Improving arm-hand skill performance is a major therapeutic target in stroke rehabilitation. Arm-hand rehabilitation may be enriched in content and variation by using technology-assisted training. Especially for people with a severely affected arm, technology-assisted training offers more challenging training possibilities. The aim of this study was to explore the feasibility of ReHab-TOAT, a “Remote Handling Based Task-Oriented Arm Training” approach featuring enriched haptic feedback aimed at improving daily activities and participation.motivation’, ‘individualization of training’, ‘potential training effects’, and ‘implementation in rehabilitation’ of patients and therapists. Five subacute or chronic stroke patients suffering moderate to severe arm-hand impairments and five rehabilitation therapists participated. All participants received 2 ReHab-TOAT sessions. Outcome measure was a bespoke feasibility questionnaire on user experiences and satisfaction regarding ‘ Both patients and therapists experienced ReHab-TOAT as being feasible. They found ReHab-TOAT very motivating and challenging. All patients perceived an added value of ReHab-TOAT and would continue the training. Small improvements regarding exercise variability were suggested. ReHab-TOAT seems to be a feasible and very promising training approach for arm-hand rehabilitation of stroke patients with a moderately or severely affected arm. Further research is necessary to investigate potential training effects of ReHab-TOAT. In addiIt has been shown that task-oriented training, i.e. repetitive training of meaningful activities in a functional context, induces changes in the cerebral cortex, supporting motor recovery based on brain plasticity , 11 and A number of systematic reviews about the effectiveness of robot-assisted therapy for upper extremities have been published in the last decade , 17. AltGiven the afore mentioned, we developed a new task-oriented arm training approach using a so-called ‘remote handling concept’ device featuring haptic feedback, to manipulate proprioception, i.e. the sense of movement, in patients with a severely affected arm-hand (UAT 1–3). This approach is called “Remote Handling concept based, Task-Oriented Arm Training” (acronym: ReHab-TOAT). The intervention consists of task-oriented training, incorporating the principles of motor learning, and is based on the training approach of two previously described and evaluated training concepts, i.e. “CARAS”  and “TOA to date , 26, 27. to date . Another to date .Therefore, the aim of the present study was to assess the feasibility of the ReHab-TOAT approach featuring enriched haptic feedback aimed at improving activities of daily living and participation in chronic and in subacute stroke patients with either a moderately or severely affected arm-hand. This was done from both the patients’ perspective and the therapists’ perspective. Special emphasis was put on motivation, individualization of training, training effects, and implementation in a rehabilitation context.2.This feasibility study was approved by the Medical Ethics Committee of Maxima Medisch Centrum in Veldhoven, the Netherlands (study code: NL70014.015.19). The study was also registered in the ISRCTN registry (ISRCTN50551089).2.1Five patients in either the subacute or chronic phase after a stroke participated. Patients were identified from the database of the department of brain injury rehabilitation of Adelante rehabilitation centre in Hoensbroek, the Netherlands. They all met the following inclusion criteria: unilateral stroke; post-stroke time between 6 and 12 weeks (subacute) or post-stroke time larger than 12 months (chronic); arm-hand motor impairment, i.e. an Utrechtse Arm/hand Test (UAT) score of 1–3 ; age > 1ore > 1+ ; non-strAlso, five therapists participated in the study. They all met the following inclusion criteria: working at a specialized rehabilitation centre, i.e. Adelante Zorggroep, Hoensbroek in the Netherlands; holding a degree in physiotherapy or occupational therapy; having at least 6 years of experience in the treatment of patients with central nervous system deficits.2.2ReHab-TOAT is a task-oriented arm training approach for stroke patients with a moderately to severely affected arm-hand  . One of The general content and time planning of each training session of the ReHab-TOAT approach is depicted in Fig. A comprehensive description of the ReHab-TOAT approach, including training session timing, training content & training build-up, and the potential for generalization of training effects, are presented in Appendix 1, 2 and 3 respectively.ReHab-TOAT was originally developed to be provided in three sessions per week, each session lasting 1.5 hours, over a training period of four weeks, based on previous research , 23. How2.3Outcome measures were user satisfaction and experiences from both patients and therapists regarding different aspects of feasibility . The out2.4The data of the quantitative part of the questionnaires from therapists and patients are reported descriptively. The answers to the open-ended questions were analysed using directed content analysis . The ini3.3.1The characteristics of the 5 stroke patients and the 5 therapists who participated in this study are shown in Table 3.2The individual results of the quantitative part of the feasibility questionnaire of the patients are presented in Table a bit boring” and “not exhausting”. In general, the patients were positive about the motivational aspect of ReHab-TOAT. They used terms like “nice” . Another patient stated the importance of the presence of a therapist during a training session “to make sure that there is therapeutic added value of the training”  of the movement possibilities of the patient during one exercise, “The individual results of the quantitative part of the feasibility questionnaire from the therapists can be found in Table Challenging and inviting to push your own limits” . However, one added value of using technology-assisted force feedback in ReHab-TOAT is that the training is made accessible to a broader group of patients. It creates the possibility to train meaningful movements even in patients with a severely affected arm-hand function or in patients with cognitive impairments.4.4In our study, both patients and therapists agreed that ReHab-TOAT has an added value for arm-hand rehabilitation of stroke patients, because of a) force feedback making therapy possible for a broader range of patients; b) the self-perceived training effects in daily life; c) the fun element; d) the challenging and motivating way of training; and e) the incorporation of self-efficacy principles and the stimulation of autonomy of the patient during training. And as therapists and patients, besides other experts, were involved in the development process of ReHab-TOAT right from the start of the project conception, their ideas and thoughts on the use of rehabilitation technology, and more specifically the use of the Dexternageable , 15.Also, in order to further optimise ReHab-TOAT, during the execution of the project several of the participants suggested to add more games and exercises, which enhances practice variability and training motivation. This, in turn, may increase generalization and transfer of training effects towards novel situations and other arm-hand skills . Therapi4.5The purpose of the present study was to assess the feasibility of ReHab-TOAT. Feasibility can be sub-classified in several key areas of interest, as described by Bowen and co-workers . We focuThe strength of this study is that ReHab-TOAT was tested by a defined group of therapists  and patients (stroke patients in chronic and subacute stage after stroke with a severely or moderately impaired arm-hand function and from different ages), in order to obtain a comprehensive set of information on the feasibility. This variety in participants was of added value, as it provided us with relevant and broad information on potential future end-users. ReHab-TOAT was tested under realistic circumstances, i.e. in regular rehabilitation conditions, in which it will also be used in the future. Furthermore, ReHab-TOAT has been developed for a wide group of patients, and contains all necessary and evidence-based training principles of current arm-hand rehabilitation regimes.During this study participants gave insights into their self-perceived performance and self-perceived training effects after only two sessions of ReHab-TOAT. Consequently, potential experienced effects that might occur after a longer use of the approach were not investigated. The same applies to aspects of the feasibility which might change during a more long-term use of the approach, especially regarding the motivation of patients. Furthermore, as none of the existing questionnaires gauging  technology was specific enough for our queries regarding ReHab-TOAT, two bespoke questionnaires were constructed, based on expert opinion from different domain experts (see Appendix 4 and 5). These questionnaires were not validated against any existing criterion or existing questionnaires.Our study did not focus on feasibility items like ‘Adaptation’, ‘Expansion’ or ‘Limited efficacy’. The latter would have necessitated a far larger number of participants in larger (and homogeneous) strata or subgroups, and a different methodological study set-up contrasting conditions and/or subgroups. No objective measures on any potential physical improvements and training effects were gauged.5.ReHab-TOAT seems to be a feasible training approach for arm rehabilitation of stroke patients with a moderately or severely affected arm. Both patients and therapists identified several benefits of ReHab-TOAT, e.g. the haptic feedback, increased motivation, enhancing self-efficacy and autonomy of the patient during the rehabilitation process, the possibility of individualizing the training (based on the patients’ needs), and the possible training effects at different levels of the ICF model. All participants wanted to continue the use of ReHab-TOAT, because they saw the high potential of gaining improvements, not only at arm-hand function level, but particularly at activity and participation level. Further research is necessary to investigate any potential training effect of ReHab-TOAT on patients’ performance at the different levels of the ICF model.This study, as part of the i2-CoRT project (www.i2-CoRT.eu), has been co-funded by the Interreg V-A Euregio Meuse-Rhine (EMR) programme under Grant EMR1. The Interreg EMR program has invested almost EUR 100 million in the development of the Interreg-region until 2020. With the investment of EU funds in Interreg projects, the European Union directly invests in the economic development, innovation, territorial development, social inclusion and education of this region.Coded data will be made available to the scientific community upon reasonable request.The supplementary files are available to download from http://dx.doi.org/10.3233/THC-220465.Click here for additional data file.


