It is also a key mediator of cell homeostasis and survival. To ensure the fine regulation of skeletal muscle processes, desmin is regulated by post-translational modifications (PTMs). It is more precisely phosphorylated by several kinases connecting desmin to intracellular processes. Desmin is also modified by O-GlcNAcylation, an atypical glycosylation. However, the functional consequence of O-GlcNAcylation on desmin is still unknown, nor its impact on desmin phosphorylation. In a model of C2C12 myotubes, we modulated the global O-GlcNAcylation level, and we determined whether the expression, the PTMs and the partition of desmin toward insoluble material or cytoskeleton were impacted or not. We have demonstrated in the herein paper that O-GlcNAcylation variations led to changes in desmin behaviour. In particular, our data clearly showed that O-GlcNAcylation increase led to a decrease of phosphorylation level on desmin that seems to involve CamKII correlated to a decrease of its partition toward cytoskeleton. Our data showed that phosphorylation/O-GlcNAcylation interplay is highly complex on desmin, supporting that a PTMs signature could occur on desmin to finely regulate its partition (i.e The major function of IFs is the maintenance of cell shape and architecture, conferring resistance of cells towards mechanical stresses. They also contribute to the organization of cells because of their interaction with organelles. Indeed, IFs interact with mitochondria and modulate energy fluxes3. Moreover, they contribute to the organization of cells into tissues as IFs interact with adhesive structures such as focal adhesions or desmosomes1. Initially considered as static structures, it is nowadays well admitted that IFs are on the contrary highly dynamic and able to respond rapidly to cellular demands. Strong regulators of IFs dynamics are post-translational modifications (PTMs), in particular phosphorylation and O-N-acetyl-β-d-glucosaminylation (O-GlcNAcylation) that modulate the IFs dynamics and interactions in response to signalling pathways7. Beyond their mechanical role, IFs are a nodal point within cells because of their interaction with a growing number of proteins, in particular proteins of signalling pathways. As a consequence, IFs are both targets and active contributors of intracellular signalling. As modifiers and organizers of signalling, IFs contribute to a dynamic cell behaviour  and in larger extent to tissue homeostasis 11.Intermediate filaments (IFs) are a major and elaborated cytoskeletal network that is at least ten-fold more abundant than microfilaments or microtubules12. Desmin interactome is vast14. This protein forms a continuous transcytoplasmic network around Z-discs through specific interactions with a plethora of structural proteins, permitting the maintenance of lateral alignment of myofibrils, the cytoskeletal lattice of muscle and the anchoring of mitochondria and nuclei16. Over the preservation of muscle integrity and the involvement in contractile activity, desmin is also a key mediator of cellular homeostasis and survival because of its implication in several cellular processes such as differentiation, apoptosis, intracellular signalisation, mechanotransduction, vesicle trafficking, organelle biogenesis and/or positioning, calcium homeostasis, protein homeostasis and the regulation of metabolism and gene expression17. Desmin bears several PTMs, in particular phosphorylation by several kinases that connect desmin to different cellular processes such as cell division, myoblast differentiation or muscle contraction18. Hence, desmin is strongly associated to the aetiology of several muscle pathologies such as dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, atypical cardiomyopathy, heart failure, sleep apnea, asthma, respiratory insufficiency, dysphagia, myofibrillar myopathies or muscle wasting21. Desmin-related myopathy and cardiomyopathy (i.e. desminopathies) could result from mutation in the desmin gene, cleavage of desmin, and/or changes on post-translational modifications, leading to an altered desmin network and the formation of toxic desmin aggregates25. As a consequence, desmin function is lost, leading to improper mitochondrial function, loss of cell adhesion and cell–cell communication, fibrosis and inflammation14. Importantly, these toxic aggregates are seeding-competent amyloid aggregates that persist in muscle fibers27, and these preamyloid and amyloid oligomers formation and deposition result from detrimental phosphorylation in heart failure29.Among the constitutive proteins of IFs, desmin is the major protein of type III IFs in striated muscles as it represents 2% and 0.35% of total proteins in heart and skeletal muscles, respectively18. However, desmin is not only phosphorylated but also modified by an atypical glycosylation: the O-N-acetyl-β-d-glucosaminylation (O-GlcNAcylation)30. The O-GlcNAcylation is an atypical glycosylation corresponding to the transfer of a unique monosaccharide, the N-acetyl-d-glucosamine, on serine or threonine hydroxyl group of a protein; O-GlcNAcylation is highly dynamic and reversible, presenting a dynamic interplay with phosphorylation31. The O-GlcNAcylation is highly abundant on cytoskeletal proteins; in particular, keratin 8 and 18 which are involved in the development and differentiation of various tissues are O-GlcNAcylated. Interestingly, while phosphorylation regulated protein interaction, ubiquitination and filament organization of keratins, O-GlcNAcylation was demonstrated to modulate the phosphorylation level of IFs33 and increase keratins solubility34, ubiquitination and proteasomal degradation of IFs proteins35. Moreover, O-GlcNAcylation was linked to the modulation of IFs, in particular vimentin6 and keratin 8/18 filaments36, with a link between O-GlcNAcylation and cell survival in stress condition34. In skeletal muscle, O-GlcNAcylation extensively modifies other nucleocytoplasmic and mitochondrial proteins and also myofibrillar proteins37. We have previously demonstrated that motor and regulatory proteins are O-GlcNAcylated modulating so the contractile properties of skeletal muscle such as calcium activation parameters41. Supporting a key role of this atypical glycosylation, the alteration in the number or the linkage of O-GlcNAc moieties is closely associated to the physiopathology of several diseases and in particular neuromuscular pathologies 42. Interestingly, some of the O-GlcNAc sites correspond to mutated sites closely associated to the development of muscle pathologies such as Laing myopathy43 or desminopathies44. The O-GlcNAc modification was also demonstrated to be involved in skeletal muscle atrophy46. Moreover, recent data from our lab underlined the crucial role of O-GlcNAcylation in the modulation of contractile activity, in the structuration of sarcomeric cytoskeleton and in the modulation of protein–protein interactions . Indeed, we have recently demonstrated that the two enzymes responsible of the O-GlcNAcylation process are located in the nodal Z-line39, and that several myofibrillar proteins are O-GlcNAc-modified, such as αB-crystallin, α-actinin or desmin among others38. We have also demonstrated in a cellular model of C2C12 skeletal muscle cells differentiated into myotubes that O-GlcNAcylation could be involved in the organization and reorganization of sarcomere. Importantly, we showed that sarcomere reorganization was linked to a modulation of several multiprotein complexes and some of them include key structural proteins, in particular desmin and its molecular chaperone, the αB-crystallin47. To support this role of O-GlcNAcylation in the modulation of desmin interactome , O-GlcNAc sites modifying both proteins are located into interaction domains44, and our data also suggest that their interaction could be impacted by O-GlcNAcylation changes47.As mentioned previously, desmin is highly post-translationally modified, especially through phosphorylationHowever, the precise impact of O-GlcNAcylation on desmin, the guardian of striated muscle integrity, remains poorly understood. The major objective of our study was to reveal the role of O-GlcNAcylation on desmin features. For this purpose, we increased or decreased the global O-GlcNAcylation level on C2C12 myotubes using pharmacological molecules inhibiting OGA or OGT (the two enzymes involved in O-GlcNAcylation and de-O-GlcNAcylation process) respectively, and investigated whether the global O-GlcNAcylation level should impact the desmin behaviour or not. We focused particularly on its expression, its partition toward cytoskeleton or insoluble fraction, and the variations of phosphorylation and O-GlcNAcylation on desmin.2 at a density of 2 × 105 cells/ml in PM. When reaching 80–90% confluence, PM was changed toward differentiation medium . This medium change corresponds to day 0 of differentiation. Cells were cultured into an incubator at 37 °C, 5% CO2 and saturated humidity, and culture media were changed every 2 days.The C2C12 mouse myoblasts  were cultured in proliferation medium . Cells were put in plate of 8.8 cm4-5S-GlcNAc 48 or 0.5 µM of Thiamet-G (thiazoline amino-ethyl gluco-configured)49 resolubilized in DMSO (dimethyl sulfoxide) and diluted in DM, to inhibit OGT and OGA respectively. Control myotubes were cultured in DM with DMSO as vehicle but without OGT or OGA inhibitors. To increase or decrease CamKII activity, we applied overnight 3 mM of caffeine50 (Thermofisher) or a co-treatment with 3 mM of caffeine added to 20 µM of KN-9351 resolubilized in water.After 5 days of differentiation, C2C12 myotubes were treated by pharmacological molecules applied overnight. We used 5 µM of AcO-(2-acetamido-2-deoxy-d-glucopyrano-silidene)-amino-N-phenyl-carbamate; Sigma), an OGA inhibitor. A recapitulative scheme of all extraction protocols is presented in Fig. Cells were rinsed three times with ice-cold PBS . Proteins were extracted by cells scraping and lysis with 150 μl of extraction buffer adapted to downstream analyses according to specific protocols defined hereinafter. All extraction buffers contained anti-proteases [Complete EDTA-Free (Roche Diagnostics)], anti-phosphatases [Phos-Stop (Roche Diagnotics)] and 50 μM of PUGNAc  was used to get whole extract. Protein content was estimated by Bradford assay.For kinase assays, cells were lysed in lysis buffer  according to manufacturer’s protocol, and after centrifugation at 13,000 rpm for 10 min at 4 °C, supernatant protein content was determined with BCA assay. For phosphatase assay, RIPA buffer without SDS and phosphatases inhibitors was used to get whole extract. Protein content was estimated by Bradford assay.52. The solution was discarded. After rinsing with 300 μl Tris/HCl buffer (50 mM Tris HCl pH 7.5), 100 μl of nuclease Buffer  supplemented with 10 U/ml of benzonase nuclease were applied for 10 min; the solution was discarded too. Cytoskeletal proteins that remained fixed on the plate were rinsed by Tris/HCl buffer and then solubilized by scrapping with 30 μl of Tris/HCl buffer supplemented by 1% SDS52. Protein content of SDS-resuspended extracts, containing cytoskeletal proteins was estimated by Lowry assay.Cells were lysed with Cell Lysis Buffer  1.5 min on ice53. Supernatant corresponds to soluble fraction; pellet, corresponding to insoluble proteins fraction, was resolubilized by 100 μl of Detergent Insoluble Buffer 53. Protein content of DSB extracts was estimated by Bradford assay while those corresponding to DIB extract was estimated by Lowry assay.Cells were lysed by Detergent Soluble Buffer  after 30 min on ice. Protein extract was centrifuged at 13,000 rpm for 10 min at 4 °C2; 1 mM MnCl2, added with anti-proteases [Complete EDTA-Free (Roche Diagnostics)], 10–20 U alkaline phosphatase (Sigma P6774) for 8 h at 37 °C. Then, proteins were denatured by boiling in Laemmli buffer.Twenty micrograms of proteins were incubated in dephosphorylation buffer  during 1 h at 4 °C for pre-clearing step. The non-retained sample was incubated with primary antibody overnight at 4 °C with gentle agitation, followed by 2 h incubation with magnetics beads (1:5). Beads were then washed sequentially using RIPA, RIPA + 0.5 M NaCl, RIPA + TNE   and lastly with TNE. Beads were finally resuspended in Laemmli buffer  and boiled during 8 min at 95 °C. The soluble fraction corresponding to the immunoprecipitated proteins was analysed by SDS-PAGE and western-blot as described below.Proteins samples were separated by SDS-PAGE on 7.5% Stain-free acrylamide or AnykD Mini-PROTEAN TGX Stain-free (SF) Precast Gels (Biorad). The electrophoretic