Haloperidol prevents the consolidation of short-term traumatic memory by modulating interleukin-1β in neural substrates

Haloperidol prevents the consolidation of short-term traumatic memory by modulating interleukin-1β in neural substrates | 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 Haloperidol prevents the consolidation of short-term traumatic memory by modulating interleukin-1β in neural substrates Pei-Jui Hung, Cai-N Cheng, Anna Kozłowska, Arthur C. Tsai, Chun-Hsiung Tseng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7497727/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The role of the dopamine system in modulating traumatic memories in posttraumatic stress disorder (PTSD) is unclear. This study investigated the effect of a dopamine D2 receptor antagonist, haloperidol, on fear responses in rats following a footshock. Fear was assessed by measuring freezing behavior during tests of short-term (STM) and long-term (LTM) memory. All rats were administered haloperidol 45 minutes before a footshock (3mA, 10 seconds) and were subsequently placed in the apparatus for two minutes daily over the next three days without a footshock. In Experiment 1, STM was tested by placing the rats back in the apparatus for a two-minute session. In Experiment 2, LTM was assessed using an identical test, which was repeated seven days later. Experiment 3 utilized the STM protocol, followed by an analysis of brain tissue for the protein interleukin-1b (IL-1b). Results demonstrated that haloperidol reduced freezing behavior in the short-term traumatic memory period. This effect was not observed a week later, indicating that haloperidol did not alter consolidated, long-term fear memories. The footshock was found to alter IL-1b expression in key fear and memory circuits. Importantly, haloperidol’s fear-reducing effect was associated with reduced IL − 1b expression in the nucleus accumbens (NAc), central amygdala (CeA), and hippocampus (CA1, CA2, CA3). These findings suggest that dopamine D2 receptor blockade could represent a new therapeutic approach to preventing the initial consolidation of traumatic memories shortly after an event, but its efficacy in treating established, long-term PTSD may be limited. Psychology Psychiatry Posttraumatic stress disorder dopamine medial prefrontal cortex amygdala hippocampus short-term memory long-term memory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Posttraumatic stress disorder (PTSD) is a severe psychiatric condition associated with learning and memory deficits [ 1 , 2 ]. Its characteristic symptoms include the persistent re-experiencing of a traumatic event, chronic emotional arousal, and the avoidance of stimuli associated with the traumatic memory [ 3 ]. This avoidance behavior is a product of classical conditioning, which involves forming an association between an environmental stimulus (conditioned stimulus, CS) and the traumatic event (unconditioned stimulus, US [ 4 , 5 ]. During this process, the CS is encoded into short-term memory (STM) and subsequently consolidated into long-term memory (LTM), forming a durable memory trace in the brain [ 6 ]. Strong evidence indicates that PTSD is highly correlated with neuroinflammation in the brain [ 7 , 8 ]. Patients with PTSD exhibit elevated levels of various neuroinflammatory biomarkers, such as interleukin-1 (IL-1), interleukin-6, tumor necrosis factor-alpha, nuclear factor-kB, and C-reactive protein [ 9 , 10 ]. Animal models often use fear conditioning tasks to mimic the clinical symptoms of PTSD [ 11 , 12 ] and show similar results, suggesting a link between PTSD or stress and increased neuroinflammation [ 13 – 16 ]. For example, studies have shown that footshock stress not only regulates aversive odor cues but also induces inflammatory cytokine responses [ 17 ]. Furthermore, optogenetic stimulation or inhibition in the basolateral amygdala (BLA) can modulate stress- and fear-memory-induced sleep alterations as well as neuroinflammatory responses in the mPFC and hippocampus [ 16 ]. These findings support the use of footshock stress in animal models mimicking the production of neuroinflammatory responses associated with the traumatic events that elicit PTSD. A recent review suggests the involvement of the dopamine system in the symptoms of PTSD and anxiety disorders [ 11 ]. For example, the dopamine D2/D3 receptor agonists rotigotine and pramipexole have been shown to attenuate freezing time in auditory fear conditioning and reduce depression responses in a single-prolonged stress model [ 18 ]. Furthermore, the 10R/10R variant of the dopamine transporter 1 (DAT1) gene is associated with a higher risk of developing PTSD, implicating it in the disorder's pathophysiology. [ 19 ]. Similarly, a variant of the dopamine D2 receptor gene is in linkage disequilibrium with the D2A1 allele, increasing the risk of PTSD, suggesting that D2 receptor signaling deficits contribute to susceptibility [ 20 ]. While it has been established that the dopamine system plays a role in PTSD symptoms, which stage of learning and memory is governed by dopamine receptors remains unclear. The present study aims to address this issue. Previous studies have demonstrated the involvement of the medial prefrontal cortex (mPFC), amygdala, and hippocampus in the fear behaviors characteristic of PTSD [ 21 , 22 ]. For example, neuroimaging research has revealed that hyperactivation in the amygdala is associated with the severe symptoms of PTSD; brain mapping evidence demonstrates that PTSD patients have a smaller and hypoactive mPFC as well as a diminished hippocampus with reduced neuronal and functional interactions [ 22 ]. To investigate the effects of haloperidol on fear-related behaviors, the present study used immunohistochemical staining to measure the levels of the neuroinflammatory protein interleukin-1b (IL-1b) in these subregions following behavioral testing. The present study has the following three aims: (1) To determine whether the administration of dopamine D2 receptor antagonist haloperidol prior to footshock stress alters freezing behavior in STM during the situational reminder and test phases. (2) To investigate whether haloperidol injections, administered after the consolidation of a fear memory, affect freezing behavior in LTM. (3) To measure the levels of the neuroinflammatory protein IL-1b in subregions of the mPFC [i.e., cingulate cortex 1 (Cg1), prelimbic cortex (PrL), and infralimbic cortex (IL)], amygdala [i.e., BLA and central amygdala (CeA)], and hippocampus [i.e., CA1, CA2, CA3, and dentate gyrus (DG)], following STM behavioral testing. 2. Methods and materials 2.1. Animals Sixty-four male Sprague–Dawley rats (weighing 250–350 g) were obtained from the National Laboratory for Animal Breeding and Research Center, Taipei, Taiwan. All rats were group-housed in cages with ad libitum access to food and water in a colony room maintained at a constant temperature on a 12 h light/dark cycle (lights on between 6:00 a.m. and 6:00 p.m.). The experiments were performed in accordance with American Psychological Association guidelines and received approval from the Fo Guang University Institutional Animal Care and Use Committee. The number of animals was limited, and every effort was made to minimize animal suffering during the experiment. 2.2. Apparatus: Inescapable footshock The inescapable footshock apparatus comprised a box with a plastic surrounding shell measuring 60 cm × 60 cm × 72 cm. The floor of the apparatus comprised metal grids (0.3 cm diameter at 0.7 cm grid intervals). 