Repeated exposure to specific phobia stimuli, Trimethylthiazoline, promoted fear extinction, hippocampal neurogenesis, and microglia activation in rats

Repeated exposure to specific phobia stimuli, Trimethylthiazoline, promoted fear extinction, hippocampal neurogenesis, and microglia activation in rats | 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 Article Repeated exposure to specific phobia stimuli, Trimethylthiazoline, promoted fear extinction, hippocampal neurogenesis, and microglia activation in rats Peter, Bohao YANG, Jackie, Ngai-Man CHAN, Timothy, Kai-Hang FUNG, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6698181/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 Specific phobia (SP) is an anxiety disorder characterized by an overwhelming and irrational fear of specific objects, situations, or activities. In clinical practice, exposure therapy is considered one of the most effective treatments for SP. This approach involves repeated exposure to fear-inducing stimuli to facilitate fear extinction. While fear extinction and systematic desensitization are learning-dependent processes, the role of hippocampal neurogenesis remains unexplored in this context. In this study, we employed an animal model of repeated exposure to Trimethylthiazoline (TMT), a predator odor derived from fox feces, to investigate the potential involvement of hippocampal neurogenesis in exposure therapy. Twenty adult male Sprague Dawley rats were randomly assigned to either a control group (exposed to distilled water) or a TMT-exposed group (exposed to TMT) repeatedly within 14 days. Serum samples were collected 2–3 days following treatment. Neurogenesis and cell proliferation in brain tissues were analyzed via immunohistochemistry. The results demonstrated that repeated TMT exposure facilitated fear extinction without inducing anxiety- or depression-like behaviors. Additionally, TMT exposure enhanced neurogenesis by promoting the differentiation of neuronal progenitors into immature neurons. Microglial activation, implicated in various stages of adult neurogenesis, also increased following TMT exposure. These findings provide preliminary evidence supporting the therapeutic mechanism, showing that repeated TMT exposure not only facilitates fear extinction but also promotes neurogenesis and microglial activation. This study suggests the potential roles of neurogenesis and microglial activation in fear extinction or systematic desensitization, and identifies novel targets for future therapeutic development in SP. Biological sciences/Neuroscience Health sciences/Health care Health sciences/Neurology Phobia anxiety depression neurogenesis microglia and astrocytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. INTRODUCTION Specific phobia (SP) is an irrational fear of a specific object or situation that poses harm to an individual 1 . Certain widespread irrational phobias, including acrophobia (fear of heights), nyctophobia (fear of darkness), and aquaphobia (fear of water), are partially attributed to genetic and evolutionary factors 2,3 . According to a population-based survey (2001–2011), cross-national lifetime prevalence rates of specific phobias were 7.4%, particularly animal phobias representing the largest proportion (3.0%; 1.4–8.7% across countries) 4 . Exposure therapy (also known as flooding therapy), a treatment that involves direct and prolonged confrontation with the feared stimulus, is one of the effective interventions for SP. This approach seeks to dismantle maladaptive fear responses by replacing, overriding, or extinguishing phobic associations 5 . To further understand the biological mechanism of SP, prior studies have established an animal model comparable to animal phobia by using Trimethylthiazoline (TMT) which is a compound originally isolated from fox feces 6 . Although laboratory rats and mice were raised in animal colonies without being exposed to predators, the fear and anxious reactions to predators and predator scents still exist and can be regarded as unconditioned or unlearned 7 . TMT evokes these unconditioned fear reactions in rodents, eliciting characteristic freezing behavior and other defensive responses 8 . Central to mediating these responses is the amygdala, a brain region widely recognized as critical for processing fear and threat-related stimuli 6 . For instance, a prior animal study demonstrated that pharmacological inactivation of the medial amygdala inhibited TMT-induced freezing behavior, while basolateral amygdala inactivation delayed the onset of freezing responses 9 . However, fear response is not limited to threat detection, it often involves associative learning processes. The hippocampus plays a key role in contextual fear conditioning by creating connections between neutral surroundings and aversive outcomes 10,11 . Although these studies clarify mechanisms of fear acquisition, critical gaps remain regarding fear extinction—the process by which learned fear diminishes when a threat-predictive stimulus is repeatedly presented without adverse consequences. Notably, it remains unknown whether innate TMT-evoked fear (unlike conditioned fear) can be extinguished through non-reinforced exposure, and whether the hippocampus engages in this process. Recent advances in neuroscience research have revealed that adult neurogenesis—defined as the process that new neurons are continuously produced and incorporated into the existing neural circuitry in the hippocampus and the subventricular zone—may be necessary for fear extinction 12,13 . For instance, prior research has indicated that neurogenesis suppression resulted in a reduced behavioral response (freezing) in transgenic rats 14 . Despite evidence may suggest a link between the hippocampal neurogenesis and fear response, it is still uncertain the effects of exposure therapy on hippocampal neurogenesis and whether neurogenesis mechanistically supports extinction in innate fear paradigms. Thus, the present study aims to investigate the effects of repeated exposure to TMT on fear extinction in an animal model. This study seeks to elucidate whether neurogenesis is involved in flooding therapy. The behaviors evaluated in this study include chronic, repeated exposure to TMT on fear response, depression-like behavior, and anxiety-like behavior. Additionally, the mechanisms underlying repeated exposure to TMT will be examined at the cellular level, with a focus on neurogenesis. We hypothesize that repeated exposure to fear stimuli will facilitate fear extinction and promote hippocampal neurogenesis. 2. METHODS 2.1 Animals and ethics declaration As much as feasible were made to reduce the quantity of animals used and their suffering. All animal experimental procedures in this study were reviewed and approved by the Animal Subjects Ethics Sub-committee of The Hong Kong Polytechnic University and conducted according to the Animals (Control of Experiments) Ordinance of Hong Kong. All methods in this study are reported in accordance with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. Adult male Sprague Dawley (SD) rats, which were six to seven weeks old, were purchased from the Hong Kong Polytechnic University's Centralized Animal Facility (CAF). The rats were housed in cages with three animals, with a temperature of 22 ± 2°C and a 12-hour light/dark cycle (with lights on at 7 a.m.). Ad libitum feed of food and tap water were allowed. 2.2 Chemical (TMT) Based on previous evidence 7 , the capability to detect TMT and other predator scents is innate. Also, innate fear response, like freezing, is a strong reaction that can be measured and correlated with the intensity of the unconditioned fear stimulus 15 . Therefore, the administration of TMT was used to induce innate fear response. One gram of TMT with a purity greater than 90% was purchased from BioSRQ. This chemical is initially a very slight yellow color that gradually darkens when exposed to oxygen. It was stored in glass vials shielded from light, between 4˚C and 6˚C. 2.3 TMT Exposure System The exposure apparatus is shown by Fig. 2 c. The exposure system and protocol were established based on a previous publication 15 . During the testing sessions, each rat was placed individually within 200 mm long by 86 mm diameter non-restrictive cylinder. At both sides of ends of the cylinder, filter papers (20 mm in length and 20 mm in breadth) soaked in distilled water or 300µM of TMT were taped so that the rat faced one of the papers. The rat is therefore unable to flee or avoid the TMT source. All animals were exposed to the same dosage of water or TMT for 10 minutes, and the actions of rats were recorded by camera for fear response and behavioral analysis. There were two different cylinders used: one was used only for vehicle exposure, while the other was used for the TMT exposure. 70% ethanol was used to clean the apparatus following each exposure session. 2.4 Experiment Design The overview of the experimental setup and study design is shown in Fig. 1 . Sprague-Dawley (SD) rats were used to perform the current study. The SD rats were randomly divided into two groups (n = 10 per group) based on the following manner: 1. Control group (Ctrl): rats were exposed to the cylinder with filter paper soaked with distilled water in the test session; 2. TMT-exposed group (TMT): rats were exposed to the cylinder with filter paper soaked with 300µM of TMT. The duration of each test session was ten minutes, and the exposure treatments were administrated for fourteen consecutive days between 11:00 and 14:00. Also, video recordings were conducted for behavioral analysis during each treatment session for fourteen consecutive days. On days 12, 13, and 14, all the animals received an intraperitoneal injection of 50 mg/kg/day bromodeoxyuridine (BrdU) to label proliferative cells in the central nervous system. From day 15 to 16, behavioral tests were carried out with recorded videos. On day 17, the plasma samples were collected, followed by animal scarification and intracardial perfusion, between 10:00–17:00, to collect brain tissue for biochemical assay. 2.5 Behavioral Test 2.5.1 Freezing behavior Freezing behavior is a strong reaction that can be measured and linked to the intensity of the unconditioned fear stimulus 7 . The assessment for fear response was carried out using the test of freezing behavior as previously described 15,16 . The videos recorded previously were analyzed by an experimenter blinded to the testing condition. The following variables were measured to evaluate fear behavior: 1. Total freezing time in TMT-exposure sessions, which is the length of time showing freezing action during the test sessions; 2. In TMT-exposure sessions, the latency to freeze refers to the interval of time between the animals being placed inside the cylinder and exhibiting freezing. A higher level of fear response was indicated by the longer freezing time and shorter latency to freeze in the 10-minute test session 7 . To determine whether freezing behavior was strengthened by repeated TMT exposure, the behavioral changes observed during the treatment were analyzed. 