SCD5A expression was found compared with SCD5B. This unequal splicing is attributed to a weaker recognition of the SCD5B-specific splicing acceptor site, based on predictions confirmed by an optimized minigene assay. The pronounced dominance of SCD5A was largely modified  or even inverted (rs1011850309_C) by natural SNVs at the TV-specific splice sites. Our results provide long missing data on the proportion of SCD5 TVs in human tissues and reveal mutation-driven changes in SCD5 AS, potentially affecting tumor-associated reprogramming of lipid metabolism, thus having prognostic significance, which may be utilized for novel and personalized therapeutic approaches.Alternative splicing (AS) is a major means of post-transcriptional control of gene expression, and provides a dynamic versatility of protein isoforms. Cancer-related AS disorders have diagnostic, prognostic and therapeutic values. Changes in the expression and AS of human stearoyl-CoA desaturase-5 (SCD5) are promising specific tumor markers, although the transcript variants (TVs) of the gene have not yet been confirmed. Our in silico, in vitro and in vivo study focuses on the distribution of SCD5 TVs (A and B) in human tissues, the functionality of the relevant splice sites, and their modulation by certain single-nucleotide variations (SNVs). An order of magnitude higher  The balance of lipid metabolism in the human body depends on an adequate supply of various saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids (FAs) of different carbon chain lengths, adjusted to the dynamically changing requirements. The MUFA-producing stearoyl-CoA desaturases (SCDs) are endoplasmic reticulum membrane-bound enzymes, which introduce the first double bond at cis-delta9 position into saturated fatty acyl-CoA molecules, mainly palmitoyl-CoA and stearoyl-CoA, to produce palmitoleyl-CoA and oleyl-CoA, respectively . Two isoSCD1 is mainly expressed in the major organs of lipid metabolism [SCD5 is highly represented in the embryonic and mature brain, pancreas and gonads instead [SCD5 gene expression may be less dependent on lipid factors compared to other mammalian desaturases. In contrast to SCD1, the promoter activity of SCD5 has been shown to be insensitive to several FAs in vitro [α, NF1, NFY) in the 5′ regulatory sequence of SCD5 [SCD5 mRNA levels. However, certain promoter polymorphisms of SCD5 have been proven to significantly reduce the promoter activity in vitro, modify TF binding sites in silico and were associated to metabolic disorders [The two human isoforms show markedly different gene expression patterns. While tabolism , SCD5 is instead ,5,6,7. I of SCD5 ,9, neith of SCD5 , changes of SCD5 , nor the of SCD5  affectedSCD5 [In recent years, the results of high-throughput whole genome, transcriptome and microRNA sequencing data have directed attention to tumor-related changes in the expression level of SCD5 ,17,18,19SCD5 . SCD5-drSCD5  and melaSCD5 ,23. FurtSCD5 , and a rSCD5 splicing event was identified as strong prognostic biomarker in a study analyzing the AS profile of kidney renal clear-cell carcinoma [Alternative splicing (AS) is a fundamental modulator of human gene expression, and it plays a significant role in expanding the diversity of functional proteins. There is increasing evidence that AS has a profound effect on cancer development. The tumor type-specific changes of the splicing process in cancer cells have significant prognostic value, can contribute to the prediction of cancer progression rate, and may be of therapeutic relevance . Along warcinoma , howeverarcinoma ,25,26.Minigene assay is a reliable in vitro tool for investigating the effect of human variations in AS . After cSCD5A and SCD5B forms and assessed their relative mRNA expression and distribution in different human cell lines and tissues. We proved the biased, SCD5A dominant acceptor site recognition of the two TVs in silico by prediction, and in vitro by generating and expressing a modified SCD5 minigene construct. Human variations affecting SCD5A- and SCD5B-specific donor and acceptor splice sites were also tested by the newly developed SCD5 minigene system.Although there are two TVs (A and B) of SCD5 in the NCBI, Ensemble and UniProt databases, they are not distinguished, examined, or even mentioned in scientific publications, with only one exception to our knowledge . TherefoSCD5 gene is located on the longer arm of chromosome 4. Its average-length exons are separated by rather long intron sequences of tens of thousands of base pairs. The first three of its six exons are identical for the two TVs, but SCD5A contains two more exons    B, wherea4B, E4B) B, which 4B, E4B) A. Accord4B, E4B) B,C.SCD5A and B TVs was tested by RT-PCR using primers specific for the common exon 2 and E5A and E4B, respectively, , thus the method proved to be suitable for quantitative comparisons .Since the two SCD5 TVs are produced from the same gene with extensively identical 5′ segments  A, their SCD5 may be due to differential recognition of A and B acceptor sites by the splicing machinery. To test this hypothesis in silico, we analyzed approximately 500-base-pair-long sequences harboring the AB donor and the A and B acceptor sites by NetGene2-2.42 online prediction program package [SCD5 pre-mRNA -based minigene system, in which the two acceptor sites can be compared simultaneously in the same cell. The schematic structure and exact sequence of the SCD5 minigene are shown in SCD5 gene, as well as the exons E4A and E5A without introns, were cloned into the expression vector. E4B was cloned together with the adjacent intron sequences. Due to their length, the entire introns 3 and 4 cannot be cloned into a vector, thus, only segments important for splicing were placed in the positions corresponding to the SCD5 gene. In the case of intron 3, a section with a length of 242 nucleotides from the 3′ end and 334 nucleotides from the 5′ end were inserted, while in the case of intron 4, the fragments were 242 and 344 nucleotides long  affecting the We examined a total of eight sequence variations of six SNVs, of which the position and main characteristics are summarized in A single missense variation  were seen to reduce the amount of SCD5A protein in the transiently transfected HEK293T cells compared with the wildtype A,C. Howe5 to 37% , and tha5 to 37% B. It is 5 to 37% C, withou5 to 37% D. The ef5 to 37% B, althousplicing C,D.SCD5B variant  or low extent  varies widely. Liver, lung, small intestine and spleen mainly express SCD1, and the pancreas, kidney and ovary are dominated by SCD5, while the two genes show balanced expression in brain, skeletal muscle and testis. Even though the SCDs catalyze the same reaction, their tissue-specific distribution and varying proportion suggest different functions, the nature of which requires further investigation.Although the characteristic gene expression of both  tissues ,5,6,7,33 results . Althoug, [The amino acid sequence of human SCD5A indicates four transmembrane domains, similarly to other SCD isoforms. The three histidine clusters considered important to fatty-acyl desaturase activity are also identified in SCD5A . It is ny 2023), ).SCD5A- and SCD5B-specific donor and acceptor splice sites, but also the effect of their SNVs on variant proportions. Our in silico and in vitro results were in very good agreement in both cases, i.e., the B acceptor site of lower-predicted probability indeed resulted in a lower level of SCD5B mRNA . Bovine serum albumin, HepG2 and HEK293T cells were purchased from Sigma-Aldrich . Polyclonal primary antibody against SCD5 was obtained from Invitrogen . Actin-specific polyclonal antibody and secondary antibodies were obtained from Cell Signaling . Human tissue RNAs were purchased from Thermo Fisher Scientific  and Zyagen Laboratories . All chemicals used in this study were of analytical