2.3. Experimental procedure The study included two behavioral experiments: the STM test (Experiment 1) and the LTM test (Experiment 2). Experiment 1 was designed to assess the effect of haloperidol (HAL) on freezing behavior and STM. Half of the rats were peritoneally injected with either 2% tartaric acid (TA) or 0.2 mg/kg HAL. After 45 min, half of the rats were given a single footshock (3mA, 10s) in the footshock apparatus for 2 min; the other half were placed in the footshock apparatus for 2 min without any footshock. On Days 2, 3, and 4, all rats were exposed to daily situational reminders, being placed in the footshock apparatus for 2 min without any footshock. Freezing behavior was tested during this time. On Day 5, the freezing behavior of all rats was measured 2 min in the footshock apparatus without administering any footshock. Experiment 2 was designed to examine the effect of HAL on freezing behavior and LTM, following the same behavioral procedure as Experiment 1. The main difference was that Experiment 2 involved testing fear behaviors across two trials, with Test 1 on Day 5 and Test 2 on Day 12. Given that Experiment 1 revealed significant differences in the effects of HAL and footshock on STM, Experiment 3 was conducted to further examine the neural substrates. Experiment 3 followed the same behavioral procedure as Experiment 1. After completing the behavioral tests, all rats were sacrificed 60 min later. Their brains were removed, and brain tissue was prepared for immunohistochemical staining to quantify levels of IL-1b proteins in the targeted regions (see Fig. 1 A). ------------------------------ Insert Fig. 1 here. ------------------------------- 2.4. Immunohistochemical staining Rats were sacrificed using a lethal dose of sodium pentobarbital. Once they were completely unresponsive, rats were perfused with 100 ml 0.9% NaCl followed by 400 ml of 4% paraformaldehyde in 0.1 M sodium phosphate-buffered saline (PBS). Their brain tissues were dissected, blocked, postfixed for 3 days. For cryoprotection, specimens were transferred to 30% sucrose for 2 days until they sank to the bottom of the solution. All section specimens were processed for c-Fos immunoreactivity staining. Free-floating brain sections were washed once in 0.1 M PBS for 10 mins, permeabilized in 3% H 2 O 2 for 1 hour, washed three times in PBS for 10 mins, and then soaked in normal goat serum for 1 hour. Sections were then washed with PBS once for 10 mins and were incubated overnight with the primary antibody. The sections were again washed once with PBS for 10 mins before incubation in a secondary antibody for 2 hours. After another 10-minute wash with PBS, the bound secondary antibody was amplified using the Vector Elite ABC kit. Positive cells were visually quantified at 20x magnification by a researcher blinded to the condition of each rat. Every third section of each brain slice was selected for counting [ 23 ]. Immunohistochemical staining was performed for IL-1b in the following specific brain areas: the mPFC’s Cg1, PrL, IL, CeA, the BLA, the NAc, and the hippocampus’s CA1, CA2, CA3, and DG. 2.5. Drugs All chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). The tartaric acid solution was prepared in normal saline; the normal saline solution was prepared by dissolving sodium chloride in distilled water. Haloperidol (0.2 mg/kg) was dissolved in the 0.2% tartaric acid vehicle solution. The haloperidol dosagee and concentration of the tartaric acid solution were based on a previous study [ 24 ]. All injections were administered intraperitoneally at a volume of 1 mL/kg. 2.6. Statistical analysis A 2 x 2 x 3 mixed three-way analysis of variance (ANOVA) was used to analyze the freezing time during the situational reminder phase. Then, a one-way ANOVA was performed to determine the freezing time for each session, with follow-up post hoc analyses with Tukey’s test when appropriate. For the test session, freezing time was analyzed using a one-way ANOVA with post hoc analyses with Tukey’s test when necessary. For the immunohistochemical data, a 2 x 2 two-way ANOVA was performed to analyze IL-1b expression in specific brain areas, including the Cg1, PrL, IL, NAc, BLA, CeA, CA1, CA2, CA3, and DG. For all analyses, p values of less than 0.05 were considered statistically significant. 3. Results Experiment 1: Haloperidol affects PTSD in short-term memory before consolidation of behaviors The effect of dopamine D2 antagonist haloperidol on PTSD freezing behavior in STM. The results revealed significant differences for haloperidol [F(1, 28) = 20.03, p < 0.05], footshock [F(1, 28) = 6.21, p < 0.05], session [F(2, 56) = 9.77, p < 0.05], and haloperidol x footshock x session [F(2, 56) = 3.26, p 0.05], session x haloperidol [F(2, 56) = 0.10, p > 0.05], and session x footshock [F(2, 56) = 1.63, p > 0.05]. A one-way ANOVA was performed to analyze the freezing time for each session. Significant differences were found among all groups in sessions 1–3 (p < 0.05). The data showed that the footshock induced severe freezing behavior over sessions 1–3, with significant differences between the TA/Footshock and TA/No footshock groups. Haloperidol administration also significantly attenuated footshock-induced freezing behaviors, as evidenced by comparing the HAL/Footshock and TA/Footshock groups over sessions 1–3 (Fig. 1 B). A one-way ANOVA was performed to evaluate freezing behavior during the test phase. The results revealed significant differences for all groups [F(3, 28) = 6.90, p < 0.05]. A subsequent post hoc with Tukey’s text analysis demonstrated that freezing behavior significantly increased in the TA/Footshock group compared to the TA/No Footshock group (p < 0.05) and that freezing time significantly decreased in the HAL/Footshock group compared to the TA/Footshock group (p 0.05). These data suggest that footshock increased freezing time, and haloperidol blunted footshock-induced freezing time. However, haloperidol alone had no effect on freezing time (Fig. 1 C). Experiment 2: Haloperidol affects PTSD in long-term memory after consolidation of behaviors A 2 x 2 x 3 mixed three-way ANOVA was performed to evaluate the effects of haloperidol on PTSD freezing behavior following consolidation in the LTM phase. Significant differences were observed for footshock [F(1, 28) = 101.31, p 0.05], session [F(2, 56) = 1.84, p > 0.05], haloperidol x footshock [F(1, 28) = 1.29, p > 0.05], session x haloperidol [F(2, 56) = 0.27, p > 0.05], session x footshock [F(2, 56) = 1.91, p > 0.05], and session x haloperidol x footshock [F(2, 56) = 0.31, p > 0.05]. Additionally, a one-way ANOVA was used to analyze freezing time among the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups for each session. The results revealed significant differences between the TA/No Footshock and HAL/No Footshock groups over sessions 1–3 (p 0.05). The data therefore indicated that footshock induced fear behaviors over sessions 1–3, consolidating the traumatic event in the LTM. However, haloperidol did not alter footshock-induced freezing behavior in the LTM phase (Fig. 2 A). ------------------------------ Insert Fig. 2 here. ------------------------------- A one-way ANOVA was performed to examine freezing behavior during the test phase in both Test 1 and Test 2. For Test 1, the results revealed significant differences among all groups [F(3, 28) = 23.48, p < 0.05]. A follow-up Tukey’s post hoc test demonstrated a significant increase in freezing time in the TA/Footshock group compared to the TA/No Footshock group (p 0.05) or the TA/Footshock and HAL/Footshock groups for Test 1 (p > 0.05; Fig. 2 B). A one-way ANOVA for Test 2 showed the same pattern of results, with a significant difference across all groups [F(3, 28) = 9.75, p < 0.05]. Subsequent Tukey’s post hoc tests revealed significant increases in freezing behavior the TA/Footshock compared to the TA/No Footshock group (p 0.05) or the TA/Footshock and the HAL/Footshock groups (p > 0.05; Fig. 2 C). Thus, while footshock-induced freezing behaviors were consolidated in the LTM phase, haloperidol did not affect these behaviors in this phase. Experiment 3: Assessment of neural substrates following haloperidol’s effect on PTSD in short-term memory before consolidation To identify which neural substrates contribute to the effects of haloperidol and footshock treatments, a 2 x 2 two-way ANOVA was performed for the target brain areas, including the subareas of the mPFC, amygdala, and hippocampus. The results revealed that, following footshock, IL-1b expression was significantly increased in the Cg1[F(1, 12) = 5.15, p < 0.05] (Fig. 3 A), PrL[F(1, 12) = 10.81, p < 0.05] (Fig. 3 B), BLA [F(1, 12) = 3.96, p = 0.07] (Fig. 4 B), CA1[F(1, 12) = 10.08, p < 0.05] (Fig. 5 A), CA2[F(1, 12) = 7.07, p < 0.05] (Fig. 8B), CA3[F(1, 12) = 21.37, p < 0.05] (Fig. 5 C), and DG [F(1, 12) = 5.76, p < 0.05] (Fig. 5 D). These findings indicate that footshock enhanced IL-1b