Nationwide, emergency response systems depend on 9-1-1 telecommunicators to prioritize, triage, and dispatch assistance to those in distress. 9-1-1 call center telecommunicators (TCs) are challenged by acute and chronic workplace stressors: tense interactions with citizen callers in crisis; overtime; shift-work; ever-changing technologies; and negative work culture, including co-worker conflict. This workforce is also subject to routine exposures to secondary traumatization while handling calls involving emergency situations and while making time urgent, high stake decisions over the phone. Our study aims to test the effectiveness of a multi-part intervention to reduce stress in 9-1-1 TCs through an online mindfulness training and a toolkit containing workplace stressor reduction resources.The study employs a randomized controlled trial design with three data collection points. The multi-part intervention includes an individual-level online mindfulness training and a call center-level organizational stress reduction toolkit. 160 TCs will be recruited from 9-1-1 call centers, complete a baseline survey at enrollment, and are randomly assigned to an intervention or a control group. Intervention group participants will start a 7-week online mindfulness training developed in-house and tailored to 9-1-1 TCs and their call center environment; control participants will be “waitlisted” and start the training after the study period ends. Following the intervention group’s completion of the mindfulness training, all participants complete a second survey. Next, the online toolkit with call-center wide stress reduction resources is made available to managers of all participating call centers. After 3 months, a third survey will be completed by all participants. The primary outcome is 9-1-1 TCs’ self-reported symptoms of stress at three time points as measured by the C-SOSI . Secondary outcomes will include: perceptions of social work environment ; mindfulness; and perceptions of social work environment and mindfulness as mediators of stress reduction.This study will evaluate the effectiveness of an online mindfulness training and call center-wide stress reduction toolkit in reducing self-reported stress in 9-1-1 TCs. The results of this study will add to the growing body of research on worksite stress reduction programs.NCT02961621 Registered on November 7, 2016 (retrospectively registered).ClinicalTrials.gov Registration Number:  Each 9-1-1 TC fields thousands of calls every year, ranging from trivial to life-threatening, while simultaneously monitoring and entering data into up to six different computer screen displays in real time during each call.The effectiveness and efficiency of an emergency response system depends on 9-1-1 telecommunicators (TCs), the emergency call receivers and dispatchers who are the Much of the research on stress in first responders has been focused on police and firefighters, and the relationship between their trauma exposures and post-traumatic stress disorder (PTSD) , 2. AlthThe adverse outcomes associated with exposure to acute and chronic occupational stressors are costly and include physical and mental diseases, and impaired work performance, which can threaten the attainment of an organization’s critical mission(s) . In addidevelop, implement, and test the effectiveness of an individual-level online mindfulness training to reduce stress in 9-1-1 telecommunicators.The objective of this study is to develop and test the effectiveness of an evidence-based, multi-level intervention program designed to reduce stress in 9-1-1 TCs. The project’s primary aim is to:develop and test the effectiveness of an organizational-level online worksite stress reduction toolkit utilized by 9-1-1 TC managers that is designed to reduce or minimize sources of stress in the 9-1-1 call center environment.test if the multi-level intervention program will lead to increases in TCs’ positive perceptions of the call center social work environment.test if participation in the mindfulness training increases mindfulness.test if increases in TC’s positive perceptions of the call center social work environment and increases in mindfulness serve as mediators of stress reduction in 9-1-1 TCs.The secondary aims are to:Borrowing from the literature on occupational stress, individual stress, and coping response we developed a conceptual framework that describes how this multilevel intervention is expected to effect stress in 9-1-1 telecommunicators  and other information  and complete the baseline survey. All participant information is stored on computers.Respondents from each enrolled call center who are eligible, have provided their informed consent, and have completed the baseline survey, will be randomized into either the mindfulness training group or wait-list control group.A short video introducing the theme for the weekA few paragraphs describing the theme and activities for the weekAn audio-guided meditative stress reduction exerciseSuggestions for brief mindfulness activities that can be performed throughout the dayA brief check-in form for participants to let study staff know how the training is goingA moderated discussion board on which participants can post questions/comments if they wishThe online mindfulness training is composed of seven modules to be completed weekly, over a 7-week time period. Each weekly online module is expected to require participants between 20 to 30 min to complete. Each individual module consists of the following components:The mindfulness training is developed by clinicians and investigators who are trained mindfulness teachers and practitioners. The training is adapted from the widely-used Mindfulness-Based Stress Reduction (MBSR) program, an evidence-based training program originally implemented as in-person training , 56. ForThe online Toolkit is comprised of several modules providing evidence-based and expert-informed content and strategies for call center managers to consider and implement in an effort to reduce select stressors and increase network support in their worksites. The specific topics were selected based upon consultations and guidance from our research program advisory board consisting of call center managers from throughout our catchment area. The on-line toolkit includes modules focused on the following content areas: Conflict management; Bullying in the Workplace; and Reducing Technostress; with additional resources on Health and Wellness. Content is specifically tailored for managers and for their utility in 9-1-1 call centers.After completing the baseline survey, participants will be assigned a study ID number and are randomized to either start the online mindfulness training (the intervention group) or are assigned to the wait-list control group.Two weeks following the end of the online mindfulness training all participants will be asked to complete the second survey. Two weeks following the end of the call-center-level toolkit availability, all participants will be asked to complete the third survey.During the 7-week training period, the intervention group will be contacted each week, receiving two emails: one containing the link to that week’s training module and one providing additional suggestions for incorporating that week’s stress reduction activities into daily living. Participants will be encouraged to complete the training on a designated weekday to allow approximately 7 days between each module. Participants will also be encouraged to practice the mindfulness skills introduced in the module throughout the week for approximately 10 min every day.Following the intervention group participants’ completion of the mindfulness training and completion of the second survey, managers of all participating call centers will be provided with a link to access the Toolkit and are asked to share the link with other administrative personnel at their call center who may be in a position to implement any of the suggested activities. Managers will have access to the Toolkit for 3 months and will receive bi-monthly emails encouraging them to use the Toolkit content and directing them to specific modules of the Toolkit. After 3 months access to the Toolkit, all participants complete the third survey.The study main outcome is the TC’s self-reported stress as measured by the Calgary Symptoms of Stress Inventory (C-SOSI), a validated 56-item scale designed to assess various subjective symptoms of stress with 8 factor derived subscales, each consisting of 6-9 items: Depression, Anger, Muscle Tension, Cardiopulmonary Arousal, Sympathetic Arousal, Neurological/GI, Cognitive Disorganization, and Upper Respiratory Symptoms . SubjectSocial Support: Perceptions regarding social support (at work with coworkers and at home with family and friends), assessed by two separate visual analogue scales (VAS). Prior research has established the reliability and concurrent validity of these measures in another first responder sample [r sample .Network Conflict: The degree of conflict experienced at home and at work, assessed by two separate VAS. Prior research has established the reliability and concurrent validity of these measures in another first responder sample [r sample .Mindfulness: The Mindful Attention Awareness Scale (MAAS) will assess attentional sensitivity to psychological, somatic, and environmental cues [tal cues . Prior rtal cues . The Fivtal cues –68. The tal cues , 68.Data regarding the following variables hypothesized to serve as mediators to the main outcome will be collected.Demographics: Participants are asked to provide their age, gender, and years of experience as 9-1-1 TCs.Overcommitment (OC): OC refers to an individual’s exhaustive coping style which can adversely impact the health and well-being. [l-being. , 70. In l-being. . OC iteml-being. . We willOvertime: 9-1-1 TCs will be asked if they have worked overtime, and if it was voluntary or mandatory.Acute stressor or stress event: Call center managers will be asked if there was an unusual organization-wide acute stressful event during the study period.Job Demands - mental: Measured with the Mental Demands subscale (5 items) from the National Institutes for Occupational Safety and Health (NIOSH) Generic Job Stress Questionnaire. [onnaire. , 72Job Demands - physical: Measured with the Job Requirements subscale (10 items) from the NIOSH Generic Job Stress Questionnaire. [onnaire. , 73Technostress: Technostress is measured using items drawn from the Techno-uncertainty and Techno-insecurity sub-scales of the Technostress creators’ Scale [s’ Scale  and we wWe will collect the following data that may serve as covariates or confounders with our main outcome.We anticipate randomizing a total of 160 9-1-1 TCs into two groups (~ 80 per group), and obtaining outcome data on survey measures from a minimum of 65 per group after accounting for attrition . A rough estimate of a clinically meaningful effect size was obtained after review of studies of mindfulness interventions that used the C-SOSI or SOSI measure as an outcome. We found seven studies –81 that The primary hypothesis for this intervention study is that TCs who are randomized to the online mindfulness training will report fewer symptoms of stress compared to TCs who are randomized to the wait-listed control group.To examine the primary hypothesis we will use repeated measures mixed effects models, with differences assessed by interaction terms between randomization group and follow-up time. The models will include fixed effects for group (2 levels) and time , site, time–by-treatment interaction, and an interaction term between the implementation of the center wide toolkit and the third time point, with a random intercept for participant. Differences between groups will be assessed using the time-by-treatment interaction term.The primary outcome variable will be the C-SOSI symptoms of stress scale. Hypotheses will be conducted at the 0.05 