grade. All experiments and measurements were carried out by using Millipore ultrapure water.SCD5 gene were included in the present study if they were identified in at least two different populations. The in silico impact of the selected sequence variants was predicted using the Variant Effect Predictor  and Netly 2022, ) online SCD5A and SCD5B were amplified from human ovary cDNA by iProof™ High-Fidelity DNA Polymerase , according to the manufacturer’s protocol. The purified DNA fragments were cloned into the pcDNA3.1(−) expression vector between the XhoI and KpnI restriction sites. The SCD5 minigene expression plasmid was generated in the pcDNA3.1(−) vector. The construct was designed to include the possible donor and acceptor sites of the third and fourth introns relevant for the alternative splicing of SCD5A and SCD5B TVs, the branch points, and their flanking sequences. A detailed description of the cloning procedure is presented in SCD5A plasmid or genomic DNA as a template. Then, the first two and the last two overlapping PCR products were combined by overlapped extension PCR. The three DNA fragments containing the three common and the three different exons, as well as the 5′ end, 3′ end and branch point regions, were successively cloned into the pcDNA3.1(−) expression plasmid using XhoI, NotI, Eco32I and KpnI restriction sites. The sequence of the cloning primers is shown in ® Site-Directed Mutagenesis Kit . The mutagenic primers are listed in The coding regions of 6 cells per well) in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution, at 37 °C in a humidified atmosphere containing 5% CO2. Cells were transfected with 1 μg SCD5 minigene plasmids using 3 μL Lipofectamine 3000, supplemented with 2 µL P3000  in 1 mL DMEM, respectively. Cells were harvested and processed 24 h after transfection.Human embryonic kidney (HEK293T) and hepatocellular carcinoma (HepG2) cells were cultured in 12-well plates  was added to each well, and the cells were scraped and briefly vortexed. After 45 min incubation on ice, the lysates were centrifuged for 15 min at maximum speed in a benchtop centrifuge at 4 °C, to remove cell debris. The protein concentration of the supernatant was measured with Pierce® BCA Protein Assay Kit , and the samples were stored at −20 °C until downstream analysis.Cell lysates were prepared for immunoblot analysis by removing the medium and washing the cells twice with PBS. The RIPA lysis buffer (100 µL)  using the SuperSignal West Pico Chemiluminescent Substrate .Aliquots of cell lysates (5 μg protein per lane) were analyzed by SDS-PAGE on 15% Tris-glycine minigels, and transferred onto Immobilon-P membranes . Primary and secondary antibodies were applied overnight at 4 °C and for 1 h at room temperature, respectively. Horseradish peroxidase (HRP)-conjugated goat polyclonal anti-actin  antibodies were used at 1:1000 dilution. SCD5 was detected with a rabbit polyclonal antibody , used at a dilution of 1:2000, followed by HRP-conjugated goat polyclonal anti-rabbit IgG  at a dilution of 1:2000. HRP was detected by C-DiGit®-4PCR Kit . cDNA samples were produced by reverse transcription of 0.5 µg DNA-free RNA, using the SensiFASTTM cDNA Synthesis Kit . Reverse-transcription PCR was performed in 10 µL final volume containing 1 µL cDNA template, 0.2 mM dNTPs, 1× PCR Buffer, 1× Q-Solution, 0.025 U/µL HotStarTaq DNA Polymerase , 1 µM forward and reverse primers. SCD5A and SCD5B sequences were amplified by a commonsense primer (5′-CGC TCT GGG TGT GAC A-3′), together with SCD5A (5′-CCC CAG CCA GCA CAT GAA AT-3′)- or SCD5B (5′-CCT CCA GGG ACA CAG AAA GAG-3′)-specific antisense primer. GAPDH cDNA was also amplified as an endogenous control using 5′-GTC CAC TGG CGT CTT CAC CA-3′ and 5′-GTG GCA GTG ATG GCA TGG AC-3′ primers. The first step of the thermocycle was an initial denaturation and enzyme activation at 95 °C for 15 min. It was followed by 20 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min. After final extension (72 °C for 10 min), the samples were separated on 1.5% agarose gel and visualized by ethidium bromide staining.Total RNA was purified from transfected HEK293T and HepG2 cells by using RNeasy Plus Mini Kit  following the manufacturer’s instruction. Possible DNA contamination was removed by DNase I treatment using RNAqueousTM SYBRTM Green Master Mix, 0.5 µM forward and reverse primers, using the QuantStudio 12K Flex Real-Time PCR System . SCD5A, SCD5B and SCD1 sequences were amplified by 5′-ATG GAA ACC GGC CCT ATG AC-3′/5′-CCC CAG CCA GCA CAT GAA AT-3′, 5′-GTG AGA TGC TTC GTG AAT GGC-3′/5′-CCT CCA GGG ACA CAG AAA GAG-3′ and 5′-CTG GCC TAT GAC CGG AAG AAA-3′, 5′-GAC CCC AAA CTC ATT CCA TAG G-3′ primer pairs, respectively. GAPDH, Actin and Tubulin cDNAs were also amplified as endogenous controls using 5′-GTC CAC TGG CGT CTT CAC CA-3′/5′-GTG GCA GTG ATG GCA TGG AC-3′, 5′-CTG GTG CCT GGG GCG-3′/5′-AGC CTC GCC TTT GCC GA-3′ and 5′-AAG TTC GCA CTG GCA CCT AC-3′/5′-AAC CAA GAA GCC CTG GAG AC-3′ primer pairs, respectively. For increased reliability, an RT negative control of each sample was also analyzed in addition to DNase I digestion. The first step of the thermocycle was an initial denaturation and enzyme activation at 95 °C for 2 min. It was followed by 40 cycles of 95 °C for 15 s, 55 °C for 15 s and 72 °C for 1 min, measurement of the fluorescent signal was carried out during annealing. Reactions were performed in triplicates, a reaction mixture with RNase-free water instead of template cDNA was employed as a non-template control. Relative expression levels were calculated as 2CT−Δ, where ΔCT values corresponded to the difference of the CT-values of the endogenous control and target genes.Quantitative qPCR assay was performed in 20 µL final volume containing 5 µL 20× diluted cDNA, 1× PowerUp® 5.2 software , and are shown as relative band densities normalized to actin as a reference or on a percentage scale. Data are presented in the diagrams as the mean values ± S.D. and were compared by ANOVA with the Tukey’s multiple comparison post hoc test, using the GraphPad Prism 6.0 software . Differences with a p < 0.05 value were considered to be statistically significant.Immunoblots were evaluated by densitometry using the Image StudioSCD5. We have demonstrated the expression of both SCD5A and SCD5B forms and the general dominance of the former in several human tissues, provided evidence for the markedly different binding probabilities of the alternative splice acceptor sites underlying the phenomenon, and revealed the impact of various human gene variants on SCD5 AS causing remarkable changes in the relative expression levels of the TVs, even in the dominance of SCD5B, in one case. We believe that our findings will facilitate further studies on tumor-related metabolic reprogramming of diagnostic, prognostic and therapeutic significance.Due to the limited expression of a few tissue types, it is possible that research on SCD5 has lagged significantly behind that of the more ubiquitous other human desaturase isoform. The existence of the two TVs of SCD5 has long been known, but remained largely disregarded in most studies. However, recent findings shed light on the importance of SCD5 expression and AS in tumor progression and metastasis, and their potential as prognostic factors and predictive biomarkers for tumor therapy. This is why we felt the need to compare the distribution of SCD5A and SCD5B in human tissues, and to elucidate the molecular background of the disproportionate AS of the primary transcript. This gap-filling research was also complemented by the investigation of certain SNVs potentially affecting the AS of