expression in the Cg1, PrL, BLA, CA1, CA2, CA3, and DG. Reduced IL-1b expression was observed in the IL [F(1, 12) = 12.63, p < 0.05] (Fig. 3 C) and NAc [F(1, 12) = 4.62, p = 0.06] following footshock treatments (Fig. 4 A). This indicates that footshock decreased IL-1b expression in the IL and NAc. No significant differences in IL-1b expression were observed in the CeA (p < 0.05; Fig. 4 C), suggesting that the CeA was not involved in IL-1b expression in response to the footshock-induced freezing behaviors. A 2 x 2 two-way ANOVA was conducted to evaluate whether haloperidol affects footshock-induced IL-1b expression. The results showed that haloperidol injections significantly attenuated footshock-induced IL-1b expression in the NAc [F(1, 12) = 7.14, p < 0.05] (Fig. 4 A), CeA[F(1, 12) = 6.91, p < 0.05] (Fig. 4 C), CA1[F(1, 12) = 4.93, p < 0.05] (Fig. 5 A), CA2[F(1, 12) = 3.67, p = 0.08] (Fig. 5 B), and CA3[F(1, 12) = 7.29, p < 0.05] (Fig. 5 C). A significant interaction effect between haloperidol injections and footshock treatments was also observed in the DG [F(1, 12) = 4.21, p = 0.06] (Fig. 5 D). ------------------------------ Insert Figs. 3 – 5 here. ------------------------------- 4. Discussion 4.1. Summary of core findings The findings of this study demonstrate that footshock-induced trauma produced neuroinflammatory responses, evidenced by increased cytokine IL-1b expression in key neural substrates of the fear circuit, including areas of the mPFC, amygdala, and hippocampus during the STM. The NAc, however, exhibited a notable downregulation of IL-1b expression. The prior administration of D2 antagonist haloperidol attenuated footshock-induced fear behavior, thereby decreasing IL-1b expression in the amygdala’s CeA (but not BLA) and the hippocampus’ CA1, CA2, and CA3 (but not DG). However, haloperidol did not affect IL-1b expression in the mPFC’s Cg1, PrL, and IL, and IL-1b expression in the NAc also remained lower. In summary, the D2 receptor plays a dual role in ameliorating fear behavior and neuroinflammation responses in this animal model of PTSD. These findings provide a new perspective on the potential of D2 antagonists as an early intervention strategy for the amelioration of PTSD symptoms. 4.2. The time-dependent role of D2 receptors in fear memory: Reconciling with the literature The precise role of the dopamine system in modulating PTSD symptoms is not fully understood. One hypothesis suggests that dopamine agonists have anti-anxiety effects and reduce fear behaviors in PTSD [ 11 ]. For example, previous research utilized a single-prolonged stress model to explore the involvement of dopamine D2 and D3 receptors in auditory fear conditioning and depression behaviors, demonstrating that D2/D3 receptor agonists rotigotine and pramipexole reduced freezing time in fear conditioning [ 18 ]. Additionally, a genetic study showed that a gene variant of the D2A1 allele induces disequilibrium, reducing dopamine D2 receptor density in the brain, increasing susceptibility to PTSD [ 20 ]. Interestingly, our study’s finding that the D2 receptor antagonist haloperidol reduced fear behaviors conflicts with the results of previous studies showing that D2 receptor agonists had anxiolytic effects. We propose that this apparent discrepancy lies in the critical role of the temporal stage of memory processing. Our results only demonstrate haloperidol’s effect on freezing behavior during the STM phase, suggesting that dopamine D2 receptor signaling is crucial for the initial formation or early stabilization of fear memory. Conversely, its role may be minimal or different during the later LTM consolidation phase, which has been the focus of many previous studies. Therefore, blocking D2 receptors immediately after a traumatic event could present a potential therapeutic window in which to prevent the consolidation of fear memory, rather than attempting to alter it once it has been consolidated. 4.3. Footshock stress and neuroinflammation Our results regarding footshock stress-induced neuroinflammatory responses are consistent with previous data demonstrating the activation of neuroimmune cells, stress-induced dysfunction of the hypothalamus–pituitary gland–adrenal gland (HPA) axis, and neuroinflammatory responses [ 7 ]. Traumatic stress events trigger nervous system defence responses, activating microglial cells and astrocytes and stimulating the immune cells in the HPA axis, ultimately resulting in neuroinflammation in many brain regions [ 7 – 9 ]. In our example, footshock stress, which mimics PTSD symptoms, induces higher levels of inflammatory cytokines, such as IL-1b, IL-6, TNF-alpha, NF-kb, and C-reactive protein [ 9 , 10 ]. Furthermore, the neuroinflammation and dysfunction of the HPA axis are associated with decreased volumes of the left amygdala [ 25 , 26 ], hippocampus [ 26 ], and ventromedial frontal cortex [ 26 ]. In summary, the present findings suggest that footshock stress produces neuroinflammatory responses, aligning with existing evidence. Building upon this, we propose that D2 receptor signaling is a critical upstream event that modulates this neuroinflammatory cascade. By blocking these receptors, haloperidol likely disrupts the initial neural encoding of fear, thereby preventing the downstream activation of immune cells and the subsequent production of IL-1b. 4.4. Experimental limitations Several limitations of the present study should be noted. First, we used only one sex (male) and one drug (haloperidol) and tested only one neuroinflammatory cytokine (IL-1b). Future studies should expand upon these parameters, for instance, by including female subjects, testing more selective D2 antagonists, and assessing a wider array of inflammatory cytokines, to provide a more comprehensive understanding of PTSD mechanisms to further inform the development of novel pharmacological treatments. Additionally, future studies should investigate the specific ways in which D2 antagonists influence neuroinflammation and the activation of microglial cells or astrocytes. 4.5. Conclusion and clinical implications This study provides compelling preclinical evidence that blocking dopamine D2 receptors within a critical short-term window following a traumatic event can both attenuate the formation of fear memory and suppress associated neuroinflammatory responses in key limbic structures. Our findings reconcile conflicting reports in the literature by highlighting a time-dependent role for D2 receptors, suggesting they play a crucial role in the initial encoding of fear rather than in its long-term consolidation. This work suggests that early pharmacological intervention targeting D2 receptors could represent a novel and promising therapeutic strategy for preventing the development of PTSD symptoms. Declarations Funding This paper was supported by funding granted to A. C. W. Huang by the National Science and Technology Council of Taiwan [NSTC 113-2410-H-431 -015 -MY2, NSTC 113-2923-H-431 -001 -MY3, and NSTC 114-2811-H-431-001]. CRediT authorship contribution statement P.J. Hung: Conceptualization, Methodology, Project administration. C.N. Cheng: Project administration, Supervision, Validation. A. Kozłowska: Resources, Conceptualization. A. C. Tsai: Methodology, Formal analysis. C.H. Tseng: Software. C.C. Chen : Conceptualization. A.C.W. Huang: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation, Funding acquisition, Conceptualization. Declaration of competing interests The authors declare no competing interests. Data availability Data will be made available upon request. References Chang SH, Yu YH, He A, Ou CY, Shyu BC, Huang ACW. BDNF Protein and BDNF mRNA Expression of the Medial Prefrontal Cortex, Amygdala, and Hippocampus during Situational Reminder in the PTSD Animal Model. Behav Neurol. 2021;2021:6657716. Giustino TF, Fitzgerald PJ, Maren S. Revisiting propranolol and PTSD: Memory erasure or extinction enhancement? Neurobiol Learn Mem. 2016;130:26–33. 