Adaptive structures based on fiber-rubber composites with integrated Shape Memory Alloys are promising candidates for active deformation tasks in the fields of soft robotics and human-machine interactions. Solid-body hinges improve the deformation behavior of such composite structures. Textile technology enables the user to develop reinforcement fabrics with tailored properties suited for hinged actuation mechanisms. In this work, flat knitting technology is used to create biaxially reinforced, multilayer knitted fabrics with hinge areas and integrated Shape Memory Alloy wires. The hinge areas are achieved by dividing the structures into sections and varying the configuration and number of reinforcement fibers from section to section. The fabrics are then infused with silicone, producing a fiber-rubber composite specimen. An existing simulation model is enhanced to account for the hinges present within the specimen. The active deformation behavior of the resulting structures is then tested experimentally, showing large deformations of the hinged specimens. Finally, the simulation results are compared to the experimental results, showing deformations deviating from the experiments due to the developmental stage of the specimens. Future work will benefit from the findings by improving the deformation behavior of the specimens and enabling further development for first applications. In recent works, Shape Memory Alloys (SMA) have proven to be highly suited for the creation of adaptive structures ,3,4,5,6.In order to further improve the deformation behavior of such soft adaptive structures, solid-body hinges can be introduced by varying the bending stiffness across the specimen ,14, thusThe presented work uses existing knitting patterns of biaxially reinforced multilayered knitted fabrics, extended towards sectioned patterns with varying numbers of reinforcement fibers to create high stiffness (HSS) and low stiffness sections (LSS) that act as solid-body hinges. Additionally, the SMA integration is extended towards sectioned patterns, enabling structures with two separately activatable hinges. The active deformation behavior of the specimens was evaluated in deformation tests, where the bending deformation was induced by thermal activation of the integrated SMA actuator. An existing mesoscopic simulation model within the framework of the finite element method (FEM) is extended with regard to the hinge sections and is calibrated and validated with experimental data.s, Af, Ms, and Mf, respectively.SMA are capable of memorizing their geometrical shape after undergoing deformation. When subjected to temperature changes after an initial deformation, they tend to return to their initial shape. At high temperatures, the crystalline configuration present within the SMA is referred to as austenite. The crystalline configuration present at low temperatures is referred to as martensite. The phase transformation from austenite to martensite is a diffusion-less solid-phase transformation leading to twinned martensite with zero stress and de-twinned martensite under significant stress influence. The austenite phase is stable at high temperatures and exhibits a cubic crystalline structure whereas martensite is stable at low temperatures with tetragonal or monoclinic crystalline structures . When heOne Way Shape Memory Effect (OWSME)Two Way Shape Memory Effect (TWSME)The change in the crystallographic structure during solid-phase transformation exhibits mechanical behavioral changes resulting in shape memory effect (SME) and pseudo-elasticity (or superelasticity). The functional properties of the SMA material can be controlled in a wide range by means of appropriate thermal/thermomechanical processing methods as well as changing the alloy composition. The shape memory effects are further categorized into two characteristics :One Way f results in austenitic (parent-memorized) phase formation, reverting the initial pseudoplastic deformation and changing its shape back to that of the unstrained state. When the SMA is cooled back to below the martensite-forming end temperature Mf, SMA reverts to twinned martensite state without further shape change. This effect is called OWSME. The temperature-induced OWSME exhibits large strains and—when the free deformation is hindered—large stresses, enabling SMAs to generate large deformations and transduce large forces for actuation tasks.For a better understanding of shape memory characteristics, it can be assumed that the SMA is existing in twinned martensite  at room temperature. Mechanically loading this SMA causes detwinning, thus, deforming the SMA pseudoplastically. Subsequent heating above the alloy-specific austenite forming end temperature AIf after cooling, the SMA reverts back to the detwinned martensite state instead of the twinned martensite phase, this effect is called TWSME. During this two-way effect, the phase transformation can be reverted to austenite when heat is supplied under constant loading. However, to obtain TWSME, the SMA material must undergo a cyclic thermomechanical treatment denoted as “training” to remember its austenitic and detwinned martensitic states. The TWSME produces less recovery strain than OWSME and the strain tends to deteriorate quickly when subjected to high temperatures, rendering TWSME less feasible for actuation tasks .f. Upon mechanical loading, the SMA undergoes a stress-induced solid-phase transformation towards Martensite, allowing large-degree deformations that are completely recovered after the load is discarded. Thus, recoverable strains of up to 10% can be utilized. Additionally, the superelastic strain exhibits a hysteresis, facilitating large energy dissipation capabilities of superelastic SMA. For more details on shape memory effects, refer to e.g., [Since superelasticity or pseudoelasticity is less relevant in this work, it is only mentioned briefly. Here, the SMA is utilized in the Austenite state above Ato e.g., ,15,16.The basic concept of active deformation of a beam sample driven by contractile SMA wires studied in this work is based on bending as the primary mode of deformation. As depicted in Upon heating by applying an electric current, the SMA wire transforms to austenite and contracts, thereby deforming the beam cf. a. Upon cAs already shown in , the geoConsequently, introducing hinges into bending structures by splitting the deforming beam into HSS and LSS can significantly increase the achievable deformations without changing length or thickness b. The LSWang et al. ,17 have Unlike thermoset FRP, which provide both high stiffness and strength, IFRC specimens employ a highly elastic rubber matrix. In combination with stiff reinforcing fibers, this results in highly anisotropic mechanical properties that allow large reversible bending deformations while maintaining high membrane stiffness. IFRC structures are therefore suitable for applications under uniaxial or planar loads such as tires, pressure hoses or conveyor belts .In this work, multilayer flat knitting technology ,20 is usIn order to support both dimensioning and provide insight in the complex material interactions of IFRC structures, using a simulation model is highly beneficial. As several authors have shown ,22,23, a® as part of a previous work by the authors [Therefore, a mesoscopic FEM modeling approach for IFRC with integrated SMA wires was developed in ANSYS authors . In meso authors , the modGeometry and mesh: The model consists of a silicone block with cavities for the reinforcement fibers and SMA wire. The reinforcement fibers in the weft direction are modeled as cylinders with elliptical cross-sections and the SMA wires are modeled as a cylinder with circular cross-sections. The thin PTFE tube  has a low stiffness section (LSS) in the middle and two high stiffness sections (HSS) at the edges. Integrated SMA wires are spanning the full plate length, changing direction in turn-around curves at the end. During activation, the LSS is more prone to deformation due to its lower stiffness and therefore acts like a solid-body hinge.Concept 2 (V2) has two LSS, each flanked by HSS. Furthermore, SMA wires are integrated in sections, so that two strands with turn-around curves span the left LSS, the next two strands span the right LSS, and so on. Thus, the SMA wires can be activated independently, deforming either left, right or both LSSs of the structure, thus enabling intermediate deformation states.Concept V1.1 with one hinge area and two layers of reinforcement fibers in the HSS in bending direction, as described in V1.2 identical to V1.1 during the knitting process but with two additional layers of reinforcement fibers in its HSS added during the manufacturing of the composite. Thus, V1.2 will have twice the amount of reinforcement fibers in its HSS compared to V1.1.In order to investigate the assumed influence of both HSS and LSS being prone to bending cf. , two varIt is expected that the bending deformation of V1.2 will be more concentrated on the LSS than in V1.1, leading to higher maximum deformation.® flat knitting machine with a segmented fabric take-up [Both concepts are realized by means of multilayer flat knitting technology. Here, one layer of the fabric holds fibers in a uniform direction without undulations, with the layers alternating between weft and warp direction. For this, a customized Aries.3 take-up  and a maTwo variants of V1 were produced, further denoted as V1.1 and V1.2. They share the same binding setup, with their distinct features being created during composite forming. Thus, subsequently, the binding setup for both variants is described jointly. The differences between V1.1 and V1.2 are described at the end of the section.I in front, weft row II in between and weft row III behind the warp rows), with warp row 1 (front) holding GF-faint yarns and warp row 2 (back) holding GF-strong yarns  are draped onto the HSS, doubling the number of GF layers and further increasing the stiffness of the section.1 and 2 being identical to V1  are placed on a metal plate and wrapped into a vacuum bag with additional layers for improving resin flow and demolding. After infusing with silicone, the specimens are cured at room temperature for 48 h. The vacuum layup is listed in Both V1 and V2 are manufactured into composite specimens with a vacuum-assisted resin infusion (VARI) process. Blanks of the fabric  based on DIN EN ISO 14,125 [3, with four GF yarns lengthwise. The specimens are tested in the weft direction, in accordance with the direction of the SMA-induced bending. The setup is displayed in In order to evaluate the bending stiffness of the specimens V1 and V2, four-point-bending tests were carried out on a Zwick and RoellO 14,125 . Sheets In order to account for the solid-body hinges present in samples V1 and V2, the existing model see  is extenAs with the existing model, symmetry is used to reduce modeling and calculation effort, by using one SMA wire only and reducing the number of weft yarn rows to six. In addition to this symmetry assumption, the structure is cut in half, reducing the model to one-half of an SMA wire and three weft yarn rows. The resulting symmetry surface is assigned the translational boundary condition described in As with the existing model, the SMA wires need to be pre-stretched in order to exhibit the shape memory effect. This is done in two preliminary load steps, with a pre-stretch force of 7.5 N per wire (reduced by a factor of 0.5 to account for the symmetry described above). In the first step, the force is ramped up, pre-stretching the wire. In the second step, the force is ramped down, recovering the elastic deformation of the SMA and leaving the SMA in a plastically deformed (detwinned) state. For V1, the free end of the SMA wire is then connected to the structure with a bonded contact.For V2, the pre-stretching process is more elaborate: SMA 2 is temporarily fixed with a