separation was done at constant voltage (300 V) for about 20 min in a tank with migration buffer . At the end of the electrophoretic separation, total proteomes were visualized after UV activation with ChemiDoc MP Imager (Imaging System) and ImageLab software (Biorad) by means of the Stain-Free technology. It is based on trihalo compound incorporation in the gel, which reacts with proteins and covalently binding to tryptophan residues, rendering them detectable after UV exposure. Proteins were then transferred on 0.2 μm nitrocellulose membrane using Trans-blot Turbo transfer system (Biorad) at 1.3 A, up to 25 V/gel during 10 min. The quality of transfer was checked by imaging of the SF profile with ChemiDoc MP Imager.55, composed of stacking gel  and resolving gel . Electrophoretic migration was performed using migration buffer as described above at constant amperage (25 mA/gel) for about 110 min. After migration, Phos-tag gels were incubated twice for 7 min in transfer buffer  supplemented with 1 mM EDTA, and then two times in transfer buffer without EDTA. Finally, proteins were transferred on 0.2 μm PVDF membrane with Trans-blot Turbo transfer system as described above. The quality of transfer was checked by imaging using the ChemiDoc MP Imager after membrane staining with red Ponceau (Thermofisher).Proteins samples were resolved on Phos-tag-PAGE gele; Desmin (Abcam ab6322) at 1/50000e; CamKII  at 1/1000e; P-CamKII (Thr286)  at 1/1000e; PAK1  at 1/1000e; P-PAK1/2 (Ser144/Ser141)  at 1/1000e in blocking solution overnight at 4 °C with gentle agitation. After three washes of 10 min in TBS-T, membranes were incubated in blocking solution containing secondary antibodies  for 1 h at RT before five washes of 10 min each in TBS-T. Signals were detected by chemiluminescence using ECL Clarity (Biorad) and ChemiDoc MP. Images were acquired and analysed using Image Lab® software.Membranes were blocked with 5% BSA for RL-2, CamKII and P-CamKII antibodies or non-fat dry milk for desmin, PAK1 and P-PAK1/2 antibodies in TBS-T . Membranes were incubated with primary antibody as follow: RL-2  at 1/2500The activity of PKA and PKC were measured using PKA and PKC activity assay kits  according to the manufacturer’s protocol. Once proteins extracted according to the protocol defined for kinases assays, 1 µg of protein extract was used for ELISA assay based on a specific phospho-peptide substrate which was phosphorylated by active PKA or PKC. The phosphorylation level of peptide substrate was determined by absorbance measurement at 450 nm on SpectraMax reader (Molecular devices).57. Native-PAGE was performed with 75 µg proteins extracted using the same procedure than SDS-PAGE except that SDS was removed from RIPA buffer, Laemmli buffer, migration buffer, 10% acrylamide/bisacrylamide [37.5:1] separating gel and 4% stacking gel. The electrophoretic separation was done under constant amperage (20 mA/gel) at 4 °C to prevent loss of phosphatase activity for about 110 min. Once the electrophoretic separation was achieved, the native gels were incubated in reaction mixture  for 15 min at 37 °C with manual shaking every minute as previously described57. Fluorescent bands corresponding to phosphatases activity were detected after UV exposition (365 nm) using ChemiDoc MP.The phosphatases activities were in-gel assayed using the fluorogenic substrate MUP (4-methyl-umbelliferyl phosphate), MUP being converted to fluorescent products once hydrolysed by phosphatase(s)The quantification of signal intensities was carried out following normalization by total protein level on Stain-Free images or by internal standards. Means of each group were related to the corresponding control mean having the value of 1. The significance of intergroup differences was determined by Kruskal–Wallis test (GraphPad Prism software). Experiments were done on 5 dishes per culture, on 3 independent cultures  and samples were distributed on several gels performed in parallel. Results were presented as mean ± SEM and outlier values were removed when existing. Significance levels were indicated by *p < 0.05, **p < 0.01 or ***p < 0.001.48 and Ac4-5S-GlcNAc49, OGA and OGT inhibitors, were used to increase and decrease the global O-GlcNAcylation level. For each treatment, we determined the differentiation status (fusion index) and cell viability (colorimetric MTT assay) which were not altered whatever the treatment applied  while Thiamet G treatment significantly increased the O-GlcNAcylation level  compared to control cells (1.00 ± 0.26).To quantify changes of the global O-GlcNAcylation level, we performed western blot using RL-2 antibody which recognizes O-GlcNAc moieties; whole protein profiles (Stain-Free) and RL-2 profiles were presented on Fig. 4-5S-GlcNAc .The desmin protein level was quantified in the whole proteome Fig. a. While 4-5S-GlcNAc treatment slightly increased desmin in insoluble fraction  compared to control (0.94 ± 0.07)  that corresponds to whole desmin protein level. Data were represented on Fig. We also studied the protein pattern of the protein extracts resulting from the differential extraction protocols using Phos-tag-PAGE. As shown on Fig. 47. This approach allowed to determine that desmin O-GlcNAcylation slightly decreased in whole extract after Thiamet G treatment  compared to control (0.93 ± 0.11)  Fig. a while iTo clarify the link between phosphorylation and O-GlcNAcylation of desmin, we immuno-precipitated O-GlcNAcylated proteins from whole protein extract of control C2C12 myotubes prior to Phos-tag-PAGE; desmin detection was carried out by western blot. As shown on Fig. 57. This approach allowed us to determine that there was no changes of global phosphatases activity whatever the treatment applied  activity but also phosphatase(s) activity. To identify without a priori the phosphatases presenting a change in their activity, we performed an in-gel protein phosphatase assay using 4-methyl-umbelliferyl phosphate substrate (MUP) Fig. a57. Thisied Fig. b; in theied Fig. .Figure 6We have demonstrated in the herein paper that increased O-GlcNAcylation level led to a significant decrease of desmin phosphorylation without alteration of phosphatases activity. To go further in the fine understanding of the interplay between phosphorylation and O-GlcNAcylation, we attempted to determine the kinase(s) involved in this decrease of phosphorylation level. We quantified PKA and PKC activities using ELISA approach and we determined the activation status of CamKII and PAK using the phosphorylation level of these enzymes, which is known to lead to their activation; the representative signals for CamKII, P-CamKII, PAK1 and P-PAK1/2 were presented in Supplemented Fig. 4-5S-GlcNAc-treated myotubes  compared to control (0.99 ± 0.01), while its activity was unmodified following the increase of global O-GlcNAcylation level in Thiamet-G-treated myotubes. The PKC activity was not modified whatever the variation of the global O-GlcNAcylation level . Our data support that O-GlcNAcylation impacts desmin protein level, its phosphorylation and O-GlcNAcylation as well as its subcellular distribution  or in heart disorders (such as heart failure), characterized by desmin aggregation62.Another important finding is that global O-GlcNAcylation impacts desmin phosphorylation. Indeed, while the Ac55; this approach was previously applied to analyse the pattern of phosphorylation of desmin in cardiomyocytes and heart tissue63. However, to our knowledge, this approach was applied for the first time in skeletal muscle cells considering cytoskeleton and soluble/insoluble material. The result obtained was intriguing since we observed a highly complex pattern of phosphorylation in insoluble material and in cytoskeleton; in parallel, we observed only one phosphorylated form of desmin in soluble fraction. These data suggest that phosphorylation, through a specific and complex pattern, could be involved in the organization of desmin IFs and not only in their disassembly. It is worth to note that a similar observation was previously done on vimentin, another type III intermediate filament protein. Indeed, it was observed that in contrast with phosphorylation by most other kinases, vimentin phosphorylation by MAPKAP kinase-2 seems to have no effect on its assembly64.While the decrease of the phosphorylation level was correlated with a decrease of the partition of desmin toward cytoskeleton, we investigated whether desmin could be differentially phosphorylated in cytoskeletal or in soluble/insoluble fractions. We thus analysed the phosphorylated pattern of desmin in each of these fractions using Phos-tag-PAGE44, but the function of the glycosylation remains undefined. The O-GlcNAcylation of IFs was previously described on the type III IFs vimentin7, and occurs essentially in the head domain65. It was shown that O-GlcNAcylation of vimentin might block filament disassembly33 and is required for vimentin IF morphology and cell migration6. However, the only one site identified to date on desmin corresponds to Ser45944, i.e. in the tail domain, suggesting thus that O-GlcNAc could exert other functions than those expected. Of course, we cannot exclude that other O-GlcNAc site(s) may modify desmin; it should be interesting to go further in the characterization of the role of O-GlcNAcylation on desmin, in particular through the identification of additional modified sites.In parallel to phosphorylation, we observe a slight decrease of desmin O-GlcNAcylation following Thiamet G treatment, while O-GlcNAcylation and phosphorylation seems to be mutually exclusive on desmin. We have demonstrated that desmin is one of the substrates of OGT66; since then, this interplay was extensively described on a large panel of proteins . Interestingly, it was also described that O-GlcNAcylation modulates kinome and phosphatome72. In addition, it was shown that OGT and OGA interact with kinases and phosphatases within multienzymatic complexes permitting the phosphorylation and/or O-GlcNAcylation of proteins of interest39. To explain the desmin phosphorylation change, we investigated whether or not kinases and phosphatases activity could be modified consecutively to OGT or OGA inhibition. The decrease of phosphorylation level could result from a decrease of kinase(s) activity and/or an increase in phosphatase(s) activity. Therefore, we applied a strategy without a priori to measure phosphatases activity57 and we didn’t quantify any changes in the activity of phosphatases. In parallel, we have selected the major kinases involved in desmin phosphorylation, i.e. PKA, PKC, PAK and CamKII18 and we determined that two kinases presented a variation of their activity and/or activation status. Thus, PKA was activated following the decrease of O-GlcNAc level (i.e. Ac4-5S-GlcNAc treatment); however, desmin phosphorylation remained unmodified. In contrast, the CamKII activation status decreased in Thiamet G-treated myotubes. This data suggest that the desmin phosphorylation decrease could result from a decrease of CamKII activity consecutively to global O-GlcNAcylation increase.Furthermore, the interplay between phosphorylation and O-GlcNAcylation was firstly mentioned in 198774. In this way, we previously observed that the interaction between α-actinin and protein partners was modulated consecutively to O-GlcNAcylation changes47. This data suggests that the modulation of CamKII activity could be much more intricate than those initially expected.However, caffeine and the subsequent activation of CamKII by calcium didn’t modify phosphorylation of desmin. In contrast, we observed a decrease of desmin phosphorylation when CamKII was inhibited by KN-93, more specifically on the phosphoform that was modified after Thiamet G treatment. Interestingly, some data argue in favour of a modulation of the activation of CamKII through interactions with proteins partners such as α-actinin in a calcium-independent mannerTo sum, we have demonstrated in the herein paper that global O-GlcNAcylation increase led to a decrease of the desmin partition towards cytoskeleton correlated to a decrease of its phosphorylation that could result from a change of CamKII activity. Our data support the fact that the subcellular localization of desmin could be finely regulated by its PTMs signature in a spatio-temporal way involving O-GlcNAcylation. It should be thus essential to clarify the PTMs signature of desmin to fully understand the fine regulation of its functions and how this signature could be changed in the physiopathology of striated muscle disorders.Supplementary Information.