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Tsai","email":"","orcid":"","institution":"Institute of Statistical Science, Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Arthur","middleName":"C.","lastName":"Tsai","suffix":""},{"id":507931390,"identity":"75c31c2d-0361-4ce0-b25e-314f29c49934","order_by":4,"name":"Chun-Hsiung Tseng","email":"","orcid":"","institution":"Department of Electrical Engineering, Yuan Ze University","correspondingAuthor":false,"prefix":"","firstName":"Chun-Hsiung","middleName":"","lastName":"Tseng","suffix":""},{"id":507931391,"identity":"454b35a3-0570-4466-aa97-c2fc0562c7a8","order_by":5,"name":"Chun Chien Chen","email":"","orcid":"","institution":"Beitou Branch, Tri-Service General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"Chien","lastName":"Chen","suffix":""},{"id":507931392,"identity":"3d60879f-0901-4105-a110-515264cdbb78","order_by":6,"name":"Andrew Chih Wei Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYHACNjDJDyISCkjRItkA0mJAihaDA2CSCPX87WfMHvPU3LHbfH514ocHBgzy/GIH8GuROJNjbsxz7FnythtvN0sAHWY4c3YCAWtu8G6T5mE7nGx24+wGkJYEg9sEtMiDtfw7nGw84+zmH0RpMQBp4W07bGfA37uNOFsMz+R/k5zbdzhBAqjXIsFAgrBf5I4fS5N48+2wPX//2c03f1TYyPNLE9ACA4kNEmCVEsQpBwF7Bv4DxKseBaNgFIyCkQUAy/5FUVz76w8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9794-7302","institution":"Fo Guang University","correspondingAuthor":true,"prefix":"","firstName":"Andrew","middleName":"Chih Wei","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-08-31 00:41:08","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7497727/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7497727/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90509369,"identity":"9a4ed7f2-05a9-413d-99ff-323c5304983a","added_by":"auto","created_at":"2025-09-03 13:20:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1347905,"visible":true,"origin":"","legend":"\u003cp\u003edepicts the flowchart for Experiments 1-3, the situational reminder for sessions 1-3, and the test phase in the short-term memory and before consolidation process. (A). The flowchart for Experiments 1–3. In Experiment 1, the rats were administered injections of either 2 % tartaric acid solution or 0.2 mg/kg haloperidol. After 45 min, half of the rats were placed in the footshock apparatus for 2 min to receive the footshock treatment (3Am, 10 second duration); the others remained in the footshock apparatus without any footshock on Day 1. On Days 2-4, all rats received a daily situational reminder. During the situational reminder phase, the rats were placed in the footshock apparatus without any footshock for 2 min. On Day 5, fear behavior was tested for 2 min. In Experiment 2, all rats experienced the same procedure as Experiment 1. The rats; fear behaviors were tested for 2 min on Day 5 for Test 1 and on Day 12 for Test 2. Experiment 3 followed the same behavioral procedure as Experiment 1. After the behavioral test and 60 min later, immunohistochemical staining was performed with IL-b protein. Note that the behavioral data from Experiment 2 did not reveal any significant differences in haloperidol injections; thus, we did not examine the neural substrates any further. (B). Mean (\u003cu\u003e+\u003c/u\u003e SEM) freezing time (Sec.) for the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups (n = 8, per group) over sessions 1–3 during the situational reminder to test freezing behavior in STM. (*) indicates p \u0026lt; 0.05. HAL: haloperidol; STM: short-term memory; TA: tartaric acid. (C). Mean (\u003cu\u003e+\u003c/u\u003e SEM) freezing time (Sec.) for the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups (n = 8, per group) in the test phase to test STM. (*) indicates p \u0026lt; 0.05. (n.s.) indicates nonsignificant differences. HAL: haloperidol; STM: short-term memory; TA: tartaric acid.\u003c/p\u003e","description":"","filename":"Fig1082725k.png","url":"https://assets-eu.researchsquare.com/files/rs-7497727/v1/1d65c8622e749ffb8e547f22.png"},{"id":90509127,"identity":"f9a39d7e-6d1a-4d22-9862-715f09136bac","added_by":"auto","created_at":"2025-09-03 13:12:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":811496,"visible":true,"origin":"","legend":"\u003cp\u003e(A). Mean (\u003cu\u003e+\u003c/u\u003e SEM) freezing time (Sec.) for the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups (n = 8, per group) over sessions 1–3 during the situational reminder to test freezing behavior in LTM. (*) indicates p \u0026lt; 0.05. HAL: haloperidol; LTM: long-term memory; TA: tartaric acid. (B)-(C). Mean (\u003cu\u003e+\u003c/u\u003e SEM) freezing time (Sec.) for the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups (n = 8, per group) in the test phase to test LTM on Days 5 and 12 for Tests 1 and 2, respectively. (*) indicates p \u0026lt; 0.05. (n.s.) indicates nonsignificant differences. HAL: haloperidol; LTM: long-term memory; TA: tartaric acid.\u003c/p\u003e","description":"","filename":"Fig2082725k.png","url":"https://assets-eu.researchsquare.com/files/rs-7497727/v1/7c4d35862318802673947ebf.png"},{"id":90507777,"identity":"20aec480-7130-4275-b079-d0b591baa5ff","added_by":"auto","created_at":"2025-09-03 13:04:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187668,"visible":true,"origin":"","legend":"\u003cp\u003eMean (+ SEM) IL-1b Expression Numbers for the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups (n = 8, per group) after behavioral tests in the STM phase for (A) Cg1, (B) PrL, and (C) IL. Cg1: cingulate cortex 1; PrL: prelimbic cortex; IL: infralimbic cortex. HAL: haloperidol; STM: short-term memory; TA: tartaric acid.\u003c/p\u003e","description":"","filename":"Fig3082725kr.png","url":"https://assets-eu.researchsquare.com/files/rs-7497727/v1/2f54457c45d6224e4fd67570.png"},{"id":90507769,"identity":"d4a6cbcf-ce68-4665-b2c8-0bb18717bb8e","added_by":"auto","created_at":"2025-09-03 13:04:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":193147,"visible":true,"origin":"","legend":"\u003cp\u003eMean (+ SEM) IL-1b Expression Numbers for the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups (n = 8, per group) after behavioral tests in the STM phase for (A). NAc, (B). BLA, and (C). CeA. NAc: nucleus accumbens; BLA: basolateral amygdala; CeA: central amygdala. HAL: haloperidol; STM: short-term memory; TA: tartaric acid.\u003c/p\u003e","description":"","filename":"Fig4082725kr.png","url":"https://assets-eu.researchsquare.com/files/rs-7497727/v1/e51f6846b6696b658f45113e.png"},{"id":90507780,"identity":"7656d051-f4d4-410d-a722-5c1be640a39a","added_by":"auto","created_at":"2025-09-03 13:04:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":188644,"visible":true,"origin":"","legend":"\u003cp\u003eMean (+ SEM) IL-1b Expression Numbers for the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups (n = 8, per group) after behavioral tests in the STM phase for (A). CA1, (B). CA2, (C). CA3, and (D). DG. DG: dentate gyrus. HAL: haloperidol; STM: short-term memory; TA: tartaric acid. (*) indicates p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig5082725kr.png","url":"https://assets-eu.researchsquare.com/files/rs-7497727/v1/26929a51fb38e2d73ec15c48.png"},{"id":90510606,"identity":"05860628-d884-472e-bfae-e6db4574268c","added_by":"auto","created_at":"2025-09-03 13:28:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2637166,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7497727/v1/bc199f3e-105c-4c7d-988a-b4270ca2d6bb.pdf"},{"id":90509130,"identity":"972dc6f0-a265-4d0c-8481-d3063760a24f","added_by":"auto","created_at":"2025-09-03 13:12:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3288697,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary\u003c/p\u003e","description":"","filename":"Supplementarymaterialsandmethods.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7497727/v1/91ecc45aa224215f11058756.