2.5.2 Open Field Test (OFT) The anxiety of rats was measured by OFT in an open field that measured 72 cm long by 72 cm wide by 40 cm deep, with 550 lux of illumination. Above the wide field, a video camera was positioned and adjusted to capture the whole test area. Each animal was placed in the arena for ten minutes, during which time it was free to roam about. The analysis was taken to comprise: Peripheral locomotor activity is the amount of time spent wandering near the walls of the test field; central locomotor activity is the amount of time spent in the middle of the field (36 cm × 36 cm region). Anxious-like behavior can be identified by an increase in the amount of time spent in the peripheral region or a decrease in time spent in the central region. 17 . 2.5.3 Forced Swimming Test (FST) The assessment of depressive-like behaviors by using FST was carried out according to previous publications 18–21 . Rats were submerged in a glass cylinder (40 cm height x 30 cm diameter) filled with water at a depth of 30 cm at room temperature. The cylinders were deep enough to prevent the rats from using the cylinders' bottoms as support. Two swimming sessions were performed: a preliminary 15-minute habituation test and a subsequent 10-minute test 24 hours later. The procedure was recorded by camera, and the period of inactivity or swimming was assessed to gauge depressive-like behavior. The FST was scored the following behaviors: (1) time spent immobile – floating in the water without struggling, with only the necessary movement to keep from drowning, (2) time spent swimming – making active motions, such as moving around in the cylinder, more than necessary to simply keep from drowning, and less movements than those shown when climbing/struggling, and (3) time spent climbing/struggling – showing vigor. Depression like behavior was indicated when rats spent more time immobile during the test 20,22 . 2.6 Animal perfusion and tissue processing Before the animals were perfused, samples of truncal blood were taken, and the serum was collected by centrifuging the samples for 15 minutes at 2000×g. Samples of serum were kept at -80°C. The concentration of Brain-derived neurotrophic factor (BDNF) was measured using biochemical analyses performed on the serum. Scarification and perfusion were carried out based on a previous publication 18 . Rats were sacrificed by receiving 200 mg/kg sodium pentobarbital by intraperitoneal (IP) injection. Immediately after sacrificing, rats were transcardially perfused with 4% paraformaldehyde (PFA), and their brains were postfixed overnight at 2–8°C and then cryoprotected in 30% sucrose. 40 µm-thick coronal brain slices were prepared by a cryostat (Leica) in a 1-in-12 series of successive sections and kept in a cryoprotectant solution at -20°C. For immunostaining, portions of the SVZ and hippocampal regions were preserved on gelatine-coated glass slides. 2.7 Immunohistochemistry 2.7.1 Immunoperoxidase staining Detection of BrdU (neuronal cell proliferation marker) positive cells followed and modified the methods in previous publications 23–25 . Six hippocampal and four subventricular sections will be affixed onto gelatin-coated slides and air dried overnight. After rehydrating the slides in 0.01 M PBS (phosphate-buffered saline), the sections were incubated in preheated citrate buffer (pH = 6.0) for antigen retrieval for 25 minutes. To denaturize the DNA, the slides were incubated in 2 N HCl for 30 minutes at 45°C, followed by acid neutralization by incubating the slides in 0.1 M borate buffer for 15 minutes at room temperature. After neutralization, the slides were washed three times with 0.01M PBS before being incubated overnight with a 1:500 mouse anti-BrdU antibody (Roche). Following incubation, the slides were washed three times with 0.01 M PBS before being incubated for two hours at room temperature with 1:200 biotinylated goat anti-mouse antibody (Dako). Following the secondary antibody incubation, three 10-minute washes with 0.01 M PBS were performed on the slides. By using diaminobenzidine hydrochloride as chromogen, signal amplification was carried out using an avidin-biotin complex system (Vector) to visualize the BrdU-labeled cells 18,25 . After being air dried and counter-stained for three minutes with 10% eosin in 70% ethanol, the immunohistostained sections were dehydrated as follows (for three minutes in each solvent): Two times at room temperature in 90% ethanol, three times in 100% ethanol, and three times in xylene. The slides were covered with DPX mounting medium (Thermo Scientific). The detection of immature neurons was conducted by immunohistostaining of doublecortin (DCX, a protein produced by immature neurons) as previously described 23–25 . The DCX immunostaining methodology is like the BrdU protocol, with the exception that the primary and secondary antibodies were rabbit anti-DCX antibody (1:200, Cell Signaling) and goat anti-rabbit biotinylated antibody (1:200, Dako) respectively, and the incubations with HCl and borate buffer were skipped. The detection of microglia and astrocytes in the hippocampus was performed by immunohistostaining of ionized calcium-binding adapter molecule 1 (IBA-1) and glial fibrillary acidic protein (GFAP) based on previous publications 26,27 . The staining protocol for IBA-1 and GFAP is same as the DCX staining protocol, which was previously mentioned, with the exception that the primary antibodies were anti-IBA-1 antibody (goat polyclonal antibody from FujiFilm, 1:200) and anti-GFAP antibody (anti-GFAP with concentration of 0.1 mg/ml from Sigma Aldrich, 1:200) respectively. 2.7.3 Immunohistoflourescent double labeling Detection of cell differentiation and colocalization was carried out by fluorescent double labeling as previously described 28,29 . To confirm if the BrdU-labeled progenitors have become immature neurons, astrocytes, or microglia cells, staining can be combined with labeling for following markers: 1. DCX labeling for immature neurons; 2. glial fibrillary acidic protein labeling (GFAP, anti-GFAP with concentration of 0.1 mg/ml from Sigma Aldrich) for astrocytes; 3. ionized calcium-binding adapter molecule 1 labeling (IBA-1, anti-IBA-1, goat polyclonal antibody from FujiFilm) for microglia cells. The procedure of fluorescent double labeling staining is similar with the BrdU peroxidase staining, with the exception that the samples were incubated by the following combinations of primary antibodies, BrdU & GFAP; BrdU & IBA-1; BrdU & DCX, and secondary antibodies were donkey anti-mouse 568 for BrdU and donkey anti- rabbits 488 for other markers. Additionally, the signal amplification and dehydration of samples were removed in immunohistoflourescent staining, and the slideFs incubated by secondary antibodies were washed by 0.01M PBS for three times following with immersing by anti-fade fluorescence mounting medium (Abcam) and attaching to glass cover slides (55 x 22mm) for observation under fluorescent microscope. 2.8 Quantification of positive cell 2.8.1 Cell counting for BrdU, DCX, IBA-1, and GFAP positive cells The BrdU and DCX positive cells are the markers of cell proliferation and immature neurons respectively. The optical fractionator probe in a Stereo Investigator System (version 11, MBF Bioscience) was used to quantify BrdU and DCX positive cells as previously mentioned 18,23,25 . At 20x magnification, the quantity of immunoreactive cells within the DG was calculated in each of the twelve sections of the brains. The settings of analysis are as follows: the dissector height is 15 µm, the guard zone height is 7.5 µm, and the counting frame size is 60 µm × 60 µm. Each animal had six coronal hippocampal sections counted. To determine the unilateral estimate of the total number of proliferative cells in the dentate gyrus, the obtained cell counts were multiplied by twelve since the systematic sampling was done on every 12th section. The total number of BrdU, DCX, IBA-1, and GFAP-positive cells was expressed as mean ± SEM. 2.8.2 Double labeling cell counting (BrdU & DCX, BrdU & IBA-1, BrdU & GFAP) The IBA-1 and GFAP-positive cells are the markers of microglia cells and astrocytes respectively. The quantification of double labeling was carried out according to a previous publication 30 . At 100x magnification, the quantity of four sorts of positive cells was counted by microscope observations by a blinded assessor. The co-expressions between BrdU and DCX, IBA-1, and GFAP were examined in 30 randomly chosen BrdU-positive cells per animal. Ratios of DCX, IBA-1, and GFAP to BrdU-labelled cells were determined as the rate of co-expression. The co-expression ratio was expressed as mean ± SEM. 2.9 Dendritic complexity of immature neurons (Sholl analysis) Sholl analysis is a methodical process that counts the number of intersections between dendrites and concentric circles drawn at predetermined distances from the soma. It is used to identify changes in the neuronal dendritic arborization 18,25,31,32 . In each animal, ten randomly chosen DCX-positive cells with tertiary dendrites in the DG were observed at 200× magnification. Microphotographs were captured and imported using the Sholl Analysis plugin (The Ghosh Lab, UCSD, La Jolla, CA, USA) into ImageJ software (National Institutes of Health, Bethesda, MD, USA). When the neurons were traced in ImageJ, concentric circles were created on cell bodies at intervals of 10 µm radius and as far out from the soma as 200 µm. The number of intersections that the dendrites and concentric circles form is used to measure the complexity of the dendritic structure. A dendritic structure that has more crossings is more complicated. 2.10 Biochemical analyses in serum (BDNF ELISA) The serum samples were collected on day 18. ELISA kits (from Invitrogen) were used to measure the serum concentration of BDNF according to the manufacturer's instructions. There were 16 animals in total. 2.11 Statistical analysis GraphPad Prism v10.4.0 software was used for statistical analysis and plotting data (San Diego, CA, USA). All data were presented as mean ± standard error of the mean (SEM). To compare differences between the TMT and Ctrl groups, an independent samples t-test was carried out. To compare differences in freezing duration between the TMT groups and Ctrl groups among pre- and post-exposure timepoints, a Two-Way ANOVA was performed. To evaluate specific differences between groups, a multiple comparisons test was performed using Tukey’s multiple comparisons test. For each test, the significance level was set at p 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. 3. RESULTS 3.1 Repeated exposure to TMT did not alter inter-group body weight but revealed differential intra-group weight dynamics On days 1, 7, and 14 of treatment, the body weight of the animals was measured. No statistical differences in body weight between groups on both day1 and day14 were found (Fig. 2 a) By checking the within group differences, the body weight of Ctrl significantly increased after sham exposure (Fig. 2 b; day1: 318.8 g ± 10.9, day14: 360.9 g ± 5.7, p = 0.0108), but no significant weight differences were found in TMT group between day1 and day14 (Fig. 2 b; day1: 310.2 g ± 11.0, day14: 343.1 g ± 7.1, p = 0.0636). 