level of significance without adjustment for multiple comparisons, with group assigned according to intent-to-treat.The effects of possible differential drop-out rates between the two groups will be assessed using sensitivity analyses under the assumption that data is not missing at random. Multiple imputation will be used to impute missing values for the outcome variable under varying levels of bias, and the resulting association between level of bias and differences between the groups estimated.Effect of the intervention on mindfulness: We will examine the effect of the intervention on mindfulness score using the same model as the primary analysis with MAAS score as the outcome.Effect of intervention on perceptions of social work environment: We will examine the effect of the intervention on perceptions of social work environment  using the same model as the primary analysis with the 4 VAS.Analysis of mindfulness and perceptions of social work environments as mediators of stress reduction: We will conduct analyses of the mediation effect of mindfulness (MAAS measures) and perceptions of social work environments  on the intervention, estimating the direct and indirect causal effects using the approach of Imai [ of Imai .Effect of training participation on stress: Training participation will be examined using two measures: number of trainings completed as measured by access logs for viewing training videos and self-report mean number of days per week on which mindfulness was practiced. The effects of level of participation will be examined using repeated measures mixed effects models, with the effect of participation level assessed by interaction terms between level of participation and follow-up time. The models will include level of participation as a covariate, fixed effect for time , site, time–by-level-of-participation interaction and a random intercept for participant.Effect of online Toolkit: A secondary aim of this study is to determine the extent to which an online Toolkit for reducing occupational stress in call centers improves call center climate four months post implementation of the Toolkit. We will use a multi-level model to examine the effects of implementation of the toolkit (as measured through the manager survey) on mean call center stress levels and call center climate, as measured by workplace support and conflict measures.The secondary outcomes examined will include mindfulness and perceptions of social work environment. We anticipate conducting the following analyses:first first-responders to assist callers contacting 9-1-1 for aid in emergencies. Research shows that this workforce is exposed to acute and chronic occupational stressors; both trauma exposures specific to emergency response, and those stressors commonly encountered by non-emergency call center workforces. This study will evaluate if an online mindfulness training and call center-wide stress reduction resources can reduce stress in 9-1-1 TCs. The results of this study will add to the growing body of research on worksite stress reduction programs, the use of online mindfulness training in workforce stress reduction, and stress reduction in 9-1-1 telecommunicators.Emergency response systems depend on 9-1-1 telecommunicators, the emergency call receivers and dispatchers who are the


Odor-preferences are usually influenced by life experiences. However, the neural circuit mechanisms remain unclear. The medial olfactory tubercle (mOT) is involved in both reward and olfaction, whereas the ventral tegmental area (VTA) dopaminergic (DAergic) neurons are considered to be engaged in reward and motivation. Here, we found that the VTA (DAergic)-mOT pathway could be activated by different types of naturalistic rewards as well as odors in DAT-cre mice. Optogenetic activation of the VTA-mOT DAergic fibers was able to elicit preferences for space, location and neutral odor, while pharmacological blockade of the dopamine receptors in the mOT fully prevented the odor-preference formation. Furthermore, inactivation of the mOT-projecting VTA DAergic neurons eliminated the previously formed odor-preference and strongly affected the Go-no go learning efficiency. In summary, our results revealed that the VTA (DAergic)-mOT pathway mediates a variety of naturalistic reward processes and different types of preferences including odor-preference in mice. Sensory perceptions are influenced by life experiences. For us, it’s a pleasure to unexpectedly smell the odor of grandmother’s cookies that we loved in our childhood, and people of different cultures may have different odor-preferences, such as blue cheese to European and stinky bean-curd to Chinese. For rodents, reward-related odor perception is critical for food foraging and reproduction . The forThe olfactory tubercle (OT) is a long and narrow tubular brain structure located at the ventral part of striatum. It belongs to the olfactory system and receives dense direct inputs from the main olfactory bulb (OB) and other olfactory cortexes such as the anterior olfactory nucleus (AON) and piriform cortex (PCX) . As a paAn earlier study showed that the VTA can modulate the activities of olfactory-related OT neurons . The VTAIn attempting to answer these questions, we first confirmed the direct DAergic projection from VTA to the mOT by using virus-based tracing system. Using fiber photometry recording, we subsequently showed that the activities of the VTA-mOT DAergic projection and the mOT can be changed by reward or odor stimulation respectively. Besides, optogenetic activation of the VTA (DAergic)-mOT pathway was able to activate multiple brain regions and form space/location preferences . Further, coupling this activation with odor stimulation simultaneously could not only generate preference for a neutral odor, but also abolished the avoidance for an aversive odor. On the other hand, inactivating the mOT-projecting VTA DAergic neurons, or blocking the dopamine receptors in the mOT, abolished the expression of previously formed odor-preferences. These results demonstrate that activation of the VTA (DAergic)-mOT pathway generates odor-preference as well as the other types of preferences. Thus, this pathway might be a key player in modulating perception of a given stimulus.To map the direct inputs of the mOT, a recombinant RV expressing green florescent protein (RV-dG-GFP) was injected into the mOT . RV-labeWe then examined the cell types of RV infected neurons in the VTA by immunochemical staining of the tyrosine hydroxylase (TH), a biomarker of DAergic neurons, and found about half of the GFP labeled cells  were TH positive .Both the OT neurons and the VTA DAergic neurons play roles in reward processing . AlthougEnergy is the basic requirement for the survival of animals, so we first detected the response of this pathway in water- or food-deprived mice to sucrose solution and solid food pellet, respectively. For this purpose, we infused AAV-DIO-GCaMP6m into the VTA of DAT-Cre mice, and recorded the fluorescence signals of the VTA-mOT DAergic terminals . When thIn addition, social interaction is critical for animals, we then investigated whether the VTA-mOT DAergic projection is involved. We examined the effect of male-female and male-male interactions, and observed reliable increases of GCaMP fluorescence individually. Because no significant difference of fluorescence changes were observed between the male-female and male-male social interactions, we pooled the data of social interaction experiments  tests revealed that mice expressing ChR2 exhibited significant preference for the light-paired chamber , compared with the control mice  which only expressed mCherry  and an aversive (TMT) odor to mice . After 2However, optical stimulation of the VTA-mOT DAergic projection might lead to the activation of the passing-by DAergic fibers in the mOT or VTA DAergic cell bodies, which may then activate afferents projecting to the other brain regions. To exclude these possibilities and address the role of the local mOT dopamine release in producing the odor-preference, we infused the D1 and D2 dopamine receptor antagonists (SCH-23390 and raclopride) 5 min before pairing the projection activation with odor stimulation . We founWe have shown that it is sufficient to generate odor-preference by activating the VTA (DAergic)-mOT pathway while exposed to the odor simultaneously, and that dopamine in the mOT is necessary for this process. However, the potentially supra-physiological activation produced by optogenetic manipulation might elicit artificial behaviors. Therefore, whether this pathway is really necessary for the formation of odor-preference under the influence of hedonic life experience still remains unclear. Further, whether or how this pathway plays roles in the perception of the preferred odors after the formation of odor-preference is also unknown. To address these questions, we decided to train water deprived mice for Go-no go task, with one of the two odors associated with water, a naturalistic reward, then examine the effects caused by the chemogenetical inactivation of the mOT-projecting VTA DAergic neurons. At first, we co-injected RV-dG-GFP into the NAc and RV-dG-dsRed into the mOT  and founMoreover, we found that dense axonal fibers were also distributed in the NAc for some mice, which was not consistent with the RV-labeled results . This miTogether, these results indicated that the VTA-mOT DAergic projection should play important roles in the formation of odor-preference under the influence of hedonic life experience.The OT has been implicated in both reward and olfactory information processing . In the Although previous study has shown that electrical stimulation of the OT promotes reward , the behLacking cellular specificity, electrical stimulation might activate local neuron population, and axon fibers projecting to the area and passing-by non-selectively. Therefore, it was difficult to decide which component(s), local neurons, afferent to or efferent from the OT, and fibers passing-by the OT, contribute to the observed effects. Our results using optogenetics proved that activation of the VTA-mOT DAergic projection contributes to the rewarding role of the mOT, although the downstream neurons in the mOT involved in this pathway still need to be elucidated. It was reported that activation of the trimeric G-protein Gs-coupled D1R increases the excitability of the D1R + neurons, and that activation of the Gi-coupled D2R decreases the excitability of the D2R + neurons . Thus, tOn one hand, artificial activation of the VTA-mOT DAergic terminals generates reward behavior; on the other hand, this pathway is activated by naturalistic rewards, including sucrose solution, solid food and social interactions. It is reported that the activities of the VTA DAergic projection are target-dependent in response to rewards and shock . For exaReward-related odor perception is critical for survival and reproduction of animals . As receIn the present study, we found that the VTA-mOT DAergic projection is activated by diverse naturalistic rewards, the activities of the mOT are altered by a wide range of odorants, and mice exhibited a preference for a given odor if that olfactory stimulant is paired with the activation of the VTA (DAergic)-mOT pathway. Our results thus provide an insight that the activation of the VTA-mOT DAergic projection by hedonic life experience might contribute to the generation of the preference for experience-related odors.We showed that it is sufficient to artificially generate odor-preference by optogenetic activation of the VTA-mOT DAergic projection . Since tIn the present study, inactivating the mOT projecting VTA neurons did not affect the learnt Go-no go performance , mice weInactivating the mOT projecting VTA neurons did not affect reward seeking, but strongly affected the learning efficiency for a new odor pair. Dopamine has been widely reported