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eHaloperidol prevents the consolidation of short-term traumatic memory by modulating interleukin-1β in neural substrates\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePosttraumatic stress disorder (PTSD) is a severe psychiatric condition associated with learning and memory deficits [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Its characteristic symptoms include the persistent re-experiencing of a traumatic event, chronic emotional arousal, and the avoidance of stimuli associated with the traumatic memory [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This avoidance behavior is a product of classical conditioning, which involves forming an association between an environmental stimulus (conditioned stimulus, CS) and the traumatic event (unconditioned stimulus, US [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. During this process, the CS is encoded into short-term memory (STM) and subsequently consolidated into long-term memory (LTM), forming a durable memory trace in the brain [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStrong evidence indicates that PTSD is highly correlated with neuroinflammation in the brain [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Patients with PTSD exhibit elevated levels of various neuroinflammatory biomarkers, such as interleukin-1 (IL-1), interleukin-6, tumor necrosis factor-alpha, nuclear factor-kB, and C-reactive protein [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Animal models often use fear conditioning tasks to mimic the clinical symptoms of PTSD [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and show similar results, suggesting a link between PTSD or stress and increased neuroinflammation [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For example, studies have shown that footshock stress not only regulates aversive odor cues but also induces inflammatory cytokine responses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, optogenetic stimulation or inhibition in the basolateral amygdala (BLA) can modulate stress- and fear-memory-induced sleep alterations as well as neuroinflammatory responses in the mPFC and hippocampus [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These findings support the use of footshock stress in animal models mimicking the production of neuroinflammatory responses associated with the traumatic events that elicit PTSD.\u003c/p\u003e\u003cp\u003eA recent review suggests the involvement of the dopamine system in the symptoms of PTSD and anxiety disorders [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For example, the dopamine D2/D3 receptor agonists rotigotine and pramipexole have been shown to attenuate freezing time in auditory fear conditioning and reduce depression responses in a single-prolonged stress model [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, the 10R/10R variant of the dopamine transporter 1 (DAT1) gene is associated with a higher risk of developing PTSD, implicating it in the disorder's pathophysiology. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Similarly, a variant of the dopamine D2 receptor gene is in linkage disequilibrium with the D2A1 allele, increasing the risk of PTSD, suggesting that D2 receptor signaling deficits contribute to susceptibility [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While it has been established that the dopamine system plays a role in PTSD symptoms, which stage of learning and memory is governed by dopamine receptors remains unclear. The present study aims to address this issue.\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated the involvement of the medial prefrontal cortex (mPFC), amygdala, and hippocampus in the fear behaviors characteristic of PTSD [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For example, neuroimaging research has revealed that hyperactivation in the amygdala is associated with the severe symptoms of PTSD; brain mapping evidence demonstrates that PTSD patients have a smaller and hypoactive mPFC as well as a diminished hippocampus with reduced neuronal and functional interactions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To investigate the effects of haloperidol on fear-related behaviors, the present study used immunohistochemical staining to measure the levels of the neuroinflammatory protein interleukin-1b (IL-1b) in these subregions following behavioral testing.\u003c/p\u003e\u003cp\u003eThe present study has the following three aims: (1) To determine whether the administration of dopamine D2 receptor antagonist haloperidol prior to footshock stress alters freezing behavior in STM during the situational reminder and test phases. (2) To investigate whether haloperidol injections, administered after the consolidation of a fear memory, affect freezing behavior in LTM. (3) To measure the levels of the neuroinflammatory protein IL-1b in subregions of the mPFC [i.e., cingulate cortex 1 (Cg1), prelimbic cortex (PrL), and infralimbic cortex (IL)], amygdala [i.e., BLA and central amygdala (CeA)], and hippocampus [i.e., CA1, CA2, CA3, and dentate gyrus (DG)], following STM behavioral testing.\u003c/p\u003e"},{"header":"2. Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Animals\u003c/h2\u003e\u003cp\u003eSixty-four male Sprague\u0026ndash;Dawley rats (weighing 250\u0026ndash;350 g) were obtained from the National Laboratory for Animal Breeding and Research Center, Taipei, Taiwan. All rats were group-housed in cages with \u003cem\u003ead libitum\u003c/em\u003e access to food and water in a colony room maintained at a constant temperature on a 12 h light/dark cycle (lights on between 6:00 a.m. and 6:00 p.m.). The experiments were performed in accordance with American Psychological Association guidelines and received approval from the Fo Guang University Institutional Animal Care and Use Committee. The number of animals was limited, and every effort was made to minimize animal suffering during the experiment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Apparatus: Inescapable footshock\u003c/h2\u003e\u003cp\u003eThe inescapable footshock apparatus comprised a box with a plastic surrounding shell measuring 60 cm \u0026times; 60 cm \u0026times; 72 cm. The floor of the apparatus comprised metal grids (0.3 cm diameter at 0.7 cm grid intervals).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Experimental procedure\u003c/h2\u003e\u003cp\u003eThe study included two behavioral experiments: the STM test (Experiment 1) and the LTM test (Experiment 2). Experiment 1 was designed to assess the effect of haloperidol (HAL) on freezing behavior and STM. Half of the rats were peritoneally injected with either 2% tartaric acid (TA) or 0.2 mg/kg HAL. After 45 min, half of the rats were given a single footshock (3mA, 10s) in the footshock apparatus for 2 min; the other half were placed in the footshock apparatus for 2 min without any footshock. On Days 2, 3, and 4, all rats were exposed to daily situational reminders, being placed in the footshock apparatus for 2 min without any footshock. Freezing behavior was tested during this time. On Day 5, the freezing behavior of all rats was measured 2 min in the footshock apparatus without administering any footshock.\u003c/p\u003e\u003cp\u003eExperiment 2 was designed to examine the effect of HAL on freezing behavior and LTM, following the same behavioral procedure as Experiment 1. The main difference was that Experiment 2 involved testing fear behaviors across two trials, with Test 1 on Day 5 and Test 2 on Day 12. Given that Experiment 1 revealed significant differences in the effects of HAL and footshock on STM, Experiment 3 was conducted to further examine the neural substrates. Experiment 3 followed the same behavioral procedure as Experiment 1. After completing the behavioral tests, all rats were sacrificed 60 min later. Their brains were removed, and brain tissue was prepared for immunohistochemical staining to quantify levels of IL-1b proteins in the targeted regions (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e------------------------------\u003c/p\u003e\u003cp\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e here.