Venous stenting could alleviate exercise intolerance associated with chronic inferior vena cava (IVC) obstruction. We describe a 36-year-old male patient with an unknown IVC-obstruction. The obstruction was discovered after a bi-iliac deep vein thrombosis (DVT). The thrombus was resolved using thrombolysis. In the chronic phase, the patient developed exercise intolerance without any leg-specific symptoms or signs. Venous stenting was performed to open the IVC-obstruction, 1 year after the acute DVT. His physical condition improved, but cardiac magnetic resonance imaging at rest did not reveal hemodynamical changes after stenting. The Short Form Health Survey (SF-36) physical and mental component summaries were increased from 40.3 to 46.1 and 42.2 to 53.7, respectively. In patients with iliocaval obstruction, improved venous flow without changes in resting hemodynamics can enhance exercise intolerance and quality of life, even in the absence of leg symptoms. Diagnostic tools performed only at rest may miss abnormalities. It is often referred to as a congenital disorder, but may also result from thrombotic events in early childhood. Assessment of IVC-anomalies, including hypoplasia or aplasia is needed in DVT patients, especially those with iliofemoral DVT. Young patients with chronic IVC-obstruction may present asymptomatic, but are at risk for acute deep vein thrombosis (DVT), presenting at a mean age of twenty-five. Patients tend to develop post-thrombotic syndrome (PTS) later in life, which may cause pain, swelling, and/or ulceration of the leg. Exercise intolerance including dyspnea, exhaustion and turning pale during physical activities, is also observed.Partial or complete inferior vena cava (IVC) obstruction has a prevalence around 0.15%. but evidence is limited supporting its use for exercise intolerance. Endovenous recanalization, followed by venous stenting, could relieve exercise intolerance in IVC-obstruction. Cardiorespiratory functioning tests can be used to evaluate exercise intolerance in IVC-obstruction and its response to treatment. However, the impact of treatment on hemodynamics and quality of life (QoL) is unclear. Venous blood return is altered in IVC-obstruction, with the azygos vein, ascending lumbar veins, and abundant communicating veins becoming more prominent. Venous recanalization presumably resolves exercise intolerance by enhanced venous blood return.Deep venous stenting is an accepted treatment for symptomatic iliofemoral obstruction,This case-report presents a patient with exercise intolerance due to IVC-obstruction, which was accidentally discovered at presentation with an acute iliofemoral DVT. One year following acute DVT, the patient underwent venous stenting. Hemodynamics, clinical features, and quality of life (QoL) were evaluated pre- and post-procedure. Informed consent was obtained from the individual described in this case-report.A 36-year-old male patient was referred to our academic hospital for endovenous treatment of acute bi-iliac DVT with coincidental finding of IVC-obstruction. One week earlier, he was admitted for iliocaval DVT and treated conservatively with a heparin pump elsewhere, subsequently replaced with a direct-oral-anticoagulant. He experienced minimal back pain and bilateral inguinal pain at discharge. However, he was readmitted after 3 days due to worsening of complaints.Upon arrival at our institution, the patient had a fever, malaise, back pain, severe bilateral groin pain, and impaired mobility. Notably, leg symptoms and/or signs were absent. Duplex ultrasound (DUS) and Computed tomography venography (CTV) revealed an extensive DVT from the femoral veins above the knee until the IVC, including a thrombosed collateral vein . From thThrombolysis successfully restored patency of both iliac veins and the most distal segment of the IVC, but was stopped due to bleeding complications before the collateral opened . RecanalDuring the 6-month-control, the patient reported exercise intolerance. His complaints consisted of reduced physical condition, dyspnea, exhaustion and paleness during physical activities such as walking and cycling, without any leg-specific symptoms or signs. These complaints were absent prior to his acute DVT. Duplex ultrasound and CTV revealed a re-occlusion of the common iliac veins and distal IVC, with patent external iliac, femoral, and popliteal veins.Venous stenting was planned to improve cardiac preload and to resolve complaints. Cardiac magnetic resonance imaging (MRI) and the Short Form Health Survey (SF-36) questionnaire were used to evaluate the effect, both pre- and post-procedure.The first attempt of recanalization failed, but in a second attempt, the IVC was successfully stented. Both femoral veins and the right jugular vein were punctured for access. The IVC was recanalized after a snaring procedure creating a through and through wire. Intravascular ultrasound (IVUS) determined stent position and length after percutaneous transluminal angioplasty . The IVC was stented right below the hepatic veins with 3 SinusXL  overlapping stents (80 × 24 mm + 80 × 24 mm + 16 × 24 mm), in which two BeYond  stents (16 × 150 mm) were placed in a kissing fashion extending into the external iliac veins, with one additional stent (16 × 120 mm) on the right side. The SF-36 is divided in physical functioning, role limitations due to physical health , bodily pain, general health, vitality, social functioning, role limitation due to emotional problems , and mental health. Each domain scores between 0 and 100, with higher scores indicating a better QoL. Domains are summarized in a mental and physical component summary.All SF-36 domains remained stable or increased after venous stenting. Disabilities before the procedure were common in the role-physical, general health, and role-emotional domains with increased mean differences between 27.0 and 76.7 post-procedure. Physical and mental component summary also increased with 5.8 and 11.5 points, respectively.We presented a young male patient with exercise intolerance due to iliocaval obstruction. Endovenous recanalization followed by venous stenting led to complete resolution of his symptoms 1 year after the initial DVT. His SF-36 scores increased and were comparable to those of an age-matched Dutch cohort. However, there were no changes in heart volumes at rest before and after venous stenting. It is not regularly evaluated in venous outflow obstruction. Currently, exercise intolerance also is not included in any PTS score. Inadequate return of venous blood to the heart may cause exercise intolerance. Reduced venous return from the lower limbs during exercise reduces stroke volume and cardiac output in iliocaval outflow obstruction. Venous stenting presumably resolves exercise intolerance, by enhancing venous return and increasing cardiac output.Exercise intolerance is often underestimated as a consequence of chronic (ilio)caval obstruction. Despite unchanged pre- and post-venous stenting heart volumes, that were within the normal range, our patient experienced an improvement, as evidenced by SF-36 scores. Cardiac volumes were also normal at rest in patients with exercise intolerance after IVC-ligation, while lower volumes were measured during exercise. Therefore, diagnostic tools may miss abnormalities if only performed at rest.Evaluating cardiac hemodynamics at rest may fail to detect venous abnormalities, as resting venous capacitance may not reveal the increased demands of venous volume during exercise.Another lesson learned is that in acute DVT resulting from chronic IVC-obstruction, the IVC may be left untreated, with only fresh clot removal followed by lifelong treatment with anticoagulants. However, if collateral vessels remain occluded, persisting symptoms and signs may necessitate invasive treatment during follow-up.This case increases awareness for symptoms and signs related to physical exercise in venous iliocaval obstruction. Future studies are needed to reveal how venous stenting affects hemodynamics during exercise in IVC-obstruction. Understanding the impact of venous stents on cardiac hemodynamics and exercise-related complaints, may improve treatment selection for IVC-obstruction.


