AT1 Receptors Mediate the Effects of Cold Stress and Amphetamine on Neurocognitive Performance

AT1 Receptors Mediate the Effects of Cold Stress and Amphetamine on Neurocognitive Performance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article AT1 Receptors Mediate the Effects of Cold Stress and Amphetamine on Neurocognitive Performance Natalia Andrea Marchese, Victoria Belén Occhieppo, Sol Micaela Angulo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6889905/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Oct, 2025 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted 7 You are reading this latest preprint version Abstract Objectives In this work, we aimed to evaluate whether Amph exposure modifies the future neurocognitive response to cold stress, an environmental cue, and the possible role of the AT1-R. Methods Male Wistar rats received AT1-R blocker (Candesartan)/Vehicle from day 0 to 5, and Amph/Saline from day 6 to 10. After seven days of withdrawal, the animals were exposed to a cold challenge (4°C for 4 h) and tested for working and long-term memory. Results Cold challenge reversed the Amph-induced working memory deficit. Previous Amph exposure blunted the interference of cold stress on long-term memory consolidation. Blood adrenaline and glucose levels were increased in the Amph-treated animals after cold stress and/or test exposure. Remarkably, the AT1-R blockade prevented all these alterations induced by Amph exposure and elicited by cold-challenge. Conclusions Our results indicate that Amph-induced neuroadaptations exhibit a wide range of responses to catecholamine-releasing stimuli, including pharmacological and non-pharmacological challenges and environmental conditions involving the AT1-R. Angiotensin II AT1-receptor Amphetamine Working memory cold stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION The role of the brain renin-angiotensin system is complex. It regulates vascular, glial, and neuronal functions through its main active peptide, angiotensin II (Ang II). Particularly, this peptide plays an important role in the modulation of central dopaminergic neurotransmission and has a crucial role in sensorial and cognitive processes through the activation of angiotensin receptors type 1 (AT1-R) (Marchese et al. 2016 , 2020 ; Basmadjian et al. 2024 ). Indeed, AT1-R is located in the soma and terminal fields of dopaminergic neurons, and there is evidence of a cross-regulation between dopamine (DA) and Ang II systems (Brown et al. 1996 ; Zhuo et al. 1998 ; Martinez-Pinilla et al. 2015 ; Basmadjian et al. 2024 ). The recreational use of amphetamine (Amph) is based on its stimulant properties over the central nervous system since it promotes mainly noradrenergic and dopaminergic neurotransmission. However, Amph exposure induces long-term changes in multiple neuronal circuits, modifying their future responses to pharmacological or non-pharmacological challenges (Marchese et al. 2016 , 2020 ). Our group previously addressed the effects of Amph exposure on learning and memory processes, along with altered neuronal connectivity. In this sense, acute Amph impairs memory retention in male rats in the one-trial inhibitory avoidance (IA) response when administered immediately post-training, partially involving AT1-R activation (Marchese et al. 2016 ). Moreover, previous exposure to Amph, followed by a withdrawal period, altered the response of the animals in the IA test concomitant with their neuronal activation pattern in the basolateral amygdala (BLA) after an Amph challenge (Marchese et al. 2016 ). The long-lasting alterations induced by Amph were also observed in the hippocampal synaptic transmission, as a lower threshold was required for LTP generation, which was prevented by the AT1-R blockade (Marchese et al. 2016 ). Long-term effects of repeated Amph exposure were also documented for executive neurocognitive tasks such as working memory, which implies active modulation of catecholaminergic levels in the Prefrontal Cortex (PFC) (Marchese et al. 2020 ; Basmadjian et al. 2024 ). Cross-effects between psychostimulants and stress have been identified in the long-term neuroadaptation involving catecholamine neurotransmission, both in animal models and humans (Nikulina et al. 2004 ; Booij et al. 2016 ; Torres-Berrio et al. 2018 ; Anderson et al. 2019 ). Considering that Amph exposure induces neuroadaptive changes, we aimed to evaluate whether Amph's previous history modifies the neurocognitive response to a non-pharmacological challenge such as cold stress. Likewise, to find out if AT1-R is involved in the possible alterations resulting from the cross-effects of exposure to Amph and cold stress. 2. MATERIALS AND METHODS 2.1. Animals A total of 269 adult male Wistar rats were obtained from the Department of Pharmacology Otto Orsingher vivarium (Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina) and randomly housed in groups of XX one week before the beginning of the experimental protocol. Throughout the experiment, animals were maintained in controlled environmental conditions (20–24°C, 12-h light/dark cycle with lights on at 07 a.m.) and had free access to food and water. All procedures were conducted with the approval of the Animal Care and Use Committee of the Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina (Res nº 46/15), following the NIH Guide for the Care and Use of Laboratory Animals. 2.2. Drugs The selective AT 1 -R antagonist, Candesartan cilexetil (CV, Laboratorios Phoenix, Buenos Aires, Argentina), was dissolved in NaHCO3 0.1N (vehicle, Veh). D-amphetamine sulfate (Amph-SIGMA, Argentina) was dissolved in 0.9% NaCl (saline). 2.3. Experimental design Animals received Veh/CV (3mg/kg by gavage) once daily for 5 days. From day 6 to 10, they were injected daily with Amph (2.5mg/kg/ Sal i.p.) and then left undisturbed in their home cages until the day of the experiment (Marchese et al. 2016 , 2020 ; Occhieppo et al. 2017 ). On day 17, animals were tested for cognitive performance in basal conditions (room temperature- RT) or after cold exposure (4h at 4°C- cold challenge). A naïve group was evaluated under the same conditions on day 17 ( Fig. 1 ) . Blood samples were obtained immediately after the cold challenge and/or the behavioral test, as indicated in Fig. 2 A, 2 C, and 5 A. Blood adrenaline was assayed by reverse-phase HPLC (Model 582 solvent delivery, ESA, Chelmsford, MA, and RP 18 Column) with electrochemical detection (ESA coulochem II) and glucose with a meter test (Optium Xceed meter, Abbott, IL, USA). Samples for immunohistochemistry were taken as indicated in Fig. 4 A. 2.4. Cognitive evaluation: working memory 2.4.1. Y-maze test Animals were placed at the center of a Y-shaped maze and left for free exploration for 8 minutes. The sequence of chosen entries was manually recorded, where three consecutive choices of three different arms were counted as an alternation, and the total possible alternations were the total number of entries minus 2. Finally, the percentage of alternations was calculated. Animals displaying 2 minutes of immobility between arms were excluded from the final analyses. 2.4.2. Holeboard test Exploratory behavior was evaluated after one 5-minute exposure to an arena with five holes. The number of heads dipping into each hole was quantified (visits to the hole). The number of visits was analyzed for each animal in the experimental groups, ordering the values from the most to the least visited hole. 2.5. Cognitive evaluation: Long-term memory The inhibitory avoidance response was determined by employing a step-through apparatus with an illuminated and a dark compartment (60×30×30cm each) connected by a guillotine door. The experiment was conducted on 2 consecutive days on the same day time. On the first day (training session), each rat was placed in the illuminated compartment, and once the rat entered completely into the dark compartment, it received 3 electric foot shocks (0.5mA, 3s each, separated by 30s). A maximum of 60 seconds was considered for the animal to enter the dark compartment; otherwise, the animals were dismissed from the experiment. On the second day (test session), the same procedure was followed (no foot-shock delivery), and the time taken to enter the dark compartment was recorded (max. 300s). This parameter is considered a measure of memory retention and is displayed in the graph as latency. In accordance with previous work (Marchese et al. 2016 ), the acute effect of cold stress on the inhibitory avoidance response was evaluated by cold exposure immediately after the training session in non-CV-treated ( Fig. 3 ) and CV-treated groups ( Fig. 4 ) . 2.6. Immunohistochemistry: c-Fos immunoreactivity Ninety minutes after the test session, animals were anesthetized and perfused transcardially with 250 mL of physiologic solution (0.9% NaCl) and heparin (200µL/L), followed by 400 mL of 4% paraformaldehyde in 0.1M phosphate buffer (PB, pH 7.4). The brains were post-fixed in the same paraformaldehyde solution overnight and stored at 4°C, 30% sucrose in PB. Coronal sections of 40 µm were cut using a freezing microtome (Leica CM15105), collected in PB 0.01M, and placed in a mixture of 10% H2O2 and 10% methanol for 2 h. Samples were incubated in 10% normal horse serum (NHS) (Natocor, Villa Carlos Paz, Córdoba, Argentina) in PB 0.1M for 2 h and immediately after the free-floating sections were incubated overnight at room temperature in a rabbit anti-c-Fos antibody (1:20,000 in 2% NHS-0.3% Triton-PB 0.1 M; Ab-5; Oncogene Science, Manhasset, NY). The sections were incubated with biotin-labeled anti-rabbit secondary antibody (1:2000 in 2% NHS-PB 0.1M; Jackson Laboratory) and avidin-biotin-peroxidase complex (1:200 in 2% NHS-PB 0.1M; Vector Laboratories) for 2 h each at room temperature. The peroxidase label was detected with diaminobenzidine hydrochloride (Sigma Chemical Co.) and intensified with 1% cobalt chloride and 1% nickel ammonium sulfate for a violet nuclear reaction product. Finally, the free-floating sections were mounted on gelatinized slides, air-dried overnight, dehydrated, cleared in xylene, and placed under a coverslip with DPX mountant for histology (Flucka Analytical). 2.7. Image processing All images were obtained by using a computerized system that included a Leica DM 4000B microscope equipped with Leica FW4000 and a DFC Leica digital camera attached to a contrast enhancement device. The images were processed using ImageJ software (U.S. National Institutes of Health, USA). The analyses were made blinded to the experimental groups. Considering our previous work (Marchese et al. 2016 , 2020 ), c-Fos immunoreactivity (c-Fos IR) assay was evaluated at the hippocampal Dentate Gyrus (DG; Bregma − 3.30/−3.60 mm) and basolateral amygdala (BLA; Bregma − 2.56/−2.80 mm), which were identified and delimited according to Paxinos and Watson atlas (Paxinos and Watson 2009 ). Image thresholds were fixed between intervals of 120–150 in black-and-white conditions; all lower values were considered background. C-Fos IR neurons were identified by dense black staining of the nucleus and counted by setting a size range for cellular nuclei (8 to 12 µm in diameter). The measurements were taken bilaterally in two sections, and the final value was obtained as the average of the four counted sections. 