As one of the core executive functions, inhibition plays an important role in human life through development. Inhibitory control is defined as the ability to suppress actions when they are unlikely to accomplish valuable results. Contemporary neuroscience has investigated the underlying neural mechanisms of inhibitory control. The controversy started to arise, which resulted in two schools of thought: a modulatory and a network account of inhibitory control. In this systematic review, we survey developmental mechanisms in inhibitory control as well as neurodevelopmental diseases related to inhibitory dysfunctions. This evidence stands against the modulatory perspective of inhibitory control: the development of inhibitory control does not depend on a dedicated region such as the right inferior frontal gyrus (rIFG) but relies on a more broadly distributed network. Inhibitory control refers to the ability to suppress prepotent actions when they are unlikely to accomplish valuable results  and is aImpaired inhibition control is a central facet of many psychiatric disorders, including attention-deficit hyperactivity disorder ADHD; , alcoholYet, understanding the developmental trends of inhibitory control is critical when aiming to reduce the adverse outcomes and burdens related to inhibition control. Further, fostering the development of inhibition in children can have positive outcomes. For example, inhibitory control plays a central role in predicting social-emotional competence. Children who had better inhibitory control abilities were more likely to have better social skills and less internalizing behaviors . MoreoveThe aim of this systematic review is to provide a qualitative summary of existing behavioral and neuroscientific investigation of inhibitory control during development. This systematic review is structured as follows: after detailing methods of conducting this systematic research and outlining the results, we list and discuss the paradigms used in inhibitory control research and review behavioral results from research focused on inhibitory control development. Next, we survey the neural basis of inhibitory control both from a modulatory and a network perspective. Then, we discuss the neuroimaging literature regarding the brain activation patterns from infants to teenagers and argue that inhibitory control is supported by domain-general regions just like other cognitive functions. After that, we considered what if inhibitory control ability goes wrong by characterizing various neurodevelopment diseases associated with inhibitory control. Finally, we conclude our study and provide directions for future research.In the literature, the Simon task , the StrSimon tasks have two very simple rules: (1) press the left button when seeing Stimulus A and (2) press the right when seeing Stimulus B. The stimuli appear one at a time, and they can be presented on either the right or left side of the screen. Participants tend to be slower when the stimulus is given on the side opposite the corresponding response. This phenomenon is also known as the Simon effect, which states that people have a strong inclination to respond on the same side as the stimulus . As a reThe Flanker task asks participants to attend to the stimulus presented in the center but to ignore the flanking stimuli close to it. In incongruent trials, where the surrounding stimuli are mapped to the opposite reaction as the stimulus in the center, people respond slower; thus, inhibitory control ability is needed to overcome the tendency to respond according to the flanking stimuli. All these abovementioned tasks measure the so-called interference inhibition.By contrast, the go/no-go task, the SST, and the masked prime task are used to measure motor inhibition . The go/1 on 14 September 2021 using the following Boolean search string: (Inhibitory control OR inhibition) AND (Development OR Infant OR Children OR Adolescent) AND . A publication year limit has been set from 2000 to 2021. All returned results were systematically identified, screened then extracted for relevant information following the PRISMA  guidelines.2A literature search was conducted on the PubMed databaseStudies that fulfilled the following criteria were included in our systematic review: (a) inhibitory control behavioral performance during development, (b) neural basis of inhibitory control during development, (c) neurodevelopmental disease related to inhibitory dysfunction.Studies falling into one or more of the following categories were excluded from further analysis: (a) not original research article , (b) used non-human organisms , and (c) not related to inhibitory control development mechanisms or inhibitory control behavioral performance during development.Among the 20,826 articles initially turned after search on PubMed, we selected 5,000 articles after initial title screening. In particular, we excluded 15,825 articles because they did not relate to inhibitory control mechanisms. Then, records left for abstract screening were 5,000. After screening, we excluded 4,945 studies because they did not relate to inhibitory control aging mechanisms, with 55 left with eligibility. We assessed the full text of these articles and excluded 25 articles because 1 of them did not use human participants, 14 of them were not original research articles, and 13 of them did not relate to inhibitory control neural mechanisms. Therefore, 27 articles were left for our systematic review . ResultsPrevious studies showed that inhibitory control develops across the lifespan. Inhibitory control develops throughout childhood . It has According to the other studies, nine-month-old infants may pick between concurrently presented places by suppressing reaction to the distractor site, similarly to adults . InhibitUnderstanding the functional brain architectures underlying human cognition is one of the major goals of modern cognitive neuroscience, which can be done by localizing different cognitive processes to distinct brain regions and their connection pathways. For example, if Brain Region A activates in Task 1, then the brain region should be considered to support Task 1 according to the localists’ view. In the context of inhibitory control, a predominant theory proposed that the rIFG/anterior insula (aIns) is dedicated to inhibitory control . EvidencIn a later review, Network perspective proposed that the inhibitory control is one of the examples of cognitive control, which relies on the same set of domain-general fronto-parietal networks see . TherefoStudies have shown that the electroencephalogram (EEG) activities recorded from frontal skeletal regions affect newborns’ inhibitory control skills . FurtherOne study examined the inhibitory control abilities in 81 toddlers aged from 24 to 27 months . SpecifiSimilar findings were obtained in studies evaluating the biobehavioral expression of inhibitory control in two groups of participants aged 4 and 4.5 years old. Children who performed better on inhibitory control tasks (such as the day/night task and the yes/no Stroop task) had changes in EEG activities in the medial frontal cortex, indicating prefrontal cortex activation in both hemispheres. Preschoolers who performed poorly on the task, on the other hand, showed no task-related increases in EEG activity , 2007.A subsequent study using the go/no-go paradigm found that successful behavioral inhibition was related with stronger activations in the prefrontal and parietal areas for children, compared with adults. To manipulate the difficulties of the task demands, Another recent study employed functional magnetic resonance imaging (fMRI) to ask children (years 10–12) and adults (ages 18–26) while they performing the marble challenge, in which they had to choose between acting on and blocking a prepotent reaction while fMRI data was recorded . The rigFurthermore, there was a link between self-reported impulsivity and deliberate inhibition. The neurological underpinnings of early childhood response inhibition were explored by At posterior electrode sites, adults display a more significant go P3 than no-go P3, indicating that they are paying attention to targets . Adults Moreover, neurological and psychiatric diseases were analyzed in other studies with health controls . For exaPost-hoc analysis revealed that younger patients made more errors than older patients and all controls. fMRI results showed preserved activation in task-relevant regions such as bilateral frontal and supplementary eye fields, putamen, and precuneus in patient and controls, with the exception of increased activation in the left posterior cingulate gyrus in patients, specifically with generalized epilepsy across neutral and reward trials.Moreover, children with deficits in behavioral inhibition face increased risk for social anxiety . HoweverInhibitory control ability is especially important in teenagers, as adolescence is a period of time that coincides with responsibility, peer interactions, and social awareness . MoreoveThere are a bulk of literature suggesting the developments in the frontal lobe maturation and white matter over the adolescence e.g., . Some ofOne longitudinal fMRI study using the AS found that the prefrontal engagement was at the max during childhood, but by adolescence, the right DLPFC engages at adult levels. The activities in the right dACC showed increased engagement throughout adolescence, which mediate behavioral developments in AS performance . Age-relSimilarly, another fMRI study on 290 participants used a go/no-go task, with 88 patients undertaking repeated scanning at 1- to 2-year intervals . One groA recent study found that inhibition and intrinsic functional brain architecture could be influenced in their relationship by age and emotional function . Using fOn the other hand, magnetoencephalography (MEG) offers a temporal resolution and the millisecond level, which allows characterization of electrophysiological activities generated by neuronal dynamics during different phases of cognitive processing. One MEG study on inhibitory control development focused on the differences in the preparatory period between adolescents and adults and found that there were significant differences in oscillatory activities in adolescents compared with adults, which also corresponds with performance . SpecifiA subsequent study used MEG to investigate the spatial temporal neural characteristics for behavioral inhibition during a go/no-go task in both teenagers and adults. A more recent study used MEG and co-registered magnetic resonance imaging (MRI) to investigate the neural basis underlying the inhibitory control abilities in teenagers with that of adults . SpecifiImpaired executive functioning is thought to be the cause of impulsivity. Impulsive behavior is described by the presence of dysfunctional inhibitory mechanisms and strong “impulsions” (or impulses), as well as being triggered and modulated by dispositional and situational factors . There wAnother important piece of evidence about the complexities of the consequences of inhibitory control deficits comes from Selective impairment of reactive inhibition seems to be a hallmark of ADHD, as found by Tourette syndrome and OCD are the other two disorders that are characterized by impaired inhibitory control. The cognitive processes underlying tic and compulsion regulation have been shown to be entirely different. A tic is characterized by a rapid, repeated, non-rhythmic muscular action or vocalization involving specific muscle units . Tic disAs these disorders have very different neural underpinnings, the deficits in inhibitory control are more consistent with the network than with the modular view of inhibitory control . In otheTo conclude, we systematically reviewed brain changes that relate to inhibitory control performance in infants, toddlers, children, and teenagers. We presented evidence against a modular account of inhibitory control and propose that inhibitory control is a broad class of cognition, which is supported by a large-scale frontal-parietal network. Specifically, we proposed that the development of inhibitory control is related to the recruitment of core inhibitory control networks rather than a single region like the rIFG. We additionally surveyed the neurodevelopment dysfunctions associated with inhibitory control across development, which was previously only noted in healthy adults.Our proposed shift to understand inhibitory control as a result of the communication of a large-scale network especially during development, which provides insights for future basic as well as clinical research. For example, subsequent research could investigate whether the performance in inhibitory control tasks could be a biomarker for various psychiatric and neurological conditions during development. In addition, future studies may also focus on two specific directions: (1) separate three processes in inhibitory control including interference resolution, action withholding, and action cancelation during development. (2) As functional and structural changes are different in inhibitory control during aging , future WK: conceptualization, writing—original draft, writing—review and editing, and funding acquisition. SH: writing—original draft. MR, KV, and AM: writing—review and editing. All authors contributed to the article and approved the submitted version.