zero-displacement boundary condition to the free end of the beam, with its free end pre-stretched towards SMA 1. SMA 1 is in turn attached to the fixed side of the beam and pre-stretched at its other end towards SMA 2. After pre-stretching, both SMA 1 and SMA 2 are connected to a central fixation element, which is in turn connected to the rubber structure. The zero-displacement boundary condition of the other end of SMA 2 is then discarded; instead, it is then connected to the fixation element at the free end of the beam with another bonded contact. The wires are then activated separately or simultaneously with temperature loads.The bending stiffness of the FRC models without SMA needs to be calibrated with experimental data of the FRC specimen in order to ensure that the stiffness behavior of the model and the real structure agree. Therefore, the mesoscopic models of hinged FRCs without SMA wires are used to replicate four-point bending tests. In order to save computing effort, symmetry is used to model only half of the geometry. The gained results are therefore multiplied by two during evaluation. The calibration is conducted by iteratively changing the Young’s modulus in the 0° direction of the fiber material model.In order to minimize the influence of gravity, specimens are clamped such that the deformation initialized due to electro-thermal activation of the integrated SMA actuator takes place parallel to the ground. The specimens are clamped on one end only. To measure the deformation, a laser scanner  with a resolution of 1.0 µm and a range of 152 to 248 mm is positioned to aim at the backside of the specimen. Upon thermal activation of the SMA actuator, the CCD line sensor of the scanner captures the surface of the deformed IFRC specimen a.For V1, the voltage is set to 16 V, resulting in a current of ~2.0 A. The cycle time is set to 5 s for activation and 10 s for deactivation, with a total of five cycles.○A, both SMA wires are activated simultaneously with 14 V, resulting in a current of 2 A due to the shorter SMA wires of V2, with 5 s activation and 10 s deactivation.In mode ○B, the SMA wires are activated consecutively. The first SMA is activated for 20 s, the second SMA for 10 s, starting 10 s after activating the first. Then, both SMA are deactivated for 10 s.In mode For V2, two modes are used:The deformation angle is obtained by first extracting the laser scanner’s data of a short section closest to the free end for each time step. A straight line is then interpolated through the measured data sets and the angle of the line relative to the starting position is calculated b. A progBolts, nuts and ring washers are used to clamp the SMA wires of V1 on the fixed side of the specimen. For V2, the SMAs on the fixed side are clamped in the same way as for V1. The SMAs protruding from the free end are fixed with nuts, bolts and ring washers to a low-weight clamping plate attached to the free end .The knitted fabrics for V1 and V2 are displayed in The observable undulations are estimated as acceptable since the optimization of the mechanical properties is not the goal of the presented work. The finalized composite specimens of V1 and V2 are displayed in Since the fibers are compressed during the VARI process, the difference in thickness of LSS and HSS is negligible. The distinct amounts of reinforcement fibers in the respective plate sections rather than their geometry induce the major change in stiffness. The geometries of the resulting composite specimen are displayed in The results of four-point bending tests for V1 and V2 are displayed in All specimen variants show the expected bending deformation behavior when activated . As in , a driftIn contrast to V1.1, the variant V1.2 with higher stiffness shows significantly increased deformation angles b. The reV2 in mode A shows simultaneous activation of both SMA sections in the specimen, while mode B displays the ability to trigger two sequential deformation states by consecutive activation of the two SMA sections. The results of the two different activation modes A and B cf.  of V2 arCompared to mode B, the maximum angles achieved with mode A are about 10° lower. This effect can be explained by settling, which is typical for SMA-driven specimens, as was observed by e.g., Ashir et al. ,12. For The specimen V1.2 in turn shows higher deformation than V2, supporting the significance of uneven deformation distribution introduced in 3, two layers of 410 tex GF fibers in the weft direction). The comparison of the maximum deformation angles reveals the difference between unhinged and hinged specimens.The results of the model calibration for V1 and V2 to the four-point bending data of both variants are displayed in It is assumed that material parameters determined for V1.1 and V2 are also suitable for V1.2. The material parameters of the orthotropic material model for the reinforcement fibers obtained from the calibration are displayed in An updated set of parameters was used for the Yeoh material model of the Sylgard rubber, providing a better fit for the experimental data than the dataset used in the previous work. The parameter set is displayed in The results of the simulations for V1.1, V1.2 and V2 are displayed in The simulations of V1.1 and V2 show by factor ~2 higher deformations than the experiments. The high deviation can be explained by the edge curling present within the specimens, consuming a significant amount of usable SMA contraction. For V1.2, the simulation shows a lower deformation angle than the experiment by a factor of 0.8. It can thus be assumed that SMA transformation is not finished at the peak temperature of 110 °C used in the simulation. However, the model is unable to converge past 110 °C. A possible explanation for the large disparity of V1.2 is the modeling approach for IFRC, which may not be suited for large bending deformations above 90°. Calibration with experimental data is similarly challenging, since to our knowledge no experimental procedure exists for such large degree bending deformations. The maximum deformation angles of simulations and experiments are displayed in Flat knitting technology is well suited for producing biaxially reinforced knitted fabrics with integrated SMA wires and sectioned high and low stiffness areas, which can then be manufactured into hinged interactive fiber-rubber composites (IFRC) with active deformation capabilities. The solid-body hinges are able to increase deformation capability by concentrating contraction of the SMA wires on a smaller section of the specimen. The higher the stiffness difference between the hinge section and non-hinge section, the larger the achievable deformations. For fiber-reinforced rubbers, the difference in stiffness can be adjusted by varying the amount of fiber material. By choosing fibers with increased/reduced fineness, the fiber content can be adapted without the need for additional textile layers. The fineness of the chosen 410 tex GF material proved to be a good choice for creating high deformation specimens. In the warp direction, higher stiffness would be more feasible for reducing curling of the free edges and thus enabling larger deformations when activated. This can be achieved by using fiber material with higher fineness in the respective warp rows, again without the need to add more layers to the knitted fabric. Alternatively, edge curling can be reduced by decreasing the diameter of the turn-around curves of the SMA wire at the end of a weft row. This was shown in V1.2, where the stiffness gradient was increased by additional fibers, improving the maximum deformations.Future work will apply hinged structures to practical applications such as grippers or locomotion scenarios, with the simulation models offering a practical tool for dimensioning and estimating structural behavior in different application scenarios. Since the hinge setup also induces local stress concentrations on both the fixation areas and the hinges, damages during cyclic actuation in these areas will be another subject of future research. Here, both damages to the IFRC and fatigue effects on the SMA are of interest.


Social interaction during adolescence strongly influences brain function and behavior, and the recent pandemic has emphasized the devastating effect of social distancing on mental health. While accumulating evidence has shown the importance of the reward system in encoding specific aspects of social interaction, the consequences of social isolation on the reward system and the development of social skills later in adulthood are still largely unknown. Here, we found that 1 week of social isolation during adolescence in male mice increased social interaction at the expense of social habituation and social novelty preference. Behavioral changes were accompanied by the acute hyperexcitability of putative dopamine (pDA) neurons in the ventral tegmental area and long-lasting expression of GluA2-lacking AMPARs at excitatory inputs onto pDA neurons that project to the prefrontal cortex. Social isolation-dependent behavioral deficits and changes in neural activity and synaptic plasticity were reversed by chemogenetic inhibition of oxytocin neurons in the paraventricular nucleus of the hypothalamus. These results demonstrate that social isolation in male mice has acute and long-lasting effects on social interaction and suggest that homeostatic adaptations mediate these effects within the reward circuit. The experience of social interaction during postnatal development and adolescence is fundamental for setting the basis for social life, and the deprivation of social experience  impacts the survival of all species, as suggested by the adverse effects of the massive social isolation imposed by the COVID-19 crisis on mental health . IdentifIn rodents, depending on the duration of juvenile social isolation, increased or decreased sociability in adulthood has been reported, suggesting that adolescence is a sensitive period for establishing social behavior later in life . Few stuSocial interaction is generally a rewarding experience with reinforcing properties. Recent studies have highlighted the necessity and sufficiency of dopamine (DA) neurons of the ventral tegmental area (VTA) to promote social interaction . Brief pDA neurons of the VTA contribute to reward-seeking behavior, motivation, and reinforcement learning. Their activity is controlled upstream by several brain structures , each ofWe first characterized the acute consequences of short-term social isolation on social interaction during adolescence. Male mice were weaned at postnatal day (P) 21 and then isolated between P28 and P35 . On the To further investigate the consequences of social isolation on different aspects of social behavior, we used a three-chamber interaction task to characterize sociability and social novelty preference . We foun+/cFOS+ double-positive cells , and we observed increased excitability compared to that of the control group .To prove the causal link between oxytocin neurons and DA neuron activity during social isolation, we crossed R26-hM4Di/mCitrine mice with Oxytocin-Ires-Cre mice to express a designer inhibitor receptor-activated exclusively by designer drugs expressed under the Cre promoter in Oxt-positive cells . ClozapiWe next investigated whether social isolation during adolescence has long-lasting consequences and the consequent neural mechanisms. After 7 days of social isolation from P28 to P35, the mice were regrouped, and social behavior was then tested during adulthood . We stilNeuronal excitability in adulthood did not differ between isolated and control mice , suggestWe then tested whether the expression of CP-AMPARs on DA neurons projecting to the PFC in adulthood is the consequence of the increased excitability of oxytocin neurons in the PVN during social isolation. We isolated Oxt-hM4Di mice during adolescence, treated them with CNO or vehicle during