to strengthen environment stimuli-encoded glutamate inputs in the NAc, thus facilitated environment cue-based reward learning . HoweverThe reasons why paired activation of the VTA-mOT DAergic projection generates odor-preference could be complex. Given that the mOT is directly innervated by mitral/tufted cells from the OB . AnotherAnimals learn to associate diverse sensory cues, not only odors, with rewards depending on their life experience. The striatum plays an important role in experience-dependent reward learning behaviors . BlockadThe OT is composed of the medial and lateral parts, which are functionally different from each other . Thus, hIt should also be pointed out that, although we intended to reveal the functions of the VTA-mOT DAergic projection in odor-preference, the border for the mOT and the lateral OT could be potentially imprecisely divided due to limited knowledge about this. Besides, it is also a difficult problem to perfectly demark the OT from the ventral pallidum. In the present work, we divided the mOT from the lateral OT at the most gyrated part of the layer 2 as we did before , and assBesides, the NAc and OT, components of the ventral striatum, mediate reward processing similarly  and bothIn summary, we have provided evidence that the VTA-mOT DAergic projection can be activated by a variety of rewards and that activation of the projection can generate robust rewarding, leading to formation of olfactory as well as spatial preferences if the corresponding stimulation is coupled with the activation of the projection simultaneously. These results may underlie a circuit mechanism for how preference is influenced by life experience.All surgical and experimental procedures were conducted in accordance with the guidelines of the Animal Care and Use Committees at the Wuhan Institute of Physics and Mathematics, the Chinese Academy of Sciences (reference number: WIPM-(2014)39). Adult male and female C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Company. DAT-Cre mice used were heterozygote and bred from DAT-internal ribosome entry site (IRES)-Cre  Bkmn/J, stock number 006660)  by matin12 genome copies per milliliter.For AAV viruses, the AAV9-EF1a-floxed-ChR2-mCherry, AAV9-EF1a-floxed-eNpHR3.0-mCherry, AAV9-EF1a-floxed-hM4Di-mCherry, AAV9-hsyn-Gcamp6s, and AAV9-EF1a-floxed-mCherry were packaged by BrainVTA Co., Ltd., Wuhan. AAV9-EF1a-DIO-GCaMP6m and AAV9-EF1a-DIO-EmGFP were gifts from Minmin Luo and constructed by replacing the coding region of ChR2-mCherry in the AAV9-EF1a-DIO-hChR2(H134R)-mCherry plasmid (constructed by K. Deisseroth) with those encoding enhanced membrane GFP (Addgene Plasmid 14757) or GCaMP6m (Addgene Plasmid 40754), respectively. All AAVs were purified and concentrated to titers of approximately 2 × 109 pfu/ml.For rabies viruses, the RV-dG-GFP and RV-dG-dsRed were packaged by standard methods as previous described  with tit12 genome copies per milliliter by BrainVTA Co., Ltd., Wuhan.To specifically inactivate the mOT-projecting VTA DAergic neurons, CAV-CMV-Cre was also packaged at titers of approximately 3 × 10All procedures on animals were performed in Biosafety level 2 (BSL2) animal facilities as we did before . BrieflyTo retrograde trace the afferent neural circuit of the mOT, RV-dG-GFP (150 nL) was unilaterally injected into the mOT of the adult male C57BL/6 mice with the following coordinates: AP, 1.20 mm; ML, 1.10 mm; and DV, −5.50 mm. Seven days after virus injection, subjected mice were sacrificed.To label the co-innervation of VTA neurons to the mOT and NAc, we co-injected 100 nL RV-dG-dsRed into the mOT and RV-dG-GFP into the NAc with the following coordinates: AP, 1.50 mm; ML, 0.7 mm; and DV, −4.50 mm. Seven days after virus injection, subjected mice were sacrificed.Φ = 200 µm; Fiblaser, Shanghai, China) or a cannula  targeted to the mOT with the following coordinates: AP, 1.20 mm; ML, 1.10 mm; and DV, −5.15 mm.To specifically manipulate and record the activities of the VTA-mOT DAergic projection, we respectively injected 300 nL of AAV9-EF1a-floxed-ChR2-mCherry, AAV9-EF1a-floxed-eNpHR3.0-mCherry or AAV9-EF1a-floxed-mCherry, AAV9-EF1a-DIO-GCaMP6m or AAV9-EF1a-DIO-EmGFP into the VTA of adult DAT-Cre mice with the following coordinates: AP, −3.20 mm; ML, 0.45 mm; and DV, −4.30 mm. Eight weeks later, subjects were ipsilaterally or bilaterally implanted with a chronic optical fiber  and followed by dental cement . After the surgery, lincomycin hydrochloride and lidocaine hydrochloride gel were applied to the incision sites to prevent inflammation and decrease pain. Mice were allowed to recover for at least 1 week before behavioral tests. Brain sections containing the mOT (AP from about 1.40 mm to 0.60 mm) were collected to confirm the site of optical fiber.To specifically inactivate the mOT-projecting VTA DAergic neurons, 250 nL of CAV-CMV-Cre was bilaterally injected into the mOT of adult male C57BL/6 mice with the same coordinate as above, and 300 nL of AAV9-EF1a-floxed-hM4Di-mCherry or AAV9-EF1a-floxed-mCherry into the VTA of the C57BL/6 mice with the following coordinates: AP, −3.20 mm; ML, 0.45 mm; and DV, −4.30 mm. About 3 weeks later, subjects were implanted with a small home-made metal bark to the skull for head-fixed Go-no go training.For the odor stimulation, isoamyl acetate, carvone, citral, and geraniol  with 5% dilutions of saturated vapors were used. And mineral oil was used as a control. Each odor was presented for four consecutive trials. The duration of the odor pulse was 2 s with an inter-trial interval of 28 s to prevent habituation. Verification of the optic fiber placement were done at the end of the experiments, using methods described in our previous publication .2 and 5% CO2. Coronal sections (300 μm thick) of the VTA were acquired using a vibratome  at 0–4°C. The slices were incubated at 34°C for 30 min and then at room temperature (22–24°C) with oxygenated artificial cerebrospinal fluid (ACSF) consisted of (in mM): 118 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 0.9 NaH2PO4, 11 D-glucose. After incubation, a slice was transferred to the recording chamber, superfused with oxygenated ACSF at a rate of 2 ml/min for recording.The DAT-Cre mice with AAV9-EF1α-DIO-ChR2-mCherry injected into the VTA were used. The mice were anesthetized with isoflurane. After rapid decapitation, the brains were quickly removed and placed into ice-cold oxygenated slicing solution saturated with mixture of 95% O2, 10 HEPES, 4 Mg-ATP, 0.4 Na-GTP, 10 Na-phosphocreatine, 0.6 EGTA . Signals were recorded with a MultiClamp 700B amplifier  and digitized at 10 kHz with DigiData 1440A . The somas of ChR2-expressing VTA DAergic neurons (mCherry-positive) were easily identified. To optically stimulate ChR2-expressing neurons, a single-wavelength LED system  with light pulses at 5 ms, 20 Hz was used. Data were analyzed using Clampfit 10.3 software and origin 9.0.Whole cell recording was performed with borosilicate glass pipettes pulled to a tip resistance of 3 to 5 MΩ . Recording electrodes were filled with intracellular solution (in mM): 140 K-gluconate, 5 KCl, 2.5 MgClvia patch cords  and a fiber-optic rotary joint , which could release the torsion of the fiber resulted from rotation of the mouse. All opto-stimulation experiments used 5 ms, 10–15 mW, 473 nm light pulses at 20 Hz via a solid-state laser for light delivery  triggered by a stimulator .For all behavioral experiments, we handled the mice for 2 days before the training experiment, 3–5 min for each day, allowing them to get used to the tethering procedure. For optogenetic stimulation, the implanted optic fiber was connected to a blue light laser The mice were placed in a behavioral arena (50 × 50 × 25 cm) for 20 min and assigned one counterbalanced side of the chamber as the stimulation side. Mice were placed in the non-stimulation side at the onset of the experiment. Laser stimulation at 20 Hz were constantly delivered when they crossed to the stimulation side, but stopped when they returned back to initial non-stimulation side. All behavioral tests were recorded using an HD digital video camera  and the offline analyses were performed by a video tracking software .The behavioral arena (47 × 32 cm) contained a red rectangle as the target area for reinforcement, located in the center of the open field. The red central rectangle, formed by border line with the height of about 1 cm, is one-tenth of the whole arena. During the experiment, the floor of the arena was covered by fresh padding, with the red central area easily seen. The behavior task consisted of three phases  and lasted for 5 consecutive days . During The pairing procedure was performed for 2 consecutive days, which was two weeks post-surgery and plate placement. On each of the pairing days, the mouse was head restrained and adapted to the holding and odorant delivery conditions for 1–2 hr. Carvone and geraniol  were randomly delivered to the mouse’s nose  with 5% of saturated vapors using a custom 8-channel olfactometer and balanced across mice. Only geraniol was delivered simultaneously with 2 s, 20 Hz trains of light pulses (473 nm). Control mice were treated identically. Furthermore, another pair of odors (v/v 5% TMT and sesame butter) was delivered in the same way, with TMT as the paired odor.In the subsequent receptor blockade experiments, saline  contained the D1R antagonist  and D2R antagonist  as blocking agent, or saline alone (200 nL) as control, was administered into the mOT through the cannula 5 min before optogenetic activation of the VTA-mOT DAergic projection paired with odor stimulation, which were described as above.The protocols were adapted from Sophie’s study  with somMice were tested with the two odors , applied by two glass sticks simultaneously, to their home cages. Home cage testing was chosen to minimize potential influences of stress and anxiety (because of the new environment/context) on the behavioral measures. The food and water were removed from cages just before tests. Tests took place during the light phase of the animals' 12/12 hr dark/light cycle. Odors were delivered for 6 min by inserting the odorized sticks from one side of the animal’s home cage. Mice were first habituated to the odor sticks for 1 min, and the time spent investigating, defined as sniffing within 1 cm of the odor stick, was recorded by a single observer blind to the experiment group over a 5-min period.After about 24 hr of water deprivation, mice were exposed to these two odors randomly. They were trained to either lick the water pipe to get the reward (a drop of water) when they smelled one odor (defined as S+ odor), or wait without any licking when presented the other odor (defined as S- odor). After several days of training, when the accuracy reached above 85%, the mice were ready for further experiments. They were intraperitoneally injected with CNO (3 mg/kg of body weight) and then tested for odor-preference blockade experiments. Moreover, in the following days, the mice were trained to distinguish another pair of odors using the same principle they learned before, until the accuracy achieved above 85% again.Mice were water deprived for 24 hr before placed in a chamber (20 × 20 × 22 cm), which was equipped with a bottle filled with 5% (w/v) sucrose solution. The lick signals were recorded by a contact lickometer connected with a Power 1401 data acquisition system.Mice were food deprived for 24 hr before placed in a chamber (20 × 20 × 22 cm). Food pellets were manually delivered to the chamber floor with only one pellet per time. Videos (10 min per session) from an overhead infrared camera were recorded. The onset of feeding was determined as the mouse picked up the pellet and started chewing.To study the effect of heterosexual interaction, the test male mouse was habituated in the dark with an optical fiber connected to the fiber ferrule on its head for 1 min. A female mouse was introduced into