Our understanding of the role of CRF1-expressing neurons in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF1+ cells in rats. Here, we describe the generation and validation of a transgenic CRF1-Cre-tdTomato rat. We report that Crhr1 and Cre mRNA expression are highly colocalized in both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in tdTomato+ neurons are similar to those previously reported in GFP+ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be targeted to CeA CRF1+ cells via virally delivered Cre-dependent transgenes, that transfected Cre/tdTomato+ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like and nocifensive behaviors. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the brain, and that the expression pattern of Cre-tdTomato cells is similar to the known expression pattern of CRF1 cells. Given the accuracy of expression in the CRF1-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF1+ neurons can now be performed in assays that require the use of rats as the model organism.Corticotropin-releasing factor type-1 (CRF Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide that acts on various cell populations in the brain and elicits stress-related behavioral (e.g. anxiety) and physiological (e.g. sympathomimetic) responses . In the 1) receptors are Gs- or Gq-coupled metabotropic receptors  neurons are densely localized to the CeAl, whereas CRF1-expressing (CRF1+) neurons are mostly localized to the CeAm   is a key nucleus in modulating affective states and behaviors related to stress, anxiety, fear, pain, and addiction . Functiothe CeAm . In addis (BNST)  and dorss (BNST) . CeA CRFbehavior , nocicepbehavior , and alcbehavior , as wellbehavior . Work usors e.g. . Howeverors e.g.  difficul1-Cre-tdTomato transgenic rat line. We show that Crhr1 and iCre mRNA are expressed in the same neurons in the CeA and BLA using hybridization histochemistry. In the CeA, we show that tdTomato+ neurons are abundant in the CeAm, and that they are surrounded and in contact with CRF-containing puncta. We recorded membrane properties, inhibitory synaptic transmission, and spontaneous firing in CeA CRF1-Cre-tdTomato cells and show that these cells are sensitive to CRF. In addition, we show that Cre-dependent DREADD receptors can be targeted for expression by CeA CRF1 cells such that DREADD stimulation of CRF1+ CeA neurons increases nociception and anxiety-like behaviors, recapitulating prior work using pharmacological strategies. Outside the amygdala, we analyzed the expression of CRF1-Cre-tdTomato cells in several brain areas across the rostrocaudal axis and show that these cells are found in brain areas known to contain CRF1-expressing cells. These anatomical, electrophysiological, and functional data support the utility of this CRF1-Cre-tdTomato transgenic rat line for the study of CRF1 neural circuit function, and will be an important new resource that will complement CRFR1:GFP and CRFR1:Cre mice  and colonies were screened by PCR for insertion of the cassette at the site of Crhr1 in the BAC. BAC DNA from a single clone that contained the correct insertionwas purified, then transformed into EL250 cells, which carry an arabinose inducible flipase construct. Cultures of EL250 carrying the inserted BAC were induced to express flp recombinase by incubating in L-arabinose for 1 hr, then selected on ampicillin (the resistance of the BAC) and screened for loss of kanamycin resistance  Transgenic Core, where BAC DNA was purified, checked for integrity by DNA laddering with EcoR1 and XbaI followed bypulsed-gel electrophoresis, and by PCR, linearized, then injected into single-cell Wistar rat oocytes.Please refer to nic mice , with thnic mice . This BAransgene . iCre isactivity . tdTomatsualized . The polsualized . To insesualized . These cTransgenic rats were generated by the UNC Transgenic Core by injecting single-cell rat oocytes with the modified BAC described above . DNA frotdTomato protein expression and Crhr1 and iCre mRNA expression within the CeA. Immunohistochemical labeling of tdTomato in the amygdala showed that tdTomato+ cells were located in the lateral, basolateral, central, and medial amygdala  that is cleaved at the 2 A site. RNAscope ISH probing of Crhr1 and iCre mRNA showed strong expression of these molecules within the CeAm .We used RNAscope ISH to test the hypothesis that the CeAm . Since pthe CeAm  and our ess iCre . There w+ cells are concentrated in the CeAl, whereas CRF1+ cells are mostly located in the CeAm (tdTomato protein in the CeA of CRF1-Cre-tdTomato rats shows that the topography of CRF+ and tdTomato+ (CRF1-Cre) cells in these rats is consistent with previous studies (Previous work showed that CeA CRFthe CeAm . Immunof studies .tdTomato+ CRF1+ neurons were identified and differentiated from unlabeled CeA neurons using fluorescent optics and brief ( < 2 s) episcopic illumination in slices from adult male and female CRF1-Cre-tdTomato rats. Consistent with our immunohistochemical studies  and this region was targeted for recordings. Passive membrane properties were determined during online voltage-clamp recordings using a 10 mV pulse delivered after break-in and stabilization. The resting membrane potential was determined online after breaking into the cell using the zero current (I = 0) recording configuration. No differences were observed between male and female membrane properties including membrane capacitance, membrane resistance, decay time constant, or resting membrane potential  revealed that CRF1+ neurons are under a significant amount of phasic inhibition  or sIPSC amplitude .These data indicate that male and female CRF1+ CeA neurons have similar basal membrane properties and are under similar levels of basal inhibitory transmission. studies , the majotential . CRF1+ Coperties . The majing type . Voltagehibition  with no hibition , left ormplitude , right.T1+ CeA neurons from male and female CRF1-Cre-tdTomato rats using the cell-attached configuration. After a stable baseline period of regular firing was established, CRF (200 nM) was focally applied and the firing activity was recorded for a sustained application period of 7–12 min. CRF1+ CeA neurons from male rats had an average baseline firing rate of 2.1 ± 0.5 Hz and focal application of CRF significantly increased the firing activity to 3.4 ± 0.7 Hz -IRES-mCitrine (Active Virus) or AAV5-hSyn-DIO-EGFP (Control Virus), rats were given an injection (i.p.) of CNO (4 mg/kg) and were sacrificed 90 min later. Brain sections containing the CeA were processed for c-Fos immunohistochemistry and the number of c-Fos+ tdTomato cells were quantified. An overwhelming majority of tdTomato cells were located in the CeAm as shown above -IRES-mCitrine. tdTomato+ and mCitrine+ neurons were identified by brief episcopic illumination using fluorescent optics and positively-identified neurons were targeted for recording in whole-cell current clamp configuration to measure changes in resting membrane potential and spontaneous firing. CNO (10 μM) significantly increased membrane potential and number of action potentials in both male -IRES-mCitrine) or control fluorophore (AAV5-hSyn-DIO-EGFP) . Behavio1+ cells . There was a significant effect of test within the CNO group only , suggesting that chemogenetic stimulation of CeA CRF1+ cells increases mechanical sensitivity. CNO treatment had no effect on paw withdrawal thresholds in rats that received the control EGFP fluorophore  expression targeted to CeA CRForophore . In the orophore .t = 2.6, p = 0.022), suggesting that chemogenetic stimulation of CeA CRF1+ cells increases anxiety-like behavior on the EPM. Control EGFP virus rats did not show differences in open arms times after CNO treatment , suggesting that chemogenetic stimulation of CeA CRF1+ cells increases anxiety-like behavior in the OF. EGFP control rats did not show differences in time spent in the center of the arena after CNO treatment  group, rats that were given CNO treatment had lower open arms time compared to rats that were given vehicle treatment , PVN, lateral hypothalamus (LHA), paraventricular thalamus (PVT), hippocampus, ventral tegmental area (VTA), periaqueductal grey (PAG), and dorsal raphe (DR)  and medial habenula (MHb) were largely devoid of tdTomato+ cells, but a small number of tdTomato cells were found in the ventricular zone of the MHb  . The denat brain  and of Cse brain ; Table 1 the MHb . The locdetected . These t1-Cre-tdTomato rat line to allow genetic manipulation and visualization of neurons that express CRF1 receptors in the rat brain. We report that, within the CeA of CRF1-Cre-tdTomato rats, CRF1-Cre-tdTomato cells are located in the medial subdivision (CeAm), consistent with previous reports of Crhr1 expression in rats ] can be targeted to CeA CRF1-Cre-tdTomato cells using a Cre-dependent expression strategy, that systemic CNO treatment induces c-Fos in hM3D(Gq)-transduced CRF1+ cells in CeA, and that CNO induces membrane depolarization and spontaneous firing activity in hM3D(Gq)-transduced CRF1+ cells in CeA. We showed that hM3D(Gq)-mediated stimulation of CeA CRF1+ cells increases anxiety-like behavior, as measured by EPM and open field tests, as well as mechanical nociception as measured by the Von Frey test. Finally, we report that Crhr1 and iCre mRNA expression are highly colocalized in the BLA, and that Cre-tdTomato is expressed in several brain areas across the rostrocaudal axis of the rat brain in expected patterns. Collectively, this work provides cellular, electrophysiological, and behavioral data demonstrating the validity and reliability of a new transgenic rat model for the identification and selective manipulation of CRF1+ neurons in the CeA.We generated a new transgenic CRF in rats  and mice in rats , and tha1-Cre-tdTomato rats were generated by modifying a BAC clone derived from F344 rats, that was injected into oocytes derived from Wistar rats. Because the originating strain of the BAC DNA differs from the strain used for transgenesis, DNA sequence differences between F344 and Wistar rats in the 200 + kb region surrounding the Crhr1 genomic locus may impact expression of iCre-2A-tdTomato in the CRF1-Cre transgenic rat. Moreover, because transgenic BAC DNA insertion was not directed, position effect, copy number, and fragmentation of the BAC insertion may possibly alter expression of reporter genes. These sources of variability were minimized by the expertise of the UNC Transgenic Core Facility, where BAC DNA was prepared, cleaved at a single site to produce full-length single stranded BAC DNA, then injected into oocytes. Two transgenic founders were isolated that contained unique DNA sequences near the Crhr1 genomic locus in the BAC, as well as DNA sequence from both terminal ends of the injected BAC, indicating insertion of full-length BAC DNA. Of these two rats, one displayed expression of tdTomato in a pattern reflecting Crhr1 expression patterns reported in rats  CeA neurons in CRF1-Cre-tdTomato rats are similar to those reported in wild-type rat CeA neurons ] to CeA CRF1-Cre cells and first tested the effects of CeA CRF1+ cell stimulation on nociception. We showed that DREADD stimulation of CeA CRF1+ cells increases mechanical sensitivity as measured by the Von Frey test, but not thermal sensitivity measured by the Hargreaves test. Numerous studies have implicated CeA CRF-CRF1 signaling in nociception. For instance, pain induced by carrageenan injection into the knee joint produces hyperactivity of CeA neurons in rats, a phenomenon that is CRF1 dependent  under basal and challenged conditions such as neuropathic or inflammatory pain, stress exposure and/or withdrawal from chronic exposure to drugs or alcohol.Our lab has shown that predator odor stress-induced hyperalgesia, as measured by the Hargreaves test, is mediated by increased CRF-CRF the CeA . Contrar1 signaling generally promotes anxiogenic responses, particularly under challenged conditions