isolation, regrouped them after 7 days until adulthood, and recorded excitatory transmission from pDA neurons projecting to the PFC from acute VTA slices . NotablyFinally, to assess the causal link between the presence of CP-AMPARs and isolation-dependent changes in social interaction, the VTA in each mouse was cannulated, and we injected a CP-AMPAR antagonist (NASPM) into the region 10 min before the direct interaction task . While NSocial isolation is an adverse experience across social species with long-lasting behavioral and physiological consequences . AlthougSocial interaction is a basic need across species, and rodents are excellent models for investigating the neural bases of social interaction and social isolation. During adolescence, social and cognitive skills depend on peer-to-peer interactions , and acuPrevious studies have indicated the role of DA neurons in the VTA in social behavior  and showFurthermore, restoration of oxytocin signaling in the VTA in an autism spectrum disorder-related mouse model was sufficient to rescue social novelty responses . Taken tWe next chose to investigate whether acute social isolation during adolescence has long-lasting consequences. Isolated mice spent more time interacting with a conspecific than control mice did 1 month after regrouping, indicating that the rebound increase in free/unrestrained social interaction in a long-lasting effect of isolation . Recently, it was reported that 2 weeks of social isolation after weaning altered the pathway from the medial PFC to the posterior paraventricular thalamus, leading to decreased sociability in adult mice . AlthougDuring adulthood, increased social interaction is accompanied and causally linked to the increase in GluA2-lacking AMPARs at excitatory inputs onto pDA neurons projecting to the PFC. GluA2-lacking AMPARs are CP ionotropic receptors with larger single-channel conductance and can undergo voltage-dependent blockade by polyamines . These rBy investigating the short-term and long-term consequences of social isolation during adolescence, our work contributes to our understanding of how social isolation impacts neural circuits and behavior. Since social isolation has a tremendous impact on mental health and increases vulnerability to psychiatric diseases, our work is relevant to identifying new therapeutic approaches to ameliorate maladaptive social behavior.tm1Ute/J, Jackson stock #026219) crossed with Oxytocin-Ires-Cre mice Dolsn/J, Jackson stock #024234) allowing thus the expression of inhibitory DREADD under Oxytocin promoter (Oxt-hM4Di mice), under 12 hr light-dark cycle (7 a.m. to 7 p.m.) with free access to food and drink. Behavioral experiments were conducted in a room with fixed low illumination (10–15 lux) and with controlled humidity (40%) and temperature (22–24°C). The experiments were always performed within a time frame that started approximately 1.5 hr after the end of the dark cycle and ended 2 hr before the start of the next dark cycle. For adolescence social isolation, mice were weaned at P21 and kept in groups until P28. Subsequently, mice were single used until P35. For 24-hr isolation, mice were single used between P34 and P35. For social isolation in adulthood, mice were kept in groups until P53 and single-housed until P60. For the long-lasting effect of SI, mice were singly housed between P28 and P35 and subsequently regrouped until P60. When group-housed, mice were housed two per cage. The experimental mice were randomly assigned to the different groups. All the procedures performed at UNIGE complied with the Swiss National Institutional Guidelines on Animal Experimentation and were approved by the Swiss Cantonal Veterinary Office Committees for Animal Experimentation.The study was conducted with male wild-type (WT), and R26-hM4Di/mCitrine mice (B6.129-Gt(ROSA)26Sor2 with transparent wall), and subsequently an unfamiliar sex-matched conspecific social stimulus  or an object was introduced, and the interaction time was recorded for 5 min. For the social interaction with a former cage mate, the arena was divided into two parts with a wall, and the experimental mouse was put on one side while the stimulus was added on the other side. After 10 min, the wall was removed, and the mice were free to interact. We used this modified protocol to reduce the number of used mice because in this way, we could have two interaction score values simultaneously for the grouped mice. All the trials were recorded with a camera placed above the arena. The interaction time was manually scored when the experimental mouse initiated the action and when the nose of the animal was oriented toward the social stimulus mouse only or toward the object.Mice were allowed to freely explore the arena for 10 min   divided into three chambers (each 20×40×22 (h) cm3). Each mouse was placed in the arena for a habituation period of 10 min when it was allowed to explore the empty arena freely. At the end of the habituation, two enclosures with vertical metal bars were placed in the center of the two outer chambers. One enclosure was empty (serving as an object), whereas the other contained a social stimulus . The experimental mouse was allowed to freely explore the apparatus and the enclosures for 10 min . Subsequently, the empty enclosure was replaced with another containing an unfamiliar conspecific social stimulus (stimulus 2). The experimental mouse was allowed to freely explore the apparatus for 10 min and the enclosures for another 10 min . The position of the empty versus social stimulus 1-containing or social stimulus 1-containing versus social stimulus 2-containing enclosures alternated and was counterbalanced for each trial to avoid any bias effects. Every session was video-tracked and recorded using ANY-maze , which provided an automated recording of the time in the compartment, and the distance moved. The time spent interacting with each enclosure was manually scored and then used to determine the preference index for the object or social target (stimulus 1 and stimulus 2). The stimulus interaction was scored when the nose of the experimental mouse was oriented toward the enclosures at a distance of approximately less than 2 cm. The arena was cleaned with 1% acetic acid solution and dried between trials. For the analysis, we calculated the ‘Preference index’ using the following formula: interaction time target 1/(interaction time target 1+interaction time target 2) or target 2/(interaction time target 1+interaction time target 2). Using this formula, the threshold is 0.5, which corresponds to the chance level to explore either target.A three-chambered social interaction assay was used, comprising a rectangular Plexiglas arena . For the long habituation task protocol, the experimental mouse was placed in the arena with a novel social stimulus . The animals were left freely to explore the cage and interact with each other for 15 min. At the end of the trial, the experimental and stimulus mice were returned to their homecage. For 4 consecutive days, the experimental mouse was exposed to the same social stimulus. For the short habituation task protocol, the experimental mouse was placed in the arena for 15 min. After a novel social stimulus  was introduced. The animals were left freely to interact with each other for 2 min. At the end of the trial, the stimulus mice were returned to their homecage, while the experimental mouse stayed in the arena alone for 5 min. At the end of the inter-trial interval, the same stimulus mouse was returned in the arena for another 2 min. The exposure was repeated four times in a row. All the trials were recorded with a camera placed above the arena. Non-aggressive behavior was manually scored when the experimental mouse initiated the action and when the nose of the animal was oriented toward the social stimulus mouse only.A clean homecage was used as an arena  for 10 min. During the familiarization phase, the animal was exposed to two identical objects (object 1 – object 2) and was let to interact for 10 min with both freely. After a retention delay of 20 min, the mice were exposed for 10 min to one of the familiar objects (object 1), while the other was replaced with a novel object (object 3). During the different phases of the test, the objects were placed on the opposite sides of the cage, alternating the position of the respective objects. Every session was video-tracked and recorded using ANY-maze . The time spent interacting with each object was manually scored and then used to determine the preference index for the different objects. The stimulus interaction was scored when the nose of the experimental mouse was oriented toward the objects at a distance of approximately less than 2 cm. The arena was cleaned with 1% acetic acid solution and dried between trials. For the analysis, we calculated the ‘Preference index’ using the following formula: interaction time target 1/(interaction time target 1+interaction time target 2) or target 2/(interaction time target 1+interaction time target 2). Using this formula, the threshold is 0.5, which corresponds to the chance level to explore either one target.A squared arena was used for the task, consisting of three phases: a first habituation phase followed by a familiarization and the actual test phase. During the habituation phase, the experimental mouse is let to freely explore the arena (40×40×40 cmThe elevated plus maze (EPM) consisted of a platform of four opposite arms (40 cm), two of them are open, and the other two are closed . The apparatus was elevated at 55 cm from the floor. Each male adult mouse was placed individually in the center of the EPM apparatus with the snout facing one of the open arms and was filmed for 5 min. Distance moved (cm) and time spent in the open and closed arms (s) of the arena were measured with ANY-maze . The apparatus was cleaned with 1% acetic acid solution and dried between trials between each session. For the analysis, we calculated the ‘Preference index’ using the following formula: interaction time target 1/(interaction time target 1+interaction time target 2) or target 2/(interaction time target 1+interaction time target 2). Using this formula, the threshold is 0.5, which corresponds to the chance level to explore either one target.2) with either gray stripes on white background or black dots on white background. The floor in each of the two chambers has different distinct textures. The two chambers are interconnected by a small corridor, with transparent walls and floor. The task is divided into three phases: Day 1, 15 min pre-TEST in which the experimental mouse is free to explore the entire apparatus; Days 2–5 conditioning phase (30 min per day for 4 consecutive days); Day 6, 15 min post-TEST, in which the experimental mouse is left freely to explore the apparatus in absence of stimuli. For the conditioning phase, one chamber was randomly assigned as the paired session chamber, with the presence of an unfamiliar conspecific mouse  and the other as the non-social session chamber . During the conditioning, for each day, the experimental mouse was left in the social chamber or in the non-social chamber for 5 min each session, alternating for six times in the two chambers (30 min of conditioning). During each conditioning session, the experimental mice were allowed to freely interact with social stimuli that remained the same one for the entire conditioning phase . The behavior of the animals was tracked automatically with the ANY-maze software , and the time spent in the two chambers was recorded for the pre- and post-TEST sessions. Preference index for social chamber was calculated as: time spent in the social chamber divided by the total time spent in the two chambers.The apparatus consists of two square-shaped chambers  containing: 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl2, 2.5 mM CaCl2, 1.0 mM NaH2PO4, 26.2 mM NaHCO3, and 11 mM glucose, bubbled