the home cage of the test male mouse. A recording session lasted for 10 min and the behavior of the test mouse was videotaped with an overhead infrared camera. In the male-male interaction sessions, a male mouse was introduced following the same procedure.The GCaMP fluorescent signals for sucrose or quinine solution, food pellets, social interactions and odors were recorded with the same setup  as previously reported .Briefly, 40-μm-thick coronal slices were obtained and the procedures for immunohistochemistry were performed similar to what we did before .To determine the input patterns of the mOT, every sixth sections of the RV-labeled brain slices were collected and stained with DAPI. Four coronal slices from the VTA  per mouse were immunohistochemically stained for the tyrosine hydroxylase .To determine the specificity of ChR2-mCherry expression in the VTA of DAT-Cre mice, 4 coronal slices from the VTA  per mouse (n = 3) were collected and immunohistochemically stained for the TH.To detect the c-fos expression evoked by optical-manipulation, mice were put in an open field and received laser illumination for 10 min and were sacrificed 1.5 hr later. Every sixth brain sections (AP from about 1.40 mm to 0.50 mm) from three mice were collected and immunohistochemically stained for c-fos .All the images were then captured and analyzed with TCS SP8 fluorescence laser scanning confocal microscope (Leica) and ImageJ software.For cell counting, the boundaries of brain regions were delineated manually with Photoshop based on the Allen Brain Atlas. The labeled neurons were quantified semi-automatically using FIJI and the cell counter plugin of ImageJ.For statistical analyses, we first analyzed data from one mouse trial by trial, then calculated the mean value and standard errors of one group, then Independent-Samples T-Test were used to determine statistical differences between groups using SPSS (version 13.0). Statistical significance was set at ***p<0.001; **p<0.01; *p<0.05. All data values were presented as mean ± s.e.m. Graphs were made using SigmaPlot (version 10.0). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Activation of the Dopaminergic Pathway from VTA to the Medial Olfactory Tubercle Generates Odor-Preference and Reward" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. All reviewers agree that showing that the DA-mOT pathway mediates reward processing and olfactory learning is indeed novel and interesting. However, the reviewers were also unanimous about the potential confounding factor of non-selective activation of DA projections to other brain areas, for example the NAc. Although the others provide some pharmacology to try to tackle this, the reviewers were not convinced by these data. The authors need to conduct some additional experiments to strengthen the study. The authors could demonstrate that the DA neurons separately project to the NAc and the mOT. If this turns out not to be the case, a combination of optogenetic stimulation of DA fibers in the mOT with pharmacological inactivation of DA cell bodies would be important. This would at least rule out the potential effect of antidromic stimulation, although the activation of axonal collaterals can still be an issue.Additionally we attach the point-by-point comments of the reviewers below.Reviewer #1:The medial olfactory tubercle (mOT) receives dense dopaminergic inputs from the ventral tegmental area and has often been considered an extension of the nucleus accumbens shell (NAc) in the ventral striatum. Although the roles of the NAc in reward processing have been the major focus of numerous research groups, the mOT has also been implicated in drug rewards. Unlike the NAc, the mOT receives strong direct olfactory inputs. Wesson and Wilson (2011) put forward some intriguing hypotheses regarding the potential functions of the mOT in olfactory processing, but progresses thus far on this mysterious structure have been very limited.Here, Zhang et al. from Fuqiang Xu's group provide several interesting observations indicating that the dopaminergic projection from the VTA to the mOT might mediate a variety of naturalistic reward processes and contributes to the formation of experience-dependent odor preference. They mapped the presynaptic partners of mOT neurons using rabies virus. By employing fiber photometry, they demonstrated that natural rewards activate dopaminergic terminals in the mOT. Optogenetically stimulating these terminals reinforces animal behavior and guides the formation of odor preference in a dopamine receptor-dependent manner. Finally, they showed that chemogenetically inactivating mOT-projecting neurons in the VTA abolishes conditioned odor preference and disrupts odor-discrimination learning using the go/no-go paradigm.I like many aspects of the study. The authors took a comprehensive approach and used several latest tools for functional dissection of neural circuits. The data are overall solid. The conclusions should be exciting to a broad audience working on reward-related behaviors and olfactory processing. I am mainly concerned that some of the behavioral effects might be produced by the projection of VTA dopamine neurons to the NAc through axonal collaterals or fibers of passage. The authors performed some experiments to address this potential caveat. For example, infusing dopamine blockers into the mOT and chemogenetically inhibiting mOT-projecting VTA neurons significantly reduce the behavioral effect of optogenetic stimulation. However, drugs may diffuse into the nearby NAc region. Similarly, injecting canine adenovirus (CAV) into the mOT might have labeled VTA neurons projecting to the NAc.The authors have several choices to address this concern. First, they could examine whether dopamine neurons projecting to the NAc and the mOT belong to separate subpopulations. Second, they could test whether the reinforcing effect of optogenetic stimulation in the mOT remains intact following lidocaine administration into the VTA. Third, they could show whether CAV-Cre injection into the mOT only drives gene expression in mOT-projecting dopamine neurons from the VTA. To the minimum, if none of these experiments work out, the authors should discuss the potential caveat and tone down some of the conclusions.Reviewer #2:This is a potentially interesting manuscript proposing that the dopaminergic innervation of the medial olfactory tubercle (mOT) by dopaminergic neurons from the ventral tegmental area (VTA) plays a role in reward. The authors show projection of dopaminergic neurons from the ventral tegmental area (VTA) to the mOT. They show that the neuronal circuit of mOT responds to reward and to odorants. They then proceed to activate or inhibit dopaminergic axons in the mOT and they argue that these manipulations elicit changes in behavior consistent with a role for mOT in reward. Unfortunately, my enthusiasm is diminished significantly by substantial problems outlined below.1) A potential major problem is that optogenetic stimulation might have resulted in direct or indirect stimulation of the nucleus accumbens (NAcc). The authors should provide evidence that the behavioral results for optogenetic stimulation of mOT are not due to direct stimulation of NAcc. After all c-Fos was increased in NAcc .2) The viral injections targeting mOT might have resulted in infection of neurons in NAcc. The figures need to be modified to clearly show that this is not the case. Was this tested for every single experiment? This criticism holds for 3) For 4) The LFP is an indirect measure of activity in the mOT. Did the authors record odorant-elicited changes in single unit spikes or neuronal activity measured with photometry? Why is the power increasing before the odorant is presented?5) The panel in 6) The authors claim that the experiment in Figure 9 shows that inactivation of DAergic neurons decreased the learned odor preference. But the viral infection resulted in expression of mCherry in a large number of non-DA neurons. Could the effect be due to GABAergic neurons projecting from VTA to mOT? Did the authors perform this experiment with halorhodopsin?7) The statistics of the manuscript are not acceptable. Which statistical test was used for Figure 7D8) For the experiment in Reviewer #3:In this manuscript the authors reported experiments aiming to address the role of the dopaminergic (DAergic) innervation of medial olfactory tubercle (mOT) from the ventral tegmental area (VTA) in the regulation of olfaction and reward. Olfactory tubercle has been implicated in both reward and olfactory information processing. However, in both the olfactory field and in the reward field, OT is a far less studied brain region compared with other olfactory regions and reward regions. The role of the VTA(DAergic)-mOT pathway in the regulation of reward and olfactory process have not been investigated in behaving animals. This work represents an important and timely study addressing fundamental questions regarding the circuit mechanisms underlying the behaviors involving olfactory processing and/or reward. With the presynaptic optogenetic activation, local administration of DA receptor antagonists in mOT, and chemogenetic silencing of mOT-projecting VTA DAergic neurons, the importance of the DAergic input from VTA to mOT in the odor-preference is reasonably well established. However, the role of this pathway in other reward processes is far less clear with the current experiments.The major issue of this study concerns the broad and non-specific effect of the optogenetic and chemogenetic manipulations. VTA DAergic neurons innervate multiple regions of the brain, and the neurons that innervate mOT likely also innervate other brain regions. Indeed, optogenetic activation of the VTA-mOT DAergic terminals was able to activate multiple brain regions . For optIn the case of the chemogenetic silencing experiments , the global inhibition of the CAV-Cre/AAV9-EF1a-floxed-hM4Di-mCherry infected VTA neurons would also abolish their function in other brain regions. Therefore, it is possible that the attenuation in the odor-cue based reward learning  may result from non-specific silencing of the projections to other brain regions. It is not clear what portion of VTA DAergic neurons are labeled by CAV-Cre/AAV9-EF1a-floxed-hM4Di-mCherry. If there were only a small portion labeled, it could be less problematic. Also, could the authors administer CNO locally at mOT to achieve local inhibition of VTA-mOT terminals?eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Minmin Luo (Reviewer #1); Diego Restrepo (Reviewer #2).Thank you for submitting your article "Activation of the Dopaminergic Pathway from VTA to the Medial Olfactory Tubercle Generates Odor-Preference and Reward" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers agree that the main concerns have been mitigated by the new experiments and there are only text corrections at this point. The authors should provide detailed parameters for the lidocaine experiment .In addition, the experiments that rule out co-lateralization should be a main figure. There are other small suggestions that the authors can address.Reviewer #1:Zhang et al. investigated the role of the dopaminergic projection from the ventral tegmental area (VTA) to the medial olfactory tubercle (mOT) in reward processing. Previously I raised the major concern that some of the behavior effects induced by optogenetic stimulation might be produced by collaterally activating dopaminergic fibers in the nucleus accumbens (NAc). In the revision, the authors performed several new experiments to address this and my other concerns. They now show that the NAc and the mOT receive largely separate dopaminergic inputs. In addition, optogenetically inhibiting dopaminergic activity in the mOT blocks many of the reward-like effects. These new results make the authors' conclusion much more convincing. The presentation of the manuscript has