such as stress or withdrawal from chronic exposure to drugs of abuse. For instance, in mice, intra-CeA infusion of a CRF1 antagonist attenuates anxiety-like behavior, as measured by open field and light-dark box tests, following immobilization stress but not under basal conditions  vivaria at UNC, LSUHSC, or UTHSC on a reverse 12 hr light/dark cycle (lights off at 7:00 or 8:00 AM) and had ad libitum access to food and water. All behavioral procedures occurred in the dark phase of the light-dark cycle. Sample sizes were estimated using published work from our labs. In situ hybridization and immunohistochemistry experiments were performed in a single pass. Behavioral experiments were performed in two independent, fully counterbalanced replicates. Slice electrophysiology experiments were conducted in a single pass, such that one experiment was performed in each slice and each experimental group contained neurons from a minimum of three rats. All procedures were approved by the Institutional Animal Care and Use Committee of the respective institutions at which procedures occurred  and were in accordance with National Institutes of Health guidelines.Adult male and female CRF13 GC/mL. At the end of surgeries, rats were monitored to ensure recovery from anesthesia and were given a minimum of 4 weeks to recover before the start of procedures. Rats were treated with the analgesic flunixin  and, in some rats, the antibiotic cefazolin  before the start of surgeries and once the following day.Rats were anesthetized with isoflurane  and mounted into a stereotaxic frame (Kopf Instruments) for all stereotaxic surgeries. The following coordinates (from bregma) were used for bilateral intra-CeA microinjections: –2.5 mm posterior, ± 4.0 mm lateral, and –8.4 mm ventral for male rats and –2.2 mm posterior, ± 4.0 mm lateral, and –8.2 mm ventral for female rats. Viral vectors for Cre-dependent expression of Gq-DREADDs or control (see below) were injected into each side of the CeA at a volume of 0.5 µL over 5 min and injectors were left in place for an additional 2 min. Viral titers were between 1.0–1.5 x 10Rats were deeply anesthetized with isoflurane and were transcardially perfused with ice-cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were extracted and post-fixed in 4% PFA for 24 hr (at 4 °C), cryoprotected in 20% sucrose for 48–72 hr (at 4 °C), and frozen in 2-methylbutane on dry ice. Coronal sections were collected using a cryostat and stored in 0.1% sodium azide in PBS at 4 °C until further processing.+ cells were quantified manually by a blinded experimenter. Four sections representative of the CeA anterior-posterior axis (~bregma –1.8––2.8) from each animal were used for analysis.Sections (40 µm) containing the CeA were washed 3 × 10 min in PBS and incubated in blocking buffer  for 2 hr at RT. Subsequently, sections were incubated in rabbit anti-c-Fos polyclonal antibody  for 48 hr at 4 °C. Sections were then washed 3 × 10 min in PBS and incubated in goat anti-rabbit Alexa Fluor 647  for 2 hr at RT. After 3 × 10 min washes in PBS, sections were mounted on microscope slides and coverslipped with Prolong Gold Antifade Reagent with DAPI . Sections were imaged using a Keyence  BZ-X800 fluorescent microscope at ×20 magnification and FosSections (40 µm) containing the CeA were washed 3 × 10 min in PBS and incubated in 3% hydrogen peroxide for 5 min. Sections were then washed 3 × 10 min in PBS and incubated in a blocking buffer containing 1% (w/v) bovine serum albumin and 0.3% Triton X-100 in PBS for 1 h at room temperature (RT). Then, sections were incubated in a rabbit anti-HA monoclonal antibody  for 48 hr at 4 °C. Sections were then washed for 10 min in TNT buffer , incubated for 30 min in 0.5% (w/v) Tyramide Signal Amplification (TSA) blocking reagent in 0.1 M Tris base, and incubated for 30 min in ImmPRESS horseradish peroxidase horse anti-rabbit antibody  at RT. Following 4 × 5 min washes in TNT buffer, sections were incubated in fluorescein TSA reagent (1:50 in TSA amplification diluent) for 10 min at RT. TSA blocking reagent, fluorescein TSA reagent, and TSA amplification diluent are part of the TSA detection kit . Sections were washed 3 × 10 min in TNT buffer, mounted on microscope slides, and coverslipped with Prolong Gold Antifade Reagent with DAPI . Sections were imaged using a Keyence BZ-X800 fluorescent microscope at ×2 and ×20 magnification.tdTomato signal degrades over time, to create a permanent set of slides for anatomical analysis, we performed immunohistochemistry to label tdTomato protein permanently. Briefly, fixed, free-floating sections (30 µm) were incubated overnight in monoclonal rabbit anti-RFP, biotin-tagged antibody . Sections were then washed 3 x with PBS and incubated in streptavidin-conjugated peroxidase (DAB-elite kit) for 1 hr. After incubation with streptavidin, sections were washed 2 x in PBS, then 2 x in 0.1 M NaOAc (pH 6.0), then stained in a solution of 0.1 M NaOAC (pH 6.0) containing nickel ammonium sulfate (3%) and 5 µl of 3% H202. Sections were stained for up to 10 min, then washed 2 x in NaOAc (pH 6.0), then in PBS, before being mounted on gelatin coated slides, dehydrated, and coverslipped in DPX. Bright-field images were acquired using a Cytation 5 imager . tdTomato and CRF immunofluorescent labeling: Tissue processing procedures were similar to the immunofluorescent procedures described above. Sections through the amygdala were incubated in antibodies against CRF  and goat-anti-RFP . Primary antibodies were detected by secondary anti-rabbit antibody conjugated with Alexa Fluor 488 and anti-goat antibody conjugated with Alexa Fluor 555 (Invitrogen), resulting in tdTomato protein being visible as red fluorescence and CRF peptide visible as green fluorescence. High-magnification images of the CeA were taken using a Leica  Sp5 confocal microscope.Because tdTomato rats  were deeply anesthetized with Avertin , then transcardially perfused with PBS followed by 4% PFA in PBS. Brains were removed, fixed in 4% PFA at 4 °C overnight, then equilibrated in 30% sucrose, sectioned into six series of sections  on a frozen sliding microtome , and stored in PBS at 4 °C. Brain slices were mounted onto glass slides, dried, and went through in situ hybridization (ISH) using a RNAscope Multiplex Fluorescent kit v2  following the manufacturer’s protocol.All solutions were prepared with DEPC treated water and all tools and surfaces were wiped with RNAzap followed by DEPC treated water. Adult Crhr1:Cre -hold = –60 mV. Cell-attached recordings were performed with no holding parameters (0 mV). All recordings were performed at room temperature. Series resistance was continuously monitored and cells with series resistance >15 MΩ were excluded from analysis. Properties of sIPSCs were analyzed and visually confirmed using a semi-automated threshold detection program . The frequency of firing discharge was evaluated and visually confirmed using threshold-based event detection analysis in Clampfit 10.2 (Molecular Devices). Experimental drugs were applied by bath application or y tube for focal application. Analysis was performed on recordings containing >60 events or that encompassed a period of 2–5 min.Following rapid decapitation, brains were extracted and sectioned as previously described . BrieflyClozapine-n-oxide  was dissolved in 5% DMSO . CRF was purchased from Tocris Bioscience , dissolved in stock solutions in ultra-pure water, and diluted to final experimental concentration in aCSF.tdTomato protein expression and Crhr1 and iCre mRNA expression within the CeA. Coronal brain sections containing the CeA were processed for immunohistochemical DAB labeling of tdTomato protein or RNAscope ISH for labeling Crhr1 and iCre mRNA, as described above. A separate set of brain sections containing CeA were processed for immunofluorescent labeling of tdTomato and CRF to map the expression pattern of these proteins in the CeA of Crhr1-Cre-tdTomato rats. Images were captured using a confocal microscope  and processed with Fiji ImageJ. Coronal sections containing the CeA were identified by neuroanatomical landmarks with reference to a rat brain atlas and captured at ×20 magnification at one single focal plane (1 µm). For analysis of Crhr1 and iCre RNAscope images, punctate signals from each channel were quantified separately following the manufacturer’s guideline (ACDbio SOP45-006). Quantification was performed by an experimenter blinded to experimental groups. Based on pilot studies, cells that had more than 3 puncta were considered positive. At least 3 sections representative of the anterior-posterior axis of the CeA were analyzed in each animal.The purpose of this experiment was to determine the pattern of 1+ neurons in the CeA. These neurons were identified using fluorescent optics and brief ( < 2 s) episcopic illumination. All labeled neurons were photographed, recorded, and saved. Intrinsic membrane properties were determined in voltage clamp configuration (Vhold = –60 mV) using pClamp 10 Clampex software. Current clamp recordings were performed to determine current-voltage (I-V) changes and the firing type of each neuron. Voltage clamp recordings of pharmacologically-isolated GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) were performed with bath application of the glutamate receptor blockers 6,7-dinitroquinoxaline-2,3-dione  and DL-2-amino-5-phosphonovalerate  and the GABAB receptor antagonist GCP55845A (1 µM). Cell-attached recordings were made in close juxtacellular  cell-attached configuration and only cells with regular spontaneous firing were included in analysis. After a stable baseline period, CRF (200 nM) was applied for a period of 7–10 min and changes in firing were measured and compared to baseline. Experiments were performed in individual slices to ensure that drug application was never repeated in the same slice.The purpose of this experiment was to characterize intrinsic properties, inhibitory synaptic transmission, and CRF sensitivity in CRF1-Cre-tdTomato cells can be activated using chemogenetics via Cre-mediated targeting of Gq-DREADD receptors [hM3D(Gq)] to these cells.The purpose of this experiment was to test if CeA CRF1-Cre-tdTomato cells, CRF1-Cre-tdTomato rats were given bilateral microinjections of AAV8-hSyn-DIO-HA-hM3D(Gq)-IRES-mCitrine  or a control virus  targeting the CeA. Four weeks later, rats were given a systemic CNO injection  and sacrificed 90 min later. Brain sections (4 sections/rat x 4 rats/group) were processed for c-Fos immunohistochemistry and the percentage of c-Fos+ tdTomato+ cells in the CeA was calculated. Cell counts were performed by an experimenter blinded to experimental groups.To test if Cre-dependent expression of Gq-DREADD receptors can stimulate CeA CRF1-Cre -tdTomato cells, CRF1-Cre-tdTomato rats were given bilateral microinjections as described above. After a minimum of 4 weeks, brain slices of CeA were prepared as described above. CeA neuronal expression of mCitrine and tdTomato were confirmed by fluorescent optics and neurons were targeted for electrophysiological recording. After a stable baseline period, CNO (10 μM) was applied and changes in membrane potential and action potential firing were measured and compared to baseline.To functionally validate Cre-dependent expression of Gq-DREADD receptors in CeA CRF1 cells on nociception and anxiety-like behavior. Rats were given intra-CeA microinjections of AAV8-hSyn-DIO-HA-hM3D(Gq)-IRES-mCitrine  or AAV5-hSyn-DIO-EGFP  and were given 4 weeks for recovery and viral expression  Hargreaves baseline session. During Von Frey and Hargreaves test sessions, rats were given CNO (4 mg/kg) or vehicle injections (i.p.) 30 min before the start of testing. All Von Frey and Hargreaves procedures occurred under regular white light illumination.Baseline paw withdrawal thresholds in the Von Frey assay and withdrawal latencies in the Hargreaves assay were measured over 3 sessions , open field (OF), and light-dark box (LD) on consecutive days . All proCrhr1 and iCre mRNA expression in a brain area outside the CeA. The basolateral amygdala (BLA) was selected for analysis because CRF1 receptors are highly expressed in, and functionally regulate the activity of pyramidal glutamatergic neurons in this area (as opposed to GABAergic neurons in CeA)  . Brain stdTomato transgene is expressed in brain areas outside of the amygdala in a pattern that is consistent with known CRF1 expression patterns. Male and female (n = 2/sex) CRF1-Cre-tdTomato rats were perfused transcardially with PBS and 4% PFA, and brains were post-fixed in 4% PFA overnight. Brains were cryoprotected, snap-frozen, and sectioned with a cryostat. Coronal sections (40 µm) were collected across the rostrocaudal axis, mounted onto microscope slides, and coverslipped with Fluorogel-II containing DAPI (Electron Microscopy Sciences). Brain sections were imaged using a Keyence BZ-X800 fluorescent microscope. For each brain region, at least three sections were analyzed.The purpose of this experiment was to examine if the Cre-Electrophysiology data on frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) were analyzed and manually confirmed using a semi-automated threshold-based detection software . Cell-attached firing discharge data were analyzed and manually confirmed using a semi-automated threshold-based detection software . Electrophysiological characteristics were determined from baseline and experimental drug application containing a minimum of 65 events each. Event data were represented as mean ± SEM or mean % change from baseline ± SEM and analyzed for independent significance using a one-sample t-test, compared by paired or unpaired t-test where appropriate. Data analysis and visualization were completed using Prism 7.0 . Behavioral data were analyzed using multifactorial repeated measures ANOVAs (for Von Frey and Hargreaves tests) or t-tests (for anxiety tests). For RM ANOVAs, between-subjects factors include sex and treatment, and the within-subjects repeated measure was test session (i.e. baseline vs. test). Data from the active and control virus groups were analyzed separately  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 4 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kate Wassum as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: James P Herman (Reviewer #2); Eric Zorrilla (Reviewer #3).Thank you for submitting your article "Cellular, physiological, and behavioral validation of a CRFR1:Cre-td Tomato rat for use in basic neuroscience research" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. (We suggest resubmitting as a Tools and Resources paper.)Essential revisions:1) As previous transgenic rat lines have shown some issues with cell specific fidelity, and since this is the initial characterization paper of the rat line, it was felt that a broader characterization of the expression in different cell types and in different brain regions was necessary. For example, the Pomerenze Crh-Cre rat was found to only express in GABAergic but not glutamatergic neurons, and so for the field to be able to understand the breadth of utility of this model and the fidelity across cell types, more characterization is required. This does not need to involve in depth electrophysiology as was done in the CeA, but could be done by mapping the expression of iCre/tdTomato in brain regions beyond the CeA, such as prefrontal cortex, hypothalamic nuclei and the BNST, where CRHR1 neurons are widely expressed. Ideally this would be compared to ISH quantification as is done in the CeA but if this is unfeasible then comparing to established in situ hybridization maps of expression of CRHR1 would still provide an idea of the fidelity with endogenous CRFR1 expression. It was felt necessary that at least in one other brain region, preferably one where CRHR1 is expressed in glutamatergic neurons, a side by side comparison of tdTomato and CRFR1 would be necessary to be sure it is not restricted to discrete cell types. It would also be good to establish if expression of tdTomato is seen in microglia or astrocytes to know if this model recapitulates the cell type specificity of CRFR1 expression.Concerns that require addressing but can be dealt with via reanalysis of existing data or modifications within the manuscript.1) The authors contextualize the necessity of developing a rat model of the CRFR1-Cre line because they state that this will allow more for the investigation of more complex behaviors then mice engage in. While this point may have some validity, it was felt that this statement and justification also required more validity. Please expand this rationale including examples of complex behavioral assays that can only be performed in rats and not mice.2) The manuscript does not discuss possible genetic strain of origin issues. The rat was generated using a BAC that involves DNA from a different strain (F344) than that bred into (Wistar), so potential strain of origin differences in regulatory or enhancer elements for the Crhr1 gene might co-segregate in early generations with the transgene. Similar considerations are true with respect to linked alleles if the strain of the gamete donors, which was not specified, differed from Wistar. Such issues would become of lesser concern with continued Wistar breeding but are not mentioned.Additionally, the location, copy #, fidelity or potential mosaicism of BAC transgene insertions after germline transmission was not mentioned. It is recommended that analysis of F1 or F2 offspring to establish location, copy #, fidelity and mosaicism of transgene insertion in order to disseminate the most desirable pedigree from this line . If already done, please add relevant findings.3) For the electrophysiology studies, adding the resting potential on the rep traces in Figure 5 and describing if Vm was adjusted for these experiments would provide clarity and is requested. Also, it could be very useful to compare intrinsic membrane results with published work on these cells to establish the normality of these measures in this line and see if the transgene expression impacted the physiological properties of these cells. It was also felt that the many of the traces used in the figures didn't seem representative and these should be modified to include genuine representative traces.4) The reviewers all appreciate the authors inclusion of both sexes, but there are some issues here that need to be addressed. First, it is likely that this study was not appropriately powered for identifying sex differences. This is recognized in some experiments, such as the anxiety behavior experiments, but it was agreed that the same level of preliminary interpretation should be exercised with the Cre fidelity data and the impacts of CRF in firing rates. However, sex differences in baseline firing rates are important and it would benefit the manuscript to discuss this in greater depth.5) The statistics on the behavior  should be checked as it was felt that repeated measures ANOVA would be more appropriate for these then t-tests.6) Consistent with IUPHAR consensus , the receptor protein should be referred to as CRF(subscript)1. Per both IUPHAR and RGD (RGD ID: 61276) nomenclature, the gene should be referred to as Crhr1.7) As the DREADD studies performed did not include an approach that inhibited these cells, the discussion should clarify that it remains to be determined if this approach will work for inhibition of cell activity using actuators like DREADDs.8) Some aspects of histochemical concordance were not provided or explored. For example, the proportion of Cre-expressing neurons that expressed CRF1 mRNA was not reported to confirm specificity (it appears to be even higher which would be good)? Cellular co-expression of Tomato with CRF1 mRNA also was not explicitly quantified.To address this, it is recommended to: (a) justify the current binary thresholds for + vs. – expression similar to PMID: 31451604, (b) analyze whether there is a semi-continuous, rather than only binary, relation between puncta expression of CRF1 vs. iCre, (c) clarify whether there are statistically significant sex differences in binary or continuous concordance (CRF1-iCre), and (d) discuss concordance of Tomato with CRF1 mRNA.9) While anxiety-like behavior was viewed as an appropriate behavioral readout for validation of the rats, it was felt that the consensus around the role of the CeA in pain was less widely accepted and had more nuance. As such, it is requested that authors to cite the work on CeA CRF and analgesia and acknowledge that their chemogenetic manipulation seem to mimic injury-induced hyperalgesic states but not stress-induced analgesic states. The authors can use this opportunity to highlight that the transgenic rat would be an excellent tool for future studies aimed at studying stress-induced analgesia.10. If you have not already done so, please attend to the important information below "Information to help as you revise your submission" including inclusions of a key resource table and statistical reporting.1-Cre transgenic rat and the role of central amygdala CRF1 receptors in nociception and anxiety-like behavior" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kate Wassum as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Generation of a CRFThe reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The authors did an adequate job in answering many of the reviewer’s concerns, however, it does seem that the author response fell short in answering two of the reviewers' concerns.Specifically, there was a consensus amongst reviewers that analysis of the penetrance and specificity of transgene across several regions should be included in this first demonstration of a new transgenic tool. While the inclusion of the BLA is an improvement, it still does not survey several regions. Rather the authors indicate that a full analysis of the specificity and penetrance of the transgene across brain regions will be reserved for a follow-up manuscript. While this may be, the reviewers agreed that it is paramount to this first publication to include more than two regions. From discussion, it was agreed amongst the reviewers that the authors should include characterization of the following brain regions: BNST, Hippocampus, VTA, LC, RN, PAG, PVN, and at least one cortical region.The second concern that does not seem to be sufficiently addressed in the revision is the need to perform experiments to determine where the BAC incorporated in the genome in the CRF1-Cre rat. This is particularly important when considering fluorescent reporter expression. The reviewers appreciated the additional discussion of potential caveats but agreed that additional experiments and analysis to validate this component of a new tool are needed. The issues that have arisen from other BAC mice and rats are believed to be a result of less than rigorous validation upon their initial introduction. As such, it seems incumbent on the authors to be as rigorous as possible in this first publication in order to avoid these recurrent issues with this particular rat tool. Essential revisions:1) As previous transgenic rat lines have shown some issues with cell specific fidelity, and since this is the initial characterization paper of the rat line, it was felt that a broader characterization of the expression in different cell types and in different brain regions was necessary. For example, the Pomerenze Crh-Cre rat was found to only express in GABAergic but not glutamatergic neurons, and so for the field to be able to understand the breadth of utility of this model and the fidelity across cell types, more characterization is required. This does not need to involve in depth electrophysiology as was done in the CeA, but could be done by mapping the expression of iCre/tdTomato in brain regions beyond the CeA, such as prefrontal cortex, hypothalamic nuclei and the BNST, where