2.8. Statistical analyses and Artwork Data were analyzed using one-way or two-way ANOVA and reported as means ± SEM. If an interaction and/or main effect was observed, comparisons were made using Bonferroni's post-test. The Unpaired t-test was used for the blood catecholamines analysis. The Kruskal-Wallis test and Dunn’s multiple comparisons were used for inhibitory avoidance analysis. A value of p < 0.05 was considered significant. The analyses were performed using GraphPad Prism® 8.02 software, and the figures were made using Inkscape®. The artwork was performed using Canva®. 3. RESULTS 3.1. Amphetamine administration induced working memory deficit via AT1-R, and the cold challenge reversed it, involving catecholamines and glucose changes. Blood adrenaline levels were evaluated after cold stress exposure and relativized to the levels of naïve rats. After the cold exposure, the Veh-Amph group showed increased adrenaline levels compared with the CV-Amph group ( Fig. 2 ; Table 1 ) . Adrenaline levels were not significantly different between the Veh-Amph and CV-Amph groups at room temperature ( Fig. 2 B; inset) . Blood glucose was evaluated before, during, and after the cold challenge and behavioral test. No significant differences were observed immediately after the cold challenge compared with the pre-cold; however, exposure to the behavioral tests significantly increased the blood glucose levels in the Veh-Amph group compared with the pre-cold and post-cold assessments. This increase was not evidenced in the Naive and CV-Amph groups ( Fig. 2 ; Table 1 ) . Table 1 Table 1 Statistical results for blood adrenaline and glucose levels. CV: AT1-R antagonist. Unpaired t-test for adrenaline and Two-way ANOVA and Bonferroni’s multiple comparisons for glucose levels. *p < 0.05. Blood adrenaline Unpaired t-test P Veh-Amph vs. CV-Amph 0.0061 Blood glucose Two-way ANOVA RM Interaction F (4, 24) = 3.260 P = 0.0286 Time F (2, 24) = 3.869 P = 0.0350 Column Factor F (2, 12) = 0.2238 P = 0.8028 Subject F (12, 24) = 1.530 P = 0.1811 Bonferroni's multiple comparisons test p Naive group "Pre-cold" time vs "Post-cold" time > 0.9999 "Test" time vs "Pre-cold" time 0.3879 "Test" time vs "Post-cold" time 0.2904 Veh-Amph group "Pre-cold" time vs "Post-cold" time > 0.9999 "Test" time vs "Pre-cold" time *0.0211 "Test" time vs "Post-cold" time *0.0348 CV-Amph group "Pre-cold" time vs "Post-cold" time > 0.9999 "Test" time vs "Pre-cold" time > 0.9999 "Test" time vs "Post-cold" time > 0.9999 In basal conditions of room temperature, the animals exposed to Amph showed decreased alternation percentage in the Y-Maze paradigm compared with the naive, and the previous CV administration prevented this effect. No significant differences were observed between the three evaluated groups after the cold challenge ( Fig. 3 ; Table 2 ) . Regarding the holeboard test, the Veh-Amph group showed a significant difference between the first and second most explored holes at basal conditions. After the cold challenge, the Veh-Amph group did not show the difference between the most explored holes, similar to the control group. The Veh-Sal and CV-Amph groups did not show this alteration at basal conditions nor after the cold challenge exposure ( Fig. 3 ; Table 2 ) . Table 2 Table 2 Statistical results for Y-Maze and Holeboard test. CV: AT1-R blockade. One-way ANOVA and Bonferroni’s multiple comparisons. *p < 0.05. Y-Maze test % Alternance Basal conditions % Alternance Cold challenge One-way ANOVA One-way ANOVA F = 7.457 P = 0.0017 F = 0.05162 P = 0.9498 Bonferroni's post-hoc p Bonferroni's post-hoc p Naive vs. Veh-Amph *0.0024 - - Naive vs. CV-Amph > 0.9999 - - Veh-Amph vs CV-Amph *0.0158 - - Total Entries Basal Conditions Total Entries Cold Challenge F = 0.9185 P = 0.4072 F = 0.09477 P = 0.9099 Holeboard test Exploration Basal conditions Exploration Cold Challenge One-way ANOVA One-way ANOVA F = 19.76 P < 0.0001 F = 13.84 P < 0.0001 Bonferroni's post-hoc p Bonferroni'spost-hocc p Naive 1 vs 2 0.1003 Naive 1 vs 2 0.1114 Veh-Amph 1 vs 2 < 0.0001* Veh-Amph 1 vs 2 0.3787 CV-Amph 1 vs 2 0.8142 CV-Amph 1 vs 2 0.0715 3.2. Amphetamine promotes resistance to cold-stress-induced memory impairment via AT1-R activation. Regarding inhibitory avoidance, Dunn´s tests indicate significant differences between animals exposed to shock and those not exposed to shock. Interestingly, exposure to cold stress interferes with memory storage, marked by similar latency times to that of animals never exposed to shock ( Fig. 4 ; Table 3 ) . Table 3 Table 3 Statistical results for the Inhibitory Avoidance test. Kruskal-Wallis test and Dunn’s multiple comparisons. *p < 0.05. Inhibitory Avoidance Kruskal-Wallis test P < 0.001 Kruskal-Wallis statistic 19.95 Dunn's multiple comparisons test p No shock vs. Shock *<0.0001 No shock vs. Shock + Cold 0.0884 Shock vs. Shock + Cold *0.0303 When animals were previously exposed to Amph, the cold challenge did not interfere with memory storage, displaying longer latency than the naive group receiving the cold challenge; whereas the animals exposed to CV/Amph showed similar latency to naive animals after the cold challenge ( Fig. 5 ; Table 4 ) . The c-Fos IR evaluated after the inhibitory avoidance test showed that the Veh/Amph group had increased c-Fos expression in the BLA, which was not observed in the CV/Amph group. No significant differences were observed in DG ( Fig. 5 ; Table 4 ) . Table 4 Table 4 Statistical results for the Inhibitory Avoidance test and neuronal activation in basolateral amygdala and dentate gyrus. Kruskal-Wallis test and Dunn’s multiple comparisons were used for the behavioral test, and One-way ANOVA and Bonferroni’s post hoc for the c-Fos IR analysis. *p 0.9999 Veh-Amph vs. CV-Amph *0.002 c-Fos immunoreactivity One-way ANOVA Basolateral amygdala F = 8.421 P = 0.0040 Bonferroni's post-hoc p Control vs. Veh-Amph *0.0043 Control vs. CV-Amph 0.7341 Veh-Amph vs. CV-Amph *0.0256 Dentate gyrus F = 0.9252 P = 0.4194 4. DISCUSSION In the present work, we extend our previous observations regarding the role of AT1-R in the Amph-induced neuroadaptations, proposing a long-term cross-talk with the catecholaminergic system, when it is activated by environmental stress. The cold exposure, probably acting as a challenge agent of the catecholaminergic system, reversed the Amph-induced working memory deficit, although this prior history with the psychostimulant blunted the interference of cold stress on long-term memory consolidation; thus mimicking previously reported effects after an Amph challenge (Marchese et al. 2016 , 2020 ). In the same direction, the AT1-R blockade prevented both alterations induced by Amph, further supporting that similar processes underpin Amph-induced adaptations when challenged by pharmacological or environmental stimuli. Working memory is a complex process that refers to temporary information storage, on a scale of seconds to minutes, necessary for the performance of a cognitive task (Cowan 2008 ; Riga et al. 2014 ). Catecholaminergic activity plays a preponderant role in working memory performance, given that moderate catecholaminergic activity in the PFC is necessary for optimal functioning. In contrast, a hypo-function or excess of dopamine/noradrenaline would be responsible for cortical dysfunction and working memory deficits. Results from our laboratory previously showed that Amph exposure induced attention memory deficits associated with structural and functional alterations in PFC, even long-term after the last Amph administration (Marchese et al. 2016 ; Basmadjian et al. 2024 ). This PFC dysfunction might be related to decreased glutamate and DA levels and diminished electrical activity in this brain area (Janetsian et al. 2015 ). In the same direction, psychostimulant users exhibit attention deficits and functional changes at the cortical level (Downey and Loftis 2014 ). According to this evidence, we previously found that Amph challenge administration (0.5mg/kg) reversed the attention memory deficit induced by previous exposure to the psychostimulant (Marchese et al. 2020 ), which could be attributed to the Amph's positive effect on catecholamine release (Arnsten 1998 ; Blaiss and Janak 2007 ). Cold stress is a well-documented stimulus for the activation of the sympathetic nervous system (Fiedler et al. 2006 ). The blood glucose level, an important marker of sympathetic activation, was found to increase in the Amph-treated group only after the working memory test. Interestingly, exposure to cold stimuli has been found to negatively affect short-term and working memory in humans, associated with reduced brain catecholamine levels, since it was found to be improved by administration of the catecholamine precursor tyrosine (Shurtleff et al. 1994 ). All this evidence points out long-term neuroadaptations and catecholaminergic misbalance induced by catecholaminergic modulators (Amph or cold stress), leading to working memory deficit. Indeed, we observed increased blood adrenaline levels only in the Veh-Amph group. Therefore, cold stress or an Amph challenge would improve the attention deficit since both stimuli trigger catecholamine release (Marchese et al. 2020 ). The limbic system activation, with single or repeated psychostimulant administration, triggers the development of neuroadaptations that can be revealed after a withdrawal period, at behavioral and neurochemical levels, and by using pharmacological or non-pharmacological challenges (Vanderschuren and Kalivas 2000 ; Marchese et al. 2016 , 2020 ). Indeed, we reported that Amph exposure induced increased hippocampal synaptic plasticity, and resistance to the interference of catecholamines in long-term memory consolidation using the same experimental protocol as in the present study (Marchese et al. 2016 ). Moreover, our previous findings indicated that repeated Amph administration did not affect the animal’s performance in the passive avoidance test, however, the repeated Amph-induced neuroadaptations were evidenced after a week of withdrawal as a resistance to the deleterious effect of the post-training Amph challenge administration (Marchese et al. 2016 ). In the same direction discussed above, it is broadly known that emotions and wakefulness influence the learning process. Moderate emotional states have reinforcing effects, while high emotional levels, such as those experienced during high-stress situations, present detrimental effects (Izquierdo and Medina 1997 ). The