Dysregulated fear reactions can result from maladaptive processing of trauma-related memories. In post-traumatic stress disorder (PTSD) and other psychiatric disorders, dysfunctional extinction learning prevents discretization of trauma-related memory engrams and generalizes fear responses. Although PTSD may be viewed as a memory-based disorder, no approved treatments target pathological fear memory processing. Hippocampal sharp wave-ripples (SWRs) and concurrent neocortical oscillations are scaffolds to consolidate contextual memory, but their role during fear processing remains poorly understood. Here, we show that closed-loop, SWR triggered neuromodulation of the medial forebrain bundle (MFB) can enhance fear extinction consolidation in male rats. The modified fear memories became resistant to induced recall  and did not reemerge spontaneously. These effects were mediated by D2 receptor signaling-induced synaptic remodeling in the basolateral amygdala. Our results demonstrate that SWR-triggered closed-loop stimulation of the MFB reward system enhances extinction of fearful memories and reducing fear expression across different contexts and preventing excessive and persistent fear responses. These findings highlight the potential of neuromodulation to augment extinction learning and provide a new avenue to develop treatments for anxiety disorders. Whether fear memories can be attenuated through on demand electrical stimulation remains unclear. Here, the authors demonstrate that fear extinction can be augmented through closed-loop stimulation of the reward system, guided by hippocampal SWRs. Extinction learning, the process of reducing the expression of learned fear responses, is essential for adaptive behavior in response to traumatic experiences.Learning unpleasant things and remembering them is advantageous for the organism for avoiding future reoccurrences. Memories that are irrelevant to survival or adaptation tend to fade away either by graceful degradation5. For example, post-traumatic stress disorder (PTSD) is a debilitating psychiatric disorder resulting from direct or indirect exposure to stressful events, threats, or life-threatening events perceived to compromise personal physical or mental safety8. Symptoms include intense feelings of unprovoked fear, panic attacks, anxiety; intrusive fear memories during wakefulness or in nightmares, fear generalization, and avoiding similar but neutral stimuli10. PTSD is highly resistant to psycho- and pharmacotherapy13.However, in some pathological scenarios, extinction learning is often impaired, leading to persistent and maladaptive fear responses14, and that closed-loop stimulation of the reward system can enhance memory consolidation15. Exposure-based extinction procedures have been found to reduce fear in a context-dependent manner, suggesting that the hippocampal representation of the extinction context drives fear attenuation16. The activity in the basolateral amygdala decreases when conditioning stimuli (CS+) are presented in the same context used for extinction but increases following non-extinction exposure to the CS+17. Furthermore, inactivation of the hippocampus has been found to enhance extinction to the CS+ and promote low fear expression in environments different from the extinction context19.Previous studies have demonstrated that hippocampal sharp-wave ripples (SWRs) play a critical role in the consolidation of fear memories20. Additionally, these neurons participate in a mutual inhibition process21. Based on these findings, we hypothesize that manipulating internal reward signals during extinction learning could facilitate the extinction of memories, thereby reducing excessive fear reactions in inappropriate contexts.Excitatory neurons in the basolateral amygdala have been shown to respond to both reward and punishment and have been proposed to be involved in mediating reward signaling induced by the omission of an unconditioned stimulus during extinctionHere, we explore whether SWR-triggered stimulation of the reward system through medial-forebrain bundle (MFB) can augment extinction learning. Our findings suggest that SWRs are crucial for mediating fear extinction, and that closed-loop neuromodulation targeting oscillatory activity related to memory processing could be a promising intervention for reducing excessive fear reactions in inappropriate contexts. Specifically, our experiments demonstrate that selective suppression of SWRs after extinction delayed fear attenuation, indicating that intact SWRs are necessary for extinction learning. Furthermore, our results show that SWR-triggered closed-loop stimulation of the reward system through MFB enhances the extinction of fearful memories, resulting in reduced fear expression across different contexts and preventing excessive and persistent fear responses. Overall, our study suggests that rewarding brain stimulation may be a promising approach to augment extinction learning, potentially beneficial to alleviate PTSD symptoms.Rats were subjected to a single session of fear conditioning  to develop PTSD-like phenotypes  to reveal the overall effect of the interventions , animals received SWR-triggered closed-loop stimulation during sleep for three consecutive days but were not exposed to the extinction paradigm Fig. .Fig. 2CoTo match the mean number of extinction sessions required for closed-loop animals to achieve the remission criterion Fig. , the numWe next tested if the SWR-triggered closed-loop stimulation interferes with already consolidated non-fear-related memories as a non-specific detrimental effect. For this purpose, the animals were trained in a spatial memory task, in which a randomly alternated visual cue indicated the correct choice in a T-maze to receive a reward (froot-loops pellet). They underwent a total of 20 trials per day until achieving 80% of correct choice. After completing the spatial memory task, the animals underwent fear conditioning, extinction, and stimulation sessions in the same way as in the previous experiment until achieving remission Fig. . During 16, required SWRs as they play a crucial role in contextual memory consolidation through cortico-hippocampal circuits23. To test this, we suppressed SWRs by ventral hippocampal commissural electrical stimulation that induces phasic silencing of hippocampal pyramidal cells and interneurons26. Since animals trained with high-intensity foot-shocks tend to resist extinction, we reduced the training intensity (5 pairings CS+US at 0.7 mA) to ensure that the extinction criterion was achieved within seven sessions in control conditions. During stimulation following each extinction, online detected SWRs triggered a single-pulse (0.5 ms) ventral hippocampal commissural stimulation ), animals received bilateral microinfusions of the Rac1 inhibitor NSC2376, D1R antagonist SCH23390, or D2R antagonist sulpiride immediately after each extinction session and before the closed-loop stimulation  introduces preset electrical stimulation in an open-loop manner, without being aligned to the internal oscillatory activity. Although DBS has been used to control fear expression in animal models36. SWRs rely on synchronous CA1 principal neuron activation mainly controlled by PV+ interneurons37. Boosting the activity of hippocampal PV+ interneurons results in selective extinction of contextual fear memory and increased SWR incidence38. However, suppression of hippocampal PV+ interneurons alters principal neuronal phase coupling to SWRs, decreasing ripple-spindle coupling and consolidation of contextual fear memory40. Our findings indicate that SWRs are necessary for the extinction of cued fear conditioning and can update the memory trace with rewarding