\u003c/p\u003e\u003cp\u003e-------------------------------\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Immunohistochemical staining\u003c/h2\u003e\u003cp\u003eRats were sacrificed using a lethal dose of sodium pentobarbital. Once they were completely unresponsive, rats were perfused with 100 ml 0.9% NaCl followed by 400 ml of 4% paraformaldehyde in 0.1 M sodium phosphate-buffered saline (PBS). Their brain tissues were dissected, blocked, postfixed for 3 days. For cryoprotection, specimens were transferred to 30% sucrose for 2 days until they sank to the bottom of the solution. All section specimens were processed for c-Fos immunoreactivity staining. Free-floating brain sections were washed once in 0.1 M PBS for 10 mins, permeabilized in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 1 hour, washed three times in PBS for 10 mins, and then soaked in normal goat serum for 1 hour. Sections were then washed with PBS once for 10 mins and were incubated overnight with the primary antibody. The sections were again washed once with PBS for 10 mins before incubation in a secondary antibody for 2 hours. After another 10-minute wash with PBS, the bound secondary antibody was amplified using the Vector Elite ABC kit. Positive cells were visually quantified at 20x magnification by a researcher blinded to the condition of each rat. Every third section of each brain slice was selected for counting [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Immunohistochemical staining was performed for IL-1b in the following specific brain areas: the mPFC\u0026rsquo;s Cg1, PrL, IL, CeA, the BLA, the NAc, and the hippocampus\u0026rsquo;s CA1, CA2, CA3, and DG.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Drugs\u003c/h2\u003e\u003cp\u003eAll chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). The tartaric acid solution was prepared in normal saline; the normal saline solution was prepared by dissolving sodium chloride in distilled water. Haloperidol (0.2 mg/kg) was dissolved in the 0.2% tartaric acid vehicle solution. The haloperidol dosagee and concentration of the tartaric acid solution were based on a previous study [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. All injections were administered intraperitoneally at a volume of 1 mL/kg.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e\u003cp\u003eA 2 x 2 x 3 mixed three-way analysis of variance (ANOVA) was used to analyze the freezing time during the situational reminder phase. Then, a one-way ANOVA was performed to determine the freezing time for each session, with follow-up post hoc analyses with Tukey\u0026rsquo;s test when appropriate. For the test session, freezing time was analyzed using a one-way ANOVA with post hoc analyses with Tukey\u0026rsquo;s test when necessary. For the immunohistochemical data, a 2 x 2 two-way ANOVA was performed to analyze IL-1b expression in specific brain areas, including the Cg1, PrL, IL, NAc, BLA, CeA, CA1, CA2, CA3, and DG. For all analyses, p values of less than 0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003eExperiment 1: Haloperidol affects PTSD in short-term memory before consolidation of behaviors\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe effect of dopamine D2 antagonist haloperidol on PTSD freezing behavior in STM. The results revealed significant differences for haloperidol [F(1, 28)\u0026thinsp;=\u0026thinsp;20.03, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05], footshock [F(1, 28)\u0026thinsp;=\u0026thinsp;6.21, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05], session [F(2, 56)\u0026thinsp;=\u0026thinsp;9.77, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05], and haloperidol x footshock x session [F(2, 56)\u0026thinsp;=\u0026thinsp;3.26, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. Nonsignificant differences were observed in haloperidol x footshock [F(1, 28)\u0026thinsp;=\u0026thinsp;0.35, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05], session x haloperidol [F(2, 56)\u0026thinsp;=\u0026thinsp;0.10, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05], and session x footshock [F(2, 56)\u0026thinsp;=\u0026thinsp;1.63, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05]. A one-way ANOVA was performed to analyze the freezing time for each session. Significant differences were found among all groups in sessions 1\u0026ndash;3 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The data showed that the footshock induced severe freezing behavior over sessions 1\u0026ndash;3, with significant differences between the TA/Footshock and TA/No footshock groups. Haloperidol administration also significantly attenuated footshock-induced freezing behaviors, as evidenced by comparing the HAL/Footshock and TA/Footshock groups over sessions 1\u0026ndash;3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eA one-way ANOVA was performed to evaluate freezing behavior during the test phase. The results revealed significant differences for all groups [F(3, 28)\u0026thinsp;=\u0026thinsp;6.90, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. A subsequent post hoc with Tukey\u0026rsquo;s text analysis demonstrated that freezing behavior significantly increased in the TA/Footshock group compared to the TA/No Footshock group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and that freezing time significantly decreased in the HAL/Footshock group compared to the TA/Footshock group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant differences were observed between the TA/No Footshock and HAL/No Footshock groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These data suggest that footshock increased freezing time, and haloperidol blunted footshock-induced freezing time. However, haloperidol alone had no effect on freezing time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003cem\u003eExperiment 2: Haloperidol affects PTSD in long-term memory after consolidation of behaviors\u003c/em\u003e\u003c/p\u003e\u003cp\u003eA 2 x 2 x 3 mixed three-way ANOVA was performed to evaluate the effects of haloperidol on PTSD freezing behavior following consolidation in the LTM phase. Significant differences were observed for footshock [F(1, 28)\u0026thinsp;=\u0026thinsp;101.31, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. However, no significant differences were found for haloperidol [F(1, 28)\u0026thinsp;=\u0026thinsp;2.57, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05], session [F(2, 56)\u0026thinsp;=\u0026thinsp;1.84, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05], haloperidol x footshock [F(1, 28)\u0026thinsp;=\u0026thinsp;1.29, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05], session x haloperidol [F(2, 56)\u0026thinsp;=\u0026thinsp;0.27, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05], session x footshock [F(2, 56)\u0026thinsp;=\u0026thinsp;1.91, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05], and session x haloperidol x footshock [F(2, 56)\u0026thinsp;=\u0026thinsp;0.31, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05]. Additionally, a one-way ANOVA was used to analyze freezing time among the TA/No Footshock, HAL/No Footshock, TA/Footshock, and HAL/Footshock groups for each session. The results revealed significant differences between the TA/No Footshock and HAL/No Footshock groups over sessions 1\u0026ndash;3 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant differences were observed for all other groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The data therefore indicated that footshock induced fear behaviors over sessions 1\u0026ndash;3, consolidating the traumatic event in the LTM. However, haloperidol did not alter footshock-induced freezing behavior in the LTM phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e------------------------------\u003c/p\u003e\u003cp\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e here.\u003c/p\u003e\u003cp\u003e-------------------------------\u003c/p\u003e\u003cp\u003eA one-way ANOVA was performed to examine freezing behavior during the test phase in both Test 1 and Test 2. For Test 1, the results revealed significant differences among all groups [F(3, 28)\u0026thinsp;=\u0026thinsp;23.48, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. A follow-up Tukey\u0026rsquo;s post hoc test demonstrated a significant increase in freezing time in the TA/Footshock group compared to the TA/No Footshock group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant differences were observed between the HAL/No Footshock and TA/No Footshock groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) or the TA/Footshock and HAL/Footshock groups for Test 1 (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eA one-way ANOVA for Test 2 showed the same pattern of results, with a significant difference across all groups [F(3, 28)\u0026thinsp;=\u0026thinsp;9.75, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05]. Subsequent Tukey\u0026rsquo;s post hoc tests revealed significant increases in freezing behavior the TA/Footshock compared to the TA/No Footshock group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with no significant differences observed between the TA/No Footshock and HAL/No Footshock (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) or the TA/Footshock and the HAL/Footshock groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Thus, while footshock-induced freezing behaviors were consolidated in the LTM phase, haloperidol did not affect these behaviors in this phase.