CRHR1 neurons are widely expressed. Ideally this would be compared to ISH quantification as is done in the CeA but if this is unfeasible then comparing to established in situ hybridization maps of expression of CRHR1 would still provide an idea of the fidelity with endogenous CRFR1 expression. It was felt necessary that at least in one other brain region, preferably one where CRHR1 is expressed in glutamatergic neurons, a side by side comparison of tdTomato and CRFR1 would be necessary to be sure it is not restricted to discrete cell types. It would also be good to establish if expression of tdTomato is seen in microglia or astrocytes to know if this model recapitulates the cell type specificity of CRFR1 expression.We now show Crhr1 and iCre co-expression in the basolateral amygdala, a brain area that contains a high density of Crhr1-expressing glutamatergic neurons . A follow-up manuscript will characterize Crhr1 and iCre co-expression throughout the rat brain.Concerns that require addressing but can be dealt with via reanalysis of existing data or modifications within the manuscript.1) The authors contextualize the necessity of developing a rat model of the CRFR1-Cre line because they state that this will allow more for the investigation of more complex behaviors then mice engage in. While this point may have some validity, it was felt that this statement and justification also required more validity. Please expand this rationale including examples of complex behavioral assays that can only be performed in rats and not mice.Please see page 4, lines 70-71, and page 8, lines 143-145.2) The manuscript does not discuss possible genetic strain of origin issues. The rat was generated using a BAC that involves DNA from a different strain (F344) than that bred into (Wistar), so potential strain of origin differences in regulatory or enhancer elements for the Crhr1 gene might co-segregate in early generations with the transgene. Similar considerations are true with respect to linked alleles if the strain of the gamete donors, which was not specified, differed from Wistar. Such issues would become of lesser concern with continued Wistar breeding but are not mentioned.Additionally, the location, copy #, fidelity or potential mosaicism of BAC transgene insertions after germline transmission was not mentioned. It is recommended that analysis of F1 or F2 offspring to establish location, copy #, fidelity and mosaicism of transgene insertion in order to disseminate the most desirable pedigree from this line . If already done, please add relevant findings.We agree that the insertion site and copy number are important considerations that may influence transgenic expression of reporter genes. We have not yet performed experiments to determine where the BAC incorporated in the genome in the CRF1-Cre rat, however, we have included these as important caveats to consider when analyzing transgenic expression, in the Discussion .3) For the electrophysiology studies, adding the resting potential on the rep traces in Figure 5 and describing if Vm was adjusted for these experiments would provide clarity and is requested. Also, it could be very useful to compare intrinsic membrane results with published work on these cells to establish the normality of these measures in this line and see if the transgene expression impacted the physiological properties of these cells. It was also felt that the many of the traces used in the figures didn't seem representative and these should be modified to include genuine representative traces.1+ CeA neurons are in line with what has previously been reported in rat CeA neurons , suggesting that the transgene did not significantly impact the overall health or activity of CRF1+ CeA neurons . The figures were edited to include traces that were more representative of the CRF1+ population in males and females .The cell-attached recordings of neuronal firing were performed with no holding parameters (0 mV) and no adjustments to Vm were made. The intrinsic properties of CRF4) The reviewers all appreciate the authors inclusion of both sexes, but there are some issues here that need to be addressed. First, it is likely that this study was not appropriately powered for identifying sex differences. This is recognized in some experiments, such as the anxiety behavior experiments, but it was agreed that the same level of preliminary interpretation should be exercised with the Cre fidelity data and the impacts of CRF in firing rates. However, sex differences in baseline firing rates are important and it would benefit the manuscript to discuss this in greater depth.1 cells that we observed should not be over-interpreted.Please see page 23, lines 330-331; page 26, lines 410-413. Given that our studies here were not sufficiently powered to conclusively determine the presence or absence of sex differences, we feel that the sex difference in baseline firing rate of CeA CRF5) The statistics on the behavior  should be checked as it was felt that repeated measures ANOVA would be more appropriate for these then t-tests.Data from Von Frey and Hargreaves tests were analyzed using repeated measures ANOVA. Data from elevated plus maze, open field, and light-dark tests were analyzed using t-tests because there are 2 between-subjects groups in these experiments. This is now clarified in the manuscript .6) Consistent with IUPHAR consensus , the receptor protein should be referred to as CRF(subscript)1. Per both IUPHAR and RGD (RGD ID: 61276) nomenclature, the gene should be referred to as Crhr1.This has been edited throughout the manuscript.7) As the DREADD studies performed did not include an approach that inhibited these cells, the discussion should clarify that it remains to be determined if this approach will work for inhibition of cell activity using actuators like DREADDs.This has been clarified in the Discussion .8) Some aspects of histochemical concordance were not provided or explored. For example, the proportion of Cre-expressing neurons that expressed CRF1 mRNA was not reported to confirm specificity (it appears to be even higher which would be good)? Cellular co-expression of Tomato with CRF1 mRNA also was not explicitly quantified.To address this, it is recommended to: (a) justify the current binary thresholds for + vs. – expression similar to PMID: 31451604, (b) analyze whether there is a semi-continuous, rather than only binary, relation between puncta expression of CRF1 vs. iCre, (c) clarify whether there are statistically significant sex differences in binary or continuous concordance (CRF1-iCre), and (d) discuss concordance of Tomato with CRF1 mRNA.tdTomato and Crhr1 mRNA expression, we probed for iCre and not tdTomato because iCre and tdTomato are expressed as a single polypeptide . Therefore, there is no reason to suspect any difference between iCre and tdTomato expression, and we probed for iCre because it is more relevant for functional studies.Please see edits on page 35, line 590 for information on our RNAscope quantification thresholding. Beyond counting cells as being positively or negatively labeled with an RNAscope probe, we did not quantify the number of puncta within each cell as this re-analysis would be difficult without specialized software. We feel that this level of analysis is beyond the scope of the current manuscript. Regarding potential sex differences, we did analyze RNAscope data with sex as a factor, using binary thresholds . Regarding concordance between 9) While anxiety-like behavior was viewed as an appropriate behavioral readout for validation of the rats, it was felt that the consensus around the role of the CeA in pain was less widely accepted and had more nuance. As such, it is requested that authors to cite the work on CeA CRF and analgesia and acknowledge that their chemogenetic manipulation seem to mimic injury-induced hyperalgesic states but not stress-induced analgesic states. The authors can use this opportunity to highlight that the transgenic rat would be an excellent tool for future studies aimed at studying stress-induced analgesia.Please see Discussion, page 24, lines 355-365.10. If you have not already done so, please attend to the important information below "Information to help as you revise your submission" including inclusions of a key resource table and statistical reporting.eLife instructions.A key resources table is now included at the beginning of the Materials and methods section (pages 28-29). Statistical reporting has been checked per The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The authors did an adequate job in answering many of the reviewer’s concerns, however, it does seem that the author response fell short in answering two of the reviewers' concerns.Specifically, there was a consensus amongst reviewers that analysis of the penetrance and specificity of transgene across several regions should be included in this first demonstration of a new transgenic tool. While the inclusion of the BLA is an improvement, it still does not survey several regions. Rather the authors indicate that a full analysis of the specificity and penetrance of the transgene across brain regions will be reserved for a follow-up manuscript. While this may be, the reviewers agreed that it is paramount to this first publication to include more than two regions. From discussion, it was agreed amongst the reviewers that the authors should include characterization of the following brain regions: BNST, Hippocampus, VTA, LC, RN, PAG, PVN, and at least one cortical region.1-GFP mice , and found that they are similar .We agree with the reviewers, and we have performed additional experiments to image and quantify Cre-tdTomato-expressing cells in multiple areas across the CRF1-Cre-tdTomato rat brain. We now show that Cre-tdTomato-expressing cells are found in brain areas known to contain CRF1 cells, including the prelimbic and infralimbic cortex, piriform cortex, bed nucleus of stria terminalis, hippocampus, paraventricular nucleus of hypothalamus, lateral hypothalamus, paraventricular thalamus, ventral tegmental area, dorsal raphe, and locus coeruleus . We compared the density of Cre-tdTomato cells in these areas to expression levels of Crhr1 mRNA in wild type rats , and density of GFP cells in CRFThe second concern that does not seem to be sufficiently addressed in the revision is the need to perform experiments to determine where the BAC incorporated in the genome in the CRF1-Cre rat. This is particularly important when considering fluorescent reporter expression. The reviewers appreciated the additional discussion of potential caveats but agreed that additional experiments and analysis to validate this component of a new tool are needed. The issues that have arisen from other BAC mice and rats are believed to be a result of less than rigorous validation upon their initial introduction. As such, it seems incumbent on the authors to be as rigorous as possible in this first publication in order to avoid these recurrent issues with this particular rat tool.We understand this concern and we performed additional experiments to try to identify the genomic insertion site of the BAC. Unfortunately, these experiments were not successful, and we were not able to conclusively determine the insertion site. We explored a commercial sequencing option, but we currently do not have the resources for this option. Given that the expression pattern of the fluorescent reporter in CRF1-Cre-tdTomato rats is consistent with the known expression pattern of CRF1 cells throughout the brain, we are confident that genomic incorporation of the transgene produced expected Cre-tdTomato expression patterns. In addition, the behavioral and physiological phenotypes of CRF1-Cre-tdTomato rats reported in this manuscript closely resemble those seen in wild-type Wistar rats, suggesting that BAC insertion into the genome did not produce observable anomalous phenotypes (added to Discussion on Pg. 24).