3.2 Repeated exposure to TMT induced fear extinction but did not change emotion-related behavior Freezing is a response associated with the intensity of the unconditioned fear stimulus. The results of the intra- and inter-group comparisons demonstrate that repeated exposure to TMT significantly decreased fear responses. On both timepoints of day 1 and day 13, statistically significant differences in freezing time were found between the TMT and Ctrl groups. On day 1, the freezing duration in TMT group (Fig. 2 d-e; 413.4 s ± 19.4) was significantly higher (p < 0.0001) than Ctrl group (120.4 s ± 19.2); on day13, statistical difference (Fig. 2 d-e; p = 0.013) was also observed between TMT (324.2 s ± 24) and Ctrl (175.8 s ± 48.2) group in which TMT group showed longer freezing time than Ctrl group. However, by comparing the freezing time between day1 and day13 within groups, a statistically significant decrease was found in TMT group (Fig. 2 e; day1: 413.4 s ± 19.4, day13: 324.2 s ± 24, p = 0.0438) but no differences were found in Ctrl group (Fig. 2 e; day1: 120.4 s ± 19.2, day13: 175.8 s ± 48.2, p = 0.2028). The results reveal that repeated exposure to TMT would help fear extinction or systematic desensitization. We carried out additional analysis, in which the within group differences between TMT and Ctrl groups were also calculated by freezing duration on day1 minus the duration on day13, and the results showed that the within group change of freezing duration in TMT group (Fig. 2 f; 89.2 s ± 21.4) was significantly higher (p = 0.0325) than Ctrl group (-55.4 ± 58.6). In OFT, no significant differences in locomotor activity between groups were found by total distance traveled in testing arena (Fig. 3 b), but marginal effect (Fig. 3 a; p = 0.0716) was found in time spent in center area among groups in which Ctrl group (16.4s ± 2.2) spent more time than TMT group (10s ± 2.5). In FST, a marginal difference (Fig. 3 c; p = 0.0642) of floating time was also found between the two groups, in which the TMT group (244.8s ± 35.3) spent more time on floating than the Ctrl group (149s ± 33.4). 3.3 Repeated exposure to TMT increased hippocampal neurogenesis and microglia activation, but decreased the total number and differentiation of astrocytes For DCX-positive cells (Fig. 4 a), which represented immature neurons, TMT treatment led to a significant decrease in their number within the hippocampus (Fig. 4 b; 14927.6 ± 903.7 cells) compared to the Ctrl group (19310.5 ± 1439.5 cells, p = 0.0164). A significant difference in the total number of BrdU-positive cells (Fig. 4 c) was observed between the Ctrl and TMT groups (Fig. 4 d; p = 0.0279). Compared to the control (Ctrl) group (16461 ± 1937.4 cells), repeated TMT exposure significantly decreased the number of BrdU-positive cells in the hippocampus (11106 ± 1006.1 cells). In terms of astrocyte numbers reflected by the GFAP marker (Fig. 5 a), showed a significant reduction (Fig. 5 b; p = 0.0157) after TMT treatment. The hippocampus of the TMT group had fewer astrocytes (12444 ± 1941.8 cells) compared to the Ctrl group (18836 ± 1036.9 cells). Regarding microglia (Fig. 5 c), as indicated by the IBA-1 marker, TMT exposure significantly increased (Fig. 5 d; p = 0.0015) the number of microglia in the hippocampus (13332 ± 403.9 cells) compared to the Ctrl group (7772 ± 875.9 cells). Furthermore, repeated TMT exposure significantly altered the ratios of DCX/BrdU (Fig. 6 a), IBA-1/BrdU (Fig. 6 c), and GFAP/BrdU (Fig. 6 e) double labelling, providing additional insights into the changes in hippocampal cell populations. DCX/BrdU ratio was significantly higher in the TMT group (Fig. 6 b; 77 ± 1.7) compared to the Ctrl group (54.2 ± 5, p = 0.0049). Conversely, the IBA-1/BrdU co-staining ratio (Fig. 6 d; 22.6 ± 2.9) and the GFAP/BrdU ratio (Fig. 6 f; 26.4 ± 1.5) were significantly decreased in the TMT group compared to the Ctrl group (IBA-1/BrdU: 34.5 ± 2.2, p = 0.0082; GFAP/BrdU: 36.1 ± 2.6, p = 0.0096). In addition, no statistically significant difference in serum BDNF level was found between groups (Fig. 7 a). Also, no significant difference between the TMT and Ctrl groups was found indicating a positive effect of repeated exposure of TMT to increase dendritic complexity (Fig. 7 b). 4. DISCUSSION The present study demonstrated that repeated exposure to a fear stimulus alleviates SP, as evidenced by the significant reduction in freezing duration. Findings in the current study align with exposure therapy, or flooding, which involves exposing the patient to fear-inducing stimuli to break the pattern of escape that maintains the fear. Despite the efficacy, previous evidence showed that the clinical application of exposure therapy to clinical practice is difficult 33 . For instance, one previous study showed that approximately 20–30% of patients with severe arachnophobia refused or prematurely terminated exposure therapy because they found it too anxiety-provoking 34 . One factor contributing to the underutilization of exposure therapy in clinical settings is repeated exposure to fear-inducing stimuli may be excessively challenging and stressful for patients. Additionally, concerns regarding the potential exacerbation of symptoms associated with specific phobias further contribute to the hesitation in employing exposure therapy 33 . To address such concerns, the present study performed anxiety- and depression- like behavior tests at the end of treatment to examine the potential adverse effects brought by repeated exposure to fear stimuli. However, no statistically significant difference in behavior tests was observed between the Ctrl and TMT groups, suggesting that repeated exposure to the fear stimuli does not induce or exacerbate anxiety and depression in animal behavioral tasks. Altogether, the current study and prior publications provide further reassurance regarding the application of exposure therapy in both preclinical and clinical practice. Accumulating evidence implicates hippocampal neurogenesis as a potential mechanism in fear extinction. For instance, a previous study highlighted that neurogenesis acts as a potential mechanism underlying exercise on adolescent fear extinction 35 . Another study also showed that mice with fewer adult-born dentate gyrus granule cells in the immature stage performed worse on extinction tasks 14 . Data in the present study align with previous observations, suggesting that neurogenesis is associated with fear extinction. We found that the total number of DCX-positive cells reduced after repeated TMT exposure. However, neuronal precursor cells begin to express DCX while actively dividing, and their neuronal daughter cells continue to express DCX for 2–3 weeks as the cells mature into neurons 36 , indicating that the period during which the typical DCX expression window is much longer than the current TMT treatment period. Given that a proportion of DCX-positive cells may pre-exist before the treatment began, a decreased total number of DCX-positive cells in the current result might only imply that repeated exposure to TMT decreased the population of immature neurons in the hippocampus. The effects of current treatment on neurogenesis still need to be confirmed. To further elucidate the effects of TMT repeated exposure on hippocampal neurogenesis, we analysed the total counts of BrdU and DCX/BrdU co-labelling to assess neurogenesis during the treatment period. Increased total number of BrdU-positive cells indicates that repeated TMT exposure enhanced cell proliferation in the hippocampus. Critically, BrdU was administered during the final three days of treatment, ensuring that labelled cells reflect proliferation events occurring specifically within this window. Similarly, DCX/BrdU co-labelled cells represent immature neurons generated during the BrdU injection period (i.e., 1–3 days before sacrifice), as DCX is transiently expressed in newly differentiated neurons. The elevated BrdU-positive cell counts and DCX/BrdU co-labelling ratio would collectively confirm that repeated exposure to TMT promoted cell proliferation and differentiation of hippocampal neurons. Furthermore, this result confirmed that the observed neurogenesis changes are caused by TMT exposure rather than pre-existing neurogenesis, resolving uncertainty associated with static DCX labelling alone. Previous studies demonstrated that microglia has an impact on each of the distinct processes involved in adult neurogenesis under physiological conditions, such as the growth of neuronal stem cells, the migration of adult neuronal cells and immature neurons, their differentiation into mature neurons, the extension of neuronal synapses, and the integration of immature neurons into an existing circuit 37–39 . To determine the involvement of microglia in neurogenesis, single and double labeling of immunohistostaining of IBA-1 were conducted in the present study. The current result is consistent with previous findings 37–39 since an increased total number of IBA-1-positive cells was observed after TMT repeated exposure. However, as IBA-1 is only expressed in macrophages and microglia, and it is increased when these cells are activated 40 . It is difficult to determine whether the increased number of IBA-1-positive cells was mainly caused by activation of existing microglia or increased differentiation from progenitors to microglia. Thus, IBA-1/BrdU co-labelling was performed in the current study, and the result showed a decreased IBA-1/BrdU ratio after treatment. This result suggests newly proliferating cells are less likely to differentiate into microglia, and existing microglia are becoming activated in response to repeated TMT exposure. Additionally, the comparison between the ratio of DCX/BrdU and IBA-1/BrdU further confirmed that more progenitor cells are differentiated into immature neurons rather than microglia. Furthermore, apart from neurogenesis, microglia can also modulate the process of fear extinction by pruning neuronal synapses 41 . It has been shown that microglia facilitate the formation, maturation, and selective elimination of immature synapses, a fundamental process for proper brain development 42 , and synapse elimination by microglia may lead to degradation of memory engrams and forgetting of previously learned contextual fear memory 41 . Therefore, the present study offered further evidence that microglia activation may promote neurogenesis in multiple stages. Regarding GFAP, the current study revealed that the total number of GFAP-positive cells and the GFAP/BrdU ratio decreased in the hippocampus after TMT exposure. By comparing with the DCX/BrdU ratio, the GFAP/BrdU ratio decreased after TMT exposure, while the DCX/BrdU ratio increased. Thus, similar to the result from IBA-1/BrdU ratio, this result further confirmed that the proliferating cells were more likely to differentiate into neurons rather than astrocytes. In addition, increased expression of GFAP as a marker of astrocyte activation and a hallmark of multiple CNS pathologies is associated with neuroinflammation 43–45 . Previous studies showed that prolonged exposure to stressful stimuli that elicit fear and anxiety activate both central and peripheral immune cells to release cytokines, such as IL-1 β , which induces astrocyte proinflammatory gene expression 45–47 . In this way, based on the reduction of the total number of GFAP-positive cells and differentiation ratio in the present study, decreased neuroinflammation may be involved in the process of fear extinction or desensitisation induced by repeated exposure to TMT. 5. Conclusion