of mice to update responding after a change in the relationship between nose poking and pellet delivery. The other aperture was occluded and not available for responding. Then, the omission procedure commenced on the available aperture as described, 76 in which case pellets were scheduled to be delivered every 20s but the counter was reset after a nose poke response, delaying delivery of the reinforcer. Thus, mice had to inhibit responding in order to receive pellets. The session was 20 min long. Then, they were trained for 5 sessions according to an RI s schedule of reinforcement. The program ended when mice had received 30 pellets or at 70 min. Mice were then tested using a progressive ratio schedule of reinforcement in which the response requirement increased by 4 with each pellet delivery i. Sessions ended at min or when mice did not respond for 5 min. Break point ratios, the highest number of responses the mice were willing to complete to receive a pellet, are reported, averaged across 3 sessions conducted on 3 consecutive days. Sucrose consumption was used to assess anhedonic-like behavior. Mice were acclimated to the cage and sucrose for 4 h and then water-restricted for 19 h. The water bottles containing the sucrose solution were returned to the cage for 1 h. The bottles were weighed immediately before and after this 1-h period. Mice were briefly anesthetized by isoflurane and euthanized by rapid decapitation 3 weeks after viral vector infusion. A single experimenter centered a 1mm corer within the ventrolateral OFC under a fluorescence dissection microscope for dissection. Tissue was homogenized by sonication, and protein content was measured by Bradford colorimetric assay. Membranes were incubated in primary antibody, Anti-GR , Abcam, ab, lot GR overnight and then in horseradish peroxidase-conjugated goat anti-rabbit Vector; secondary antibody. Immunoreactivity was assessed using a chemiluminescence substrate Pierce and measured using a ChemiDoc Imager Bio-rad. All gels were normalized to their corresponding total protein. The signals were normalized to the control sample mean from the same membrane to control for variance between gels. Samples were run twice to ensure replication. Mice were deeply anesthetized and euthanized by intracardiac perfusion 4 days after the conclusion of behavioral experiments. Viral vector infusion sites were verified and characterized by imaging mCherry. Brains were prepared as above. In another experiment, mice were euthanized 1 h after the choice test, and c-Fos was visualized in memory trace neurons, identified by viral-mediated mCherry expression. Uniform exposure parameters were used throughout, and anatomical landmarks were used to ensure images were similarly localized. When normalized, values were normalized to the control sample mean. For c-Fos quantification, analyses were performed using CellProfiler. The analysis pipeline included thresholding Otsu method and identifying primary objects, 77 defined as viral transduced neurons. Mice were deeply anesthetized and euthanized by intracardiac perfusion 3 weeks after viral vector infusion. Analyses were performed using CellProfiler. YFP-expressing mice were deeply anaesthetized and euthanized by intracardiac perfusion 4 days after the conclusion of behavioral experiments. YFP in these mice labels layer V neurons. Z-stacks of dendritic segments were acquired using a 0. The location of the imaged segments within target regions was confirmed by zooming out to a low magnification. A single blinded user generated all images. Dendritic spine head location was manually indicated, and FilamentTracer processing algorithms were used to calculate morphological parameters. Morphological classification of dendritic spines was determined using parameters modified from ref. A single blinded individual quantified all dendritic spines within a given experiment. For analyses of responding across the instrumental omission session, simple linear regression was also used to generate line of best fit. Comparisons were made by unpaired t-tests. For GR immunofluorescence, the control viral vector was infused into the VLO of one hemisphere and the Cre-expressing viral vector into the other hemisphere. Comparisons were made by paired t-tests across hemispheres. Dendritic spine morphometric analyses were conducte by Kolmogorov-Smirnov K-S comparisons. Comparisons were 2-tailed except for the western blot validating Nr3c1 knockdown, which was 1-tailed based on the a priori hypothesis that gene silencing would reduce protein levels. Group sizes were determined based on power analyses and preexisting datasets. All experiments were conducted at least twice. We thank Dr. Brian Dias for generously sharing resources. We thank Aylet Allen for assistance with the devaluation test and graphical abstract. Conceptualization and methodology, M. 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Cocaine disrupts action flexibility via glucocorticoid receptors

How can I buy cocaine online in Bielefeld

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How can I buy cocaine online in Bielefeld