Sustained peripheral somatosensory stimulations, such as high-frequency repetitive somatosensory stimulation (HF-RSS) and vibrated stimulation, are effective in altering the balance between excitation and inhibition in the somatosensory cortex (S1) and motor cortex (M1). A recent study reported that whole-hand water flow (WF) stimulation induced neural disinhibition in the M1. Based on previous results, we hypothesized that whole-hand WF stimulation would lead to neural disinhibition in the S1 because there is a strong neural connection between M1 and S1 and aimed to examine whether whole-hand WF stimulation would change the neural balance between excitation and inhibition in the S1. Nineteen healthy volunteers were studied by measuring excitation and inhibition in the S1 before and after each of the four 15-min interventions. The excitation and inhibition in the S1 were assessed using somatosensory evoked potentials (SEPs) and paired-pulse inhibition (PPI) induced by single- and paired-pulse stimulations, respectively. The four interventions were as follows: control, whole-hand water immersion, whole-hand WF, and HF-RSS. The results showed no significant changes in SEPs and PPI following any intervention. However, changes in PPI with an interstimulus interval (ISI) of 30 ms were significantly correlated with the baseline value before whole-hand WF. Thus, the present findings indicated that the whole-hand WF stimulation had a greater decreased neural inhibition in participants with higher neural inhibition in the S1 at baseline. Considering previous results on M1, the present results possibly show that S1 has lower plasticity than M1 and that the duration (15 min) of each intervention may not have been enough to alter the balance of excitation and inhibition in the S1. Somatosensory function based on somatosensory information processing in the central nervous system plays a crucial role in our daily lives. Several brain regions are involved in somatosensory function, such as the primary somatosensory cortex (S1), posterior parietal cortex, presupplementary motor area, and basal ganglia . In partSustained peripheral somatosensory stimulation is effective for altering the balance between excitation and inhibition , such asIn vivo experiments identified the anatomic substrate for this cross-systemic plasticity as topographically and functionally specific reciprocal connections between the primary motor cortex (MI) and the primary somatosensory cortex (SI)  stimulation in which sustained somatosensory stimulation by water flow to a large site of the hand is applied; this induces neural disinhibition in the primary motor cortex (M1) . In vivotex (SI) . Other atex (SI) . In humatex (SI) . Additiotex (SI) . Therefotex (SI) , which mThe paired-pulse paradigm has become a standard procedure for evaluating the balance between excitation and inhibition in human participants. It involves the application of pairs of stimuli in close succession (paired-pulse stimulation), which can be used as a marker of intracortical excitability in the S1. In the case of the S1, paired-pulse inhibition (PPI) expresses the result of paired-pulse peripheral stimulations with a short interstimulus interval (ISI), in which the cortical responses to the second stimulus are more significantly suppressed compared to the first stimulus. The PPI is quantified by calculating the ratio of the amplitude of the second response divided by the amplitude of the first response. Small ratios indicate higher PPI, whereas large ratios indicate lower PPI, which is used as a marker for enhanced excitation.The present study aimed to clarify the effect of whole-hand WF on the balance between excitation and inhibition in the S1 using a paired-pulse paradigm. As previous evidence has shown that whole-hand WF produces simultaneous somatosensory stimulation to a large area of the hand , we hypoNineteen healthy right-handed participants  participated in the present study. None of the participants had a history of neurological or psychiatric diseases. All participants signed a written informed consent form, which provided a full explanation of this study and methods before the experimental session. This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Niigata University of Health and Welfare (18712-210823).°C ± 1°C, and the water temperature was 33°C ± 1°C. The ambient and water temperatures were modulated to avoid changing the skin temperature. The experimental protocol is shown in The interventions (15 min) were as follows: non-immersed control (CON), whole-hand water immersion (WI), whole-hand WF stimulation (WF), and HF-RSS. In all interventions, the participants were instructed to place their right hand in the sluicing device, keeping it relaxed. The left hand was placed on a soft support beside the body and was kept relaxed. The hands were fixed in the same position for whole-hand WF interventions, using a belt to avoid muscle contractions. For whole-hand WF, WF was applied to the palm of the right hand using a sluicing device  at ∼40 L/min . In all 2 area, with the anode located on the distal phalanx of the right index finger and the cathode located on the proximal phalanx of the same finger. The intensity was set to the maximal intensity tolerated without pain minus 0.1 mA  through the isolator  to surface adhesive electrodes of an approximately 1 cms 0.1 mA .Somatosensory input from peripheral nerves activates several cortical areas, and this modulation can be evaluated using somatosensory evoked potentials (SEPs). We measured SEPs before and after each intervention, according to a previously described methodology . SEPs weSingle-pulse SEP and PPI were measured to assess the balance between cortical excitation and inhibition in the S1. Paired-pulse electrical stimulation of the median nerve was applied with ISIs of 5 ms and 30 ms in combination with SEP recording. The ISIs were determined in two separate sequences based on the methods reported in previous studies , 2017. PThe peak-to-peak amplitudes of the cortical N20 and P25 response components for the first and second paired-pulse stimulations were analyzed. In the paired-pulse trial, the responses after the second response were obtained by subtracting a single SEP . Each PPSingle-pulse SEP amplitudes , latency , PPI ratio , and sensory thresholds were analyzed using statistical software .The distributions of all data were confirmed by the Shapiro–Wilk test. Sensory threshold was entered into a Friedman test with “intervention”  as the within-subject factor. Other parameters were entered into a generalized linear mixed model (GLMM) with “intervention” and “time” (pre and post) as the within-subject factors.p < 0.05. All data are expressed as the mean ± standard deviation.Pearson or Spearman correlation analysis was performed to assess the relationship between PPI at pre-intervention and the change in PPI with each ISI. Statistical significance was set at 2 = 5.654, p = 0.13).For single-pulse SEP, GLMM revealed no significant interactions and main effect in N20, P25, and N20-P25 peak-to-peak, except for the main effect of “intervention” in N20-P25 peak-to-peak .F  = 2.319, p = 0.086, η2p = 0.120; PPI_30 ms: F  = 0.792, p = 0.504, η2p = 0.045]. There was no main effect of “intervention”  and “time” .r = –0.589, p = 0.010).The present data showed that 15-min whole-hand WF, HF-RSS, whole-hand WI, and CON did not affect the balance between excitation and inhibition in the S1, as measured by the N20-P25 amplitude of SEP and PPI. Additional findings revealed that the change in PPI with longer ISI (30 ms) was negatively correlated with the baseline value in whole-hand WF.via N-methyl-D-aspartate receptors located within layers IV/V of SI -expressing inhibitory interneurons inhibit pyramidal cells within the microcolumn through synapses on superficial dendrites . Additio/V of SI . These P/V of SI . The inh/V of SI . PPI at /V of SI . At long/V of SI . TherefoPaired-pulse evoked inhibition has been reported to occur regardless of modality in the motor, visual, auditory, and somatosensory cortices . In the A possible explanation for this is the difference in neural plasticity between S1 and M1. A previous study demonstrated that quadripulse transcranial magnetic stimulation (QPS), which is the most powerful and reliable non-invasive brain stimulation method to induce neural plasticity in humans , was appInterestingly, we found that the higher the PPI_30 ms at pre-intervention, the more the disinhibition after whole-hand WF intervention, but not after other interventions. The results, in which the participants with higher PPI_30 ms at pre-intervention had more disinhibition after whole-hand WF, could be explained by the relationship between the cortical inhibition and neural plasticity. Previous studies have reported that lower cortical inhibition, when assessed by a paired-pulse paradigm, was related to lower neural plasticity in S1 . CorticaThe sensorimotor cortex hyperexcitability represents an important pathogenic mechanism underlying several neurological diseases such as familial adult myoclonic epilepsy type 2 (FAME2) , ALS Ra, dystoniThe present study has several limitations. First, the sample size is small; it was determined on the basis of previous studies, whereas it should have been based on a power analysis. Second, we measured only SEP and PPI to evaluate S1 excitability. To clarify the change in neural balance between excitation and inhibition in the S1 in greater detail, it would be preferable to investigate other biomarkers of inhibitory function such as early and late components of SEP high-frequency oscillation (e- and l-HFO) , which rThe present data showed that 15-min whole-hand WF, HF-RSS, whole-hand WI, and CON did not affect the balance between excitation and inhibition in the S1, as measured by the N20-P25 amplitude of SEP and PPI. Additional findings revealed that the change in PPI with longer ISI (30 ms) was negatively correlated with the baseline value in whole-hand WF. These results indicated that even a 15-min whole-hand WF can induce intracortical disinhibition in the participants with high inhibition in S1, which raises the possibility that whole-hand WF could be a useful tool for improving somatosensory function.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Niigata University of Health and Welfare. The patients/participants provided their written informed consent to participate in this study.DS and KY contributed to conception and design of the study. DC, KI, and DS performed the experiment and statistical analysis. DC and DS wrote the first draft of the manuscript. TF and GO wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.