The current study provides novel insights into the mechanisms underlying fear extinction induced by repeated exposure to TMT. Our behavioral results reinforced the efficacy of repeated fear stimulus exposure in reducing phobia-like freezing behavior, aligning with the principles of exposure therapy. Notably, the absence of exacerbation in anxiety- or depression-like behavioral tasks may address a critical clinical concern, offering preclinical evidence that repeated fear exposure, under controlled conditions, does not amplify negative affective states. This finding supports the safety and feasibility of exposure-based interventions, potentially encouraging broader clinical adoption. At the cellular level, reduced DCX-positive immature neurons and increased BrdU-labelled proliferating cells highlighted the dynamic nature of hippocampal neurogenesis during fear extinction. The preferential differentiation of BrdU-positive cells into newborn neurons (over microglia or astrocytes) further suggests that fear extinction may actively transfer progenitor cells toward a neuronal fate. Also, the present study advances understanding of glial contributions to fear extinction: increased total number of IBA-positive cells, coupled with reduced IBA-1/BrdU co-labelling, implies microglial activation, rather than differentiation, facilitates neurogenesis and synaptic remodelling, aligning with their roles in synaptic pruning and circuit refinement. Collectively, our findings proposed a potential treatment model in which repeated exposure to fear stimuli diminishes the fear responses through promoting hippocampal neurogenesis and microglial activation. However, the current study lacks mechanistic evidence linking hippocampal neurogenesis directly to behavioral outcomes and microglia activation. Thereby, future studies could further confirm the causality by pharmacologically inhibiting neurogenesis or microglial activation to investigate. Declarations ACKNOWLEDGEMENT We acknowledge Miss XU Yi, who offered support in preparation of the manuscript. DATA AVAILABILITY STATEMENT All data generated during this study are available from the corresponding author on request. ADDITIONAL INFORMATION Competing interests The authors declare no competing interests. FUNDING SOURCE This study is supported by the General Research Fund (15105621) by University Grant Council of Hong Kong SAR. Author Contribution Conceptualization: PBY, JNMC, TKHF, and BWML; Methodology: PBY, JNMC, TKHF, and BWML; Investigation: PBY, JNMC, and TKHF; Writing: PBY; Funding Acquisition: BWML; Resources: BWML and SPCN; Supervision: WKH, WKWL, JWHL, KFS, SPCN, and BWML. References LeBeau, R. T. et al. Specific phobia: a review of DSM-IV specific phobia and preliminary recommendations for DSM‐V. Depression and anxiety 27 , 148–167 (2010). Davey, G. C. L. Psychopathology and treatment of specific phobias. Psychiatry 6 , 247–253 (2007). https://doi.org/https://doi.org/10.1016/j.mppsy.2007.03.007 Garcia, R. Neurobiology of fear and specific phobias. 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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-6698181","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":468775720,"identity":"efab8b02-2a7b-48b8-a3a0-24ef4a7dd41e","order_by":0,"name":"Peter, Bohao YANG","email":"data:image/png;base64,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","orcid":"","institution":"Department of Rehabilitation Sciences, The Hong Kong Polytechnic University","correspondingAuthor":true,"prefix":"","firstName":"Bohao","middleName":"YANG","lastName":"Peter","suffix":""},{"id":468775721,"identity":"1aa7d93b-4989-48de-b042-01ea517fcc3b","order_by":1,"name":"Jackie, Ngai-Man CHAN","email":"","orcid":"","institution":"Department of Rehabilitation Sciences, The Hong Kong Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Ngai-Man","middleName":"CHAN","lastName":"Jackie","suffix":""},{"id":468775722,"identity":"c87d63ff-9b4a-4fe4-8e36-c071b626d296","order_by":2,"name":"Timothy, Kai-Hang FUNG","email":"","orcid":"","institution":"Department of Rehabilitation Sciences, The Hong Kong Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Kai-Hang","middleName":"FUNG","lastName":"Timothy","suffix":""},{"id":468775723,"identity":"03b39af6-c8f9-4eb2-b0f3-85ddaa708462","order_by":3,"name":"Wai-Kai HOU","email":"","orcid":"","institution":"Department of Psychology, Centre for Psychosocial Health, The Education University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Wai-Kai","middleName":"","lastName":"HOU","suffix":""},{"id":468775724,"identity":"7a761e1e-2b33-4d62-a14f-c587374e4f35","order_by":4,"name":"Way, Kwok-Wai LAU","email":"","orcid":"","institution":"School of Nursing and Health Studies, Hong Kong Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Kwok-Wai","middleName":"LAU","lastName":"Way","suffix":""},{"id":468775725,"identity":"7b939de3-422b-4c37-a669-0fe32160c5a3","order_by":5,"name":"Joseph, Wai-Hin LEUNG","email":"","orcid":"","institution":"Program in Neuroscience and Mental Health, SickKids Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Wai-Hin","middleName":"LEUNG","lastName":"Joseph","suffix":""},{"id":468775726,"identity":"d454df8d-7054-43d0-b652-6c9622e8d34d","order_by":6,"name":"Kwok-Fai SO","email":"","orcid":"","institution":"Key Laboratory of CNS Regeneration (Ministry of Education), Guangdong-Hong Kong- Macau Institute of CNS Regeneration, Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Kwok-Fai","middleName":"","lastName":"SO","suffix":""},{"id":468775727,"identity":"634684d4-415a-4325-99c2-1bc008cfad8f","order_by":7,"name":"Shirley, Pui-Ching NGAI","email":"","orcid":"","institution":"Department of Rehabilitation Sciences, The Hong Kong Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Pui-Ching","middleName":"NGAI","lastName":"Shirley","suffix":""},{"id":468775728,"identity":"64780ed1-f1ac-43c2-a302-ddb057f4ba0f","order_by":8,"name":"Benson, Wui-Man LAU","email":"","orcid":"","institution":"Department of Rehabilitation Sciences, The Hong Kong Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Wui-Man","middleName":"LAU","lastName":"Benson","suffix":""}],"badges":[],"createdAt":"2025-05-19 10:53:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6698181/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6698181/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84311045,"identity":"427a2263-017e-49c0-acee-8e87c3db4481","added_by":"auto","created_at":"2025-06-10 12:27:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19881,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of experimental setup. Two cohorts of animals received either sham or TMT treatment for fourteen consecutive days. Video recordings for behavioral analysis were conducted during each treatment session throughout this period. Bromodeoxyuridine (BrdU) injections were administered from Day 12 to Day 14. Subsequent behavioral tests were performed on Days 15 and 16, and the animals were sacrificed on Day 17 for brain tissue collection and biochemical assays.\u003c/p\u003e","description":"","filename":"TMTFigure11.png","url":"https://assets-eu.researchsquare.com/files/rs-6698181/v1/f1b754ab42d3799a17831c6b.png"},{"id":84311049,"identity":"d73c1a15-6906-40d1-bd83-e0b64c00f10a","added_by":"auto","created_at":"2025-06-10 12:27:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":382107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eBody weight was measured on day1, 7, and 14 of treatment; \u003cstrong\u003e(b) \u003c/strong\u003ea significant increase in body weight was found within Ctrl group by comparing the weight on day1 and day14 whereas no significant body weight increase was found in TMT group; \u003cstrong\u003e(c) \u003c/strong\u003eillustration of exposure apparatus; \u003cstrong\u003e(d) \u003c/strong\u003efreezing behavior was recorded and analysed on day 1, 3, 5, 7, 9, 11, and 13 during treatment; \u003cstrong\u003e(e)\u003c/strong\u003e comparison of freezing duration between groups revealed that the TMT group exhibited significantly longer freezing durations than the Ctrl group on both day 1 and day 13; \u003cstrong\u003e(e\u0026amp;f) \u003c/strong\u003ea significant decrease in freezing duration from day1 to day13 was found in TMT group, while no significant change was found in Ctrl group; results were expressed as Mean ± SEM; ns: p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"TMTFigure12.png","url":"https://assets-eu.researchsquare.com/files/rs-6698181/v1/71e1032847cb3b6aefde5204.png"},{"id":84311046,"identity":"6d62fff4-61bd-46d6-ba5c-8852c6ec2a3d","added_by":"auto","created_at":"2025-06-10 12:27:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":23903,"visible":true,"origin":"","legend":"\u003cp\u003eNo statistically significant difference was found in all emotion-related behavior tests; \u003cstrong\u003e(a\u0026amp;c) \u003c/strong\u003emarginal differences were found in time spent in center of OFT (p = 0.0716) and floating duration of FST (p = 0.0642); \u003cstrong\u003e(b) \u003c/strong\u003eno significant difference was found in total distance travelled in OFT between Ctrl and TMT group; results were expressed as Mean ± SEM; ns: p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"TMTFigure13.png","url":"https://assets-eu.researchsquare.com/files/rs-6698181/v1/4158047e4a6192ffc311eb98.png"},{"id":84312374,"identity":"11c6b514-f739-4600-b2a3-791845ce5a7a","added_by":"auto","created_at":"2025-06-10 12:43:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2323139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRepresentative images of the effect of the TMT exposure on the total number of DCX positive cells in the hippocampus; \u003cstrong\u003e(b) \u003c/strong\u003erepeat exposure by TMT significantly reduced the total number of DCX positive cells in hippocampus; \u003cstrong\u003e(c) \u003c/strong\u003erepresentative images of the effect of the TMT exposure on the total number of BrdU positive cells in the hippocampus; \u003cstrong\u003e(d) \u003c/strong\u003erepeat exposure by TMT significantly increased the total number of BrdU positive cells in hippocampus; results were expressed as Mean ± SEM; ns: p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"TMTFigure14.png","url":"https://assets-eu.researchsquare.com/files/rs-6698181/v1/80b7624e8ea9803f39385cf4.png"},{"id":84311475,"identity":"114fc193-90b3-4e31-b058-248dd1002d4e","added_by":"auto","created_at":"2025-06-10 12:35:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3296422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRepresentative images of the effect of the TMT exposure on the total number of GFAP positive cells in the hippocampus; \u003cstrong\u003e(b) \u003c/strong\u003erepeat exposure by TMT significantly reduced the total number of GFAP positive cells in hippocampus; \u003cstrong\u003e(c) \u003c/strong\u003erepresentative images of the effect of the TMT exposure on the total number of IBA-1 positive cells in the hippocampus; \u003cstrong\u003e(d) \u003c/strong\u003erepeat exposure by TMT significantly increased the total number of IBA-1 positive cells in hippocampus; results were expressed as Mean ± SEM; ns: p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"TMTFigure15.png","url":"https://assets-eu.researchsquare.com/files/rs-6698181/v1/0aca16a6a5aec1410754a2ac.png"},{"id":84311473,"identity":"2e580f60-8f0a-41ec-a8cd-c1dd2e07e31e","added_by":"auto","created_at":"2025-06-10 12:35:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2204845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRepresentative images of BrdU \u0026amp; DCX double labeling; \u003cstrong\u003e(b) \u003c/strong\u003erepeat exposure by TMT significantly increased the DCX/BrdU ratio in hippocampus; \u003cstrong\u003e(c) \u003c/strong\u003erepresentative images of BrdU \u0026amp; IBA-1 double labeling; \u003cstrong\u003e(d) \u003c/strong\u003erepeat exposure by TMT significantly reduced IBA-1/BrdU ratio in hippocampus; \u003cstrong\u003e(e) \u003c/strong\u003erepresentative images of BrdU \u0026amp; GFAP double labeling;\u003cstrong\u003e (f) \u003c/strong\u003erepeat exposure by TMT significantly reduced GFAP/BrdU ratio in hippocampus; results were expressed as Mean ± SEM; ns: p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"TMTFigure16.png","url":"https://assets-eu.researchsquare.com/files/rs-6698181/v1/870621da52dd7a88270a50b0.png"},{"id":84311047,"identity":"1b80b4df-f98d-4573-846a-02d837f8e7a1","added_by":"auto","created_at":"2025-06-10 12:27:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":28181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eno significant difference was found in BDNF level in serum sample between groups; \u003cstrong\u003e(b) \u003c/strong\u003eno statistically significant difference was observed in the number of intersections among the Ctrl and TMT groups; results were expressed as Mean ± SEM; ns: p \u0026gt; 0.05; *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"TMTFigure17.png","url":"https://assets-eu.researchsquare.com/files/rs-6698181/v1/c248ed151fe022e4f5adf2f8.png"},{"id":85329907,"identity":"38f7f01f-bb99-4721-9181-6b0f449c9188","added_by":"auto","created_at":"2025-06-24 17:46:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9827584,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6698181/v1/e74fd7ce-abde-481a-8c4b-8ae88517350f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Repeated exposure to specific phobia stimuli, Trimethylthiazoline, promoted fear extinction, hippocampal neurogenesis, and microglia activation in rats","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eSpecific phobia (SP) is an irrational fear of a specific object or situation that poses harm to an individual \u003csup\u003e1\u003c/sup\u003e. Certain widespread irrational phobias, including acrophobia (fear of heights), nyctophobia (fear of darkness), and aquaphobia (fear of water), are partially attributed to genetic and evolutionary factors \u003csup\u003e2,3\u003c/sup\u003e. According to a population-based survey (2001\u0026ndash;2011), cross-national lifetime prevalence rates of specific phobias were 7.4%, particularly animal phobias representing the largest proportion (3.0%; 1.4\u0026ndash;8.7% across countries) \u003csup\u003e4\u003c/sup\u003e. Exposure therapy (also known as flooding therapy), a treatment that involves direct and prolonged confrontation with the feared stimulus, is one of the effective interventions for SP. This approach seeks to dismantle maladaptive fear responses by replacing, overriding, or extinguishing phobic associations \u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo further understand the biological mechanism of SP, prior studies have established an animal model comparable to animal phobia by using Trimethylthiazoline (TMT) which is a compound originally isolated from fox feces \u003csup\u003e6\u003c/sup\u003e. Although laboratory rats and mice were raised in animal colonies without being exposed to predators, the fear and anxious reactions to predators and predator scents still exist and can be regarded as unconditioned or unlearned \u003csup\u003e7\u003c/sup\u003e. TMT evokes these unconditioned fear reactions in rodents, eliciting characteristic freezing behavior and other defensive responses \u003csup\u003e8\u003c/sup\u003e. Central to mediating these responses is the amygdala, a brain region widely recognized as critical for processing fear and threat-related stimuli \u003csup\u003e6\u003c/sup\u003e. For instance, a prior animal study demonstrated that pharmacological inactivation of the medial amygdala inhibited TMT-induced freezing behavior, while basolateral amygdala inactivation delayed the onset of freezing responses \u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, fear response is not limited to threat detection, it often involves associative learning processes. The hippocampus plays a key role in contextual fear conditioning by creating connections between neutral surroundings and aversive outcomes \u003csup\u003e10,11\u003c/sup\u003e. Although these studies clarify mechanisms of fear acquisition, critical gaps remain regarding fear extinction\u0026mdash;the process by which learned fear diminishes when a threat-predictive stimulus is repeatedly presented without adverse consequences. Notably, it remains unknown whether innate TMT-evoked fear (unlike conditioned fear) can be extinguished through non-reinforced exposure, and whether the hippocampus engages in this process.\u003c/p\u003e \u003cp\u003eRecent advances in neuroscience research have revealed that adult neurogenesis\u0026mdash;defined as the process that new neurons are continuously produced and incorporated into the existing neural circuitry in the hippocampus and the subventricular zone\u0026mdash;may be necessary for fear extinction \u003csup\u003e12,13\u003c/sup\u003e. For instance, prior research has indicated that neurogenesis suppression resulted in a reduced behavioral response (freezing) in transgenic rats \u003csup\u003e14\u003c/sup\u003e. Despite evidence may suggest a link between the hippocampal neurogenesis and fear response, it is still uncertain the effects of exposure therapy on hippocampal neurogenesis and whether neurogenesis mechanistically supports extinction in innate fear paradigms.\u003c/p\u003e \u003cp\u003eThus, the present study aims to investigate the effects of repeated exposure to TMT on fear extinction in an animal model. This study seeks to elucidate whether neurogenesis is involved in flooding therapy. The behaviors evaluated in this study include chronic, repeated exposure to TMT on fear response, depression-like behavior, and anxiety-like behavior. Additionally, the mechanisms underlying repeated exposure to TMT will be examined at the cellular level, with a focus on neurogenesis. We hypothesize that repeated exposure to fear stimuli will facilitate fear extinction and promote hippocampal neurogenesis.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals and ethics declaration\u003c/h2\u003e \u003cp\u003eAs much as feasible were made to reduce the quantity of animals used and their suffering. All animal experimental procedures in this study were reviewed and approved by the Animal Subjects Ethics Sub-committee of The Hong Kong Polytechnic University and conducted according to the Animals (Control of Experiments) Ordinance of Hong Kong. All methods in this study are reported in accordance with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. Adult male Sprague Dawley (SD) rats, which were six to seven weeks old, were purchased from the Hong Kong Polytechnic University's Centralized Animal Facility (CAF). The rats were housed in cages with three animals, with a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and a 12-hour light/dark cycle (with lights on at 7 a.m.). \u003cem\u003eAd libitum\u003c/em\u003e feed of food and tap water were allowed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Chemical (TMT)\u003c/h2\u003e \u003cp\u003eBased on previous evidence \u003csup\u003e7\u003c/sup\u003e, the capability to detect TMT and other predator scents is innate. Also, innate fear response, like freezing, is a strong reaction that can be measured and correlated with the intensity of the unconditioned fear stimulus \u003csup\u003e15\u003c/sup\u003e. Therefore, the administration of TMT was used to induce innate fear response. One gram of TMT with a purity greater than 90% was purchased from BioSRQ. This chemical is initially a very slight yellow color that gradually darkens when exposed to oxygen. It was stored in glass vials shielded from light, between 4˚C and 6˚C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 TMT Exposure System\u003c/h2\u003e \u003cp\u003eThe exposure apparatus is shown by Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. The exposure system and protocol were established based on a previous publication \u003csup\u003e15\u003c/sup\u003e. During the testing sessions, each rat was placed individually within 200 mm long by 86 mm diameter non-restrictive cylinder. At both sides of ends of the cylinder, filter papers (20 mm in length and 20 mm in breadth) soaked in distilled water or 300\u0026micro;M of TMT were taped so that the rat faced one of the papers. The rat is therefore unable to flee or avoid the TMT source. All animals were exposed to the same dosage of water or TMT for 10 minutes, and the actions of rats were recorded by camera for fear response and behavioral analysis. There were two different cylinders used: one was used only for vehicle exposure, while the other was used for the TMT exposure. 70% ethanol was used to clean the apparatus following each exposure session.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experiment Design\u003c/h2\u003e \u003cp\u003eThe overview of the experimental setup and study design is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Sprague-Dawley (SD) rats were used to perform the current study. The SD rats were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;10 per group) based on the following manner: 1. Control group (Ctrl): rats were exposed to the cylinder with filter paper soaked with distilled water in the test session; 2. TMT-exposed group (TMT): rats were exposed to the cylinder with filter paper soaked with 300\u0026micro;M of TMT. The duration of each test session was ten minutes, and the exposure treatments were administrated for fourteen consecutive days between 11:00 and 14:00. Also, video recordings were conducted for behavioral analysis during each treatment session for fourteen consecutive days. On days 12, 13, and 14, all the animals received an intraperitoneal injection of 50 mg/kg/day bromodeoxyuridine (BrdU) to label proliferative cells in the central nervous system. From day 15 to 16, behavioral tests were carried out with recorded videos. On day 17, the plasma samples were collected, followed by animal scarification and intracardial perfusion, between 10:00\u0026ndash;17:00, to collect brain tissue for biochemical assay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Behavioral Test\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Freezing behavior\u003c/h2\u003e \u003cp\u003eFreezing behavior is a strong reaction that can be measured and linked to the intensity of the unconditioned fear stimulus \u003csup\u003e7\u003c/sup\u003e. The assessment for fear response was carried out using the test of freezing behavior as previously described \u003csup\u003e15,16\u003c/sup\u003e. The videos recorded previously were analyzed by an experimenter blinded to the testing condition. The following variables were measured to evaluate fear behavior: 1. Total freezing time in TMT-exposure sessions, which is the length of time showing freezing action during the test sessions; 2. In TMT-exposure sessions, the latency to freeze refers to the interval of time between the animals being placed inside the cylinder and exhibiting freezing. A higher level of fear response was indicated by the longer freezing time and shorter latency to freeze in the 10-minute test session \u003csup\u003e7\u003c/sup\u003e. To determine whether freezing behavior was strengthened by repeated TMT exposure, the behavioral changes observed during the treatment were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Open Field Test (OFT)\u003c/h2\u003e \u003cp\u003eThe anxiety of rats was measured by OFT in an open field that measured 72 cm long by 72 cm wide by 40 cm deep, with 550 lux of illumination. Above the wide field, a video camera was positioned and adjusted to capture the whole test area. Each animal was placed in the arena for ten minutes, during which time it was free to roam about. The analysis was taken to comprise: Peripheral locomotor activity is the amount of time spent wandering near the walls of the test field; central locomotor activity is the amount of time spent in the middle of the field (36 cm \u0026times; 36 cm region). Anxious-like behavior can be identified by an increase in the amount of time spent in the peripheral region or a decrease in time spent in the central region. \u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Forced Swimming Test (FST)\u003c/h2\u003e \u003cp\u003eThe assessment of depressive-like behaviors by using FST was carried out according to previous publications \u003csup\u003e18\u0026ndash;21\u003c/sup\u003e. Rats were submerged in a glass cylinder (40 cm height x 30 cm diameter) filled with water at a depth of 30 cm at room temperature. The cylinders were deep enough to prevent the rats from using the cylinders' bottoms as support. Two swimming sessions were performed: a preliminary 15-minute habituation test and a subsequent 10-minute test 24 hours later. The procedure was recorded by camera, and the period of inactivity or swimming was assessed to gauge depressive-like behavior. The FST was scored the following behaviors: (1) time spent immobile \u0026ndash; floating in the water without struggling, with only the necessary movement to keep from drowning, (2) time spent swimming \u0026ndash; making active motions, such as moving around in the cylinder, more than necessary to simply keep from drowning, and less movements than those shown when climbing/struggling, and (3) time spent climbing/struggling \u0026ndash; showing vigor. Depression like behavior was indicated when rats spent more time immobile during the test \u003csup\u003e20,22\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Animal perfusion and tissue processing\u003c/h2\u003e \u003cp\u003eBefore the animals were perfused, samples of truncal blood were taken, and the serum was collected by centrifuging the samples for 15 minutes at 2000\u0026times;g. Samples of serum were kept at -80\u0026deg;C. The concentration of Brain-derived neurotrophic factor (BDNF) was measured using biochemical analyses performed on the serum. Scarification and perfusion were carried out based on a previous publication \u003csup\u003e18\u003c/sup\u003e. Rats were sacrificed by receiving 200 mg/kg sodium pentobarbital by intraperitoneal (IP) injection. Immediately after sacrificing, rats were transcardially perfused with 4% paraformaldehyde (PFA), and their brains were postfixed overnight at 2\u0026ndash;8\u0026deg;C and then cryoprotected in 30% sucrose. 40 \u0026micro;m-thick coronal brain slices were prepared by a cryostat (Leica) in a 1-in-12 series of successive sections and kept in a cryoprotectant solution at -20\u0026deg;C. For immunostaining, portions of the SVZ and hippocampal regions were preserved on gelatine-coated glass slides.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunohistochemistry\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Immunoperoxidase staining\u003c/h2\u003e \u003cp\u003eDetection of BrdU (neuronal cell proliferation marker) positive cells followed and modified the methods in previous publications \u003csup\u003e23\u0026ndash;25\u003c/sup\u003e. Six hippocampal and four subventricular sections will be affixed onto gelatin-coated slides and air dried overnight. After rehydrating the slides in 0.01 M PBS (phosphate-buffered saline), the sections were incubated in preheated citrate buffer (pH\u0026thinsp;=\u0026thinsp;6.0) for antigen retrieval for 25 minutes. To denaturize the DNA, the slides were incubated in 2 N HCl for 30 minutes at 45\u0026deg;C, followed by acid neutralization by incubating the slides in 0.1 M borate buffer for 15 minutes at room temperature. After neutralization, the slides were washed three times with 0.01M PBS before being incubated overnight with a 1:500 mouse anti-BrdU antibody (Roche). Following incubation, the slides were washed three times with 0.01 M PBS before being incubated for two hours at room temperature with 1:200 biotinylated goat anti-mouse antibody (Dako). Following the secondary antibody incubation, three 10-minute washes with 0.01 M PBS were performed on the slides. By using diaminobenzidine hydrochloride as chromogen, signal amplification was carried out using an avidin-biotin complex system (Vector) to visualize the BrdU-labeled cells \u003csup\u003e18,25\u003c/sup\u003e. After being air dried and counter-stained for three minutes with 10% eosin in 70% ethanol, the immunohistostained sections were dehydrated as follows (for three minutes in each solvent): Two times at room temperature in 90% ethanol, three times in 100% ethanol, and three times in xylene. The slides were covered with DPX mounting medium (Thermo Scientific).\u003c/p\u003e \u003cp\u003eThe detection of immature neurons was conducted by immunohistostaining of doublecortin (DCX, a protein produced by immature neurons) as previously described \u003csup\u003e23\u0026ndash;25\u003c/sup\u003e. The DCX immunostaining methodology is like the BrdU protocol, with the exception that the primary and secondary antibodies were rabbit anti-DCX antibody (1:200, Cell Signaling) and goat anti-rabbit biotinylated antibody (1:200, Dako) respectively, and the incubations with HCl and borate buffer were skipped.\u003c/p\u003e \u003cp\u003eThe detection of microglia and astrocytes in the hippocampus was performed by immunohistostaining of ionized calcium-binding adapter molecule 1 (IBA-1) and glial fibrillary acidic protein (GFAP) based on previous publications \u003csup\u003e26,27\u003c/sup\u003e. The staining protocol for IBA-1 and GFAP is same as the DCX staining protocol, which was previously mentioned, with the exception that the primary antibodies were anti-IBA-1 antibody (goat polyclonal antibody from FujiFilm, 1:200) and anti-GFAP antibody (anti-GFAP with concentration of 0.1 mg/ml from Sigma Aldrich, 1:200) respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3 Immunohistoflourescent double labeling\u003c/h2\u003e \u003cp\u003eDetection of cell differentiation and colocalization was carried out by fluorescent double labeling as previously described \u003csup\u003e28,29\u003c/sup\u003e. To confirm if the BrdU-labeled progenitors have become immature neurons, astrocytes, or microglia cells, staining can be combined with labeling for following markers: 1. DCX labeling for immature neurons; 2. glial fibrillary acidic protein labeling (GFAP, anti-GFAP with concentration of 0.1 mg/ml from Sigma Aldrich) for astrocytes; 3. ionized calcium-binding adapter molecule 1 labeling (IBA-1, anti-IBA-1, goat polyclonal antibody from FujiFilm) for microglia cells. The procedure of fluorescent double labeling staining is similar with the BrdU peroxidase staining, with the exception that the samples were incubated by the following combinations of primary antibodies, BrdU \u0026amp; GFAP; BrdU \u0026amp; IBA-1; BrdU \u0026amp; DCX, and secondary antibodies were donkey anti-mouse 568 for BrdU and donkey anti- rabbits 488 for other markers. Additionally, the signal amplification and dehydration of samples were removed in immunohistoflourescent staining, and the slideFs incubated by secondary antibodies were washed by 0.01M PBS for three times following with immersing by anti-fade fluorescence mounting medium (Abcam) and attaching to glass cover slides (55 x 22mm) for observation under fluorescent microscope.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Quantification of positive cell\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Cell counting for BrdU, DCX, IBA-1, and GFAP positive cells\u003c/h2\u003e \u003cp\u003eThe BrdU and DCX positive cells are the markers of cell proliferation and immature neurons respectively. The optical fractionator probe in a Stereo Investigator System (version 11, MBF Bioscience) was used to quantify BrdU and DCX positive cells as previously mentioned \u003csup\u003e18,23,25\u003c/sup\u003e. At 20x magnification, the quantity of immunoreactive cells within the DG was calculated in each of the twelve sections of the brains. The settings of analysis are as follows: the dissector height is 15 \u0026micro;m, the guard zone height is 7.5 \u0026micro;m, and the counting frame size is 60 \u0026micro;m \u0026times; 60 \u0026micro;m. Each animal had six coronal hippocampal sections counted. To determine the unilateral estimate of the total number of proliferative cells in the dentate gyrus, the obtained cell counts were multiplied by twelve since the systematic sampling was done on every 12th section. The total number of BrdU, DCX, IBA-1, and GFAP-positive cells was expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Double labeling cell counting (BrdU \u0026amp; DCX, BrdU \u0026amp; IBA-1, BrdU \u0026amp; GFAP)\u003c/h2\u003e \u003cp\u003eThe IBA-1 and GFAP-positive cells are the markers of microglia cells and astrocytes respectively. The quantification of double labeling was carried out according to a previous publication \u003csup\u003e30\u003c/sup\u003e. At 100x magnification, the quantity of four sorts of positive cells was counted by microscope observations by a blinded assessor. The co-expressions between BrdU and DCX, IBA-1, and GFAP were examined in 30 randomly chosen BrdU-positive cells per animal. Ratios of DCX, IBA-1, and GFAP to BrdU-labelled cells were determined as the rate of co-expression. The co-expression ratio was expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Dendritic complexity of immature neurons (Sholl analysis)\u003c/h2\u003e \u003cp\u003eSholl analysis is a methodical process that counts the number of intersections between dendrites and concentric circles drawn at predetermined distances from the soma. It is used to identify changes in the neuronal dendritic arborization \u003csup\u003e18,25,31,32\u003c/sup\u003e. In each animal, ten randomly chosen DCX-positive cells with tertiary dendrites in the DG were observed at 200\u0026times; magnification. Microphotographs were captured and imported using the Sholl Analysis plugin (The Ghosh Lab, UCSD, La Jolla, CA, USA) into ImageJ software (National Institutes of Health, Bethesda, MD, USA). When the neurons were traced in ImageJ, concentric circles were created on cell bodies at intervals of 10 \u0026micro;m radius and as far out from the soma as 200 \u0026micro;m. The number of intersections that the dendrites and concentric circles form is used to measure the complexity of the dendritic structure. A dendritic structure that has more crossings is more complicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Biochemical analyses in serum (BDNF ELISA)\u003c/h2\u003e \u003cp\u003eThe serum samples were collected on day 18. ELISA kits (from Invitrogen) were used to measure the serum concentration of BDNF according to the manufacturer's instructions. There were 16 animals in total.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism v10.4.0 software was used for statistical analysis and plotting data (San Diego, CA, USA). All data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). To compare differences between the TMT and Ctrl groups, an independent samples t-test was carried out. To compare differences in freezing duration between the TMT groups and Ctrl groups among pre- and post-exposure timepoints, a Two-Way ANOVA was performed. To evaluate specific differences between groups, a multiple comparisons test was performed using Tukey\u0026rsquo;s multiple comparisons test. For each test, the significance level was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and presented in figures as ns: p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; *: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e \u003cb\u003e3.1 Repeated exposure to TMT did not alter inter-group body weight but revealed differential intra-group weight dynamics\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOn days 1, 7, and 14 of treatment, the body weight of the animals was measured. No statistical differences in body weight between groups on both day1 and day14 were found (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) By checking the within group differences, the body weight of Ctrl significantly increased after sham exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb; day1: 318.8 g\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9, day14: 360.9 g\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7, p\u0026thinsp;=\u0026thinsp;0.0108), but no significant weight differences were found in TMT group between day1 and day14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb; day1: 310.2 g\u0026thinsp;\u0026plusmn;\u0026thinsp;11.0, day14: 343.1 g\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1, p\u0026thinsp;=\u0026thinsp;0.0636).\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Repeated exposure to TMT induced fear extinction but did not change emotion-related behavior\u003c/h2\u003e \u003cp\u003eFreezing is a response associated with the intensity of the unconditioned fear stimulus. The results of the intra- and inter-group comparisons demonstrate that repeated exposure to TMT significantly decreased fear responses. On both timepoints of day 1 and day 13, statistically significant differences in freezing time were found between the TMT and Ctrl groups. On day 1, the freezing duration in TMT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-e; 413.4 s\u0026thinsp;\u0026plusmn;\u0026thinsp;19.4) was significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) than Ctrl group (120.4 s\u0026thinsp;\u0026plusmn;\u0026thinsp;19.2); on day13, statistical difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-e; p\u0026thinsp;=\u0026thinsp;0.013) was also observed between TMT (324.2 s\u0026thinsp;\u0026plusmn;\u0026thinsp;24) and Ctrl (175.8 s\u0026thinsp;\u0026plusmn;\u0026thinsp;48.2) group in which TMT group showed longer freezing time than Ctrl group. However, by comparing the freezing time between day1 and day13 within groups, a statistically significant decrease was found in TMT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ee; day1: 413.4 s\u0026thinsp;\u0026plusmn;\u0026thinsp;19.4, day13: 324.2 s\u0026thinsp;\u0026plusmn;\u0026thinsp;24, p\u0026thinsp;=\u0026thinsp;0.0438) but no differences were found in Ctrl group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ee; day1: 120.4 s\u0026thinsp;\u0026plusmn;\u0026thinsp;19.2, day13: 175.8 s\u0026thinsp;\u0026plusmn;\u0026thinsp;48.2, p\u0026thinsp;=\u0026thinsp;0.2028). The results reveal that repeated exposure to TMT would help fear extinction or systematic desensitization. We carried out additional analysis, in which the within group differences between TMT and Ctrl groups were also calculated by freezing duration on day1 minus the duration on day13, and the results showed that the within group change of freezing duration in TMT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ef; 89.2 s\u0026thinsp;\u0026plusmn;\u0026thinsp;21.4) was significantly higher (p\u0026thinsp;=\u0026thinsp;0.0325) than Ctrl group (-55.4\u0026thinsp;\u0026plusmn;\u0026thinsp;58.6).\u003c/p\u003e \u003cp\u003eIn OFT, no significant differences in locomotor activity between groups were found by total distance traveled in testing arena (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), but marginal effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; p\u0026thinsp;=\u0026thinsp;0.0716) was found in time spent in center area among groups in which Ctrl group (16.4s\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2) spent more time than TMT group (10s\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5). In FST, a marginal difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec; p\u0026thinsp;=\u0026thinsp;0.0642) of floating time was also found between the two groups, in which the TMT group (244.8s\u0026thinsp;\u0026plusmn;\u0026thinsp;35.3) spent more time on floating than the Ctrl group (149s\u0026thinsp;\u0026plusmn;\u0026thinsp;33.4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Repeated exposure to TMT increased hippocampal neurogenesis and microglia activation, but decreased the total number and differentiation of astrocytes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor DCX-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), which represented immature neurons, TMT treatment led to a significant decrease in their number within the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; 14927.6\u0026thinsp;\u0026plusmn;\u0026thinsp;903.7 cells) compared to the Ctrl group (19310.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1439.5 cells, p\u0026thinsp;=\u0026thinsp;0.0164). A significant difference in the total number of BrdU-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) was observed between the Ctrl and TMT groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed; p\u0026thinsp;=\u0026thinsp;0.0279). Compared to the control (Ctrl) group (16461\u0026thinsp;\u0026plusmn;\u0026thinsp;1937.4 cells), repeated TMT exposure significantly decreased the number of BrdU-positive cells in the hippocampus (11106\u0026thinsp;\u0026plusmn;\u0026thinsp;1006.1 cells). In terms of astrocyte numbers reflected by the GFAP marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), showed a significant reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; p\u0026thinsp;=\u0026thinsp;0.0157) after TMT treatment. The hippocampus of the TMT group had fewer astrocytes (12444\u0026thinsp;\u0026plusmn;\u0026thinsp;1941.8 cells) compared to the Ctrl group (18836\u0026thinsp;\u0026plusmn;\u0026thinsp;1036.9 cells). Regarding microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), as indicated by the IBA-1 marker, TMT exposure significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed; p\u0026thinsp;=\u0026thinsp;0.0015) the number of microglia in the hippocampus (13332\u0026thinsp;\u0026plusmn;\u0026thinsp;403.9 cells) compared to the Ctrl group (7772\u0026thinsp;\u0026plusmn;\u0026thinsp;875.9 cells).\u003c/p\u003e \u003cp\u003eFurthermore, repeated TMT exposure significantly altered the ratios of DCX/BrdU (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), IBA-1/BrdU (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), and GFAP/BrdU (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee) double labelling, providing additional insights into the changes in hippocampal cell populations. DCX/BrdU ratio was significantly higher in the TMT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb; 77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7) compared to the Ctrl group (54.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5, p\u0026thinsp;=\u0026thinsp;0.0049). Conversely, the IBA-1/BrdU co-staining ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed; 22.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9) and the GFAP/BrdU ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef; 26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5) were significantly decreased in the TMT group compared to the Ctrl group (IBA-1/BrdU: 34.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2, p\u0026thinsp;=\u0026thinsp;0.0082; GFAP/BrdU: 36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6, p\u0026thinsp;=\u0026thinsp;0.0096).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, no statistically significant difference in serum BDNF level was found between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Also, no significant difference between the TMT and Ctrl groups was found indicating a positive effect of repeated exposure of TMT to increase dendritic complexity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe present study demonstrated that repeated exposure to a fear stimulus alleviates SP, as evidenced by the significant reduction in freezing duration. Findings in the current study align with exposure therapy, or flooding, which involves exposing the patient to fear-inducing stimuli to break the pattern of escape that maintains the fear. Despite the efficacy, previous evidence showed that the clinical application of exposure therapy to clinical practice is difficult \u003csup\u003e33\u003c/sup\u003e. For instance, one previous study showed that approximately 20\u0026ndash;30% of patients with severe arachnophobia refused or prematurely terminated exposure therapy because they found it too anxiety-provoking \u003csup\u003e34\u003c/sup\u003e. One factor contributing to the underutilization of exposure therapy in clinical settings is repeated exposure to fear-inducing stimuli may be excessively challenging and stressful for patients. Additionally, concerns regarding the potential exacerbation of symptoms associated with specific phobias further contribute to the hesitation in employing exposure therapy \u003csup\u003e33\u003c/sup\u003e. To address such concerns, the present study performed anxiety- and depression- like behavior tests at the end of treatment to examine the potential adverse effects brought by repeated exposure to fear stimuli. However, no statistically significant difference in behavior tests was observed between the Ctrl and TMT groups, suggesting that repeated exposure to the fear stimuli does not induce or exacerbate anxiety and depression in animal behavioral tasks. Altogether, the current study and prior publications provide further reassurance regarding the application of exposure therapy in both preclinical and clinical practice.\u003c/p\u003e \u003cp\u003eAccumulating evidence implicates hippocampal neurogenesis as a potential mechanism in fear extinction. For instance, a previous study highlighted that neurogenesis acts as a potential mechanism underlying exercise on adolescent fear extinction \u003csup\u003e35\u003c/sup\u003e. Another study also showed that mice with fewer adult-born dentate gyrus granule cells in the immature stage performed worse on extinction tasks \u003csup\u003e14\u003c/sup\u003e. Data in the present study align with previous observations, suggesting that neurogenesis is associated with fear extinction. We found that the total number of DCX-positive cells reduced after repeated TMT exposure. However, neuronal precursor cells begin to express DCX while actively dividing, and their neuronal daughter cells continue to express DCX for 2\u0026ndash;3 weeks as the cells mature into neurons \u003csup\u003e36\u003c/sup\u003e, indicating that the period during which the typical DCX expression window is much longer than the current TMT treatment period. Given that a proportion of DCX-positive cells may pre-exist before the treatment began, a decreased total number of DCX-positive cells in the current result might only imply that repeated exposure to TMT decreased the population of immature neurons in the hippocampus. The effects of current treatment on neurogenesis still need to be confirmed.\u003c/p\u003e \u003cp\u003eTo further elucidate the effects of TMT repeated exposure on hippocampal neurogenesis, we analysed the total counts of BrdU and DCX/BrdU co-labelling to assess neurogenesis during the treatment period. Increased total number of BrdU-positive cells indicates that repeated TMT exposure enhanced cell proliferation in the hippocampus. Critically, BrdU was administered during the final three days of treatment, ensuring that labelled cells reflect proliferation events occurring specifically within this window. Similarly, DCX/BrdU co-labelled cells represent immature neurons generated during the BrdU injection period (i.e., 1\u0026ndash;3 days before sacrifice), as DCX is transiently expressed in newly differentiated neurons. The elevated BrdU-positive cell counts and DCX/BrdU co-labelling ratio would collectively confirm that repeated exposure to TMT promoted cell proliferation and differentiation of hippocampal neurons. Furthermore, this result confirmed that the observed neurogenesis changes are caused by TMT exposure rather than pre-existing neurogenesis, resolving uncertainty associated with static DCX labelling alone.\u003c/p\u003e \u003cp\u003ePrevious studies demonstrated that microglia has an impact on each of the distinct processes involved in adult neurogenesis under physiological conditions, such as the growth of neuronal stem cells, the migration of adult neuronal cells and immature neurons, their differentiation into mature neurons, the extension of neuronal synapses, and the integration of immature neurons into an existing circuit \u003csup\u003e37\u0026ndash;39\u003c/sup\u003e. To determine the involvement of microglia in neurogenesis, single and double labeling of immunohistostaining of IBA-1 were conducted in the present study. The current result is consistent with previous findings \u003csup\u003e37\u0026ndash;39\u003c/sup\u003e since an increased total number of IBA-1-positive cells was observed after TMT repeated exposure. However, as IBA-1 is only expressed in macrophages and microglia, and it is increased when these cells are activated \u003csup\u003e40\u003c/sup\u003e. It is difficult to determine whether the increased number of IBA-1-positive cells was mainly caused by activation of existing microglia or increased differentiation from progenitors to microglia. Thus, IBA-1/BrdU co-labelling was performed in the current study, and the result showed a decreased IBA-1/BrdU ratio after treatment. This result suggests newly proliferating cells are less likely to differentiate into microglia, and existing microglia are becoming activated in response to repeated TMT exposure. Additionally, the comparison between the ratio of DCX/BrdU and IBA-1/BrdU further confirmed that more progenitor cells are differentiated into immature neurons rather than microglia. Furthermore, apart from neurogenesis, microglia can also modulate the process of fear extinction by pruning neuronal synapses \u003csup\u003e41\u003c/sup\u003e. It has been shown that microglia facilitate the formation, maturation, and selective elimination of immature synapses, a fundamental process for proper brain development \u003csup\u003e42\u003c/sup\u003e, and synapse elimination by microglia may lead to degradation of memory engrams and forgetting of previously learned contextual fear memory \u003csup\u003e41\u003c/sup\u003e. Therefore, the present study offered further evidence that microglia activation may promote neurogenesis in multiple stages.\u003c/p\u003e \u003cp\u003eRegarding GFAP, the current study revealed that the total number of GFAP-positive cells and the GFAP/BrdU ratio decreased in the hippocampus after TMT exposure. By comparing with the DCX/BrdU ratio, the GFAP/BrdU ratio decreased after TMT exposure, while the DCX/BrdU ratio increased. Thus, similar to the result from IBA-1/BrdU ratio, this result further confirmed that the proliferating cells were more likely to differentiate into neurons rather than astrocytes. In addition, increased expression of GFAP as a marker of astrocyte activation and a hallmark of multiple CNS pathologies is associated with neuroinflammation \u003csup\u003e43\u0026ndash;45\u003c/sup\u003e. Previous studies showed that prolonged exposure to stressful stimuli that elicit fear and anxiety activate both central and peripheral immune cells to release cytokines, such as IL-1\u003cem\u003eβ\u003c/em\u003e, which induces astrocyte proinflammatory gene expression \u003csup\u003e45\u0026ndash;47\u003c/sup\u003e. In this way, based on the reduction of the total number of GFAP-positive cells and differentiation ratio in the present study, decreased neuroinflammation may be involved in the process of fear extinction or desensitisation induced by repeated exposure to TMT.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe current study provides novel insights into the mechanisms underlying fear extinction induced by repeated exposure to TMT. Our behavioral results reinforced the efficacy of repeated fear stimulus exposure in reducing phobia-like freezing behavior, aligning with the principles of exposure therapy. Notably, the absence of exacerbation in anxiety- or depression-like behavioral tasks may address a critical clinical concern, offering preclinical evidence that repeated fear exposure, under controlled conditions, does not amplify negative affective states. This finding supports the safety and feasibility of exposure-based interventions, potentially encouraging broader clinical adoption. At the cellular level, reduced DCX-positive immature neurons and increased BrdU-labelled proliferating cells highlighted the dynamic nature of hippocampal neurogenesis during fear extinction. The preferential differentiation of BrdU-positive cells into newborn neurons (over microglia or astrocytes) further suggests that fear extinction may actively transfer progenitor cells toward a neuronal fate. Also, the present study advances understanding of glial contributions to fear extinction: increased total number of IBA-positive cells, coupled with reduced IBA-1/BrdU co-labelling, implies microglial activation, rather than differentiation, facilitates neurogenesis and synaptic remodelling, aligning with their roles in synaptic pruning and circuit refinement. Collectively, our findings proposed a potential treatment model in which repeated exposure to fear stimuli diminishes the fear responses through promoting hippocampal neurogenesis and microglial activation. However, the current study lacks mechanistic evidence linking hippocampal neurogenesis directly to behavioral outcomes and microglia activation. Thereby, future studies could further confirm the causality by pharmacologically inhibiting neurogenesis or microglial activation to investigate.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Miss XU Yi, who offered support in preparation of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study are available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING SOURCE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by the General Research Fund (15105621) by University Grant Council of Hong Kong SAR.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: PBY, JNMC, TKHF, and BWML; Methodology: PBY, JNMC, TKHF, and BWML; Investigation: PBY, JNMC, and TKHF; Writing: PBY; Funding Acquisition: BWML; Resources: BWML and SPCN; Supervision: WKH, WKWL, JWHL, KFS, SPCN, and BWML.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeBeau, R. 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In clinical practice, exposure therapy is considered one of the most effective treatments for SP. This approach involves repeated exposure to fear-inducing stimuli to facilitate fear extinction. While fear extinction and systematic desensitization are learning-dependent processes, the role of hippocampal neurogenesis remains unexplored in this context. In this study, we employed an animal model of repeated exposure to Trimethylthiazoline (TMT), a predator odor derived from fox feces, to investigate the potential involvement of hippocampal neurogenesis in exposure therapy. Twenty adult male Sprague Dawley rats were randomly assigned to either a control group (exposed to distilled water) or a TMT-exposed group (exposed to TMT) repeatedly within 14 days. Serum samples were collected 2\u0026ndash;3 days following treatment. Neurogenesis and cell proliferation in brain tissues were analyzed via immunohistochemistry. The results demonstrated that repeated TMT exposure facilitated fear extinction without inducing anxiety- or depression-like behaviors. Additionally, TMT exposure enhanced neurogenesis by promoting the differentiation of neuronal progenitors into immature neurons. Microglial activation, implicated in various stages of adult neurogenesis, also increased following TMT exposure. These findings provide preliminary evidence supporting the therapeutic mechanism, showing that repeated TMT exposure not only facilitates fear extinction but also promotes neurogenesis and microglial activation. This study suggests the potential roles of neurogenesis and microglial activation in fear extinction or systematic desensitization, and identifies novel targets for future therapeutic development in SP.\u003c/p\u003e","manuscriptTitle":"Repeated exposure to specific phobia stimuli, Trimethylthiazoline, promoted fear extinction, hippocampal neurogenesis, and microglia activation in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 12:27:48","doi":"10.21203/rs.3.rs-6698181/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":"02544c74-4943-4bcf-889b-7c4c11c3c615","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49770135,"name":"Biological sciences/Neuroscience"},{"id":49770136,"name":"Health sciences/Health care"},{"id":49770137,"name":"Health sciences/Neurology"}],"tags":[],"updatedAt":"2025-06-24T17:38:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 12:27:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6698181","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6698181","identity":"rs-6698181","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}