with 95% O2 and 5% CO2 at room temperature. Whole-cell voltage-clamp or current-clamp electrophysiological recordings were conducted at 35–37° in aCSF . Recording pipette contained the following internal solution: 140  mM K-Gluconate, 2  mM MgCl2, 5  mM KCl, 0.2  mM EGTA, 10  mM HEPES, 4  mM Na2ATP, 0.3  mM Na3GTP, and 10  mM creatine-phosphate. The cells were recorded at the access resistance from 10 to 30 MΩ. Resting membrane potential (in mV) was read using the Multiclamp 700B Commander (Molecular Devices) while injecting no current (I=0) immediately after breaking into a cell. Action potentials (APs) were elicited in current-clamp configuration by injecting depolarizing current steps  from 0 to 400 pA in presence.Horizontal midbrain slices 200-μm thick containing the VTA or coronal midbrain slices 250-μm thick containing PVN were prepared. Brains were sliced by using a cutting solution containing: 90.89 mM choline chloride, 24.98 mM glucose, 25 mM NaHCOAR antagonist picrotoxin (100 µM). Recording pipette contained the following internal solution: 130 mM CsCl, 4 mM NaCl, 2 mM MgCl2, 1.1 mM EGTA, 5 mM HEPES, 2 mM Na2ATP, 5 mM sodium creatine phosphate, 0.6 mM Na3GTP, 0.1 mM spermine, and 5 mM lidocaine N-ethyl bromide (QX-314). Access resistance (10–30 MΩ) was monitored by a hyperpolarizing step of –4 mV at each sweep, every 10 s. Data were excluded when the resistance changed >20%. The AMPA/NMDA was calculated by subtracting the mixed EPSC (+40 mV), the non-NMDA component isolated by D-APV (50 µM at +40 mV) bath application. The ratio values may be underestimated since it was calculated with spermine in the pipette. The RI of AMPARs is the ratio of the chord conductance calculated at a negative potential (–60 mV) divided by the chord conductance at positive potential (+40 mV). For sIPSCs were recorded from VTA pDA neurons in the presence of 3 mM kynurenic acid. The patch pipettes were filled with (in mM): 30 K-gluconate, 100 KCl, 10 creatin-phosphate, MgCl2, 3.4 Na2ATP, 0.2 Na3GTP, 1.1 EGTA, 5 HEPES, pH adjusted to 7.3 with NaOH, osmolarity to 289 mOsm. All the synaptic responses were collected with a Multiclamp 700B-amplifier , filtered at 2.2  kHz, digitized at 5  kHz, and analyzed online using Igor Pro software .For VTA excitability, pDA neurons were identified accordingly to their position , morphology (large soma), and cell capacitance -Alexa Fluor 488 (Thermo Fisher Scientific #C34775) conjugated were performed in WT and Oxt-hM4Di mice at P21 or P45–P50. Mice were anesthetized with a mixture of oxygen (1 L/min) and isoflurane 3%  and placed in a stereotactic frame . The skin was shaved, locally anesthetized with 40–50 µl lidocaine 0.5%, and disinfected. Unilateral or bilateral craniotomy (1 mm in diameter) was then performed at following stereotaxic coordinates: NAc ML ±0.85 mm, AP +1.3 mm, DV –4.5 mm from Bregma; mPFC (four injection site) position 1 ML ±0.27 mm, AP +1.5 mm, DV −3, –25 mm, position 2 ML ±0.27 mm, AP +1.75 mm, DV −3, –2.5 mm, position 3 ML ±0.27 mm, AP +2 mm, DV −2.6, –2.2 mm, position 4 ML ±0.27 mm, AP +2.25 mm, DV –2 mm from Bregma; VTA AP –3, ML ±0.5, DV –4.3 from bregma. The CTB was injected via a glass micropipette  either into the NAc, mPFC, and VTA at the rate of 100 nl/min for a total volume of 200 nl on each side. For NASPM experiments, unilateral implantations of stainless steel 26-gauge cannula  were performed on WT mice at P54. Mice were anesthetized and placed in a stereotactic frame as previously described. Unilateral craniotomy (1 mm in diameter) was performed over the VTA at the following stereotactic coordinates: ML: ± 0.9 mm, AP: –3.2 mm, DV: –3.95 mm from Bregma. The cannula was implanted at a 10° angle, placed above the VTA, and fixed on the skull with dental acrylic. A removable cap protected the cannula. All animals underwent behavioral experiments 1–2 weeks after surgery.Isolated or grouped mice were treated for 1 week with Clozapine N-oxide . CNO was dissolved in the drinking water at 5 mg/200 ml in 4% sucrose and 0.2% saccharin solution. Mice received either CNO solution or sugar solution only as control. The solutions were prepared fresh daily. For the acute effects of SI, after 1 week of treatment, mice underwent a direct free interaction task or were used for whole-cell patch-clamp recordings. For the long-lasting effect of SI, CNO treatment was stopped at P35, mice were regrouped until P60, and normal water was given. Mice underwent direct free interaction tasks or were used for whole-cell patch-clamp recordings. For the experiments with 1-Naphthylacetyl spermine trihydrochloride (NASPM), mice were infused using a Minipump injector . Ten minutes before each trial, mice were either infused with four µg of NASPM dissolved in 500 L of aCSF  or aCSF only (vehicle). After infusion, mice underwent to direct free interaction task.For the staining in For the staining in Tissue images of PVN were acquired using a confocal laser-scanning microscope LSM700 (Zeiss). Images were analyzed with ImageJ software. The region of interest was delimited around PVN, and the number of Oxytocin, cFOS, and AVP positive cells were counted manually for each slice using separate color channels. Subsequently, channels merge images were created for Oxytocin-cFOS, Oxytocin-AVP, and AVP-cFOS, and double-positive cells were manually counted for each merged image.Horizontal midbrain slices 200-μm thick containing the VTA were prepared as described before. Whole-cell voltage-clamp electrophysiological recordings were conducted at 35–37° in aCSF . Putative VTA-DA neurons were identified for larger soma compared to the other cells and a capacitance >28 pF. Cells with capacitance lower than 27 pF were not considered putative-DA neurons . RecordiStatistical analysis was conducted with GraphPad Prism 9 . Statistical outliers were identified with the ROUT method  (Q=1) an This paper evaluated the lasting effects of acute social isolation on future social interactions in juvenile mice, revealing a compelling oxytocin-mediated mechanism. A clear hypothesis has been laid out within a defined anatomical framework, and social interactions were evaluated using appropriate behavioral paradigms, chemogenetic, and pharmacological tools. The work provides new insights on oxytocin signaling as a key regulator of the neural substrates underlying enduring effects of social interaction. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) 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 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kate Wassum as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Michy Kelly (Reviewer #1).Thank you for submitting your article "Oxytocin neurons control the effects of social isolation via the mesocortical pathway" 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. In addition to addressing the concerns raised in the public review, please address each of the essential revisions provided below.Essential revisions:1) All reviewers noted that the lack of females profoundly lessen the impact of the findings. Ideally, all experiments would need to be conducted in females. However, the reviewers understand that this may not be feasible depending on the availability of animals and staffing levels. If this is the case, it is strongly recommended that the authors at the least acknowledge this significant shortcoming and add extensive discussion related to the effect of oxytocin in females .2) There was consensus among the reviewers that the statistical analyses are not appropriate for the experimental design and this also needs to be addressed thoroughly. In particular:a. Parametric statistics are based on assumptions of normality and equal variance, yet only a test of normality is mentioned and only for t-tests and one way ANOVAs. It is stated that normality was assumed for datasets requiring multifactorial ANOVAs. Please assess each dataset for both normality and equal variance before using parametric statistics. 2way ANOVAs frequently fail one or the other assumption, thus necessitating the employ of multiple non-parametric tests to assess the various factors . This is a particular concern because in many datasets, variance appears to systematically increase with increasing mean .b. Throughout the manuscript, 2-way ANOVAs are reported for behavioral tasks where 2-way repeated measure ANOVAs should have been used . In the ephys data, 2 WAY RM ANOVA were used as appropriate; however, it is surprising there was not a significant interaction for many of those experiments  given the distinct overlap at "0". Was the "0" data not included in the RM ANOVA?c. Throughout the manuscript, only main effects are reported when a significant interaction between effects would be required for the post hoc testing conducted and conclusions drawn. For example, in Figure 1G, the main effect of chamber that is reported says that across the experimental groups, novel > familiar. To statistically make a conclusion that the group vs isolated mice performed differently, you would need a significant interaction between chamber x housing condition to justify the post hoc tests examining the effect of chamber within group housed and chamber within isolated mice. In Figure 1J as well, you are trying to conclude the housing conditions perform differently across days. But to say that, the 2way rm would need to yield a significant interaction of housing condition x day to warrant a post hoc analysis of days within group housed and days within isolated. Again, this is not limited to Figure 1, I just use them here as examples.d. Many 2-way ANOVAs have fractional df's reported. In the case of linear models, it is my understanding df's should be whole numbers, so it is not clear why dfs are presented in many 2way analyses as decimals . I believe in more complicated analyses, unequal variances can yield fractional df's, but in the case of unequal variance, a non-parametric statistic should be employed. Would you please clarify or revise?e. The method of data presentation in Figure 1 S2 is not clear. Typically, "preference index" is a single number that incorporates relative preference for a novel object as a function of total time of exploration (=n-f/n+f). I do not believe it would be statistically appropriate to compare normalized data for ob1 vs ob2 because the combined value of both factors would be the same between groups . The more typical way of analyzing these data is to compare the preference index for the novel object between the group vs. isolated mice to determine if there is a group effect, and then to conduct a one-sample t-test for each group's index vs.5 to determine if they exhibited significant memory . The authors may refer to the following paper for guidance:https://www.sciencedirect.com/science/article/pii/S1074742714000070. If you were to analyze the raw seconds of exploration, then you could do a paired analyses of O1 vs O2, that said, the analysis should be a 2factor RM with housing condition vs object as factors. The same applies to the analyses of closed vs open arms.f. Legend for Figure 3L is missing the post hoc analyses to say which group is different from which. Further, it seems equal variance could possibly fail given that variability increases in parallel with the increases mean .g. In Figure 3 S1B, was there not a significant interaction? It seems there is an effect of increasing intensity within the no CNO group, but not the CNO group. Note that a significant interaction overrides main effects as it states that the effect of one factor depends on the level of the other factor.3) It is not clear how oxytocin neurons were identified in Figure 3 S1. With current details provided, it appears that recordings of all PVN neurons (non-oxytocin neurons as well) were obtained in mice; please clarify. Moreover, cFOS+ and Oxt+ coexpression images are needed to relate the increase in the activity of PVN (via the IEG proxy) and the density of