evidence converges on the role of catecholamines in wakefulness, emotion, and stress response; as well as in the presence of its receptors in areas involved in the processing of long-term memories with emotional valences, such as PFC, hippocampus (HPC), and BLA (Izquierdo et al. 2006 ; Nieh et al. 2013 ). Since we found that the previous history with Amph blunted the cold-stress effect over long-term memory, we propose that the Amph administration induced endurable neuroadaptations changing the brain scenario at the time of the cold-stress exposure. Curiously, we found increased c-Fos positive cells in the BLA but none in the DG. In this sense, the long-term neuroadaptations induced by Amph were also evaluated by c-Fos IR, a recognized tool that provides a pattern of ongoing neuronal activation in the central nervous system (Morgan and Curran 1991 ; Herdegen and Leah 1998 ; Marchese et al. 2016 ). Previously, we found that c-Fos IR cells in these regions were increased synchronously in response to memory reactivation in the passive avoidance test (Marchese et al. 2016 ). Results from the presented evidence suggest that a lower number of c-Fos positive cells in BLA is found in control animals after the Amph challenge, which also displayed reduced performance in the passive avoidance test. Our previous and present results are evidence of a resistant effect to the reduced neuronal activation only in BLA induced by acute catecholaminergic-modulating challenge, however, in the HPC, the neuronal activity remains reduced even with previous Amph exposure. Using a similar Amph administration protocol and withdrawal schedule, Tse et al. (Tse et al. 2011 ), found electrophysiological evidence showing resistance to the acute effects of Amph over BLA excitatory and inhibitory evoked responses in PFC. Indeed, we have previously reported, using the same experimental protocol, an increased synaptic transmission (lower threshold to generate LTP) within the DG in the repeated Amph group without challenge (Marchese et al. 2016 ). It is important to highlight that acute or repeated Amph administration modifies the excitatory and inhibitory neurotransmission in the BLA–PFC pathway in different ways since it is under dopaminergic activity modulation (Tse et al. 2011 ). Interestingly, the same response was reported during withdrawal of repeated cocaine administration (Perez et al. 2010 ; Gabach et al. 2013 ). In the present work, the described resistant effect of repeated Amph over decreased BLA neuronal activity and the previously observed increased hippocampal synaptic transmission evidenced the neuroadaptive changes induced by Amph. These changes may underlie the lack of impairment in memory task performance in passive avoidance after cold stress. The endurable effects of Amph exposure on working and long-term memories are mediated by the AT1-R. The AT1-R blockade prevents the long-term neuroadaptations underlying the resistance to the deleterious effect on memory induced by repeated Amph administration and unmasked by cold stress (present work) or Amph exposure (Marchese et al. 2016 , 2020 ). The AT1-R was found to play a functional role in the development of these altered responses in PFC, BLA, and HPC since their blockade prevented the long-term changes induced by repeated Amph exposure concerning c-Fos IR in PFC and BLA and synaptic transmission efficacy in HPC (Marchese et al. 2016 , 2020 ). Still, the AT1-R blocker per se did not affect the behavioral or neurochemical responses (Marchese et al. 2016 , 2020 ; Occhieppo et al. 2017 ; Basmadjian et al. 2024 ). Broad evidence from our lab and other authors supports the close relationship between DA and Ang II via AT1-R (Labandeira-Garcia et al. 2011 , 2014 ; Marchese et al. 2016 , 2020 ; Occhieppo et al. 2017 ; Rico et al. 2017 ; Basmadjian et al. 2024 ). The AT1-R/DA/Amph cross-talk may be underlying the neuroadaptive changes observed in BLA and HPC, as well as the behavioral output since the blockade of the AT1-R blunted the development of these Amph-induced alterations. This idea goes along with the results reported by our lab (Paz et al. 2011 , 2014 ; Casarsa et al. 2015 , 2025 ; Marchese et al. 2016 , 2020 ; Occhieppo et al. 2017 ; Basmadjian et al. 2024 ). On the other hand, it is important to notice that acute and chronic cold stress increases Ang II levels in plasma and brain tissue (Yang et al. 1993 ; Peng et al. 2002 ). The Ang II plasma levels have been found to increase after 4 hr of cold exposure, returning to control levels twenty-four hours later (Cassis et al. 1998 ). Recently, we showed that a challenge of Ang II administered intracerebrally induced working memory deficits in naive animals but not in animals previously exposed to Amph (Casarsa et al. 2015 , 2025 ). Remarkably, animals who received Amph and the AT1-R blocker showed working memory deficits similar to the control groups (Casarsa et al. 2015 , 2025 ). Considering the close relationship between the catecholamines and Ang II (reviewed by ), all the evidence converges again to catecholamines' role in memory processing, highlighting the AT1-R modulatory role of these neurotransmitter systems. Altogether, our results suggest that Amph-induced neuroadaptations show a broad spectrum response to different catecholamine-releasing stimuli, including pharmacological and non-pharmacological ones, which are modulated by the AT1-R. Declarations Ethical Approval All procedures were carried out following the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and the EU (Eighth Edition, 2011) and approved by the Animal Care and Use Committee, School of Chemical Sciences (Res HCD n° 46/15), National University of Cordoba. Experiments were made to minimize the number of animals used and their suffering. Consent to participate Not applicable Consent to publish Not applicable Data Accessibility Statement The authors confirm that all data underlying the findings are fully available at https://rdu.unc.edu.ar/ (Repositorio Digital de la Universidad Nacional de Córdoba). Author contributions Investigation and experiments: NAM; Conceptualization and Methodology: NAM, VBO, SMA, MFP, and CB; Data curation and formal analysis: NAM, VBO, and CB; Writing – original draft: NAM, VBO, and SMA; Writing – review and editing: MFP and CB revised it. Funding acquisition and project administration: CB. Funding This study was supported by grants from Fondo para la Investigación Científica y Tecnológica (Foncyt) BID PICT 2016 N0403, Secretaría de Ciencia y Tecnología (Secyt)-UNC, and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) PIP 2022 to Dr. Bregonzio. Conflict of interest statement The authors declare that they have no competing interests. References Anderson EM, McFadden LM, Matuszewich L (2019) Interaction of stress and stimulants in female rats: Role of chronic stress on later reactivity to methamphetamine. Behav Brain Res 376:. https://doi.org/10.1016/J.BBR.2019.112176 Arnsten AFT (1998) Catecholamine modulation of prefrontal cortical cognitive function. 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Brain Res Rev 28:370–490. https://doi.org/10.1016/S0165-0173(98)00018-6 Izquierdo I, Bevilaqua LRM, Rossato JI, et al (2006) Different molecular cascades in different sites of the brain control memory consolidation. Trends Neurosci 29:496–505. https://doi.org/10.1016/J.TINS.2006.07.005 Izquierdo I, Medina JH (1997) Memory formation: the sequence of biochemical events in the hippocampus and its connection to activity in other brain structures. Neurobiol Learn Mem 68:285–316. https://doi.org/10.1006/NLME.1997.3799 Janetsian SS, Linsenbardt DN, Lapish CC (2015) Memory impairment and alterations in prefrontal cortex gamma band activity following methamphetamine sensitization. Psychopharmacol 232:2083–2095. https://doi.org/10.1007/s00213-014-3840-7 Labandeira-Garcia JL, Garrido-Gil P, Rodriguez-Pallares J, et al (2014) Brain renin-angiotensin system and dopaminergic cell vulnerability. Front Neuroanat 8:67. https://doi.org/10.3389/fnana.2014.00067 Labandeira-Garcia JL, Rodriguez-Pallares J, Villar-Cheda B, et al (2011) Aging, Angiotensin system and dopaminergic degeneration in the substantia nigra. Aging Dis 2:257–274 Marchese NA, Artur de laVillarmois E, Basmadjian OM, et al (2016) Brain Angiotensin II AT1 receptors are involved in the acute and long-term amphetamine-induced neurocognitive alterations. Psychopharmacol 233:795–807. https://doi.org/10.1007/s00213-015-4153-1 Marchese NA, Occhieppo VB, Basmadjian OM, et al (2020) Angiotensin II modulates amphetamine-induced glial and brain vascular responses, and attention deficit via angiotensin type 1 receptor: Evidence from brain regional sensitivity to amphetamine. Eur J Neurosci 51:1026–1041. https://doi.org/10.1111/ejn.14605 Martinez-Pinilla E, Rodriguez-Perez AI, Navarro G, et al (2015) Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum. Biochem Pharmacol 96:131–142. https://doi.org/10.1016/j.bcp.2015.05.006 Morgan JI, Curran T (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu Rev Neurosci 14:421–451. https://doi.org/10.1146/ANNUREV.NE.14.030191.002225 Nieh EH, Kim SY, Namburi P, Tye KM (2013) Optogenetic dissection of neural circuits underlying emotional valence and motivated behaviors. Brain Res 1511:73–92. https://doi.org/10.1016/J.BRAINRES.2012.11.001 Nikulina EM, Covington HE, Ganschow L, et al (2004) Long-term behavioral and neuronal cross-sensitization to amphetamine induced by repeated brief social defeat stress: Fos in the ventral tegmental area and amygdala. Neuroscience 123:857–865. https://doi.org/10.1016/j.neuroscience.2003.10.029 Occhieppo VB, Marchese NA, Rodriguez ID, et al (2017) Neurovascular unit alteration in somatosensory cortex and enhancement of thermal nociception induced by amphetamine involves central AT1 receptor activation. Eur J Neurosci. https://doi.org/10.1111/ejn.13594 Paxinos G, Watson C (2009) The Rat Brain in stereotaxic coordinates, 6th edn. Elsevier, Oxford Paz MC, Assis MA, Cabrera RJ, et al (2011) The AT(1) angiotensin II receptor blockade attenuates the development of amphetamine-induced behavioral sensitization in a two-injection protocol. Synapse 65:505–512. https://doi.org/10.1002/syn.20868 Paz MC, Marchese NA, Stroppa MM, et al (2014) Involvement of the brain renin-angiotensin system (RAS) in the neuroadaptive responses induced by amphetamine in a two-injection protocol. Behav Brain Res 272:314–323. https://doi.org/10.1016/j.bbr.2014.07.021 Peng JF, Kimura B, Phillips MI (2002) The predominant role of brain angiotensinogen and angiotensin in environmentally induced hypertension. Regul Pept 110:25–32. https://doi.org/10.1016/S0167-0115(02)00156-8 Perez MF, Gabach LA, Almiron RS, et al (2010) Different chronic cocaine administration protocols induce changes on dentate gyrus plasticity and hippocampal dependent behavior. Synapse 64:742–753. https://doi.org/10.1002/syn.20788 Rico AJ, Dopeso-Reyes IG, Martinez-Pinilla E, et al (2017) Neurochemical evidence supporting dopamine D1-D2 receptor heteromers in the striatum of the long-tailed macaque: changes following dopaminergic manipulation. Brain Struct Funct 222:1767–1784. https://doi.org/10.1007/s00429-016-1306-x Riga D, Matos MR, Glas A, et al (2014) Optogenetic dissection of medial prefrontal cortex circuitry. Front Syst Neurosci 8:. https://doi.org/10.3389/FNSYS.2014.00230 Shurtleff D, Thomas JR, Schrot J, et al (1994) Tyrosine reverses a cold-induced working memory deficit in humans. Pharmacol Biochem Behav 47:935–941. https://doi.org/10.1016/0091-3057(94)90299-2 Torres-Berrio A, Cuesta S, Lopez-Guzman S, Nava-Mesa MO (2018) Interaction Between Stress and Addiction: Contributions From Latin-American Neuroscience. Front Psychol 9:. https://doi.org/10.3389/FPSYG.2018.02639 Tse MTL, Cantor A, Floresco SB (2011) Repeated amphetamine exposure disrupts dopaminergic modulation of amygdala-prefrontal circuitry and cognitive/emotional functioning. J Neurosci 31:11282–11294. https://doi.org/10.1523/JNEUROSCI.1810-11.2011 Vanderschuren LJ, Kalivas PW (2000) Alterations in dopaminergic and glutamatergic transmission in the induction and expression of behavioral sensitization: a critical review of preclinical studies. Psychopharmacol 151:99–120 Yang G, Xi ZX, Wan Y, et al (1993) Changes in circulating and tissue angiotensin II during acute and chronic stress. Biol Signals 2:166–172 Zhuo J, Moeller I, Jenkins T, et al (1998) Mapping tissue angiotensin-converting enzyme and angiotensin AT1, AT2 and AT4 receptors. J Hypertens 16:2027–2037 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 20 Oct, 2025 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted Editorial decision: Revision requested 28 Aug, 2025 Reviews received at journal 01 Jul, 2025 Reviewers agreed at journal 26 Jun, 2025 Reviewers invited by journal 24 Jun, 2025 Editor assigned by journal 16 Jun, 2025 Submission checks completed at journal 16 Jun, 2025 First submitted to journal 13 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6889905","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476257758,"identity":"e0ab590c-ddf2-4409-a686-ce3bc0fb574f","order_by":0,"name":"Natalia Andrea Marchese","email":"","orcid":"","institution":"Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET), Universidad Nacional de Córdoba","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"Andrea","lastName":"Marchese","suffix":""},{"id":476257759,"identity":"32d0fad7-90b1-4bca-99df-b288752f940a","order_by":1,"name":"Victoria Belén Occhieppo","email":"","orcid":"","institution":"Instituto de Farmacología Experimental Córdoba (IFEC-CONICET), Universidad Nacional de Córdoba","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"Belén","lastName":"Occhieppo","suffix":""},{"id":476257760,"identity":"5d88b2ad-0eb9-4734-8e63-2c6a78d12cd3","order_by":2,"name":"Sol Micaela Angulo","email":"","orcid":"","institution":"Instituto de Farmacología Experimental Córdoba (IFEC-CONICET), Universidad Nacional de Córdoba","correspondingAuthor":false,"prefix":"","firstName":"Sol","middleName":"Micaela","lastName":"Angulo","suffix":""},{"id":476257762,"identity":"b0f5bbf3-8071-4b6d-b1c1-4898a6115520","order_by":3,"name":"Mariela Fernanda Pérez","email":"","orcid":"","institution":"Instituto de Farmacología Experimental Córdoba (IFEC-CONICET), Universidad Nacional de Córdoba","correspondingAuthor":false,"prefix":"","firstName":"Mariela","middleName":"Fernanda","lastName":"Pérez","suffix":""},{"id":476257765,"identity":"2a8320b8-85e9-489f-945b-95bfcbd17dd4","order_by":4,"name":"Claudia Bregonzio","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDACdgYGZhj7AEMFhCGBVwszipYzpGphYGwjQgt/M/Mz6cI9Nnb90u0PD3ycdziPv4H54G0eBrt8XFokDrOZSc94lpY8c86BhIMztx0uljjAlmzNw5Bs2YBDiwEzg5k0z4HDyQY3Eg4c5t12OHEDAw9QhIHZAJctBszs34Ba/gO1JDYc5p0D0sIPFGGox6MFZOaBA3YGN5IZDvM2gG1hA2o5jFOLxGGeYusZB5ITJGekMRyccSw9ccZhNmPLOQbHcWrhb2/feLvggJ09v0T64w8faqwT+9ubH954U1GNUwsMJDbAmeBoIqiBgcGesJJRMApGwSgYsQAAwaBQ74n1RjUAAAAASUVORK5CYII=","orcid":"","institution":"Instituto de Farmacología Experimental Córdoba (IFEC-CONICET), Universidad Nacional de Córdoba","correspondingAuthor":true,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Bregonzio","suffix":""}],"badges":[],"createdAt":"2025-06-13 16:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6889905/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6889905/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00210-025-04741-4","type":"published","date":"2025-10-20T16:16:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85488848,"identity":"2ae7cd22-77f6-4c4a-9803-f2211c0d5a54","added_by":"auto","created_at":"2025-06-26 12:36:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55173,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of the drug administration experimental protocol used in the present work. By day 10, three experimental groups were defined (Naive/ Veh-Amph and CV-Amph) and evaluated 7 days after (day 17) in basal condition or after a cold-stress challenge. Veh: Vehicle; CV: AT1-R antagonist Candesartan; Amph: Amphetamine. RT: room temperature\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6889905/v1/7e36e720dd900d2e4d0fe0f7.png"},{"id":85488853,"identity":"03fcc86a-c066-4999-b296-0bdc6f2c52a6","added_by":"auto","created_at":"2025-06-26 12:36:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119151,"visible":true,"origin":"","legend":"\u003cp\u003eCold stress modifies adrenaline and glucose levels. Scheme of the experimental protocols for the assessment of adrenaline and glucose blood levels (A and C). Adrenaline was measured immediately after the cold challenge, showing a significant increase in the Amph-exposed group (B). Basal levels of adrenaline were not affected by previous treatments (inset) (B). Blood glucose was analyzed three times: before and after the cold challenge exposure, and after the behavioral test, in the three experimental groups, a significant effect on blood glucose was found after the behavioral test in Amph-treated animals. Data are expressed as Mean ± SEM. *p\u0026lt;0.05. Veh: Vehicle; CV: AT1-R antagonist Candesartan; Amph: Amphetamine. BS: Blood sampling.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6889905/v1/c19ca23b80ce2b9acd86e99a.png"},{"id":85488851,"identity":"1afea986-0a58-476b-9489-8cc9970551f5","added_by":"auto","created_at":"2025-06-26 12:36:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212229,"visible":true,"origin":"","legend":"\u003cp\u003eThe previous Amph administration induced a working memory deficit, and the cold challenge reversed it. A) Scheme of the experimental protocol at room temperature. Behavioral assessment of working memory performance at basal conditions by the Y-maze (B) and Holeboard (C) tests. D) Scheme showing experimental protocol with cold challenge exposure. Y-maze (E) and Holeboard (F) tests after the cold challenge exposure, with the inset showing the total exploration. In the holeboard, the holes are ordered from the most explored to the least explored hole. Data are expressed as Mean ± SEM. *p\u0026lt;0.05. Veh: Vehicle; CV: AT1-R antagonist Candesartan; Amph: Amphetamine. BS: Blood sampling.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6889905/v1/bd8fcbc66c387f89b97af895.png"},{"id":85489985,"identity":"fe6df7c3-8f0c-4f59-833e-756d035a7f15","added_by":"auto","created_at":"2025-06-26 12:44:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97243,"visible":true,"origin":"","legend":"\u003cp\u003eA) Inhibitory avoidance protocol. B) Latency observed in Inhibitory avoidance. Data are expressed as Mean ± IQ range. *p\u0026lt;0.05 significant difference from the other groups.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6889905/v1/e05c2162bf0b2887c48a7274.png"},{"id":85489987,"identity":"11972b57-0929-48ef-854b-bac779c8c184","added_by":"auto","created_at":"2025-06-26 12:44:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":180815,"visible":true,"origin":"","legend":"\u003cp\u003ePrevious Amph administration blunted the cold-stress effect on long-term memory consolidation. A) Inhibitory avoidance protocol. B) Latency observed in Inhibitory avoidance. Data are expressed as Mean ± IQ range. C-D) c-Fos IR (number of c-Fos positive cells in 0.1 mm2) in the BLA and DG, respectively. Data are expressed as Mean ± SEM. *p\u0026lt;0.05 significant difference from the other groups.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6889905/v1/175a4925648c9deb6bf80aa9.png"},{"id":85488854,"identity":"9de27160-cff8-46e5-90b6-b1bcf09c1357","added_by":"auto","created_at":"2025-06-26 12:36:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":278991,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation summarizing the results obtained previously and in the present work. Catecholamines are critical players in the cognitive process, and their hypo- or hyperfunctioning leads to a cognitive deficit. Other authors reported that repeated Amph administration decreases catecholamine levels in the prefrontal cortex while an acute administration of this psychostimulant or cold stress increases it (colored arrow). We showed that repeated Amph exposure altered the catecholamines and the cognitive performance via AT1-R, and it was reversed by Amph challenge or cold stress (black arrows).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6889905/v1/27acfbbd379c1e56248c18ca.png"},{"id":94490367,"identity":"ac2caf88-546a-41dd-81bb-4499090b9d78","added_by":"auto","created_at":"2025-10-27 17:09:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1747946,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6889905/v1/781034da-b149-4007-9698-3f31c7d56caa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"AT1 Receptors Mediate the Effects of Cold Stress and Amphetamine on Neurocognitive Performance","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe role of the brain renin-angiotensin system is complex. It regulates vascular, glial, and neuronal functions through its main active peptide, angiotensin II (Ang II). Particularly, this peptide plays an important role in the modulation of central dopaminergic neurotransmission and has a crucial role in sensorial and cognitive processes through the activation of angiotensin receptors type 1 (AT1-R) (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Basmadjian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Indeed, AT1-R is located in the soma and terminal fields of dopaminergic neurons, and there is evidence of a cross-regulation between dopamine (DA) and Ang II systems (Brown et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Zhuo et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Martinez-Pinilla et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Basmadjian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe recreational use of amphetamine (Amph) is based on its stimulant properties over the central nervous system since it promotes mainly noradrenergic and dopaminergic neurotransmission. However, Amph exposure induces long-term changes in multiple neuronal circuits, modifying their future responses to pharmacological or non-pharmacological challenges (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our group previously addressed the effects of Amph exposure on learning and memory processes, along with altered neuronal connectivity. In this sense, acute Amph impairs memory retention in male rats in the one-trial inhibitory avoidance (IA) response when administered immediately post-training, partially involving AT1-R activation (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, previous exposure to Amph, followed by a withdrawal period, altered the response of the animals in the IA test concomitant with their neuronal activation pattern in the basolateral amygdala (BLA) after an Amph challenge (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The long-lasting alterations induced by Amph were also observed in the hippocampal synaptic transmission, as a lower threshold was required for LTP generation, which was prevented by the AT1-R blockade (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Long-term effects of repeated Amph exposure were also documented for executive neurocognitive tasks such as working memory, which implies active modulation of catecholaminergic levels in the Prefrontal Cortex (PFC) (Marchese et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Basmadjian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCross-effects between psychostimulants and stress have been identified in the long-term neuroadaptation involving catecholamine neurotransmission, both in animal models and humans (Nikulina et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Booij et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Torres-Berrio et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Anderson et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Considering that Amph exposure induces neuroadaptive changes, we aimed to evaluate whether Amph's previous history modifies the neurocognitive response to a non-pharmacological challenge such as cold stress. Likewise, to find out if AT1-R is involved in the possible alterations resulting from the cross-effects of exposure to Amph and cold stress.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eA total of 269 adult male Wistar rats were obtained from the Department of Pharmacology Otto Orsingher vivarium (Facultad de Ciencias Qu\u0026iacute;micas, Universidad Nacional de C\u0026oacute;rdoba, Argentina) and randomly housed in groups of XX one week before the beginning of the experimental protocol. Throughout the experiment, animals were maintained in controlled environmental conditions (20\u0026ndash;24\u0026deg;C, 12-h light/dark cycle with lights on at 07 a.m.) and had free access to food and water.\u003c/p\u003e \u003cp\u003e All procedures were conducted with the approval of the Animal Care and Use Committee of the Facultad de Ciencias Qu\u0026iacute;micas, Universidad Nacional de C\u0026oacute;rdoba, Argentina (Res n\u0026ordm; 46/15), following the NIH Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Drugs\u003c/h2\u003e \u003cp\u003eThe selective AT\u003csub\u003e1\u003c/sub\u003e-R antagonist, Candesartan cilexetil (CV, Laboratorios Phoenix, Buenos Aires, Argentina), was dissolved in NaHCO3 0.1N (vehicle, Veh). D-amphetamine sulfate (Amph-SIGMA, Argentina) was dissolved in 0.9% NaCl (saline).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design\u003c/h2\u003e \u003cp\u003eAnimals received Veh/CV (3mg/kg by gavage) once daily for 5 days. From day 6 to 10, they were injected daily with Amph (2.5mg/kg/ Sal i.p.) and then left undisturbed in their home cages until the day of the experiment (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Occhieppo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On day 17, animals were tested for cognitive performance in basal conditions (room temperature- RT) or after cold exposure (4h at 4\u0026deg;C- cold challenge). A na\u0026iuml;ve group was evaluated under the same conditions on day 17 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBlood samples were obtained immediately after the cold challenge and/or the behavioral test, as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. Blood adrenaline was assayed by reverse-phase HPLC (Model 582 solvent delivery, ESA, Chelmsford, MA, and RP 18 Column) with electrochemical detection (ESA coulochem II) and glucose with a meter test (Optium Xceed meter, Abbott, IL, USA). Samples for immunohistochemistry were taken as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cognitive evaluation: working memory\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Y-maze test\u003c/h2\u003e \u003cp\u003eAnimals were placed at the center of a Y-shaped maze and left for free exploration for 8 minutes. The sequence of chosen entries was manually recorded, where three consecutive choices of three different arms were counted as an alternation, and the total possible alternations were the total number of entries minus 2. Finally, the percentage of alternations was calculated. Animals displaying 2 minutes of immobility between arms were excluded from the final analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Holeboard test\u003c/h2\u003e \u003cp\u003eExploratory behavior was evaluated after one 5-minute exposure to an arena with five holes. The number of heads dipping into each hole was quantified (visits to the hole). The number of visits was analyzed for each animal in the experimental groups, ordering the values from the most to the least visited hole.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cognitive evaluation: Long-term memory\u003c/h2\u003e \u003cp\u003eThe inhibitory avoidance response was determined by employing a step-through apparatus with an illuminated and a dark compartment (60\u0026times;30\u0026times;30cm each) connected by a guillotine door. The experiment was conducted on 2 consecutive days on the same day time. On the first day (training session), each rat was placed in the illuminated compartment, and once the rat entered completely into the dark compartment, it received 3 electric foot shocks (0.5mA, 3s each, separated by 30s). A maximum of 60 seconds was considered for the animal to enter the dark compartment; otherwise, the animals were dismissed from the experiment. On the second day (test session), the same procedure was followed (no foot-shock delivery), and the time taken to enter the dark compartment was recorded (max. 300s). This parameter is considered a measure of memory retention and is displayed in the graph as latency. In accordance with previous work (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the acute effect of cold stress on the inhibitory avoidance response was evaluated by cold exposure immediately after the training session in non-CV-treated \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e and CV-treated groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Immunohistochemistry: c-Fos immunoreactivity\u003c/h2\u003e \u003cp\u003eNinety minutes after the test session, animals were anesthetized and perfused transcardially with 250 mL of physiologic solution (0.9% NaCl) and heparin (200\u0026micro;L/L), followed by 400 mL of 4% paraformaldehyde in 0.1M phosphate buffer (PB, pH 7.4). The brains were post-fixed in the same paraformaldehyde solution overnight and stored at 4\u0026deg;C, 30% sucrose in PB. Coronal sections of 40 \u0026micro;m were cut using a freezing microtome (Leica CM15105), collected in PB 0.01M, and placed in a mixture of 10% H2O2 and 10% methanol for 2 h. Samples were incubated in 10% normal horse serum (NHS) (Natocor, Villa Carlos Paz, C\u0026oacute;rdoba, Argentina) in PB 0.1M for 2 h and immediately after the free-floating sections were incubated overnight at room temperature in a rabbit anti-c-Fos antibody (1:20,000 in 2% NHS-0.3% Triton-PB 0.1 M; Ab-5; Oncogene Science, Manhasset, NY). The sections were incubated with biotin-labeled anti-rabbit secondary antibody (1:2000 in 2% NHS-PB 0.1M; Jackson Laboratory) and avidin-biotin-peroxidase complex (1:200 in 2% NHS-PB 0.1M; Vector Laboratories) for 2 h each at room temperature. The peroxidase label was detected with diaminobenzidine hydrochloride (Sigma Chemical Co.) and intensified with 1% cobalt chloride and 1% nickel ammonium sulfate for a violet nuclear reaction product. Finally, the free-floating sections were mounted on gelatinized slides, air-dried overnight, dehydrated, cleared in xylene, and placed under a coverslip with DPX mountant for histology (Flucka Analytical).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Image processing\u003c/h2\u003e \u003cp\u003eAll images were obtained by using a computerized system that included a Leica DM 4000B microscope equipped with Leica FW4000 and a DFC Leica digital camera attached to a contrast enhancement device. The images were processed using ImageJ software (U.S. National Institutes of Health, USA). The analyses were made blinded to the experimental groups. Considering our previous work (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), c-Fos immunoreactivity (c-Fos IR) assay was evaluated at the hippocampal Dentate Gyrus (DG; Bregma \u0026minus;\u0026thinsp;3.30/\u0026minus;3.60 mm) and basolateral amygdala (BLA; Bregma \u0026minus;\u0026thinsp;2.56/\u0026minus;2.80 mm), which were identified and delimited according to Paxinos and Watson atlas (Paxinos and Watson \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Image thresholds were fixed between intervals of 120\u0026ndash;150 in black-and-white conditions; all lower values were considered background. C-Fos IR neurons were identified by dense black staining of the nucleus and counted by setting a size range for cellular nuclei (8 to 12 \u0026micro;m in diameter). The measurements were taken bilaterally in two sections, and the final value was obtained as the average of the four counted sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analyses and Artwork\u003c/h2\u003e \u003cp\u003eData were analyzed using one-way or two-way ANOVA and reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. If an interaction and/or main effect was observed, comparisons were made using Bonferroni's post-test. The Unpaired t-test was used for the blood catecholamines analysis. The Kruskal-Wallis test and Dunn\u0026rsquo;s multiple comparisons were used for inhibitory avoidance analysis. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. The analyses were performed using GraphPad Prism\u0026reg; 8.02 software, and the figures were made using Inkscape\u0026reg;. The artwork was performed using Canva\u0026reg;.