information. Closed-loop disruption of SWRs delayed but did not block extinction since 80% of animals still achieved the remission criterion, consistent with the contextual dependence of fear extinction19, although cued fear conditioning is amygdala- dependent43. Our initial hypothesis that SWRs encode contextual features of ‘safety’ during the extinction is supported by the decreased time to achieve remission but does not explain the fear reduction to CS+.SWRs encode and consolidate spatial memory and are involved in fear memory processing. Selective pre or post-training inactivation of CA3 disrupts the acquisition and consolidation of contextual fear memory by reducing the number and dominant frequency of CA1 ripples and shifting underlying CA1 ensemble activity44 indicating that during SWRs, replay, and information integration involve the contextual features of an engram and the corresponding emotional memory traces. The multiple roles of SWRs and hippocampal place cells in processing contingencies beyond spatial localization support this idea46. Thus, the SWR-triggered closed-loop MFB stimulation and the resulting reward signal coincide with the widespread ongoing brain network activity orchestrating the consolidation of fear extinction47 during SWR events. Neuronal activity in the BLA increases during SWRs49 and coordinated reactivation between the dorsal hippocampus and BLA during offline aversive memory processing peaks around the SWRs50. Therefore, the SWR-triggered closed-loop neuromodulation may provide a reward safety signal to a consolidating aversive memory51 and/or enhance the network activity that encodes fear extinction52. Since SWRs are also important in encoding context, it cannot be excluded that the enhancement shown in this study might also influence spatial or contextual learning. While we demonstrated that the closed-loop SWR-triggered MFB stimulation does not interfere with already consolidated spatial memories, revealing any effects on their acquisition or extinction may require further studies.SWRs play a critical role in establishing temporally precise ‘windows’ for integrating information across neocortical and subcortical structures. A widespread increase in neocortical activity precedes SWRs56 characterized by high temporal and neurochemical precision. This hypothesis is supported by the absence of closed-loop effect when animals are not exposed to the extinction learning. In such cases, the reward signal triggered by MFB stimulation does not coincide with extinction-contingent SWRs, which prevent the enhancement of fear attenuation.The potential mechanism underlying the closed-loop neuromodulation of SWRs and reward signaling resembles a counterconditioning process by memory updating with contrasting emotional valence57. This increase in activity has a positive correlation with extinction learning. In addition, optogenetic excitation of VTA dopaminergic neurons at the time of the US omission accelerates fear extinction58. These results suggest that dopaminergic activity during extinction encodes prediction error or mismatch between expectancies59. This system is more active during the initial phase (unexpected omission) compared to late phase (expected omission) of extinction learning58. Together our results from MFB closed-loop neuromodulation, combined with the assumption that US omission may be rewarding itself60, suggest that dopaminergic signaling plays a crucial role in the consolidation of extinction during offline states, particularly during SWRs.Interestingly, when the US is unexpectedly omitted during extinction, there is an increase in the activity of dopaminergic neurons in the VTA61. A cluster of dopaminergic neurons in the anterior VTA/SNc directly connect with CA162. A global manipulation of the reward system through MFB deep brain stimulation can ameliorate depression-like behaviors in animal models and depression symptoms in human patients63. We found that temporally precise electrical stimulation in these circuits during SWRs may scaffold the extinction enhancement. We argue for a dopamine-dependent mechanism, since previous studies have shown that MFB stimulation leads to an increase in dopamine release in BLA66 and the effects of closed-loop neuromodulation were prevented by a selective local antagonism of D2 but not D1 receptors. Moreover, our stimulation protocol was able to induce conditioned place preference.The idea that dopaminergic signaling is essential for extinction consolidation is supported by the fact that MFB fibers, which connect nodes involved in reward and emotional processing, play a critical role in this process. The VTA sends dopaminergic axons to the NAc, amygdala and PFC via the MFB67 and fear extinction can revert the enhanced activity of these neurons and decrease AMPAR expression induced by fear conditioning68. Dopamine enhances the excitability of BLA projection neurons, and D1 and D2 receptor activation increase excitability and input resistance, respectively69.Multiple lines of evidence supports that fear conditioning induces long-term potentiation of amygdala principal neurons70. Fear memories and extinction are encoded by different BLA neuronal populations. Rather than overwriting the original fear learning engrams, extinction engrams can suppress the activity of neurons that were initially engaged in fear learning. Furthermore, since neurons that mediate extinction learning also overlap with those involved in reward processing, the activation of these neurons could also signal reward20.Indeed, dopamine release in the BLA during fear learning is controlling the saliency of the footshock and the extinction through prediction error signaling of non-reinforced CS+ presentation71, supporting the idea that dopamine release is modulated by SWRs. Dopaminergic projections from VTA innervate PV+ interneurons expressing D2 receptors, contributing to the suppression of BLA principal neurons72. The suppression of feed-forward inhibition can induce LTP at excitatory afferent synapses in the BLA, an effect also mediated by D2 receptors73. Although the initial fear generalization phenotype was not evaluated after our closed-loop intervention, there is evidence that cue fear generalization is promoted by high-intensity training74 and is a limiting factor for extinction75. Generalization may be mediated by the temporal proximity between CS+ and CS-, linking memory traces by neuronal co-allocation to overlapping engrams76. Given this scenario, it is expected that closed-loop MFB stimulation would impact not only the CS+ trace but also the overlapping CS-trace. This hypothesis should be addressed in future studies.Our experimental design cannot differentiate whether post-extinction SWRs are related to the reactivation of the original fear memory or represent the consolidation of the extinction. However, increased dopamine release during SWRs could change the emotional valence of an engram replay or directly suppress neurons engaged in fear learning. Reward-responsive VTA neuronal activity is coupled to SWRs during quiet wakefulness2. Inhibition of Rac1 activity in the dHPC impairs extinction of contextual fear memories77 and photoactivation of Rac1 in the motor cortex suppresses motor learning29.Dopamine stimulation of engram cells may enhance forgetting by activating Rac1/Cofilin, which modulates actin cytoskeleton and cellular morphology77 and RAC1 activation is required for plasticity-related mechanism during fear extinction78. Since the disruption in fear attenuation was more pronounced under closed-loop stimulation, the synaptic plasticity may differ between normal and enhanced extinction. Additional work is required to determine the mechanisms of interaction between dopamine receptors and Rac1 modulation during fear extinction.Our findings suggest three sequential mechanisms underpinning closed-loop extinction