\u003c/p\u003e\u003cp\u003e\u003cem\u003eExperiment 3: Assessment of neural substrates following haloperidol\u0026rsquo;s effect on PTSD in short-term memory before consolidation\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo identify which neural substrates contribute to the effects of haloperidol and footshock treatments, a 2 x 2 two-way ANOVA was performed for the target brain areas, including the subareas of the mPFC, amygdala, and hippocampus. The results revealed that, following footshock, IL-1b expression was significantly increased in the Cg1[F(1, 12)\u0026thinsp;=\u0026thinsp;5.15, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), PrL[F(1, 12)\u0026thinsp;=\u0026thinsp;10.81, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), BLA [F(1, 12)\u0026thinsp;=\u0026thinsp;3.96, p\u0026thinsp;=\u0026thinsp;0.07] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), CA1[F(1, 12)\u0026thinsp;=\u0026thinsp;10.08, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), CA2[F(1, 12)\u0026thinsp;=\u0026thinsp;7.07, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;8B), CA3[F(1, 12)\u0026thinsp;=\u0026thinsp;21.37, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and DG [F(1, 12)\u0026thinsp;=\u0026thinsp;5.76, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These findings indicate that footshock enhanced IL-1b expression in the Cg1, PrL, BLA, CA1, CA2, CA3, and DG.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eReduced IL-1b expression was observed in the IL [F(1, 12)\u0026thinsp;=\u0026thinsp;12.63, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and NAc [F(1, 12)\u0026thinsp;=\u0026thinsp;4.62, p\u0026thinsp;=\u0026thinsp;0.06] following footshock treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This indicates that footshock decreased IL-1b expression in the IL and NAc. No significant differences in IL-1b expression were observed in the CeA (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), suggesting that the CeA was not involved in IL-1b expression in response to the footshock-induced freezing behaviors.\u003c/p\u003e\u003cp\u003eA 2 x 2 two-way ANOVA was conducted to evaluate whether haloperidol affects footshock-induced IL-1b expression. The results showed that haloperidol injections significantly attenuated footshock-induced IL-1b expression in the NAc [F(1, 12)\u0026thinsp;=\u0026thinsp;7.14, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), CeA[F(1, 12)\u0026thinsp;=\u0026thinsp;6.91, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), CA1[F(1, 12)\u0026thinsp;=\u0026thinsp;4.93, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), CA2[F(1, 12)\u0026thinsp;=\u0026thinsp;3.67, p\u0026thinsp;=\u0026thinsp;0.08] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and CA3[F(1, 12)\u0026thinsp;=\u0026thinsp;7.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). A significant interaction effect between haloperidol injections and footshock treatments was also observed in the DG [F(1, 12)\u0026thinsp;=\u0026thinsp;4.21, p\u0026thinsp;=\u0026thinsp;0.06] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e------------------------------\u003c/p\u003e\u003cp\u003eInsert Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e here.\u003c/p\u003e\u003cp\u003e-------------------------------\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Summary of core findings\u003c/h2\u003e\u003cp\u003eThe findings of this study demonstrate that footshock-induced trauma produced neuroinflammatory responses, evidenced by increased cytokine IL-1b expression in key neural substrates of the fear circuit, including areas of the mPFC, amygdala, and hippocampus during the STM. The NAc, however, exhibited a notable downregulation of IL-1b expression. The prior administration of D2 antagonist haloperidol attenuated footshock-induced fear behavior, thereby decreasing IL-1b expression in the amygdala\u0026rsquo;s CeA (but not BLA) and the hippocampus\u0026rsquo; CA1, CA2, and CA3 (but not DG). However, haloperidol did not affect IL-1b expression in the mPFC\u0026rsquo;s Cg1, PrL, and IL, and IL-1b expression in the NAc also remained lower. In summary, the D2 receptor plays a dual role in ameliorating fear behavior and neuroinflammation responses in this animal model of PTSD. These findings provide a new perspective on the potential of D2 antagonists as an early intervention strategy for the amelioration of PTSD symptoms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4.2. The time-dependent role of D2 receptors in fear memory: Reconciling with the literature\u003c/h2\u003e\u003cp\u003eThe precise role of the dopamine system in modulating PTSD symptoms is not fully understood. One hypothesis suggests that dopamine agonists have anti-anxiety effects and reduce fear behaviors in PTSD [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For example, previous research utilized a single-prolonged stress model to explore the involvement of dopamine D2 and D3 receptors in auditory fear conditioning and depression behaviors, demonstrating that D2/D3 receptor agonists rotigotine and pramipexole reduced freezing time in fear conditioning [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, a genetic study showed that a gene variant of the D2A1 allele induces disequilibrium, reducing dopamine D2 receptor density in the brain, increasing susceptibility to PTSD [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInterestingly, our study\u0026rsquo;s finding that the D2 receptor antagonist haloperidol reduced fear behaviors conflicts with the results of previous studies showing that D2 receptor agonists had anxiolytic effects. We propose that this apparent discrepancy lies in the critical role of the temporal stage of memory processing. Our results only demonstrate haloperidol\u0026rsquo;s effect on freezing behavior during the STM phase, suggesting that dopamine D2 receptor signaling is crucial for the initial formation or early stabilization of fear memory. Conversely, its role may be minimal or different during the later LTM consolidation phase, which has been the focus of many previous studies. Therefore, blocking D2 receptors immediately after a traumatic event could present a potential therapeutic window in which to prevent the consolidation of fear memory, rather than attempting to alter it once it has been consolidated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Footshock stress and neuroinflammation\u003c/h2\u003e\u003cp\u003eOur results regarding footshock stress-induced neuroinflammatory responses are consistent with previous data demonstrating the activation of neuroimmune cells, stress-induced dysfunction of the hypothalamus\u0026ndash;pituitary gland\u0026ndash;adrenal gland (HPA) axis, and neuroinflammatory responses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Traumatic stress events trigger nervous system defence responses, activating microglial cells and astrocytes and stimulating the immune cells in the HPA axis, ultimately resulting in neuroinflammation in many brain regions [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In our example, footshock stress, which mimics PTSD symptoms, induces higher levels of inflammatory cytokines, such as IL-1b, IL-6, TNF-alpha, NF-kb, and C-reactive protein [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, the neuroinflammation and dysfunction of the HPA axis are associated with decreased volumes of the left amygdala [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], hippocampus [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and ventromedial frontal cortex [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In summary, the present findings suggest that footshock stress produces neuroinflammatory responses, aligning with existing evidence.