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e \u003cem\u003e3.1. Amphetamine administration induced working memory deficit via AT1-R, and the cold challenge reversed it, involving catecholamines and glucose changes.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBlood adrenaline levels were evaluated after cold stress exposure and relativized to the levels of na\u0026iuml;ve rats. After the cold exposure, the Veh-Amph group showed increased adrenaline levels compared with the CV-Amph group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Adrenaline levels were not significantly different between the Veh-Amph and CV-Amph groups at room temperature \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; \u003cb\u003einset)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eBlood glucose was evaluated before, during, and after the cold challenge and behavioral test. No significant differences were observed immediately after the cold challenge compared with the pre-cold; however, exposure to the behavioral tests significantly increased the blood glucose levels in the Veh-Amph group compared with the pre-cold and post-cold assessments. This increase was not evidenced in the Naive and CV-Amph groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical results for blood adrenaline and glucose levels. CV: AT1-R antagonist. Unpaired t-test for adrenaline and Two-way ANOVA and Bonferroni\u0026rsquo;s multiple comparisons for glucose levels. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eBlood adrenaline\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnpaired t-test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVeh-Amph vs. CV-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.0061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBlood glucose\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwo-way ANOVA RM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF (4, 24)\u0026thinsp;=\u0026thinsp;3.260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.0286\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF (2, 24)\u0026thinsp;=\u0026thinsp;3.869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.0350\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColumn Factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF (2, 12)\u0026thinsp;=\u0026thinsp;0.2238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.8028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubject\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF (12, 24)\u0026thinsp;=\u0026thinsp;1.530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.1811\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBonferroni's multiple comparisons test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNaive group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Pre-cold\" time vs \"Post-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Test\" time vs \"Pre-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.3879\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Test\" time vs \"Post-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.2904\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eVeh-Amph group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Pre-cold\" time vs \"Post-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Test\" time vs \"Pre-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e*0.0211\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Test\" time vs \"Post-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e*0.0348\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCV-Amph group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Pre-cold\" time vs \"Post-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Test\" time vs \"Pre-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\"Test\" time vs \"Post-cold\" time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn basal conditions of room temperature, the animals exposed to Amph showed decreased alternation percentage in the Y-Maze paradigm compared with the naive, and the previous CV administration prevented this effect. No significant differences were observed between the three evaluated groups after the cold challenge \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eRegarding the holeboard test, the Veh-Amph group showed a significant difference between the first and second most explored holes at basal conditions. After the cold challenge, the Veh-Amph group did not show the difference between the most explored holes, similar to the control group. The Veh-Sal and CV-Amph groups did not show this alteration at basal conditions nor after the cold challenge exposure \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical results for Y-Maze and Holeboard test. CV: AT1-R blockade. One-way ANOVA and Bonferroni\u0026rsquo;s multiple comparisons. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eY-Maze test\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e% Alternance Basal conditions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e% Alternance Cold challenge\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eOne-way ANOVA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eOne-way ANOVA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;7.457\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.0017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;0.05162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.9498\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBonferroni's post-hoc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBonferroni's post-hoc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaive vs. Veh-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*0.0024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaive vs. CV-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVeh-Amph vs CV-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*0.0158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTotal Entries Basal Conditions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eTotal Entries Cold Challenge\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;0.9185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.4072\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;0.09477\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.9099\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHoleboard test\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eExploration Basal conditions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eExploration Cold Challenge\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eOne-way ANOVA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eOne-way ANOVA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;19.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;13.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBonferroni's post-hoc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBonferroni'spost-hocc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaive 1 vs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNaive 1 vs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1114\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVeh-Amph 1 vs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVeh-Amph 1 vs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3787\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCV-Amph 1 vs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCV-Amph 1 vs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0715\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Amphetamine promotes resistance to cold-stress-induced memory impairment via AT1-R activation.\u003c/h2\u003e \u003cp\u003eRegarding inhibitory avoidance, Dunn\u0026acute;s tests indicate significant differences between animals exposed to shock and those not exposed to shock. Interestingly, exposure to cold stress interferes with memory storage, marked by similar latency times to that of animals never exposed to shock \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical results for the Inhibitory Avoidance test. Kruskal-Wallis test and Dunn\u0026rsquo;s multiple comparisons. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eInhibitory Avoidance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eKruskal-Wallis test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKruskal-Wallis statistic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDunn's multiple comparisons test p\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo shock vs. Shock\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u0026lt;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo shock vs. Shock\u0026thinsp;+\u0026thinsp;Cold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0884\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShock vs. Shock\u0026thinsp;+\u0026thinsp;Cold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*0.0303\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhen animals were previously exposed to Amph, the cold challenge did not interfere with memory storage, displaying longer latency than the naive group receiving the cold challenge; whereas the animals exposed to CV/Amph showed similar latency to naive animals after the cold challenge \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe c-Fos IR evaluated after the inhibitory avoidance test showed that the Veh/Amph group had increased c-Fos expression in the BLA, which was not observed in the CV/Amph group. No significant differences were observed in DG \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical results for the Inhibitory Avoidance test and neuronal activation in basolateral amygdala and dentate gyrus. Kruskal-Wallis test and Dunn\u0026rsquo;s multiple comparisons were used for the behavioral test, and One-way ANOVA and Bonferroni\u0026rsquo;s post hoc for the c-Fos IR analysis. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eInhibitory Avoidance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eKruskal-Wallis test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKruskal-Wallis statistic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDunn's multiple comparisons test p\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl vs. Veh-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*0.0124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl vs. CV-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVeh-Amph vs. CV-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ec-Fos immunoreactivity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eOne-way ANOVA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBasolateral amygdala\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;8.421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.0040\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBonferroni's post-hoc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl vs. Veh-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*0.0043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl vs. CV-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7341\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVeh-Amph vs. CV-Amph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*0.0256\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDentate gyrus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;0.9252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.4194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eIn the present work, we extend our previous observations regarding the role of AT1-R in the Amph-induced neuroadaptations, proposing a long-term cross-talk with the catecholaminergic system, when it is activated by environmental stress. The cold exposure, probably acting as a challenge agent of the catecholaminergic system, reversed the Amph-induced working memory deficit, although this prior history with the psychostimulant blunted the interference of cold stress on long-term memory consolidation; thus mimicking previously reported effects after an Amph challenge (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the same direction, the AT1-R blockade prevented both alterations induced by Amph, further supporting that similar processes underpin Amph-induced adaptations when challenged by pharmacological or environmental stimuli.\u003c/p\u003e \u003cp\u003eWorking memory is a complex process that refers to temporary information storage, on a scale of seconds to minutes, necessary for the performance of a cognitive task (Cowan \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Riga et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Catecholaminergic activity plays a preponderant role in working memory performance, given that moderate catecholaminergic activity in the PFC is necessary for optimal functioning. In contrast, a hypo-function or excess of dopamine/noradrenaline would be responsible for cortical dysfunction and working memory deficits. Results from our laboratory previously showed that Amph exposure induced attention memory deficits associated with structural and functional alterations in PFC, even long-term after the last Amph administration (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Basmadjian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This PFC dysfunction might be related to decreased glutamate and DA levels and diminished electrical activity in this brain area (Janetsian et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the same direction, psychostimulant users exhibit attention deficits and functional changes at the cortical level (Downey and Loftis \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). According to this evidence, we previously found that Amph challenge administration (0.5mg/kg) reversed the attention memory deficit induced by previous exposure to the psychostimulant (Marchese et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which could be attributed to the Amph's positive effect on catecholamine release (Arnsten \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Blaiss and Janak \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Cold stress is a well-documented stimulus for the activation of the sympathetic nervous system (Fiedler et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The blood glucose level, an important marker of sympathetic activation, was found to increase in the Amph-treated group only after the working memory test. Interestingly, exposure to cold stimuli has been found to negatively affect short-term and working memory in humans, associated with reduced brain catecholamine levels, since it was found to be improved by administration of the catecholamine precursor tyrosine (Shurtleff et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). All this evidence points out long-term neuroadaptations and catecholaminergic misbalance induced by catecholaminergic modulators (Amph or cold stress), leading to working memory deficit. Indeed, we observed increased blood adrenaline levels only in the Veh-Amph group. Therefore, cold stress or an Amph challenge would improve the attention deficit since both stimuli trigger catecholamine release (Marchese et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe limbic system activation, with single or repeated psychostimulant administration, triggers the development of neuroadaptations that can be revealed after a withdrawal period, at behavioral and neurochemical levels, and by using pharmacological or non-pharmacological challenges (Vanderschuren and Kalivas \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Indeed, we reported that Amph exposure induced increased hippocampal synaptic plasticity, and resistance to the interference of catecholamines in long-term memory consolidation using the same experimental protocol as in the present study (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, our previous findings indicated that repeated Amph administration did not affect the animal\u0026rsquo;s performance in the passive avoidance test, however, the repeated Amph-induced neuroadaptations were evidenced after a week of withdrawal as a resistance to the deleterious effect of the post-training Amph challenge administration (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the same direction discussed above, it is broadly known that emotions and wakefulness influence the learning process. Moderate emotional states have reinforcing effects, while high emotional levels, such as those experienced during high-stress situations, present detrimental effects (Izquierdo and Medina \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The evidence converges on the role of catecholamines in wakefulness, emotion, and stress response; as well as in the presence of its receptors in areas involved in the processing of long-term memories with emotional valences, such as PFC, hippocampus (HPC), and BLA (Izquierdo et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Nieh et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Since we found that the previous history with Amph blunted the cold-stress effect over long-term memory, we propose that the Amph administration induced endurable neuroadaptations changing the brain scenario at the time of the cold-stress exposure. Curiously, we found increased c-Fos positive cells in the BLA but none in the DG. In this sense, the long-term neuroadaptations induced by Amph were also evaluated by c-Fos IR, a recognized tool that provides a pattern of ongoing neuronal activation in the central nervous system (Morgan and Curran \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Herdegen and Leah \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Previously, we found that c-Fos IR cells in these regions were increased synchronously in response to memory reactivation in the passive avoidance test (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Results from the presented evidence suggest that a lower number of c-Fos positive cells in BLA is found in control animals after the Amph challenge, which also displayed reduced performance in the passive avoidance test. Our previous and present results are evidence of a resistant effect to the reduced neuronal activation only in BLA induced by acute catecholaminergic-modulating challenge, however, in the HPC, the neuronal activity remains reduced even with previous Amph exposure. Using a similar Amph administration protocol and withdrawal schedule, Tse et al. (Tse et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), found electrophysiological evidence showing resistance to the acute effects of Amph over BLA excitatory and inhibitory evoked responses in PFC. Indeed, we have previously reported, using the same experimental protocol, an increased synaptic transmission (lower threshold to generate LTP) within the DG in the repeated Amph group without challenge (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is important to highlight that acute or repeated Amph administration modifies the excitatory and inhibitory neurotransmission in the BLA\u0026ndash;PFC pathway in different ways since it is under dopaminergic activity modulation (Tse et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Interestingly, the same response was reported during withdrawal of repeated cocaine administration (Perez et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gabach et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the present work, the described resistant effect of repeated Amph over decreased BLA neuronal activity and the previously observed increased hippocampal synaptic transmission evidenced the neuroadaptive changes induced by Amph. These changes may underlie the lack of impairment in memory task performance in passive avoidance after cold stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eThe endurable effects of Amph exposure on working and long-term memories are mediated by the AT1-R.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe AT1-R blockade prevents the long-term neuroadaptations underlying the resistance to the deleterious effect on memory induced by repeated Amph administration and unmasked by cold stress (present work) or Amph exposure (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The AT1-R was found to play a functional role in the development of these altered responses in PFC, BLA, and HPC since their blockade prevented the long-term changes induced by repeated Amph exposure concerning c-Fos IR in PFC and BLA and synaptic transmission efficacy in HPC (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Still, the AT1-R blocker per se did not affect the behavioral or neurochemical responses (Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Occhieppo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Basmadjian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Broad evidence from our lab and other authors supports the close relationship between DA and Ang II via AT1-R (Labandeira-Garcia et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Occhieppo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rico et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Basmadjian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The AT1-R/DA/Amph cross-talk may be underlying the neuroadaptive changes observed in BLA and HPC, as well as the behavioral output since the blockade of the AT1-R blunted the development of these Amph-induced alterations. This idea goes along with the results reported by our lab (Paz et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Casarsa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Marchese et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Occhieppo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Basmadjian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, it is important to notice that acute and chronic cold stress increases Ang II levels in plasma and brain tissue (Yang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The Ang II plasma levels have been found to increase after 4 hr of cold exposure, returning to control levels twenty-four hours later (Cassis et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Recently, we showed that a challenge of Ang II administered intracerebrally induced working memory deficits in naive animals but not in animals previously exposed to Amph (Casarsa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Remarkably, animals who received Amph and the AT1-R blocker showed working memory deficits similar to the control groups (Casarsa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Considering the close relationship between the catecholamines and Ang II (reviewed by ), all the evidence converges again to catecholamines' role in memory processing, highlighting the AT1-R modulatory role of these neurotransmitter systems.