enhancement: (1) SWRs reactivate the memory trace in BLA. (2) Closed-loop MFB stimulation promotes concurrent dopamine release in BLA. (3) BLA dopamine release can induce D2 receptor-mediated plasticity processes culminating in Rac1 activation. Blocking Rac1 signaling prevents spontaneous or closed-loop neuromodulation-induced fear reduction during renewal. However, Rac1 inhibition without closed-loop neuromodulation did not extend the number of sessions required for successful fear extinction using the remission criterion. This partial disruption without electrical stimulation is expected since previous studies have shown that RAC1 inhibition impairs the extinction of contextual fear memories79. It should be noted that in addition to the MFB, other components of the reward system, as the nucleus accumbens (NAc), may also be viable targets for closed-loop neuromodulation. This is supported by evidence demonstrating that NAc-DBS can elicit striatal dopamine release in humans80. Although in our experiments the detection of SWRs was invasive, alternatively, cortical slow-waves and spindles concurring with SWRs in animals82 may be detected non-invasively to align stimulation. Thus, closed-loop stimulation triggered by cortical EEG activity could replace SWRs detection. Further, non-invasive techniques  could stimulate reward-associated cortical areas instead of penetrating electrodes. Importantly, our experiments were performed in male animals only. Considering sex differences in the renewal and the context-dependence of extinction in rodents83 as well as the higher risk of women to develop anxiety-related disorder compared to men85, future experiments are needed to assess if the closed-loop neuromodulation approach can be extended to females.Our results suggest a novel translational treatment of fear-related disorders. The US Food and Drug Administration (FDA) approved MFB stimulation for treatment-resistant depression in clinical trials, with promising efficacy87, our intervention avoids the side effects of systemic treatments . Coupling between SWRs, cortical slow-waves, and spindles may offer a potential way to translate our approach towards a non-invasive therapy in the future.Our framework to study and attenuate fear-related memories relies on closed-loop stimulation guided by classical biomarkers of memory consolidation. Closed-loop stimulation can reduce the side effects from chronic and excessive stimulation of DBS approaches. Temporally precise manipulation of the reward system during SWRs overcomes the resistance to extinction in an animal model with key features of PTSD. Moreover, SWRs are critical for extinction learning. Although dopaminergic agonists can enhance fear extinctionRats  were kept in a 12-hour light/ dark cycle. All experiments were performed in accordance with the European Union guidelines (2003/65/CE) and the National Institutes of Health Guidelines for the Care and Use of Animals for Experimental Procedures. The experimental protocols were approved by the Ethical Committee for Animal Research at the Albert Szent-Györgyi Medical and Pharmaceutical Center of the University of Szeged (XIV/218/2016 and XIV/824/2021).88 targeting the anterior cingulate cortex (ACC) , bilateral BLA  and the bilateral CA1 subfield of the dorsal hippocampus . To improve DH- SWRs detection, a custom-built microdrive89 was used in some experiments, allowing the vertical adjustment over the CA1 subfield. A custom-built bipolar electrode consisting of two insulated (except 200 µm at the tip) Tungsten wires  was implanted in the left medial-forebrain bundle . LFP electrodes and the base of the microdrive were secured to the skull with dental acrylic . Two stainless-steel screws above the cerebellum served as ground and reference for the recordings, respectively. A Faraday cage was built using copper mesh and dental acrylic on the skull around the implanted electrodes.The animals were anesthetized with 2% isoflurane and craniotomies were performed according to stereotaxic coordinates. Intracortical electrode triplets In experiments involving concomitant electrophysiological recording and local pharmacological infusion, in addition to electrodes, rats were bilaterally implanted with 25-gauge guide cannulas above the BLA . Cannulae were fixed to the skull with dental acrylic (Unifast Trad). Caps were used to cover cannulae to avoid any accidental occlusion.ad libitum. All recording sessions took place in the same room using 12/12 h light/dark cycle with light onset/offset at 7 h/19 h The multiplexed signals were acquired at 500 Hz per channel for closed-loop neuromodulation experiments88. The neuronal signals were preamplified , multiplexed on head, and stored after digitalization at 20 kHz sampling rate per channel . During home cage stimulation, preamplified signals were analyzed online by a programmable digital signal processor  using a custom-made sharp-wave ripple detection algorithm, as follows.Rats were housed individually in Plexiglass home cages . LFP recordings were performed in the home cage and the fear conditioning box (see below). Recording and stimulation sessions for closed-loop or open-loop interventions were performed during the first hour following the extinction protocol. To avoid any twisting and over-tension of the recording cables, a bore-through electrical commutator  was used. Food and water were available Two LFP signals were used for real-time SWRs detection. For ripple detection, a channel from the tripolar electrodes from CA1 pyramidal layer with the largest ripple amplitude was selected and band-pass filtered (150–250 Hz), and root-mean-square (RMS) power was calculated in real-time for ripple detection. For noise detection, manual inspection from channels of the ACC, VHC, or AMY was performed to select the signal with no ripple-like activity and lower noise incidence to enhance signal-to-noise ratio during detection. In case of the ACC, signal was filtered between 80 and 500 Hz. SWRs were defined as events crossing the ripple thresholds in the absence of the noise signal. Amplitude threshold for ripple was adjusted for each animal before fear conditioning training. SWRs were defined as events crossing ripple thresholds in the absence of the noise signal in the neocortical site. Threshold crossings triggered a stimulation train lasting 100 ms and composed of fourteen 1-ms long, 100 µA square-wave pulses at 140 Hz) in the MFB or single pulse   depending on the experiment performed. MFB stimulation was performed under current mode and VHC stimulation in voltage-controlled mode. The threshold of the detection algorithm was set for each rat separately. Behavioral  effect of MFB stimulation was confirmed with a place preference task (see below).https://github.com/buzsakilab/buzcode) was employed combined with post manually corrections. Time-frequency spectrum was calculated in MATLAB using Multitaper Spectral Estimation from the Chronux Toolbox (http://chronux.org/). A 2 s sliding window with a 50% overlap, a time-bandwidth product of 5 and tapers of 3 were chosen.The offline ripples were analyzed using custom-made MATLAB  routines. Raw signals were down sampled from 20 kHz to 500 Hz and bandpass filtered in the ripple band (150–250 Hz) of hippocampal channels. Normalized squared signal was calculated. Putative SWRs events were defined as those where the beginning/end cutoffs exceeded 2 SDs and the peak power 3 SDs. The detection window was set in 150 ms. SWR duration limits were set to be between 20 and 200 ms, otherwise the events were excluded to minimize artifacts. All ripple events were drawn out for manually speculations after offline detection. The closest stimulation onset from the digital channel was selected for further analysis. Then calculated the time delay between the successfully detected ripples events and the stimulation time. For