\u003c/p\u003e\u003cp\u003eBuilding upon this, we propose that D2 receptor signaling is a critical upstream event that modulates this neuroinflammatory cascade. By blocking these receptors, haloperidol likely disrupts the initial neural encoding of fear, thereby preventing the downstream activation of immune cells and the subsequent production of IL-1b.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Experimental limitations\u003c/h2\u003e\u003cp\u003eSeveral limitations of the present study should be noted. First, we used only one sex (male) and one drug (haloperidol) and tested only one neuroinflammatory cytokine (IL-1b). Future studies should expand upon these parameters, for instance, by including female subjects, testing more selective D2 antagonists, and assessing a wider array of inflammatory cytokines, to provide a more comprehensive understanding of PTSD mechanisms to further inform the development of novel pharmacological treatments. Additionally, future studies should investigate the specific ways in which D2 antagonists influence neuroinflammation and the activation of microglial cells or astrocytes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.5. Conclusion and clinical implications\u003c/h2\u003e\u003cp\u003eThis study provides compelling preclinical evidence that blocking dopamine D2 receptors within a critical short-term window following a traumatic event can both attenuate the formation of fear memory and suppress associated neuroinflammatory responses in key limbic structures. Our findings reconcile conflicting reports in the literature by highlighting a time-dependent role for D2 receptors, suggesting they play a crucial role in the initial encoding of fear rather than in its long-term consolidation. This work suggests that early pharmacological intervention targeting D2 receptors could represent a novel and promising therapeutic strategy for preventing the development of PTSD symptoms.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis paper was supported by funding granted to A. C. W. Huang by the National Science and Technology Council of Taiwan [NSTC 113-2410-H-431 -015 -MY2, NSTC 113-2923-H-431 -001 -MY3, and NSTC 114-2811-H-431-001].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP.J. Hung:\u003c/strong\u003e Conceptualization, Methodology, Project administration. \u003cstrong\u003eC.N. Cheng:\u0026nbsp;\u003c/strong\u003eProject administration, Supervision, Validation. \u003cstrong\u003eA. Kozłowska:\u003c/strong\u003e Resources, Conceptualization. \u003cstrong\u003eA. C. Tsai:\u003c/strong\u003e Methodology, Formal analysis. \u003cstrong\u003eC.H. Tseng:\u003c/strong\u003e Software. \u003cstrong\u003eC.C. Chen\u003c/strong\u003e: Conceptualization. \u003cstrong\u003eA.C.W. Huang:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Investigation, Formal analysis, Data curation, Funding acquisition, Conceptualization. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChang SH, Yu YH, He A, Ou CY, Shyu BC, Huang ACW. BDNF Protein and BDNF mRNA Expression of the Medial Prefrontal Cortex, Amygdala, and Hippocampus during Situational Reminder in the PTSD Animal Model. Behav Neurol. 2021;2021:6657716.\u003c/li\u003e\n\u003cli\u003eGiustino TF, Fitzgerald PJ, Maren S. 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Neuropsychopharmacology. 2000;23(1):79\u0026ndash;88.\u003c/li\u003e\n\u003cli\u003eLatagliata EC, Valzania A, Pascucci T, Campus P, Cabib S, Puglisi-Allegra S. Stress-induced activation of ventral tegmental mu-opioid receptors reduces accumbens dopamine tone by enhancing dopamine transmission in the medial pre-frontal cortex. Psychopharmacology (Berl). 2014;231(21):4099\u0026ndash;108.\u003c/li\u003e\n\u003cli\u003eAdkins AM, Colby EM, Kim WK, Wellman LL, Sanford LD. Stressor control and regional inflammatory responses in the brain: regulation by the basolateral amygdala. J Neuroinflammation. 2023;20(1):128.\u003c/li\u003e\n\u003cli\u003eArakawa H, Arakawa K, Blandino P, Jr., Deak T. The role of neuroinflammation in the release of aversive odor cues from footshock-stressed rats: Implications for the neural mechanism of alarm pheromone. Psychoneuroendocrinology. 2011;36(4):557\u0026ndash;68.\u003c/li\u003e\n\u003cli\u003eMalikowska-Racia N, Salat K, Nowaczyk A, Fijalkowski L, Popik P. 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Ann N Y Acad Sci. 2006;1071:67\u0026ndash;79.\u003c/li\u003e\n\u003cli\u003eHuang AC, Shyu BC, Hsiao S, Chen TC, He AB. Neural substrates of fear conditioning, extinction, and spontaneous recovery in passive avoidance learning: a c-fos study in rats. Behav Brain Res. 2013;237:23\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eHuang AC, Hsiao S. Haloperidol attenuates rewarding and aversively conditioned suppression of saccharin solution intake: reevaluation of the anhedonia hypothesis of dopamine blocking. Behavioral Neuroscience. 2002;116:646\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eStarcevic A, Postic S, Radojicic Z, Starcevic B, Milovanovic S, Ilankovic A, et al. Volumetric analysis of amygdala, hippocampus, and prefrontal cortex in therapy-naive PTSD participants. Biomed Res Int. 2014;2014:968495.\u003c/li\u003e\n\u003cli\u003eMorey RA, Haswell CC, Hooper SR, De Bellis MD. Amygdala, Hippocampus, and Ventral Medial Prefrontal Cortex Volumes Differ in Maltreated Youth with and without Chronic Posttraumatic Stress Disorder. Neuropsychopharmacology. 2016;41(3):791\u0026ndash;801.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Fo Guang University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Posttraumatic stress disorder, dopamine, medial prefrontal cortex, amygdala, hippocampus, short-term memory, long-term memory","lastPublishedDoi":"10.21203/rs.3.rs-7497727/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7497727/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe role of the dopamine system in modulating traumatic memories in posttraumatic stress disorder (PTSD) is unclear. This study investigated the effect of a dopamine D2 receptor antagonist, haloperidol, on fear responses in rats following a footshock. Fear was assessed by measuring freezing behavior during tests of short-term (STM) and long-term (LTM) memory. All rats were administered haloperidol 45 minutes before a footshock (3mA, 10 seconds) and were subsequently placed in the apparatus for two minutes daily over the next three days without a footshock. In Experiment 1, STM was tested by placing the rats back in the apparatus for a two-minute session. In Experiment 2, LTM was assessed using an identical test, which was repeated seven days later. Experiment 3 utilized the STM protocol, followed by an analysis of brain tissue for the protein interleukin-1b (IL-1b). Results demonstrated that haloperidol reduced freezing behavior in the short-term traumatic memory period. This effect was not observed a week later, indicating that haloperidol did not alter consolidated, long-term fear memories. The footshock was found to alter IL-1b expression in key fear and memory circuits. Importantly, haloperidol\u0026rsquo;s fear-reducing effect was associated with reduced IL\u0026thinsp;\u0026minus;\u0026thinsp;1b expression in the nucleus accumbens (NAc), central amygdala (CeA), and hippocampus (CA1, CA2, CA3). These findings suggest that dopamine D2 receptor blockade could represent a new therapeutic approach to preventing the initial consolidation of traumatic memories shortly after an event, but its efficacy in treating established, long-term PTSD may be limited.\u003c/p\u003e","manuscriptTitle":"Haloperidol prevents the consolidation of short-term traumatic memory by modulating interleukin-1β in neural substrates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 13:04:03","doi":"10.21203/rs.3.rs-7497727/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff265012-a9bc-427b-92cc-79a570efc8a0","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54136904,"name":"Psychology"},{"id":54136905,"name":"Psychiatry"}],"tags":[],"updatedAt":"2025-09-03T13:04:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 13:04:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7497727","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7497727","identity":"rs-7497727","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}