also been substantially improved.Reviewer #2:Outstanding; the authors have addressed the questions thoroughly.Reviewer #3:In the revised manuscript the authors conducted two sets of experiments, as per reviewer 1's suggestion, to address the major concern raised by all reviewers concerning the potential non-specific effect of the optogenetic and chemogenetic manipulations. In one, the authors conducted dual tracing experiments by injecting in the same animal distinct retrograde labeling viral vectors to mOT and NAc, respectively. They found that there was only a very small percentage (< 2%) of VTA neurons being double labeled . The data suggest that VTA comprises separate populations of neurons innervating mOT and NAc. This is a significant finding. The result provides a strong support for their interpretation about the observed behavioral outcome being the specific effect of manipulation of VTA-mOT pathway. With this piece of data, the major concern on the previous submission of this manuscript should be satisfactorily addressed. I would suggest including this piece of data in the main body of paper, not as a figure supplement.In another set of experiments, authors conducted real-time place preference test upon VTA administration of lidocaine and optogenetic stimulation of VTA-mOT projecting terminals . The autIn addition, In the figure legend for Figure supplement 4, "(F) The magnification of VTA labeled neurons in B." should be "(F) The magnification of VTA labeled neurons in E". Reviewer #1:[…] The authors have several choices to address this concern. First, they could examine whether dopamine neurons projecting to the NAc and the mOT belong to separate subpopulations. Second, they could test whether the reinforcing effect of optogenetic stimulation in the mOT remains intact following lidocaine administration into the VTA. Third, they could show whether CAV-Cre injection into the mOT only drives gene expression in mOT-projecting dopamine neurons from the VTA. To the minimum, if none of these experiments work out, the authors should discuss the potential caveat and tone down some of the conclusions.We deeply appreciate your pertinent concerns and constructive suggestions. We have attempted to follow your suggestions closely to revise the manuscript.To exclude the potential caveat, we have done a series of new experiments you have suggested (2 of the 3) and an extra one not suggested.1) Virus tracing experiment. We co-injected RV-dG-dsRed into the mOT and RV-dG-GFP into the NAc, respectively, and found that the co-labeled rate of the VTA neurons were negligible (1.65%), which implied that it may not be common for a VTA DAergic neuron to innervate both the NAc and the mOT . However2) We have conducted additional experiments on pharmacological inhibition of the VTA cell bodies, followed by optogenetic stimulation of the VTA-mOT DAergic projection. First, we found that the acute place preference induced by activation of the VTA-mOT DAergic projection were still retained even though the VTA cell bodies were inhibited by lidocaine , suggestReviewer #2:[...] 1) A potential major problem is that optogenetic stimulation might have resulted in direct or indirect stimulation of the nucleus accumbens (NAcc). The authors should provide evidence that the behavioral results for optogenetic stimulation of mOT are not due to direct stimulation of NAcc. After all c-Fos was increased in NAcc .We agree with this suggestion, therefore we have performed a series of new experiments to exclude this possibility, from three different aspects . The detailed information is presented above (Reply for major comments of reviewer #1).2) The viral injections targeting mOT might have resulted in infection of neurons in NAcc. The figures need to be modified to clearly show that this is not the case. Was this tested for every single experiment? This criticism holds for Thanks for the reviewer's suggestion. We have re-done the injection experiments and modified the figures. The injection site for every mouse was examined at the end of the experiments. The results are shown in Figure supplement 4 in the revised version.3) For We deeply agree with the reviewer’s suggestion. We have re-done the injecting experiments and replaced 4) The LFP is an indirect measure of activity in the mOT. Did the authors record odorant-elicited changes in single unit spikes or neuronal activity measured with photometry? Why is the power increasing before the odorant is presented?We agree with the reviewer's concern. Therefore, we have done new experiments to record odorant-elicited neuronal activity with fiber photometry. We have added this result in the revised version in 5) The panel in We agree with the reviewer. This discordant may be caused by the bad image quality. We have counted more cells, and drawn a new pie chart accordingly. We have performed more immunostaining experiments and obtained images with much improved quality. The new results have been presented in 6) The authors claim that the experiment in Figure 9 shows that inactivation of DAergic neurons decreased the learned odor preference. But the viral infection resulted in expression of mCherry in a large number of non-DA neurons. Could the effect be due to GABAergic neurons projecting from VTA to mOT? Did the authors perform this experiment with halorhodopsin?We have re-done the staining experiment with a higher staining quality and acquired the image with higher resolutions. It turned out that most of the mCherry+ neurons are also TH positive. We pulled the data together and found that about 90.86%  mCherry labeled neurons were dopaminergic. Moreover, we have performed an inactivation experiment by injecting AAV-DIO-eNpHR in DAT-cre mice, and performed an inhibition experiment with halorhodopsin and found that the S+ odor preference can’t be established after odor- food associative learning. These data has been added to the revised version .7) The statistics of the manuscript are not acceptable. Which statistical test was used for We agree with the suggestion, and have added the specific statistical tests for 8) For the experiment in Thanks for the helpful suggestion. As shown in Reviewer #3:In this manuscript the authors reported experiments aiming to address the role of the dopaminergic (DAergic) innervation of medial olfactory tubercle (mOT) from the ventral tegmental area (VTA) in the regulation of olfaction and reward. Olfactory tubercle has been implicated in both reward and olfactory information processing. However, in both the olfactory field and in the reward field, OT is a far less studied brain region compared with other olfactory regions and reward regions. The role of the VTA(DAergic)-mOT pathway in the regulation of reward and olfactory process have not been investigated in behaving animals. This work represents an important and timely study addressing fundamental questions regarding the circuit mechanisms underlying the behaviors involving olfactory processing and/or reward. With the presynaptic optogenetic activation, local administration of DA receptor antagonists in mOT, and chemogenetic silencing of mOT-projecting VTA DAergic neurons, the importance of the DAergic input from VTA to mOT in the odor-preference is reasonably well established. However, the role of this pathway in other reward processes is far less clear with the current experiments.We deeply appreciate your valuable comments and helpful suggestions. We have attempted to follow your suggestions closely to revise the manuscript. The focal point of this study was that the VTA-mOT DAergic projection plays important roles in the formation of odor-preference and place/location preference. The role of this pathway in other reward processes should be an interesting project for the future.The major issue of this study concerns the broad and non-specific effect of the optogenetic and chemogenetic manipulations. VTA DAergic neurons innervate multiple regions of the brain, and the neurons that innervate mOT likely also innervate other brain regions. Indeed, optogenetic activation of the VTA-mOT DAergic terminals was able to activate multiple brain regions . For optThanks for the reviewer's suggestion. However, the antagonist may easily diffuse into the nearby NAc region through mOT infusing, as the other reviewers have pointed out. So, we have conducted the optogenetic RTPP tests when the VTA cell bodies were inhibited by lidocaine. We found that activation of the VTA (DAergic)- mOT pathway produced place preference even though the VTA cell bodies were inhibited .In the case of the chemogenetic silencing experiments , the global inhibition of the CAV-Cre/AAV9-EF1a-floxed-hM4Di-mCherry infected VTA neurons would also abolish their function in other brain regions. Therefore, it is possible that the attenuation in the odor-cue based reward learning  may result from non-specific silencing of the projections to other brain regions. It is not clear what portion of VTA DAergic neurons are labeled by CAV-Cre/AAV9-EF1a-floxed-hM4Di-mCherry. If there were only a small portion labeled, it could be less problematic. Also, could the authors administer CNO locally at mOT to achieve local inhibition of VTA-mOT terminals?Thanks for the reviewer’s suggestion. The problem you are concerned with is similar to reviewers #1&2. We have performed a series of new experiments to exclude this possibility. The detailed information is presented above (please see Reply for major comment of reviewer #1). We have counted the infected VTA DA neurons and the whole VTA DA neurons, and found that there were only a small portion of the VTA DA neurons projecting to the mOT (37.20%), which should make the “axonal collaterals” less problematic. As for the local administration of CNO at mOT, a paper showed in Nature Neuroscience 17, 577–585 (2014) that it could be used to manipulate axonal terminals of DREADD-expressing neurons, we believe that local administration of CNO at mOT could inhibit the VTA- mOT terminals. However, CNO may also diffuse, as the same with dopamine blockers, into the nearby NAc region.[…] Reviewer #3:In the revised manuscript the authors conducted two sets of experiments, as per reviewer 1's suggestion, to address the major concern raised by all reviewers concerning the potential non-specific effect of the optogenetic and chemogenetic manipulations. In one, the authors conducted dual tracing experiments by injecting in the same animal distinct retrograde labeling viral vectors to mOT and NAc, respectively. They found that there was only a very small percentage (< 2%) of VTA neurons being double labeled . The data suggest that VTA comprises separate populations of neurons innervating mOT and NAc. This is a significant finding. The result provides a strong support for their interpretation about the observed behavioral outcome being the specific effect of manipulation of VTA-mOT pathway. With this piece of data, the major concern on the previous submission of this manuscript should be satisfactorily addressed. I would suggest including this piece of data in the main body of paper, not as a figure supplement.We deeply appreciate and agree with your positive comments. We have transferred the original Figure supplement 4C-G into the main body of the revised manuscript as new In another set of experiments, authors conducted real-time place preference test upon VTA administration of lidocaine and optogenetic stimulation of VTA-mOT projecting terminals . The autWe deeply appreciate your constructive suggestions, and have deleted this piece of data and text accordingly in the revised version.In addition, We agree with this helpful suggestion. We have combined the original In the figure legend for Figure supplement 4, "(F) The magnification of VTA labeled neurons in B." should be "(F) The magnification of VTA labeled neurons in E".Thank you so much for your careful reading, we have revised it in the revised version.


Background: The modulation efficacy of Transcranial magnetic stimulation (TMS) on consciousness improvement of patient with disorder of consciousness (DOC) has not been definitely confirmed.Objective: This study proposes TMS-EEG to assess effects of repetitive TMS (rTMS) on brain modulation of DOC.Methods: Twenty sessions of 10 Hz rTMS were applied over the dorsolateral prefrontal cortex for a patient with DOC. Measures of Coma Recovery Scale-Revised (CRS-R) score, TMS-evoked potential (TEP), perturbation complexity index (PCI), and global mean field power (GMFP) were used to evaluate the consciousness level of the patient at three intervals: before the rTMS protocol (T0), immediately after one session rTMS (T1), and immediately after 20 sessions (T2).Results: It was found that the patient was diagnosed of a minimally conscious state minus (MCS-) by means of CRS-R at the interval of T0, however the TEP and PCI indicated the patient was vegetative state (VS). At the interval of T1, there was not any clinical behavioral improvement in CRS-R, but we could find significant changes in TEP, PCI, and GMFP. At the interval of T2 there was a significant increase of consciousness level according by CRS-R score, PCI value, TEP, and GMFP after 20 sessions of 10 Hz rTMS on the patient with DOC.Conclusions: We demonstrated that TMS-EEG might be an efficient assessment tool for evaluating rTMS protocol therapeutic efficiency in DOC. Although, some studies have attempted to demonstrate some pharmacologic or nonpharmacologic effects, until now there were no evidence-based guidelines regarding the treatment of patients with DOC  between the patient with DOC and controls, five age matched female healthy subjects age 43–50 participated to this study to obtain the mean control TEP. Written informed consent to subjects in the study was obtained. The present study was approved by ethics committee of Beijing Army General Hospital.R2 stimulator with a 70 mm figure-of-eight coil . Stimulation intensity varied across this experiment was determined relative to the resting motor threshold (RMT), defined as the lowest TMS intensity which could evoke at least 5 out of 10 EMG with an amplitude >50 μV peak-to-peak in the relaxed first dorsal interosseous muscle of the right hand. To avoid contamination of TMS-evoked potential by auditory potentials evoked by the click associated with the TMS discharge, patient was wearing inserted earplugs continuously playing a masking noise.The process of the intervention and evaluation could be found in Figure The CRS-R is a tool to characterize the level of consciousness and to monitor neuro-behavioral recovery in patients with DOC , EEG was continuously acquired from 62 channels with positions of the international 10–10 system. The equipment used TMS-compatible sintered Ag/AgCl-pin electrodes. We set a band-pass filtered at DC to 1000 Hz in the recorder, and the EEG signal was digitized at a sampling rate of 2.5 kHz. During the experiment, the skin/electrode impedance was maintained below 5 kΩ. EEG was recorded in day 1 and day 20. As shown in Figure Off-line analysis was performed with EEGLAB 12.0.2.5b, running in a MATLAB environment . The continuous EEG signal was segmented into epochs starting the TMS pulse onset and ending 300 ms ] which describes the spatiotemporal pattern of activation caused by the TMS perturbation. At last, the Lempel-Ziv complexity index was used to compress the matrix. The PCI index was calculated as the normalized Lempel-Ziv complexity. The PCI is expected to be low if there is reduced interaction among cortical areas and will be high if interaction of the cortical areas increased. As suggested , was applied to evaluate the consciousness level of the patient. The PCI was proposed before  was calculated with the multichannel average signals as follows , Beijing Municipal Science & Technology Commission (No. Z141107002514111), Beijing Municipal Commission of Education and Innovation Cultivation Fund of the PLA Army General Hospital (No. 2015-LC-09).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both groups were trained 5 days per week for 3 weeks. The primary outcome measure was Fugl-Meyer Assessment-Upper Extremity (FMA-UE) motor score, and the secondary outcomes were Wolf Motor Function Test-Functional Ability Scale (WMFT-FAS) and Barthel index (BI). The AA group, in comparison to the Control group, showed significantly greater increases in FMA-UE score  and WMFT-FAS score  after 3 weeks of treatment, whereas the increase in BI was not significant . There were no adverse events. We conclude that arm training using the AA robotic device is safe and able to reduce motor deficits more effectively than matched conventional arm training in subacute phase of stroke. The study has been registered at the ClinicalTrials.gov, ID: NCT02729649.The ArmAssist is a simple low-cost robotic system for upper limb motor training that combines known benefits of repetitive task-oriented training, greater intensity of practice, and less dependence on therapist assistance. The aim of this preliminary study was to compare the efficacy of ArmAssist (AA) robotic training against matched conventional arm training in subacute stroke subjects with moderate-to-severe upper limb impairment. Twenty-six subjects were enrolled within 3 months of stroke and randomly assigned to the AA group or Control group ( Stroke is the leading cause of long-term disability in the industrialized world . Up to 8The main principle of contemporary rehabilitation is a task-specific practice with a large number of repetitions, which is a potent stimulus for promoting motor learning , 6. StudVarious robot-assisted systems have been developed for enhancing arm motor recovery, including unilateral, bilateral, proximal arm and distal arm training devices. Robots encourage high repetition of movements with minimal supervision in a highly motivating environment. Robotic devices should provide therapy relevant to users and conform to the principles of conventional therapy. They should be easy to set up, appeal to users, require minimal supervision from therapists, and ideally be able to assess motor performance and therapy outcomes , 8. A reA few simple and low-cost robotic devices have been evaluated in stroke patients with moderate arm weakness. Improvements in kinematic and clinical outcomes were reported in moderately disabled chronic stroke patients who participated in a telerehabilitation program using a simple robotic device . The stuArmAssist (AA) is a low-cost robotic system developed for shoulder and elbow rehabilitation after stroke . It is a modular system that combines a portable device for providing arm support over a table with interactive games operating on a web-based platform . The AA The aim of this study was to determine preliminary efficacy of the AA robotic device in comparison to the matched conventional arm training in subacute stroke patients undergoing rehabilitation. The primary outcome measure was Fugl-Meyer Assessment-Upper Extremity (FMA-UE) motor score, and the secondary outcomes were Wolf Motor Function Test-Functional Ability Scale (WMFT-FAS) and Barthel index (BI).NCT02729649) prior to participant enrollment. All participants signed the informed consent form and all research procedures were performed in accordance with the Declaration of Helsinki.We conducted a single-blind , two-arm parallel, randomized controlled trial in subacute stroke patients with equal allocation to the two groups. The study was approved by the Ethics Committee of the Clinic for Rehabilitation “Dr. Miroslav Zotović” affiliated with the Faculty of Medicine, University of Belgrade. The study was registered at the ClinicalTrials.gov .n = 13) or the Control group (n = 13).Twenty-six hemiparetic subacute stroke subjects were recruited for this study between January 2015 and March 2016 from the pool of waitlisted patients scheduled for inpatient rehabilitation. During the screening process, all potential participants were interviewed and examined by the study investigator with more than 5 years of clinical experience in neurological rehabilitation. The inclusion criteria were (a) unilateral paresis as a result of first ischemic or hemorrhagic stroke confirmed by computed tomography or MRI that occurred less than 3 months before enrollment, (b) the ability to understand and follow simple instructions, and (c) the ability to perform some active movements in the shoulder and/or elbow joints in the sitting position, allowing for trunk compensation if needed. The exclusion criteria were (a) multiple strokes, (b) bilateral impairment, (c) severe sensory deficits in the paretic upper limb, (d) the inability to provide informed consent, and (e) medical conditions that could interfere with treatment . Baseline FMA in the participants with severe motor impairments was ≤25 and in those with moderate impairments 26 to 50 out of 66 . The parR Studio software v. 0.98.976 , SPSS 17.0 , and G ∗Power 3.1. The minimal clinically important difference for FMA-UE was estimated previously at 10% of the maximum score (6.6 points) [The sample size calculations were performed using  points)  and adopThe AA group received a conventional rehabilitation with an additional 30 minutes of the AA training. The Control group received the same conventional rehabilitation and an additional 30 minutes of occupational therapy that was matched in its structure and amount to the AA training as close as possible. The AA or matched arm training was administered over 15 sessions each lasting 30 minutes, scheduled 5 days per week (Monday–Friday) for 3 weeks, at least 24 hours apart, and at about the same time of day. Both types of arm training were provided by a physiotherapist with a graduate degree and vast experience in neurological rehabilitation, who was not involved in the assessment. The arm training was carried out in the same therapy room and separated from other treatments by at least a 30-minute break. To assess adverse events, the subjects were asked at the end of each session to report any new symptoms.The conventional rehabilitation was provided 5 days per week for 3 weeks divided into two 30-minute sessions of occupational therapy and physiotherapy. Occupational therapy for the paretic upper limb included passive stretching within submaximal ranges of motion to inhibit spasticity, active-assisted movements, functional tasks, and activities of daily living, all progressed individually. Physiotherapy consisted of range of motion exercises for upper and lower extremities, gentle stretching, splinting/casting, facilitation of active voluntary movement, and exercises to improve endurance, balance, strength, and gait. If necessary, speech therapy was provided three to five times per week.The AA training consists of self-directed active movements through interactive gaming with focus on reaching. The movements required for playing the games are horizontal abduction-adduction in the shoulder and flexion-extension in the elbow with the hand in neutral position. The number of reaching movements in each game ranges from 2 to 8 per minute, depending on the cognitive load of the game. In this study, the subjects performed, on average, 4–5 repetitions per minute or about 120–150 movements per session.The subjects were seated in a chair with a back support and two adjustable shoulder straps to prevent compensatory trunk movements. The straps were adjusted if necessary to allow limited movement of the trunk for comfort. The subject was positioned in the semicircular opening of the table . The chaThis training consisted of exercises mirroring the structure and amount of AA training as close as possible . The setThe primary outcome measure was FMA-UE motor score, which assesses the degree of synergistic movements in the paretic upper limb. Individual items pertaining to the shoulder/elbow and hand segments are scored on a 3-point ordinal scale and summed for a maximum possible score of 66 , 16.The secondary outcomes were WMFT and BI. The WMFT is an activity-based test that evaluates upper extremity performance via timed and functional tasks. Each item is rated on a 6-point Functional Ability Scale (FAS) and summed into total WMFT-FAS score. We used a 17-item WMFT, consisting of 15 function-based tasks and two strength tasks, each scored from 0 to 5 for a maximum score of 75 points . The BI All outcomes were evaluated at baseline and after 3 weeks of intervention by a physiotherapist experienced in neurological rehabilitation, who was blinded to the group allocation . t-test (continuous data) and Chi-square or Fisher's test . The main outcome measures did not deviate significantly from normality . For the main analysis, the before-after differences were calculated and compared between the two groups using the independent sample t-test. The effect size was calculated using Cohen's d coefficient  to infer the magnitude of changes. The level of statistical significance was set at p < 0.05 (two-tailed).Data were analysed using SPSS v17.0  and R-studio (2014) v0.98.976 , following an intention-to-treat analysis using the last forward method. For descriptive purposes, the demographics and baseline outcome measures were compared between the two groups using the independent sampleSeventy-three consecutive patients from the waitlist were screened of whom 26 met the eligibility criteria . None of the 26 recruited subjects dropped out. There were no significant differences in baseline demographic and clinical parameters between the two groups Tables  and 2. Np = 0.002) and FMA-UE shoulder/elbow score (p = 0.006) in the AA group compared to the Control group. The improvements in WFMT-FAS (p = 0.025) and shoulder/elbow portion of WFMT (p = 0.010) were also significantly greater in the AA group. All effect sizes were large.The changes in primary and secondary outcomes at the end of treatment are shown in In this study, we compared the efficacy of the AA robotic arm training against the matched conventional arm training in subacute stroke patients with moderate-to-severe arm motor impairment. The results showed that 15 sessions of the AA training resulted in comparably greater reduction in the upper limb motor impairment. The increases in FMA-UE score in both the AA group (18 points) and the conventional group (9 points) were greater than the adopted 6.6-point minimal clinically important difference, suggesting that these gains may be considered meaningful. Moreover, in contrast to most of the previous studies that examined outcomes after robot-assisted arm training , our resNumerous studies have shown benefits of various robotic devices for improving function of the paretic arm after stroke. Some earlier studies of the robot-assisted arm therapy included chronic and mildly disabled stroke patients , 21, 22,Our findings are in agreement with the results of previous studies examining effects of low-cost robotic therapy during the subacute phase of recovery from stroke. For example, significant improvements in the upper limb motor performance, as measured by FMA and the Motricity Index, were reported after 2 weeks of robot-assisted treatment in 25 subacute stroke patients . SimilarIn contrast to the FMA-UE and WMFT, we found no significant differences in BI between the two groups. This may be due to a short duration of treatment since BI reflects global physical abilities, which depend on restoration of many other functions and associated comorbidities . No signThe improvements after AA training may be due to greater engagement and cognitive demands required for successfully completing virtual gaming tasks. Also, the AA group was required to convert motor action into visual spatial coordinates through interactions with the computer screen. Increased cognitive demands were found to modulate activity in multiple brain motor networks and may enhance motor learning .This study has several limitations consistent with its preliminary nature. We did not consider other factors that could have influenced the outcomes, such as type and location of stroke, motivation, and cognitive abilities. Also, we were able to report an approximate number of repetitions only, whereas the two training protocols were closely matched on several other characteristics of executed movements, such as the type (reaching), plane of action , goal (aiming for targets), and constraints (self-directed). No reports of even mild adverse events  with the AA training may reflect an interviewer or response bias. Another limitation is a lack of follow-up to determine durability of effects. Contextual factors such as patient and caregiver impressions should be evaluated in the future. Finally, kinematic analyses are necessary to distinguish improved performance due to arm motor recovery from compensatory movements.Arm training using the AA robotic device reduced impairment and activity-related motor deficits more effectively than matched conventional arm training in subacute phase of recovery from stroke. There were no (serious) adverse events. These preliminary results support further research aimed at identifying most suitable candidates and most optimal timing of the AA robotic training as well as feasibility and potential benefits of administration in home settings.


Resin cements have led to great advances in dental ceramic restoration techniques because of their ability to bond to both dental structures and restorative materials.  The aim of this study was to assess the performance of resin cements when different curing modes are used, by evaluating the degree of conversion and bond strength to a ceramic substrate. Three resin cements were evaluated, two dual-cured (Variolink II and RelyX ARC) and one light-cured (Variolink Veneer). The dual-cured resin cements were tested by using the dual activation mode  and light-activation mode (base paste only). For degree of conversion (DC) (n=5), a 1.0 mm thick feldspathic ceramic disc was placed over the resin cement specimens and the set was light activated with a QTH unit. After 24 h storage, the DC was measured with Fourier transform infrared spectroscopy (FTIR). For microshear bond strength testing, five feldspathic ceramic discs were submitted to surface treatment, and three cylindrical resin cement specimens were bonded to each ceramic surface according to the experimental groups. After 24 h, microshear bond testing was performed at 0.5 mm/min crosshead speed until the failure. Data were submitted to one-way ANOVA followed by Tukey test (p<0.05). Scanning electron microscopy (SEM) was used for classifying the failure modes. Higher DC and bond strength values were shown by the resin cements cured by using the dual activation mode. The Variolink II group presented higher DC and bond strength values when using light-activation only when compared with the Variolink Veneer group. The base paste of dual-cured resin cements in light-activation mode can be used for bonding translucent ceramic restorations of up to or less than 1.0 mm thick. Conservative ceramic restorations for changing the position, shape, or color of anterior teeth have been widely used in modern dental practice1. The success of ceramic veneers has been attributed to the establishment of a durable bond among tooth tissues, luting composite and ceramic substrate18.The ability of ceramics to match natural dentition, due to their good physical and optical properties, makes them the material of choice for patients with high esthetic expectations9. Commercially available resin cements for luting ceramic veneers can be light activated or dual-cured, depending on the opacity and thickness of the ceramic restoration28. Light-cured resin cements allow extended working time and removal of the excess around the restoration before light-activation, reducing the time needed for finishing after restorations have been luted20. In addition to these features, light-cured resin cements have the great advantage of improved color stability, since no tertiary amines are used as chemical activator, which could cause color change over the time28.Resin-based cements are the materials most commonly used for luting dental ceramic restorations, mainly because of their improved physicochemical properties. In general, these materials are composed of a polymeric matrix based on dimethacrylate monomers, filler particles and pigments1. These systems are composed of a catalyst paste consisting of benzoyl peroxide (initiator) and base paste, containing a light-cured resin cement, in an endeavor to increase free radical concentration even under thick or opaque restorations28. The free radicals formed by the chemical reaction (tertiary amines/benzyl peroxide) would compensate for the lack of those that result from the physical initiation system activated by light 11. However, dual-cured resin cements may have reduced color stability, due to the possibility of some degree of oxidation occurring in the components involved in chemical curing (tertiary amines)16.Dual-cured resin cements were introduced to combine the advantages of light- and chemically-cured resin cements and are the most common luting materials used nowadays29. Nonetheless, some dual-cured resin cement manufacturers offer a light-cured option for their systems in the absence of a light-cured resin cement, and indicate that dental practitioners should use the base paste only in a light-activation mode. However, there is still no evidence that the light-cured components of dual-cured resin cements have the ability to reach good physical and mechanical properties when used alone.Therefore, light-cured resin cements may be a more suitable option when luting thin translucent ceramic veneers11. As seen, inadequate curing with a reduced degree of conversion may cause changes in the physical characteristics of resin-based components, affecting their mechanical properties, altering dimensional stability, decreasing bonding to tooth structures, capable of resulting in the unsatisfactory clinical performance of these materials25.In addition to formulation, the success of ceramic restorations depends on optimal cure of resin cements. The degree of conversion is crucial in determining the mechanical performance of resin-based materials and their biocompatibility. The strength, elastic modulus, hardness and solubility of composite resins are directly related to the degree of conversionTherefore, the aim of this study was to assess the performance of resin cements when using different curing modes, by evaluating the degree of conversion and bond strength to a ceramic substrate. The hypothesis was that dual-cured resin cements used in the dual activation mode with base and catalyst pastes, would behave differently when using these cements in the light-activation mode with base paste only. A resin cement with a light-curing option only was also used for the comparisons.For this study, three resin cements were evaluated, two dual-cured , and one light-cured . Dual-cured resin cements were tested in the dual activation mode by mixing the base and catalyst pastes (BC), or in the light-activation mode, by using the base paste only (B). The light-cured resin cement was tested only in the light-activation mode. Thus, five experimental groups were defined.Twenty-six feldspathic ceramic discs  were obtained, one of which was used to perform the degree of conversion test and 25 were used for microshear bond strength testing. The materials information is summarized in 4. The ceramic disc and the tip of the light-curing unit were positioned over the resin cement specimens with the help of a Teflon ring. The diameter of the light wand tip was exactly the same diameter as that of the disc/sample. For the dual activation mode, the base and catalyst pastes of dual-cured resin cements were mixed according to the manufacturer’s directions and then three minutes were waited for the self-cure reaction to occur. After this, all the groups were light-cured through the ceramic discs using a quartz-tungsten halogen (QTH) light-curing unit  at 800 mW/cm2 output for 40 s , resin cements were put into vinyl molds (1 mm thick x 2 mm inner diameter x 10 mm outer diameter). A Teflon mold was placed around the vinyl molds. Mylar strips  were used on bottom and top surfaces of the mold to ensure smoothness of the specimens, allow the ceramic disc to be placed over the resin cement and to avoid inhibition of polymerization by oxygenfor 40 s . All spe(C=C)Ab(C=O) / Ab(C=C)Aa(C=O))]% DC = 100 [1 -  with distilled water for 5 min. After drying the specimens, a layer of silane coupling agent  was applied and left to react for 1 min.Twenty-five ceramic discs were embedded in PVC cylinders using polyester resin . After divesting, the ceramic disc surfaces were sequentially polished using silicon-carbide papers , mounted on a water-cooled polisher . The discs were submitted to surface treatment with 10% hydrofluoric acid  for 2 min24. The specimens were light-cured by using the QTH unit at 800 mW/cm2 output for 40 s, with the light source placed at the top of the specimens, and within a Teflon ring, thus light-curing all the resin cylinders simultaneously  were placed in parallel on each ceramic disc and filled with resin cement. For the dual activation mode, the cement was light-activated three minutes after it was insertedaneously . All speMicroshear testing (n=5) was performed in a mechanical testing machine , in which each ceramic disc was attached to a flat base by means of cyanoacrylate-based glue . A stainless steel wire (0.2 mm in diameter)  was placed around the resin cement cylinder at its base, as close as possible to the ceramic/resin cement interface. The test was performed at a crosshead speed of 0.7 mm/min until failure of the specimens.The failure mode of the specimens was analyzed by scanning electron microscopy (SEM)  at 200x magnification, at an acceleration voltage of 25 kV. The failure modes were classified as: I- adhesive failure; II- cohesive failure at resin cement; III- cohesive failure at ceramic substrate; IV- mixed failure with predominance of resin cement; V- mixed failure with predominance of ceramic substrate. Adhesive failure was characterized as a fault at the junction between the ceramic and the resin cement; cohesive failure when there was a fracture in the body of the cement or the ceramic; and mixed failure when there was cohesion failure in both materials, simultaneously.post hoc tests were performed to determine differences among groups. Statistical significance was set at α=0.05. Descriptive analysis was used for failure mode.Degree of conversion and microshear bond strength data were individually analyzed by one-way analysis of variance (ANOVA). Tukey The mean degree of conversion (%) and standard deviation obtained for the experimental groups are shown in The mean bond strength values and standard deviation are shown in The failure mode analysis shown in Figures 4 and 5 showed prevalence of cohesive failures in the ceramic substrate (Type III) for the resin cements used in the dual activation mode as well as for the Variolink Veneer light-cured cement. A higher percentage of cohesive failures in resin cement (Type II) or mixed failures with predominance of resin cement (Type IV) were observed for RelyX ARC when using the light-activation mode. No dominant failure mode was verified for the Variolink II in the light-activation mode.10.The hypothesis tested was accepted. The dual-cured resin cements showed a higher degree of conversion (DC) and at least similar bond strength values when used in the dual activation mode  when compared with the resin cements used in the light-activation mode (base paste only). The base paste of both the dual-cured resin cements presented higher DC and bond strength values than the light-cured resin cement tested. Moreover, it is important to note that the base paste of Variolink II showed higher bond strength values than the base paste of RelyX ARC; and to consider that not all resin cements are equal in formulation and may present different curing properties14. Thus, when the manufacturer informs that a cement is dual-cured, it is imperative to respect the instruction to perform the double polymerization process, in order to achieve higher DC values and improved properties in daily practice.When comparing the resin cements using only the base paste in the light-activation mode, there were no significant differences between the DC of RelyX ARC and Variolink II. However, the DC of both resin cements was reduced in the light-activation compared with the dual activation mode. Therefore, the importance of a chemical activation in addition to light curing in dual-cured resin cements must be emphasized. Previous studies have shown the importance of dual-curing resin cements for clinical situations in which the light from the curing unit is not capable of fully reaching all regions of the cavity or preparation, such as in the apical region of root canals during adhesive post luting or the deep internal areas of preparations for indirect adhesive restorations21. The inadequate polymerization of resin cements, represented by a low DC, may result in increased dissolution of some components in the oral cavity, which may cause pulp sensitivity, in addition to a faster marginal degradation process and early loss of the indirect restoration22. A high DC ensures the best chemical and physical properties of resin cements26.Composite-based restorative materials are constantly submitted to agents present in saliva, food and drinks, which may help to degrade the organic matrix25. Thus, for the DC methodology of this study, a feldspathic ceramic disc was interposed between the resin cement and the light source during curing of the specimens, seeking to emulate what happens in the oral cavity during clinical practice. In this study, it was shown that for cases in which a light-curing resin cement is required, the base pastes of the dual-cured resin cements evaluated used in the light-activation mode showed suitable properties that may allow their use for luting thin ceramic restorations.Variables such as color, thickness and type of ceramic for indirect restorations may interfere in the amount of light reaching the resin-based luting material, influencing the DC19.Variolink II activated in both dual and light-activation modes showed the highest bond strength values, followed by RelyX ARC in the dual activation mode. In turn, when used in the light-activation mode with the base paste only, RelyX ARC presented values close to the lowest bond strength values, but these were statistically similar to those verified for the light-cured resin cement Variolink Veneer. Despite presenting a suitable bond strength to dental substrates, it is also important for resin cements to offer adequate bond strength to indirect restorations. Bonding to ceramic restorations follows similar principles as those used for bonding to dental tissues. The adhesive or resin cement infiltrates into the roughness created on the surface of these materials, thus enabling micro-mechanical retention7. Additionally, to assure interaction between the ceramic and resin cement, the application of a coupling agent based on methacryloxypropyltrimethoxysilane allows the formation of covalent bonds between the silica from glass ceramics and the organic matrix from resin cements8. The silane coupling agent is also capable to increase wettability and surface energy on the ceramic surface23.For this purpose, acid-sensitive ceramics are submitted to surface etching with hydrofluoric acid to increase the surface roughness, improve wettability, surface free energy, and to expose a silicon oxide networkRegarding the failure mode, dual-activated resin cements (VL-BC and RX-BC) and light-cured resin cement (VL-V) predominantly presented cohesive failures in the ceramic surface of the specimens, characterized as a body failure of the restorative material. This probably occurred because a conventional feldspathic ceramic without any reinforcement was used as the substrate material for bonding tests and also due to the higher bond strengths achieved in these specimens. RX-B specimens presented more cohesive failures involving the resin cement, with body failures in the luting agent. VL-B exhibited heterogeneous failure modes that were not conclusive. No classical adhesive failures were observed for the experimental groups. Therefore, despite the mechanical properties evaluated in this study showing that there were no significant differences between light-cured resin cement and dual-cured resin cements used in the light-activation mode, no favorable failure mode was observed for RX-B and VL-B. Thus, more studies are necessary to evaluate other properties that justify their use in clinical practice.Although further studies are still needed, the use of dual-cured resin cements used only in the light-activation mode may be an alternative for luting translucent ceramic restorations up to or less than 1.0 mm thick. The degree of conversion and bond strength showed similar behavior between base paste of dual-cured materials and light-cured resin cement. Furthermore, it is important to understand the chemical and mechanical properties of resin cements and the strategies capable of improving these properties, to ensure the success of restorative procedure.