\u003c/p\u003e \u003cp\u003eAltogether, our results suggest that Amph-induced neuroadaptations show a broad spectrum response to different catecholamine-releasing stimuli, including pharmacological and non-pharmacological ones, which are modulated by the AT1-R.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were carried out following the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and the EU (Eighth Edition, 2011) and approved by the Animal Care and Use Committee, School of Chemical Sciences (Res HCD n\u0026deg; 46/15), National University of Cordoba. Experiments were made to minimize the number of animals used and their suffering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Accessibility Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all data underlying the findings are fully available at\u0026nbsp;https://rdu.unc.edu.ar/ (Repositorio Digital de la Universidad Nacional de C\u0026oacute;rdoba).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigation and experiments: NAM; Conceptualization and Methodology: NAM, VBO, SMA, MFP, and CB; Data curation and formal analysis: NAM, VBO, and CB; Writing \u0026ndash; original draft: NAM, VBO, and SMA; Writing \u0026ndash; review and editing: MFP and CB revised it. Funding acquisition and project administration: CB.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from Fondo para la Investigaci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (Foncyt) BID PICT 2016 N0403, Secretar\u0026iacute;a de Ciencia y Tecnolog\u0026iacute;a (Secyt)-UNC, and Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (CONICET) PIP 2022 \u0026nbsp;to Dr. Bregonzio.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnderson EM, McFadden LM, Matuszewich L (2019) Interaction of stress and stimulants in female rats: Role of chronic stress on later reactivity to methamphetamine. 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Psychopharmacol 229:41\u0026ndash;50. https://doi.org/10.1007/s00213-013-3084-y\u003c/li\u003e\n\u003cli\u003eHerdegen T, Leah JD (1998) Inducible and constitutive transcription factors in the mammalian nervous system: Control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res Rev 28:370\u0026ndash;490. https://doi.org/10.1016/S0165-0173(98)00018-6\u003c/li\u003e\n\u003cli\u003eIzquierdo I, Bevilaqua LRM, Rossato JI, et al (2006) Different molecular cascades in different sites of the brain control memory consolidation. Trends Neurosci 29:496\u0026ndash;505. https://doi.org/10.1016/J.TINS.2006.07.005\u003c/li\u003e\n\u003cli\u003eIzquierdo I, Medina JH (1997) Memory formation: the sequence of biochemical events in the hippocampus and its connection to activity in other brain structures. Neurobiol Learn Mem 68:285\u0026ndash;316. https://doi.org/10.1006/NLME.1997.3799\u003c/li\u003e\n\u003cli\u003eJanetsian SS, Linsenbardt DN, Lapish CC (2015) Memory impairment and alterations in prefrontal cortex gamma band activity following methamphetamine sensitization. Psychopharmacol 232:2083\u0026ndash;2095. https://doi.org/10.1007/s00213-014-3840-7\u003c/li\u003e\n\u003cli\u003eLabandeira-Garcia JL, Garrido-Gil P, Rodriguez-Pallares J, et al (2014) Brain renin-angiotensin system and dopaminergic cell vulnerability. Front Neuroanat 8:67. https://doi.org/10.3389/fnana.2014.00067\u003c/li\u003e\n\u003cli\u003eLabandeira-Garcia JL, Rodriguez-Pallares J, Villar-Cheda B, et al (2011) Aging, Angiotensin system and dopaminergic degeneration in the substantia nigra. Aging Dis 2:257\u0026ndash;274\u003c/li\u003e\n\u003cli\u003eMarchese NA, Artur de laVillarmois E, Basmadjian OM, et al (2016) Brain Angiotensin II AT1 receptors are involved in the acute and long-term amphetamine-induced neurocognitive alterations. Psychopharmacol 233:795\u0026ndash;807. https://doi.org/10.1007/s00213-015-4153-1\u003c/li\u003e\n\u003cli\u003eMarchese NA, Occhieppo VB, Basmadjian OM, et al (2020) Angiotensin II modulates amphetamine-induced glial and brain vascular responses, and attention deficit via angiotensin type 1 receptor: Evidence from brain regional sensitivity to amphetamine. Eur J Neurosci 51:1026\u0026ndash;1041. https://doi.org/10.1111/ejn.14605\u003c/li\u003e\n\u003cli\u003eMartinez-Pinilla E, Rodriguez-Perez AI, Navarro G, et al (2015) Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum. Biochem Pharmacol 96:131\u0026ndash;142. https://doi.org/10.1016/j.bcp.2015.05.006\u003c/li\u003e\n\u003cli\u003eMorgan JI, Curran T (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu Rev Neurosci 14:421\u0026ndash;451. https://doi.org/10.1146/ANNUREV.NE.14.030191.002225\u003c/li\u003e\n\u003cli\u003eNieh EH, Kim SY, Namburi P, Tye KM (2013) Optogenetic dissection of neural circuits underlying emotional valence and motivated behaviors. Brain Res 1511:73\u0026ndash;92. https://doi.org/10.1016/J.BRAINRES.2012.11.001\u003c/li\u003e\n\u003cli\u003eNikulina EM, Covington HE, Ganschow L, et al (2004) Long-term behavioral and neuronal cross-sensitization to amphetamine induced by repeated brief social defeat stress: Fos in the ventral tegmental area and amygdala. Neuroscience 123:857\u0026ndash;865. https://doi.org/10.1016/j.neuroscience.2003.10.029\u003c/li\u003e\n\u003cli\u003eOcchieppo VB, Marchese NA, Rodriguez ID, et al (2017) Neurovascular unit alteration in somatosensory cortex and enhancement of thermal nociception induced by amphetamine involves central AT1 receptor activation. Eur J Neurosci. https://doi.org/10.1111/ejn.13594\u003c/li\u003e\n\u003cli\u003ePaxinos G, Watson C (2009) The Rat Brain in stereotaxic coordinates, 6th edn. Elsevier, Oxford\u003c/li\u003e\n\u003cli\u003ePaz MC, Assis MA, Cabrera RJ, et al (2011) The AT(1) angiotensin II receptor blockade attenuates the development of amphetamine-induced behavioral sensitization in a two-injection protocol. 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Pharmacol Biochem Behav 47:935\u0026ndash;941. https://doi.org/10.1016/0091-3057(94)90299-2\u003c/li\u003e\n\u003cli\u003eTorres-Berrio A, Cuesta S, Lopez-Guzman S, Nava-Mesa MO (2018) Interaction Between Stress and Addiction: Contributions From Latin-American Neuroscience. Front Psychol 9:. https://doi.org/10.3389/FPSYG.2018.02639\u003c/li\u003e\n\u003cli\u003eTse MTL, Cantor A, Floresco SB (2011) Repeated amphetamine exposure disrupts dopaminergic modulation of amygdala-prefrontal circuitry and cognitive/emotional functioning. J Neurosci 31:11282\u0026ndash;11294. https://doi.org/10.1523/JNEUROSCI.1810-11.2011\u003c/li\u003e\n\u003cli\u003eVanderschuren LJ, Kalivas PW (2000) Alterations in dopaminergic and glutamatergic transmission in the induction and expression of behavioral sensitization: a critical review of preclinical studies. Psychopharmacol 151:99\u0026ndash;120\u003c/li\u003e\n\u003cli\u003eYang G, Xi ZX, Wan Y, et al (1993) Changes in circulating and tissue angiotensin II during acute and chronic stress. Biol Signals 2:166\u0026ndash;172\u003c/li\u003e\n\u003cli\u003eZhuo J, Moeller I, Jenkins T, et al (1998) Mapping tissue angiotensin-converting enzyme and angiotensin AT1, AT2 and AT4 receptors. J Hypertens 16:2027\u0026ndash;2037\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Angiotensin II, AT1-receptor, Amphetamine, Working memory, cold stress","lastPublishedDoi":"10.21203/rs.3.rs-6889905/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6889905/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eIn this work, we aimed to evaluate whether Amph exposure modifies the future neurocognitive response to cold stress, an environmental cue, and the possible role of the AT1-R.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMale Wistar rats received AT1-R blocker (Candesartan)/Vehicle from day 0 to 5, and Amph/Saline from day 6 to 10. After seven days of withdrawal, the animals were exposed to a cold challenge (4\u0026deg;C for 4 h) and tested for working and long-term memory.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCold challenge reversed the Amph-induced working memory deficit. Previous Amph exposure blunted the interference of cold stress on long-term memory consolidation. Blood adrenaline and glucose levels were increased in the Amph-treated animals after cold stress and/or test exposure. Remarkably, the AT1-R blockade prevented all these alterations induced by Amph exposure and elicited by cold-challenge.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results indicate that Amph-induced neuroadaptations exhibit a wide range of responses to catecholamine-releasing stimuli, including pharmacological and non-pharmacological challenges and environmental conditions involving the AT1-R.\u003c/p\u003e","manuscriptTitle":"AT1 Receptors Mediate the Effects of Cold Stress and Amphetamine on Neurocognitive Performance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 12:36:37","doi":"10.21203/rs.3.rs-6889905/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-28T10:19:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-01T15:41:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192667686397375118214680793405193261172","date":"2025-06-26T08:47:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-24T08:17:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-16T04:30:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-16T04:29:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2025-06-13T16:45:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"94e4c518-fe05-43f7-9616-6ca9415127f3","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:24:01+00:00","versionOfRecord":{"articleIdentity":"rs-6889905","link":"https://doi.org/10.1007/s00210-025-04741-4","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2025-10-20 16:16:35","publishedOnDateReadable":"October 20th, 2025"},"versionCreatedAt":"2025-06-26 12:36:37","video":"","vorDoi":"10.1007/s00210-025-04741-4","vorDoiUrl":"https://doi.org/10.1007/s00210-025-04741-4","workflowStages":[]},"version":"v1","identity":"rs-6889905","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6889905","identity":"rs-6889905","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}