Oxt+ cells in PVN . Also, please clarify whether neurons are increasing Oxt production to become more efficiently labeled or changing identity of their synthesized neuropeptides?4) Although it is reasonable to suggest that the lack of preference for the novel mouse and the failure to habituate to a mouse could reflect a reduced preference for novelty . This could also/alternatively reflect a social learning and/or memory deficit (they fail to form a memory of the first-presented mouse). To disentangle these seemingly opposite consequences, typically habituation sessions are conducted for a longer period of time and behavior examined across time within the same day .5) The extensive and definitive use of the term "social craving" throughout is unjustified based on the data presented . Craving implies having experienced something that is rewarding and wanting more of it; however, these mice never experienced any social interactions with non-cagemates, so how can they crave it? To this end, it might be informative to test these behaviors using cage mates as stimulus mice.6) Regarding the statement "Indeed, we could speculate that social isolation during adolescence results in an intense urge to interact with whatever conspecific is present." This can be tested in a straightforward fashion by using cage mates as stimulus mice, or measuring interactions in the home cage, instead of using novel mice.7) It is important to clarify why stimulus mice were 1 week younger and whether this is this true for adolescent and adult studies? This is of particular relevance because in the adult study illustrations, a small (seemingly juvenile) mouse is drawn--which would suggest much more than 1 week younger in the case of testing adults. If adults were tested with juvenile mice, as the illustration implies, it complicates interpretation of the adult isolation study since the differential effect of isolation may not be related to the period of isolation but rather a fundamentally different preference between adolescent and adult mice for other juvenile mice.8) The authors identified putative DA neurons based on position, morphology, and capacitance, while providing sparse details in the methods. This section should contain detailed information and/or a small post hoc immunohistochemistry validated dataset.9) The authors have exclusively shown scaled-up excitatory responses by pharmacologically blocking GABAA receptors, determining synaptic scaling after social isolation without considering potential effects on inhibitory post synaptic responses. Please provide prediction for the latter. Furthermore, it is not clear if synaptic scaling reported in Figure 5 induced by adolescent isolation only emerges slowly in the adult, or it is already acutely happening at the end of isolation at p35. Please discuss in detail.10) It is unclear how oxytocin neuron activity contributes to synaptic plasticity onto VTA-NAc projections. One possibility is that oxytocin expressing PVN neurons projecting to VTA also release glutamate and contribute to the synaptic plasticity. It would be informative to optogenetically examine the synaptic connectivity between PVN->VTA projections and VTA->NAc projection neurons.eLife. Your revised article has been evaluated by Kate Wassum (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Oxytocin neurons mediate the effect of social isolation via the VTA circuits" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:1) Please acknowledge the lack of females as a shortcoming of the study and provide more discussion of this limitation; e.g., that differences in the system in males vs females and how these findings here may only apply to the male brain. Please also state the sex of the subjects in the abstract.2) The post hoc analyses referred to in 2f of the letter are still missing. The statistical interaction was reported, but the post hoc tests were not, nor were significance markers added to the figure to indicate at which pA the groups differ.3) Please make sure all statistics are reported. For instance, in some cases a 2 way ANOVA was used but only one main effect is reported.https://reviewer.elifesciences.org/author-guide/full "Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05."4) As isolated mice show increased social interaction during direct social interaction, but not during the 3 chamber sociability assay, it is important to more thoroughly explore why isolated mice show different outcomes between these two tests. To better understand the cause of increased direct social interaction , it would be informative to conduct additional behavioral testings from isolated mice . If new behavior testing data such as social CPP or aggression assay (resident intruder etc) is not possible, at the very least a more detailed behavioral characterization such as chasing during free social interaction test is needed to address this concern and better distinguish craving vs aggressive encounter. Overall, more careful consideration is required to interpret the nature social behavior in isolated mice captured by free social interaction and 3 chamber task.5) The response to essential review 2c to statistically compare between groups was not fully addressed. [Results] page 1, lines 18-19 and Figure 1EF. To evaluate if the house condition modifies the sociability, it is essential to run these statistics for Figure 1HI. Related to this, the 3 chamber testing data should be presented and analyzed in a similar way across different sets of data. E.g., Figure 1 HI are missing center chamber time and the preference index data while all the rest of the 3 chamber data include these.In general, more clarity on whether or not the 3-chamber social preference test results do or do not support the conclusion of social isolation leading to an increase in social interaction would help. E.g., by clarifying whether isolation leads to an increase in social interaction specifically in "free/unrestrained"social interactions in adulthood. Essential revisions:1) All reviewers noted that the lack of females profoundly lessen the impact of the findings. Ideally, all experiments would need to be conducted in females. However, the reviewers understand that this may not be feasible depending on the availability of animals and staffing levels. If this is the case, it is strongly recommended that the authors at the least acknowledge this significant shortcoming and add extensive discussion related to the effect of oxytocin in females .We thank the reviewers for this comment. We agree that results from the female cohort would increase the impact of the findings; however, these experiments would take a considerable amount of time and be a matter of future study. As suggested by the reviewers, we have now added a comment about this point (p 9 lines 8 to 15)2) There was consensus among the reviewers that the statistical analyses are not appropriate for the experimental design and this also needs to be addressed thoroughly. In particular:We apologize for the inappropriate statistical analysis. In the new version of the paper we have revised all the statistical analysis and for each “Source data” file, we have now added the output of all statistical tests that were used.a. Parametric statistics are based on assumptions of normality and equal variance, yet only a test of normality is mentioned and only for t-tests and one way ANOVAs. It is stated that normality was assumed for datasets requiring multifactorial ANOVAs. Please assess each dataset for both normality and equal variance before using parametric statistics. 2way ANOVAs frequently fail one or the other assumption, thus necessitating the employ of multiple non-parametric tests to assess the various factors . This is a particular concern because in many datasets, variance appears to systematically increase with increasing mean .We thank the reviewer for the comment. We now used a parametric test only if both normality and equal variance were not violated in the revised statistical analysis. If the contrary, we used a non-parametric test. Regarding multifactorial ANOVAs, we first checked normality, and all the analysis were then performed assuming no sphericity, which implies Geisser-Greenhouse correction. We have now included these details in Material and methods, and all the stats have been upgraded in the figures and legends.b. Throughout the manuscript, 2-way ANOVAs are reported for behavioral tasks where 2-way repeated measure ANOVAs should have been used . In the ephys data, 2 WAY RM ANOVA were used as appropriate; however, it is surprising there was not a significant interaction for many of those experiments  given the distinct overlap at "0". Was the "0" data not included in the RM ANOVA?We apologize for the mistake, and we have now used the appropriate statistical test for each dataset. In all the figures we have now reported the F value for the “interaction” between variables, and as written before, each “Data source” file contains all statistical analysis outputs.c. Throughout the manuscript, only main effects are reported when a significant interaction between effects would be required for the post hoc testing conducted and conclusions drawn. For example, in Figure 1G, the main effect of chamber that is reported says that across the experimental groups, novel > familiar. To statistically make a conclusion that the group vs isolated mice performed differently, you would need a significant interaction between chamber x housing condition to justify the post hoc tests examining the effect of chamber within group housed and chamber within isolated mice. In Figure 1J as well, you are trying to conclude the housing conditions perform differently across days. But to say that, the 2way rm would need to yield a significant interaction of housing condition x day to warrant a post hoc analysis of days within group housed and days within isolated. Again, this is not limited to Figure 1, I just use them here as examples.We have now modified the statistical analysis according to what we have recently published :3199-3211. doi: 10.1111/ejn.15179). Since the aim of the 3-chamber task is to evaluate if a mouse prefers a social stimulus over an object and a novel social stimulus over a familiar one, we decided to perform a stand-alone analysis for each group of mice. In particular, we used paired t-test analysis for interaction time , One-way RM ANOVA for time in the chamber , and unpaired samples t-test for the distance moved . The 3-chamber task for Figure1-Supplement 2 and Figure4-Supplement 1 were analyzed in the same way.Regarding the habituation task , we now have included the interaction effect for the 2-way RM ANOVA and we added new panels comparing the interaction time between DAY1 (or TRIAL1) and DAY4 (or TRIAL4).d. Many 2-way ANOVAs have fractional df's reported. In the case of linear models, it is my understanding df's should be whole numbers, so it is not clear why dfs are presented in many 2way analyses as decimals . I believe in more complicated analyses, unequal variances can yield fractional df's, but in the case of unequal variance, a non-parametric statistic should be employed. Would you please clarify or revise?We thank the reviewer for this comment. The presence of fractional df’s is due to the Geisser-Greenhouse correction. We now added all the statistical details in the material and methods section.e. The method of data presentation in Figure 1 S2 is not clear. Typically, "preference index" is a single number that incorporates relative preference for a novel object as a function of total time of exploration (=n-f/n+f). I do not believe it would be statistically appropriate to compare normalized data for ob1 vs ob2 because the combined value of both factors would be the same between groups . The more typical way of analyzing these data is to compare the preference index for the novel object between the group vs. isolated mice to determine if there is a group effect, and then to conduct a one-sample t-test for each group's index vs.5 to determine if they exhibited significant memory . The authors may refer to the following paper for guidance:https://www.sciencedirect.com/science/article/pii/S1074742714000070. If you were to analyze the raw seconds of exploration, then you could do a paired analyses of O1 vs O2, that said, the analysis should be a 2factor RM with housing condition vs object as factors. The same applies to the analyses of closed vs open arms.We thank the reviewer for the comment; however we would like to point out that using “n/(n+f)” (the one that we use in our paper) or “(n-f)/(n+f)” as preference index will give the same statistically results. Indeed, the latter is a mathematical transposition of the first equation. However, since a mouse has a 50% of chance to explore one or the other object, we believe that our way to calculate preference index is more intuitive because if a mouse explores for the same amount of time the two objects, the preference index will be equal to 0.5, which correspond to the chance level. Our analysis aimed to demonstrate that both groups of mice were able to prefer a novel object over a familiar one and not to state that one group performed better compared to the other. Indeed, we first show no bias toward one of the two objects during the familiarization phase. Certainly, the paired t-test between the preference index for objects one and two is not significantly different. Our analysis mathematically gives the same result as calculating a one-sample t-test against the chance level. Since the social novelty was impaired in the three-chamber task in social isolated mice, our analysis of the NORT wants to determine if isolated mice discriminate a novel versus a familiar object and not compare performances between groups. We applied the same analysis for the EPM.f. Legend for Figure 3L is missing the post hoc analyses to say which group is different from which. Further, it seems equal variance could possibly fail given that variability increases in parallel with the increases mean .We revised the statistical analysis, and we decided to use a three-way RM ANOVA since we have three variables . Moreover, we slightly increase the n for some groups to have a more homogeneous number.g. In Figure 3 S1B, was there not a significant interaction? It seems there is an effect of increasing intensity within the no CNO group, but not the CNO group. Note that a significant interaction overrides main effects as it states that the effect of one factor depends on the level of the other factor.We are sorry for the lack of information. We now added all the statistical analysis details.3) It is not clear how oxytocin neurons were identified in Figure 3 S1. With current details provided, it appears that recordings of all PVN neurons (non-oxytocin neurons as well) were obtained in mice; please clarify. Moreover, cFOS+ and Oxt+ coexpression images are needed to relate the increase in the activity of PVN (via the IEG proxy) and the density of Oxt+ cells in PVN . Also, please clarify whether neurons are increasing Oxt production to become more efficiently labeled or changing identity of their synthesized neuropeptides?We are sorry for the lack of information in the previous version of the manuscript. We have now added in the material and methods section the relative details. The OXT cells expressing hM4Di channel can be recognized thanks to the mCitrine reporter present on the hM4Di transgene Thus, in new Figure 3S1 J-L, we report excitability recorded from identified OXT neurons. Moreover, in the new figure 3-S1, we now added the immunohistochemical validation of the OXT-hM4Di mouse line (new figure 3-S1I).For the cFOS/OXT analysis, we have now repeated the experiment, and we performed triple staining with three different antibodies against cFOS, OXT, and AVP, and analyzed the relative and absolute expression of the three proteins as well as the co-expression of cFOS-OXT, cFOS-AVP and OXT-AVP . Our new analysis shows an increase in the absolute number of OXT+ and cFOS + cells in isolated mice and an increase in the double-positive cells (OXT+ /cFOS + cells). Furthermore, since no differences were found in AVP expression or OXT-AVP colocalization, the results suggest that PVN neurons from isolated mice produced oxytocin more efficiently.4) Although it is reasonable to suggest that the lack of preference for the novel mouse and the failure to habituate to a mouse could reflect a reduced preference for novelty . This could also/alternatively reflect a social learning and/or memory deficit (they fail to form a memory of the first-presented mouse). To disentangle these seemingly opposite consequences, typically habituation sessions are conducted for a longer period of time and behavior examined across time within the same day .We thank the reviewer for the comment. To prove that social isolation does not induce social learning or memory deficits, we performed another habituation task presenting the same social stimulus for 4 consecutive trials for 2 minutes with an intertrial interval of 5 minutes. We scored the interaction time for each trial. We observed that both Grouped and Isolated mice habituate to the same social stimulus with these settings. Indeed, the interaction time on Trial 4 is statistically different from Trial 1 for both groups (new figure 1-S1I-M). These results demonstrate that isolated mice can form short-time memory of the social stimulus while long term memory is impaired (new figure 1I-M)5) The extensive and definitive use of the term "social craving" throughout is unjustified based on the data presented . Craving implies having experienced something that is rewarding and wanting more of it; however, these mice never experienced any social interactions with non-cagemates, so how can they crave it? To this end, it might be informative to test these behaviors using cage mates as stimulus mice.6) Regarding the statement "Indeed, we could speculate that social isolation during adolescence results in an intense urge to interact with whatever conspecific is present." This can be tested in a straightforward fashion by using cage mates as stimulus mice, or measuring interactions in the home cage, instead of using novel mice.We thank the reviewer for the comment. We now performed free-social interaction after social isolation using former cage mates as stimuli as suggested by the reviewer (new figure 1E-F). We found that isolated mice interact more compared to grouped mice even when stimulus mice are former cage-mate.7) It is important to clarify why stimulus mice were 1 week younger and whether this is this true for adolescent and adult studies? This is of particular relevance because in the adult study illustrations, a small (seemingly juvenile) mouse is drawn--which would suggest much more than 1 week younger in the case of testing adults. If adults were tested with juvenile mice, as the illustration implies, it complicates interpretation of the adult isolation study since the differential effect of isolation may not be related to the period of isolation but rather a fundamentally different preference between adolescent and adult mice for other juvenile mice.We thank the reviewer for the comment. As stated in the material methods, stimuli mice are always 1-week younger compared to the experimental mice thus they are P28 for adolescent experimental mice, and P50-53 for adult experimental mice. We have now modified the illustration in figure 1-S2B to be more in line with the age of the mice.8) The authors identified putative DA neurons based on position, morphology, and capacitance, while providing sparse details in the methods. This section should contain detailed information and/or a small post hoc immunohistochemistry validated dataset.We are sorry for the lack of information. We have provided all the details now in the material and method session. Moreover, we now added a post-hoc immunohistochemistry validation (new figure 5 – Supplement 2)9) The authors have exclusively shown scaled-up excitatory responses by pharmacologically blocking GABAA receptors, determining synaptic scaling after social isolation without considering potential effects on inhibitory post synaptic responses. Please provide prediction for the latter. Furthermore, it is not clear if synaptic scaling reported in Figure 5 induced by adolescent isolation only emerges slowly in the adult, or it is already acutely happening at the end of isolation at p35. Please discuss in detail.We thank the reviewer for the comment. We have now performed new experiments and patched DA neurons projecting the PFC or NAc obtained from adolescent mice immediately after the social isolation. We found an increase in rectification index when recoding from DA neurons projecting to PFC at P35 (new figure 5-supplement 1A-K). Moreover, we now recorded sIPSCs from VTA DA neurons, and we found no differences in amplitude, frequency, rise, and decay time of the inhibitory postsynaptic currents, suggesting thus that inhibitory responses are not affected by adolescent social isolation.10) It is unclear how oxytocin neuron activity contributes to synaptic plasticity onto VTA-NAc projections. One possibility is that oxytocin expressing PVN neurons projecting to VTA also release glutamate and contribute to the synaptic plasticity. It would be informative to optogenetically examine the synaptic connectivity between PVN->VTA projections and VTA->NAc projection neurons.We thank the reviewer for the comment. As suggested by the reviewer, we injected ChR2 in the PVN at PND5 and CholeraToxin in the mPFC at PND21. We then isolated the mice for seven days (P28 and P35), and we recorded from DA neurons projecting to the PFC while optogenetically stimulating PVN inputs in slices. Unfortunately, the injection of ChR2 in the PVN at PND5 resulted very challenging and we were not able to measure any current due to the lack of infection of the PVN.1) Please acknowledge the lack of females as a shortcoming of the study and provide more discussion of this limitation; e.g., that differences in the system in males vs females and how these findings here may only apply to the male brain. Please also state the sex of the subjects in the abstract.We thank the reviewer for the comment. We have now clearly stated in the abstract that we used only male, and we add a discussion about the limitation of the study in the discussion session (lines265-275).2) The post hoc analyses referred to in 2f of the letter are still missing. The statistical interaction was reported, but the post hoc tests were not, nor were significance markers added to the figure to indicate at which pA the groups differ.We are sorry for the lack of information. All the statistical parameters are now reported in the legend. Post-hoc analyses have now been reported. For figure 3K (former figure 3L) we reported in the figure the post-hoc analysis of isolated group versus all other groups while reported in the source file all the rest of the comparison.3) Please make sure all statistics are reported. For instance, in some cases a 2 way ANOVA was used but only one main effect is reported. https://reviewer.elifesciences.org/author-guide/full "Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05."We are sorry for the lack of statistical details that we have now added all the statistical information in the revised version of the manuscript. Moreover, all the statistical data are also reported in the source files.4) As isolated mice show increased social interaction during direct social interaction, but not during the 3 chamber sociability assay, it is important to more thoroughly explore why isolated mice show different outcomes between these two tests. To better understand the cause of increased direct social interaction , it would be informative to conduct additional behavioral testings from isolated mice . If new behavior testing data such as social CPP or aggression assay (resident intruder etc) is not possible, at the very least a more detailed behavioral characterization such as chasing during free social interaction test is needed to address this concern and better distinguish craving vs aggressive encounter. Overall, more careful consideration is required to interpret the nature social behavior in isolated mice captured by free social interaction and 3 chamber task.(30)=2.167, p=0.0383).We thank the reviewer for the comment. As suggested, we performed the social conditioning place preference task after isolation. The new results  show that both Grouped and Isolated mice show preference for the compartment paired with social interaction. Concerning figure 1H, since the ANOVA does not show interaction for social preference, we are not allowed to perform multiple comparison. However, we would like to report that if we perform an unpaired t-test between interaction time stimulus 1 for grouped and isolated mice, we found a significant difference (t5) The response to essential review 2c to statistically compare between groups was not fully addressed. [Results] page 1, lines 18-19 and Figure 1EF. To evaluate if the house condition modifies the sociability, it is essential to run these statistics for Figure 1HI. Related to this, the 3 chamber testing data should be presented and analyzed in a similar way across different sets of data. E.g., Figure 1 HI are missing center chamber time and the preference index data while all the rest of the 3 chamber data include these.In general, more clarity on whether or not the 3-chamber social preference test results do or do not support the conclusion of social isolation leading to an increase in social interaction would help. E.g., by clarifying whether isolation leads to an increase in social interaction specifically in "free/unrestrained"social interactions in adulthood.We thank the reviewer for the comment. The data regarding the time in chamber and preference index for figure 1 were already present in Figure 1 – supplementary 1. Since figure 1 has many panels, we decided to include in the main figure only the interaction time.In regards of the statistical analysis for the three-chamber test throughout the manuscript, in this new version of the manuscript we revised all the statistic such as:– For the interaction time, we first performed a paired t-test within single groups. This statistical test is important for the sociability test to verify that control group prefer stimulus over object. Indeed, the absence of sociability in this group would drop all conclusions. As now requested by the reviewer, we also performed RM 2-way ANOVA. Now all the statistical analysis have been reported in the figures, legends, and source data files. Multiple comparisons were made when interaction between variables was significant.– For time in chamber, One-way RM ANOVA– For preference index, One-sample t-test against chance level.We now show that during the social novelty phase (figure 1I) there is an interaction between the house condition and the interaction time confirming that social isolation induces an alteration of the social novelty preference.Moreover, as suggested, we added in the text “in free/unrestrained social interaction” to better clarify the effect of social isolation in adulthood.


Background: Lateral flow immunoassays (LFIAs) are able to achieve affordable, large scale antibody testing and provide rapid results without the support of central laboratories. As part of the development of the REACT programme extensive evaluation of LFIA performance was undertaken with individuals following natural infection. Here we assess the performance of the selected LFIA to detect antibody responses in individuals who have received at least one dose of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine.Methods: This was a prospective diagnostic accuracy study. Sampling was carried out at renal outpatient clinic and healthcare worker testing sites at Imperial College London NHS Trust. Two cohorts of patients were recruited; the first was a cohort of 108 renal transplant patients attending clinic following two doses of SARS-CoV-2 vaccine, the second cohort comprised 40 healthcare workers attending for first SARS-CoV-2 vaccination and subsequent follow up. During the participants visit, finger-prick blood samples were analysed on LFIA device, while paired venous sampling was sent for serological assessment of antibodies to the spike protein (anti-S) antibodies. Anti-S IgG was detected using the Abbott Architect SARS-CoV-2 IgG Quant II CMIA. A total of 186 paired samples were collected. The accuracy of Fortress LFIA in detecting IgG antibodies to SARS-CoV-2 compared to anti-spike protein detection on Abbott AssayResults: The LFIA had an estimated sensitivity of 92.0%  and specificity of 93.6%  using the Abbott assay as reference standard (using the threshold for positivity of 7.10 BAU/ml)Conclusions: Fortress LFIA performs well in the detection of antibody responses for intended purpose of population level surveillance but does not meet criteria for individual testing. This approach, using antibody lateral flow devices, has been used across England in the REACT2 , identifying one for subsequent use. The test selected  detects antibody against the spike protein of the virus  and would therefore be expected to detect vaccine induced antibody responses. This study examined the accuracy of the Fortress LFIA device in detecting antibodies in two cohorts of vaccinated individuals and explored the relationship between LFIA results and viral neutralisation.Prior to the scale up of antibody testing for surveillance, extensive clinical and laboratory evaluation of diagnostic accuracy following natural infection was performed on a range of LFIA antibody teststh May 2021. Samples were collected from two groups: renal transplant patients (cohort 1) and healthcare workers (cohort 2).This was a prospective diagnostic accuracy study conducted between 20th December 2020 and 26Every attempt was made to address potential sources of bias. All eligible participants were offered enrolment where practical and every effort was made to ensure understanding of the participant information sheet (PIS) and study procedure, using translation services where necessary. Potential participants were given time to consider participation and trained research staff were able to answer questions relating to the study.Eligibility for both cohorts was defined as:1)Adult (>/=18 years old)2)Able to understand and consent to study3)Received either one or two doses of any UK approved vaccine for COVID-194)Able to comply with study procedure/ study not thought to be risk to patientsample size required is 106 cases and 76 controls. Patients were pragmatically enrolled to ensure minimum sample size achieved.Sample size was computed based on an expected sensitivity of 90% and specificity of 95%, with a minimal acceptable lower confidence limit of -10% for both estimates. Under power 1 – β = 0.85 and α = 0.05, the minimumParticipants. Participants were recruited between 1st February 2021 and 26th May 2021.Those recruited were 108 renal transplant recipients who were attending clinic at Hammersmith Hospital 28 days  following a second dose of a SARS-CoV-2 vaccine, . Participants were recruited directly from clinic by trained medical and nursing staff who explained the study and provided with PIS and informed consent form (ICF). Participants underwent a finger-prick capillary blood draw for immediate testing on the LFIA device and, at the same time, gave a venous blood samples for later serology testing. Clinical characteristics were obtained from electronic medical records  detects IgG and IgM to the S1 subunit of the spike protein. Participants were also provided with verbal instructions on how to use the test by a member of the research team, prior to performing self-testing, with support provided where necessary. The LFIA result was assessed independently by two observers. The results were reported by the colour intensity of the IgG band, and documented as either a positive or negative result. Participants. Participants were recruited between 20th December 2020 and 31st January 2021. Overall, 39 healthcare workers were consented when attending for first vaccination with BNT162b2 Pfizer-BioNTech. Participants were approached by trained members of the research team at the vaccination centre and provided with a PIS and ICF with explanation of the study. Of these participants, 38 had repeat samples taken at 21 days post vaccination and one further participant had samples taken at 21 days who had not had day 0 samples. In total there were 40 participants. Data was collected on age and gender. Medical records of participants were not accessed for this cohort.Lateral flow immunoassay testing. Participants were supported in capillary blood draw for use with the Fortress LFIA devices as described above. Results were reviewed and recorded by the study team.Serological testing. Serological assessment was performed on the Abbott Architect SARS-CoV-2 IgG Quant II CMIA which reports a quantitative anti-Spike antibody titre. This assay has previously been reported to have specificity of 100% on stored pre-covid serum samples and sensitivity of 93.9% for samples ≥14 days post symptom onset. The threshold value for positivity of 7.10 binding antibody units (BAU)/ml. At the time of the study in the healthcare worker cohort (cohort 2), quantitative antibody titres were reported in AU/ml. To allow combination with cohort 1 data, these were converted to BAU/ml by multiplying by 0.142. Double antigen binding assay (DABA) testing for discordant results  was performed on available stored samples from cohort 1. Detailed methodology of DABA has been described previously. Briefly, the Imperial Hybrid DABA is a sequential two step double binding assay for the detection and measurement of antibody directed to the receptor binding domain of SARS-CoV-2. The proteins employed were expressed and gifted by the Crick Institute, London. In order to evaluate specificity the Hybrid DABA was tested on stored plasma and serum samples predating the SARS-CoV-2 outbreak (n=825) in which 0 samples tested positive, giving a specificity of 100%.2 for 42 h before fixing cells in 4% PFA . Cells were treated with methanol 0.6% H2O2 and stained for 1h with a 1:3000 dilution of 40143-R019 rabbit mAb to SARS-CoV-2 nucleocapsid protein . A 1:3000 dilution of sheep anti-rabbit HRP (horseradish peroxidase) conjugate (Sigma) was then added for 1 h. TMB  substrate (Europa Bioproducts) was added and developed for 20 mins before stopping the reaction with 1M hydrogen chloride (HCl). Plates were read at 450nm and 620nm and the concentration of serum needed to reduce virus signal by 50% was calculated to give NT50 values.In addition, for cohort 2, individuals provided blood for assessment of neutralisation assays. The ability of sera to neutralise the SARS-CoV-2 virus was assessed by neutralisation assay on Vero cells. Sera were serially diluted in OptiPRO SFM (Life Technologies) and incubated for 1h at room temperature with 100 TCID50/well of SARS-CoV-2/England/IC19/2020 and transferred to 96-well plates pre-seeded with Vero-E6 cells. Serum dilutions were performed in duplicate. Plates were incubated at 37°C, 5% COThe primary outcome of the study was sensitivity and specificity of the LFIA device in detecting SARS-CoV-2 IgG antibodies identified by the Abbott platform.A secondary analysis was conducted using reference standard as either Abbott or, for discordant results (positive LFIA negative serology) in cohort 1 using in house DABA as reference standard for serological positivity.MedCalc diagnostic test evaluation calculator (version 20.015). Graphical analyses was performed usingGraphPad Prism 9.1.2 software. An open-source alternative isR.Outcomes are presented with the corresponding binomial exact 95% confidence interval (95% CI). Statistical analyses (specificity and sensitivity) were performed with open access online websiteEthics approvals were sought for each cohort prior to commencement of the study.The renal cohort ethics were obtained from Health Research Authority, Research Ethics Committee .For the healthcare worker cohort, we got ethics from the South Central-Berkshire B Research Ethics Committee (IRAS ID: 283805), and Medicines and Healthcare products Regulatory Agency approval for use of the LFIA for research purposes only..The characteristics of the participants are described inThe combined results describe both cohort 1 and cohort 2 . Using the threshold value for positivity on serological testing of ≥7.10 BAU/ml, the LFIA had an estimated sensitivity of 92.0%  and specificity of 93.6%  using the Abbott assay as reference standard.Secondary analysis (Abbott or DABA as reference standard). Using the threshold vale for positivity on serological testing of ≥7.10 BAU/ml (n=183) on Abbott Architect Assay and confirmatory DABA result for available discordant samples (n=3) as the reference standard, the overall performance of the test in these combined cohorts produce an estimate of sensitivity of 92.0%  and specificity of 95.1% . Results were similar when analysing cohort 1 and cohort 2 individually  were significantly higher in those with positive LFIA compared to those without . Only on, though did not meet Medicines and Healthcare products Regulatory Agency (MHRA) criteria for individual use which recommend antibody tests should have a sensitivity of >98% (95% CI 96% to 100%) and specificity of >98% on a minimum of 200 known negative controls. The test has undergone extensive evaluation for home self-testing and has since been used widely in community studies of antibody prevalence in England.This study demonstrates that the Fortress LFIA device performs well in detecting IgG antibodies in vaccinated individuals when comparing against a serological assay widely used in routine practice. LFIAs have been a helpful tool the assessment of population antibody prevalence of SARS-CoV-2, and can play a role in informing vaccination strategy going forwards. The Fortress LFIA has been assessed previously for its performance following natural infectionThe performance of the LFIA in the cohorts of vaccinated individuals here demonstrates slightly higher sensitivity than previously reported for natural infection, though this difference is not significant. This is likely to reflect higher background titres of antibody following vaccination, particularly after second doses, when compared to natural infection in the community, at least in the healthcare worker cohort. The LFIA device does not detect very low levels of antibody which may still correlate with protection from severe disease and/ or hospitalisation. However, in the general population, the number of such individuals with low titres following two vaccinations will be low .A small number of LFIA tests appear to produce false positive results (n=4) with undetectable antibodies in the commercial laboratory assay. To understand whether these were genuine false positives, these four samples were tested with a second sensitive assay (DABA). Only one if these discordant samples tested positive. . Although the LFIA studied has a threshold below which it can’t detect Spike specific antibody that is present, that threshold is close to the level at which neutralising antibody can be reliably measured  there is a place for further studies with larger sample sizes in the community.As a result of this pragmatic study design, some participants (13) in the HCW cohort provided two samples, at different time points, which were analysed within the study. As it is likely that there are differences in both the quantitative and qualitative immune responses in these samples, we analysed these samples independently given the primary purpose of the study was to evaluate the diagnostic accuracy of the LFIA compared to gold. If the 13 participants who provided two samples were removed from the calculations, the estimated sensitivity of the LFIA would be 87.7% with a wider (95%-CI 75.8- 97.1) for HCW cohort and to 91% (95%-CI 84.1- 95.6) overall.The performance of the LFIA evaluated is sufficiently good that it can continue to play a helpful role in the assessment of population antibody responses resulting from widespread infection and high levels vaccination coverage, particularly given the correlation of LFIA results with the functional measure of live virus neutralisation. Over time, antibody titres will begin to wane and ongoing population surveillance can play a helpful role in informing decisions on policy for subsequent vaccination programmes, the targeting of booster vaccines. Rapid antibody testing may prove useful in initial screening of patients to receive monoclonal antibody therapy as lab methods may cause a delay in therapy to potentially eligible patients.https://doi.org/10.7910/DVN/KCDZIN.Harvard Dataverse Repository: "Replication Data for: SARS-CoV-2 Antibody Lateral Flow Assay for Possible Use in Seroprevalence Surveys: a Diagnostic Accuracy Study",This project contains the following underlying data:- Anti Spike Protein LFIA For HCW Cohort.tab- Anti Spike Protein LFIA For Renal Cohort.tab- Neutralisation Titres for HCW Cohort.tab- DABA testing of LFIA Positive Abbott Negative Renal Transplant Samples.tabCreative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the Thank you for addressing my previous comments for version 1 of this manuscript. I have no further comments.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Diagnostics; statisticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I'm happy with the revisions, I have no further comments.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Diagnostics, Infectious DiseasesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This study determines the diagnostic accuracy of the Fortress LFIA for SARS-CoV-2 IgG antibodies after vaccination against an established reference assay. Specificity and sensitivity are estimated from samples of two cohorts and pooled estimates are reported as the main result. The authors provide an in-depth analysis of false-positive results and report additional results of neutralisation assays for one cohort. The results show lower sensitivity and higher specificity compared to the two cited previous evaluations of this LFIA, but both differences are not statistically significant. Overall, the study is well documented and the availability of raw data allows easy reproduction of the results. My only concern is the sample design and analysis of the HCW cohort. The calculations assume that the 78 samples are independent, but they are in fact pairs from 39 individuals measured before and after vaccination. I would sympathize with neglecting this issue for cases with a negative result at day 0 followed by a positive result at day 21, which one might treat as ‘sufficiently’ independent; however, one is not surprised to find the 13 positive samples of day 0 to also test positive at day 21. An ad-hoc remedy for this double-counting is to remove 13 of these 26 samples from the calculation as they provide no new information and artificially inflate the sample size. The estimated sensitivity is then reduced to 87.7% with a wider 95%-CI of  (compare to the reported 92.3%) for that cohort, and to 91.0%  instead of 92.0% for the pooled analysis. Specificities are of course unaffected. The equally problematic case of negative results on both occasions does not occur in these data. Please comment on the sample design of the HCW cohort and its consequences.A significance level of 15% is reported for the sample size calculation; statistical folklore and the reported sample size suggest it to mean 5%.In Table 2, you report N=39 for the D21 seropositive column, but males and females add to 40.Please add a label for the vertical axis of Figure 1. Three minor corrections:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Diagnostics; statisticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Many thanks for this comment. We acknowledge as a result of the pragmatic study design, some participants in the HCW cohort (13) have provided two samples to the study, at different time points (>21 days apart), which have been treated as independent samples for the purpose of testing the accuracy of the LFIA devices against gold-standard. We agree that if this study was looking at individual differences in participants, it would not be appropriate to include both sets of samples as this would introduce bias. In individuals who have provided two samples, it is likely that both the quantitative and qualitative immune responses will be different. As such, given that the primary outcome for this study is to test the diagnostic accuracy of the LFIA device against the gold-standard Abbott titre we feel it is helpful to include these samples in the analysis. Acknowledging the reviewer's comment, we have included a section addressing this in the discussion section and have shown the proposed sensitivity analysis removing the duplicate participants' samples’ in the supplement.My only concern is the sample design and analysis of the HCW cohort. The calculations assume that the 78 samples are independent, but they are in fact pairs from 39 individuals measured before and after vaccination. I would sympathize with neglecting this issue for cases with a negative result at day 0 followed by a positive result at day 21, which one might treat as ‘sufficiently’ independent; however, one is not surprised to find the 13 positive samples of day 0 to also test positive at day 21. An ad-hoc remedy for this double-counting is to remove 13 of these 26 samples from the calculation as they provide no new information and artificially inflate the sample size. The estimated sensitivity is then reduced to 87.7% with a wider 95%-CI of  (compare to the reported 92.3%) for that cohort, and to 91.0%  instead of 92.0% for the pooled analysis. Specificities are of course unaffected. The equally problematic case of negative results on both occasions does not occur in these data. Please comment on the sample design of the HCW cohort and its consequences. This was a mistake and has been edited in the paper. The significance level is 5%. In Table 2, you report N=39 for the D21 seropositive column, but males and females add to 40. This has been changed. A significance level of 15% is reported for the sample size calculation; statistical folklore and the reported sample size suggest it to mean 5%.Label added. Please add a label for the vertical axis of Figure 1.   This study assesses the diagnostic accuracy of the Fortress LFIA for SARS-CoV-2 anti-S Ab compared to a lab-based serological assay reference standard. The study is appropriately conducted, based on two different cohorts but giving similar results. The authors have attempted to understand if FP results are due to FN results from the reference standard by using an additional assay for discordant results. Comments that need addressing:DiscussionThe small sample size leads to wide CIs - this needs to be emphasised in the discussion/limitations section.Please state what the previously reported diagnostic accuracy for natural infection was for this assay.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Diagnostics, Infectious DiseasesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for this comment. We have addressed this in the discussion/limitations section. Please state what the previously reported diagnostic accuracy for natural infection was for this assay.We have included information and the reference paper on the reported diagnostic accuracy for natural infection for this assay.The small sample size leads to wide CIs - this needs to be emphasised in the discussion/limitations section.