the brain states classifications (SWS/REM), SleepScoreMaster toolbox from Buzcode (The Rac1 inhibitor NSC2376 (10 µg/µl), D1 dopamine receptor antagonist SCH23390 (0.50 µg/µl), and D2 dopamine receptor antagonist sulpiride (1 µg/µl) were dissolved in sterile physiological saline (0.9% NaCl). NSC2376, SCH23390, and sulpiride were infused bilaterally into the BLA using a 33 G gauge injectors connected to Hamilton syringes via 20-gauge plastic tubes. The infusion injectors tip protruding 2.0 mm below the tip of the cannula and aimed the BLA center. A total volume of 0.5 μl per side was infused by a microinfusion pump at a rate of 0.125 μl/min. Injectors were left in place for an additional minute to ensure proper drug diffusion. All drugs were infused after the extinction sessions.The experiments were carried out in a fear conditioning apparatus comprising three contextual Plexiglas boxes  placed within a soundproof chamber. Four different contextual configurations were used (Habituation and Test Context (A): square configuration, white walls with black vertical horizontal lines, white smooth floor, washed with 70% ethanol; Training Context (B): square configuration, gray walls, metal grid on black floor, washed 30% ethanol; Extinction Context (C): rectangular configuration, white walls with black dots, white smooth floor; and Renewal and Remote/Reinstatement context (D): hybrid context comprising a square configuration, gray walls from training context, white smooth floor, washed with 70% ethanol. All sessions were controlled using a MATLAB custom script.On day 1, animals were exposed to the habituation session in context A. After 2 min of contextual habituation, they were exposed to 5 alternating presentations of two different tones . Tone time intervals were randomized (30–40 s) during the session. No behavioral differences were detected under exposition to the two frequencies.On day 2, cue fear conditioning was performed in context B. After 2 min of contextual habituation, animals received 5 trials of one tone (CS+: 7.5 kHz) immediately followed by a 2 s long footshock as unconditioned stimulus . The other tone (CS−: 2.5 kHz) was presented 5 times intermittently but never followed by the US.On day 3, animals underwent fear retrieval in context A. After 2 min of contextual habituation, rats were exposed to presentations of the CS+ or CS- in two different sessions. Each session consisted of a block of five tones. The order of the CS+ and the CS− in each session was randomized. Sessions were repeated every 4–6 h.In context C, from day 5 until reaching the remission criterion (see below), rats received extinction training consisting of twenty CS+ presentations without the US (unreinforced tones). Tones were repeated with randomized intervals (30–40 s) during the session.90. The block of the first five tones during each extinction session was assessed to determine fear reduction level of the given day. Considering individual differences under fear conditioning92 fear reduction during extinction was expressed as a fraction of the percentage of freezing expressed during the CS+ test (Day 3) (% Freezing Reduction = Freezing extinction × 100/Freezing test CS+). Fear remission was considered achieved when animals expressed <20% of the initial freezing during the first block of the day . Extinction training was repeated for maximum 7 days.We used an extinction threshold criterion to assess the efficacy of fear reduction after extinction sessions similar toTwenty-four hours or 25 days after achieving the remission, animals were exposed to context D (Hybrid context) as a renewal or remote test, respectively. In each test, rats were exposed to a block of five CS+ presentations after 2 min of contextual habituation. Time intervals between tones were randomized (30–40 s) during the session.To promote fear recovery, animals were placed in a neutral environment outside the conditioning box and received an unconditioned footshock after 30 s contextual exposition, with the same intensity used during fear conditioning. The animals were returned to their home cage 30 s following the footshock.Animals were submitted to a reinstatement test in context D 24 hours after the immediate footshock. Rats were exposed to a block of 5 CS+ presentations after 2 min of contextual habituation. Time intervals between tones were randomized during the session.Freezing behavior was used as a memory index in the fear conditioning task. Freezing was analyzed offline using Solomon software , for behavioral coding by an experienced observer that was blinded to the experimental group. Freezing was defined as the absence of all movements, except those related to breathing, while the animal was alert and awake.The conditioning box consisted of three chambers, two for the conditioning session having the same dimensions (24 × 40 × 50 cm), and the other serving as a central/start chamber (10 × 40 × 50 cm). Each chamber was employed with contextual cues and floor texture to distinguish them.Conditioned place preference test consisted of three phases: pre-conditioning (day 1), conditioning (days 2–6), and test (day 7). The pre-conditioning session (15-min) was intended to reduce novelty and determine initial preferences for any of the two chambers by assessing the time spent in each compartment. Conditioning always took place in the initially less preferred chamber. Conditioning sessions were performed during the following five days. Animals underwent two conditioning sessions each day with 6–8 h intervals between sessions. In one session, animals were placed in the initially less preferred compartment and received MFB stimulation . During the other session, the animals were placed in the opposite compartment without stimulation. The order of the sessions was randomized between animals and days. A 15 min place preference test was conducted in the absence of stimulation 24 h after the last conditioning day. The video of the animal behavior was recorded and analyzed offline using the ANY-Maze  video tracking software.Animals on food restriction (no less than 85% of their baseline weight) were habituated to the T-maze during 5 days before the training. The T-maze was constructed from black acrylic, with 80 cm long and 30 cm wide alleys and 40 cm high walls. Two removable doors closed the side alleys. During training, a light cue indicated the correct arm to receive a reward (froot-loops pellet). A total of 20 trials per day were performed until achieving 80% of correct choice. A removable door in the central arm was used to confine the animal at the starting point during cue presentation. After 3 min, the alley was removed, and the animal allowed to run in the maze. After arm selection, the alley was closed and the animal remains additional 3 min in the maze before next trial. Afterwards, fear conditioning, extinction, and stimulation sessions started. Animals were tested in the T-maze after the extinction sessions to verify any disruption of the consolidated spatial memory. Extinction and stimulation sessions and T-maze tests were separated by five hours and the order of the behavioral tasks were randomized each day.Following the termination of the experiments, animals were deeply anesthetized with 1.5 g/kg urethane (i.p.), and the recording sites of each electrode were lesioned with 100 µA anodal direct current for 10 s (Supplemental Fig. p < 0.05. Data were analyzed using two-tailed Mann–Whitney U test, Kruskal–Wallis test, or Mixed ANOVA followed by Dunn’s post hoc or Bonferroni’s multiple comparisons test. Data are expressed and visualized as median ± IQR, individual data points are also shown where applicable. Detailed statistics are shown in Supplementary Data Statistical analyses were performed using GraphPad Prism 8 software. Significance was set at Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary