Chemogenetic modulation of CRF neurons in the BNST compensates for phenotypic behavioral differences in fear extinction learning of 5-HT2C receptor mutant mice.

Chemogenetic modulation of CRF neurons in the BNST compensates for phenotypic behavioral differences in fear extinction learning of 5-HT2C receptor mutant mice. | 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 Chemogenetic modulation of CRF neurons in the BNST compensates for phenotypic behavioral differences in fear extinction learning of 5-HT2C receptor mutant mice. Hannah Schulte, Hanna Böke, Patricia Lössl, Maria Worm, Ida Siveke, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5604701/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jan, 2026 Read the published version in Translational Psychiatry → Version 1 posted You are reading this latest preprint version Abstract Psychopharmacotherapy is often used to treat anxiety- and stress-associated psychiatric disorders, including posttraumatic stress disorder (PTSD). Adjunctive therapy is most typically used with medications that influence serotonin balance, such as selective serotonin reuptake inhibitors (SSRIs). Contrary to expectations, SSRIs show an anxiety-increasing effect during the initial treatment phase. Among the 14 different serotonin receptor subtypes, pharmacological studies have demonstrated that 5-HT2C receptors (5-HT2CRs) in the bed nucleus of the stria terminalis (BNST) play a significant role in the anxiogenic effect of acute SSRI treatment. Although numerous studies have confirmed the role of the 5-HT2CR in anxiety behavior, little is known about its involvement in learned fear and fear extinction. In particular, fear extinction is considered a central neural mechanism in the treatment of PTSD patients. Recent results from 5-HT2CR knockout mice (2CKO) revealed that global loss of 5-HT2CRs enhances fear extinction, without affecting fear acquisition. Here, we implemented a chemogenetic approach to examine the neuronal substrate which underlies this extinction-enhancing effect in 2CKO mice. DREADD-activation of BNST CRF neurons promotes fear extinction in 5-HT2C WT mice, whereas DREADD-inactivation of BNST CRF neurons impairs fear extinction in 2CKO mice. Thus, using activating and inactivating DREADDs, we were able to directionally modulate fear extinction. These findings provide a possible explanation for the fear extinction-enhancing effect in 2CKO mice with relevance for the treatment of PTSD patients. Biological sciences/Neuroscience Health sciences/Diseases/Psychiatric disorders/Schizophrenia Figures Figure 1 Figure 2 Introduction Fear and anxiety are closely related emotions. Anxiety is characterized by persistent, excessive worries that are pursued even in the absence of an immediate threat. In contrast, fear is a natural response to a specific, known danger 1 . Although anxiety and fear are fundamentally different emotional states, they may share common neural and behavioral mechanisms 2 . Dysfunctions in the underlying neural networks may lead to anxiety, fear, and stress-related disorders, such as post-traumatic stress disorder (PTSD) 3 , 4 . From a neurobiological perspective, the development of PTSD is linked to classical fear conditioning, while its persistence is attributed to a malfunction in extinction learning 3 , 4 . Individuals with PTSD often struggle to extinguish the learned association between a neutral environmental stimulus and the fear response, which can impair the efficacy of therapeutic interventions. In laboratory settings, fear conditioning (FC) and fear extinction (FE) paradigms are commonly used experimental approaches to study the neurobiological features of aversive learning that contribute to PTSD 5 . FC involves repeatedly pairing a neutral, non-threatening stimulus, like a tone (conditioned stimulus, CS), with an aversive stimulus, such as a mild foot shock (unconditioned stimulus, US), until the animal begins to display a fear response not only to the shock but also to the tone alone (conditioned response, CR) 5 , 6 . On the other hand, extinction learning is an important biological adaptation mechanism that allows old behavior patterns to be unlearned when they are linked to invalid information. It is important to note that extinction learning suppresses the originally acquired fear association without erasing or overwriting it 7 , 8 . Extinction learning is achieved by repeatedly presenting the CS in the absence of the US, reducing the CR, and weakening the previously learned association between a stimulus and a defensive reaction 8 . Studying the cognitive mechanisms behind the formation and extinction of fear contributes to a better understanding of the emergence, maintenance, and treatment of PTSD. In this context, the amygdala is the crucial brain region responsible for forming the association between the signal of an aversive event and the aversive stimulus, giving them emotional significance 9 . Convergent evidence has implicated the amygdala in the extinction of fear 10 , while more recent work also points toward a key role for the BNST in threat anticipation and fear modulation 11 – 16 . The BNST is highly interconnected with the central amygdala (CeA) 17 , 18 , however, early research on the rodent BNST revealed a functional division between the amygdala and the BNST, supported by human fMRI data. These findings imply that the BNST is implicated in mediating specific fear and anxiety-like behaviors that are communicated without reference to the physical CeA circuit 19 . Within this context, the BNST mediates more diffuse, prolonged fear or unconditioned anxiety states, while the amygdala mediates impending, phasic fear experiences 19 . More recent research, however, suggests that the BNST is also involved in processing phasic, discrete stimuli 11 , 14 , 20 , 21 . Early BNST lesion experiments demonstrated that fear responses were reduced when directly exposed to an aversive stimulus, such as predator scents 22 . Recent electrophysiological recordings in rodents demonstrated enhanced activity of BNST neurons during fear acquisition and CS-dependent fear recall 11 , 15 , 23 , 24 . Another study demonstrated that chemogenetic activation of GABAergic neurons in the BNST during fear conditioning or memory consolidation using a designer receptor exclusively activated by designer drugs (DREADD), which activates the G q -pathway, increased CS fear recall without affecting fear expression during conditioning or recall. This implies a modulatory role of the BNST in fear memory formation 11 . Additionally, studies have shown that the BNST is also highly involved in PTSD symptoms like altered hypervigilance, arousal states, and increased sensitization to the environment 19 , 25 . Despite the role of the BNST in anxiety behavior, the knowledge about how it affects fear learning - particularly concerning explicit cues and fear extinction - is still emerging. Serotonin (5-HT) plays a pivotal role in anxiety- and fear-related behaviors through its effects in the BNST 26 . The BNST contains various serotonin receptor subtypes, which are expressed differently across neuron types 27 , 28 . Depending on the subtype involved, serotonin can elicit either inhibitory or excitatory neuronal responses 27 . Among the 14 serotonin receptor subtypes, the 5-HT1A and 5-HT2C receptors in the BNST have received particular attention in recent years, as their activation has opposing effects on fear behavior. In the anterodorsal subregion of the BNST (BNSTad), 5-HT1A receptor (5-HT1AR) activation has anxiolytic (anxiety-reducing) effects, while 5-HT2C receptor (5-HT2CR) stimulation increases anxiety 14 , 29 , 30 . Optogenetic and chemogenetic approaches revealed that 5-HT input to the BNST from the DRN elicits anxiogenic behavior and increased fear learning via 5-HT2CR signaling 14 , 31 . Although numerous studies in recent years have examined the role of the BNST in the acquisition, expression, and recurrence of fear, data on fear extinction are completely lacking. In our previous study, we discovered that the BNSTov and the BNSTad play opposing roles in fear extinction, with WT mice showing an extinction-associated reduction in BNSTov activity and an increase in BNSTad 32 . Optogenetic interventions produced comparable outcomes in anxiety tasks, showing that the BNSTov has an anxiogenic role, whereas the BNSTad has been described as anxiolytic 33 . Similarly, we discovered altered activity in the dorsal BNST in 2CKO mice in an anxiolytic and extinction-supporting direction, even under basal conditions 32 . 2CKO animals had previously been characterized as having an anxiolytic phenotype, with reduced cFos expression in corticotropin-releasing factor (CRF)-expressing neurons in response to anxiety-inducing stimuli 34 . Building on our previous research, we used a chemogenetic approach to investigate the neuronal network in the BNSTad underlying the extinction-enhancing effect in 2CKO mice. Our results revealed that chemogenetic modulation of BNST CRF neurons produces bidirectional effects on the fear extinction phenotype. Activation of BNST CRF neurons using a G q -coupled (hM3Dq) DREADD promotes fear extinction in 5-HT2CR WT mice, while inactivation of BNST CRF neurons with a G i -coupled (hM4Di) DREADD impairs fear extinction in 2CKO mice. The findings reported here, provide a mechanistic explanation for the extinction-enhancing effect in 2CKO mice. The effect that we observed may be a key mechanism behind SSRI-induced anxiolysis, since the anxiolytic effects of systemic long-term SSRI administration are associated with 5-HT2CR desensitization 35 . Materials and Methods Subjects Adult male mice (9–16 weeks of age) were used for all experiments. To generate a transgenic CRF-ires-Cre/5-HT2CR mouse line, CRF-ires-Cre mice (B6(Cg)- Crh tm1(cre)Zjh /J, stock no. 012704, Jackson Laboratory) were bred with 5-HT2CR knockout (KO) mice KO (B6.129-Htr2c tm1Jul /J, stock no. 002627, Jackson Laboratory). All mice had either a hemizygous (2CKO) or wild-type (WT) background for the 5-HT 2C R allele and were homozygous for the CRF-ires-Cre background. Mice were group-housed (2–5 individuals per cage) with a constant room temperature and a 12 h light/dark cycle while food and water were provided ad libitum. All experiments were performed during the light period. The experiments were conducted with the approval of the local ethics committee (Bezirksamt Arnsberg) and the animal care committee of North-Rhine-Westphalia (LANUV; Landesamt für Umweltschutz, Naturschutz und Verbraucherschutz Nordrhein-Westfalen, Germany; AZ. 81-02.04.2021.A412). Studies were conducted in compliance with the 2010 European Communities Council Directive (2010/63/EU) for care of laboratory animals and supervised by the animal welfare commission of the Ruhr-University Bochum. Viral constructs and Stereotaxic DREADD injections Double-floxed adeno-associated viruses (AAVs) were used to express Gq-coupled (AAV1.pAAV.hSyn.DIO.hM3D(Gq)mCherry, Titer: 2.2 x 10 13 , #44361, Addgene) or Gi-coupled (AAV1.pAAV.hSyn.DIO.hM4D(Gi)-mCherry, Titer: 2.3 x 10 13 , #44362 Addgene) DREADDs in the BNSTad. The animals were anesthetized with isoflurane (initially at 5% (v/v) and then maintained at 1.5–2.5% (v/v)) and positioned in a stereotaxic frame (Stoelting). For analgesia, subcutaneous injections of buprenorphine (0.1 mg/kg) and carprofen (2 mg/kg) were administered. The scalp was treated with lidocaine as a local anesthetic. The viral constructs were bilaterally injected into the BNSTad through pressure injection utilizing custom-pulled glass pipettes (tip diameter 5–10 µm). The following stereotactic coordinates relative to bregma were used: AP + 0.26, ML +/- 0.75, DV -4.1 32 . AAV-injected animals were kept in their home cages for three weeks to allow for sufficient virus expression. Drug application DREADD activation within the BNSTad was achieved by i.p. injection of the designer receptor ligand clozapine-N-oxide (CNO; Tocris Bioscience; Cat. No: 6329). CNO was administered at a dose of 1 mg/kg, 40 minutes prior to the extinction session. In addition to the genotype groups, the DREADD-injected 2CKO and WT mice were randomly assigned to treatment groups. Control groups (WT Saline/ 2CKO Saline) received an i.p. injection of 0.3 ml 0.9% saline 40 min prior to extinction. Behavioral testing To reduce stress related to cage changes and enable acclimatization to the experimenter, each individual was handled with a tunnel on four to five consecutive days before the fear conditioning experiment 36 . In addition, the mice were restrained during the last two to three days of the handling procedure to familiarize them with the i.p. injections. The fear conditioning apparatus followed the design and specifications as previously described by Süß et al. (2022). On day 1 of the fear conditioning and extinction protocol, mice were placed into context A characterized by white walls, an electrified grid floor, white LED illumination (250 lx), 30% fan intensity, and 70 %(v/v) ethanol as a background odor, for a 10-minute habituation without stimulus presentation (Fig. 1 a, 2 a). The apparatus was cleaned with soap water between subjects. Fear conditioning occurred in context A on day 2. After a 2-minute baseline period (Bl), mice were given five pairings of a 30-second tone (conditional stimulus, CS, 7.5 kHz, 60 dB) with a foot shock (unconditional stimulus, US, 0.35 mA) that co-terminated with the last second of the CS. Between stimulus presentation intertrial intervals (ITIs), ranging from 30 to 120 s were implemented. After the final CS/US pairing, mice were maintained in the chamber for additional 60 s (post-stimulus time, PST). On day 3, fear retrieval and extinction took place in context B, consisting of a perforated floor plate, black and white striped walls, 100 %red LED illumination (28 lx), 100 %fan intensity and 1 %(v/v) acetic acid as background odor. 40 min prior to fear retrieval and extinction, mice received an i.p. injection of CNO (1 mg/kg) or 0.3 ml 0.9 %saline solution as control. Injected individuals were moved to a separate waiting cage to reduce stress for the experimental group in the home cage. After a 2 min Bl period, mice were exposed to 14 CS presentations separated by ITIs (30–120 s), followed by a 60-second PST. Behavioral analysis As previously described by Süß et al. (2022), a custom-made software written in MATLAB (MathWorks) was used for video recording and stimulus presentation. Subsequently, all behavioral parameters were analyzed using EthoVision XT 15 tracking software (11.5, Noldus). The following behavioral parameters were analyzed: total distance moved (in cm), maximum velocity (in cm/s), and freezing behavior (in %). Freezing behavior was defined as the absence of any movement other than respiration for more than two seconds. The percentage of freezing behavior was binned for two CS presentations (CS 1–7). The first bin (bin1) represents fear retrieval. Individuals that did not exhibit freezing behavior were excluded from data analysis. Immunohistochemistry and verification of virus expression Following the fear conditioning and extinction procedure, mice were deeply anesthetized and transcardially perfused with ice-cold PBS (1x), followed by ice-cold paraformaldehyde (4% PFA in PBS (w/v), pH 7.4, Sigma Aldrich). Brains were post fixed in 4% PFA and cryoprotected in 30% sucrose solution in PBS at 4°C (w/v, Sigma Aldrich). Coronal sections of 30 µm were cut with a cryostat (CM3050 S, Leica) and collected in 24-well plates filled with PBS for subsequent free-floating antibody staining (see also Süß et al.,2022). Non-specific binding sites were blocked in 10% normal donkey serum (NDS, v/v, Merck Millipore) in 0.3% PBS-Triton X-100 (PBS-T, v/v, Sigma Aldrich) for 1 h at RT, followed by incubation with the primary antibody (rabbit anti PKCδ (1:1000, ab182126, Abcam) in 3% NDS in 0.3% PBS-T overnight at 4°C. Protein kinase delta (PKCδ) staining was performed as a marker for the BNSTov 37 , 38 . Secondary antibody solution included donkey anti rabbit DyLight® 488, (SA5-10038, Thermo Fisher Scientific) in 3 % ND in 0.3% PBS-T for 1.5 h at RT. Overview images of the immunostained sections in the caudal (AP 0.00), medial (AP + 0.15), and rostral (AP + 0.30) BNST regions were captured with a Leica fluorescence microscope (M205 FCA, Leica Microsystems) to confirm the exact virus placement. Images were processed and analyzed with ImageJ software 39 . The viral pattern indicated by the expression of mCherry was redrawn, slightly colored and overlayed using CorelDRAW® Graphic Suite software (Corel GmbH). Representative confocal microscopic images (20x/0.7 NA objective) of the BNSTad region from individuals subjected to fear conditioning were taken using a Leica laser-scanning confocal microscope (TCS SP5II, Leica Microsystems). Mice lacking bilateral viral expression within the BNSTad were excluded from data analysis. Statistics Graphs were created using SigmaPlot 12.5 (Systat Software) and the data were analyzed utilizing IBM SPSS Statistics (Version 29.0) software. Normality was tested before each analysis (Shapiro-Wilk test). Further, homoscedasticity (Levene’s test) was assessed, while sphericity (Mauchly’s test) was verified for cases of repeated measures. A two-way analysis of variance (ANOVA) or a three-way repeated measures ANOVA (RM-ANOVA) was used to calculate statistical significance. Groups were compared with Bonferroni’s post hoc test. Data violating any assumption were analyzed using a Kruskal-Wallis Test, followed by Dunn’s post hoc test. Friedman’s test was applied in cases of repeated measures. Statistical significance was determined using a critical alpha level of 0.05 (p < .05). All statistical information is displayed in appendix (Table 1), while the most relevant results are presented in the text as mean ± SEM (p-value). Results Chemogenetic inhibition of BNST CRF neurons impairs fear extinction in 2CKO mice We applied cell type-specific chemogenetic inhibition of BNST CRF neurons using CRF-ires-Cre/2CKO mice to assess if a subset of neurochemically distinctive CRF neurons in the BNSTad promotes fear extinction in these mice (Fig. 1 a and b). We will refer to these mice as 2CKO and WT mice, respectively. The accuracy of virus injection and the extent of viral spread were confirmed for each individual following the FC paradigm. (Fig. 1 c and d). Confocal images revealed hM4Di-mCherry spread (magenta) within the dBNST was predominantly restricted to the BNSTad, almost entirely avoiding the BNSTov (BNSTad and BNSTov boundaries exemplarily outlined by dashed lines, Fig. 1 c). In addition, the distribution of the virus was assessed in the rostral, medial, and caudal BNST regions of each mouse that underwent FC. The more intense blue color indicates viral expression primarily in the dorsal BNST region. In contrast, the ventral BNST region exhibited lower virus expression, as reflected by the lighter color intensity (Fig. 1 d). To investigate how BNST inactivation influences fear extinction, we chemogenetically inactivated BNST CRF neurons during fear extinction training (Fig. 1 a and e). An i.p. dose of CNO (1 mg/kg) 40 minutes before the onset of extinction training was used to activate hM4Di (Fig. 1 a). The control group was administered saline solution (0.3ml, i.p.) 40 min prior to the fear extinction training. To examine the effects of CRF neuron inactivation in the BNST on extinction learning, freezing behavior was measured during CS intervals in both conditioning and extinction sessions (Fig. 1 e). Statistical analysis of the data revealed no significant alterations in freezing behavior during fear conditioning between WT and 2CKO mice for saline and CNO-treated groups (Fig. 1 e). During the fear retrieval test (Fig. 1 e bin1 and j), CS-induced fear memory recall was significantly higher in WT saline mice compared to 2CKO saline mice. In the further course of the extinction training, WT animals maintained a high level of freezing behavior compared to 2CKO animals (Figure, 1e). Between-group comparisons revealed significant genotype differences, confirming the previously published accelerated fear extinction phenotype in 2CKO mice 32 . Notably, chemogenetic inhibition of BNST CRF neurons in 2CKO mice resulted in a markedly increased CS-induced fear recall with significantly impaired fear extinction phenotype compared to the saline-treated 2CKO group (Fig. 1 e). After the inactivation of BNST CRF neurons in 2CKO animals, the previously observed phenotypic differences were no longer significant compared to saline-treated WT animals. Beside freezing, no statistically significant differences were observed in locomotor activity during the habituation phase, suggesting that 2CKO animals did not exhibit hyperactivity (Fig. 1 f). Additionally, by examining the maximal movement velocity (Fig. 1 g), we investigated the animal's reaction to the US 40 . The maximal movement velocity in all groups was considerably higher during the CS/US pairings than throughout the baseline period (Bl) (Fig. 1 g). Contrary to our previous results, we could not detect increased affected responses in both 2CKO groups. We examined the overall distance traveled and maximum velocity during the BL period of the extinction session (Fig. 1 h, i) and found no significant effects of CNO/saline injection on locomotor behavior.We also examined the overall distance traveled and the maximum velocity throughout the BL period of the extinction session to rule out any potential effects of CNO/saline injection on locomotor activity (Fig. 1 h and i). Significant changes in motor behavior brought on by CNO/saline injection per se were not detected. Taken together, our behavioral findings suggest that silencing of CRF neurons in the BNSTad region enhances CS-induced fear recall while delaying extinction in 2CKO mice. This indicates that a subset of these GABAergic CRF neurons in the BNSTad is significantly involved in the manifestation of the fear extinction phenotype in 2CKO mice. Conversely, the activation of CRF neurons in 2CKO mice contributes to accelerated fear extinction. Chemogenetic activation of BNST CRF neurons accelerates fear extinction in wild-type mice Since chemogenetic inactivation of CRF neurons enhanced fear recall in 2CKO mice and impaired extinction learning, we hypothesized that chemogenetic activation of these neurons in WT animals could have the opposite effect. Therefore, 2CKO and WT mice were bilaterally injected with a double-floxed stimulating hM3Dq-mCherry DREADD (Fig. 2 a and b). Subsequently, mice were exposed to the same auditory fear conditioning and extinction paradigm and hM3Dq was activated with CNO (1mg/kg) 40 minutes before fear extinction on day 3 (Fig. 2 a). Again, freezing behavior was analyzed during the Bl and for CS time bins during fear conditioning and fear extinction (Fig. 2 e). Virus spreading was validated in all animals. The viral construct was injected into the BNSTad, however hM3Dq virus expression was also observed in the ventral BNST (vBNST) across the rostral, middle, and caudal regions (Fig. 2 d). During fear conditioning, there were no significant changes in freezing behavior for CS presentations between groups. (Fig. 2 e). However, during extinction learning, we observed substantial genotype and treatment effects. Initially, WT mice exhibited considerably higher Bl freezing compared to 2CKO animals in the saline-treated control group. DREADD activation via CNO significantly reduced Bl freezing in WT mice to levels comparable to those of 2CKO animals. Additionally, statistical analysis revealed genotype and treatment effects in the subsequent extinction learning process. In the saline-treated hM3Dq-expressing control group, 2CKO and WT mice exhibited notable differences in freezing behavior. Unlike the hM4Di-expressing control groups in the first experiment, no changes in freezing behavior associated with CS-induced fear retrieval were observed this time (Fig. 2 e and j). Furthermore, WT mice maintained a consistently high level of freezing behavior with a slower decline over time compared to 2CKO animals (Fig. 2 e). Similar to the control groups in the previous hM4Di-DREADD experiment, the virus injection and he experimental procedure had only minimal impact on the genotype differences between WT and 2CKO animals. Again, no obvious genotype effects were detected for the total distance moved during the habituation session and maximal movement velocity during conditioning (Fig. 2 f and g). Consequently, all groups displayed comparable locomotor activity and shock responsivity. However, during the Bl of the fear extinction session, significant genotype differences in locomotor activity were observed between WT and 2CKO mice, with the 2CKO control group exhibiting an increased total distance moved (Fig. 2 h). Further, the maximal movement velocity was significantly increased in 2CKO mice in the initial Bl of extinction learning (Fig. 2 i). Noteworthy, CNO-induced DREADD activation significantly influenced the progression of fear extinction in WT animals. WT mice expressing hM3Dq exhibited a non-significant trend toward reduced fear retrieval (Fig. 2 e and j) and a significantly faster decline in freezing behavior over time due to CNO activation (Fig. 2 e). Additional statistical analysis confirmed the absence of phenotypic differences in fear extinction between CNO-treated WT mice expressing hM3Dq and saline-treated 2CKO mice expressing hM3Dq (Fig. 2 e). As no significant effect of CNO activation on any locomotor parameters was observed (Fig. 2 h and i), the effects must be attributed to reduced fear behavior. These results suggest that BNST CRF neurons are engaged in the phenotypic differences during extinction learning associated with 5-HT2CR knockout. Chemogenetic activation of distinctive BNST CRF neurons was sufficient to facilitate fear extinction in WT mice, while chemogenetic inactivation of these neurons in 2CKO mice enhanced CS-induced fear retrieval and delayed fear extinction. Discussion The study is based on our previous findings that mice constitutively lacking the 5-HT2CR display enhanced fear extinction in an auditory fear conditioning paradigm 32 . However, the network and specific neuronal cell type mainly responsible for the differences in extinction learning phenotypes between 2CKO and WT animals could only be partially identified. Therefore, this study has been designed to further analyze the neuronal substrate underlying the extinction-supporting phenotype of 2CKO mice. Our results demonstrate that this phenotype could be modified in two ways via chemogenetic modulation of CRF neurons in the BNST. Earlier research has highlighted that GABA and glutamate serve as the primary inhibitory and excitatory neurotransmitters in the BNST. The ratio of GABAergic to glutamatergic neurons differs among BNST subregions 18 , with GABAergic neurons accounting for approximately 70–90% of the cells in the BNSTad. The dBNST is highly heterogeneous, with GABAergic neurons expressing various neuropeptides, including CRF 18 . Based on previous studies, we hypothesized that CRF neurons in the BNSTad could be a promising target for extinction-associated differences related to our mouse model, given their central role in anxiety-related behaviors mediated by the BNST 41 . Within the BNST, the anterolateral group is believed to have the highest expression levels of extrahypothalamic CRF neurons 42 , although expression levels vary across the region. The highest density of CRF-expressing neurons in the BNST is found in the BNSTov 18 , 43 . BNST-CRF neurons primarily use GABA as a co-transmitter, leading to an overall inhibitory effect 43 , 44 . While CRF neurons in the BNST have traditionally been associated with anxiogenic effects 45 – 48 , more recent studies have revealed that BNST-CRF neurons also project to the VTA and LH in an anxiolytic manner 14 , 24 , with approximately 58% of BNST-CRF output neurons forming anxiolytic connections to the VTA or LH 14 . Building on the anxiolytic phenotype observed in 5-HT2CR KO mice, characterized by reduced cFos expression (a marker of neuronal activity) in BNST CRF neurons 34 , Marcinkiewicz et al. hypothesized that serotonergic input from the DRN modulates BNST CRF neuron activity via 5-HT binding at 5-HT2CRs 14 . Their findings showed that approximately 70% of dBNST CRF neurons express 5-HT2CRs, indicating that 5-HT signaling regulates their activity. Our results revealed that in 2CKO mice cFos levels are increased in an extinction-supporting direction in the BNSTad 32 . Consequently, we anticipated that the absence of the 5-HT2CR on local CRF neurons might impair the inhibition of anxiolytic projection neurons, thereby accelerating extinction. Additionally, we proposed that chemogenetic inactivation of CRF neurons might counteract the disinhibition caused by the absence of the 5-HT2CR in knockout mice. Indeed, we demonstrated that chemogenetic inactivation of BNST CRF neurons during extinction learning enhances fear recall and impairs extinction learning in 2CKO mice. This key finding aligns with earlier studies reporting that a subset of CRF-expressing, GABAergic neurons with long-range projections within the extended amygdala exerts anxiolytic effects by enhancing dopamine release into the VTA 13 , 24 . The same study also reported that mice with chronic CRH deficiency exhibited elevated anxiety levels and increased freezing behavior in both cued and contextual fear conditioning paradigms 13 . Other studies examining the impact of biochemically distinct neuron populations in the BNST on fear behavior have yielded somewhat contradictory results. In a cued fear conditioning paradigm, Bruzsik et. al. reported that neither chemogenetic inhibition nor activation of BNST CRF neurons altered contextual or CS-induced fear recalls 11 . The differing results may be due to the varying experimental timelines. While Bruzsik et al. modulated CRF neurons chemogenetically during acquisition and consolidation, our modulation occurred 24 hours after consolidation, just before extinction learning. Interestingly, inhibition of CRF neurons in the central amygdala (CeA) immediately after cued fear acquisition has been shown to enhance fear extinction 49 . Additionally, ectopic excitation of CRF + neurons in the CeA impairs fear memory acquisition and facilities extinction, whereas CRF + neuron inhibition impairs extinction memory 50 . While these findings are specific to CeA CRF neurons, they point to a possible involvement of BNST CRF signaling in fear extinction processes. Consistent with the results in 2CKO mice, DREADD-dependent CNO activation of BNST CRF neurons in WT mice led to a faster fear extinction, as indicated by an accelerated decline in the freezing response. Previous studies have shown that stimulation of CRF neurons in the anterior BNST is crucial for fear learning, particularly in relation to prolonged fear responses and contextual fear conditioning 14 , 15 . At the first glance it may seem surprising for BNST CRF stimulation to decrease fear responses, given that intra-BNST CRF administration typically enhances both fear and anxiety by modifying neuronal circuits involved in continuous threat monitoring and stress-induced behavioral alterations 12 , 42 . Importantly, the specific outcomes can vary depending on the subregion stimulated, the behavioral paradigm used, and the functional subset of neurons involved 11 , 32 . According to the proposed microcircuit by Marcinkiewicz et al., the BNST contains distinct populations of CRF neurons 14 . Given that the chemogenetic method used in this study activated all CRF neurons in the BNSTad without distinguishing between interneurons and projection neurons, the overall excitation could produce a net anxiolytic effect. This is likely because the anxiolytic impact of the projection neurons probably outweighs the inhibitory effects of the interneurons. Consistent with these findings, impaired fear extinction observed in WT saline mice was ameliorated by chemogenetic activation of BNST CRF neurons using hM3Dq. In addition, optogenetic activation of BNST VTA-projecting GABA neurons resulted in an anxiolytic behavioral phenotype 24 . Another study, focusing on corticotropin-releasing factor receptor type 2 (CRFR2) neurons in the posterior BNST (pBNST) demonstrated that optogenetic stimulation of these neurons could reduce anxiety, attenuate the stress response, and improve stress-induced anxiety 51 . It is important to note that the BNST is a complex structure composed of multiple functional subregions, each of which can affect anxiety-related behaviors in different ways 21 . In this context, the observed behavioral changes cannot be solely attributed to the chemogenetic modulation of CRF neurons in the BNSTad, as increased viral expression was also detected in the vBNST and juxtacellular BNST (BNSTjc). The vBNST is known to be a highly heterogeneous structure 27 that innervates the VTA 24 , 52 . Neurons in the vBNST exhibit varied responses to aversive stimuli, with activation of glutamatergic vBNST projection neurons leading to aversive and anxiogenic reactions, while activation of GABAergic projection neurons tends to produce anxiolytic effects 24 . Additionally, GABAergic CRF neurons of the BNSTjc have strong connections to the CeA and project to the LH and CRF neurons of the ovBNST 42 , 53 . Chemogenetic modulation of CRF projection neurons in the BNSTjc could also affect CRF neurons of the BNSTov. Therefore, the modulation of CRF neurons in the BNST and the resulting behavioral changes are not limited to the BNSTad, but may also involve the vBNST, BNSTjc, and, secondarily, the BNSTov. Nonetheless, the most significant viral expression was observed in the BNSTad. Beyond the observed differences in the fear extinction phenotype, we identified another notable distinction between WT and 2CKO mice. Specifically, WT mice demonstrated heightened fear generalization in a safe context relative to 2CKO mice, as evidenced by the occurrence of increased Bl freezing behavior on day 2. While the genotype effect achieved statistical significance only in the hM3Dq-expressing cohort, CNO-induced DREADD activation of BNST CRF neurons significantly diminished fear generalization, indicated by reduced Bl freezing in WT mice. These results are in accordance with other studies highlighting the contribution of BNST CRF neurons in sustained fear, which can lead to fear generalization 12 . Interestingly, CRF knockdown in the BNST can promote fear generalization, especially following partially reinforced fear conditioning in females 12 . Our results indicate that the 5-HT2CR in the BNST must be partially involved in fear generalization, as the absence of this receptor dampens fear generalization to ambiguous cues. The examination of certain locomotor parameters revealed partially significant differences between WT and 2CKO mice. However, these differences lacked consistency across cohorts and deviated from previous observations 32 . Earlier studies suggest that results regarding locomotor effects due to 5-HT2CR knockout are often variable and highly experiment-dependent 54 . Consistently, no significant differences were detected between treatment groups in the analyzed parameters during habituation and conditioning. This suggests an absence of pre-existing heterogeneity among groups prior to DREADD activation. Minor alterations in locomotor behavior compared to previous studies may be partially attributed to the incorporation of the CRF-ires-Cre mouse line, although this cannot be fully excluded as a contributing factor. In summary, serotonin in the BNST plays a complex role in anxiety modulation, primarily through its actions on different receptor subtypes. The balance between activation of anxiolytic (e.g., 5-HT1A) and anxiogenic (e.g., 5-HT2C) receptors, which are located on functionally distinct subsets of BNST CRF neurons, appears to be significant for the predominant effect on anxiety-like behaviors 14 , 29 , 55 . Pharmacological studies have shown that 5-HT2CRs in the BNST are crucial in mediating the anxiogenic effects of acute SSRI treatment 56 , 57 . 5-HT2CR activation in turn induces aversive behavior by activating BNST CRF neurons, which inhibit presumed GABAergic (anxiolytic) outputs from the BNST to the VTA and LH. This effect can be blocked by 5-HT2CR antagonism 14 , 57 . The absence of 5-HT2CRs on local BNST CRF neurons redirects 5-HT action towards 5-HT1ARs, resulting in local disinhibition of anxiolytic BNST CRF VTA/LH-projecting neurons. Consequently, in 2CKO mice, neural activity in the BNST is shifted towards anxiolytic VTA/LH projections supporting accelerated extinction learning 32 . By employing chemogenetic modulation of BNST CRF neurons in both 2CKO and WT mice, we were able to bi-directionally alter the fear extinction phenotype, thus supporting this hypothesis. Our results indicate that understanding serotonin's role in the BNST could lead to new therapeutic approaches for stress- and anxiety-related disorders. In this context, the 2CKO mice could serve as an important preclinical model for further exploration of the underlying neural mechanisms. Declarations Conflict of interest The authors declare no conflict of interests. Author contributions K.S., I.S. and S.H. designed the experiments. H.S., H.B., and P.L. performed experiments. H.S., H.B. and M.W. analyzed the data and performed statistics. H.S., H.B., M.W., K.S. and S.H. wrote the manuscript. Acknowledgments We thank Stefan Dobers, Winfried Junke, Stefan Rasche, Margareta Möllmann, Manuela Schmidt, Elli Buschtöns and Gina Hillgruber for technical support. This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants: Project ID 316803389 - SFB 1280, K.S. and S.H. (Subproject A07); Project number 492434978 - GRK 2862/1, Sub-projects (01, S.H.; 07, K.S.;09, I.S.) 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S01-04-5-HT 2C Receptor Activation Inhibits Stress-Induced Increase in 5-HT Transmission: Relevance to the Effects of Antidepressant Drugs. Eur. psychiatr. 2010; 25. Pelrine E, Pasik SD, Bayat L, Goldschmiedt D, Bauer EP. 5-HT2C receptors in the BNST are necessary for the enhancement of fear learning by selective serotonin reuptake inhibitors. Neurobiology of learning and memory 2016; 136: 189–195. Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files SupplementaryStatisticsTable.pdf Supplementary Statistics Cite Share Download PDF Status: Published Journal Publication published 10 Jan, 2026 Read the published version in Translational Psychiatry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-5604701","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":399532156,"identity":"b8fd4ebb-7e8c-4c79-a618-a6dcd8627f8e","order_by":0,"name":"Hannah Schulte","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Schulte","suffix":""},{"id":399532157,"identity":"2b8312bc-70f2-44e4-81b7-869518a2c153","order_by":1,"name":"Hanna Böke","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hanna","middleName":"","lastName":"Böke","suffix":""},{"id":399532159,"identity":"a1d4b041-8a0b-454c-988b-f5704358dd79","order_by":2,"name":"Patricia Lössl","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"","lastName":"Lössl","suffix":""},{"id":399532158,"identity":"cc784f7f-9377-4162-968b-d0e23b0e24b8","order_by":3,"name":"Maria Worm","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Worm","suffix":""},{"id":399532160,"identity":"40ba967b-4f60-4118-9ad0-ae5d8783a01c","order_by":4,"name":"Ida Siveke","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ida","middleName":"","lastName":"Siveke","suffix":""},{"id":399532161,"identity":"fdf822fa-1722-46ef-a9a2-9baf9aec4644","order_by":5,"name":"Stefan Herlitze","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Herlitze","suffix":""},{"id":399532155,"identity":"9ae316b8-d2a7-4a4c-bb4d-b67db17485f4","order_by":6,"name":"Katharina Spoida","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACCQYGNggFBgbMDPxgETZ8WpjRtEg2EKcFDpgZDA4Q0CLZfv7Yg597LPL4G9ifPfhQYC1vfPuM2QOGMhucWqR5ktkNe55JFEsc4DE3nGGQbrjtXI65AcO5NJxa5BiS2SR4DkgkNhzgYZPmMTjMuO0Mj5kEY9th3Fr4H7NJ/gFqmX+A/RlIi/3mHrCW/7gdJpEMNByoZcMBBjOQlsQNPGAtB3B7f8ZjM2kZoJaNhyF+SZ5xhq3cIOFcMk4tEucTn0m+OVCXOO94OzDE/ljb9vcwb3vwocwOpxYEYEaOiwQiNIAAnugbBaNgFIyCEQ0AHpRL8kXpYVYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4132-0655","institution":"Ruhr University Bochum","correspondingAuthor":true,"prefix":"","firstName":"Katharina","middleName":"","lastName":"Spoida","suffix":""}],"badges":[],"createdAt":"2024-12-08 22:05:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5604701/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5604701/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41398-025-03799-1","type":"published","date":"2026-01-10T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77888483,"identity":"e48f02d9-806a-4c17-b105-f9c429c7e1bf","added_by":"auto","created_at":"2025-03-06 13:30:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2708737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemogenetic inactivation of CRF neurons in the BNSTad impairs fear extinction.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e\u0026nbsp;Schematic representation of the stereotaxic AAV injection encoding the inhibitory hM4D(Gi) DREADD, followed by a three-day fear conditioning and extinction paradigm, using CRF-ires-cre/5-HT\u003csub\u003e2C\u003c/sub\u003eR-KO (2CKO) mice and their wild-type (WT) littermates. DREADD activation was achieved by i.p. injection (CNO; 1 mg/kg) 40 min prior to fear extinction. \u003cstrong\u003eb)\u003c/strong\u003e Illustration of the viral construct encoding hM4Di-mCherry, under control of the Cre/\u003cem\u003eLoxP\u003c/em\u003e system, driven by Human synapsin promotor (hSyn). \u003cstrong\u003ec)\u003c/strong\u003e Representative image of the BNST area, including the BNSTov and BNSTad, with DREADD expression localized exclusively in the BNSTad. Scale bar = 200 µm \u003cstrong\u003ed)\u003c/strong\u003e Redrawing and overlay of viral spreading within the BNST region. \u003cstrong\u003ee)\u003c/strong\u003e Freezing behavior was analyzed during baseline period (Bl) and conditioned stimulus (CS) presentation on the days of fear conditioning (day 2) and extinction (day 3). Freezing behavior decreased over time in all groups (\u003csup\u003e++\u003c/sup\u003ep \u0026lt; .010; \u003csup\u003e+++\u003c/sup\u003ep \u0026lt; .001; Friedman’s test). 2CKO mice showed less freezing behavior during fear extinction compared to WT littermates (\u003csup\u003e##\u003c/sup\u003ep \u0026lt; .010; \u003csup\u003e###\u003c/sup\u003ep \u0026lt; .001; Kruskal-Wallis test followed by Dunn’s post-hoc test). Chemogenetic inactivation of BNSTad CRF neurons enhanced freezing behavior of 2CKO mice (*p \u0026lt; .050; Kruskal-Wallis test followed by Dunn’s post-hoc test). Data are shown as means ± SEM. \u003cstrong\u003ef) \u003c/strong\u003eTotal distance moved during habituation (day\u0026nbsp;1) did not differ between groups. \u003cstrong\u003eg)\u003c/strong\u003e Maximum velocity increased in all groups during CS and unconditioned stimulus (US) pairings on conditioning day (day 2) compared to the Bl period (\u003csup\u003e+\u003c/sup\u003ep \u0026lt; .050; \u003csup\u003e++\u003c/sup\u003ep \u0026lt; .010; Friedman’s test). \u003cstrong\u003eh)\u003c/strong\u003e The total distance moved during the Bl period of fear extinction did not differ between groups. \u003cstrong\u003ei)\u003c/strong\u003e There were no group variations in maximum velocity during the Bl period of fear extinction. \u003cstrong\u003ej)\u003c/strong\u003e Freezing behavior during fear retrieval (average freezing during the first two CS presentations on fear extinction day; bin1) revealed significant differences between genotypes (\u003csup\u003e##\u003c/sup\u003ep \u0026lt; .050) and treatment groups (*p \u0026lt; .010; Kruskal-Wallis test followed by Dunn’s post-hoc test). For figures f-j) boxes represent the interquartile range (IQR), including the 1\u003csup\u003est\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e quartiles, line within boxes indicates the median and range (whiskers) extend to the upper and lower quartiles within the 1.5 IQR.\u003c/p\u003e","description":"","filename":"Figure1Gi.png","url":"https://assets-eu.researchsquare.com/files/rs-5604701/v1/b08c45072099deb7a5443c3a.png"},{"id":77888479,"identity":"0cdd15ae-c3ad-4dde-9eeb-4eb1001217d0","added_by":"auto","created_at":"2025-03-06 13:30:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2731287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemogenetic activation of CRF neurons in the BNSTad facilitates fear extinction.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e\u0026nbsp;Schematic representation of stereotaxic AAV injection encoding the excitatory hM3D(Gq) DREADD and the subsequent three-day fear conditioning and extinction paradigm, using CRF-ires-cre/5-HT\u003csub\u003e2C\u003c/sub\u003eR-KO (2CKO) mice and their wild-type (WT) littermates.\u0026nbsp; DREADD activation was achieved by i.p. injection (CNO; 1 mg/kg) 40 min prior to fear extinction. \u003cstrong\u003eb)\u003c/strong\u003e Illustration of the viral construct encoding hM3Dq-mCherry, under control of the Cre/\u003cem\u003eLoxP\u003c/em\u003e system, driven by Human synapsin promotor (hSyn). \u003cstrong\u003ec)\u003c/strong\u003e Representative image of the BNST area, including the BNSTov and BNSTad, with DREADD expression exclusively localized in the BNSTad. PKCδ staining was used as a marker for the BNSTov. Scale bar\u0026nbsp;=\u0026nbsp;200 µm \u003cstrong\u003ed)\u003c/strong\u003e Redrawing and overlay of viral spreading within the BNST region. \u003cstrong\u003ee)\u003c/strong\u003e Freezing behavior was analyzed during baseline period (Bl) and conditioned stimulus (CS) presentation on the days of fear conditioning (day 2) and extinction (day 3). Freezing behavior decreased over time in all groups (\u003csup\u003e++\u003c/sup\u003ep \u0026lt; .010; \u003csup\u003e+++\u003c/sup\u003ep \u0026lt; .001; Friedman’s test).\u0026nbsp; 2CKO mice showed less freezing behavior on the day of fear extinction compared to WT mice (\u003csup\u003e#\u003c/sup\u003ep \u0026lt; .050; \u003csup\u003e##\u003c/sup\u003ep \u0026lt; .010; Kruskal-Wallis test followed by Dunn’s post-hoc test). Chemogenetic activation of BNSTad CRF neurons reduced freezing behavior of WT mice (*p \u0026lt; .050; **p \u0026lt; .010; Kruskal-Wallis test followed by Dunn’s post-hoc test). Data are shown as means\u0026nbsp;±\u0026nbsp;SEM. \u003cstrong\u003ef)\u003c/strong\u003e Total distance moved during habituation (day\u0026nbsp;1) did not differ between groups. \u003cstrong\u003eg)\u003c/strong\u003e Maximum velocity increased in all groups during CS and unconditioned stimulus (US) pairings on conditioning day (day 2) compared to the Bl period (\u003csup\u003e+\u003c/sup\u003ep \u0026lt; .050; \u003csup\u003e++\u003c/sup\u003ep \u0026lt; .010; Friedman’s test). \u003cstrong\u003eh)\u003c/strong\u003e Total distance moved during the Bl of fear extinction revealed a genotype specific difference (\u003csup\u003e##\u003c/sup\u003ep \u0026lt; .010; two-way ANOVA). \u003cstrong\u003ei)\u003c/strong\u003e\u0026nbsp;Maximum velocity during the Bl of fear extinction revealed genotype specific differences (\u003csup\u003e#\u003c/sup\u003ep \u0026lt; .050; two-way ANOVA). j) Freezing behavior during fear retrieval (average freezing during the first two CS presentations on fear extinction day; bin1) showed no significant differences between groups. For figures f-j) boxes represent the interquartile range (IQR), including the 1\u003csup\u003est\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e quartiles, line within boxes indicates the median and range (whiskers) extend to the upper and lower quartiles within the 1.5 IQR.\u003c/p\u003e","description":"","filename":"Figure2Gq.png","url":"https://assets-eu.researchsquare.com/files/rs-5604701/v1/c10ee4fdc62e887b16cc19e1.png"},{"id":101935865,"identity":"412f9b7f-5032-4134-a03d-ac389316cfe1","added_by":"auto","created_at":"2026-02-05 08:29:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5763193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5604701/v1/ff60ecde-92bf-432b-be3c-54ad40ef7b59.pdf"},{"id":77888477,"identity":"c9b5842d-343c-49d6-a8b3-6fe4024eaaf7","added_by":"auto","created_at":"2025-03-06 13:30:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":122802,"visible":true,"origin":"","legend":"Supplementary Statistics","description":"","filename":"SupplementaryStatisticsTable.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5604701/v1/3c280f93be36d69e9c5a0dc3.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Chemogenetic modulation of CRF neurons in the BNST compensates for phenotypic behavioral differences in fear extinction learning of 5-HT2C receptor mutant mice.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFear and anxiety are closely related emotions. Anxiety is characterized by persistent, excessive worries that are pursued even in the absence of an immediate threat. In contrast, fear is a natural response to a specific, known danger \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although anxiety and fear are fundamentally different emotional states, they may share common neural and behavioral mechanisms \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Dysfunctions in the underlying neural networks may lead to anxiety, fear, and stress-related disorders, such as post-traumatic stress disorder (PTSD) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFrom a neurobiological perspective, the development of PTSD is linked to classical fear conditioning, while its persistence is attributed to a malfunction in extinction learning \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Individuals with PTSD often struggle to extinguish the learned association between a neutral environmental stimulus and the fear response, which can impair the efficacy of therapeutic interventions.\u003c/p\u003e \u003cp\u003eIn laboratory settings, fear conditioning (FC) and fear extinction (FE) paradigms are commonly used experimental approaches to study the neurobiological features of aversive learning that contribute to PTSD \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. FC involves repeatedly pairing a neutral, non-threatening stimulus, like a tone (conditioned stimulus, CS), with an aversive stimulus, such as a mild foot shock (unconditioned stimulus, US), until the animal begins to display a fear response not only to the shock but also to the tone alone (conditioned response, CR) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. On the other hand, extinction learning is an important biological adaptation mechanism that allows old behavior patterns to be unlearned when they are linked to invalid information. It is important to note that extinction learning suppresses the originally acquired fear association without erasing or overwriting it \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Extinction learning is achieved by repeatedly presenting the CS in the absence of the US, reducing the CR, and weakening the previously learned association between a stimulus and a defensive reaction \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStudying the cognitive mechanisms behind the formation and extinction of fear contributes to a better understanding of the emergence, maintenance, and treatment of PTSD. In this context, the amygdala is the crucial brain region responsible for forming the association between the signal of an aversive event and the aversive stimulus, giving them emotional significance \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Convergent evidence has implicated the amygdala in the extinction of fear \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, while more recent work also points toward a key role for the BNST in threat anticipation and fear modulation \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The BNST is highly interconnected with the central amygdala (CeA) \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, however, early research on the rodent BNST revealed a functional division between the amygdala and the BNST, supported by human fMRI data. These findings imply that the BNST is implicated in mediating specific fear and anxiety-like behaviors that are communicated without reference to the physical CeA circuit \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Within this context, the BNST mediates more diffuse, prolonged fear or unconditioned anxiety states, while the amygdala mediates impending, phasic fear experiences \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. More recent research, however, suggests that the BNST is also involved in processing phasic, discrete stimuli \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Early BNST lesion experiments demonstrated that fear responses were reduced when directly exposed to an aversive stimulus, such as predator scents \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Recent electrophysiological recordings in rodents demonstrated enhanced activity of BNST neurons during fear acquisition and CS-dependent fear recall \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Another study demonstrated that chemogenetic activation of GABAergic neurons in the BNST during fear conditioning or memory consolidation using a designer receptor exclusively activated by designer drugs (DREADD), which activates the G\u003csub\u003eq\u003c/sub\u003e-pathway, increased CS fear recall without affecting fear expression during conditioning or recall. This implies a modulatory role of the BNST in fear memory formation \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Additionally, studies have shown that the BNST is also highly involved in PTSD symptoms like altered hypervigilance, arousal states, and increased sensitization to the environment \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Despite the role of the BNST in anxiety behavior, the knowledge about how it affects fear learning - particularly concerning explicit cues and fear extinction - is still emerging.\u003c/p\u003e \u003cp\u003eSerotonin (5-HT) plays a pivotal role in anxiety- and fear-related behaviors through its effects in the BNST\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The BNST contains various serotonin receptor subtypes, which are expressed differently across neuron types \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Depending on the subtype involved, serotonin can elicit either inhibitory or excitatory neuronal responses \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Among the 14 serotonin receptor subtypes, the 5-HT1A and 5-HT2C receptors in the BNST have received particular attention in recent years, as their activation has opposing effects on fear behavior. In the anterodorsal subregion of the BNST (BNSTad), 5-HT1A receptor (5-HT1AR) activation has anxiolytic (anxiety-reducing) effects, while 5-HT2C receptor (5-HT2CR) stimulation increases anxiety \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOptogenetic and chemogenetic approaches revealed that 5-HT input to the BNST from the DRN elicits anxiogenic behavior and increased fear learning via 5-HT2CR signaling \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Although numerous studies in recent years have examined the role of the BNST in the acquisition, expression, and recurrence of fear, data on fear extinction are completely lacking. In our previous study, we discovered that the BNSTov and the BNSTad play opposing roles in fear extinction, with WT mice showing an extinction-associated reduction in BNSTov activity and an increase in BNSTad \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Optogenetic interventions produced comparable outcomes in anxiety tasks, showing that the BNSTov has an anxiogenic role, whereas the BNSTad has been described as anxiolytic \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Similarly, we discovered altered activity in the dorsal BNST in 2CKO mice in an anxiolytic and extinction-supporting direction, even under basal conditions \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. 2CKO animals had previously been characterized as having an anxiolytic phenotype, with reduced cFos expression in corticotropin-releasing factor (CRF)-expressing neurons in response to anxiety-inducing stimuli \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBuilding on our previous research, we used a chemogenetic approach to investigate the neuronal network in the BNSTad underlying the extinction-enhancing effect in 2CKO mice. Our results revealed that chemogenetic modulation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons produces bidirectional effects on the fear extinction phenotype. Activation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons using a G\u003csub\u003eq\u003c/sub\u003e-coupled (hM3Dq) DREADD promotes fear extinction in 5-HT2CR WT mice, while inactivation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons with a G\u003csub\u003ei\u003c/sub\u003e-coupled (hM4Di) DREADD impairs fear extinction in 2CKO mice.\u003c/p\u003e \u003cp\u003eThe findings reported here, provide a mechanistic explanation for the extinction-enhancing effect in 2CKO mice. The effect that we observed may be a key mechanism behind SSRI-induced anxiolysis, since the anxiolytic effects of systemic long-term SSRI administration are associated with 5-HT2CR desensitization \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003eAdult male mice (9\u0026ndash;16 weeks of age) were used for all experiments. To generate a transgenic CRF-ires-Cre/5-HT2CR mouse line, CRF-ires-Cre mice (B6(Cg)-\u003cem\u003eCrh\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1(cre)Zjh\u003c/em\u003e\u003c/sup\u003e/J, stock no. 012704, Jackson Laboratory) were bred with 5-HT2CR knockout (KO) mice KO (B6.129-Htr2c \u003csup\u003etm1Jul\u003c/sup\u003e/J, stock no. 002627, Jackson Laboratory). All mice had either a hemizygous (2CKO) or wild-type (WT) background for the 5-HT\u003csub\u003e2C\u003c/sub\u003eR allele and were homozygous for the CRF-ires-Cre background. Mice were group-housed (2\u0026ndash;5 individuals per cage) with a constant room temperature and a 12 h light/dark cycle while food and water were provided \u003cem\u003ead libitum.\u003c/em\u003e All experiments were performed during the light period. The experiments were conducted with the approval of the local ethics committee (Bezirksamt Arnsberg) and the animal care committee of North-Rhine-Westphalia (LANUV; Landesamt f\u0026uuml;r Umweltschutz, Naturschutz und Verbraucherschutz Nordrhein-Westfalen, Germany; AZ. 81-02.04.2021.A412). Studies were conducted in compliance with the 2010 European Communities Council Directive (2010/63/EU) for care of laboratory animals and supervised by the animal welfare commission of the Ruhr-University Bochum.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eViral constructs and Stereotaxic DREADD injections\u003c/h3\u003e\n\u003cp\u003eDouble-floxed adeno-associated viruses (AAVs) were used to express Gq-coupled (AAV1.pAAV.hSyn.DIO.hM3D(Gq)mCherry, Titer: 2.2 x 10\u003csup\u003e13\u003c/sup\u003e, #44361, Addgene) or Gi-coupled (AAV1.pAAV.hSyn.DIO.hM4D(Gi)-mCherry, Titer: 2.3 x 10\u003csup\u003e13\u003c/sup\u003e, #44362 Addgene) DREADDs in the BNSTad. The animals were anesthetized with isoflurane (initially at 5% (v/v) and then maintained at 1.5\u0026ndash;2.5% (v/v)) and positioned in a stereotaxic frame (Stoelting). For analgesia, subcutaneous injections of buprenorphine (0.1 mg/kg) and carprofen (2 mg/kg) were administered. The scalp was treated with lidocaine as a local anesthetic. The viral constructs were bilaterally injected into the BNSTad through pressure injection utilizing custom-pulled glass pipettes (tip diameter 5\u0026ndash;10 \u0026micro;m). The following stereotactic coordinates relative to bregma were used: AP\u0026thinsp;+\u0026thinsp;0.26, ML +/- 0.75, DV -4.1 \u003csup\u003e32\u003c/sup\u003e. AAV-injected animals were kept in their home cages for three weeks to allow for sufficient virus expression.\u003c/p\u003e\n\u003ch3\u003eDrug application\u003c/h3\u003e\n\u003cp\u003eDREADD activation within the BNSTad was achieved by i.p. injection of the designer receptor ligand clozapine-N-oxide (CNO; Tocris Bioscience; Cat. No: 6329). CNO was administered at a dose of 1 mg/kg, 40 minutes prior to the extinction session. In addition to the genotype groups, the DREADD-injected 2CKO and WT mice were randomly assigned to treatment groups. Control groups (WT Saline/ 2CKO Saline) received an i.p. injection of 0.3 ml 0.9% saline 40 min prior to extinction.\u003c/p\u003e\n\u003ch3\u003eBehavioral testing\u003c/h3\u003e\n\u003cp\u003eTo reduce stress related to cage changes and enable acclimatization to the experimenter, each individual was handled with a tunnel on four to five consecutive days before the fear conditioning experiment \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In addition, the mice were restrained during the last two to three days of the handling procedure to familiarize them with the i.p. injections. The fear conditioning apparatus followed the design and specifications as previously described by S\u0026uuml;\u0026szlig; et al. (2022). On day 1 of the fear conditioning and extinction protocol, mice were placed into context A characterized by white walls, an electrified grid floor, white LED illumination (250 lx), 30% fan intensity, and 70 %(v/v) ethanol as a background odor, for a 10-minute habituation without stimulus presentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The apparatus was cleaned with soap water between subjects. Fear conditioning occurred in context A on day 2. After a 2-minute baseline period (Bl), mice were given five pairings of a 30-second tone (conditional stimulus, CS, 7.5 kHz, 60 dB) with a foot shock (unconditional stimulus, US, 0.35 mA) that co-terminated with the last second of the CS. Between stimulus presentation intertrial intervals (ITIs), ranging from 30 to 120 s were implemented. After the final CS/US pairing, mice were maintained in the chamber for additional 60 s (post-stimulus time, PST). On day 3, fear retrieval and extinction took place in context B, consisting of a perforated floor plate, black and white striped walls, 100 %red LED illumination (28 lx), 100 %fan intensity and 1 %(v/v) acetic acid as background odor. 40 min prior to fear retrieval and extinction, mice received an i.p. injection of CNO (1 mg/kg) or 0.3 ml 0.9 %saline solution as control. Injected individuals were moved to a separate waiting cage to reduce stress for the experimental group in the home cage. After a 2 min Bl period, mice were exposed to 14 CS presentations separated by ITIs (30\u0026ndash;120 s), followed by a 60-second PST.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eBehavioral analysis\u003c/h3\u003e\n\u003cp\u003eAs previously described by S\u0026uuml;\u0026szlig; et al. (2022), a custom-made software written in MATLAB (MathWorks) was used for video recording and stimulus presentation. Subsequently, all behavioral parameters were analyzed using EthoVision XT 15 tracking software (11.5, Noldus). The following behavioral parameters were analyzed: total distance moved (in cm), maximum velocity (in cm/s), and freezing behavior (in %). Freezing behavior was defined as the absence of any movement other than respiration for more than two seconds. The percentage of freezing behavior was binned for two CS presentations (CS 1\u0026ndash;7). The first bin (bin1) represents fear retrieval. Individuals that did not exhibit freezing behavior were excluded from data analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and verification of virus expression\u003c/h2\u003e \u003cp\u003eFollowing the fear conditioning and extinction procedure, mice were deeply anesthetized and transcardially perfused with ice-cold PBS (1x), followed by ice-cold paraformaldehyde (4% PFA in PBS (w/v), pH 7.4, Sigma Aldrich). Brains were post fixed in 4% PFA and cryoprotected in 30% sucrose solution in PBS at 4\u0026deg;C (w/v, Sigma Aldrich). Coronal sections of 30 \u0026micro;m were cut with a cryostat (CM3050 S, Leica) and collected in 24-well plates filled with PBS for subsequent \u003cem\u003efree-floating\u003c/em\u003e antibody staining (see also S\u0026uuml;\u0026szlig; et al.,2022). Non-specific binding sites were blocked in 10% normal donkey serum (NDS, v/v, Merck Millipore) in 0.3% PBS-Triton X-100 (PBS-T, v/v, Sigma Aldrich) for 1 h at RT, followed by incubation with the primary antibody (rabbit anti PKCδ (1:1000, ab182126, Abcam) in 3% NDS in 0.3% PBS-T overnight at 4\u0026deg;C. Protein kinase delta (PKCδ) staining was performed as a marker for the BNSTov \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Secondary antibody solution included donkey anti rabbit DyLight\u0026reg; 488, (SA5-10038, Thermo Fisher Scientific) in 3 % ND in 0.3% PBS-T for 1.5 h at RT. Overview images of the immunostained sections in the caudal (AP 0.00), medial (AP\u0026thinsp;+\u0026thinsp;0.15), and rostral (AP\u0026thinsp;+\u0026thinsp;0.30) BNST regions were captured with a Leica fluorescence microscope (M205 FCA, Leica Microsystems) to confirm the exact virus placement. Images were processed and analyzed with ImageJ software \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The viral pattern indicated by the expression of mCherry was redrawn, slightly colored and overlayed using CorelDRAW\u0026reg; Graphic Suite software (Corel GmbH). Representative confocal microscopic images (20x/0.7 NA objective) of the BNSTad region from individuals subjected to fear conditioning were taken using a Leica laser-scanning confocal microscope (TCS SP5II, Leica Microsystems). Mice lacking bilateral viral expression within the BNSTad were excluded from data analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eGraphs were created using SigmaPlot 12.5 (Systat Software) and the data were analyzed utilizing IBM SPSS Statistics (Version 29.0) software. Normality was tested before each analysis (Shapiro-Wilk test). Further, homoscedasticity (Levene\u0026rsquo;s test) was assessed, while sphericity (Mauchly\u0026rsquo;s test) was verified for cases of repeated measures. A two-way analysis of variance (ANOVA) or a three-way repeated measures ANOVA (RM-ANOVA) was used to calculate statistical significance. Groups were compared with Bonferroni\u0026rsquo;s post hoc test. Data violating any assumption were analyzed using a Kruskal-Wallis Test, followed by Dunn\u0026rsquo;s post hoc test. Friedman\u0026rsquo;s test was applied in cases of repeated measures. Statistical significance was determined using a critical alpha level of 0.05 (p\u0026thinsp;\u0026lt;\u0026thinsp;.05). All statistical information is displayed in appendix (Table\u0026nbsp;1), while the most relevant results are presented in the text as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (p-value).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemogenetic inhibition of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons impairs fear extinction in 2CKO mice\u003c/h2\u003e \u003cp\u003eWe applied cell type-specific chemogenetic inhibition of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons using CRF-ires-Cre/2CKO mice to assess if a subset of neurochemically distinctive CRF neurons in the BNSTad promotes fear extinction in these mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). We will refer to these mice as 2CKO and WT mice, respectively. The accuracy of virus injection and the extent of viral spread were confirmed for each individual following the FC paradigm. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d). Confocal images revealed hM4Di-mCherry spread (magenta) within the dBNST was predominantly restricted to the BNSTad, almost entirely avoiding the BNSTov (BNSTad and BNSTov boundaries exemplarily outlined by dashed lines, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In addition, the distribution of the virus was assessed in the rostral, medial, and caudal BNST regions of each mouse that underwent FC. The more intense blue color indicates viral expression primarily in the dorsal BNST region. In contrast, the ventral BNST region exhibited lower virus expression, as reflected by the lighter color intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). To investigate how BNST inactivation influences fear extinction, we chemogenetically inactivated BNST\u003csup\u003eCRF\u003c/sup\u003e neurons during fear extinction training (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and e). An i.p. dose of CNO (1 mg/kg) 40 minutes before the onset of extinction training was used to activate hM4Di (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The control group was administered saline solution (0.3ml, i.p.) 40 min prior to the fear extinction training. To examine the effects of CRF neuron inactivation in the BNST on extinction learning, freezing behavior was measured during CS intervals in both conditioning and extinction sessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Statistical analysis of the data revealed no significant alterations in freezing behavior during fear conditioning between WT and 2CKO mice for saline and CNO-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). During the fear retrieval test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee bin1 and j), CS-induced fear memory recall was significantly higher in WT saline mice compared to 2CKO saline mice. In the further course of the extinction training, WT animals maintained a high level of freezing behavior compared to 2CKO animals (Figure, 1e). Between-group comparisons revealed significant genotype differences, confirming the previously published accelerated fear extinction phenotype in 2CKO mice \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Notably, chemogenetic inhibition of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons in 2CKO mice resulted in a markedly increased CS-induced fear recall with significantly impaired fear extinction phenotype compared to the saline-treated 2CKO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). After the inactivation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons in 2CKO animals, the previously observed phenotypic differences were no longer significant compared to saline-treated WT animals. Beside freezing, no statistically significant differences were observed in locomotor activity during the habituation phase, suggesting that 2CKO animals did not exhibit hyperactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Additionally, by examining the maximal movement velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), we investigated the animal's reaction to the US \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The maximal movement velocity in all groups was considerably higher during the CS/US pairings than throughout the baseline period (Bl) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Contrary to our previous results, we could not detect increased affected responses in both 2CKO groups. We examined the overall distance traveled and maximum velocity during the BL period of the extinction session (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh, i) and found no significant effects of CNO/saline injection on locomotor behavior.We also examined the overall distance traveled and the maximum velocity throughout the BL period of the extinction session to rule out any potential effects of CNO/saline injection on locomotor activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and i). Significant changes in motor behavior brought on by CNO/saline injection per se were not detected.\u003c/p\u003e \u003cp\u003eTaken together, our behavioral findings suggest that silencing of CRF neurons in the BNSTad region enhances CS-induced fear recall while delaying extinction in 2CKO mice. This indicates that a subset of these GABAergic CRF neurons in the BNSTad is significantly involved in the manifestation of the fear extinction phenotype in 2CKO mice. Conversely, the activation of CRF neurons in 2CKO mice contributes to accelerated fear extinction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eChemogenetic activation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons accelerates fear extinction in wild-type mice\u003c/h2\u003e \u003cp\u003eSince chemogenetic inactivation of CRF neurons enhanced fear recall in 2CKO mice and impaired extinction learning, we hypothesized that chemogenetic activation of these neurons in WT animals could have the opposite effect. Therefore, 2CKO and WT mice were bilaterally injected with a double-floxed stimulating hM3Dq-mCherry DREADD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). Subsequently, mice were exposed to the same auditory fear conditioning and extinction paradigm and hM3Dq was activated with CNO (1mg/kg) 40 minutes before fear extinction on day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Again, freezing behavior was analyzed during the Bl and for CS time bins during fear conditioning and fear extinction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Virus spreading was validated in all animals. The viral construct was injected into the BNSTad, however hM3Dq virus expression was also observed in the ventral BNST (vBNST) across the rostral, middle, and caudal regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). During fear conditioning, there were no significant changes in freezing behavior for CS presentations between groups. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). However, during extinction learning, we observed substantial genotype and treatment effects. Initially, WT mice exhibited considerably higher Bl freezing compared to 2CKO animals in the saline-treated control group. DREADD activation via CNO significantly reduced Bl freezing in WT mice to levels comparable to those of 2CKO animals. Additionally, statistical analysis revealed genotype and treatment effects in the subsequent extinction learning process. In the saline-treated hM3Dq-expressing control group, 2CKO and WT mice exhibited notable differences in freezing behavior. Unlike the hM4Di-expressing control groups in the first experiment, no changes in freezing behavior associated with CS-induced fear retrieval were observed this time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and j). Furthermore, WT mice maintained a consistently high level of freezing behavior with a slower decline over time compared to 2CKO animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Similar to the control groups in the previous hM4Di-DREADD experiment, the virus injection and he experimental procedure had only minimal impact on the genotype differences between WT and 2CKO animals. Again, no obvious genotype effects were detected for the total distance moved during the habituation session and maximal movement velocity during conditioning (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and g). Consequently, all groups displayed comparable locomotor activity and shock responsivity. However, during the Bl of the fear extinction session, significant genotype differences in locomotor activity were observed between WT and 2CKO mice, with the 2CKO control group exhibiting an increased total distance moved (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Further, the maximal movement velocity was significantly increased in 2CKO mice in the initial Bl of extinction learning (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Noteworthy, CNO-induced DREADD activation significantly influenced the progression of fear extinction in WT animals. WT mice expressing hM3Dq exhibited a non-significant trend toward reduced fear retrieval (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and j) and a significantly faster decline in freezing behavior over time due to CNO activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Additional statistical analysis confirmed the absence of phenotypic differences in fear extinction between CNO-treated WT mice expressing hM3Dq and saline-treated 2CKO mice expressing hM3Dq (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). As no significant effect of CNO activation on any locomotor parameters was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh and i), the effects must be attributed to reduced fear behavior.\u003c/p\u003e \u003cp\u003eThese results suggest that BNST\u003csup\u003eCRF\u003c/sup\u003e neurons are engaged in the phenotypic differences during extinction learning associated with 5-HT2CR knockout. Chemogenetic activation of distinctive BNST\u003csup\u003eCRF\u003c/sup\u003e neurons was sufficient to facilitate fear extinction in WT mice, while chemogenetic inactivation of these neurons in 2CKO mice enhanced CS-induced fear retrieval and delayed fear extinction.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study is based on our previous findings that mice constitutively lacking the 5-HT2CR display enhanced fear extinction in an auditory fear conditioning paradigm \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, the network and specific neuronal cell type mainly responsible for the differences in extinction learning phenotypes between 2CKO and WT animals could only be partially identified. Therefore, this study has been designed to further analyze the neuronal substrate underlying the extinction-supporting phenotype of 2CKO mice. Our results demonstrate that this phenotype could be modified in two ways via chemogenetic modulation of CRF neurons in the BNST.\u003c/p\u003e \u003cp\u003eEarlier research has highlighted that GABA and glutamate serve as the primary inhibitory and excitatory neurotransmitters in the BNST. The ratio of GABAergic to glutamatergic neurons differs among BNST subregions \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, with GABAergic neurons accounting for approximately 70\u0026ndash;90% of the cells in the BNSTad. The dBNST is highly heterogeneous, with GABAergic neurons expressing various neuropeptides, including CRF \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Based on previous studies, we hypothesized that CRF neurons in the BNSTad could be a promising target for extinction-associated differences related to our mouse model, given their central role in anxiety-related behaviors mediated by the BNST \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Within the BNST, the anterolateral group is believed to have the highest expression levels of extrahypothalamic CRF neurons \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, although expression levels vary across the region. The highest density of CRF-expressing neurons in the BNST is found in the BNSTov \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. BNST-CRF neurons primarily use GABA as a co-transmitter, leading to an overall inhibitory effect \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile CRF neurons in the BNST have traditionally been associated with anxiogenic effects \u003csup\u003e\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, more recent studies have revealed that BNST-CRF neurons also project to the VTA and LH in an anxiolytic manner \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, with approximately 58% of BNST-CRF output neurons forming anxiolytic connections to the VTA or LH \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Building on the anxiolytic phenotype observed in 5-HT2CR KO mice, characterized by reduced cFos expression (a marker of neuronal activity) in BNST\u003csup\u003eCRF\u003c/sup\u003e neurons \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, Marcinkiewicz et al. hypothesized that serotonergic input from the DRN modulates BNST\u003csup\u003eCRF\u003c/sup\u003e neuron activity via 5-HT binding at 5-HT2CRs\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Their findings showed that approximately 70% of dBNST\u003csup\u003eCRF\u003c/sup\u003e neurons express 5-HT2CRs, indicating that 5-HT signaling regulates their activity. Our results revealed that in 2CKO mice cFos levels are increased in an extinction-supporting direction in the BNSTad \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Consequently, we anticipated that the absence of the 5-HT2CR on local CRF neurons might impair the inhibition of anxiolytic projection neurons, thereby accelerating extinction. Additionally, we proposed that chemogenetic inactivation of CRF neurons might counteract the disinhibition caused by the absence of the 5-HT2CR in knockout mice. Indeed, we demonstrated that chemogenetic inactivation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons during extinction learning enhances fear recall and impairs extinction learning in 2CKO mice. This key finding aligns with earlier studies reporting that a subset of CRF-expressing, GABAergic neurons with long-range projections within the extended amygdala exerts anxiolytic effects by enhancing dopamine release into the VTA \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The same study also reported that mice with chronic CRH deficiency exhibited elevated anxiety levels and increased freezing behavior in both cued and contextual fear conditioning paradigms \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Other studies examining the impact of biochemically distinct neuron populations in the BNST on fear behavior have yielded somewhat contradictory results. In a cued fear conditioning paradigm, Bruzsik et. al. reported that neither chemogenetic inhibition nor activation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons altered contextual or CS-induced fear recalls \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The differing results may be due to the varying experimental timelines. While Bruzsik et al. modulated CRF neurons chemogenetically during acquisition and consolidation, our modulation occurred 24 hours after consolidation, just before extinction learning. Interestingly, inhibition of CRF neurons in the central amygdala (CeA) immediately after cued fear acquisition has been shown to enhance fear extinction \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Additionally, ectopic excitation of CRF\u003csup\u003e+\u003c/sup\u003e neurons in the CeA impairs fear memory acquisition and facilities extinction, whereas CRF\u003csup\u003e+\u003c/sup\u003e neuron inhibition impairs extinction memory \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. While these findings are specific to CeA\u003csup\u003eCRF\u003c/sup\u003e neurons, they point to a possible involvement of BNST\u003csup\u003eCRF\u003c/sup\u003e signaling in fear extinction processes.\u003c/p\u003e \u003cp\u003eConsistent with the results in 2CKO mice, DREADD-dependent CNO activation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons in WT mice led to a faster fear extinction, as indicated by an accelerated decline in the freezing response. Previous studies have shown that stimulation of CRF neurons in the anterior BNST is crucial for fear learning, particularly in relation to prolonged fear responses and contextual fear conditioning \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. At the first glance it may seem surprising for BNST\u003csup\u003eCRF\u003c/sup\u003e stimulation to decrease fear responses, given that intra-BNST\u003csup\u003eCRF\u003c/sup\u003e administration typically enhances both fear and anxiety by modifying neuronal circuits involved in continuous threat monitoring and stress-induced behavioral alterations \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Importantly, the specific outcomes can vary depending on the subregion stimulated, the behavioral paradigm used, and the functional subset of neurons involved \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. According to the proposed microcircuit by Marcinkiewicz et al., the BNST contains distinct populations of CRF neurons \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Given that the chemogenetic method used in this study activated all CRF neurons in the BNSTad without distinguishing between interneurons and projection neurons, the overall excitation could produce a net anxiolytic effect. This is likely because the anxiolytic impact of the projection neurons probably outweighs the inhibitory effects of the interneurons. Consistent with these findings, impaired fear extinction observed in WT saline mice was ameliorated by chemogenetic activation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons using hM3Dq. In addition, optogenetic activation of BNST VTA-projecting GABA neurons resulted in an anxiolytic behavioral phenotype \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Another study, focusing on corticotropin-releasing factor receptor type 2 (CRFR2) neurons in the posterior BNST (pBNST) demonstrated that optogenetic stimulation of these neurons could reduce anxiety, attenuate the stress response, and improve stress-induced anxiety \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. It is important to note that the BNST is a complex structure composed of multiple functional subregions, each of which can affect anxiety-related behaviors in different ways \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In this context, the observed behavioral changes cannot be solely attributed to the chemogenetic modulation of CRF neurons in the BNSTad, as increased viral expression was also detected in the vBNST and juxtacellular BNST (BNSTjc). The vBNST is known to be a highly heterogeneous structure \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e that innervates the VTA \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Neurons in the vBNST exhibit varied responses to aversive stimuli, with activation of glutamatergic vBNST projection neurons leading to aversive and anxiogenic reactions, while activation of GABAergic projection neurons tends to produce anxiolytic effects \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Additionally, GABAergic CRF neurons of the BNSTjc have strong connections to the CeA and project to the LH and CRF neurons of the ovBNST \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Chemogenetic modulation of CRF projection neurons in the BNSTjc could also affect CRF neurons of the BNSTov. Therefore, the modulation of CRF neurons in the BNST and the resulting behavioral changes are not limited to the BNSTad, but may also involve the vBNST, BNSTjc, and, secondarily, the BNSTov. Nonetheless, the most significant viral expression was observed in the BNSTad.\u003c/p\u003e \u003cp\u003eBeyond the observed differences in the fear extinction phenotype, we identified another notable distinction between WT and 2CKO mice. Specifically, WT mice demonstrated heightened fear generalization in a safe context relative to 2CKO mice, as evidenced by the occurrence of increased Bl freezing behavior on day 2. While the genotype effect achieved statistical significance only in the hM3Dq-expressing cohort, CNO-induced DREADD activation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons significantly diminished fear generalization, indicated by reduced Bl freezing in WT mice. These results are in accordance with other studies highlighting the contribution of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons in sustained fear, which can lead to fear generalization \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Interestingly, CRF knockdown in the BNST can promote fear generalization, especially following partially reinforced fear conditioning in females \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Our results indicate that the 5-HT2CR in the BNST must be partially involved in fear generalization, as the absence of this receptor dampens fear generalization to ambiguous cues.\u003c/p\u003e \u003cp\u003eThe examination of certain locomotor parameters revealed partially significant differences between WT and 2CKO mice. However, these differences lacked consistency across cohorts and deviated from previous observations \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Earlier studies suggest that results regarding locomotor effects due to 5-HT2CR knockout are often variable and highly experiment-dependent \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Consistently, no significant differences were detected between treatment groups in the analyzed parameters during habituation and conditioning. This suggests an absence of pre-existing heterogeneity among groups prior to DREADD activation. Minor alterations in locomotor behavior compared to previous studies may be partially attributed to the incorporation of the CRF-ires-Cre mouse line, although this cannot be fully excluded as a contributing factor.\u003c/p\u003e \u003cp\u003eIn summary, serotonin in the BNST plays a complex role in anxiety modulation, primarily through its actions on different receptor subtypes. The balance between activation of anxiolytic (e.g., 5-HT1A) and anxiogenic (e.g., 5-HT2C) receptors, which are located on functionally distinct subsets of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons, appears to be significant for the predominant effect on anxiety-like behaviors \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Pharmacological studies have shown that 5-HT2CRs in the BNST are crucial in mediating the anxiogenic effects of acute SSRI treatment \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. 5-HT2CR activation in turn induces aversive behavior by activating BNST\u003csup\u003eCRF\u003c/sup\u003e neurons, which inhibit presumed GABAergic (anxiolytic) outputs from the BNST to the VTA and LH. This effect can be blocked by 5-HT2CR antagonism \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The absence of 5-HT2CRs on local BNST\u003csup\u003eCRF\u003c/sup\u003e neurons redirects 5-HT action towards 5-HT1ARs, resulting in local disinhibition of anxiolytic BNST\u003csup\u003eCRF\u003c/sup\u003e VTA/LH-projecting neurons. Consequently, in 2CKO mice, neural activity in the BNST is shifted towards anxiolytic VTA/LH projections supporting accelerated extinction learning \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. By employing chemogenetic modulation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons in both 2CKO and WT mice, we were able to bi-directionally alter the fear extinction phenotype, thus supporting this hypothesis. Our results indicate that understanding serotonin's role in the BNST could lead to new therapeutic approaches for stress- and anxiety-related disorders. In this context, the 2CKO mice could serve as an important preclinical model for further exploration of the underlying neural mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eK.S., I.S. and S.H. designed the experiments. H.S., H.B., and P.L. performed experiments. H.S., H.B. and M.W. analyzed the data and performed statistics. H.S., H.B., M.W., K.S. and S.H. wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank Stefan Dobers, Winfried Junke, Stefan Rasche, Margareta M\u0026ouml;llmann, Manuela Schmidt, Elli Buscht\u0026ouml;ns and Gina Hillgruber for technical support. This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants: Project ID 316803389 - SFB 1280, K.S. and S.H. (Subproject A07); Project number 492434978 - GRK 2862/1, Sub-projects (01, S.H.; 07, K.S.;09, I.S.)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSteimer T. The biology of fear- and anxiety-related behaviors. Dialogues in clinical neuroscience 2002; 4: 231\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin LM, Liberzon I. The neurocircuitry of fear, stress, and anxiety disorders. Neuropsychopharmacology official publication of the American College of Neuropsychopharmacology 2010; 35: 169\u0026ndash;191.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlechert J, Michael T, Vriends N, Margraf J, Wilhelm FH. Fear conditioning in posttraumatic stress disorder: evidence for delayed extinction of autonomic, experiential, and behavioural responses. 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Neurobiology of learning and memory 2016; 136: 189\u0026ndash;195.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5604701/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5604701/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePsychopharmacotherapy is often used to treat anxiety- and stress-associated psychiatric disorders, including posttraumatic stress disorder (PTSD). Adjunctive therapy is most typically used with medications that influence serotonin balance, such as selective serotonin reuptake inhibitors (SSRIs). Contrary to expectations, SSRIs show an anxiety-increasing effect during the initial treatment phase. Among the 14 different serotonin receptor subtypes, pharmacological studies have demonstrated that 5-HT2C receptors (5-HT2CRs) in the bed nucleus of the stria terminalis (BNST) play a significant role in the anxiogenic effect of acute SSRI treatment. Although numerous studies have confirmed the role of the 5-HT2CR in anxiety behavior, little is known about its involvement in learned fear and fear extinction. In particular, fear extinction is considered a central neural mechanism in the treatment of PTSD patients. Recent results from 5-HT2CR knockout mice (2CKO) revealed that global loss of 5-HT2CRs enhances fear extinction, without affecting fear acquisition. Here, we implemented a chemogenetic approach to examine the neuronal substrate which underlies this extinction-enhancing effect in 2CKO mice. DREADD-activation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons promotes fear extinction in 5-HT2C WT mice, whereas DREADD-inactivation of BNST\u003csup\u003eCRF\u003c/sup\u003e neurons impairs fear extinction in 2CKO mice. Thus, using activating and inactivating DREADDs, we were able to directionally modulate fear extinction. These findings provide a possible explanation for the fear extinction-enhancing effect in 2CKO mice with relevance for the treatment of PTSD patients.\u003c/p\u003e","manuscriptTitle":"Chemogenetic modulation of CRF neurons in the BNST compensates for phenotypic behavioral differences in fear extinction learning of 5-HT2C receptor mutant mice.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-06 13:30:35","doi":"10.21203/rs.3.rs-5604701/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"526c273a-834d-468f-9df3-16a3bfd7a0b3","owner":[],"postedDate":"March 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42572321,"name":"Biological sciences/Neuroscience"},{"id":42572322,"name":"Health sciences/Diseases/Psychiatric disorders/Schizophrenia"}],"tags":[],"updatedAt":"2026-02-05T08:28:52+00:00","versionOfRecord":{"articleIdentity":"rs-5604701","link":"https://doi.org/10.1038/s41398-025-03799-1","journal":{"identity":"translational-psychiatry","isVorOnly":false,"title":"Translational Psychiatry"},"publishedOn":"2026-01-10 05:00:00","publishedOnDateReadable":"January 10th, 2026"},"versionCreatedAt":"2025-03-06 13:30:35","video":"","vorDoi":"10.1038/s41398-025-03799-1","vorDoiUrl":"https://doi.org/10.1038/s41398-025-03799-1","workflowStages":[]},"version":"v1","identity":"rs-5604701","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5604701","identity":"rs-5604701","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}